The causal role of human papillomavirus infections in cervical cancer has been documented beyond reasonable doubt. The association is present in virtually all cervical cancer cases worldwide. It is the right time for medical societies and public health regulators to consider this evidence and to define its preventive and clinical implications. A comprehensive review of key studies and results is presented.
Keywords: human papillomavirus, cervical cancer, causality, review
During the 1990s, epidemiological studies, supported by molecular technology, provided evidence on the causal role of some human papillomavirus (HPV) infections in the development of cervical cancer. This association has been evaluated under all proposed sets of causality criteria and endorsed by the scientific community and major review institutes. The finding is universally consistent, and to date there are no documented alternative hypotheses for the aetiology of cervical cancer.
HPV has been proposed as the first ever identified, “necessary cause” of a human cancer. In practical terms, the concept of a necessary cause implies that cervical cancer does not and will not develop in the absence of the persistent presence of HPV DNA.
Cervical cancer is still the second most common cancer in women worldwide, although it is a theoretically preventable disease.
In developed parts of the world, and in populations where cytology based programmes are established, it would be beneficial to add HPV testing to the screening protocol. HPV testing was shown by several studies, including one randomised trial, to be of help in solving the ambiguous cases generated by cytology reading.
In populations where cytology programmes are either not in place or are not efficient, HPV testing should now be considered and evaluated as an alternative test for primary screening.
Prevention of exposure to high risk HPV types by vaccination may prove to be the most efficient and logistically feasible preventive intervention for cervical cancer.
At this stage of development, regulatory agencies are requested to evaluate the scientific evidence and weigh its implications in relation to costs, public health investments, and policy. This is a subjective evaluation that could be guided by a careful description of the most relevant studies and findings.
A major discovery in human cancer aetiology has been the recognition that cervical cancer is a rare consequence of an infection by some mucosatropic types of HPV. In public health terms, this finding is equally important as the discovery of the association between cigarette smoking and lung cancer, or between chronic infections with hepatitis B virus (HBV) or hepatitis C virus and the risk of liver cancer. Moreover, as in the HBV disease model, intense efforts are currently going into the development and testing of vaccines that may prevent the relevant HPV infections, and presumably, cervical cancer.
By the year 2000, the epidemiological evidence included a large and consistent body of studies indicating, beyond any reasonable doubt, strong and specific associations relating HPV infections to cervical cancer. The observations have been reported from all countries where investigations have taken place. Studies include prevalence surveys, natural history investigations, case–control studies and, more recently, a randomised intervention trial. Natural history and follow up studies have clearly shown that HPV infection preceded the development of cervical cancer by several years and confirmed that sexual transmission is the predominant mode of HPV acquisition. These studies satisfied, in biological terms, the long known clinical and epidemiological observations that cervical cancer displayed the profile of a sexually transmitted disease (STD). Case–control studies, case series, and prevalence surveys have unequivocally shown that HPV DNA can be detected in adequate specimens of cervical cancer in 90–100% of cases, compared with a prevalence of 5–20% in cervical specimens from women identified as suitable epidemiological controls.
The association has been recognised as causal in nature by several international review parties since the early 1990s, and the claim has been made that this is the first necessary cause of a human cancer ever identified.
The implications of the recognition that, in the absence of viral DNA, cervical cancer does not develop, are of considerable practical importance. On the one hand, the concept of risk groups comes into focus. High risk women can now be sharply redefined as the group of persistent HPV carriers. Operatively, this represents substantial progress from previous versions of the high risk group that identified women by their exposure to a constellation of ill defined factors (low socioeconomic status, high number of sexual partners, smoking, use of oral contraceptives, history of STDs, and any combination of the above). Most of these factors are now viewed either as surrogates of HPV exposure or as relevant cofactors given the presence of HPV DNA. On the other hand, if indeed HPV is a necessary cause of cervical cancer, the implication is that specific preventive practices targeting some putative non-HPV related cervical cancer cases are no longer justified. Finally, technology is now available to screen HPV DNA positive women in the general population. Therefore, the final consideration on the nature of the association between HPV and cervical cancer is of considerable public health relevance. Research at the population level has largely accomplished its task by providing an exhaustive body of evidence. It is now time for public health institutions to evaluate these achievements, consider the costs and benefits involved, and apply this knowledge to their guidelines, recommendations, and policy.
Research in relation to the aetiology of cervical cancer has made substantial progress in the past two decades, both in scientific and operational terms. For decades, the epidemiological profile of women with cervical cancer was recognised as suggestive of a sexually transmitted process, and several infectious agents were proposed over the years including syphilis, gonorrhea, and type 2 herpes simplex virus (HSV-2).
The development of technology to test for the presence of HPV DNA in cellular specimens in the early 1980s and the multidisciplinary collaboration within the field made possible the establishment of a definite aetiological role for HPV in cervical cancer. Evidence is also accumulating for HPV involvement in a considerable proportion of cancers of the vulva, vagina, anal canal, perianal skin, and penis. The association of HPV with cervical cancer has provided the background and the justification for improving screening programmes and for developing HPV vaccines.
Figure 1 is a schematic view of the time scale of this dynamic process. It includes an indication of the results obtained as technology evolved in sensitivity expressed as the per cent of cervical cancer cases that were found to contain viral DNA. The figure also indicates the types of HPV tests that were predominantly used and an estimate of the periods in which the key types of studies were initiated.
Evolution of epidemiological research on human papillomavirus (HPV) and cervical cancer in the past two decades. FISH, filter in situ hybridisation; GP-PCR, general primer PCR; HC I–II, hybrid capture first and second generation; PCR, polymerase...
Figure 2 displays the approximate number of scientific papers identified by Medline searches on HPV and on HPV and cervical cancer, and the number of research abstracts presented at the major annual papillomavirus conferences (http://www.ipvsoc.org). The 1980s generated a rapidly increasing number of publications on HPV DNA prevalence in cervical cancer and reports on validation of the available detection methods. The 1990s produced the key results of case–control and cohort studies, and beginning in the late 1990s there was an increasing number of publications on the clinical uses of HPV testing in screening and triage.
Scientific publications on human papillomavirus (HPV) identified by Medline and the number of research abstracts presented at the annual Papillomavirus International Conferences.
CAUSALITY CRITERIA IN HUMAN CANCER RESEARCH
Epidemiological studies are essential to establish the association between risk factors and cancer and to qualify the nature of the association. Traditionally, these include case series, case–control studies, cohort studies, and intervention studies.
