MONOGRAPH ON HUMAN PAPILLOMAVIRUSES

denatured VLPs or with VLPs from animal papillomaviruses. HPV 16 antibodies were slow to develop, with a median latency of 6–12 months and titres were...

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HUMAN PAPILLOMAVIRUSES 1.

Human Papillomavirus (HPV) Infection

1.1

Evolution, structure and molecular biology

1.1.1

Introduction

Papillomaviruses are small, non-enveloped, epitheliotropic, double-stranded DNA viruses that infect mucosal and cutaneous epithelia in a wide variety of higher vertebrates in a species-specific manner and induce cellular proliferation. Only bovine papillomaviruses (BPVs) 1 and 2 are known to infect mesenchymal tissues and to show crossspecies transmission. More than 100 types of human papillomaviruses (HPVs) have been identified and approximately half of them infect the genital tract. Many types of HPV have been found in cervical cancers, while others are found rarely or not at all in large series of cancers, which gives rise to the nomenclature of ‘high-’ and ‘low-risk’ HPVs. These other types are associated with other anogenital and oropharyngeal cancers. A number of HPVs have been found to be present in skin cancers in patients who have epidermodysplasia verruciformis (EV); these types are also found in both non-melanoma skin cancers and normal skin. The potential associations of HPVs with these and other cancers are discussed in other sections. All papillomaviruses share a common genetic structure that is distinct from that of polyomaviruses. A double-stranded circular DNA genome encodes approximately eight open-reading frames (ORFs). Similarly, all papillomaviruses have a non-enveloped icosahedral capsid. Understanding of the biology of papillomavirus infection was hindered by the lack of tissue culture systems to propagate the viruses, the lack of animal models for HPVs and difficulties in finding animal models of natural infection. The advent of molecular cloning of HPV genomes in the early 1980s provided the first opportunity to study individual viral genes. However, only in the late 1990s did propagation of viruses in organotypic cultures make the first attempts at viral genetics possible. The availability of complete and partial genomic sequences from a wide variety of HPV types has enabled the establishment of a new taxonomic structure and has provided a window to study the co-evolution of papillomaviruses with their primate hosts. Early evidence suggests that HPV types, as defined by DNA sequencing, also remain serologically distinct. –47–

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Molecular studies now provide a coherent picture of the mechanisms that regulate viral gene expression and replication; nevertheless, gaps in the understanding of HPV biology remain. Striking progress has been made in defining the activities of viral oncoproteins from high-risk genital HPVs, in particular HPVs 16 and 18, that promote the disruption of normal cell-cycle control. The ability to target the retinoblastoma (Rb) family of proteins and p53 and to induce telomerase are some of the critical events that contribute to the development of malignancy. 1.1.2

Structure of the viruses (a)

Viral components and physical properties

Papillomaviruses are small, non-enveloped, icosahedral DNA viruses that have a diameter of 52–55 nm. The viral particles consist of a single double-stranded DNA molecule of about 8000 base-pairs (bp) that is bound to cellular histones and contained in a protein capsid composed of 72 pentameric capsomers. The capsid contains two structural proteins — late (L)1 (55 kDa in size; 80% of total viral protein) and L2 (70 kDa) — which are both virally encoded. Virus-like particles (VLPs) can be produced by the expression of L1, alone or in combination with L2, in mammalian or non-mammalian expression systems. The intact virion has a density of 1.34 g/mL in cesium chloride and a sedimentation coefficient (S20, W) of 300 (Kirnbauer et al., 1992; Hagensee et al., 1993a). (b)

HPV genome, proteins and life cycle

The genomes of all HPV types contain approximately eight ORFs that are all transcribed from a single DNA strand. The ORF can be divided into three functional parts: the early (E) region that encodes proteins (E1–E7) necessary for viral replication; the late (L) region that encodes the structural proteins (L1–L2) that are required for virion assembly; and a largely non-coding part that is referred to as the long control region (LCR), which contains cis elements that are necessary for the replication and transcription of viral DNA. The viral E proteins are transcribed from the early promoter (e.g. P97 in HPV 31) whereas the L proteins are transcribed principally from the late promoter (P742 in HPV 31) (see Figure 1) (Fehrmann & Laimins, 2003). The E1 and E2 proteins of HPV act as factors that recognize the origin of replication; E2 protein is also the main regulator of viral gene transcription. E4, despite its name, is believed to be involved in the late stages of the life cycle of the virus and E5 may function during both early and late phases. The E6 and E7 proteins target a number of negative regulators of the cell cycle, primarily p105Rb and p53, respectively. During the viral life cycle, E6 and E7 facilitate stable maintenance of viral episomes and stimulate differentiating cells to re-enter the S phase. The L1 and L2 proteins assemble in capsomers, which form icosahedral capsids around the viral genome during the generation of progeny virions (Fehrmann & Laimins, 2003).

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Figure 1. The genome of the high-risk HPV 31

Modified from Fehrmann & Laimins (2003) The diagram indicates the ORFs of the early (E) and late (L) genes, the long control region (LCR), the two major promoters that drive viral expression (P97 and P742) and the two polyadenylation sites (AE4140 and AL7227).

Papillomaviruses are highly epitheliotropic; specifically, they establish productive infections only within stratified epithelia of the skin, the anogenital tract and the oral cavity. The viral life cycle is linked to the differentiation of the infected epithelial cell (see Figures 2 and 3). The life cycle is thought to be initiated by the infection of basal epithelial cells, presumably at sites of injury. Although several potential receptors have been reported, it is unclear which of them is of physiological importance (see Section 1.1.5(g)). Basal cells comprise the proliferating cellular component of stratified epithelia, in which the viral genome is established when a low copy number, nuclear plasmid and early genes are expressed preferentially although at low levels (Stoler & Broker, 1986; Schneider et al., 1987; Frattini et al., 1996; Oguchi et al., 2000). The ability of HPVs to establish their genome in basal cells relies upon the E1 (Hubert & Laimins, 2002), E2 (Stubenrauch et al., 1998), E6 (Thomas et al., 1999) and in some cases E7 (Thomas et al., 1999; Flores et al., 2000) genes. Normally, when basal cells undergo cell division, the daughter cell that loses contact with the basement membrane and migrates into the suprabasal compartment withdraws from the cell cycle and initiates a programme of terminal differentiation. However, in HPV-positive human keratinocytes and cervical epithelial cells, the suprabasal cells fail to withdraw from the cell cycle and continue to support DNA synthesis and express markers for cell proliferation (Jeon et al., 1995; Flores et al., 1999). HPV 16 E7 has been shown to be necessary and sufficient to induce suprabasal DNA synthesis (Flores et al., 2000). In addition, the E5 oncoprotein contributes quantitatively

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Figure 2. Schematic representation of abnormal epithelial differentiation induced by HPV infection

Modified from Fehrmann & Laimins (2003) Normal and HPV-infected epithelia are compared, and differentiation-dependent viral functions are listed.

to this property both in HPV 16 (Genther et al., 2003) and HPV 31 (Fehrmann et al., 2003). Within this suprabasal compartment, cells support the amplification of the viral genome, expression of capsid genes and assembly of progeny virus (Peh et al., 2002). The cottontail rabbit papillomavirus (CRPV) E4 gene, which is detected preferentially in the differentiated compartment of infected tissue, is required for viral DNA amplification and expression of the L1 capsid gene (Peh et al., 2004). Encapsidation of HPV DNA within capsids to generate progeny virus within the terminally differentiated cell compartment is quantitatively dependent upon L2, the minor capsid protein (Holmgren et al., 2005). L2 is also required for the infectivity of HPV 16 (Yang, R. et al., 2003a) and HPV 31 (Holmgren et al., 2005) virions. L2 may play a role in the cell-surface binding of HPV 16 virions (Yang, R. et al., 2003a), intracellular transport of the HPV 33 virion (Florin et al., 2002a) and localization of viral DNA within the nucleus (Day et al., 2004). In the context of HPV-associated cervical cancer, the viral life cycle is perturbed in two fundamental ways. First, the progressive histopathological changes that arise in the cervical epithelium include the loss of terminal differentiation. This inhibition of the differentiation process leads to a cellular state that cannot support the full viral life cycle. Second, the circular viral DNA genome, which normally resides as a nuclear plasmid, often becomes integrated into the host genome and thereby becomes disrupted and its replication defective. Whether any property of the virus drives this integration event or whether it reflects random recombination events remains unclear; however, two consequences of integration can be the selective up-regulation of the viral oncogenes E6 and E7 and a selective growth advantage over cells that harbour the viral genome as a nuclear plasmid (Jeon & Lambert, 1995; Jeon et al., 1995). Integration events that are found in

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Figure 3. Replication cycle of a papillomavirus

Modified from Howley & Lowy (2001) To establish a wart or papilloma, the virus must infect a basal epithelial cell. Knowledge of the initial steps in the replication cycle such as attachment (1), uptake (2), endocytosis (3) and transport to the nucleus and uncoating of the viral DNA (4) is limited. E-region transcription (5), translation of the E proteins (6) and steadystate viral DNA replication (7) all occur in the basal cell and in the infected suprabasal epithelial cell. Events in the viral life cycle that lead to the production of virion particles occur in the differentiated keratinocyte: vegetative viral DNA replication (8), transcription of the L region (9), production of the capsid proteins L1 and L2 (10), assembly of the virion particles (11), nuclear breakdown (12) and release of virus (13).

cervical cancer lead to the selective expression of E6 and E7 (Schwarz et al., 1985; Yee et al., 1985), which is a hallmark of cervical cancers. Whether viral integration alters cellular gene expression in any biologically relevant manner remains unclear. In a recent review, more than 190 reported integration loci were analysed with respect to changes in the viral structure and the targeted genomic locus. The results confirmed that HPV integration sites are randomly distributed over the whole genome with a clear predilection for fragile sites. There was no evidence for targeted disruption or functional alteration of critical cellular genes by the integrated viral sequences (Wentzensen et al., 2004). A more complete assessment of the role of HPV integration in carcinogenesis is provided in Section 4.1.4.

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Classification of papillomaviruses

Papillomavirus isolates are traditionally described as ‘types’, and types have been detected in all carefully examined mammals and birds, with the possible exception of laboratory mice. In the only host that has been studied extensively — humans — more than 100 HPV types have been described based on the isolation of complete genomes; a yet larger number is presumed to exist based on the detection of subgenomic amplicons. Many of these HPV types have been shown to be ubiquitous and distributed globally. Over the last 30 years, the taxonomy of papillomaviruses, which was initially based on genomic cross-hybridizations and restriction patterns, has been changed to a system based on phylogenetic algorithms that compares either whole viral genome sequences or subgenomic segments. This scientific progress has led to a refinement but never to contradictions of previous taxonomies. There is also strong evidence that papillomavirus genomes are very static, and sequence changes by mutation or recombination are very rare events. Mutational changes apparently occur at frequencies that do not differ greatly from those of the DNA genomes of the infected host organism. Papillomaviruses had originally been grouped together with polyomaviruses in one family, the Papovaviridae. This was based on similar, non-enveloped capsids and the common circular double-stranded DNA genomes. Because it was later recognized that the two groups of viruses have different genome sizes, completely different genome organizations and no similarities in major nucleotide or amino acid sequences, they are now officially recognized by the International Committee on the Taxonomy of Viruses (ICTV) as two separate families — Papillomaviridae and Polyomaviridae. A modified taxonomy and nomenclature has recently been proposed (de Villiers et al., 2004a). The L1 ORF is the most conserved region within the genome and has therefore been used for the identification of new papillomavirus types over the past 15 years. A new papillomavirus isolate is recognized if the complete genome has been cloned and the DNA sequence of the L1 ORF differs by more than 10% from the closest known type. Differences in homology of between 2% and 10% define a subtype and those of less than 2% define a variant. A few hundred putative new papillomavirus types have been identified since the advent of the polymerase chain reaction (PCR) and application of degenerate or consensus primers. Amplification of conserved regions, mostly within the L1 ORF, has been used. These partial fragments are usually labelled by using the initials of an individual or laboratory, followed by a laboratory number (see, e.g., Chow & Leong, 1999). A number of these short fragments constitute partial sequences of later defined HPV types (de Villiers et al., 2004a). Recently, instead of primary cloning of a complete papillomavirus genome, PCR amplification of overlapping fragments has been used to assemble a full-length genome. Such isolates are termed HPVcand(number) (de Villiers et al., 2004a). An understanding of the relationship between papillomavirus types based on a comparison of nucleotide sequences began to emerge more than 10 years ago (Chan et al., 1992a,b; van Ranst et al., 1992a,b). Continued research based on these principles has led

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to the taxonomic groupings, which today are widely accepted. Phylogenetic assemblages occasionally coincide with biological and pathological properties, but often diverge. The closely related HPV types 2 and 27, 6 and 11, and 16 and 31, which cause common warts, genital warts and cervical cancer, respectively, are three excellent cases of the numerous consistencies between phylogeny and pathology. However, there are also some discrepancies: the phylogenetic group of genital HPV types, which incorporates all HPV types found in genital lesions, also contains some HPV types that are mostly found in cutaneous lesions, such as HPV 2. Also, highly unrelated viruses, such as HPV 2 (genus alpha) and HPV 4 (genus gamma), can cause similar cutaneous papillomas (de Villiers et al., 2004a). The evolution of papillomaviruses has often been debated (de Villiers et al., 2004a). Comparative studies that used the E6, L1 or the combined E6–E7–L1 ORFs (van Ranst et al., 1992a,b; Myers et al., 1994; Chan et al., 1995), however, have resulted in phylogenetic trees that establish similar or even identical relationships. A frequently used 291-bp amplicon, a small segment of the L1 gene, suffices as a foundation to generate highly informative phylogenetic comparisons (Bernard et al., 1994). Sequence comparisons of the complete genomes of 118 papillomaviruses reveal a high diversity, but a distribution similar to that found when L1 ORF sequences were compared. A cladogram based on the complete L1 ORF of 96 HPV types and 22 animal papillomavirus types is presented in Figure 4. The frequency distribution of pairwise identity percentages from sequence comparisons of the L1 ORF demonstrates three taxonomic levels, on the basis of comparison of both complete genomes and L1 genes, namely genera, species and types (Figure 5) (de Villiers et al., 2004a). Extensive sequence comparisons using the L1 ORF of 96 HPV types and 22 animal papillomaviruses led to the establishment of the following classifications. Higher-order clusters of HPV types (e.g. the genital HPVs) had previously been called ‘supergroups’ or ‘major branches’ (Myers et al., 1994; Chan et al., 1995). For these taxa, the new term ‘genus’ was introduced. Different genera share less than 60% nucleotide sequence identity in the L1 ORF. Full-length sequences of complete genomes have more than 23% but less than 43% nucleotide sequence identity when compared with genera of the Papillomaviridae. Lower-order clusters of HPV types (e.g. HPV types 6, 11, 44 and 55) had been called ‘groups’, ‘subgroups’ or ‘minor branches’. For these taxa, the new term ‘species’ was introduced. Such species within a genus share between 60 and 70% nucleotide sequence identity. The traditional papillomavirus types within a species share between 71 and 89% nucleotide sequence identity within the complete L1 ORF (de Villiers et al., 2004a). The introduction of the term ‘genus’ is useful, as this concise term will now replace the somewhat vague expressions of ‘major branches’ or ‘supergroups’. Throughout all biology, including virology, specific genera typically unite species, which are clearly phylogenetically related but are often biologically quite diverse. The same applies to papillomavirus genera. A summary of the biological properties known for each genus is presented in Table 2, together with specific characteristics of the organization of its genome in cases where this differs from the typical pattern. The introduction of the term

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Figure 4. Phylogenetic tree containing the sequences of 118 papillomavirus types

Modified from de Villiers et al. (2004a) The L1 ORF sequences were used in a modified version of the Phylip version 3.572 and based on a weighted version of the neighbour-joining analysis. The tree was constructed using the Treeview programme of the University of Glasgow. The numbers at the ends of each of the branches identify an HPV type; c-numbers refer to candidate HPV types. All other abbreviations refer to animal papillomavirus types. For the meaning of each abbreviation, please refer to Table 1. The outermost semicircular symbols identify papillomavirus genera, e.g. the genus alpha-papillomavirus. The number at the inner semicircular symbol refers to papillomavirus species. To give an example taken from the upper part of the figure, the HPV types 7, 40, 43, and cand91 together form the HPV species 8 in the genus alpha-papillomavirus.

‘species’ is biologically useful, as these are natural taxa based on the close phylogenetic relationship of certain types and because such species typically assemble papillomavirus types that have common biological and pathological properties, a requirement of the ICTV guidelines. To give examples, all HPV types that form a species with HPV 2 are typically found in common skin warts, and all HPV types that form a species with HPV 16 are ‘high-risk’ HPV types that are found in cervical cancer and its precursor lesions. More detailed information about each species and papillomavirus types within a genus is presented in Table 1. The type species have been chosen either because they are the most comprehensively investigated type, because they best represent the species or because

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Table 1. Characteristics of species within specific genera Other papillomavirus types

Comments

Alpha-papillomavirus

1

HPV 32 (X74475)

HPV 42 (M73236)

More frequently in benign lesions (low risk); oral or genital mucosa; third ORF in ELR

2

HPV 10 (X74465)

More frequently cause cutaneous than mucosal lesions; low risk; E5 biologically different

3

HPV 61 (U31793)

4

HPV 2 (X55964)

HPV 3 (X74462) HPV 28 (U31783) HPV 29 (U31784) HPV 78 HPV 94 (AJ620211) HPV 72 (X94164) HPV 81 (AJ620209) HPV 83 (AF151983) HPV 84 (AF293960) candHPV 62 candHPV 86 (AF349909) candHPV 87 (AJ400628) candHPV 89 (AF436128) HPV 27 (X73373) HPV 57 (X55965)

5

HPV 26 (X74472)

HPV 51 (M62877)

HPV 53 (X74482)

HPV 69 (AB027020) HPV 82 (AB027021) HPV 30 (X74474)

6

Mucosal lesions; lower risk

Common skin warts; frequently in benign genital lesions in children; several larger uncharacterized ORFs scattered throughout genome; E5 ORF biologically different High-risk mucosal lesions, also in benign lesions

High-risk mucosal, but also in benign lesions

HPV 56 (X74483) HPV 66 (U31794)

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Type species

Other papillomavirus types

Comments

7

HPV 18 (X05015)

HPV 39 (M62849) HPV 45 (X74479) HPV 59 (X77858) HPV 68 (X67161) HPV 70 (U21941) candHPV85 (AF131950)

High-risk mucosal lesion

8

HPV 7 (X74463)

HPV 40 (X74478) HPV 43 (AJ620205) candHPV 91 (AF131950)

Low-risk mucosal and cutaneous lesions; HPV 7 also known as butcher’s wart virus — often in mucosal and skin lesions in HIVinfected patients

9

HPV 16 (K02718)

HPV 31 (J04353) HPV 33 (M12732) HPV 35 (X74476) HPV 52 (X74481) HPV 58 (D90400) HPV 67 (D21208)

High-risk — malignant mucosal lesions

10

HPV 6 (X00203)

Mostly associated with benign mucosal lesions; low risk; reports of HPV 6 in verrucous carcinoma

11

HPV 34 (X74476)

HPV 11 (M14119) HPV 13 (X62843) HPV 44 (U31788) HPV 74 (U40822) PcPV (X62844) HPV 73 (X94165)

12 13

RhPV 1 (M60184) HPV 54 (U37488)

– –

Mucosal genital lesions in rhesus monkeys Low-risk mucosal

14

candHPV 90 (AY057438)



Low-risk mucosal

15

HPV 71 (AB040456)

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Table 1 (contd)

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Mucosal lesions — high risk

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Other papillomavirus types

Comments

Beta-papillomavirus

1

HPV 5 (M17463)

HPV 8 (M12737) HPV 12 (X74466) HPV 14 (X74467) HPV 19 (X74470) HPV 20 (U31778) HPV 21 (U31779) HPV 25 (U74471) HPV 36 (U31785) HPV 47 (M32305) HPV 93 (AY382778)

Most frequently cause cutaneous lesions, but reports of DNA in mucosa; commonly associated with lesions in EV or immunosuppressed patients; mostly benign lesions, but reported in malignant lesions, also in immunocompetent patients

2

HPV 9 (X744464)

Most frequently cause cutaneous lesions, but reports of DNA in mucosa; commonly associated with lesions in EV or immunosuppressed patients; mostly benign lesions, but reported in malignant lesions, also in immunocompetent patients

3

HPV 49 (X74480)

4

1

HPVcand92 (AF531420) HPVcand96 (AY382779) HPV 4 (X70827)

HPV 15 (X74468) HPV 17 (X74469) HPV 22 (U31780) HPV 23 (U31781) HPV 37 (U31786) HPV 38 (U31787) HPV 80 (Y15176) HPV 75 (Y15173) HPV 76 (Y15174) –

2 3 4

5 Gamma-papillomavirus

Benign cutaneous lesions Pre- and malignant cutaneous lesions Pre- and malignant cutaneous lesions Cutaneous lesions; histologically distinct homogenous intracytoplasmic inclusion bodies Cutaneous lesions

HPV 50 (U31790) HPV 60 (U31792)

– –

Cutaneous lesions Cutaneous lesions

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HPV 48 (U31790)

HPV 65 (X70829) HPV 95 (AJ620210) –

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Table 1 (contd)

Comments

Delta-papillomavirus

5 1

HPV 88 EEPV (M15953)

– RPV (AF443292)

2

DPV (M11910)



Cutaneous lesions E9 gene within ELR with transforming properties E9 gene within ELR with transforming properties

3

OvPV-1 (U83594)

OvPV-2 (U83585)

4

BPV-1 (X02346)

BPV-2 (M20219)

Epsilon-papillomavirus

1

BPV-5 (AF457465)



Zeta-papillomavirus Eta-papillomavirus

1 1

EqPV, AF498323 FcPV, AY957109

– –

Theta-papillomavirus Iota-papillomavirus

1 1

PePV, AF420235 MnPV (U01834)

– –

Kappa-papillomavirus

1

CRPV (K02708)

2

ROPV (AF227240)

1

COPV (L22695)



ELR, 1500 bp in length

2 1

FdPV (AF377865) HPV 1 (V01116)

– –

ELR, 1271 bp in length Histologically distinct heterogenous intracytoplasmic inclusion bodies; LCR, 982 bp in length

Lambdapapillomavirus Mu-papillomavirus

E5 gene in ELR with transforming properties; trans-species infection causing sarcoids in horses

High divergence within the E6 and E7 ORFs described for different isolates; associated with cutaneous lesions Associated with oral lesions

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Table 1 (contd)

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Table 1 (contd) Genus

Comments

2

HPV 63 (X70828)



Nu-papillomavirus

1

HPV 41 (X56147)



Histologically distinct filamentous intracytoplasmic inclusion bodies; LCR, 558 bp in length Several larger uncharacterized ORFs scattered throughout the genome; ELR only 17 nucleotides; all E2-BSs in LCR modified

Xi-papillomavirus

1

BPV-3 (AF486184)

BPV-4 (X05817) BPV-6 (AJ620208)

Omikronpapillomavirus

1

PsPV (AJ238373)

Pi-papillomavirus

1

HaOPV (E15110)

E8 gene within E6 region of BPV-4 has transforming properties similar to E5 of BPV-1 E7 ORF absent; several larger ORFs in L1 ORF region



No ELR; partial overlap between E2 and L2 ORFs

From de Villiers et al. (2004a) The table shows division of the Papillomaviridae into genera and species, following the phylogenetic tree shown in Figure 5. For each species, the table lists a type species, other papillomavirus types that belong to these species and biological and pathological properties of each species. bp, base pair; BS, binding site; BPV, bovine papillomavirus; candHPVs, candidate HPVs, cloned and characterized from PCR products; COPV, cannine oral papillomavirus; CRPV, cottontail rabbit papillomavirus; DPV, deer papillomavirus; DPV, deer papillomavirus; EEPV, European elk papillomavirus; ELR, region between early and late genes; EqPV, Equus caballas (horse) papillomavirus; EV, epidermodysplasia verruciformis; FcPV, Fringilla coelebs (chaffinch) papillomavirus; FdPV, Felis domesticus (cat) papillomavirus; HaOPV, hamster oral papillomavirus; HIV, human immunodeficiency virus; HPV, human papillomavirus; MnPV, Mastomys natalensis papillomavirus; ORF, openreading frame; OvPV, ovine papillomavirus; PePV, Psittacus erithacus timneh (parrot); PsPV, Phocoena spinipinnis papillomavirus; ROPV, rabbit oral papillomavirus; RPV, reindeer papillomavirus

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Figure 5. Frequency distribution of pairwise identity percentages from nucleotide sequence comparison of the L1 ORFs of 118 papillomavirus types

Modified from de Villiers et al. (2004a)

there is only one type in that taxon. Table 1 is an important reference that groups together (with the type species in many type-rich taxa) all those HPV types that belong to the same species and will presumably have properties similar or identical to the type species, but cannot be studied (for purposes of basic research, drug development and vaccination) as intensely as the type species. As an example, species No. 9 groups — with the type species HPV 16 — the HPV types 31, 33, 35, 52, 58 and 67, which have been studied to a lesser extent (with the exception of HPV 31) but which probably have similar biological and pathological properties as HPV 16. Several hundred papillomavirus types have been partially identified in the form of short DNA fragments, but interest in isolating full-length genomes appears to be declining. The number of HPV types isolated and fully characterized now exceeds 100. A regulated taxonomic description of non-human papillomaviruses is particularly necessary because it is extremely probable that only a tiny fraction of all animal papillomavirus types have been identified or isolated. The present methodology used for the detection of papillomavirus types is very limiting, as it is based on the information available from known types. Hopefully, future efforts will be directed towards identifying additional types that are very distantly related to the known genera. An example of the large diversity of animal papillomaviruses are the two recently described types from birds, both of which lack traditional E6 and E7 ORFs (Tachezy et al., 2002a,b; Terai et al., 2002) and are less closely related to any mammalian papillomavirus type than they are to one another. Several of the papillomavirus types that presently appear as single species within a genus have in the past been identified only because of the availability of lesions that harbour

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Table 2. Biological properties and characteristics of organization of genome for each genus

Alpha-papillomavirus

Mucosal and cutaneous lesions in humans and primates High- and low-risk classification based on molecular biological data: high-risk types (pre- and malignant lesions) immortalize human keratinocytes; low-risk types (benign lesions) do not. Recent compilations of epidemiological data demonstrate more frequent association of specific species at high-risk types.

Conserved with an E5 ORF within the ELR (∼300–500 bp); ORFs in ELR from different species may be divided into three groups: classical E5 ORF; closer related to the ungulate E5 ORF; putative ORF with distinct conserved motives

Beta-papillomavirus

Cutaneous lesions in humans Infections exist in latent form in general population, activated under conditions of immune suppression. Also referred to as EV–HPV types due to close association with disease EV Cutaneous lesions in humans Histologically distinguishable by intracytoplasmic inclusion bodies specific for type species

ELR generally < 100 nucleotides in length; E5 ORF absent

Lesions in ungulates Induces fibropapillomas in the respective host. Trans-species transmission occurs inducing sarcoids. BPV; cutaneous papillomas in cattle

ORFs located in ELR have transforming properties.

Cutaneous lesions in horses Avian papillomaviruses Cutaneous lesions in host Avian papillomaviruses Cutaneous lesions in host

Undefined ORF overlapping with L2 ORF E4 and E5 ORFs absent; no typical E6 ORF, but an ancestral E7 ORF with partial E6 characteristics E4 and E5 ORFs absent; no typical E6 ORF, but an ancestral E7 ORF with partial E6 characteristics

Gamma-papillomavirus

Delta-papillomavirus

Epsilon-papillomavirus Zeta-papillomavirus Eta-papillomavirus Theta-papillomavirus

ELR < 100 nucleotides in length; E5 ORF absent

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Iota-papillomavirus

Rodent papillomaviruses Cutaneous lesions Isolated from rabbits Cutaneous and mucosal lesions

E5 ORF absent; E2 ORF considerably larger than in other genera E6 ORF larger than in other papillomaviruses; harbours an uncharacterized E8 ORF within the E6 ORF region.

Lambda-papillomavirus

Animal papillomaviruses Benign mucosal and cutaneous lesions

ELR region exceptionally large (1500 bp and 1271 bp in two known species)

Mu-papillomavirus

Human papillomaviruses Cutaneous lesions Histologically distinguishable by intracytoplasmic inclusion bodies specific for type species

LCR relatively large (982 bp and 558 bp in two known species)

Nu-papillomavirus

Human papillomavirus Benign and malignant cutaneous lesions

Several larger uncharacterized ORFs scattered throughout genome; E2 BSs in LCR all modified

Xi-papillomavirus

Bovine papillomaviruses Induce true papillomas in host Cutaneous or mucosal lesions Isolated from genital warts in cetaceans

Characteristic E6 ORF absent; E8 ORF (located in E6 ORF region) with properties similar to E5 ORF of BPV-1 E7 ORF absent; several larger unidentified ORFs located in L1 ORF region ELR absent with E2 and L2 ORFs partially overlapping

Kappa-papillomavirus

Omikron-papillomavirus Pi-papillomavirus

Isolated from hamsters Mucosal lesions

From de Villiers et al. (2004a) BPV, bovine papillomavirus; BS, binding site; ELR, region between the early and late genes of the papillomavirus genome; EV, epidermodysplasia verruciformis; LCR, long control region; ORF, open-reading frame

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Genus

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Table 2 (contd)

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many viral particles or from which substantial amounts of circular double-stranded DNA could be purified. Subtypes of papillomaviruses are defined as being 2–10% genomically different from any papillomavirus type. This term originally had a different meaning, and was used when different isolates of the same type differed partially in their restriction enzyme cleavage patterns, such as HPV 2a, HPV 2b and HPV 2c. It later became clear that these subtypes should rather fall under the category ‘variants’. Other misclassifications, which were originally based on hybridization data, include the classification of papillomaviruses as types that now fall under the subtype classification. The HPV 55 genome shares 95% homology with that of HPV 44 and therefore constitutes a subtype of HPV 44. The same classification applies to HPV 64 which is a subtype of HPV 34 and HPV 46 which is a subtype of HPV 20. The numbers HPV 46, HPV 55 and HPV 64 will remain vacant to avoid any future confusion with published data. Also, comparing published data of the L1 ORF between the pygmy chimpanzee papillomavirus and the common chimpanzee papillomavirus showed 93% similarity. The latter is therefore a subtype of the pygmy chimpanzee papillomavirus (de Villiers et al., 2004a). As the search for new papillomaviruses identified so few genomes that diverged by 2–10% from defined types, it can be concluded that papillomavirus types are clearly natural taxa. It is unclear why genomes that are intermediate to closely related papillomavirus types are so rare (de Villiers et al., 2004a). Most HPV types have been isolated repeatedly in a large number of clinical studies, and the sequences of these isolates have been compared. As may be expected, most of these isolates differ from one another. It should be stressed, however, that there is no rapid diversification as in certain RNA viruses, since most HPV types could be re-isolated in the form of only 10–100 different genomic variants that normally showed approximately 1–2% sequence diversity. The phylogenetic implications of this, namely the slow, linked evolution of host and virus, have been discussed extensively while the clinical implications, i.e. pathological diversity within individual HPV types, are still under investigation (De Villiers et al., 2004a). 1.1.4

Evolution of papillomaviruses

Papillomaviruses are an ideal model system for the study of the evolution of DNA viruses. On several levels, phylogenetic trees of papillomaviruses reflect the relationship of their hosts. One branch of HPVs includes one ape and two monkey papillomaviruses, possibly because the diversification of the viruses predated the separation of the infectedprimate taxa. This hypothesis predicts that the root of the evolution of some if not all HPV types should point to Africa, since humans evolved from non-human primates in this continent.

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(a)

Non-human primates

To understand the mode and time scale of the evolution of papillomaviruses, 326 genital samples from rhesus monkeys and long-tailed macaques were examined with a PCR protocol optimized to detect genital HPV types. In 28 of the samples, amplicons were found that were derived from 12 different and novel viral genomes — rhesus monkey papillomaviruses (RhPV)-a to RhPV-m, with the probable taxonomic status of ‘type’. This frequency of novel RhPVs suggests that rhesus monkeys may play host to papillomaviruses with a diversity similar to that of HPVs. In phylogenetic trees, all 12 novel RhPVs and the previously described type RhPV-1 were members of the genital HPV supergroup and formed three minor branches that were distinct from the 11 branches formed by genital HPVs. It appears that the evolution of primate lineages that lead to the genus Macaca and to humans created transmission barriers for papillomaviruses, which resulted in a viral evolution that was closely linked to the host. Additional support for the hypothesis of linked evolution derives from the phylogenetic association of two other ape and monkey viruses with genital HPVs: the supergroup formed by at least seven ungulate papillomaviruses and the isolated phylogenetic position of the only bird papillomavirus known at that time (Chan, S.Y. et al., 1997a). Portions of the genome from two different papillomaviruses of the Abyssinian Colobus monkey were sequenced and analysed phylogenetically. This revealed that the major evolutionary separation between genital and EV-associated papillomaviruses, hitherto found only in humans, also exists in animals. The sequence of the LCR of Colobus monkey papillomavirus type 2 (CgPV-2) revealed extensive conservation of functional elements that are typical of the EV-associated viruses, which suggests that CgPV-2 could be a model to study human skin cancer in relation to EV-associated HPVs. Although isolated from the same monkey species, the other Colobus monkey virus, CgPV-1, is a typical genital virus as shown by comparison of E and L gene sequences. The presence of these two major phylogenetic divisions of papillomaviruses in both human and monkey hosts strongly suggests that this diversification predated the evolutionary split between monkeys and apes. This would imply that at least two different groups of papillomavirus have evolved separately in their respective primate hosts for more than 22 million years with only moderate sequence changes since their genesis (Chan, S.Y. et al., 1997b). (b)

Humans

Genomic segments of 118 HPV type 16 (Chan et al., 1992a) isolates from 76 cervical biopsies, 14 cervical smears, three vulval biopsies, two penile biopsies, two anal biopsies and one vaginal biopsy were amplified, cloned and sequenced. The specimens were taken from patients in Brazil, Germany, Singapore and Tanzania. The sequence of a 364-bp fragment of the LCR of the virus revealed 38 variants, most of which differed by one or several point mutations. In the phylogenetic trees that were constructed, two branches could be distinguished. Nearly all of the variants from Tanzania were assigned to one

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(African) branch and all of the German and most of the Singaporean variants were assigned to the other (Eurasian) branch. While some German and Singaporean variants were identical, each group also contained variants that formed unique branches. In contrast to the internal homogeneity within the groups of the Singaporean, German and Tanzanian variants, the Brazilian variants were clearly divided between the two branches. Exceptions to this were the seven Singaporean isolates with mutational patterns typical of the Tanzanian isolates. The data suggest that HPV 16 evolved separately over a long period in Africa and Eurasia. Representatives of both branches may have been transferred to Brazil through past colonial immigration. The comparable efficiencies of transfer of the African and the Eurasian variants to South America suggest the pandemic spread of HPV 16 in past centuries. Representatives of the African branch were possibly transferred to the Far East along old Arab and Indonesian sailing routes. The data indicate that HPV 16 is a well-defined virus type, since the variants show only a maximal genomic divergence of about 5%. The small amount of divergence in any one geographical location and the lack of marked divergence between the Tanzanian and Brazilian African genome variants two centuries after their probable introduction into South America suggest a very slow rate of viral evolution. The phylogenetic tree, therefore, probably represents a minimum of several centuries of evolution, if not an age equal to that of the respective human races. The diversity of a hypervariable 364-bp segment from the HPV 16 LCR genome was investigated in 301 virus isolates collected from 25 different ethnic groups and geographical locations. Altogether, 48 variants could be distinguished that had diversified from one another along five phylogenetic branches. Variants from two of these branches were nearly completely confined to Africa. Variants from a third branch were the only variants identified in Europeans but occurred at lower frequency in all other ethnic groups. A fourth branch was specific for Japanese and Chinese isolates. A small fraction of all isolates from Asia and from indigenous as well as immigrant populations in the Americas formed a fifth branch. Important patterns of HPV 16 phylogeny suggested coevolution of the virus with people of the three major human races, namely, Africans, Caucasians and East Asians. However, several minor patterns are indicative of smaller bottlenecks of viral evolution and spread, which may correlate with the migration of ethnic groups in prehistoric times. The colonization of the Americas by Europeans and Africans is reflected in the composition of their HPV 16 variants. The HPV 16 genomes of today represent a degree of diversity that may have evolved over a large time span, probably exceeding 200 000 years, from a precursor genome that may have originated in Africa (Ho et al., 1993a). In a similar study, the genomic sequences of HPV type 18 isolates from four continents were compared. Diversity within HPV 18 correlates with patterns of human evolution and the spread of Homo sapiens: HPV 18 variants, similarly to HPV 16 variants, are specific for the major human races, with maximal diversity in Africa. African HPV 18 variants are at the root of the phylogenetic tree. The identification of an African HPV 45 isolate further reduces the evolutionary distance between HPV 18 and HPV 45. HPV 18

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variants from Amazonian Indians are the closest relatives to those from Japanese and Chinese patients and suggest that a single point mutation in the phylogenetically evaluated genomic segment represents at least 12 000 years of evolution. The diversity within HPV 18, and probably within other HPV types, is estimated to have evolved over a period of more than 200 000 years and diversity between HPV types may have evolved over several million years (Ong et al., 1993). The host specificity and the benign nature of most papillomavirus infections suggest that these viruses are extremely well adapted parasites. It has been proposed that this could be indicative of host–virus co-evolution (Chan et al., 1992b), but it is more probable that the evolution of papillomaviruses is dominated by unilateral host selection, as adjustment to the molecular mechanism of the host cell had to be made (Shadan & Villarreal, 1993). 1.1.5

Function of viral proteins

The functions of the papillomavirus proteins are discussed below and summarized in Table 3. Unless otherwise stated, the description of protein functions refers to HPV proteins. When individual proteins from different papillomaviruses have a common characteristic, they are designated with the generic heading of ‘papillomavirus’. (a)

E1

The 73-kDa viral protein E1 is required for viral replication; it binds to a specific DNA sequence (E1 binding site; E1BS) in the viral origin of replication and assembles into hexameric complexes with the aid of a second viral protein, E2 (Frattini & Laimins, 1994). The resultant complex has helicase activity (first predicted from similarities in amino acids to SV40 large-T antigen) and initiates DNA unwinding to provide the template for subsequent synthesis of progeny DNA (Wilson et al., 2002). The functional domains of the E1 protein have been characterized for several papillomaviruses. The carboxyl terminal half has adenosine triphosphatase (ATPase) helicase activities and is necessary and sufficient for oligomerization. A change in amino acids in the ATPase domain (Pro-479 to Ser), which is predicted to inactivate adenosine triphosphate (ATP) binding, impaired the activity of E1 (Hughes & Romanos, 1993). This domain also interacts with E2 protein and the DNA polymerase α subunit p70 (Masterson et al., 1998), but is not sufficient to support replication (Amin et al., 2000). A segment of approximately 160 amino acid residues immediately upstream of the ATPase/helicase domain (from approximately residue 190 to residue 350) is the DNA-binding domain (DBD; Titolo et al., 2000; White et al., 2001; Titolo et al., 2003). A stretch of about 50 amino acids within the amino terminus of E1 acts as a localization regulatory region (LRR), that contains a dominant nuclear export sequence (NES) and a nuclear localization signal (NLS), both of which are regulated by phosphorylation (Sun et al., 1998; Amin et al., 2000; Deng et al., 2004).

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Table 3. Functions of papillomavirus proteinsa E1

Adenosine triphosphatase (ATPase) and DNA helicase; recognizes and binds to the viral origin of DNA replication as a hexameric complex; necessary for viral DNA replication.

E2

Main regulator of viral gene transcription; binds the viral transcriptional promoter as a dimer; involved in viral DNA replication; interacts with and recruits E1 to the origin.

E4

Acts late in the viral life cycle; interacts with the keratin cytoskeleton and intermediate filaments; localizes to nuclear domain 10; induces G2 arrest; believed to facilitate virus assembly and release. Induces unscheduled cell proliferation; interacts with 16k subunit c of vacuolar ATPase; may activate growth factor receptors and other protein kinases; inhibits apoptosis; inhibits traffic of major histocompatibility complexes to the cell surface.

E5

E6

Induces DNA synthesis; induces telomerase; prevents cell differentiation; interacts with four classes of cellular proteins: transcriptional co-activators, proteins involved in cell polarity and motility, tumour suppressors and inducers of apoptosis, primarily p53, and DNA replication and repair factors.

E7

Induces unscheduled cell proliferation; interacts with histone acetyl transferases; interacts with negative regulators of the cell cycle and tumour suppressors, primarily p105Rb. Major viral structural protein; assembles in capsomeres and capsids; interacts with L2; interacts with cell receptor(s); encodes neutralizing epitopes. Minor viral structural protein; interacts with DNA; interacts with nuclear domain 10s; believed to facilitate virion assembly; may interact with cell receptor(s); encodes linear virus neutralizing epitopes.

L1 L2

a

Some of the activities of the viral proteins have been described in cultured cells or other experimental systems; some have been observed in vivo. For references, see text and Sections 4.1.2 and 4.1.3.

E1 also interacts with replication protein A (RPA), which results in the rapid stabilization of single-stranded DNA generated by E1 helicase activity (Han et al., 1999; Loo & Melendy, 2004). Interaction with H1 histone may play a role in unravelling the viral chromatin by removing H1 histones before unwinding the DNA (Swindle & Engler, 1998). (b)

E2

The E2 gene encodes a product of around 40–45 kDa, depending on the papillomavirus. The protein is tripartite. First, in the carboxyl terminus, a dimerization domain results in the formation of homodimers that recognize and bind 12-bp palindromic DNA sequences (ACCGNNNNCGGT) within the LCR, defined as E2-BSs (Desaintes & Demeret, 1996). Second, the middle region of E2 — the hinge — has a rather indeterminate function, although it is important for regulating the stability of some E2 proteins and determining their nuclear localization in others (Zou et al., 2000). Third, the amino

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terminal domain is essential for regulation of transcription and viral DNA replication through the interaction with E1 protein (Desaintes & Demeret, 1996). The majority of studies have demonstrated that expression of HPV E2 protein at various levels in human cells results in the repression of transcription from the viral promoter. In one study, low levels of HPV 16 E2 were shown to activate transcription in primary human epithelial cells, but repression occurred at high levels (Bouvard et al., 1994a). One of the proposed mechanisms for repression by E2 is that it binds to the E2BS adjacent to the TATA box of the LCR and thus interferes sterically with the binding of the TATA-binding protein (TBP) to the same site as has been shown for BPV-1 E2 (Dostatni et al., 1991) and HPV 18 E2 (Steger & Corbach, 1997). In support of this hypothesis, mutation of the E2-BS adjacent to TATA partially relieves repression of transcription by E2 (Dostatni et al., 1991). Low levels of E2 appear to activate transcription from the viral LCR, whereas higher levels operate solely as a transcriptional repressor. This would provide a negative feedback loop to control the levels of E6 and E7 oncoproteins. Disruption or silencing of the E2 gene leads to the elevated levels of E6 and E7 observed in cell transformation. Conversely, overexpression of BPV-1 E2 in cell lines derived from HPV-induced cervical cancers results in suppression of HPV E6 and E7 expression and promotes the reactivation of the p53 and p105Rb pathways and the consequent senescence of cells (Goodwin et al., 1998). E2 plays an important role in the segregation of newly replicated viral DNA with mitotic chromosomes, which ensures a similar distribution of viral genomes in the daughter cells. During mitosis, E2 is associated with viral DNA and with cell centrosomes and the mitotic spindle via its carboxyl terminal domain; this association is thought to be responsible for partitioning the viral genome into daughter cells (Van Tine et al., 2004). E2 interacts with the minor viral structural protein L2, which leads to inhibition of the transactivation but not the replication function of E2 for both BPV and HPV proteins (Heino et al., 2000; Okoye et al., 2005). This may be a mechanism whereby, at late stages of the viral life cycle, the functions of E2 are withdrawn from transcription and directed towards the amplification of viral DNA to facilitate the production of new viral progeny. E2 also interacts with numerous cellular proteins in cultured cells. Amongst these, three proteins are of particular interest as they are involved in the DNA damage response: topoisomerase II beta-binding protein 1 (TopBP1) (Boner et al., 2002), breast cancer type 1 (BRCA1) tumour suppressor protein (Kim, J. et al., 2003) and poly(ADP-ribose) polymerase 1 (PARP1) (Lee et al., 2002). These interactions may be involved in the regulation of viral DNA replication and also in the protection of the viral genome when the cell is damaged. The recruitment of the viral genome to sites of DNA damage through an interaction with TopBP1 or BRCA1 may provide a quick means of repairing the viral genome and suppressing replication when the cell is exposed to DNA-damaging agents. [The Working Group noted that it has not been proven that the interactions between E2 and cellular proteins established in cultured cells take place in vivo.]

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E4

The HPV E4 gene is located in the E region and overlaps with E2 but is transcribed in a different reading frame. The E4 protein is heterogeneous with the major form; it is a fusion product with a 5-amino acid sequence from the N-terminus of E1 (E1^E4). Despite its genomic location and its ‘E’ name, the E4 protein is expressed primarily at later stages and is the most abundant viral protein expressed during the virus life cycle. Its expression coincides almost exactly with the onset of vegetative viral DNA replication; however, although the protein is detected in cells in which viral DNA replication is ongoing and in highly differentiated cells that express the capsid genes and synthesize new progeny virions, E4 is not found in virion particles. It aggregates through sequences at its C terminus, and these aggregates are found in both the cytoplasm and the nucleus of the infected cell (Doorbar et al., 1991, 1997; Roberts et al., 1997). The functions of the E4 protein appear to be regulated partly by post-translational modification — oligomerization, phosphorylation and proteolytic cleavage — as in the case of interference by E4 in the cell cycle. The functions of E4 have been suggested to play a role in facilitating and supporting viral genome amplification, the regulation of late gene expression, the control of virus maturation and the mediation of virus release. The HPV E4 protein plays no role in cell transformation as has been shown for BPV-1 E4 (Neary et al., 1987), and its expression is progressively lost from neoplastic lesions during their progression to cancer (Crum et al., 1990). E4 interacts with and disrupts the organization of intermediate filaments, the cornified cell envelope (CCE), mitochondria and ND10 domains. It also interferes with the cell cycle. (i) E4 and intermediate filaments A leucine-rich motif (LLXLL) at the N-terminus of most E4 proteins is responsible for the association of E4 with the keratin cytoskeleton (Roberts et al., 1994) and a hydrophobic sequence at the protein C terminus mediates disruption of the cytoskeleton (Roberts et al., 1997). In cultured epithelial cells, the keratin cytoskeleton is often collapsed in a perinuclear bundle (Doorbar et al., 1991; Roberts et al., 1993, 2003), but perinuclear bundles of E4 and keratins are also observed in epithelial cells in vivo (Wang, Q. et al., 2004). The ability of E4 to disrupt the cytoskeleton might compromise the structural integrity of infected cells in the upper layers of warts, and enable these cells to rupture readily and release newly synthesized virus particles into the environment (Doorbar et al., 1991). (ii) E4 and CCE The role of E4 in aiding virus release is supported by the association of E4 with the CCE, which is a highly resistant structure beneath the plasma membrane of differentiated keratinocytes in the stratum corneum. It comprises cross-linked proteins, including loricrin, involucrin, small proline-rich proteins, cytokeratin 10 and other proteins that are covalently linked through transglutamination. Newly synthesized papillomavirions have to pass this resistant cell envelope before release into the environment. The CCE from

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HPV 11-infected genital epithelium is abnormal and more fragile than that of uninfected tissue (its thickness is ∼65% that of uninfected epithelium) and its association with this compromised CCE suggests that E4 could interfere with the normal assembly of CCE and aid the release of progeny virus (Brown & Bryan, 2000). (iii) E4 and mitochondria In epithelial cell lines, E4 also binds mitochondria through its N-terminal domain and causes their redistribution from the microtubule networks to E4-containing bundles. This redistribution of mitochondria causes a reduction in their membrane potential and eventually cell apoptosis (Raj et al., 2004). These observations confirm the hypothesis that E4 facilitates virus release through disruption of the cytokeratin network and the CCE and through induction of apoptosis. (iv) E4 and nuclear domain 10 Nuclear domain (ND) 10s are nuclear structures that contain numerous proteins, among which promyelocytic leukaemia protein (PML) is necessary for their integrity. Studies with other viruses have shown that ND10s are associated with virus replication and transcription, and that many viral proteins induce the reorganization or disruption of ND10s (Everett et al., 1999). In HPV 1-induced warts, PML is relocated from ND10s to the periphery of nuclear aggregates of full-length E4; a similar redistribution is found in keratinocytes that express E4 alone (Roberts et al., 2003). The E4 of HPV 16 is probably similarly responsible for the disruption of ND10s. It is still not clear why viruses need to disperse ND10s, but this process may be linked to a switch between early and later stages of replication of a virus, and would be in accordance with the role of E4 in the late stages of HPV replication. Dispersal of ND10s by E4 may also be relevant to virion assembly, as the structural proteins L1 and L2 are recruited to ND10s in both BPV and HPV (Day et al., 1998; Florin et al., 2002a,b). (v) E4 and the cell cycle The expression of several different E4 proteins, including E4 from HPV 16 and 18, induces G2 arrest in the cell cycle in keratinocytes. G2 arrest is mediated by a proline-rich sequence near to the N-terminus of E4 (Davy et al., 2002; Nakahara et al., 2002; Knight et al., 2004). G2 arrest is due to the sequestration and retention of activated cyclin B1 complexes to ‘collapsed’ E4–keratin bundles in the cytoplasm of epithelial cells (Knight et al., 2004; Wang, Q. et al., 2004). However, E4-induced G2 arrest is not dependent on the binding of E4 to keratins (Davy et al., 2002). It is not clear how relevant these activities of E4 in tissue cultures are to virus maturation and production. It has been hypothesized that suprabasal cells, driven into S phase by E7, are maintained in this phase by E4 to maximize viral genome amplification. However, continuous unscheduled replication of the host DNA would limit the availability of precursor nucleotides and replication enzymes to the virus. By inhibiting cellular DNA synthesis, E4 would make replication factors available for viral DNA replication.

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Thus, the effect of E4 would be to keep the infected cell in a metabolically active state without competing with host DNA synthesis, and so boost virus genome replication. The E5, E6 and E7 proteins are described only briefly here as they are discussed in greater detail in Sections 4.1.2 and 4.1.3. (d)

E5

Not all HPVs have an E5 ORF. The E5 ORF and the protein that it encodes vary in length among papillomaviruses. The hydrophobic nature of the protein is conserved but not the primary amino acid sequence (DiMaio & Mattoon, 2001). E5 from HPVs is considered to be a transforming protein because it transforms cultured murine fibroblasts and keratinocytes (Chen & Mounts, 1990; Leptak et al., 1991), enhances the immortalization potential of E6 and E7 (Stöppler et al., 1996) and, in cooperation with E7, stimulates the proliferation of mouse primary cells (Bouvard et al., 1994b; Valle & Banks, 1995). When HPV 16 E5 was expressed from a heterologous promoter in cultured cells, it enhanced the activity of epidermal growth factor receptor (EGFR) in the presence of ligand (Leechanachai et al., 1992; Pim et al., 1992; Crusius et al., 1998); co-immunoprecipitation studies have indicated that HPV 16 E5 can also form a complex with EGFR when both proteins are overexpressed (Hwang et al., 1995). Through activation of EGFR, E5 can interfere with several signal transduction pathways, including the mitogen-activated protein (MAP) kinase pathway (Crusius et al., 1997). However, similarly to BPV E5 (Faccini et al., 1996; Ashrafi et al., 2000), HPV 16 E5 inhibits gap-junction intercellular communication (Oelze et al., 1995) and withdraws transformed cells from the homeostatic control of neighbouring cells. Also similarly to BPV E5 (Goldstein et al., 1991; Faccini et al., 1996), HPV 16 E5 binds the 16k protein subunit c of the vacuolar H+ATPase (v-ATPase) (Conrad et al., 1993; Adam et al., 2000). The interaction between BPV E5 (and HPV 16 E5) and the 16k subunit c is considered to be responsible for the lack of acidification of the cellular endomembrane compartments, including the Golgi apparatus (Schapiro et al., 2000) and endosomes (Straight et al., 1995), and, as a consequence, the impeded transport of proteins as is the case for major histocompatibility complexes (MHC) class I and II (Ashrafi et al., 2002; Zhang et al., 2003; Ashrafi et al., 2005). HPV 16 E5 can also inhibit apoptosis induced by Fas-ligand and tumour necrosis factorrelated apoptosis-inducing ligand (TRAIL) (Kabsch & Alonso, 2002) and by ultraviolet (UV) light (Zhang et al., 2002). (e)

E6

The best known property of the E6 proteins of high-risk HPVs is the inability to bind and degrade the tumour-suppressor protein p53 through the recruitment of the protein ligase, E6-associated protein (E6-AP) (Scheffner et al., 1990; Huibregtse et al., 1993). This results in inhibition of the transcriptional activity of p53 (Lechner et al., 1992; Mietz et al., 1992) and the abrogation of p53-induced apoptosis, including apoptosis induced by E7 through the destabilization of p105Rb (Jones et al., 1997a). In addition, E6 binds to numerous other cellular proteins that can be divided into four broad classes: transcrip-

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tional co-activators, proteins involved in cell polarity and motility, tumour suppressors and inducers of apoptosis, and DNA replication and repair factors. Several proteins belong to more than one class. Proteins that belong to the first class are p300 (Patel et al., 1999; Zimmermann et al., 1999), myc (Gross-Mesilaty et al., 1998) and interferon regulatory factor 3 (Ronco et al., 1998); those that belong to the second are paxillin (demonstrated for BPV 1 E6; Tong & Howley, 1997; Tong et al., 1997; Vande Pol et al., 1998), the mammalian homologue of Drosophila discs large tumour-suppressor gene product (Kiyono et al., 1997; Lee et al., 1997; Gardiol et al., 1999), Scribble (Nakagawa & Huibregtse, 2000), membrane-associated guanylate kinase with inverted orientation (MAGI-1) (Glaunsinger et al., 2000) and multiple PD2 protein 1 (MUPP1) (Lee et al., 2000); those that belong primarily to the third group are p53 (Scheffner et al., 1990) and Bak (Thomas & Banks, 1999); and those that belong to the fourth class are mcm7 (Kühne & Banks, 1998; Kukimoto et al., 1998), XRCC1 (Iftner et al., 2002) and O6-methylguanine–DNA methyltransferase (Srivenugopal & Ali-Osman, 2002). Additional proteins that interact with E6 have been described by Mantovani and Banks (2001). E6 induces the expression and activity of telomerase (Klingelhutz et al., 1996; Gewin & Galloway, 2001; Oh et al., 2001; Veldman et al., 2001); this activation of telomerase has been purported to be responsible for cell immortalization by E6, although the precise mechanism by which E6 achieves this effect is still unclear (see Section 4.1.3). Through the interactions described above, E6 can affect transcriptional pathways, disrupt cell adhesion and architecture, inhibit apoptosis, abrogate DNA damage responses, induce genome instability and immortalize cells. (f)

E7

The biochemical and biological properties of the E7 protein of HPV are described in detail in Sections 4.1.2 and 4.1.3 and in Zwerschke and Jansen-Dürr (2000) and Münger et al. (2001). The main cellular partner of E7 is the tumour-suppressor protein p105Rb (Dyson et al., 1989; Münger et al., 1989a). Association of E7 with p105Rb causes its degradation (Boyer et al., 1996), and leads to the loss of p105Rb control over E2F transcription factors (Phelps et al., 1991; Chellappan et al., 1992). In addition to binding p105Rb, E7 can bind to p107 and p130, two other members of the family of pocket proteins (Dyson et al., 1992; Davies et al., 1993). E7 complexes with cyclins (Dyson et al., 1992; Arroyo et al., 1993; Tommasino et al., 1993; McIntyre et al., 1996) and inactivates the cyclin-associated kinase inhibitors p21cip1 and p27kip1 (Funk et al., 1997; Jones et al., 1997b; Zerfass-Thome et al., 1996). The interactions with pocket proteins underlie the ability of E7 to immortalize cells and to abrogate normal responses to DNA damage (Helt et al., 2002); in addition, interaction with negative cell cycle regulators leads to unscheduled cell proliferation (Malanchi et al., 2004). Other partners of E7 include the S4 subunit of the 26 S proteasome (Berezutskaya & Bagchi, 1997), Mi2beta, a component of the nuclease remodelling and deacetylase (NURD) histone complex (Brehm et al., 1998, 1999), the fork head domain transcription factor, MPP2 (Lüscher-Firzlaff et al., 1999), the

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transcription factor, activator protein 1 (AP-1) (Antinore et al., 1996), insulin-like growth factor binding protein 3 (Mannhardt et al., 2000), TBP (Massimi et al., 1997; Phillips & Vousden, 1997), TBP-associated factor-110 (Mazzarelli et al., 1995) and a novel human DnaJ protein, hTid-1 (Schilling et al., 1998). Another important aspect of the biology of E7, independent from p105Rb binding, is its ability to destabilize centrosomes, which causes mitotic defects and genome instability (Duensing & Münger, 2001, 2003). These interactions contribute to the interference of E7 in transcription and signal transduction pathways and in DNA repair. ( g)

L1

L1 is the major structural protein of papillomaviruses. The conformation of L1 in the virion has largely been elucidated through the use of VLPs (Zhou et al., 1992; Kirnbauer et al., 1992; Hagensee et al., 1993). VLPs are empty capsids that are assembled in tissue culture cells through the overexpression of either L1 alone or L1 plus L2. HPV 16 L1 assembles into regular 72-pentamer T=7 capsids and complex loops protrude from the surface of the capsomer structure (Chen, X.S. et al., 2000). Virions or VLPs bind to cells but dissociated capsomeres do not, which implies that interactions between capsomeres are necessary for receptor binding (Volpers et al., 1995). The binding of HPV VLPs or BPV virions to a variety of cell lines of different origin from a broad range of animal species suggests that the cell surface receptor for papillomavirus is widely expressed and evolutionarily conserved (Roden et al., 1994; Müller et al., 1995; Volpers et al., 1995). The strict host range and tissue specificity of the papillomaviruses led to the original hypothesis that an epithelium-restricted receptor existed. The promiscuity of virus binding suggests that specificity is determined by some post-binding event. However, the above results do not rule out the presence of a secondary receptor that confers specificity to a generic primary receptor, and evidence suggests that L2 may bind to a secondary viral receptor (Kawana et al., 2001; Yang, R. et al., 2003a). The cell receptor is (or has) probably a protein component, because treatment of the cell surface with trypsin prevents binding to VLPs (Müller et al., 1995; Roden et al., 1995; Volpers et al., 1995), but its nature is still elusive. Several candidate receptors have been proposed, such as integrin α6β1, integrin α6β4 (Evander et al., 1997; McMillan et al., 1999; Yoon et al., 2001) or the Ig receptor, FcγRIII (CD16) (Da Silva et al., 2001a). However, subsequent studies have not confirmed a prerequisite role for α6 integrin in papillomavirus infection (Sibbet et al., 2000; Giroglou et al., 2001a; Shafti-Keramat et al., 2003), and the role of CD16 as a papillomavirus receptor needs confirmation. Also cell-surface glycosaminoglycans (GAGs) have been suggested to be the primary receptors of papillomaviruses (Joyce et al., 1999). Sequence comparison between L1 of different papillomaviruses suggests a conserved heparin-binding domain at the C-terminus and cleavage of this domain from L1 prevents its binding to both heparin and human keratinocytes. In addition, GAGs are critical for papillomavirus infection: Chinese hamster ovary cells deficient in GAG synthesis bind VLPs very poorly, and K562 cells, which

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express very little surface GAG, bind small amounts of VLPs but bind larger amounts of VLP when they express exogenous syndecan (Joyce et al., 1999; Giroglou et al., 2001a; Selinka et al., 2002; Drobni et al., 2003; Shafti-Keramat et al., 2003). GAGs therefore appear to be the best candidates for the primary papillomavirus receptor. L1 VLPs are highly immunogenic (Kirnbauer et al., 1992), present conformational virus-neutralizing epitopes (Ludmerer et al., 1997; White et al., 1998; Carter et al., 2003) and can be used to detect HPV antibodies in the sera of patients with high specificity (Kirnbauer et al., 1994) (see Section 1.2.1). (h)

L2

L2 is the minor capsid protein of papillomaviruses. Despite the paucity of L2 in the virion, this protein has recently been shown to have many more functions than a purely structural role. L2 contributes to the binding of virions to the cell receptor(s), facilitates virion uptake and transport to the nucleus, delivers the viral DNA to replication centres, helps the packaging of the viral DNA into capsids and, by virtue of the presence of a neutralization epitope common to L2 proteins of many papillomaviruses, may be instrumental in conferring immunity across different types of virus. L2 contributes to the interaction of the virion with the cell surface. Two distinct regions in the N-terminal portion of L2 interact with the cell surface; in one case, interaction takes place after binding of the capsid (Kawana et al., 2001; Yang, R. et al., 2003a). These results suggest that multiple cell receptors for papillomaviruses exist and that, after an initial low-specificity interaction between L1 and the cell surface, a conformational switch takes place in the capsid that allows exposure of L2 epitopes and interaction with a more specific secondary receptor. The hypothesis of a conformational change in the capsid is supported by the observation that L2 from animal and human papillomaviruses induces neutralizing antibodies as a linear protein but not when assembled in the capsid (Christensen et al., 1991; Lin et al., 1992; Chandrachud et al., 1995; Campo et al., 1997; Kawana et al., 1999; Roden et al., 2000). The L2 neutralizing epitope is conserved among papillomaviruses, which raises the possibility of its use in cross-protective vaccines (Kawana et al., 2003). HPV 16 L1/L2 VLPs or VLPs of L1 alone are taken up by the cell with similar kinetics. However, L1 VLPs remain widely distributed in the cytoplasm whereas L1/L2 VLPs exhibit a radial distribution across the cytoplasm and accumulate in the perinuclear region, suggesting that L2 helps the transport of the capsids across the cytoplasm. This transport was inhibited by cytochalasin B, an actin-depolymerizing agent, and an Nterminal peptide of L2 binds directly to actin, which raises the possibility that papillomavirus capsids travel along actin cables (Yang, R. et al., 2003b). Other possibilities have, however, been considered; one is that the capsids infect cells via a clathrin-dependent pathway (Day et al., 2003). Input L2 deposits viral DNA at ND10, an event that is critical for the efficient transcription and replication of the viral genome (Day et al., 2004) and is supported by the recruitment of E2 to ND10s (Day et al., 1998). At later stages of the virus life cycle, the

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binding of newly synthesized L2 to viral DNA (Zhou et al., 1994) and dispersal of ND10s by E4 (Roberts et al., 2003) would facilitate capsid assembly. 1.1.6

Regulation of gene expression

The regulation of gene expression in genital human papillomaviruses has been reviewed (Bernard, 2002). (a)

Organization of the LCR

The regulation of gene expression in papillomaviruses is controlled by cellular and viral transcription factors, different promoters, differential splicing, differential transcription termination signals and the stability of different viral mRNAs. In order to be successful — from a viral point of view — the process of gene regulation should achieve: (a) epithelial-specific transcription; (b) differential expression of virus-specific genes during differentiation of squamous epithelia, in particular the switch from early to late genes; (c) feedback control by viral gene products, which may play an important role in the persistence of papillomavirus infection; and (d) response to physiological factors of the infected host on viral gene expression. Many or all of these phenomena are deregulated during malignant progression of virus-induced lesions. Most of the regulatory events mentioned above are controlled by protein factors that are bound to cis-responsive elements in the LCR of the virus. The LCRs of most genital HPVs range in size from 800 to 900 bp (about 12% of the viral genome) and have a similar organization of cis-responsive elements (Stünkel & Bernard, 1999). Figure 6 is a schematic representation of the LCR of HPV type 16: the four E2 protein-binding sites are typical for the LCRs of all genital HPVs. The first and second E2 binding sites from the 5′ end divide the LCR into three functionally distinct segments (O’Connor et al., 1996). (i) The 5′ segment The 5′ segment of the LCR is about 300 bp long and is flanked by the translation termination codon of the L1 gene and the first E2 binding site. It contains a nuclear matrix attachment region (Stünkel et al., 2000), transcription termination and polyadenylation sites for late transcripts, as well as a negative regulatory element that acts at the level of late mRNA stability (Kennedy et al., 1991). The central segment functions as an epithelialspecific transcription enhancer; it fails to activate transcription from heterologous promoters in non-epithelial cell types (Gloss et al., 1987; Cid et al., 1993; Taniguchi et al., 1993). This is probably an important mechanism for the epithelial tropism of HPVs. This enhancer is modulated by physiological factors such as steroid hormones and by intracellular signalling pathways downstream of membrane-bound receptors. A large number of cellular transcription factors have been reported to bind to about 20 different sites in this part of the LCR.

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Figure 6. A schematic representation of the HPV 16 LCR, which can be considered as a model for the LCRs of all genital HPVs

Modified from O’ Connor et al. (1996) Four E2 binding sites serve as landmarks, and two of them divide the LCR into functionally distinct segments, which have been called the 5′, the central and the 3′ segments. The 5′ segment contains the transcription termination signal, denoted ‘pA’; the central segment contains the epithelial specific enhancer that constitutes the majority of transcription factor binding sites; and the 3′ segment contains the origin of replication and the E6/E7 promoter. All the transcription factor binding sites are denoted by the abbreviation used in the text with the exception of TEF-1 which is denoted TF1.

Epithelial specificity refers to the capacity of viruses or genomic constructs to stimulate strongly homologous and heterologous promoters in cells that express epithelial markers such as certain keratin genes. This activity is similar in cells that derive from cutaneous, squamous mucosal and mucosal epithelia. The same constructs demonstrate very little activity in endothelial or hepatic cells in spite of their capacity to express keratin, and no activity in cells of other differentiation types, such as fibroblasts or lymphoid cells (Cripe et al., 1987; Gloss et al., 1987; Chong et al., 1991). Epithelial specificity of genital HPVs is regulated by epithelial-specific transcription factors that bind to specific sites in the LCR. Non-genital HPVs have much lower enhancer activity. The activity of the enhancers is counterbalanced by silencers, which are located between the enhancer and the promoter. Their principal function appears to be repression of transcription in the basal layer of infected epithelia. The low transcriptional activity of the virus in these cells reflects the low level of gene expression required during most of its life cycle (Sailaja et al., 1999). The enhancers of many genital HPVs are activated by glucocorticoid and progesterone receptors (Gloss et al., 1987; Pater et al., 1988; Chan et al., 1989; Cid et al., 1993) which result in increased expression of the E6 and E7 genes. Mechanistically, gluco-

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corticoid and progesterone act through the same cis-responsive elements. Different elements that might mediate responses to estrogen, testosterone or retinoids have not been determined to date. A repressive effect of retinoids on HPV gene expression has been observed (Bartsch et al., 1992). (ii) The 3′ segment The 3′ segment of the LCR is the region between the second E2 binding site and the translation start codon of the E6 gene. It is about 140 bp long and contains a single E1 binding site, which identifies the origin of replication. The transcription start site, which is only about 5 bp upstream of the ATG codon of E6, is located about 90 bp downstream of the E1 binding site. A segment of about 45 bp within these 90 bp contains an Sp1 transcription factor binding site, two E2 binding sites and a TATA box (O’Connor et al., 1996; Stünkel & Bernard, 1999). Together, these sites provide a complex system that can modulate the promoter activity of E6/E7. The factor Yin Yang 1 (YY1) can both repress and stimulate a number of viral and cellular promoters (Shi et al., 1997). Each of the three segments of the LCR of HPV 16 and 18, and possibly of all genital HPVs, has one or multiple YY1 binding sites (Bauknecht et al., 1992; May et al., 1994; Bauknecht et al., 1995; Lee et al., 1998). Some of these binding sites repress E6/E7 transcription. Repression is relieved by mutational change of some YY1 sites in vivo, which results in mutant genomes with increased carcinogenicity (May et al., 1994). Regulation of expression of the late genes in genital HPVs is not well understood. The analysis of late gene expression was greatly facilitated by the availability of organotypic raft cultures that mimic differentiating epithelium. Exposure of CIN612 cells that contain episomal copies of HPV 31 to activators of protein kinase C in raft culture led to the induction of a bi-cistronic E1^E4-L1 RNA from a newly identified differentiation-dependent promoter at position 742 within the E7 ORF (Hummel et al., 1995). Time-dependent expression profiling analysis revealed a peak of late RNA expression at day 12 after exposure of the raft culture to the air–liquid interface (Ozbun & Meyers, 1997). Similarly, a differentiation-dependent late promoter has been identified at position 670 within the E7 ORF of HPV 16 (Grassmann et al., 1996). A promoter (P7535) has been located in the 5′ part of the LCR of HPV 8 and has been shown to give rise to transcripts that encode late genes. Surprisingly, this promoter is stronger in transient transfections in tissue culture than the E6/E7 promoter of this virus (Stubenrauch & Pfister, 1994). 1.1.7

Methylation status of cytosine in CpG sequences in the viral genome

Little is known about epigenetic factors that are associated with the progression of HPV infection from the subclinical stage to invasive carcinoma. In the context of the viral life cycle, there is evidence of de-novo mechanisms of methylation at cytosine residues in CpG sequences within the viral LCR in poorly differentiated cervical epithelial cells obtained from a grade 1 cervical intraepithelial neoplasia (CIN1) lesion. These cells

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harbour the viral DNA as a nuclear plasmid; this methylation is lost as cells differentiate and the viral genome is amplified. The methylation pattern seen in poorly differentiated cells includes methylation of E2 binding sites, which probably suppresses E2-mediated transcriptional regulation of the viral genes (Kim, K. et al., 2003b). The recognition by E2 of its cognate DNA binding site is sensitive to CpG methylation (Thain et al., 1996), which could explain why expression of genes from the viral genomes in these cells is unresponsive to exogenous E2 (Bechtold et al., 2003). However, in derivative cell lines that contain viral DNA in the integrated form, viral genes regain responsiveness to exogenous E2 protein (Bechtold et al., 2003), which raises the possibility that the methylation pattern of the viral genome is altered upon integration. Consistent with this prediction, the LCR is hypomethylated in the single integrated copy of HPV 16 in SiHa cells. Furthermore, in an analysis of 81 patients from two different cohorts, the LCR of HPV 16 DNA was hypermethylated in 52% of asymptomatic smears, 21.7% of precursor lesions and only 6.1% of invasive carcinomas. This suggests that neoplastic transformation is inversely correlated with methylation of CpG, and that demethylation occurs before or concomitantly with neoplastic progression (Badal et al., 2003). A similar study with HVP 18 gave comparable results (Badal et al., 2004). 1.1.8

Replication

The replication of papillomavirus DNA has been reviewed (Lambert, 1991; Melendy et al., 1995; Wilson et al., 2002; Longworth & Laimins, 2004). After initiation at a single site within the LCR, replication of papillomavirus DNA proceeds bi-directionally (Yang & Botchan, 1990; Flores & Lambert, 1997). E1 is the essential origin-recognition protein for papillomavirus replication. In-vitro studies have shown that replication starts at a single E1 binding site that is located in the 3′ segment of the LCR (see Section 1.1.6). In genital HPVs, it lies approximately half way between the two E2 binding sites near the promoter and the single E2 binding site on the 5′ side of this segment; it is an A/T-rich region with only low sequence conservation (Mohr et al., 1990; Lu et al., 1993). E1 protein forms heteromers with E2 protein in solution. These heteromers stimulate initiation of replication by modulating recognition of the E1 binding site through binding of E2 to either of two flanking sites (Sverdrup & Kahn, 1995). The resultant helicase complex initiates the unwinding of DNA at the origin of replication to provide the template for subsequent synthesis of progeny DNA (Rocque et al., 2000). Due to the overlap of alternative cis-responsive elements involved in E1/E2 binding to DNA, replication can repress transcription from the E6 promoter (Sandler et al., 1993). The E1 protein–DNA complex initiates replication and requires additional cellular factors similar to those required for the replication of SV40 (Seo et al., 1993). These factors include replication protein A, replication factor C, proliferating-cell nuclear antigen (PCNA) and DNA polymerase alpha-primase and DNA polymerase delta. Both polymerases (also known as phosphocellulose column fraction IIA) are essential for the replication of viral DNA in vitro (Melendy et al., 1995).

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Papillomaviruses control the copy number of their genomes in infected cells, which is a prerequisite for episomal maintenance during persistent infection. This process is not under cellular control but involves the viral sequence-specific DNA-binding E2 activator and E8^E2C repressor proteins. E2 repressor proteins have been demonstrated to counteract transcriptional activation by E2 and to inhibit the E1/E2-dependent replication of papillomavirus origins (Lambert et al., 1990; Bouvard et al., 1994a; Stubenrauch et al., 2000). All E2 repressor proteins lack the amino-terminal domain of E2 that is responsible for activation of transcription and DNA replication but retain the carboxy-terminal domain that mediates specific DNA recognition and dimerization among E2 proteins (McBride et al., 1991). The E8^E2C repressor proteins consist of the peptide sequence from the small E8 ORF fused to the C-terminus of E2. E8^E2C transcripts were shown to be present throughout the entire replication cycle of HPV 31. The E8^E2C protein of HPV 31 strongly repressed the basal activity of the major viral early promoter P97 independently of E2. Mutation in the E8 gene and disruption of the fusion protein led to a 30–40-fold increase in the transient DNA replication levels in both normal and immortalized human keratinocytes. The results suggest that the E8^E2C protein plays a role in the control of copy numbers (Zobel et al., 2003). In addition to its role in modulating viral gene expression and DNA replication, E2 also plays an important role in the efficient segregation of papillomaviral replicons to daughter cells during cell division through its capacity to bind its cognate E2 binding sites in the viral genome (Piirsoo et al., 1996). E2 is thought to tether the viral genome to the host chromosomes during mitosis (Calos, 1998; Lehman & Botchan, 1998; Skiadopoulos & McBride, 1998; Ilves et al., 1999; Bastien & McBride, 2000). This is probably mediated by the interaction of E2 with a cellular Brd4 bromodomain protein (You et al., 2004; Brannon et al., 2005). 1.2

Serological response

The study of immunity to HPV has long been hampered by the difficulty in obtaining HPV virions from cell cultures since production of the infectious virus is strictly linked to epithelial cell differentiation. Initial studies used virions obtained from pooled material derived from warts. Experimental inoculation of BPV, CRPV or HPV (mainly HPV 1) virions into animals has indicated that denaturated virions elicit antibodies that are broadly cross-reactive among papillomavirus types, whereas intact virions induce antibodies that are largely typespecific. Determination of the DNA sequence of several HPV types has allowed molecular biologists and immunologists to clone specific viral genes, to produce structural and regulatory viral proteins by the use of various expression vectors, and to design synthetic peptides. The experimental production of HPV 11 virions in nude mice also provided a new source of antigen. Data from these initial studies showed little sero-reactivity to denaturated virions or denaturated viral proteins, which suggests that antibodies produced

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by HPV-infected patients mostly recognize conformational epitopes on the surface of the virus (Galloway, 1992, 1994). The discovery that L1 protein can assemble into VLPs that are structurally and immunochemically indistinguishable from authentic virions (Kirnbauer et al., 1992; Hagensee et al., 1994) has provided a valuable tool for the characterization of conformational HPV surface epitopes and strongly stimulated studies aimed at the design of prophylactic and therapeutic vaccines (for review, see Breitburd & Coursaget, 1999; Lowy & Schiller, 2006). 1.2.1

Antigenic properties of HPV virion proteins

Papillomavirus capsids are non-enveloped icosahedrons that comprise a major capsid protein, L1, and a minor capsid protein, L2 (Orth & Favre, 1985). L1 can assemble on its own into pentameric structures or capsomers, 72 of which in turn assemble into capsids or VLPs that are structurally and immunochemically indistinguishable from authentic virions (Kirnbauer et al., 1992; Hagensee et al., 1994). The repetitive structure of the capsids is highly immunogenic. Vaccination with L1 VLPs generates high-titre antibodies that are neutralizing and can protect against infection (Breitburd et al., 1995; Suzich et al., 1995; Kirnbauer et al., 1996; Koutsky et al., 2002). Recently, the crystallographic structure of a T=1 L1 VLP was determined (Chen, C.H. et al., 2000). An alignment of 49 HPV L1 gene sequences showed that residues exposed on the surface were not conserved between types and were located on hypervariable loops (see Figure 7). In contrast, highly conserved residues of L1 were located below the surface of the capsomer. Consistent with this observation, neutralizing antibodies have been shown to react with conformational epitopes of L1 that are predominantly type-specific (Hines et al., 1994; Roden et al., 1996a; Carter et al., 2000). Conformation-dependent neutralizing epitopes are present not only on capsids or VLPs, but are also retained on individual capsomers (Rose et al., 1998; Yuan et al., 2001). A revised model for HPV VLPs was proposed by Modis et al. (2002). In this model, the C-terminal extension adopts a conformation similar to that in the T1 structure but, instead of returning to the capsomer of origin, the arm is displaced onto, and ultimately invades, a neighbouring capsomer. A consequence of the invading arm model is that residues on the C-terminal arm would be accessible on the surface. It was noted that several amino acids in this C-terminal region are divergent among HPV types and, thus, may be important for recognition by type-specific antibodies. A broadly cross-reactive epitope is also found on L1 molecules, but this is folded within the virion and is only immunoreactive when denatured L1 is used as the immunogen (Firzlaff et al., 1988; Jin et al., 1990). Antibodies raised against denatured L1 proteins have been useful in immunohistochemical assays to detect HPV infection. L2 is incorporated into capsids, probably at the 12 pentavalent vertices (Trus et al., 1997). While L2 is not necessary for capsid formation, it is essential for genome encapsidation and infectivity (Roden et al., 1996a). Although much of it remains inside the capsid, a small segment of L2 is exposed on the surface and can induce neutralizing anti-

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Figure 7. Molecular structural model of the HPV 6 major capsid protein L1

From Orozco et al. (2005); see cover Surface-exposed loops are indicated by arrows.

bodies (Christensen et al., 1991; Campo et al., 1997b; Kawana et al., 1999, Roden et al., 2000). Neutralizing antibodies directed against L2 tend to be much less potent than those generated against L1 (Christensen et al., 1991; Roden et al., 2000). The immunogenic epitopes along the L1 and L2 proteins have been determined in two ways: first, by generating murine monoclonal antibodies to either denatured L1 or L2 proteins or to VLP proteins and, second, by mapping immunogenic epitopes that arise as a consequence of natural infection. The generation of monoclonal antibodies to VLPs has given rise to a variety of antibody types including those that were conformation-dependent and type-specific, those that were both type-specific and cross-reactive to linear epitopes and a few that were cross-reactive with intact VLPs to varying extents (Christensen et al., 1990; Sapp et al., 1994; Christensen et al., 1996a). [The Working Group noted that inoculation of experimental animals with large amounts of VLPs, some of which could be improperly folded, may result in antibody types that are not usually produced in natural infection.] All conformation-dependent type-specific monoclonal antibody epitopes identified to date have been found to reside on one or more hypervariable loops on the surface of VLPs (see Figure 7 and Table 4). H16.V5 was characterized as a complex epitope composed of multiple regions, the FG and HI loops (Christensen et al., 2001). It was further shown that both of these loops were necessary for the transfer of HPV 16-specific binding onto HPV 31 chimeric VLPs (Carter et al., 2003). It has been proposed that the F50L point mutation disrupts the binding of H16.V5 and H16.E70 (White et al., 1999) by altering the conformation of residues on the FG loop (Chen, C.H. et al., 2000). An alternative hypothesis is that this mutation changes the conformation of the BC loop to which it is adjacent. To address this question, hybrid VLPs were created in which the HPV 52 BC loop

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Table 4. Location of conformational L1 epitopes recognized by monoclonal antibodies raised against HPV 6, 11, 16 and 31 VLPs and/or virions Antibody

Regiona

Amino-acid position

Region(s) required to transfer binding

H6.B10

BC, EF

49–54, 170–179

BC, EF

H6.M48 H6.N8

49–54, 170–179 49–54

BC, EF BC

H11.A3

BC, EF BCb and FGc or DEc BC, EF

49–54, 170–179

BC, EF

H11.B2

DE, FG

131, 132, 246, 278

DE, FG

H11.H3

DEb, FG, HI

132, 246, 346

HI

H16.V5

FG, HI

260–290, 345–363

FG, HI

H16.E70

DE, FG

130–143, 260–290

H16.U4

C-terminal arm EF

425–445

H31.A4

Required for binding

175–186

EF

Reference

Christensen et al. (1996a); Wang, S.S. et al. (2003) Christensen et al. (1996a) Christensen et al. (1996a); Wang, S.S. et al. (2003) Christensen et al. (1990a); Ludmerer et al. (1997) Christensen et al. (1990a); Christensen et al. (1996a,b); Ludmerer et al. (1996) Christensen et al. (1990a); Ludmerer et al. (1996) White et al. (1999); Christensen et al. (2001) Christensen et al. (1996b); White et al. (1999) Christensen et al. (2001); Carter et al. (2003) Carter et al. (2006)

VLP, virus-like particle a See Figure 7 b Mutations in this region resulted in a partial reduction in binding. c Mutations in this region showed a reduction in binding only when combined with other mutations.

was substituted onto the HPV 16 L1 backbone and the HPV 16 BC loop onto the HPV 52 L1 backbone. HPV 16 VLPs with an F50L mutation were shown to be degraded by trypsin, which indicates a failure to fold correctly; thus F50 is probably not part of the epitope (Carter et al., 2003). Residues at both ends of the FG loop were shown to be involved in the binding of H16.V5 and H16.E70. To determine which residues were important for antibody binding, a series of point mutations and smaller regional mutations along the FG loop were examined. VLPs with four intertypic substitutions between amino acids 260 and 273 (16:260–273) and VLPs with three substitutions between positions 285 and 290 (16:285–290) showed substantial loss of reactivity to H16.V5 and H16.E70 (Carter et al., 2003). Previous studies had shown that residues 266 and 282 were important for H16.E70 binding but not for H16.V5 binding (Roden et al., 1997a; White et al., 1999). None of the point mutations tested (A266T, N270S, N285T, S288N, N290T) were found to be essential for H16.V5 binding. H16E.70 binding was more sensitive to point mutations in the FG loop; the greatest loss of binding was to VLPs with substitutions at positions 285, 288 and 266.

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A polar residue at position 270 was important for both H16.V5 and H16.E70 binding because substitution of Asn270 with Ala strongly reduced antibody reactivity (Carter et al., 2003). Both H16.V5 and H16.E70 showed reduced binding to 16:N270S VLPs, but binding to 16:N270A VLPs was more strongly reduced. Although Ser and Ala are amino acids of similar size (somewhat smaller than Asn), Ser has a polar side-chain that can participate in a hydrogen bond similarly to Asn. Thus, the data suggest that Asn270 participates in a hydrogen bond that is important for antibody recognition of the FG loop by both H16.V5 and H16.E70. H16.E70 was found to be a complex epitope because both the FG and DE loops were necessary for binding. The DE loop has also been shown to be essential for binding to HPV 11 by several monoclonal antibodies (Ludmerer et al., 1996, 1997). However, Christensen et al. (2001) found that H16.E70 binding could be transferred to HPV 11/16 hybrid VLPs that did not contain the HPV 16 DE loop but possessed the HPV 16 C-terminus from residue 172 onward. A new antibody-binding site was discovered on the C-terminal arm of L1 between positions 427 and 445 (Carter et al., 2003). Recognition of these residues by the H16.U4 antibody suggests that this region is exposed on the surface and supports a recently proposed molecular model of HPV VLPs (Modis et al., 2002). 1.2.2

Immune response to papillomavirus infection

Generally there is little evidence of cross-reactive papillomavirus antibodies in human sera. Three lines of evidence support the notion that antibody responses to HPV infection are type-specific: first, reaction of a collection of sera against a panel of HPV 6, HPV 16 and HPV 18 capsids showed that individual sera reacted differently to the three capsids (Carter et al., 2000); second, pre-adsorption experiments suggest that sera that react with multiple HPV capsids contain multiple type-specific antibodies, rather than cross-reactive antibodies; third, there is a stronger correlation between seropositivity to a specific HPV capsid and detection of that type of HPV DNA than detection of any other type of HPV DNA (Kirnbauer et al., 1994; Carter et al., 2000). The most consistent result from studies that investigated the immune response to HPV infection was the finding that the presence of antibodies to HPV 16 E7 protein was associated with cervical cancer at relative risks that ranged from 2.5 to 30 (JochmusKudielka et al., 1989; Mann et al., 1990; Mandelson et al., 1992; Müller et al., 1992; Hamsikova et al., 1994; Sun et al., 1994a), and with oral and oropharyngeal squamouscell carcinomas (Zumbach et al., 2000a; Herrero et al., 2003). Antibodies to E6 protein were also found to be elevated in cervical, oral and oropharyngeal cancer patients compared with controls (Meschede et al., 1998; Zumbach et al., 2000a,b; Herrero, 2003), as were antibodies to HPV 18 E6 and E7 in some reports. Among cases whose tumours contained HPV 16 DNA, seropositivity ranged from 25 to 50%. There was no elevation of seropositivity among individuals with preneoplastic lesions such as carcinoma in situ, and some studies even observed the strongest association with late-stage cervical cancer

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(Fisher et al., 1996; Baay et al., 1995, 1997). This has led to the hypothesis that antibodies to E6 or E7 develop as a consequence of prolonged exposure to the tumour. However, antibodies to E6 or E7 do not serve as prognostic markers for progression (Park et al., 1998a; Lehtinen et al., 2003); nor do they predict poor survival, irrespective of the stage (Silins et al., 2002). Antibodies to E2 or E4 have also been associated with cervical cancer and CIN in some studies (Dillner et al., 1989; Jochmus-Kudielka et al., 1989), but not in others (Mann et al., 1990; Mandelson et al., 1992). In rabbits infected with CRPV, antibodies to E2 but not E4 were found in about one-third of animals bearing either papillomas or carcinomas (Lin et al., 1993). Human serum antibodies that react with fusion proteins or synthetic peptides of HPV have been found in individuals without genital tract cancers in a number of studies (Dillner, 1990; Jenison et al., 1990; Köchel et al., 1991). The major antigen targets appear to be the capsid proteins, in particular 6 L1, 6 L2, 16 L2 and 18 L2. Antibodies to E2 and E7 were less frequently and those to E4 were occasionally observed. Some studies found interesting correlations between seropositivity and HPV-related disease or detection of HPV DNA (Van Doornum et al., 1994; Wikstrom et al., 1995). However, in other studies, the prevalence of HPV antibodies was not strongly associated with other parameters of HPV infection (Jenison et al., 1990; Köchel et al., 1991). To date, only a few seroepidemiological studies have used assembled HPV 1 VLPs. Carter et al. (1994) examined the prevalence of HPV 1 antibodies in 91 college women of whom 60% were seropositive. Among those with a history of foot warts, 89% were seropositive. The level of reactivity to HPV 1 was higher among subjects for whom foot warts were reported recently and lower among those who reported having foot warts 5–10 years previously. HPV 6 or 11 VLPs have been used to measure seroreactivity in several studies (Carter, J.J. et al., 1995; Wikstrom et al., 1995; Eisemann et al., 1996; Carter et al., 2000). In general, there was a strong association between the detection of HPV 6/11 antibodies in individuals in whom HPV 6 DNA or genital warts were detected. The strongest association between seropositivity and genital warts was seen among women with recurrent warts. This may suggest that repeated or prolonged exposure to HPV antigens is necessary to develop a detectable antibody response. Enzyme-linked immunosorbent assay (ELISA) seropositivity was not correlated with past or present genital warts among men, in spite of higher mean ELISA values for men with genital warts versus men without genital warts (Carter, J.J. et al., 1995). Men have been shown to have lower levels of seropositivity to other sexually transmitted diseases and this may reflect a reduced expression of viral antigens or less accessibility to the immune system. More studies in men are needed to confirm these observations. A large number of studies have examined seroreactivity using HPV 16 VLPs. A comparison of the percentage of positive results among these studies is difficult because of differences in the choice of the cut-off points. Seropositivity to HPV 16 L1 was first examined in 122 women who attended health clinics for women and students (Kirnbauer

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et al., 1994). Using a cut-off point based on women with no detectable HPV DNA in the genital tract, 6% of women with no HPV DNA were seropositive compared with 59% of women with HPV 16 DNA, 31% of women with HPV 18 DNA and 38% of women with HPV 31 DNA. The strongest associations were seen in women with evidence of high levels of HPV 16. For example, women who had DNA detectable by both ViraType and PCR were twice as likely to be seropositive than women in whom HPV 16 DNA was detectable by PCR only (67% versus 33%). Dysplasia was also strongly associated with seropositivity (45–75%). In another study, in which the cut-off point was chosen by selecting an optimum optical density on the basis of the specificity and sensitivity of the results, HPV 16 L1 seropositivity was examined in subjects who were enrolled in case–control studies of CIN3 and invasive cervical cancer in Spain and Colombia (Nonnenmacher et al., 1995). All cases were selected on the basis of having detectable HPV 16 DNA in the cervix. Seropositivity among cases of CIN3 was 73% and 81% in Spain and Colombia, respectively; that among cases of cervical cancer was 59% and 51%, respectively. The fact that the percentage of seropositivity was higher among cases of CIN3 may reflect the more frequent and abundant expression of L1 in premalignant lesions, although age-associated effects were not examined. In another study that examined cases of anogenital cancers, HPV 16 seropositivity ranged from 50% in HPV 16-positive vaginal cancers to 70% in HPV 16-positive vulvar cancers in situ (Carter et al., 2001), which also supports the hypothesis that intraepithelial neoplasias that probably express high levels of L1 elicit a measurable antibody response. The control populations from Colombia showed higher levels of reactivity (43 and 22% for CIN3 and cancer, respectively) than those from Spain (10 and 3%, respectively), a finding that parallels the increased risk for cervical cancer found in Colombia. The high level of HPV 16 L1 seropositivity in the Colombian controls probably reflects the high level of previous HPV infections in this group (Nonnenmacher et al., 1995). A similar result was observed when seropositivity to HPV 16 was compared between blood donors in the USA and those in Jamaica, where the rate of cervical cancer is three times higher. Jamaican blood donors had a 4.2-fold greater probability of having HPV 16 antibodies than blood donors in the USA (Strickler et al., 1999a). In many studies, monogamous women have been found to have low seroprevalences (between 2 and 7%) (Andersson-Ellström et al., 1994; Carter et al., 1996; Dillner et al., 1996; Wideroff et al., 1996; Viscidi et al., 1997; Kjellberg et al., 1999). Large-scale surveys among children under 13 years of age found seroprevalences of the order of 2% (Mund et al., 1997; af Geijersstam et al., 1999). Although there is consensus that carcinogenic genital HPVs are mainly sexually transmitted, controversial data exist regarding whether non-sexual transmission occurs. The specificity of HPV capsid serology for sexually transmitted HPV infections is at least 98% and it may be even higher if some non-sexually transmitted infections occurred among control groups of sexually inexperienced subjects.

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The natural history of HPV 16 serum immunoglobulin (Ig) G antibodies has been examined in several large studies (Carter et al., 1996, 2000; Wang et al., 2003; Ho et al., 2004; Viscidi et al., 2004; Wang, S.S. et al., 2004). In spite of differences in the populations examined, the study designs, the methodology and the choice of serological cutoff points, an overall consistent picture has emerged. HPV 16 antibodies are type-specific as shown by the fact that women with cervico-vaginal HPV 16 DNA were 8–10-fold more likely to seroconvert than women with no or other types of HPV DNA. The antibodies recognized conformational epitopes on the HPV 16 VLPs, since sera did not react with denatured VLPs or with VLPs from animal papillomaviruses. HPV 16 antibodies were slow to develop, with a median latency of 6–12 months and titres were low. Development of antibodies did not occur in all women in whom incident HPV 16 infection could be documented. Two studies found that 73% (Carter et al., 2000) and 56.7% (Ho et al., 2004) of women with incident HPV 16 infections seroconverted. The acquisition of HPV antibodies was most strongly associated with persistent infection. Persistence of antibodies generally lasted a few years, but results from long-term follow-up studies are not yet available. There is no evidence that antibodies modulate the state of disease, and it has been difficult to show that antibodies protect against re-infection, perhaps because it is difficult to distinguish between first infection or re-activation of infection. A number of cross-sectional studies have demonstrated that IgA responses, specific for HPV VLPs correlate with IgG responses or with the detection of HPV DNA of the same specific type (Heim et al., 1995; Wang, Z. et al., 1996; Sasagawa et al., 1998). Only a few longitudinal studies have been conducted (Bontkes et al., 1999; Hagensee et al., 2000). In a recent study (Onda et al., 2003) that examined the appearance of IgA antibodies following incident HPV 16 infection, the median time to antibody detection from the primary detection of HPV 16 DNA was 10.5 months for IgA in cervical secretions and 19.1 months for serum IgA. Serum IgA antibody conversion was observed less frequently and occurred later than IgA conversion in cervical secretions or serum IgG conversion. Loss of IgA antibodies was rapid — 12.0 months for IgA in cervical secretions and 13.6 months for serum IgA — whereas approximately 20% of women with serum IgG antibodies reverted within 36 months. In conclusion, the development of immune responses to HPV antigens is not well understood. This is in part due to the fact that different fusion proteins or peptides have been used in various studies, which has resulted in a lack of consistency. Because several studies have not found strong associations with disease, these approaches are receiving less attention than the VLP-based ELISAs. Seropositivity to E6 and E7 is clearly a consequence of tumour development, but it is not known whether factors other than prolonged exposure to antigen influence seropositivity.

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Methods for the detection of HPV infection

1.3.1

Non-molecular techniques for the detection of genital HPV infection

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The methods described in this section — visual inspection, colposcopy, cytology and histology — do not detect the factual presence of HPV, but are indirect methods that detect the clinical sequelae of an HPV infection, i.e. the presence of a clinically and/or histologically diagnosed CIN lesion or cancer. Consequently, estimates of sensitivity and specificity address the characteristics of the clinical and not the analytical test performance. Cytology and histology are restricted to a correlation with the presence of HPV. The use of cytology as a screening tool for cervical cancer has been reviewed (IARC, 2005). (a)

Visual inspection techniques

Direct visual inspection (DVI; also known as visual inspection with acetic acid (VIA) or with Lugol’s iodine (VILI)) requires that a woman lie in the lithotomy or supine position, a speculum is passed to visualize the cervix and the cervix is then washed with a dilute solution (3–5%) of acetic acid or with Lugol’s iodine. Thereafter, the cervix is examined with the naked eye or with a hand-held magnifying device (usually 4 × magnification) and an adequate light source. The acetic acid causes ‘whitening’ (known as ‘acetowhitening’) of epithelial cells with a high nuclear cytoplasmic ratio. The exact reason for the acetowhitening effect is not known. A range of epithelial changes appear acetowhite after the application of acetic acid, which include immature squamous metaplasia, infection of the cervix with HPV (both low- and high-risk types) and true precursors of cervical cancer. Iodine darkens the glycogen that is stored in cervical epithelial cells. Areas of immature metaplasia, neoplasia, atrophia and condyloma stain only partially or not at all. DVI has been evaluated in a number of large clinical trials, either alone or in comparison with cytology and HPV DNA testing. Definitions of a positive DVI test and training techniques have varied. Most studies have been cross-sectional in nature and have been limited by verification bias, since the ‘gold standard’ (usually colposcopy and/or biopsy) has only been applied to women with positive tests, which makes the diagnosis of disease in women with negative screening tests impossible. Verification bias tends to overestimate the specificity of the test. Most studies have used high-grade precursors of cervical cancer and/or cancer as the outcome measure. High-grade precursors of cervical cancer are known as CIN grades 2 and 3 or high-grade squamous intraepithelial lesions (HSIL), which encompasses the diagnoses of CIN2 or -3. In some of the larger cross-sectional studies, colposcopy and/or biopsy were used to establish the presence of high-grade precursors of cervical cancer or cancer (Ottaviano & La Torre, 1982; Cecchini et al., 1993; Megevand et al., 1996; Sankaranarayanan et al., 1998, 1999; University of Zimbabwe/JHPIEGO Cervical Cancer Project, 1999; Denny et al., 2000; Belinson et al., 2001a; Denny et al., 2002; Cronjé et al., 2003). A relatively

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wide range of estimated sensitivities and specificities have been reported; although all studies showed sensitivities of more than 60%, most reported relatively low specificities and positive predictive values. However, all of them reported high negative predictive values, which has important implications for national screening programmes. One very large (n > 50 000) study compared VILI with VIA (Sankaranarayanan et al., 2004a,b; IARC, 2005) and found that VILI was more sensitive than VIA and equally specific. For low-resource countries, DVI has several potential advantages, the most important of which are the simplicity of the test, its low cost, the fact that primary health care providers can be trained to perform the test in a relatively short period of time and that an immediate result is provided, which avoids the inevitable loss to follow-up that occurs when the results of the test or treatment of lesions is delayed (Sankaranarayanan et al., 1998, 1999; Denny et al., 2002; Sankaranarayanan et al., 2004a). A disadvantage of DVI is the difficulty of standardizing quality control, which is particularly important because of the subjective nature of the test. Standardization of a positive test is hindered by its subjective nature and, unlike cytology, there is no permanent record of the appearance of the cervix to allow screeners and their trainers to review the diagnosis (b)

Colposcopy

Colposcopy is a procedure that allows illuminated stereoscopic and magnified (typically × 6–40) viewing of the cervix. The woman is placed in the lithotomy position; the cervix is exposed by insertion of a bivalve speculum and various solutions (normal saline, 3–5% dilute acetic acid and Lugol’s iodine) are applied to the cervical epithelium in sequence. The aim of colposcopy is to examine the transformation zone and find areas of abnormality. The latter is defined and graded according to morphological features, namely, acetowhiteness, margins, blood vessels and iodine uptake. Terminology to describe the morphological findings in a standard fashion has evolved over the years and a grading system has been proposed (IARC, 2005). Although colposcopy continues to be used routinely as part of a standard gynaecological examination by many clinicians in some European and Latin–American countries, in the English-speaking world, it is selectively applied for diagnosis of women who are referred because of an abnormal cytological test. For this reason, studies that assess colposcopy as a diagnostic procedure are susceptible to bias and the performance of colposcopy when used for diagnostic purposes may exceed its accuracy and reproducibility when it is used as a screening tool (see Table 5). Two meta-analyses have been performed on the accuracy of diagnostic colposcopy applied to women referred with abnormal cytology. Mitchell et al. (1998) performed a systematic review of 86 articles published between 1960 and 1996, nine of which met the inclusion criteria and eight of which were eligible for meta-analysis. At the cut-off level of normal versus abnormal on colposcopy, the average weighted sensitivity, specificity and area under the receiver operating characteristic curve of histological CIN2 or more were 96%, 48% and 80%, respectively. At the cut-off level of normal and low-grade SIL

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Table 5. Sensitivity and specificity of diagnostic and screening colposcopy for the detection of HPV-related neoplastic lesions (≥ CIN2 and cancer) No. of patients

Sensitivity (%)

Specificity (%)

Reference

Diagnostic colposcopy Meta-analysis Meta-analysis

96 24–90

48 67–97

Mitchell et al. (1998) Olaniyan (2002)

Screening colposcopy 196 163 4761 1997

76 90.7 13.2 81

96 NA 99.2 77

Davison & Marty (1994) Hilgarth & Menton (1996) Schneider et al. (2000) Belinson et al. (2001b)

CIN, cervical intraepithelial neoplasia; NA, not available

(LSIL) versus HSIL and cancer on colposcopy, the corresponding results were 85%, 69% and 82%. This suggests that, independent of prevalence and compared with low-grade lesions, high-grade lesions and cancer are diagnosed with higher sensitivity. Olaniyan (2002) reviewed publications from 1966 to 2000 and the results of his meta-analysis, based on eight studies, seven of which were also included in the previous meta-analysis, were similar. A few studies have assessed the performance of colposcopy as a screening tool. In a cross-sectional study, 1997 unscreened Chinese women (aged 35–45 years) were first assessed by VIA performed by one gynaecologist, after which a second gynaecologist (blinded to the VIA results) performed colposcopy and took direct biopsies from abnormal areas (Belinson et al., 2001b). All women also had a biopsy taken from each of the four quadrants (and all had had an endocervical curettage [ECC]) in order to estimate the performance of colposcopy in a screening setting. Sensitivity and specificity of colposcopy and direct biopsy for high-grade CIN or cancer were 81% (95% confidence interval [CI], 72–89%) and 77% (95% CI, 75–78%) compared with the combined histological findings from the direct, four-quadrant and ECC specimens. A similar study in Germany enrolled 4761 women aged 18–70 years who had visited one of 10 gynaecologists for standard care. They were screened by conventional cytology (obtained under colposcopic vision), colposcopy and HPV testing of cervicovaginal samples by PCR with probes for 13 high-risk types (Schneider et al., 2000). Biopsies and ECC were performed where appropriate and, if colposcopy was normal, two biopsies and ECC were obtained. The sensitivity and specificity of screening colposcopy for detecting at least CIN2, with histological confirmation, were 13.3% (95% CI, 7.0–20.5) and 99.3% (95% CI, 99.0–99.6), respectively.

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(i)

Genital HPV infections other than HPV-associated cervical neoplasia Both the male and female genital tracts are sites where clinically overt HPV infection can occur. Genital condylomas (warts) are easily detected with the naked eye. Bright lighting is essential and a hand-held magnifying glass is helpful. A variation on the technique of cervical colposcopy, known as high-resolution anoscopy (HRA), has been used to assess anal intraepithelial neoplasia (AIN) in the anal canal and perianal region (Jay et al., 1997) using 3% acetic acid, Lugol’s solution and magnification. HRA is used to guide selection of tissues from which a biopsy should be taken for the diagnosis of AIN or anal cancer. Although most authorities agree that this test is insensitive and nonspecific (Beutner et al., 1998a), colposcopy with or without the application of acetic acid can be helpful for the detection of smaller lesions or subclinical disease in the vagina, vulva, penis, anus and perianal skin and can help guide biopsy, especially for lesions that are suspected of being SIL or malignant. Few studies were able to correlate the clinical or subclinical appearance of HPVinduced lesions with the presence of the virus at the molecular level. In men, Bleeker et al. (2005a) correlated the prevalence and size of flat condylomata, as detected by colposcopy and washing with 3% acetic acid, with penile scrapes that were positive for PCR-detected HPV and viral load: higher loads reflected higher prevalence and larger size of penile lesions. (ii) Non-genital HPV infection One earlier study (Panici et al., 1992) evaluated the ability of colposcopy to detect clinical manifestations of HPV in the oral cavity in 101 male and female patients with genital condylomata who practiced orogenital sex; most of the patients (83%) had oral condylomata that could not be seen by the naked eye. Colposcopically, the oral lesions appeared as filiform (50%), moruloid (26%) and mixed (24%). HPV DNA was detected by filter in-situ hybridization in 45% of the 20 patients sampled. (c)

Cytology and histology

Reliable detection of cytological evidence of an HPV infection is notoriously difficult. The best evaluated sign of an HPV infection is koilocytosis or koilocytotic atypia, which is the combination of nuclear atypia and the formation of a perinuclear halo (Koss & Durfee, 1955). The link between the presence of koilocytes in cervical smears and HPV was established in the mid 1970s by histological and cytological investigations (Meisels & Fortin, 1976; Purola & Savia, 1977; Della Torre et al., 1978). With the advent of molecular techniques to detect the HPV genome, it became evident that cytological and histological features are not sensitive indicators of the presence of HPV. In a majority of women who are positive for HPV DNA, no cytological or histological correlates of HPV infection can be detected (Bauer et al., 1991; Rozendaal et al., 2000). Other cytological signs such as atypia that are indicative of the presence of (precursors of) cervical cancer do not provide a diagnostic tool for HPV infection per se.

