2. Studies of Cancer in Humans
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P_179_278.qxp 30/11/2007 09:40 Page 179 HUMAN PAPILLOMAVIRUSES 179 2. Studies of Cancer in Humans 2.1 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 appro- priate. 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 surro- gate 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 end- points. 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 P_179_278.qxp 30/11/2007 09:40 Page 180 180 IARC MONOGRAPHS VOLUME 90 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 surro- gate 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 repre- sent 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 neo- plasia, 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 suffi- cient 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 limi- tations of statistical power. At present, type-specific prospective evidence of carcino- genicity 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 scree- ning 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 vali- dated. 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 life- time 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 esti- mation 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. P_179_278.qxp 30/11/2007 09:40 Page 181 HUMAN PAPILLOMAVIRUSES 181 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 DNA- positive. 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 co- transmitted 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 co- infection 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 statis- tical power to evaluate all combinations adequately. This problem is most apparent when assessing the possible carcinogenicity of uncommon types that occur mainly in combi- nation 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 diffe- rential 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 P_179_278.qxp 30/11/2007 09:40 Page 182 182 IARC MONOGRAPHS VOLUME 90 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 equi- vocal or lacking. Moreover, the interpretations of mild and equivocal HPV-related cyto- logy 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 require- ment 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.