Comparisons of exposure between patients with cervical cancer and their relevant controls were initially established using questionnaires. Most studies conducted before the availability of HPV DNA detection systems identified as key risk factors several variables related to the sexual behaviour of the women and of their sexual partners. The most frequently reported risk factors included the number of sexual partners, an early age at first intercourse, or any previous STD.1
Once the relevant biomarkers were validated, in this case the presence of HPV DNA in exfoliated cervical cells, it became possible to advance over questionnaire based studies and establish biologically sound comparisons between patients and controls. In epidemiological terms, these comparisons would analyse cervical cells from women with cervical cancer and from otherwise comparable women without cervical cancer (case–control studies), or from cohorts of women tested for viral DNA (cohort studies). To characterise the link between HPV and invasive cervical cancer, case–control studies proved to be the key study design and the only ones ethically acceptable in human populations. Typically in such a study and at the time of fieldwork, controls are selected to match the age distribution of the cases and, as much as possible, the general characteristics of the cases (place of residence, socioeconomic status, health plan, etc). To characterise the association, all study participants are requested to comply with a questionnaire to assess their individual exposure to any known or suspected risk factor for the disease. The information is then used to estimate the odds ratios (ORs) of disease related to any given exposure. Multivariate analyses have the ability to compare (through statistical adjustment) strictly equivalent groups of women in relation to any of the exposures of interest. The adjusted differences (ratios) in the prevalence in HPV markers between cases and controls are then obtained after having eliminated the effects of any other differences in exposure. Likewise, comparisons of cases and controls in relation to other variables of interest will provide estimates of the relevance of other factors (oral contraceptives (OCs) or smoking) and identify the variables that merely reflect HPV exposure (surrogate variables).
When the technology to detect HPV DNA in samples of DNA extracted from exfoliated cervical cells became available, it was relatively easy to show that most of the sexual behaviour variables were in fact surrogate measures of HPV exposure, reflecting the predominant pathway of acquisition of HPV. As methods became more sensitive, the parameters that merely expressed the probability of HPV (or any other STD) infection, such as number of sexual partners, became statistically irrelevant.2–5
Causality in public health requires a judgment based on scientific evidence from human and experimental (animal) observations. As such, only the latter may benefit from the most stringent criteria of causality; that is, the repeated induction of the disease by exposure to the relevant agent(s) compared with the “spontaneous” occurrence of the same disease in unexposed and yet comparable groups of animals. All causal associations of human cancers have been recognised based on educated judgment of the results of epidemiological studies at the level already available for HPV and cervical cancer. Final proof can only be confirmed by intervention (preventive) trials, in which a reduction of the disease burden (incidence or mortality) is observed following the introduction of a preventive practice in strictly controlled conditions. These studies typically include as controls populations to whom the existing standard of preventive care is being offered.
Table 1 displays some of the criteria that have been proposed to evaluate the nature of the associations encountered by epidemiological studies. This is particularly relevant when causality is being proposed because, as a consequence, preventive or clinical recommendations are made.
Epidemiological considerations important for causal inference
In addition to the criteria listed in table 1, some additional contributions might be worth discussing. In 1976, Evans reviewed the history of the causality criteria in infectious disease models and adapted the early postulates of Henle-Koch to both the viral origin of acute diseases and to the relation between viral infections and cancer.14 The human models that inspired most of the latter included two examples: Epstein-Barr virus (EBV) infections and Burkitt's lymphoma, and HSV-2 viral infections and cervical cancer. The technology that was discussed was largely based on antibody detection and the studies involved were seroepidemiological surveys and case–control studies. Antibody measurements were the methods of choice for the assessment of exposure. Evans proposed a unified scheme for causation that included most of the criteria mentioned in table 1.14 In 1976, Rothman15 introduced the concepts of “necessary and sufficient causes”. This model is useful to accommodate the growing evidence of the multifactorial origin of human cancer in many instances. Finally, several authors have defined criteria to evaluate the findings of molecular technology that provided the basis of the studies of HPV and cervical cancer.16,17
Because of its wider acceptance, we will discuss in detail the criteria proposed by Hill, and its version adopted by the International Agency for Research on Cancer (IARC) monograph programme, in addition to the model on necessary and sufficient causes proposed by Rothman in 1995.18
In brief, the criteria proposed by Hill8 as summarised by Rothman19 include the following:
Hill suggested that the following aspects of an association should be considered when attempting to distinguish causal from non-causal associations: (1) strength, (2) consistency, (3) specificity, (4) temporality, (5) biological gradient, (6) plausibility, (7) coherence, (8) experimental evidence, and (9) analogy.
By “strength of association”, Hill means the magnitude of the ratio of incidence rates. Hill's argument is essentially that strong associations are more likely to be causal than weak associations because if they were the result of confounding or some other bias, the biasing association would have to be even stronger and would therefore presumably be evident. Weak associations, on the other hand, are more likely to be explained by undetected biases. Nevertheless, the fact that an association is weak does not rule out a causal connection.
Consistency refers to the repeated observation of an association in different populations under different circumstances.
The criterion of specificity requires that a cause should lead to a single effect, not multiple effects. However, causes of a given effect cannot be expected to be without other effects on any logical grounds. In fact, everyday experience teaches us repeatedly that single events may have many effects.
Temporality refers to the necessity that the cause should precede the effect in time. The temporality of an association, is a sine qua non: if the “cause” does not precede the effect that is indisputable evidence that the association is not causal.
Biological gradient refers to the presence of a dose–response curve. If the response is taken as an epidemiological measure of effect, measured as a function of comparative disease incidence, then this condition will ordinarily be met.
Plausibility refers to the biological plausibility of the hypothesis, an important concern but one that may be difficult to judge
Taken from the Surgeon General's report on Smoking and Heath (1964)9: “The term coherence implies that a cause and effect interpretation for an association does not conflict with what is known of the natural history and biology of the disease.”
Such evidence is seldom available for human populations. In human data, the experimental criterion takes the form of preventive interventions and explores whether there is evidence that a reduction in exposure to the agent is associated with a reduction in risk.
The insight derived from analogy seems to be handicapped by the inventive imagination of scientists, who can find analogies everywhere. Nevertheless, the simple analogies that Hill offers—if one drug can cause birth defects, perhaps another can also—could conceivably enhance the credibility that an association is causal.
As is evident, these nine aspects of epidemiological evidence offered by Hill to judge whether an association is causal are saddled with reservations and exceptions; some may be wrong (specificity) or occasionally irrelevant (experimental evidence and perhaps analogy). Hill admitted that: “none of my nine viewpoints can bring indisputable evidence for or against the cause and effect hypothesis and none (except temporality) can be required as a sine qua non”.
The IARC in its monograph programme largely adopted the causality criteria proposed by Hill and established rules to decide on the carcinogenicity of a given exposure, particularly when human data are scarce and must be combined with experimental data. However, the final qualification of the carcinogenicity of any given substance being evaluated is taken by vote of the external (non-IARC) participants.
The monograph programme and its criteria has been reviewed and accepted by most scientists in the field of human carcinogenesis. To date, 77 monographs have been published, of which five involve biological agents such as HPV.12 In its preamble, the monograph programme establishes guidelines to qualify an epidemiological observation as causal, and also defines rules to be followed when human data suggest lack of carcinogenicity potential. These criteria are useful to challenge any aetiological hypothesis when the epidemiological studies are inconsistent or when only weak associations are reported.