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In histological sections, the presence of koilocytes may be difficult to diagnose since fixation artefacts or poor dehydration can result in the presence of cells with perinuclear halos giving the cells a ‘koilocyte-like’ appearance. Anal cytology may also be used to diagnose AIN similarly to the use of cervical cytology to diagnose CIN (Palefsky et al., 1997a,b). Anal cytology may be classified using Bethesda criteria similar to those for cervical cytology (ASCCP guidelines, discussed in Wright et al., 2002). 1.3.2

Detection of HPV proteins in infected tissues

Immunological detection of HPV in human cells or tissues is often hindered for two main reasons: first, the late capsid proteins are only expressed in productive infections (Shah, 1992); and second, the early proteins are usually expressed in small amounts in infected tissues; in addition, the production of specific antibodies to be used for immunochemistry has long been hampered due to the lack of a suitable in-vitro culture system to obtain HPV virions (see Section 1.2). Molecular biological methods to express individual HPV antigens from any HPV type redefined the approach to produce HPV antibodies (reviewed in Galloway, 1992). Bacterial fusion proteins had several advantages: they provided an inexpensive, plentiful and reproducible source of the early and late viral antigens from any HPV type. The main disadvantage was that most fusion proteins are insoluble and had to be used in western blot assays under denaturing conditions that provide only linear epitopes. A series of type-specific antibodies have been generated from HPV recombinant proteins expressed in different heterologous systems. These antibodies can be used to demonstrate the expression of HPV proteins in biological samples using different methodologies including direct visualization in cells or tissues (immunohistochemistry) or in protein extracts (western blots and immune precipitation assays). Recently, the expression of HPV L1 protein was assessed by immunocytochemistry, using monoclonal antibodies against L1 of HPV 16 only or L1 from a pool of high-risk HPV types, in cervical smears diagnosed with LSIL or HSIL and compared with the presence of HPV DNA: 59% of the LSIL smears contained high-risk HPV DNA (types 16, 18, 33, 39, 45, 56 and 58) and 44% stained with the antibody against high-risk HPV capsid proteins; in contrast, only 33% of the HSIL were immunostained with the same antibodies while 93% were positive for HPV DNA (Melsheimer et al., 2003). This suggested that loss of L1 expression in high-grade lesions, as measured with these antibodies, could be used as a prognostic marker for cervical neoplasia. Detection of HPV early proteins is difficult due to the low expression levels generally observed in cells or tissues derived from HPV-positive lesions. Antibodies against E5, E6 or E7 are available but their use is mostly restricted to in-vitro assays (Chang et al., 2001; Fiedler et al., 2004). However, a polyclonal rabbit antiserum was recently raised by immunization with highly purified native HPV 16 E7 protein. Using this serum, HPV16 E7 could be detected by immunohistochemical staining of paraffin sections of biopsies of cervical HSIL and cervical cancer tissues (Fiedler et al., 2004).

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Since HPV infections supersede cell cycle controls, the immune detection of cell proteins that are differentially expressed in infected cells is currently being considered for use as tumour and prognostic marker, as well as for application in different modalities of cervical cancer screening (IARC, 2005). For instance, the level of expression of the cyclin-dependent kinase inhibitor p16INK4a was recently evaluated. An inverse relationship was found between the expression of p16INK4a and the presence of the normal retinoblastoma protein (pRB) in cancer cell lines in which the p16INK4a protein is detectable when pRB is mutated, deleted or inactivated, and is markedly reduced or absent in cell lines that contain normal pRB (Li et al., 1994). pRB was shown to act as a negative regulator of p16INK4a gene transcription via repression of E2F activity (Li et al., 1994; Khleif et al., 1996). Because the E7 protein of high-risk mucosal HPVs inactivates pRB, the resulting overexpression of p16INK4a may be a good marker for infection by these HPV types. A monoclonal antibody to p16INK4a has been developed that can detect p16INK4a protein in tissue sections (Klaes et al., 2001). In an immunohistological study, the antibody staining was restricted to tissues from CIN2/CIN3, from CIN1 associated with highrisk HPV or from cervical cancer. Immunostaining of p16INK4a allowed precise identification of even small CIN or cervical cancer lesions in biopsy sections and helped reduce inter-observer variation in the histopathological interpretation of cervical biopsy specimens. Thus, p16 immunohistochemistry may reduce false-negative and falsepositive biopsy interpretation and thereby significantly improve cervical (pre)-cancer diagnosis (Klaes et al., 2002). Further studies are needed, however, to assess the value of p16INK4a immunostaining in the diagnosis of CIN and in cervical cancer screening. 1.3.3

Detection of HPV nucleic acids

Direct detection of HPV genomes and their transcripts can be achieved with hybridization procedures that include southern and northern blots, dot blots, in-situ hybridization, Hybrid CaptureTM and DNA sequencing. A variety of signal detection procedures are available, which can further increase the sensitivity of these assays. Viral DNA and RNA can also be detected by a series of assays based on PCR. In this case, the viral genomes are selectively amplified by a series of polymerization steps, which result in an exponential and reproducible increase in HPV nucleotide sequences present in the biological specimen. Currently, the two methodologies most widely used for the detection of genital HPV types are Hybrid CaptureTM version 2 and PCR with generic primers. These assays have equivalent sensitivities and specificities and both are suitable for highthroughput testing and automated processing and reading, which are necessary steps for their use in large epidemiological studies and in clinical settings. The only procedure that is potentially capable of recognizing all HPV types and variants present in a biological specimen is DNA sequencing of an amplimer obtained by PCR with consensus primers, either after cloning into plasmids or by direct sequencing of the PCR fragment. This methodology, however, is at present labour-intensive and requires

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expensive equipment. Moreover, direct sequencing does not appear to be suitable for the identification of specimens that contain multiple HPVs, since it preferentially detects the over-represented type (Vernon et al., 2000). Recent results obtained with multiple primer sequencing (Rady et al., 1995; Gharizadeh et al., 2003) and general primer-denaturing high-performance liquid chromatography (Li, J. et al., 2003) suggest that it is possible to overcome this problem. The performance of these new methodologies requires confirmation in studies with large numbers of clinical samples. The sensitivity and specificity of the various methods available vary largely but have improved considerably over the last decade, due to better quality and stability of the reagents and the accessibility to equipment that was once considered to be sophisticated. The characteristics of these assays are summarized in Table 6. Important elements to consider are collection procedure, specimen storage and sample preparation. In general, tests that use no primary amplification step, such as Hybrid CaptureTM 2, are less affected by most of these variables, whereas PCR-based procedures tolerate impurities less well because of their enzymatic nature. Therefore, it is desirable to use sampling devices that allow the collection of a large cell sample and storage/transport media that not only preserve cell morphology but also stabilize DNA as well as RNA. Although a large variety of instruments for taking cervical swabs is available, further development of devices for the self-collection of vaginal samples is ongoing (Gravitt et al., 2001). Procedures and devices to collect samples from men are currently being evaluated. (a)

PCR-based methods

HPV DNA can be amplified selectively by a series of reactions that lead to an exponential and reproducible increase in viral sequences present in the biological specimen. Analysis of the amplified products is generally performed by dot-blot, line-strip hybridization or restriction-fragment length polymorphism that can ultimately be coupled with direct DNA sequencing. The commonly used PCR-based methods for HPV detection in clinical samples are presented in Tables 7 and 8. The sensitivity and specificity of PCRbased methods vary, depending mainly on the primer set, the size of the PCR product, the reaction conditions and efficacy of the DNA polymerase used in the reaction, the spectrum of HPV types amplified, the ability to detect multiple types and the availability of a type-specific assay. PCR can theoretically produce 109 copies from a single doublestranded DNA molecule after 30 cycles of amplification. Therefore, care must be taken to avoid false-positive results derived from cross-contaminated specimens or reagents. Several procedures are available to avoid the potential problems of using PCR protocols for HPV DNA detection. The most widely used protocols use consensus primers that are directed at a highly conserved region of the L1 gene, since they are potentially capable of detecting all mucosal HPV types. Among these are the single pair of consensus primers GP5/6 (Van den Brule et al., 1990) and its extended version GP5+/6+ (Jacobs et al., 1995; de Roda Husman et al., 1995) and the MY09/11 degenerate primers (Manos et al., 1989) and its modified version, PGMY09/11 (Gravitt et al., 1998, 2000). Identification of more than 30 types can be

Based on cell morphology

Pap smears/tissues Colposcopy Visual inspection

Not applicable Not applicable Not applicable

Low/high Moderate/low Low/low

Limited because of their low sensitivities

Detection of HPV proteins

Immunocyto/histochemistrya Electron microscopya Western blota

Low/high Low/high Low/high

Low/low Low/low Low/moderate

Highly dependent on sampling and tissue preservation Cannot type HPV

Southern blota,b In-situ hybridizationa,b Dot blot

Moderate/high Moderate/moderate Low/high

Moderate/moderate Moderate/moderate Low/high

Hybrid capturec,d,e

High/high

High/moderate

PCR Real-time PCRd,e

High/high Very high/high

Very high–high/moderate–high Very high/ND

ELISA Peptides VLPs Fused E6/E7

Low/low Moderate/high High/moderate

Low/low Low/low Low–moderate/high

Detection of HPV genomes Direct methods

Signal amplification Target amplification Detection of anti-HPV antibodies

c,d,e

CIN, cervical intraepithelial neoplasia; ELISA, enzyme-linked immunosorbent assay; ND, No data available; PaP, Papanicolaou test; PCR, polymerase chain reaction; VLPs, virus-like particles a Technically cumbersome and/or time-consuming b Requires DNA and tissue preservation c Less dependent on sampling; can be done in crude samples d Suitable for high-throughput testing and automation e Provides information on viral load

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Test

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Table 6. Characteristics of HPV test technologies

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Table 7. Commonly used polymerase chain reaction (PCR)-based methods for HPV detection in clinical samples: description of the main primer sets used in PCR amplification Characteristics

Amplified fragment length

Specificity

Reference

MY09/11

Amplify a highly conserved L1 region

∼450 bp

Mucosal HPVs

Manos et al. (1989)

WD72, 76, 66, 67, 154 GP5/6

Amplify consensus region in the E6 gene

∼240–250 bp

Resnick et al. (1990)

Amplify a highly conserved L1 region

∼150 bp

Mucosal HPVs (HPV 6, 11, 16, 18, 31, 33, 39, 42, 45, 52...) Mucosal HPVs

CPI/CPIIG

Degenerate primers in the E1 gene

∼188 bp

HMB01

Primer analogous to MY09

Broad spectrum, Mucosal HPVs (HPV 16, 18, 31, 33, 45, 51...) Cutaneous HPVs (HPV 1, 2, 3, 4, 5, 7, 8, 10, 14, 19, 20, 21, 22, 23, 24, 25, 36, 37, 46, 49...) Specific for HPV 51

HD primers

Set of 18 different degenerate primer combinations

All known as well as unknown HPVs

L1C1/L1C2

Amplify a highly conserved L1 region

∼244–256 bp

CPI/CPIIS

Degenerate primers of the E1 gene Amplify same region than CPI/CPIIG primer set

∼188 bp

CP65/CP70

Degenerate primers in EV-HPV L1 region

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Primer sets

Tieben et al. (1994)

Berkhout et al. (1995)

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EV-HPVs

Hildesheim et al. (1994) Shamanin et al. (1994a,b, 1996); de Villiers et al. (1997, 1999a) Shidara et al. (1994)

HUMAN PAPILLOMAVIRUSES

Mucosal HPVs (HPV 6, 11, 16, 18, 31, 33, 52, 58 and more..) Broad spectrum of mucosal and cutaneous HPVs similar to CPI/CPII G

Van den Brule et al. (1990) Smits et al. (1992)

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Specificity

Reference

GP5+/6+

Extended version of GP5/6

∼150 bp

Mucosal HPVs

Jacobs et al. (1995); de Roda Husman (1995)

pU-31B/2R

Amplify a consensus region within E6 and E7 genes

∼228 bp

HPV 6 and 11

Sano et al. (1995)

pU-1M/2R

Amplify a consensus region within E6 and E7 genes

∼231–268 bp

Mucosal HPVs (HPV 16, 18, 31, 33, 52b, 58 and more..)

Sano et al. (1995)

IU/IUDO CP66/CP69

Amplify a consensus region in E1 gene Degenerate primers in EV-HPV L1 region used for a nested amplification following PCR reaction with the CP65/CP70

∼188 bp

Mucosal HPVs EV-HPVs

Paz et al. (1997) de Villiers et al. (1997)

PGMY09/11

Modified version of MY09/11

Mucosal HPVs

Gravitt et al. (1998, 2000)

SPF-PCR

Amplify a smaller region of L1; several primer sets have been designed

∼65 bp

Mucosal HPVs

Kleter et al. (1998)

FAP59/64

Degenerate primers in EV-HPV L1 region

∼480 bp

Cutaneous HPVs including EVHPVs

Forslund et al. (1999, 2003a,b)

bp, base-pair; EV, epidermodysplasia verruciformis Note: It is important to stress that, although highly sensitive and specific, these primer sets may differ considerably in their abilities to amplify specific types present in multiple infections (see Table 8 and Section 1.3).

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Table 7 (contd)

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Table 8. Commonly used polymerase chain reaction (PCR)-based methods for HPV detection in clinical samples: detection of the PCR-amplified products Potential highthroughput setting

Reference

Southern blot

PCR products are separated by electrophoresis on agarose gels then transferred onto nylon membranes; membranes are then hybridized with type specific probes.

Yes

No

Pfister & Haneke (1984)

Type-specific PCR

Following PCR amplification with consensus or degenerate primer sets, HPV amplicons are submitted to a second PCR run using type-specific primers.

Yes

Yes

Van den Brule et al. (1990)

Dot-blot

PCR products are denaturated and applied to replicate nylon membranes with dot-blot apparatus; membranes are then hybridized with type-specific probes. An aliquot of PCR amplification products is digested with a pool of restriction enzymes and the resultant restriction pattern is analysed on gel electrophoresis.

Yes

No

Bauer et al. (1991)

Relative typing

No

Bernard et al. (1994a)

ELISA or EIA

Following PCR amplification with biotin-labelled consensus primers, HPV amplicons are captured on streptavidin-coated microwell plates and detected with a digoxigenin-labelled HPV generic probe mix.

No

Yes

Jacobs et al. (1997); Kornegay et al. (2001)

Reverse line blot or LiPA

Following PCR amplification with biotin-labelled consensus primers, PCR products are hybridized to specific HPV probes immobilized on a plastic-backed nylon membrane strip.

Yes

Yes

Gravitt et al. (1998); Kleter et al. (1999); Van den Brule et al. (2002)

RFLP

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Method

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Table 8 (contd) HPV typing

Potential highthroughput setting

Reference

SSCP

Following PCR amplification with radioactive consensus primers, PCR products are separated by electrophoresis on a non-denaturing polyacrylamide gel; typing is made by comparing the migration band patterns obtained with those observed for HPV control types.

Relative typing

No

Picconi et al. (2000)

Sequencing

Sequencing of the PCR products can be done either directly following PCR reaction or after cloning of the amplified fragments; this is the most accurate technique for HPV typing.

Yes

Yes

Asato et al. (2004)

LiPA, reverse line-blot hybridization; ELISA, enzyme-linked immunosorbent assay; EIA, enzyme immunoassay; RFLP, restriction fragment length polymorphisms; SSCP, single-strand conformational polymorphisms

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achieved by hybridization with type-specific probes that can be performed in different formats and analysis of restriction-fragment length polymorphism by gel electrophoresis (Bernard et al., 1994a), dot-blot hybridization (Bauer et al., 1991), line-strip assays (Gravitt et al., 1998) and microtitre-plate assays (Jacobs et al., 1997; Kornegay et al., 2001) which can be automated. Another pair of consensus primers is available that amplifies a smaller fragment of the L1 gene (65 bp compared with 150 bp for the GP primers and 450 bp for MY09/11). This short PCR fragment (SPF)-PCR is designed to discriminate between a broad spectrum of HPVs in an ELISA format (Kleter et al., 1998) or in reverse line-blot hybridization (LiPA) (Kleter et al., 1999; Van den Brule et al., 2002). The SPF and GP5+/6+ systems are widely used in epidemiological studies and have been adapted to formats for high-throughput testing. It is important to stress that, although the analytical sensitivity and specificity of these methods have been thoroughly compared (see below), they may differ considerably in their ability to detect specific types present in multiple infections. For instance, Qu et al. (1997) observed a 3-log decrease in the amplification of HPV 35 by MY09/11–PCR and that of HPV types 53 and 61 by GP5+/6+–PCR. In another comparison study, van Doorn et al. (2002) observed that the PGMY09/11–line blot assay system detected HPV 42, 56 and 59 more frequently, whereas SPF–LiPA detected HPV types 31 and 52 more frequently. This differential ability to detect specific HPV types was observed with MY09/11 and PGMY09/11 when performed with Taq Gold DNA polymerase: infections with HPV types 6, 16, 51, 53, 58, 61 and Pap 291 were detected more frequently with MY09/11–PCR while types 40, 52, 56 and 59 were detected more frequently with PGMY09/11 (Castle et al., 2002a). The first commercially available PCR-based HPV diagnostic kit for multiple types is the Amplicor™ Human Papillomavirus test kit. This assay is based on a non-degenerate pool of primers to amplify a short fragment of the L1 gene of 13 high-risk genotypes (170 bp, compared with the 450 bp obtained with PGMY09/11; see above). The amplicon is immobilized using a pool of capture molecules bound to the wells of a microtitre plate and visualized by colorimetric detection by Roche Amplicor™ chemistry. Moreover, a new test has been developed to use TaqGold™ DNA polymerase, which minimizes the amount of non-specific amplification and increases the sensitivity of the test. Because it amplifies a shorter fragment, it is considered to have a higher analytical sensitivity and a lower clinical specificity and to be adaptable for less well-preserved specimens. This system has been licensed in Europe since 2003. A PCR-based linear array HPV product, which exploits the PGMY09/11 amplification system and is capable of identifiying 37 HPV genotypes, including all high- and low-risk genotypes in the human anogenital region, is also being developed. A fast and reliable HPV typing method has been developed using non-radioactive reverse line blotting (RLB) of GP5+/6+ PCR-amplified HPV genotypes. In this way, 40 HPV-positive clinical samples can be typed simultaneously for 37 HPV types (14 highrisk and 23 low-risk types) (Van den Brule et al., 2002). A nested PCR approach has been developed that is capable of detecting all EV-associated HPV types (Berkhout et al., 1995). This methodology has been shown to be reliable

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in detecting very high frequencies of known as well as new EV-HPV types in cutaneous lesions of renal transplant recipients. An alternative PCR approach (primers FAP59/64) that is targeted to cutaneous HPV amplifies a broad spectrum of these HPV types from clinical samples, including new types, such as HPV 92 (Forslund et al., 1999, 2003a,b). Recently, PCR protocols based on a 5′-exonuclease assay and real-time detection of the accumulation of fluorescence were developed and named real-time PCR. The release of fluorescence at each amplification cycle is directly proportional to the amount of amplicon generated and is therefore considered to be an accurate method for estimating viral load. A Taqman quantitative PCR system has been reported to assess HPV viral load, while controlling for variation in the cellular content of the sample by quantification of a nuclear gene. Several reports indicated that a higher risk for cervical neoplasia was associated with higher viral loads of high-risk HPV types, in particular HPV 16 (Swan et al., 1997; Joseffson et al., 1999; Ylitalo et al., 2000a; van Duin et al., 2002). Other studies have evaluated the viral loads of different HPV types using either real-time PCR (Tucker et al., 2001; Moberg et al., 2004) or a low-stringency consensus PCR method (Schlecht et al., 2003a). Although they showed that the risk for cervical neoplasia is associated with higher copy numbers of different HPV types (Gravitt et al., 2003; Prétet et al., 2004), the variability in copy numbers is too great for viral load to be used as a predictor of CIN lesions (Sherman et al., 2003a). It is preferable to conclude that low viral copy numbers are associated with a low risk for developing CIN. However, further studies are warranted. Quantitative PCR for cutaneous HPV types 5, 8, 15, 20, 24 and 36 has been developed. Using this technique, variable but low HPV DNA copy numbers were found in HPV DNA-positive non-melanoma skin cancer and actinic keratosis tissues, with a median value of 1 HPV DNA copy per 344 cells (Weissenborn et al., 2005). An HPV oligonucleotide microarray-based detection system has been developed by immobilizing HPV type-specific oligonucleotide probes and a control (β-globin probe) on an aldehyde-derivatized glass slide. Target DNA is submitted to standard PCR in the presence of fluoresceinated nucleotides (Cy5 or Cy3) using primers for both the β-globin (PC03/04) and L1 regions (modified GP5/6 primers) of several HPV types. Randomly labelled PCR products are then hybridized onto the chip, which is then scanned by laser fluorescence. In the case of multiple infections, multiple hybridization signals can be seen (Kim, C.J. et al., 2003). This HPV DNA Chip® was shown to perform well in a prevalence study of HPV DNA (Hwang et al., 2003, 2004). The performance of another chip (GeneTrack® HPV DNA chip) which allows the detection of 12 low-risk and 15 high-risk HPV types was successfully evaluated in HPV-positive cell lines and a small series of normal and tumour biopsies from patients with cancer of the tonsil (Oh, T.J. et al., 2004). Despite its potential for further development, the utility of this system has not yet been demonstrated. It is now being considered whether HPV RNA is an important target for the molecular diagnosis of HPV infections. The aim of testing for viral RNA is to evaluate the expression of HPV genomes (and hence their activity in infected cells) unlike HPV DNA

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assays that detect only the presence of viral genomes. This is important for the identification of clinically relevant HPV infections. HPV 16 E6 and E7 transcripts can be detected with a high degree of sensitivity in clinical specimens using PCR-based methods including reverse transcriptase-PCR (RT-PCR) (Sotlar et al., 1998), quantitative RT-PCR (Culp & Christensen, 2003) and real-time PCR (Lamarcq et al., 2002; Wang-Johanning et al., 2002). Recent studies showed that testing for E6/E7 transcripts of HPV types 16, 18, 31, 33 and 45 was more specific for discerning individuals who developed high-grade cervical disease than the detection of HPV DNA by PCR with GP5+/6+ consensus primers (Molden et al., 2005). Moreover, the detection of such transcripts identified which high-risk HPV infections persisted without having to perform repeat testing (Cuschieri et al., 2004a). The latter studies were performed with the PreTect HPVProofer™, a commercially available RNA-based real-time nucleic acid sequence based amplification kit. This reaction generates single-stranded RNAs to which specific molecular beacon probes can hybridize simultaneously to produce a fluorescent signal. The formation of newly generated RNA molecules is determined in real-time PCR by continuous monitoring of fluorescence in a fluorescent reader. Another important application for studies of HPV RNA has been suggested by Klaes et al. (1999) who developed a method to amplify papillomavirus oncogene transcripts to differentiate between episomal and integrated HPV genomes. The rationale behind this method is that HPV genomes are often integrated into the host chromosomes in cervical cancers while, in normal and premalignant tissues, viral DNA is usually kept as episome. Using this assay, a strong correlation was shown between detection of integrated high-risk HPV transcripts and the presence of high-grade cervical neoplasia (Klaes et al., 1999). This assay could provide a tool to predict disease progression and to monitor the efficacy of therapy (Ziegert et al., 2003). The main problem with these techniques is that RNA is more prone to degradation than DNA and is therefore less available in most biological specimens, depending on the time and type of storage conditions (Habis et al., 2004). For this reason, there is great interest in collection media that can preserve both DNA and RNA molecules. It was shown that the routine collection of specimens in liquid-based cytology solutions allows both morphological and immunohistochemical evaluations, and DNA and RNA studies can be performed for at least 14 days following sampling (Tarkowski et al., 2001; Cuschieri et al., 2004a; Nonogaki et al., 2004; Cuschieri et al., 2005). Testing for the presence of more than one HPV type in a biological specimen preferentially uses PCR-based methods, since Hybrid Capture 2 does not discriminate between HPV types. In general, it appears that PCR systems that use multiple primers such as PGMY09/11 and SPF-PCR are more effectual at detecting multiple infections than those that use single consensus primers, such as GP5+/6+. This may be especially true in cases of mixed infections where one type is present in large amounts. Since more accurate tools are being developed for identifying multiple infections, it should be established whether the presence of multiple infections/lesions would be a useful marker for persistent infection and onset or progression of disease.

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(b)

Commercial nucleic acid hybridization methods (Hybrid Capture™)

This is the only commercially available assay for the detection of HPV DNA that has been approved by the Food and Drug Administration in the USA. The two previous versions that had a low sensitivity have now been replaced by Hybrid Capture 2, one of the most extensively used HPV tests in both epidemiological settings and clinics. Hybrid Capture 2 is based on hybridization in solution of long synthetic RNA probes that are complementary to the genomic sequence of 13 high-risk (16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59 and 68) and five low-risk (6, 11, 42, 43 and 44) HPV types and that are used to prepare high- (B) and low- (A) probe cocktails, which are applied in two separate reactions. DNA present in the biological specimen is then hybridized in solution with each of the probe cocktails to allow the formation of specific HPV DNA–RNA hybrids, which are then captured by antibodies that are bound to the wells of a microtitre plate and that recognize them specifically. The immobilized hybrids are detected by a series of reactions that give rise to a luminescent product that can be measured in a luminometer. The intensity of emitted light, expressed as relative light units, is proportional to the amount of target DNA present in the specimen and provides a semiquantitative measure of the viral load. Hybrid Capture 2 is currently available in a 96-well microplate format, is easy to perform in clinical settings and can be automated. Furthermore, Hybrid Capture 2 does not require special facilities to avoid cross-contamination, because it does not rely on target amplification to achieve high sensitivity, as do PCR protocols. Often, only the high-risk cocktail is used; this reduces both the duration and cost of the test. The Food and Drug Administration has recommended a cut-off value for test-positive results of 1.0 relative light unit (equivalent to 1 pg HPV DNA per 1 mL of sampling buffer). Peyton et al. (1998) found that Hybrid Capture 2 with the high-risk probe at a 1.0-pg/mL cut-off detected HPV types 53, 66, 67 and 73, as well as other undefined types; raising the cut-off to 10.0 pg/mL did not eliminate the cross-reactivity to types 53 and 67, which may decrease the specificity of the test (Castle et al., 2002a). A newly modified, experimental Hybrid Capture assay named Hybrid Capture 3 uses RNA probes, as in Hybrid Capture 2, but in combination with biotinylated capture oligonucleotides that are directed to unique sequence regions within the desired target to increase test specificity (Lorincz & Anthony, 2001). The assay has been developed further to reduce cross-reactivity while maintaining sensitivity and for use either on DNA or RNA as targets. A recent comparison study concluded that, at the optimal cut-off points, Hybrid Capture 2 and 3 had similar screening performance characteristics for high-grade lesions diagnosed at the enrolment visit (Castle et al., 2003a). (c)

Southern and northern blot hybridization

For the analysis of HPV genomes, hybridization procedures in solid phase, such as southern blot for DNA and northern blot for RNA molecules, are excellent and can generate high-quality information; however, they are time-consuming and require large amounts of highly purified nucleic acids. Moreover, they require well-preserved, full-size

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molecules and therefore cannot be carried out on all biological specimens, particularly not those derived from fixed tissues in which degradation of DNA is often observed. They are also technically cumbersome and are not suitable for large-scale population studies. In these techniques, high-molecular-weight, highly purified DNA is digested with different restriction endonucleases and is submitted to electrophoresis on agarose gels. After denaturation, the DNA molecules are transferred to nitrocellulose or nylon filters, fixed and submitted to hybridization with specific HPV probes. Depending on the label incorporated in the probes, different signal detection systems can be used. To increase the sensitivity of the test, radioactively labelled probes are commonly used, which limits the application of southern blot to certain laboratory conditions. Despite the stringent requirements, southern blot is considered to be the golden standard for the evaluation of HPV genomes, since it can identify HPV genomes in a specimen accurately and specifically; moreover, it determines the physical status of the genomes (episomal or integrated) and gives a semiquantitative measure of viral load. Several studies have described the presence of HPV DNA in human tissues and cell lines by southern blot (Dürst et al., 1985; Lorincz et al., 1992; Matsukura & Sugase, 2001). Because of the relatively lower analytical sensitivity of this test compared with target (PCR) or signal (Hybrid Capture) amplification procedures, discrepancies in HPV DNA prevalence and type distribution in cervical tumours have been reported (Matsukura & Sugase, 2004) (see the comparison of HPV testing methods in Table 9). (d)

In-situ hybridization

In-situ hybridization is a technique by which specific nucleotide sequences are identified in cells or tissue sections with conserved morphology, which allows the precise spatial localization of target genomes in the biological specimen. One great advantage of in-situ hybridization is that it can be applied to routinely fixed and processed tissues, which overcomes the relatively low analytical sensitivity of this method. Moreover, the integration status of HPV genomes can be inferred from the signal distribution in the nuclei of infected cell (Mincheva et al., 1987; Berumen et al., 1995). In-situ hybridization has been used to detect messenger RNA (mRNA) as a marker of gene expression when levels of viral proteins are low (Stoler et al., 1989). The sensitivity of this method can be increased by combining it with PCR, a procedure known as in-situ PCR (Nuovo et al., 1991a,b,c), but this is a difficult technique that has not been used widely. The major limitation of in-situ hybridization is the potential for errors in HPV typing because of probe cross-hybridization, but recent improvements enabled its use for the detection of HPV DNA and RNA in tissues with high sensitivities and specificities (Birner et al., 2001; Kenny et al., 2002). Moreover, detection of HPV 16 in cervical metastatic lymph nodes of head and neck cancer patients by in-situ hybridization was highly correlated with the localization of the primary tumour (Begum et al., 2003). [The Working Group noted that this methodology can clearly provide important information on HPV-mediated pathogenesis; however, its technical complexity and the requirement for intact tissue samples make in-situ hybridization inadequate for large epidemiological investigations.]

No. of samples

M2+/ M1+

Kappaa

Comments

Cervicovaginal lavages Cervical scrapes

MY09/11b + dot blot

GP5+/6+b (dot blot)

94.6%

0.8

GP5+/6+ detected fewer multiple infections; differences in the detection systems for types 35, 53 and 61

SPF-PCR EIAc

GP5+/6+ (southern blot)

70.6%

0.65

HC2 (HR)d cut-off e 1.0 pg/mL GP5+/6+g

72%

0.58

69.0%

0.77

MY09/11 + line blot assay

87.7%

0.83

0.53

Qu et al. (1997)

208

Kleter et al. (1998)

534

Peyton et al. (1998)

208

Cervical scrapes

MY09/11 + dot blotb

Kleter et al. (1999) Gravitt et al. (2000)

766

Cervical Scrapes Cervicovaginal lavages

SPF-PCR LiPAf PGMY09/11 + line blot assayh

Castle et al. (2003a)

4345i

Cervicovaginal lavages

HC3 (HR)d (prototype) cut-off 0.6 g/mL

HC2 (HR) cut-off 1.0 pg/mL

89%

Hesselink et al. (2004)

76

Cervical smears

GP5+/6+ EIA + rev. line blotj

ISH (HR)k

62%

Kulmala et al. (2004)

1511

Cervical smears

GP5+/6+ (dot blot)

HC2 (HR) cut-off 1.0 pg/mL

92%

247

When the analysis was restricted to HPV types detected by both assays, agreement between methods was greater than 90%. HPV types 34, 53, 70 and 74 not represented in the GP5+/6+ system

HC3 was slightly more sensitive to detect CIN3+ than HC2; HC3 results were more concordant with MY09/11 PCR results than HC2 (1247 specimens). Increased viral loads measured by both methods were associated with high-grade CIN, but the sensitivity of ISH to detect these lesions was too low.

0.67

Slightly higher sensitivities for detection of HSIL by HC2

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Table 9. Inter-assay comparisons of technologies for the detection of HPV DNA in clinical samples

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Reference

Comments

7%

0.48

73%

0.7

Negative samples were re-amplified in a nestedPCR with GP5+/6+; positivity increased further in oral but not in cervical samples.

Method 1

Method 2

M2+/ M1+

106

Oral scrapes

GP5+/6+

MY09/11

56

Cervical scrapes

GP5+/6+

MY09/11

See Table 7 for a description of the primers used. CIN, cervical intraepithelial neoplasia; EIA, enzyme immunoassay; HC, hybrid capture; HR, high-risk mucosal HPV types; HSIL, high-grade squamous intraepithelial lesion; ISH, in-situ hybridization; LiPA, reverse hybridization line probe assay; SPF, short PCR fragment a Agreement between positives b 39 HPV types detected c 43 HPV types detected d 13 HPV types detected e Relative light units/positive control f 20 HPV types detected g 14 HPV types detected h 27 HPV types detected i From a cohort of more than 20 000 women j 37 HPV types detected k BenchMark ISH View Blue Detection Kit for HPV (Ventana Med.Systems; AZ, USA)

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Type of specimen

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Remmerbach et al. (2004)

No. of samples

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(e)

Comparison of HPV testing methods

Table 9 presents a comparison of HPV detection assays in clinical samples. In general, there are good to excellent rates of agreement between tests performed with Hybrid Capture 2 and those with generic PCR systems that employ MY09/11 and GP5+/6+, which emphasizes the availability of several viable HPV tests. An analysis of the intra- and interlaboratory variability of these two PCR protocols (Jacobs et al., 1999) showed excellent agreement between laboratories that used standardized methods. Therefore, validated protocols, reagents and reference samples assure the best test performance in different settings. It is very important to stress, however, that the analytical sensitivities and specificities of HPV tests vary largely, depending on assay characteristics, the type and quality of the biological specimen and the type and quality of the reagents used, including the use of different DNA polymerases that can affect test performance (Castle et al., 2002a). Moreover, caution should be used to interpret such comparisons, because the assays differ in their ability to detect different HPV types (Kleter et al., 1998) either as single or multiple infections. Current commercially available tests have been developed to detect the most common high-risk HPV types, as confirmed by a large series of epidemiological studies that included people from all over the world. Adaptation of the assays to include HPV types according to their geographical distribution should be considered as a means of increasing test specificity. Although the analytical sensitivity of some HPV detection assays can be very high, which is valuable in addressing the burden of HPV infections epidemiologically, its corresponding clinical significance is not so evident (Iftner & Villa, 2003; Snijders et al., 2003). This is because several HPV infections do not persist and therefore do not lead to clinically relevant disease. Approaches to increase the clinical sensitivity of HPV assays that are being considered include: (a) testing only for the clinically relevant high-risk HPV types, (b) adding a viral load measure and (c) testing for high-risk HPV E6 and E7 transcripts. Several studies have evaluated these and other possibilities, some of which are presented here. Continuous assessment and validation of current and new methodologies is essential for the evaluation of the carcinogenic risk of certain HPVs to humans. 1.3.4

Detection of HPV infections and HPV-associated cancers by serological assays

The antibody response to papillomaviruses is a key determinant of protective immunity. HPV serology is also an important epidemiological tool for the assay of past and present HPV infections and for the prediction of HPV-associated cancers and their precursor lesions. Antibody responses to the HPV capsid are used as a marker of cumulative exposure to HPV while antibodies to E6 and E7 have been shown to be markers of malignant HPV-associated cervical or oropharyngeal disease. The antibody responses to HPV infections and in HPV-associated disease are discussed in detail in Section 1.2.

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The development of serological assays was hampered initially by the lack of suitable cell culture systems to propagate papillomaviruses and to prepare infectious virions. This has been overcome by recombinant DNA technologies that have allowed the generation of VLPs that display conformational, type-specific epitopes of purified, correctly folded early proteins such as E6 and E7 and of infectious pseudovirions that are suitable for neutralization assays. (a)

Detection of capsid antibody

It has been shown by several groups that infection of cells with recombinant vaccinia viruses or baculoviruses that express the Ll with or without the L2 ORFs of HPV types 1, 6, 11 and 16 (Zhou et al., 1992; Hagensee et al., 1993; Kirnbauer et al., 1993; Rose et al., 1993) leads to accumulation in the nucleus of what appeared to be HPV capsids. HPV 1 particles analysed by cryoelectron microscopy at a resolution of 3.5 nm were found to be indistinguishable from HPV virions purified from foot warts (Hagensee et al., 1994). Such empty capsids (also referred to as VLPs) were then used to develop ELISAs to detect antibodies in human sera and mucosal secretions for HPV types 1, 6, 11, 16 and 18 (Hagensee et al., 1993; Rose et al., 1993; Carter et al., 1994; Hines et al., 1994; Le Cann et al., 1994). For these assays, VLPs are usually produced by baculovirus expression in insect cells, purified by one or more rounds of equilibrium density or other ultracentrifugations, adsorbed to plastic surfaces and used as antigens to bind capsid-specific antibodies. ELISAs for VLPs have now become the most widely used and accepted method to analyse HPV capsid-specific antibodies. In addition, VLP-based ELISAs have been established for other mucosal high-risk HPV types 31, 33, 35 and 45 (Sapp et al., 1994; Marais et al., 2000a; Giroglou et al., 2001b; Combita et al., 2002) and for cutaneous HPV types 5, 8, 15, 20, 24 and 38 (Favre et al., 1998a; Stark et al., 1998; Wieland et al., 2000; Feltkamp et al., 2003). Alternative methods for the detection of antibodies to HPV VLP have been developed. To increase the specificity of VLP-based ELISAs, competitive binding assays have been established for HPV types 6, 11, 16 and 18 (Palker et al., 2001; Opalka et al., 2003). In these tests, human antibodies compete for binding to VLPs that are adsorbed on plastic surfaces with a radio- or fluorescence-labelled monoclonal HPV type-specific reporter antibody directed to a dominant conformational epitope on the VLPs. However, such competitive assays usually have lower analytical sensitivity compared with direct binding assays. In other approaches, monoclonal antibodies that recognize conformational VLP epitopes (Hagensee et al., 2000) or heparin-sulfate (cross-linked to bovine serum albumin) to which intact VLPs bind specifically (Wang et al., 2005) are adsorbed on a plastic surface to capture selectively L1 that displays conformational epitopes. Finally, inhibition of VLPmediated haemagglutination has been described for HPV types 6, 11, 16, 18, 33 and 45 (Roden et al., 1996b). HPV L1 expressed in bacteria as the glutathione-S transferase (GST) fusion protein has been shown to form capsomers spontaneously, to display most epitopes defined on VLPs and to be suitable as an antigen for the detection of HPV capsid antibody (Rose

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et al., 1998, Yuan et al., 2001). To circumvent the tedious procedures of production and purification and the varying yields and quality of VLPs from different HPV types, an alternative ELISA for HPV capsid antibody has been developed based on the affinity of GST–L1 fusion proteins purified on glutathione-coated plastic surfaces. It has been shown to have similar analytical sensitivity and specificity for HPV 16 and 18 as the conventional VLP-based ELISA (Sehr et al., 2002). Recently, this type of assay has been adapted to fluorescent bead technology which allows the fast analysis of antibodies against many different (theoretically up to 100) proteins in parallel using only minute amounts of serum (Chen et al., 2005). In view of the many papillomavirus types that potentially infect humans, this assay type could be of value in sero-epidemiological studies that analyse type-specific seroprevalences for large groups of HPV types simultaneously. Several years of research were required to validate VLP-based ELISAs, and validation was laborious in the HPV system because: (a) early methods for the detection of HPV DNA were inaccurate, to the extent that misclassification seriously flawed early epidemiological studies of HPV (Franco, 1992); (b) many of the more than 100 different HPV types are not associated with malignancy and are not sexually transmitted, which renders serological cross-reactions difficult to predict on the basis of DNA homology; (c) most HPV infections are rapidly cleared spontaneously. In follow-up studies of HPV DNA-positive women, some 70% cleared their HPV DNA within 12 months (see also Section 1.2.2). Thus, many people who test negatively for HPV DNA may have had a previous infection; (d) seroconversions can appear many months after infection (see Section 1.2.2), and many people with a recently acquired HPV infection may not have seroconverted; and (e) testing for the HPV genome in samples taken from the uterine cervix will not detect infections at other body sites. In spite of these major theoretical difficulties, serology with viral capsids has shown an amazing concordance with detection of viral DNA at the cervix for several HPV types. In the original report, serum IgG antibodies against capsids of HPV 16 of a wild-type strain were found in 59% of women who tested positively for cervical HPV 16 DNA, whereas only 6% and 9% of women who tested negatively for cervical HPV DNA or positively for the benign HPV types 6 and 11, respectively, had these antibodies (Kirnbauer et al., 1994). Human antibodies mostly recognize conformational epitopes on the capsid surface. HPV capsids can be disrupted, usually by treatment with high pH carbonate buffer, to destroy the type-specific epitopes; this results in the loss of type-specific serological reactivity, whereas cross-reactive antibody responses remain unaffected (Carter et al., 1993; Dillner et al., 1995a). Similar results were obtained previously using purified virions isolated directly from lesions (Steele & Gallimore, 1990; Bonnez et al., 1991). It was also shown that neutralizing antibodies to HPV type 11 virions recognized conformational epitopes on synthetic HPV type 11 capsids. An alternative method for assaying type-specific antibodies is based on the fact that they are usually present at higher titres than crossreactive antibodies. By assigning a ‘cut-off’ value that classifies low-titred reactivity as negative, specific results can be also obtained without a negative control or confirmatory

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assays (Wideroff et al., 1995). Human anti-capsid antibody responses were found to be directed against epitopes on the Ll protein, because addition of L2 protein did not augment the association between HPV infection and antibody reactivity (Carter et al., 1993). The sensitivity of assays is measured using panels of serum samples obtained from individuals with a documented infection with the virus in question, i.e. by detection of the viral genome. State-of-the-art detection of viral DNA is not entirely straightforward, and misclassification is most commonly due to the inability to distinguish between some of the many viral genotypes, to contamination in PCR assays and to inadequate sampling. Whereas there is good to excellent agreement between laboratories for certain assays such as the PCR–ELISA system based on the general primers GP5+/GP6+, there is poor agreement between different assays for the detection of HPV DNA (Jacobs et al., 1999). In general, studies of the sensitivity of HPV capsid serology that have used state-of-theart methodology for the detection of HPV DNA have found a sensitivity of 50% or more (Andersson-Ellström et al., 1994; Kirnbauer et al., 1994; Wideroff et al., 1995; Carter et al., 1996; Wideroff et al., 1996a; Kjellberg et al., 1999). In a large population-based study that used nested PCR technology, sensitivity was found to be 65–75% (Kjellberg et al., 1999). Persistence is a covariate of HPV seropositivity that may result from misclassification or may be a biological phenomenon. The clearly detectable presence of HPV DNA is more commonly associated with HPV seropositivity than its weakly detectable presence (Viscidi et al., 1997). A heavy infection may produce more viral protein that may induce a more effective antibody response. Alternatively, a weakly detectable presence of HPV DNA may be more commonly misclassified and not be due to true infection. The persistent presence of HPV DNA in samples taken at two different occasions from the same woman is more commonly associated with seropositivity than a transient presence of HPV DNA that was not detectable in a second sample taken from the same woman (Wideroff et al., 1995). Transient infections may not be present in the body long enough to evoke an antibody response. Alternatively, detection of HPV DNA that could not be repeated in a second sample may have been misclassified or may have reflected the presence of viral genomes that never resulted in an infection. The HPV virion is stable and resistant to desiccation and remains extracellularly viable for at least 1 week (Roden et al., 1997b). Specificity is assayed by comparing serum samples taken from women infected with the same HPV type, women infected with other HPV types and women not exposed to HPV. Comparisons with women infected with other types of HPV are confounded by the fact that different carcinogenic genital types are transmitted similarly and that women in the high-risk group currently infected with a certain HPV type may have had previous infections with other HPV types. All serological studies of type specificity of the HPV capsid have found a strong type-restricted component, and, in a large population-based study performed in a population with a modest number of lifetime sexual partners, no covariation with the presence of other HPV types was found, which indicated type specificity (Kjellberg et al., 1999). Type specificity of HPV capsid-based assays is also supported by

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a very large number of experimental studies on immunological cross-reactivity of monoclonal antibodies against HPV capsids. Whereas disrupted or partially disrupted viruses expose epitopes that are broadly cross-reactive or even group specific (Jenson et al., 1980; Dillner et al., 1991), conformationally dependent epitopes on intact capsids have generally been HPV type-specific (Christensen et al., 1996b). The exceptions are HPV 6 and 11 that have been shown to contain shared epitopes and type-specific epitopes on intact capsids (Christensen et al., 1994, 1996b). The specificity of HPV capsid serology is also indicated by the fact that panels of serum samples taken from subjects with no or little sexual experience have very low seroprevalences (see Section 1.2.2). Seroprevalence from different studies and laboratories must be compared with caution due to interlaboratory variation in assays and different definitions of cut-off. Interlaboratory agreement between three laboratories has been assessed in one study that determined seropositivity for HPV 16 by VLP-based ELISA. Variation coefficients of 0.61 to 0.8 were found (Strickler et al., 1997). Especially important factors include the use of different groups of sera as a basis for determination of cut-off and different mathematical definitions of cut-off. WHO is currently developing serological reference reagents for the major HPV types. (b)

Neutralization assays

Neutralization assays are thought to be more type-specific than antibody-binding assays. Many neutralization assays are based on infectious pseudovirions (Table 10). While initial assays were technically complex and tedious, and were therefore restricted to the analysis of only small numbers of sera, they allowed the definition of neutralizing epitopes by monoclonal antibodies (see also Section 1.2.1). Recent developments suggest that the high-throughput analysis that is needed for large epidemiological and vaccination studies may be feasible. (c)

Detection of antibodies to E6 and E7

Antibodies to E6 and E7 proteins of HPV types 16 and 18 are markers of HPV-associated malignant disease but, since not all patients with tumours show such antibodies, they cannot be used as diagnostic markers. The association of E6 and E7 antibodies with cervical cancer was already apparent in initial studies that analysed only linear epitopes by either peptide ELISA or western blot analysis, despite the low sensitivity and specificity of these assays. Methods that apply full-length E6 or E7 proteins that present conformational epitopes, i.e. immunoprecipitation assays with in-vitro transcribed and translated HPV 16 E6 or E7 proteins (Stacey et al., 1992, 1993; Viscidi et al., 1993; Nindl et al., 1994; Sun et al., 1994b; Chee et al., 1995; Nindl et al., 1996), showed higher sensitivity and specificity. ELISAs that use yeast-expressed biochemically purified and renatured full-length HPV 16 and 18 E6 and E7 proteins have been shown to be more specific and equally sensitive compared with radioimmunoprecipitation assays (Meschede et al., 1998). These

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Table 10. HPV neutralization assays Type and source of infectious particles

Read-out

Reference

HPV 11; virions from athymic mouse xenograft HPV 11; virions from athymic mouse xenograft BPV1; virions from lesions

Xenografted human foreskin transformation RT-PCR of HPV 11 mRNA in xenografted human foreskin C127 mouse fibroblast focus formation Abortive rabbit cell infection

Christensen & Kreider (1990) Bonnez et al. (1992) Christensen et al. (1995)

HPV 16; pseudovirions generated from HPV 16 L1 and L2 expressed from Semliki Forest viruses vector and carrying BPV1 genome, in cultured hamster cells harbouring autonomously replicating BPV-1 genome (BPHE-1 cells)

C127 mouse fibroblast focus formation

Roden et al. (1996a)

HPV 33; pseudovirions carrying β-galactosidase marker plasmid and generated form L1 and L2 expressed by vaccinia-virus in COS-7 cells

β-Galactosidase expression in COS-7 cells

Unckell et al. (1997)

HPV 11; virions from athymic mouse xenograft

RT-PCR of viral mRNA in infected Leiserowitz cultured neonatal human foreskin et al. (1997) keratinocytes or immortalized human adult skin cell line HaCaT

HPV 16; virions from SCID mouse xenograft

RT-PCR of viral mRNA in infected immortalized human adult skin cell line HaCaT β-Galactosidase expression in infected COS-7 cells

CRPV; virions from lesions

HPV 16 and 6; pseudovirions assembled in vitro from L1/L2 VLPs produced in insect cells and β-galactosidase marker plasmid

White et al. (1998) Kawana et al. (1998); Matsumoto et al. (2000) Yeager et al. (2000)

HPV 6, 11, 16 and 18; pseudovirions generated by coupling of β-lactamase marker plasmid to L1/L2 VLPs produced in yeast

β-Lactamase activity in infected C33A cervical carcinoma cell line

HPV 6, 16 and 31b; virions from cultured trophoblast cell line 3A

HPV DNA replication and/or gene expression in infected 3A cells

HPV 16 and 31; pseudovirions generated by coupling of luciferase marker plasmid to L1 VLPs produced in insect cells

Luciferase activity in infected COS-7 cells

HPV 16 and 45; virions generated in raft cultures

RT-PCR of viral mRNA in infected McLaughlinimmortalized human adult skin cell Drubin et al. line HaCaT (2003, 2004)

Liu et al. (2001a); You et al. (2003) Bousarghin et al. (2002)

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Table 10 (contd) Type and source of infectious particles

Read-out

HPV 16 and 18; pseudovirions carrying Quantification of secreted alkaline secreted alkaline phosphatase marker plasmid phosphatase activity and generated from expression of codon modified L1 and L2 genes in 293T cells

Reference Pastrana et al. (2004)

BPV, bovine papillomavirus; CRPV, cottontail rabbit papillomavirus; RT-PCR, reverse transcriptasepolymerase chain reaction; VLP, virus-like particle

ELISAs have been used to demonstrate the association of antibodies for HPV 16 and 18 E6 and E7 proteins with cervical cancer (Meschede et al., 1998; Zumbach et al., 2000b) and also oral cancer (Zumbach et al., 2000a; Herrero et al., 2003). Recently, ELISAs based on the expression of affinity-purified HPV 16 and 18 E6 and E7 in bacteria as GST fusion proteins have been developed, which appear to be of greater sensitivity (Sehr et al., 2001). Epidemiological studies using these assays have not yet been published. (d)

Detection of antibodies to E1, E2, E4 and E5

From studies that used linear epitopes as antigens in either peptide ELISA or western blot analysis, there is some indication that antibodies to E2 and E4 or some specific linear sequences of these proteins are associated with cervical cancer, but no consistent picture has emerged. As seen for antibodies to L1 and also to E6 and E7 proteins, assays that use proteins that also present conformational epitopes need to be developed before this question can be analysed appropriately. 1.4

Natural history and epidemiology of HPV infection

1.4.1

Introduction

HPV is a prevalent pathogen, the epidemiology of which has mostly been studied in the uterine cervix and the vagina. This section is therefore restricted to the natural history of genital HPV types. The cervical transformation zone can be considered as a ring of tissue that is susceptible to the carcinogenicity of HPV. Cervical HPV infection can be assessed visually, microscopically (via cytology or histology) and by molecular detection methods. The basic steps that lead from the normal cervix to cancer are well established (see Figure 8). To a large extent, these are probably also valid for the natural history of HPV in lesions at other anogenital sites; however, the molecular epidemiology of HPV infection at these sites is not as well characterized as that in the uterine cervix.

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Figure 8. Natural history of preclinical abnormalities of the cervix

From IARC (2005) a Classical histological features of CIN1 are uncommon among women who have transient infections. b This entity is not as well defined as CIN3.

The major steps known to be necessary for cervical carcinogenesis include HPV infection, persistence of that infection, progression to precancerous lesions and eventually invasion. Provided that the latter step has not taken place, this process is reversible by the clearance of HPV infection and regression of precancer, which happen in many women who have ever experienced HPV infection. As discussed below, HPV infection might usefully be separated into low-viral load infections that engender no microscopically evident abnormalities and higher-viral load infections that do. As described in Section 1.1, over 100 types of HPV exist, of which more than 40 are mucosotropic viruses that infect the anogenital and upper aerodigestive tracts (de Villiers et al., 2004a). Among the latter, approximately 15 are considered to be high-risk types. The various HPV types do not all occur in different populations at the same rate; therefore, although much is known about the epidemiology and natural history of HPV infections, little is known about the long-term characteristics of infections at the type-specific level, e.g. the assessment of viral persistence. Most knowledge refers to HPV 16, which is the type most frequently found in tumours in the general population, and is discussed separately below. 1.4.2

Transmission and acquisition (a)

Horizontal transmission

The most common mode of horizontal transmission of anogenital HPV is by sexual activity through contact with infected cervical, vaginal, vulvar, penile or anal epithelium. In the early 1950s, Barrett et al. (1954) reported that genital warts developed within 4–6

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weeks in wives of servicemen who had returned from overseas and who had had genital warts. Oriel (1971) reported that 64% of sexual partners of individuals who had genital warts developed genital warts themselves after a mean interval of 2–3 months. Similar results have been reported by others (Teokharov, 1969; Barrasso et al., 1987). There is now overwhelming epidemiological evidence for the role of sexual activity in the transmission of anogenital HPV (Franco et al., 1995; Bosch et al., 1996; Dillner et al., 1999; Bleeker et al., 2002; Castellsagué et al., 2003; Sellors et al., 2003). Studies among initially virginal women strongly confirm the sexually transmitted nature of HPV infection (Rylander et al., 1994; Kjaer et al., 2001). Sexual contact with an infected partner is necessary for transmission, presumably through microscopic abrasions in the mucosa or skin, and HPV infections are easily transmitted; however, on the basis of data on lesbians, it appears that intromissive intercourse in which an infected penis enters the vagina is not strictly necessary (Marrazzo et al., 2001). Moreover, transmission may take place in one anogenital site, such as the introitus, and the infection may be spread by self-inoculation to another site (Winer et al., 2003). As a group, anogenital HPVs are the most common sexually transmitted infections but there is some evidence that the degree of sexual transmissibility may vary among types and across populations (Franco et al., 1995; Kjaer et al., 1997; Rousseau et al., 2000). In addition to the sexual behaviour of women, epidemiological studies suggest that age, both of women and their partners, genetic and environmental susceptibility factors, use of barrier contraceptives, co-infections, male sexual behaviour and male circumcision are related to the prevalence of HPV (reviewed by Schiffman & Kjaer, 2003). A series of studies has also established that the sexual behaviour of and HPV infection in the male partner significantly increase the risk whereas circumcision of the male partner was associated with a significant reduction in risk for invasive cervical cancer among women (Castellsagué et al., 2002). Although fewer studies have been conducted on the prevalence of HPV infection among men than among women, HPV infections also appear to be common in men (Baldwin et al., 2004; Shin et al., 2004; Weaver et al., 2004). In the few studies that have evaluated factors associated with infection in men, sexual history, age and possibly condom use are associated with the prevalence of HPV (Baldwin et al., 2004; Shin et al., 2004; Weaver et al., 2004). Published data on the natural history of HPV in men are scarce; however, several large prospective studies of HPV infection in men are currently being carried out. As with any other sexually transmitted infection, prevention of HPV infection would greatly benefit from a better understanding of the determinants of transmission and infection among men. HPV infections can be transmitted not only by peno-vaginal intercourse, but also by other sexual practices, e.g. oral sex, peno-anal intercourse, digital–vaginal sex and use of insertive sex toys (Edwards & Carne, 1998; Sonnex et al., 1999; Gervaz et al., 2003). Marrazzo et al. (2000) reviewed genital HPV infection in women who had sex with women. This review suggested that sexual practices between female sexual partners could result in transmission of HPV. Hand carriage of genital HPV types in patients with genital

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warts was identified by Sonnex et al. (1999); their findings supported the possibility of HPV transmission by digital–genital contact. The non-sexual mode of transmission of genital HPV remains a controversial issue. Most studies among sexually inexperienced young women (Andersson-Ellström et al., 1994; Dillner et al., 1999) demonstrated that non-sexual transmission of HPV is uncommon. However, a number of studies (Pao et al., 1992; Cason et al., 1995; Winer et al., 2003) reported that HPV might occasionally be transmitted through modes other than sexual activity. The possible non-sexual routes include vertical transmission, fomites and skin contact (Mindel & Tideman, 1999; Frega et al., 2003). (b)

Vertical transmission

Vertical transmission occurs when a parent conveys an infection to its unborn offspring, including a special form of vertical transmission — perinatal infection. Vertical transmission of HPV from mother to child was first suggested in the 1950s (Hajek, 1956) and was subsequently supported by several other studies (Cason et al., 1995; Puranen et al., 1997; Tseng et al., 1998). Rare cases of anogenital warts in newborns have been reported (Tang et al., 1978) and HPV DNA has been detected in mucosal scrapes and washes obtained from infants (Roman & Fife, 1986; Jenison et al., 1990; Fredericks et al., 1993; St Louis et al., 1993). HPV DNA was rarely detected even among babies born to HPV-infected mothers (Watts et al., 1998). Results from studies of transmission in infants are not consistent, and do not provide a clear indication of the rate of infection among neonates who are exposed perinatally. Differences in samples and techniques may be the reasons for the variability and inconsistency in these results. Tenti et al. (1999) investigated HPV type-specific concordance between mother–infant pairs and found that HPV-positive newborns carried HPV types identical to those found in their mothers. However, discordant mother–newborn pairs have been reported in several studies, as well as HPV-positive babies born to HPV-negative mothers and transmission of HPV by the transplacental route before delivery (Puranen et al., 1996). Perinatal transmission of HPV has been demonstrated unequivocally for the rare disease juvenile respiratory papillomatosis (Dillner et al., 1999). Earlier studies of juvenile-onset recurrent respiratory papillomatosis in infants and young children indicated that HPV infections may be transmitted from mother to infant, probably at the time of delivery. Age of the mother, birth order of the infant and mode of delivery are considered to be important determinants of transmission. Most infants who develop juvenile-onset recurrent respiratory papillomatosis are the first-born single or twin infant of women who tend to be younger than other mothers who gave birth at the same institutions (Kashima et al., 1992a), and many are delivered vaginally rather than by caesarean section (Shah et al., 1986). Cesarean delivery is generally thought to protect against perinatal transmission of HPV (Tseng et al., 1998) but, as shown by other studies among children delivered by cesarean section, some of them can be HPV-positive (Chatterjee et al., 1998). Kosko and Derkay (1996) and Summersgill et al. (2001) postulated a very limited role for cesarean section in the prevention of transmission of HPV.

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Despite the evidence for vertical transmission, its overall importance in terms of public health may not be as great as that suspected by patients and health care providers (Winer & Koutsky, 2004). It would be particularly valuable to confirm the prevalence of established HPV infections in babies after vaginal birth in the absence of convincing seroconversions (using assays that provide specific although insensitive biomarkers of infection) (Dillner et al., 1999). Even if anogenital infections with high viral load are rare in babies, exposure at birth could influence immune response later in life at the time of sexual exposure (Mant et al., 2000), but rigorous assessment of such a theoretical effect will require very complex study designs. There seems to be consensus, however, that perinatal transmission is generally a rare event (Winer & Koutsky, 2004). (c)

Issues in assessing transmission

Assessment of type-specific concordance between genital HPV infections in heterosexual couples has been addressed in several studies as further proof of the principle of sexual transmissibility of HPVs. Although some studies (Ho et al., 1993b; Baken et al., 1995) found good agreement among the couples studied, most demonstrated a relatively poor correlation between HPV-positivity and types in cervical and penile samples (Strand et al., 1995; Castellsagué et al., 1997), even among couples where both the wife and husband reported only one lifetime sexual partner (Franceschi et al., 2002). The possible explanations of HPV discordance include problems related to the sensitivity of the detection method, inadequate sampling techniques, the timing of the sampling of penile and cervical specimens, multiple partners of men or women in some couples and different rates of spontaneous regression of HPV infection in men and in women. New epidemiological studies have begun to focus on the dynamics of HPV infection in men and on the actual characteristics of transmission in heterosexual couples. Because the basic tenet of analytical epidemiology is the observation of individual subjects, several methodological challenges need to be overcome in studies of couples or of infection that begins with an index subject and is eventually transmitted to partners and spread from that point. These studies are very important because they can estimate the probabilities of infective contact per sexual act and partner. These estimates are fundamental for models of transmission of infection that are used to assess the potential impact of HPV vaccination and the cost-effectiveness of different preventive strategies, because, to date, such models have had to make simplified assumptions concerning the parameters of sexual transmission (Hughes et al., 2002). Measurements of HPV infection in men and women are prone to error, which emphasizes the difficulties of ascertaining infection in the context of multiple types and even molecular variants and makes the distinction between persistence, recurrence and acquisition very difficult. Studies that could detect incident HPV infections among virgins who were being initiated in sexual intercourse would be useful, because the earliest aspects of transmission and immune response have not been clarified adequately by long-term cohort studies. It is uncertain whether sexual intercourse near menarche is uniquely prone to establishing infection (or persistence and progression). The proximity of first intercourse to

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menarche does not appear to increase the risk for HPV infection (Collins et al., 2005). The apparently limited protective role of condoms should be better estimated to guide the debate on this issue, and the possible role of susceptibility in the acquisition of multiple HPV types has not been assessed adequately. The currently available, limited data suggest that HPV types, although probably sexually co-transmitted, influence the transmission of each other to a minimal extent if at all (Thomas et al., 2000; Liaw et al., 2001; Rousseau et al., 2001). The type specificity of serological responses supports this conclusion (Wideroff et al., 1996a; Carter et al., 2000). Recently, studies of sexual couples revealed a beneficial effect of condoms on the regression of flat penile lesions (Bleeker et al., 2003). This effect was only demonstrable in couples who showed a concordance of HPV type and was associated with the maintenance of flat penile lesions or the development of new penile lesions in the areas surrounding existing penile lesions (Bleeker et al., 2005b). This suggests re-infection and the development of new penile lesions in men who are susceptible to the same HPV type as that harboured by the female partner. However, further studies of multiple infections could be important to guide strategies on vaccines. For instance, it would be useful to know whether the prevention of HPV 16 infection would affect the epidemiological niche occupied by other HPV types in various populations. In summary, improvement in our knowledge of the transmission of HPV has a significant implication for the prevention of HPV infection and also for reducing the incidence of precancerous lesions. Sexual transmission of genital HPV has been demonstrated unequivocally. However, further epidemiological studies are required to enhance the understanding of HPV transmission by non-sexual routes and to provide empirically valid parameters of sexual transmissibility to address health promotion, the (cost-)effectiveness of which will have to be evaluated. Detection of HPV mRNA may provide confirmatory evidence of infection rather than evidence of contamination or whether viral DNA is being transcribed. Large prospective cohort studies with repeated measurements of viral endpoints would be informative on the long-term persistence of HPV infection in children, since current data are usually obtained from cross-sectional studies. 1.4.3

Prevalence of HPV infection

The age-specific prevalence curve of cervical (and vaginal) HPV infection, as measured by HPV DNA, has a large peak that follows typical population norms of sexual initiation, which confirms sexual transmission (Burk et al., 1996). In some populations, age-specific prevalences decline sharply and reach very low levels at older ages, which is consistent with viral transience as well as lower incidence at older ages (see Figure 9). However, in populations in India (Franceschi et al., 2005) and sub-Saharian Africa (Thomas et al., 2004), the prevalence of HPV never falls substantially. The age curve of HPV infection tends to rise again in middle age in some populations, notably in Latin America (Lazcano-Ponce et al., 2001; Herrero et al., 2005). The incidence rates of invasive cervical cancer tend to peak about 20–25 years after the peak age for HPV infection prevalence, and the incidence of CIN peaks in between.

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Figure 9. Prevalence of high-risk types of human papillomavirus (HPV)a among sexually active and cytologically normal women aged ≥ 15 years, in different countries. IARC multi-centre HPV prevalence surveys

Modified from Anh et al. (2003), Matos et al. (2003), Molano et al. (2003), Thomas et al. (2004) a Includes HPV 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, 68, 73 and 82.

Table 11 lists the most relevant studies of the prevalence of HPV in cytologically negative women (also excluding atypical squamous cells of undetermined significance [ASCUS]; see the footnote for exceptions) for several populations worldwide with various age ranges. The restriction of surveys on the prevalence of type-specific HPV DNA to cytologically negative women was intended to minimize any influence of longer duration of lesions related to specific types. The selected studies were population–surveillancebased, the study population consisted of about 350 women or more and the test used was type-specific PCR and HPV genotyping. Different primers were included and varied to some extent in type-specific sensitivity. The population-wide prevalence of HPV in women varies from 1.5% in Spain to 39% in Honduras and Kenya, although careful attention needs to be given to the age distribution of the population being studied, as the prevalence of HPV is strongly age-related. In general, the prevalence is highest in Africa and South America, lowest in Europe and intermediate in Asia. However, observed rates vary within the regions (7.8% in Italy versus 1.5% in Spain; 2.0% in Hanoi versus 10.9% in Ho Chi Minh, Viet Nam). The high variability might also reflect differences in the selection of the women, although prevalence varies remarkably even across the study centres coordinated by IARC.

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Table 11. Rates of detection of HPV DNA by polymerase chain reaction (PCR) amplification among women with cytologically negative Papanicolaou smears Reference, study area

Primer system

Age range No. (years)

MY09-11

Franco et al. (1999), Brazil

MY09-11

Liaw et al. (1999), USA

11

16

18

31

33

35

39

45

51

52

56

58

59

68

73

82





1.3

0.7

0.9

0.7























1.8

10.9 4.1

3.4

0.7

0.2

0.0

0.0

0.0

0.2

0.0

1.8

0.0

0.0

0.0

0.0

1818

3.5

20–65

438

39.0

0.2

18–60

1425b

13.8

0.5c 0.5c 2.7

0.8

1.1

0.4

0.2

0.1

0.5

0.7

0.6

0.6

1.2

0.1

0.4

0.2

0.1

MY09-11 > 16

991

15.8

0.6c 0.6c 2.5

0.8

0.9

0.6

0.3

0.7

0.9

1.8

0.8

0.5

0.9

0.7

0.2

0.0

0.0

Herrero et al. (2000), Costa Rica

MY09-11

18–94

305

11.0

0.7

0.0

1.0

1.0

0.3

0.7

0.3

0.7

0.0

0.3

1.0

0.0

1.6

0.0

0.3

0.7

0.3

Lazcano-Ponce et al. (2001), Mexico

BGH 20, BPCO4

15–69

1248

13.5

0.5

1.0

1.8

1.1

1.5

1.0

0.3

1.0

0.6

0.8

0.8

0.3

1.0

0.2

0.3

0.1

0.3

Sasagawa et al. (2001), Japan

LCR-E7

16–72

1562

9.7

0.0

0.1

1.2

0.8

0.5

0.4

0.2

0.1

0.1

0.4

1.0

0.3

0.4

0.1

0.1

0.1

0.0

Forslund et al. MY09-11 32–38 (2002), Sweden

6123b

6.8





2.1

0.6

1.1

0.4

0.3

0.2

0.8

0.4

0.3

0.5

0.3

0.1







Maehama et al. (2002), Japan

L1C1/C2

30–85

3963b

10





0.3

0.1

0.3

0.3

0.6











0.2









Anh et al. (2003), Ho Chi Minh,

GP5+/6+

15–69

922b

10.9

0.0

0.0

3.3

1.2

0.8

1.1

0.3

0.9

0.7

0.8

1.1

1.1

1.5

0.0

0.7

0.1

0.0

De Vuyst et al. (2003), Kenya

SPF10

25/55

369

38.8

0.5

0.5

3.5

2.2

3.3

1.9

2.7

1.4

1.6

1.1

6.2

1.4

2.7

0.3

1.6





Page 119

Ferrera et al. (1999), Honduras

6

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TSa 20–45 16, 18, 31, 35

Specific HPV type (%)

HUMAN PAPILLOMAVIRUSES

Cuzick et al. (1995), United Kingdom

Overall HPV

119

Primer system

Age range No. (years)

Overall HPV

Specific HPV type (%) 18

31

33

35

39

45

51

52

56

58

59

68

73

82

16.7

0.1

0.3

4.0

1.9

1.8

1.4

1.9

1.0

1.1

0.4

1.2

0.9

1.3

0.8

0.8

0.2

0.1

909b

1.5

0.1

0.0

1.0

0.0

0.4

0.0

0.5

0.1

0.0

0.4

0.0

0.2

0.1

0.2

0.2

0.0

0.0

994b

2.0

0.0

0.0

0.2

0.2

0.1

0.0

0.0

0.1

0.0

0.0

0.0

0.2

0.2

0.2

0.1

0.0

0.0

20–74

821

8.5

0.4

0.0

0.7

0.4

0.0

1.1

0.1

0.5

0.2

0.1

0.5

0.6

0.5

0.2

0.1

0.0

0.0

Sukvirach et al. GP5+/6+ (2003), Lampang and Songkla, Thailand

15–69

1673

4.8

0.0

0.0

0.7

0.3

0.3

0.5

0.2

0.3

0.1

0.2

0.3

0.2

0.4

0.1

0.2

0.0

0.0

Xi et al. (2003), MY09-11 Senegal

> 35

1639

12.5

0.2

0.0

1.0

0.9

0.4

0.7

0.0

0.1

0.2

0.3

0.5

0.3

0.7

0.4

0.1

0.3

0.1

Asato et al. (2004), Japan

L1C1/C2

21–93

3049

10.2

0.1

0.0

0.5

0.2

0.3

0.4

0.8

0.1

0.0

0.9

1.2

0.6

0.2

0.2

0.5

0.0

0.0

Cuschieri et al. GP5+/6+ (2004b), Scotland, United Kingdom

17–78

3089

12.7





3.4

1.4

0.7

0.5

0.3

0.4

0.9

0.9

0.8

0.6

0.7

0.7

0.2

0.8

0.1

Ferreccio et al. (2004), Chile

15–69

921

11.2

0.2

0.4

2.2

0.4

0.5

0.1

0.3

0.7

0.7

0.7

0.8

1.3

1.0

0.9

0.0

0.2

0.0

Shin et al. SPF10 (2004), Republic of Korea

16–29

672b,d 15.2

0.7

0.3

1.3

1.2

0.7

0.4

0.3

0.9

0.1

1.8

1.3

1.5

0.7

0.4

0.5

0.5

0.0

Thomas et al. (2004), Nigeria

> 15

844

0.4

0.4

3.0

1.7

2.6

0.6

3.0

0.4

2.1

1.1

1.5

2.1

2.5

0.6

0.2

0.5

0.4

de Sanjosé et al. GP5+/6+ 15–69 (2003), Spain Hanoi, Vietnam GP5+/6+ 15–69 Shin et al. GP5+/6+ (2003), Republic of Korea

Matos et al. (2003), Argentina

GP5+/6+ 15–69

GP5+/6+

GP5+/6+

987

24.8

Page 120

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Reference, study area

P_115_178.qxp

120

Table 11 (contd)

P_115_178.qxp 30/11/2007 09:26

Primer system

Age range No. (years)

Overall HPV

Specific HPV type (%) 6

11

16

18

31

33

35

39

45

51

52

56

58

59

68

73

82

Franceschi et al. GP5+/6+ (2005), South India

16–59

1799

14.0

0.2

0.0

2.8

0.8

0.8

0.8

0.8

0.6

0.3

0.4

0.7

1.1

0.2

0.7

0.0

0.2

0.2

Herrero et al. (2005), Costa Rica

MY09-11

> 17

7459

22.4

0.4

0.2

2.2

1.1

1.1

0.5

0.2

0.4

0.5

1.5

1.1

0.5

1.3

0.3

0.2

0.3

0.3

Ronco et al. (2005), Italy

GP5+/6+

25–64

997

7.8

0.1

0.2

2.7

0.1

0.3

0.1

0.1

0.3

0.6

0.1

0.3

0.4

0.4

0.1

0.2

0.0

0.0

See Table 7 for a description of the primers used. a TS, type specific b A small number of women with abnormal cytology included c Estimated d 75% virgins

HUMAN PAPILLOMAVIRUSES

Reference, study area

Page 121

Table 11 (contd)

121

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IARC MONOGRAPHS VOLUME 90

HPV type 16 had the highest prevalence in all European studies (Cuzick et al., 1995; Forslund et al., 2002; de Sanjosé et al., 2003; Cuschieri et al., 2004b; Ronco et al., 2005) and also in most of the other studies. Examples of exceptions are a study from Kenya (6.2% HPV 52 versus 3.5% HPV 16) and one from Nigeria (Thomas et al., 2004) (3% HPV 35 versus 3% HPV 16). In all but one study (Asato et al., 2004), HPV 16 was either first or second in rank, and no other type consistently dominated. However, among the HPV-positive women, the percentage with HPV 16 varied from 8 to 66%. Types 6, 11, 59, 68, 73 and 82 were consistently rare in all of the studies. Table 12 summarizes the prevalence of HPV in cervical specimens among commercial sex workers. Using PCR-based methods for the detection of HPV DNA, overall prevalence of all HPV types that were tested varied by region and ranged from 14.4% in Singapore to 77.4% in Belgium. Infection with a high-risk HPV type was more common: HPV 16 had the highest prevalence that ranged from 4.3 to 13.9% . Commercial sex workers had a higher prevalence of HPV infection compared with women who were not involved in such occupations (Juárez-Figueroa et al., 2001; Thomas et al., 2001a; Mak et al., 2005). A study of the determinants of regional variation in age-specific HPV prevalence will help an understanding of viral persistence, clearance and possibly latency. Some studies of highly exposed women such as prostitutes (Kjaer et al., 2000) have shown a significant decrease in the prevalence of HPV with age, despite continuously high sexual activity, and indicate loss of viral detection and type-specific immunity to re-infection. In contrast, a study of sexually active human immunodeficiency virus (HIV)-negative men who had sex with men (Chin-Hong et al., 2004) showed that the prevalence of anal HPV infection was high among men under 30 years of age (approximately 60%) but remained high in all age groups studied. These data suggest that repeated exposures may contribute to high prevalence over a wide age range, at least in the anal canal. Studies that focus on older women and their male partners are also needed, particularly cohort studies with repeated measurements that assess male and female sexual practices and immunity. The changes in sexual mores that began in the mid-1960s would have been expected to lead to an increase in the prevalence of HPV infection over time in most western populations. The extremely high prevalence of HPV in young women in North America (Winer et al., 2003) and the United Kingdom (Peto et al., 2004) supports the existence of a strong cohort effect. Confirmation of this hypothesis, however, would require that preserved specimens of representative samples from different eras be tested at the same time with the same sensitive testing technology, a proposition that could not be easily implemented. There is, however, limited evidence from seroepidemiological studies that the prevalence of antibodies against certain HPV types may have increased. For instance, in Finland, seropositivity for HPV 16 among women aged 23–31 years increased from 17% in 1983–85 to 24% in 1995–97 (Laukkanen et al., 2003). In contrast, the prevalence of HPV 16 and HPV 11 was stable between the two periods at 9–12%.

P_115_178.qxp

Reference, study area

Age range (years)

Method of detection

Prevalence (%)

Kjaer et al. 182 (2000), Denmark

20–45

GP5+/6+ primers

32.4

Chan et al. 187 (2001), Singapore

19–71

PVCOU/PVCOD concensus primers with probing for 11/16/18

14.4

12.3

JuárezFigueroa et al. (2001), Mexico

495

18–62

MYBO9/MYB11/ HMBB01 L1 concensus primers

48.9

Thomas et al. (2001a), Thailand

251

15–35

Choi et al. (2003), Republic of Korea

417

Ford et al. (2003), Indonesia

Overall

High risk

Prevalence of specific high-risk types (%) Low risk

16

18

31

39

45

Prevalence of specific low-risk types (%) 51

52

56

58

59

66 6

11

34

40

42

43

0

0

6.7

2.4

1.4

3.2

1.1

1.1

53

54

73

1.1

43

24.6

11.1

3.6 11.1

MY09/MY11 primers 47 with probing for 6/11/ 16/18/31/33/35/39/45

36.3

10.8

13.9

6

15–51

Hybrid Capture 2 47 detection; genotyping by DNA oligonucleotide microarray with MY09/11 primers

64

9

11.5

3.6

614

14–47

Oligoprobes specific for 16, 18, 31, 33, 35, 45, 52, 6, 11 and a probe with a mixture of 16, 18, 31, 52

38.4

14.5

3.5

Tideman et al. (2003), Australia

288

16–36

MY09/MY11 primers 31.6

12.2

Mak et al. (2005), Belgium

93

17–58

SPF10

55.9

0.53

3.2

0.8

1.6

5.7

13.9a

1.4

6.6c

1.9

4.6

4.7

0.53 1.6

5.5

4.4

4.9

7.9

2.7

3.6

4

6.3

9.5

5.3 5.9

6.5

3.2 10.8g

0.53

3.4

10.8b

2.4

2.9

3

5.5d

09:26

2.7

HUMAN PAPILLOMAVIRUSES

4.3

4

4

3.1

4

2.4

1

2.4e

1.2

0.2 3.1

3.5b

17

12.9f

3.2

–f

6.5

3.2

7.5

9.7

1.1

–f

6.5

2.2

4.3

3

5.4

2.2 1.1

123

See Table 7 for a description of the primers used. a HPV 31/33/35/39 b HPV 6 /11 c HPV 16/18 d HPV 31/33/35 e HPV 45/52 f HPV 16/31/52 g May show cross reactivity with HPV 68

35

9.9

2.7

77.4

33

Page 123

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Table 12. Prevalence of cervical HPV detected by polymerase chain reaction (PCR) among commercial sex workers

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Incidence, persistence and clearance

Many prospective epidemiological studies published since the last evaluation (IARC, 1995) provide data on incident infection (although such events may represent latent infections that for some reason become detectable again) and duration of infections by different types. Tables 13 and 14 show the main characteristics of these studies and illustrate the estimates of incidence and duration by type, respectively. Table 13 summarizes the incidence of type-specific HPV infection (infection per 100 person–years). Based on these data, approximately 5–15% of HPV-negative women are infected each year with any of the high-risk types of HPV (Franco et al., 1999; Moscicki et al., 2001; Richardson et al., 2003; Sellors et al., 2003; Muñoz et al., 2004). The incidence of infection with high-risk HPV types tends to be higher than that with low-risk types (Moscicki et al., 2001; Richardson et al., 2003; Muñoz et al., 2004). The most common types of incident infection tend to include HPV 16, 18, 31, 33 and 51; one of the highest type-specific infections among the studies is HPV 16 (Ho et al., 1998a; Franco et al., 1999; Woodman et al., 2001; Giuliano et al., 2002a; Richardson et al., 2003; Winer et al., 2003; Harper et al., 2004; Muñoz et al., 2004). In addition, rates of high-risk HPV infection tend to be greater among younger than older women (Franco et al., 1999; Muñoz et al., 2004), although median duration of infection appears to be comparable by age (Muñoz et al., 2004). Only limited prospective data are available on the duration of HPV infection with age, as determined by related longitudinal measurements of type-specific HPV DNA. One of two studies (Muñoz et al., 2004) suggested that duration of HPV infection increases with age (Castle et al., 2005). It is widely accepted that persistence of HPV infection is essential for the development of cervical precancerous lesions and cancer. Fortunately, most HPV infections are transient and become undetectable within 1–2 years even by sensitive PCR assays (Ho et al., 1998a; Franco et al., 1999; Molano et al., 2003a; Richardson et al., 2003; Muñoz et al., 2004). Consequently, anogenital HPV infections tend to resolve spontaneously, as do warts anywhere on the body. Presumably, they are cleared completely by the cell-mediated immune system, are self-limited or are suppressed into long-term latency. Knowledge of how often HPV transience in the short term represents successful immune clearance versus a selflimited infection would be useful. However, this question cannot easily be answered by the measurement technologies currently available to epidemiologists. A major unresolved question regarding the natural history of HPV is the extent to which viral infections are cleared. Even when no HPV DNA is detectable by conventional molecular tests, small foci of cells that maintain infection at low DNA copy numbers could exist, and they may explain the results of studies in immunosuppressed individuals. However, it is not known how frequently this occurs in immunocompetent individuals, how long it lasts, what causes re-emergence into a detectable state or what fraction of cancers arises after a period of latency. Answers to these questions will greatly affect prevention strategies that rely on the detection of HPV DNA.

P_115_178.qxp 30/11/2007

Table 13. Incident cervical HPV infection as detected by HPV DNA among women who were HPV-negative at baseline Setting

No. at risk

Mean/ median follow-up (years)

Test method

Type-specific incidence rate of HPV (per 100 person–years) 16

18

31

33

35

39

45

51

52

0.8

0.8

1.1

0.9

3.7

1.3

Ho et al. (1998a)a, USA

University students

608

2.2

PCR and southern blot

25–49

20

3.4

1.9

1.0

Franco et al. (1999), Brazil

Lowincome maternal and child health programme

1425

0.8

MY09/11 PCR

26–39; 33.3

33.3

1.6

0.3

1.1

Moscicki et al. (2001)a, USA

Family planning clinic

105

1.9

PCR with dot blot

13–21; 20.0

20

Woodman et al. (2001)a, United Kingdom

Family planning clinic

1075

2.4

GP5+/6+ PCR

15–19, 18g

15–19

4.2

2.5

1.1

1.4

Giuliano et al. (2002), USA

Family planning clinic

331

0.8

MY09/11 PCR

18–35; 24.2

24.2

7.1

1.0

3.0

1.5

Koutsky et al. (2002), USA

Placebo arm of HPV vaccine trial

765

1.5

Typespecific PCR

16–23; 20.1

16–23

3.8

Richardson et al. (2003), Canada

University health clinics

621

1.8

MY09/11 PCR

17–42; 23

17–42

6.2

2.3

2.0

Sellors et al. (2003)a, Canada

Medical practices

253

1.2

HC2h

15–49; 32.7

32.7

56

58

59

53

66

1.3

1.1

1.6

1.8

3.3

1.4

1.1

HR

11

0.8c

1.5

5.5

2.2

0

4.1

4.1

3.5

1.0

1.8

1.5

10.9d

16.1

5.9f

26.8

1.7c

1.0

4.0

4.0

3.0

2.0

1.0

16.8

2.7

Any

19.9

15.8e

0.4

LR

2.5

8.1b

1.9

6

15.7

1.4

35.2

14.9

22.8

9.5

Page 125

Incidenc e rate by age

HUMAN PAPILLOMAVIRUSES

Age in years (range; mean)

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30/11/2007

Reference, study area

Setting

No. at risk

Test method

Age in years (range; mean)

Incidence rate by age

Type-specific incidence rate of HPV (per 100 person–years) 16

18

31

33

2.4

6.0i

444

3.4

MY09/11 PCR

18–20; 19.2

18–20

5.5

2.1

Harper et al. (2004), North America, Brazil

Placebo arm of HPV vaccine trial

553

2.3

SPF10 PCR

15–25

15–25

2.4

1.4

Muñoz et al. (2004), Colombia

Cervical cancer screening center and familyplanning clinics

1610

4.1

GP5+/6+ PCR

15–85; 32.3g

15–85 15–19m 20–24m 25–29m 30–44m ≥ 45m 15–19 20–24 25–29 30–44 ≥ 45 < 35 ≥ 35

1.0 3.7 2.3 2.0 1.3 0.0

0.7 1.3 2.2 1.3 0.9 0.0

0.7 3.0 1.1 0.7 0.4 0.0

1.9 1.2

0.6 0.0

1.6 0.4

45

51

0.7

4.5j

52

56

58

59

53

66

HR

4.2

6

11

LR

Any

3.9

0.5

4.1k

0.2 1.2 0.6 0.0 0.1 0.0

0.2 0.6 0.5 0.0 0.3 0.0

2.0 2.6 3.4 3.7 1.8 0.7

6.2 17.2 11.3 9.5 5.4 1.4

10.8 d 11.0 d

17.9 13.8

3.4l

0.4 1.2 0.0 0.7 0.2 0.0

0.5 2.5 0.5 1.0 0.5 0.0

0.5 1.8 0.0 0.3 0.6 0.0

2.0 0.2

1.7 0.8

0.5 1.2 0.0 0.0 0.2 0.0

0.7 3.0 1.1 0.0 0.6 1.3

1.4 0.6

2.2 1.4

See Table 7 for a description of the primers used. HR, high-risk HPV types (16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 68 and others); HC, Hybrid Capture; LR, low-risk HPV types (6, 11, 26, 40, 42, 53, 54, 55, 57, 66, 73, 82, 83, 84, 73 and others) a Calculated estimate of incidence rate from reported data: incidence rate per 100 person–years = number of events/(no. at risk × mean follow-up) × 100 b 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 68 c 6, 11 d 6/11, 26, 32, 34, 40, 42, 44, 53, 54, 55, 57 59, 62, 64, 66, 67, 69, 70, 72, 73 e 16, 18, 31/33/35, 39, 45, 51, 52, 56, and 58 f 6/11/42/44 g Median age reported h Hybrid Capture includes HPV types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59 and 68. i 33, 35, 39 j 51, 52, 55, 58 k 40, 42, 53, 54 l Infection with either HPV-16 or -18 m Reported cumulative risk at 1 year

5.0 17.4 9.5 6.9 4.1 0.7 21.4 7.6 7.1 9.3 6.6 10.6b 4.8b

1.2c 0.4c

Page 126

University students

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Winer et al. (2003), USA

35

09:26

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Table 13 (contd)

P_115_178.qxp 30/11/2007

Table 14. Duration of cervical HPV infection as detected by HPV DNA among women who were HPV-negative at baseline Reference, study area

Setting

No. at risk

Testing method

Median duration (months) of infection by specific HPV type 16

18

31

33

35

39

45

51

52

608

2.2

PCR and southern blot

20

11

12

6

7

6

6

6

7

7

University students

Franco et al. (1999), Brazil

Low-income maternal and child health programme

1425a

0.8

MY09/11 PCR

26–39; 33.3

Woodman et al. (2001), United Kingdom

Family planning clinic

1075

2.4

GP5+/6+ PCR

15–19; 18d

Giuliano et al. (2002), USA

Family planning clinic

331a

0.8

MY09/11 PCR

24.2

Richardson et al. (2003), Canada

University health clinics

621

1.8

MY09/11 PCR

17–42; 23

19.4

9.4

20.0

Muñoz et al. (2004), Colombia

Cervical cancer screening centre and familyplanning clinics

1610

4.1

GP5+/6+ PCR

15–85; 32.3d

13.7

11.9

16.5

58

59

53

66

6

6

8

6

HR

6

6

13.5b

10.3

7.8

8.6

9.0

13.0

8.2c

9.8

13.4

9.0

12.2

8.4

9.7

14.6

13.9

14.8

13.2

Any

8

9.4e

11.0

8.5

8.0

LR

13.7

4.3

6.4

14.8

12.3

11.1

HUMAN PAPILLOMAVIRUSES

Ho et al. (1998a), USA

56

See Table 7 for a description of the primers used. HR, high-risk HPV types (16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 68 and others); LR, low-risk HPV types (6, 11, 26, 40, 42, 53, 54, 55, 57, 66, 73, 82, 83, 84, 73 and others); PCR, polymerase chain reaction a Duration calculated for prevalent cases of infection b 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 68 c 6/11, 26, 32, 34, 40, 42, 44, 53, 54, 55, 57 59, 62, 64, 66, 67, 69, 70, 72, 73 d Median age reported e 6, 11

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Persistence (i.e. long-duration of detectable HPV infection) is uncommon compared with clearance. From a practical point of view, persistence can be defined as the detection of the same HPV type (or, with a higher degree of certainty, the same intratypic variant) two or more times over a certain period. There is no consensus as to the length of time that implies persistence, but at least 6 months to 1 year is the time frame that is usually chosen. Although commonly adopted, this definition of convenience does not correspond to the understanding of the natural history of HPV. The median duration of type-specific HPV infection in several prospective studies is summarized in Table 14. Duration tends to be longer for high-risk HPV types compared with low-risk types (Franco et al., 1999; Giuliano et al., 2002a; Muñoz et al., 2004). This approach is complemented by a study of longer-term infection with a median follow-up of 5.1 years (Schiffman et al., 2005) which showed particularly pronounced persistence of HPV 16 compared with any other HPV type. In contrast to other short-term studies, the persistence of high-risk types other than HPV 16 was not much longer than that for many low-risk types (Schiffman et al., 2005). The longer duration of infection with high-risk types of HPV may have implications for the pathogenesis of CIN3 and cancer. It is of practical importance that epidemiologists agree on a uniform definition of HPV persistence, for example, taking into account whether analysis of viral variant is required as an extra level of taxonomic detail to ascertain this phenomenon (Franco et al., 1994). Proof that this safeguard is essential for studies of low-risk populations has yet to be obtained; however, it seems that persistent infections tend to maintain the same original variant, at least in the case of HPV 16 or 18 (Villa et al., 2000). There is considerable uncertainty concerning the importance of measurements of viral load or the presence of associated, microscopically evident abnormalities with respect to the duration of persistence. More data are needed, particularly to clarify whether infections with different types of HPV act independently on the cervix, with regard to both immunology and direct interaction. The sparse data are conflicting as to whether the presence or the absence of any one type alters the duration of any other type-specific infection (analogous to whether types influence the acquisition of each other as mentioned above) (Thomas et al., 2000; Liaw et al., 2001; Rousseau et al., 2001). Persistence of high-risk HPV DNA after local ablation or excision of high-grade CIN is predictive of failure of treatment, whereas clearance of HPV predicts success of treatment (reviewed in Arbyn et al., 2004a,b; Zielenski et al., 2004). 1.4.5

Microscopic abnormalities

Microscopic abnormalities are diagnosed in only a minority of women who have HPV that is detectable by DNA assays. The fraction depends on the thresholds of the molecular and microscopic tests and clinical specimens examined, and can range widely from 5 to 30% (Schiffman & Kjaer, 2003). Microscopic diagnoses are prone to subjectivity and lack of interobserver reproducibility, particularly when mild or equivocal changes are involved. Therefore, misclassification is always a major concern when epidemiologists contemplate

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how best to consider HPV infection as a transition state in multistage models such as that shown in Figure 8. It is important to develop a rational classification for HPV infections that arise in a prospective epidemiological study. The persistence of at least one carcinogenic HPV type is the necessary state for the emergence of precancer. However, other aspects of infection may contribute to the likelihood that an infection will progress, such as type, load and concurrent abnormalities. Even among the carcinogenic types, HPV 16 is uniquely associated with risk for cancer and, even for HPV 16 (and other carcinogenic types), variants are relevant to the natural history. Low viral loads detectable only by PCR (not the commercially available Hybrid Capture) are associated with microscopic normalcy and with low risk of subsequent precancer and/or cancer. Viral load is clearly associated with concurrent disease (Cuzick et al., 2003), but the value of increasing loads with respect to subsequent prediction of lesions has not been established (Lorincz et al., 2002; Schlecht et al., 2003a,b). It is still not known whether microscopically evident abnormalities represent a stage in the natural history that is separate from HPV detected by DNA testing alone (Castle et al., 2002b). In a recent 24-month prospective follow-up of women with carcinogenic HPV DNA, the presence or absence of mild histological abnormalities did not materially affect the risk for subsequent precancer (Cox et al., 2003). Observations suggest that a fraction of precancers arise from HPV infections in the absence of mild or even equivocal microscopically evident abnormalities (Koutsky et al., 1992; Cuzick et al., 1995). This might also represent a misclassification of cytology or histology or rapid transit through the mildly abnormal phase. It has been proposed that precancers develop in HPV-infected mucosa independent from and adjacent (internal) to CIN1 rather than being an internal subclonal event (Kiviat et al., 1992). These hypotheses can be addressed only through very intensive longitudinal studies that combine visual, microscopic and molecular measurements. 1.4.6

Progression to precancer

HPV infections (even with carcinogenic types) are so common that becoming infected is not the limiting factor in cervical carcinogenesis. The critical step for most women might be whether a precancerous lesion develops as an uncommon outcome of infection (Figure 8). The first difficult task is to define ‘precancer’ on the basis of histology, i.e. that an intraepithelial lesion is destined to progress, although latency may be very long. There is substantial heterogeneity in the microscopic diagnosis and biological meaning of CIN2 lesions in particular. Some certainly represent acute HPV infections of particularly bad microscopic appearance that are destined, however, to regress, whereas others are incipient precancers that are destined to persist with a high risk of invasion. Some non-carcinogenic HPV infections can produce lesions that are diagnosed as CIN2, which shows that this level of abnormality is not a sufficient surrogate for cancer risk. CIN3 should be used as a surrogate for precancer and CIN2 as a buffer zone of equivocal diagnosis, similarly to

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ASCUS or more minor cytological abnormalities. [The Working Group generally agreed that a combination of CIN2 and CIN3 as high-grade CIN is a sub-optimal end-point for intervention studies due to the potential for misclassification of CIN2.] In studying the transition from HPV infection to precancer, attention should be restricted to women with carcinogenic types of HPV (unless a particular controlled comparison is being made). Within this group, viral characteristics, host factors and behavioural co-factors that increase the risk of progression or decrease the probability of viral clearance need to be determined. Persistence of HPV (defined at the type-specific level) is by far the most important determinant of progression (Nobbenhuis et al., 1999) but there has been considerable heterogeneity in the way in which epidemiological studies have determined persistence and the time to ascertainment of lesion outcomes (reviewed in Schiffman & Kjaer, 2003). The time between the occurrence of HPV infection in the late teens or early twenties and the peak of precancer at around 30 years of age is about 7–10 years. More rapid progression does occur and should be studied, but it may not be possible to study the full extent of the latency process prospectively. Using cytological end-points, it is clear that presence of carcinogenic HPVs in a specimen carries a prognostic value. The mean time to progression from ASCUS to LSIL or worse and from LSIL to HSIL or worse is significantly shorter in women who have carcinogenic HPV types than in women who have no HPV infection (e.g. mean times for ASCUS progression are 67.0 and 88.0 months, respectively, in women with carcinogenic HPV and no HPV; difference, 21.0 months; 95% CI, 11.3–30.7 months). In general, cervical abnormalities persist longer and progress more quickly in women who have carcinogenic HPV infections than in women who have noncarcinogenic infections or no HPV (Schlecht et al., 2003b). 1.4.7

Progression of lesions

Several natural history studies have analysed the risks for progression beginning at different points in the continuum of pre-invasive lesions. For a balanced interpretation of these data, the following caveats must be considered for most of these studies: the small sample size, the highly selected study population, the insufficient follow-up time, the reporting of crude rates of progression and regression without a precise actuarial analysis of cumulative risk over time and the variability of methods to detect the development of lesions during follow-up. In particular, detection methods that use cytology cannot provide reliable estimates of rates of lesions and those that use histology may have altered the course of the natural history of the disease because frequent cervical biopsies may remove the entire lesion. Overall, these problems tend to affect the comparability of results across studies. These drawbacks notwithstanding, the following conclusions can be drawn from natural history studies: (a) the vast majority of CIN2 are transient and regress to normal within relatively short periods, although some may progress to CIN3 or to cancer over variable periods of time; and (b) in contrast, CIN3 carries a much greater probability of progression to invasion, although many such lesions may eventually regress.

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Östör (1993) conducted a pooled analysis of studies published from 1950 to 1992 to derive average estimates of regression and progression by grade of CIN. The average probabilities of regression were 57% for CIN1, 43% for CIN2 and 32% for CIN3. The equivalent probabilities of progression to carcinoma in situ were 11% for CIN1 and 22% for CIN2, and those of progression to invasion were 1% for CIN1, 5% for CIN 2 and 12% for CIN3. A substantial proportion of lesions were biopsied, including cone biopsies, and were classified as persistent without further qualification as to the duration of the sojourn time within each grade, i.e. 32%, 35% and 56% for grades 1, 2 and 3, respectively. Mitchell et al. (1994) conducted a similar meta-analysis but modified the method for ascertaining lesions during follow-up in order to stratify the estimates. By considering only studies with cytological follow-up and all grades of CIN combined, the probabilities of regression, persistence and progression to any higher-grade lesion were 34%, 41% and 25%, respectively. Regarding the latter progression figure, 10% of the lesions progressed to carcinoma in situ and 1% to invasive cancer. The equivalent cumulative probabilities for all grades of CIN that had been followed by both cytology and biopsy were 45%, 31% and 23% for regression, persistence and progression, respectively. Within the latter probability, progression to carcinoma in situ was 14% and that to invasive cancer was 1.4%. Progression rates to invasive cancer for studies that followed up only patients with carcinoma in situ by biopsy ranged from 29 to 36%. In a meta-analysis of studies published since 1970 that included more than 27 000 patients who were followed without treatment, Melnikow et al. (1998) calculated the following weighted average rates of progression to HSIL at 24 months according to baseline cytological abnormality: ASCUS, 7.1%, LSIL, 20.8%, and HSIL (persistence), 23.4%. Cumulative progression rates to invasive cancer at 24 months by cytological abnormality were 0.3% for ASCUS, 0.2% for LSIL, and 1.4% for HSIL. The following average rates of regression to a normal Pap smear were estimated: 68.2% for ASCUS, 47.4% for LSIL, and 35.0% for HSIL. [The Working Group noted that none of the CINs was tested for HPV DNA in the above three studies.] CIN3 lesions tend not to regress over short-term follow-up; however, risk for and timing of invasion versus eventual regression follow stochastic processes that are mediated by biological variables. The median age at diagnosis of women with precancer (CIN3) in many countries that carry out screening is approximately 30 years, whereas the median age of women with invasive cancers is skewed towards much older ages. The age of women who have screen-detected invasive cancer tends to be more than 10 years older on average than women with CIN3, which suggests a long average sojourn time in the precancer state. The size of the precancerous lesion can be used as a proxy for risk of invasion but prospective proof cannot be obtained for obvious ethical reasons. Epidemiological studies have not been able to suggest risk factors for invasion. The frequently-discussed phenomenon of HPV DNA integration is associated with invasion, but it is difficult to prove that it is causal.

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Accuracy and reliability of measurements

Advances in the understanding of the natural history of HPV have followed intensive methodological efforts to standardize accurate and reliable measurements of HPV DNA. In most cases, the incoherent results from the late 1980s and early 1990s were caused by unsuspected misclassification of HPV status in the first large-scale molecular epidemiological studies of HPV and cervical cancer (reviewed by Franco, 1991, 1992; Schiffman & Schatzkin, 1994). Improvements in cytology and serology have been less extensive but very important. In future cohort studies that multiply the number of measurements taken over time, the importance of optimized methods will be even greater if observation and interpretation of the patterns of viral clearance, persistence, possible recurrence and progression are to be anticipated. 1.4.9

Serology

Serology of VLPs by ELISA methods is a very useful epidemiological tool for defining past and cumulative exposure to HPV infection. The assays are reasonably type-specific and are usually negative in individuals who have never been infected (Dillner, 1999; Kjaer et al., 2001). This specificity is useful for the definition of HPV-infected cohorts, in whom etiological co-factors can be studied. For example, serology can be used to define HPV-exposed individuals among control subjects in case–control studies that emphasize analyses only among the exposed. However, only about half of the women with currently detectable infections of the same type (with the use of DNA and microscopy) are seropositive, which suggests that the current techniques to measure the serological response are still not sufficiently sensitive. Therefore, HPV seronegativity does not exclude exposure, partly because current assays for seropositivity do not cover more than a few types of HPV. To date, serological assays have not proved to be useful in defining immunological responses related to the natural history of HPV infection. Two important caveats must be recognized for the interpretation of sero-epidemiological studies. The first is the cross-reactivity and relatively low sensitivity in terms of types and the second is the fact that infections in other mucosal sites of the body (e.g. the mouth) also elicit antibody responses that cannot be distinguished from those arising in the anogenital area. 1.4.10

Other sites (see Table 15)

A few studies have addressed the prevalence of HPV in smears from the vagina, vulva, foreskin, anus and urethra from the general population. From these results, it has been suggested that the prevalence of any HPV-type infection in the vagina and vulva is in the same range as that of the cervix. The prevalence of HPV among men (penis and urethra) varied from less than 10% to about 50%. Prevalence in neonates and primary school children (anal smear or foreskin) showed very low percentages (< 1%).

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Table 15. Prevalence of HPV infection at sites other than the cervix Reference, location

Site

Primer

Overall HPV (%)

Specific HPV type (%) 6

11

2.9

8.6

105

18–23

Army conscripts (men)

Typespecific primers

17.1

Oral epithelial scrapes

62

Not reported

Healthy young adult volunteers

Type 16specific

44.0

Lawton et al. (1992), Australia

Oral mucosa

60

Not reported

PCR

60.0

Chen et al. (1993), Australia

Foreskin

92



Neonates

L1 consensus primers

0

Eike et al. (1995), Denmark

Oral smear

61

20–79

Normal individuals

Consensus primer

0

Nasal smear

48

Koch et al. (1997), Denmark

Anal smear

706

Oral smear

482

Bowden et al. (1999), Australia

Vagina

1090

Morin et al. (2000), Canada

Vulva

322

2.1 0–17

Nurseries, kindergarten, primary school

PCR + dot-blot

0.5

12–73

Two communitybased studies using selfadministered tampon

PCR

42.0

22.5 ± 4.0

Gynaecological clinic

PCR

23.9

16 1.9

18

31

33

35

39

45

51

52

56

58

59

73

82

5.7

44.0

0.2

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Kataoka et al. (1991), Sweden

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Reference, location

Site

Oral cavity

Kojima et al. (2003), Japan

Primer

Overall HPV (%)

Specific HPV type (%) 6

11

16

18

31

33

35

39

45

51

52

56

58

59

73

82

1.8a

40.5 (29–65)

Populationbased oesophageal cancer screening programme

GP5+/6+

7.0

268

≤ 20

Infants, children, adolescents

MY09/11

6.0

Oral cavity

77

3–5

Nursery school

L1 C1/C2 1003/1004

48.1

de Lima Soares et al. (2003), Brazil

Vagina

341

≥ 15

Rural area

HC

26.6

Kreimer et al. (2004), USA

Oral rinse

590

40.6 (18–85)

PGMY09/ 11

12.2

1.7

0.7

0.7

0.3

0.2

0.2

0.5

0.3

0.7

0.3

0.8

Tonsil brush biopsy

583

Oral cancer screening programme

3.1

1.0

0.2

0.2

0.0

0.2

0.0

0.0

0.0

0.2

0.0

0.0

Kurose et al. (2004), Japan

Oral mucosa

668

0–60+

Healthy volunteers

MY09/11

0.6

Lambropoulos et al. (1997), Greece

Oral cavity

169

14–85

Asymptomatic subjects

PCR + southern blot

9.5

2.4

0.0

0.7

4.0

0.4

29.7

4.1

0.6

2.7

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No.

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Table 15 (contd)

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Site

No.