Finally, another useful way of examining the nature of an association was provided by a model system that proposed that any given disease would occur as a consequence of human exposure to a “sufficient cause”.18 A sufficient cause is described, in its simplest model, as the concurrence in a given individual of a constellation of factors (called the components of the sufficient cause), following which the disease will develop. Each given disease will have its own sufficient cause or sets of sufficient causes (lung cancer may have a sufficient cause that involves cigarette smoking, but another sufficient cause that does not include smoking, such as intense radon exposure in non-smokers). According to the model, a necessary cause is described as a component of a sufficient cause that is part of all the sufficient causes described. To prevent disease it is not necessary to identify all the components of a sufficient cause, or to remove them all: it is sufficient to remove one component from each sufficient cause, that is to remove, if it exists, the necessary cause.
COMPLIANCE OF THE CAUSALITY CRITERIA IN THE HPV AND CERVICAL CANCER MODEL
In the following sections, we will review several studies that have provided evidence of the association between HPV and cervical cancer, using the criteria outlined in table 1. For purposes of clarity, we shall concentrate the discussion on the criteria that have proved to be of greater value in the evaluation of human carcinogens and on the studies that focused on invasive cervical cancer.
Strength of the association
This criterion is usually discussed by examining the magnitude of the relative risk (RR), or the OR, which is the estimate of the RR in case–control studies. We shall use as the primary example the results of the IARC multicentre case–control study on invasive cervical cancer, as presented at international scientific meetings, and either published or at different stages of preparation for publication. In brief, this project included nine case–control studies in different parts of the world, mostly in high risk countries. HPV DNA testing was done in two central research laboratories using the MYO9/1120 and the general primer (GP) GP5+/6+21,22 polymerase chain reaction (PCR) testing systems. The published results have reported ORs for cervical cancer in the range of 50 to 100 fold for HPV DNA. ORs for specific associations (such as HPV-16 and squamous cell cancer and HPV-18 and cervical adenocarcinomas) range between 100 and 900. These estimates lead to calculations of attributable fractions (AF) for the entire study greater than 95%.23
Table 2 shows the size of the multicentre case–control study and the prevalence of HPV DNA in each relevant group. Figure 3 displays the HPV DNA prevalence in eight countries in cervical cancer cases and controls. It is noteworthy that the first two studies conducted in Spain and Colombia (fig 3) used early versions of the MYO9/11 PCR system that identified HPV DNA in approximately 75% of the cases. The rest of the studies were analysed using the GP5+/6+ PCR system and its modifications, which resulted in an almost 20% increase in the HPV DNA detection rate.
Prevalence of human papillomavirus (HPV) DNA in cases and controls in the IARC multicentre case–control study.24–30
Size of the IARC multicentre case–control study and human papillomavirus (HPV) DNA prevalence
Table 3 shows the corresponding estimates of the RR (OR and 95% confidence interval (CI)). Results are presented separately for squamous cell carcinomas and adenocarcinomas of the cervix. Given the case–control design of the study, these very high ORs reflect the risk in relation to existing HPV DNA in cervical cells (HPV DNA point prevalence), not in relation to “ever” being infected with HPV (cumulative lifetime exposure). Furthermore, if HPV shedding was intermittent among controls, their corresponding HPV prevalence would have been underestimated, resulting in an inflation of the ORs observed. It is usually interpreted that the HPV DNA point prevalence at advanced age (over 40 years of age) reflects viral persistency. However, much research is still devoted to defining viral persistency and its prognosis accurately, a crucial definition for the clarification of the uses of HPV testing in screening and patient management.31
Odds ratio for the association of human papillomavirus (HPV) DNA and cervical cancer in the IARC multicentre case–control study: preliminary data23,32,33
Most of the discussion in the text uses HPV DNA as a generic marker that includes any positive result for several HPV types. It is now possible to provide estimates of the RR for at least 10 different HPV types showing that there are no significant differences in the risk of cervical cancer in relation to the HPV types most commonly found in these lesions. The preliminary results of the IARC multicentre case–control were pooled and summarised by Muñoz et al in 2000,32 at the HPV 2000 Papillomavirus Conference (www.hpv2000.com). These analyses indicated that for squamous cell carcinomas, the age and centre adjusted OR was 83.3 (95% CI, 54.9 to 105.3). The prevalence of the four most common HPV types and their ORs among 1545 cases with single infections were: HPV-16, 59% (OR = 182); HPV-18, 12% (OR = 231); HPV-45, 4.8% (OR = 148); and HPV-31, 3.7% (OR = 71.5). Other less common HPV types showing equally high ORs were: HPV-33, OR = 77.6; HPV-35, OR = 34.8; HPV-51, OR = 42.7; HPV-52, OR = 145.7; HPV-58, OR = 78.9; and HPV-59, OR = 347.3.
The most common types among cases were also the most common types among HPV positive control women: HPV-16, 30.3%; HPV-18, 8.2%; HPV-31, 4.8%; and HPV-45, 3.9%. These findings indicate that in addition to HPV-16 and HPV-18, HPV types 31, 33, 35, 45, 51, 52, 58, and 59 should be considered as human carcinogens.
The HPV type distribution in the population and in patients with cervical cancer shows a seemingly modest geographical variability that has not been fully described (J Kornegay, personal communication, 2001).34–36 The description and the implications of such variability for HPV testing and HPV vaccination are to be determined.
The results of the multicentre study are consistent with findings from other countries that have generated recent data on invasive cervical cancer and preinvasive disease in Costa Rica,37 Thailand,38 Norway,39 Denmark,40 and virtually all other countries in which these studies have been conducted.
Multiple HPV types were detected in the multicentric study on average in 7.3% of the cases and 1.9% of the controls, and did not show a significantly increased risk (OR = 54.5; 95% CI, 35.5 to 83.6) over women positive for only one HPV type (OR = 86.6; 95% CI, 68.2 to 110).
The proportion of multiple types in a given specimen varies across studies and particularly in relation to the HPV detection method used. Table 4 provides an indication of the proportion of specimens from cases and from the general population that showed multiple types. The table suggests that populations at high risk of cervical cancer and with high rates of human immunodeficiency virus (HIV) positivity tend to show higher proportions of multiple types than do populations not belonging to these risk groups. Longitudinal studies have suggested that the one time, cross sectional detection of type specific HPV may underestimate the cumulative lifetime diversity of exposure to HPV.31 However, in all studies of invasive carcinoma, the risk linked to multiple HPV types does not vary significantly from the risk linked to single HPV types.
Prevalence of multiple human papillomavirus (HPV) types in patients with cervical cancer and women without cervical cancer
The similarity in the prognostic value of detection of any of the 10 high risk HPV types, in addition to any combination of them, clearly indicates that group testing for high risk HPVs would be sufficient in the context of clinical and screening protocols.
Figure 4 shows, for comparison purposes, some estimates of the strength of associations between environmental factors and human cancer that were recognised as causal in nature by epidemiological studies and subsequently proved in human populations by intervention studies. The figure includes risk (RRs or ORs) as the measurement of the strength of the associations and AFs representing the proportion of disease that is attributable to (caused by) the exposure. Below the reference line the risk column displays its reverse estimate as a less than one (protective) OR or RR and the protective fractions (PF%) in the right hand column show results that have already been achieved in disease reduction after specific exposure reduction interventions.
Selected examples of the strength of the associations (RR/ OR) between risk factors and human cancer; estimates of the attributable fraction (AF%) and of the protective fraction (PF%). Refs: the Philippines,28 Costa Rica,37 Bangkok,5 Taiwan,43 Greece,...
Strength of association. Evaluation
The association between HPV DNA in cervical specimens and cervical cancer is one of the strongest ever observed for a human cancer. HPV-16 accounts for almost 50% of the types identified in cervical cancer. The cancer risk for any one of at least 10 HPV types or for any combination of HPV types does not differ significantly.
There is a striking consistency between the results of the multicentre case–control study and over 50 other studies conducted in other countries, under different protocols and HPV DNA testing systems. Figures 5, 6, and 7 summarise the results of studies that compared the prevalence of HPV DNA in patients with cervical cancer and controls. Some of the studies used the prevalence of HPV-16 DNA to calculate ORs and some reported results for HPV DNA (all types combined). Some studies focused on invasive cervical cancer, whereas others used preinvasive lesions as the definition of cases. When indicated, separate analyses are presented for squamous cell carcinomas and for adenocarcinomas. Studies that have compared risk factors for cervical intraepithelial neoplasia stage 3 (CIN 3) and invasive cancer have not reported any significant differences in their associations with HPV or with their epidemiological profile.38,49
Odds ratios (OR) and 95% confidence intervals for associations found in case–control studies using PCR methods between human papillomavirus 16 (HPV-16) (or its nearest surrogate) and invasive cervical cancers. *The OR estimate is ∞ owing...
Odds ratios (OR) and 95% confidence intervals for associations found in case–control studies using non-PCR methods between human papillomavirus 16 (HPV-16) (or its nearest surrogate) and invasive cervical cancers. *The OR estimate is ∞...
Odds ratios (OR) and 95% confidence intervals for associations found in case–control studies after the year 2000. HPV, human papillomavirus.
Apart from confirming the high ORs shown in figs 5 and 6, fig 7 also demonstrates the consistency of results between squamous cell carcinomas and adenocarcinomas, the consistency of findings between preinvasive disease and invasive cancer, and the consistency of findings between risk estimates for HPV DNA (all types considered) and risk estimates restricted to high risk types.
The association between HPV DNA in cervical specimens and cervical cancer is consistent in a large number of investigations in different countries and populations. There are no published studies with observations challenging the central hypothesis on causality.
Specificity, as defined by Hill, tended to be relegated to a secondary level for cancer causality evaluation once it became clear that carcinogenic exposures are usually complex (for example, cigarette smoke) and can induce cancer in different organs and even cancers of different histological profile in the same organ.
In the case of HPV, the complexity of the association is being unveiled. The HPV family includes over 100 HPV types, of which 30–40 are mucosatropic and at least 15 types have been clearly linked to cervical cancer. In addition, some of these types are also related to other cancers of the genital tract (vulvar cancer, vaginal cancer, and cancers of the anal canal, perianal skin, and the penis) and perhaps to cancers of other organs (such as oropharyngeal and skin cancer).
To examine the association of HPV and human cancer in light of the specificity criteria, we shall widen the original scope (one exposure/one disease) to verify whether a more complex model involving multiple HPV types and several cancer sites seems to occur with frequencies suggesting a consistent departure from a random model.
About 15 HPV types are involved in over 95% of the cervical cancer cases. HPV-16 and HPV-18 are the most common types identified and represent 50% and 10%, respectively, of the viral types involved in invasive cancer. Figure 8 shows the cumulative prevalence of five HPV types in cervical carcinomas by histological type in 2400 cases included in the multicentre case–control study. It clearly shows that these five HPV types comprise 80–95% of the viral types identified in carcinomas.
Cumulative prevalence of human papillomavirus (HPV) types in cervical cancer. Taken from the IARC multicentre case–control study; preliminary data.23
Adenocarcinomas and adenosquamous cell carcinomas are more closely related to HPV-18 and its phylogenetically related family (HPV types 39, 45, and 59) than are squamous cell carcinomas, which in turn are closely linked to HPV-16 and its phylogenetically related family (HPV types 31, 35, and 52).34,87 The reasons for such specificity are unknown.
Cancers of the vulva and vagina are closely related to HPV-16. Approximately 40–50% of vulvar cancer shows HPV DNA, and in several series HPV-16 is by far the predominant type in more than 80% of cases.88–90
Cancer of the tonsil is closely related to HPV-16, whereas other cancers of the oral cavity show inconsistent and lower prevalences of HPV DNA.91–94
Skin cancers related to the epidermodysplasia verruciformis condition are related to a restricted number of dermatotrophic HPV types. These are also recovered from basal cell carcinomas and squamous cell carcinomas of the skin in immunosuppressed and immunocompetent individuals.95
Other associations, reported in a small number of cases, seem to occur with some specificity. For example HPV-16 and cancers of the conjunctiva96 and HPV-16 and cancers of the ungueal bed.
Studies on HPV variants (variation within HPV types at the single nucleotide level) are beginning to unveil risk differences.97–99 The geographical distribution of HPV variants and its relevance for HPV testing and for vaccine development are still uncertain.
HPV has been excluded as a likely cause or even as a risk factor for other human cancers. A large number of investigations (largely unpublished) have not provided support to the hypothesis of the involvement of these viruses in the causation of cancers of the endometrium, ovary, prostate, or other sites (reviewed by Shah and Howley16 and Syrjänen and Syrjänen100).
The association of type specific HPV DNA and cervical cancer is significantly different from random. Systematic patterns of HPV type and cervical cancer histology suggest a fair degree of specificity. Patterns are also observed when the scope of HPV and cancer expands to include the full spectrum of HPV types and the large number of addi-tional cancer sites that have been investigated.
In conclusion, although the specificity criteria can be viewed as of secondary applicability, the global picture indicates that HPV types are not randomly associated with human cancer. A fair degree of specificity is consistently reported, even if the complexities of the type specific viral properties and of the organ/cell susceptibility have not been fully disclosed.
Of the criteria outlined by Hill and repeatedly endorsed by the IARC monograph programme and other bodies, the demonstration that exposure has occurred before the diagnosis is considered a “sine qua non” condition for a risk factor and for establishing causality. Five groups of studies have contributed data relevant to the temporality criterion.
Cross sectional studies have repeatedly reported that subclinical HPV infections are highly prevalent in young individuals, whereas invasive cervical cancer typically develops in the third decade and later (fig 9). The cross sectional prevalence of HPV DNA decreases spontaneously to a background level of 2–8% in most populations in groups that are 40 years old and above. In countries where intensive screening of young women takes place, part of the HPV prevalence reduction could be attributable to aggressive treatment of HPV related cervical lesions. Women who remain chronic HPV carriers are currently described as the true high risk group for cervical cancer. In some populations, a second mode of HPV DNA prevalence has been observed for older women (50 years and above), with uncertain relevance in relation to the risk of cervical cancer.36,37,101 In all settings investigated, the point prevalence of HPV DNA in the young age groups is strongly related to the sexual behaviour patterns that are dominant in each population.102–107
Age specific prevalence (%) of high rish (HR) human papillomavirus (HPV) DNA in 3700 women entering a screening programme and age specific incidence rate (x105) (ASIR) of cervical cancer in the Netherlands. Adapted from Jacobs et al and Parkin et al....