Age (years)

Setting

Primer

Rosenblatt et al. (2004), Brazil

Penis

90

Not reported

Partners of women joining cervical cancer screening programme

HC

Shin et al. (2004), Republic of Korea

Penis

381

16–23 (21.3 ± 2.2)

University students

SPF10 primer

Smith, E.M. et al. (2004a,b), USA

Oral rinse

333

≥ 18

Routine screening visits in hospital

MY09/11

Overall HPV (%)

Specific HPV type (%) 6

11

0.5

0.3

16

18

31

33

35

39

45

51

52

0.8

0.3

0.8

0.8

56

58

51.1

8.7

18.3

0.5

10.0

0.5

< 1.0

0.3

< 1.0

0.3

< 1.0

59

73

82

HUMAN PAPILLOMAVIRUSES

Reference, location

See Table 7 for a description of the primers used. HC, Hybrid Capture; PCR, polymerase chain reaction a HPV 16/18

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Prevalence studies that address the oral mucosa in adults showed very diverse results that ranged from 0 to 60%. This was also true for the few studies among children. It has been suggested that HPV type 16 is by far the most prevalent type, and that HPV 6 and 11 are much less prevalent. The reason for the diversity between the studies needs to be explored. One study on the oesophagus showed a prevalence of HPV infection of 7.0% (type 16 or 18 had a prevalence of 1.8%) (Peixoto Guimaraes et al., 2001). 1.5 1.5.1

Pathology of HPV infection of the genital tract and evidence therefrom for progression to malignancy Evolution of concepts and terminology (a)

Dysplasia and carcinoma in situ

By the late 1800s, the histological changes that occurred at the margins of invasive squamous-cell cancers of the cervix had been recognized and described by Williams (1888). Their significance was not appreciated at the time, but these changes were later called carcinoma in situ and described precursors of cervical cancer. Reagan and Hamonic (1956) introduced the term ‘dysplasia’ to designate cervical epithelia that contained cytologically atypical cells but lacked the full-thickness of differentiation. Dysplasias were further divided into mild, moderate and severe, depending on their degree of differentiation. From this terminology, it was implicit that the higher the grade, the closer the lesion was in aggregate to invasion. This assumption was based upon the observation that higher-grade dysplasias resembled carcinoma in situ and invasive cancer more closely than those of a lower grade. However, carcinoma in situ remained in the minds of clinicians as the only true precursor of cancer. Patients with this disease were generally treated by total hysterectomy and those with lesser degrees of epithelial change — dysplasias — were either treated by cervical conization or followed prospectively without treatment (reviewed in Younge, 1965). (b)

Cervical intraepithelial neoplasia (CIN)

With continuing clinical experience, it became obvious both to pathologists and clinicians that there was extremely poor inter- and intra-observer reproducibility in the differentiation of carcinoma in situ from dysplasia. It was particularly difficult for pathologists to distinguish between severe dysplasia and carcinoma in situ, and clinicians became increasingly sceptical of the rationale for therapy that was dictated by the classification system for dysplasia–carcinoma in situ. In view of this, and after the completion of a number of laboratory and clinical studies that were begun in the 1960s, it became apparent that severe dysplasia and carcinoma in situ could not be distinguished reproducibly at any level and that the lesser degrees of atypia — particularly mild and moderate dysplasia — merged imperceptibly in objective measurements with the higher-grade lesions (Richart, 1987). These observations led to the introduction of the term ‘cervical intraepithelial neoplasia’ (CIN) to designate the spectrum of cervical diseases that were thought to play a role

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in cervical carcinogenesis. The reasoning behind the terminology of CIN was that a continuum of change began with mild dysplasia and ended with invasive cancer after passing progressively through the intermediate stages of intraepithelial disease. The clinical impact of this new terminology was that presumed precursor lesions should be treated based on their size and location. In CIN1 (mild dysplasia), neoplastic basaloid cells occupy the lower third of the epithelium; in CIN2 (moderate dysplasia), neoplastic basaloid cells and mitotic figures occupy the lower two-thirds of the epithelium; and in CIN3, mitotic figures and basaloid cells can be found throughout the whole thickness of the epithelium. In the grading of CIN lesions, CIN3 included severe dysplasia and carcinoma in situ and, in terms of treatment, less emphasis was placed on hysterectomy in favour of outpatient-directed methods and conservation of the uterus (Richart, 1987). As molecular data accumulated, it became apparent that the spectrum of atypical epithelial changes that occurred in the female lower genital tract and that were etiologically related to HPV could best be described as a two-tiered, rather than a three-tiered disease process, and the CIN classification was modified accordingly (Richart, 1990). Those lesions commonly referred to as mild dysplasia, flat condyloma or CIN1, which were thought to be the result of a productive HPV infection, were designated low-grade CIN. Those lesions that contained more severe cytological atypia (CIN2 and CIN3), which were thought to be true potential precursors of cancer and to require treatment, were designated high-grade CIN. The distinction between low-grade CIN and high-grade CIN was based upon an assessment of cytological atypia and the presence or absence of abnormal mitotic figures. However, it was emphasized that the diagnostic decision should be taken at an operational level as well as at a morphological level so that the clinician could infer accurately from the diagnosis whether the pathologist believed that the lesion being diagnosed was a true precursor of cancer or not. Several publications have questioned whether high-grade CIN develops from existing low-grade CIN or develops de novo (Koutsky et al., 1992, Kiviat & Koutsky, 1993). The current commonly held opinion is that CIN3 can develop either via the sequence of CIN1 and CIN2 into CIN3 or directly from a high-risk HPV infection with no demonstrable stages of CIN1 or CIN2 (Kiviat et al., 1992; Park et al., 1998b; Nobbenhuis et al., 1999; Winer et al., 2005). (c)

Squamous intraepithelial lesions (SILs)

Because of the problems caused by an extremely low degree of intra- and interobserver reproducibility in cytological diagnoses, a group was convened in Bethesda, MD (USA), to devise a uniform cytological terminology (National Cancer Institute Workshop, 1989; Luff, 1992). This meeting concluded that molecular data are more consistent with a two-tiered, rather than a three-tiered system. This new nomenclature known as ‘The Bethesda System’ introduced the terms ‘low-grade squamous intraepithelial lesion’ (LSIL) and ‘high-grade squamous intraepithelial lesion’ (HSIL) (see Table 16). LSIL includes CIN1 or mild dysplasia, koilocytosis, koilocytotic atypia and flat condyloma. HSIL includes CIN2 and CIN3, or moderate and severe dysplasia, and carcinoma in situ.

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Negative

AGC

Negative

HSIL

Borderline and mild

Moderate

HSIL Severe

SCC

In situ

dysplasia/dyskaryosis Netherlands/CISOE-Aa

Description

Pap 1

Pap 2

Pap 3a1

Normal

Atypia

Mild

Pap 3a2

Pap 3b

Pap 4

Pap 5

Moderate

Severe

In situ

Invasive

dysplasia/dyskaryosis Histology

CIN SIL

Grade 0

Grade 1 LSIL

Invasive

carcinoma

Grade 2

carcinoma Grade 3

HSIL

From Bulkmans et al. (2004) ADC, adenocarcinoma; AGC, atypical glandular cells; ASC-H, atypical squamous cells that cannot exclude HSIL; ASCUS, atypical squamous cells of undetermined significance; CIN, cervical intraepithelial neoplasia; HSIL, high-grade squamous intraepithelial lesion; LSIL, low-grade squamous intraepithelial lesion; Pap, Papanicolaou test; SIL, squamous intraepithelial lesion; SCC, squamous-cell carcinoma a C stands for compositon of the smear, I for inflammatory changes, S for squamous epithelium, O for other and endometrium, E for endocervical columnar epithelium and A indicates whether or not the smear is adequate.

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ADC

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AGC favour neoplastic

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Table 16. Relationship between histological classification of CIN and SIL and cytomorphological Pap classification

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In order to evaluate and update the 1991 Bethesda classification, the 2001 Bethesda system was established. The terminology used was agreed after a review process in which more than 400 cyto-/histopathologists, gynaecologists, cytotechnologists, epidemiologists, health physicians and lawyers were involved. The dichotomous division of SIL into LSIL and HSIL was based on virological, molecular and clinical observations that LSIL is more frequently a result of a transient HPV infection whereas HSIL is more frequently associated with viral persistence and high risk for progression. LSIL includes changes that mainly reflect HPV infection, which eliminates the distinction between condylomatous atypia and CIN1, whereas HSIL includes higher-risk lesions, including precursors of cancer (Solomon et al., 2002). In addition, equivocal interpretations called atypical squamous cells of undetermined significance (ASCUS) are more common than definite lesions; approximately half of these lesions are related to HPV infection. In addition, HSIL is usually associated with high-risk HPV types and is monoclonal and aneuploid in contrast to LSIL (Fu et al., 1983; Lungu et al., 1992; Park et al., 1998b; Hering et al., 2000). Data from the ASCUS/LSIL Triage Study confirm that (a) LSIL is a fairly reproducible break-point compared with HSIL; (b) the subdivision of cytological HSIL into moderate and severe dysplasia or CIN2 and CIN3 is not very reproducible; and (c) the cytopathological effects of HPV cannot be reliably separated from those of CIN1 or mild dysplasia (Bulkmans et al., 2004; Schiffman & Adrianza, 2000). However, despite the moderate reproducibility of diagnoses into three CIN grades, pathologists in several European countries still use the three-tiered designation. They noted that (a) separation into CIN1, CIN2 and CIN3 correlates to a general extent with rates of progression and/or regression of the lesions (Mitchell et al., 1996); (b) the use of MIB-1, an antibody directed against cell proliferation-associated Ki-67 antigen that stains cells in the G2M phase, increases the reproducibility of the CIN classification (Bulten et al., 1996; Kruse et al., 2001); and (c) with regard to microscopic morphological interpretation, poor reproducibility does not denigrate clinical value (Renshaw et al., 2003). The terminology of CIN is especially helpful to correlate cytopathological and histopathological findings and to manage individual patients based on the finding that moderate dysplasia (CIN2) has characteristics more similar to mild dysplasia (CIN1) than to severe dysplasia/carcinoma in situ (CIN3) (Östör, 1993; Nobbenhuis et al., 1999). CIN or dysplasia can be substituted for SIL or used as an additional descriptor (Table 16; Solomon et al., 2002; Bulkmans et al., 2004). A good example of such an approach that allows easy translation to the Bethesda 2001 system is the CISOE-A classification that is used in The Netherlands (Bulk et al., 2004), in which C stands for composition of the smear, I for inflammatory changes, S for squamous epithelium, O for other and endometrium and E for endocervical columnar epithelium; A determines whether or not the smear is adequate. (d)

Adenocarcinoma in situ

Adenocarcinoma in situ is characterized by a complex gland formation that arises within the normal endocervical glands, cytological atypia, an increased mitotic rate and a

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gland-within-gland pattern. High-risk HPVs are found in nearly all adenocarcinomas in situ and in adenocarcinomas of the cervix. HPV 18 is more frequent in this disease than in squamous-cell carcinoma (Zielinski et al., 2003). (e)

Intraepithelial neoplasms of other organs in the male and female anogenital tract

Intraepithelial lesions of the vagina, penis and anus are generally diagnosed using a modification of the CIN terminology system and are also graded into three groups. The presumed precursor lesions for these organs are referred to as vaginal intraepithelial neoplasia (VAIN), vulvar intraepithelial neoplasia (VIN), penile intraepithelial neoplasia (PIN) and anal intraepithelial neoplasia (AIN) (Zbar et al., 2002). Similarly to CIN lesions, it is believed that these lesions progress via increasing degrees of intraepithelial involvement. However, their progression rate is rather slow. Follow-up data on PIN1 lesions indicate that transition to high-grade PIN is a rare event (Bleeker et al., 2003). 1.5.2

Temporal and spatial relationships between precursors of cervical cancer and invasive cancer (a)

Histological observations

The original observations that suggested the existence of a precursor of cervical cancer and that led to the term carcinoma in situ were made by pathologists who noted that the epithelium overlying or adjacent to cervical cancers contained cytological alterations that were similar to those found in invasive cancers. This simple but important observation led to the concept that cancers were preceded by a precursor state that could be recognized histologically. The invention of the colposcope by Hinselmann (1925) allowed gynaecologists to recognize clinically alterations in the cervical epithelium that could be diagnosed by punch biopsy as carcinoma in situ. These alterations could then be treated to prevent the development of invasive cancer. However, it was not until Papanicolaou and Traut (1943) published their observations on exfoliated cells that it was discovered that these early histological and colposcopical observations could be used as part of mass screening programmes and be translated into schemes for cancer prevention. Subsequent observers noted that the mean age at diagnosis of mild, moderate and severe dysplasia, carcinoma in situ and invasive cancer increased progressively and that this increase was accompanied by an increase in the size of the lesion. This increase was in turn found to be accompanied by an increase in gland and canal involvement; in addition, the larger lesions were more likely to contain areas of invasion. These observations lent strong support to the hypothesis of the progression of CIN to cancer (Jones, 2006). Retrospective analysis of lesions that were diagnosed as co-existing LSIL and HSIL in relation to the presence of high-risk HPV revealed which of the lesions (which span two grades: CIN1 and CIN2) most probably represented morphological progression from a single infection (Park et al., 1998b). However, lesions that contain CIN1 and CIN3 may be attributed either to progression of the lesion or to two coincidental infections. Further-

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more, in retrospective studies that analysed previous smears from women with cervical cancer for the presence of HPV and of abnormal cells, it was noted that (a) many women had smears with abnormal cells that had been overlooked by the cytopathologist/cytotechnician; and (b) the same high-risk HPV type was present both in the cervical carcinoma biopsy and in the previous smear (Walboomers et al., 1995; Wallin et al., 1999; Zielinski et al., 2001a,b). This temporal relationship between cervical precursor lesions and cervical cancer in the presence of the same high-risk HPV type indicates the progression of such precursor lesions to cervical cancer. (b)

Microinvasive and early invasive cervical cancers

The most important direct pathological evidence that putative precursors are in fact precancerous lesions was the histological observation of invasion arising from such lesions. Tongues of invasion that range from only one or two cells to larger lesions are seen to arise directly from surface CIN lesions or from intraepithelial lesions that involve the endocervical glands. These tongues of microinvasive carcinoma may be single or multiple and are generally accompanied by a local inflammatory infiltrate and a desmoplastic response. In the cervix, the risk of metastasis depends upon the degree of stromal penetration. Microinvasive cancer with a stromal penetration of ≤ 3 mm and a length of ≤ 7 mm (FIGO [International Federation of Gynaecology and Obstetrics] stage Ia1) rarely metastasizes and can be treated conservatively. Invasive lesions with a depth of stromal penetration > 3 but < 5 mm and < 7 mm in length (FIGO stage Ia2) have a minimum risk of metastasis and can be treated conservatively if the woman wants to preserve functional integrity. Invasive lesions with a depth of stromal penetration > 5 mm or > 7 mm in length (FIGO stage IB1 or more advanced stage) are treated radically (Lécuru et al., 1997). (c)

Clinical and epidemiological observations

Smith and Pemberton (1934) drew attention to the fact that patients who had invasive cervical cancer were commonly found to have had carcinoma in situ in their biopsies; when patients whose carcinoma in situ had been diagnosed by biopsy were followed without treatment, a significant number developed invasion. Similar observations were made by Kottmeier (1961) who followed 31 women with carcinoma in situ prospectively for at least 12 years; 72% of these women developed invasive cancer. In a similar study in New Zealand (McIndoe et al., 1984), 131 patients with persistently abnormal Papanicolaou (Pap) smears were followed for 4–23 years; 22% developed invasive carcinoma of the cervix or vaginal vault and 69% had persistent carcinoma in situ, which was treated subsequently. These observations of the natural history of carcinoma in situ suggest that, in the majority of the patients, once this disease is established, it rarely regresses spontaneously. There is therefore a discrepancy between the cumulative incidence of carcinoma in situ observed in the natural history studies conducted in British Columbia (Canada), The Netherlands and Denmark, which suggested that a high proportion of carcinomas in situ do regress without treatment, and the cumulative incidence of invasive cancer seen in earlier observational studies (Smith & Pemberton, 1934; Kottmeier, 1961; McIndoe et al., 1984; Miller, 1992).

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An explanation might be that, in these observational studies, carcinoma in situ was diagnosed at a relatively late phase and thus represented large lesions, whereas, in the nationwide screening programmes, much smaller lesions were diagnosed as having a lower viral load and a higher tendency for regression. The natural history of cervical precursor lesions of a lower histological grade than carcinoma in situ has been studied by Ho et al. (1998a) and Nobbenhuis et al. (1999) and has been reviewed extensively by Östör (1993). 1.5.3

Histological changes in HPV-related lesions of the lower female genital tract

The natural history of an HPV infection is age-dependent; a dramatic increase in the detection of cervical HPV DNA occurs after the initiation of sexual activity (Koutsky et al., 1992; Melkert et al., 1993; Hildesheim et al., 1994; Ho et al., 1998a; Kjaer et al., 2001, Winer et al., 2003). High-risk HPV is usually assumed to enter the cells of the basal and parabasal layers at sites of minor trauma or where the anatomical architecture provides easy access. Depending on host and cellular factors, the infection can be cleared spontaneously and quickly (transient HPV infection): this happens in about 70% of women within 1 year (Ho et al., 1998a; Woodman et al., 2001); the remaining 30% develop detectable CIN lesions. Subject to their stage and the immune status, CIN lesions may regress after the HPV infection has been cleared (Nobbenhuis et al., 2001). About 50% of low-grade lesions regress within 1 year, while a smaller proportion of high-grade lesions regress. The persistence of high-risk HPV infection is prerequisite for progressive CIN. (a)

Latent HPV infection

HPV genomes are present in the basal layers of infected epithelia and differentiation is required for the production of virions. The latency of HPV can be defined as a state in which viral DNA is maintained in the absence of virion production. Latent HPV infection is operationally defined as an infection in which the replication of viral DNA is synchronized with the cell cycle but in which none of the cytopathogenic effects of HPV can be detected. Although no direct evidence for a solely latent HPV infection has been found, a number of clinical observations suggest that it may occur. (i) HPV DNA can be detected in apparently normal cervical epithelium, and several studies have shown that the risk for women with a high-risk HPV-positive, cytomorphologically normal smear to develop an abnormal smear within 2 years or CIN3 within 4 years is substantially increased (Koutsky et al., 1992; Hildesheim et al., 1994; Liaw et al., 1999; Rozendaal et al., 2000). (ii) A common observation is that women who have no clinical or cytological evidence of HPV while in the interpartum state may develop HPV-related lesions during the relatively immunocompromised state of pregnancy and that such lesions regress without treatment post partum (Nobbenhuis et al., 2002). (iii) Women who take immunosuppressive therapy for renal transplantation and those with HIV infection have a higher incidence of CIN and cervical cancer (Klein

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et al., 1994; Williams et al., 1994; Wright et al., 1994; Cappiello et al., 1997; Sun et al., 1997; Cu-Uvin et al., 1999; Ellerbrock et al., 2000). (iv) Patients in whom HPV-related lesions have been treated may have detectable HPV DNA despite normal cytological, colposcopical and histological findings. Such patients are at increased risk for recurrence compared with HPV DNAnegative controls (Koutsky et al., 1992; Nobbenhuis et al., 2001). (b)

Low-grade CIN

A number of studies reported that it was possible to distinguish between virus-containing flat condyloma and a true ‘virus-free’ CIN lesion (Meisels & Fortin, 1976). However, subsequent studies found that the distribution of HPV types in those lesions designated as flat condyloma and CIN was indistinguishable (Kadish et al., 1986; Willet et al., 1989) and that, due to this lack of consistent morphological features, the ability to make such distinctions has extremely low inter- and intra-observer reproducibility. In addition, no differences in nuclear DNA content was observed, as both have diploid/polypoid DNA distribution patterns (Fu et al., 1983; Fujii et al., 1984). It is therefore not thought to be possible to separate flat condylomas from low-grade CIN or SIL lesions. Low-grade CIN is, by definition, a lesion that is well differentiated but abnormal and contains alterations that are characteristic of the cytopathogenic effects of a replicative HPV infection. Operationally, it is a lesion that is thought by pathologists to be the result of a productive viral infection and not to represent a true precursor of cancer. Low-grade CIN lesions can arise through infection by any of the anogenital HPV types. It supposedly arises from HPV-infected basal cells, which may gain the capacity to multiply the virus to very high copy numbers. However, this productive stage is restricted to postmitotic, differentiated cells in the suprabasal layers of the epithelium that are withdrawn from the cell cycle. Detailed in-situ hybridization and immunohistochemical studies have shown that a high expression level of viral genes, multiplication of the viral genome, synthesis of early (E6, E7, E2 and E4) and late gene products (L1 and L2), encapsulation of the HPV genome and release of virion particles together with the exfoliation of upper epithelial layers are strictly linked to terminal differentiation of the infected epithelia. The cytopathogenic effects of one HPV type compared with those of another are generally reported to be indistinguishable under light microscopy; however, some investigators have reported that HPV 16-induced lesions are more pleiomorphic than those induced by other HPV types (Crum et al., 1991). Most low-grade CIN lesions have a thickened epithelium due to the acanthosis that accompanies epithelial hyperplasia and many also have papillomatosis. The basal and parabasal layers characteristically have little cytological atypia, are arranged in a uniform fashion on the basal lamina and are not highly disorganized. As viral replication begins in the upper parabasal and lower intermediate layers of the epithelium, it is accompanied by the characteristic cytopathogenic effects of HPV infection that include cytological and organizational binucleation, perinuclear cytoplasmic cavitation with a thickened cytoplasmic membrane and, most importantly, nuclear atypia. The expression of E4-encoded

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proteins in squamous epithelial cells causes the cytokeratin matrix to collapse due to a specific binding to cytokeratin proteins (Doorbar et al., 1991) and possibly leads to the typical perinuclear cavitation, which is a feature of productive HPV infection. The combination of nuclear atypia and perinuclear halo formation is referred to as koilocytosis or koilocytotic atypia (Koss & Durfee, 1955). These koilocytotic cells are the principal hallmark of productive HPV infection of the cervical, vaginal or vulvar mucous membrane. It should be emphasized that perinuclear halos may be produced as a result of other cervical or vaginal infections or may accompany repair or metaplastic processes. The most characteristic histological feature of anogenital HPV infection, and that which is most useful diagnostically, is nuclear atypia. HPV-related nuclear atypia is due to heteroploidy (Fu et al., 1981), which appears to result from mitotic spindle abnormalities and leads to DNA replication without cytokinesis. The result of this interference with the mitotic process is the formation of bi- and multinucleated cells and enlarged atypical nuclei, accompanied by heteroploidization. In low-grade lesions, the nuclei are principally diploid and polypoid. Mitotic figures are generally increased in low-grade lesions but are mainly confined to the lower third of the epithelium, as are undifferentiated or basal-type cells, and are characteristically absent from the upper layers of the epithelium. Most of the mitotic figures have a normal appearance, but cells with tripolar mitosis or tetraploid-dispersed metaphases may also be seen (Winkler et al., 1984). These two types of abnormal mitotic figure are also commonly found in polyploid lesions in other organs. (c)

High-grade CIN

High-grade CIN lesions (CIN2 and -3) are substantially more atypical cytologically than low-grade CIN, have a higher degree of disorganization and have undifferentiated cells that extend beyond the lower third of the epithelium. This is reflected in the spectrum of HPV types found in low-grade CIN, which differs substantially from that found in high-grade CIN lesions (Matsukura & Sugase, 1995). In high-grade CIN, nuclear crowding, substantial pleomorphism, loss of both tissue organization and cellular polarity occur, and mitotic figures are characteristically found in the middle and upper thirds of the epithelium in addition to those in the lower third. The cytological atypia that is found in high-grade CIN lesions differs substantially from that seen in the low-grade lesions. The nuclei in high-grade CIN are generally larger, their nuclear membranes are more prominent and tend to be convoluted and distorted, and the nuclear chromatin pattern is characteristically clumped, coarsely granular and contains prominent chromo-centres. As the nuclei enlarge, the nuclear cytoplasmic ratio is altered in favour of the nucleus and the cell borders, which commonly contain visible desmosomes in low-grade lesions, become indistinct and difficult to define. In contrast to low-grade lesions, expression of the viral oncogenes E6 and E7 in high-grade lesions also occurs in the dividing, immature, metaplastic basal stem cells. It has been reported that the E6 protein in particular but also the E7 protein of HPV 16 induce chromosomal aberrations (White et al., 1994; Duensing

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& Munger, 2002). The characteristic koilocyte of low-grade CIN is generally absent or markedly attenuated in high-grade lesions. One of the most important features that distinguishes high-grade CIN from low-grade CIN is the presence of abnormal mitotic figures (Winkler et al., 1984). Although many different types of abnormal mitotic figure are found in high-grade CINs, the most characteristic is the three-group metaphase (i.e. chromosomal material on either side of the equatorial chromosomes in the metaphase) (Claas et al., 1992). Other abnormal mitotic figures that are commonly seen include the two-group metaphase, multipolar mitoses in excess of three, lagging metaphase chromosomes, coarsely clumped chromosomes and highly abnormal, bizarre mitotic figures. Abnormal mitotic figures are found in aneuploid lesions (aneuploidy is a marker for cancer or precancer) and have been reported to be the histological marker that best predicts the biological behaviour of CIN (Fu et al., 1981). As they are an excellent surrogate marker for aneuploidy (Bergeron et al., 1987a,b; Fu et al., 1988), these mitotic abnormalities serve as a useful objective marker to distinguish between lowgrade and high-grade CIN. In the presence of an abnormal mitotic figure, a lesion is consistently aneuploid and is a true precursor of cancer. In the absence of abnormal mitotic figures, other histological features commonly used to classify these lesions should be taken into account. (d)

Microinvasive and invasive squamous-cell cancer of the cervix

Microinvasive squamous-cell cancer of the cervix consists of a single (or multiple) irregular tongue(s) of neoplastic squamous epithelium that breaks through the plane of the basal lamina and invades the cervical stroma or epithelial lamina propria. Characteristically, areas of microinvasion are better differentiated than those of high-grade CIN from which they most commonly arise. They lack the smooth contour and crisp demarcation from the subjacent stroma that is found in both surface high-grade CIN and high-grade CIN with glandular involvement. Areas of microinvasion infiltrate in an irregular fashion and split collagen bundles. Microinvasive foci are commonly accompanied by an inflammatory and desmoplastic response. Microinvasion is defined as a lesion that invades the cervical stroma to a depth of no more than 5 mm; frank invasive cancer has a histological appearance similar to that of microinvasive cancer but has invaded more than 5 mm into the cervical stroma. No convincing evidence has been found that the histological appearance of invasive cancer or the prognosis of the patient can be predicted from the HPV type that has produced the lesion (van Bommel et al., 1993; Pilch et al., 2001). (e)

Adenocarcinoma in situ and adenocarcinoma of the cervix

Adenocarcinoma in situ is mainly localized in the endocervical canal; representative cells are therefore rare or absent in cytological specimens, and cytology rarely results in diagnosis. Moreover, because of the incomplete overview of the endocervical canal and the poorer prognosis of adenocarcinoma of the cervix compared with squamous-cell carcinoma, clinicians always remove intraepithelial lesions of the glandular cells and data on the natural history of these lesions are therefore lacking (Boon et al., 1981; Ruba et al.,

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2004). Whereas SIL occurs on the squamous side of the cervical squamo-columnar junction, adenocarcinomas in situ and adenocarcinomas occur on the columnar side. They are commonly associated with CIN lesions, particularly those that are high grade (Luesley et al., 1987). The endocervical epithelium does not appear to sustain productive HPV infections, and low-risk HPV types have not been found in endocervical neoplasia (Higgins et al., 1992a). Adenocarcinoma in situ is characterized by a complex gland formation in the distribution of the normal endocervical glands, cytological atypia, an increased mitotic rate and a gland-within-gland pattern. Cytological alterations similar to those seen in other aneuploid cell populations are present and abnormal mitotic figures are common. Adenocarcinoma in situ is distinguished from invasive adenocarcinoma by virtue of its pattern and lack of demonstrable invasion. Similarly to CIN3 and squamous-cell carcinomas, highrisk HPV is found in nearly all adenocarcinomas in situ and adenocarcinomas of the cervix (Zielinski et al., 2003). HPV 18 is found more commonly in these adenocarcinomas and some studies have described a poorer prognosis for these tumours (Walker et al., 1989; Schwartz et al., 2001). (f)

Condyloma acuminatum, intraepithelial neoplasia and cancer of the vagina

The histological changes in the vaginal mucous membrane that are associated with HPV infection and HPV-induced neoplasia are similar to those that are seen in the cervical mucous membrane. Thus, condylomata acuminata and VAIN may be present. Similarly to CIN, VAIN can be separated into three histological grades. The progression to vaginal cancer appears to be slow and the tumours have the morphology of a squamous-cell carcinoma. HPV 16 is the most prevalent type described in these lesions. (g)

Condyloma acuminatum, intraepithelial lesions and cancer of the vulva

The most characteristic HPV-related lesions found on the vulva are acuminate warts. Condyloma acuminatum, which is nearly always caused by HPV 6 or 11 (Gissmann et al., 1982a; Nuovo et al., 1990; Matsukura & Sugase, 1995), is an exophytic lesion. It has cytological and histological features and organizational alterations similar to those seen in the cervical and vaginal mucous membranes, except for the presence of substantial acanthosis and papillomatosis. Condylomata acuminata that occur on the mucous membranes characteristically have the full constellation of HPV-related cytopathogenic effects, including koilocytosis. Warts that occur in the keratinizing epithelium, however, commonly contain minimal cytological atypia, and koilocytes may be difficult to identify, particularly in clinically older lesions. The intraepithelial lesions of the vulvar skin (VIN) have a much more complicated histological pattern than those of the mucous membranes of the cervix and vagina. It is common to distinguish three different types of VIN histologically — basaloid, warty and

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well-differentiated. High-risk HPV types are found principally in the warty and basaloid types of VIN and are uncommon in the well-differentiated type (van Beurden et al., 1995). The basaloid type is composed generally of small, fairly uniform cells that are hyperchromatic and contain alterations in the distribution pattern of nuclear chromatin. These cell types tend to have low mitotic activity, and abnormal mitotic figures are seldom encountered. Warty-type VIN is generally a highly pleomorphic lesion with multinucleated cells, cytological atypia, coarse chromatin clumping, large numbers of mitoses and abnormal mitotic figures. It is commonly associated with koilocytosis, and adjacent condylomatous-type changes are frequently seen. The well-differentiated type of VIN is characteristically composed of a complex proliferative lesion that is only minimally altered in pattern and contains minimal nuclear atypia. Dyskeratosis is a common feature. VIN can be present as either a solitary patch or as multifocal lesions. Irrespective of this presentation, progression of VIN3 to vulvar carcinoma is rarer than was previously assumed (van Beurden et al., 1995) and radical vulvectomy has been replaced by more conservative treatments (van Seters et al., 2002). Carcinomas of the vulva are also of the basaloid, warty and well-differentiated types and have the same association with high-risk HPV as VIN. About 40% of vulvar carcinomas are high-risk HPV-positive; they occur in younger women and tend to have a more benign behaviour pattern than HPV-negative tumours (Al-Ghamdi et al., 2002; Gualco et al., 2003). Recently, a modified terminology based on morphological criteria only and not on HPV type or clinical appearance has been proposed for squamous VIN (Sideri et al., 2005). (h)

Condyloma acuminatum, intraepithelial lesions and carcinomas of the anus and penis

Condylomata acuminata of the anus and penis have the same histological appearance and contain the same HPV types as those in the cervix. Squamous neoplasms of the anus are similar morphologically to those that arise in other keratinizing epithelia, including HPV-related lesions of the vulva. The anus has a squamo-columnar junction and a transformation zone similar to that seen in the cervix. Squamous-cell cancers and their precursors develop at the squamo-columnar junction and in the transformation zone of the anus, as in the cervix. Anal canal tumours are histologically more similar to squamous carcinomas of the cervix, whereas perianal tumours more closely resemble those in the vulva and are of the basaloid, warty and more highly differentiated type. The association with high-risk HPV is strong (Frisch et al., 1997): more than 90% of tumours in the anal canal contain high-risk HPV, mostly type 16, whereas those in the perianal canal region contain slightly lower levels of high-risk HPV and again HPV 16 is the most dominant type. Receptive anal intercourse, especially starting at a younger age, is an important risk factor (Frisch et al., 1997; Gervaz et al., 2003). Squamous neoplasms of the penis are similar to those of the vulva with respect to the diversity of histological types and association with HPV (Ferreux et al., 2003). Most penile cancers are basaloid, warty, verrucous or keratinizing squamous-cell cancers. As in the

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vulva, basaloid and warty cancers are more strongly associated with HPV (primarily HPV 16) than squamous-cell cancers. The histology of PIN resembles intraepithelial neoplasia at other genital sites and ranges from grade 1 to grade 3. The appearance of PIN varies considerably depending on the circumcised status of the patient and location of the lesion. PIN1 lesions have been shown to have high copy numbers of HPV DNA (Bleeker et al., 2003) and form the main reservoir of HPV in men. 1.5.4

Pathology of cutaneous HPV infection and non-melanoma skin cancer (a)

Cutaneous HPV infection

Skin warts differ in clinical morphology and histological pattern depending on the HPV type by which they are induced. Cutaneous warts include common warts (verruca vulgaris; mainly associated with HPV 2, 4, 7 and 57), deep plantar and palmar, myrmecial warts (HPV 1), plane warts (verruca planar; HPV 3, 10 and 41), intermediate warts (mixtures of common and flat warts; HPV 26, 27, 28 and 29) and cystic or punctate, mainly plantar warts (HPV 60, 63 and 65) (reviewed by Jablonska et al., 1997). Skin warts are benign, show limited growth and often regress spontaneously. Common histological features comprise papillomatosis, acanthosis and parakeratosis to varying degrees. Virus-specific cytopathogenic effects are most prominent in the granular layer of the epithelium, where mature virus particles appear and spread throughout the nuclei or in paracrystalline arrays. HPV of the beta genus induce red-brown plaque-like lesions and achromic, scaly, pityriasis versicolor-like lesions only in EV patients (see Section 2.7.1) and exceptionally in immunosuppressed patients (Orth, 1986; Majewski et al., 1997). These HPV types are therefore referred to as EV-HPV (Orth et al., 2001). They are also highly prevalent in the general population (Boxman et al., 1997; Astori et al., 1998; Boxman et al., 1999; Antonsson et al., 2000; Forslund et al., 2003c), but do not induce the characteristic pathology. The histology of pathognomonic EV lesions reveals large cells with pale-stained cytoplasm in the spinous and granular layers. This specific cytopathic effect is linked to high levels of viral replication in differentiating keratinocytes. Common warts, plantar warts and genital warts are rare in EV patients. However, such patients are not infrequently infected by HPV 3 and 10 that induce flat warts, as in the general population, and occasionally confluent, elevated brownish plaques mainly on the extremities and the face (Majewski et al., 1997). In some EV patients, the lesions are highly proliferative, with features of papilloma or seborrheic keratoses (Jacyk et al., 1993a; Tomasini et al., 1993). This cytopathic effect depends on the association of these lesions with EV-HPV or HPV 3 (Majewski et al., 1997). Cutaneous warts develop in up to 90% of transplant recipients who survive the onset of immunosuppression by more than 5 years (Leigh et al., 1999). Two or more distinct HPV types were co-detected in most of these warts, and, in addition to the HPV types responsible for warts in the general population, EV-HPV and genital HPV DNA were also

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detected. However, no EV phenotype was expressed in most of these cases (Obalek et al., 1992; Harwood et al., 1999). A causative role of HPV in seborrheic keratoses has been speculated because of their histological similarity to warts, in that they display papillomatosis, acanthosis and hyperkeratosis. Mucosal HPV was detected in 20% of non-genital seborrheic keratoses in one study (Tsambaos et al., 1995) but not in others (Lee, E.S. et al., 2001). EV-HPV DNA was detected in small copy numbers in 76% of non-genital seborrheic keratoses (Li et al., 2004). EV-HPV DNA and HPV 16 DNA were also detectable by PCR in lesions of a case of stucco keratosis (Stockfleth et al., 2000), a skin disorder with multiple warty lesions that show papillomatous acanthokeratosis on histopathology. In view of the small copy numbers and not infrequently multiple genotypes in one specimen, it remains doubtful that HPVs are causative factors (Li et al., 2004). In two cases of Darier disease, which is characterized by crusted papules, plaques and verrucous lesions on nearly all parts of the body and histologically shows suprabasal lacunae and dyskeratosis, papillomatous proliferation and vacuolated keratinocytes in the upper stratum malpighii, HPV 5, 8, 36 and 38 from genus beta were detected by nested PCR whereas PCRs for mucosotropic HPV were negative (Li, Y.H. et al., 2002). In psoriasis, low levels of EV-HPV DNA can be detected in up to 90% of lesions and skin scrapings (Favre et al., 1998; Weissenborn et al., 1999; Mahé et al., 2003). The significantly higher prevalence of antibodies against capsid proteins of HPV 5 and 8 in patients with psoriasis compared with healthy donors (Favre et al., 1998; Stark et al., 1998) points to increased levels of productive infection in this extensive epidermal proliferation that is mediated by T-cell activation. It has been speculated that EV-HPV may contribute to the pathogenesis of psoriasis through enhancement of epidermal proliferation by early proteins and stimulation of T lymphocytes with the late, structural proteins (Majewski & Jablonska, 2003). (b)

Non-melanoma skin cancer

Non-melanoma skin cancer refers to basal-cell and squamous-cell carcinoma and includes the precancerous lesions, actinic keratoses and Bowen disease. Actinic keratosis is in essence a cutaneous counterpart of SIL in the genital mucosa (Fu & Cockerell, 2003). Keratoacanthoma, a common cutaneous lesion that broadly resembles a squamous-cell carcinoma, displays benign biological behaviour. In about half of the patients with EV, premalignant actinic keratosis and squamous-cell carcinoma arise in the lesions of this disease, mainly on parts of the body that are exposed to the sun, more than 25–30 years after its onset. The carcinomas are locally destructive but their invasive and metastatic potential is very low (Majewski et al., 1997). The cytopathic effect of EV-HPV is already absent by the onset of actinic keratosis. Some carcinomas in EV patients are typical basaliomas. In immunosuppressed transplant patients, both the clinical and histopathological features of non-melanoma skin cancer differ. Such patients have an up to 100-fold increased risk for squamous-cell carcinoma and a 10-fold increased risk for basal-cell carcinoma. It is

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not possible to distinguish reliably between keratoacanthoma and squamous-cell carcinoma in transplant recipients and, for management and classification purposes, they are referred to collectively as squamous-cell carcinomas. Similarly, actinic keratoses, intraepidermal carcinoma and Bowen disease in transplant recipients are not distinct entities and, since they are all thought to be dysplastic precancerous lesions, are referred to collectively as verrucous keratoses (Blessing et al., 1989). Squamous-cell carcinomas appear to arise from these verrucous lesions, which contain multinucleated cells and large numbers of atypical mitoses, koilocytes and parakeratotic peaks (Price et al., 1988; Blessing et al., 1989; Glover et al., 1995). These histopathological features have been cited to support a putative role of HPV in these lesions. Non-melanoma skin cancers of EV patients were consistently found to harbour large numbers of copies of extrachromosomal HPV DNA (EV-HPV types 5, 8, 17, 20 or 47) (Orth, 1987). In non-EV patients, highly sensitive detection techniques, such as nested PCR, are necessary to identify mostly EV-HPV DNA in up to 85% of actinic keratoses (Pfister et al., 2003), in 25–55% of basal- and squamous-cell carcinomas of immunocompetent individuals and in up to 90% of squamous-cell carcinomas in organ transplant recipients (reviewed in Harwood & Proby, 2002; Pfister, 2003; Harwood et al., 2004). A diverse spectrum of HPV types was detected and no single type predominated. Infections with several types were frequently noted in immunosuppressed patients. The small amounts of HPV DNA in skin cancers of non-EV patients suggest that only a minority of the tumour cells contain HPV DNA. In quantitative PCR studies, copy numbers varied from 50 HPV DNA copies per cell to 1 copy per 14 000 cells, with a median of 1 copy per 324 cells. In-situ hybridization identified only a few HPV DNA-positive nuclei per section (Weissenborn et al., 2005). An exception to this picture is skin carcinomas on the fingers, which appear to be strongly associated with genital HPV types (mostly HPV 16) (Alam et al., 2003). HPV 16 transcripts have also been detected in these carcinomas (Sanchez-Lanier et al., 1994). The rate of recurrence of HPV-associated digital squamous-cell carcinomas after surgical treatment greatly exceeds that for cutaneous cancer in general (Alam et al., 2003). 1.6

Non-malignant clinical lesions (other than precursors of cancer) of established HPV etiology

Genital HPVs cause condylomata, laryngeal papillomas and some papillomas at other mucosal sites, e.g. the oral or sinonasal cavity and conjunctiva. Cutaneous HPV types and EV HPV types cause skin lesions. HPVs have been reported to be associated with many other conditions, but the significance of these observations is as yet unclear (Shah & Howley, 1996). This section addresses only benign conditions that are clearly associated with HPV.

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Anogenital area

The terms condyloma acuminatum and genital wart are synonyms. For many years, exophytic warts were the only recognized HPV-associated manifestations of HPV infection in the genital tract. Increasing attention to the lower female genital tract with the extensive use of acetic acid, colposcopy, histology and molecular analysis revealed the presence of a spectrum of manifestations of anogenital HPV infection. Flat lesions, also called flat warts, are the most commonly reported manifestation of HPV infection that is not clinically overt. Flat warts are not easily seen by the naked eye, but application of acetic acid opacifies the thickened epithelium in contrast to the surrounding normal skin or mucosa and makes them visible, particularly under a magnifying glass or through the colposcope. Flat lesions can be found in most areas that exhibit exophytic warts. It has been estimated that flat lesions are at least twice as common as exophytic warts in the anogenital region (Koutsky et al., 1988; Beutner et al., 1998a; Wiley et al., 2002). Flat lesions frequently cluster in multiple lesions that are often confluent. Most probably, there is a continuum between normal skin or mucosa with detectable HPV DNA (i.e. latent infection) and overt anogenital warts that are clinically evident. Estimates of the prevalence of condylomata vary from 0.24 to 13% depending mainly on the risk of sexually transmitted diseases and age distribution in the population examined (Kjaer & Lynge, 1989). The prevalence in patients at clinics for sexually transmitted disease was 11% compared with 2% in college students and was highest in the group aged 16–24 years (Kiviat et al., 1989). Positivity for HPV DNA, which may reflect subclinical disease, was more than twice as common as clinical disease in 377 first attendees at such a clinic; 15% had genital warts, compared with 35% who were positive for HPV by ViraPap/ViraTypeTM (Borg et al., 1993). In women, the vulva, vestibule, vagina, perineum and perianal region are the most common sites for condylomata acuminata. HPV 6 and 11 were detected by southern blot hybridization in up to 95% of condylomata acuminata (Gissmann et al., 1982a; Johnson et al., 1991; Nuovo et al., 1991b). Several studies have investigated the relationship between vulvar vestibulitis, vestibular papillomatosis and HPV infection (Growdon et al., 1985; Moyal-Baracco et al., 1990; Costa et al., 1991; Umpierre et al., 1991; Wilkinson et al., 1993; Bornstein et al., 1996, 1997; Origoni et al., 1999; Morin et al., 2000). However, conflicting results were reported, probably because of the different populations studied and the different techniques used to reveal vulvar HPV infection. The most recent reports seem to exclude a direct role for HPV in the genesis of vulvar pain syndromes, even if a co-causal role cannot be excluded. Studies that included healthy subjects for comparison with cases found that a high percentage of asymptomatic women harbour HPV DNA in the vulvo-vestibular area (Handsfield, 1997). Condylomata acuminata are rarely detected on the uterine cervix. HPV 6 and 11 were identified in 65% and HPV 16 and 18 in 8% of these lesions by southern blot hybridization (Mitrani-Rosenbaum et al., 1988). Cervical condylomata may be hyperkeratotic

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and are sometimes confused with cancer owing to a bizarre pattern of vessels (Coppleson, 1991). The major capsid protein, L1, is detected more frequently and in greater quantities in condylomata acuminata of the uterine cervix than in similar lesions of the penis or the vulva (35% compared with 12% in a total of 95 cases), which indicates a higher content of virus particles (Wools et al., 1994). Genital warts are rarely observed in children. In addition to HPV 6 and 11, HPV 2 has also been detected in children and the route of transmission is through either the hands or auto-inoculation since all children with HPV 2-positive condylomata also had common cutaneous warts (Obalek et al., 1993). In men, penile and urethral condylomata show a distribution of HPV types similar to that of genital warts in women. In a series of 108 male patients, condylomata were located on the penile shaft in 51%, on the shaft and perianal region in 14%, on the shaft and scrotum in 2%, on the shaft and urethral meatus in 15% and on the urethral meatus alone in 18% (Rosemberg, 1991). Several authors have described the papular and macular aspects of the lesions (Barrasso et al., 1987; Del Mistro et al., 1987; Labropoulou et al., 1994). Recently, Bleeker et al. (2003) classified penile lesions into condylomata acuminata, papular lesions and flat lesions. Flat lesions are associated with mainly high-risk types of HPV and high viral loads, and form the reservoir of HPV in men (Bleeker et al., 2003, 2005a). While this information is very helpful to study viral transmission and spread between individuals, from a clinical viewpoint, routine use of 3% acetic acid, HPV typing or histology are unnecessary because these lesions do not necessitate cytodestructive treatment. Anal condyloma is one of the most common diseases of the anal canal and perianal region (for a review, see Vukasin, 2002). Together with AIN, anal condyloma is one of the primary clinical manifestations of HPV infection in the anal canal and on the perianal skin. It is usually found in conjunction with HPV 6 or 11, but HPV types known to be associated with anal cancer, such as HPV 16 or 18 (Syrjanen et al., 1987a; Bradshaw et al., 1992; Soler et al., 1992; Caruso & Valentini, 1999), or very rarely cutaneous HPV types may also be found (Soler et al., 1992; Strand et al., 1999). Typical perianal condylomata have a papillary appearance and may be highly keratotic, may be single or multiple and may be discrete or become confluent. Lesions may be asymptomatic or may be associated with burning or itching. Condylomata in the perianal region may also be flat and hyperpigmented, although a biopsy should be obtained in the latter case to exclude high-grade AIN. Bushke-Löwenstein tumours, also known as giant condylomata, may also occur in the perianal region. These usually contain HPV 6 or 11 but may also harbour carcinogenic HPV types such as HPV 16 (Kibrité et al., 1997; for a review, see Trombetta & Place, 2001). Anal condylomata are often seen inside the anal canal, where they may be associated with spontaneous bleeding or bleeding with bowel movements or anal intercourse. Inside the anal canal, the lesions may be papillary or flat. The manifestations and natural history of anal warts may differ between HIV-positive and HIV-negative patients. In HIV-negative patients, anal condylomata, typically associated with HPV 6 or 11, rarely progress to cancer although this has been documented in

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a few cases (Metcalf & Dean, 1995). However, the proportion of patients with anal condyloma who also have high-grade AIN is greater in HIV-positive patients than in HIV-negative patients (Anderson et al., 2004), and progression from low-grade lesions is more common in HIV-positive than in HIV-negative patients (Palefsky et al., 1998a,b; Anderson et al., 2004). Progression from anal condyloma to invasive anal cancer, particularly in immunosuppressed patients, has also been reported (Byars et al., 2001). 1.6.2

Upper respiratory tract

Recurrent respiratory papillomatosis is a relatively rare disease caused by members of the HPV family (Gissmann et al., 1982b; Mounts et al., 1982; Mounts & Kashima, 1984). HPV 11 is the most prevalent type (50–84%) found in laryngeal papillomas (Gissmann et al., 1983; Ushikai et al., 1994). When analysis is restricted to adult papillomas, HPV 16 is found most commonly (Corbitt et al., 1988). Although recurrent respiratory papillomatosis can be found anywhere in the aerodigestive tract, there appears to be a predilection for areas where there is a junction of squamous and ciliary epithelium. This includes the limen vestibuli (junction of the nasal vestibule and the nasal cavity proper), nasopharyngeal surface of the soft palate, mid-zone of the laryngeal surface of the epiglottis, upper and lower margins of the ventricle, undersurface of the vocal folds and the carina and bronchial spurs (Mounts & Kashima, 1984; Kashima et al., 1992a,b). HPV is also detected in the normal mucosa adjacent to lesions. Recurrent respiratory papillomatosis has a worldwide distribution, although it is more prevalent in some countries and areas than in others (Shykhon et al., 2002). It is a disease of both children and adults and exhibits a bimodal age distribution. The first peak occurs at less than 5 years of age and the second between the ages of 20 and 30 years (Kashima & Shah, 1982; Gissmann et al., 1983; Irwin et al., 1986), with incidences in the USA of 4.3 and 1.8 per 100 000, respectively (Shykhon et al., 2002). Boys and girls appear to be nearly equally affected by juvenile-onset recurrent respiratory papillomatosis in contrast with adult-onset recurrent respiratory papillomatosis, which preferentially affects men over women at a ratio of approximately 3:2 (Kashima et al., 1992b; Padyachee & Prescott, 1993; Doyle et al., 1994). This difference reflects the different mode of acquisition: by vertical transmission for the juvenile form and by sexual contact for the adult form. Vertical transmission of juvenile-onset recurrent respiratory papillomatosis from an active or latent maternal anogenital HPV infection was first recognized in 1956; a later prospective study showed that 50% of infants born to mothers with cervical HPV during pregnancy carried HPV in their nasopharynx (Sedlacek et al., 1989). It has been estimated that 10–25% of women of child-bearing age have evidence of latent or active HPV in cervical swabs and HPV DNA has been found in one-third to one-half of aerodigestive tract swabs of children born to affected mothers. However, only one in 400 infants delivered to these women is estimated to be at risk for subsequent recurrent respiratory papillomatosis (Bauman & Smith, 1996). In adults with recurrent respiratory papillomatosis, biopsies of normal mucosa adjacent to the papillomatosis were HPV DNApositive in a majority of patients (Steinberg et al., 1983; Rihkanen et al., 1993, 1994).

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Distal disease can develop and portends a poorer prognosis owing to its inaccessibility. HPV 11 is believed to have a greater propensity for distal pulmonary spread and a poorer prognosis for ultimate remission (Bauman & Smith, 1996). Distal bronchial obstruction can also result in post-obstructive pneumonia. Tracheal involvement occurs in 2–17% of patients without tracheostomies and appears as cobblestoning of the mucosa coupled with the presence of papillomas; more distal bronchopulmonary involvement is reported in 4–11% of children with long-standing disease (Shykhon et al., 2002). Although recurrent respiratory papillomatosis is considered to be a benign condition, the disease may undergo malignant degeneration. 1.6.3

Oral cavity

Numerous HPV types (including subtypes 1, 2, 4, 6, 7, 11 and 13) have been detected in benign lesions of the oral cavity (Garlick & Taichman, 1991; Flaitz & Hicks, 1998). Oral HPV-related benign verrucal-papillary lesions are clinically subdivided into verruca vulgaris, condyloma acuminatum, multiple and single papillomas and focal epithelial hyperpasia (Scully et al., 1985). Verruca vulgaris is induced by HPV 2 and 4. All 10 verrucae vulgares from the lip in one series were positive for HPV 2 DNA (Eversole et al., 1987a). Condyloma acuminatum and oral squamous papillomas are associated with HPV 6 and 11. Studies have detected the HPV capsid immunohistochemically in 10 and 22% of oral condylomatous and hyperkeratotic papillomas, respectively (Madinier & Monteil, 1987). More sensitive techniques such as southern blotting, however, have detected HPV 6 and 11 DNA in up to 85% of cases (Eversole et al., 1987b). Patients with genital condyloma have a high incidence of HPV-induced oral lesions; up to 50% of individuals with widespread genital condyloma have oral condyloma acuminatum (Eversole et al., 1987b). Of 202 cases of benign oral leukoplakia, 2.5% was positive for HPV 6 and 11 and 3.5% for HPV-16 by in-situ hybridization (Gassenmaier & Hornstein, 1988). One study on a gingival subset of oral proliferative verrucous leukoplakia, an oral lesion characterized as a solitary, recurring, progressive white patch that develops a verruciform architecture, showed no association with HPV (Fettig et al., 2000). HPV 13 (Pfister et al., 1983a) and HPV 32 (Beaudenon et al., 1987) are associated with focal epithelial hyperplasia of the oral mucosa (Heck disease), which is very rare in Europe and appears to be linked to certain ethnic groups, such as Inuits, native Americans, South African blacks (Cape coloureds) and individuals of Turkish or North African extraction. Clinically, the lesions are mostly flat and of the same colour as the surrounding mucosa, have a smooth surface and do not undergo malignant conversion. In 22 Mexican patients, human leukocyte antigen (HLA) DR4 (DRB1*0404) was significantly increased (odds ratio, 3.9; 95% CI, 1.86–8.03; p < 0.001); 17 of 20 patients (85%) were infected with HPV 13 (Garcia-Corona et al., 2004).

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Conjunctiva

Conjunctival papilloma is a benign and common tumour of the stratified squamous epithelium of the conjunctiva (Santos & Gómez-Leal, 1994). Conjunctival papillomas are known to occur in both children and adults, but they are most common among people aged 20–39 years (Sjö et al., 2000) with a slight preponderance among men (60%). Conjunctival papillomas are positive for genital HPV types 6, 11 and 16, which have been identified by in-situ hybridization or PCR (Naghashfar et al., 1986; Mäntyjärvi et al., 1989; Saegusa et al., 1995). The largest PCR-based study found 92% HPV DNA positivity; most of the 52 cases examined were HPV 6- or 11-positive and only one showed a multiple infection that included HPV 16 (Sjö et al., 2001). Only one report investigated normal conjunctival tissue and found HPV 16 and 18 at a frequency of 32% (Karcioglu & Issa, 1997). The access of HPV to the conjunctiva is still under investigation. Transmission to the conjunctiva may occur as a result of fetal passage through an infected birth canal or by ocular contact with contaminated hands or objects (Bailey & Guethlein, 1990). The presence of HPV 6 and 11 in adult conjunctival papillomas may reflect either activation of a latent HPV infection acquired at birth or an infection acquired later in life by transmission from other mucosal sites through either of the latter mechanisms (Naghashfar et al., 1986; McDonnell et al., 1987). 1.6.5

Skin

The skin of both healthy populations and immunosuppressed patients harbours a very large spectrum of HPV genotypes that includes EV-HPVs (Antonsson et al., 2000). Skin warts are clearly associated with HPV and are classified according to macroscopic and microscopic morphological criteria. Infection with specific HPV types can be broadly correlated with these lesions (Gross et al., 1982; Jablonska et al., 1997). Typical common or mosaic warts, i.e. rough keratotic papules or nodules, on the hands, knuckles or periungual areas contain HPV 2, 4, 7, 26, 27, 28 or 29. Using PCR on specimens obtained from 111 immunocompetent patients, HPV 2a was found in 15% of the warts, HPV 2c in 24% (now known to be HPV 27; Chan et al., 1994), HPV 57 in 12%, a variant of HPV 57 in 13% and HPV 4 only in one endophytic common hand wart (Rübben et al., 1993). Mucosal HPV 35 was found once in a periungueal wart of a patient with HPV 35-positive Bowenoid papulosis of the anogenital area (Rüdlinger et al., 1989). Butchers warts have the clinical appearance of common warts but occur on the hands of those who work with raw meat, fish and poultry. Using southern blot hybridization among 60 butchers, HPV 1 was found in 6.7% of warts examined, HPV 2 in 45%, HPV 3 in 15%, HPV 4 in 10% and HPV 7 in 23% (Orth et al., 1981). A similar distribution of HPV types was seen with PCR analysis; 23/26 lesions were positive for HPV DNA: 7.5% for HPV 2, 11.5% for HPV 4, 27% for HPV 7 and 42% for unidentified HPV types (possibly containing HPV 1 or 3) (Melchers et al., 1993). In another series, HPV 7 was

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found by PCR in 74/112 (66%) warts of men who worked in meat-processing plants (abattoir workers and butchers) (Keefe et al., 1994). Filiform or papillomatous common warts that are found most frequently on the face, lips, eyelids or nares contain HPV 1, 2 or 7 (Jablonska et al., 1985; Egawa et al., 1993a). HPV 7 was found in two individuals with generalized or extensive facial warts with filiform appearance (de Villiers et al., 1986a). Flat or plane warts, which can appear at different locations on the body and can form a linear arrangement (i.e. Koebner warts), are associated with HPV 2, 3, 10, 26, 27, 28, 29 or 41 (Melton & Rasmussen, 1991). Deep plantar warts, i.e. hyperkeratotic plaques or nodules on the plantar surface of the foot, are usually positive for HPV 1 or 4 (Rübben et al., 1993). HPV-associated epidermal cysts of the sole of the feet from 32 Japanese patients contained HPV 60 (Kato & Ueno, 1992; Egawa et al., 1994). HPV 1 and 63 were present in the same nucleus of one plantar wart (Egawa et al., 1993b). The morphological and virological findings of skin lesions in immunocompromised patients after transplants or in patients with EV are discussed in Section 2.7. EV-HPVs such as HPV 5 or 36 are often detected in patients with burns, cutaneous auto-immune bullous diseases or psoriatic lesions in which epidermal repair processes are very active (Favre et al., 1998a, 2000). Recently, it was suggested that these viruses are commensal in healthy individuals (Antonsson et al., 2000). 1.7

Therapy and vaccination

1.7.1

Therapy of benign disease (a)

Mucosal and cutaneous warts

Warts are the clinical manifestation of a benign productive HPV infection that can be cleared spontaneously. However, cytoreductive treatment is generally indicated to help the immune system to clear the infection more quickly and is aimed at the removal of all visible clinical lesions. This can be accomplished by medical or surgical methods, none of which is capable of removing the virus. Since this is the causative agent of the disease, the possibility of transmission and recurrence is not eliminated. Surgical methods for the treatment of genital and cutaneous warts include cryotherapy, electrodesiccation, surgical excision and laser–ablation techniques. Current therapies for HPV-related warts and neoplasia are summarized in Table 17 (modified from Zanotti & Belinson, 2002). An overview of the efficacy of different HPV treatment regimens is given in Table 18 (modified from Rivera & Tyring, 2004; for further reviews, see Jablonska, 1998; Gibbs et al., 2002; Torrelo, 2002; Gunter, 2003; Stanley, 2003; Bernard, 2004; Kodner & Nasraty, 2004; Fox & Tung, 2005).

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Table 17. Current therapies for HPV-related warts and neoplasia Cytotoxic agents Trichloroacetic acid

For the destruction of genital warts An 80–90% solution is applied directly to the wart in the clinic, and causes chemical destruction of wart epithelium. Treatment is repeated weekly. It is not absorbed systemically and can be used in pregnancy. It may cause burning of the surrounding skin.

Podophyllin

For the destruction of genital warts A plant compound that works by arresting cells in mitosis, it is applied weekly to warts at a concentration of 10–25% in a compound of tincture of benzoin in the clinic and should be washed off after 1–4 h. Applications should be less than 0.5 mL. This compound is absorbed in the systemic circulation and should not be used in pregnancy. Excessive exposure can cause bone-marrow depression.

Podofilox

For the destruction of genital warts A 0.5% solution is applied twice a day for 3 days, followed by 4 days without treatment. It is designed for self-application to reduce the number of clinic visits. Not to be used in pregnancy

5-Fluorouracil

For the treatment of multifocal or extensive VIN or VAIN An antimetabolite, it is applied as a 5% cream. A thin layer of cream is usually spread over lesions one to three times per week, but regimens may vary. It is designed for self-application. It causes tissue destruction by interfering with DNA and RNA synthesis and may cause significant local irritation. Not to be used in pregnancy.

Physical ablation Laser ablation

For the destruction of extensive genital warts or treatment of multifocal or extensive VIN or VAIN Carbon dioxide laser uses intense focal heat to vaporize tissue. This is a destructive method that does not permit pathological assessment of involved tissue. Usually, general anaesthesia is required. Postprocedural discomfort may be significant.

Excision Surgical excision

For large exophytic condylomata or confluent VIN or VAIN Surgical excision with re-approximation and closure using absorbable suture enables the pathological assessment of diseased tissue. Multifocal disease may not be amenable to this form of therapy. General anaesthesia is usually required. Postprocedural discomfort is generally less than that with laser treatment.

Loop electrode excision procedure

Primarily used to excise CIN It may also be used to excise genital warts or VIN or VAIN. The depth of excision may be difficult to control for vulvar and vaginal excision. It uses a radiofrequency alternating current passed along a thin wire loop to excise lesions with minimal thermal artefact.

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Table 17 (contd) Immuno-modulation Imiquimod

For the treatment of genital warts Recent evidence in small case series also suggests efficacy in VAIN and anal dysplasia (Davis et al., 2000; Pehoushek & Smith, 2001). It modifies the immune response, is a potent inducer of IFN-α and enhances cell-mediated cytological activity against viral targets. Applied topically, it induces local production of IFN and other cytokines that can be important mediators of viral clearance. It is designed for self-application as primary or adjuvant therapy of genital warts and is not recommended for mucosal surfaces, such as the vagina. A 5% cream is applied to warts overnight three times per week for up to 16 weeks; this regimen has led to complete clearance of genital warts in more than 30–60% of patients (Beutner et al., 1998b; Gollnick et al., 2001). Mild to moderate local inflammation is the most common side-effect, but the drug is well tolerated; no systemic side-effects have been reported.

Interferons (IFNs)

These have both immunomodulatory and direct antiviral activity. Routes of administration include intralesional injection, topical and systemic; for recombinant IFN-α or -β, intralesional injections are given at the base of each wart three times a week for 3 weeks; topical creams have little reported success; intramuscular or subcutaneous administration of IFV-γ is associated with a 30–50% clearance rate (Kirby et al., 1988; Bornstein et al., 1997). Systemic adverse effects, such as flu-like symptoms and leukopenia, are substantial, even with intralesional use. Despite its marked promise, IFN has never been widely used for primary therapy of genital warts because it has to be given via injection and produces systemic side-effects.

From Zanotti & Belinson (2002) CIN, cervical intraepithelial neoplasia; IFN, interferon; VAIN, vaginal intraepithelial neoplasia; VIN, vulvar intraepithelial neoplasia

(i) Pharmacological therapies Pharmacologically induced cytodestruction of virus-infected tissue has been achieved by the application of a wide variety of chemicals: podophyllin resin, podophyllotoxin, organic acids, such as salicylic acid, trichloroacetic acid and bichloroacetic acid, and cytostatic agents, such as bleomycin, cidofovir and 5-fluorouracil. More recently, immunomodulating compounds with antiviral properties, such as interferon (IFN)-α and imiquimod, have demonstrated potential efficacy. Cytodestructive drugs Podophyllin resin and its purified derivative podophyllotoxin belong to the lignan family of natural products that have important antineoplastic and antiviral properties. These compounds destroy virus-associated lesions by inducing tissue necrosis. The mechanism by which podophyllotoxin blocks cell division is related to its inhibition of microtubule assembly in the mitotic apparatus that results in cell-cycle arrest at metaphase (Manso-Martinez, 1982).

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Table 18. Efficacy of treatment regimens for HPV-related warts and neoplasia Maximum duration

Clearance

Recurrencea

Podophyllin resin (Edwards et al., 1988; Lacey et al., 2003)

P

Once or twice weekly

6 weeks

30–60%

30–70%

Podophyllotoxin (Lacey et al., 2003)

S

Three consecutive days alternating with 4 days of rest

6 weeks

45–75%

30–70%

Salicylic acid (Gibbs et al., 2002; Rivera & Tyring, 2004; Fox & Tung, 2005)

S

Soak in water for 5 min and dry; file wart; the solution and gel are applied two to three times daily and allowed to dry; discs are applied and covered for 48 h before removal.

20 weeks

48–87%

Insufficient data

Tri- and bichloroacetic acid (Godley et al., 1987; MenendezVelazquez et al., 1993; Fox & Tung, 2005)

P

Once weekly

‘Several’ weeks

60–81%

36%

5-Fluorouracil, topical (Pride, 1990)

S

Apply a thin layer one to three times each week and wash with soap and water after 3–10 h

6–8 weeks

47–68%

10–70%

5-Fluorouracil, intralesional (Swinehart et al., 1997a,b)

P

Injection once weekly

6 weeks

39–77%

58–70%

Bleomycin (Munn et al., 1996)

P

Intralesional injection; a variety of techniques available

4 injections

33–92%

Insufficient data

Cryotherapy (Jablonska, 1998; Rivera & Tyring, 2004; Fox & Tung, 2005)

P

Anaesthetic followed by freezing of the lesion and 1–2 mm of surrounding healthy tissue for 20–30 sec

6 weeks

50–96%

20–70%

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Table 18 (contd) Regimen

Maximum duration

Clearance

Recurrencea

Electrosurgery or laser (Bergman & Nalick, 1991; Ferenczy, 1991; Jablonska, 1998; von Krogh, 2001; Maw, 2004; Fox & Tung, 2005)

P

Carbon dioxide or Nd:YAG; exact regimen varies according to lesion.

3 weeks

≥ 90%

6–51%

IFN, intralesional (Friedman-Kien, 1995; Syed et al., 1995; Monsonego et al., 1996; Bornstein et al., 1997; Cox et al., 2004)

P

Two to three times weekly

8 weeks

36–75%

0–32%

Imiquimod (Beutner et al., 1998b,c; Edwards et al., 1998; Moore et al., 2001; Hengge & Cusini, 2003)

S

Three times weekly for 6–10 h

16 weeks

37–50%

13–19%

IFN, interferon; Nd:YAG, neodymium/yttrium/aluminium garnet laser; P, applied by physician; S, self-applied by patient Variable follow-up

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A 0.5% solution of podophyllotoxin (podophilox) applied topically reduced the mean number of anogenital warts from 6.3 to 1.1, destroyed about 70% of all warts and totally cleared warts in 29–50% of patients (Bonnez et al., 1994). In a comparative study, a 0.5% podophyllotoxin lotion totally cleared 81% of warts compared with a 61% clearance by 25% podophyllin (p < 0.001) (Kinghorn et al., 1993). In spite of this potency, the use of these products is no longer recommended because they engender a large variety of adverse effects and recurrence rates of up to 65% (Wiley et al., 2002). In addition, podophyllin and its derivatives are teratogens and should not be used in pregnant patients (von Krogh & Longstaff, 2001). Salicylic acid, in the form of a solution, a gel or a disc soaked with solution, is commonly used for the treatment of non-genital warts in adults and children with clearance rates of up to 75% (Rivera & Tyring, 2004). Other keratolytic compounds, such as glycolic acid, pyruvic acid, formic acid and glutaraldehyde, have also been used, particularly for the treatment of viral warts in children (reviewed by Torrelo, 2002). Pooled data from six placebo-controlled trials, in which 15–60% salicylic acid was used to treat cutaneous warts, showed a cure rate of 75% (144/191) in cases and 48% (89/185) in controls (odds ratio, 3.9; 95% CI, 2.4–6.4) (reviewed by Gibbs et al., 2002). Trichloroacetic acid and bichloroacetic acid have been used as an alternative to podophyllin. These compounds induce a massive coagulation of proteins, which results in destruction of the wart. They are applied topically as 50–85% solutions and can be selfadministered (Godley et al., 1987). In a more recent study, the clinical cure rate of an 85% solution of trichloroacetic acid in pregnant patients with cervical condylomata was 83% (Menendez Velazquez et al., 1993). However, trichloroacetic acid must be applied with extreme care in order to prevent acid burn to the surrounding skin (Fox & Tung, 2005). Bleomycin is a chemotherapeutic drug that interferes with DNA synthesis and causes necrosis of lesions. It is usually given by subdermal injection, but lateral injection, topical application and pricking with a bifurcated needle have also been used. Clearance rates of 33–92% have been reported; in particular, the multipuncture method has resulted in clearance rates of over 90% (Munn et al., 1996). 5-Fluorouracil is not known to have a specific molecular target in the HPV life cycle, but has been reported to be effective against genital HPV precursor lesions (Krebs, 1991; Syed et al., 2000). Reported clearance rates are 39–77%, but recurrence rates can be as high as 58% at 3 months and 70% at 6 months after treatment (Swinehart et al., 1997a,b). 5-Fluorouracil is contra-indicated in pregnant women. Immunomodulating agents In contrast to surgical and cytodestructive therapies of cutaneous and genital warts, the goal of recently developed treatments with antiviral and immunomodulating agents is not simply to remove the lesion, but also to reduce the amount of latent and subclinical viral infection sufficiently in order to diminish the rate of recurrence. This is achieved by mobilizing the so-called ‘innate immunity’, which recognizes stress signals and activates adaptive immunity in a targeted, appropriate and effective response. Pharmacological

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agents that modulate the function of dendritic cells and macrophages could play a role in this process and, therefore, could have important therapeutic value. All IFNs have anti-HPV activity, although the specific interferon response-mediator, double-stranded RNA, is not known to occur in the HPV life cycle. Partial and total remission of laryngeal papillomas as well as cutaneous and anogenital warts have been achieved with topical, intralesional and systemic administration of IFN. Combined therapies, such as surgery in combination with IFN or podophyllin in conjunction with IFN α-n1, were proposed as the most efficacious therapies (Weck et al., 1986). The antiviral effects of IFN on infected cells within the lesion do not cause damage to the surrounding tissue. In general, treatment with intralesional IFN-α appears to be equally as effective as traditional therapies, and it may be particularly useful in the treatment of lesions that have failed to respond to other modalities (Browder et al., 1992). Although IFN-α has been approved by the Federal Drug Administration for clinical treatment of genital warts in the USA, it is not generally recommended due to dose-limiting sideeffects (Wiley et al., 2002). Imidazoquinolines induce immunomodulating cytokines, partly through the activation of Toll-like receptors (TLRs)1. The imidazoquinoline, imiquimod, and its homologues activate macrophages and other cells and thus induce secretion of pro-inflammatory cytokines — predominantly IFN-α in plasmacytoid dendritic cells, and tumour necrosis factor (TNF) α and interleukin (IL)-12 in myeloid dendritic cells. These locally generated cytokines induce a Th1 cell-mediated immune response and the production of cytotoxic effectors (Stanley, 2002). Imiquimod directly enhances the immune response to HPV and thereby reduces the viral load. The compound was the first imidazoquinoline to be used for the treatment of anogenital warts and approved by the Federal Drug Administration in the USA: application of imiquimod cream (5%) three times a week overnight for up to 16 weeks is effective and safe, and the recurrence rate is low (Cox et al., 2004). (ii) Surgical treatments The most frequently used surgical therapies for the treatment of HPV-related mucocutaneous lesions include cryotherapy, laser surgery, electrodesiccation/fulguration and surgical excision. These treatments are generally equivalent in terms of clearance rates of the warts but are associated with high rates of recurrence (Maw, 2004). In early studies, cryotherapy or carbon dioxide laser therapy led to the complete cure of genital warts after several sessions in the majority of patients (Rosemberg, 1991). Cryotherapy Cryotherapy destroys warts by freezing the tissue. Results show that cryosurgery of HPV lesions is only moderately traumatic and gives good aesthetic and functional results

1

Named after the Toll pathway in Drosophila melanogaster, which controls resistance to fungal and grampositive bacterial infections (Hoffmann & Reichart, 2002; Janssens & Beyaert, 2003)

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(Kourounis et al., 1999). In addition, large lesions may be treated and the depth of cryonecrosis is more suitably adapted (Scala et al., 2002). Laser surgery The carbon dioxide laser is a high-precision, non-blood-letting light scalpel used for the incision and excision of tissues and to seal small blood vessels. Healing occurs by granulation and the post-operative period is relatively painless for the patient. The risk for post-operative morbidity and complications is low (Bar-Am et al., 1993). Hyperthermia induced by a neodymium:yttrium–aluminium garnet (Nd:YAG) laser or a 585-nm pulsed dye laser has been used for the treatment of condylomata (Volz et al., 1994; El-Tonsy et al., 1999; Kenton-Smith & Tan, 1999). Photodynamic therapy Photodynamic therapy with topical application of amino-laevulinic acid followed by irradiation with light of different wavelengths has been used for some time for the treatment of superficial premalignant and malignant skin tumours (reviewed in Roberts & Cairnduff, 1995). This therapy was later shown to be effective against recalcitrant warts (Stender et al., 1999). Surgical excision Scissors or a scalpel can be used to excise genital warts. Superficial scissor excision is useful when only a few lesions are present. Extensive intra-anal warts are most conveniently removed under general anaesthesia by a proctologist. General anaesthesia may also be preferred for surgical procedures for children and sensitive patients with extensive warts on the vulvo-anal area (von Krogh, 2001). (b)

Recurrent respiratory papillomatosis

HPV infections of the mother can be transmitted to the respiratory tract of the newborn child, which may result in juvenile-onset recurrent respiratory papillomatosis, the most common benign neoplasm of the larynx in children (Kimberlin, 2004; see Section 1.6.2). The risk factors for this vertical transmission have not been well identified. The role of caesarean section in preventing the transmission of HPV-associated disease from mother to child may be limited, as infection via amniotic fluid has also been reported to occur (Kosko & Derkay, 1996; Bandyopadhyay et al., 2003). Although their histology is benign, the epithelial proliferations observed in respiratory papillomatosis may result in progressive hoarseness, stridor (the sound produced by turbulent flow of air through a narrowed segment of the respiratory tract, which is a sign of airway obstruction in a child), obstruction of the airways and respiratory distress. In addition, the papillomas are characterized by multiple recurrences despite surgical removal. Additional treatments to contain the virus and growth of the papillomas include cidofovir, indole-3-carbinol, di-indolylmethane, IFN and photodynamic therapy. However, no single modality of treatment seems to be effective in eradicating this disease (Auborn, 2002).

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Radiotherapy is not recommended because it can cause malignant transformation of laryngeal warts. (i)

Pharmacological therapies

Cidofovir Cidofovir is an acyclic nucleoside phosphonate that has been identified as an antiviral drug that specifically inhibits viral DNA polymerases, but does not affect cellular enzymes. The strong activity of cidofovir against HPV lesions (Stragier et al., 2002) is unexpected, because the virus does not encode polymerase and the anti-HPV function of the drug apparently depends on other activities. A phase II trial revealed a clearance rate for HPV of 47% with minimal adverse reactions (Snoeck et al., 2001). Cidofovir is approved for intralesional application in laryngeal papillomas (Coulombeau et al., 2002). Nephrotoxicity is the dose-limiting side-effect for cidofovir when it is used intravenously (5 mg/kg) (De Clercq, 2003). (ii) Surgical treatments Surgery remains the first choice for the treatment of recurrent respiratory papillomatosis. The main goals of surgical resection are to assure an adequate airway, to improve the voice and to facilitate remission of disease while reducing morbidity. Traditionally, cryosurgery, suction diathermy and ultrasonography have been used. At present, surgical procedures that use cold steel, carbon dioxide laser and a laryngeal shaver blade are the most common (Shykhon et al., 2002). Cold-steel surgery The use of traditional surgical tools (‘cold steel’) to remove papillomas from the vocal cords is still preferred over the laser technique in some cases, because the latter burns healthy tissue and creates a vapour plume that may cause viral infection in the trachea or lungs. In contrast, cold-steel surgery causes loss of blood and infected tissue, which may contaminate the lower airways (Shykhon et al., 2002). A relatively novel device used in the surgical removal of papillomas is the powered laryngeal shaver blade, which is claimed to be safer and more accurate than traditional tools, and only causes injury to the superficial mucosa (Shykhon et al., 2002). Carbon dioxide laser vaporization Carbon dioxide laser vaporization is widely used to treat recurrent respiratory papillomatosis. Care must be taken to avoid airway fire (Varcoe et al., 2004) and to protect medical personnel, as viral particles are released in the laser plume (Ferenczy et al., 1990; Calero & Brusis, 2003). Nd:YAG laser Besides surgical resection and the established carbon dioxide laser treatment, laser surgery by the use of a fibre-guided Nd:YAG laser light promises to be an effective and only minimally traumatic treatment for recurrent respiratory papillomatosis. A novel

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fibre-guidance instrument was developed for endolaryngeal laser surgery of this disease. Five patients (aged 4–8 years) were treated with fibre-guided Nd:YAG continuous-wave laser light (wavelength, 1064 nm; power, 10 W; irradiance, 3.5 kW/cm2). By 12 months after treatment, all patients showed regression of the disease. Nd:YAG laser surgery seems to prevent a rapid recurrence of juvenile respiratory papillomatosis (Janda et al., 2004) Photodynamic therapy Photodynamic therapy of recurrent respiratory papillomatosis involves administration to the patient of a photosensitizing agent that concentrates in rapidly growing tissues. The lesions are then excised with a tuneable laser, which preferentially destroys the cells that accumulated the dye. The technique does not eradicate the virus, but may reduce the growth rate of the papillomas by 50% and may be particularly useful for the treatment of endobronchial lesions. The main side effect is photosensitivity, which lasts for weeks to months, and has sometimes led to hospitalization for cutaneous burns (Shykhon et al., 2002). 1.7.2

Therapy of precancerous lesions (a)

Therapy of CIN

Treatment of pre-invasive disease of the cervix is based on local control and prevention of progression. When abnormal cells are detected in a cervical smear, a thorough evaluation includes colposcopy to detect the lesions, direct biopsy and removal of the lesion, where appropriate, with minimal associated morbidity. However, since cervical precancer is an HPV-induced disease, spontaneous regression is also possible. (i) Surgical techniques Two categories of treatment are available: destructive and excision techniques. The success rates for ablative or excisional techniques is > 90%. While precancer is cured in most of the treated patients, eradication of HPV from the genito-urinary tract is not always possible with currently available techniques (Cirisano, 1999); thus the possibility of persistence of the virus and recurrence of the disease remains. Destructive techniques Techniques that involve destruction of the whole atypical transformation zone can be applied only if strict criteria are employed to ensure that no evidence of an invasive cervical cancer lesion is present; a pretreatment biopsy is therefore mandatory. These techniques, which include carbon dioxide vaporization, cryotherapy, electrocauterization and cold (thermo) coagulation, all have success rates of approximately 90%. A meta-analysis found that there is very little difference between these techniques with regard to the success of treatment or the occurrence of complications (Cirisano, 1999).

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Excision techniques Excision techniques that involve surgical removal (followed by histological analysis) range from carbon dioxide laser excision to the cold-steel technique to the rare application of hysterectomy. However, the loop electrosurgical excision procedure (LEEP) or large loop electrosurgical excision of the transformation zone (LLETZ) using an electrosurgical unit are now the most common techniques. They must be performed after a comprehensive colposcopic examination and the intention is to remove the entire lesion (LEEP) or the whole transformation zone (LLETZ) with an adequate margin of normal squamous epithelium surrounding the abnormal area and with minimal artefactual damage (Prendiville, 2005). (ii) Pharmacological treatments Imiquimod, a non-specific modulator of immune response, has been used in limited trials to treat low-grade lesions. Results suggest a variable clinical response but with associated systemic side-effects (Diaz-Arrastia et al., 2001). HPV vaccines have been used to treat low-grade lesions in limited trials (see Section 1.7.4). (iii) Follow-up after treatment of CIN There is a well-recognized risk of recurrence of CIN and rarely of invasive cancer following both its ablative and excision treatment. Follow-up can be carried out by colposcopy, cytology or HPV DNA testing, or by a combination of any of these. Two large metaanalyses showed that the combination of cytology and HPV testing increased the sensitivity to detect persistent or recurrent CIN and the negative predictive value to identify women at little or no risk for persistence or recurrence. Cytology and colposcopy may still be needed in order to rule out false-positive and false-negative results (Paraskevaidis et al., 2004; Zielinski et al., 2004). (b)

Therapy of VIN

Therapy of VIN is aimed at the removal of a cancer precursor lesion; however, treated patients are still at increased risk for developing invasive vulvar cancer and require longterm follow-up. Treatment modalities can be surgical or pharmacological; however, the real possibility of preventing invasive disease in patients affected by VIN by the use of extensive vulvar surgery is questioned, because relapses frequently occur and treatmentrelated sequelae associated with wide excisional therapy have a high psychological impact on the body image of the treated patients. However, surgery is still the preferred option in the therapy of VIN. (i) Surgical techniques The aims of the surgical approach are full histological assessment of the affected tissue combined with complete elimination of the precancerous lesion. Surgical therapies include excisional and destructive methods; excisional methods are preferred, since occult

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invasion has been reported in more than 10% of cases with a pre-operative biopsy that showed VIN3. Cold-steel surgery, laser excision and laser evaporation are effective modes of treatment. The treatment can be frequently completed without hospitalization and only under local infiltration of anaesthetics. No substantial difference in the various techniques has been reported. (ii) Pharmacological treatments Topical treatment is attractive, since it can be applied directly by the patient and is easily monitored for efficacy. Unfortunately, study results have been disappointing, with only few responses and high rates of complication and recurrence. In addition, with this therapy, diagnosis has to rely on the biopsy only, with the risk that an early invasive lesion may be overlooked. Reported pharmacological treatments include 5-fluorouracil, topical bleomycin, IFN-α, cidofovir, photodynamic therapy and imiquimod. Results on the treatment of VIN with imiquimod were first published in a report of four cases (Davis et al., 2000). Several small series of patients with high response rates to imiquimod have been described since that time (Diaz-Arrastia et al., 2001; Jayne & Kaufman, 2002; van Seters et al., 2002). Another series of patients demonstrated a clinical improvement in only 27%. Local side-effects limited the frequency of application, which might explain this low response rate (Todd et al., 2002). (c)

Therapy of VAIN

As the vagina connects the cervix and the vulva, treatment of VAIN is affected mainly by the presence of associated cervical or vulval lesions. VAIN can have different clinical presentations and treatment is tailored to the individual patient. The aim of the treatment is to remove the lesion; this can be accomplished by either pharmacological or surgical therapy, depending on the site and the size of the disease, the presence or absence of the cervix, and the age and clinical history of the patient. Pharmacological treatment includes cytostatic drugs, such as bleomycin and 5-fluorouracil, and immunomodulants, such as imiquimod. Surgical treatments include cold-steel surgery and carbon dioxide laser therapy; the latter is associated with minimal morbidity but has a low success rate with up to 50% of recurrences (Murta et al., 2005). Endovaginal brachyradiotherapy is also used for VAIN3 lesions (Fine et al., 1996). 1.7.3

Therapy of invasive cancer (a)

Cervical cancer

Although cervical cancer is preventable, once an invasive lesion occurs, it carries a substantial risk of death. The clinical stage of the disease at presentation is the single most important predictor of long-term survival (see FIGO Staging Classification for Cervical Cancer in Table 19). Recurrences more than 5 years after treatment are extremely rare. Hence, 5-year survival is a good indicator of a cure. When treated appropriately, 5-year survival exceeds 80% for patients with stage I disease, exceeds 70% for patients with

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Table 19. FIGO staging classification for cervical cancer Stage I Stage I is carcinoma that is strictly confined to the cervix; extension to the uterine corpus should be disregarded. The diagnosis of both stages IA1 and IA2 should be based on microscopic examination of removed tissue, preferably a cone, which must include the entire lesion. Stage IA: Invasive cancer identified only microscopically. Invasion is limited to measured stromal invasion with a maximum depth of 5 mm and no wider than 7 mm. Stage IA1: Measured invasion of the stroma no greater than 3 mm in depth and no wider than 7 mm in diameter Stage IA2: Measured invasion of stroma greater than 3 mm but no greater than 5 mm in depth and no wider than 7 mm in diameter Stage IB: Clinical lesions confined to the cervix or preclinical lesions greater than stage IA. All gross lesions, even with superficial invasion, are stage IB cancers. Stage IB1: Clinical lesions no greater than 4 cm in size Stage IB2: Clinical lesions greater than 4 cm in size Stage II Stage II is carcinoma that extends beyond the cervix, but does not extend to the pelvic wall. The carcinoma involves the vagina, but not as far as the lower third. Stage IIA: No obvious parametrial involvement; involvement of up to the upper twothirds of the vagina. Stage IIB: Obvious parametrial involvement, but not to the pelvic sidewall Stage III Stage III is carcinoma that has extended to the pelvic sidewall. On rectal examination, there is no cancer-free space between the tumour and the pelvic sidewall. The tumour involves the lower third of the vagina. All cases with hydronephrosis or a non-functioning kidney are Stage III cancers. Stage IIIA: No extension to the pelvic sidewall, but involvement of the lower third of the vagina Stage IIIB: Extension to the pelvic sidewall or hydronephrosis or non-functioning kidney Stage IV Stage IV is carcinoma that has extended beyond the true pelvis or has clinically involved the mucosa of the bladder and/or rectum. Stage IVA: Spread of the tumour into adjacent pelvic organs Stage IVB: Spread to distant organs FIGO, International Federation of Gynaecology and Obstetrics From Alliance for Cervical Cancer Prevention (2004)

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stage IIA disease, is approximately 40–50% for patients with stage IIB and stage III disease and is less than 10% in patients with stage IV disease (Sankaranarayanan et al., 1995; Yeole et al., 1998; Alliance for Cervical Cancer Prevention, 2004). Treatment of cervical cancer is mainly through radiotherapy; five recent studies have demonstrated that chemoradiation improves survival compared with radiotherapy alone; surgery alone or in association with radiotherapy can also be used in early-stage disease. Chemotherapy with platin compounds is used in combination with radiotherapy or surgery, or is used alone as palliation in advanced or recurrent disease (Ryu, 2002). Treatment options depending on the stage of cancer are described below and summarized in Table 20. The strengths and limitations of these treatment methods are listed in Table 21. (i) FIGO stage IA1 Stage IA1 disease (depth of invasion, < 3 mm; width, < 7 mm) has a risk of metastasis to regional lymph nodes of 1.2% and with a death rate of less than 1% (Benedet & Anderson, 1996). When preservation of fertility is important, a cone biopsy may be considered as a therapeutic procedure provided that (a) the woman is available for long-term follow-up, (b) the cervix is amenable to cytological and colposcopic evaluation, (c) the margins of the cone biopsy are free of both intraepithelial and invasive changes and (d) there is no evidence of lymphatic or vascular invasion. (ii) FIGO stage IA2 Stage IA2 (depth of invasion, 3–5 mm; width, < 7 mm) has a risk of metastasis to regional lymph nodes of nearly 8% and a mortality rate of 2.4% (Benedet & Anderson, 1996). The recommended treatment is modified radical hysterectomy and bilateral pelvic lymphadenectomy. If preservation of fertility is important, a large cone biopsy with nodal dissection or trachelectomy with nodal dissection (extraperitoneal or laparoscopic) may be considered (Dargent et al., 2000; Shepherd et al., 2001). (iii) FIGO stage IB Treatment strategies for stage IB invasive cancer include primary radiation therapy with external beam radiation and either high- or low-dose rate brachytherapy or primary surgery with radical hysterectomy and pelvic lymphadenectomy. Published observational data indicate a 5-year survival rate of 87–92% for either approach (Waggoner, 2003). Stage IB1 The treatment of stage IB1 cervical cancer (tumour diameter of < 4 cm confined to the cervix) depends on the resources and type of oncology services available and on the age and general health of the woman. Dual treatments (surgery and radiotherapy) are more harmful, more expensive and associated with a higher rate of complications. Therefore, primary therapy should aim to use only one radical treatment — either surgery or radiation with or without concurrent chemotherapy; concurrent chemotherapy usually comprises treatment with cisplatin during external beam therapy. Five-year survival rates of 80–90%

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Radical surgery

Chemotherapy

Radiotherapy Intracavitary (brachytherapy)

External beam (teletherapy)

Involves delivery of radiation using radioactive sources in special applicators placed in the cervical canal and vaginal fornices. Two types: low dose-rate, e.g. cesium-137 (treatment takes 1–3 days) and high dose-rate, e.g. iridium-192 (treatment takes a few minutes)

Involves delivery of a radiation beam to the cancer from an external source, i.e. the teletherapy machine. Telecobalt machines or linear accelerators can be used to deliver external beam radiotherapy.

The most common agents are cisplatin or carboplatin given as intravenous infusions.

Indication

Early stages (stage I and selected cases of stage IIA)

All stages, including palliative care

All stages, including palliative care

Advanced stages (in combination with radiotherapy) Palliative care Recurrent disease

Level of facility

Treatment for cancer is centralized and provided in tertiary-level facilities. Radical surgery is possible in some secondary-level hospitals.

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Major surgical procedure performed under general anesthesia that involves removal of cervix, uterus (with or without ovaries), parametrial tissue, upper part of the vagina, and lymph nodes in the pelvis. Requires careful dissection of both ureters.

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Description

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Table 20. Options for the treatment of cervical cancer

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Chemotherapy

Strengths

Surgery performed by skilled and experienced surgeons is effective in the treatment of early stage (stage I and selected stage IIA) disease. Allows preservation of ovaries in young women and avoids vaginal stenosis (narrowing). Limited capital investment is required for development of surgical services compared with radiotherapy services.

Used in the treatment of all stages of cervical cancer as well as other kinds of cancer (e.g. breast, head and neck). Effectiveness varies with the stage of the disease.

Can be combined with radiotherapy for the management of locally advanced cancer. Can be used in the management of very advanced cervical cancer.

The role of curative surgery diminishes in patients with cervical cancer that has spread beyond the cervix into the surrounding tissues. Requires skilled and experienced gynaecologists. Requires a stay in hospital (10–14 days). Complications include pelvic sepsis, pelvic thrombosis and postoperative pneumonia. Ureterovaginal or vesicovaginal fistula can occur as a post-operative complication in < 1% of patients.

Requires trained and skilled radiation oncologists, medical physicists and radiotherapy technicians to provide the treatment and to operate and maintain the equipment. Requires expensive equipment and supply of radioactive sources. Service contracts and spare parts are also necessary. If utilization is low, the cost per patient increases since the machine must be maintained and the radioactive source changed periodically, regardless of how many patients are treated. Requires a reliable power supply. Acute side-effects include radiation-induced inflammation of the rectum (proctitis) and urinary bladder (cystitis). Late complications, such as bowel obstruction and rectovaginal and vesicovaginal fistula formation, may occasionally occur. Low dose-rate brachytherapy requires an operating room and anaesthesia services to place the intrauterine catheter and vaginal ovoids. However, this machine can only be used to treat gynaecological cancers.

Limitations

Requires trained and experienced medical oncologists. Chemotherapeutic agents are expensive, making them inaccessible and not widely available in many countries. Not effective as firstline treatment.

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From Alliance for Cervical Cancer Prevention (2002)

Radiotherapy is the only realistic treatment once the disease has spread beyond stage IIA, when surgery is neither feasible nor effective. It is commonly used for less extensive tumours when surgical expertise is not available. Survival rates are equal to those of surgery in early-stage cancers. Suitable alternative option for women with early disease but at high risk for surgery. Mainly provided as an outpatient/ambulatory service.

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Table 21. Strengths and limitations of methods of treatment of cervical cancer

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following either radical surgery or radical radiation as primary therapy have generally been reported (Hopkins & Morley, 1991; Landoni et al., 1997; Waggoner, 2003). Stage IB2 For stage IB2 disease (tumour diameter of > 4 cm confined to the cervix), 5-year survival rates are reduced to approximately 65–75% (Hopkins & Morley, 1991; Sankaranarayanan et al., 1995). Para-aortic nodes are commonly involved in this stage, as well as an increase in central and distant features associated with recurrence. Options for treatment include (a) primary chemoradiation therapy alone (Rose et al., 1999), (b) primary radical hysterectomy with bilateral regional lymph node dissection, usually followed by radical adjuvant radiation (with or without concurrent chemotherapy) which is determined by pathological criteria such as disease-free margins, lymph–vascular space involvement and metastases to lymph nodes (Keys et al., 1999) and (c) neo-adjuvant chemotherapy, followed by radical surgery as described above and the possible use of post-operative radiation (Sardi et al., 1993). (iv) Advanced disease (FIGO stages II, III and IV) The standard treatment of advanced cervical cancer is primary radical radiation with a combination of external beam and intracavitary brachytherapy and concurrent chemoradiation therapy (Keys et al., 1999; Morris et al., 1999; Rose et al., 1999; Whitney et al., 1999). (v) Recurrent disease Recurrent cervical cancer may be in the pelvis, at distant sites or both. The majority of recurrences occur within 2 years of diagnosis; the prognosis is poor and most patients die from the disease. Management of women with distant metastases and advanced recurrent cervical cancer requires the efforts of a multidisciplinary team, and includes palliative use of anticancer therapies (chemotherapy, radiation therapy for treatment of symptoms and surgery such as colostomy for relief of symptoms related to recto-vaginal fistulae), control of symptoms (pain, bleeding, discharge and symptoms related to specific metastases) and emotional, psychological and spiritual support of the patient and her family (Alliance for Cervical Cancer Prevention, 2004). (b)

Vulvar cancer

Invasive vulvar cancer has been treated surgically for many years. The standard radical operation consisted of radical vulvectomy with bilateral inguinofemoral lymphadenectomy. Over the last 20 years, treatment of this cancer has changed dramatically, with a progressive decrease in surgical aggressiveness and the introduction of more conservative and personalized surgery. The treatment has evolved from a single type of operation to a philosophy of individualization, conservation and restoration. Changes from the standard approach include limited resection of the primary tumour and inguinofemoral lymphadenectomy that is carried out by a separate groin incision to decrease the associated morbidity of more extensive surgery.

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Vulvar surgery inevitably results in mutilation of the female genitalia and thus has a considerable psychological impact on the patient. Plastic surgery of the vulvar area is therefore more frequently used to cope with the problem of vulvar reconstruction and female body image. The status of the lymph nodes is the most important prognostic factor in squamous-cell vulvar cancer and recurrence in an undissected groin invariably has a fatal outcome for the patient. Complete inguinofemoral lymphadenectomy is required in lesions with more than 1 mm depth of invasion (FIGO stage IB and higher). In primary tumours < 2 cm in diameter and with a depth of invasion ≤ 1 mm (FIGO stage A), dissection of groin nodes can be omitted. Patients with negative nodes and lesions of < 8 cm in diameter have a good prognosis, with a 5-year survival rate of more than 80%. Conversely, metastasis to groin nodes carries a substantial risk for recurrence and death from the disease, and requires additional radiation treatment of the inguinopelvic areas. Recently, a technique to determine the pathological status of early-stage vulvar cancer was introduced that limits lymphadenectomy to the sentinel nodes (De Cicco et al., 2000; de Hullu et al., 2000). The results of an ongoing multicentric observational study on the safety of this new surgical technique are awaited before the introduction of this conservative treatment into clinical practice. Verrucous carcinoma of the vulva is an unusual variant of squamous-cell carcinoma that shows local malignancy. Treatment is based on wide local excision; since it rarely metastasizes to regional lymph nodes, the surgical step of inguinofemoral lymphadenectomy can be omitted. Radiation therapy is contra-indicated because it has been reported to render the tumour more aggressive and lead to the development of distant metastasis. (c)

Vaginal cancer

Primary vaginal squamous-cell cancer is a rare occurrence that comprises 1–2% of all gynaecological cancers. Radical radiotherapy is the main form of treatment, and includes external beam radiation and endovaginal brachytherapy; supplementation with concomitant chemotherapy with cisplatin is an option based on several factors that include the extent of the disease and the clinical condition of the patient. Radical surgery can be used in early lesions located in the upper third of the vagina; adjuvant radiation treatment is indicated in the presence of pathological risk factors for recurrence, such as positive pelvic lymph nodes or surgical margins close to the tumour. Pelvic exenteration is an option in selected primary or recurrent cases that are surgically suitable for such an extensive procedure (Berek et al., 2005). 1.7.4

Therapeutic vaccination

Therapeutic vaccination would be the most obvious strategy, since host immunity plays an important role in viral clearance. Several kinds of vaccine strategies are currently under investigation.

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The aim of therapeutic vaccines is to eradicate infected cells or reduce their number. Initial strategies were targeted to eliminate residual malignant cells in patients with cervical cancer, although the prevention of progression of HSIL, LSIL or even cytologically normal HPV-infected cells are all possible end-points. Therapeutic vaccines have also been used as an approach to eradicate genital warts. Once HPV infection has been established, it is improbable that antibodies play a role in the eradication of infected cells. Cytotoxic T lymphocytes (CTL) are the primary effectors of tumour eradication. Many strategies for the generation of CTL involve the stimulation of antigen-presenting cells (to process the tumour or viral antigens, and present them in the context of the MHC receptor) and adhesion of co-stimulatory molecules to produce anti-tumour lymphocytes. In many cases, HPV-associated tumours express only the E6 and E7 oncoproteins; thus, most efforts have focused on eliciting CTLs directed against E6 or E7. These viral proteins are also expressed throughout the epithelium that is undergoing lytic viral replication. It is not entirely certain, however, that these proteins are expressed in basal cells. Since basal cells are capable of proliferation, it is possible that only E1 and E2 are expressed to maintain the viral genome. CTLs that are reactive against the capsid antigens may play a role in reducing the extent of infection but would not be effective in targeting neoplastic cells. There is a considerable amount of literature on approaches that have been used to generate HPV-specific CTL and to kill tumours in preclinical models (Da Silva et al., 2001b) but this is not reviewed here. Only agents that are currently being or will shortly be used in clinical trials are discussed. Many groups have considered the use of HPV peptides because they are relatively inexpensive and are well tolerated. Much effort has been made to map HLA class Irestricted epitopes of HPV 16 and 18 E6 and E7 (Kast et al., 1993; Beverley et al., 1994) and clinical trials have been carried out on patients whose HLA genotype (usually A*0201) and HPV tumour type matched the viral peptide epitopes. In one trial with 15 HPV 16positive, A*0201-positive cancer patients, no CTLs were detected nor was there evidence of clinical benefit (Ressing et al., 2000). A similar trial with 19 cervical cancer patients used two E7-A*0201 epitope peptides and a helper peptide and showed little evidence of clinical improvement (Van Driel et al., 1999). However, a similar approach was used in a trial with 18 women who had HSIL of the cervix or vulva: 10 mounted CTL responses to the E7 peptide and three of the 10 had a complete clinical response (Muderspach et al., 2000). Preclinical data have suggested that longer peptides that contain a helper T-cell epitope linked to the CTL epitope are more efficient at eliciting CTLs than the minimal epitope; this effect is enhanced further by mixing the peptide with a dendritic cell-activating adjuvant (Zwaveling et al., 2002). Peptide vaccines are well tolerated and immunologists are making advances to understanding the mechanisms that result in robust generation of CTLs. The data suggest, however, that vaccination of peptides may be most efficient in individuals who have pre-invasive disease and are not immunocompromised (Steller, 2002). An additional problem with the use of peptides is that the HLA genotype of the patient and the HPV genotype of the tumour must be known. This has prompted many investi-

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gators to consider full-length E6 and/or E7 proteins, or fusion products with other proteins. One on-going trial is examining the safety and immunogenicity of an E6/E7 fusion protein in a saponin-based adjuvant among women with cervical HSIL (Steller, 2002). To increase the immunogenicity of the E7 protein, it has been fused to heat-shock proteins of Mycobacterium tuberculosis (hsp70) (Chen, C.H. et al., 2000) or to hsp65 of Calmette-Guerin bacillus (Goldstone et al., 2002). This fusion product has been used in an open-label trial to immunize men with anal HSIL, some of whom also had anogenital warts. Of 14 patients with warts, three had complete resolution of warts and 10 had a 70–95% reduction in the size of the warts. A fusion protein of HPV 6 L2/E7 was developed for the treatment of genital warts. Twenty-seven subjects with genital warts were treated in an open-label trial (Lacey, C.J.N. et al., 1999; Thompson et al., 1999). All 27 developed L2 and/or E7 antibodies and 19/25 subjects tested had proliferative responses. By 8 weeks after vaccination, the warts of five subjects had completely cleared and the remaining subjects were offered conventional therapy. Of the 13 whose warts eventually cleared, none showed any recurrence. Similarly, an L2/E7 fusion protein of HPV 16 was designed for the treatment of anogenital dysplasia. In a trial in women with VIN/VAIN3, immunogenicity was demonstrated but no clinical response (Smyth et al., 2004). Preclinical studies have shown that dendritic cells play a critical role in antigen presentation in vivo. These cells can be loaded with peptide epitopes: when mixed with proteins, they engulf the protein and process fragments through the class I antigen presentation pathway. Dendritic cells can also be transfected or transduced by nucleic acids that encode the desired antigens. Several studies have shown that peptide- or protein-pulsed dendritic cells are much more effective in eliciting anti-tumour CTLs than peptides alone (Schoell et al., 1999). In the context of HPV immunotherapy, monocytes were taken from the peripheral blood of cervical cancer patients and differentiated in culture using IL-4 and granulocyte macrophage colony-stimulating factor; the dendritic cells were mixed with a HLA-A*0201 E7 epitope and used to sensitize the autologous peripheral blood mononuclear cells from the cancer patients (Steller et al., 1998; Santin et al., 1999). A case report of a woman who had an adenocarcinoma that contained HPV 18 and who was treated over 10 months with dentritic cells that had been pulsed with HPV 18 E7 protein suggested that metastatic disease was inhibited for a period of time (Santin et al., 2002). Other small clinical studies have also used autologous dendritic cells pulsed with peptides or proteins as immunogens (Adams et al., 2001; Ferrara et al., 2003). The use of dendritic cells will probably play an important role in future vaccine strategies. In addition to being potent elicitors of antibodies, VLPs can also induce T-cell responses. Vaccination of subjects with HPV 16 VLPs was shown to induce both CD4+ and CD8+ T-cell responses (Pinto et al., 2003). In a trial in men with genital warts, HPV 6 VLPs induced antibodies and a delayed-type hypersensitivity response with complete regression in 25/33 patients; however, no placebo group was included (Zhang et al., 2000). To enhance their immunogenicity and, in particular, to stimulate a mucosal immune response, VLPs have been engineered to encapsidate a plasmid that expresses IL-2 (Oh, Y.K. et al., 2004).

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As discussed in Section 1.8, chimeric VLPs that contain a linked segment of E7 have been developed, and have been shown to induce specific HLA T cells in humans after in-vitro vaccination (Kaufmann et al., 2001). The use of viral vectors to introduce genes for vaccination is an effective way to stimulate many branches of the immune system. Recombinant vaccinia viruses, which have the advantage of being able to carry large inserts and not persisting in the host, have been widely used. The disadvantage of this method is that older individuals may have a pre-existing immunity to vaccinia virus which reduces the response; in addition, vaccinia virus may pose a risk to immunosuppressed recipients. A recombinant vaccinia virus that expresses the HPV 16 and 18 E6 plus E7 genes was created. In order to circumvent the potential problem of introducing oncogenes, the E6 and E7 proteins were mutated to block their binding to key tumour suppressors (Boursnell et al., 1996). In an initial study, the vaccine was found to be safe when administered to nine patients with late-stage cervical cancer; as most of the patients were immunosuppressed, only one developed CTLs but she also had clinical remission (Borysiewicz et al., 1996). In a more recent trial, 29 patients with stage IB or IIA cervical cancer were vaccinated (Kaufmann et al., 2002). After a single vaccination, four patients developed CTLs and eight developed serological responses to the HPV proteins. Two recent studies have tested a single dose of TA-HPV, a recombinant vaccinia virus that encodes modified HPV 16 and 18 E6 and E7, in patients with VIN (Baldwin et al., 2003; Davidson et al., 2003). Davidson et al. (2003) vaccinated 18 women who had HPV 16-positive high-grade VIN with a single dose of TA-HPV, which resulted in a reduction in the size of the lesion by at least 50% in eight patients, and a further four patients showed significant relief of symptoms. A second vaccination formulation, HPV 16 L2E6E7 fusion protein, has been tested in 10 patients with highgrade VIN who had previously been primed with TA-HPV. All but one demonstrated HPV 16-specific proliferative T-cell and/or serological responses following vaccination. However, no direct correlation between immunological and clinical responses was seen (Davidson et al., 2004). This approach is promising but emphasizes the difficulty of achieving immunotherapeutic responses in immunocompromised patients. Clinical trials of other viral delivery systems, including recombinant adenoviruses (Tobery et al., 2003), adeno-associated virus (Liu et al., 2000) and RNA-based poliovirus (van Kuppeveld et al., 2002) and alphavirus (Velders et al., 2001) vaccines, which have all been constructed to express E7 or poly-epitope proteins should begin soon. DNA has emerged as an attractive candidate for a vaccine because it is inexpensive and does not require a cold chain. DNA uptake by antigen-presenting cells results in the expression of the encoded antigen, and induction of both antibodies and CTLs. In a phase I trial, a plasmid-encoding multiple HLA A2 epitope of HPV 16 E7 was encapsulated in biodegradable polymer microparticles. Twelve HLA- and HPV-matched subjects with anal HSIL were vaccinated: 10/12 exhibited an increased immune response and three showed partial histological responses (Klencke et al., 2002). Enhancement of DNA vaccines by coexpression of cytokine genes such as granulocyte macrophage colony-stimulating factor has also been tested (Leachman et al., 2000).

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177

Prophylaxis1

The discovery that the major capsid protein L1 can assemble into VLPs that are structurally and immunogenically indistinguishable from authentic virions and studies aimed at the characterization of HPV conformational epitopes that induce neutralizing antibodies that can block new infection have had a considerable impact on the development of prophylactic vaccines (see Section 1.2). This section highlights some important innovations in prophylaxis that have occurred since the Working Group was convened, in 2005. To date, two prophylactic vaccines have been developed and tested in large multicentric trials (Harper et al., 2004; Villa et al., 2005; Harper et al., 2006; FUTURE II Study Group, 2007; Garland et al., 2007). Both are based on the recombinant expression and self-assembly of the viral protein L1 into VLPs. The HPV VLPs contain no DNA and hence are noninfectious. Injection of the HPV VLPs elicits a strong and sustained type-specific response. One of the vaccines, Gardasil® (Merck & Co.), protects against HPV 6, 11, 16 and 18 (quadrivalent) and the other, Cervarix® (GlaxoSmithKline), protects against HPV 16 and 18 (bivalent). The expected outcome of prophylactic vaccination is a reduction in the incidence of HPV-related genital diseases, including cervical, penile, vulvar, vaginal and anal cancer and precancerous lesions. In addition, a reduction in the incidence of the genital warts has been observed among those who received the quadrivalent vaccine and a reduction in laryngeal papillomatosis can be anticipated among their children (Arbyn & Dillner, 2007). Since 8 June 2006, the quadrivalent vaccine has been licensed for use in females 9–26 years of age in the USA by the Food and Drug Administration which recognized the indications of safe and strong protection against cervical cancer, genital warts, cervical adenocarcinoma in situ, CIN grades 1, 2 and 3 and VIN grades 2 and 3 that are caused by HPV 6, 11, 16 and 18 and stated that the vaccine is effective if administrated before HPV infection (Dillner et al., 2007). The Advisory Committee of Immunization Practices and the American Cancer Society recommend routine vaccination of girls aged 11–12 years, and the vaccine may be administered to girls as young as 9 years old. Vaccination is also recommended for girls and young women aged 13–26 years who have not been vaccinated previously (Markowitz et al., 2007; Saslow et al., 2007). On 20 September 2006, the European Medicine Evaluation Agency officially authorized the marketing of the quadrivalent vaccine Gardasil® in the European Union. An application has also been made to this Agency for a licence for the bivalent vaccine, Cervarix®. Although their high efficacy has clearly been shown, it is important to recognize the limitations of currently available vaccines and available data: (a) these vaccines do not protect against all high-risk HPV types; (b) they do not treat existing HPV infections; (c) the long-term duration of protection and the required length of protection to prevent cancer are unknown; [It should be noted, however, that follow-up of young women did not 1 This

section was updated by the IARC Secretariat after the Working Group meeting, and the text was reviewed by three members of the Working Group.