These population studies provide support for the concept that HPV infections precede the development of cervical cancer by some decades. In fact, from most cancer registries, including the USA based registries, it is well established that the age specific incidence of cervical cancer has a rising trend in the age interval 20–40, and shows a plateau or continues to increase smoothly after that age. Only occasionally do cases of invasive disease occur at earlier ages. Figure 9 shows the age specific, cross sectional prevalence of high risk HPV DNA in a screening programme in the Netherlands, and the corresponding age specific incidence rates of cervical cancer in that country. The distributions shown in fig 9 are highly reproducible in studies in other settings in high and low risk countries.3,24,106,108 However, the age specific incidence rates of invasive cervical cancer are strongly influenced by the local impact of screening programmes in each country.3,24,106,108
Follow up studies
For cervical cancer, compliance with the temporality criteria has been established by numerous cohort studies that monitored women from cytological normalcy to the stage of high grade cervical intraepithelial neoplasia (high grade squamous intraepithelial lesions (HSIL) or CIN 2/3). Monitoring of women to invasive disease is not acceptable on ethical grounds and thus that information is not available.
Repeated sampling of women being followed for viral persistence and cervical abnormalities has shown that the median duration of the infections is around eight months for high risk HPV types, compared with 4.8 months for the low risk HPV types. In two unrelated studies, the time estimates were fairly consistent. In one study in a high risk population in Brazil, the mean duration of HPV detection was 13.5 months for high risk HPV types and 8.2 months for the non-oncogenic types. HPV-16 tended to persist longer than the average for high risk types other than HPV-16.109 The results were remarkably similar in a student population in the USA and in the UK.31,110 The self limiting course of most HPV infections is consistent with the cross sectional profile displayed in fig 9. However, the currently observed time intervals may still suffer from imprecision in the estimates of time at first exposure, from the variability in the endpoint definition, and from censoring as a result of treatment of the early lesions.
Follow up studies of women with and without cervical abnormalities have indicated that the continuous presence of HR-HPV is necessary for the development, maintenance, and progression of progressive CIN disease.110–114 A substantial fraction (15–30%) of women with HR-HPV DNA who are cytomorphologically normal at recruitment will develop CIN 2 or CIN 3 within the subsequent four year interval.111,115,116 Conversely, among women found to be HR-HPV DNA negative and cytologically identified as either atypical squamous cells of undetermined significance (ASCUS) or borderline or mild dysplasia, CIN 2/3 is unlikely to develop during a follow up of two years, and their cytology is likely to return to normal.117,118 Women found positive for low risk HPVs rarely become persistent carriers and their probability of progression to CIN 2/3 is extremely low.117,119
As ongoing cohorts expand their follow up time, more precise estimates are being provided on the predictive value of viral persistence as defined by repeated measurements of viral types and variants. One such cohort in Sao Paulo has shown that the incidence of cervical lesions in women who were HPV negative twice was 0.73/1000 women months. The corresponding incidence among women with repeated HPV-16 or HPV-18 positive results was 8.68, a 12 fold increased incidence. The OR for HPV persistence among women who were twice HPV positive for the same oncogenic types was OR = 41.2 (95% CI, 10.7 to 158.3).120 Retrospective assessment of HPV status using archival smears from cases of cervical cancer and controls has provided evidence that HPV DNA preceded the development of invasive disease, and showed its value in signalling false negatives smears.117 An interesting observation from the same group suggests that the clearance of HR-HPV in otherwise established cytological lesions is a marker associated with the regression of CIN lesions.118,121 Finally, the persistence of HPV DNA after treatment for CIN 2/3 is an accurate predictor of relapse, and is at least as sensitive as repeated vaginal cytology.122
These results are useful in defining the clinical role of HPV testing. However, most observations on preinvasive disease have limitations for making inferences on cervical cancer causality. This is because even in controlled settings, observations are not allowed to continue beyond the stage of HSIL/CIN 3 or carcinoma in situ.
A particularly interesting approach to conducting follow up studies of invasive cancer (as opposed to studies of CIN 3) without ethical and time constraints is provided by so called “nested case–control studies”. These are studies initiated several years in the past that assembled and stored large banks of biological specimens from healthy individuals. Linkage studies can then identify cases of cervical cancer (or any other condition) that have occurred in the interval and the original specimens can then be analysed for the presence of HPV biomarkers. HPV DNA prevalence can then be compared with the corresponding prevalence in specimens of epidemiologically sound controls (individuals from the same cohort who did not develop the condition under otherwise equivalent exposures). These studies have documented the existence of HPV exposure years before the development of the disease, thus reproducing the conditions of a longitudinal study. With this approach, a RR estimate of 16.4 (95% CI, 4.4 to 75.1) was seen for invasive cervical cancer in Sweden using DNA extracted from stored Papanicolaou (Pap) smears123 and a RR of 32 (95% CI, 6.8 to 153) was seen in the Netherlands.117 In a similar study design, an OR of 2.4 (95% CI, 1.6 to 3.7) was obtained using serological markers of HPV exposure.124
Since the late 1980s, multiple studies have evaluated HPV testing as an adjunct to cytology in screening programmes. These have considered HPV testing either as a triage test in cases of mild abnormalities125–127 or as a primary screening test.128–130 It is not the purpose of this paper to review this literature and excellent summaries are being regularly produced and updated (see later). In brief, triage studies have shown that HPV testing is more sensitive than repeated cytology in identifying underlying high grade lesions in women with ASCUS.114,119,121,131,132 Studies that reflect primary screening conditions (in the absence of fully randomised trials) have shown that the sensitivity of HPV tests is higher than standard cytology in detecting high grade lesions, whereas the specificity is age dependent. HPV tests show lower specificity than cytology in younger women, accounting for the bulk of transient infections, whereas in older women (ages 30–35 and above) specificities tend to be similar for both tests.107,133,134
In terms of causality assessment, these studies showed that it is possible to predict the concurrent presence of neoplastic disease (usually HSIL, CIN 2–3, or severe dyskaryosis), or the risk of developing it, by means of HPV DNA detection. This property of the HPV test offers an indirect measurement of the strength of the association and of the temporal sequence of the events.
Determinants of HPV infection
Epidemiological studies investigating risk factors for HPV infection clearly and consistently have shown that the key determinants among women are the number of sexual partners, the age at which sexual intercourse was initiated, and the likelihood that each of her sexual partners was an HPV carrier.103,105,135–141 These are lifelong behavioural traits, thus clearly preceding the development of cervical cancer.