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detect evidence of waning immunity over 5 years (Harper et al., 2006; Villa et al., 2006) and that the quadrivalent vaccine was shown to induce immune memory (Olsson et al., 2007).] and (d) the cost of the primary vaccination, the recommended three-dose injection schedule and the possible need for additional booster vaccinations will probably limit the use of vaccine among medically underserved and uninsured populations. In addition, it will be important to evaluate the impact of the HPV VLP vaccines on other genital and nongenital HPV-associated tumours and in other populations such as individuals at high risk for anal cancer (e.g. men who have sex with men). Further, much research is needed to develop and evaluate alternative vaccine approaches to reduce the cost and expand the coverage of vaccination. It is also crucial to ensure the introduction and success of HPV vaccination programmes in developing countries (Saslow et al., 2007). Several additional approaches to prophylactic vaccines have been considered (for a review, see Breitburd & Coursaget, 1999; Schiller & Nardelli-Haegliger, 2006). Neutralization epitopes are not only present on VLPs; advances in purifying bacterially expressed L1 proteins have shown that they can assemble into pentameric structures, such as capsomers that contain neutralizing epitopes (Li et al., 1997).Vaccination of dogs with these capsomers in the canine oral papillomavirus model was fully protective (Yuan et al., 2001). Capsomers may therefore offer a simplified, economical alternative to VLPs. Other approaches to provide low-cost systems that generate conformationally correct L1 protein include expression in plants, which can potentially lead to development of edible vaccines (Biemelt et al., 2003; Warzecha et al., 2003). Immunization with naked DNA has the theoretical advantage of simple production. Naked DNA vaccination with L1 expression plasmids can induce antibody responses in animal models that are increased if codon-modified genes are used (Mossadegh et al., 2004). Delivery of naked DNAs can be facilitated by their incorporation into recombinant viruses. Viral vectors could not only deliver the L1 gene more efficiently but in many cases would be compatible with needle-free mucosal delivery. HPV 16 L1 recombinants of two DNA viruses, adenovirus 5 (Berg et al., 2005) and adeno-associated virus (Kuck et al., 2006), have been developed as candidate prophylactic vaccines. Several other attractive RNA viral vectors, including alphavirus vectors, are also currently under investigation (Vajdy et al., 2004). Live bacteria vaccines are potentially simple and inexpensive to manufacture, and can also be relatively inexpensive to deliver if administered mucosally. Four distinct L1 recombinant bacteria vaccines have been developed and tested for immunogenicity in animal models (Schiller & Nardelli-Haegliger, 2006). Among them, L1 recombinant clones of attenuated Salmonella enterica serovar Typhimurium and Typhi strains were shown to induce strong neutralizing antibody responses after a single intranasal or oral application in mice (Baud et al., 2004). This was the case for the attenuated Ty21 strain Vivotif that expresses L1. This strain has an excellent safety record, based on its use as an oral vaccine to prevent typhoid fever in tens of millions of individuals worldwide. Therefore, this clone could potentially serve as a combined HPV/typhoid fever vaccine (Schiller & Nardelli-Haegliger, 2006).

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The minor capsid structural viral protein L2 has been shown to elicit antibodies that neutralize both homologous and heterologous HPV types (Kawana et al., 1999; Roden et al., 2000). VLPs that consist of L1 proteins fused to L2 epitopes appear to be promising, since the presence of L2 conveys epitopes that cross-neutralize with a broad range of HPV types and was also shown to increase the yield of VLP production compared with L1-only VLPs (Slupetsky et al., 2007). In order to obtain combined prophylactic/therapeutic vaccines, ways to stimulate the cell-mediated immune response against viral non-structural proteins and neutralizing antibody production have been explored. The most advanced candidates for this type of vaccines are chimeric VLPs that incorporate peptides of early proteins as fusions of L1 or L2. To date, two chimeric VLPs have been tested in clinical trials: an HPV L1–E7 chimeric VLP that targets HPV 16-associated high-grade cervical dysplasia (Schäfer et al., 1999; Schreckenberger & Kaufmann, 2004) and an HPV 16 L2–E6–E7 chimera with a potential to induce cross-neutralizing antibodies (de Jong et al., 2002). Taken together, there is a great hope for a reduction in the morbidity and mortality associated with HPV-related anogenital diseases in populations who receive the available prophylactic vaccines. The promising outcome of prophylactic vaccines from a broad public health perspective, however, can only be attained if vaccination can be achieved for those groups of women for whom access to cervical cancer screening services is most problematic. For these reasons, the development of second-generation vaccines that are expected to be cheaper, easy to deliver and/or to provide T-cell response to cure preexisting HPV infections is highly desirable.

2. 2.1

Studies of Cancer in Humans

Methodological concerns (a)

Choice of disease end-point

To obtain epidemiological evidence of the risk for cervical cancer due to a specific type of human papillomavirus (HPV), the choice of disease end-point must be appropriate. The risk for invasive cancer is examined optimally by a case–control design or among historical cohorts in which archived specimens are tested. Prospective studies that follow women forward in time must ethically rely on surrogate end-points, the choice of which is critical. For studies of HPV infection, invasive cancer and grade 3 cervical intraepithelial neoplasia (CIN3; which subsumes diagnoses of severe dysplasia and carcinoma in situ) are considered to be the primary disease endpoints. The inclusion of CIN3 as a surrogate for invasive cancer permits prospective studies that would otherwise be unethical, because it is a condition that often requires medical treatment, whith thus interrupts the natural history of the disease. CIN3 is the immediate precursor of invasive cervical cancer, and the two diseases share a similar

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cross-sectional virological and epidemiological profile (except for an earlier average age at diagnosis of CIN3) and demonstrate good histopathological reproducibility (Shah et al., 1980; Walker et al., 1983; Muñoz et al., 1992, 1993). Therefore, CIN3 is a practical surrogate end-point for cervical cancer, although a proportion of cases of CIN3 regress rather than invade. However, in cohort studies, new diagnoses of small CIN3 lesions may represent diseases that were missed at the time of enrolment when HPV was assayed. This may lead to misclassification bias and spurious risk estimates. While the choice of CIN3 is imperfect, less severe and more common grades of neoplasia, particularly CIN1, are clearly unreliable and are too closely linked to newly acquired infections with a broad range of HPV types to serve as surrogate end-points of cervical cancer. CIN2 is probably the result of a mixture of newly acquired infections and incipient CIN3; it is often treated, but can represent ‘over-called’ low-grade lesions. Virologically, the persistence of HPV for several years cannot be used as an accurate surrogate of type-specific carcinogenicity because persistence is a necessary but not sufficient characteristic of carcinogenicity (Ho et al., 1995; Nobbenhuis et al., 1999). (b)

Impact of study design

When an etiological fraction of cervical cancers that can be attributed to a specific HPV type is small, it is more difficult to conduct prospective studies because of limitations of statistical power. At present, type-specific prospective evidence of carcinogenicity is readily available for HPV 16 and, to a lesser extent, for HPV 18. Because of the latency between average age at first HPV infection (late teens to early twenties) and average age at diagnosis of CIN3 (approximately 25–30 years of age), large longitudinal cohorts are only now attaining sufficient follow-up time to permit a reasonable assessment of a few additional HPV types. To overcome this limitation, some of the longest-term studies published to date have been based on HPV typing of archived slides from screening programmes, using a nested case–control approach. However, techniques for assaying the full spectrum of HPV types in these old specimens have not been fully validated. Since each individual HPV type is relatively uncommon, most prospective studies have combined all putative carcinogenic HPV types to assess the possible clinical utility of a pooled-probe HPV test. As a result, epidemiologists often rely on cross-sectional designs to estimate the risk for individual HPV types. Estimation of the absolute risk, incidence rate and even the lifetime cumulative incidence rate of cervical cancer among infected compared with uninfected women and among women infected with each type of HPV alone would be ideal. The risk associated with each HPV type could then be estimated with adjustment for potential confounding due to co-infection with other HPV types. However, the correct estimation of these risks would require lifetime longitudinal follow-up of huge cohorts of women, while cross-sectional designs that use prevalence risk estimates suffer from unavoidable limitations due to the lack of a reliable measurement of lifetime exposure to HPV infection.

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Case–control designs typically rely on the assessment of HPV DNA at the time of diagnosis for cases and at a similar age for controls. Since persistence of HPV DNA is a hallmark of cervical cancer/CIN3, the vast majority of cases are found to be HPV DNApositive. In contrast, the low prevalence in controls reflects both recently acquired infections and an unknown, small fraction of infections from previous years (most of which proved to be transient after 1–2 years). (c)

Problem of multiple infections

HPV 16 and HPV 18 were classified previously as Group 1 carcinogens (IARC, 1995). In an assessment of whether additional types are also carcinogenic, possible confounding must be taken into account because different HPV types are frequently cotransmitted sexually. Multiple infections (i.e. infections with more than one HPV type) have been found in more than 25% of infected women in many surveys, but available polymerase chain reaction (PCR) assays are less sensitive and reproducible for the detection of multiple-type rather than single-type infections. When considering the possible carcinogenicity of a specific HPV type, any association with cancer due to coinfection with HPV 16 or HPV 18 must be ruled out. Possible strategies to address this type of confounding include the exclusion of all HPV 16- or HPV18-infected individuals, stratification for HPV type or group and multivariate statistical modelling. However, these strategies are constrained by the need to investigate concurrently approximately 40 relatively rare anogenital types of HPV. Thus, even very large studies typically lack statistical power to evaluate all combinations adequately. This problem is most apparent when assessing the possible carcinogenicity of uncommon types that occur mainly in combination with other HPV types. (d)

Choice of method for HPV testing

DNA testing is the reference standard for the detection of current HPV infection. There is a wealth of evidence from case–control studies that putative carcinogenic types assessed as a pooled group are associated with an increase in the risk for invasive cancer and CIN3. The collective strength of this evidence led the Food and Drug Administration in the USA to license Hybrid Capture 2, which allows the detection of 13 high-risk and five low-risk HPV types, as an adjunctive screening method. Similarly, several studies have employed PCR-based methods that still pool putative carcinogenic types, which prevents individualized assessment of the carcinogenicity of specific types. Thus, the assessment of type-specific carcinogenicity relies exclusively on studies that employ PCR with probes for individual types. However, each PCR-based system has selective differential sensitivity for individual HPV types; this could affect the risk estimates because of differential misclassification of cases and controls. Infections in cases result in lesions that are the site of viral replication, and tend to have higher viral loads than infections that do not cause obvious lesions such as those that occur in controls. Thus, a PCR system with relatively low sensitivity for a given HPV type tends to detect infections of that type

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differentially in cases compared with controls, and thus overestimates the odds ratio for that specific type. Serological data have yielded useful information for the assessment of exposure to HPV. HPV serology based on virus-like particles (VLPs) is a relatively type-specific but insensitive measure of exposure. Therefore, seropositive women appear to have been truly exposed to HPV, although the anatomical site of infection cannot be ascertained. Extremely large archives of serum specimens have permitted nested case–control studies of cervical cancer and CIN3 with an exceptional statistical power that is currently lacking in studies of DNA. Serology is included here to define HPV-exposed study populations for the consideration of etiological co-factors such as tobacco smoking or Chlamydia trachomatis. Finally, serology is discussed with reference to sites other than the cervix for which valid comprehensive DNA sampling is problematic. (e)

Heterogeneity of definitions of initial cytomorphology

Cohort and case–control studies have previously emphasized the distinction among HPV-infected women between those with normal versus mildly abnormal cytology. However, different types of HPV infection cause overlapping and pleiomorphic cellular changes that are sometimes pathognomonic (e.g. koiloctytotic atypia) but are often equivocal or lacking. Moreover, the interpretations of mild and equivocal HPV-related cytology differ greatly between geographical regions and assessors. A normal Papanicolaou (Pap) test in one geographical region might be called equivocal or even a low-grade squamous intraepithelial lesion (LSIL) in another region (Scott et al., 2002a). Thus, for epidemiological studies to assess HPV type-specific carcinogenicity, a necessary requirement is that the study subjects are tested for a specific type of HPV and that cases have a confirmed diagnosis of CIN3 or cancer. For type-specific analyses, it is not necessary to focus excessively on the subtler, variable issues of whether control or cohort subjects had completely normal cytology. (f)

HPV types, cervical cancer and cancers at other sites

Since the association between HPV and cervical cancer is well established, the sections on cervical cancer focus on evaluating specific HPV types. In these sections, a limited number of highly stringent HPV DNA detection techniques were considered to be adequate to provide evidence of an association. For cancers at sites other than the cervix, their relationship with HPV is not well established. There are fewer studies on the association between HPV and cancers at sites other than the cervix, and the number of cases reported is much smaller. To allow for a preliminary assessment of the association between HPV and these cancers, a wider variety of techniques and methods were considered to be acceptable for presentation in their respective sections.

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Cancer of the cervix

2.2.1

Historical perspective

183

Early studies on HPV and cervical cancer reported largely on HPV 16 and 18, which were the first two cancer-associated types that were isolated and used to design the initial testing systems (IARC, 1995). Developments in the technology used in large epidemiological and clinical studies evolved in two directions. Clinically designed testing systems generated cocktails of HPV probes, with the understanding that the individual risk for any HPV type of the high-risk group was clinically equivalent. Typically, these studies reported on the presence or absence of HPV DNA of the high-risk or low-risk cocktails (Peyton et al., 1998; Vernon et al., 2000; Castle et al., 2002b). Research-oriented testing systems developed type-specific procedures and these were used to refine the understanding of type-specific risk and to make advancements in studies of HPV transmission, in the definition of HPV persistence and in investigations of HPV DNA at other organ sites (Manos et al., 1989; Jacobs et al., 2000). As described in Section 1, the ability to identify multiple types of HPV in one specimen and the presence of some cross-reactivity has introduced some additional variability in the interpretation of the available data. The literature that related HPV and HPV types to cervical cancer up to 1994–95 was reviewed previously (IARC, 1995). It was concluded that there was sufficient evidence for the carcinogenicity of HPV types 16 and 18 (Group 1), HPV types 31 and 33 were classified as probably carcinogenic to humans (Group 2A) and an undefined group of other HPVs were evaluated as possibly carcinogenic to humans (Group 2B). Some evidence was suggestive that the same association existed for other HPV types, although the number of studies was limited. The only correlation observed between HPV type and clinical outcome was an increased relative frequency of HPV 18 in cervical adenocarcinoma compared with the more common squamous-cell carcinoma. The case–control study in Spain and Colombia coordinated by IARC was instrumental in showing highly significant and high odds ratios with three different HPV DNA testing methods, namely the southern blot hybridization procedure, which was considered as the standard at the time, the first version of a testing cocktail intended for clinical use (Virapap) and the initial PCR systems based on the MY09/11 primers (Bosch et al., 1992; Muñoz et al., 1992). HPV type-specific risk estimates were provided for HPV 16, 18, the combination of HPV 31, 33 and 35 and for unidentified HPV types (Bosch et al., 1992; Muñoz et al., 1992; IARC, 1995). The high prevalence of HPV DNA among cases triggered intense research into the viral status of the apparently HPV-negative cases. Stringent laboratory analyses of case series of cervical cancer led to the conclusion that HPV is a necessary cause of cervical cancer (IARC, 1995; Walboomers et al., 1999). These prompted analyses restricted to HPV-positive women for the evaluation of other risk factors. The study also showed that the risk factor profiles of the apparently HPVnegative and HPV-positive cervical cancer cases were notably similar, that the risk profile was identical for the established pre-invasive and invasive conditions and that the results

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were consistent in two countries with a contrasting incidence of cervical cancer (Bosch et al., 1992; Muñoz et al., 1993; Moreno et al., 1995). 2.2.2

Data on pooled HPV types (a)

Cross-sectional studies and studies with short-term follow-up

(i) Risk for ≥ CIN2/3 from primary screening data Table 22 presents data from primary cervical cancer screening studies on the association between HPV positivity and the risk for ≥ CIN2/3. In some studies, verification of high-grade disease by colposcopy and histology was restricted to women who had positive cytological or HPV test results. In particular, all women screened in three studies were verified independently of screen test results and were therefore theoretically free from verification bias (Belinson et al., 2001b; Blumenthal et al., 2001; Sankaranarayanan et al., 2004b) whereas verification bias was at least partially corrected in the statistical analyses in two studies (Schneider et al., 2000; Kulasingam et al., 2002). Assessment of outcome is potentially hampered by the misclassification that is inherent to the use of an imperfect diagnostic gold standard. Nevertheless, relative risks associated with HPV status can be computed with acceptable reliability as the ratio of the risk for ≥ CIN2/3 in HPV-infected versus non-HPV-infected subjects. This relative risk is equivalent to the ratio of positive predictive value over the complement of the negative predictive value (relative risk = positive predictive value/(1-negative predictive value)). The relative risk for ≥ CIN2 that is associated with HPV positivity varied from 5.6 (Blumenthal et al., 2001) to 256 (Cuzick et al., 1999). In one study in which verification bias was not taken into account, the relative risk was infinite (Clavel et al., 2001) since the Hybrid Capture 2 result predicted all detected cases of ≥ CIN2. The relative risk was higher when the outcome of ≥ CIN3 was considered, and varied from 27 (Kulasingam et al., 2002) to 530 (Petry et al., 2003). (ii)

Triage of women with atypical squamous cells of undetermined significance (ASCUS) or LSIL Using a meta-analytical approach, Arbyn et al. (2002, 2004a,b, 2005) documented the diagnostic performance of two management options for women with an equivocal Pap smear to detect women who need follow-up — reflexive high-risk HPV DNA testing versus repeat cytology. Two different triage groups were considered: (a) women with equivocal Pap smears, reported as ASCUS or borderline; and (b) women with LSIL or mild dyskaryosis. In these meta-analyses, diagnostic accuracy for the outcome of histologically confirmed CIN2 or CIN3 or worse was the focus of interest. The selection of studies considered was restricted to those in which ≥ CIN3 was the reported outcome and sensitive HPV DNA detection systems were used such as the Hybrid Capture 2 and PCR tests. Relative risks were computed as for the primary screening data. The inter-study variation of the relative risks and the pooled measures are displayed graphically by the forest plots in Figures 10 for ASCUS and 11 for LSIL and are further

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Table 22. Association between ≥ CIN2/3 and HPV status in women participating in cervical cancer screening Method of detection

Outcome

No. of women

Test posivity rate

Proportion of ≥ CIN2/3 HPV+

HPV–

Relative riska

Verification bias

09:40

Reference, study location

≥ CIN2

1703

0.073

0.160

0.001

255.84

Not corrected

Schiffman et al. (2000), Costa Rica

HC2

≥ CIN2 ≥ CIN3

8554 8554

0.139 0.075

0.102 0.096

0.002 0.001

45.09 109.05

Not corrected Not corrected

Schneider et al. (2000), Germany

HC2b

≥ CIN2

4761

0.104

0.204

0.003

68.90

Partially corrected

Wright et al. (2000), South Africa

HC2

≥ CIN2

1365

0.208

0.137

0.007

19.36

Not corrected

Belinson et al. (2001b), China

HC2

≥ CIN2

1997

0.228

0.180

0.002

73.35

None

Blumenthal et al. (2001), Zimbabwe

HC2

≥ CIN2

2073

0.429

0.189

0.034

5.59

None

Clavel et al. (2001), France

HC2

≥ CIN2

5671

0.134

0.094

0.000

939.20c

Kulasingam et al. (2002), USA

HC2

≥ CIN2 ≥ CIN3

4075 4075

0.219 0.279

0.096 0.018

0.015 0.001

6.28 27.31

Salmerón et al. (2003), Mexico

HC2

≥ CIN2 ≥ CIN3

7732 7732

0.094 0.079

0.129 0.120

0.001 0.001

130.86 215.76

Not corrected Not corrected

Petry et al. (2003), Germany

HC2

≥ CIN3

7592

0.047

0.073

0.000

530.44

None corrected

Not corrected Partially corrected Partially corrected

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185

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Table 22 (contd)

Sankaranarayan et al. HC2 (2004b), India

Outcome

≥ CIN2 ≥ CIN3

No. of women

18085 18085

Test posivity rate

Proportion of ≥ CIN2/3 HPV+

HPV–

0.070 0.070

0.128 0.089

0.005 0.002

Relative riska

Verification bias

28.32 55.27

None None

CIN, cervical intraepithelial neoplasia; HC2, Hybrid Capture 2: targets HPV types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59 and 68; +, positive; –, negative a Relative risks were calculated by the Working Group using data from Lörincz and Richart (2003), IARC (2005) and from the original publications or requested directly from the authors. b PCR system that includes identification of 14 types (13 types as in HC2 + HPV 66) c RR computed using Yates correction, by adding 0.5 to each cell of the 2 × 2 contingency table. This correction is required for studies where the risk for ≥ CIN2/3 in the HPV-negative group is zero. Risk for ≥ CIN2/3 if HPV positive corresponds with the positive predictive value of HPV testing for the presence of underlying ≥ CIN2/3. Risk for ≥ CIN2/3 if HPV negative corresponds with 1–negative predictive value.

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Reference, study location

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Figure 10. Meta-analysis of the prediction of histologically confirmed ≥ CIN3 in women with an index Pap smear that showed ASCUS: relative risk for HPV-positive women versus HPV-negative women Risk ratio (95% CI)

Study

Lin et al. (2000)

26.10 (1.61,421.97)

Zielinski et al. (2001)

16.80 (0.92,307.85)

Kulasingam et al. (2002)

10.52 (1.38,80.37)

Pretorius et al. (2002)

16.81 (7.75,36.45)

Sherman et al. (2002)

21.12 (8.67,51.44)

Lonky et al. (2003)

16.41 (2.19,123.06)

Wensveen et al. (2003)

8.44 (0.44,160.58)

Overall (95% CI)

17.47 (10.45,29.22)

.01

.1

1

10

100 Risk ratio

The meta-analysis is restricted to studies in which Hybrid Capture 2 or sensitive PCR was used to detect HPV DNA. ASCUS, atypical squamous cells of undetermined significance; CIN, cervical intraepithelial neoplasia; Pap, Papanicolaou test

documented in Tables 23 and 24, respectively. Overall, HPV-positive women with ASCUS from seven pooled studies had a risk for ≥ CIN3 that was 17.47 times (95% confidence interval [CI], 10.45–29.22) higher than that of HPV-negative women (Figure 10). The relative risk was substantially lower in LSIL triage settings (4.30; 95% CI, 2.71–6.82) (Figure 11). Relative risks were pooled using random-effect metaanalytical models (Dersimonian & Laird, 1986; Sutton et al., 2000). The risk for ≥ CIN3 in some studies was equal to zero in the HPV-negative group, which yielded a relative risk of infinity (∞). Such studies cannot be incorporated into a meta-analysis. For these studies, 0.5 was added to the nominator and 1 to the denominator in both the HPV-negative and HPV-positive groups. This correction yielded a considerable underestimate of the relative risk, especially in small studies. Nevertheless, the underestimation in the pooled relative risk was generally smaller than when studies with a relative risk of ∞ should have been discarded.

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Figure 11. Meta-analysis of the prediction of histologically confirmed ≥ CIN3 in women with an index Pap smear showing LSIL; relative risk for HPV-positive women versus HPV-negative women Risk ratio (95% CI)

Study

Bavin et al. (1993)

3.20 (1.71,6.01)

Chesebro et al. (1997)

15.04 (1.95,116.13)

Lin et al. (2000)

8.57 (0.55,134.03)

Zielinski et al. (2001)

2.38 (0.14,41.59)

Kulasingam et al. (2002)

5.66 (0.35,91.65)

Pretorius et al. (2002)

5.79 (1.87,17.90)

Sherman et al. (2002)

5.26 (1.69,16.36)

Overall (95% CI)

4.30 (2.71,6.82)

.01

.1

1

10 100 Risk ratio

CIN, cervical intraepithelial neoplasia; LSIL, low-grade squamous intraepithelial lesion; Pap, Papanicolaou test

Most triage studies were cross-sectional in design or involved a follow-up time that only lasted from the assessment of HPV status to the verification of outcome. The largest triage study (Castle et al., 2005) included 5060 women who had ASCUS or LSIL. Oncogenic HPV-positive women who had ASCUS or LSIL had a 2-year absolute risk for CIN3 of approximately 15% or 17%, respectively. Women who had ASCUS or LSIL cytology who were HPV 16 DNA-positive at baseline had a 2-year cumulative absolute risk for ≥ CIN3 of 32.5% (95% CI, 28.4–36.8%) and 39.1% (95% CI, 33.8–44.7), respectively, thus the risk estimates seemed to be substantially lower than the cross-sectional relative risks. The difference in cross-sectional and longitudinal cumulative relative risks might indicate that cross-sectional studies overestimate a surplus of prevalent disease which ultimately regresses. This time-dependent effect was also observed in large cohort studies that focused on the natural history of HPV infection and precancerous cervical lesions (Liaw et al., 1999; Kjaer et al., 2002; Schlecht et al., 2003c). Increased risk for severe dysplasia was associated with a high risk for ≥ CIN2. The largest contribution to the relative risk derives from HPV 16 infection (Castle et al., 2005)

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Table 23. Triage of ASCUS: short-term outcome of ≥ CIN3 in high-risk HPV-positive versus HPV-negative women with ASCUS Follow-up period

HPV test method

HPV types targeted

No. of women

Test posivity rate

Proportion of ≥ CIN3 HPV+

HPV–

Relative risk

Relative riska

13 HR types (2)

74

0.527

0.359

0.000



26.1

Zielinski et al. (2001a)b

0–4.5 years

HC2

13 HR types (2)

213

0.347

0.054

0.000



16.8

Kulasingam et al. (2002)

Nested within primary screening setting, short follow-up

HC2

13 HR types (2)

270

0.511

0.080

0.008

10.5

Pretorius et al. (2002)

Short follow-up

HC2

13 HR types (2)

949

0.322

0.183

0.011

16.8

Sherman et al. (2002)

RTS, short follow-up

HC2

13 HR types (2)

2198

0.540

0.104

0.005

21.1

Lonky et al. (2003)

Short follow-up

HC2

13 HR types (2)

278

0.460

0.109

0.007

16.4

Wensveen et al. (2003)b

Cross-sectional

HC2

13 HR types (2)

148

0.453

0.045

0.000



8.4

ASCUS, atypical squamous cells of undetermined significance; CIN, cervical intraepithelial neoplasia; HC2, Hybrid Capture 2; HR, high-risk; RTS, randomized triage study; +, positive; –, negative a Relative risk corrected by adding 0.5 to each cell that contributes to the computation of the relative risk b With Yates correction (+ 0.5)

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Reference

Follow-up period

HPV types targeted

No. of women

Test posivity rate

Proportion of ≥ CIN3 HPV+

HPV–

Relative risk

Relative riska

PCRb

HPV 16

179

0.374

0.343

0.107

3.2

Chesebro et al. (1997)

Cross-sectional

HC2

9 HR types (1)

159

0.799

0.134

0.009

15.0

Lin et al. (2000)a

Cross-sectional

HC2

13 HR types (2)

45

0.756

0.353

0.000



8.6

HC2

13 HR types (2)

65

0.800

0.077

0.000



2.4 5.7

Zielinski et al. (2001a)a Kulasingam et al. (2002)a

Nested within primary screening setting, short follow-up

HC2

13 HR types (2)

125

0.832

0.125

0.000



Pretorius et al. (2002)

Short follow-up

HC2

13 HR types (2)

283

0.763

0.259

0.045

5.8

Sherman et al. (2002)

RTS, short follow-up

HC2

13 HR types (2)

849

0.848

0.122

0.023

5.3

ASCUS, atypical squamous cells of undetermined significance; CIN, cervical intraepithelial neoplasia; HC2, Hybrid Capture 2; HR, high-risk; LSIL, low-grade squamous intraepithelial lesion; PCR, polymerase chain reaction; RTS, randomized triage study; +, positive; –, negative a With Yates correction (+ 0.5) b PCR targeting HPV 16 (medium/high signal)

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Table 24. Triage of LSIL: short-term outcome of ≥ CIN3 in high-risk HPV-positive versus high-risk HPV-negative women with ASCUS

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(see Table 25). In HPV 16-positive women with ASCUS, the risk for ≥ CIN2 within 2 years was 16.1 (95% CI, 12.0–21.7) times higher than that in high-risk HPV-negative women. Positivity for other high-risk types was associated with a relative risk of 6.1 (95% CI, 4.5–8.3), which was similar to that associated with ASCUS that was unqualified by HPV. The relative risk associated with HPV positivity was lower in LSIL patients than in ASCUS patients, but was significantly higher when women were infected with HPV 16 compared with women infected with other high-risk HPV types (Castle et al., 2005).

Table 25. Two-year cumulative risk for ≥ CIN2 according to initial high-risk HPV status (positivity for HPV 16 and for other high-risk HPV types) in women with ASCUS or LSIL compared with high-risk HPV-negative women No.

Absolute risk (%)

Relative risk

95% CI

ASCUS HC2-negative All ASCUS ASCUS HPV16-positive ASCUS other high-risk HPV-positive, HPV16-positive

1559 3488 443 1245

3.0 15.3 48.5 18.4

1.0 5.1 16.1 6.1

– 3.8–6.8 12.0–21.7 4.5–8.3

LSIL HC2-negative All LSIL LSIL HPV 16-positive LSIL other high-risk HPV-positive, HPV 16-positive

237 1572 310 931

8.4 25.4 51.1 22.7

1.0 3.0 6.1 2.7

– 2.0–4.1 3.9–8.2 1.7–3.7

Adapted from Castle et al. (2005) ASCUS, atypical squamous cells of undetermined significance; CI, confidence interval; CIN, cervical intraepithelial neoplasia; HC2, Hybrid Capture 2; LSIL, low-grade squamous intraepithelial lesion

(b)

Prospective studies

Since the previous review (IARC, 1995), a few large prospective studies have shown that HPV infection, as assessed by DNA testing for a group of putative high-risk types, predicted an increased risk for subsequent development of CIN3 or invasive cancer. Although such studies do not add to the assessment of type-specific carcinogenicity, they are noted for completeness. Nobbenhuis et al. (1999) conducted a follow-up study of 353 women aged 18–55 years who had been referred because of cervical abnormalities for a median of 33 months (range, 2–72 months) without taking any biopsies until the clinical appearance of ≥ CIN3 or until the end of study. Two hundred and ninety-seven women (87%) had mild or moderate dyskaryosis at baseline; among them, 182 (61%) were high-risk HPV-positive (defined as harbouring one or more of the following types: 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59,

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66 or 68, using PCR with GP5+/6+ primers). Sixty-nine (31.9%) of these high-risk HPVpositive women developed ≥ CIN3 compared with only three (2.6%) of the 115 women that were high-risk HPV-negative at baseline, corresponding to a relative risk of 14.5 (95% CI, 4.7–44.8). A longer persistence of high-risk HPV-positivity was associated with an increase in the relative risk for the development of CIN3. Two of the three initially high-risk HPVnegative cases who developed CIN3 acquired HPV types during follow-up, which subsequently persisted until assessment of the outcome. Using MY09/11 PCR with TaqGold followed by dot-blot detection of the 13 types targeted by Hybrid Capture 2, Ferreccio et al. (2003) examined the association of histologically confirmed CIN3 that occurred within 2 years and cancer that occurred within 7 years among 8551 women in Guanacaste, Costa Rica, who represented a mixed prevalent/incident case group. Ninety cases of CIN3 (mean age, 36.9 years) and 20 cases of invasive cancer (mean age, 43.4 years) were detected by multi-technique screening and not from symptoms. The cumulative incidence among HPV-positive women (unadjusted for loss to follow-up) was 8.6% while the comparable incidence among HPV-negative women was only 0.2%. Sherman et al. (2003b) performed a 10-year follow-up of 20 810 women (mean age, 35.9 years) who were screened with a Pap smear and HPV testing from 1989 to 1999 at the Kaiser Permanente Center, Portland, USA. Among 171 women who had CIN3 or cancer diagnosed during the follow-up period, 123 (71.9%) had baseline Pap results of atypical squamous cells or worse and/or a positive HPV test, 102 (86.4%) of whom were diagnosed within the first 45 months of follow-up. During this 45-month period, the cumulative incidence of CIN3 or cancer was 4.54% among women with a Pap test result of atypical squamous cells or worse, positive HPV tests or both compared with 0.16% among women with negative Pap and HPV tests; thus, negative baseline Pap and HPV tests were associated with a lower risk for CIN3 or cancer in the subsequent 45 months. Clavel et al. (2004) followed 4401 cytologically negative women for a median period of 34 months primarily to estimate the negative predictive value of Hybrid Capture 2negativity for histologically confirmed CIN2 or CIN3 (combined). Five cases were observed and none was Hybrid Capture 2-positive at enrollment. 2.2.3

Data on type-specific HPV (a)

Case series

The cervix uteri of women with normal Pap smears or with mild cytological abnormalities (e.g. LSIL) harbour a broad spectrum of HPV types. Herrero et al. (2000) tested 3024 women in Guanacaste, Costa Rica, for 40 different HPV types and detected 34 different HPV types in women with normal cytological findings or LSIL. Franceschi et al. (2005) tested 1891 women in Dindigul District, India, for 44 different HPV types and detected 36 different HPV types in either single- or multiple-type infections among women with normal cytological findings or LSIL.

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As the severity of cervical lesions increases, not only does the overall prevalence of HPV rise greatly, but the relative frequency of different HPV types also changes substantially. This ‘enrichment’ of certain HPV types, together with the depletion of others across the spectrum of cervical neoplasias, is well illustrated by the findings of three large systematic reviews carried out at the IARC on the distribution of HPV types in LSIL (8308 women from 50 studies; Clifford et al., 2005), high-grade squamous intraepithelial lesions (HSIL; 4338 women from 52 studies; Clifford et al., 2003a) and squamous-cell cervical carcinoma (10 058 women from 85 studies; Clifford et al., 2003b). The three IARC reviews were carried out according to the same protocol: articles that included HPV type-specific prevalence data were identified and key information (e.g. country of sample, sample size, type of cervical specimen and PCR primers used to detect HPV-positive samples) was extracted. Published findings did not generally allow the distinction of single-type from multiple-type infections and, therefore, the prevalence of each individual HPV type was evaluated independently of whether other types were detected. The three reviews were limited to studies that (a) included a minimum of 20 cases of LSIL, HSIL or cervical cancer and (b) reported type-specific prevalence of at least one HPV type other than HPV 6, 11, 16 or 18. When study methods suggested that additional type-specific data were available, these data were requested from the authors. All five continents were represented, although to varying extents. Other case series that described the distribution of HPV types in invasive cancer have been published since the IARC systematic review, including some in previously unstudied populations (Cuzick et al., 2000; Bachtiary et al., 2002; Dybikowska et al., 2002; Mortazavi et al., 2002; Nakagawa et al., 2002; Pegoraro et al., 2002; Alonio et al., 2003; Gao et al., 2003; Hwang et al., 2003; Kay et al., 2003; Plunkett et al., 2003; Rabelo-Santos et al., 2003; Stanczuk et al., 2003; Tran-Thanh et al., 2002; Tsuda et al., 2003; Widschwendter et al., 2003; Xi et al., 2003; Silins et al., 2004; Schellekens et al., 2004). Overall HPV prevalence was 71% among LSILs, 84% among HSILs, 88% among squamous-cell carcinomas and 77% among cervical adeno- or adenosquamous carcinomas. HPV types in Table 26 were grouped into: (a) HPV 16 and 18 (i.e. the types most frequently detected in cervical cancer worldwide); (b) other high-risk or probably highrisk types, 11 of which (i.e. HPV 31, 33, 35, 39, 45, 51, 52, 56, 58, 59 and 68) are currently included in the Hybrid Capture 2 DNA test approved by the US Food and Drug Administration as an adjunct to primary cytological screening and for triage of women with equivocal cytology (Wright et al., 2004), and five types (i.e. HPV 26, 53, 66, 73 and 82) that have been considered for inclusion in the HPV DNA test (Muñoz et al., 2003); and (c) the three most common low-risk HPV types (i.e. HPV 6, 11 and 70). Figure 12 gives a graphical representation of the data of Table 26. HPV 16 was 2.5 times more prevalent in HSIL than in LSIL. Other types showed either a similar prevalence in LSIL and HSIL or a substantially higher prevalence in LSIL than in HSIL. Most importantly, HPV 16 and 18 were found three- and two times, respectively, more frequently in squamous-cell carcinoma than in LSIL, whereas HPV 26, 39, 51, 56 and 73 were at least 10-times and HPV 53 and 66 were approximately 30-times more

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Table 26. Distribution of HPV types across cervical lesions of increasing severity HPV type

16 18

LSIL

HSIL

ADC

LSIL: LSIL: SCC ADC ratio ratio

No.

%

No.

%

No.

%

No.

%

8308 8308

18.7 6.1

4338 4338

45.0 7.1

1464 1455

31.3 37.7

8594 8502

54.3 12.6

0.5 0.7

0.8 0.2

1090 1331 985 716 755 693 757 693 811 681 452

1.7 0.9 0.8 0.1 5.8 0.1 0.5 0.0 0.5 0.7 0.2

7204 8449 6223 3899 5174 4580 5304 4493 5646 4488 4148

4.2 4.3 1.0 0.4 4.2 0.6 2.5 0.7 3.0 0.8 0.5

2.7 1.7 5.7 19.0 1.2 17.7 3.6 13.6 2.8 7.6 6.6

6.8 8.2 7.1 76.0 0.8 106.0 18.0

Other high-risk or possibly high-risk 31 8155 8.2 4036 8.8 33 8078 5.3 4302 7.2 35 6395 4.3 2690 4.4 39 4301 5.8 1841 1.1 45 4748 3.7 2214 2.3 51 4721 8.0 2171 2.9 52 4380 6.7 2153 5.2 56 4431 7.2 2110 3.0 58 4498 6.3 2175 6.9 59 4281 4.6 1636 1.5 68 4292 2.5 1763 1.0 26a 53a 66a 73a 82a

SCC

3506 3358 4135 3432 2923

1.0 7.6 6.5 2.4 1.9

806 1589 1778 1364 812

0.6 2.3 2.1 1.0 0.5

362 381 508 377 219

0.0 0.0 0.2 0.0 0.0

3728 3053 4799 2844 2526

0.1 0.2 0.2 0.2 0.4

13.0 51.0 43.0 16.0 6.0

Low-risk 6 4696 11 4525 70 1114

6.2 3.2 2.2

3015 3015 1031

1.9 1.3 1.6

1049 1000 493

0.1 0.1 0.0

6569 6578 3122

0.6 0.3 0.2

13.3 13.7 17.0

16.8 8.7 16.5

43.0

80.0 41.0

ADC, adenocarcinoma or adenosquamous carcinoma; HSIL, high-grade squamous intraepithelial lesion; LSIL, low-grade squamous intraepithelial lesion; SCC, squamous-cell carcinoma a Not currently included in the US Food and Drug Administration-approved Hybrid Capture 2 HPV DNA test

common in LSIL than in squamous-cell carcinoma. A ratio of approximately 10 between LSIL and squamous-cell carcinoma was also found for low-risk types HPV 6, 11 and 70. The type-specific findings for adeno- or adenosquamous carcinoma were consistent with those observed for squamous-cell carcinoma except for the more marked enrichment of HPV 18 from LSIL to adeno- or adenosquamous than to squamous-cell carcinoma. Comparisons of HPV distribution in international cross-sectional studies face several problems, including differences in the accuracy in cytological/histological classification and viral detection, as well as non-negligible heterogeneity in the distribution of HPV types across different populations.

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Figure 12. Prevalence of HPV types in cervical lesions of increasing severity

Modified from Franceschi & Clifford (2005b)

Of particular note since the previous review (IARC, 1995), a high prevalence of HPV 35 has been reported in invasive cancer from previously unstudied regions in East Africa (19%) (Naucler et al., 2004) and India (6%) (Castellsagué et al., 2001; Franceschi & Clifford, 2005). Furthermore, a failure in the sensitivity of MY09/11 PCR primers to detect HPV 35 has also been identified, so that the prevalence of HPV 35 may have been underestimated in some of the previous case series (Iftner & Villa, 2003).

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Nevertheless, the picture that emerges from the IARC systematic reviews suggests that: HPV 16 and 18 are substantially enriched in squamous-cell carcinoma compared with LSIL; some high-risk types are approximately equally represented (HPV 33 and 45) or moderately over-represented (HPV 31, 52 and 58) in LSIL than in squamous-cell carcinoma; and HPV 26, 53, 66, 73 and 82, which are not currently included in the DNA tests approved by the US Food and Drug Administration, are extremely rare in squamouscell carcinoma, but this is also the case for some of the types that are currently included (e.g. HPV 39, 51 and 56). In conclusion, the available evidence from cross-sectional comparisons of the distribution of HPV types in cervical lesions of increasing severity lends strong support to the notion that the risk that a woman will develop HSIL or cervical cancer varies substantially according to the specific HPV type with which she is infected. (b)

Case–control studies

Since the last review (IARC, 1995), a number of larger case–control studies have been completed that allow a more accurate evaluation of the type-specific risk of a number of additional HPV types. Only studies that reported HPV DNA results by type, as assessed by PCR, and by case and control status and included histologically confirmed end-points are reviewed and evaluated separately by disease end-point. Over the past 10 years, several specific and sensitive PCR-based methods of HPV detection have been used in epidemiological studies, and it is important to highlight that the various PCR systems differentially amplify different HPV types in disease and non-disease samples. Therefore, caution must be taken in interpreting the relative strength of the association between specific HPV types and risk for disease across studies. Due to the relative infrequency of some HPV types, smaller case–control studies have reported unstable risk estimates for certain HPV types. Greater emphasis is therefore given to larger studies and those that reported pooled data in relation to the risk associated with types other than HPV 16 and 18. As far as possible, the risk estimates presented here focus on those associated with single HPV infections only. The risk estimates published by the authors are presented where these are available by HPV type. When raw data were available from the individual publications, these were used to generate the crude odds ratio by HPV type. Finally, due to the problems of type specificity in seroepidemiological studies, those reports that only provided data on seroprevalence are not included. In the mid-1990s, a growing interest in the risk associated with different HPV types came from examination of data from case series that indicated a relatively high prevalence of HPV types other than HPV 16 and 18 in cervical tumours (Bosch et al., 1995; Huang et al., 1997). Table 27 summarizes the results of case–control studies of HPV-specific infection and pre-invasive and invasive lesions of the cervix. In Honduras, Ferrera et al. (1999) conducted a population-based case–control study (149 cases of CIN3 or invasive cervical cancer and 438 controls) to investigate risk factors for cervical cancer. HPV was detected using general primer-mediated MY09/11 PCR

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Table 27. Characteristics of case–control studies on HPV-specific infection and pre-invasive and invasive lesions of the cervix HPV types tested

No. and type of cases

No. and type of controls

Ferrera et al. (1999), Honduras

Cervical screening

General primer-mediated PCR+ MY09/11 PCR sequencing

16, 18, 45, 33, 59, 31, 35, 52, 58, 56, 66, 11, 53, 70, 6, 22, 55, 62, 21

45 CIN3, 104 ICC

438 hospitalbased

Hwang et al. (1999), Korea

Hospital-based

Consensus primer PCR; RFLP analysis

16, 18, 31, 33, 35, 52, 58

35 CIN, 41 ICC

130 healthy women

Sasagawa et al. (2001), Japan

Screening

LCRF1 to –4+ E7 primer; RFLP analysis

16, 18, 45, 33, 31, 35, 52, 58, 56, 51

145 LSIL, 137 HSIL, 72 SCC, 12 ADC, 16 condyloma

1562 normal cytology

Thomas et al. (2001b), Thailand

Hospital-based

MY09/11 PCR; generic + type-specific oligonucleotide probed for hybridization

16, 18, 31, 33, 35, 39, 45

42 ADC, 190 SCC

291 otolaryngological and general wards

Thomas et al. (2001c), Thailand

Hospital-based

General PCR; type-specific PCR

16, 18

190 ICC

75 CIS

Altekruse et al. (2003), USA

Populationbased

PGMY-based 27-type reverse line blot detection

6, 11, 16, 18, 26, 31, 33, 35, 39, 40, 42, 45, 51, 52, 53, 54, 55, 56, 57, 58, 59, 66, 68, 73, 82, 83, 84

139 SCC, 124 ADC

307 populationbased

Franceschi et al. (2003), India

Hospital-based

GP5+/6+ PCR; ETA with HPV specific oligoprobe cocktails

16, 18, 45, 33, 59, 31, 52, 58, 73, 56, 51, 66, 11, 70, 40, 42, 72, 81

193 ICC, 12 ADC

213

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HPV types tested

No. and type of cases

No. and type of controls

Muñoz et al. (2003), Multicentre

Populationand hospitalbased

E7 primer PCR for biopsies, for smears, MY0G/11 or GP5 +/6+ PCR; oligohybridization

16, 18, 26, 45, 33, 59, 31, 35, 52, 58, 66, 73, 39, 56, 26, 51, 68, 11, 53, 6, 81, 82

1918 prevalent SCC and ADC

1928

Asato et al. (2004), Japan

Populationand hospitalbased

L1 consensus primer PCR; nucleotide sequencing

16, 18, 45, 33, 59, 31, 35, 52, 58, 73, 39, 56, 51, 66, 68, 82, 90, 91, 54, 53, 70, 6, 61, 71, 32, 42, 67, 72, 84, 86

356 SCC

3249 hospitalbased

Hammouda et al. (2005), Algeria

Hospital-based

GP5+/6+ PCR; EIA with type-specific oligoprobe cocktail detecting 36 types and southern blot

16, 18, 45, 33, 31, 35, 52, 73, 39, 56, 51, 66, 42, 84

198 SCC

202

Herrero et al. (2005), Costa Rica

Populationbased

MY09/11 PCR; dot blot with type-specific oligoprobes

16, 18, 45, 33, 59, 31, 35, 52, 58, 73, 39, 56, 26, 51, 66, 68, 82, 11, 54, 53, 70, 6, 61, 71, 22, 32, 40, 42, 55, 62, 67, 72, 81, 84, 74, 83, 85, 89, 21

73 CIN3, 35 SCC

8374 normal equivocal and lowgrade dysplasia

See Table 7 for a description of the primers used. ADC, adenocarcinoma; CIN, cervical intraepithelial neoplasia; CIS, carcinoma in situ; EIA, enzyme immunoassay; HSIL, high-grade squamous intraepithelial lesions; ICC, invasive cervical carcinoma; LSIL, low-grade squamous intraepithelial lesions; PCR, polymerase chain reaction; RFLP, restriction fragment length polymorphism; SCC, squamous-cell carcinoma

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Table 27 (contd)

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followed by PCR-based sequencing that detected several different HPV types. However, due to the rarity of most HPV types, risk estimates for disease could only be generated for HPV types 16, 18, 31, 33, 52 and 58. The prevalence of any HPV type was 95% in cases of invasive cervical cancer, 87% in cases of CIN and invasive cervical cancer and 39% in controls. Compared with normal cytology, the odds ratio for invasive cervical cancer, associated with HPV 16-related types was 14.88 (95% CI, 5.12–43.25) and that for invasive disease associated with HPV 18-related types was 74.66 (95% CI, 7.77–717.62). Significantly elevated risks for invasive cervical cancer compared with normal cytology were also observed for HPV types 31 [odds ratio, 3.4], 33 [odds ratio, 34], 52 [odds ratio, 12.8] and 58 [odds ratio, 11.2]. In addition to the HPV types for which risk estimates could be generated, the authors observed HPV 45 infection among six cases and HPV 59 in one case, with none in the corresponding controls. A statistically significant association with HPV was observed for CIN2 and -3 and invasive cancer that showed an upward trend to more severe lesions and was more pronounced for HPV 16 and related types. A significantly elevated risk for CIN3 was observed with HPV 16 [odds ratio, 19], 18 [odds ratio, 8.9], 31 [odds ratio, 21.4], 33 [odds ratio, 71.5], 52 [odds ratio, 53.6] and 58 [odds ratio, 26.3]. HPV types 53, 66 and 70 were identified among controls but not among cases. Hwang (1999) conducted a case–control study that included 130 healthy women, 35 patients with CIN and 41 patients with invasive cervical carcinoma in the Republic of Korea. HPV was detected by PCR followed by type-specific analyses by restriction fragment length polymorphism (RFLP). Significantly elevated risks for invasive cervical cancer were observed for HPV 16 [odds ratio, 146.3], 18 [odds ratio, 156], 52 [odds ratio, 39] and 58 [odds ratio, 78]. In Japan, Sasagawa et al. (2001) estimated the risk of HPV infection for biopsyconfirmed cervical malignancies by testing cell samples from 366 women with abnormal cytology and 1562 women with normal cytology for HPV with the long control region (LCR)-E7 PCR method that can amplify the E6–E7 DNA of more than 36 mucosal types of HPV. The prevalence of HPV infection was 9.7% in controls, 91% in HSIL and 93% in invasive cervical cancer. For HSIL and invasive squamous-cell carcinoma, the highest odds ratios were observed with HPV 16 (odds ratio, 43; 95% CI, 1.24–75 and 69; 95% CI, 36–131, respectively). For adenocarcinoma, the highest odds ratio was seen with HPV 18 (odds ratio, 94; 95% CI, 28–317). In addition to HPV types 16 and 18, elevated risks were observed for the association between HPV types 11, 31, 51, 52, 53 and 58 and squamous-cell carcinoma with magnitudes > 5. Thomas et al. (2001b) studied women in Thailand who had been diagnosed with preinvasive or invasive cervical cancer. PCR-based assays that used MY09/11 were carried out to determine HPV DNA in cervical scrapings from 232 diagnosed cases (190 women with squamous-cell carcinoma and 42 women with adenocarcinoma) and 291 hospitalized controls in Bangkok. HPV types 16, 18 and 45 were determined individually and a combined measurement of HPV types 31, 33, 35 and 39 was conducted. Only risk estimates for HPV 16 and 18 were reported separately. The prevalence of HPV types 16 and 18 was

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72.4% in cases and 14.0% in controls. The 168 women with HPV 16- and 18-positive cervical cancers were compared with 250 HPV-negative controls. The odds ratio for HPV 16 was 83 (95% CI, 39–232) for squamous-cell cancer and 24 (95% CI, 8.7–76) for adenocarcinoma. In addition, HPV 45 was observed in one case but not in controls. In another report by Thomas et al. (2001c) in Bangkok, Thailand, 190 women with invasive cervical cancer from the previous report were compared with 75 women with insitu disease. HPV DNA testing of cervical scrapings showed high-risk types in 79% of invasive and 57% of intraepithelial tumours. The 291 hospital-based controls for invasive cervical cancer and 124 controls for carcinoma in situ had HPV prevalences of 6.9% and 10.4%, respectively. Types 16 and 18, but not types 31/33/35/39, were more common in invasive than in intraepithelial tumours, and untyped HPV DNA was more common in insitu lesions. The odds ratio for invasive carcinoma in situ with HPV types 16 and 18 was reported to be 11.0 (95% CI, 3.9–33.0) and 10.0 (95% CI, 1.2–86.0), respectively. In the northeastern USA, Altekruse et al. (2003) conducted a case–control study that included 124 women with cervical adenocarcinoma, 139 with cervical squamous-cell carcinoma and 307 control subjects to determine HPV genotypes and sexual and reproductive risk factors using a PCR-based reverse line blot detection system (MY09/11 L1 consensus primer system). Specimens were grouped hierarchically by HPV genotype: 18, 16, 18-related (39, 45, 59 and 68), other high-risk (26, 31, 33, 35, 51, 52, 55, 56 and 58) and low-risk (6, 11, 40, 42, 51, 53, 54, 57, 66, 73, 82, 83 and 84). HPV 18 was associated most strongly with adenocarcinoma (odds ratio, 11.9; 95% CI, 3.6–39.5) and HPV 16 was associated most strongly with squamous-cell carcinoma (odds ratio, 10.5; 95% CI, 5.2–21.2). The relative importance of HPV genotypes 16 and 18 and the differences in reproductive co-factors suggest distinctly separate causes for cervical adenocarcinoma and squamous-cell carcinoma. Franceschi et al. (2003) evaluated the role of HPV and other risk factors in the etiology of invasive cervical carcinoma in a hospital-based case–control study in Chennai, southern India. A total of 205 cases of invasive cervical cancer (including 12 adenocarcinomas) and 213 frequency- and age-matched control women were included. HPV DNA in cervical cells was evaluated by a PCR assay (GP5+/6+). HPV infection was detected in all but one case of invasive cervical cancer and in 27.7% of control women. HPV 16 was the most common type in both cases and controls (60.2% and 17.4%, respectively), followed by HPV 18 and 33. Compared with women who were infected by HPV 16, those infected with HPV 18 showed an increased odds ratio of 3.9 (95% CI, 0.9–17.4). In this study, multiple HPV infections did not yield a higher odds ratio for invasive cervical cancer than single infections. One of the largest studies that has contributed to an understanding of the association between infection by individual HPV types and cervical cancer is the analysis of data pooled from nine case–control studies of invasive cervical cancer conducted by the IARC in Brazil, Colombia, Mali, Morocco, Paraguay, Peru, the Philippines, Spain and Thailand, the results of which were reported by Muñoz et al. (2003). Detection of HPV DNA in cervical scrapings (exfoliated cells) and biopsy specimens was performed blindly in

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central laboratories using PCR-based assays. PCR primers for the L1 gene, MY09/11, were used in the Colombian and Spanish studies and the GP5+/6+ general primer system was used in the remaining studies. A total of 1918 cases and 1928 controls were included in the pooled analysis. Overall, the prevalence of HPV infections was 90.7% in cases and 13.4% in controls and the pooled odds ratio for any HPV type for cervical cancer was 158.2 (95% CI, 113.4–220.6). The authors concluded that, in addition to HPV types 16 (odds ratio, 435) and 18 (odds ratio, 248), HPV types 31 (odds ratio, 124), 33 (odds ratio, 374), 35 (odds ratio, 74), 39 (odds ratio, ∞), 45 (odds ratio, 198), 51 (odds ratio, 67), 52 (odds ratio, 200), 56 (odds ratio, 45), 58 (odds ratio, 115), 59 (odds ratio, 419), 68 (odds ratio, 54), 73 (odds ratio, 106) and 82 (odds ratio, ∞) should be considered as carcinogenic. In addition, HPV 53 was found in one case of invasive cervical cancer only. No significant associations were reported for HPV 6 and 11. In Okinawa, Japan, Asato et al. (2004) conducted a case–control study to determine the association between HPV infections and invasive cervical cancer. The study included 356 women who had been newly diagnosed with squamous-cell carcinoma of the cervix and 3249 controls. Cervical swabs taken before any treatment was started were analysed using a consensus primer pair to amplify DNA from the L1 region of HPV by PCR. This method would, however, underestimate the prevalence of HPV in multiple infections. Direct sequencing of PCR products resulted in the identification of nucleotide sequences of 30 HPV DNA genotypes. Overall, 87.4% of cases and 10.2% of controls were HPV DNA-positive. Among cases, 84.5% were HPV-positive for types 16, 18, 31, 33, 35, 52 and 58. The odds ratio associated with being positive for HPV 16 was the highest (534.6). Significantly elevated risks for invasive cervical cancer were also associated with HPV 18 (odds ratio, 259), 31 (odds ratio, 137), 33 (odds ratio, 151), 35 (odds ratio, 31), 51 (odds ratio, 9), 52 (odds ratio, 36), 53 (odds ratio, 14), 54 (odds ratio, 22), 56 (odds ratio, 25), 58 (odds ratio, 180), 59 (odds ratio, 52), 66 (odds ratio, 65), 68 (odds ratio, 12), 70 (odds ratio, 32) and 82 (odds ratio, 65). HPV 45, 73 and 82 were each detected in a single case. This study is one of the first large case–control studies in which HPV genotyping was based completely on nucleotide sequencing which allowed the investigators to estimate the risks for cervical cancer associated with previously uncharacterized genotypes. Hammouda et al. (2005) conducted a case–control study in Algiers, Algeria, that included a total of 198 cases of cervical carcinoma and 202 age-matched control women. HPV infection was detected in 97.7% of cases and 12.4% of controls (odds ratio, 635). HPV 16 was the most common type in both cases and controls, followed by HPV 18 and 45. Twelve types (HPV 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 66 and 73) were found as single infections in cases. Significantly elevated risks for invasive cervical cancer were observed for HPV 16 [odds ratio, 503], 18 [odds ratio, 572], 31 [odds ratio, ∞], 33 [odds ratio, ∞], 35 [odds ratio, ∞], 39 [odds ratio, ∞], 51 [odds ratio, ∞], 56 [odds ratio, ∞], 66 [odds ratio, ∞], 73 [odds ratio, ∞] and 45 [odds ratio, 159]. In addition, HPV 52 was found in one case. Multiple HPV infections did not yield a higher odds ratio for cervical carcinoma than single infections. The distribution of HPV types in cases of cervical carcinoma

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and controls in Algeria was observed to be more similar to that found in Europe than to that found in sub-Saharan Africa, where HPV 16 is less prevalent. Herrero et al. (2005) presented the results of their prevalent case–control analysis of the enrolment visit of the cohort study in Guanacaste, Costa Rica. In this study, a population-based cohort of 8514 sexually active women was tested for individual HPV types and screened by cytology for CIN and cancer. An expert panel of pathologists histologically confirmed all the lesions detected. The overall prevalence of HPV was 26.5%, and HPV 16 was the type most commonly detected (3.6% of the population). High-risk HPV infection was strongly associated with risk for all grades of CIN and cancer. HPV 16, 58 and 18 were the most common types in women diagnosed with CIN3 and cancer. Significantly increased risks for CIN3 were found for HPV types 16 [odds ratio, 272], 31 [odds ratio, 83.3], 56 [odds ratio, 46.3], 58 [odds ratio, 73.5] and 68 [odds ratio, 96.1]. HPV types 16 [odds ratio, 504], 18 [odds ratio, 595], 45 [odds ratio, 390], 52 [odds ratio, 149], 39 [odds ratio, 202], 58 [odds ratio, 184] and 66 [odds ratio, 312] were significantly associated with an elevated risk for invasive cervical cancer. HPV types 59, 35, 56, 68, 53, 54, 26 and 73 were detected in controls only. Multiple-type infections were associated with an increased risk compared with single-type infections for all grades of CIN and cancer, except for HPV 16-positive CIN3 and cancer. (c)

Cohort studies

(i) Prospective studies with data on DNA Table 28 summarizes the results of cohort studies of HPV type-specific infection and pre-invasive and invasive carcinoma. Koutsky et al. (1992) conducted the first prominent cohort study of HPV infection with some type specificity and a disease end-point of CIN2 or CIN3. In the 24 months following study entry, the relative risk for women who were infected with HPV 16 or HPV 18 (combined) compared with HPV DNA-negative women was 11 (95% CI, 4.6–26). Using the stored collection of cytological slides from Swedish women who participated in a multi-decade screening programme, Wallin et al. (1999) examined type-specific persistence of HPV DNA before the development of invasive cervical cancer. Using two different PCR techniques and DNA sequencing, HPV in cells scraped from cytological smears was typed. A total of 118 women in whom invasive cancer developed on average 5.6 years later (range, 0.5 months to 26.2 years) were compared with 118 women who remained healthy during a similar length of time. In addition to testing the cytological slides, the important issue was addressed of whether the HPV type in the pre-morbid cytological sample matched the diagnostic type among cases who had available histology blocks. There was a clear excess of HPV 16 persistence associated with the risk for developing cancer (16 cases, no controls). There was also a non-significant excess of HPV 18 persistence (four cases, no controls), and single cases with persistence of HPV 31, 33 and 73. In a series of publications from the same project, Josefsson et al. (2000) and Ylitalo et al. (2000a,b) measured HPV 16 viral load by applying quantitative PCR to cell

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Table 28. Cohort studies on HPV-specific infection and pre-invasive and invasive lesions of the cervix Reference, study location

Method of detection (types included)

No. and type of cases

Odds ratio (95% CI)

Koutsky et al. (1992), USA

Dot filter hybridization, Virapap specific primers (16, 18, 6, 11, 31, 33, 35)

28 CIN

HPV 16/18 11 (4.6–26)

Wallin et al. (1999), Sweden

PCR MY09/MY1GP5+/6+ DNA sequencing

118 ICC

HPV 16/18/31/33/73 16.4 (4.4–75.1)

Josefsson et al. (2000); Ylitalo et al. (2000a,b), Sweden

Quantitative PCR (16)

478 CIS

High viral load 25 (12.4–31.8) Medium viral load 6.6 (1.7–11.2)

Woodman et al. (2001), United Kingdom

PCR GP5+/GP6+, MY09/11 and specific primers (16, 18, 31, 33, 52, 58)

23 CIN2/3

HPV 6/11 3.8 (1.5–9.8) HPV 16 8.5 (3.7–19·2) HPV 18 3.3 (1.4–8.1)

Zielinski et al. (2001a,b), The Netherlands

PCR GP5+/6+ using a cocktail of HPV type-specific oligoprobes (16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, 68)

57 ICC

No risk or p-value reported

van Duin et al. (2002), The Netherlands

PCR (16)

12 CIN2/3

7.7 (1.6–33)

van der Graaf et al. (2002), The Netherlands

Short-fragment PCR 10 general primer set (6, 11, 16, 18, 31, 33, 34, 35, 39, 40, 42, 43, 44, 45, 51, 52, 53, 54, 56, 58, 59, 66, 68/73, 70, 74)

77 CIN3, SCC

HPV 16 104.8 (29.5–372.7) HPV 18, 31, 33 10.8 (4.3–27.2)

Kjaer et al. (2002), Denmark

PCR GP5+/6+ (high risk 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, 68, low risk 6, 11, 40, 42, 43, 44)

112 CIN2/3

See Table 29

Xi et al. (2002), USA

MY09/11, HMB01, human β-globin primers (6, 11, 16, 18, 31, 33, 35, 39, 45, 56, 40, 42, 53, 54, 51, 52, 55, 58)

6 CIN 2/3

HPV 16 nonprototype-like variants 3.5 (1.0–11.8)

Schiffman et al. (2005), Costa Rica

PCR MY09/11 PCR (2, 6, 11, 13, 16, 18, 26, 31–35, 39, 40, 42–45, 51–59, 61, 62, 64, 66–74v, 81–85, 82v (AE2), 89, AE9, AE10)

8 ICC 61 CIN3

No risk or p-value reported

See Table 7 for a description of the primers used. CI, confidence interval; CIN, cervical intraepithelial neoplasia; CIS, carcinoma in situ; ICC, invasive cervical carcinoma; PCR, polymerase chain reaction; SCC, squamous-cell carcinoma

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scrapings of archival cytology slides from the Swedish screening programme. A total of 2081 smears from 478 cases of carcinoma in situ and 1754 smears from 608 controls were tested. Elevated HPV 16 viral loads were observed among cases compared with controls starting at 13 years before diagnosis, although the smears were considered to be cytologically normal (Ylitalo et al., 2000b). Thus, detection of HPV 16 DNA predicted a risk for a diagnosis of carcinoma in situ many years later. About 25% (95% CI, 12.4–31.8) of women infected with a high viral load before the age of 25 years developed cervical carcinoma in situ within 15 years. Women with a medium viral load had an absolute risk of 6.6% (95% CI, 1.7–11.2) after 15 years (Ylitalo et al., 2000a). Women with low viral loads were at marginally elevated risk compared with HPV 16-negative women (Ylitalo et al., 2000a). The median latency between the initial HPV 16 infection and diagnosis of carcinoma in situ was estimated to be between 7 and 12 years but may be up to two decades for some women (Ylitalo et al., 2000b). These estimates are concordant with the time between modal ages of HPV infection and CIN3 observed in population-based crosssectional studies. Woodman et al. (2001) studied the natural history of incident cervical infection with HPV 16, 18, 31, 33, 52, 58 or 6/11 in relation to the development of CIN2 or CIN3 among 1075 British women aged 15–19 years who had recently become sexually active. The median duration of follow-up of the cohort was 29 months; thus, the few cases represented the leading edge of the incidence curve of CIN2 (14 cases) or CIN3 (14 cases). Specifically, among the 23 cases for whom HPV DNA was detected, the median time from first detection to diagnosis of CIN2 or CIN3 was 26 months (range, 0–69 months). The univariate relative risks for CIN2 or CIN3 were elevated for HPV 6/11 (3.8; 95% CI, 1.5–9.8), 16 (8.5; 95% CI, 3.7–19.2), 18 (3.3; 95% CI, 1.4–8.1), 31 (3.5; 95% CI, 1.0–11.8), 52 (2.3; 95% CI, 0.3–17.2) and 58 (2.9; 95% CI, 0.8–10.1), but not for HPV 33 (0.6; 95% CI, 0.1–4.4). The evidence of type-specific carcinogenicity afforded by these data is weakened by the short follow-up, the incomplete typing with possibility of type–type confounding and the inclusion of CIN2 in the disease group. Zielinski et al. (2001b) conducted a retrospective case–control study of type-specific DNA detection (by GP5+/6+ PCR) in the last normal cervical smears archived for 57 women who developed cervical cancer approximately 8 years later compared with 114 controls matched on age and date of screening. The types found in the subsequent smears and diagnostic biopsies of the case women were the same as those detected at the baseline smear. The only statistically significant difference was for HPV 16 (29/57 cases, 2/114 controls). Other types showed non-significant excesses among cases: HPV 18 (three cases, two controls), 31 (three cases, two controls), 45 (one case, one control) and 33 (one case, no control). Most of the smears that were originally interpreted as normal were reinterpreted, blinded to other study information, as abnormal for cases but not for controls. Re-interpretation as abnormal was strongly linked to HPV DNA positivity. This study demonstrates the difficulty of determining the true cytological state at baseline in longitudinal studies.

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Using quantitative PCR, van Duin et al. (2002) tested the viral load of archived HPV 16 DNA-positive specimens from a Dutch cohort that included 12 women who subsequently developed CIN2 or CIN3 and 47 controls who developed ≤ CIN1. All baseline smears were considered to be normal, although that interpretation does not rule out the possibility of neoplasia being missed by cytology. Over the average of almost 3 years of follow-up, an association was observed between high versus low baseline viral load of HPV 16 and risk for CIN2 or CIN3 (odds ratio, 7.7; 95% CI, 1.6–33). van der Graaf et al. (2002) tested scraped cells from archived cytological smears in a nested case–control study within the Dutch (Utrecht) mass-screening programme, using a short fragment PCR 10 amplification system and typing by a reverse hybridization line probe. After exclusions for missing slides, the case group included 62 women with CIN3 and 15 with micro- or gross invasion. The 270 controls were matched to cases on age and follow-up time. During an average follow-up period of 5.6 years, 29 cases compared with three controls had slides that contained only HPV 16 (odds ratio, 104.8; 95% CI, 29.5–372.7). The presence of HPV 18, 31 and/or 33 (not distinguished individually) was associated with an odds ratio of 10.8 (95% CI, 4.3–27.2). Kjaer et al. (2002) conducted a prospective cohort study among more than 10 000 women aged 20–29 years in Copenhagen, Denmark, and tested baseline specimens for type-specific DNA using GP5+/GP6+ PCR. In order to limit the study to incident cases, women with a history of cervical neoplasia, abnormal baseline cytology or abnormalities diagnosed within 9 months from baseline were excluded. A total of 112 cases of CIN2 or CIN3 were observed at the follow-up visits approximately 2 years after baseline. Because of the relatively short follow-up, some of these cases were possibly present but were missed at baseline. With this caveat, elevated univariate odds ratios were found for most putative carcinogenic HPV types, including HPV 16, 18, 31, 33, 45, 51, 52, 58 and 66 (Table 29). Increased risk estimates based on one or two cases were associated with HPV 35, 39, 56 and 59. Based on a single case each, elevated univariate risk estimates were seen for HPV 6 and 11 which are associated with condyloma acuminatum. Other identified types were rare or absent in cases and controls. However, this analysis did not take into account the possible confounding influences of multiple infections, which were found in 25% of HPV-positive cases and 12% of HPV-positive controls. In a 5-year longitudinal cohort study among female university students in Seattle, USA, Xi et al. (2002) observed that incident infections with non-prototype-like HPV 16 variants conferred a 3.5 (95% CI, 1.0–11.8) increase in risk for histological CIN2 or CIN3 compared with prototype-like HPV 16 variants. Of the 48 women with incident HPV 16 prototype-like variants, six developed CIN2 or CIN3, while six of 14 women with non-prototype-like variants developed these diseases. The difference in risk was not mediated by a difference in average length of viral persistence, ethnicity or current use of oral contraceptives. Schiffman et al. (2005) conducted a population-based prospective study of HPV infection and subsequent development of CIN3 and cancer in a cohort of 10 000 women in Guanacaste, Costa Rica. They tested for more than 40 types of HPV DNA using MY09/11 PCR with TaqGold polymerase, and followed more than 7000 sexually active

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Table 29. Distribution of HPV types among cases and cytologically normal women who were positive for HPV at enrolment HPV type

Cases (n = 115) of high-grade lesions (%)

Controls (n = 100) (%)

Odds ratioa

6 11 16 18 31 33 35 39 42 44 45 51 52 56 58 59 66 X

1 (0.9) 1 (0.9) 43 (37.4) 10 (8.9) 15 (13.0) 7 (6.1) 1 (0.9) 2 (1.8) 0 0 5 (4.5) 6 (5.3) 5 (4.5) 2 (1.8) 5 (4.5) 2 (1.8) 4 (3.6) 6 (5.3)

5 (0.8) 1 (0.2) 27 (4.1) 11 (1.7) 7 (1.1) 11(1.7) 1 (0.2) 2 (0.3) 1 (0.2) 1 (0.2) 1 (0.2) 1 (0.2) 2 (0.3) 1 (0.2) 5 (0.8) 0 1 (0.2) 22 (3.4)

1.2 5.9 14.4 5.7 14.3 3.9 5.9 5.9 – – 30.5 57.0 15.2 11.9 6.0 – 24.1 1.6

From Kjaer et al. (2002) Figures are numbers (percentages of women). a Crude odds ratios calculated by the Working Group

women who had no evident prevalent CIN2, CIN3 or cancer and no hysterectomy for an average of over 5 years. Cases of incident histologically confirmed CIN3 and cancer were considered to be caused by a particular type of HPV only if that type was found both at study enrolment and at the time of diagnosis. The results confirmed the case–control literature, and were consistent with the data of Kjaer et al. (2002). The risks for cancer and CIN3 with HPV types were clearly associated with their phylogenetic relatedness. Compared with other types, HPV 16 was the most likely to persist for 5 years and, when persistent, to be linked to CIN3 and cancer. HPV 16-related infections also tended to lead to incident CIN3/cancer. Most of the other cancer-associated types were phylogenetically related to HPV 18. Some HPV species (clades; e.g. A3) showed virtually no association with CIN3 or cancer despite a relatively high prevalence and a tendency of some types in those clades to persist.

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(ii) Prospective studies with serological data (Table 30) Studies of archived sera permit the assessment of exposure to HPV before a diagnosis of CIN3 or cancer. The assays themselves tend to be type-specific, although the possibility of confounding remains because genital HPV infections are transmitted by a common sexual route and few types are assessed in any study. Lehtinen et al. (1996) focused on HPV 16 serology within a cohort of Finnish women that was followed for up to 23 years. The odds ratio for HPV 16 seropositivity was 12.5 (95% CI, 2.7–57) among 72 cases (27 with cancer and 45 with carcinoma in situ) and 143 matched controls. The risk estimates were increased for both short and long lapses of time from sampling to diagnosis. Dillner et al. (1997) compared 182 women who had developed cervical cancer with 538 controls matched on age and time of enrolment into a joint Nordic cohort. Sera were tested for antibodies to HPV 16, 18 and 33. HPV 16 seropositivity was associated prima-

Table 30. Prospective serological studies on HPV-specific infection and pre-invasive and invasive lesions of the cervix Reference, study location

Method of detection (types included)

No. and type of cases

Odds ratio (95% CI)

Shah et al. (1997), USA

ELISA (16, 6, 6b)

14 ICC, 28 CIN3

HPV 16 3.9 (1.4–10.7)

Lehtinen et al. (1996), Finland

ELISA (16)

27 ICC, 45 CIS

12.5 (2.7–57)

Dillner et al. (1997) Finland, Norway, Sweden

ELISA (16, 18, 33)

182 ICC

HPV 16 3.2 (1.7–6.2)

Luostarinen et al. (1999), Finland, Norway, Sweden

ELISA (16, 18, 33, 6/11)

182 ICC

No signifiant association

Vonka et al. (1999), Czech Republic

ELISA (16, 18, 33)

43 dysplasies, 19 CIS, 5 ICC

HPV 16 3.85 (1.11–13.91)

Wallin et al. (2000), Sweden

ELISA (73)

41 CIN2/3

1.5 (0.35–6.65)

Hisada et al. (2001), USA

ELISA (16)

52 ICC, 47 CIS

2.0 (1.0–3.4)

Sigstad et al. (2002), Norway, Finland, Sweden

ELISA (16, 18, 33)

127 ICC

HPV 16 4.4 (2.2–8.8) HPV 18 17 (2.1–140)

CI, confidence interval; CIN, cervical intraepithelial neoplasia; CIS, carcinoma in situ; ELISA, enzyme-linked immunosorbent assay ; ICC, invasive cervical carcinoma

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rily with an increased risk for squamous-cell carcinoma (odds ratio, 3.2; 95% CI, 1.7–6.2) while HPV 18 seropositivity tended to be associated with a higher risk for cervical adenocarcinomas (odds ratio, 3.4; 95% CI, 0.8–14.9). HPV 33 seropositivity was not significantly associated with either squamous-cell or adenocarcinoma (odds ratio, 1.6 and 1.7, respectively). Shah et al. (1997) tested pre-diagnostic sera from 14 cases of invasive cancer and 28 cases of CIN3 and compared them with those from 83 matched controls. The odds ratio for antibodies to HPV 16 VLPs was 3.9 (95% CI, 1.4–10.7), but HPV 6 antibodies were not associated with the subsequent occurrence of cervical cancer or CIN3. Luostarinen et al. (1999) further tested the same Nordic subjects followed by Dillner et al. (1997) for HPV 6/11 to assess the joint effect of simultaneous exposure with carcinogenic (HPV 16, 18 and 33) and non-carcinogenic HPV types on the risk for subsequent development of cancer. HPV 6/11 seropositivity was not strongly associated with risk for cancer. However, there was evidence of an antagonistic modification of effect for the combination of HPV 16 seropositivity and HPV 6/11 seropositivity (but not other combinations). The authors suggested that HPV 6/11 seropositivity might reflect cross-protective immunity. Vonka et al. (1999) re-tested stored sera from the Prague cohort that was originally assembled in the 1970s to study herpes simplex virus (HSV) and cervical cancer (Vonka et al., 1984) for HPV 16, 18 and 33 VLP antibodies. While the original study failed to find an association of HSV seropositivity with subsequent cervical cancer, the re-analysis of 67 prospective cases and 129 matched controls showed an elevated relative risk for all three HPV types. The case group included 43 moderate or severe dysplasias, 19 carcinomas in situ and five invasive carcinomas. The relative risks were 3.85 (95% CI, 1.11–13.91) for HPV 16, 2.70 (95% CI, 0.87–8.55) for HPV 18 and 1.51 (95% CI, 0.55–4.13) for HPV 33. Wallin et al. (2000) explored the possible carcinogenicity of HPV 73 in a serological study of stored blood from 41 cases of CIN2–3 in northern Sweden and 82 matched controls. The odds ratio for HPV 73 antibodies preceding case diagnosis by an average of 2–3 years was 1.5 (95% CI, 0.35–6.65). Hisada et al. (2001) tested precancer sera archived from pregnant women in a Californian cohort study and observed an age- and race-adjusted odds ratio of 2.0 (95% CI, 1.0–3.4) for the association of seropositivity to HPV 16 VLPs and subsequent risk for invasive (52 cases) or in-situ (47 cases) cervical cancer. In a retrospective cohort study based on archived blood and cervical tissue, Sigstad et al. (2002) confirmed that pre-diagnostic HPV 16 and HPV 18 seropositivity was linked to a risk for subsequent cervical cancer and corresponded to the same HPV DNA types found in the tumours. A total of 127 cases of invasive cancer in the large Nordic Janus cohort and 376 controls matched on age, country and time of blood collection were studied. HPV 16-seropositive women had a relative risk of 4.4 (95% CI, 2.2–8.8) for developing invasive cancer containing HPV 16 DNA, but had no excess risk for developing other cancers. Similarly, HPV 18 seropositivity predicted a risk (odds ratio, 17; 95% CI, 2.1–140) only for cancers that contained HPV 18, of which 10/20 were

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adenocarcinomas. The results for HPV 33 were not type-specific; tumours that contained HPV 33 were very rare and HPV 33 seropositivity was associated with the development of cancers that contained HPV 16. In prospective studies of individual HPV types, the data from DNA-based and serological studies are concordant although absolute risks associated with DNA positivity are higher than those associated with seropositivity. HPV 16 is clearly carcinogenic and persists longer than other carcinogenic types. Because of its persistence, HPV 16 is apparently more closely linked to malignant transformation. There is also some prospective evidence for the carcinogenicity of HPV 18. The evidence for other types, although scant, is concordant with the more statistically powerful case–control literature. 2.3

Cancer at other anogenital sites

2.3.1

Cancer of the vulva

Vulvar cancer has two distinct histopathological types and sets of risk factors. Only a limited number of studies have characterized the prevalence of HPV DNA by histological type, but the results have been consistent. Keratinizing vulvar cancer is associated with a low prevalence of HPV (generally less than 10%), occurs in older women and is associated with lichen planus. In contrast, HPV DNA is found in a high proportion of basaloid and warty vulvar cancers (> 55%), which occur in younger women than keratinizing cancers and are associated with classical risk factors for the acquisition of HPV (Schiffman & Kjaer, 2003). These cancers are often associated with overlying vulvar intraepithelial neoplasia (VIN), which in turn has a strong association with HPV infection. The data suggest two distinct sets of cancer: one that is associated with HPV and may be preceded by VIN, and another that is not clearly associated with HPV and whose precancerous natural history is poorly understood. (a)

Case series

Table 31 presents series of more than 10 cases of cancer of the vulva or VIN3. Among the HPV DNA-positive vulvar cancers, HPV 16 is the most common type, followed by HPV 18 at a much smaller percentage. The proportion of HPV-positive vulvar cancers that contained HPV 31 or HPV 33 was variable in the small number of studies that specifically probed for these types. One study (Iwasawa et al., 1997) found a 1.4% prevalence of HPV 33 but did investigate HPV 31. Another small study of 11 cases of vulvar carcinomas (Abdel-Hady et al., 2001) found HPV 33 in two of three HPV-positive cases. Similar to the data on vulvar cancer, case series on the prevalence of HPV DNA show that a high proportion (> 70%) of VIN3 are positive (Table 31) and that the most common type is HPV 16. Two studies (Junge et al., 1995; Van Beurden et al., 1998) also showed that a small proportion of lesions (< 11%) are DNA-positive for HPV 33. Two studies

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Reference, study location

Type-specific HPV positivity (%) 6

11

Hørding et al. (1994), Denmark

E6/E7 primers (6, 11, 16, 18, 33)

51 keratinizing 17 warty 10 basaloid

3.9 70.5 100

0 0 0

0 0 0

Trimble et al. (1996), USA

ISH (6/11/16/18/ 31/33/35/42/43/4 4/45/51/52/56)

21 basaloidwarty 48 keratinizing

85.7

Iwasawa et al. (1997), Finland

MY09/11 typespecific (6, 11, 16, 18, 33)

74

36.5

Madeleine et al. (1997), USA

MY09/11 (6/11, 16, 18/45, 31/33/52) or RFLP

55

50.9

Abdel-Hady et al. (2001), United Kingdom

GP5+/6+ (6/11, 16, 18, 31, 33)

11

Carter et al. (2001), USA

MY09/11 and RFLP; typespecific for 16, 18

E6/E7 primers (6/11, 16, 18, 31, 33); ISH (6/11, 16/18, 31/33)

Vulvar cancer

VIN3 Junge et al. (1995), Denmark

}

16

18

28.2

0 0 0

31

}

NR

33

}

Others (type)

Multiple infections (%)

Comments

1.3

Paraffin-embedded tissue; HPV DNA found in 81% of cancers with overlying VIN3, 9% of tissues with overlying VIN1–2 and 0% of tissues with adjacent lichen sclerosis

3.8

Paraffin-embedded tissue; basaloid or warty carcinoma but not keratinizing squamous carcinoma associated with classical risk factors for cervical cancer

6.3

0

0

25.7

12.2

43.6

1.8b

27.3

27.3

9.0

38

79

55.3

2.6

62 PCR [n = 55] ISH [n = 58]

89.1

1.8

0 0

0 0

[78.2] 0 [36.2]

1.4

3.6c

1.8d

18.1

13.2e

0

7.9f

[10.9] [5.2]

4.0

Paraffin-embedded tissue; 65 of 74 cases were women 61 years of age or older; histological type not specified

1.8

Paraffin-embedded tissue; histological type not specified

18

Paraffin-embedded tissue; histological type not specified

0

Paraffin-embedded tissue; histological type not specified

NR

Paraffin-embedded tissue; cases of severe dysplasia and carcinoma in situ

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Method of detectiona (types included)

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Table 31. Prevalence of HPV DNA in case series of vulvar cancer (≥ 10 cases) and grade 3 vulvar intraepithelial neoplasia (VIN3) (≥ 9 cases)

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Table 31 (contd) Overall HPV positivity (%)

Trimble et al. (1996), USA

ISH for 6/11/16/18/ 31/33/35/42/43/44 /45/51/52/56

54 basaloid or warty

88.9

Madeleine et al. (1997), USA

MY09/11 probes (6/11, 16, 18/45, 31/33/52) or RFLP

253

71.5

Van Beurden et al. (1998), Netherlands

CPI and CPIIG and sequencing

27

Abdel-Hady et al. (2001), United Kingdom

GP5+/6+ and type-specific for 6/11, 16, 18, 31, 33

32

71.9

Carter et al. (2001), USA

MY09/11 and RFLP; typespecific for 16, 18

181

91.2

Todd et al. (2004), United Kingdom

GP5/6 and typespecific for 16, 18, 31, 33

9

Type-specific HPV positivity (%) 6

11

18

31

33

Others (type)

Comments

Paraffin-embedded tissue; squamous hyperplasia (VIN2) and basaloid or warty VIN (VIN3) combined 5.5

100

[88.8]

16

Multiple infections (%)

61.7

5.9b

5.9c

92.6

[18.8]

3.7

2.8d

9.5

Paraffin-embedded tissue; histological type not specified

3.7 (45)

0

Paraffin-embedded tissue; histological type not specified; patients with pre-existing, concomitant or subsequent cervical or vaginal neoplasm

NR

Paraffin-embedded tissue; cases were VIN2 or VIN3, predominantly (26/32) warty or mixed wartybasaloid type.

6.1

Paraffin-embedded tissue; carcinoma in situ

0

Paraffin-embedded tissue; 77.8% of cases had prior history of CIN.

46.8

74.6

6.6

[66.7]

0

8.8e

0

7.2f

[22.2]

See Table 7 for a description of the primers used. CIN, cervical intraepithelial neoplasia; ISH, in-situ hybridization; NR, not reported; PCR, polymerase chain reaction; RFLP, restriction fragment length polymorphism a Unless otherwise specified, the method is PCR; ‘;’ denotes independent methods whereas ‘and’ denotes subsequent steps. b 18 or 45 c 31, 33 or 52 d Unknown e 31, 33, 35 or 39 f HPV 6, 45, 52, 54, 58, 66, 72, 73 or unknown types

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(Madeleine et al., 1997; Carter et al., 2001) showed the presence of HPV 18 or 45 in 5.9% and that of HPV 18 in 6.6% of lesions, respectively. (b)

Case–control studies

Results from case–control studies of vulvar cancer and VIN that used serological assays as detection method are consistent with the findings on DNA. Sun et al. (1996) examined HPV-specific antibodies in the sera of patients in the USA who had basaloid or warty squamous-cell vulvar cancer, keratinizing vulvar cancer and VIN to determine the association between these conditions and the presence of antibodies to HPV 16. The study included a total of 54 cases (14 basaloid or warty cancers, 18 keratinizing cancers and 22 VIN) for whom serological specimens were available and 44 controls. The prevalence of antibodies to HPV 16 VLPs was significantly higher in HPVassociated VIN (59.1%) and basaloid or warty cancers (50.0%) than in keratinizing cancers (22.2%) or controls (18.2%). The odds ratios were 5.4 (95% CI, 1.7–18) for VIN and 4.5 (95% CI, 1.2–16) for basaloid and warty cancers. For keratinizing cancers, the odds ratio was not statistically significant at 1.3 (95% CI, 0.32–4.9). A seroepidemiological nested case–control study from Finland and Norway (Bjørge et al., 1997a) showed that HPV 16 seropositivity was associated with an increased risk for vulvar and vaginal cancers combined (odds ratio, 4.5; 95% CI, 1.1–22) and a strongly increased risk for pre-invasive vulvar and vaginal lesions (odds ratio, ∞; 95% CI, 3.8–∞). Seropositivity for HPV 18 was associated with an increased risk for pre-invasive lesions (odds ratio, 12; 95% CI, 1.2–590) but not for invasive cancer (odds ratio, 1.5; 95% CI, 0.3–7.5). Hildesheim et al. (1997a) studied 142 histologically confirmed cases of VIN3 and invasive vulvar cancer and 126 community controls in the USA. Sera were tested for immunoglobulin G (IgG) antibodies against HPV 16 L1/L2 VLPs. Overall, 44.4% of cases and 11.9% of controls were HPV 16-seropositive. A stronger association between HPV 16 seropositivity and disease was observed for VIN3 (odds ratio, 13.4; 95% CI, 3.9–46.5) than for invasive cancer (odds ratio, 2.9; 95% CI, 0.94–8.7), although there was a suggestion that the association was stronger among women who had been diagnosed with basaloid or warty cancer (odds ratio, 3.8; 95% CI, 0.76–18.9) than among those with keratinizing cancer (odds ratio, 1.6; 95% CI, 0.35–7.4). Madeleine et al. (1997) conducted a population-based case–control study in the USA to examine the association between HPV positivity, cigarette smoking, HSV-2 infection and the risk for vulvar cancer. The study included 400 in-situ and 110 invasive tumours of the vulva diagnosed among women who lived in the Seattle area from 1980 to 1994. In most analyses, cases were compared with 1043 controls. Serum samples were analysed for antibodies against HPV 6, 16 and 18. The prevalence of seropositivity to HPV 16 was 53.3% in in-situ cases, 43.8% in invasive cases and 22.2% in controls. HPV 16 seropositivity was associated with an increased risk for in-situ and invasive vulvar cancers (odds ratio, 3.6; 95% CI, 2.6–4.8; and 2.8; 95% CI, 1.7–4.7, respectively).

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Overall, the data indicate that HPV 16 is the predominant HPV type in VIN3 and vulvar cancer, particularly basaloid and warty cancer. In vulvar cancers, HPV 18, 45, 31 or 33 may play a smaller role. 2.3.2

Cancer of the vagina (a)

Case series

The number of case series of vaginal cancer (Table 32) has remained small since the previous review (IARC, 1995). In two studies (Carter et al., 2001; Daling et al., 2002), the majority of vaginal cancers were positive for HPV DNA (90.7% of 54 cases and 64.0% of 25 cases, respectively). HPV 16 was the most common type and was found in at least 70% of HPV-positive tumours. HPV 6 or 11 was found in two cases of vaginal cancer in one study. HPV 18 or 45 and HPV 31, 33, 35 or 39 were also found in a small number of cases. Similarly to the vaginal cancers, a high proportion of grade 3 vaginal intraepithelial neoplasia (VAIN3) tissues were also positive for HPV DNA and again the most common type was HPV 16 (Table 32). HPV 6 or 11, HPV 18 or 45 and HPV 31, 33 or 35 were found in a small number of cases (Van Beurden et al., 1998; Daling et al., 2002). (b)

Case–control studies

As with the studies of vulvar cancer, case–control studies of VAIN and vaginal cancer that used serological assays as the detection method are consistent with the findings on HPV DNA. Hildesheim et al. (1997b) conducted a case–control study of VAIN and vaginal cancer in the USA. The study included 23 histologically confirmed cases of in-situ and invasive vaginal cancer and 28 community controls. Blood samples were collected from participants and tested for the presence of antibodies to HPV 16 VLPs, HSV-2 and C. trachomatis. Overall, 50% of cases and 25% of controls were positive for HPV 16 VLP antibodies. Women positive for HPV 16 VLP antibodies were at a 3.5-fold increased risk for vaginal neoplasia (95% CI, 0.97–13) and those with high antibody levels (high optical density) were at a 33-fold increased risk for the disease (95% CI, 2.5–430). The risk estimate was not affected by adjustment for HSV-2 or C. trachomatis seropositivity. The association was stronger for in-situ neoplasia than for invasive cancer, with relative risks of 5.4 (95% CI, 0.93–31) and 1.7 (95% CI, 0.22–14), respectively. Daling et al. (2002) conducted a population-based case–control study that included 156 women with in-situ or invasive vaginal cancer diagnosed between January 1981 and June 1998 and 2041 control women identified through random-digit dialling in western Washington State, USA. Antibodies to HPV-16 L1 were strongly related to risk for vaginal cancer (odds ratio, 4.3; 95% CI, 3.0–6.2). Women with vaginal cancer were more likely to have had five or more lifetime sexual partners (odds ratio, 3.1; 95% CI, 1.9–4.9), to have an early age at first intercourse (< 17 years; odds ratio, 2.0; 95% CI, 1.2–3.5) and to be current smokers at diagnosis (odds ratio, 2.1; 95% CI, 1.4–3.1) than control women.

90.7

25

64.0

6

11

8.0

16

63.0

5.6

56.0

12.0d

Southern blot with PBM-58

3

100

66.6

van Beurden et al. (1998), Netherlands

CPI and CPIIG and sequencing

8

100

75.0

Daling et al. (2002), USA

MY09/11 and probing for 6/11, 16, 18/45, 31

74

82.4

8.1

18

54.1

31

33

3.7b

Others (type)

27.8c

0

0

0

33.3 (51) 12.5

8.1d

5.4e

See Table 7 for a description of the primers used. CIN, cervical intraepithelial neoplasia; PCR, polymerase chain reaction; RFLP, restriction fragment length polymorphism a Unless otherwise specified, the method is PCR; (;) denotes independent methods whereas ‘and’ denotes subsequent steps. b 31, 33, 35 or 39 c 6, 45, 52, 54, 58, 66, 72, 73 or unknown HPV types d 18 or 45 e 31, 33 or 35

Multiple infections (%)

Comments

9.3

Paraffin-embedded tissue

[12.0]

Paraffin-embedded tissue

0

Fresh tissue

Paraffin-embedded tissue; VAIN2 and 3 with simultaneous CIN lesions or invasive cervical neoplasia Paraffin-embedded tissue

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VAIN 3 Sugase & Matsukura (1997), Japan

MY09/11 and probing for 6/11, 16, 18/45, 31

54

Type-specific HPV positivity (%)

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Overall HPV positivity (%)

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Vaginal cancer Carter et al. MY09/11 and (2001), USA RFLP; type-specific for 16, 18

No. of cases

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Methoda of detection and types tested

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Table 32. Prevalence of HPV DNA in case series of vaginal cancer and grade 3 vaginal intraepithelial neoplasia (VAIN3 ≥ 3 cases) (≥

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Approximately 30% of cases had been treated for a prior anogenital tumour, most often of the cervix. 2.3.3

Cancer of the penis

Similarly to vulvar cancer, the prevalence of HPV DNA in penile cancer varies with histological type. Most case series do not specify the histological type (Table 33), but for those that do (Gregoire et al., 1995; Bezerra et al., 2001a,b; Rubin et al., 2001; Ferreux et al., 2003), warty and basaloid carcinomas of the penis in general had a higher prevalence of HPV infection than verrucous and keratinizing carcinoma. These studies mostly included a small number of cases and the range of HPV prevalence in the tissues was wide. Basaloid and warty carcinomas may be preceded by penile intraepithelial neoplasia (PIN), which is also associated with HPV infection (Aynaud et al., 1994; Rubin et al., 2001). Among the HPV-positive penile cancers, HPV 16 was the most common type. However, the majority of studies included at least one case of cancer with HPV 6 or 11, which in several studies were more common than HPV 18 (Levi et al., 1998; Rubin et al., 2001). HPV 31 or 33 were detected only rarely. Overall, similarly to other anogenital cancers, HPV 16 is the predominant HPV type and, similarly to vulvar cancer, warty and basaloid carcinomas tended to be those cancers with the highest proportion of HPV DNA positivity. However, the relationship between histopathology and HPV prevalence is not as clear as that in cancer of the vulva. Also, HPV 6 or 11 appear to play a more prominent role in penile cancers than in other cancers of the anogenital region. 2.3.4

Cancer of the anus

[For anal cancer in HIV-positive patients, see Section 2.8.3(b).] (a)

Case series

Case series of cancer of the anus are presented in Table 34. Anal cancer resembles cervical cancer more than the other anogenital cancers with respect to overall prevalence of HPV positivity. The prevalence of HPV DNA in anal cancer in different case series varies widely, but most studies that used MY09/MY11 or GP5+/GP6+ primers showed a prevalence of 80% or above. Basaloid cancers are similar to squamous-cell carcinomas with respect to prevalence of HPV DNA and are more probably a histological variant rather than a separate entity. HPV 16 is the most common type in squamous-cell cancers (76%) followed by HPV 18 with a much smaller percentage (9%). Most studies that included a broad range of HPV type-specific probes showed a low prevalence of HPV 31, 33 or 6 or 11.

p. 216.qxp

No. and type of lesions

Southern blot

Cupp et al. (1995), USA

MY09/11 and typespecific for 16, 18

42 SCC 13 carcinoma in situ

54.8 92.3

Gregoire et al. (1995), USA and Paraguay

Type-specific for 6, 11, 16, 18 and primer for wide range including 16, 18, 31, 33, 35, 52

45 typical 12 basaloid 10 papillary 9 warty 6 verrucous 19 mixed, warty or basaloid 8 mixed, other

11.1 75.0 0 22.2 0 47.4

Levi et al. (1998), Brazil

Type-specific for 6/11, 16, 18 MY09/11 and probing for 6, 11, 16, 18, 31

64 carcinoma

28.1

50 carcinoma

56.0

Buonaguro et al. (2000), Uganda

Southern blot for 16; PCR for 16 and sequencing

13 SCC

38.4

Picconi et al. (2000), Argentina

GP5/6 and SSCP for 6, 11, 16, 18, 31, 33

34 SCC

70.6

Bezerra et al. (2001a,b), Brazil

L1 consensus primers and probing for 6/11/ 16/18/31/33/34/35/39/ 40/42/43/44/45/51/52/ 54/56/58

60 SCC 11 warty carcinoma

26.7 45.5

100

Type-specific HPV positivity (%) 6

11

0a

16

18

100

0

40.5 84.6

0.9

0.9

31

33

Others

0

11.9c 0

4.8 15.4

Multiple infections (%)

Comments

0

Frozen tissue

7.7 2.4

0.9c

0

3.1

9.4c

1.6

Paraffin-embedded tissue

6.0

c

4

Frozen tissue; no comments on histopathology

21.1

0 4.7

14.1

12.0

32.0

12.0

38.4

[5.9]

18.2

0

[23.5]

15 27.3

Paraffin-embedded tissue; HPV positivity associated with aggressive, higher-grade tumours; logistic regression showed that the only association with HPV positivity was tumour histopathology.

Frozen tissue

[11.8]

5

0

0

3.3d

8.8c

0

Paraffin-embedded tissue; histological type not specified

3.3e

0 0

Paraffin-embedded tissue

Page 216

Aynaud et al. (1994), France

4 PIN3

Overall HPV positivity (%)

16:16

Method of detection and types tested

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Reference, study location

06/12/2007

216

Table 33. Prevalence of HPV DNA in case series of penile cancer ((J J 13 cases) and penile intraepithelial neoplasia ((J J 5 cases)

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Table 33 (contd)

Carter et al. (2001), USA

MY09/11 and RFLP; type-specific for 16/18

Rubin et al. (2001), USA and Uruguay

SPF10 and LiPA for 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, 68, 70 and 6, 11, 34, 40, 42, 43, 44, 53, 54, 74 GP5+/6+ EIA and type-specific for 37 types; ISH

Ferreux et al. (2003), Netherlands

No. and type of lesions

33 SCC

Overall HPV positivity (%)

6

11

81.8

106 keratinizing 5 warty 15 basaloid 30 PIN

34.9 100 80.0 90.0

55 SCC 48 SCC-NOS 2 warty 2 sarcomatoid 1 verrucous

37.7 35.4 100 0 100

Comments

Others

Multiple infections (%)

12.1c

9.1

Paraffin-embedded tissue; no comments on histopathology

20.8g 0 20.0h 16.7j

NR

Paraffin-embedded tissue; no difference in prevalence between samples from Uruguay and the USA

3.7k 4.2k 0 0 0

NR

Snap-frozen samples

Type-specific HPV positivity (%) 16

69.7

3.8 0 0 20.0

5.6 2.1 50 0 100

0 0 0 3.3

17.9 100 66.7 36.7

28.3 29.2 50 0 0

18

3

0.9 0 0 0

31

33

6.0f

0 0 0 0 [6.7] 0 i 30.0

See Table 7 for a description of the primers used. EIA, enzyme immunoassay; ISH, in-situ hybridization; LiPA, line blot hybridization; NOS, not otherwise specified; NR, not reported; PCR, polymerase chain reaction; PIN, penile intraepithelial neoplasia; RFLP, restriction fragment length polymorphism; SCC, squamous-cell carcinoma; SSCP, single-strand conformational polymorphism a 6, 11 or 42 b 31, 33 or 35 c 31, 33, 35 or 39 d HPV 35, 45, 52, 68, 51/70/74 and unknown types e 31, 33, 39, 44, 51, 52, 58 or 66

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Method of detection and types tested

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Reference, study location

217

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Method of detection and types tested

No. and type of lesions

Overall HPV positivity (%)

Type-specific HPV positivity (%) 6

11

16

Others

41.1

39.3

Shroyer et al. (1995), USA

MY09/11 and probing for 6/11, 16, 18, 33 ISH for 6/11, 16/18, 31/33/35

11 basaloid 16 non-basaloid

90.9 75.0

0 12.5

9 basaloid 11 non-basaloid

66.7 72.7

0 0

Ramanujam et al. (1996), USA

ISH for 6, 11, 16, 18, 31, 33, 35

53 (37 women, 16 men)

34.0

VincentSalomon et al. (1996), France

Southern blot for 6/11/42, 16/18/33, 31/35/39 PCR specific for 6/11, 16, 18, 33

15 SCC 9 basaloid

46.7 55.5

0 0

0 0

33.3 44.4

0 0

18 SCC 9 basaloid

66.7 77.8

0 0

0 0

66.7 55.5

0 22.2

ISH for 6, 11, 16, 18

35 invasive

68.6

0

0

68.6

0

38.0c

4.0

63.6 62.5

9.1 12.5 44.4 72.7

7.4 4.0

0 0

0 0

9.1 12.5

33.3 0

Paraffin-embedded tissue; in five samples, HPV was detected with only one of the two detection methods. Paraffin-embedded archival tissue

22.2 0 Paraffin-embedded tissue

0 0

0 0

13.3d 11.1d

0 0

0 0

Frozen tissue

0 0 0

Paraffin-embedded tissue

Page 218

54 invasive and 2 in-situ 50

6.0

33

Comments

IARC MONOGRAPHS VOLUME 90

ISH for 6/11, 16/18 and 31/33/35 MY09/11 and typespecific for 6, 16, 18

Williams et al. (1996), United Kingdom

31

1.8b

Noffsinger et al. (1995a,b), Canada, China and USA

46.0c

5.6

18

Multiple infections (%)

09:40

Reference, study location

P_179_278.qxp

218

≥ 5 cases) Table 34. Prevalence of HPV DNA in case series of anal cancer (≥

P_179_278.qxp 30/11/2007

Table 34 (contd) Method of detection and types tested

No. and type of lesions

388 (304 women, 84 men)

Unger et al. (1997), USA

ISH for 6/11, 16/18, 31/33/35

3 SCC from HIV-positive patient and 3 SCC from HIVnegative patient

Cuesta et al. (1998), USA

ISH for 6/11, 16/18, 31/33/35

6 verrucous carcinomas from HIV-positive patients

Lai et al. (1998), China

MY09/11 and probing for 6, 11, 16, 18, 33

19 SCC 8 cloacogenic cancers 23 adenocarcinomas 6 adenosquamous carcinomas

87.6

6

11

16

1.3

0

72.9

5.7

83.3f

16.7f

100

16.7

83.3

66.7

18

31 0.8

33

Others

5.9

1.8e

16.7f

Multiple infections (%)

Comments

Paraffin-embedded tissue

16.7f

33.3

Paraffin-embedded tissue

Paraffin-embedded tissue

5.3 0

0 0

0 0

5.3 0

0 0

0 0

0

0

0

0

0

0

0

0

0

0

0

0

0

Paraffin-embedded archival tissue

Page 219

GP5+/6+ and probes for HR types (16/18/ 31/33/35/39/45/51/ 52/56/58/59/66/68) and LR types (6/11/ 40/42/43/44); typespecific for 6, 11, 16, 18, 31, 33

Type-specific HPV positivity (%)

HUMAN PAPILLOMAVIRUSES

Frisch et al. (1997), Denmark and Sweden

Overall HPV positivity (%)

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Reference, study location

Method of detection and types tested

No. and type of lesions

Type-specific HPV positivity (%) 11

16

PU-1M for 16/18/ 31/33/52/58 and PU31B primers for 6/11

33

39.4

Indinnimeo et al. (1999), Italy

Type-specific for 6/11, 16, 18, 31/33

7 squamocellular 7 cloacogenic

71.4

28.6

57.1

57.1

Frisch et al. (1999), Denmark and Sweden

GP5+/6+ and probes for HR types (16/18/ 31/33/35/39/45/51/52 /56/58/59/66/68) and LR types (6/11/40/42/ 43/44); type-specific for 16, 18, 31, 33

331 (253 women, 78 men)

Carter et al. (2001), USA

MY09/11 and RFLP; specific primers for 6, 18

64 (45 women, 38 men)

93.8

31

33

Others

36.4g

Poletti et al. (1998), Switzerland

HR: [84] Men: 63 Women: 90 LR: [4.5] Men: 6.4 Women: 4.0

18

3.03

Paraffin-embedded tissue 42.9

[73]

[6]

79.7

9.4

[1]

Comments

[5]

6.2i

Paraffin-embedded tissue [2]h

[2.7]

10.8j

10.9

Paraffin-embedded tissue

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Table 34 (contd) Reference, study location

Type-specific HPV positivity (%) 6

11

16

18

92.2 97.6 40.0

76.0 95.1 15.0

8.9 0 5.0

94.4 81.8 92.6

80.6 66.7

5.6 7.6

91.2 88.4 91.8

67.7 75.9

8.8 6.3

31

33

Others

Multiple infections (%)

Comments

Paraffin-embedded tissue

See Table 7 for a description of the primers used. HIV, human immunodeficiency virus; ISH, in-situ hybridization; PCR, polymerase chain reaction; RFLP, restriction fragment length polymorphism; SCC, squamous-cell carcinoma a Unless specified otherwise, the method is PCR; ‘;’ denotes independent methods whereas ‘and’ denotes subsequent steps. b 31, 33 or 35 c The Working Group noted some discrepancies between text and table in the number of HPV-positive tumours detected by PCR: the text reported 23/50 HPV-positive tumours (46%) whereas the table presented only 22 positive cases (44%); the text reported 21 lesions positive for HPV 16 by PCR and the table reported only 19 HPV 16positive samples. d Unknown e 40/42/43/44 f One tissue contained 16, 18 and 33. g 16, 18, 31, 33, 52 or 58 h Untyped high risk i 31, 33, 35 or 39 j 6, 45, 52, 54, 58, 66, 72, 73 or unknown types

HUMAN PAPILLOMAVIRUSES

179 SCC 41 basaloid 20 adenocarcinoma Men 36 in-situ 76 invasive All Women 34 in-situ 112 invasive All

Overall HPV positivity (%)

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No. and type of lesions

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Method of detection and types tested

221

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(b)

Case–control studies

Frisch et al. (1999) studied the prevalence of HPV DNA in cancers of the anal canal and of the perianal skin. Anal cancers in women and homosexual men were more frequently HPV-positive for high-risk types (p < 0.01) and located in the anal canal (p ≤ 0.01) than were cancers in heterosexual men. In both women and men, cancers of the anal canal contained high-risk HPV DNA more often than perianal skin cancers and increased high-risk HPV DNA positivity was seen with higher localization in the anal canal: 95 and 83% of cancers that involved the anal canal in women and men, respectively, were HPV-positive for highrisk types versus 80 and 28% of perianal skin cancers (p for trend < 0.001). Basaloid features, adjacent and anal intraepithelial neoplasia (AIN), poor or absent keratinization and a predominance of small or medium neoplastic cells were all strongly associated with positivity for high-risk HPV types. The relationship between HPV infection and the subsequent risk for anal and perianal skin cancer was also studied in a case–cohort study among subjects who developed anal and perianal skin cancer during follow-up (median time, 10 years). Twenty-eight cases and 1500 controls were analysed for the presence of antibodies against HPV 16, 18, 33 or 73. An increased risk for developing anal and perianal skin cancer was observed among subjects who were seropositive for HPV 16 (odds ratio, 3.0; 95% CI, 1.1–8.2) and HPV 18 (odds ratio, 4.4; 95% CI, 1.1–17). The highest risks were seen for HPV 16-seropositive patients over the age of 45 years at serum sampling and for patients with a lag time of less than 10 years (Bjørge et al., 2002). Daling et al. (2004) measured antibodies to HPV 16 in cases of anal cancer and controls. HPV seropositivity was found in 51% of heterosexual male cases, 49% of not exclusively heterosexual male cases, 16% of heterosexual male controls, 42% of female cases and 15% of female controls. In summary, cancer of the anal canal resembles cervical cancer in its high prevalence of HPV 16 and, to a lesser extent, other high-risk HPV types. Among these, HPV 18 is the next most common. HPV 6 or 11, 31 and 33 are uncommon but are found in a small proportion of tumours. In contrast, cancer of the perianal skin resembles vulvar and penile cancers, with a lower prevalence of HPV DNA positivity overall. [This may in part reflect the distance of the tumour from the anal verge, representing a mixture of true anal cancers and of skin cancers that are generally negative for infection with genital HPV types (see Section 2.5).] 2.4

Cancer of the upper aerodigestive tract

2.4.1

Cancer of the oral cavity

Cancer of the oral cavity (including tumours of the tongue, floor of the mouth, gum, palate and other sites of the mouth) is strongly associated with tobacco smoking (IARC, 2004) or chewing (IARC, 2007) and alcoholic beverage drinking (IARC, 1988), with attributable fractions in the order of 90%. However, some tumours occur in subjects who

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223

are not exposed to known risk factors, and only a fraction of exposed subjects develop tumours, which suggests that other exposures may be independently involved or act as cofactors. HPV is known to infect the oral cavity of healthy individuals, and several HPVrelated lesions have been characterized. However, most of the epidemiology and natural history of oral HPV infection remains to be elucidated (Herrero, 2003). (a)

Case series

Numerous studies have investigated the prevalence of HPV in tumour specimens of subjects with cancer of the oral cavity. Reported estimates have ranged from 0 to 100% (reviewed by Franceschi et al., 1996; Gillison & Shah, 2001; Kreimer et al., 2005). Table 35 presents series that included more than 40 cases of cancer of the oral cavity and evaluated the presence of HPV using PCR methods. The prevalence of HPV in these studies ranged from 4 to 80%. For instance, in the large IARC multicentric study, the prevalence of HPV in these cancers was 3.9% (Herrero et al., 2003); in a recent systematic review that pooled HPV DNA results from oral squamous-cell cancer tissue specimens (Kreimer et al., 2005), the overall prevalence in 2642 cases was 23.5% (95% CI, 21.9–25.1%). The wide variation in prevalence estimates is probably related to differences in populations, HPV testing methods, prevalence of other risk factors and the combination of specific topographical locations included in the studies. In general, studies from western countries indicate lower prevalence than those conducted in India, China or Japan. The type most commonly reported in all studies was HPV 16, which was detected in more than 60% of HPV-positive tumours in two-thirds of the studies and in more than 80% of the tumours in half of the studies (Table 35). In the meta-analysis by Kreimer et al. (2005), 68.2% of the positive cancers were positive for HPV 16. The second most common type was HPV 18, with occasional reports of HPV 33, 6 and 11. Several studies have compared the prevalence of HPV DNA in cases of oral cancer and tissues from individuals without oral cancer. Ostwald et al. (1994) used PCR methods to detect HPV DNA in biopsies from 26 oral squamous-cell carcinomas and exfoliated cells of the buccal mucosa from 97 healthy volunteers. HPV was detected in 61.5% of cancers and 1% of volunteers. In a study in India, frozen biopsies of 83 patients with cancer of the oral cavity were compared with exfoliated cells from 102 volunteers from a dental clinic (Koppikar et al., 2005). HPV was detected by PCR in 38.6% of cancer patients and 5% of the control group. HPV 8, 16 and 18 were the most common types found in these cancers, but other types, including several from the genus beta-papillomavirus, were also detected. The latter were the only types detected in normal subjects. Zhang et al. (2004) reported a study of 73 cases of cancer of the oral cavity and 40 specimens from patients with benign tissue biopsies in China. PCR methods were used to detect HPV DNA for types 16 and 18, which were detectable in 74% of the cases and 55% of the non-cancer patients (p = 0.04).

30/11/2007

Sites included

Overall HPV positivity (%)

Type-specific HPV positivity (%)

MY09/11; GP5/6; type-specific for 6/11, 16, 18

Buccal mucosa, tongue, floor of mouth, lower alveolus, other

Shindoh et al. (1995), Japan

Type-specific for 16, 18, 33 and dot blot hybridization

Paz et al. (1997), USA

MY09/11; IU/IWDO; typespecific primers for 6, 16, 18, 31/33/35/ 44/45/56

6

11

16

18

91

73.6

13.2

19.8

41.7

Tongue, gingiva, floor of mouth, maxillary, mouth, palate, retromolar

77

31.2

31.2

Tongue, floor of mouth, oral cavity

64

12.5

Wen et al. (1997), China

Type-specific for 16, 18 and southern blot

45

31.1

D’Costa et al. (1998), India

MY09/11 and southern blot for 6, 11, 16, 18, 33

Buccal mucosa, lower alveolus, tongue, floor of mouth, hard palate, maxilla

100

15.0

0c

Schwartz et al. (1998), USA

MY09/11; typespecific primers for 6, 11, 16, 18

Tongue, gum, floor of mouth, other parts of mouth, NOS

186

20.4

6*

3.1

Multiple infections (%)

Comment

47.3

40.7

Fresh frozen and paraffin-embedded tissue

1.3

1.3

Paraffin-embedded tissue

31

33

1.6d

7.8

0c

20.0

24.4

15.0

0

∼11*

Others (type)

Fresh frozen tissue

13.3

0c

Paraffin-embedded tissue Fresh frozen tissue

Paraffin-embedded tissue; *read from graph

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Balaram et al. (1995), India

No. of cases

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224 Table 35. Prevalence of HPV DNA in case series of cancer of the oral cavity (> 40 cases) detected by the polymerase chain reaction (PCR) methoda

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Table 35 (contd) Method of detection and types tested

Sites included

No. of cases

Overall HPV positivity (%)

Gillison et al. (2000), USA

MY09/MY11; typespecific primers for 33 types

Oral cavity

84

11.9

Tsuhako et al. (2000), Japan

Type-specific for 6, 11, 16, 18

Tongue, mouth floor, buccal mucosa, lower gum, maxilla, lip

83

57.8

Mork et al. (2001), Finland, Norway and Sweden

GP5+/6+; CPI/CPIIG

Tongue, floor of mouth, oral cavity NOS

59

Premoli-dePercoco & Ramirez (2001), Venezuela

Type-specific for 6, 11, 16, 18

Tongue, buccal mucosa, floor of the mouth, others

50

60.0

van Houten et al. (2001), Netherlands

GP5+/6+

NOS

45

4.4

4.4

Snap-frozen tissue

Ringström et al. (2002), USA

MY09/11

NOS

41

4.9

4.9

Snap-frozen tissue

Shin et al. (2002), Republic of Korea

Type-specific for 16, 18, 33

Oral cavity, salivary glands

76

14.5

5.3

Type-specific HPV positivity (%) 6

11

∼11e

1.2

33.7

37.3

31

33

Snap-frozen tissue

1.2d

[7]

0

0

50.0

Others (type)

Paraffin-embedded tissue Paraffin-embedded tissue

16.0

10.5

0

0

2.6

Paraffin-embedded; women only

Tissue collection not specified

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1.2

18

Comment

HUMAN PAPILLOMAVIRUSES

15.7

16

Multiple infections (%)

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225

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Table 35 (contd) Sites included

No. of cases

MY09/11; GP5+/6+ with typing by sequencing and gene chip hybridization

Tongue, mouth, gingiva, lip, palate, floor of mouth

103

Herrero et al. (2003), Multicentric, 9 countries

GP5+/6+ and southern blot for 6, 11, 16, 18, 31, 33

Base of tongue, other parts of tongue, gum, floor of mouth, palate, mouth

Kansky et al. (2003), Slovenia

MY09/11; GP5+/6+; multiple types of primer sets and RFLP

Kojima et al. (2003), USA

Type-specific HPV positivity (%) 6

11

16

18

49.5

28.2

26.2

766

3.9

∼4

Tongue, floor of mouth, rectomolar trigonum, buccal mucosa

55

5.5

1.8

PCR and DNA sequencing; ISH; immunohistochemistry for 38

Tongue, buccal mucosa, maxillary and mandibular gingiva, hard palate, floor of mouth

53

66.0

Ostwald et al. (2003), Germany

Type-specific primers for 6/11, 16, 18

Oral cavity, lip

118

43.2

Sugiyama et al. (2003), Japan

Type-specific for 16, 18

Gingiva, tongue, oral floor, cheek, lip, palate

86

34.9

4.2

31

33

Others (type)

Multiple infections (%)

Comment

Paraffin-embedded tissue

1.0

Snap-frozen tissue

1.8

29.7

13.6

34.9

2.3

1.8 (58)

Paraffin-embedded tissue

66.0 (38)

Paraffin-embedded tissue

Snap-frozen tissue

2.3

Paraffin-embedded tissue

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Chang et al. (2003), Taiwan, China

Overall HPV positivity (%)

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Table 35 (contd) Sites included

Dahlgren et al. (2004), Sweden

GP5+/GP6+ or CPI/CIIPG and sequencing; typespecific PCR for 16, 18, 33

All tumours were from the oral tongue.

Smith, E.M. et al. (2004a), USA

MY09/11 and dot blot and sequencing

Tongue, floor of mouth, gingiva, hard palate, lip, major salivary glands, other oral mucosa

No. of cases

85 mobile 25 base 126

Overall HPV positivity (%)

Type-specific HPV positivity (%) 6

11

16

18

31

33

2.4

2.4

0

0

40.0

28.0

0

4.0

10

7.9

2.3

See Table 7 for a description of the primers used. EIA, enzyme immunoassay; ISH, in-situ hybridization; NOS, not otherwise specified; RFLP, restriction fragment length polymorphism a Only studies that used PCR as the method of detection of HPV were included. b ‘;’ denotes independent methods whereas ‘and’ denotes subsequent steps. c Subgroup of 70 samples examined d Untyped e 90% of the 253 tumour specimens analysed in the study were positive for HPV 16.

Others (type)

Multiple infections (%)

Comment

Paraffin-embedded tissue 4.0 (35) Paraffin-embedded tissue

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Method of detection and types tested

HUMAN PAPILLOMAVIRUSES

Reference, study location

227

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(b)

Case–control studies

Table 36 presents case–control studies of cancer of the oral cavity that used exfoliated cells or immunological markers to determine the presence of HPV. The study by Schwartz et al. (1998) included 284 cases of cancer of the oral cavity and oropharynx combined and 477 controls. Results for an HPV 16 capsid antibody response were available for 259 cases and 446 controls. Antibodies against HPV 16 L1 VLPs were equally prevalent in tumours of the floor of the mouth and in controls (odds ratio, 1.1; 95% CI, 0.5–2.5), but were more frequent in cancers of the tongue (odds ratio, 2.4; 95% CI, 1.5–3.8) after adjustment for age, sex, tobacco smoking and alcoholic beverage drinking. In a joint Nordic cohort, serum samples were collected from almost 900 000 individuals who were then followed through record linkage with tumour registries. Mork et al. (2001) evaluated the association of serum levels of antibodies against HPV 16 capsids with the occurrence of different cancers of the head and neck in a case–control study nested within this cohort. Elevated odds ratios were detected for cancers of the tongue (2.8; 95% CI, 1.2–6.6) and of the oral cavity not otherwise specified (3.6; 95% CI, 0.5–23.6). [The Working Group noted that this study adjusted for serum levels of cotinine and is important because of its prospective nature.] In a case–control study by Dahlstrom et al. (2003), antibodies against HPV 16 L1 VLPs were detected at similar levels in 36 cases of squamous-cell carcinoma of the oral cavity and 120 cancer-free controls selected from a managed care organization (8.3 versus 9.2%, respectively). Herrero et al. (2003) conducted a multicentre case–control study in Australia, Canada, Cuba, India, Italy, Northern Ireland, Poland, Spain and the Sudan from April 1996 to December 1999. The study included 1670 cases (1415 with cancer of the oral cavity and 255 with cancer of the oropharynx) and 1732 controls. Oral exfoliated cells and blood were obtained from all participants and fresh biopsy specimens from cases. HPV DNA was detected by PCR followed by enzyme immunoassay or southern blot hybridization. Antibodies against HPV 16 L1, E6 and E7 proteins in plasma were detected by enzymelinked immunosorbent assay (ELISA). Detection of HPV DNA in exfoliated cells was performed in only less than 50% of samples and did not correlate with detection of HPV DNA in biopsy specimens. HPV DNA was detected in biopsy specimens of 3.9% (95% CI, 2.5–5.3%) of 766 cancers of the oral cavity with valid PCR results. Antibodies against HPV 16 L1 were associated with increased odds ratios for cancers of the oral cavity (1.5; 95% CI 1.1–2.1), as were antibodies against either HPV 16 E6 or E7 (2.7; 95% CI, 1.6–4.7) after adjusment for country, age, sex, tobacco smoking, alcoholic beverage drinking and paan chewing. Van Doornum et al. (2003) reported the prevalence of antibodies against HPV 16 E7 to be 5.3% among 56 cases of carcinoma of the tongue and 2% among 100 non-cancer controls. Smith, E.M. et al. (2004b) reported a case–control study that included 130 cases of cancer of the oral cavity and 333 control subjects, frequency-matched for age and sex.

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Table 36. Case–control studies of HPV prevalence and cancer of the oral cavity Reference, study location

No. of controls

446

38 107 237

446 446 435 pop. controls Cohort of ∼950 000 residents

Mork et al. (2001), Finland, Norway and Sweden

Tongue, floor of mouth, oral cavity NOS Tongue

19

Herrero et al. (2003), Multicentric, 9 countries

Base of tongue, other parts of tongue, gum, floor of mouth, palate, mouth

511

613

1299

1527

1319

1581

201

333

130

333

Smith et al. (2004b), USA

Oral cavity and oropharynx combined (see Table 38) Oral cavity: lip vermillion and inner mucosa, tongue, gingiva, floor of mouth, hard palate, other oral mucosa, parotid gland, submandibular gland

57

Antibodies against HPV 16 L1 VLPs

PCR in exfoliated cells Antibodies against HPV 16 L1 VLPs

HPV prevalence (%)

Odds ratio (95% CI)

Comments/adjustments

Adjusted for age, sex, tobacco smoking and alcoholic beverage drinking *Read from graph

Cases

Controls

51.4

35.0

2.3 (1.6–3.3)

26* 20* 9.3

35.0 35.0 9.2

1.1 (0.5–2.5) 2.4 (1.5–3.8) 0.9 (0.5–1.6)

11

2

3.6 (0.5–26.3)

16

7

2.8 (1.2–6.6)

PCR of exfoliated cells Antibodies against HPV 16 L1 VLPs Antibodies against E6 and E7 proteins

4.7

6.9

0.6 (0.3–1.1)

8.9

6.0

1.5 (1.1–2.1)

4.2

1.5

2.7 (1.6–4.7) 4.3 (0.8–23.2) if both positive

PCR and sequencing of oral exfoliated cells

28.4

18.3

1.8 (1.1–2.7)

15

18.3

NR

Seropositivity for HPV 16

Adjusted for country, sex, age, tobacco smoking, alcoholic beverages, paan chewing

HUMAN PAPILLOMAVIRUSES

259

Method of detection

Adjusted for age, sex, tobacco smoking, alcoholic beverage consumption

229

CI, confidence interval; NR, not reported; PCR, polymerase chain reaction; VLP, virus-like particles a Detection of HPV DNA in exfoliated cells was considered to be an inadequate indication of HPV infection in this study as it did not correlate with detection of HPV DNA in tumours.

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Oral cavity and oropharynx combined; see Table 35 for organs included and Table 38 Floor of mouth Tongue

No. of cases

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Sites included

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Oral exfoliated cells and tumour tissue were analysed for HPV content by PCR and dot blot hybridization and for HPV type by DNA sequencing. HPV DNA was detected in oral cells from 15% of cases of cancer of the oral cavity and 18.3% of controls. Risk estimates restricted to cancers of the oral cavity were not presented. Studies of non-genital sites present a special challenge for the assessment of exposure to HPV because oral exfoliated cells do not appear to demonstrate infection adequately. As an alternative, serological markers of HPV exposure or expression (e.g. antibodies against HPV L1 VLPs or against E6 or E7 proteins) have been used in some recent studies (Schwartz et al., 1998; Herrero et al., 2003; see above). Moreover, it is not always feasible to define clearly the precise anatomical location of the primary tumour, and multiple locations are frequent. Misclassification of the primary tumour site can introduce distortions in prevalence estimates for individual sites. For example, the base of the tongue is not classified consistently in the different studies when sites are grouped as oral cavity or oropharynx. A recent, carefully conducted study (Dahlgren et al., 2004) indicated that the base of the tongue has an HPV prevalence of 40% compared with 2.3% in the oral tongue. 2.4.2

Cancer of the oropharynx and tonsil

Similar to cancers of the oral cavity, cancers of the oropharynx and tonsil are strongly associated with tobacco smoking and alcoholic beverage drinking, but other etiological agents may play an independent role or act as co-factors. (a)

Case series

A series of studies have reported prevalence of HPV in cancers of the oropharynx and tonsil. Those that used PCR as the method of detection and included more than 40 cases are summarized in Table 37. The prevalence of HPV in the selected studies ranged from 14 to 57%, and the largest study to date (Herrero et al., 2003) reported a prevalence of 18%. In a systematic review that pooled HPV DNA results from 969 cases of squamouscell cancer of the oropharynx (Kreimer et al., 2005), the overall prevalence of HPV was 35.6% (95% CI, 32.6–38.7%). Among HPV-positive tumours, there was a marked and very consistent predominance of HPV 16. In all studies included in Table 37, HPV 16 was present in at least 78% of HPV-positive tumours and, in the review by Kreimer et al. (2005), the corresponding figure was 87%. HPV 18 is almost never present and other types (HPV 6, 11, 31 and 33) are only detected sporadically. In the studies that presented separate estimates for cancer of the tonsil, this anatomical site consistently showed the highest prevalence of HPV. Using in-situ hybridization with probes for HPV 6, 11 and 16 under high stringency, Niedobitek et al. (1990) tested 28 tonsillar carcinomas and 30 tonsils removed because of chronic inflammation. Six of the cases and none of the controls were HPV 16-positive [p < 0.001].

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Reference, study location

Methoda of detection and types tested

No. and type of lesions

Overall HPV positivity (%) 36.4 44b

Gillison et al. (2000), USA

MY09/MY11/HMB01; mixture for 16/18/51/66 and probing with 33 type-specific probes

60 oropharynx 52 tonsil 8 others

56.7 62 25

Mellin et al. (2000), Sweden

GP5+/6+; type-specific for 16, 33

60 tonsil carcinomas

43.3

Lindel et al. (2001), Switzerland

SPF 1/2 for high-risk types and sequencing

99 oropharynx 40 tonsil

Strome et al. (2002), USA

MY09/11, others; typespecific for 16

Herrero et al. (2003), Multicentric, 9 countries

0 10

18

31

33

Others (type)

36.4 34.1 1

51

Paraffin-embedded tissues; *read from graph 1

1

3

Fresh-frozen tissues; type distribution for combination of cases of the oral cavity and oropharynx (n = 253)

43.3

1.7

Paraffin-embedded tissues

14.1 15

11.1

1.0

52 tonsil carcinoma

46.2

GP5+/6+ and EIA for 6, 11, 16, 18, 31, 33

142 tonsil

Li, W. et al. (2003), Australia China

GP5+/6+ and other consensus primers; typespecific for 16

Smith et al. (2004a), USA

MY09/11/HMB01; dot blot and sequencing

1.0 (35) 1.0 (45)

Paraffin-embedded tissues

40.4

3.8 (12)

Paraffin-embedded tissues

18.3 26

∼17

[< 1]

Snap-frozen tissues

67 tonsil carcinoma

46.3

44.7

16 tonsil carcinoma

0

31 tonsil 5 oropharynx

58.1 0

Paraffin-embedded tissues

Paraffin-embedded tissues 51.6

3.2

3.2

Paraffin-embedded tissues

231

See Table 7 for a description of the primers used. EIA, enzyme immunoassay; PCR, polymerase chain reaction; a Only those studies where HPV was detected by PCR were included; ‘;’ denotes independent methods whereas ‘and’ denotes subsequent steps.

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11 oropharynx 44 tonsil

16

09:40

MY09/11; type- specific for 6, 11, 16, 18

11

HUMAN PAPILLOMAVIRUSES

Schwartz et al. (1998), USA

Comment

Type-specific HPV positivity (%) 6

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Table 37. Prevalence of HPV DNA in case series of oropharyngeal and tonsillar cancer (> 40 cases)

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Snijders et al. (1992a,b) used PCR and southern hybridization techniques to test 10 cases of carcinoma of the tonsil and seven control patients with tonsillitis. All cases tested positive for HPV versus none of the controls [odds ratio, ∞; 95% CI, 7.5–∞]. The presence of HPV in cancer cells was confirmed by RNA in-situ hybridization. In Japan, Watanabe et al. (1993) tested 12 cases of carcinoma of the oropharynx, hypopharynx and tonsil and 28 control specimens from patients with chronic tonsillitis. Three methods for HPV testing were used — dot-filter, southern hybridization and PCR. The results from PCR showed a prevalence of HPV DNA of 25% among cases and 4% among controls. (b)

Case–control studies

Table 38 presents case–control studies of cancer of the oropharynx that used exfoliated cells or immunological markers to determine the presence of HPV. In the study by Schwartz et al. (1998), the odds ratio for cancer of the tonsil associated with the detection of antibodies against HPV 16 L1 was 3.9 (95% CI, 2.0–7.8) after adjustment for age, sex, tobacco smoking and alcoholic beverage drinking. In the nested case–control study reported by Mork et al. (2001), the risk for developing cancer of the oropharynx associated with pre-diagnostic detection of antibodies against HPV 16 L1 VLPs was 14.4 (95% CI, 3.6–58.1). In the serological case–control study of Dahlstrom et al. (2003), HPV 16 L1 VLP seropositivity was associated with a 60-fold (95% CI, 5.7–620) increase in risk for oropharyngeal squamous-cell carcinoma (70 cases) compared with 120 cancer-free, clinicbased controls, following adjustment for reported alcoholic beverage use and serum cotinine level, which is a biomarker of recent exposure to tobacco. In the IARC multicentre study (Herrero et al., 2003), there was no difference in the prevalence of HPV DNA in exfoliated cells from cancers of the oropharynx and those from controls. However, the odds ratio for antibodies against HPV 16 L1 VLPs was 3.5 (95% CI, 2.1–5.9), and that for antibodies against HPV 16 E6 or E7 proteins was 4.5 (95% CI, 2.0–10.1). When both antibodies were detected, the odds ratio was 67.1 (95% CI, 12.9–348). These estimates were adjusted for country, tobacco smoking, alcoholic beverage drinking and paan chewing. In another case–control study of 48 cases of oropharyngeal carcinoma and 100 cancer-free controls, levels of HPV 16 L1 antibodies were significantly elevated in cases compared with controls (33 versus 18%; p = 0.04); however, seroprevalence of HPV E7 was similar (4% versus 2% for cases and controls, respectively) (Van Doornum et al., 2003). Smith, E.M. et al. (2004a) reported an adjusted odds ratio for oropharyngeal cancer of 3.6 (95% CI, 1.8–7.1) associated with detection of HPV in exfoliated cells. The oropharynx, and in particular the tonsils, are the extragenital sites where the role of HPV is most clear for a defined subset of tumours. Similarly to vulvar cancer, a dual etiology has been postulated for oropharyngeal tumours (Herrero et al., 2003). One subset includes smoking-related cancers, with squamous histological features and p53 mutations.

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Table 38. Case–control studies of HPV prevalence and cancers of the oropharynx and tonsil No. of cases

No. of controls

Method of detection

35.0

2.3 (1.6–3.3)

44.0

35.0

3.9 (2.0–7.8)

Antibodies against HPV 16 L1 VLPs

38

10

14.4 (3.6–58.1)

Seropositivity for HPV 16

58.6

9.2

59.5 (5.7–620)

Adjusted for cotinine, alcoholic beverages, matching variables; prevalence in tonsil cancers, 59.4%

8.9

6.9

1.0 (0.4–2.5)a

13.4

6.0

3.5 (2.1–5.9)

5.3

1.5

4.5 (2.0–10.1) 67.1 (12.9–348) if both positive

Adjusted for country, sex, age, tobacco smoking, alcoholic beverages, paan chewing

L1, 18 E7, 2

L1, 2.25 (1.0–4.9)

Antibodies against HPV 16 L1 VLPs

49

446

Mork et al. (2001), Finland, Norway and Sweden

Oropharynx (ICD 145)

26

Dahlstrom et al. (2003), USA

Base of tongue, tonsil, other oropharynx

70

120

Antibodies against HPV 16 L1 VLPs

Herrero et al. (2003), Multicentric, 9 countries

Oropharynx and tonsil

90

613

238

1527

243

1581

PCR in oral exfoliated cells Antibodies against HPV 16 L1 VLPs Antibodies against E6 and E7 proteins

48

100

Antibodies against HPV 16 L1 VLPs and E7 proteins

L1, 33 E7, 4

HUMAN PAPILLOMAVIRUSES

Adjusted for age, sex, tobacco smoking, alcoholic beverage drinking

51.4

446

Oropharynx

Comments/adjustments

Controls

259

Van Doornum et al. (2003), Netherlands

Odds ratio (95% CI)

Cases Oral cavity and oropharynx combined Tonsil

Cohort of ∼900 000 residents

HPV prevalence (%)

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Sites included

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Oral cavity and oropharynx combined (see Table 36) Oropharynx: base of tongue, soft palate, uvula, palatine tonsil fossa, pillar and overlapping regions, oropharynx, oropharynx NOS

No. of cases

No. of controls

201

333

71

333

Method of detection

PCR of oral exfoliated cells and sequencing

HPV prevalence (%)

Odds ratio (95% CI)

Cases

Controls

28.4

18.3

1.8 (1.1–2.7)

38.0

18.3

NR

Comments/adjustments

CI, confidence interval; NOS, not otherwise specified; NR, not reported; PCR, polymerase chain reaction; VLP, virus-like particles a Detection of HPV DNA in exfoliated cells was considered to be an inadequate indication of HPV infection as it did not correlate with the detection of HPV DNA in tumours.

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The other includes HPV-related tumours (reviewed by Gillison & Shah, 2001), which have been shown to have basaloid histological features, to occur more frequently in nonsmokers, to be less frequently associated with p53 mutations (Braakhuis et al., 2004; Dai et al., 2004), to be associated with distinct patterns of genetic alterations (Braakhuis et al., 2004) and possibly to have a better prognosis (Pintos et al., 1999). 2.4.3

Cancer of the oesophagus

The possibility that HPV infection may play a role in the etiology of squamous-cell carcinoma of the oesophagus has been proposed, but the data have not been consistent (reviewed by Gillison & Shah, 2003). The epithelium of the oesophagus is similar to that of the oral cavity, and papillomas have been described at this anatomical site although they are rare, and HPV is not consistently detected therein. (a)

Case series

Table 39 presents series of cancer of the oesophagus that studied more than 40 cases; the 19 studies show great heterogeneity with regard to the prevalence of detection of HPV DNA, which ranged from 0 to 55%. Five studies from France (Benamouzig et al., 1995), Italy (Talamini et al., 2000), Japan (Saegusa et al., 1997), the Netherlands (Kok et al., 1997) and Slovenia (Poljak et al., 1998), which included more than 45 cases and used PCR methods with common consensus primers, showed 0% prevalence of HPV in oesophageal cancers. In contrast, many studies from China, an area with a high incidence of cancer of the oesophagus, reported a high overall prevalence of HPV that was generally around 50%. The largest study, also from China and which included 700 cases, showed an overall HPV prevalence of 17% using in-situ hybridization (Chang et al., 2000). In oesophageal cancers, the predominance of HPV 16 is less marked than that at other sites of the head and neck, and HPV 18, 6 and 11 are detected commonly. A recent study by de Villiers et al. (2004b) among patients with other head and neck cancers detected HPV DNA in 67% of the premalignant or malignant oesophageal tissue biopsies, which pointed to a particular subgroup of cancers of the oesophagus. In the study by Benamouzig et al. (1992), the presence of oesophageal HPV infection was studied in endoscopic biopsies of 12 patients with oesophageal squamous-cell carcinoma, 24 control patients exposed to similar known risk factors (alcoholic beverages and tobacco) and seven non-exposed controls. Five of 12 patients with oesophageal carcinoma had HPV infection in the normal oesophagus tissue using dot blot hybridization (three of these also had HPV-positive tumour tissue). Only one of the controls had an oesophageal HPV infection (p < 0.01). HPV 16 and 18 were the types most frequently detected. (b)

Case–control studies

Case–control studies that assessed HPV prevalence by serology have reported contradictory results. A carefully conducted population-based case–control study in Sweden

Methoda of detection and types tested

No. of cases

Type-specific HPV positivity (%) 6

11

16

18

5.9

13.7

17.6

13.7

51

49.0

Toh et al. (1992), Japan

Consensus PCR for 16/18/31/33/52/58, 6/11/16/18/31/33 and type-specific primers for 16, 18

45

6.7

2.2

4.4

Togawa et al. (1994), International

Consensus primers and RFLP for 6, 11, 16, 18

72

23.6

12.5

1.4

Benamouzig et al. (1995), France

MY09/11 and typespecific for 6/11, 16/18, 31/33; dot blot for 6/11, 16/18

75

0

Suzuk et al. (1996), China

MY09/11 Type-specific for 6, 16, 18

70 70

0 4.3

Lam et al. (1997), Hong Kong, SAR

Type-specific for 16, 18 and southern blot with consensus probes

70

Turner et al. (1997), Canada, USA

MY09/11 type-specific primers for 16, 18/33

Kok et al. (1997), Netherlands

MY09/11, GP5+/6+, CPI/CPIIG, CPI/CPIIS

Others (type)

2.0

Paraffin-embedded tissue Fresh tissue

9.7 (unknown)

Paraffin-embedded or frozen tissue Frozen tissue

2.9

Paraffin-embedded tissue

8.6

8.6

Snap-frozen tissue

51

2.0

2.0

Paraffin-embedded tissue

63

0

1.4

Paraffin-embedded tissue

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33

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31

Multiple infections (%)

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Table 39. Prevalence of HPV DNA in case series of oesophageal carcinomas (> 40 cases)

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Table 39 (contd) Reference, study location

Methoda of detection and types tested

No. of cases

Type-specific HPV positivity (%) 6

11

16

18

31

33

Others (type)

Multiple infections (%)

Comments

09:40

Overall HPV positivity (%)

Mizobuchi et al. (1997), Japan

Type-specific for 16, 18 and southern blot

41

7.3

Saegusa et al. (1997), Japan

L1C1/L1C2 for 6/11/16/18/31/33/42/ 52/58; pU-1M/pU-2R for 16/18/31/33/ 52/58

92

0

Paraffin-embedded tissue

Poljak et al. (1998), Slovenia

MY09/11, GP5+/6+, WD; nested PCR; typespecific PCR for 6, 16, 18

120

0

Paraffin-embedded tissue

de Villiers et al. (1999a), China

Degenerate primer set HD; CP; GP5+/6+

70

4.3

Lavergne & de Villiers (1999), China and South Africa

Degenerate PCR and sequencing

63

30.2

Chang et al. (2000), China

ISH with broadspectrum probe and type-specific probes for 6/11, 16, 18, 30, 53

700

16.6

Talamini et al. (2000), Italy

MY09/11, GP5+/6+

42

0

7.3

1.1

Fresh frozen tissue

0

1.6

25.3 (untyped)

3.0

1.7

12.0 (untyped)

6.3

Snap-frozen tissue

Paraffin-embedded tissue

Paraffin-embedded tissue

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9.5

1.4

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Snap-frozen tissue

237

Methoda of detection and types tested

No. of cases

Overall HPV positivity (%)

Type-specific HPV positivity (%) 6

11

16

18

31

33

Others (type)

Multiple infections (%)

Comments

22.7

Snap-frozen tissue

Li, L.T. et al. (2001, 2002), China

Type-specific for 16, 18

62

62.9

Paraffin-embedded tissue

Matsha et al. (2002), South Africa

MY09/11, GP5+/6+

50

46.0

Shen et al. (2002), China

Consensus primers PCR for 6/11, 16/18

55 44

65.5

PCR for 16, 18

77

54.5

39.0

22.1 1.9

4.0

34.1

14.0 (39), 2.0 (52), 4.0 (untyped)

Paraffin-embedded tissue

43.2

Si et al. (2003), Hong-Kong, SAR

MY09/11 and typespecific for 16, 18

319

13.5

12.2

Lu et al. (2004), China

Type-specific for 16

104

52.9

52.9

Katiyar et al. (2005), India

MY09/11 Type-specific for 16, 18

101 60

26.7 66.7

16.8

Fresh tissue 10.4 Paraffin-embedded and snap-frozen tissues Paraffin-embedded tissue 2.0

See Table 7 for a description of the primers used. ISH, in-situ hybridization ; PCR, polymerase chain reaction a Unless otherwise specified, the method is PCR; ‘;’ denotes independent methods whereas ‘and’ denotes subsequent steps.

Snap-frozen tissue

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22.0

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Table 39 (contd)

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(Lagergren et al., 1999) showed no association of antibodies against HPV 16 or HPV 18 L1 VLPs with either squamous-cell or adenocarcinoma of the oesophagus after adjustment for age, sex, smoking status, alcoholic beverage intake, education and the presence of the other HPV type. Another serology-based study from the Netherlands (Van Doornum et al., 2003) reported 17% HPV 16 seropositivity among cases of oesophageal carcinoma compared with 18% among cancer-free controls. No cases were seropositive for HPV 16 E7 compared with 2% of controls. In contrast, a hospital-based study in China assessed HPV16 VLPs in cases of oesophageal cancer (95% of which were squamous-cell carcinomas) and cancer-free controls and reported that higher antibody levels significantly increased the relative risk for oesophageal cancer (odds ratio, 4.5; 95% CI, 1.8–11.9), after adjustment for age and sex (Shen et al., 2002a). [The Working Group noted that the study lacked the ability to adjust further for potentially important confounders, such as tobacco use, alcoholic beverage consumption, dietary patterns or sexual behaviour.] (c)

Cohort studies

Prospective seroepidemiological studies in Scandinavian cohorts point to an association between HPV and oesophageal cancer. An initial study in Finland (Dillner et al., 1995b) indicated a 14-fold increase in risk for oesophageal cancer associated with prediagnostic detection of HPV 16 capsid antibodies, and another study in Norway indicated a 6.2-fold increase in risk after adjustment for cotinine levels as markers of tobacco exposure (Bjørge et al., 1997b). 2.4.4

Cancer of the larynx

The laryngeal epithelium is known to be susceptible to HPV infection because of the well-established association of HPV types 6 and 11 with juvenile- and adult-onset laryngeal papillomatosis. A few retrospective case series have reported laryngeal squamouscell carcinoma among patients with a history of laryngeal papillomatosis; HPV 11 DNA was most commonly detected in these cancer specimens (Shen et al., 1996; Reidy et al., 2004). However, laryngeal papillomatosis is not a precursor for most laryngeal cancers. Similarly to cancers of the oral cavity and pharynx, the main risk factors for laryngeal squamous-cell carcinoma are tobacco use and alcoholic beverage consumption and the attributable fraction for these exposures is large. (a)

Case series

Table 40 presents studies of at least 40 cases of squamous-cell carcinomas of the larynx that employed PCR-based detection methods. The overall HPV prevalence ranged from 7% in a study in the USA to 59% in a large study in China (Ma et al., 1998). HPV 16 was the predominant type detected, and accounted for approximately all [74%] HPVpositive laryngeal squamous-cell carcinomas. HPV 18 was the second most commonly detected type; HPV 6, 11 and 33 were detected in a few cases, and no other carcinogenic HPV types were reported. [The Working Group noted that the number of specimens exa-

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Type-specific for 11 and 16 and dot blot

48

54.2

Shidara et al. (1994), Japan

L1C1/L1C2 and RFLP

45

24.4

Suzuki et al. (1994), Japan

L1C1/L1C2 and RFLP

41

26.8

Fouret et al. (1995), France

Primers WD 72, 76, 66, 67, 154 and southern blot for 6, 11, 16, 18, 31, 33

59

5.1

Almadori et al. (1996), Italy

PCR for 6, 11, 16, 18 and southern blot

45

20.0

Paz et al. (1997), USA

MY09/11, IU/IWDO, typespecific for 6, 16, 18, probing for 31, 33, 35, 44, 45, 56

49

8.2

Overall HPV positivity (%)

Type-specific HPV positivity (%) 6

0

11

16

0

54.2

0

18

31

33

Others (type)

Multiple infections (%)

Comment

Snap-frozen tissues

20.0

4.4

22.0

4.9

Paraffin-embedded tissues 0

0

Paraffin-embedded tissues Paraffin-embedded tissues

4.4

0

20.0

0

6.1

0

Snap-frozen tissues

2.0 (unknown)

Snap-frozen tissues

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Pérez-Ayala et al. (1990), Spain

No. of cases

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Table 40. Prevalence of HPV DNA in case series of laryngeal cancer (> 40 cases)

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Table 40 (contd)

MY09/11 and EIA for 6, 11, 16, 18, 31

75

Hoffmann et al. (1998), Germany

Type-specific primers for 6, 11, 16, 18, 33 and southern blot; consensus primers and southern blot for type-specific negative samples

Ma et al. (1998), China

Overall HPV positivity (%)

Type-specific HPV positivity (%) 6

11

16

18

29.3

12.0

10.7

29

20.7

6.8

pU-1M/pU-2R for 6/11 and pU-31B/pU-2R for 16/18/31/33/52/58

102

58.8

Mineta et al. (1998), Japan

Type-specific for 16, 18

42

31.0

Gorgoulis et al. (1999), Greece

MY09/11 and GP5/6; typespecific for 6, 11, 16, 18, 31, 33, 35

91

20.9

Pintos et al. (1999), Canada

GP5+/6+ and southern blot

52

15.4

Gillison et al. (2000), USA

MY09/11/HMBO1, TS for 16/18/51/66, southern blot with 33 type-specific probes and sequencing; type-specific for 16 and 18

86

18.6

25.5

3.3

2.0

0

31

33

Others (type)

1.3

5.3 (untyped)

Multiple infections (%)

1.3

Comment

Snap-frozen tissues Snap-frozen tissues

29.4

21.6

26.2

4.8

14.3

3.3

0

1.0

19.6

Paraffin-embedded tissues Snap-frozen tissues

0

3.3

3.3

Snap-frozen tissues

Paraffin-embedded tissues ∼17

1.2 (untyped)

Fresh frozen tissues; type-specific data in combination with other sites of head and neck cancers

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Cattani et al. (1998), Italy

No. of cases

09:40

Methoda of detection and types tested

HUMAN PAPILLOMAVIRUSES

Reference, study location

241

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Methoda of detection and types tested

Jacob et al. (2002), India

Type-specific for 16 and 18

44

34.1

34.1

Báez et al. (2004), Puerto Rico

Type-specific for 6

52

46.2

46.2

No. of cases

Overall HPV positivity (%)

Type-specific HPV positivity (%) 6

11

16

18

31

33

Others (type)

0

See Table 7 for a description of the primers used. EIA, enzyme immunoassay; PCR, polymerase chain reaction a Unless otherwise specified, the method is PCR; ‘;’ denotes independent methods whereas ‘and’ denotes subsequent steps.

Multiple infections (%)

Comment

Paraffin-embedded tissues Snap-frozen tissues

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mined in each study was small (< 105 cases), which may in part explain the large variability in the prevalence estimates.] A relatively low prevalence of overall HPV DNA, coupled with the high prevalence of HPV16 and the lack of other HPV types among HPVpositive laryngeal carcinomas, have been consistently reported in the more recent studies of HPV and laryngeal squamous-cell carcinoma. Similar results were reported in a large systematic review of published studies that met the same inclusion criteria of adequate sample size and PCR-based HPV detection methods (Kreimer et al., 2005). Of 1435 laryngeal and hypopharyngeal squamous-cell carcinomas, the pooled HPV DNA prevalence was 24% (95% CI, 22–26%). In the type-specific analysis among HPV-positive laryngeal squamous-cell carcinomas, HPV16 accounted for 66% of infections, and HPV18 and HPV6 accounted for 16 and 14% of infections, respectively. (b)

Case–control studies

Table 41 reports the results of case–control studies. One early study (Brandsma & Abramson, 1989) reported similarly low prevalence of HPV in both cases (5%) and controls with benign disease or structural abnormalities (4%), which suggested no association between HPV and laryngeal cancer. Smith et al. (2000) investigated HPV DNA in 44 patients with laryngeal or hypopharyngeal squamous-cell carcinoma and 12 controls with benign laryngeal conditions. HPV DNA was collected from biopsy material from the cases and from upper respiratory tract brushings from the controls. HPV DNA was detected in 25.0% (11/44) of cases and in 16.7% (2/12) of controls. The prevalence of carcinogenic HPV types (HPV 16, 18, 31, 45 and 70 were detected) was similar in cases (18.2%) and controls (16.7%). However, after adjustment for tobacco, alcoholic beverage consumption and age, the presence of carcinogenic HPV DNA elevated the relative risk for laryngeal squamous-cell carcinoma threefold (odds ratio, 3.0) (although not significantly) compared with controls. One prospective serological study that included laryngeal squamous-cell carcinoma (Mork et al., 2001) investigated the seroprevalence of HPV16 L1 VLP in a nested case–control study of 76 cases of laryngeal cancer and 411 controls using several Nordic cancer registries linked with serum banks. HPV seropositivity increased the risk for developing laryngeal cancers 2.4-fold (95% CI, 1.0–5.6), following adjustment for serum cotinine levels, a biological marker for tobacco exposure. [The serological samples were collected on average 9.4 years before the diagnosis of cancer, and thereby provide evidence of exposure to HPV that preceded the development of disease. However, this study lacked additional information on potential confounding factors, such as alcoholic beverage use and sexual behaviour.] One serological case–control study detected HPV16 L1 VLPs in 35.7% of 14 laryngeal squamous-cell carcinomas (Dahlstrom et al., 2003), which was a higher prevalence than that detected in controls (9.2% of 120). In contrast, another serological study of 127 cases of laryngeal cancer and 100 cancerfree controls reported prevalences of HPV 16 of 20 versus 18%, respectively (Van Doornum et al., 2003).

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Reference, study location

No. of cases

No. of controls

Method of detection and types tested

HPV prevalence (%) Cases

Southern blot

García-Milían et al. (1998), Cuba

33 SCC

25

Nishioka et al. (1999), Japan

27

Smith et al. (2000), USA

NR

HPV-positive tumours harboured DNA sequences related to HPV 11.

p < 0.05

Matched for age

5

4

PCR with MY09/11

48

16

35

PCR for 16/18 and dot blot for 16 and 18

19

6

3.75 (0.72–19.67)

Matched for age and sex; adjusted for tobacco smoking

44 SCC

12 patients with benign laryngeal conditions

PCR with MY09/11 or MY09/GP5+ and sequencing

18a

17a

3.0 (CI not given)

DNA from oral source, laryngeal source and biopsies; adjusted for age and alcoholic beverage and tobacco consumption

Mork et al. (2001), Norway, Finland and Sweden

76 SCC

411

Antibodies against HPV 16 L1 VLP

12

5

2.4 (1.0–5.6)

Adjusted for cotinine levels

Dahlstrom et al. (2003), USA

14 SCC

120

Antibodies against HPV 16 VLP

35.7

9.2

NR

Van Doornum et al. (2003), Netherlands

127

100

Antibodies against HPV 16 L1 VLP and E7 protein

L1, 20 E7, 2

L1, 18 E7, 2

p = 0.876 NR

See Table 7 for a description of the primers used. CI, confidence interval; NR, not reported; PCR, polymerase chain reaction; SCC, squamous-cell carcinoma; VLP, virus-like particles a The high-risk types found in the study and accounted for were 16, 18, 31, 45 and 70.

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Controls

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60 SCC

Comments/adjustments

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Odds ratio (95% CI)

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Table 41. Case–control studies of prevalence of HPV and laryngeal cancer

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Altogether, the evidence suggests that HPV may be involved in the development of some laryngeal cancers, but the associations documented to date are not as clear nor as strong as those observed at other upper aerodigestive sites, such as the tonsils and the oropharynx (Herrero, 2003). If HPV DNA causes a subset of laryngeal cancers, it is probably a smaller subset than that documented for other sites of the head and neck. However, HPV 16 predominates over other HPV types among HPV-associated laryngeal cancers. There is some evidence against an association between HPV and laryngeal squamous-cell carcinoma: (a) cell lines derived from laryngeal tumours contain low viral load, which probably reflects the absence of a clonal relationship (Atula et al., 1999); (b) the presence of HPV DNA is not limited to the tumour specimen and is also found in normal surrounding tissue (Venuti et al., 2000); and (c) HPV DNA is not consistently detected in premalignant lesions of laryngeal squamous-cell carcinoma (Fouret et al., 1995; Poljak et al., 1997; Smith et al., 2000), which suggests a lack of continuity of HPV infection throughout the malignant process. In the oral region, a clear lack of concordance of HPV prevalence between biopsy tissue and oral specimens has been demonstrated (Herrero, 2003). To take samples from the the larynx of healthy controls is practically impossible; therefore, new methods to assess exposure to HPV must be considered. Immunological markers (e.g. antibodies against HPV E6 and E7) may also help distinguish cancers in which HPV has played an etiological role. 2.5

Cancer of the skin and conjunctiva

2.5.1

Cancer of the skin

In contrast to the papillomavirus types found in the cancers of the mucosae which are classified in the genus alpha of the papillomaviruses, the papillomavirus types found in skin cancers mainly belong to the genus beta of the family also termed as epidermodysplasia verruciformis (EV)-HPV (see Section 1.1.3). This can be explained by a different tropism of the two genera, because the skin is histologically distinct from the mucosae. The pathology of skin lesions is described in Section 1.5.4. (a)

Case series

Early studies of skin cancer that used southern blot or PCR for the detection of mucosal HPV types suggested a low prevalence of HPV in skin cancers except for those that occur at periungual and palmoplantar sites. In these rare tumours (occasionally found in patients who also have HPV 16/18-positive cervical lesions), the high prevalence of HPV 16/18 DNA suggests possible transmission of HPV infection from genital sites (IARC, 1995). Since the development of highly sensitive, partially nested PCRs designed to detect epidermodysplasia verruciformis (EV)-related and cutaneous HPV types (Berkhout et al., 1995; Shamanin et al., 1996; see Section 1.3.3), a much higher prevalence of HPV DNA

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has been found in non-melanoma skin cancers (Tables 42 and 43). However, HPV DNA is also frequently detected in specimens of normal skin and in plucked hairs (see Section 2.5.1(b)). A diverse spectrum of HPV types, including HPV 20, 38, 41 and 48, have been detected, and many new partial HPV DNA sequences (350–430 nucleotides from the L1 gene) have been identified. Most of them have been assigned to genera beta (including EV-associated HPV) and gamma (see Section 1.1.3) (Berkhout et al., 1995; Shamanin et al., 1996; Bens et al., 1998; Forslund et al., 2003a). The need for highly sensitive detection techniques can be explained by the very small amounts of HPV DNA present in skin tumours. When HPV DNA was determined by quantitative, type-specific real-time PCR in precancerous actinic keratoses and non-melanoma skin cancers that were positive in nested PCR, viral loads ranged from 1 HPV DNA copy per 14 200 cell equivalents to 50 HPV DNA copies per 1 cell equivalent (Weissenborn et al., 2005). The HPV DNA load was significantly higher in actinic keratoses than in squamous-cell carcinomas and Bowen disease. In most cases, probably not every tumour cell harbours an HPV genome, which is supported by in-situ hybridization that shows only a few HPV DNA-positive cell nuclei per section. The genus- and species-specific PCRs used differ in sensitivity towards individual types, and thus affect the spectrum of HPV types identified in cases in which DNA levels are close to the limit of detection (Pfister, 2003). When HPV 5 and 8-specific nested PCR was added to an EV-specific nested PCR with degenerate primers, for example, these types were shown to be more prevalent than had been anticipated previously (Meyer et al., 2000). Using HPV 38-specific, hot-start PCR to amplify part of the E6 open-reading frame (ORF), HPV 38-related DNA was detected in 55% of basal-cell carcinomas, 46% of squamous-cell carcinomas and 10% of healthy skin samples (Caldeira et al., 2003). (i) Squamous-cell carcinoma and keratoacanthoma Nearly all studies published after 1996 revealed HPV prevalences in squamous-cell carcinoma of 27–60% (Table 42). The prevalence of EV-associated beta-HPV was generally in the range of 30–50%; probably as a result of the primers employed for the PCR, it was only 12% or lower in two studies (Shamanin et al., 1996; Iftner et al., 2003). HPV 16 and related high-risk mucosal HPV were occasionally detected in some case series; however, the high prevalence (28%) of HPV 16 in a French study (Cairey-Remonnay et al., 2002) could not be confirmed by others even when highly sensitive mucosal HPV-specific PCR was used. HPV 4 was found in one of 26 and nine of 72 squamous-cell carcinomas in two case series (Shamanin et al., 1996; Iftner et al., 2003) and HPV 41 in two of 10 squamous-cell carcinomas in another (Grimmel et al., 1988). The original findings of a high prevalence of HPV 16/18 in periungual squamous-cell carcinomas (Moy et al., 1989; Eliezri et al., 1990) were confirmed by numerous case reports and more recent surveys of digital squamous-cell carcinomas (Forslund et al., 2000; Alam et al., 2003; Table 44). HPV 16 and occasionally HPV 31, 35 and 73 were detected in up to 90% of these tumours (Alam et al., 2003). Of 72 cases of digital squamous-cell carcinomas, 10% had an antecedent genital dysplasia or carcinoma that contained the same HPV type as

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Table 42. Prevalence of HPV DNA in case series of squamous-cell carcinoma (SCC) and keratoacanthoma (KA) of the skin Reference, study location

Method of detection and types tested

Overall HPV positivity (%)

Type-specific HPV positivity (%)

Comment

16/18, related typesa

Pfister et al. (1986), Germany

Southern blot and restriction digest

6 KA

33.3

Scheurlen et al. (1986), Germany

Dot blot hybridization and restriction digest

7 KA

14.3

Grimmel et al. (1988), Germany

Southern blot for 41

6 KA 10 SCC

0 20.0

Eliezri et al. (1990), USA

ISH for 6/11, 16/18, 31/33/35, 42/43/44, 45/56, 51/52

16 SCC

0

Kawashima et al. (1990), Poland

Dot blot for 5/8/14, 17/20/23/24, 6/11, 16/18/33, 2/3, 1/4/7, 3/10/28; PCR and Southern blot for 11/16

33 KA 51 SCC (NOS) 25 SCC (lip)

0 2.0 4.0

4.0*

Pierceall et al. (1991), USA

PCR for 6/11, 16, 18

21 SCC

19.0

19.0*

Shamanin et al. (1996), Germany

PCR with broad range degenerate primers [not specific for EV]

26 SCC

30.8

4 KA

50.0

25-related (2)

Frozen tissue; HPV 9 and 37 in a single KA present at ~10 copies/cell

41 (2)

Frozen tissue. HPV 41 not found in any of 44 melanomas or 47 non-malignant skin lesions in other patients Tissue stored at –20 °C; see also Tables 43 and 44

Untyped (1)

11.5

3.8

Frozen tissue.; no HPV DNA found in any of 14 cutaneous horns in control patients (see also Tables 43 and 46) *HPV 16

HUMAN PAPILLOMAVIRUSES

14.3

Fresh or frozen tissue; no HPV DNA found in the normal skin biopsies from the HPV 16- positive tumour patients; *all HPV 16 4, 32, 51 (1 each), 42 (2) 6 (1), 34 (1)

Frozen tissue

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EV-, betaHPV

Other types detected (no.)

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No. and type of lesions

247

Method of detection and types tested

No. and type of lesions

Overall HPV positivity (%)

Type-specific HPV positivity (%)

Comment

16/18, related typesa

30 KA

26.7

Paraffin-embedded tissue; in non-lesional skin samples from reduction mammoplasty specimens or uninvolved skin from melanoma patients, 1/26 was positive for HPV DNA.

Harwood et al. (2000), United Kingdom

PCR with 9 degenerated primers for EV, cutaneous, and mucosal HPV

22 SCC

27.2

27.2

Meyer et al. (2000), Germany

Nested PCR with mucosal-, cutaneous-, and EV-HPVspecific degenerate and typespecific (5, 8) primers, followed by RFLP analysis

10 SCC

50.0

40.0

6 (1), untyped (1)

O’Connor et al. (2001a), Ireland

Nested PCR with MY09/11 and EV-HPV-specific primers and sequencing

12 SCC

83.3

58.3

Unknown (3)

Three of 20 normal skin samples from controls positive in a nested PCR with EV-HPV-specific primers

CaireyRemonnay et al. (2002), France

PCR with MY09/11 and FAPprimers; hybridization for 6, 11, 16, 18, 31, 33, 35, 45, 51, 52, 58, 68

51 SCC

37.2

Untyped cutaneous (6)

Paraffin-embedded tissue

Forslund et al. (2003a), Norway

PCR with FAP primers

12 KA

33.3

Forslund et al. (2003c), Australia

PCR with FAP and HPV 38specific primers, cloning, and sequencing

12 SCC

33.3

0

29.4

Frozen tissue

Frozen tissue

33.3

Tissue stored at –20°C; 92% of perilesional and 63% of buttock swabs from same patients were HPV-positive.

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Hsi et al. (1997), USA

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EV-, betaHPV

Other types detected (no.)

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Table 42 (contd)

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Table 42 (contd) Method of detection and types tested

No. and type of lesions

Type-specific HPV positivity (%) EV-, betaHPV

16/18, related typesa

Other types detected (no.)

Comment

Iftner et al. (2003), Germany, USA

PCR with broad range degenerate primers

72 SCC

59.7

5.6

12.5

4 (9)

Frozen tissue; other types reported in combination with basal-cell carcinomas

Meyer et al. (2003), Germany

(Nested) PCR with mucosalcutaneous-, and EV-HPVspecific degenerate primers

15 SCC

46.7

46.7

6.7

6 (1)

Frozen tissue; 2/13 normal skin samples from patients with non-melanoma skin cancers (see Table 46) contained HPV DNA.

Pfister et al. (2003), Poland

Nested PCR with group-specific (mucosal, EV) and type-specific (HPV 8) primers and sequencing

20 SCC

45.0

45.0

5.0

Paraffin-embedded tissue

HUMAN PAPILLOMAVIRUSES

Overall HPV positivity (%)

See Table 7 for a description of the primers used. EV, epidermodysplasia verruciformis; ISH, in-situ hybridization; NOS, not otherwise specified; PCR, polymerase chain reaction a Alpha 7- and alpha 9-HPV

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Reference, study location

249

Method of detection and types tested

No. of lesions

Overall HPV positivity (%)

Type-specific HPV positivity (%)

Comment

16/18, related typesa

13

0

Frozen tissue

Eliezri et al. (1990), USA

In-situ hybridization (probe specificity not reported)

26

3.8

Paraffin-embedded tissue; see also Tables 42 and 44

Kawashima et al. (1990), Poland

Dot blot for 5/8/14, 17/20/23/ 24, 6/11, 16/18/33, 2/3, 1/4/7, 3/10/28; PCR and southern blot for 11/16

53

1.9

Pierceall et al. (1991), USA

PCR for 6/11, 16, 18

16

18.8

Nahass et al. (1992), USA

PCR [with consensus primers] and dot blot hybridization

3 scrotal

0

Fixed tissue

Zhu et al. (1993a,b), USA

PCR with MY09/11 and southern blot

13

0

Fresh tissue stored at 4 °C

Shamanin et al. (1996), Germany

PCR with broad range degenerate primers

11

36.4

27.3

Biliris et al. (2000), Greece

Multiplex, type-specific PCR for 1, 2, 5, 8, 11, 16, 18, 33

72

30.6

26.4

Harwood et al. (2000), United Kingdom

PCR with EV, cutaneous and mucosal HPV-specific primers

30

36.7

33.3

1.9

Frozen tissue; see also Tables 42 and 46

18.8*

Fresh or frozen tissue. No HPV DNA was found in the normal skin biopsies from the HPV-positive tumour patients; *all HPV 16

4 (1), 6 (1), 7 (1) 13.9*

Frozen tissue

Frozen tissue; *all HPV 18 Alpha 2- (1), mucosal (1)

Frozen tissue

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Grimmel et al. (1988), Germany

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EV-, betaHPV

Other types detected (no.)

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Table 43. Prevalence of HPV DNA in case series of basal-cell carcinoma of the skin

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Table 43 (contd) Method of detection and types tested

No. of lesions

Type-specific HPV positivity (%) EV-, betaHPV

Other types detected (no.)

Comment

6, 28, 34 (1 each)

Snap frozen; 8 of 31 (26%) perilesional skin tissues were HPV DNA-positive.

16/18, related typesa

Wieland et al. (2000), Germany and Poland

(Nested) PCR with genital/mucosal-, cutaneousand EV-specific primers

69

43.4

40.6

0

Forslund et al. (2003c), Australia

PCR with FAP and HPV 38specific primers, and sequencing

19

21.1

21.1

0

Iftner et al. (2003), Germany

PCR with broad range degenerate primers

18

27.8

0

16.7

Tissue stored at –20 °C; 52% of perilesional and 57% of buttock swabs from same patients were HPV-positive. 27 (1), untyped (1)

Frozen tissue

HUMAN PAPILLOMAVIRUSES

Overall HPV positivity (%)

See Table 7 for a description of the primers used. EV, epidermodysplasia verruciformis; PCR, polymerase chain reaction a Alpha 7- and alpha 9-HPV

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Reference, study location

251

No. and type of lesions

Overall HPV positivity (no. positive/ totala)

Type-specific HPV positivity (no. positive/total)a 6/11

Other types detected (no.)

Comment

30/11/2007

Method of detection and types tested

16/18

8/10

6/10

Frozen tissue; episomal HPV 16 found in 4/6 HPV 16-positive specimens

Ostrow et al. (1989a), USA

Southern blot for 2, 6, 16, 18, 31

1 digital

1/1

1/1

Episomal and integrated HPV 16 demonstrated in tumour tissue by twodimensional gel electrophoresis

Eliezri et al. (1990), USA

ISH (NA)

12 periungual

9/12

7/12

Guitart et al. (1990), USA

ISH for 6/11, 16/18, 31/33/35

9 nail bed SCC

1/9

1/9*

Paraffin-embedded tissue; *HPV 16; the patient also had HPV 16 in cervical tissue.

Ashinoff et al. (1991), USA

PCR for 16/18 and dot blot

2 periungual

2/2

2/2*

Paraffin-embedded tissue; *HPV 16 in both lesions

Moy & Quan (1991), USA

Dot blot for 6/11, 16, 18

1 digital

1/1

1/1*

Frozen tissue; *HPV 16

Sánchez-Lanier et al. (1994), USA

Southern blot for 6/11, 16, 18

4 digital

4/4

4/4*

Expression of unspliced E6 and spliced E6I transcripts in all patients; *all HPV 16

McHugh et al. (1996), USA

RT, in-situ PCR

1 metastatic digital

1/1

35 (1)

HPV 35 RNA in axillary lymph node metastases

Downs et al. (1997), United Kingdom

PCR with L1 consensus primers

1 digital

1/1

Untyped (1)

Subungual SCC in Darier disease

Forslund et al. (2000), Sweden

PCR with neighbour primers, type-specific for 16 and consensus primers

15 digital

67%

53%

73 (2)

Paraffin-embedded tissue; study investigating link with genital SCC

Alam et al. (2003), USA

NA

[∼25] digital

∼90%

[∼65%]

31 (1), 35 (1), unknown (5)

History of cervical SCC in 2 HPV-positive tumour patients

ISH, in-situ hybridization; NA, not available; PCR, polymerase chain reaction; RT, reverse transcriptase a Unless otherwise specified

Untyped (2)

Paraffin-embedded tissue; see also Tables 42 and 43

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Dot blot for 6/11, 16, 18

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Moy et al. (1989), USA

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Table 44. Prevalence of HPV DNA in case series of periungual and palmar squamous-cell carcinoma (SCC) of the skin

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253

the digital tumour (Alam et al., 2003), which underlines the possibility of genital–digital spread. It should be emphasized that tumours at this site are extremely rare. Whereas HPV was found in only a few keratoacanthomas when detected by southern blot hybridization (Pfister et al., 1986; Scheurlen et al., 1986), studies that used sensitive PCR reported a prevalence of about 30% in such lesions (Table 42). (ii) Basal-cell carcinoma In broad-spectrum PCR-based studies, the prevalence of HPV in basal-cell carcinoma was 21–44% in series of 11–72 cases (Table 43). EV-associated HPV usually predominated. (iii) Verrucous carcinoma (epithelioma cuniculatum) Individual reports have documented HPV 1, 6/11 and 16/18 in single cases of verrucous carcinoma (Garven et al., 1991; Noel et al., 1993; Sasaoka et al., 1996), a rare, indolent (typically non-metastasizing) tumour that occurs at acral sites. Verrucous carcinomas also develop in the anogenital region, where they may be found in conjunction with Buschke-Löwenstein tumours, and are usually associated with HPV 6 or 11. Verrucous carcinomas may also be found in the anogenital region in the absence of BuschkeLöwenstein tumours (see Section 2.3.3). However, the relationship between HPV infection and verrucous carcinomas in the anogenital region is poorly understood. HPV was not identified in any of 11 tumours in one case series (Petersen et al., 1994) (Table 45). (iv)

Premalignant cutaneous disease (Bowen disease and actinic keratoses) As for squamous-cell carcinoma, early southern blot-based studies indicated a very low prevalence of HPV in Bowen disease and actinic keratoses (Table 46), except for the rarely occurring periungual and palmoplantar Bowen disease; HPV 16/18 was found in 57–70% of these lesions in several case series (Table 47). When broad-range, sensitive PCRs were used, HPV DNA was generally detected in 35–85% of actinic keratoses and Bowen disease (Table 46). EV-associated HPV predominated in several cases series, whereas, in one study, they were found in only 7% of premalignant lesions in which a broad spectrum of cutaneous as well as high- and low-risk mucosal HPV were detected (Iftner et al., 2003). In one comparative study, the prevalence of EV-HPV DNA was similar in both low- and highgrade actinic keratoses (Pfister et al., 2003). (v) Malignant melanoma There is little evidence for an association between HPV and malignant melanoma. HPV 17 and HPV 38 were found in a superficial spreading malignant melanoma of an immunosuppressed patient but not in 35 other malignant melanomas (Scheurlen et al., 1986). HPV DNA related to EV-associated HPV 5 and 20 was detected in two of 15 melanoma biopsies (13%) in one study in which HPV DNA was also found in seven of 20 normal skin samples (35%) from randomly selected patients who were undergoing cosmetic surgery (Astori et al., 1998). In another case series, beta-HPV were identified in

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Table 45. Prevalence of HPV DNA in case series of verrucous carcinoma (VC) or epithelioma cuniculatum (EC) of the skin Method of detection and types tested

No. and type of lesions

6/11 Knobler et al. (1989), Austria

Dot blot (6, 11, 16/18)

1 EC, lower leg

1/1

1/1

Garven et al. (1991), USA

ISH for 6/11, 16, 18/31/33

1 VC, leg

1/1

1/1

Noel et al. (1993), Belgium

ISH for 1, 2, 3, 4, 11, 16, 18

1 VC, leg

1/1

Petersen et al. (1994), Denmark

PCR with MY09/11

11 CC, site not specified

0/11

Sasaoka et al. (1996), Japan

PCR with consensus primers, restriction mapping and southern blot for 16

2 VC, foot

2/2

Other types detected (no.)

Comment

16/18 Frozen tissue; the tumour contained HPV 11. 1/1

Paraffin-embedded tissue; the tumour contained HPV 11 and 18. 1 (1)

Paraffin-embedded tissue

Paraffin-embedded tissue

2/2

CC, carcinoma cuniculatum [belongs to the group of verrucous carcinoma]; PCR, polymerase chain reaction

Both tumours contained HPV 16.

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Type-specific HPV positivity (no. positive/total)

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Overall HPV positivity (no. positive/total)

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Table 46. Prevalence of HPV DNA in case series of Bowen disease (BD) and actinic keratoses (AK) of the skin Reference, study location

Method of detection and types tested

No. and type of lesions

Type-specific HPV positivity (no. positive/total)a EV-, betaHPV

1/3

Pfister & Haneke (1984), Germany

Southern blot for 1, 3, 6, 8, 11, 13

1 BD, dorsal hand

1/1

Kawashima et al. (1986), Poland

Southern blot for 1/2/4/7, 3/10/28, 5/8/14, 17/20/23/24

12 BD

0

Stone et al. (1987), USA

Southern blot for 1, 4, 6, 11, 16, 18

1 BD, foot

1/1

Grimmel et al. (1988), Germany

Southern blot for 41

6 AK

1/6

GuerinReverchon et al. (1990), France

ISH for 1, 2, 5, 6/11, 16/18

11 BD, leg, face, hand

5/11

Kawashima et al. (1990), Poland

Dot blot for 5/8/14, 17/20/ 23/24, 6/11, 16/18/33, 2/3, 1/4/7, 3/10/28; PCR and southern blot for 11/16

83 non-genital BD 55 AK

2/83 3/55

Kettler et al. (1990), USA

ISH for 1, 6, 11, 16, 18

25 non-genital BD

6/25

Frozen tissue; thumb not further defined

1/3

2 (1)

Tissue type not reported

Frozen tissue

1/1

2/11*

2/55

[4]/25

Fresh tissue 41 (1)

Frozen tissue

2 (1), untyped (3)

Paraffin-embedded tissue; *1 lesion with HPV 2 and 16, 1 lesion with HPV 16 and 18; no HPV DNA in 4 samples of normal skin or 6 samples of unrelated skin disease

34 (2) Untyped (1)

Frozen tissue; see also Tables 42 and 43

Paraffin-embedded tissue

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3 BD, thumb, lower leg, dorsal hand

16/18, related typesb

255

Southern blot for 16

Comment

HUMAN PAPILLOMAVIRUSES

Ikenberg et al. (1983), Germany

Other types detected (no.)

09:40

Overall HPV positivity (no. positive/ total)a

Method of detection and types tested

No. and type of lesions

Overall HPV positivity (no. positive/ total)a

Type-specific HPV positivity (no. positive/total)a

Comment

16/18, related typesb

09:40

EV-, betaHPV

Other types detected (no.)

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Table 46 (contd)

1 BD forearm (6-year-old boy)

1/1

1 (1)

Harwood et al. (2000), United Kingdom

PCR with EV-, cutaneousand mucosal-HPV-specific degenerate primers

11 carcinoma in situ (BD) and AK

6/11

3/11

Forslund et al. (2003c), Australia

PCR with FAP and type 38specific primers, cloning, and sequencing

10 AK

7/10

7/10

Iftner et al. (2003), Germany

PCR with broad range degenerate primers

71 AK 20 BD

58% 70%

}7%

}11%

1 (5), 6 (3), 27 (6), 2, 3, 4, 7, 34, 57, 73 (1 each)

Frozen tissue

Meyer et al. (2003), Germany

(Nested) PCR with mucosalcutaneous-, and EV-HPVspecific degenerate primers

36 AK/BD

36%

22%

8%

6 (1)

Frozen tissue; 2/13 normal skin samples from patients with nonmelanoma skin cancers (see Table 42) contained HPV DNA.

Pfister et al. (2003), Poland

Nested group-specific (mucosal, EV) and typespecific PCR for type 8

54 AK 60 AK 18 BD

85% 67% 33%

80% 40% 33%

3/10/28/77 (2); 6/11/16/66 (1)

Paraffin-embedded tissue

Frozen tissue

Tissue stored at –20 °C; 80% of perilesional and 60% of buttock swabs from the same patients were HPV-positive.

0% 0% 11%

See Table 7 for a description of the primers used. EV, epidermodysplasia verruciformis; ISH, in-situ hybridization; NA, not available; PCR, polymerase chain reaction a Unless otherwise specified b Alpha 7- and alpha 9-HPV

Frozen tissue Paraffin-embedded tissue Paraffin-embedded tissue

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ISH for 1, 6, 11, 16, 18

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Inaba et al. (1993), Japan

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Table 47. Prevalence of HPV DNA in case series of periungual and palmoplantar Bowen disease of the skin Method of detection and types tested

No. and type of lesions

Type-specific HPV positivity (no. positive/ total) 6/11

Other types detected (no.)

Comments

16/18

Southern blot hybridization

1 periungual

1/1

34 (1)

Frozen tissue

Rüdlinger et al. (1989), Switzerland

Southern blot hybridization for 1–5, 7, 10, 27, 30, 31, 33–38

1 periungual

1/1

35 (1)

Bowenoid lesion on vulva also contained HPV 35.

Kettler et al. (1990), USA

ISH for 1, 6, 11, 16, 18

5 palmoplantar

4/5

4/5

Paraffin-embedded tissue; 3/4 contained HPV 16, 1/4 contained HPV 16-related type.

Ashinoff et al. (1991), USA

PCR for 16/18 and dot-blot

5 periungual

3/5

3/5

Fixed tissue; all HPV 16

McGrae et al. (1993), USA

PCR and dot blot for 6, 11, 16, 18, 31, 33, 39, 45

3 periungual (1 patient)

3/3

3/3

All contained HPV 16; the patient had HPV 6-containing condylomata of the penis.

Nordin et al. (1994), Sweden

PCR for 16

1 digital

1/1

1/1

HPV 16 also found in vulvar and cervical dysplastic tissue from this patient

Sau et al. (1994), USA

ISH for 6/11, 16/18, 31/33/ 51

7 nail bed

4/7

4/7

Paraffin-embedded tissue

?

HUMAN PAPILLOMAVIRUSES

Kawashima et al. (1986), Poland

ISH, in-situ hybridization; PCR, polymerase chain reaction

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Overall HPV positivity (no. positive/ total)

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257

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four of 54 malignant melanomas (7%). Samples of normal skin of the patients were not available (Miracco et al., 2001). (b)

Case–control studies

A few studies compared the prevalence of HPV DNA in skin cancer tissue with that in healthy skin samples from control subjects or in swabs from perilesional skin. Depending on the sensitivity of the detection systems, prevalence rates of 22 and 86% were found in cancer tissue from immunocompetent patients compared with 8 and 22% in uninvolved skin, respectively (Stark et al., 1994; O’Connor et al., 2001a). In a study that compared the prevalence of EV-HPV DNA in 91 cases of solar keratosis or Bowen disease, 72 squamouscell carcinomas and 106 normal skin samples, adjusted odds ratios for the presence of EVHPV DNA were 9.2 (95% CI, 1.0–80) for solar keratosis and Bowen disease and 9.6 (95% CI, 0.9–100) for squamous-cell carcinoma (Iftner et al., 2003). In another study in Poland, very little difference between the positivity rates of basal-cell carcinomas and paired healthy skin (32% and 26%) was observed (Wieland et al., 2000). The prevalence of HPV DNA was much higher in perilesional swabs (92% and 52%, respectively) than in squamous-cell carcinomas (33%) and basal-cell carcinomas (21%) of patients in Australia (Forslund et al., 2003a). The high prevalence in swabs may reflect contamination of the skin surface by HPV rather than infection (Forslund et al., 2004). A significant association between the presence of EV-HPV DNA and skin cancer was observed in punch biopsies from the clinically normal skin of 38 immunosuppressed renal transplant recipients and 39 immunocompetent individuals (odds ratio for both groups combined, 6.41; 95% CI, 1.79–22.9) (Harwood et al., 2004). Conversely, no association was found between the presence of cutaneous or mucosal HPV types and skin cancer. A slightly positive, non-significant association was found between EV-HPV DNA in plucked eyebrow hairs and squamous-cell carcinoma in a population in subtropical Australia (odds ratio, 2.00; 95% CI, 0.50–8.0) (Boxman et al., 2000). However, a strong association was observed between EV-HPV DNA and solar keratoses among men (odds ratio, 3.40; 95% CI, 1.77–6.53) but not among women (Boxman et al., 2001). In a Dutch population, HPV DNA in eyebrow hairs (all EV-HPV types except for HPV 2 in one individual) was associated with a history of squamous-cell carcinoma (odds ratio, 1.7; 95% CI, 1.1–2.7) (Struijk et al., 2003). Positive associations were observed for the individual EV-HPV types 5, 8, 15, 20, 24 and 38 and the adjusted odds ratios for HPV 5, 15 and 20 were statistically significant. Two seroepidemiological case–control studies evaluated HPV infection as a risk factor for cutaneous squamous-cell carcinoma in immunocompetent individuals (Feltkamp et al., 2003; Masini et al., 2003). Infection with EV-related HPV types (5, 8, 15, 20, 23, 24, 36 and 38) was assessed by serology using a L1-VLP ELISA method. In Italy (Masini et al., 2003), positive serology for HPV 8 was associated with an odds ratio for cutaneous squamous-cell cancer of 3.2 (95% CI, 1.3–7.9). Other variables significantly associated with this tumour were family history of non-melanoma skin cancer, high professional or recreational exposure to the sun, light eye colour, large number of solar

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keratoses and seborrheic keratoses on the body surface and residence in buildings that emit radon. In the Netherlands (Feltkamp et al., 2003), the estimated relative risk for squamous-cell carcinoma was significantly increased in HPV 8- and HPV 38-seropositive subjects after adjusting for age and sex (odds ratio, 14.7; 95% CI, 1.6–135; and 3.0; 95% CI, 1.1–8.4, respectively). The estimated relative risk for nodular and superficial multifocal basal-cell carcinoma was also significantly increased in HPV 8-positive subjects (odds ratio, 9.2; 95% CI, 1.1–78.2; and 17.3; 95% CI, 2.1–143, respectively) and to a lesser extent in HPV 20-seropositive subjects (odds ratio, 3.2 and 3.4, respectively). No associations were found for HPV 16. The relative risk for developing malignant melanoma was not increased among HPV-seropositive individuals. Available studies do not allow an evaluation of whether exposure to the sun confounds or modifies the effect of HPV on skin cancer. 2.5.2

Cancer of the eye and conjunctiva

Early case reports and case series on the prevalence of HPV in eye lesions have been reviewed previously (IARC, 1995). HPV (mostly type 16) was found by PCR in both intraepithelial neoplasia of the conjunctiva (80–100%) and in nearly all invasive carcinomas of the conjunctiva, eyelid and lacrimal sac that were tested. In studies published since that time, the detection rate of HPV has varied widely. In a study in subtropical Tanzania, most cases of conjunctival epithelial dysplasia and epithelial neoplasms were found to be HPV-positive by in-situ hybridization (Moubayed et al., 2004). Using PCR-RFLP and in-situ hybridization methods, Saegusa et al. (1995) detected HPV 16 in two of four dysplasias (50%) and one of four squamous-cell carcinomas (25%) but in no basal-cell epithelioma of the conjunctiva. No other HPV types were found. Nakamura et al. (1997a) detected HPV 16 in two of four dysplasias and HPV 18 in one case of severe dysplasia and in one of four carcinomas of the conjunctiva. Eng et al. (2002) failed to detect DNA of HPV types 6, 11, 16 or 18 in any of 20 formalin-fixed, paraffinembedded malignant epithelial tumours of the conjunctiva. HPV DNA was detected in about 30% of non-familial sporadic retinoblastoma in two studies (Orjuela et al., 2000; Palazzi et al., 2003). In spite of a similar overall prevalence of HPV, the spectrum of types differed between the two studies, e.g. for HPV 16 (10% versus 23%) and for HPV 18 (28% versus 0%). It has been proposed that HPV infection represents an alternative carcinogenic mechanism to retinoblastoma gene inactivation but there was no significant correlation between the detection of HPV DNA and immunohistochemical detection of the retinoblastoma protein (Orjuela et al., 2000). HPV 16 DNA and E6-specific mRNA were detected by in-situ hybridization and reverse transcriptase in-situ PCR, respectively, in five of 10 conjunctival intraepithelial neoplasias; HPV 18 DNA and mRNA were present in the other five specimens (Scott et al., 2002b). Neither HPV DNA nor mRNA were detected in clinically uninvolved con-

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junctival specimens from the same patients or from five age-matched control subjects (p < 0.001). In contrast to these findings, other studies detected HPV not only in epithelial neoplasms but also in non-neoplastic lesions as well as in apparently healthy conjunctiva. In one study, HPV 16 DNA was found by PCR with consensus primers and dot blot hybridization using 28 type-specific probes, in two of 10 invasive cancers of the conjunctiva, and in the normal muscosa of one of 30 age- and sex-matched controls (Palazzi et al., 2000). In another study, HPV 16 infection was found by PCR in seven of 20 samples from carcinomas (35%) and in two of six samples from conjunctivitis (Waddell et al., 1996). Karcioglu and Issa (1997) identified HPV 16 and 18 DNA by PCR in eight of 14 (57%) in-situ squamous-cell carcinomas, in 17 of 31 (55%) invasive squamous-cell carcinomas, in four of 20 (20%) samples of climatic droplet keratopathy, in 11 of 31 (35%) samples of scarred corneas and in six of 19 (32%) samples of normal conjunctival tissue obtained during routine cataract extractions. HPV DNA was not detected by PCR with MY09/11 primers in any of 28 pathological specimens that ranged from intraepithelial neoplasia to invasive squamous-cell carcinoma or in 23 disease-free, age- and sex-matched patients (Tulvatana et al., 2003). The weak association between infections with genital HPV types and carcinoma of the conjunctiva is supplemented by the lack of a statistically significant association between anti-HPV 16 antibody status and the risk for conjunctival neoplasia (Newton et al., 2002; Waddell et al., 2003). In a pilot study of 21 squamous-cell carcinomas of the conjunctiva and 22 conjunctival samples of control subjects from Uganda, broad-spectrum and EV-specific PCRbased assays detected EV-HPV types in 86% of the cases and in 36% of controls (odds ratio after adjustment for exposure to the sun, 22.7; 95% CI, 1.7–312) (Ateenyi-Agaba et al., 2004). No mucosal HPV types were found in either cases or controls by genus alpha- and type 16-, 18-, and 45-specific PCR. As human immunodeficiency virus (HIV) serology was not available for the study patients, the strong association between EV-HPV and carcinoma of the conjunctiva could not be adjusted for a possible immunosuppression due to HIV infection, which appeared to be strongly associated with conjunctival cancer in Uganda (Newton et al., 2002). 2.6

Cancer at other sites

2.6.1

Cancer of the nose and nasal sinuses

Inverted papillomas are rare tumours of the nasal cavity and paranasal sinuses. Although commonly benign, they frequently reveal signs of invasive growth and convert into malignant tumours in up to 13% of cases (Bernauer et al., 1997). They are therefore discussed jointly with carcinomas of the nasal cavity. The presence of HPV infection in an extensive squamous-cell papilloma of the nasal cavity was first detected by immunoperoxidase staining of group-specific antigens (Syrjänen et al., 1983). A further study analysed 14 patients with 13 inverted papillomas

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and three squamous-cell carcinomas that extended to several sites of the nasal cavity and paranasal sinuses by indirect immunoperoxidase staining (Syrjänen et al., 1987b). Seven of the 25 papilloma biopsies analysed expressed HPV antigens. By in-situ hybridization with probes for HPV 6, 11 and 16, nine lesions in seven patients were shown to contain HPV 11 DNA. The three carcinomas tested were positive for HPV 16 DNA. In another study that used only in-situ hybridization to detect HPV 6 and 11, a high prevalence of both HPV 6 and 11 was noted in the 21 inverted papillomas analysed (Weber et al., 1988). A new virus type, HPV 57, was subsequently isolated from an invasively growing inverted papilloma of the maxillary sinus (de Villiers et al., 1989). When type-specific primers were used, this virus was subsequently identified in six of eight inverted nasal papillomas, in one of three inverted papillomas with dysplasia and in two of four inverted papillomas with carcinoma (Wu et al., 1993). HPV 57 was also detected in a further case of inverted papilloma (Ogura et al., 1996). Most other studies that used type-specific primers detected HPV 11 and HPV 6 in inverted papillomas and in some malignant tumours, and a limited number of cases were reported to be positive for HPV 18 and 16 DNA. Two inverted nasal papillomas contained HPV 11 DNA, one of which had a 500-base-pair insertion (Respler et al., 1987). In another study, HPV 6 DNA was identified by southern blot in one of seven inverted papillomas (Ishibashi et al., 1990). Kashima et al. (1992b) found HPV 11 in five of 29 inverted papillomas; two other papillomas contained HPV 6 DNA, and one of 24 squamous carcinomas contained HPV 18 DNA. A specific search for HPV 16 and 18 sequences by PCR in nasal carcinomas found that six of 49 cases were positive for HPV 16 and one for HPV 18 DNA (Furuta et al., 1992). Bernauer et al. (1997) detected HPV DNA in seven of 21 inverted papillomas; one lesion, which was associated with a squamous-cell carcinoma, was positive for HPV 18. One of two carcinomas that occurred within papillomas of the nasal septum contained HPV 6/11 DNA and the other contained HPV 18 DNA (Buchwald et al., 1997). Expression of E6/E7 genes of HPV 6 was detected in an inverted papilloma (Harris et al., 1998a). HPV 11 was found by in-situ hybridization and by PCR in one inverted papilloma (Kraft et al., 2001). Among 28 cases of squamous-cell carcinoma associated with inverted papillomas, four were HPV-positive: one for HPV 6/11, one for HPV 16/18, one for HPV 6/11 and 16 and one for HPV 18 (Buchwald et al., 2001). In summary, it appears that inverted papillomas are frequently positive for HPV 11, 6 and 57 DNA, whereas a small percentage (~5–15%) of carcinomas arising at the same sites contain HPV 18, 16, 11, 6 and 57 DNA at decreasing frequency. 2.6.2

Cancer of the lung

A number of studies have investigated the prevalence of HPV DNA in lung cancer tissues in patients with juvenile-onset recurrent respiratory papillomatosis and in women with a history of CIN3. [It must be noted that almost all studies are case series and laboratory personnel were not blinded as to the nature of the specimens; other aspects of proper epidemiological design were also lacking.]

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Table 48 summarizes the prevalence of HPV detected by PCR in studies of lung cancer that involved at least 20 subjects. HPV DNA prevalence in lung tumours ranged from zero (Shamanin et al., 1994a; Szabó et al., 1994; Welt et al., 1997; Wistuba et al., 1998; Gorgoulis et al., 1999) to very high levels, especially in studies conducted in Asia. Many of the positive reports that come from Asia are from Taiwan, China (Cheng et al., 2001, 2004; Wu et al., 2005) and Okinawa, Japan (Hirayasu et al., 1996; Tsuhako et al., 1998; Iwamasa et al., 2000; Miyagi et al., 2000, 2001). A recent study in Taiwan, China, that tested non-cancer lung specimens as controls (Cheng et al., 2001) found that 77 (55%) of 141 lung cancers were positive for HPV 16/18 by nested PCR and in-situ hybridization, and that the detection of HPV 16 and HPV 18 was more common in tumour specimens from women than in those from men (p < 0.0001), in those from nonsmokers than in those from smokers (p < 0.001) and in adenocarcinomas than in squamous-cell carcinomas (p < 0.03). Furthermore, specimens from cases were significantly more likely to be HPV DNA-positive than lung tissue from non-cancer patients (55% versus 27%; p < 0.001). From these results, it was suggested that HPV infection may play a role in lung carcinogenesis among Taiwanese nonsmoking women: only 10% of lung cancer cases in women occur in patients with a history of cigarette smoking, and adenocarcinomas constitute 59% of lung cancers among women compared with 31% among men (Chen et al., 2004). In two other studies that involved many of the same patients, HPV 11 was also reported in 13% of lung specimens from non-cancer patients (Cheng et al., 2004) and HPV 16 was reported in 13% of peripheral blood specimens from non-cancer patients (Chiou et al., 2003). [Although the high prevalence of HPV DNA in normal control specimens could reflect false-positive results, a high concordance with in-situ hybridization was demonstrated, and the prevalence of HPV was higher in specimens obtained from lung cancer patients than in those from non-cancer patients in each of these studies.] Similarly, in tumours collected during 1993 in Okinawa, Japan, 34 (79%) of 43 squamous-cell carcinomas tested were positive for HPV DNA when type-specific E6/E7 PCR for HPV 16, 18, 6 and 11 was used (Hirayasu et al., 1996). Subsequently, it was reported that the prevalence of HPV-positive squamous-cell carcinomas steadily decreased in specimens obtained after 1995 (Miyagi et al., 2000): 68% were HPV DNApositive in 1995, 35% in 1996, 23% in 1997 and 24% in 1998. The decreasing prevalence of HPV-positive specimens correlated with a marked fall in the number of squamous-cell carcinomas of the lung in Okinawa during the same time frame, and a concordance of nearly 100% between PCR and in-situ hybridization results was shown. A separate evaluation of adenocarcinomas showed that 78% of 23 case specimens contained HPV DNA (Tsuhako et al., 1998). Cases of HPV DNA-positive squamous-cell carcinoma that had high infiltration with Langerhans cells were found to have a better prognosis (Miyagi et al., 2001). Normal control tissue specimens were not tested. In spite of the above results, several large case series failed to detect HPV DNA in any lung cancer specimens tested or detected it in a very small percentage among a total of 290 cases (Shamanin et al., 1994a; Hiroshima et al., 1999; Clavel et al., 2000).

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Table 48. Prevalence of HPV DNA in case series of lung cancer detected by the polymerase chain reaction (PCR) method Reference, study location

Method of detection and types tested

No. of cases

Type of lesion

Overall HPV positivity (%)

Type-specific HPV positivity (%) 6

11

16

18

Others (type)

Multiple infections (%)

Comment

29

SCC

10.3

Liu et al. (1994), China

PCR for 11, 16; ISH for 11, 16

49

SCC

14.3

Shamanin et al. (1994a,b), Germany

L1 consensus primers and 4/60/65 + southern blot

85

SCC (40%), ADC (15%), others

0

Frozen samples

Szabo et al. (1994), Japan

PCR

47

SCC (85%), large cell carcinoma

0

Paraffin-embedded tissue

Xing et al. (1994), China [cited in Syrjänen (2002)]

PCR, ISH

49

SCC

Al-Ghamdi et al. (1995), United Kingdom

E1 consensus primers and type-specific for 6, 7, 11, 16, 18

66

SCC (50%), others

9.1

Kinoshita et al. (1995), Japan

PCR for 16, 18, 33 and southern blot for 18; ISH for 18

36

ADC (61%), SCC (28%), small cell carc.

8.3

Li et al. (1995), China

Dot blot for 16, 18; PCR for 16, 18

50

SCC (54%), ADC (32%), small cell (4%)

32.0

Nuorva et al. (1995), Finland

MY09/11 and nested PCR for 6, 11, 16, 18, 31, 33; ISH for 6, 11, 16, 18, 31, 33

22

Bronchioalveolar carcinoma

36.4

Zhang, Z.F. et al. (1995), China

PCR for 6/11, 16, 18

34

[SCC]

11.8

Da et al. (1996), China

Consensus primer; ISH for 16/18

40

SCC (40%), ADC (30%); small-cell carcinoma (23%)

PCR, 55.0 ISH, 25.0

10.3 12.2

8.2

1.5

4.1

8.2

4.5

Snap-frozen tissue 2.0

2

1.5

1

36.1

9.1

13.6

Paraffin-embedded archival tissue

44.0

12.0

4.5

4.5

Paraffin-embedded tissue

Frozen (PCR, southern blot) or fixed (ISH) samples

22.7 (31); 22.7 (33)

2.0

Paraffin-embedded (90%) or frozen tissue (10%)

31.8

Paraffin-embedded tissue

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Ogura et al. (1993), Japan

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Hirayasu et al. (1996), Japan

Method of detection and types tested

No. of cases

Type of lesion

Type-specific HPV positivity (%)

58.9

15.1

6

11

18

37.0

43.8

Others (type)

Comment

30.1

Paraffin-embedded tissue

PCR for 6, 11, 16, 18 and southrn blot

73

ISH for 6/11, 16/18, 31/33/51

94

Noutsou et al. (1996), Greece

Consensus and mixed type-specific primers for 11/16/18/33, and RFLP

99

SCC (41%), ADC (41%)

15.2

Soini et al. (1996), Finland

MY09/11 and nested PCR for 6, 11, 16, 18, 31, 33; ISH for 6, 11, 16, 18, 31, 33

43

SCC (65%), ADC (26%), small-cell carcinoma (7%)

30.2

Welt et al. (1997), Germany

ISH for 6, 11, 16, 18; PCR with MY09/11 and nested with CN3/MY09

32

SCC

0

Bohlmeyer et al. (1998), USA

MY09/11, southern blot, and dot blot for 6, 11, 16, 18, 33

34

SCC

5.9

Papadopoulou et al. (1998), Greece

MY09/11 and Southern blot for 6/11, 16/18

52

SCC

69.2

11.5

Tsuhako et al. (1998), Japan

ISH; PCR for 6, 11, 16, 18, 31/33/51

23

Adenosquamous carcinoma

78.3

13.0

Wistuba et al. (1998)

PCR for 16, 18, 31, 33

35

Small cell (40%), SCC (31%), ADC (29%)

0

Paraffin-embedded archival tissue

Gorgoulis et al. (1999), Greece

MY09/11, nested GP5/6 and type-specific for 6, 11, 16, 18, 31, 33, 35 and dot-blot

68

SCC (46%), ADC (47%), large cell carcinoma

0

Frozen and paraffinembedded tissue

Hennig et al. (1999a), Norway

GP5+/6+ and probing for 6, 11, 16, 18; ISH for 6, 11, 16, 18

75

ADC (37%), SCC (24%), small-cell carcinoma, others

49.3

28.7

0

16

Multiple infections (%)

9.6

7.4

4.0

8.1

14.0

18.6

16.3

2.0

20.9 (31); 20.9 (33)

Paraffin-embedded tissue

25.6

Paraffin-embedded tissue

Paraffin-embedded tissue 5.9

17.3

21.2

13.0

1.3

52.2

33.3

Paraffin-embedded tissue 13.5

30.4

1.3

5.8

Paraffin-embedded tissue

30.4

Paraffin-embedded tissue

9.3

Paraffin-embedded tissue; patients with history of CIN3

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Overall HPV positivity (%)

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Table 48 (contd)

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Table 48 (contd) Method of detection and types tested

No. of cases

Type of lesion

Overall HPV positivity (%)

AdC

0.4

Clavel et al. (2000), France

Hybrid Capture II

185

SCC (55%), AdC (32%)

2.7

Iwamasa et al. (2000), Japan 1993 1998

PCR for 6, 11, 16, 18 and southern blot

Miyagi et al. (2000), Japan

18

Others (type)

Paraffin-embedded tissue

0.4 2.7*

Snap-frozen tissue; *oncogenic types in Hybrid Capture II assay Storage of tissue not reported; *read from graph

43 21

SCC SCC

80* 24*

PCR for 6, 11, 16, 18 and southern blot; ISH for 6, 11, 16, 18

157

SCC

51.0

Cheng et al. (2001), Taiwan, China

MY09/11 and typespecific for 16, 18; ISH for 16, 18

141

54.6

60

SCC (41%), AdC (59%) Normal biopsies

Miyagi et al. (2001), Japan

PCR for 6, 11, 16, 18 and southern blot; ISH for 6, 11, 16, 18

59 62

SCC AdC

49.2 19.4

10.2 1.6

Cheng et al. (2004), Taiwan, China

MY09/11 and typespecific for 6, 11; ISH for 6, 11

141

SCC (41%), AdC (59%) Normal biopsies

38.3

28.4

9.9

15.0

1.7

13.3

Zafer et al. (2004), Turkey

MY09/11 and RFLP for 16, 18

40

SCC (63%), ADC (33%)

5.0

Brouchet et al. (2005), France

ISH; immunohistochemistry with VP1 antibody for 6/11/16/18/31/33/42/51/ 52/56/58

122

SCC (33%), ADC (25%)

0

Wu et al. (2005), Taiwan, China

Not reported

166

SCC (43%), ADC (57%)

54.8

60

16

14.0

8.9

26.7

24.2

25.5

35.5

41.1

15.0

11.7

18.6 6.4

20.3 11.3

18.5

Paraffin-embedded tissue Tissue section

Paraffin-embedded tissue Tissue section

5.0

Tissue stored at –20 °C Paraffin-embedded tissue

Frozen tissue

265

See Table 7 for a description of the primers used. ADC, adenocarcinoma; CIN, cervical intraepithelial neoplasia; ISH, in-situ hybridization; RFLP, restricted fragment length polymorphism; SCC, squamous-cell carcinoma * Three additional cases with HPV-31/33/35 a ‘;’ denotes independent methods whereas ‘and’ denotes subsequent steps.

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PCR for 16, 18, 33 and southern blot

6

Multiple infections (%)

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Hiroshima et al. (1999), Japan

Type-specific HPV positivity (%)

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Reference, study location

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At multiple locations in the respiratory tract, including the bronchial spurs, squamocolumnar junctions are found, which are types of tissue that may be particularly prone to HPV-associated tumorigenesis (Syrjänen, 2002). Juvenile-onset recurrent respiratory papillomatosis, an HPV-associated lesion that predominantly contains HPV 6 and 11, may spread to the trachea and bronchi, and solitary squamous-cell papillomas of the bronchi have been reported (Syrjänen, 2002). Some of these papillomas have been found to contain HPV, mostly types 6 and 11 (Flieder et al., 1998; Syrjänen, 2002). Although HPV 6 and 11 are thought to cause primarily benign lesions in anogenital epithelium, a recent study detected HPV 11 DNA in three lung cancers in patients with juvenile-onset respiratory papillomatosis; HPV 6, 16 or 18 were not detected. In one tumour specimen for which adequate material was available for testing, the HPV 11 genome was found to be integrated into the host genome (Reidy et al., 2004). Several other studies of lung cancer in patients with respiratory papillomatosis also detected HPV 11 in tumour specimens (Byrne et al., 1987; Guillou et al., 1991; Rady et al., 1998; Cook et al., 2000). In one case, HPV 6 was found (DiLorenzo et al., 1992). Together, these data suggest that, within the setting of juvenile-onset recurrent respiratory papillomatosis, HPV 11 and, to a lesser extent HPV 6, may on rare occasions (estimated to occur in less than 1% of patients) be associated with the development of lung cancer (Cook et al., 2000; Reidy et al., 2004). An increased risk for lung cancer has been reported in women who have been diagnosed with anogenital cancer or CIN3 (Frisch & Melbye, 1995). It was hypothesized that the relationship was due to a mutual association of lung cancer and anogenital tumours with tobacco smoking, but a possible connection with HPV infection has also been suggested (Hennig et al., 1999a). There is at least one well-documented case of an HPV 16-positive anaplastic lung carcinoma in a woman who had had a cervical cancer 9 years previously, although the possibility of a late metastasis could not be fully excluded (Stremlau et al., 1985). Furthermore, in 75 women with bronchopulmonary cancer following a diagnosis of CIN3 (with no radiotherapy), 37 (49%) were shown to be HPVpositive by PCR with GP5+/GP6+ primers (18 for HPV 16, 12 for HPV 6, five for HPV 16/6, one for HPV 16/11 and one for HPV 16/18), and the overall concordance between the HPV types in the lung tumours and in CIN3 specimens was greater than 60% (Hennig et al., 1999a). [It was not stated whether this was more than a chance finding, in view of the high prevalence of HPV 16 in lung tumours and the predominance of HPV 16 in CIN3.] Of 22 cases of bronchopulmonary cancers who did not have a history of CIN (controls), three were HPV 6-positive and none contained HPV 16, 11 or 18. In contrast, no HPVpositive bronchopulmonary carcinomas were detected when tested by in-situ hybridization. In two studies summarized earlier in this section (Chiou et al., 2003; Cheng et al., 2004), it was speculated that haematogenous spread of HPV 16 and 18 from the cervix may explain some HPV-associated cancer in the lung. In this connection, another study reported the detection of HPV DNA sequences in 52% of peripheral blood mononuclear cells from patients with genital HPV infections, but not in the 19 control subjects with no HPV infection (Pao et al., 1991).