The role of men as possible vectors of HPV was measured in the early epidemiological studies by questionnaires that asked about the sexual behaviour of the husbands or sexual partners of patients with cervical cancer and controls. In addition, more recent studies had the ability to measure HPV DNA in exfoliated cells from the penile shaft, the coronal sulcus, and the distal urethra.142–146
These and other studies consistently showed that the risk of cervical cancer for a given woman can be predicted by the sexual behaviour of her husband as much as her own sexual behaviour. In populations where female monogamy is dominant, the population of female sex workers plays an important role in the maintenance and transmission of HPV infections. Moreover, the probability that a woman is an HPV carrier and her risk of developing cervical cancer have been shown to be related to the presence of HPV DNA in the penis or the urethra of her husband or sexual partner.104,147–149 More recently, it has been possible to confirm that male circumcision protected men from being HPV carriers and their wives from developing cervical cancer.150 These observations confirmed, in terms of HPV infections, observations made over a century ago151 and a scientific hypothesis formulated almost 30 years ago that male sexual behaviour is a central determinant of the incidence of cervical cancer.152,153
In conclusion, the natural history studies of HPV infections satisfy in biological terms most of the observations that were historically linked to cervical cancer. In the past two decades, the cervical cancer puzzle has become a coherent description that includes the identification of HPV as the sexually transmitted aetiological agent and the characterisation of the major determinants of HPV acquisition.154
HPV infections precede cervical precancerous lesions and cervical cancer by a substantial number of years. The epidemiology and the dynamics of HPV infection in populations satisfy previous observations that related cervical cancer to a sexually transmitted disease.
This refers to the presence of a dose–response curve indicating that the magnitude of the exposure is related to the risk of disease. This requirement, largely supported by chemically induced models of carcinogenesis, is difficult to apply in models of viruses and cancer. For HPV DNA, it is difficult to measure viral load in relation to the DNA of the cancer cells in the specimen, although early studies tended to show a correlation between HPV DNA amount and disease status.127 Some recent publications have provided relevant evidence using real time PCR methods. A study that used a nested case–control design found that cases consistently had higher viral loads for HPV-16 than controls, and that high viral loads could be detected up to 13 years before the diagnosis of cervical cancer.155 Women with high viral loads for HPV-16 had a 30 fold greater risk of developing cervical cancer than did HPV negative women. This also applied to women under the age of 25. A related paper using the same population showed that the 20% of the population with the highest viral loads for HPV-16 had a 60 fold higher risk of developing carcinoma in situ when compared with HPV negative women.85 Of importance for clinical and screening purposes, another study confirmed that high viral loads predicted cervical lesions and, more interestingly, that the reduction of viral load or clearance of viral DNA in repeated visits predicted regression of CIN lesions to normalcy.156 These studies suggest that measuring viral load, at least of HPV-16, may distinguish between clinically relevant infections and those that are unlikely to progress. However, in contrast to the above results one large prospective study in Portland USA, using quantitative hybrid capture, did not find viral load to be a determinant of risk of future CIN 3 (A Lorincz et al, unpublished data, 2002). More research is needed to validate these methods and the results need to be extended and confirmed in clinical studies.157
Biological gradient. Evaluation
The risk of cervical cancer may be related to estimates of viral load. The technology to estimate viral load is being developed and compliance with the biological gradient requirement needs to be further validated.
Biological plausibility and coherence
The mechanisms by which HPV induces cancer in humans and the molecular genetics of the process are being investigated intensively and excellent reviews are readily available.12,16,17,158–161 These investigations provide additional evidence on the causality of the association by describing viral and host interactions leading to cell transformation and malignancy. Of the criteria outlined in table 1, both the “biological plausibility” and understanding of the “mechanisms” are in rapid expansion as a consequence of developments in molecular methods and technology.
Figure 10 shows in a schematic manner some of the major components of the transition from HPV infection to cervical cancer. Whereas transient infections are largely subclinical, progression is closely related to the persistence of viral DNA. This process goes frequently with viral disruption in the E1/E2 regions and integration into the cellular DNA. E2 disruption releases the viral promoters of E6 and E7 and increases the expression of these transforming genes. The E6 and E7 viral proteins are capable of selectively degrading the p53 and retinoblastoma gene (RB) products, respectively, leading to inactivation of two important cellular negative regulatory proteins.
Mechanisms of human papillomavirus (HPV) carcinogenesis. HSIL, high grade squamous intraepithelial lesion; LSIL, low grade squamous intraepithelial lesion; RB, retinoblastoma gene.
Some characteristics that provide support for the role of HPV in the induction of cervical cancer were recently outlined.17 Accordingly, the causal nature of this association is indicated by: (1) the regular presence of HPV DNA in the neoplastic cells of tumour biopsy specimens; (2) the demonstration of viral oncogene expression (E6 and E7) in tumour material (but not in stromal cells); (3) the transforming properties of these genes (E6 and E7); (4) the requirement for E6 and E7 expression to maintain the malignant phenotype of cervical carcinoma cell lines; (5) interaction of viral oncoproteins with growth regulating host cell proteins; and (6) epidemiological studies pointing at these HPV infections as the major risk factors for cervical cancer development.
In their review, Shah and Howley16 provided references to some of the key experiments that exemplify most of the requirements indicated by zur Hausen,17 namely: (1) The genomes of HPV-16 and HPV-18 are capable of immortalising human keratinocytes in cell culture, whereas the DNA of the low risk HPV types (6/11) do not.162 (2) In raft cultures, the oncogenes of the high risk HPV types induce morphological changes that closely resemble preinvasive cervical lesions.163,164 (3) In HPV associated lesions, the viral genome is present in every cell and is always transcriptionally active.165 (4) The viral genome is present in the original tumour and in metastases.166 (5) Most of the cell lines established from cervical cancer contain either HPV-16 or HPV-18 genomes.167 (6) The pattern of transcription changes as the lesion increases in severity. All open reading frames (ORFs) are expressed in early lesions but the expression of ORFs E4 and E5 is not found in many invasive cancers.165 (7) The E6 and E7 ORFs contain the transforming ability of HPV. These are always intact and are consistently expressed in cervical cancer cell lines, in cells transformed by HPV, and in HPV associated cancer tissue. They are transcribed at higher levels in high grade lesions than in low grade lesions.165,168 (8) In most cell lines and in many HPV associated cancers, the HPV DNA is integrated into the cellular DNA. HPV-18 is nearly always integrated, whereas HPV-16 can be found episomally or in the integrated form.76,169,170
In reviewing work on the molecular genetics of cervical carcinoma, Lazo indicated different mechanisms of cancer induction. The effects of E6 and E7 on host regulatory proteins can be considered to be HPV related mechanisms. An additional effect could be expected from the consequences of viral integration and the specific impact on the integration sites. The third mechanism, which may or may not be related to HPV, is the accumulation of the cellular genetic damage needed for tumour development. The existence of this mechanism is strongly suggested by the observations of recurrent losses of heterozygosity and by recurrent amplifications in a large fraction of cervical carcinomas.160 The role of non-identified tumour suppressor genes is also suggested by experiments showing that the tumorigenicity of HeLa cells could be suppressed by fusion with normal fibroblasts or keratinocytes, and that the tumorigenicity of SiHa cells was suppressed by the introduction of chromosome 11 via microcell transfer technology.171–173 Similarly, the immortality of Hela and Sitta cells was suppressed by the introduction of chromosomes 3, 4, and 6.