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In summary, with the exception of the studies in Taiwan, China, there is a paucity of data in non-cancer lung specimens, which greatly limits the interpretation of the large number of studies that have been reported to date. In those studies that did test non-cancer lung specimens, the high prevalence of HPV DNA reported was unexpected. [Simultaneous testing of normal human tissues, for which there is broad agreement that the prevalence of HPV is very low (in addition to cancer and non-cancer lung specimens), is necessary before the specificity of the assay results reported can be accepted entirely.] 2.6.3

Cancer of the colon and the rectum

Cancers of the colon and the rectum are biologically distinct from cancer of the anus. Thus, data that combine anal and rectal cancers should be interpreted with caution and the term anorectal cancers should be avoided. Whereas anal cancer has a strong association with HPV (see Section 2.3), the relationship between HPV and cancers of the colon and rectum has not been established. Most studies that analysed a possible role of HPV in cancer of the colon were carried out in the early 1990s. A study performed in former Czechoslovakia that tested 13 adenocarcinomas and 10 adenomas of the colon for HPV 2, 6, 16 and 18 by southern blot hybridization failed to find any evidence of HPV DNA (Boguszakova et al., 1988). Similarly, Gilbert et al. (1991) failed to find HPV 16 DNA in eight adenocarcinomas of the anus, rectum or sigmoid colon in patients with Crohn disease. Shroyer et al. (1992) analysed 22 colon adenocarcinomas by PCR for HPV 6, 11, 16, 18 and 33 and by in-situ hybridization for HPV types 6/11, 16/18 and 31/33/35. None of the colon cancers revealed a positive reaction. Similar results were reported by Shah et al. (1992) who found no HPV DNA in 19 primary tumours of the colon using PCR with the MY09/MY11 consensus primers and southern blot hybridization with a generic probe for HPV 16/18. A more recent study (Audeau et al., 2002) also reported negative results after testing 20 colorectal cancers by immunohistochemistry staining with a monoclonal antibody that reacts with HPV 6, 11, 16 and 18. The number of positive reports is small and some were based mainly on immunohistochemistry. Kirgan et al. (1990) detected the presence of HPV antigen in 29/30 (97%) invasive carcinomas, in 18 of 30 adenomas and in seven of 30 biopsies (23%) of normal mucosa of the colon. Two years later, it was reported that 13 of 38 (32%) carcinomas, 8/21 (38%) adenomas and two of 24 (8%) normal biopsies of the colon contained the HPV L1 region as demonstrated by PCR amplification (McGregor et al., 1993). A study from Taiwan, China, reported the presence of both HPV 16 and HPV 18 DNA in three cell lines derived from colorectal adenocarcinomas (Cheng et al., 1991); subsequently, it was found that NIH3T3 cells transformed with colonic cancer cells contained HPV 16 DNA (Cheng et al., 1993). A study from Turkey reported HPV 18 and 33 infections in 39 and 30, respectively, of 51 colon cancers using PCR and direct sequencing (Sayhan et al., 2001). In a retrospective case–control study of 55 cases of colorectal cancer, the same number of tissues adjacent to the tumour and 10 control specimens were tested by nested

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PCR and in-situ PCR for the presence of HPV DNA (Bodaghi et al., 2005). In this series, 23 of 55 (42%) samples of colorectal cancer tissue and 15 of 52 (29%) samples of tissues adjacent to the tumour were positive for HPV DNA; 31 had HPV 16, five were positive for HPV 18 and two contained HPV 45 DNA. Ten samples contained HPV DNA in both the tumour and adjacent tissues and five contained HPV DNA only in the tissues adjacent to the tumour. None of the control tissues was HPV-positive. The findings were confirmed by in-situ hybridization, although the HPV DNA copy number was generally low. In view of the limitations of the techniques used in most of the studies and in the absence of larger studies that include more case–control analyses, the observed positive reports require a cautious interpretation. 2.6.4

Cancer of the breast

Data on HPV in malignant tumours of the breast are controversial. In a series of 15 breast cancers analysed by low-stringency filter hybridization, Ostrow et al. (1987) failed to find evidence of HPV DNA. In studies that used HPV 16- and 18-specific primers, Wrede et al. (1992) and Gopalkrishna et al. (1996) also failed to find HPV DNA in 80 and 30 breast cancer biopsies, respectively. Similarly, Bratthauer et al. (1992) were unable to detect HPV 6, 11, 16 or 18 in 15 intraductal papillomas, 15 papillary carcinomas and 13 infiltrating ductal carcinomas of the breast. The analysis of paraffin sections from 20 nipples with Paget’s disease (10 central intraductal and 10 invasive carcinomas) by PCR with MY09/MY11 consensus primers and by dot blot hybridization for HPV 6/11, 16/18 or 31/33/35 provided no evidence of HPV DNA (Czerwenka et al., 1996). These negative findings contrasted with several positive reports: Di Lonardo et al. (1992) detected HPV 16 DNA sequences by PCR and southern blot hybridization using specific primers in ten of 40 breast carcinomas and some axillary lymph node metastases. HPV 11 and 18 were not detected in any sample. More recently, Hennig et al. (1999b) examined 41 breast carcinomas from 38 patients with a history of CIN3 by PCR with specific primers and detected HPV 16 DNA in 19 cases (46%). Only one of these tumours was also positive by in-situ hybridization. A study conducted in China reported the presence of HPV 33 DNA in 14/32 cases of invasive ductal carcinomas of the breast, detected by PCR and Southern blot hybridization (Yu et al., 2000). Liu et al. (2001) examined 17 breast cancer samples using broad spectrum PCR, cloning and sequencing and by Southern blot hybridization for HPV 16, 18 and 31. Six (35%) of the biopsies were positive for HPV types 16, 18 and 31, and viral DNA was largely episomal. Another report from China found 19 of 28 (68%) breast carcinoma samples to be HPV-positive (Li, T. et al., 2002). In a study that compared cancer tissue with normal biopsies, Damin et al. (2004) analysed 20 specimens of reduction mammoplasty, 21 fibroadenomas and 101 breast carcinomas using specific primer sets that target the E6 region of HPV 16 or 18. Twenty-five (24.7%) of the carcinomas, but none of the other biopsies, were found to be HPV 16- (15 cases) or HPV 18- (11 cases) positive. One specimen contained sequences of both virus types. Recently, de Villiers et al. (2005) cloned several HPV types from 25 of 29 (86%)

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breast carcinomas and 20 of 29 (69%) corresponding samples of the mamilla from the same patient. Many ductal areas of the mamillae revealed condyloma-like histological patterns. The most prevalent HPV type in carcinomas and nipples was HPV 11, followed by HPV 6. A number of additional types were found, including those commonly detected in mucosal and cutaneous lesions, such as HPV 16, 23, 27 and 57 (nipples and carcinomas), 20, 21, 32, 37, 38, 66 and GA3-1 (nipples only) and 3, 15, 24, 87 and DL473 (carcinomas only). [It is plausible that surface areas may be infected frequently by a variety of different HPV types (see Section 2.5); thus the significance of these findings is currently difficult to assess.] 2.6.5

Cancer of the ovary

HPV was not detected in eight case series of cancer of the ovary from North America and Europe (de Villiers et al., 1986b; Ostrow et al., 1987; Leake et al., 1989; McLellan et al., 1990; Beckmann et al., 1991; Trottier et al., 1995; Anttila et al., 1999; Chen et al., 1999). Initial results that showed the presence of HPV 6 in 10/12 samples of epithelial ovarian carcinomas using in-situ hybridization (Kaufman et al., 1987) could not be reproduced in later analyses by either in-situ or southern blot hybridization or by PCR of the same and additional ovarian carcinoma specimens (Kaufman et al., 1990). In two studies of ovarian cancer from Taiwan, China, HPV 16 DNA was found in 50 and 8% and HPV 18 DNA in 17 and 2% of 18 and 60 cases, respectively, using PCR (Lai et al., 1994; Ip et al., 2002). Quantitative real-time PCR of the samples from the latter study revealed a prevalence of HPV 16 in 18 of 56 cases and between less than one and four HPV 16 DNA copies per cellular genome (Yang, H.J. et al., 2003). Only one ovarian cancer contained several copies per genome. One study from China compared the prevalence of HPV 16 detected by in-situ hybridization with an E6-specific probe in 50 ovarian epithelial cancers and in 30 non-malignant ovarian tissues, most of which had been removed for uterine pathology (Wu, Q.-J. et al., 2003). Twenty-six (52%) of the cancers were positive compared with two (7%) of the controls (odds ratio, 16.7; 95% CI, 3.2–71.4). In rare cases, squamous-cell carcinoma of the ovary may originate from cervical squamous-cell carcinoma in situ. Pins et al. (1997) described a case of CIN3 with contiguous upward spread to the endometrium, fallopian tubes and ovaries, focal invasion and HPV 16 DNA in all tumours detected by PCR. Upward spread of HPV 16-positive CIN may also explain the HPV 16-positivity of primary squamous-cell carcinoma of the ovary in two further cases (Mai et al., 1996; Manolitsas et al., 1998). 2.6.6

Cancer of the prostate

A possible association of prostatic cancer with sexual behaviour and exposure to sexually transmitted infection has been reported (Hayes et al., 2000; Rosenblatt et al., 2001; Strickler & Goedert, 2001). Furthermore, men with anal cancer, a disease that has

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been associated with HPV, have an increased risk for developing subsequent prostatic cancer (Rabkin et al., 1992). Table 49 presents case series of cancer of the prostate and benign prostatic hypertrophy in association with HPV prevalence. A few studies found an association of HPV with prostatic cancer (McNicol & Dodd, 1990a; Anwar et al., 1992a; Serth et al., 1999; Carozzi et al., 2004). In three of these studies, specimens of non-cancerous prostate were also found to have a substantial, although lower, prevalence or copy number of HPV DNA (McNicol & Dodd, 1990a; Serth et al., 1999; Carozzi et al., 2004). Other studies reported that HPV DNA was equally prevalent in cancers, benign prostatic hypertrophy and normal prostatic tissue (McNicol & Dodd, 1990b, 1991; Ibrahim et al., 1992; Dodd et al., 1993; Wideroff et al., 1996b). The majority of studies did not detect HPV in prostatic cancer specimens (Masood et al., 1991; Effert et al., 1992; Serfling et al., 1992; Anderson et al., 1997; Gherdovich et al., 1997; Noda et al., 1998; Strickler et al., 1998a; Saad et al., 1999). A few studies have reported the detection of HPV DNA in specimens of benign hypertrophic prostate tissue and prostate cancer using non-amplification methods that are less prone to false-positive test results than PCR (McNicol & Dodd, 1990a). Seroepidemiological studies of exposure to HPV have similarly reported conflicting findings. Four cross-sectional studies failed to detect a relationship between the presence of HPV antibodies and prostatic cancer (Strickler et al., 1998a,b; Hayes et al., 2000; Rosenblatt et al., 2003), and another cross-sectional study found a borderline association of prostatic cancer with antibodies to HPV 33 (odds ratio, 1.6; 95% CI, 1.0–2.7) but not to HPV 16 or 18 (Adami et al., 2003). In contrast, two nested case–control studies reported odds ratios for the association between HPV 16 antibodies and prostatic cancer greater than 2.5 (Dillner et al., 1998; Hisada et al., 2000). These latter findings have been interpreted by some authors as evidence that prospective HPV serological data reflect the strong association of HPV antibodies with sexual behaviour and the relationship of sexual behaviour with the risk for prostatic cancer. Such an association is not found in crosssectional studies because, by the time prostatic cancer occurs, men have aged sufficiently that some have lost detectable levels of HPV antibody (Strickler & Goedert, 2001). Overall, the failure of most studies that used sensitive PCR methods to detect HPV in prostatic cancer or, when detected, to find similar or higher prevalence of HPV DNA in non-cancer than in cancer tissues does not support a role of HPV in prostate carcinogenesis. 2.6.7

Cancer of the urinary bladder and urethra

The majority of bladder cancers that occur in the developed world are transitional-cell carcinomas (approximately 90%), and the proportion of squamous-cell carcinomas ranges from 3 to 10%. In contrast, in countries where schistosomes are endemic, the majority of bladder cancers are squamous-cell cancers (60–80%) (Cooper et al., 1997). The prevalence of HPV DNA in case series of cancers of the urinary bladder is summarized in Table 50. [For these tumours, contamination from the lower genital tract during acquisition of tissues is a particular concern.] In studies of transitional-cell carcinomas, or

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Table 49. Prevalence of HPV DNA in case series of prostate cancer and benign prostatic hypertrophy Reference, study location

Method of detection and types tested

Type of lesion

Overall HPV positivity (%)

Type-specific HPV positivity (%) 6

16

Comment

18

4 12

Cancer Benign hypertrophy

75.0 33.3

Mostly TURP, also SPP; frozen tissue

McNicol & Dodd (1990b), Canada

PCR for E6 HPV 16, 18

4 15 5

Cancer Benign hypertrophy Normal autopsies

100 93.3 20.0

Masood et al. (1991), USA

ISH for 6/11, 16, 18/31, 33/35

20 20

Cancer Benign hypertrophy

0 0

McNicol & Dodd (1991), Canada

PCR for E6 HPV 16, 18

27 56

Cancer Benign hypertrophy

51.9 62.5

51.9 60.7

3.7 5.4

Anwar et al. (1992a), Japan

PCR for E6 HPV 16, 18, 33

68 10 10

Cancer Benign hypertrophy Normal autopsies

41.2 0 0

16.2

25.0

Effert et al. (1992), USA

‘Differential’ PCR for 16, 18 and southern blot

30

Cancer

Ibrahim et al. (1992), USA

PCR and dot-blot for 6, 11, 16, 18, 31, 33, 35, 39, 45

40 12 17

Cancer Benign hypertrophy Normal tissue

Rotola et al. (1992), Italy

PCR for E6 HPV 6/11, 16

8 17

Cancer Benign hypertrophy

Serfling et al. (1992), USA

PCR with L1 consensus primer and Southern blot

30

Cancer

Dodd et al. (1993)c, Canada

RT-PCR for E6/E7 mRNA of HPV 16

7 10

Cancer Benign hypertrophy

100 93.3 20.0

Mostly TURP, also SPP and autopsy; frozen samples Biopsies and TURP; paraffinembedded tissue

0

Mostly TURP, also SPP; frozen samples TURP, SPP and autopsy; paraffinembedded tissue Collection not specified; frozen tissue

15.0 0 11.8

15.0

Biopsies/TURP/SPP; paraffinembedded and frozen samples

11.8 50.0b 64.7b

75.0 82.3

0

42.9 50.0

20.0

Collection not specified; frozen samples Collection not specified; frozen samples

42.9 50.0

Collection not specified; frozen samples

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McNicol & Dodd (1990a), Canada

NS [> 75] NS [≥ 82]

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No. of cases

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Reference, study location

Method of detection and types tested

Type of lesion

Overall HPV positivity (%)

Type-specific HPV positivity (%) 6

16

Comment

09:40

No. of cases

18

2.3 5.9 0

2.3

Carcinoma Adenoma

52.9 31.8

52.9 31.8

51

Cancer

15.7

15.7

PCR with L1 and E6 consensus primers and dot blot for 6, 11, 16, 18, 31, 33, 45

56 42

Cancer Benign hypertrophy

Anderson et al. (1997), United Kingdom

PCR with E2- and E6specific primers for 16, and E1 consensus primer

14 10

Cancer Benign hypertrophy

0 0

TURP; frozen tissue

Gherdovich et al. (1997), Italy

PCR with MY09/11; nested PCR

5 60

Cancer Benign hypertrophy

0 0

Surgery; frozen tissue

23

Cancer and intraepithelial neoplasia

Tu et al. (1994), USA

PCR with L1 consensus primer and Southern blot for 16, 18

43 17 1

Cancer Metastases in lymph nodes Normal tissue

Moyret-Lalle et al. (1995), France

PCR for E6 of 16, 18 and hybridization

17 22

Suzuki et al. (1996), Japan

PCR with L1 consensus primer and RFLP

Wideroff et al. (1996b), USA

L1, 12.5 L1, 9.5

0 0

0 0

Surgical, not TURP; paraffinembedded tissue

5.9

Surgical not TURP; tumours, paraffin embedded; metastases, frozen samples

0 0

Collection not specified; frozen samples Surgery or autopsy; frozen tissue

0 0

TURP, surgery and biopsy; paraffin-embedded tissue

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13.0d

Sarkar et al. (1993), USA

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Table 49 (contd)

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Reference, study location

PCR for E6 HPV 16 (2 probes) and for L1 HPV 6/11/16/18/33

No. of cases

Type of lesion

Benign hypertrophy

37

Normal tissue

16 3.8 18.9

18 Radical retropubic resection of the prostate; paraffin-embedded tissues from 41 patients

9.5 33.3 2.7 13.5

Transperineal biopsy fixed in 10% formalin

See Table 7 for a description of the primers used. ISH, in-situ hybridization; NS, not specified; PCR, polymerase chain reaction; RFLP, restriction fragment length polymorphism; RT, reverse transcriptase; SPP, suprapubic resection of the prostate; TURP, transurethral resection of the prostate a ‘;’ denotes independent methods whereas ‘and’ denotes subsequent steps. b 6 or 11 c All samples also reported in study by McNicol & Dodd (1991) d Samples positive only after Southern blot analysis e HPV positivity determined in the preservation fluid after 1–2 h of storage of the biopsies

Noda et al. (1998), Japan

Nested PCR with consensus primer and RFLP for 16, 18, 31, 33, 35, 52, 58

38 71

Cancer Benign hypertrophy

0 4.2

Strickler et al. (1998a), Italy and USA

PCR with MY09/11 and GP5+/6+

63 61

Cancer Benign hypertrophy

0 0

Mostly TURP, also SPP; frozen samples

Saad et al. (1999), Canada

PCR with MY09/11 and southern blot

40

Cancer

0

Collection not specified; fresh samples

Carozzi et al. (2004), Italy

PCR with consensus primer and primer for 16/18/31/33/35/45/ 52/58, and typing for 6, 11, 16, 18, 31, 33, 35, 45, 52, 58

26 25

Cancer Benign hypertrophy

65.4 48.0

4.2

Surgical and TURP; paraffinembedded tissue

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21

E6a, 3.8 E6b, 18.9 L1, 0 E6a, 9.5 E6b, 33.3 L1, 0 E6a, 2.7 E6b, 13.5 L1, 0

6

Comment

273

Cancer

Type-specific HPV positivity (%)

HUMAN PAPILLOMAVIRUSES

53

Overall HPV positivity (%)

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Method of detection and types tested

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Table 49 (contd)

30/11/2007

Reference, study location, year(s) of study

Methoda of detection and types tested

15.8 0

Kerley et al. (1991), USA, NR

PCR for 6, 11, 16, 18 and restriction digest

18 TCC 4 SCC 5 controls

0 25.0 0

Anwar et al. (1992b), Japan, NR

PCR for 6, 11, 16, 18, 33 and dot-blot

46 TCC 2 SCC 21 controls

82.6 50.0 33.3

6

11

16

Other types (n)

Multiple infections (%)

Tissue collection; storage

18 15.8 Transurethral resection; paraffin-embedded specimens Paraffin-embedded specimens

25.0 47.8b 19.0

Knowles (1992), United Kingdom, NR

PCR with GP5/6 and Southern blot

100 TCC

0

Furihata et al. (1993), Japan, 1981–92

ISH for 16, 18, 33

90 TCC

31.1

Chang et al. (1994), Finland, 1966–87

PCR with MY09/11

108 TCC

0

Maloney et al. (1994), USA, 1979–92

PCR with GP5/6 and typespecific for 16, 18

20 TCC 22 SCC

0 4.5

Kamel et al. (1995), Finland, 1987–92

ISH for 6, 11, 16, 18, 31, 33

40 TCC

60.0

30.0

7 SCC

42.9

14.3

28.3 38 50.0c 9.5

33 (14)

60

33 (2)

5

21.1

33 (16)

19

18.9

Paraffin-embedded archival specimens; type-specific positivity combines TCC and SCC. Cystoscopic biopsies; frozen samples Cystectomy; paraffinembedded specimens Paraffin-embedded archival specimens Cystectomy; paraffinembedded specimens

4.5 25.0

20.0 28.6

40.0

31 (16); 33 (12) 31 (3); 33 (1)

45 29

Paraffin-embedded archival specimens

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76 TCC 3 SCC

Type-specific HPV positivity (%)

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Overall HPV positivity (%)

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Bryant et al. (1991), United Kingdom, NR

No. and type of lesions

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Table 50. Prevalence of HPV DNA in case series of cancer of the urinary bladder

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Table 50 (contd) Methoda of detection and types tested

No. and type of lesions

Overall HPV positivity (%)

Type-specific HPV positivity (%) 6

11

16

71 TCC 8 controls

39.4 0

Lopez-Beltran & Muñoz (1995), Spain, NR

PCR for 6, 11, 16, 18; ISH for 6/11, 16/18, 31/33/35

76 TCC

PCR, 9.2 ISH, 5.3

Sano et al. (1995), Japan, 1989–93

PCR with pU-31B/2R, pU1M/2R, L1C1/L1C2 and type-specific for 16, 18

80 TCC 11 SCC

Smetana et al. (1995), Israel, 1986–90

ISH for 6/11, 16/18, 31/33/35; PCR with E1 consensus primer and southern blot for 6/11, 16/18

110 TCC 41 controls

Boucher et al. (1996), United Kingdom, NR

Southern blot for 6/11, 16

54 TCC, 1 SCC

0

Mvula et al. (1996), Japan, NR

PCR with L1 consensus primer and type-specific for 16, 18

34 TCC 2 SCC

8.8 0

2.9

Tenti et al. (1996), Italy, NR

PCR with MY09/11 and type-specific primers and southern blot for 6/11, 16, 18, 33

79 TCC

32.9

29.1

1.4

1.3

18 Transurethral resection or cystectomy; frozen specimens

38.0

9.2 5.3

1.3

0 0 25.5 4.9

Tissue collection; storage

Transurethral resection; paraffin-embedded specimens Paraffin-embedded archival specimens

16.4 2.4

9.1

Paraffin-embedded specimens and control biopsies

2.4

Radical cystectomy or transurethral resection; formalin-fixed specimens? Paraffin-embedded archival specimens 12.7

8.9

Transurhetral resection or cystectomy; paraffinembedded specimens

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Multiple infections (%)

HUMAN PAPILLOMAVIRUSES

LaRue et al. (1995), Canada, NR

Other types (n)

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Reference, study location, year(s) of study

Methoda of detection and types tested

No. and type of lesions

Type-specific HPV positivity (%) 6

11

16

Other types (n)

Multiple infections (%)

Tissue collection; storage

18

25 SCC

0

Paraffin-embedded archival specimens from patients infested with Schistosoma haematobium

Lu et al. (1997), United Kingdom, 1987–94

ISH for 16, 18

22 TCC 5 SCC

0 0

Paraffin-embedded archival specimens

Aynaud et al. (1998), France, NR

PCR with MY09/11 and type-specific for 6, 11, 16, 18, 33

58 TCC

0

Transurethral excision; frozen specimens

Simoneau et al. (1999), Canada, 1990–92

PCR with MY09/11 and dot-blot for 6, 11, 16, 18, 33; PCR for 16

187 TCC

9

Sur et al. (2001), South Africa, 1994–96

PCR with GP5+/6+

64 TCC

1.6

Paraffin-embedded archival specimens

Westenend et al. (2001), Netherlands, NR

ISH for 6/11, 16/18 31/33/51

16 SCC

0

Biopsy or cystectomy; paraffin-embedded archival specimens

0.5

4.8

2.1

1.1

See Table 7 for a description of the primers used. ISH, in-situ hybridization; NR, not reported; PCR, polymerase chain reaction; SCC, squamous-cell carcinoma; TCC, transitional-cell carcinoma a ‘;’ denotes independent methods whereas ‘and’ denotes subsequent steps. b Not clear from article if 22 or 28 HPV 6/11 positive samples (47.8 and 60.9%, respectively) c 16, 18 or 33

Transurethral resection; frozen specimens

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Cooper et al. (1997), South Africa, NR

2.1

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Table 50 (contd)

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in a few studies in which the type of bladder cancer was not specified and that were conducted in countries where schistosomes are not endemic, no HPV DNA was found in a total of 430 bladder cancers using PCR (Kerley et al., 1991; Knowles, 1992; Saltzstein et al., 1993; Sinclair et al., 1993; Chang et al., 1994; Maloney et al., 1994; Sano et al., 1995; Aynaud et al., 1998) or in a total of 91 bladder cancers using in-situ hybridization or Southern blot technique (Ostrow et al., 1987; Boucher et al., 1996; Lu et al., 1997). In other PCR-based studies, HPV was detected in 3–80% of the samples of transitionalcell carcinomas: in about 3% in the studies by Chetsanga et al. (1992; 1/44), Mvula et al. (1996; 1/34), Tekin et al. (1999; 2/42), Sur et al. (2001; 1/91) and Fioriti et al. (2003; 1/32); in 9% in the studies by Lopez-Beltran and Muñoz (1995; 7/76) and Simoneau et al. (1999; 16/187); in 20% in the study by Gopalkrishna et al. (1995; 2/10); in about 30% in the studies by Smetana et al. (1995; 20/59), Tenti et al. (1996; 26/79), Chan et al. (1997; 6/20) and Gazzaniga et al. (1998; 11/35); and in 39%, 50% and 83% in the studies by LaRue et al. (1995; 28/71), Aglianò et al. (1994; 23/46) and Anwar et al. (1992b; 38/46), respectively. Widely variable prevalences of HPV positivity were also determined using less sensitive methods such as in-situ or southern blot hybridization: 5% (Lopez-Beltran & Muñoz, 1995; 4/76), 16% (Bryant et al., 1991; 12/76), 20% (Shibutani et al., 1992; 4/20), 31% (Furihata et al., 1993; 28/90), 40% (de Gaetani et al., 1999; 17/43) and 60% (Kamel et al., 1995; 24/40). Only genital (low- and high-risk) HPV types have been assessed in these studies and HPV 16 and 18 were detected most frequently. Relatively few studies addressed HPV positivity in squamous-cell carcinoma of the bladder using PCR (Kerley et al., 1991; Anwar et al., 1992b; Maloney et al., 1994; Sano et al., 1995; Mvula et al., 1996) or in-situ or southern hybridization (Bryant et al., 1991; Kamel et al., 1995; Boucher et al., 1996; Lu et al., 1997; Westenend et al., 2001) (see Table 50). From these studies, six of 73 (8%) squamous-cell carcinomas were found to be positive for HPV 6, HPV 11 or high-risk HPV. HPV 6 was reported in another squamouscell carcinoma of the bladder and was identified by southern blot, PCR and in-situ hybridization (Wilczynski et al., 1993). No HPV DNA was detectable by PCR in 25 schistosomiasis-associated bladder cancers (Cooper et al., 1997). In carcinomas of the urethra, HPV 6 or 11 has been detected in two individual cases and in three of a series of four cases (Grussendorf-Conen et al., 1987; Mevorach et al., 1990; Alonso et al., 1997). HPV 16 has been detected in eight of 17 invasive cancers (47%) and in four of six metastases in women with urethral carcinoma (Wiener & Walther, 1994), as well as in four of 14 squamous-cell cancers of the urethra (29%) in men (Wiener et al., 1992). In a study that investigated the location within the male urethra, HPV 16 DNA was found in six squamous-cell carcinomas of the pendulous urethra, but in none of the primary cancers of the bulbous (six) and posterior (two) urethra (Cupp et al., 1996).

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Co-factors of HPV in cervical cancer

It is now clear that HPV infection is causally related to cervical cancer and its precursor lesions (IARC, 1995). However, because of the high prevalence of HPV infection in the general female population and the relatively low rate of cervical cancer, environmental and host genetic factors may influence the progression from infection to cancer (Santos et al., 2001; Castellsagué & Muñoz, 2003; Wang & Hildesheim, 2003). To evaluate the co-factors of HPV, it is important to note the complex and multifactorial origin of cancer. One or more co-factors could substantially contribute to HPV-initiated cervical carcinogenesis depending on its (their) prevalence in the population studied. For example, in female populations whose use of tobacco use is low to non-existent, significant associations between tobacco smoking and disease are unlikely to be detected. Similarly, variations in the prevalence of hormonal contraceptive use in populations by region and country result in the differences in risk estimates for this factor that are observed across countries. These population differences need to be taken into account when interpreting the strength of the association observed between any putative HPV cofactor and cervical cancer. Evidence for co-factors of HPV derives from two types of study design: prospective cohort studies in which exposure is assessed before the occurrence of disease and case–control studies in which exposure is ascertained after the diagnosis of disease. Moreover, several different analytical approaches may be used in the statistical analyses of these data. Since cervical cancer only develops in women who are infected with HPV, the most appropriate analytical approach is to restrict all statistical analyses to HPV-positive women; that is, women who are at risk for development of the disease. The ideal controls or comparison group would be women who were infected with HPV at the same time as the case women, but who did not develop the disease. Methodologically, this is difficult to achieve; therefore, one approach has been simply to restrict analyses to both cases and controls who were infected with either any HPV type or carcinogenic HPV types. It is important to note that the use of this method of restriction involves a potential for disease misclassification that would bias the risk estimate observed towards the null. Therefore, the studies summarized below may have reported underestimates of the true association between, for example, tobacco smoking and disease. 2.7.1

Non-infectious co-factors for cervical cancer

A complete summary of possible co-factors for cervical cancer and its precursor lesions has not been attempted here because many potential co-factors have not been evaluated rigorously in epidemiological studies, and the focus has therefore been placed on tobacco smoking, hormonal contraceptive use, parity and nutritional and genetic factors.

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(a)

279

Tobacco smoking

The role of tobacco smoking in the etiology of cervical cancer has been a topic of debate for many years (Layde, 1989). In an extensive review of the literature through to 1985, Winkelstein (1986) stated that 15 of 18 studies reported a significantly increased risk for cervical cancer in tobacco smokers. In another review of the literature that covered the years 1986–89 (Winkelstein, 1990), 11 of 15 studies reported significant positive associations of different magnitude between tobacco smoking and risk for cervical cancer. Despite this evidence, there have still been doubts as to whether tobacco smoking is truly associated with the risk for cervical cancer. The discrepancy is based on the problem of misclassification with respect to exposure to HPV. Moreover, most studies conducted before 1990 did not measure HPV status or control for sexual activity, which is known to differ according to smoking status. Since 1990, most studies have shown a significant association between tobacco smoking and cervical cancer and its precursor lesions after either adjustment for HPV infection in the analyses or restriction of the analyses to HPVpositive women. Evidence for the association between tobacco smoking and cervical cancer reported here derives from the most rigorously designed studies that restricted statistical analyses to HPV-positive women. (i) Case–control studies Over the past few decades, numerous case–control studies have been conducted worldwide to quantify the association between tobacco smoking and the risk for cervical cancer (reviewed by Haverkos et al., 2003). Muñoz et al. (1993) first reported an analysis of the association between tobacco smoking and the risk for CIN3/carcinoma in situ that was restricted to 218 HPV-positive women who participated in a case–control study in Colombia and Spain. No significant association was observed, but the study was hampered by low statistical power. Studies on tobacco smoking and cervical cancer that have been published subsequently are summarized in Table 51. In a case–control study conducted in Denmark (Kjaer et al., 1996) that included 141 prevalent cases, current and former smokers had a twofold increased risk for SIL or ASCUS compared with those who had never smoked. In an update of the same study, the risk appeared to be higher (Kruger-Kjaer et al., 1998). In a comparison of cases of CIN3 and CIN1 in the USA, Ho et al. (1998b) observed a significant dose–response in the increase in risk among women who smoked 10 or more cigarettes per day and had a smoking history of more than five pack–years compared with those who had never smoked or former smokers. No association with CIN2 was observed. Among HPV 16-positive women, Olsen et al. (1998a) observed an increase in risk of 4.6 for CIN2/3 among those who had ever smoked tobacco in a population-based case– control study conducted in Norway. Ylitalo et al. (1999) conducted a case–control study among women who were selected from those registered in the Uppsala county cervical cytology programme in Sweden. Cases and controls were matched by HPV status and selected on the basis of cytology

Intensity/duration of smoking

Odds ratio (95% CI)

Detection method and comments

Kjaer et al. (1996), Denmark

141 ASCUS and SIL combined

153 normal cytology

Former Current

2.3 (0.8–6.6) 1.9 (1.2–3.2)





PCR GP5+/6+; adjusted for age

Ho et al. (1998b), USA

44 CIN3

163 CIN1

Former Current

1.8 (0.64–5.22) 2.1 (1.09–5.15)

≤ 10 cigarettes/day* > 10 cigarettes/day*

1.49 (0.61–3.67) 3.35 (1.22–9.15) p trend = 0.018 1.75 (0.71–4.31) 2.66 (1.15–6.15) p-trend = 0.019

PCR and southern blot; no association with CIN2 (n = 52); adjusted for age, education, ethnicity, no. of Pap smears in past 3 years, high-risk versus lowrisk HPV infection; *reference category is never or former smokers.





PCR GP5+/6+; adjusted for age, years of sex life without barrier contraceptive, partner’s education, marital status [follow-up of Kjaer et al. (1996)]

≤ 5 pack–years* > 5 pack–years*

Kruger-Kjaer et al. (1998), Denmark

82 ASCUS 86 LSIL 71 HSIL

155 normal cytology

ASCUS LSIL HSIL Current versus former ASCUS LSIL HSIL

4.2 (1.4–12.6) 2.5 (0.8–8.0) 3.2 (0.9–11.4) 1.9 (1.0–3.4) 1.5 (0.8–2.7) 1.9 (1.0–3.8)

Olsen et al. (1998a), Norway

60 CIN2/3, histologically confirmed

14 with no dysplasia

Ever versus never smoker

4.6 (0.9–22.9)

Former smoker 1–10 cigarettes/day > 10 cigarettes/day

4.2 (0.5–37.9) 3.3 (0.5–20.8) 5.9 (1.0–35.6)

PCR; population-based controls; adjusted for age

Ylitalo et al. (1999), Sweden

178 CIS HPV 16/18-positive

178 HPV16/18positive; no history of in-situ or ICC and no hysterectomy

Former Current

2.1 (1.0–4.3) 2.3 (1.3–4.3)

1–9 years 10–19 years ≥ 20 years

2.3 (1.1–5.2) 2.5 (1.3–4.7) 1.8 (0.8–4.1) p trend = 0.62 2.3 (1.1–4.8) 2.4 (1.6–7.3) 1.6 (0.8–3.2) p-trend = 0.17

PCR; adjusted for marital status, OC use, age at sexual debut, no. of sexual partners, age at menarche, parity, years in school

346 cytologically normal

Never and party smokers Former Current

1.0

PCR; adjusted for HPV DNA, age

Kjellberg et al. (2000), Sweden

122 CIN2/3 histologically confirmed

0.15–3.95 pack–years 4.00–7.95 pack–years ≥ 8.00 pack–years 1.0 2.3 (1.0–5.6) 2.6 (1.2–5.6)

Never and party smokers 1–4 cigarettes/day 5–14 cigarettes/day ≥ 15 cigarettes/day

0.5 (0.1–1.9) 3.2 (1.2–8.4) 5.8 (1.7–19.4) p-trend < 0.001

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Table 51. Case–control studies of tobacco smoking and pre-invasive and invasive cervical cancer restricted to HPV-positive women

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Table 51 (contd) Smoking statusa

Odds ratio (95% CI)

Intensity/duration of smoking

Odds ratio (95% CI)

Detection method and comments

Hildesheim et al. (2001), Costa Rica

136 HSIL/cancer high-risk HPVinfected, histologically confirmed

624 high-risk HPV

Former Current

2.4 (1.2–5.1) 2.3 (1.3–4.3)

1–5 cigarettes/day ≥ 6 cigarettes/day < 10 years ≥ 10 years

2.3 (1.3–3.9) 2.7 (1.1–6.7) 2.6 (1.2–5.3) 2.2 (1.2–4.2) p-trend = 0.003

HC2 + PCR; no association with passive exposure; adjusted for age, no. of pregnancies, HPV types

Lacey et al. (2001), USA

58 ADC

49 healthy community members

Ever Former Current

0.7 (0.3–1.5) 1.0 (0.4–2.5) 0.5 (0.2–1.1)

< 1 pack/day ≥ 1 pack/day

1.2 (0.5–2.9) 0.4 (0.2–1.0) p-trend = 0.10 0.9 (0.4–4.0) 1.3 (0.4–2.1) 0.4 (0.1–4.0) p-trend = 0.25

PCR MY09/11; adjusted for age, ethnicity

≤ 10 years 11–20 years > 20 years 70 SCC

Ever Former Current

1.5 (0.7–3.0) 1.2 (0.5–3.1) 1.6 (0.7–3.5)

< 1 pack/day ≥ 1 pack/day ≤ 10 years 11–20 years > 20 years

1463 squamous ICC 211 CIS

254 hospitaland populationbased

Ever Former Current

Giuliano et al. (2004), Mexico border

35 ASCUS/AGUS 25 LSIL 19 HSIL cytology-based diagnosis

201 normal cytology

ASCUS/AGUS Former Current LSIL Former Current HSIL Former Current

2.08 (1.33–3.27) 1.80 (0.95–3.44) 2.30 (1.31–4.04)

1.57 (0.52–4.76) 0.75 (0.29–1.92)

≤ 5 cigarettes/day > 6 cigarettes/day < 20 years ≥ 20 years

1.89 (1.05–3.41) 2.23 (1.18–4.20) 2.36 (1.30–4.29) 1.85 (0.97–3.51)

PCR MY09/11; pooled analysis of 10 case–control studies; adjusted for age, centre, education, no. of sexual partners, age at first intercourse, OC use, parity, screening





PCR MY09/11; adjusted for country, parity, C. trachomatis infection, Pap smear in the past 3 years, age

0.44 (0.05–3.78) 2.19 (0.82–5.85) 0.43 (0.05–3.63) 0.61 (0.16–2.30)

281

Plummer et al. (2003), 4 continents

1.6 (0.6–3.9) 1.3 (0.6–3.0) p-trend = 0.49 1.2 (0.5–3.0) 3.2 (1.0–9.7) 0.8 (0.3–2.7) p-trend = 0.57

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Smoking statusa

Harris et al. (2004), USA

High-risk HPVpositive 137 CIN 1 143 CIN 2/3 histologically confirmed

181 ≤ ASCUS and high-risk HPV-positive

Odds ratio (95% CI)

Intensity/duration of smoking

Odds ratio (95% CI)

CIN1 Former Current

1.7 (0.8–3.6) 1.8 (1.1–3.1)

1–10 cigarettes/day > 10 cigarettes/day 0.1–5 pack–years > 5 pack–years

1.4 (0.9–2.5) 2.5 (1.2–5.3) 1.7 (1.0–2.8) 2.1 (1.0–4.5)

CIN2/3 Former Current

2.0 (0.9–4.1) 1.6 (1.0–2.7)

1–10 cigarettes/day > 10 cigarettes/day 0.1–5 pack–years > 5 pack–years

1.4 (0.8–2.4) 2.6 (1.3–5.5) 1.4 (0.8–2.4) 2.6 (1.3–5.2)

Detection method and comments

PCR MY09/11; adjusted for age, no. of HPV types

ADC, adenocarcinoma; AGUS, atypical glandular cells of undetermined significance; ASCUS, atypical squamous cells of undetermined significance; CI, confidence interval; CIN, cervical intraepithelial neoplasia; CIS, carcinoma in situ; HC2, Hybrid Capture 2; HSIL, high-grade intraepithelial lesion; ICC, invasive cervical cancer; LSIL, low-grade squamous intraepithelial lesion; OC, oral contraceptive; Pap, Papanicolaou test; PCR, polymerase chain reaction; SCC, squamous-cell carcinoma; SIL, squamous intraepithelial cells The reference category is never smokers, if not otherwise specified.

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from the most recent Pap smear. A significant approximately twofold increase in risk for carcinoma in situ was observed for both former and current smokers compared with those who had never smoked. Among women who participated in a study in Västerbotten County, Sweden, ever having used tobacco was associated with a strong dose–response in the increase in risk for CIN2/3 (Kjellberg et al., 2000). Among women with high-risk HPV infection, Hildesheim et al. (2001) observed a significant approximately twofold increase in risk for HSIL and cervical cancer among former and current smokers. In a multicentric case–control study among women in the USA, Lacey et al. (2001) did not observe a significant increase in risk for adenocarcinoma or squamous-cell carcinoma of the cervix with smoking. Plummer et al. (2003) pooled data from eight IARC case–control studies of invasive cervical cancer and two studies of carcinoma in situ, including the study of Muñoz et al. (1993). More than 1600 cases were included in the pooled analysis. Ever and current smokers had a twofold higher risk for cervical cancer compared with those who had never smoked. Increasing intensity and duration of smoking did not confer substantial additional risk. In a study conducted at the USA–Mexico border among Hispanic women, Giuliano et al. (2004) did not observe a significant association between tobacco smoking and the risk for LSIL or HSIL. Harris et al. (2004) reported an increase in risk for ≥ CIN1 among women in the USA who were positive for high-risk HPV-type infections and who were current smokers. A dose–response in the increase in risk for ≥ CIN1 was observed for intensity of smoking. (ii) Prospective studies Several large prospective cohort studies have published risk estimates for the association between tobacco smoking and risk for cervical cancer (Table 52). Deacon et al. (2000) reported a significant increase in risk among participants in a cohort study of 61 570 women in Manchester, United Kingdom. In this nested case– control study, 199 histologically confirmed incident cases of CIN3 were compared with 181 HPV-positive controls (women with normal to CIN2 lesions). In adjusted analyses, ever use of tobacco was associated with a significant more than twofold increase in risk, as was smoking one or more packs of cigarettes per day. Among participants of the Kaiser Permanente Cohort in Portland, Oregon (USA), Castle et al. (2002b) reported a significant increase in risk of at least twofold for incident CIN3 among former smokers and current smokers compared with nonsmokers, regardless of the intensity of smoking and analytical method used to assess the association (Kaplan Meier estimates versus logistic regression) among women infected with high-risk HPV types. Other prospective studies supported a role for tobacco smoking in the modulation of the natural history of HPV infections. In a prospective study in the USA, Giuliano et al.

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No. and type of controls

Smoking status

Odds ratio (95% CI)

Intensity/ duration of smoking

Odds ratio (95% CI)

Detection method and comments

Deacon et al. (2000), United Kingdom

61 570

199 incident CIN3, histologically confirmed

181 normal and < CIN3

Ever Former

2.20 (1.44–3.35) 1.69 (0.76–3.75)

< 1 pack/day ≥ 1 pack/day

1.48 (0.79–2.76) 2.57 (1.49–4.45) p trend < 0.001

PCR MY09/11; adjusted for age at first intercourse, total no. of sex partners, years since start of last regular relationship, history of spontaneous abortion

Castle et al. (2002b), USA

20 759 women of whom 1812 high-risk HPVpositive

58 incident CIN3 10 incident cancers (highrisk HPVpositive only)

1790 with normal cytology

Former

2.1 (1.1–3.9)a

Former

3.3 (1.6–6.7)b

< 1 pack/day ≥ 1 pack/day < 1 pack/day ≥ 1 pack/day

2.2 (1.2–4.2)a 2.9 (1.5–5.6)a 2.9 (1.4–6.1)b 4.3 (2.0–9.3)b

HC2; 10 years follow-up; matched by cytologic interpretation of baseline Pap smears, age, screening behaviour a Kaplan Meier analysis b Logistic model

See Table 7 for a description of the primers used. CI, confidence interval; CIN, cervical intraepithelial neoplasia; HC2, Hybrid Capture 2; Pap, Papanicolaou test; PCR, polymerase chain reaction

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(2002a) observed a longer duration of high-risk HPV infections and a lower probability of clearing these infections among women who had ever smoked. Similarly, in a cohort of HIV-positive and -negative women, Minkoff et al. (2004) observed a significant increase in the incidence of persistent HPV infections among current smokers. (b)

Hormonal contraceptive use

The use of hormonal contraceptives, most commonly combined oral contraceptive formulations of estrogen and progesterone, has been hypothesized to be associated with development of pre-invasive and invasive cervical lesions (Castellsagué & Muñoz, 2003). In addition to the limitations in design of research conducted without taking HPV status appropriately into account, the investigation of hormonal contraceptives relative to the risk for cervical cancer has been limited by confounding with Pap smear history, and formulation and dose of hormonal contraceptives. (i) Case–control studies (Table 53) Several case–control studies evaluated the risk for pre-invasive and invasive cervical cancer associated with the use of exogenous hormones, either for contraception or control of menopausal symptoms, among HPV-positive women (Kjaer et al., 1996; Kruger-Kjaer et al., 1998; Lacey, J.V. et al., 1999; Ylitalo et al., 1999; Lacey et al., 2000; Hildesheim et al., 2001; Moreno et al., 2002; Berrington et al., 2002; Smith et al., 2003; Giuliano et al., 2004; Shields et al., 2004). Of these, four observed significant associations with invasive lesions (Ylitalo et al., 1999; Berrington et al., 2002; Moreno et al., 2002; Smith et al., 2003) and one with adenocarcinoma in situ (Lacey et al., 1999). In a case–control study in a Danish population reported by Kjaer et al. (1996) and updated by Kruger-Kjaer et al. (1998), no association with preneoplastic disease was observed for current users of oral contraceptives or current users with a long duration of use. In contrast, Ylitalo et al. (1999) reported a significantly increased cervical cancer risk of 2.65 for current oral contraceptive use in Sweden. The risk was significant after 2 years of use. Studies conducted among populations in the USA have failed to detect associations with either pre-invasive or invasive squamous-cell carcinoma despite adjustment for Pap smear screening history (Lacey, J.V. et al., 1999; Giuliano et al., 2004). However, Lacey, J.V. et al. (1999) detected a significant positive association between current oral contraceptive use and adenocarcinoma in situ. In the same study population, no association between non-contraceptive hormonal use and either adenocarcinoma or squamous-cell carcinoma of the cervix was detected (Lacey et al., 2000). In a prevalent case–control study conducted by Hildesheim et al. (2001) in Costa Rica, a significant increase in risk for HSIL/cancer was observed only among oral contraceptive users with a duration of use of 5 years or more who had had three or fewer pregnancies. In a meta-analysis of studies restricted to HPV-positive women, Smith et al. (2003) observed a significantly increased risk (odds ratio, 2.5; 95% CI, 1.6–3.9) for CIN3 and invasive cervical cancer only for long-term oral contraceptive use (≥ 10 years). Shields et al. (2004) found no significant association between endogenous

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OC use status

Kjaer et al. (1996), Denmark

141 HSIL and SIL combined

153 cytologically normal

Never users ≥ 20 years 17–19 years ≤ 16 years

Kruger-Kjaer et al. (1998), Denmark

82 ASCUS 86 LSIL 71 HSIL

155 cytologically normal

No association reported

Lacey, J.V. (1999), USA

48 squamous CIS 91 squamous ICC 33 ADC in situ 91 ADC

48 healthy population-based

ADC in situ Ever Former Current Invasive ADC Ever Former Current

Squamous-cell CIS Ever Former Current

Squamous ICC Ever Former Current

Odds ratio (95% CI)

Intensity/ duration of use

Odds ratio (95% CI)

Detection method and comments





PCR GP5+/6+; adjusted for age

NR





PCR GP5+/6+; adjusted for age, years of sex life without barrier contraceptive, partner’s education, marital status; [update of the study by Kjaer et al. (1990)]

5.4 (0.7–43.4) 3.1 (0.4–27.5) 17.1 (1.5–188.2)

≤ 2 years 2–6 years > 6 years

4.0 (0.4–44.3) 4.8 (0.4–51.9) 6.2 (0.7–52.7) p-trend = 0.12

1.3 (0.4–4.4) 1.3 (0.4–4.1) 2.1 (0.4–11.9)

≤ 2 years 2–6 years > 6 years

1.5 (0.3–6.6) 1.1 (0.2–5.2) 1.0 (0.2–4.2) p-trend = 0.88

1.7 (0.5–6.2) 1.8 (0.5–6.7) 1.6 (0.3–8.5)

≤ 2 years 2–6 years > 6 years

1.4 (0.3–7.2) 3.8 (0.7–19.3) 1.1 (0.3–5.0) p-trend = 0.85

1.2 (0.4–3.8) 1.0 (0.3–3.2) 0.7(0.1–3.6)

≤ 2 years 2–6 years > 6 years

1.1 (0.3–4.2) 1.9 (0.4–8.4) 0.9 (0.2–3.7) p trend = 0.99

0.5 (0.2–1.4) 0.6 (0.3–1.3) 0.8 (0.4–1.7)

PCR MY09/11; multicentre study; adjusted for age, ethnicity, income, lifetime no. of sexual partners, no. of Pap smears; current use is defined as use 12 months before diagnosis for cases and at reference date for controls.

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No. and type of controls

OC use status

Odds ratio (95% CI)

Intensity/ duration of use

Odds ratio (95% CI)

Detection method and comments

Ylitalo et al. (1999), Sweden

178 CIS

178 HPV16/18positive

Former Current

1.54 (0.76–3.12) 2.65 (1.06–6.67)

< 2 years 2–< 10 years ≥ 10 years

1.55 (0.65–3.70) 2.23 (1.02–4.86) 2.79 (1.14–6.87)

PCR; adjusted for marital status, smoking, age at sexual debut, no. of sexual partners, age at menarche, parity, years in school

Lacey et al. (2000), USA

139 SCC 124 ADC

49 healthy community members matched by age, ethnicity, residence

Ever use ADC 1.1 (0.31–3.9) SCC

0.49 (0.13–1.9)

< 3 months ≥ 3 months < 3 months ≥ 3 months

1.4 (0.23–8.4) 0.9 (0.17–4.7) 0.8 (0.13–5.1) 0.3 (0.04–1.9)

PCR MY09/11; multicentric study; non-contraceptive hormone use; no association with either age at first use or whether estrogen was opposed or unopposed.

Hildesheim et al. (2001), Costa Rica

136 HSIL/cancer high-risk HPVpostive histologically confirmed

624 high-risk HPV

Former Current

0.93 (0.55–1.6) 1.5 (0.83–2.8)

< 5 years ≥ 5 years

0.99 (0.58–1.7) 1.30 (0.70–2.3)

HC2 + PCR; adjusted for age, no. of pregnancies, cigarettes/day; no statistical interaction with parity; however, significantly elevated risk observed among women with OC use ≥ 5 years and having < 3 pregnancies (odds ratio, 3.1; 95% CI, 1.1–1,9)

Berrington et al. (2002), United Kingdom

221 ICC

393 from general practitioners’ records





0 year 1–4 years 5–9 years ≥ 10 years

1.00 (0.1–7.8) 1.74 (0.8–3.8) 0.76 (0.3–2.1) 3.92 (1.1–14.1)

Serology; odds ratio calculated as floating absoluted risk with floating CI

Moreno et al. (2002), 4 continents

1676 squamous ICC and CIS

255 population- or hospital-based

Ever

1.42 (0.99–2.04)

1 year 2–4 years 5–9 years ≥ 10 years

0.67 (0.41–1.08) 0.80 (0.51–1.24) 2.82 (1.46–5.42) 4.03 (2.09–7.79)

PCR MY09/11; pool of 10 case– control studies (8 ICC and 2 CIS); adjusted for age, centre, education, no. of sex partners, age at first intercourse, parity, no. of Pap smears in life

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287

Shapiro et al. (2003), South Africa

484 SCC 40 ADC

254

Injectable (progesterone) Ever

0.9 (0.6–1.5)

< 1 year 1–4 years 5–9 years 10–14 years ≥ 15 years

0.9 (0.5–1.6) 0.9 (0.6–1.6) 0.8 (0.5–1.4) 1.1 (0.6–2.2) 0.8 (0.4–1.7)

Combined OC use Ever

0.9 (0.7–1.3)

< 1 year 1–4 years ≥ 5 years

0.8 (0.5–1.2) 0.9 (0.6–1.6) 1.3 (0.6–2.7)

< 5 years 5–9 years ≥ 10 years

0.9 (0.7–1.2) 1.3 (1.0–1.9) 2.5 (1.6–3.9)

Smith et al. (2003), 4 continents

1279 cases CIN 3 and ICC

265

NR

Giuliano et al. (2004), USA– Mexico Border

35 ASCUS/AGUS 25 LSIL 19 HSIL cytology–based diagnosis

201

HSIL OC use Former Current Injectable use Former Current ASCUS/AGUS OC use Former Current Injectable use Former Current LSIL Former Current Injectable use Former Current

Odds ratio (95% CI)

0.50 (0.15–1.66) 0.66 (0.17–2.59) 0.63 (0.16–2.54) 1.75 (0.40–7.65)

0.87 (0.53–1.42) 1.38 (0.81–2.36) 0.86 (0.49–1.52) 0.90 (0.41–2.00) 0.59 (0.28–1.24) 0.35 (0.11–1.11) 0.86 (0.33–2.22) 1.61 (0.54–4.82)

Intensity/ duration of use

Odds ratio (95% CI)

Detection method and comments

Study population had high exposure to hormonal contraceptives and high rate of disease; adjusted for age, ethnicity, age at first intercourse, lifetime no. of sex partners, education, smoking, rural/urban residence, no. of previous Pap smears

Meta-analysis of 5 case–control studies including Lacey et al. (1999), Deacon et al. (2000), Hildesheim et al. (2001), Berrington et al. (2002) and Moreno et al. (2002). Each study adjusted for different potential confounding factor. PCR PGMY09/11; adjusted for country, parity, C. trachomatis infection, Pap smears in the past 3 years, age

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Table 53 (contd)

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No. and type of controls

Shields et al. (2004), Costa Rica

67 ≥ CIN2 premenopausal 43 ≥ CIN2 postmenopausal

134 premenopausal 86 post-menopausal

OC use status

Odds ratio (95% CI)

No association

Intensity/ duration of use

Odds ratio (95% CI)

Detection method and comments

No associations between sex hormone binding globulins, estradiol, free estradiol, estrone, estrone sulfate, or dehydroepiandrosterone and disease regardless of menopausal status; adjusted for menopausal status, age, days since last menses or years since menopause

See Table 7 for a description of the primers used. ADC, adenocarcinoma; AGUS, atypical glandular cells of undetermined significance; ASCUS, atypical squamous cells of undetermined significance; CI, confidence interval; CIN, cervical intraepithelial neoplasia; CIS, carcinoma in situ; HC2, Hybrid Capture 2; HSIL, high-grade intraepithelial lesion; ICC, invasive cervical cancer; LSIL, low-grade squamous intraepithelial lesion; NR, not reported; OC, oral contraceptive; Pap, Papanicolaou test; PCR, polymerase chain reaction; SCC, squamous-cell carcinoma; SIL, squamous intraepithelial cells

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circulating hormone concentrations and risk for HSIL among the same Costa Rican study population. In summary, no study reported a significant increased risk for invasive cervical cancer in ever versus never users of oral contraceptives. When data were pooled across the studies, a significant elevation in risk was only observed among women who had used oral contraceptives for 5 or more years and the risk increased further among those with a duration of use of 10 or more years (Smith et al., 2003). Among a South African population that has a high burden of cervical cancer, a high prevalence of hormonal contraceptive use and a low prevalence of Pap smear screening, Shapiro et al. (2003) observed no significant associations with hormonal contraceptive use regardless of the formulation (combined oral estrogen and progesterone or injected progesterone) or duration of use. (ii) Prospective studies (Table 54) Two prospective studies evaluated the association between hormonal contraceptive use and incidence of CIN among HPV-positive women. Deacon et al. (2000) reported no significant increase in risk among current users of oral contraceptive in a study conducted in the United Kingdom. Similarly, Castle et al. (2002b) reported no association with current oral contraceptive use among women aged 16 and older in the USA. (c)

Parity

(i) Case–control studies (Table 55) For several decades, high parity has been suspected to increase the risk for in-situ carcinoma and cancer of the cervix. Unfortunately, many of the studies on cervical cancer either did not measure HPV or did not control for HPV infection or other variables in sexual history that are potential confounders of the association between parity and risk for cervical cancer. The few studies that restricted their statistical analyses to HPV-positive women are reviewed below. In Denmark, Kjaer et al. (1996) observed an increased risk of borderline significance of 1.9 between one or more live births and the risk for ASCUS and SIL combined compared with women who reported no previous pregnancies. However, in a later update of the study by Kruger-Kjaer et al. (1998), no significant associations were observed when the cytological categories ASCUS, LSIL and HSIL were examined separately. Hildesheim et al. (2001) observed a significant elevation in risk for HSIL/cancer among women who had had four to five live births among participants in Costa Rica. However, a linear increase in risk with increasing parity was not observed. The strongest evidence for an association between parity and risk for cervical cancer is from the pooled analysis conducted at IARC on 10 case–control studies by Muñoz et al. (2002). Although only two of the eight individual studies of invasive cervical cancer and one of the two studies of carcinoma in situ observed significant associations with parity, when the studies were pooled, an odds ratio of 1.81 was observed for women who reported one to two full-term pregnancies compared with none. The risk estimate increased to 3.82 among women who had had seven or more full-term pregnancies. While no statistically

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No. and type of cases

No. and type of controls

OC use status

Odds ratio (95% CI)

Intensity/ duration of OC use

Odds ratio (95% CI)

Detection method and comments

Deacon et al. (2000), United Kingdom

61 570

199 incident CIN3 histologically confirmed

181 < CIN3

Former Current

1.15 (0.63–2.10) 1.28 (0.66–2.50)

1–47 months 48–95 months ≥ 96 months

1.19 (0.58–2.43) 0.76 (0.38–1.53) 1.52 (0.80–2.88)

PCR MY09/MY11; no age restriction

Castle et al. (2002b), USA

20 759 women of whom 1812 highrisk HPVpositive

58 incident CIN3 10 incident cancers (highrisk HPVpositive only)

1790 with normal cytology

Current

0.84 (0.49–1.5)a 0.61 (0.32–1.1)b

HC2; 10 years followup; no adjustment a Kaplan Meier b Conditional logistic model

HUMAN PAPILLOMAVIRUSES

Reference, study location

See Table 7 for a description of the primers used. CI, confidence interval; CIN, cervical intraepithelial neoplasia; HC2, Hybrid Capture 2; PCR, polymerase chain reaction

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No. of full term pregnancies

Odd ratio (95% CI)

Detection method and comments

Kjaer et al. (1996), Denmark

141 ASCUS and SIL combined

153 cytologically normal

0 ≥1

0.9 (0.5–1.5) 1.9 (1.0–4.4)

PCR GP5+/6+; adjusted for age

Kruger–Kajer et al. (1998), Denmark

82 ASCUS 86 LSIL 71 HSIL

155 cytologically normal

HSIL 0 ≥1

0.8 (0.4–1.7) 1.8 (0.3–2.3)

Hildesheim et al. (2001), Costa Rica

136 HSIL/cancer highrisk HPV-positive, histologically confirmed

624 high-risk HPV

0–1 2 3 4–5 6–8 ≥9

1.0 1.0 (0.48–2.2) 1.5 (0.73–3.2) 3.5 (1.7–7.2) 2.2 (0.98–5.0) 1.4 (0.56–3.4) p trend = 0.04

PCR GP5+/6+; adjusted for age, years of sex life without barrier contraceptive, partner’s education, marital status; no association with either ASCUS or LSIL; follow-up of the study by Kjaer et al. (1996) HC2 + PCR; no association with passive exposure; adjusted for age, no. of pregnancies, cigarettes/day

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Table 55. Case–control studies of parity and pre-invasive and invasive cervical cancer restricted to HPVpositive women

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Table 55 (contd) No. of full term pregnancies

Muñoz et al. (2002), 4 continents

1676 squamous CIS and ICC 124 adeno CIS and ICC

255

Squamous CIS/ ICC 0 1–2 3–4 5–6 ≥7

Giuliano et al. (2004), USA– Mexico Border

35 ASCUS/AGUS 25 LSIL 19 HSIL cytology-based diagnosis

201

Odd ratio (95% CI)

1.00 (0.55–1.81) 1.81 (1.31–2.52) 2.55 (1.95–3.34) 2.83 (2.02–3.96) 3.82 (2.66–5.48) p trend = 0.0002

Adeno CIS/ICC 0 1–2 3–4 5–6 ≥7

1.00 (0.21–4.86) 3.47 (1.80–6.70) 2.90 (1.77–4.75) 2.47 (1.44–4.23) 2.92 (1.55–5.50)

0 1–2 3–4 ≥5

1.0 0.38 (0.08–1.88) 2.14 (0.60–7.64) 0.81 (0.06–10.65)

Detection method and comments

PCR MY09/11; pool of 10 case– control studies (8 ICC and 2 CIS); adjusted for age, centre, education, no. of sex partners, age at first intercourse, OC use, smoking, history of Pap smears; CIs were estimated by treating the relative risk as a floating absolute risk.

PGMY 09/11; adjusted for country, parity, C. trachomatis infection, Pap smears in the past 3 years, age

See Table 7 for a description of the primers used. AGUS, atypical glandular cells of undetermined significance; ASCUS, atypical squamous cells of undetermined significance; CI, confidence interval; CIS, carcinoma in situ HC2, Hybrid Capture 2; HSIL, high-grade intraepithelial lesion; ICC, invasive cervical cancer; LSIL, low-grade squamous intraepithelial lesion; OC, oral contraceptive; Pap, Papanicolaou test; PCR, polymerase chain reaction; SIL, squamous intraepithelial cells

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significant interaction was detected, there appeared to be a higher risk among women with high parity and young age at first full-term pregnancy, and high parity and 5 or more years of oral contraceptive use. A significant increase in risk for adenocarcinoma was also detected among women with one to two full-term pregnancies, although this did not increase linearly with increasing parity. In a study of women residing along the USA–Mexico border by Giuliano et al. (2004), no significant association between SIL and parity was observed. (ii) Prospective studies (Table 56) The number of prospective studies that have evaluated the association between parity and risk for cervical cancer among HPV-positive women is limited. Deacon et al. (2000) observed no increase in risk with increasing parity among women participating in a cohort study in Manchester, United Kingdom. Similarly, Castle et al. (2002b) found no increase in risk for CIN3 or cancer among participants in the cohort study in Guanacaste, Costa Rica. However, this study is limited by the fact that parity was assessed only at baseline with no further assessment throughout the 10-year follow-up period. (d)

Nutrients

Over the past few decades, numerous studies have examined the association between risk for cervical cancer and dietary intake or serological measures of nutrient concentrations. However, most of these studies have methodological limitations that include lack of measurement or adequate consideration of HPV infection in the analyses. Of the studies that did measure HPV infection, very few restricted their analyses to HPV-positive women. Therefore, only a small proportion of all studies are reviewed here. In addition to the problems of assessment of HPV infection, a drawback that is common to all studies that attempt to examine associations between nutrients and disease is the incomplete consideration of confounding factors, such as tobacco smoking or oral contraceptive use, which are associated with both cervical cancer and nutritional status. In addition, most studies were conducted before the availability of reliable laboratory methods for separating and quantifying the major carotenoids and their geometric isomers in serum. Finally, due to the significant disparity in the content of nutrients in foods by region and variety, the data on carotenoid, selenium and folate content in the food supply are liable to be imprecise. The net result of these limitations is a significant exposure misclassification that results in attenuation of the true association. (i) Case–control studies (Table 57) Of the studies that restricted analyses to HPV-positive women, only two assessed the association between dietary intake of nutrients and the risk for cervical cancer or CIN (Wideroff et al., 1998; Rajkumar, 2003). Wideroff et al. (1998) examined the association between vitamins A, C and E, β-carotene, folate and zinc and the risk for incident HSIL among women resident in Portland, Oregon (USA). Although the risk appeared to be lower among those who consumed higher concentrations of β-carotene, folate and zinc, none of

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Table 56. Prospective studies of parity and pre-invasive cervical cancer restricted to HPV-positive women No. and type of cases

No. and type of controls

Number of full term pregnancies

Odds ratio (95% CI)

Detection method and comments

Deacon et al. (2000), United Kingdom

61 570

199 incident CIN3 histologically confirmed

181 (includes < CIN 3) stratified random sampling

0 1 2 ≥3

1.0 1.57 (0.88–2.77) 1.13 (0.64–1.99) 1.90 (0.94–3.85)

PCR MY09/MY11

Castle et al. (2002b), USA

20 759 women of whom 1812 high-risk HPV-positive women

58 incident CIN3 10 incident cancers (high-risk HPVpositive only)

1790 with normal cytology

0 1–2 ≥3 0 1–2 ≥3

1.0 1.1 (0.64–1.7)a 0.7 (0.31–1.6)a 1.0 1.2 (0.67–2.1)b 0.7 (0.24–1.9)b

HC2; parity assessed at enrolment only, not throughout the 10 year follow–up period a Kaplan Meier analysis b Logistic regression models

See Table 7 for a description of the primers used. CI, confidence interval; CIN, cervical intraepithelial neoplasia; HC2, Hybrid Capture 2; PCR, polymerase chain reaction

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44 CIN3

163 CIN1

CIN3 versus CIN1 Vitamin C 2.86 (0.61–13.52) Log α-toc. 0.63 (0.04–9.01) Log β-car. 0.49 (0.13–1.82) Retinol 1.01 (1.00–1.03)

PCR MY09/11 ; adjusted for age, education, ethnicity, no. of Pap smears in past 3 years, high-risk versus low-risk HPV, smoking status; odds ratio for a unit increase in micronutrient level

Ho et al. (1998c), USA

262 histologically confirmed ≥ CIN1

80 normal cytology with no history of abnormal cytology

CIN1–3 versus normal Vitamin C 0.34 (0.13–1.00) Log α-toc 0.25 (0.04–1.66) Log β-car. 0.88 (0.33–2.33) Retinol 0.99 (0.98–1.00) Vitamin C < 0.8 mg/dL 1.00 ≥ 0.8 mg/dL 0.41 (0.19–0.89)

PCR MY09/11; adjusted for age, ethnicity, income, smoking status; odds ratio for a unit increase in micronutrient level

Wideroff et al. (1998), USA

68 high-risk HPV-positive HSIL

69 high-risk HPV-positive

Diet + supplements Vitamin A quartiles 1 1.0 2 1.9 (0.6–5.5) 3 1.0 (0.3–2.8) 4 1.4 (0.5–4.2) β-Carotene quartiles 1 1.0 2 0.6 (0.2–2.0) 3 0.8 (0.2–2.3) 4 0.6 (0.2–2.0)

PCR and dot blot hybridization; adjusted for age

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Detection method and comments

Vitamin C quartiles 1 1.0 2 1.9 (0.7–5.6) 3 1.0 (0.4–2.8) 4 1.3 (0.4–3.6) Vitamin E quartiles 1 1.0 2 0.8 (0.2–2.2) 3 0.6 (0.2–1.7) 4 1.0 (0.4–2.6) Folate quartiles 1 1.0 2 0.7 (0.2–1.9) 3 0.7 (0.2–2.2) 4 0.7 (0.3–2.1) Zinc quartiles 1 1.0 2 1.0 (0.3–2.9) 3 1.0 (0.4–2.9) 4 0.8 (0.3–2.2)

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Odds ratio for diet (95% CI)

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Table 57 (contd)

673 HIVinfected normal cytology

Retinol ≥ 1.1 μmol/L 1.0 < 1.1 μmol/L 1.8 (1.1–1.3)

PCR MY09/11; analyses adjusted for age, race/ethnicity, CD4+ cell count, HIV type 1 RNA, body mass index, serum albumin

Goodman et al. (2001), USA

150 histologically confirmed SIL

179 normal cytology

MTHFR variants CC 1.0 CT 2.0 (1.1–3.7) TT 2.9 (1.0–8.8) p trend = 0.02

PCR MY09/11 and dot-blot hybridization; adjusted for age, ethnicity, tobacco, alcohol, no. of sex partners before age 20 years, HPV infection

297

208 HIVinfected SIL

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75 (serum)/63 (RBC folate) histologically confirmed ICC

Weinstein et al. (2001b) USA

Rajkumar et al. (2003), Chennai, India

Odds ratio for diet (95% CI)

Odds ratio for serum/plasma levels (95% CI)

Detection method and comments

27 (serum)/23 (RBC folate) HPV 16seropositive

Serum folate High 1.0 Low 2.4 (0.8–7.4) RBC folate High 1.0 Low 1.4 (0.5–4.8)

Serology ELISA; adjusted for ethnicity, study site

183 ICC

79 HPV 16seropositive

Homocysteine quartiles 1 1.00 2 2.45 (0.9-7.1) 3 3.81 (1.3-11.2) 4 1.93 (0.6-5.9) p trend = 0.42

Serology only; adjusted for age, ethnicity, study site, no. of sex partners, age at first intercourse, years since last Pap smear, no. of pregnancies, smoking, OC use, education, income

190 ICC

51 cytologically normal

Vegetable and fruit intake (servings/week) < 6 1.00 6 0.88 (0.26–3.03) ≥ 7 0.37 (0.11–1.22) p trend = 0.08

PCR GP5+/6+; adjusted for age, area of residence, occupation, marital status, age at first marriage, no. of pregnancies, husband’s extramarital affairs, body mass index, chewing habit

See Table 7 for a description of the primers used. β-car., β-carotene; CI, confidence interval; CIN, cervical intraepithelial neoplasia; ELISA, enzyme-linked immunosorbent assay; HSIL, high-grade intraepithelial lesion; ICC, invasive cervical cancer; MTHFR, methylene tetrahydrofolate reductase; OC, oral contraceptive; Pap, Papanicolaou test; PCR, polymerase chain reaction; RBC, red blood cell; α-toc., α-tocopherol

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Table 57 (contd)

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these associations reached statistical significance. Among women resident in Chennai, India, Rajkumar et al. (2003) observed a non-significant inverse association between consumption of vegetables and fruit and the risk for invasive cervical cancer. Serum retinol has been examined in two studies that restricted their analyses to HPVpositive women. Ho et al. (1998b,c) did not observe significant associations between serum retinol and CIN1–3 among women in the USA. This was the only study that examined the association between serum carotenoids, α-tocopherols and vitamin C concentrations and risk for CIN1–3 combined or considered separately. Only serum vitamin C was significantly associated with a reduced risk for disease (odds ratio, 0.41 for ≥ 0.8 mg/dL versus < 0.8 mg/dL), and the association was limited to the comparison between women with CIN1–3 and those with normal cytology. Among women infected with HIV in the USA, French et al. (2000) observed significant associations between higher serum retinol concentrations and the risk for SIL. Two studies examined the concentration in serum or red blood cells of nutrients that are involved in one-carbon methyl transfer reactions, such as folate and vitamin B12, or the accumulation in serum of homocysteine, a biomarker of insufficient one-carbon nutrient status (Goodman et al., 2001; Weinstein et al., 2001a,b). Weinstein et al. (2001a,b) observed elevated risks for invasive cervical cancer among women in the USA who had low serum and red blood cell concentrations of folate, although these associations did not reach statistical significance. In the same study, an increase in risk for invasive cervical cancer with elevated levels of serum homocysteine was observed, but no significant trend in risk. Goodman et al. (2001) observed a significant increase in risk for SIL among women with single nucleotide variants in the methylenetetrahydrofolate reductase gene, which is involved in the methylation of homocysteine to methionine. The analysis was not restricted to HPV-positive women but HPV status was controlled for in the statistical analysis. (ii) Prospective studies (Table 58) Of the prospective studies that restricted analyses to HPV-positive women, only one examined risk for invasive cervical cancer (Lehtinen et al., 1999) and the others examined the risk for persistent HPV infection (Giuliano et al., 1997; Sedjo et al., 2002a,b; Giuliano et al., 2003; Sedjo et al., 2003a,b). Lehtinen et al. (1999) examined the association between serum retinol and α-tocopherol and the risk for invasive cervical cancer among women who were resident in Finland and Sweden. No significant associations were observed, although retinol levels appeared to interact with HPV status. Giuliano et al. (1997) observed significant associations among Hispanic women resident in the USA between serum β-carotene, β-cryptoxanthin, lutein and α-tocopherol when those who were transiently infected with HPV were compared with those who were persistently infected with high-risk HPV over a 3-month period. The Young Women’s Health Study reported a decreased risk for HPV persistence for the highest versus the lowest tertile of serum levels of vitamin B12 (odds ratio, 0.40) and cis-

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Giuliano et al. (1997), USA

33 persistently HPV-positive women

Lehtinen et al. (1999), Finland and Sweden

Sedjo et al. (2002a), USA

Odds ratio for diet (95% CI)

Odds ratio/p value for serum/ plasma levels (95% CI)

Detection method and comments

32 intermittently HPV-positive women

α-Carotene β-Carotene Lycopene β-cryptoxanthin Lutein α-Tocopherol Vitamin C

HC 1 high-risk probe; p-value for difference in adjusted mean (μM) value; Mexican American women; adjusted for age, age at first intercourse, no. of pregnancies, duration of OC use

34 ICC

105 matched controls

Retinol Low 1.0 High 1.19 (0.55–2.58) α-Tocopherol Low 1.0 High 1.52 (0.70–3.30)

HPV 16-, 18-, 33-seropositive

131 persistent HPV (diet) 109 persistent HPV (nutrient biomarkers)

70 intermittent HPV (diet) 60 intermittent HPV (biomarkers)

Folate tertiles 1 1.00 2 0.95 (0.39–2.29) 3 1.20 (0.51–2.78) p-trend = 0.662 Vitamin B12 tertiles 1 1.00 2 0.57 (0.24–1.32) 3 0.40 (0.17–0.96) p trend = 0.037

HC 2 Probe B; risk of persistent infection; adjusted for age, age at first intercourse, marital status, smoking, race, body mass index

Folate tertiles 1 1.00 2 0.63 (0.29–1.38) 3 0.52 (0.23–1.18) p trend = 0.109 Vitamin B12 tertiles 1 1.00 2 1.47 (0.68–3.19) 3 0.68 (0.30–1.54) p trend = 0.439 Vitamin B6 tertiles 1 1.0 2 0.92 (0.43–2.00) 3 0.61 (0.27–1.39) p trend = 0.254

0.028 0.179 0.351 0.010 0.034 0.001 0.556

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Table 58 (contd) Odds ratio for diet (95% CI)

Odds ratio/p value for serum/ plasma levels (95% CI)

Detection method and comments

Sedjo et al. (2002b), USA

131 persistent HPV (diet) 101 persistent HPV (nutrient biomarkers)

69 intermittent HPV (diet) 58 intermittent HPV (biomarker)

Fruit tertiles 1 (low) 1.00 2 0.89 (0.42–1.87) 3 (high) 0.59 (0.27–1.30) p trend = 0.206 Vegetable tertiles 1 (low) 1.00 2 0.38 (0.17–0.84) 3 (high) 0.46 (0.21–0.97) p trend = 0.033 Lutein tertiles 1 (low) 1.00 2 0.37 (0.13-0.82) 3 (high) 0.50 (0.24–1.07) p trend = 0.054

trans-Lycopene tertiles 1 (low) 1.00 2 0.49 (0.21–1.14) 3 (high) 0.78 (0.35–1.78) p trend = 0.496 cis-Lycopene tertiles 1 (low) 1.00 2 0.57 (0.25–1.29) 3 (high) 0.44 (0.19–1.01) p trend = 0.046

HC 2 Probe B; adjusted for age, race, smoking, body mass index; risk of persistent infection; no significant association detected with plasma vitamin A, α- and β-carotene, β-cryptoxanthin, lutein

Sedjo et al. (2003a), USA

84 high-risk HPV-positive with persistent infection

trans-Lycopene tertiles 1 (low) 1.00 2 3.03 (1.02–7.65) 3 (high) 2.79 (1.17–6.66) p trend = 0.025 cis-Lycopene tertiles 1 (low) 1.00 2 3.50 (1.51–8.08) 3 (high) 2.92 (1.28–6.63) p trend = 0.010

PCR PGMY 09/11; Cox proportional hazard model adjusted for age, ethnicity, no. of new male partners, marital status; hazard ratios were estimated for the association of HPV clearance to each nutrient tertile; no significant association detected with αand β-carotene, β-cryptoxanthin, lutein/zeaxanthin, tocopherols, folate, vitamin B12

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Table 58 (contd) Odds ratio for diet (95% CI)

Giuliano et al. (2003a), Brazil

185 HPV typespecific persistent infection

248 transiently HPV-infected

β-Cryptoxanthin quartiles 1 (low) 1.00 2 0.60 (0.33–1.09) 3 0.48 (0.27–0.87) 4 0.47 (0.26–0.85) p trend = 0.007 Lutein/zeaxanthin quartiles 1 (low) 11.00 2 20.58 (0.32–1.05) 3 30.44 (0.24–0.78) 4 40.49 (0.27–0.87) p trend = 0.066 Vitamin C quartiles 1 1.00 2 0.63 (0.35–1.15) 3 0.84 (0.47–1.48) 4 0.50 (0.27–0.92) p trend = 0.66

Odds ratio/p value for serum/ plasma levels (95% CI)

See Table 7 for a description of the primers used. CI, confidence interval; HC, Hybrid Capture; OC, oral contraceptive; PCR, polymerase chain reaction

Detection method and comments

PCR MY09/11; risk of persistent infection; adjusted for kcal, income, education, no. of persons in household, no. of sex partners during past 5 years, total no. of pregnancies; significant inverse association also detected with papaya and orange consumption; no significant association detected with α- and βcarotene

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lycopene (odds ratio, 0.44) (Sedjo et al., 2002a,b). In the same study, the authors reported an approximately threefold higher probability of oncogenic HPV clearance among women in the highest compared tertile of both trans- and cis-lycopene concentrations (Sedjo et al., 2003a). Increasing levels of dietary vegetables decreased the risk for persistent HPV infection (Sedjo et al., 2002b). Giuliano et al. (2003) assessed the association between dietary nutrient intake and risk for HPV persistence among women who participated in the Ludwig-McGill HPV Natural History Study in Sao Paolo, Brazil. Dietary intakes of βcryptoxanthin, lutein/zeaxanthin and vitamin C were significantly inversely associated with risk for persistent type-specific HPV infection. In addition, consumption of papaya was inversely associated with persistent HPV infection in this population. (iii) Clinical trials (Table 59) In this section, all nutrient-based clinical trials are evaluated, although several did not measure HPV infection and none limited their analyses to HPV-positive women. Randomization assures that the proportion of HPV-positive women is comparable between treatments and that the majority of women who had cervical preneoplastic lesions were HPV-positive. Although randomized clinical trials are considered to be the gold standard to demonstrate the efficacy of a chemopreventive agent, intervention studies of nutrients and risk for cervical cancer present unique problems. First, the widespread adoption of the use of supplemental vitamins and minerals in the USA, where most chemoprevention studies have been carried out to date, can adversely affect the feasibility of testing the efficacy of a single nutrient to prevent cervical cancer. Unlike pharmaceutical agents that proceed through phase I and II trials to determine the safe and optimal dose, a ‘best-guess’ estimate has been used to choose doses for phase III nutrient chemoprevention trials. Information on duration of treatment and length of follow-up that is needed to demonstrate an effect has also been lacking. Finally, the statistical power of a study to assess efficacy is based on the accumulation of an adequate number of events (e.g. regression or progression of CIN). Non-compliance to the study regimen and high rates of spontaneous regression of lesions such as CIN1 (> 80% spontaneous regression) combine to reduce the power of any study to detect differences between treatment and control groups. As a consequence of the above limitations, only one of 10 clinical trials conducted has shown significant protective effects of the nutrient that was tested, which was topical all-trans-retinoic acid (Meyskens et al., 1994). Folate trials Two phase II trials of folic acid for the prevention of cervical cancer have been completed (Butterworth et al., 1992; Childers et al., 1995). Both found no significant effect of treatment on the regression or progression of lesions. In Alabama, USA, Butterworth et al. (1992) found no protective effect of a daily dose of 10 mg folate on either regression or progression of cervical lesions after 6 months of treatment. However, the majority of participants entered the trial with CIN1 lesions that have a high rate of spontaneous

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No. of subjects enrolled

No. of subjects completing trial

Entry diagnosis

End-point

Detection method of primary end-point

Dose

Duration

Results

Folic acid Butterworth et al. (1992), USA

235

199

CIN1/2

Lesion regression and HPV DNA

Biopsy and PCR HPV 16

10 mg/day

6 months

No effect

331

262

89% KA/CIN1 11% CIN2

Lesion regression

Colposcopy

5 mg/day

6 months

No effect

278

278

28% CIN1 42% CIN2 30% CIN3

Lesion regression

Cytology, colposcopy

10 mg/day

3 months

No effect

Fairley et al. (1996), Australia

114

111

5% Atypia 62% HPV 23% CIN1 10% CIN2

Lesion regression and HPV DNA

Cytology and PCR and HC HPV DNA

30 mg/day

12 months

No effect on either lesion regression or HPV positivity

Romney et al. (1997), USA

98

69

51% CIN1 46% CIN2 39% CIN3

Lesion regression and HPV 16

Biopsy and PCR HPV 16

30 mg/day

9 months

Decreased regression in β-carotene arm

Mackerras et al. (1999), Australia

147

141

100% minor atypia or CIN1

Lesion regression

Cytology

30 mg/day β-carotene 500 mg/day vitamin C

24 months

No effect of either β-carotene or vitamin C

Keefe et al. (2001), USA

103

78

43% CIN2 57% CIN3

Lesion regression and HPV

Biopsy and PCR HPV DNA

30 mg/day

24 months

No effect on regression

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Table 59 (contd) No. of subjects completing trial

Entry diagnosis

End-point

Detection method of primary end-point

Dose

Duration

Results

Retinoic acid Meyskens et al. (1994), USA

301

232

50% CIN2 50% CIN3

Lesion regression

Biopsy

1 mL 0.375% topical all trans-retinoic cream

Treated at 1, 3, 6 months; endpoint assessed at 15 months

Increased regression of CIN2 at 15 months

36 at 6 months 30 at 12 months

33% CIN2 67% CIN3

Lesion regression and HPV DNA

Biopsy and HC

200 mg/day oral N-(4hydroxyohenyl) retinamide

6 months

No effect

104

39% CIN2 61% CIN3

Lesion regression

Cytology

25 or 50 mg aliretinoin or placebo daily

3 months

No effect

Follen et al. (2001), USA

39

Alvarez et al. (2003), USA

114

CIN, cervical intraepithelial neoplasia; HC, Hybrid Capture; KA, koilocytic atypia; PCR, polymerase chain reaction

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regression. In Arizona, USA, Childers et al. (1995) similarly found that 5 mg folic acid per day had no significant effect on cervical lesions after 6 months of treatment. Again, the majority of participants entered the study with CIN1 lesions. β-Carotene trials Five phase II/IIII trials of β-carotene supplements have been conducted, none of which demonstrated an increase in regression or a decrease in progression of any preneoplastic lesion. De Vet et al. (1991) found no effect of treatment with 10 mg per day β-carotene for 3 months among women in The Netherlands. A longer duration of treatment (9–24 months) with higher doses (30 mg) was also ineffective in altering rates of regression of lesions in studies conducted by Fairley et al. (1996) in Australia, Romney et al. (1997) in the USA, Mackerras et al. (1999) in Australia and Keefe et al. (2001) in the USA. Retinoic acid trials Three trials tested different formulations of retinoic acid (Meyskens et al., 1994; Follen et al., 2001; Alvarez et al., 2003). In the only placebo-controlled trial of a topical retinoid (all-trans-retinoic acid), Meyskens et al. (1994) found a significant effect of three administrations of the compound on the regression of CIN2 lesions after 15 months. In comparison, two studies of oral doses of retinoids failed to demonstrate an effect (Follen et al., 2001; Alvarez et al., 2003). In a small trial conducted by Follen et al. (2001), daily oral doses of 200 mg N-(4-hydroxyohenyl) retinamide had no effect on regression of CIN2/3 lesions after 6 months of treatment. Similarly, daily administration of 25 or 50 mg aliretinoin had no effect on the regression of CIN2/3-lesions after 3 months of treatment (Alvarez et al., 2003). Most of the studies were small and high rates of spontaneous regression of lesions were observed. As a result, these studies had insufficient power to test adequately the efficacy of the selected agents. Since effective ablative treatments are available for CIN2/3, it is questionable whether continued efforts should be made to find chemopreventive alternatives to the current standard of care. Future efforts in this area will require multisite collaboration to ensure that an adequate sample size of women with histologically confirmed CIN2/3 lesions are enrolled and complete the treatment protocol. (e)

Genetic factors

Familial clustering of cervical cancer has been explored as a potential marker of inherited genetic susceptibility. Population-based studies that used routinely collected data and record linkage in Sweden, Iceland and America consistently found moderately increased risk estimates for carcinoma in situ and invasive carcinoma among women who had a first- or a seconddegree relative with cervical cancer (Amundadottir et al., 2004; de Zelmanowicz et al., 2005; Couto & Hemminki, 2006). However, these investigations did not fully take into account relevant covariates such as HPV status or screening practices.

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Heritability estimates from the Swedish Cancer Registry suggest that genes are responsible for less than 30% of cervical tumours (Hemminki et al., 1999; Magnusson et al., 2000; Couto & Hemminki, 2006). In a multicentric case–control study in Latin America that included 481 patients with invasive cervical cancer and 801 population controls, Brinton et al. (1987) found a familial tendency for all cell types of cervical cancer. Women who had a family history of cervical cancer had a 2.49-fold higher risk for adenocarcinoma (based on one case), a 9.93-fold higher risk for adenosquamous carcinoma (based on 2 cases) and a 3.11-fold higher risk for squamous cell carcinoma (based on 13 cases). In the Republic of Korea, Yoo et al. (1997) conducted a case–control study that included 203 cases of invasive cervical cancer and reported a 2.20-fold (95% CI, 1.21–4.01) increase in risk associated with a family history of cervical cancer. Cusimano et al. (1989) reported an odds ratio of 2.87 (95% CI, 1.05–7.83) for a family history of cervical cancer in Sicily. In a case–control study in the USA, Hildesheim et al. (1999) reported a significantly increased risk for rapid onset of cervical cancer among young women whose mothers had a history of cervical cancer. Familial studies are limited in their capacity to separate fully the effects that can be attributed to genetic susceptibility from those that are related to common environmental and behavioural traits. However, reports have consistently suggested that a familial risk exists and further studies aimed at identifying relevant biomarkers would be pertinent. (f)

Human leukocyte antigen (HLA) polymorphisms and risk for cervical cancer

Major histocompatibility gene products that are complexed with peptides derived from viral antigens can induce T-cell responses on the surface of antigen-presenting cells that clear viral infections. HLA class II genes (DR, DQ and DP) are expressed on B cells and macrophages, where they present antigen fragments to CD4+ T cells. Although they are not usually expressed on epithelial cells, expression of HLA class II genes is increased in cervical cancer cells (Glew et al., 1992). CD4+ T cells have been reported to have killer activity and could thus potentially kill cervical cancer cells directly (de Jong et al., 2004; Steele et al., 2005); however, CD4+ T cells more commonly provide helper functions that assist the maturation of CD8+ T cells. CD8+ T cells recognize peptides in conjunction with the more ubiquitously expressed HLA class I genes. These genes (A, B and C) are highly polymorphic and present antigens on most cells, including cervical epithelial cells. Although class I and II gene products were initially defined using serological reagents, it has more recently been possible to characterize the HLA genotype using PCR-based methods with specific primers. Several studies have examined the association between HLA genotypes and the risk for cervical cancer. Most of these focused on class II alleles because the methods for genotyping these genes were developed earlier than those for the detection of class I alleles. Class II DR and DQ alleles are co-dominantly expressed. The large number of polymorphisms of these alleles lead to variations in the antigen recognition site on the cell

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surface, which may confer susceptibility or resistance to HPV infection and neoplastic progression. Malignant transformation and regression of cottontail rabbit papillomavirusinduced lesions were clearly shown to be associated with class II DR and DQ genes (Han et al., 1992). The results of several selected studies are summarized in Table 60. The first associations between HLA class II genes and cervical cancer were reported with DR5, DR6 and DQ3 (Wank & Thomssen, 1991). A second report on the same samples employed HLA typing using DNA-based methods and assigned the increase in risk to DQB1*0301/0303 (Wank et al., 1993). A number of studies that used different ethnic populations confirmed this association (Helland et al., 1992; Gregoire et al., 1994; Nawa et al., 1995; Duggan-Keen et al., 1996) but others did not find statistically significant associations for these alleles (Glew et al., 1992; Apple et al., 1994; Allen et al., 1996; Lin et al., 2001). Other allele groups that have been reported to confer risk include DRB1*11 (DugganKeen et al., 1996), DRB1*15 (Cuzick et al., 2000; Maciag et al., 2000; Beskow et al., 2001) and DQB1*06 (Gregoire et al., 1994; Beskow et al., 2001) and the related haplotypes DRB1*0401–DQB1*0301 (Cuzick et al., 2000), DRB1*1101–DQB1*0301 (Lin et al., 2001) and DRB1*1501–DQB1*0602 (Apple et al., 1994). In addition, DRB1*13 and the DRB1*13–DQB1*06 haplotype have been reported to confer protection against the development of cervical cancer (Apple et al., 1995; Sastre-Garau et al., 1996). Class II DRB1 and DQB1 genes were examined in 315 women who had invasive squamous-cell cervical cancer and 381 population-based controls who were residents of the metropolitan area of Seattle, WA (USA) (Madeleine et al., 2002). An increased risk for squamous-cell cancer was associated with two DRB1 alleles (DRB1*1001 and DRB1*1101) and one DQ allele (DQB1*0301). Decreased risks for squamous-cell cancer were associated with DRB1*0301 and DRB1*13. The relative risks for squamous-cell cancer that contains HPV 16 were different from those for squamous-cell cancers that contain other high-risk HPV types for three alleles: DRB1*0401, DRB1*07 and DQB1*06. The increased risks associated with the DQB1*0301 allele were specific to the DRB1*0401–DQB1*0301 (odds ratio, 1.9; 95% CI, 1.2–3.1) and DRB1*1101–DQB1*0301 (odds ratio, 2.9; 95% CI, 1.6– 5.2) haplotypes. Similarly, the associations with disease for the DRB1*0301 and DRB1*13 alleles were specific to the DRB1*0301–DQB1*02 (odds ratio, 0.7; 95% CI, 0.5–1.0) and DRB1*13–DQB1*06 (odds ratio, 0.6; 95% CI, 0.4–0.9) haplotypes. Fewer studies have examined the association between class I alleles and cervical cancer. In some cases, the risk was associated with the genotypes A2 or A*3303 (Wang et al., 2002a), although the association with A*3303 was seen only in a population of women from Portland (OR, USA) and not in two other populations that were examined. In contrast, the same study found a protective effect for CW*0202 in all three populations, and a protective effect for CW*0401 for both populations in the USA. The B*07 allele has been examined frequently and some (Hildesheim et al., 1998; Wang et al., 2002b), but not all (Gostout et al., 2003) studies found that it conferred risk. In the progression of HPVrelated dysplasia, the B*44 allele was associated with a strong risk (odds ratio, 9.0) in one

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Table 60. Association of human leukocyte antigen (HLA) class II alleles with the risk for cervical cancer Reference, study location

Madeleine et al. (2002), USA

HLA groups/allele(s)

DRB1* DRB1*07

Total no. of cases

Type of cases

p Value or odds ratio (95% CI)

315

HPV 16 SCC

1.5 (1.0–2.3)

Allen et al. (1996), Sweden

DRB1*08 DRB1*0802

150

Cervical cancer

p = 0.005

Madeleine et al. (2002), USA

DRB1*10

315

SCC HPV SCC HPV SCC

5.6 (1.2–26.1) 7.3 (1.5–36.7) 7.3 (1.5–36.7)

66

Invasive cancer

p = 0.009

Cervical cancer

NS

Invasive carcinoma

1.0 (0.4–2.2)

SCC SCC HPV 16 SCC

1.8 (1.2–2.9) 2.4 (1.4–4.2) 2.4 (1.3–4.6)

DRB1*1001 Wank & Thomssen (1991), USA

DR5 (DRB1*11, DRB1*12)

Duggan-Keen et al. (1996), United Kingdom

DRB1*11

150

Lin et al. (2001), Senegal

DRB1*11

55

Madeleine et al. (2002), USA

DRB1*11 DRB1*1101

Wank & Thomssen (1991), USA Apple et al. (1994, 1995), USA

DR6 (DRB1*13, DRB1*14) DRB1*13

315

66

p = 0.004

98

SCC and ADC

0.3 (0.1–0.7)

Sastre-Garau et al. (1996), France

DRB1*1301/1302

126

Invasive cancer

p = 0.0004

Cuzick et al. (2000), United Kingdom

DRB1*1301

116

SCC

NS

Invasive cancer

0.5 (0.2–1.1)

Lin et al. (2001), Senegal

DRB1*13

55

Krul et al. (1999), Netherlands

DRB1*13

172

SCC

NS

Madeleine et al. (2002), USA

DRB1*13

315

SCC HPV 16 SCC

0.6 (0.4–0.9) 0.6 (0.3–0.9)

DRB1*1301

SCC HPV 16 SCC

0.6 (0.4–1.0) 0.6 (0.3–1.1)

DRB1*1302

SCC HPV 16 SCC

0.6 (0.3–1.1) 0.4 (0.2–1.0)

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Table 60 (contd) Reference, study location

HLA groups/allele(s)

Total no. of cases

Type of cases

p Value or odds ratio (95% CI)

Maciag et al. (2000), Brazil

DRB1*15

161

SCC

2.2 (1.3–3.9)

Krul et al. (1999), Netherlands

DRB1*15

172

SCC

NS

Madeleine et al. (2002), USA

DRB1*15

315

SCC

1.0 (0.7–1.4)

Beskow et al. (2001), Sweden

DRB1*1501

440

CIS

p = 0.027

Madeleine et al. (2002), USA

DRB1*1501

315

SCC

1.0 (0.7–1.4)

Cuzick et al. (2000), United Kingdom

DRB1*1501

116

HPV 16 SCC

p = 0.05

127

Cervical cancer

p = 0.023

161

SCC

0.6 (0.4–0.9)

315

SCC

1.2 (0.9–1.7)

CIS

p = 0.028

HPV 16 SCC

0.9 (0.6–1.4)

Invasive carcinoma

0.6 (0.1–2.7)

Gostout et al. (2003), USA

Maciag et al. (2000), Brazil Madeleine et al. (2002), USA

DR2 (1501) DQB1* DQ1 (DQB1*05, DQB1*06) DQB1*05 DQB1*05

Beskow et al. (2001), Sweden

DQB1*0602

Madeleine et al. (2002), USA

DQB1*0602

315

Lin et al. (2001), Senegal

DQB1*0602

55

Sastre-Garau et al. (1996), France

DQB1*0603

126

Invasive cancers

Madeleine et al. (2002), USA

DQB1*0603

315

SCC

0.6 (0.4–1.0)

Gregoire et al. (1994), USA

DQB1*0604

66

SCC

p = 0.02

Helland et al. (1992), Norway

DQB1*0604

158

SCC

0.4 (0.1–1.1)

Madeleine et al. (2002), USA

DQB1*0604

315

SCC

0.6 (0.3–1.3)

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Table 60 (contd) Reference, study location

HLA groups/allele(s)

Total no. of cases

Wank & Thomssen (1991), USA

DQ3 (DQB1*07, DQB1*08, DQB1*09)

Helland et al. (1992), Norway

DQ3

158

Nawa et al. (1995), Japan

DQ3

23

Sastre-Garau et al. (1996), France

DQ3

Allen et al. (1996), Sweden

66

Type of cases

p Value or odds ratio (95% CI)

Invasive cancer

p = 0.0009 2.0 (1.3–3.2)

SCC

p = 0.0003

126

Invasive cancer

p = 0.03

DQ3

150

Cervical cancer

p = 0.11

Apple et al. (1994), USA

DQ3

28

SCC and ADC

NS

Krul et al. (1999), The Netherlands

DQ3

172

SCC

NS

Glew et al. (1992), United Kingdom

DQ3

53

SCC

NS

315

SCC HPV 16 SCC

1.6 (1.2–2.2) 1.5 (1.0–2.2)

Madeleine et al. (2002), USA

DQB1*07

Duggan-Keen et al. (1996), United Kingdom

DQB1*0301

150

Cervical cancer

p = 0.04

Cuzick et al. (2000), United Kingdom

DQB1*0301

116

SCC

p = 0.02

Helland et al. (1992), Norway

DQB1*0301

158

SCC

1.8 (1.1–3.0)

Madeleine et al. (2002), USA

DQB1*0301

315

SCC HPV 16 SCC

1.6 (1.2–2.2) 1.5 (1.0–2.2)

Gregoire et al. (1994), USA

DQB1*0301

66

SCC

NS

Wank et al. (1993), USA

DQB1*0301

8.71 p = 0.0001

Vandenvelde et al. (1993), Belgium

DQB1*0301/0302

NS

Lin et al. (2001), Senegal

DQB1*0301/0302

Madeleine et al. (2002), USA

DQB1*09 DQB1*0303

55 315

Invasive cancer

0.8 (0.4–1.9)

SCC SCC

1.3 (0.8–2.1) 0.9 (0.6–1.3)

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Table 60 (contd) Reference, study location

HLA groups/allele(s)

Gregoire et al. (1994), USA

DQB1*0303

Wank et al. (1993), USA

DQB1*0303

Total no. of cases

66

p Value or odds ratio (95% CI)

SCC

2.7 4.50 p = 0.0012

DRB1*:DQB1* DRB1*0401–DQB1*0301

116

Madeleine et al. (2002), USA

DRB1*0401–DQB1*0301

315

Allen et al. (1996), Sweden

DRB1*0401–DQB1*0301

150

Lin et al. (2001), Senegal

DRB1*1101–DQB1*0301

55

Madeleine et al. (2002), USA

DRB1*1101–DQB1*0301

Allen et al. (1996), Sweden

Cuzick et al. (2000), United Kingdom

Type of cases

p = 0.02 HPV 16 SCC

1.9 (1.2–3.1) p = 0.01

Invasive carcinoma

2.6 (1.0–7.1)

315

HPV 16 SCC

2.9 (1.6–5.2)

DRB1*1101–DQB1*0301

150

Cervical cancer

NS

Duggan-Keen et al. (1996), United Kingdom

DRB1*07–DQB1*0201

150

Allen et al. (1996), Sweden

DRB1*0802–DQB1*0402

150

Cervical cancer

p = 0.001

Maciag et al. (2000), Brazil

DRB1*15–DQB1*0602

161

SCC

2.0 (1.2–3.6)

Cuzick et al. (2000), United Kingdom

DRB1*1501–QB1*0602

116 HPV 16

NS 1.8 (1.0–3.3)

Apple et al. (1994, 1995), USA

DRB1*1501–DQB1*0602

98

SCC and ADC HPV 16

2.9 (1.3–6.7) 4.8 (1.9–11.8)

Madeleine et al. (2002), USA

DRB1*1501–DQB1*0602

315

HPV 16 SCC HPV 16

1.0 (0.7–1.4) 0.9 (0.6–1.4)

Allen et al. (1996), Sweden

DRB1*1501–DQB1*0602

150

Cervical cancer

NS

Madeleine et al., USA

DRB1*0301–DQB1*02 DRB1*13–DQB1*06

315

HPV 16 SCC

0.7 (0.5–1.0) 0.6 (0.4–0.9)

Apple et al. (1994, 1995), USA

DRB1*13–DQB1*0603

98

NS

0.3 (0.1–0.8)

ADC, adenocarcinoma; CIS, carcinoma in situ; NS, not significant; SCC, squamous-cell carcinoma

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study (Bontkes et al., 1998) and the B*07–DQB1*0302 haplotype was associated with a strong risk (odds ratio, 8.2) in another (Wang et al., 2002b). Epidemiological studies in different populations have found various relationships between risk for cervical cancer and HLA polymorphisms. Comparisons are hampered by issues such as small sample sizes, inappropriate controls and chance findings. The probability of chance findings is increased by multiple comparisons that are often made between the extensive number of polymorphisms and the disease. Ethnic admixture within study groups of one race also may contribute to differences in the distribution of HLA polymorphisms between seemingly homogeneous populations. This heterogeneity may influence risk estimates by masking true effects. 2.7.2

Infectious co-factors

The central etiological role of HPV (a sexually transmitted infection) in cervical tumorigenesis has led to hypotheses that other microbial agents that also infect the cervicovaginal epithelium could act as co-factors and increase or decrease the risk for cervical cancer in the presence of a high-risk HPV infection (Lacey, 1992). The proposed mechanisms of co-factors (described in Section 4.1.5(a)) include direct biological interactions, such as viral co-activation of HPV replication, and indirect effects, such as damage to the epithelial barrier that protects against HPV infection. In this section, special emphasis has been placed on studies of co-factors that controlled for the effects HPV infection in the analyses. Two sources of bias were common concerns in the studies reviewed: (a) the inability to assess temporality; many studies assessed exposure using serological assays that do not distinguish between current and past infections. Moreover, most studies that tested directly for infections in the cervix were cross-sectional. Therefore, it was generally not known whether the presumptive infectious co-factor was present concurrently with a high-risk HPV infection, or whether it preceded or followed the development of cervical neoplasia. This most probably caused bias towards the null. (b) residual confounding by HPV; detection of a sexually transmitted infection, even among women who are all currently positive for HPV DNA, may be a surrogate marker for high-risk behaviours or high-risk sexual partners and a consequently greater cumulative lifetime exposure to high-risk HPV. Because of the very strong association of HPV with cervical cancer, a small degree of residual confounding by HPV could account for moderate effects that are putatively associated with a sexually transmitted infection. (a)

Herpes simplex virus (HSV)

Genital HSV infection is one of the microbial agents that is most frequently studied as a potential co-factor for cervical cancer. Before the causal role of HPV in the development of cervical cancer was firmly established, HSV was itself regarded as a candidate etiological agent — one that, similarly to HPV, could help explain the association of cervical cancer with sexual behaviour (Brinton, 1992). Although in-vitro studies conducted during

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the 1970s demonstrated the carcinogenic potential of HSV (Duff & Rapp, 1971a,b; Duff, 1975), HSV DNA was not consistently detected in cervical cancer specimens (Brinton, 1992). An increased understanding of the causal role of HPV and the publication of a large prospective study that found no association of HSV-2 seroantibodies with incident cervical cancer (Vonka et al., 1984) shifted the focus away from HSV-2 as an etiological agent (Brinton, 1992; Ferrera et al., 1997a; Lehtinen et al., 2002). In retrospect, the study by Vonka et al. (1984) may have lacked statistical power because of the small number of cases observed during follow-up, and concerns were expressed regarding over-matching (Brinton, 1992). Moreover, laboratory studies demonstrated that HSV-2 DNA does not need to persist for HSV-2 to play a role in the transformation of cervical epithelial cells (i.e. a possible ‘hit and run’ mechanism) (Galloway & McDougall, 1983; Jones, 1995). HSV-2 has a much greater tropism for genital tissue and recurs with greater frequency in the genital tract than HSV-1 (Engelberg et al., 2003; Sacks et al., 2004). Therefore, seroepidemiological studies conducted during or after the 1990s, when assays that could distinguish between HSV-2 and HSV-1 infection came into use, are of most interest (Ashley & Wald, 1999). The majority of seroepidemiological studies found a moderate or no association between HSV-2 antibodies and cervical neoplasia (Table 61). Lehtinen et al. (2002) pooled data and specimens from three population-based Nordic cohorts to form a collective population of more than 500 000 women. No difference was found between the baseline seroprevalence of HSV-2 among 178 incident cervical cancer cases and 525 controls after adjustment for antibodies to HPV 16/18/33 VLPs and cigarette smoking (odds ratio, 1.0; 95% CI, 0.6–1.7). A study by the IARC (Smith et al., 2002a) that pooled data from seven separate case– control investigations conducted in Brazil, Colombia, Morocco, Peru, the Philippines, Spain and Thailand found a higher seroprevalence of HSV-2 in 1158 cases of squamouscell carcinoma and 105 cases of adeno-/adenosquamous carcinoma than in 1117 controls; after limiting the analysis to HPV DNA-positive subjects and adjusting for seropositivity to C. trachomatis, the associations of HSV-2 seroantibodies with squamous-cell carcinoma (odds ratio, 2.2; 95% CI, 1.4–3.4) and adeno-/adenosquamous carcinoma (odds ratio, 3.4; 95% CI, 1.5–7.7) were still significant. Several additional investigations reported significant associations of HSV-2 seroantibodies with cervical cancer (Hildesheim et al., 1991; Jha et al., 1993; Becker et al., 1994; Koffa et al., 1995; Daling et al., 1996; Thomas et al., 2001b, c) or cervical neoplasia (Olsen et al., 1998b), but most of these studies either did not control statistically for HPV infection despite having tested for HPV (Becker et al., 1994; Thomas et al., 2001b,c) or observed an association only among HPV-negative women (Jha et al., 1993; Koffa et al., 1995; Daling et al., 1996). Studies that tested for HSV DNA in the cervix also gave conflicting results. A high prevalence of HSV DNA was detected in neoplastic cervical specimens using sensitive PCR methods in some studies (Koffa et al., 1995; Han et al., 1997) but these data were not confirmed by others (Vecchione et al., 1994; Tran-Thanh et al., 2003). After laboratory data

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No. and type of controls

HSV-2 seroprevalence

Hildesheim et al. (1991), Costa Rica, Panama, Colombia, Mexico

Case–control

766 ICC

1532 normal cytology

HPV 16/18-positive Cases Controls

Peng et al. (1991), China

Case–control

101 ICC

146 normal cytology

Koutsky et al. (1992), USA

Prospective cohort of cytologically normal women enrolled through an STD clinic

28 CIN2/3

Jha et al. (1993), United Kingdom

Case–control

Becker et al. (1994), USA

Case–control

%

Best epidemiological control for HPV and method of HPV detection

Comments

57 39

HPV 16/18 1.6 (1.3–1.9) HPV 16/18 and HSV-2 8.8 (5.9–13.0)

Filter in-situ hybridization for HPV 16/18/6/11

An insensitive and nonspecific assay for HPV, complicating the interpretation of these data.; results suggest interaction between HPV 16/18 and HSV-2.

ICC Controls

42 29

ICC 1.3 (0.7–2.3)

Adjusted for HPV 16 and 33 DNA by PCR

HPV DNA assay detected few high-risk types.

213 who did not develop CIN2/3

Cases Controls

45 43

CIN2/3 1.0 (0.5–2.3)

Adjustment for HPV by Virapap or dot-filter hybridization

HPV assays were insensitive but few HPV-negative women developed CIN2/3; no details of HSV assay given

219 ICC

387 normal cytology

Invasive cancer Controls

11 5

HPV-adjusted 2.2 (1.1–4.5) HPV-positive 1.8 (0.4–8.6) HPV-negative 3.0 (1.5–6.0)

Adjusted by antibodies to HPV 16/18 E7

Insensitivity of HPV 16/18 E7 assay increases change of residual confounding by HPV; HSV-2 ELISA may not have been type-specific; blood collected years after diagnosis

128 Hispanic and 73 nonHispanic CIN2/3

216 Hispanic and 121 nonHispanic with normal cytology

All subjects CIN2/3 Controls

All subjects 1.3 (0.8–1.9) Hispanic 3.1 (1.6–6.2) Non-Hispanic 0.7 (0.4–1.2)

Tested for HPV by PCR

No adjustment for HPV

36 29

315

Association or odds ratio (95% CI)

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No. and type of controls

HSV-2 seroprevalence

%

Association or odds ratio (95% CI)

Best epidemiological control for HPV and method of HPV detection

Comments

Dillner et al. (1994), Finland

Case–control

94 ICC

188 normal cytology

IgA in cases IgG in cases Data for controls not reported

28 92

IgA 1.0 (0.6–1.8) IgG 1.1 (0.4–2.6)

Tested for multiple HPV 16 peptide antibodies

No adjustment for HPV

de Sanjosé et al. (1994), Spain and Columbia

Case–control

Spain 249 CIN3 223 ICC

Spain 242 matched to CIN3 238 matched to cancer

Spain CIN3 Controls ICC Controls

14 11 26 12

Spain CIN3 1.2 (0.7–2.0) ICC 1.1 (0.6–1.7)

Adjusted for HPV DNA by PCR

HSV assay may have had some crossreactivity between HSV1 and HSV-2 (Lacey, 1992).

Columbia 276 CIN 3 150 ICC

Columbia 270 matched to CIN 3 149 matched to cancers

Columbia CIN3 Controls ICC Controls

61 50 73 60

Columbia CIN3 1.1 (0.7–1.7) ICC 1.1 (0.6–2.1) No association

Adjusted for penile HPV DNA by PCR; HPV in husbands strongly associated with case status

Husbands’ HSV-2 seroprevalence data are indirect evidence for or against HSV-2 as a cofactor.

All subjects 1.2 (0.8–1.8) HPV sero- and DNAnegative 3.6 (1.6–8.0)

Stratified and/or adjusted for HPV 16 serology (all subjects), HPV DNA (cases) by PCR

Only 77% of cases were HPV DNA-positive and given HPV serology insensitivity, residual HPV confounding was a concern.

Bosch et al. (1996), Spain

Case–control of patients’ male partners

306 husbands of women with CIN3 or SCC

327 husbands of women with normal cytology

Data not shown

Daling et al. (1996), USA

Case–control

264 SCC

541 normal cytology

Squamous carcinoma Controls

37 26

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Table 61 (contd)

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Table 61 (contd) HSV-2 seroprevalence

%

Association or odds ratio (95% CI)

Best epidemiological control for HPV and method of HPV detection

Comments

Lehtinen et al. (1996), Finland

Nested case– control

27 ICC 72 in-situ cancer

143 cancerfree women

Cancer Controls

15 26

Statistical adjustment for HPV 16 VLP antibodies

Analysis controlled for detection of HPV 16 antibodies

Muñoz et al. (1996a), Colombia

Case–control of patients’ male partners

210 husbands of women with CIN3 92 husbands of women with SCC

262 husbands of women with normal cytology

Data not shown

No association

Tested for penile HPV DNA by PCR

Husbands’ HSV-2 seroprevalence data are indirect evidence for or against HSV-2 as a cofactor.

Ferrera et al. (1997a), Honduras

Case–control

25 CIN3 48 ICC

50 normal cytology matched to CIN3, 93 matched to ICC

All subjects tested

92

No significant association

Statistical adjustment for HPV DNA by PCR

Few details of the HSV2 assay were provided, and its specificity could not be confirmed.

Olsen et al. (1998), Norway

Case–control

94 CIN2/3

228 normal cytology

CIN2/3 Controls

41 25

2.6 (1.0–6.3)

Adjusted for HPV DNA by PCR and antibodies to HPV 16 VLPs

Stronger association among HPV 16 DNApositive subgroup of cases and controls. HSV-1 antibodies also associated with CIN2/3

Yoshikawa et al. (1999), Japan

Cross-sectional

94 CIN1 40 CIN2 33 CIN3

130 normal cytology

Any CIN Controls

79 72

2.0 (0.7–5.9)

Adjusted for HPV DNA by PCR

Details of HSV assay not reported

Thomas et al. (2001c), Thailand

Case–control

190 SCC

291 normal cytology

Squamous carcinoma Controls

58 50

1.4 (1.0–2.0)

0.6 (0.2–1.4)

Tested for HPV DNA by PCR, but no control for HPV in analyses

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HSV-2 seroprevalence

%

Lehtinen et al. (2002), Finland, Norway and Sweden

Nested case– control set in three cohorts

178 cancers identified through cancer registries

527 cancerfree women

Controls Cases

12 15

Smith et al. (2002a), Brazil, Columbia, Morocco, Peru, Philippines and Thailand

Pooled analysis of multiple case– control studies

1158 SCC 105 adenocarcinomas

1117 normal cytology

Controls Squamous carcinoma Adenocarcinoma

26 44 44

Castle et al. (2003b), Jamaica

Cross-sectional

92 CIN3/ICC 117 CIN2

201 CIN1

CIN1 CIN2 CIN3/cancer

Yokoyama et al. (2003), Japan

Cohort

41 CIN1 and 43 CIN2 that persisted or progressed

73 CIN1 and 28 CIN2 that regressed

Regressed Persisted/progressed

Association or odds ratio (95% CI)

Best epidemiological control for HPV and method of HPV detection

Comments

Statistical adjustment for HPV 16/18/31 by VLP serology

A meta-analysis of prior longitudinal studies also reported in this paper found no HSV-2 effect; total cohort size, 550 000

Squamous carcinoma 2.2 (1.4–3.4) Adenocarcinoma 3.4 (1.5–7.7)

Analysis limited to HPV DNApositive by PCR

Based on comparison with the 164 HPV DNApositive normal controls

61 62 74

CIN3/cancer 1.2 (0.6–2.3) CIN2 0.8 (0.5–1.5)

Analysis limited to HPV DNApositive by PCR

79 77

Regressed 1.1 (0.6–1.9)

Adjusted for HPV DNA by PCR

1.0 (0.6–1.7)

Details of HSV assay not reported

CI, confidence interval; CIN, cervical intraepithelial neoplasia; ELISA, enzyme-linked immunosorbent assay; ICC, invasive cervical cancer; Ig, immunoglobulin; SCC, squamous-cell carcinoma; STD, sexually transmitted disease; VLP, virus-like particles

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had shown that selected HSV-2 oncogenes (e.g. Xho-2) may be integrated into and persist in cervical cancer cells, Tran-Thanh et al. (2003) tested 200 cervical cancer specimens and 244 normal specimens and failed to detect these or other HSV-2 sequences in any cervical specimens. Molecular epidemiological data have provided only inconsistent support for the hypothesis that HSV-2 is a co-factor in cervical tumorigenesis. Null results were often observed despite the potentially positive bias related to residual confounding by HPV, especially since the chronic and recurrent nature of genital HSV-2 infection would suggest that studies of HSV-2 might be less affected than studies of transient (e.g. bacterial) infections by an inability to address temporality. (b)

Other herpes viruses

All three β-herpesviruses, cytomegalovirus (CMV), human herpes virus (HHV)-6 and HHV-7 have been detected in cervical specimens (Han et al., 1997; Chan et al., 2001). While several studies that used sensitive assays to test for viral DNA have suggested that these viruses may be by-standers rather than co-factors in cervical tumorigenesis (Thompson et al., 1994; Boyle & Smith, 1999; Chan et al., 2001; Tran-Thanh et al., 2002), a few small studies reported associations between CMV and cervical neoplasia (Koffa et al., 1995), between HHV-6 and cervical cancer (Chen et al., 1994; Yadav et al., 1996) and between HHV-7 and high-grade cervical neoplasia (Lanham et al., 2001). Serological studies have also given conflicting results. Yokoyama et al. (2003) found that higher CMV IgG seroantibody titres were significantly associated with a greater risk of persistent CIN and Koutsky et al. (1992) reported a higher incidence of CIN2/3 in young patients with sexually transmitted diseases who were CMV-seropositive. Other studies, however, observed no association of CMV seroantibodies with cervical neoplasia (Jha et al., 1993; Dillner et al., 1994; de Sanjosé et al., 1994; Ferrera et al., 1997a; Yoshikawa et al., 1999). Epstein-Barr virus (EBV), a γ-herpesvirus, can infect epithelial cells and may play a role in nasopharyngeal carcinoma. However, the role of EBV in cervical tumorigenesis is controversial and consideration must be given to the fact that positive PCR results can occur due to the ability of EBV to infect infiltrating immune cells (Shoji et al., 1997; Boyle & Smith, 1999). Several studies using standard PCR (Koffa et al., 1995; Voog et al., 1997; Ammatuna et al., 2000; Lanham et al., 2001) and studies that used in-situ PCR and in-situ hybridization to identify the specific infected cells (Payne et al., 1995; O’Leary et al., 1997; Shoji et al., 1997; Elgui de Oliveira et al., 1999) found no association of EBV with the presence of cervical neoplasia, whereas positive findings were reported by others using similar methods (Landers et al., 1993; Se Thoe et al., 1993; Sasagawa et al., 2000). In two seroepidemiological studies, serum EBV IgG antibodies were not associated with cervical cancer (Jha et al., 1993; Dillner et al., 1994). (c)

Chlamydia trachomatis

C. trachomatis is the most common sexually transmitted bacterial infection; it is an obligate intracellular bacterium that has prevalence rates of 3–10% among young sexually

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active women in the general community, which can rise to 24% in high-risk populations (Burstein et al., 1998; Stamm, 1999; Burstein et al., 2001; Turner et al., 2002). Because 85–90% of C. trachomatis infections are asymptomatic, many remain undiagnosed (Turner et al., 2002) and untreated, and can persist for several months or even years (Stamm, 1999; Peipert, 2003; Stephens, 2003). C. trachomatis infection may also recur, or even possibly be reactivated, similarly to viral infections (Stephens, 2003; Hogan et al., 2004). Infection with C. trachomatis is associated with squamous metaplasia and hypertrophic ectopy and, when infection is chronic, may lead to sequelae, including pelvic inflammatory disease (Paavonen et al., 1988; Stamm, 1999). Therefore, although C. trachomatis is a treatable bacterial infection, it is an important cause of chronic intracellular cervical infection and, as a result, longterm co-infection with high-risk HPV may not be uncommon. C. trachomatis has also been studied extensively as a potential co-factor in cervical tumorigenesis. In comparison with HSV-2, many more epidemiological studies have reported a positive association between this bacterium and high-grade cervical neoplasia and/or invasive cervical cancer (Table 62). Two large epidemiological investigations observed highly significant associations between C. trachomatis seroantibodies and cervical squamous-cell carcinoma (Koskela et al., 2000; Smith, J.S. et al., 2004). The Nordic nested case–control study found that 30% of 149 squamous-cell carcinoma cases compared with 13% of 442 controls were seropositive for C. trachomatis at baseline, a difference that persisted after adjustment for detection of antibodies to HPV 16, 18 and/or 31 VLPs (odds ratio, 2.2; 95% CI, 1.3–3.5) (Koskela et al., 2000). In the IARC study, 53% of 1139 squamous-cell carcinoma cases but only 31% of 1100 controls were seropositive for C. trachomatis and the strength of this association increased with increasing C. trachomatis antibody titre (p for trend < 0.001) (Smith, J.S. et al., 2004). No association with adeno-/adenosquamous carcinoma was observed in either study. The Nordic nested case–control study also reported a possible association of C. trachomatis serovar with risk for cervical cancer (Anttila et al., 2001); this association was not found in the IARC study (Smith, J.S. et al., 2004). Similar associations between antibodies to C. trachomatis and cervical neoplasia were observed in several other seroepidemiological studies that controlled for HPV (Koutsky et al., 1992; de Sanjosé et al., 1994; Smith et al., 2002b; Matsumoto et al., 2003). Two cross-sectional studies observed a significant association between C. trachomatis antibodies in men and CIN3 or cancer in their wives (Bosch et al,. 1996; Muñoz et al., 1996a). Only a minority of studies did not detect a significant association between C. trachomatis antibodies and cervical neoplasia (Lehtinen et al., 1996; Ferrera et al., 1997a). Furthermore, detection of C. trachomatis DNA in the cervix was also associated with neoplasia in several studies. A nested case–control investigation based on the Swedish contingent of the Nordic cohort found that C. trachomatis DNA in prediagnostic (still normal) Pap smears was strongly related to the risk for subsequent cervical cancer (odds ratio, 17; 95% CI, 2.6–∞) (Wallin et al., 2002). A cross-sectional study showed an association between HSIL (odds ratio, 5.8; 95% CI, 1.5–22) but not squamous cancer (odds ratio, 2.1; 95% CI, 0.36–12) and cervical C. trachomatis DNA in HPV DNA-positive subjects

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Table 62. Selected epidemiological studies (that assessed exposure to HPV) on the association between Chlamydia trachomatis (CT) infection, cervical neoplasia and cervical cancer No. and type of controls

Prevalence of Chlamydia

Koutsky et al. (1992), USA

Prospective cohort of cytologically normal women enrolled through an STD clinic

28 incident CIN2/3

213 who did not develop CIN2/3

CT culture Cases Controls CT seroprevalence Cases Controls

Jha et al. (1993), United Kingdom

Case–control

219 ICC

387 normal cytology

ICC Controls

17 7

Becker et al. (1994), USA

Case–control

128 Hispanic and 73 nonHispanic CIN2/3

216 Hispanic, and 121 non-Hispanic women with normal cytology

CT seroprevalence CIN2/3 Controls

85 82

de Sanjosé et al. (1994), Spain and Columbia

Case–control

Spain 249 CIN3 223 ICC

Spain 242 normal cytology matched to CIN3 cases 238 normal cytology matched to cancer cases Columbia 270 normal cytology matched to CIN3 cases 149 normal cytology matched to cancer cases

CT seroprevalence Spain CIN3 Controls ICC Controls

29 11 28 16

Columbia CIN3 Controls ICC Controls

48 25 53 41

327 husbands of normal cytology women

CT seroprevalence Case husbands Control husbands

21 8

Columbia 276 CIN3 150 ICC

Bosch et al. (1996), Spain

Case–control of patients’ male partners

306 husbands of women with CIN3 or SCC

%

21 13

Association(s) and/or odds ratio (95% CI)

Best epidemiological control for HPV and method of HPV detection

Comments

CT culture 1.1 (0.5–2.8) CT seroprevalence 2.4 (1.0–5.7)

Adjustment for HPV by Virapap or dotfilter hybridization

HPV assays used were insensitive, but few HPVnegative women developed CIN2/3; no details of the CT seroassay provided

HPV-adjusted 2.2 (1.2–3.9) HPV-positive 1.5 (0.4–5.6) HPV-negative 3.1 (1.7–5.5)

Adjusted or stratified by antibodies to HPV 16/18 E7

Insensitivity of HPV 16/18 E7 assay increases chance that CT could be surrogate for HPV; blood collected years after diagnosis

Tested for HPV DNA by PCR, but no control for HPV in analyses

No adjustment for HPV

Adjusted for HPV DNA status determined using consensus PCR

Increasing CT titre had stronger association with case status, but assay specificity was not optimal (Smith, J.S. et al., 2004).

Adjusted for penile HPV DNA by PCR

Husbands’ CT seroprevalence data are indirect evidence of CT as a co-factor; HPV in husbands was strongly associated with cancer in wives.

75 55

1.1 (0.7–1.9) Spain CIN3 2.2 (1.1–4.6) ICC 1.7 (0.9–3.1)

Columbia CIN3 1.8 (1.1–2.9) ICC 0.9 (0.5–1.8)

2.6 (1.4–4.6)

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Prevalence of Chlamydia

%

Association(s) and/or odds ratio (95% CI)

Lehtinen et al. (1996), Finland

Nested case– control

72 in-situ cancer or ICC

143 matched controls from same cohort

CT seroprevalence Cancer Controls

10 4

3.0 (0.7–13.4)

Muñoz et al. (1996a), Colombia

Case–control of patients’ male partners

210 husbands of women with CIN3 92 husbands of women with SCC

262 husbands of normal cytology women

CT seroprevalence Case husbands Control husbands

29 15

2.4 (1.4–4.1)

Muñoz et al. (1996b), Spain, Colombia

Case–control

85 HPV DNAnegative and normal cytology

725 HPV DNAnegative and normal cytology

CT seroprevalence HPV-negative HPV-positive

20 40

2.3 (1.2–4.2)

Ferrera et al. (1997a), Honduras

Case–control

25 CIN3 50 ICC

50 normal cytology matched to CIN3, 95 matched to ICC

CT seroprevalence ICC Controls CIN 3 Controls

70 62 80 68

Lehmann et al. (1999), Germany

Cross-sectional

29 HPV DNApositive

115 HPV DNAnegative

CT DNA Any HPV HPV-negative

10 2

Koskela et al. (2000), Finland, Norway and Sweden

Nested case– control

149 SCC 32 ADC

442 women from same cohorts who remained cancerfree, matched to SCC and 94 to ADC

CT seroprevalence SCC Controls ADC Controls CT DNA prevalence SCC

30 13 9 7 10

ICC 0.95 (0.36–2.5) CIN3 2.0 (0.5–7.9)

Best epidemiological control for HPV and method of HPV detection Statistical adjustment for HPV 16 VLP antibodies Tested for penile HPV DNA by PCR, but this was not associated in wives.

Husbands’ CT seroprevalence data are indirect evidence of CT as a co-factor, but null association of HPV DNA with cancer may imply residual HPV confounding.

HPV DNA by PCR

Some data incorporated in Smith, J.S. et al. (2004)

Statistical adjustment for HPV DNA by PCR

HPV DNA by PCR

No control for shared sexual risk factors between CT and HPV

Adjustment for HPV 1618/33 by VLP serology

Some data reported earlier (Dillner et al., 1994, 1997); current data later re-analysed to assess effect of CT serovar (Anttila et al., 2001) and possible interactions between HPV 16 and CT (Hakama et al., 2000; Luostarinen et al., 2004)

p < 0.05

SCC 2.2 (1.3–3.5) ADC 0.4 (0.1–1.7)

Comments

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Prevalence of Chlamydia

Giuliano et al. (2001, 2002b), USA– Mexico border

Cross-sectional

259 high-risk HPV 65 low-risk HPV

2153 HPVnegative

CT DNA HPV-negative Any HPV High-risk HPV Low-risk HPV

8 16 18 9

Smith et al. (2002b), Africa, Asia, South America, Spain

Case–control

455 SCC 44 ADC

539 normal cytology

CT seroprevalence Controls SCC ADC

Tamim et al. (2002), Lebanon

Cross-sectional

61 HPV DNApositive 49 HPV DNApositive

478 HPV DNAnegative 80 HPV DNAnegative

HPV-negative HPV-positive CT DNA HPV-negative HPV-positive

Wallin et al. (2002), Sweden

Nested case– control

118 ICC

118 cancer-free women from same cohorts

CT DNA prevalence Controls Invasive cancers

Castle et al. (2003b), Jamaica

Cross-sectional

117 CIN2 92 CIN3

201 CIN1

CT DNA CIN1 CIN2 CIN3 CT seroprevalence CIN1 CIN2 CIN3

Matsumoto et al. (2003), Japan

Cross-sectional

80 CIN1 34 CIN2 27 CIN3

109 normal cytology

CT seroprevalence Any CIN Control

%

Best epidemiological control for HPV and method of HPV detection

Comments

Any HPV 1.8 (1.2–2.7) High-risk HPV 2.1 (1.4–3.2) Low-risk HPV 1.2 (0.5–3.2)

HPV DNA by PCR

Adjusted for sexual behaviour, age, other risk factors

22 48 30

SCC 2.1 (1.1–4.0) ADC Not significant

Cancer analysis limited to HPV DNA-positive by PCR

Some data incorporated in Smith, J.S. et al. (2004)

22 26

HPV DNA by PCR 1.4 (0.7–2.7)

No control for shared sexual risk factors between CT and HPVpositive. CT associated with ‘abnormal cytology’ in HPV-negative strata. Updates Finan et al. (2002).

Adjusted analysis for detection of HPV by PCR

CT DNA and HPV DNA detected in baseline specimens, obtained years before cancer

By laboratory method CT DNA p trend = 0.96 CT serology p trend = 0.61

Analysis limited to HPV DNA-positive by PCR

Assessed CT gradient by grade of neoplasia; association of CT with HPV DNA not assessed, no comparison of CIN with normal controls

Any CIN adjusted for: HPV DNA and age 1.7 (0.7–4.3) HPV serology and age 2.7 (1.3–6.0)

Adjusted for HPV DNA by PCR, and/or HPV 16/52/58 by VLP serology

Disparity in results raises concern that falsenegative HPV serology resulted in residual HPV confounding; some data reported in Yoshikawa et al. (1999)

13 59

0 8

9 10 9 20 17 22 23 11

10.2 (4.2–24.3)

17.1 (2.6–∞)

323

Association(s) and/or odds ratio (95% CI)

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Prevalence of Chlamydia

Molano et al. (2003b), Colombia

Cross-sectional

216 high-risk HPV-positive 52 low-risk HPV-positive 9 other HPV

1536 HPVnegative

CT DNA HPV-negative > 1 HPV Any HPV High-risk HPV

Yokoyama et al. (2003), Japan

Cohort

41 CIN1 and 43 CIN2

73 CIN1 and 28 CIN2 that regressed

CT IgA Seroprevalence Regressed Persisted/progressed

Giuliano et al. (2004), USA– Mexico border

Cross-sectional

30 HSIL

1876 normal cytology

da Silva et al. (2004), Brazil

Case–control

26 HPV DNApositive

Smith, J.S. et al. (2004), Brazil, Colombia, Morocco, Peru, Philippines, Spain, Thailand

Pooled analysis of multiple case–control studies

Golijow et al. (2005), Argentina

Cross-sectional

%

5 10 7 7

Association(s) and/or odds ratio (95% CI)

Best epidemiological control for HPV and method of HPV detection

Comments

> 1 HPV 2.5 (1.1–5.9) Any HPV 1.3 (0.8–2.4) High-risk HPV 1.3 (0.7–2.4)

HPV DNA by PCR

Adjusted for no. of regular and casual sex partners, age at first intercourse, condom use; no association of CT with abnormal Pap test, but analysis combined ASCUS and SIL.

Adjusted for HPV DNA by PCR

Tested for CT IgA (rather than IgG — as in most studies); no report of assay sensitivity or specificity

Analysis limited to HPV DNA-positive by PCR

Subjects same as those in study of Giuliano et al. (2001)

HPV DNA by PCR

No control for shared sexual risk factors

24 29

0.8 (0.5–1.3)

CT DNA Control HSIL

7 13

1.1 (0.3–4.1)

26 HPV DNAnegative

CT DNA HPV-negative HPV-positive

8 35

6.4 (1.1–55.4)

1139 SCC 99 ADC

1100 normal cytology

CT seroprevalence Controls SCC ADC

31 53 39

SCC 1.8 (1.2–2.7) ADC 1.0 (0.5–2.0)

Analysis limited to HPV DNA-positive by PCR

Based on comparison with 164 HPV DNA-positive normal controls; some data from Smith et al. (2002b) and de Sanjosé et al. (1994) included; association of SCC increased with CT titre.

75 HSIL 35 SCC

79 normal cytology

CT DNA Control HSIL SCC

11 47 20

HSIL 5.8 (1.5–22) SCC 2.1 (0.36–12)

Analysis limited to HPV DNA-positive by PCR

Based on comparison with 24 HPV DNA-positive normal controls

ADC, adenocarcinoma; ASCUS, atypical squamous cells of undetermined significance; CIN, cervical intraepithelial neoplasia; HSIL, high-grade squamous intraepithelial lesion; ICC, invasive cervical cancer; Ig, immunoglobulin; PCR, polymerase chain reaction; SCC, squamous-cell carcinoma; SIL, squamous intraepithelial lesion

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(Golijow et al. 2005). Another cross-sectional study that did not find an association between C. trachomatis DNA and HSIL (Giuliano et al., 2004) reported an association between the detection of HPV and C. trachomatis DNA after adjustment for sexual behaviour (Giuliano et al., 2001, 2002b). Several other studies (Muñoz et al., 1996b; Lehmann et al., 1999; Tamim et al., 2002; Molano et al., 2003b; da Silva et al. 2004), but not all (Smith, J.S. et al., 2002b, 2004; Golijow et al., 2005), also observed an association between C. trachomatis (DNA or antibodies) and detection of HPV. Only limited data, however, address the more specific question of the stage at which C. trachomatis might have its effects in the multistage process of HPV-associated cervical tumorigenesis and whether it has an effect on (a) the risk for HPV infection, (b) the persistence of HPV, (c) the development of neoplastic cervical lesions and/or (d) the persistence and progression of cervical lesions after their development. Most of the data that are available do not support a role of C. trachomatis infection in the progression of cervical neoplasia. C. trachomatis was not associated with the persistence or progression of lesions in a study of 114 prevalent CIN1 and 71 CIN2 that used C. trachomatis IgA seroantibodies as markers of active infection (Yokoyama et al., 2003) or in follow-up studies conducted during the 1980s that detected C. trachomatis by culture (Syrjanen, K. et al., 1986, 1987; Yliskoski et al., 1992). Similarly, a cross-sectional investigation found no differences in the detection of C. trachomatis DNA or C. trachomatis IgG seroantibodies by grade of neoplasia in colposcopy patients with CIN1 (n = 201), CIN2 (n = 117) or CIN3 (n = 92) (Castle et al., 2003), and at least one study that had detected a greater prevalence of C. trachomatis DNA in SIL relative to normal specimens did not find differences in prevalence between LSIL, HSIL and cancer (Golijow et al., 2005). Overall, the data reported to date provide initial evidence of a possible epidemiological association between C. trachomatis and cervical neoplasia. Although the possibility of residual confounding by HPV can not be excluded, the frequent null results reported for HSV-2 and other sexually transmitted infections make it more difficult to attribute the association of C. trachomatis and cervical neoplasia entirely to their shared sexual risk factors. The exact stage(s), however, of the multistage process of HPV-associated tumorigenesis that might be affected by C. trachomatis has not been examined carefully and remains uncertain. (d)

Other non-viral infections

Several other non-viral infectious agents have been postulated as co-factors for cervical cancer. An association of Trichomonas vaginalis with cervical neoplasia was observed in several studies (Zhang & Begg, 1994), including a few large prospective cohort investigations (Gram et al., 1992; Zhang et al., 1995; Viikki et al. 2000). However, these studies did not control appropriately for HPV and were noted to have other limitations (Boyle & Smith, 1999; Watts et al., 2005). In contrast, two recent cross-sectional investigations (Becker et al., 1994; Schiff et al., 2000) and one prospective cohort study (Watts et al., 2005) found no relation between T. vaginalis and CIN2/3 or incident SIL, respectively, and neither was bacterial vaginosis found to be associated with cervical neoplasia in several

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recent investigations (Peters et al., 1995; Frega et al., 1997; Castle et al., 2001; Boyle et al., 2003; Watts et al., 2005). Two studies that did report a significant association between bacterial vaginosis and cervical neoplasia did not control for possible confounding factors (Platz-Chrisensen et al., 1994; Schiff et al., 2000). Studies of the relationship between bacterial vaginosis and HPV infection gave conflicting results (Peters et al., 1995; Sikstrom et al., 1997; Castle et al., 2001; Jamieson et al., 2002; Mao et al., 2003; Watts et al., 2005). A few epidemiological investigations reviewed by Boyle and Smith (1999) have assessed the possible association of Neisseria ghonnorrhoeae with cervical neoplasia and most reported no association (Takac, 1998; Boyle & Smith, 1999). Among the studies that reported a positive association, a large case–control study found some increase in risk for CIN3 or cancer with seroantibodies to N. ghonnorrhoeae in the women (de Sanjosé et al., 1994) but no association with seroantibodies in their husbands (Bosch et al., 1996; Muñoz et al., 1996a). A prospective study of young patients at clinics for sexually transmitted disease reported an increased risk for incident CIN2/3 among culture-positive women, but no information on other possibly correlated risk factors was available (Koutsky et al., 1992). There is also little evidence to suggest that Candida albicans (Becker et al., 1994; Takac, 1998; Schiff et al., 2000) or Treponema pallidum (de Sanjosé et al., 1994; Bosch et al., 1996; Muñoz et al., 1996a; Ferrera et al., 1997a; Schiff et al., 2000; Thomas et al., 2001c) are co-factors for cervical tumorigenesis. (e)

Inflammation caused by various infections

Although only infection with C. trachomatis has been consistently associated with cervical neoplasia in epidemiological studies, it has been suggested that cervical inflammation in general, regardless of the specific microbial agent involved, may be a risk factor for progression of HPV infection (Castle & Giuliano, 2003). If this assumption is correct, it might help to explain some of the variable findings reviewed above. Consistent with this hypothesis, a cross-sectional study observed that the specific level of inflammation (graded by the number of invading neutrophils) was directly associated with a risk for CIN2/3 (Castle et al., 2001). In other studies, CIN2/3 was found to be more strongly associated with ‘any’ cervical co-infection than with co-infection assessed on an agent-specific basis (Schiff et al., 2000), and data from two independent studies suggested that the risk for cervical cancer increased with increasing numbers of possible co-infections detected (Schmauz et al., 1989; Dillner et al., 1994). Moreover, a small cohort study observed that variations in the detection of HPV DNA over time were related to the presence of any one of a number of cervicovaginal infections rather than to one specific infectious agent (McNicol et al., 1994). (f)

Possible protective effects of adeno-associated virus

Adeno-associated virus (AAV) is the one infectious agent that may reduce the risk for cervical neoplasia. AAV can suppress papillomavirus replication and cellular transformation in vitro (Hermonat, 1992, 1994a). However, although a few epidemiological studies

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found that detection of AAV DNA was associated with a decreased risk for the presence and/or grade of neoplasia (Walz et al., 1997; Coker et al., 2001), most studies did not (Strickler et al., 1999b; Odunsi et al., 2000; Lanham et al., 2001; Ahn et al., 2003; Grce et al., 2004). Serological studies have also given conflicting results. Although several earlier seroepidemiological studies found possible inverse associations of AAV antibodies with cervical neoplasia (Sprecher-Goldberger et al., 1971; Mayor et al., 1976; Georg-Fries et al., 1984; Tobiasch et al., 1994), two subsequent serological studies gave negative results. The first found no association of AAV antibodies with cervical neoplasia or invasive cervical cancer in two separate, independent populations of patients (Strickler et al., 1999). The second study found a non-significant inverse association with cervical cancer (odds ratio, 0.4; 95% CI, 0.1–1.6) after controlling for HPV, and no association with CIN3 (odds ratio, 1.4; 95% CI, 0.3–6.8) (Smith et al., 2001). 2.8

Special populations

2.8.1

Skin cancer in patients with epidermodysplasia verruciformis (EV) and HPV infection

EV is a very rare, inherited condition that was first described by Lewandowsky and Lutz (1922). During the following 60 years, approximately 250 cases were reported worldwide (Lutzner & Blanchet-Bardon, 1985). The condition is usually recognized before puberty and is characterized by widespread HPV infection and the later development of multiple cutaneous squamous-cell carcinomas, predominantly at sites that are exposed to the sun. Although basal-cell carcinomas have been described in EV patients, they appear to be rare, and there are no reports of increased risk for malignant melanoma in EV patients (Orth et al., 1980; Orth, 1986, 1987; Majewski et al., 1997; Pfister, 2003). Two cases of eccrine carcinoma and a single case of a malignant proliferating trichilemmal tumour have been described in EV patients (Motegi et al., 2003). No published standardized mortality ratios (SMRs) are available for skin cancer in EV patients, but a squamous-cell carcinoma:basalcell carcinoma ratio of 16:1 was reported in one study of 66 EV patients (Tanigaki et al., 1986). About half of these patients had developed warts by the age of 10 years, whilst squamous-cell carcinoma developed between the ages of 30 and 50 years. The average time lag between onset of EV-type skin warts and squamous-cell carcinoma was 24.5 years (Tanigaki et al., 1986). Other virus-associated cancers, including liver cancer associated with chronic hepatitis B virus infection (van Voorst Vader et al., 1986; see also IARC, 1994), genital carcinoma, tonsillar carcinoma and Burkitt lymphoma, have rarely been described in EV patients (Lutzner & Blanchet-Bardon, 1985; Ishiji et al., 2000). The limited epidemiological data on EV have pointed to a considerable time lag between primary HPV infection and the development of HPV-associated changes. HPV infection is probably acquired during the first days after birth, particularly in EV families; even in the general population, 45% of skin swab samples were positive for HPV DNA shortly after birth (Antonsson et al., 2003). There is one report of possible vertical

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transmission in an EV patient (Favre et al., 1998b). The same HPV types as those found in the skin lesions of the mother were detectable in her amniotic fluid, placenta and genital scrapes. In contrast, EV-type skin warts never start to appear before 4–5 years of age and carcinomas develop much later. Majewski and Jablonska (1997) observed EV patients with skin autografts from the uninvolved internal aspect of the arm that covered areas of the forehead that had been excised for carcinomas. Within the grafted skin, benign lesions started to develop only several years after transplantation. No carcinoma developed for up to 20 years of graft life, whereas premalignant and malignant changes appeared around the grafts. This suggests that HPV-associated skin carcinogenesis is a very slow process. The high level of consanguinity in EV families suggests an autosomal recessive mode of inheritance (Lutzner, 1978; Tanigaki et al., 1986) but, in one family, the inheritance appeared to be X-linked (Androphy et al., 1985). Genetic linkage analyses of consanguineous EV families identified susceptibility loci on chromosomes 17q25 and 2p21-p24 (Ramoz et al., 1999, 2000). A more detailed analysis of three Algerian and two Colombian EV1 (17q25)-linked families revealed homozygous nonsense mutations in one of two adjacent novel genes, EVER1 and EVER2, in affected family members (Ramoz et al., 2002). No such mutations were observed in 90 unrelated individuals. The predicted fulllength EVER1 and EVER2 proteins share 28.4% of their amino acids. They have features of integral membrane proteins and are localized in the endoplasmic reticulum. The immunogenetic background responsible for defects in immunosurveillance of EVHPV infections and the inability to eliminate EV-HPV-infected cells remains poorly defined (Majewski et al., 1997). A comparison of the prevalence of interleukin (IL) 10 promoter polymorphisms in 22 Brazilian EV patients and 27 healthy individuals indicated that genotypes that determine a low IL10 production are significantly increased in EV patients (de Oliveira et al., 2003). This is not easy to interpret because IL10 is generally regarded as immunosuppressive, although it has been speculated that low levels of IL10 would allow higher production of tumour necrosis factor-α (TNFα), which could impede antigen-presenting Langerhans cells. Mutations in exons 5–8 of the TP53 gene were detected in one of three benign lesions, two of five actinic keratoses, three of nine Bowen carcinomas in situ and five of eight squamous-cell carcinomas of two EV patients (Padlewska et al., 2001). Five of nine mutations characterized by sequencing were C→T transitions at dicytidine sites that are considered to be ultraviolet (UV) signature mutations. The other four mutations could be caused by reactive oxygen species that result from exposure to the UVA component of sunlight or from oxidative metabolism. The importance of sunlight (see also IARC, 1992) in the development of EV-associated squamous-cell carcinomas is suggested by the fact that, although skin warts are found on all body sites, the carcinomas occur almost exclusively on sites exposed to the sun (Tanigaki et al., 1986). Furthermore, squamous-cell carcinomas appear to develop more frequently in Caucasian EV patients who live in subtropical and tropical climates than in those who live in temperate climates, and are rare in black EV patients. Only two of 33 (6%) black South African EV patients developed squamous-cell carcinomas (van Voorst Vader et al., 1987)

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compared with 40–50% of Caucasian patients who lived in Europe (Orth et al., 1979), 58% of Japanese patients who lived in Japan (Tanigaki et al., 1986) and 100% of patients who lived in South America (Rueda & Rodriguez, 1976). HPV types in warts and skin cancers in EV patients (a)

Skin warts

EV patients develop a variety of skin warts: common warts (verruca vulgaris), consistently plane warts (verruca planar, that are usually somewhat flatter than plane warts in the general population) and frequently the so-called EV-specific lesions, namely, red plaquelike lesions and scaly, pityriasis versicolor-like lesions (Orth et al., 1979; Orth, 1987; Majewski et al., 1997). Table 63 summarizes studies of HPV typing of EV-associated skin warts, all of which used restriction enzyme cleavage and hybridization methods without amplification. Reports are based on a limited number of specimens, often from single patients, and only one study included information on control material (Jacyk et al., 1993a). Some studies did not specify the type of skin wart examined and many did not specify the number of samples examined. A large number of HPV types has been found in plane warts and EV-specific lesions. The phylogenetically related types HPV 5, 8, 9, 12, 14, 15, 17, 19, 20, 21, 22, 23, 24, 25, 36, 38, 47 and 50 all belong to the papillomavirus genus beta according to the new nomenclature (de Villiers et al., 2004a). They are widely referred to as EV-HPV because they were originally found only in EV patients. In addition to EV-HPV, HPV 3 and 10, which are found in plane warts in the general population, were frequently detected. A very similar spectrum of HPV types has been identified in five EV patients by PCR with degenerate HPV primers (HPV 8, 19, 20, 24, 38, 5-related and 9-related from genus beta, and HPV 2 and 57 that are found in common warts in the general population) (Harris et al., 1997; Surentheran et al., 1998). Cutaneous lesions induced by EV-HPV are highly polymorphic, and include verruca planar, red plaque-like lesions, pityriasis versicolor-like lesions and lesions similar to seborrheic keratoses. However, they share a specific cytopathic effect that is identical for various EV-HPVs, the intensity of which depends only on the viral load and the activity of the disease (Majewski et al., 1997). The cytopathic effect is characterized by large clear cells with clear nucleoplasma and cytoplasmic keratohyaline granules. A keratinocyte differentiation-dependent viral transcription pattern has been revealed by in-situ hybridization of benign HPV 5-induced lesions, which is characteristic of productive warts with strong E4-specific signals almost throughout the epithelium and L1/2-specific signals in the superficial layers of the stratum granulosum (Haller et al., 1995). HPV 3 and 10 are predominantly associated with plane warts, which may be large, pigmented and confluent. The cytopathic effect is again specific with a ‘bird’s eye-like’ appearance of cells due to vacuolization around pyknotic dark nuclei (Majewski et al., 1997).

Types included

No. of cases

Clinical description

+

Ostrow et al. (1982), USA

5

2

NA

+

Pfister et al. (1983a) [Turkish patient]

1, 2, 3, 4, 5, 6, 8, 10, 11, 20, 25, 29

1

NA

+

Kremsdorf et al. (1984), France

14a, 14b, 15, 17a, 17b, 19, 20, 21, 22, 23, 24

8

VV/VP PV

Lutzner et al. (1984), France

2, 3, 5, 8, 9, 14, 15, 17, 20, 21, 22, 23, 24

11

NA

van Voorst Vader et al. (1986), Netherlands

5, 8, 17, 19, 20, 24

1

NA

Kanda et al. (1989), Japan

1, 2, 3, 5, 8, 12, 14, 17, 20, 21, 38

12

VP RP PV

VP, VV PV RP RP

+ +

20

2, 9 9, 12 12

+

+

+

+

+

+c

19c, 25c, one type not specified

Frozen tissue

+

14a, 14b, 15, 21 19, 21, 23, 24

Frozen tissue

+

2, 14, 22, 9, 9related

Frozen tissue

19, 24

Frozen tissue

14, 38 12, 14, 38 (multiple HPV types in some lesions) 14, 21

Frozen tissue

+

+ +

Frozen and paraffinembedded tissue Frozen tissue

+ +

Othersa

+

+

+

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+ +

1, 2, 3, 4, 5, 8, 9, 12

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5

Orth et al. (1979)b, Europe

14

Comments

HPV type-specific positivity 1–3, 10

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Table 63. HPV types detected by southern blot in skin warts in epidermoplasia verruciformis (EV) patients with multiple lesions

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Table 63 (contd) Reference, study location

Types included

20 not specified

5

VP PV Seborrheic keratoses PV VP

Jacyk et al. (1993b), South Africa

20 not specified

1

PV VV

Yutsudo et al. (1994), Japan

3, 17, 20, 38 and others not specified

1 1

PV VV, VP

Adachi et al. (1996), Japan

3, 5, 8, 9, 12, 14, 17, 20, 25, 47; type-specific primers for HPV 5, 14, 21, 47

1

NA

1–3, 10

5

+

+ +

8

17

20

Frozen tissue +

+

+

+ +

Othersa

+

+

+

No HPV found in 10 keratoses from non-EV patients

Frozen tissue; includes information on controls

9 4, 9

Frozen tissue

38

Fresh tissue

14, 21, 47

Paraffin-embedded tissue

+ +

NA, not available; PV, pityriasis versicolor-like lesions; RP, red plaque-like lesions; VP, verruca planar; VV, verruca vulgaris a Of those types tested b EV-HPV were named HPV 4 in the original paper and further differentiated and later renamed as HPV 5, 8, 9 and 12 (Orth et al., 1980; Kremsdorf et al., 1984). c Specified later (Gassenmaier et al., 1984)

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Jacyk et al. (1993a), South Africa

20

Comments

HPV type-specific positivity

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20 not specified

Clinical description

HUMAN PAPILLOMAVIRUSES

Jacyk & de Villiers (1993), South Africa

No. of cases

331

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(b)

HPV types in squamous-cell carcinoma of EV patients

Table 64 summarizes data on HPV types in EV-associated skin cancer, which are derived mostly from single case reports. All studies were performed using hybridization methods without amplification. In the limited number of invasive squamous-cell carcinomas analysed, HPV 5, 8, 14, 17, 20 and 47 have been identified. HPV-21 DNA was disclosed by PCR amplification and sequence analysis in a malignant proliferating trichilemmal tumour (Motegi et al., 2003). The dominance of HPV-5 and -8 in malignant tumours from Europe and the USA is in contrast to the presence of multiple EV-HPV types in benign lesions of the same patients (Pfister et al., 1983b; Van Voorst Vader et al., 1986; reviewed in Orth, 1987), which may point to an increased carcinogenic potential. However, due to the overall small number of patients, data on prevalence should still be regarded as preliminary. In contrast, HPV 3 or 10 were never detected in EV-associated squamous-cell carcinomas and no malignant conversion was observed in EV patients only infected by HPV 3 (Majewski et al., 1997). HPV 5, 8, 17, 20 and 47 DNAs have been found in squamous-cell carcinomas as episomal oligomers and monomers, some in concatemeric form (approximately 100 copies/cell) (Pfister et al., 1983b; Yutsudo et al., 1985; Deau et al., 1991; Yutsudo et al., 1994; Adachi et al., 1996). This is in contrast to the frequently observed integration of HPV DNA in cervical cancers. HPV 14 was identified only rarely in a skin carcinoma and appeared to be integrated into the cellular DNA (Orth, 1987). In another case, HPV 5 DNA was integrated in a metastasis whereas viral genomes persisted extrachromosomally in the corresponding primary tumour (Yabe et al., 1989, 1991, 1999). Transcripts of HPV 5, 17 and 20 have been demonstrated in squamous-cell carcinomas (Orth, 1987; Yutsudo & Hakura, 1987; Yutsudo et al., 1994). Both wild-type HPV genomes and those with deletions have been found in primary and metastatic tumours (Ostrow et al., 1982; Yabe et al., 1989; Deau et al., 1991). Some cancers contain mostly or only viral DNA with deletions, which primarily affect the late genes but may extend into the non-coding genome region. In addition, sequence variants of the HPV 5 and 8 E6 gene have been demonstrated in some EV-associated cancers (Deau et al., 1991). The significance of these findings and their role in transformation remains unclear. 2.8.2

Studies of the incidence of HPV-associated neoplasia in transplant patients

A consistent increase in the incidence of malignancies in organ transplant recipients has been attributed to the effect of chronic immunosuppression that is required to prevent rejection of the transplanted tissue. Immunocompromised individuals are at increased risk for HPV-associated anogenital and cutaneous cancers compared with age-matched healthy individuals. Organ transplant recipients have an almost 100-fold increased risk for squamous-cell skin carcinoma and a 10-fold increased risk for basal-cell carcinoma (Leigh et al., 1999). In Australia, where exposure to UV light is high, the cumulative incidence of skin cancer increased progressively from 7% after 1 year of immunosuppression to 40%

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Table 64. HPV types detected by southern blot in skin cancer in epidermoplasia verruciformis (EV) patients Reference, study location

Types included

No. of cases

HPV type-specific positivity 5

8

17

Comments

20

+

Frozen tissue; HPV 5 found in PV lesions, primary SCC and metastatic SCC in same patient; wild-type and sub-genomic HPV 5 found in primary and metastatic tumour

Pfister et al. (1983b) [Turkish patient]

1, 2, 3, 4, 5, 6, 8, 10, 11, 20, 25, 29

1

+

Frozen tissue; 100 copies/cell; oligomeric DNA, some persisting in concatemeric form

Lutzner et al. (1984), France

1, 2, 3, 5, 8, 9, 14, 15, 17, 20, 21, 22, 23, 24

5

+

Yutsudo et al., (1985), Japan

17

1

Orth (1986, 1987), International

1, 2, 3, 4, 5, 8, 9, 12, 14a/b

van Voorst Vader et al. (1986), Netherlands

+

Frozen tissue; HPV 14 found in one SCC; the seven skin cancers include three Bowen disease and four SCC. +

Fresh tissue; 100 copies/cell; episomal DNA as oligomers and monomers; HPV transcripts in SCC suggests infection.

14

+

1, 2, 3, 4, 5, 8, 9, 10, 12, 14, 15, 17, 19–24

1

+

Frozen tissue; HPV 17 and 24 found in peri-lesional tissue

Yabe et al. (1989, 1991, 1999), Japan

5

1

+

Tissue storage not specified; HPV 5 found in benign lesions; deleted forms of HPV 5 found in both primary and metastatic tumours from same patient

Yutsudo et al. (1994), Japan

3, 17, 20, 38 and others not specified

1

Adachi et al. (1996), Japan

3, 5, 8, 9, 12, 14, 17, 20, 25, 47; type-specific primers for 5, 14, 21, 47

1

Ishiji et al. (2000), Japan

3, 5, 16, 20, 57, 58, 60

1

Frozen tissue; HPV-14b (1 tumour) (approximately 100–300 copies/cell)

+

Fresh tissue; 100 copies/cell; episomal DNA as oligomers and monomers; HPV transcripts in SCC suggests infection. Paraffin-embedded tissue; HPV 47 episomal DNA as oligomers and monomers

+

Paraffin-embedded tissue; HPV 20 DNA also disclosed by in-situ hybridization in the nuclei of some cancer cells

333

PV, pityriasis versicolor-like lesion; SCC, squamous-cell carcinoma

+

Page 333

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Ostrow et al. (1982), USA

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after 9 years and 70% after 20 years (Bouwes Bavinck et al., 1996). These data suggest the interplay of UV light and immunosuppression as risk factors in the development of skin cancer (IARC, 1992). The exact role of immunosuppression in conferring increased risk is not known. Current data suggest that it is most strongly associated with the early stages of dysplasia, and that progression to cancer per se is not associated with immunosuppression. Similarly, the biology of HPV infection among immunocompromised individuals is not yet known in detail. Also, questions remain about the biology of HPV infection among transplant patients compared with immunocompromised individuals who are HIV-positive (Palefsky & Holly, 2003). The first reports of cutaneous and anogenital lesions as a result of HPV infection in immunosuppressed transplant recipients appeared in the 1980s. Lutzner et al. (1980) described two immunosuppressed renal allograft recipients who developed skin lesions. In both patients, structural antigens of HPV 5 were identified in these lesions by immunofluorescence. The histological and ultrastructural features observed were similar to those previously seen in patients with HPV 5-associated EV, the only condition in which this HPV type had been detected until that time. The data suggested a role of this potentially high-risk virus in skin cancers that are known to occur with increased frequency in immunosuppressed allograft recipients. Lower genital cytopathology was evaluated in 105 immunosuppressed renal transplant recipients (Halpert et al., 1986). Evidence of HPV infection was found in 17.5% and lower genital neoplasia was observed in 9.5% of the patients. The rate of viral infection in the immunosuppressed patients was ninefold greater than that in a general population and 17-fold greater than that in a matched immunocompetent population. The rate of cervical neoplasia was 16-fold greater than that in the general population and nine-fold greater than that in a matched immunocompetent population. In one-third of patients with HPV lesions and one-half of patients with neoplastic lesions, multiple lower genital sites were also involved. Of the risk factors evaluated, only the number of sexual partners was associated with the development of HPV-related lower genital neoplasia. Studies on the incidence of HPV-associated cancer in transplant patients have been reviewed previously (IARC, 1995). A number of more recent studies are reviewed below. (a)

HPV infection, CIN and invasive cervical and anogenital carcinomas in transplanted patients

Studies up to 1994 on the prevalence of anogenital and cervical lesions and/or HPV infection in immunosuppressed women following transplantation were reviewed previously (IARC, 1995) and are summarized in Table 65. At least seven studies of transplanted patients have been published subsequently. To test the hypothesis that renal allograft recipients are at high risk for anal HPV infection and AIN, 133 renal allograft recipients and 145 control patients underwent anoscopy and biopsy (Ogunbiyi et al., 1994). PCR was used to detect HPV 16 DNA in biopsy samples. A histological diagnosis of anal HPV infection or AIN was made in 32 allograft recipients: HPV infection was detected in five patients, 20 had AIN1, three had AIN2,

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Reference, study location

No. and % with HPV or lesion Controls

No.

No.

%

Comments

9a (3.4–20.2)

Frozen tissue

17b (5.0–50.6)

Frozen tissue

09:55

Transplanted patients

Relative risk (95% CI) or p value %

8.5

Halpert et al. (1986), USA

Cytology

18/81

22.8

2/81

MacLean et al. (1986), New Zealand

Cytology

5/24

21



Alloub et al. (1989), United Kingdom

DNA hybridization HPV 6/11 HPV 16/18 Mixed 6/11 and 6/18

22/49 9/49 10/49 3/49

45 18.4 20.4 6.1

26/69 22/69 4/69 –

Gentile et al. (1991), Italy

Cytology/histology

12/39

31



Frozen tissue

Gitsch et al. (1992), Germany

Histology (condyloma)

7/23

30



Frozen tissue

Fairley et al. (1994a), Australia

PCR (L1 consensus primers)

15/69

22

1/22

4.5

p = 0.05

Frozen tissue; the p value was obtained using a Fisher’s exact test.

Ogunbiyi et al. (1994), United Kingdom

Anoscopy/biopsy

5/133

0

0



Frozen tissue

3.8

– 2.5

Frozen tissue

38 32 6

Paraffin-embedded tissue p = 0.36 p < 0.005

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335

Cytology (koilocytotic atypia)

HUMAN PAPILLOMAVIRUSES

HPV infection Schneider et al. (1983), USA

Method of detection

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Table 65. Prevalence or risk of cervical HPV infection, cervical intraepithelial neoplasia (CIN) and invasive carcinoma of the cervix and anogenital carcinomas in transplanted patients

Method of detection

No. and % with HPV or lesion Transplanted patients

Controls

No.

No.

%

%

Relative risk (95% CI) or p value

Comments

4.2







Frozen tissue

Roka et al. (2004), Austria

Hybrid Capture 2 with high-acid low-risk probes

14/60

23.3







Frozen tissue; kidney and liver; transplanted patients

6/132

4.5



Frozen tissue

0



Frozen tissue; mean time since transplant, 61 months

Invasive carcinoma Schneider et al. Cytology (1983), USA MacLean et al. (1986), New Zealand

Cytology

0/24

Fairley et al. (1994b), Australia and New Zealand

Cytology

12 cases

NA



3.3 (1.7–5.8)

Frozen tissue; mean follow-up, 5.8 years; SIR comparing patients on dialysis with transplanted patients

Birkeland et al. (1995), Denmark, Finland, Norway and Sweden

Histology

28 cases

NA



8.6 (5.7–13)

Frozen tissue; mean follow-up, 4.8 years; SIR; populationbased cancer registry data

Ozsaran et al. (1999), Turkey

Histology

20/48

Brown et al. (2000), USA

PCR MY11/09 and specific primers for 6, 11, 16, 18

13/16

4.7 81









8/13

62

p = 0.02

Paraffin-embedded tissue

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Ozsaran et al. (1999), Turkey

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Table 65 (contd)

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Reference, study location

No. and % with HPV or lesion Controls

No.

No.

%

[14 (2.8–40)]

Frozen tissue; mean follow-up, 3.6 years

%

3/131



Cordiner et al. (1980), United Kingdom

Cytology/histology

5/26

19



Frozen tissue; after a mean of 3.8 years of immunosuppression

Ingoldby et al. (1980), United Kingdom

Cytology

0/50

0



Paraffin-embedded tissue; 3 years of follow-up

Schneider et al. (1983), USA

Cytology

6/132

4.5



Frozen tissue; mean time to CIN since transplant, 38 months

Halpert et al. (1986), USA

Cytology

10/81

12

2/81

Alloub et al. (1989), United Kingdom

Histology

24/49

49

7/69

Gentile et al. (1991), Italy

Cytology/histology

1/39

2.6



Frozen tissue; mean time since transplant, 77 months

Gitsch et al. (1992), Austria

Histology

2/23

8.7



Frozen tissue

David et al. (1993), Germany

Cytology/histology

5/58

8.6



Frozen tissue

2.5 10

[5.6 (1.1–38)]

Frozen tissue; mean time since transplant, 47 months

[8.5 (3.0–25)]

Paraffin-embedded tissue

337

Cytology

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2.3

Comments

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Transplanted patients

Relative risk (95% CI) or p value

HUMAN PAPILLOMAVIRUSES

CIN Porreco et al. (1975), USA

Method of detection

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Table 65 (contd)

Method of detection

Relative risk (95% CI) or p value

No. and % with HPV or lesion

No.

%

7.2

0/22

0

20.3

1/145

0.68

p < 0.05

Frozen tissue

4.9

0/3000

0



Frozen tissue

5/69

Ogunbiyi et al. (1994), United Kingdom

Anoscopy/biopsy

27/133

Longuet et al. (1996), France

Southern blot hybrid

4/81

Sasadeusz et al. (2001), Australia

Cytology

Before transplant 7/77 After transplant 11/87

9.1



2.2 (1.1–4.2)

12.6



7.0 (4.8–10.2)

17/166

10.2



Cytology

Frozen tissue

Includes four high-grade smears; the risks are for transplanted women compared with the general population.

Includes CIN3; incidence of CIN3 lesions in lung transplanted patients versus women screened from the New South Wales cytology registry

[ ] Calculated by the Working Group CI, confidence interval; CIN, cervical intraepithelial neoplasia; PCR, polymerase chain reaction; SIR, standardized incidence ratio a Compared with the general population b Compared with the matched immunocompetent population

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Cytology

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No.

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Controls

Fairley et al. (1994a), Australia

Malouf et al. (2004), Australia

Comments

Transplanted patients %

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Reference, study location

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Table 65 (contd)

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three had AIN3 and one patient had anal cancer. One subject with AIN was detected in the control group. HPV 16 DNA was detected in 47% and 12.4% of anal biopsies in the transplant recipients and the controls, respectively. Renal allograft recipients were found to be at high risk for anal HPV infection and neoplasia (p < 0.05). The presence of HPV and the ensuing risk for CIN were studied in 48 renal transplant patients who received immunosuppressive therapy (Özsaran et al., 1999). Cervical smears were analysed and colposcopy was conducted. Genital neoplasia was found in 20 of the patients. Koilocytosis developed in six of eight (75%) patients who received highdose immunosuppressive therapy that was necessitated by rejection of the transplant. HPV was found in two of 48 patients, both of whom had koilocytosis in their cervical biopsies. The data show that renal transplant patients who receive immunosuppressive therapy are at increased risk for CIN. The prevalence of anal HPV infection was studied in organ transplant patients before immunosuppressive therapy (Roka et al., 2004). Patients (40 men, 20 women) who underwent solid-organ transplantation (kidney, liver) for the first time were routinely screened for anal HPV infection. Anal swabs were obtained within 24 h after transplantation and analysed for the presence of mucosal-type HPV DNA by liquid DNA/RNA hybridization. Some type of HPV DNA was detected in 14 patients (23.3%), nine patients (15%) were positive for high-risk HPV, eight (13.4%) were positive for low-risk HPV and three (5%) were positive for both types. The prevalence of HPV infection tended to be higher in liver transplant than in kidney transplant recipients (29.4% versus 20.9%), but the difference was not significant. The prevalence of previous HPV infection (23.3%) before immunosuppressive therapy was started was higher than that found in previous studies or in a control group. In particular, the rate of infection with high-risk HPV types was 15%. HPV types were analysed in lower genital tract neoplasms of renal transplant recipients and compared with virus types found in immunocompetent patients who had similar neoplasms and in normal immunocompetent controls (Brown et al., 2000). Twenty specimens from lower genital tract neoplasms of 16 renal transplant patients, 13 specimens from 13 immunocompetent patients with similar histology and samples from 13 patients with normal lower genital-tract histology were analysed by PCR for the presence of HPV infection. Primers included the L1 region consensus primers and primers specific for the HPV E6 region for subtypes 6, 11, 16 and 18. HPV infection was detected in 21 of 46 specimens tested. Thirteen of the HPV-positive specimens were from transplant patients and eight were from immunocompetent patients (five with and three without disease). This difference was statistically significant between the transplant and immunocompetent group (p = 0.02). Although no difference in HPV 6 and/or 11 was detected between the two groups, the difference in HPV types 16 and/or 18 approached statistical significance (p = 0.06). High-risk HPV 16 and/or 18 were found at a higher rate in transplant patients than in their immunocompetent counterparts. The combination of reduced immune function and increased HPV 16 and/or 18 infection rate places these patients at increased risk for aggressive lower genital tract neoplastic progression.

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The genome of a novel HPV type was cloned from an iatrogenically immunosuppressed woman with persistent low-grade vaginal AIN (Longuet et al., 1996). HPV 74 was found to be phylogenetically related to the low-risk HPV types 6, 11, 44 and 55. HPV 74 or a variant of this type was found in specimens from three additional immunosuppressed women but not in about 3000 anogenital specimens from immunocompetent patients. Immunocompromised patients, such as renal allograft recipients, have a higher rate of cytological abnormalities following infection with HPV. This is thought to be due to prolonged persistence of the virus because of impaired clearance by the immune system. A retrospective review was conducted of the cervical cytology of women who underwent bone-marrow transplantation and who had had cervical smears performed in the period 1990−98 (Sasadeusz et al., 2001). The number of cytological abnormalities was significantly higher than that in the general population, both before (age-adjusted odds ratio, 2.2; p = 0.02) and after bone-marrow transplantation (odds ratio, 7.0; p < 0.0001). After transplantation, allogeneic recipients had more abnormalities than autologous patients (odds ratio, 2.6; p = 0.02) although only allogeneic recipients had a higher level of abnormalities after transplantation than before (allogeneic odds ratio, 6.8; p = 0.004). These observations suggested that pre-transplant disease and treatment factors increase the risk for cytological abnormalities and that transplant-related factors such as conditioning therapy and immunosuppression further increase this risk. The incidence and outcomes of HPV infection and cervical abnormalities after lung transplantation were investigated in a retrospective cross-sectional study of 166 female recipients who underwent transplantation between February 1989 and June 2001 (Malouf et al., 2004). The incidences of low-grade epithelial abnormality of the cervix, CIN1 and the earliest pre-cancerous changes of the cervical epithelial cells (CIN3) in the post-transplant cohort were 42.2 and 30, respectively, per 1000 women screened. In a large reference population of 20−69-year-old women, these figures were 8.3 and 6.2, respectively, per 1000 women screened. It was concluded that the incidence of cervical abnormalities in lung transplant recipients is about five times higher than that in the general population. (b)

HPV DNA in transplant-associated skin lesions (i)

Skin warts

Individual case reports documented the presence of multiple HPV types, including EV-associated types (Soler et al., 1992; Purdie et al., 1993), in skin warts of transplant recipients. HPV 27 (Ostrow et al., 1989b) and HPV 49 (Favre et al., 1989) were first identified in warts of transplant recipients and HPV 26 in warts of a patient with an unusual immune deficiency syndrome (Ostrow et al., 1984). Table 66 summarizes the results of larger studies (i.e. that include more than five lesions) of transplant-associated viral warts. In most studies that used in-situ hybridization or southern blot, the overall detection rate of HPV DNA ranged from 60 to 90%. Three studies that used PCR amplification with several sets of genus- and species-specific, degene-

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Table 66. Prevalence of HPV DNA in skin warts of transplant recipients Overall HPV positivity (%)a

HPV type-specific positivity

Southern blot (1, 2, 3, 4, 5/8, 16/18)

Rüdlinger et al. (1986), United Kingdom

Comments

1–4, related typesb

EV-, betaHPV

6/11, 16/18

Other types (no. of lesions)

16

8/16 (50)

6/16

1/16

1/16



Paraffin-embedded tissue

Southern blot (1–4, 10, 5, 6/11, 16)

54

39/54 (72)

39/54

0/54

0/54



Frozen tissue; multiple HPV types found in single lesions; no control warts examined

van der Leest et al. (1987), USA

Southern blot (1–6)

32 (44)

39/44 (89)

50/44*

4/44*

0/44



Frozen tissue; *double infections in 15 cases

Barr et al. (1989), United Kingdom

Dot blot and southern blot (1, 2, 4, 5/8)

77

NA

NA

12/77





Frozen tissue; no control warts examined

Wilson et al. (1989), United Kingdom

Southern blot (1, 2, 3, 4, 5, 8)

18

13/18 (72)

9/18

0/18



4/18 not further characterized

Frozen tissue; viral genome in HPV 2 warts showed polymorphism at PvuII and PstI sites.

Blessing et al. (1990), United Kingdom

ISH (4, 5, 8)

20

4/20 (20)

0/20

4/20





Frozen tissue; simple warts, dysplastic warts and EV-like lesions (3) studied; no specimen contained > 1 HPV type; no control wart samples

Obalek et al. (1992), France and Poland

Southern blot (1–7, 10, 16, 18, 28, 41, 50)

56 (82)

72/82 (88)

72/82

10/82

0/82

2/82 detected under nonstringent hybridization conditions

Frozen tissue; EV HPV always co-detected with HPV 3 or related types.

Page 341

Gassenmaier et al. (1986), Germany

No. of cases (warts)

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Method of detection (types included)

HUMAN PAPILLOMAVIRUSES

Reference, study location

341

HPV type-specific positivity

ISH (1a, 2a, 5, 16/18)

Soler et al. (1993), France

Comments

1–4, related typesb

EV-, betaHPV

6/11, 16/18

Other types (no. of lesions)

17

14/17 (82)

9/17

0/17

10/17



Frozen and paraffin-embedded tissue; multiple HPV types found in single lesions; no control warts examined

Southern blot, ISH and PCR (5, 6/11, 16/18, 1a, 2a)

18 transplant 3 non-transplant

11/18 (61) 0/3 (0)

1/18 0/3

1/18 0/3

4/18 0/3



Frozen tissue

Trenfield et al. (1993), Australia

Southern blot (1, 2, 3, 4, 5/8, 10, 11, 16/18, 41)

18

5/18 (28)

5/18

0/18

0/18



Frozen tissue

Hepburn et al. (1994), New Zealand

Dot blot (1–5, 6/11, 8, 41, 48, 49)

36 (44)

19 (43)

26/44

4/44

5/44

41 (1)

Multiple types found in some lesions

Pélisson et al. (1994), France

ISH (1a, 2a, 5, 6a, 11a, 16 and 18)

8 transplant 7 non-transplant 7 non-transplant normal skin

5/8 (63) 4/7 (57)

4/8 4/7

1/8 0/7

4/8 0/7

– –

Frozen tissue; simple warts examined; multiple HPV types found in single lesions

0/7 (0)

0/7

0/7

0/7



Shamanin et al. (1994b), United Kingdom

PCR and direct sequencing (1–4, 10, 5/8, 6/11, 16/18 and others)

50

28 (60)

15/50

6/50

1/50

Uncharacterized (14)

Frozen tissue; benign warts and EV-like lesions (3) studied; no control warts examined

Stark et al. (1994), United Kingdom

Southern blot and PCR (1, 2, 5/8, 6/11, 16/18 and others)

18 transplant 6 non-transplant

10/18 (55) 2/6 (33)

4/18 2/6

3/18 0/6

3/18 0/6

0/18 0/6

Frozen tissue

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Overall HPV positivity (%)a

09:55

Euvrard et al. (1993), France

No. of cases (warts)

30/11/2007

Method of detection (types included)

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Reference, study location

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Table 66 (contd)

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Table 66 (contd) Overall HPV positivity (%)a

HPV type-specific positivity

Nested PCR (2 sets of degenerate primers)

Harwood et al. (1999), United Kingdom

Comments

1–4, related typesb

EV-, betaHPV

6/11, 16/18

Other types (no. of lesions)

8 (15)

1/15 (93)

10/15

2/15

0/15



Frozen tissue.

Several nested PCR with degenerate, cutaneous, mucosal, EV-HPV-specific primers and direct sequencing

23 (51)

51/51 (100)

47/51

41/51

14/51

7 (4), 41 (5)

Co-detection of two or more distinct HPV types in 94% of lesions

Berkhout et al. (2000), Netherlands

Nested PCR with degenerate primers, EV, alpha 2/4, direct sequencing

12 VV 7 VP 16 VS

9/12 (75) 6/7 (86) 10/16 (63)

6/12 6/7

2, 6, 3*/12 0, 1, 2*/7

4/16

7, 7, 5*/16

O’Connor et al. (2001b), Ireland

Nested PCR with mucosal and EVHPV-specific degenerate primers

11

11/11 (100)

6/11

10/11

Frozen tissue; *subgroups of beta-HPV, multiple HPV types in some lesions 0/11



Co-detection of two or more distinct HPV types in 6 warts

EV, epidermodysplasia verruciformis; ISH, in-situ hybridization; NA, not available; PCR, polymerase chain reaction; VP, verruca plana; VS, verruca seborrheica; VV, verruca vulgaris a Of those types tested b Alpha 2-, alpha 4-, gamma- and mu-HPV

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de Villiers et al. (1997), United Kingdom

No. of cases (warts)

09:55

Method of detection (types included)

HUMAN PAPILLOMAVIRUSES

Reference, study location

343

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rate primers detected HPV DNA in more than 90% of skin warts (de Villiers et al., 1997; Harwood et al., 1999; O’Connor et al., 2001b). Common skin-associated HPV types from the genera alpha2, alpha4, gamma and mu, including HPV 1, 2, 3, 4, 10, 27, 28, 57 and 77, were the most common types to be identified in studies in which appropriate probes were used (83–92%). Depending on the genusspecific sensitivity of the detection system, EV-HPV types (genus beta) were found in 10–90% of the warts. In earlier studies, mucosal HPV types 6/11 and 16/18 were found in 23 of 199 (11%) transplant samples and in none of 16 (0%) controls when probes that detect these HPV types were employed [p = 0.32]. A large PCR-based study of 51 warts identified mucosal types in 14 of these (27%). This study employed the most comprehensive set of PCR primers, which allowed co-detection of two or more distinct HPV types in 94% of the lesions (Harwood et al., 1999). Mixed infections were seen predominantly with cutaneous and EV-HPV types. In contrast, in immunocompetent individuals, single HPV types only were detected with the same complex PCR system in all but one of 20 warts from 15 immunocompetent individuals (Harwood et al., 1999). It should be emphasized that no EV phenotype was usually expressed in mixed infections of skin warts of transplant patients (van der Leest et al., 1987; Obalek et al., 1992; Harwood et al., 1999). However, Morrison et al. (2002) described verrucae planae with the histological diagnosis of EV in eight of 17 patients who had had organ transplants (HPV 11) or acquired immunodeficiency syndrome (AIDS) (HPV 6) and identified HPV 8 and HPV 5 by in-situ hybridization. (ii) Verrucous keratoses (precancerous lesions) Table 67 summarizes the prevalence of HPV DNA in case series (of more than five lesions) of verrucous keratoses. HPV DNA detection rates in transplant-associated verrucous keratoses were approximately 20–30% in most studies that used southern blot or in-situ hybridization without amplification. In early PCR-based studies, detection rates were between 24 and 48% (Shamanin et al., 1994b; Stark et al., 1994; Tieben et al., 1994), which increased to 49–88% by the use of more sophisticated and comprehensive primer systems (de Jong-Tieben et al., 1995; de Villiers et al., 1997; Berkhout et al., 2000; Harwood et al., 2000). HPV DNA was also found in 80% of precancerous lesions by a nested PCR with degenerate primers (de Jong-Tieben et al., 1995) but in no more than 30% by single-step PCRs that had been developed earlier (Tieben et al., 1994). This underlines the importance of assay sensitivity and indicates that most HPV DNA persists at low copy levels in precancerous skin lesions and in skin cancers (see Sections (iii) and (iv)) of transplant recipients. This notion corresponds to observations of skin cancers in the general population. In contrast, the prevalence of HPV DNA in skin warts was similar when determined by southern blot (60–90%) and highly sensitive PCRs (100%). The combination of data from studies that used probes designed to detect type-specific HPV, albeit with different methodologies, showed that overall common skin-associated HPV types were found in 27 of 219 (12%) transplant samples compared with four of 23 (17%) control samples [p = 0.68]. These types were found in 11 of 17 lesions (65%) by broad-spectrum PCR assays. When comparing studies with similar methodologies, common

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Table 67. Prevalence of HPV DNA in verrucous keratoses of transplant recipients Method of detection (types included)

No. of cases (lesions)

Overall HPV positivity (%)a

HPV-type specific positivity

Comments

1–4, related typesb

EV-, betaHPV

6/11, 16/18

Other types (no. of lesions)

09:55

Reference, study location

11

1/11 (9)

1/11

0/11

0/11



Frozen tissue; no control samples examined

Barr et al. (1989), United Kingdom

Dot blot (1, 2, 4, 5/8)

NA

NA

NA

7/44

NA

NA



Blessing et al. (1990), United Kingdom

ISH (4, 5/8)

19

5/19 (26)

2/19

3/19





Frozen tissue; no control samples examined

Euvrard et al. (1991), France

ISH (1, 2, 5, 16/18)

0/7 (0)

0/7

0/7

0/7



Frozen tissue

Viac et al. (1992), France

ISH (multiple probes)

11

4/11 (36)

2/11

0/11

0/11

Uncharacterized (2/11)

Frozen tissue

Euvrard et al. (1993), France

ISH (1, 2, 5, 16/18)

21

5/21 (24)

5/21

1/19

3/21



Multiple HPV types identified in single lesions; no control tissue examined

Soler et al. (1993), France

Southern blot, ISH and PCR (1, 2, 3, 4, 5/8, 6/11, 16/18)

18

11/18 (61)

4/18

1/18

15/18



Frozen tissue; multiple HPV types found in single lesions

Trenfield et al. (1993), Australia

Southern blot (1, 2, 3, 4, 5/8, 11, 16/18)

26

4/26 (15)

3/26

1/26

0/26



Frozen tissue

7

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ISH (1a, 2, 3, 4, 5/8, 6/11, 16)

HUMAN PAPILLOMAVIRUSES

Rüdlinger et al. (1986), United Kingdom

345

30/11/2007

Reference, study location

Method of detection (types included)

No. of cases (lesions)

HPV-type specific positivity

Comments

1–4, related typesb

EV-, betaHPV

6/11, 16/18

Other types (no. of lesions)

09:55

Overall HPV positivity (%)a

31 transplant 13 non-transplant

0/31 (0) 0/13 (0)

– –

0/31 0/13

0/31 0/13



Paraffin-embedded tissue

Pélisson et al. (1994), France

ISH (1, 2a, 3, 4, 5, 6a/11a, 16/18)

10 transplant 2 non-transplant

4/10 (40) 0/2 (0)

2/10 0/2

1/10 0/2

4/10 0/2



Frozen tissue; multiple HPV types found in single lesions

Shamanin et al. (1994b), United Kingdom

Southern blot and PCR (1–4, 10, 5/8, 6/11, 16/18 and others)

40

19/40 (48)

6/40

6/40

0/40

Uncharacterized (7/40)

Frozen tissue; no control samples studied

Stark et al. (1994), United Kingdom

Southern blot and PCR (1, 2, 3, 4, 5/8, 6/11, 16/18)

46 transplant 21 non-transplant

11/46 (24) 4/21 (19)

5/46 3/21

2/46 2/21

1/46 0/21

Unknown (3/46)

Frozen tissue; no control samples examined

Tieben et al. (1994), Netherlands

PCR and direct sequencing (multiple probes)

10

3/10 (30)

1/10

1/10

0/10

Uncharacterized (1/10)

Frozen tissue; no control samples

de Jong-Tieben et al. (1995), Netherlands

PCR (degenerate nested primers, direct sequencing)

15 AK 5 BD

14/15 (93) 2/5 (40)

de Villiers et al. (1997), United Kingdom

Nested PCR (2 sets of degenerate primers, sequencing)

12 (17)

11/17 (65)

1/17

6/17

Frozen tissue; frequently more than one HPV type detected 7 (1)

Frozen tissue

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PCR (5/8, 6/11, 16/18)

IARC MONOGRAPHS VOLUME 90

McGregor et al. (1994), United Kingdom

14/15 2/5

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Table 67 (contd)

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Table 67 (contd) Method of detection (types included)

No. of cases (lesions)

1–4, related typesb

EV-, betaHPV

6/11, 16/18

2/1

Comments Other types (no. of lesions)

Harwood et al. (2000), United Kingdom

Degenerate PCR for EV, cutaneous, and mucosal HPV

9 (17)

15/17 (88)

11/17

12/17

Berkhout et al. (2000), Netherlands

Nested PCR with degenerate primers, EV, alpha 2/4, direct sequencing

56

38/56 (68)

NA/56

21/56

Frozen tissue

de Jong-Tieben et al. (2000), Netherlands

PCR (degenerate nested primers, direct sequencing)

37 AK 11 BD

18/37 (49) 8/11 (73)

18/37 8/11

Three of 28 (11%) clinically normal skin samples EV-HPV-positive

Forslund et al. (2003a), Australia

PCR with FAP- and HPV 38-specific primers, cloning and sequencing

6

2/6 (33)

2/6

Frozen tissue; 67% of perilesional and buttock swabs were HPVpositive; 70% of 10 lesions of immunocompetent patients HPVpositive (see Table 24?)

1/6

Frozen tissue; 55% of 11 lesions of immunocompetent patients HPVpositive (see Table 24?)

AK, actinic keratoses; BD, Bowen’s disease; EV, epidermodysplasia verruciformis; ISH, in-situ hybridization; NA, not available; PCR, polymerase chain reaction a Of those tested b Alpha 2-, alpha 4-, gamma- and mu-HPV

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HPV-type specific positivity

HUMAN PAPILLOMAVIRUSES

Overall HPV positivity (%)a

09:55

Reference, study location

347

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cutaneous HPV types are clearly less prevalent in precancerous lesions than in skin warts. Mucosal HPV types were found in about 10% of transplant samples. The high prevalence of HPV now detected in precancerous lesions is due to the frequent detection of EV-HPV types in an average of 65% of transplant samples compared with 6% in earlier studies. (iii) Squamous-cell carcinoma Table 68 summarizes the prevalence of HPV DNA in case series of transplant-associated squamous-cell carcinoma (see also Table 21 in IARC, 1995 for comparison). Rates of detection of HPV DNA in these tumours in studies that used southern blot and in-situ hybridization and in early studies that used PCR varied extremely from 0 to 100%. In one case–control study that used PCR and multiple probes, HPV DNA was found in two of nine (22%) control squamous-cell carcinomas compared with 10 of 30 (33%) transplantassociated squamous-cell carcinomas (Stark et al., 1994). Consistently high rates of detection of HPV DNA ranging from 54 to 91% have been found since 1995 by the use of sophisticated and comprehensive primer systems. Direct comparisons within individual studies consistently showed higher HPV DNA prevalences in squamous-cell carcinomas of immunosuppressed patients than in those of immunocompetent patients (Harwood et al., 2000; Meyer et al., 2000; O’Connor et al., 2001b; Forslund et al., 2003b). The combination of the data from studies before 1995 showed that common skin-associated HPV types were found in 34 of 452 (7%) transplant and one of nine (11%) control samples. These types were found in 24 of 44 carcinomas (55%) by the broadest PCR spectrum employed (Harwood et al., 2000). Mucosal HPV types were found in about 10% of transplant-associated squamous-cell carcinomas. As in the case of precancerous lesions, the high prevalence of HPV found in squamous-cell carcinoma during the past 10 years is due to the frequent detection of a broad spectrum of beta-HPV, including EV-HPV and related types, in 70–80% of the cases. HPV 8 was detected in a primary squamous-cell carcinoma from the arm and its lymph node metastasis (Morrison et al., 2002). (iv) Basal-cell carcinoma Table 69 summarizes the prevalence of HPV DNA in case series of basal-cell carcinoma. In studies from 2000 and later, the overall rates of detection were between 33 and 80%. Combining the data from these four studies, the overall detection rate was 30 of 52 (57%); common skin-associated HPV types were found in 23%, beta-HPV in 44% and mucosal HPV in 4%. (v) Multiple lesions In patients from whom multiple lesions were analysed, certain HPV types were found to prevail in both benign, precancerous and malignant lesions located on different anatomical sites, and partially removed on different occasions up to 7 years apart (Höpfl et al., 1997; de Villiers et al., 1997; Harwood et al., 2000). However, overall no single HPV type seemed to prevail in cutaneous precancerous lesions or skin cancers of transplant recipients.

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Table 68. Prevalence of HPV DNA in squamous-cell carcinoma (SCC) of transplant recipients Reference, study location

Method of detection (types included)

No. of cases (lesions)

HPV type-specific positivity

Comments

1–4, related typesb

EV-, betaHPV

6/11, 16/18

Other types (no. of lesions)

09:55

Overall HPV positivity (%)a

16/25 (64)

1/25

15/25





Frozen tissue

Magee et al. (1989), USA

ISH (1–4, 16/18, 6/11)

8

8/8 (100)

0/8



8/8





Blessing et al. (1990), United Kingdom

ISH (4, 5/8)

11

2/11 (18)

2/11

0/11





Frozen tissue

Dyall-Smith et al. (1991), United Kingdom

PCR amplification (1–4, 5, 7, 9, 11, 16/18, 19, 25)

188

0/188 (0)

0/188

0/188

0/188



Frozen tissue; no control SCC studied

Viac et al. (1992), France

ISH (multiple probes)

2/8 (25)

1/8

0/8

1/8





Euvrard et al. (1993), France

ISH (1, 2, 5, 16/18)

46

25/46 (54)

20/46

2/46

15/46



Frozen tissue; multiple HPV types found in single lesions; no control samples studied

Purdie et al. (1993), United Kingdom

Dot blot and Southern blot (1–4, 10, 5/8, 6/11, 16/18)

10

6/10 (60)

2/10

2/10

0/10

Unknown (4/10)



Smith et al. (1993), Australia

PCR amplification (probes not specified)

20

0/20 (0)











Soler et al. (1993), France

Southern blot, PCR and ISH (1–4, 5/8, 6/11, 16/18)

26

21/26 (81)

0/26

6/26

20/26



Frozen tissue; multiple HPV types found in single lesions

Trenfield et al. (1993), Australia

Southern blot (multiple probes)

40

2/40 (5)

1/40

1/40

0/40



Frozen tissue

8

Page 349

25

349

Dot blot (1, 2, 4, 5/8)

HUMAN PAPILLOMAVIRUSES

Barr et al. (1989), United Kingdom

Method of detection (types included)

No. of cases (lesions)

HPV type-specific positivity

Comments

1–4, related typesb

EV-, betaHPV

6/11, 16/18

Other types (no. of lesions)



0/14

0/14



Paraffin-embedded material

09:55

Overall HPV positivity (%)a

30/11/2007

Reference, study location

14 transplant 22 nontransplant

0/14 (0) 0/22 (0)



0/22

0/22

Pélisson et al. (1994), France

ISH (1a, 2a, 5, 6a/11a, 16/18)

13

8/13 (62)

3/13

1/13

7/13



Frozen tissue; no control SCC studied

Shamanin et al. (1994b), United Kingdom

Southern blot and PCR (1, 2, 3, 5, 7, 10, 37, 40)

23

13/23 (57)

4/23

0/23

0/23

41 (1); unknown (8)

Frozen tissue; no control samples examined

Stark et al. (1994), United Kingdom

Southern blot and PCR (1–4, 5/8, 6/11, 16/18)

30 transplant patients 9 controls

10/30 (33)

3/30

0/30

2/30

Unknown (6)

Frozen samples

2/9 (22)

1/9

1/9

0/9

Tieben et al. (1994), Netherlands

PCR and direct sequencing (multiple probes)

24

5/24 (21)

1/24

3/24

0/24

Unknown (2)

Frozen tissue

Berkhout et al. (1995), Netherlands

PCR (degenerate nested primer, direct sequencing)

53

43/53 (81)

0/53

43/53

0/53

Shamanin et al. (1996), United Kingdom

Broad-range PCR with degenerate primers

20

13/20 (65)

7/20

2/20

1/20

de Villiers et al. (1997), United Kingdom

Nested PCR (2 sets of degenerate primers)

22 11 intraepidermal carcinomas

20/22 (91) 10/11 (91)

9/28

> 70%

1*

Multiple HPV types found in some lesions Frozen tissue

Frozen tissue; *HPV 11 in a SCC on the thumb

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McGregor et al. (1994), United Kingdom

41, 54, 61 (1 each), 69 (4)

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Table 68 (contd)

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Table 68 (contd) Reference, study location

Method of detection (types included)

HPV type-specific positivity

Comments

1–4, related typesb

EV-, betaHPV

6/11, 16/18

Other types (no. of lesions)

4//44

66 (1)

Degenerate PCR for EV, cutaneous, and mucosal HPV

18 (44)

37/44 (84)

24/44

33/44

Berkhout et al. (2000), Netherlands

Nested PCR with degenerate primers, EV, alpha 2/4, direct sequencing

81

63/81 (78)

18/81

32,41, 23*/81

Frozen tissue; *subgroups of betaHPV, multiple HPV types in some lesions. 10/31 clinically normal skin samples HPV-positive

de Jong-Tieben et al. (2000), Netherlands

PCR (degenerate nested primers, direct sequencing)

50

34/50 (68)

34/50

Three of 24 (13%) clinically normal skin samples EV-HPVpositive

Meyer et al. (2000), Germany

Nested PCR with mucosa, cutaneous, and EV-HPVspecific degenerate and type-specific (5, 8) primers

9

6/9 (67)

5/9

O’Connor et al. (2001b), Ireland

Nested PCR with mucosa and EV-HPV-specific degenerate primers

9

8/9 (89)

0/9

8/9

0/9



Forslund et al. (2003a), Norway

PCR with FAP-specific primers

60

33/60 (55)

NA

NA

NA

NA

One HPV 10 and mostly beta-HPV in 8 patients

Forslund et al. (2003c), Australia

PCR with FAP- and HPV38-specific primers, cloning and sequencing

11

6/11 (54)

2/11

5/11





Frozen tissue; 91% of perilesional and 73% of buttock swabs were HPV-positive.

70 (1)

Frozen tissue; multiple HPV types found in single lesions

Frozen tissue

351

EV, epidermoplasia verruciformis; ISH, in-situ hybridization; KA, keratoacanthoma; NA, not available; PCR, polymerase chain reaction a Of those types tested b Alpha 2-, alpha 4-, gamma- and mu-HPV

HUMAN PAPILLOMAVIRUSES

Harwood et al. (2000), United Kingdom

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Overall HPV positivity (%)a

09:55

No. of cases (lesions)

Method of detection (types included)

No. of cases (lesions)

Overall HPV positivity (%)a

Comments

HPV type-specific positivity EV-, betaHPV

6/11, 16/18

Other types

09:55

1–4, related typesb 1

0/1 (0)

0/1

0/1

0/1





Obalek et al. (1988), Poland

Southern blot (1, 4, 5, 10, 11, 16/38)

2

2/2 (100)

2/2

0/2

0/2





Euvrard et al. (1993), France

ISH (mixed probe)

2

0/2 (0)

0/2

0/2

0/2





Trenfield et al. (1993), Australia

Southern blot (1–4, 5/8, 11, 16/18)

11

1/11 (9)

1/11

0/11

0/11





McGregor et al. (1994), United Kingdom

PCR amplification (5/8, 6/11, 16/18)

11 (transplant) 15 (non-transplant)

0/11 (0) transplant 0/15 (0) non-transplant



0/11 0/15

0/11 0/15



Paraffin embedded tissue

Pélisson et al. (1994), France

ISH (1, 2, 5, 6/11, 16/18)

4

3/4 (75)

1/4

0/4

3/4



Frozen tissue; no control BCC samples

Tieben et al. (1994), Netherlands

PCR (four consensus primers designed to detect cutaneous HPV types)

4

0/4 (0)

0/4

0/4

0/4

0/4

Frozen tissue

Shamanin et al. (1996), United Kingdom

Broad-range PCR with degenerate primers

5

3/5 (60)

3/5

0/5

0/5

51, 56

Frozen tissue; multiple HPV types in two cases

Harwood et al. (2000), United Kingdom

Degenerate PCR for EV-, cutaneous and mucosal HPV

18/24 (75)

9/24

13/24

2/24

Frozen tissue

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Rüdlinger et al. (1986), United Kingdom

15 (24)

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Table 69. Prevalence of HPV DNA in basal-cell carcinoma (BCC) of transplant recipients

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Table 69 (contd) Method of detection (types included)

No. of cases (lesions)

Overall HPV positivity (%)a

Nested PCR with degenerate primers, EV, alpha 2/4, direct sequencing

14

5/14 (36)

de Jong-Tieben et al. (2000), Netherlands

PCR (degenerate nested primers, direct sequencing)

9

3/9 (33)

Forslund et al. (2003c), Australia

PCR with FAP- and HPV 38- specific primers, cloning and sequencing

5

4/5 (80)

1–4, related typesb

EV-, betaHPV

2/14

0,3, 4*/14

*Subgroups of beta-HPV; multiple HPV types in some lesions

3/9

Three of 24 (13%) clinically normal skin samples EVHPV-positive

3/5

Frozen tissue; 83% of perilesional and 60% of buttock swabs were HPVpositive.

1/5

6/11, 16/18

Other types

HUMAN PAPILLOMAVIRUSES

Berkhout et al. (2000), Netherlands

Comments

HPV type-specific positivity

See Table 7 for a description of the primers used. EV, epidermodysplasia verruciformis; ISH, in-situ hybridization; PCR, polymerase chain reaction a Of those types tested b Alpha 2-, alpha 4-, gamma- and mu-HPV

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353

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(c)

HPV infection and cancer at other sites in transplant patients

(i) Head and neck region Three cases of head and neck squamous-cell carcinoma were reported in patients who were 18, 29 and 53 years of age at the time of tumour diagnosis after renal, cardiac or bone-marrow transplantation (Bradford et al., 1990). Time from transplant to diagnosis of tumour ranged from 7 months to 12 years. Only the youngest patient had no history of exposure to the traditional pre-disposing factors, tobacco and alcohol use. Histopathology of all three tumours showed features of koilocytosis with hyperkeratosis and parakeratosis suggestive of HPV infection (Bradford et al., 1990) [the Working Group noted that this paper does not provide specific data on the presence of HPV]. A 36-year-old renal transplant recipient who took cyclosporin A presented with bilateral nasal polypoid lesions that involved the nasal septum and lateral nasal walls (Harris et al., 1998b). Pathological findings from surgical excision demonstrated an inverted papilloma with focal atypia and mild dysplasia. DNA from the tissue was tested by PCR and revealed the presence of HPV type 6. Analysis of RNA showed transcription in the tissue of the HPV early proteins E6 and E7. Histologically normal nasal tissue from the same patient contained HPV DNA and transcripts similar to those described in the inverted papilloma specimen (Harris et al., 1998b). HPV infection in oral cyclosporin-induced gingival overgrowth was investigated in renal transplant recipients by assessing morphological changes and by the use of in-situ hybridization with HPV-specific probes (Bustos et al., 2001). Biopsies of gingival overgrowth lesions from 13 renal transplant recipients and four samples of healthy mucosa from these patients were analysed. The pathologist was not aware of the HPV result. Twelve of the 13 samples studied (92.3%) contained HPV, of which four tested positive for HPV 6/11 and one for HPV 16. In 11 of the HPV-positive cases, koilocytotic atypia was found. The four biopsies of normal mucosa from gingival overgrowth patients also contained HPV DNA. These data show that suppression of T-cell function by cyclosporin therapy can result in an increase in HPV infection and add to the growth-stimulating activity of cyclosporin in the oral mucosa. A total of 10 paraffin-embedded biopsy specimens of epithelial tumours from six heart transplant recipients were studied for the presence of HPV (Auvinen et al., 2002). These cases included all epithelial cancer cases among the malignancies seen in 249 heart transplant patients at Helsinki University. HPV DNA was amplified by PCR. A specimen from one patient revealed the presence of HPV 16. In this patient, who had received a heart transplant in 1991 and subsequent chemotherapy including cyclosporin, a tonsillar tumour (epidermoid carcinoma) was discovered in 1997. (ii) Urinary tract Three cases of de-novo lower urinary tract carcinoma in renal transplant recipients were reported, which showed the potential for unusually rapid urothelial extension and invasion in chronically immunosuppressed individuals (Lemmers & Barry, 1990). Two patients had a history of perianal condylomata acuminata. Tumours from one of these

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355

harboured the genetic sequences of HPV type 6. One patient had multiple manifestations of cyclophosphamide-related urothelial injury, including bladder carcinoma. One report described two cases of rapidly progressive, multifocal transitional-cell carcinomas of the bladder that developed in two patients after renal and cardiac transplantation, respectively (Noel et al., 1994). In both cases, HPV 16 DNA was detected using the PCR amplification method. This HPV type has not been previously described in this type of tumour in transplant recipients. HPV infection may play a role in the development of rapidly progressive multifocal transitional-cell carcinoma in the bladder of immunosuppressed patients. 2.8.3

Studies in human immunodeficiency virus (HIV)-infected persons (a)

Studies of the uterine cervix

(i) Prevalence of cervical HPV infection and SIL (Table 70) In a study from Italy that included 221 women at high risk for HIV, among the 121 HIV-positive women, 58 (47%) had HPV lesions, 23 (40%) of whom had CIN1–3. In the 100 HIV-negative women, 23 (23%) had HPV lesions; among these 23 women, six (26%) had CIN1–3. These findings suggest that HIV infection is associated with HPV lesions and that cervical cytological abnormalities develop in this situation (Branca et al., 1995). Murphy et al. (1995) performed a retrospective study of 136 HIV-positive women who attended an inner city ambulatory HIV clinic over a 6-year period between 1987 and 1992 in Dublin, Ireland. During this time, a total of 165 HIV-infected women attended for management of their HIV disease. The results of cervical cytological specimens (smears) were available for 136 (82.4%) women. Forty-one (30.1%) women had mild dysplasia/ CIN1, 21 (15%) had CIN2 and 17 (12.5%) had CIN3. The overall prevalence of dysplasia was 58.1%. Twenty-seven (34.2%) of the women with CIN had cytological evidence of HPV infection. No association between the clinical stage of HIV disease and the presence or degree of SIL was observed (Murphy et al., 1995). Sun et al. (1995) compared the prevalence of HPV infection and CIN in more than 650 HIV-positive and HIV-negative women from the New York area: 60% of HIV-positive and 36% of HIV-negative women had detectable cervical HPV DNA (p < 0.001); 27% of HIV-positive women had HPV 16, 24% had HPV 18 and 51% had more than one type. Of the HIV-negative women, 17% had HPV 16, 9% had HPV 18 and 26% had multiple types. Latent HPV infection was defined as the presence of HPV in the absence of disease. Among 208 HIV-positive women with HPV infection, 126 (61.6%) had no evidence of CIN compared with 97.3% of HIV-negative women with HPV infection. Thus, HPV infection in HIV-positive women appears to be more probably associated with CIN than that in HIV-negative women. Bongain et al. (1996) studied 111 HIV-positive women in Nice, France, 39 of whom were pregnant. Each participant underwent four cervical biopsies: 9.9% had CIN2 and 8.2% had CIN3. No significant differences in the prevalence of CIN were noted between

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transmission group, Centers for Disease Control stage of disease, CD4+ cell count and pregnancy. Langley et al. (1996) studied 68 HIV-1-positive, 58 HIV-2-positive, 14 HIV-1-positive/HIV-2-positive and 619 HIV-negative women who attended clinics for sexually transmitted diseases in Senegal. HPV was detected in 43% of women by PCR and in 7% by southern transfer hybridization; 7.4% of all women had SIL. Both HIV-1 and HIV-2 were associated with HPV infection. HIV-2 was also associated with SIL but the association between HIV-1 and SIL did not reach statistical significance. Most lesions were LSIL. HIV-positive women who had SIL had a lower ratio of CD4:CD8 cells than HIV-positive women without SIL (p = 0.003). Petry et al. (1996) compared the prevalence of cervical HPV infection and CIN among 62 HIV-positive women and 77 HIV-negative women who were immunosuppressed due to other causes. HIV-positive women had a higher prevalence of cervical HPV infection and CIN than HIV-negative immunosuppressed women. Sopracordevole et al. (1996) assessed the relationship between HIV status, level of CD4+ cells and SIL in 51 HIV-positive women in Aviano, Italy. Thirty of 51 patients (58.8%) had confirmed SIL. There was no significant difference in the CD4+ cell count between women with or without SIL, which suggests that the expression of HPV-related dysplasia is a complex process. Cappiello et al. (1997) assessed the association between different HPV genotypes, HIV infection and CIN in a multisite study carried out in Italy. The women were intravenous drug users or sexual partners of intravenous drug users. CIN was detected in 36% of HIVpositive women and in 9% of HIV-negative women. The prevalence of HPV did not differ significantly between HIV-positive and HIV-negative women. The most frequently detected genotypes in both groups were HPV 16 and HPV 18 and were similar between HIV-positive and HIV-negative women. HIV-positive women showed a wider spectrum of HPV genotypes, including low-risk and rare types. Chiasson et al. (1997) studied the prevalence of CIN and vulvovaginal lesions in a group of HIV-positive and HIV-negative women from the New York area, USA. Vulvovaginal condylomata acuminata were found in 5.6% of HIV-positive and 0.8% of HIVnegative women. Multicentric disease was more common among HIV-positive than HIVnegative women and HIV-positive women with vulvovaginal disease were more likely to have CIN than those without (odds ratio, 2.9; 95% CI, 1.1–74). In a multivariate analysis, HIV positivity (adjusted odds ratio, 5.3; 95% CI, 1.3–35.3) and HPV infection (adjusted odds ratio, 6.1; 95% CI, 1.7–39.4) were associated with the detection of vulvovaginal condylomata. Drapkin et al. (1997) performed a retrospective chart review of 89 HIV-positive and 100 HIV-negative women who attended Duke University clinics in southeastern USA. SIL was found in 49.4% of HIV-positive and in 23.0% of HIV-negative women (odds ratio, 3.3; 95% CI, 1.7–6.1).

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Table 70. Prevalence of cervical HPV infection and squamous intraepithelial lesions (SIL) in HIV-positive and HIV-negative women No. of HIVnegative controls

Branca et al. (1995), Italy

No HPV DNA detection

121

100

Murphy et al. (1995), Ireland

No HPV DNA detection

136

Sun et al. (1995), USA

MY09/MY11 primers with RFLP typing and E6 primers (16, 18)

344

Bongain et al. (1996), France

325

HPV prevalence %

60.4 HIV+, 35.7 HIV–

Odds ratio (95% CI)/ p value

p < 0.001

111

Langley et al. (1996), Senegal

PCR with SBH

Petry et al. (1996), Germany

Viratype

68 HIV-1, 58 HIV-2, 14 HIV-1 and HIV-2

619

62

77 allograft recipients, 19 immunosuppressed

57.1 HIV-1, 50.0 HIV-2, 75.0 HIV-1 and HIV-2, 40.1 HIV– 50.0 HIV+, 19.5 HIV– allograft recipients, 31.6 HIV– immunosuppressed

Pathology reading

Comments

23.2 HIV+, 11.0 HIV–

Cytology, histology

HIV– women were at high risk for HPV infection.

58.1 SIL HIV+, 27.9 HSIL

Cytology

Retrospective chart review; 80.9% of women acquired HIV through IDU.

LSIL 14.2 HIV+, 2.8 HIV– HSIL 4.9 HIV+, 0.6 HIV–

Cytology, histology

HPV testing performed on cervicovaginal lavage specimen

6.1 CIN1 9.9 CIN2 8.2 CIN3

Histology

Of 111 participants 39 were pregnant at time of study; each patient had 4 cervical biopsies.

Cytology, histology

Adjusted odds ratio; women enrolled from STD clinics in Senegal; HPV testing performed on endocervical swabs

Cytology, histology

Cervical swab material used for HPV testing

Cervical abnormality

2.9 (1.7–4.9) 1.7 (1.0–2.9) 4.9 (0.8–10.3)

%

SIL 7.5 HIV-1, 11.1 HIV-2, 16.7 HIV-1 and HIV-2, 6.8 HIV– 46.8 HIV+, 16.9 HIV– allograft recipients, 31.6 HIV– immunosuppressed

Odds ratio (95% CI)/ p-value

1.8 (0.7–4.7) 2.9 (1.2–7.2)

HUMAN PAPILLOMAVIRUSES

No. of HIVpositive cases

5.2 (1.4–19.6)

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Method of detection (types included)

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Reference, study location

357

Method of detection (types included)

Sopracordevole et al. (1996), Italy

No HPV DNA detection

51

Cappiello, et al. (1997), Italy

MY09/MY11 PCR and RFLP (specific types not specified)

135

101

40.3 HIV+, 29.6 HIV–

1.61 (0.89– 1.21)

35.8 HIV+, 9.2 HIV–

5.52 (2.43– 12.91)

Cytology, histology

HPV typing performed on cells scraped from glass slides; participants or their sexual partners were IDUs.

Chiasson et al. (1997), USA

PCR on cervicovaginal lavage specimen using L1 consensus primers

396

375

61.7 HIV+, 35.7 HIV–

p < 0.001

5.6 HIV+, 0.8 HIV–

p < 0.001

Cytology, histology

Study examined prevalent condyloma of vulva, vagina and perianal region and multicentric condyloma

Drapkin et al. (1997), USA

No HPV DNA detection

89

100

SIL 49.4 HIV+, 23.0 HIV–

3.3 (1.7–6.1)

Cytology

Retrospective chart review of women in southeastern USA

Ferrera et al. (1997b), Honduras

GP5/GP6 PCR with SB analysis (6, 11, 16, 18, 31, 33)

23

28

No. of HIVnegative controls

HPV prevalence %

Cervical abnormality

56.5 HIV+, 18.0 HIV–

135

Calore et al. (1998), Brazil

No HPV DNA detection

82

Cytology, histology

6.0 (1.5–26.7)

Study of prostitutes in Tegucigalpa, Honduras; HPV testing on wooden spatula specimen 23 genital condyloma, 47.3 cervical SIL on cytology, 2 vaginal SIL

101

40.0 HIV+, 31.7 HIV–

p = 0.19

LSIL 35.6 HIV+, 8.9 HIV– LSIL, 19.5 HIV+ HSIL, 6.1 HIV+

Cytology

Study of women hospitalized with HIV infection at YaleNew Haven Hospital from October 1994 to April 1995

Cytology

HPV testing performed on cervical cytobrush sample

Cytology

Women aged 13–21 years

5.64 (2.5– 13.2)

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MY09/MY11 primers with RFLP (16, 18, 31, 33, 35, 53, 58)

Odds ratio (95% CI)/ p-value

58.8 SIL

55 with adequate cytology results

Rezza et al. (1997), Italy

%

Comments

09:55

Odds ratio (95% CI)/ p value

Pathology reading

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Frankel et al. (1997), USA

No. of HIVpositive cases

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Table 70 (contd)

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Table 70 (contd)

MY09/ MY11 primers with RFLP analysis for typing

No. of HIVnegative controls

HPV prevalence %

Odds ratio (95% CI)/ p value

151 HIV+ and HIV– women with LSIL 60 HIV+ and HIV– women with HSIL

151 HIV+ and HIV–

68.2 LSIL 30.5 LSIL controls

4.4 (2.6–7.4)

240 HIV+ and HIV–

81.7 HSIL 20.4 HSIL controls

14.3 (6.6– 30.9)

p < 0.0001

Maiman et al. (1998), USA

MY09/MY11 primers with probing (6, 11, 32, 40, 42, 43, 44, 53, 54, 55, 61, 70, Pap 155, Pap 291, AE2, 16, 18, 26, 31, 33, 35, 39, 45, 51, 52, 56, 58, 68, 73)

253

220

75.1 HIV+, 46.8 HIV–

Rezza et al. (1998), Italy

MY09/MY11 primers with RFLP typing (16, 18, 31, 33, 35, 53, 58, 11, 44, 46, 54, 59, 66, CP 6108, CP 8304)

135

101

40.0 HIV+, 31.7 HIV–

Cervical abnormality %

LSIL 15.4 HIV+, 3.6 HIV–, HSIL 7.9 HIV+, 1.6 HIV– SIL 50.8 HIV+, CD4 < 200; 28.3 HIV+, CD4 < 200–499; 24.6 HIV+, CD4 > 500

Pathology reading

Comments

Cytology, histology

Cervical Viva-brush specimen used for HPV testing; controls were chosen at random among women shown not to have cervical lesions; adjusted odds ratio

Cytology, histology

Cervicovaginal lavage specimen used for HPV testing

Odds ratio (95% CI)/ p-value

p < 0.0001

HPV testing performed on cytobrush specimen

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La Ruche et al. (1998), Cote d’Ivoire

No. of HIVpositive cases

09:55

Method of detection (types included)

HUMAN PAPILLOMAVIRUSES

Reference, study location

359

30/11/2007

Uberti-Foppa et al. (1998), Italy

Cu-Uvin et al. (1999), USA

No. of HIVnegative controls

HPV prevalence %

Odds ratio (95% CI)/ p value

%

Odds ratio (95% CI)/ p-value

Viratype Plus with typing (16, 18, 31, 35, 45, 52, 56 and 6, 11, 42, 43, 44)

224

257

HR type 15.8 HIV+, 3.6 HIV– LR type 5.4 HIV+, 8.3 HIV–

5.0 (1.8–14.6)

SIL 10.6 HIV+, 2.5 (HIV–)

5.3 (2.0–15.2)

SBH (6/11/42, 16/18/33, 31/35/39) and MY09/MY11 PCR with probing (16, 18, 33) and GP1/GP2 primers with probing (all other HPV types)

253

HC2 and MY09/MY11 primers with typing by RFLP

168

MY09/MY11/ HMBO1 L1 PCR

851

Cervical abnormality

434

Comments

Cytology

Women were sexual partners of men with HIV-1 infection.

Cytology (with biopsy for HSIL)

HPV testing on cervical cytobrush sample; 278 women were followed prospectively at 6-month intervals for 1 year; reference category was HIV– women.

Cytology, histology

Collection method for HPV testing not specified; 91 HIV+ women acquired HIV through heterosexual contact and 74 through IDU.

1.8 (0.6–4.9)

160

100

Pathology reading

SIL 265 HIV+, 7.5 HIV– Among HIV+ 14.9, CD4 > 500; 26.0, CD4 200– 500; 38.7, CD4 < 200 HC 66.1 HIV+, 15.0 HIV– PCR 91 HIV+, 48 HIV–

p < 0.001

64.3 HIV+, 27.6 HIV–

4.7 (3.7–6.1)

p < 0.001 –

1.5 (0.5–4.2) 3.4 (1.4–8.3) 4.4 (1.7–11.4)

HERS population; HPV testing in cervicovaginal lavage specimen

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Rugpao et al. (1998), Thailand

Six et al. (1998), France

No. of HIVpositive cases

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Table 70 (contd)

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Table 70 (contd) No. of HIVpositive cases

No. of HIVnegative controls

MY09/MY11 PCR with RFLP typing (more than 44 types)

141

65.7 vaginal samples, 64.4 cervical samples, 47.4 perianal samples

Hankins et al. (1999), Canada

MY09/MY11 primers with probing (14 different types)

375

67.2 HIV+

Kapiga et al. (1999), Tanzania

No HPV DNA detection

691

Leroy et al. (1999), Rwanda

No HPV DNA detection

103

Luque et al. (1999), USA

HC2

93

%

Cervical abnormality Odds ratio (95% CI)/ p value

2.57 (1.29– 13.56)

Comments

Cytology, histology

HPV testing on cytobrush specimens from cervical, vaginal and perianal areas

Cytology

Women were participants in the Canadian Women’s HIV Study.

Odds ratio (95% CI)/ p-value

10.9 confirmed SIL HIV+

107

47.3 overall, 21.5 HR, 12.9 LR

%

Pathology reading

SIL 2.9 HIV+; CD4 > 500, 2.1; CD4 200–500, 2.3; CD4 > 500, 8.8

p for trend = 0.02

Cytology

Participants were HIV+ pregnant women; cytology collected 3–6 months after delivery

24.3 HIV+, 6.5 HIV–

4.6 (1.8–12.3)

Cytology

All women were pregnant.

51.3 HIV VL > 10 000 copies/mL, 24.3 HIV VL < 10 000 copies/mL

2.11* (1.12– 10.19)

Cytology

Sampling from Cervex brush; *relative risk

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Goncalves et al. (1999), Brazil

HPV prevalence

09:55

Method of detection (types included)

HUMAN PAPILLOMAVIRUSES

Reference, study location

361

30/11/2007

No. of HIVnegative controls

Massad et al. (1999), USA

MY09/MY11/HMB01 with probing (6, 11, 16,18, 26, 31, 32, 33, 35, 39, 40, 45, 51, 52, 53, 54, 55, 56, 58, 59, 61, 66, 68, 69, 70, 73, Pap 155, Pap 291, AE2)

1713

482

Palefsky et al. (1999), USA

MY09 MY11/HMB01 with probing (6, 11, 16,18, 26, 31, 32, 33, 35, 39, 40, 45, 51, 52, 53, 54, 55, 56, 58, 59, 61, 66, 68, 69, 70, 73, Pap 155, Pap 291, AE2)

1778

Stratton et al. (1999), USA

No HPV DNA detection

452 pregnant 126 nonpregnant

Temmerman et al. (1999), Kenya

GP5+/GP6+ primers with probing (6, 11, 16, 18, 31, 33)

51

500

HPV prevalence %

63.4 HIV+, 29.8 HIV–

Cervical abnormality Odds ratio (95% CI)/ p value

%

Odds ratio (95% CI)/ p-value

LSIL 14.9 HIV+, 2.3 HIV– HSIL 2.5 HIV+, 1.2 HIV–

8.9 (4.81– 16.4)

41.2 HIV+, 14.3 HIV–

4.08 (3.29– 5.05)

3.91 (2.00– 7.65)

17.6 HIV+, 5.1 HIV–

Comments

Cytology

Women were participants in the WIHS; HPV testing was performed on a cervicovaginal lavage specimen.