Cytology-based cervical cancer screening has greatly reduced the incidence and death of cervical cancer in developed countries. However, its suboptimal sensitivity and negative predictive value have not been overcome, despite the application of liquid-based cytology techniques and the Bethesda diagnostic classification. The fact that high-risk human papillomavirus (HPV) infection is a necessary event for cervical cancer development determines the value of HPV testing in cervical cancer screening. Compared with cytology, HPV DNA testing has a higher sensitivity and negative predictive value (NPV), providing more reassurance for women with a negative result. The American Society of Colposcopy and Cervical Pathology (ASCCP) initially recommended HPV testing as a triage for cytology diagnosed as atypical squamous cells of unknown significance (ASC-US) in 2001  and further recommended HPV testing for co-testing with cytology in 2006 . The European Research Organization on Genital Infection and Neoplasia (EUROGIN) then proposed HPV testing as a primary screening approach in 2008 . Since then, HPV-based screening has been performed in many European and Asian countries. Recently, the United States revealed the baseline data and three-year follow-up data of ATHENA (Addressing the Need for Advanced HPV Diagnostics) in 2011–2015, which was the largest prospective clinical study of HPV-based primary screening in the US, confirming that primary HPV testing screening was safer and more effective than primary cytology [4-7]. Based on those results, the US Food and Drug Administration (FDA) approved Roche Cobas HPV testing alone for cervical cancer screening in women over 25 years of age in 2014. Additionally, the ASCCP published interim guidelines in 2015 and recommended that primary HPV testing screening be considered as an alternative to current cytology-based cervical cancer screening methods .
As the largest developing country, China possesses the most cervical cancer patients with 98,900 new cases and 30,500 death cases in 2015 . Because of a severe lack of cytologists, cytology-based cervical cancer screening methods are restricted in China as well as in other developing countries. Compared with other HPV tests, Cobas® HPV testing using the real-time fluorescent PCR technique can simultaneously detect HPV 16/18 and another 12 high-risk HPV genotypes. With HPV 16/18 genotyping, the strategy of primary HPV screening with type 16/18 genotyping triage further reduces the necessary cytology tests and dependence on cytologists, which facilitates cervical cancer screening in developing countries. However, there are still no up-to-date data on the suitability of HPV testing with type 16/18 genotyping in Chinese women. Therefore, we assessed the effectiveness of HPV testing with type 16/18 genotyping and HPV-based program to detect high-grade cervical intraepithelial neoplasia (CIN) or cancer in 11,064 Chinese women through a population-based cross-sectional study. The aim of the study is to provide evidence of the suitability of an HPV-based screening strategy in China.
Materials and Methods
This study is a prospective population-based cancer screening trial. We chose Longyou County in Zhejiang Province, China as a candidate screening locale. A total of 332 administrative villages and communities were randomly selected, and large-scale cervical cancer screening was not performed in the past three years in these communities. All women were recruited by local community staff and doctors in the Longyou County Maternal and Child Health-Care Center according to household registry. Approximately 20% of women did not respond to the screening invitation because of outgoing for work or study, and other reasons. A total of 11,356 women aged 21–65 years participated in this cervical cancer screening program. All participants had a sexual history.
Those women were excluded: pregnancy or within 6 weeks or less of the post-partum period; previous total hysterectomy; a history of CIN or worse, vulvar intraepithelial neoplasia or worse, or vaginal intraepithelial neoplasia or worse; a history of other malignancies; a history of cervical cancer screening or physical cervical therapy in the past three years; serious autoimmune disease or uremia; and vaccinated or planned to vaccinate against HPV infection in the near future.
The study was in line with the 2013 Declaration of Helsinki and was approved by the Ethics Committee of Women's Hospital, Zhejiang University School of Medicine.
Each woman who met the inclusion criteria without meeting the exclusion criteria signed the informed consent form, provided a brief medical history, and underwent speculum examination by a gynecologist. According to instruction, samples of cervical exfoliate cells were collected using a cytology brush (Hologic, Bedford, MA) and stored in the tubes with preservation solution for the Thinprep cytology test (TCT, Hologic, Bedford, MA) and HPV DNA test (Cobas® 4800 Test, Roche Molecular Systems, Pleasanton, CA), respectively. All samples were collected during April to May of 2015.
The results of HPV testing were divided into the following: HPV-, HPV16/18+ (result positive for either genotype 16 or 18, with or without 12 other types), and HPV non-16/18+ (result negative for genotype 16/18 and positive for 1 or more of 12 other high-risk types). Cytology slides were read by two pathologists of our hospital, and 5674 cases were read with computer-aided reading (Hologic, MA) prior to examination by cytologists. Cytology results were reported according to the Bethesda 2014 classification . The cytology diagnoses were divided into negative for intraepithelial lesion or malignancy (NILM), atypical cells of undetermined significance (ASC-US), low-grade squamous intraepithelial lesion (LSIL), atypical squamous cells–cannot exclude high-grade squamous intraepithelial lesion (ASC-H), high-grade squamous intraepithelial lesion (HSIL), atypical glandular cells (AGC), and cervical cancer cells. If the diagnoses given by two cytologists were concordant, they were reported as the cytology diagnosis; otherwise, a third pathologist was consulted to reach a consensus diagnosis (2 of 3 agreements).
All women with positive HPV testing or abnormal cytology (ASC-US or worse) were referred to colposcopy; meanwhile, 4.5% of women with both negative tests were randomly selected and referred to colposcopy. Colposcopy was performed by colposcope specialists of our hospital. A woman underwent further cervical biopsy and/or endocervical curettage (ECC) if her colposcopy diagnosis was a high-grade lesion or a non-high-grade lesion with one of the following conditions: cytology LSIL or worse; cytology ASC-US and HPV+; HPV 16/18+; AGC; and unsatisfactory colposcopy. Tissues were paraffin embedded and slides were routinely HE stained. Two pathologists at our hospital separately made the diagnosis. If the diagnoses were concordant, they were reported as the pathologic diagnosis; otherwise, a panel of pathologists was consulted to reach a consensus diagnosis. The pathologic diagnosis standard was the 2014 WHO Classification of Tumors of the Female Genital Tract [11, 12]. The histological diagnoses of cervical lesions were divided into normal, low-grade squamous intraepithelial lesion (LSIL/CIN1, including the condylomatous variant), high-grade squamous intraepithelial lesion (HSIL)/CIN2, HSIL/CIN3 (including adenocarcinoma in situ), and carcinoma (squamous cell carcinoma or adenocarcinoma). Due to ethical considerations, most of the women with both negative HPV and cytology results were not referred to colposcopy and biopsy/ECC but were all regarded as LSIL or less. All women with pathologic abnormalities were processed according to the newest ASCCP guidelines . The screening process is shown in Figure 1. HPV testing, cytology, and pathologic examination were performed with blinding to the results of each test.