Cytology

Women were participants in the WIHS; HPV testing was performed on a cervicovaginal lavage specimen.

Cytology

Women were participants in the WITS; 240 women had a Pap smear post partum.

Cytology

HPV testing on endocervical brush specimens; women recruited from a family planning clinic in Nairobi

2.68 (1.13– 6.34)

LSIL 17.0 pregnant, 23.8 nonpregnant HSIL 2.0 pregnant, 2.4 non-pregnant 469

Pathology reading

4.77 (1.84– 12.36)

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Table 70 (contd)

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Table 70 (contd) No. of HIVnegative controls

HPV prevalence

Ahdieh et al. (2000), USA

MY09/MY11/HMB01 PCR primers

184

84

69.6 HIV+, 75.7 CD4+ < 200, 68.5 CD4+ ≥ 200, 26.2 HIV–

Ammatuna et al. (2000), Italy

HC2 and 2-step PCR combining MY09/ MY11 primers with GP5+/GP6+ PCR

110

Branca et al. (2000), Italy

MY09/MY11 PCR with RFLP analysis (16, 18, 31, 33, 35, 53, 58)

266

Cubie et al. (2000), Scotland, United Kingdom

HC2

63

%

60.9 HIV+

193

12.5 HIV VL < 500, 15.4 HIV VL 500– 5000, 32.4 HIV VL 5000– 50 000, 52.9 HIV VL > 50 000

Cervical abnormality Odds ratio (95% CI)/ p value

%

Pathology reading

Comments

Odds ratio (95% CI)/ p-value Women were participants in the ALIVE cohort and followed semi-annually; HPV was tested on cervicovaginal lavage specimen.

53.6 HIV+

3.55 (1.96– 6.48)

Cytology, histology

HPV typing performed on Ayre’s spatula specimen

LSIL 21.8 HIV+, 6.6 HIV– HSIL 7.6 HIV+, 3.4 HIV–

3.9 (2.2–7.0) for SIL

Cytology

Women were participants in the DIANAIDS cohort; HIV– women were at high risk for HPV infection; an Ayre’s spatula specimen was used for HPV testing.

Cytology, histology

Sampling from Cervex brush; women followed at 6month intervals

25.0 HIV VL < 500, 23.1 HIV VL 500–5000, 37.8 HIV VL 5000– 50 000, 52.9 HIV VL > 50 000

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30/11/2007

MY09/MY11/HMB01 (6, 11, 16,18, 26, 31, 32, 33, 35, 39, 40, 45, 51, 52, 53, 54, 55, 56, 58, 59, 61, 66, 68, 69, 70, 73, Pap 155, Pap 291, AE2)

Heard et al. (2000), France

No. of HIVnegative controls

HPV prevalence

Cervical abnormality

%

Odds ratio (95% CI)/ p value

1314

66.2 overall, 96.2 SIL, 61.3 normal cytology

11.45 (5.51– 23.82)

MY09/MY11 primers (16, 18, 33) plus SBH with probing (6/11/42, 16/18/33, 31/35/39, other types) and sequencing of unidentifiable types

307

49.5 by PCR 55.5 by SBH

Marais et al. (2000), South Africa

MY09/MY11 primers and an HPV 16specific primer set

47

52

85.1 HIV+, 42.3 HIV–

p = 0.00001

Moscicki et al. (2000), USA

MY09/MY11 primers (6, 11, 42, 44, 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58)

133

55

77.4 HIV+, 54.5 HIV– HR 54.9 HIV+, 29.1 HIV–

1.4 (1.1–1.8)

%

13.7 CIN1 13.3 CIN2/3

1.8 (1.2–2.7)

Pathology reading

Comments

Cytology

Women were participants in the WIHS; HPV testing was performed on a cervicovaginal lavage specimen; women in this analysis had measurement of serum retinol; multivariate odds ratio

Cytology, histology

HPV testing on cotton swab and cervical spatula specimens

Odds ratio (95% CI)/ p-value

HPV testing performed on cervicovaginal lavage specimens; participants were commercial sex workers. SIL 33.1 HIV+, 10.9 HIV–

Cytology

Young women aged 13–18 years were participants in the REACH cohort; HPV testing was performed on a cervicovaginal lavage specimen; most of the women had high CD4+ levels, with 50% having CD4+ > 500/mm3.

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Table 70 (contd) No. of HIVnegative controls

HPV prevalence

Torrisi et al. (2000), Italy

MY09/MY11 PCR with restriction endonuclease typing

104

106 previously normal (Group 2), 112 previously abnormal cytology (Group 3)

53.8 HIV+, 6.6 Group 2, 41.9 Group 3

Womack et al. (2000), Zimbabwe

HC2

249

217

64.3 HIV+, 27.6 HIV–

Duerr et al. (2001), USA

SBH/HC2 and MY09/MY11 primers (6, 11, 16, 18, 26, 31, 33, 35, 39, 40, 42, 45, 51, 52, 53, 54, 55, 56, 58, 59, 66, 68, 70, Pap 155, Pap 291, W13B)

709

341

64.5 HIV+, 29.2 HIV–

Hameed et al. (2001), USA

HC

209

%

48 HIV+

Pathology reading

Comments

SIL 50 HIV+, 5.7 Group 2, 56.3 Group 3

Cytology, histology

HPV performed on cervicovaginal lavage specimen

17.3 HIV+, 5.9 HIV–

Cytology, histology

HPV typing performed on Cyto-soft cervical brush specimen; primary care setting in Harare, Zimbabwe

Cervical abnormality Odds ratio (95% CI)/ p value

p < 0.001

%

18.8 HIV+, 5.3 HIV–

11 LSIL 0.1 HSIL

Odds ratio (95% CI)/ p-value

p < 0.001

Cervicovaginal lavage specimen used for HPV testing; women were participants in the HERS.

Cytology

HUMAN PAPILLOMAVIRUSES

No. of HIVpositive cases

Study of HPV testing and cytology among women being followed for HIV infection; HPV testing performed on cervical swab material

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No. of HIVnegative controls

HPV prevalence

Mayaud et al. (2001), Tanzania

Thomas et al. (2001a), Thailand

%

Odds ratio (95% CI)/ p value

MY09/MY11 primers with reverse line blot assay and specific priming (6, 11, 16, 18, 31)

100

555

Age < 20 years 41 HIV+, 34 HIV– 20–29 years 33 HIV+, 36 HIV– ≥ 30 years 33 HIV+, 25 HIV–

MY09/MY11 PCR (6/11, 16, 18, 31, 33, 35, 39, 45)

37

214

6/11 27.0 HIV+, 8.4 HIV– 16 24.3 HIV+, 12.1 HIV– 31/33/35/39 27.0 HIV+, 11.7 HIV–

Volkow et al. (2001), Mexico

MY09/MY11 primers and GP5/GP6 primers, specific priming of HPV 16 E6/E7 and HPV 18 LCR

85

44 with HIV+ male partner, 55 commercial sex workers

Chirenje et al. (2002), Zimbabwe

No HPV DNA detection

207

355

68.7 HIV+, 28.6 HIV–

Pathology reading

Comments

1.02 (0.6–16)

Cytology

Pregnant women in Tanzania studied; endocervical cytobrush specimen used for HPV testing

1.1 (0.4–2.9)

Cytology

Study of commercial sex workers in Bangkok; HPV testing performed on cervical samples obtained with a Teflon-coated swab

Cervical abnormality %

Odds ratio (95% CI)/ p-value

1.2 (0.4–3.2)

1.2 (0.5–3.3)

SIL 17.8 HIV+, 12.5 HIV– HSIL 8.2 HIV+, 1.8 HIV–

p < 0.05

Cytology, histology

HPV testing performed on cervical cytobrush

25.6 HIV+, 6.7 HIV–

p < 0.001

Cytology, histology

Women aged 18–50 years recruited from family health centres and family planning clinics in Harare

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Table 70 (contd) No. of HIVnegative controls

HPV prevalence %

Odds ratio (95% CI)/ p value

Jamieson et al. (2002), USA

SBH (11, 16, 18, 51, 52, 53) and MY09/MY11 primers (6, 11, 16, 18, 26, 31, 33, 35, 39, 40, 42, 45, 51, 52, 53, 54, 55, 56, 58, 59, 66, 68, 70, Pap 155, Pap 291, W13B)

767

390

63.7 HIV+, 27.4 HIV–

2.3 (2.0–2.8)

Levi et al. (2002), Brazil

SPF10 primers with reverse line blot (6, 11, 16, 18, 31, 33, 34, 35, 39, 40, 42, 43, 44, 45, 51, 52, 53, 54, 56, 58, 59, 66, 68/73, 70, 74)

208

Tate & Anderson (2002), USA

No HPV DNA detection

43

103

Hawes et al. (2003), Senegal

MY09/MY11 primers (16, 18, 31, 33, 35, 45, 51, 52, 56)

335 HIV-1 only, 69 HIV-2 only, 29 HIV-1 and HIV-2

3686

98.1 HIV+; most common types: 6, 39.2; 51, 31.9; 11, 26.0; 18, 24.0; 16, 22.5

Cervical abnormality %

69.1 HIV-1 only, 61.8 HIV-2 only, 67.9 HIV-1 and 2, 25.3 HIV–

SIL/ICC 17.2 HIV-1 only, 19.5 HIV-2 only, 34.4 HIV-1 and 2, 4.0 HIV–

Comments

Cytology

Cervicovaginal lavage specimen used for HPV testing. Women were participants in the HER.

Cytology, histology

Cervical cytobrush specimens were used for HPV testing.

Odds ratio (95% CI)/ p-value

13.4% Pap III on cytology

73 HIV+, 27 HIV–

Pathology reading

p = 0.019

2.2 (1.0–4.8)

Cytology, histology Cytology, histology

Cervical cell samples obtained for HPV testing; instrument not specified

6.0 (2.1–17.1) 8.0 (2.0–3.15)

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No. of HIVnegative controls

HPV prevalence

Baay et al. (2004), Zimbabwe

GP5+/GP6+ primers with probing for HR types (16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, 68) and LR types (6, 11, 34, 40, 42, 43, 44, 54)

61

174

54.0 HIV+ 27.0 HIV–

3.18 (1.67– 6.10)

15.3 HIV+, 6.0 HIV–

Branca et al. (2004), Italy

MY09/MY11 PCR plus type-specific priming (E6/E7) (6/11, 16, 18, 31, 33, 35, 45, 52, 53, 58, 66)

17

227

35.7 HIV+, 29.2 HIV–

p = 0.43

HSIL on Pap 50 HIV+, 18.2 HIV–

Levi et al. (2004), Brazil

HC2 and PGMY primers with line blot probing

255

36

87 HIV+, 100 HIV–

%

Pathology reading

Comments

p = 0.037

Cytology

Cervicovaginal lavage samples used for HPV testing

4.5 (1.08– 18.8)

Cytology, histology

Women were patients referred for assessment of abnormal Pap smears; exocervical and endocervical specimens used for HPV testing

Cytology, histology

Cervical brush samples used for HPV testing; HIV+ women were enrolled during routine gynaecological visit; HIV–controls were all referred to the gynaecologist due to suspicion of CIN or condyloma.

Cervical abnormality Odds ratio (95% CI)/ p value

%

Odds ratio (95% CI)/ p-value

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Strickler et al. (2005), USA

MY09/MY11/HMB01 (6, 11, 16, 18, 26, 31, 32, 33, 35, 39, 40, 45, 51, 52, 53, 54, 55, 56, 58, 59, 61, 66, 68, 69, 70, 73, Pap 155, Pap 291, AE2)

No. of HIVpositive cases

No. of HIVnegative controls

1848

514

HPV prevalence %

Cervical abnormality Odds ratio (95% CI)/ p value

p < 0.001 incident HPV detection versus persistence of HPV infection

%

Pathology reading

Comments

Cytology

Women were participants in the WIHS; HPV testing was performed on a cervicovaginal lavage specimen; relationship between HIV viral load, CD4+ level and prevalence, incidence and persistence of HPV infection or SIL examined; interaction was stronger for prevalent and incident infection or SIL than persistent infection or SIL.

Odds ratio (95% CI)/ p-value

See Table 7 for a description of the primers used. ALIVE, AIDS Link to Intravenous Drug Experience; CI, confidence interval; CIN, cervical intraepithelial neoplasia; DIANAIDS, Italian collaborative study on HIV/HPV; HC2, Hybrid Capture 2; HERS, HIV Epidemiology Research Study; HIV, human immunodeficiency virus; HIV+, HIV-positive; HIV–, HIV-negative; HR, high-risk; HSIL, high-grade squamous intraepithelial lesion; ICC, invasive cervical cancer; IDU, intravenous drug user(s); LCR, long control region; LR, low-risk; LSIL, low-grade squamous intraepithelial lesion; Pap, Papanicolaou test; PCR, polymerase chain reaction; REACH, Reaching for Excellence in Adolescent Care and Health; RFLP, restriction fragment length polymorphism; SBH, Southern blot hybridization; STD, sexually transmitted disease; VL, viral load; WIHS, Women’s Interagency HIV Study; WITS, Women and Infant Transmission Study

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Ferrera et al. (1997b) studied the prevalence of HPV infection in 23 HIV-positive and 28 HIV-negative prostitutes in Tegucigalpa, Honduras: 56.5% of HIV-positive and 18.0% HIV-negative women were positive for HPV DNA (odds ratio, 6.0; 95% CI, 1.5–26.7). Frankel et al. (1997) characterized the prevalence of SIL among a group of HIVpositive women admitted to Yale-New Haven Hospital for an HIV-related illness. Of these, 55 women had cytology adequate for interpretation and 47.3% had evidence of cervical SIL. Rezza et al. (1997) studied 135 HIV-positive and 101 HIV-negative women in Italy. SIL was diagnosed in 35.6% of HIV-positive and 8.9% of HIV-negative women (odds ratio, 5.64; 95% CI, 2.48–13.17). HPV DNA was detected in 72% of women with SIL and in 25% of women without SIL. HPV DNA was detected more often among HIV-positive (40%) than among HIV-negative (32%) women (not statistically significant). Multivariate analysis of risk factors for SIL included the use of oral contraceptives (adjusted odds ratio, 2.64; 95% CI, 1.16–6.00), being HIV-positive with < 200 CD4+ cells (adjusted odds ratio, 7.29; 95% CI, 2.12–24.02) and having a high-risk HPV type (adjusted odds ratio, 17.53; 95% CI, 6.25–49.23). In a young population (13–21 years) of 82 HIV-positive women in Brazil (Calore et al., 1998), 21 (26%) showed characteristic features of HPV-infection and SIL. La Ruche et al. (1998) reported on 2170 HIV-positive and HIV-negative women in Abidjan, Cote d’Ivoire. Women with LSIL, HSIL or cervical cancer were enrolled as were controls without cervical disease. Risk factors for cervical lesions that were studied included cervical HPV infection and HIV-1 and HIV-2 positivity. In a multivariate analyses, LSIL was associated with HPV positivity (adjusted odds ratio, 4.4; 95% CI, 2.6–7.4), HIV-1 positivity (adjusted odds ratio, 2.1; 95% CI, 1.2–3.7) and parity of more than three children (adjusted odds ratio, 2.1; 95% CI, 1.1–3.9). Risk factors for HSIL in the multivariate analysis included HPV positivity (adjusted odds ratio, 14.3; 95% CI, 6.6–30.9), HIV-1 positivity (adjusted odds ratio, 2.3; 95% CI, 1.1–4.7), tobacco chewing (adjusted odds ratio, 5.4; 95% CI, 1.3–22.9) and illiteracy (adjusted odds ratio, 2.1; 95% CI, 1.0–4.3). The only risk factor for cervical cancer that was significant was HPV positivity (adjusted odds ratio, 13.3; 95% CI, 3.2–55.5). A wide diversity of HPV types was found in LSIL and HSIL, but HPV 16 was the most common. Maiman et al. (1998) examined risk factors for SIL in 253 HIV-positive and 220 HIVnegative women in the New York area, USA, and evaluated the sensitivity and specificity of cervical cytology in HIV-positive women. The sensitivity and specificity of cytology for all grades of CIN were 0.60 and 0.80 and those for high-grade CIN were 0.83 and 0.74, respectively. Abnormal cytology was found in 32.9% of HIV-positive women and 7.6% of HIV-negative women. In a multivariate analysis, risk factors for abnormal cytology were HIV infection with > 500 CD4+ cells (p = 0.002), 200–499 CD4+ cells (p < 0.001) and < 200 CD4+ cells (p < 0.001) and HPV infection (p < 0.001). Rezza et al. (1998) studied risk factors for cervical HPV infection in 135 HIV-positive and 101 HIV-negative Italian women at risk for HIV infection (intravenous drug users or sexual partners of men at risk for HIV infection). Women who were intravenous drug users

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were at nearly threefold higher risk for HPV infection than heterosexual women (adjusted odds ratio, 2.7; 95% CI, 1.4–5.0), and this difference was not influenced by HIV serostatus. Rugpao et al. (1998) studied 224 HIV-positive and 257 HIV-negative women who were partners of men with HIV-1 infection in Chiang Mai, Thailand. HIV-positive women were five times more likely to have an infection with a high-risk HPV type and cervical SIL. The prevalence of SIL increased with decreasing CD4+ cell count, but the trend was not significant. Six et al. (1998) investigated the impact of HIV infection on the prevalence, incidence and short-term prognosis of CIN in a prospective study among women from France and French Guyana with a 1-year follow-up. Prevalence of CIN was 7.5% among HIV-negative women and 26.5% among HIV-positive women. Factors associated independently with prevalence of CIN were lower level of CD4+ cells, infection with HPV 16, 18 and 33 and related types, infection with HPV 31, 35 and 39 and related types, lifetime number of sexual partners, younger age, past history of CIN and lack of past cervical screening. Of 344 women, 278 were followed for 1 year at 6-month intervals. Incidence of CIN ranged from 4.9% in HIV-negative women to 27% in HIV-positive women with < 500 × 106/L CD4+ cells (p < 0.001). Progression from LSIL to HSIL during follow-up was detected in 38.1% of HIV-positive women with ≤ 500 × 106/L CD4+ cells but not in HIV-negative women or HIV-positive women with > 500 × 106/L CD4+ cells. HPV 16, 18 and 33 and related types were also associated with higher incidence of CIN and progression from low-grade to highgrade CIN. Uberti-Foppa et al. (1998) studied the prevalence of HPV and cytological abnormalities in HIV-positive women in Milan, Italy. Using Hybrid Capture 2, 66% of HIV-positive women and 15% of HIV-negative women were positive for HPV DNA (p < 0.0001). PCR gave positive results for HPV DNA in 91% and 48%, respectively (p < 0.001). No significant difference was observed with respect to prevalence of HPV between women who acquired it through intravenous drug use and women who acquired HIV through heterosexual contact (p = 0.09). LSIL and HSIL were both more common among intravenous drug users than among women who acquired HIV through sexual contact. Cu-Uvin et al. (1999) examined the prevalence of cervical HPV infection in 851 HIVpositive women and 434 HIV-negative women at high risk in the HIV Epidemiology Research Study. HPV infection was more prevalent among HIV-positive women (64% versus 28%). This study showed no statistically significant difference in the prevalence of lower genital tract infections other than HPV between HIV-positive and HIV-negative women. Goncalves et al. (1999) studied cervical cytology and HPV from cervical, vaginal and perianal scrapes from 141 HIV-positive women in Santos City, Brazil. One or more specimens that were positive for HPV DNA were found in 80.8% of patients. Two or more HPV types were detected in 45% of the samples. The most frequent HPV types detected were 16 and 18 (30.5%) and, overall, 34.8% had high-risk types; 65.7% of vaginal samples, 64.4% of cervical samples and 47.4% of perianal samples were positive for HPV DNA.

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Hankins et al. (1999) examined risk factors for prevalent HPV infection in 375 women who participated in the Canadian Women’s HIV Study: 67.2% of the women were positive for HPV DNA and 10.9% had SIL. Women with SIL were more likely to have HPV infection than those without SIL (p = 0.002). In a multivariate analysis, risk factors for HPV infection included CD4+ cell count < 0.20 × 109/L (adjusted odds ratio, 1.99; 95% CI, 1.17–3.37), non-white race (adjusted odds ratio, 2.00; 95% CI, 1.17–3.42), inconsistent use of condoms in the 6 months before study entry (adjusted odds ratio, 2.02; 95% CI, 1.16–3.50) and lower age; women aged 30–39 years (adjusted odds ratio, 0.51; 95% CI, 0.30–0.87) and 40 years or older (adjusted odds ratio, 0.52; 95% CI, 0.26–1.0) had lower risks than women aged < 30 years. Kapiga et al. (1999) studied the prevalence of and risk factors for cervical SIL among 691 HIV-positive women who attended antenatal clinics in Dar es Salaam, Tanzania. Cytology was collected 3–6 months post partum. Mid-upper arm circumference was measured as an indicator of body wasting. The prevalence of SIL in the study population was 2.9%; 55% of the lesions were LSIL and 45% were HSIL. Risk factors for SIL in multivariate analysis included having a CD4+ cell count < 200/mm3 (odds ratio, 6.15; 95% CI, 1.19–41.37) and decreased by 68% for each 5-cm increase in mid-upper arm circumference (odds ratio, 0.32; 95% CI, 0.10–0.93), which indicated that more advanced HIVrelated immunosuppression and body wasting were the primary risk factors for SIL in this population. Leroy et al. (1999) studied the prevalence of SIL and its association with HIV-1 infection among 103 HIV-positive and 107 HIV-negative pregnant women in Kigali, Rwanda. The prevalence of SIL was higher in HIV-positive women than in HIV-negative women: 24.3% versus 6.5% (odds ratio, 4.6; 95% CI, 1.8–12.3); that of LSIL was 14.6% in HIV-negative and 4.6% in HIV-positive women; and that of HSIL was 9.7% and 1.9%, respectively. Luque et al. (1999) included 93 HIV-positive women in upstate New York (USA) in a cross-sectional study to evaluate the relationship between plasma HIV-1 RNA levels and cervical HPV infection. HIV-1 RNA plasma levels of > 10 000 copies/mL were associated with the detection of high-risk HPV DNA types in cervical specimens (relative risk, 2.57; 95% CI, 1.29–13.56). In addition, similar HIV-1 RNA plasma levels were associated with abnormal Pap smears (relative risk, 2.11; 95% CI, 1.12–10.19). Massad et al. (1999) studied the prevalence of and risk factors for abnormal cervical cytology among 1713 HIV-positive women and 482 risk-matched HIV-negative control women who participated in the Women’s Interagency HIV Study. Cervical cytology was abnormal in 38.3% of HIV-positive women and 16.2% of HIV-negative women. HSIL was found in only 2.5% of the HIV-positive women. In a multivariate analysis, risk factors for abnormal cytology included HIV infection, lower CD4+ cell count, higher level of HIV RNA, HPV positivity, previous history of abnormal cytology, being employed and the number of male sex partners within 6 months of enrolment. Having more than one abortion was associated with a decreased risk for cytological abnormality.

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Palefsky et al. (1999) characterized the prevalence of 39 different HPV types in 1778 HIV-positive and 500 HIV-negative women participating in the Women’s Interagency HIV Study. HIV-positive women had an increased risk for HPV infection (odds ratio, 4.08; 95% CI, 3.29–5.05). The distribution of HPV types was wide in both HIV-positive and HIVnegative women. HPV 16 was found in 5.2% of HIV-positive and 2.0% of HIV-negative women (p < 0.001). The prevalence of most HPV types increased with progressively lower CD4+ cell strata. HPV 16 was not one of these types (see comments by Strickler et al., 2003; Section 2.8.3(a)(ii)). In a multivariate analysis, HIV-positive women with a CD4+ cell count < 200/mm3 were at highest risk for HPV infection compared with HIV-negative women, regardless of HIV RNA load (odds ratio, 10.13; 95% CI, 7.32–14.04), followed by women with a CD4+ cell count > 200/mm3 and an HIV RNA load > 20 000 copies/mL (odds ratio, 5.78; 95% CI, 4.17–8.08) and those with a CD4+ cell count > 200/mm3 and an HIV RNA load < 20 000 copies/mL (odds ratio, 3.12; 95% CI, 2.36–4.12), after adjustment for other factors. Other risk factors for HPV infection among HIV-positive women included racial/ ethnic background (African-American versus Caucasian; odds ratio, 1.64; 95% CI, 1.19– 2.28), current tobacco smoking (odds ratio, 1.55; 95% CI, 1.20–1.99) and younger age (age < 30 years versus ≥ 40 years; odds ratio, 1.75; 95% CI, 1.23–2.49). Stratton et al. (1999) studied the prevalence of SIL in a cohort of 452 pregnant and 126 non-pregnant HIV-positive women who participated in the Women and Infant Transmission Study. The prevalence of SIL was similar for pregnant (17.0%) and non-pregnant women (23.8%) (p = 0.09). In a multivariate analysis, a lower percentage of CD4+ cells (p < 0.001), HSV infection (p = 0.03) and inflammation on the Pap smear (p < 0.001) were all associated with SIL, but pregnancy status was not. Temmerman et al. (1999) studied 51 HIV-positive and 469 HIV-negative women at a family planning clinic in Nairobi, Kenya. In a multivariate analysis, detection of HPV was associated with HIV-1 infection (odds ratio, 3.9; 95% CI, 2.0–7.7) and the number of pregnancies (for ≥ 3 pregnancies compared with 0 or 1; odds ratio, 0.4; 95% CI, 0.2–0.9). HPV infection was strongly associated with high-grade CIN (odds ratio, 14.9; 95% CI, 6.8–32.8); 17.6% of HIV-positive and 5.1% of HIV-negative women had CIN as detected by a Pap test (odds ratio, 4.77; 95% CI, 1.84–12.36). In a multivariate model, predictors of high-grade CIN included HIV-1 positivity (odds ratio, 4.8; 95% CI, 1.8–12.4), the number of lifetime sexual partners (for ≥ 4 partners compared with 0 or 1; odds ratio, 3.8; 95% CI, 1.1–13.5) and education (for secondary compared with primary; odds ratio, 0.38; 95% CI, 0.17–0.88). Ahdieh et al. (2000) studied 184 HIV-positive and 84 HIV-negative women who participated in the ATDS Link to Intravenous Drug Experience cohort and were followed semi-annually over a 6-year period. Of the 187 participants who were positive for HPV at least once, the probability of subsequent HPV positivity was 47.5% for HIV-negative women, 78.7% for HIV-positive women with CD4+ cell counts ≥ 200 and 92.9% for HIVpositive women with CD4+ cell counts < 200 cells/μL (p < 0.001). Compared with HIVinfected participants, the relative incidence of HPV clearance was 0.29% and 0.10%

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among HIV-positive women with CD4+ cell counts ≥ 200 and < 200 cells/μL (p < 0.001), respectively. Ammatuna et al. (2000) studied the presence of HPV DNA in cervical scrapings from 110 HIV-positive women. Using PCR, HPV DNA was found in 60.9% of the samples. Using Hybrid Capture 2, low-risk HPV types were found in 19.4% of the patients, highrisk HPV types in 41.8% and both low-risk and high-risk types in 38.8%. CIN was found in 53.6% of the women. HPV was associated with the detection of CIN (odds ratio, 3.55; 95% CI, 1.96–6.48). Branca et al. (2000) studied 266 HIV-positive and 193 HIV-negative women at high risk in Italy. HIV-positive women were more likely to have SIL (odds ratio, 3.9; 95% CI, 2.2–7.0), most of which were low-grade, while a high prevalence of HPV DNA PCR genotypes was observed in both groups: 48.5% of HIV-positive women and 52% of HIVnegative women had one or more high-risk HPV types detected by PCR. In a study from Scotland, United Kingdom (Cubie et al., 2000) that included 63 HIVinfected women, high-risk HPV types were detected in 25% of those with normal cytology, while over 80% of women with abnormal cytology were high-risk HPV-positive. French et al. (2000) explored the relationship between vitamin A (retinol) deficiency and SIL in 1314 HIV-positive women who participated in the Women’s Interagency HIV Study. At the baseline visit, 15.5% had retinol concentrations consistent with deficiency (< 1.05 μmol/L). In a multivariate model, SIL was associated with retinol concentrations < 1.05 μmol/L (odds ratio, 1.62; 95% CI, 1.02–2.58) together with HPV infection (odds ratio, 11.45; 95% CI, 5.51–23.82), older age (per 10 years; odds ratio, 0.57; 95% CI, 0.44–0.76), being Hispanic/Latin American (odds ratio, 1.86; 95% CI, 1.03–3.37), higher CD4+ levels (per 100 cells/mm3; odds ratio, 0.80; 95% CI, 0.72–0.88), a body mass index < 18.5 (odds ratio, 2.16; 95% CI, 1.05–4.47) or a body mass index > 25.0 (odds ratio, 0.66; 95% CI, 0.46–0.93). Heard et al. (2000) studied risk factors for CIN in 307 HIV-positive women. CIN was diagnosed in 27.0% and HPV infection in 52.8% of the women. Among all HPV-positive women, high HPV load was found in 55.6%. High HPV viral load was more common among women with CD4+ cell counts < 200/μL compared with those with CD4+ cell counts > 200/μL (p = 0.002). High HPV viral load was also associated with an increased risk for CIN in a multivariate analysis in comparison with HPV-negative women (adjusted odds ratio, 16.8; 95% CI, 7.0–40.3). Low HPV viral load was a risk factor for CIN only in women with CD4+ cell counts < 200/μL (adjusted odds ratio, 7.4; 95% CI, 1.3– 43.0). Marais et al. (2000) studied the prevalence of HPV infection in 47 HIV-positive and 52 HIV-negative sex workers in South Africa as well the prevalence of antibodies to HPV 16 VLP by ELISA in cervicovaginal lavage and serum specimens. HIV-positive women had a significantly higher prevalence of HPV DNA than HIV-negative women (85% versus 42%; p = 0.00001). They also had a lower rate of positivity than HIV-negative women for serum IgA antibodies (p = 0.012) but a higher rate of positivity for cervical anti-VLP 16 IgG antibodies (p = 0.002).

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Moscicki et al. (2000) studied the prevalence of cervical HPV infection and SIL in 133 HIV-positive and 55 HIV-negative women aged 13–18 years who participated in the Reaching for Excellence in Adolescent Care and Health cohort. Few of the HIV-infected women (6.6%) had CD4+ cell levels < 200/mm3. HPV infection was found in 77.4% of HIV-positive and 54.5% of HIV-negative women (relative risk, 1.4; 95% CI, 1.1–1.8). Among those with HPV infection, 70.1% of the HIV-positive and 30% of the HIV-negative women had abnormal cytology (p < 0.001). In a multivariate analysis, HIV positivity was a significant risk factor for both HPV infection (odds ratio, 3.3; 95% CI, 1.6–6.7) and SIL (odds ratio, 4.7; 95% CI, 1.8–14.8). CD4+ cell count and HIV viral load were not associated with HPV infection or SIL. Torrisi et al. (2000) examined the prevalence of HPV in 104 HIV-positive women, 106 HIV-negative women with previously normal cytology (Group 2) and 112 HIV-negative women with previously abnormal cytology (Group 3). SIL was found in 50% of HIVpositive versus 5.66% of HIV-negative Group 2 (p < 0.001) and 56.3% of HIV-negative Group 3 women (p = 0.433). HPV DNA positivity was found in 53.8% of HIV-positive, 6.6% of HIV-negative Group 2 and 42% HIV-negative Group 3 women. Multiple HPV types were found in 21.4% of HIV-positive women. Womack et al. (2000) characterized cervical HPV infection in 466 women at high risk for HIV infection during primary cervical cancer screening in Zimbabwe. Compared with HIV-negative women, HIV-positive women had a more than twofold prevalence of HPV (64.3% versus 27.6%), a nearly threefold higher prevalence of high-grade CIN (17.3% versus 5.9%) and more than sevenfold the amount of HPV DNA. The amount of HPV DNA increased with severity of disease in both HIV-negative and HIV-positive women. Duerr et al. (2001) examined risk factors for SIL among 709 HIV-positive and 341 HIV-negative women who participated in the HIV Epidemiology Research Study. SIL was more common among HIV-positive than among HIV-negative women (18.8% versus 5.3%; p < 0.001) as was HPV infection (64.5% versus 29.2%; p < 0.001). In a multivariate analysis, the association with SIL was higher for high-risk HPV types (adjusted prevalence ratio, 27.0; 95% CI, 12.5–58.4) than for low-risk types (adjusted prevalence ratio, 10.5; 95% CI, 4.5–24.6). Intermediate-risk types showed little difference from high-risk types (adjusted prevalence ratio, 25.0; 95% CI, 11.6–54.2). Lower CD4+ cell levels were also associated with SIL but more weakly than HPV infection (CD4+ < 200; adjusted prevalence ratio, 1.9; 95% CI, 1.2–3.0; CD4+ 200–500; adjusted prevalence ratio, 1.6; 95% CI, 1.0–2.5). Hameed et al. (2001) studied 209 HIV-positive women for whom Hybrid Capture and cytology data were available. One hundred and one women (48%) were positive for HPV subtypes by DNA typing by this method; 19/9% had SIL according to cytology, most of which were low-grade. Mayaud et al. (2001) studied the relationship between HPV infection, HIV infection and SIL in 100 HIV-positive and 555 HIV-negative pregnant women in Tanzania. There was no association between HPV and HIV (odds ratio, 1.02; 95% CI, 0.6–1.6). SIL was

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associated with HPV (odds ratio, 3.66; 95% CI, 1.9–7.0), but not with HIV (odds ratio, 1.54; 95% CI, 0.7–3.4). Thomas et al. (2001a) reported no significant association between HPV and HIV infection in a study of sex workers in Bangkok. Volkow et al. (2001) studied the prevalence of HPV infection and SIL in 85 HIV-positive and 99 HIV-negative women at high risk in Mexico. Cases included women who were positive for HIV and accepted to participate. HPV DNA was detected by PCR in 69% of HIV-positive women and 29% of HIV-negative women (p < 0.0001). Chirenje et al. (2002) performed a cross-sectional study of the prevalence of CIN among 207 HIV-positive women and 355 HIV-negative women who attended a family health centre and family planning clinics in Harare, Zimbabwe. Cervical cytology was abnormal in 25.6% of HIV-positive women compared with 6.7% of HIV-negative women (p < 0.001). Jamieson et al. (2002) examined risk factors for HPV infection and its association with cytological abnormalities at baseline in 767 HIV-positive women and 390 HIV-negative women in the HIV Epidemiology Research Study. HIV-positive women were more likely to have HPV infection than HIV-negative women (prevalence ratio, 2.3; 95% CI, 2.0–2.8). The distribution of HPV types was similar between the HIV-positive and HIVnegative women. HPV viral loads as measured by PCR dot blot signal strength were higher among HIV-positive than among HIV-negative women as was the proportion of HPV-positive women with multiple HPV types. Among women with high HPV viral load, HIV infection was not associated with SIL. Levi et al. (2002) examined the prevalence of HPV infection and multiplicity of HPV types in 208 HIV-positive women in Brazil. Almost all women (98%) were HPV-positive; 78.9% had multiple HPV types with an average of three per patient. HPV 6 was the most common genotype (39.2%) followed by types 51 (31.9%), 11 (26.0%), 18 (24.0%) and 16 (22.5%); 28 patients (13.4%) had a Pap III score. The prevalence of high-risk genotypes increased with the cytological classification. There were no significant associations between the number of HPV genotypes, abnormal cytology, HIV viral load and CD4+ cell count. Tate and Anderson (2002) compared recurrence rates of CIN after ablation and hysterectomy in 43 HIV-positive women with those in 103 HIV-negative women. All patients were followed up for at least 24 months. Recurrence was greater in the HIV-positive women for all treatment modalities (73% versus 27%; p = 0.019). Higher recurrence rates were seen in women with CD4+ cell counts < 200 cells/mm3 compared with women with CD4+ cell counts > 200/mm3 (55% versus 26%; p = 0.002). The mean HIV viral load was also higher among women who had recurring disease than among those who did not (18 384 versus 3892; p = 0.002). Hawes et al. (2003) studied 4119 women who attended an outpatient clinic in Senegal, an area in which both HIV-1 and HIV-2 are highly prevalent in the population. Among women infected with high-risk HPV, those with HIV-1 (odds ratio, 2.2; 95% CI, 1.0–4.8), HIV-2 (odds ratio, 6.0; 95% CI, 2.1–17.1) or both HIV-1 and HIV-2 (odds ratio, 8.0;

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95% CI, 2.0–31.5) were more likely to have HSIL or cervical cancer than HIV-negative women. This relationship was not detected among women without high-risk HPV infection. HIV-2-positive women were more likely to have HSIL (odds ratio, 3.3; 95% CI, 0.9–12.4) or cervical cancer (odds ratio, 7.9; 95% CI, 1.1–57) than HIV-1-positive women. The authors hypothesized that the increase in risk associated with HIV-2 infection may reflect the longer periods of mild immunosuppression than are typically seen with HIV-1, and this may be relevant to the effect of highly active antiretroviral therapy (HAART) on the natural history of CIN. Baay et al. (2004) studied the prevalence of cervical HPV infection in a population of women from rural Zimbabwe. The prevalence of HPV was higher in HIV-positive (54%) than in HIV-negative women (27%) (odds ratio, 3.18; 95% CI, 1.67–6.10). The most common HPV types in HIV-positive women were 33 (5.2%), 35 (4.6%), 45 (4.6%) and 58 (4.6%); HPV 16 was found in only 3.4%. Among HIV-negative women, the most common types were HPV 35 (11.5%), 6 (9.8%) and 58 (8.2%); HPV 16 was found in only 3.3%. Branca et al. (2004) assessed risk factors and HPV-related mechanisms of CIN in 17 HIV-positive and 227 HIV-negative women in Italy. HPV prevalence was 36% in HIVpositive and 29% in HIV-negative women. HIV-positive women had more frequent HSIL Pap tests (p = 0.04), CIN2 or higher in cervical biopsy (p = 0.049) and external genital warts (p = 0.019). Levi et al. (2004) studied HIV-positive women from Sao Paulo, Brazil. HPV-DNA prevalence was 87% in HIV-positive women, and 45% were infected by more than two types, compared with 8.3% in HIV-negative women. HPV 16 was the most common type found in HIV-positive women (30.9%) followed by types 52 (22.8%) and 59 (20.6%). In HIV-negative women, the most common types were HPV 51 (19.4%), 16 (16.7%) and 73 (16.7%). The number of HPV types detected among HIV-positive women increased, but not significantly, with increasing grade of Pap smear, whereas HPV viral load as measured by Hybrid Capture 2 (Group B high-risk types) was significantly increased (p < 0.001). Strickler et al. (2005) studied the effect of HIV RNA level and CD4+ cell count on the natural history of type-specific HPV infection in 1848 HIV-positive and 514 HIVnegative women who participated in the Women’s Interagency HIV Study cohort. A strong interaction between the CD4+ cell count and plasma HIV viral load was found for both prevalent (p = 0.002) and incident (p = 0.001) detection of HPV. The hazard ratio for incident HPV detection was highest among women with a CD4+ cell count < 200/mm3 (hazard ratio range, 4.0–5.0) or an HIV RNA level > 100 000 copies/mL; the relationship was weaker for persistent HPV infection. Although incident HPV detection was associated with the number of recent sexual partners (p for trend < 0.001), 22% of sexually inactive HIV-positive women with a CD4+ cell count < 200/mm3 also had at least one incidentally detected HPV type. There was strong interaction between the effects of HIV RNA and CD4+ cell count on incident SIL, but only a weak effect on its persistence. The weak effect of HIV RNA viral load and CD4+ cell count on HPV and persistence of

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SIL may explain the limited number of HIV-positive women who develop HSIL and cervical cancer. The data on sexual activity and incident detection of HPV are consistent with the possibility that at least some of the HPV detected in HIV-positive women reflects reactivation of previously acquired HPV infection rather than a newly acquired infection. (ii) Natural history of cervical HPV infection and SIL (Table 71) Heard et al. (1995) followed 43 HIV-positive women who had normal cytology or SIL at baseline every 6 months for up to 18 months; 18 of 19 (95%) women who had SIL at baseline and who were not treated and eight of 13 (61%) women who were treated with surgery had persistent lesions. Spinillo et al. (1996) studied 48 HIV-positive and 38 HIV-negative women with a history of intravenous drug use who attended an antenatal clinic during their first trimester of pregnancy. Participants were re-examined during their second and third trimesters and 8–12 weeks post partum: 27.1% of HIV-positive women and 7.9% of HIV-negative women had CIN at their baseline visit (p = 0.027). None of the lesions progressed throughout pregnancy in either HIV-positive or HIV-negative women. Sun et al. (1997) compared the persistence of cervical HPV infection among 220 HIVpositive and 231 HIV-negative women in the New York City area, USA. HPV DNA was detected at baseline in 56% of the HIV-positive and 31% of the HIV-negative women. After four examinations, the cumulative prevalence of HPV infection was 83% in the HIV-positive and 62% in the HIV-negative women (p < 0.001). Twenty per cent of the HIV-positive and 3% of the HIV-negative women had persistent infections with high-risk types (18 or 45) (p < 0.001). The detection of HPV DNA in women who had previously had negative tests was not associated with sexual activity during the interval since the preceding examination, which suggests the possibility of reactivation of a previously acquired HPV infection as an explanation for the detection of at least some of these infections. The risk for persistent HPV infection in HIV-seropositive compared with HIV-negative women was 7.5 (95% CI, 3.6–16). Minkoff et al. (1998) characterized the relationship between HIV status and infection with high-risk HPV types in 268 HIV-positive and 265 HIV-negative women in the New York area, USA. The prevalence at baseline of any HPV type was 73% among HIVpositive and 43% among HIV-negative women (p < 0.0001). The respective prevalence of high-risk HPV types was 32.5 and 17.0% (p < 0.001). The rate of detection of new highrisk HPV types was almost three times higher among HIV-positive than among HIV-negative women (p < 0.01). However, there was no difference in the rate of loss of detection of high-risk HPV types. Eckert et al. (1999) compared the prevalence and type of HPV infection in the genital tract of 23 HIV-positive and 23 HIV-negative women who were matched for cytology. After matching, the groups had a similar prevalence of HPV DNA and of high-risk HPV types at baseline. On follow up, HIV-positive women were more likely to develop SIL (38% versus 10%; p = 0.03), to have visits at which HPV DNA was detected (68% versus

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Table 71. Natural history of cervical HPV infection and squamous intraepithelial lesions (SIL) in HIV-positive and HIV-negative women No. of HIVpositive cases

No. of HIVnegative controls

No HPV DNA detection

43 SIL

Spinillo et al. (1996), Italy

ISH (6/11, 16/18, 31/33/35)

48 CIN1–3

38 CIN1–3

NR

Sun et al. (1997), USA

MY09/MY11 PCR and type-specific primers (16 and 18)

220

231

At baseline 56 HIV+, 31 HIV– Persistent HPV infection 83 HIV+, 62 HIV–

7.5 (3.6–16)

Minkoff et al. (1998), USA

MY09/MY11 PCR primers (2, 6, 11, 13, 26, 32,34, 40, 42, 53, 54, 55, 57, 59, 61, 62, 64, 66, 69, 70, 72, Pap 155, Pap 291, AE2, AE5-8, W13B, 16, 18, 26, 31, 33, 35, 39, 45, 51, 52, 56, 58, 68, 73)

268

265

73 HIV+, 43 HIV– HR HPV 32.5 HIV+, 17.0 HIV–

p < 0.0001

Eckert et al. (1999), USA

MY09/MY11 PCR primers (6/11, 31/33/35/39, 16/16/45)

23

73.9 HIV+, 65.2 HIV–

p = 0.4

%

Cervical abnormality Odds ratio (95% CI)/ p-value

%/incidence

23

Comments

Cytology, histology

Participants followed every 6 months with a Pap smear for at least 18 months

Biopsy, cytology, histology

Participants were HIV+ women with history of IDU being seen for antenatal care in their first trimester; re-examined in second and third trimester and 8–12 weeks post partum.

Odds ratio (95% CI)/ p-value

Untreated patients, 95; treated patients, 61 27.1 HIV+, 7.9 HIV–

Pathology reading

p = 0.027

Persistent HPV infection defined as detection of the same type of HPV at 2 or more examinations during the follow-up period of 3–12 months; HPV testing performed on cervicovaginal lavage; almost all CIN were low-grade.

NR

Cytology, histology

HPV testing performed on cervicovaginal lavage specimen; women followed every 6 months

Cytology, histology

Dacron swabs used to measure HPV in the cervix/ectocervix and vaginal wall; participants matched by cytology results at baseline and followed every 4 months for 56 (HIV+) and 53 visits (HIV–)

p < 0.001

Baseline 39 HIV+, 39 HIV– Follow-up 38 HIV+, 10 HIV–

p = 0.03

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No. of HIVnegative controls

HPV prevalence %

Odds ratio (95% CI)/ p-value

%/incidence

Odds ratio (95% CI)/ p-value

La Ruche et al. (1999), Cote d’Ivoire

MY09/MY11 PCR

38

56

83.3 HIV+ 58.8 HIV–

p = 0.015

Persistent CIN 76 HIV+, 18 HIV– Progression to high-grade CIN 18 HIV+, 0 HIV–

4.3* (2.4– 7.7)

Petry et al. (1999), Germany

HC1

138

NR

15.9 CIN1, 12.3 CIN2 or -3

Cubie et al. (2000), Scotland, United Kingdom

HC for HR HPV, HC2 with probes (16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 68)

63

Normal cytology, 25; abnormal cytology, 8.0; persistent HPV infection, 42.9

NR

Ellerbrock et al. (2000), USA

MY09/MY11 PCR primers with RFLP typing and E6 primers (16 and 18)

328

325

Cervical abnormality

91% HIV+, 75% HIV–

Pathology reading

Comments

Cytology, histology on women with highgrade cytology referred for colposcopy

HPV testing using cervical VibaBrush specimen; women followed after a median of 5 months from the initial smear; *relative risk

Cytology, histology

3.2 (1.7– 61)

Cytology, histology

Women had no SIL at enrollment; HPV testing performed on cervicovaginal lavage specimen; Women followed for approximately 30 months

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Table 71 (contd)

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Table 71 (contd)

MY09/MY11/ HMB01 PCR primers (16/18/31/45, 33/35/39/51/52/ 56/58/59/68, 6/11/26/40/42/53/54/55/ 66/73/82/ 83/84)

Calore et al. (2001), Brazil

No. of HIVnegative controls

HPV prevalence

871

439

Cumulative pre-valence of HPV infection: increase from 73.4 to 90.2 HIV+ women with CD4+ < 200, increase from 28.1 to 54.0 HIV–

%

Cervical abnormality Odds ratio (95% CI)/ p-value

1587 (baseline), 409 (followup)

Cohn et al. (2001), USA

HC 2, HC RLU

103

Massad et al. (2001), USA

MY09/MY11/ HMB01 PCR (6, 11, 16, 18, 26, 31, 32, 33, 35, 39, 40, 45, 51, 52, 53, 54, 55, 56, 58, 59, 61, 66, 68, 69, 70, 73, Pap 155, Pap 291, AE2)

1639

66 HIV+, 56.3 with HR types HIV+

452

NR

p value = 0.0006

%/incidence

Pathology reading

Comments

Cytology

Women were participants in the HERS and were followed at 6-month intervals for assessment of typespecific HPV infection; Increased HPV viral load using PCR as indicated by increased dot blot signal strength from 1 to 4

Odds ratio (95% CI)/ p-value

12.6 SIL or cervical cancer

Cytology

20 CIN after 1 year of follow-up

Cytology, biopsy

Women were participants in the American Foundation for AIDS Research Community Based Clinical Trials Network in 6 US cities; Cervical Dacron swabs used for HPV testing; Women studied at baseline, 6 months and 12 months; Cases had CD4+ ≤ 500/mm3.

Cytology

Women were participants in the WIHS; HPV testing performed on a cervicovaginal lavage specimen; Women had measurement of serum retinol AND were followed every 6 months; Median follow-up, 4.0 years

73 HIV+, 42 HIV–

4.0 (2.6– 6.1)

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PCR using L1 consensus primers and RFLP typing analysis; some samples studied with E6-specific primers (16 and 18)

Silverberg et al. (2002), USA

MY09/MY11 PCR primers (6, 11, 16, 18, 26, 31, 32, 33, 35, 39, 40, 45, 51, 52, 53, 54, 55, 56, 58, 59, 61, 66, 68, 69, 70, 73, 83, 84)

No. of HIVnegative controls

HPV prevalence %

Odds ratio (95% CI)/ p-value

%/incidence

Odds ratio (95% CI)/ p-value

481 (baseline), 385 (incidence analysis)

437 (baseline), 341 (incidence analysis)

54 HIV+, 32 HIV–

p < 0.001

Vulvovaginal and perianal condyloma and dysplasia 6.2 HIV+, 0.9 HIV– Incident condyloma (all sites) 7 HIV+, 1 HIV–

p < 0.0001

551 (WIHS)

HPV 6/11 5.0 HIV+, 0.9 HIV– Other HPV 58.7 HIV+, 29.0 HIV– HPV 6/11 4.3 HIV+, 1.2 HIV– Other HPV 59.9 HIV+, 26.0 HIV–

2032 (WIHS)

863 (HERS)

420 (HERS)

Cervical abnormality

p < 0.001

p < 0.001

Genital warts WIHS 9.8 HIV+, 3.1 HIV– HERS 13.6 HIV+, 5.0 HIV–

Pathology reading

Comments

Cytology, histology

Study examined incident condyloma of vulva, vagina and perianal region and multicentric lesions; HPV testing performed on cervicovaginal lavage specimen

Cytology, physical examination

Women were participants in the WIHS and HERS and were followed every 6 months.

13.8 (10.9– 17.3)

p < 0.001 p < 0.001

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Table 71 (contd)

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Table 71 (contd)

MY09/MY11 PCR primers with RFLP genotyping and confirmation of typing by sequencing

No. of HIVnegative controls

HPV prevalence

89

48

At baseline 38.6 HIV+, 27.1 HIV– New infection during follow-up 27.1 HPV/HIV+, 3.1 (HPV/HIV+) Cleared infection during follow-up 22.8 HPV+/HIV+, 69.2 HPV+/HIV–

%

Ford et al. (2003), Indonesia

PCR (primers not specified) with hybridization (6, 11, 16, 18, 31, 33, 35, 45, 52)

631 (baseline), 618 (18 months)

38.3 at baseline, 29.7 at 18 months

Strickler et al. (2003), USA

PCR MY09/MY11/ HMB01 PCR (6, 11, 16,18, 26, 31, 32, 33, 35, 39, 40, 45, 51, 52, 53, 54, 55, 56, 58, 59, 61, 66, 68, 69, 70, 73, Pap 155, Pap 291, AE2)

2929

HPV 6 HPV 11 HPV 16 HPV 18 HPV 31

Cervical abnormality Odds ratio (95% CI)/ p-value

%/incidence

Pathology reading

Comments

Odds ratio (95% CI)/ p-value

NR

Women were participants in the DIANAIDS project and were followed for a mean of 14 months.

NR

Study of female sex workers in Bali, Indonesia; HPV testing performed at baseline and 18 months later; HPV testing performed on cervical swab specimen

8.8 (1.20– 64.6)

0.33 (0.16– 0.67)

4.9 (2.0– 12.02) 2.05 (0.93– 4.50) 1.69 (1.01– 2.81) 2.24 (1.23–4.08) 3.07 (1.55– 6.07)

NR

Cytology

Hazard ratio estimates for association between CD4+ < 200 versus ≥ 500 with incident detection of specific HPV types; women were participants in the WIHS and HERS; HPV testing was performed on a cervicovaginal lavage specimen.

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No. of HIVnegative controls

HPV prevalence %

Odds ratio (95% CI)/ p-value

Massad et al. (2004a), USA

PCR MY09/ MY11/HMB01 PCR (6, 11, 16, 18, 26, 31, 32, 33, 35, 39, 40, 45, 51, 52, 53, 54, 55, 56, 58, 59, 61, 66, 68, 69, 70, 73, Pap 155, Pap 291, AE2)

202

21

HR types 49.5 HIV+, 19.1 HIV– LR types 19.8 HIV+, 9.5 HIV–

p < 0.001

Moscicki et al. (2004), USA

PCR MY09/ MY11/HMB01 PCR primers (16-like, 16, 31/33/35, 52, 58, 67, 18like, 18, 39, 45, 59/68/70, 56-like, 56, 53/66, 26/69, 51, 6/11/42/43/44, 54/40, 13/32, 67/72, 2/57, 55)

172

84

NR

Cervical abnormality

Pathology reading

Comments

%/incidence

Odds ratio (95% CI)/ p-value

Progression of CIN 1 3.8 HIV+, 0 HIV–

0.4 (0.25– 0.66)

Cytology, histology

Women were participants in the WIHS. HPV testing performed on a cervicovaginal lavage specimen; women with a histological diagnosis of CIN1 were included in the analysis; women were followed for a mean of 3.3 years.

LSIL at baseline 22.5 HIV+, 10.2 HIV– HSIL at baseline 5.3 HIV+, 0 HIV–

p < 0.002

Cytology

Cervicovaginal lavage samples used for HPV testing; participants were women aged 13–18 years in the REACH cohort.

p = 0 .13

See Table 7 for a description of the primers used. CI, confidence interval; CIN, cervical intraepithelial neoplasia; DIANAIDS, Italian collaborative study on HIV/HPV; HC, Hybrid Capture; HERS, HIV Epidemiology Research Study; HIV, human immunodeficiency virus; HIV–, HIV-negative; HIV+, HIV-positive; HR, high-risk; HSIL, high-grade squamous intraepithelial lesion; IDU, intravenous drug user(s); ISH, in-situ hybridization; LR, low-risk; LSIL, low-grade squamous intraepithelial lesion; NR, not reported; Pap, Papanicolaou test; PCR, polymerase chain reaction; REACH, Reaching for Excellence in Adolescent Care and Health; RFLP, restriction fragment length polymorphism; RLU, relative light unit; WIHS, Women’s Interagency HIV Study

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Table 71 (contd)

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40%; p = 0.04) and to have more visits at which multiple HPV DNA types were detected (18% versus 0%; p = 0.02) than HIV-negative women. La Ruche et al. (1999) performed a short-term prospective study of CIN in Abidjan, Cote d’Ivoire. Of 94 women with a cytological diagnosis of SIL, 36 were infected with HIV-1 and two with HIV-2. The average follow-up period after the initial smear was 5 months. HIV-positive women had a higher percentage of persistent CIN (76%) than HIVnegative women (18%) (relative risk, 4.3; 95% CI, 2.4–7.7). Progression to high-grade CIN occurred more frequently among HIV-positive (18%) than HIV-negative women (0%). In a multivariate analysis, persistence of lesions was associated with HIV positivity and an undetermined grade of CIN at baseline; HPV infection was not a significant risk factor after adjustment for the other factors in the model. Petry et al. (1999) studied the role of HPV testing as a screening tool for incident SIL among 138 HIV-positive women in Germany. The prevalence of high-grade neoplasia (≥ CIN2) was 12.3% (17/138), and the total prevalence of cervical neoplasia including CIN1 was 26.8% (37/138). Cubie et al. (2000) performed a prospective observational cohort study of 63 HIVpositive women in Edinburgh, United Kingdom. Abnormal cervical cytology, particularly that of low grade, was common in these HIV-infected women. Using Hybrid Capture, high-risk HPV types were detected in 25% of the women with normal cytology. Over 80% of those with abnormal cytology of any grade were positive for HPV. Persistent high-risk HPV infection, as defined by two or more consecutive HPV-positive results, was common and found in 27 of 63 (42.9%) women from whom multiple samples were obtained. Progression of cervical disease, even among the more strongly immunosuppressed women, was a rare event in this cohort. Ellerbrock et al. (2000) studied risk factors for incident SIL in 328 HIV-positive and 325 HIV-negative women in the New York area, USA. During follow-up, the incidence of SIL was 8.3 cases per 100 person–years in HIV-positive and 1.8 cases per 100 person–years in HIV-negative women (p < 0.001). Of the incident SILs, 91% were LSIL in HIV-positive women and 75% in HIV-negative women. In a multivariate analysis, risk factors for incident SIL included HIV infection (relative risk, 3.2; 95% CI, 1.7–6.1), transient HPV DNA detection (relative risk, 5.5; 95% CI, 1.4–21.9), persistent infection with HPV DNA types other than 16 or 18 (relative risk, 7.6; 95% CI, 1.9–30.3), persistent infection with HPV DNA types 16 and 18 (relative risk, 11.6; 95% CI, 2.7–50.7) and younger age (< 37.5 years versus ≥ 37.5 years of age; relative risk, 2.1; 95% CI, 1.3–3.4). Ahdieh et al. (2001) performed a prospective study of HPV infection in women who participated in the HIV Epidemiology Research Study. In a multivariate analysis, increased signal strength on dot blot, but not viral risk category, was independently associated with persistence of HPV infection among HIV-positive women (odds ratio, 2.5; 95% CI, 2.1– 2.9). Persistence was 1.9 (95% CI, 1.5–2.3) times more common among women with a CD4+ cell count < 200 cells/μL compared with those with a count > 500 cells/μL. Among the HPV types examined, HPV 16 had the highest incidence rate among HIV-negative women (1.67 per 100 person–years). Among HIV-positive women, HPV 18 had the highest

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incidence rate among the high-risk types (2.61 per 100 person–years) but low-risk HPV 53 had the highest incidence rate overall (6.23 per 100 person–years). Calore et al. (2001) studied cytological specimens from 1587 HIV-positive women in Brazil: 12.6% had SIL or cervical cancer in at least one specimen; 24 women progressed from normal to LSIL within 3 years and 11 progressed from normal to HSIL within 3 years. Cohn et al. (2001) studied the 1-year incidence of CIN in 103 women who participated in the AIDS Research Community Based Clinical Trials Network in six cities in the USA. Higher HPV viral loads as measured by higher Hybrid Capture RLU ratios were associated with high-grade CIN (p = 0.0006). Incident CIN occurred in 20% of women during 1 year of follow-up, and was associated with higher HPV RLU ratios at baseline (p = 0.03). Massad et al. (2001) studied the incidence, progression and regression rates of abnormal cervical cytology among 1639 HIV-positive and 452 HIV-negative women who participated in the Women’s Interagency HIV Study. At least one abnormal smear was found during the whole follow-up among 73.0% of HIV-positive and 42.3% of HIV-negative women (p < 0.001). The incidence of HSIL was low among HIV-positive women and only 5.9% ever developed HSIL during follow-up. The incidence of SIL was 8.9/100 person–years among HIV-positive and 2.2/100 person–years among HIV-negative women (relative risk, 4.0; 95% CI, 2.6–6.1). Progression 6 months after an abnormal smear was found in 14% of HIV-positive women. HIV positivity, HPV positivity, lower CD4+ cell count and higher HIV RNA level predicted the incidence of abnormal cytology. HPV positivity and higher HIV RNA level predicted the progression of abnormalities found at baseline. HPV negativity, higher CD4+ lymphocyte count and lower HIV RNA level predicted regression of disease. Conley et al. (2002) studied the incidence of vulvovaginal and perianal condylomata acuminata and intraepithelial neoplasia in 925 HIV-positive and HIV-negative women in the New York area, USA. Vulvovaginal and perianal condylomata acuminata or intraepithelial neoplasia were found in 6% of HIV-positive and 1% of HIV-negative women at baseline. Among women without lesions at enrolment, 9% of HIV-positive and 1% of HIV-negative women developed vulvovaginal or perianal lesions over a median followup of 3.2 years. Risk factors for incident disease included HIV-1 infection (p = 0.013), HPV infection (p = 0.0013), lower CD4+ cell counts (p = 0.0395) and history of frequent intravenous drug use (p = 0.02). Silverberg et al. (2002) examined the relationship between HIV infection and incidence of genital warts and infection with HPV 6 or 11 in both the Women’s Interagency HIV Study and HIV Epidemiology Research Study populations. The prevalence of HPV 6 or 11 was 5.6 times higher and that of genital warts was 3.2 times higher in HIV-positive than in HIVnegative women in the Women’s Interagency HIV Study and 3.6 times and 2.7 times higher, respectively, in the HIV Epidemiology Research Study. In the former, the risk for HPV 6 or 11 infection increased from 5.1 (95% CI, 2.9–8.8) among HIV-negative women to 8.8 (95% CI, 6.1–12.8) among HIV-positive women with CD4+ cell counts > 200/mm3 and to 12.8 (95% CI, 8.8–18.8) among HIV-positive women with CD4+ cell counts ≤ 200/mm3. In this study, infection with HPV 6 or 11 was associated with an increased risk for genital warts

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compared with HPV negativity in HIV-negative women (odds ratio, 2.7; 95% CI, 1.6–4.6), HIV-positive women with CD4+ cell counts > 200/mm3 (odds ratio, 4.9; 95% CI, 3.2–7.7) and HIV-positive women with CD4+ cell counts ≤ 200/mm3 (odds ratio, 5.3; 95% CI, 3.3–8.5). The incidence of infection with HPV 6 or 11 and the incidence of genital warts measured in cases per 100 person–years were higher among HIV-positive women than among HIV-negative women in both of these studies. Branca et al. (2003) studied the natural history of cervical HPV infection in 89 HIVpositive and 48 HIV-negative women who participated in the DIANAIDS study. New HPV infections during follow-up were more common among HIV-positive than HIVnegative women (odds ratio, 8.8; 95% CI, 1.2–64.6), and clearance of HPV infection at baseline was less frequent among HIV-positive than HIV-negative women (odds ratio, 0.33; 95% CI, 0.16–0.67). In a multivariate analysis, risk factors for HPV positivity at the end of the study included HIV positivity (p < 0.001), PCR positivity at entry (p = 0.009), p53 polymorphism at aa-72 (p = 0.01), high-risk HPV type (p = 0.02) and significant Pap smear at entry (p = 0.04). Ford et al. (2003) studied female sex workers in Bali, Indonesia, for cervical HPV infection at baseline and again 18 months later. The prevalence of HPV infection was 38.3% at baseline, which declined to 29.7% after 18 months. The prevalence of HPV infection declined with age (p < 0.01). Infection with N. gonorrhoeae was associated with HPV infection at baseline (p = 0.03). HPV infection declined in the study area that had the more intensive educational programme (p < 0.01). Strickler et al. (2003) examined the relationship between prevalence and incidence of specific HPV types in HIV-positive women who participated in the Women’s Interagency HIV and HIV Epidemiology Research Studies. In a cross-sectional analysis of data from the first study, HPV 16 had a weaker association with more advanced immune status as measured by CD4+ cell counts than other HPV types. This largely reflected the observation that the prevalence of HPV 16 was higher among women with higher CD4+ cell levels than that of other HPV types, the proportional prevalence of which was increased at lower CD4+ cell strata. A summary prevalence ratio and incidence hazard ratio were estimated for each HPV type. Using data from both studies, the prevalence ratio for HPV 16 was low compared with that of other HPV types at every visit in both populations. The prevalence ratio was smallest for HPV 16 compared with that of all HPV types measured (1.25; 95% CI, 0.97–1.62; p = 0.01). The association of CD4+ T-cell stratum with incidence of HPV 16 was also among the smallest measured. HPV types that had small summary prevalence ratios also had small incidence hazard ratios. The investigators concluded that the prevalence and incidence of HPV 16 is more weakly associated with immune status as measured by CD4+ cell level in HIV-positive women than that of other HPV types, which suggests that HPV 16 may be more resistant to the effects of immune surveillance. Massad et al. (2004a) studied the natural history of histologically confirmed CIN1 in 202 HIV-positive and 21 HIV-negative women who participated in the Women’s Interagency HIV Study. The prevalence of high-risk HPV in HIV-infected women was 49.5% compared with 19.1% in HIV-negative women. Progression occurred in eight (3.8%) HIV-

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positive women (incidence density, 1.2/100 person–years) but not in HIV-negative women. Regression occurred more often in HIV-negative than in HIV-positive women (relative risk, 0.40; 95% CI, 0.25–0.66; p < 0.001). In a multivariate analysis, regression was associated with HPV infection (hazard ratio for low-risk HPV, 0.28; 95% CI, 0.13–0.61; hazard ratio for high-risk HPV, 0.34; 95% CI, 0.20–0.55 versus no HPV detected) and Hispanic ethnicity (hazard ratio, 0.48; 95% CI, 0.23–0.98). The lesions of HIV-positive women with HPV infection at the time of diagnosis of CIN were less likely to regress than those of HIVnegative women (hazard ratio, 0.18; 95% CI, 0.05–0.62). Moscicki et al. (2004) studied the incidence of HSIL diagnosed cytologically among HIV-positive and HIV-negative adolescent girls who participated in the Reaching for Excellence in Adolescent Care and Health cohort. The incidence of HSIL at the end of follow-up was higher for HIV-positive girls (21.5%) than for HIV-negative girls (4.8%). In a multivariate analysis, use of hormonal contraceptives (hazard ratio, 2.60; 95% CI, 1.25–5.40), high concentrations of IL12 in cervical mucous (hazard ratio, 2.28; 95% CI, 1.17–4.43), persistent LSIL diagnosed cytologically (hazard ratio, 1.67; 95% CI, 1.29–2.18) and infection with HPV type 16 only (hazard ratio, 3.69; 95% CI, 1.06–12.80), HPV type 18 only (hazard ratio, 4.49; 95% CI, 1.16–17.42) and multiple high-risk HPV types (16, 18 and 56) (hazard ratio, 3.69; 95% CI, 1.07–12.71) were significantly associated with the development of HSIL. (iii)

Effect of highly active antiretroviral therapy (HAART) on CIN (Table 72) Heard et al. (1998) examined the natural history of cervical lesions in 49 HIV-positive women in Paris, France, before and 5 months after initiation of HAART. They examined the prevalence of HPV using southern blot hybridization (to define high-level infection) and PCR (to identify low-level infection). The prevalence of SIL decreased from 69 to 53% during follow-up (p < 0.0001). Two women with HSIL regressed to LSIL and one regressed to normal, and nine of 21 (43%) women with LSIL regressed to normal. In all patients except one, the prevalence of HPV infection detected by southern blot hybridization and PCR did not change. There was a greater increase in the absolute number of CD4+ cells in the subgroup of patients whose lesions regressed (99 versus 50 × 106/L; p = 0.03) compared with those whose lesions did not regress. Delmas et al. (2000) evaluated the natural history of SIL in HIV-positive women in a cohort that was followed in 12 European countries. Women with lower CD4+ cell counts (< 200 × 106/L) had twice the prevalence and incidence of SIL than women with higher CD4+ cell counts (> 500 × 106/L), and fewer had regression of CIN1. Regression of SIL in women with a low CD4+ cell count was lower (20.5%) in those who received antiretroviral therapy than in those who did not (31.4%), but the difference was not significant (p = 0.30). Lillo et al. (2001) studied the effect of HAART on high-risk HPV infections and related cervical lesions in 163 HIV-positive women in Italy. High prevalences of both high-risk HPV infection (68%) and SIL diagnosed cytologically were found at baseline

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Table 72. Effect of highly active antiretroviral therapy (HAART) on cervical intraepithelial neoplasia (CIN) in HIV-positive women No. of HIVpositive cases

MY09/MY11 PCR primers (16, 18, 33) plus SBH (6/11/42, 16/18/33, 31/35/39, other types), sequencing of unidentifiable types

49

Delmas et al. (2000), 12 European countries

SBH (6, 11, 42, 16, 18, 31, 33, 35, 39), PCR MY09/MY11 (16, 18, 33), GP1/GP2 primers to detect other types

467 (baseline), 229 (followup), 115 CIN1 (followup)

Lillo et al. (2001), Italy

HC 2 and MY09/ MY11 PCR with E6 and L1 priming (6, 11, 16, 18, 31, 33, 35, 45)

163

HPV prevalence %

Cervical abnormality Odds ratio (95% CI)/ p value

HIV+ Before HAART 81 by PCR, 54 by SBH After HAART 93 by PCR, 56 by SBH

68 HR HPV; persistent HPV infection in HAARTtreated versus non-treated

1.18 (0.63– 3.46)

Pathology reading

Comments

%

Odds ratio (95% CI)/ p value

9/21 women with LSIL at baseline regressed to normal cytology (3 with persistent colposcopic abnormalities); 2/13 women with HSIL at baseline regressed to LSIL and one regressed to normal.

p < 0.0001

Cytology, histology

HPV testing on cervical cotton swab and wooden spatula; women were followed prospectively at a median of 5 months after initiation of HAART.

SIL 24.2 HIV+ at baseline, 23.6 at 1 year, 29.5 at 18 months Progression of LSIL to HSIL 8.1 at 1 year Regression of LSIL to normal 30.9 at 1 year Cumulative regression rate among women with CD4+ < 200 20.5 with HAART, 31.4 without HAART

p = 0.30

Cytology

Women were participants in a European cohort on natural history of HIV infection; HPV testing was performed on cervical brush specimens; women with normal cytology at baseline were followed every 6 months for a median of 2 years; women diagnosed with CIN1 were followed for a median of 18 months without treatment.

6.2 HSIL, 20.2 LSIL; progression of cytological changes in HAART-treated versus non-treated

2.01 (0.44– 9.20)

Cytology, histology

Participants followed at 6month intervals; HPV infection defined as having the same type at baseline and follow-up, transitory as having a change in HPV status and HPV– as having consistently negative HPV tests; HPV samples obtained by brushing squamocolumnar junction

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MY09/MY11/ HMB01 PCR (6, 11, 16, 18, 26, 31, 32, 33, 35, 39, 40, 45, 51, 52, 53, 54, 55, 56, 58, 59, 61, 66, 68, 69, 70, 73, Pap 155, Pap 291, AE2)

507

No HPV DNA detection

56

Heard et al. (2002), France

Regression to normal cytology in HAARTversus non-HAARTtreated

168 CIN (96 HAARTtreated)

No. of HIVnegative controls

HPV prevalence %

NR

62

NR

Cervical abnormality Odds ratio (95% CI)/ p value

%

Odds ratio (95% CI)/ p value

Regression of cytological abnormalities 35.3 with HAART, 29.5 without HAART Progression of cytological abnormalities 15.6 with HAART, 22.3 without HAART

1.4 (1.04– 1.82)

Disease persistence among women not treated within 6 months of diagnosis 60 HIV+, 32 HIV– Persistent or recurrent CIN after therapy 17.6 with HAART, 70.3 without HAART

p < 0.05

Regression to normal, 34.1 Progression to highgrade, 22.7

1.93 (1.14– 3.29)

Pathology reading

Comments

Cytology

Women were participants in the WIHS; HPV testing performed on a cervicovaginal lavage specimen; women had measurement of serum retinol and were followed every 6 months; all had HR HPV infection; progression and regression were measured using consecutive Pap smear pairs.

0.68 (0.52– 0.88)

Retrospective chart review of women treated for CIN; cervical conization or loop electrosurgical excision therapy used

p < 0.05

Cytology, histology

Women were followed at 6month intervals; 37 were treated with HAART at the time of study initiation, 59 initiated HAART after the baseline visit.

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Table 72 (contd)

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No. of HIVpositive cases

Moore, A.L. et al. (2002), United Kingdom

No HPV DNA detection

71

Schuman et al. (2003), USA

MY09/MY11 PCR primers (6, 11, 16, 18, 26, 31, 33, 35, 39, 40, 42, 45, 51, 52, 53, 54, 55, 56, 58, 59, 66, 68, 70, Pap 155, Pap 291, W13B)

774

UbertiFoppa et al. (2003), Italy

HC2 PCR MY09/ MY11 and SPF10

83 stable with no or standard HAART, 71 more potent HAART

No. of HIVnegative controls

391

HPV prevalence %

Cervical abnormality Odds ratio (95% CI)/ p value

%

Odds ratio (95% CI)/ p value

Prevalence of CIN 55 before HAART, 62 after HAART Regression, 13

p = 0.20

64.0 HIV+, 28.7 HIV–

NR

SIL baseline end-point Standard HAART LSIL, 30.1–13.3 HSIL, 14.5–1.2 More potent HAART LSIL, 15.5–24 HSIL, 12.7–2.8

p < 0.0001

Pathology reading

Comments

Cytology, histology

Women attended a gynaecology clinic, required cytology before HAART initiation and another at least 6 months after HAART initiation; median time between pre- and postHAART smears, 10 months.

Cytology

Cervicovaginal lavage specimen used for HPV testing; women were participants in the HERS and were followed every 6 months.

Histology

Mean age of patients was 32.3 ± 5.2 years (range, 21–45 years); histology classified according to the Bethesda system

p = 0.06

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No. of HIVpositive cases

MY09/MY11/ HMB01 PCR (6, 11, 13, 16, 18, 26, 31, 32, 33, 35, 39, 40, 45, 51, 52, 53, 54, 55, 56, 58, 59, 61, 66, 68, 69, 70, 73, Pap 155, Pap 291, AE2)

141 with regression of SIL, 171 with no regression of SIL

Massad et al. (2004b), USA

PCR MY09/MY11 and specific probes (16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 68, 73 and other low-risk types)

15 621

469

HPV prevalence %

Cervical abnormality Odds ratio (95% CI)/ p value

%

Odds ratio (95% CI)/ p value

NR

Incidence of regression 0 (95% CI, 0–2.4)/100 person–years before HAART, 12.5 (95% CI, 9.9– 15.1)/ 100 person–years after HAART

p = 0.002

NR

HAART versus nonHAART Genital warts VIN1 VIN2–3

Pathology reading

Comments

Cytology

Participants in the WIHS; HPV testing performed on a cervicovaginal lavage specimen; women had measurement of serum retinol and were followed every 6 months; all had HR HPV infection?; progression and regression were measured among women with a normal Pap smear at enrolment who developed SIL during at least 7 years of follow-up.

Cytology

Follow-up of 8 years

0.76 (0.58– 0.99) 0.65–0.49– 0.88) 0.64 (0.40– 1.04)

See Table 7 for a description of the primers used. CI, confidence interval; HC, Hybrid Capture; HERS, HIV Epidemiology Research Study; HIV, human immunodeficiency virus; HIV+, HIV-positive; HIV–, HIV-negative; HR, high-risk; HSIL, high-grade squamous intraepithelial lesion; ISH, in-situ hybridization; LSIL, low-grade squamous intraepithelial lesion; NR, not reported; Pap, Papanicolaou test; PCR, polymerase chain reaction; SBH, southern blot hybridization; SIL, squamous intraepithelial lesion; VIN, vulvar intraepithelial neoplasia; WIHS, Women’s Interagency HIV Study

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Table 72 (contd)

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(LSIL, 20.2%; HSIL, 6.2%). The women were followed every 6 months for a mean observation time of 15.4 months (range, 6–24 months). Although CD4+ cell counts increased significantly in subjects who received HAART, persistence of high-risk HPV infection (adjusted odds ratio, 1.18; 95% CI, 0.63–3.46) and progression of SILs (adjusted odds ratio, 2.01; 95% CI, 0.44–9.20) were comparable among those who were and those who were not treated with HAART. Minkoff et al. (2001) examined the relationship between the use of HAART and progression or regression of cervical cytology in the Women’s Interagency HIV Study. Women with persistent HPV positivity were more likely to have lesions that progressed: 16.2% progressed with HPV positivity at one of three visits, 23.6% progressed with positivity at two of three visits and 24.6% progressed with positivity at all three visits (p < 0.001). After adjustment for CD4+ cell count and cytological status, women who took HAART were more likely to experience regression (odds ratio, 1.4; 95% CI, 1.04–1.82) and less likely to show progression (odds ratio, 0.68; 95% CI, 0.52–0.88). Robinson et al. (2001) performed a retrospective chart review of 56 HIV-positive and 62 HIV-negative women to determine the rates of recurrence, persistence and progression of CIN after excisional therapy with and without HAART. Among those who were not treated within 6 months of diagnosis, persistence occurred in 60% of HIV-positive and 32% of HIV-negative women (p < 0.05). Risk factors for recurrence among HIV-positive women after treatment included margin involvement of specimens obtained by loop electrosurgical excision (p < 0.05). Disease in women who took HAART that included protease inhibitors was less likely to recur or persist after treatment than that in HIV-positive women who did not take HAART (p < 0.05). Heard et al. (2002) studied 168 HIV-positive women, of whom 37 were taking HAART at the time of study initiation and 59 began taking HAART after baseline, every 6 months. Overall, regression of CIN was observed in 39.9% of the women. The risk for regression of CIN was nearly twice as high in women who received HAART than in those who did not (relative hazard, 1.93; 95% CI, 1.14–3.29). HAART had a similar effect on the regression of low-grade CIN to normality (30/88 women; 1.99; 95% CI, 0.94–4.18) and on the reversion of high-grade to low-grade CIN (31/174 women). Moore, A.L. et al. (2002) studied the prevalence of CIN in 71 HIV-positive women 10 months after initiation of HAART. The prevalence of CIN before HAART was 55%; a median of 10 months after treatment with HAART, this had increased to 62% (p = 0.20). Thirteen per cent of patients experienced regression of a CIN lesion, which was associated with a greater but non-statistically significant increase in CD4+ cell count. Schuman et al. (2003) examined risk factors for the progression and regression of cytological abnormalities in 774 HIV-positive and 391 HIV-negative women who participated in the HIV Epidemiology Research Study. HAART was not significantly associated with the probability of progression of cervical lesions. Uberti-Foppa et al. (2003) assessed the long-term effect of HAART by comparing HPV and cytology/histology results at the beginning and end of a study of 83 women who were stable without HAART or who were taking HAART but did not change their therapy

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(Group S), and 71 women who changed to a more potent HAART regimen due to failure of the treatment (Group W). Participants were followed at 6–12-month intervals for a mean of 36 months. HPV infection was defined as positivity for the same type at baseline and at follow-up; infection was described as transitory when a change in HPV status occurred; and HPV status was considered to be negative when HPV tests were consistently negative. Although treatment with HAART increased the CD4+ cell level among women in both groups, HAART-associated increases in CD4+ cell level did not affect the persistence of HPV. However, women who took HAART in Group S had fewer HPV-positive low-grade biopsies at the end of the study compared with baseline (30.1% versus 13.3%; p = 0.00004). No significant reduction was seen in Group W. The data suggest that long-term use of HAART may be beneficial in the regression of low-grade lesions. Ahdieh-Grant et al. (2004) studied 312 HIV-positive women in the Women’s Interagency HIV Study who had normal cervical cytology at baseline, who developed incidental SIL during 7 years of follow-up and who could be monitored for regression or progression of lesions in relation to the use of HAART. Of these, 141 (45.2%) had lesions that regressed to normal with a median time to regression of 2.7 years. The incidence of regression increased (p for trend = 0.002) after HAART was introduced. Women whose lesions did not regress had lower CD4+ cell levels than those whose lesions regressed (p < 0.01). However, the majority of cervical lesions among HIV-positive women, whether they took HAART or not, did not regress to normal. Massad et al. (2004b) followed the incidence and predictors of genital warts and VIN among HIV-positive and HIV-negative women at high risk who participated in the Women’s Interagency HIV Study for up to 8 years. In a multivariate analysis, warts were associated with HAART (relative hazard, 0.76; 95% CI, 0.58–0.99), CD4+ cell count (relative hazard, 0.91/100 cell/cm2 increase; 95% CI, 0.86–0.96), history of AIDS (relative hazard, 1.25; 95% CI, 1.00–1.57), abnormal Pap test results (relative hazard, 2.18; 95% CI, 1.73–2.75), high- or medium-risk HPV types (relative hazard, 1.91; 95% CI, 1.48–2.47), low-risk HPV types (relative hazard, 1.48; 95% CI, 1.10–2.00), tobacco smoking (relative hazard, 1.43; 95% CI, 1.09–1.88), having one child (relative hazard, 1.54; 95% CI, 1.11–2.13) and age (relative hazard, 0.74/10 years; 95% CI, 0.64–0.86). VIN of any grade was linked to HAART (relative hazard, 0.65; 95% CI, 0.49–0.88), CD4+ cell count (relative hazard, 0.92; 95% CI, 0.87–0.97), abnormal Pap test results (relative hazard, 16.03; 95% CI, 11.33–22.69), high- or medium-risk HPV types (relative hazard, 1.37; 95% CI, 1.06–1.77) and age (relative hazard, 0.85/10 years; 95% CI, 0.73–0.99). While HAART was associated with a reduced relative hazard for incidental genital warts and VIN of any grade, it was not significantly associated with a reduced relative hazard for VIN2–3 in a multivariate analysis (relative hazard, 0.64; 95% CI, 0.40–1.04). Two studies analysed the incidence of cervical cancer before and after the introduction of HAART. The International Collaboration on HIV and Cancer (2000) examined the relationship between HAART and incidence of cancer in HIV-positive adults. Rate ratios were estimated by comparing incidence rates from 1997 to 1999 with those from 1992 to 1996. The

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rate ratio for cervical cancer was 1.87 (95% CI, 0.77–4.56), which indicated that there had been no significant change in the incidence of cervical cancer since the introduction of HAART. Dorrucci et al. (2001) analysed the incidence of cervical cancer in Italian women before and after the introduction of HAART. They estimated the incidence per 1000 person–years of cervical cancer as a first AIDS-defining disease for the periods 1981–91, 1992–95 and 1996–98 in 483 women with a median follow-up of 7 years. Compared with 1981–95, the hazard ratio for cervical cancer for 1996–98 was 7.41 (95% CI, 1.21–45.44). After adjustment for age at HIV seroconversion, the hazard ratio decreased to 4.75 (95% CI, 0.80– 28.24). The incidence of cervical cancer had not declined after the introduction of HAART in this population. (b)

Studies of the anorectal region

(i) Prevalence of anal HPV infection and anal SIL (Table 73) Carter, P.S. et al. (1995) studied 90 HIV-positive men, 77 HIV-negative men and 43 men of unknown HIV status who attended a genitourinary medicine clinic in London, United Kingdom. The relative risk for AIN for HIV-positive men was 1.58 (95% CI, 1.01–2.48) compared with HIV-negative men. The relative risk for developing AIN for those with anal warts compared with those without anogenital warts was 4.70 (95% CI, 1.81–12.20). Hillemans et al. (1996) examined the prevalence of anal HPV infection and anal disease in 102 HIV-positive and 96 HIV-negative women at high risk in the New York metropolitan area, USA. Using Hybrid Capture 2 on anal swabs, HPV DNA was found in 29.4% of HIV-positive and 2.1% of HIV-negative women. Anal cytological abnormalities were low-grade or atypical, and were found in 27.3% of HIV-positive and 6.4% of HIVnegative women with satisfactory smears. Of 33 women with anal cytological abnormalities, 19 (58%) had anal HPV DNA compared with 13 (8%) of 160 women without cytological abnormalities (p < 0.001). In a multivariate logistic regression analysis, HPV infection was the only risk factor associated with anal cytological abnormalities (adjusted odds ratio, 16.0; 95% CI, 8.9–3.2). In a logistic regression model of risk factors for anal cytological abnormalities that did not include HPV DNA positivity, HIV-positive women who had a CD4+ cell count < 200/mm3 were at higher risk than HIV-positive women who had a CD4+ cell count > 200/mm3 (adjusted odds ratio, 7.3; CI, 1.8–49.2). HIV positivity was found to be an independent risk factor for HPV infection and, similarly to anal cytological abnormalities, the strength of the association with anal HPV infection among HIVpositive women was greater in those who had CD4+ T-lymphocyte counts < 200/mm3 than in those who had CD4+ counts > 200/mm3 (adjusted odds ratio, 11.6; 95% CI, 2.1–64.5). Melbye et al. (1996) studied 81 HIV-positive and 70 HIV-negative women in Copenhagen and Aarhus, Denmark. Using PCR, anal HPV was detected in 78% of HIVpositive women and 60% of HIV-negative women. Abnormal anorectal smears were found in 19% of HIV-positive women and in none of the HIV-negative women.

No. of HIVnegative controls

Men

90

77 HIV–, 43 unknown HIV status

Hillemans et al. (1996), USA

Women

HC2 with typing of HC2-positives using MY09/MY11 PCR

102

96

Melbye et al. (1996), Denmark

Women

MY09/MY11 PCR (6, 11, 16, 18, 31, 33, 35, 45, 39, 51, 52) and HC

81

70

Palefsky et al. (1997a), USA

Men

MY09/MY11 PCR (39 different HPV types) and HC

129

Friedman et al. (1998), USA

Men

HC2 and MY09/ MY11/HMB01 PCR

184

79

HPV prevalence (%)

Anorectal abnormality (%)

Pathology reading

Comments

AIN 41.1 HIV+, 26.0 HIV–, 39.5 unknown HIV status Anogenital warts 58.9 (HIV+), 79.2 (HIV–), 95.3 unknown HIV status

Histology

Men were recruited from a genitourinary medicine clinic.

29.4 HIV+ 2.1 HIV–

27.3 HIV+, 6.4 HIV–

Cytology

HPV testing on anal swab material

PCR 78 HIV+, 60 HIV– HC 38 HIV+, 7 HIV–

19 HIV+, 0 HIV–

Cytology

HPV testing on anal Dacron swab material and cervical cotton swab material; women were recruited from HIV screening clinics.

PCR, 93.2 HIV+ HC, 84.5 HIV+

35 ASCUS, 12 LSIL, 1 HSIL

Cytology

Testing on anal swab material; study subjects were MSM with CDC group IV HIV disease.

90.4 HIV+, 69.6 HIV–

60.0 HIV+, 29.6 HIV–

Cytology

HPV testing on anal swab material; patients were MSM.

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396 Table 73. Prevalence of anal HPV infection and anal squamous intraepithelial lesions (SIL) in HIV-positive and HIV-negative individuals

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Table 73 (contd) Sex

Method of detection (types included)

No. of HIVpositive cases

No. of HIVnegative controls

Palefsky et al. (1998a), USA

Men

No HPV DNA detection

346

262

MY09/MY11 PCR (39 different HPV types) and HC2

346

262

PCR 93.1 HIV+, 61.0 HIV– HC 87.3 HIV+, 37.3 HIV–

NR

Same cohort as above; testing on anal swab material; patients were MSM.

232 (181 MSM), 51 heterosexual)

NR

AIN in satisfactory smears 30.0 HIV+ MSM, 4.7 HIV– MSM, 0 HIV– heterosexual

Anal cytology obtained with a cytobrush

Men

PCR with E6 primers (6, 11, 16, 18)

66

Goldstone et al. (2001), USA

Men

No HPV DNA detection

131

Holly et al. (2001), USA

Women

MY09/MY11 PCR (39 types)

251

68

Pathology reading

Abnormal cytology 45.9 HIV+, 9.9 HIV– AIN by cytology or histology 35.8 HIV+, 7.3 HIV–

Cytology, histology

Comments

Biopsy 60.0 high-grade AIN, 3 invasive anal cancer

Cytology, histology, biopsy

Patients referred for condyloma or other presumably benign anorectal disease

Anal cytology 26.0 HIV+, 8.2 HIV–

Cytology, histology

WIHS study

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Anorectal abnormality (%)

HUMAN PAPILLOMAVIRUSES

Palefsky et al. (1998b), USA

HPV prevalence (%)

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397

No. of HIVpositive cases

No. of HIVnegative controls

HPV prevalence (%)

Anorectal abnormality (%)

Pathology reading

Comments

Palefsky et al. (2001), USA

Women

MY09/ MY11 PCR (39 types and HC2)

251

68

76.2 HIV+, 42.1 HIV– Anal HPV 79 HIV+ 63 HIV–

Sobhani et al. (2001), France

Men and women

In-situ PCR (6, 11, 16/18, 31, 33)

116

60

Oncogenic HPV 27 HIV+, 1.3 HIV–

Condyloma recurrence 75 HIV+, 6 HIV–

Drobacheff et al. (2003), France

Men and women

HC2

50

50

58.0 HIV+, 6.0 HIV–

4 HIV+, 0 HIV–

Moscicki et al. (2003), USA

Men and women

MY09/MY11 PCR for HPV LR (6/11/42/44) and HR (16/18/31/33/ 35/39/42/51/52/56/5 8)

241

107

Boys 48.3 HIV+, 36.0 HIV– Girls 32.2 HIV+, 13.4 HIV–

Boys 52.5 HIV+, 16.7 HIV– Girls 21.3 HIV+, 5.7 HIV–

Cytology

Subjects were adolescents participating in the REACH cohort; HPV testing on anal swab material

Piketty et al. (2003), France

Men

MY09/MY11 PCR (39 different HPV types)

50 with no history of RAI, 67 with history of RAI

No RAI 46.0 any infection RAI 85

No RAI 16 LSIL, 19 HSIL RAI 49 LSIL, 18 HSIL

Cytology, histology

HPV testing from anal swab specimen

HPV testing on anal swab specimens

Eight women were diagnosed with condyloma at entry; fixed anal biopsy specimens; ISH or in-situ PCR for EBV and HSV; immunohistochemistry for CMV HPV testing on anal swab material; the risk for HPV infection in HIV+ compared with HIV– was 9.7 (95% CI, 3.2–29.7).

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Table 73 (contd)

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Sex

Method of detection (types included)

Chin-Hong et al. (2004), USA

Men

MY09/ MY11 PCR (39 different HPV types)

No. of HIVpositive cases

No. of HIVnegative controls

HPV prevalence (%)

Anorectal abnormality (%)

1218

56.8, 25.6 HR types, 26.4 LR types

NR

Pathology reading

Comments

Study of sexually active MSM in 4 US cities participating in the EXPLORE cohort

See Table 7 for a description of the primers used. AIN, anal intraepithelial neoplasia; ASCUS, atypical squamous cells of undetermined significance; CDC, Centers for Disease Control; CI, confidence interval; CMV, cytomegalovirus; EBV, Epstein–Barr virus; EXPLORE, collaborative study among homosexual men in six US cities; HC2, Hybrid Capture 2; HIV, human immunodeficiency virus; HIV–, HIV-negative; HIV+, HIV-positive; HR, high-risk; HSIL, high-grade squamous intraepithelial lesion; HSV, herpes simplex virus; ISH, in-situ hybridization; LR, low-risk; LSIL, low-grade squamous intraepithelial lesion; MSM, men who had sex with men; NR, not reported; PCR, polymerase chain reaction; RAI, receptive anal intercourse; REACH, Reaching for Excellence in Adolescent Care and Health

HUMAN PAPILLOMAVIRUSES

Reference, study location

Page 399

Table 73 (contd)

399

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Palefsky et al. (1997a) characterized the prevalence of anal HPV infection and anal lesions in 129 HIV-positive men who had sex with men and had Centers for Disease Control group IV HIV disease. Abnormal anal cytology was detected in 39% of subjects and anal HPV infection, as measured by PCR, in 93%. Risk factors for abnormal cytology in a multivariate analysis included HPV 16/18 infection (relative risk, 2.1; 95% CI, 1.2–3.5) and intravenous drug use (relative risk, 1.8; 95% CI, 1.2–2.7). Infection with HPV 6/11 also had significantly elevated relative risks in a separate model. Anal cytological abnormalities and HPV infection are common among homosexual/bisexual men with group IV HIV disease. Friedman et al. (1998) studied the prevalence of anal HPV infection, using Hybrid Capture 2 and PCR, and anal disease, using cytology, in a cohort of HIV-positive and HIV-negative men who had sex with men. HPV was found more commonly in HIV-positive men (90.4% versus 69.6%), among whom the number of HPV types detected and Hybrid Capture RLU ratios were higher. Among HIV-positive men, the quantity of HPV DNA as measured by Hybrid Capture RLU ratio was inversely associated with CD4+ cell count. Anal cytological abnormalities were more common among HIV-positive than HIVnegative men (60% versus 29.6%) and, among HIV-positive men, AIN was found more frequently among those with a CD4+ cell count < 200/μL. Men with a high Hybrid Capture RLU ratio were 2.8 times more likely to have AIN (95% CI, 1.1–7.3) than those with samples that were both PCR- and Hybrid Capture 2-negative. Palefsky et al. (1998a) examined the prevalence of AIN in a cohort of 346 HIV-positive and 262 HIV-negative men who had sex with men. Abnormal cytology was detected in 45.9% of HIV-positive and 9.9% of HIV-negative men. Using cytology or histology, AIN was diagnosed in 36% of HIV-positive and 7% of HIV-negative men (relative risk, 5.7; 95% CI, 3.6–8.9). High-grade AIN was found in 17 HIV-positive men (5%) and in only one HIV-negative man. Among HIV-positive men, the relative risk for AIN increased with lower CD4+ cell levels but was elevated even in men with CD4+ cell levels > 500/mm3 (relative risk, 3.8; 95% CI, 2.1–6.7) compared with HIV-negative men. Highlevel HPV infection, as measured by detection of both low-risk and high-risk types using Hybrid Capture, was another significant risk factor for AIN in both HIV-positive (relative risk, 8.8; 95% CI, 2.3–35) and HIV-negative men (relative risk, 20; 95% CI, 5.5–71) compared with HPV-negative men. Palefsky et al. (1998b) also studied the prevalence of anal HPV infection in the cohort described above. Anal HPV DNA was detected by PCR in 93% of HIV-positive and 61% of HIV-negative men. The spectrum of HPV types was similar in HIV-positive and HIVnegative men, and HPV 16 was the most common type. Infection with multiple HPV types was found in 73% of HIV-positive and 23% of HIV-negative men. Among HIV-positive men who were positive by Hybrid Capture for low-risk and high-risk types, lower CD4+ cell levels were associated with higher levels of high-risk HPV DNA (p = 0.004) but not low-risk HPV DNA. These data suggest increased replication of the higher-risk HPV types with more advanced immunosuppression. For HIV-positive men, risk factors for the presence or absence of HPV DNA could not be assessed because most were positive for

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HPV DNA using PCR or Hybrid Capture. Among the risk factors examined in univariate analyses for the HIV-negative men, the relative risks for HPV infection detected by PCR were: lifetime rectal drug use, 1.4 (95% CI, 1.1–1.7); lifetime history of rectal discharge, 1.3 (95% CI, 1.0–1.7); and lifetime level of receptive anal intercourse compared with no receptive anal intercourse: low, 1.3 (95% CI, 0.97–1.7); medium or high, 1.5 (95% CI, 1.1–2.1; p for trend = 0.03). The relative risks for HPV DNA positivity using Hybrid Capture were similar to those using PCR. Sayers et al. (1998) studied 66 HIV-positive and 181 HIV-negative men who had sex with men and 51 HIV-negative heterosexual men who attended a genitourinary medicine clinic in Edinburgh, United Kingdom. AIN was noted in 30% of satisfactory anal smears from HIV-positive men, in 4.7% of satisfactory smears from HIV-negative men who had sex with men and in no smear from HIV-negative heterosexual men. There was no significant difference in the detection of HPV types 6, 11, 16 and 18 between HIV-positive and HIV-negative men. Goldstone et al. (2001) determined the prevalence of anal HSILs and anal squamouscell cancer in 131 HIV-positive and 69 HIV-negative men who had sex with other men. Ninety-three per cent had abnormal anal cytology. Biopsy results revealed that 60% of patients had high-grade AIN and 3% had invasive anal cancer. Four of five men with anal squamous-cell cancer were HIV-positive. Holly et al. (2001) examined the prevalence of anal lesions in 251 HIV-positive and 68 HIV-negative women at high risk who participated in the San Francisco section of the Women’s Interagency HIV Study. Abnormal anal cytology was diagnosed in 26% of HIVpositive and 8% of HIV-negative women. High-grade AIN was detected by histology or cytology in 6% of HIV-positive and 2% of HIV-negative women. HIV-positive women had an increased risk for anal disease as their CD4+ cell counts decreased (p < 0.0001) and as their plasma HIV RNA viral load increased (p = 0.02). HIV-positive women with abnormal cervical cytology had an increased risk for abnormal anal cytology at the same visit (relative risk, 2.2; 95% CI, 1.4–3.3). In a multivariate analysis, an HIV viral load of > 100 000 copies/mL (relative risk, 2.4; 95% CI, 1.1–3.9), history of anal intercourse (relative risk, 2.3; 95% CI, 1.2–3.6) and concurrent abnormal cervical cytology (relative risk, 2.1; 95% CI, 1.0–3.6) were significantly associated with abnormal anal cytology. Palefsky et al. (2001) studied the same population of women for the presence of anal HPV using PCR and Hybrid Capture 2 (relative risk, 1.8; 95% CI, 1.3–2.5). Among 200 women for whom there were concurrent data on anal and cervical HPV, anal HPV was more common than cervical HPV in both HIV-positive (79% versus 53%) and HIV-negative (43% versus 24%) women. In a multivariate analysis of HIV-positive women, CD4+ cell counts ≤ 200 cells/mm3 compared with counts > 500 cells/mm3 (relative risk, 1.4; 95% CI, 1.1–1.5) and cervical HPV infection (relative risk, 1.3; 95% CI, 1.1–1.4) were associated with anal HPV infection. Women over 45 years of age had a lower risk than women under 36 years of age (relative risk, 0.80; 95% CI, 0.50–0.99), and African-American women had a lower risk (relative risk, 0.86; 95% CI, 0.72–1.0) than Caucasian women.

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Sobhani et al. (2001) determined the prevalence of anal dysplasia and cancer in patients with anal condyloma with respect to HIV status and HPV positivity. The most important factors that differed significantly between HIV-positive and HIV-negative patients were the prevalence of oncogenic HPV and other current infections (44% versus 0%). During follow-up, condylomas recurred in 75% of HIV-positive patients but in only 6% of HIV-negative patients. Drobacheff et al. (2003) studied 50 HIV-positive and 50 HIV-negative men and women in France. Using Hybrid Capture 2 on anal swab material, HPV DNA was found in 58% and 6% of the samples, respectively. There was no difference in the prevalence of high-risk HPV types between men with and without a history of anal intercourse. Risk factors for HPV infection were CD4+ cell counts < 500/μL (relative risk, 2.13; 95% CI, 1.0–4.7) and history of anogenital warts (relative risk, 2.36; 95% CI, 1.2–4.6). A very low prevalence of anal lesions was found in this study but the authors did not mention the use of high-resolution anoscopy to detect lesions. Using the Hybrid Capture RLU ratio, HPV load was greater in patients with CD4+ cell counts ≤ 500/μL than in patients with CD4+ cell counts > 500/μL (p < 0.04). Similar to the results of Palefsky et al. (2001), HIV-positive women in this study had a rate of anal HPV infection that was similar to that of HIVpositive men. Overall, the data showed that HIV-positive patients with low CD4+ cell counts have anal HPV infection, regardless of the route of HIV transmission. One study reported anal HPV and cytology data among HIV-positive and HIV-negative adolescent boys with a history of sex with men and girls who participated in the Reaching for Excellence in Adolescent Care and Health cohort (Moscicki et al., 2003). The prevalence of anal HPV infection was similar in HIV-infected [28/58 (48%)] and uninfected [9/25 (36%)] boys (p = 0.3), but was greater in HIV-positive girls [59/183 (32%)] than in HIV-negative girls [11/82 (13%)] (p < 0.001). Abnormal anal cytology was more common among boys (41.6%) than girls (16.5%; p < 0.001). Independent risk factors for abnormal anal cytology in boys included infection with low-risk HPV types, infection with high-risk HPV types, infection with unknown HPV types and HIV positivity. Among girls, independent risk factors for abnormal anal cytology included infection with high-risk or unknown HPV types and number of sexual partners within the past 3 months. The results suggest that anal cytology screening should be considered in HIV-positive homosexual/bisexual boys and possibly HIV-positive girls. Piketty et al. (2003) compared the prevalence of anal HPV infection and SIL among 67 HIV-positive men who had sex with men and 50 HIV-positive heterosexual male intravenous drug users with no history of receptive anal intercourse; 46% of the heterosexual intravenous drug users had anal HPV infection and 18% had anal HSIL. Among the 67 men who had sex with men, 85% had anal HPV infection and 18% had anal HSIL. The data showed that anal HPV infection and anal SIL may be acquired in the absence of anal intercourse in HIV-positive men. Chin-Hong et al. (2004) studied the age-related prevalence of anal HPV infection among 1218 sexually active, HIV-negative men who had sex with men aged 18–89 years who participated in the EXPLORE study in four cities in the USA. HPV DNA was found

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in the anal canal of 57% of the study participants. The prevalence of anal HPV infection did not change with age or geographical location. In a multivariate analysis, anal HPV infection was associated with receptive anal intercourse during the preceding 6 months (odds ratio, 2.0; 95% CI, 1.5–2.8; p < 0.0001) and with having 6–30 sexual partners during the preceding 6 months (odds ratio, 1.4; 95% CI, 1.1–1.9), and more than 30 partners (odds ratio, 2.3; 95% CI, 1.5–3.6). (ii) Natural history of anal HPV infection and anal SIL (Table 74) In a prospective study of 158 HIV-positive and 147 HIV-negative men who had sex with men and did not have anal disease at baseline, high-grade AIN developed in 15.2 and 5.4%, respectively (Critchlow et al., 1995). High-grade AIN among HIV-positive men was associated with the detection of high levels of HPV 16 or 18, detection of HPV types other than 16 or 18, CD4+ cell count ≤ 500 × 106/L and the number of positive HPV tests. To characterize the natural history of anal HPV infection, Critchlow et al. (1998) followed 287 HIV-negative and 322 HIV-positive men who had sex with men who attended a community-based clinic. Anal HPV DNA was detected at study entry in 91.6% of HIV-positive and 65.9% of HIV-negative men. Detection of HPV was associated with lifetime number of sexual partners and recent receptive anal intercourse (HIV-negative men), lower CD4+ lymphocyte count (HIV-positive men) and anal warts (all men). Among the men who were negative for HPV at study entry, detection of HPV during follow-up was associated with HIV, unprotected receptive anal intercourse and any sexual contact since the last visit. Becoming HPV-negative during follow-up was less common among men with HIV infection or high levels of HPV at study entry. Men with low-risk HPV types at entry were more likely to become HPV-negative than men with intermediate- or high-risk HPV types (p ≤ 0.05). In a prospective study of the incidence of anal HSIL among 346 HIV-positive and 262 HIV-negative men who had sex with men, Palefsky et al. (1998a) showed that HIV-positive men were more likely to develop HSIL than HIV-negative men (relative risk, 3.7; 95% CI, 2.6–5.7). A life-table estimate of the 4-year incidence of HSIL was 49% (95% CI, 41–56%) among HIV-positive and 17% (95% CI, 12–23%) among HIVnegative men. The incidence of high-grade AIN within 2 years of follow-up was 20% in HIV-positive and 8% in HIV-negative men who were normal at baseline; 62% of HIVpositive and 36% of HIV-negative men with low-grade AIN at baseline progressed to high-grade AIN. Of the HIV-positive men who had ASCUS at baseline, 70.3% were diagnosed with low- or high-grade AIN within 2 years, as were 30.8% of HIV-negative men. Overall, the relative risk for progression of anal disease in HIV-positive men was 2.4 (95% CI, 1.8–3.2) compared with HIV-negative men. The relative risk increased to 3.1 (95% CI, 2.3–4.1) in HIV-positive men with CD4+ cell counts < 200/mm3. Infection with multiple HPV types was a risk factor for progression of anal disease in both HIVpositive (relative risk, 2.0; 95% CI, 1.0–4.1) and HIV-negative (relative risk, 5.1; 95% CI, 2.3–11) men. The incidence of anal HSIL and progression of LSIL to HSIL within 2 years

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No. of HIVpositive cases

No. of HIVnegative controls

HPV prevalence (%)

Anorectal abnormality (%)

Pathology reading

Comments

Critchlow et al. (1995), USA

SBH and MY09/ MY11 PCR

158

147

NR

High-grade AIN, 15.2 HIV+, 5.4 HIV–

Cytology, histology

Dacron anal swab specimens used for HPV testing. Participants had no a