The study initially assessed the effectiveness of HPV testing, with cytology testing as a control, for identifying high-grade CIN and then compared the effectiveness of four strategies that are currently used for cervical cancer screening. Strategy 1 (Co-testing) primarily screens women with both cytology and HPV testing, and then refers those with cytology ASC-US/HPV+ or LSIL or worse to colposcopy. Strategy 2 (Primary cytology with triage by HPV testing) primarily screens women with cytology alone and then refers those with cytology LSIL or worse to colposcopy, triages those with ASC-US by reflex HPV testing, and refers HPV+ women to colposcopy. Strategy 3 (Primary HPV testing with triage by cytology) primarily screens women with HPV testing alone, triages HPV+ women with cytology, and refers those with cytology ASC-US or worse to colposcopy. Strategy 4 (Primary HPV testing with type 16/18 genotyping) primarily screens women with HPV testing plus type 16/18 genotyping, refers HPV 16/18+ women to colposcopy, triages HPV non-16/18+ women with cytology and refers those with cytology ASC-US or worse to colposcopy. A detailed flow chart is shown in Figure 2.
Abnormal rates of cytology and HPV testing were calculated based on all participants with cytology and HPV testing results. The effectiveness of detecting high-grade CIN was assessed using the sensitivity, specificity, positive predictive value (PPV), NPV, positive likelihood ratio (PLR), and negative likelihood ratio (NLR). Both raw and adjusted data were analyzed. When adjusted data were analyzed, the numbers of participants with pathological examination diagnosed as CIN2 or worse were used as a positive base to calculate the likely number of high-grade CIN cases that would have been found if all participants were referred to colposcopy for different cytology and HPV testing results. For screening strategies, the sensitivity, specificity, PPV, and NPV as well as the numbers of colposcopy cases and of screening tests needed to detect one case of high-grade CIN were calculated. All data were analyzed by SPSS 19.0 and medcalc 15.6 software. A P value less than 0.05 (two-sided) was considered statistically significant.
Comparison between HPV testing and cytology to identify high-grade CIN
Of 11,356 participants, 247 women met the exclusion criteria and 45 had invalid HPV results/invalid or unsatisfactory cytology, and they were excluded. As a result, 11,064 women were included in the analysis.
The total positivity rate of HPV testing was 9.8%. The positivity rates of HPV 16, HPV 18 and other high-grade genotypes were 1.8%, 0.6% and 7.4%, respectively. The HPV positivity rate had two age peaks, 21–24 (15.4%) and 60–65 (14.4%) years, as shown in Figure 3. The total abnormal cytology rate was 6.1%, with frequencies of ASC-US of 2.5%, AGC of 0.05%, LSIL of 2.4%, ASC-H of 0.6%, HSIL of 0.5%, and cervical cancer of 0.1%, respectively. A total of 1750 women were referred to colposcopy; of those, 567 were pathologically abnormal, including 427 cases with CIN1, 53 with CIN2, 75 with CIN3 (including 2 adenocarcinomas in situ), and 12 with cervical squamous carcinoma.
The effectiveness of cytology and HPV testing to identify high-grade CIN is shown in Table 1. Before verification bias correction, compared with cytology (LSIL or worse), HPV testing had a higher sensitivity (90.0% vs. 66.4%, P = 0.000) and NPV (98.1% vs. 96.6%, P = 0.050), while it had a significantly lower specificity (44.5% vs. 82.4%, P = 0.000), for detecting CIN2+. After verification of bias correction, the sensitivities of HPV testing and cytology were 90.0% and 66.7%, but the specificities were 91.3% and 97.2%, respectively, and the difference was significant. The AUC of HPV testing was larger than that of cytology (0.91 vs. 0.82) for detecting CIN2+. With CIN3+ as the identifying target, HPV testing and cytology demonstrated similar results.
|Sensitivity||126/140 (90.0%, 83.8–94.4)||93/140 (66.4%, 58.0–74.2)||P = 0.000||135/150 (90.0%, 84.0–94.3)||100/150 (66.7%, 58.5–74.1)||P = 0.000|
|Specificity||716/1610 (44.5%, 42.0–46.9)||1326/1610 (82.4%, 80.4–84.2)||P = 0.000||9966/10914 (91.3%, 90.8%–91.8%)||10611/10914 (97.2%, 96.9%–97.5%)||P = 0.000|
|PPV||126/1020 (12.4%, 10.4–14.5)||93/377 (24.7%, 20.4–29.3)||P = 0.000||135/1083 (12.5%, 10.6–14.6)||100/403 (24.8%, 20.7–29.3)||P = 0.000|
|NPV||716/730 (98.1%, 96.8–99.0)||1326/1373 (96.6%, 95.5–97.5)||P = 0.050||9966/9981 (99.9%, 99.8–99.9)||10611/10661 (99.5%, 99.4–99.7)||P = 0.000|
|PLR||1.62 (1.51–1.74)||3.77 (3.22–4.41)||10.36 (9.56–11.23)||24.0 (20.5–28.1)|
|NLR||0.22 (0.14–0.37)||0.41 (0.32–0.52)||0.11 (0.07–0.18)||0.34 (0.27–0.43)|
|Sensitivity||79/87 (90.8%, 82.7–96.0)||64/87 (73.6%, 63.0–82.5)||P = 0.009||84/92 (91.3%, 83.6–96.2)||68/92 (73.9%, 63.7–82.5)||P = 0.006|
|Specificity||722/1663 (43.4%, 41.0–45.8)||1350/1663 (81.2%, 79.2–83.0)||P = 0.000||9973/10972 (90.9%, 90.3–91.4)||10637/10972 (97.0%, 96.6–97.3)||P = 0.000|
|PPV||79/1020 (7.8%, 6.2–9.6)||64/377 (17.0%, 13.3–21.2)||P = 0.000||84/1083 (7.8%, 6.2–9.5)||68/403 (16.9%, 13.4–20.9)||P = 0.000|
|NPV||722/730 (98.9%, 97.9–99.5)||1350/1373 (98.3%, 97.5–98.9)||P = 0.294||9973/9981 (99.9%, 99.8–99.9)||10637/10661 (99.8%, 99.7–99.9)||P = 0.008|
|PLR||1.60 (1.48–1.74)||3.91 (3.33–4.59)||10.03 (9.20–10.93)||24.21 (20.61–28.43)|
|NLR||0.21 (0.11–0.41)||0.33 (0.23–0.46)||0.10 (0.05–0.19)||0.27 (0.19–0.38)|
All subjects were divided into three age-groups: 21–24 years, 25–49 years, and 50–65 years. We compared the effectiveness of HPV testing for detecting high-grade CIN in different age-groups. The 21- to 24-year group was not included in the statistics because there were too few samples. With CIN2+ as the identifying target, after verification bias correction, the sensitivity of HPV testing in the 50- to 65-year group was higher than that in the 25- to 49-year group (97.6% vs. 87.2%), but the effect was not significant, and the NPV was similar (99.97% vs. 99.8%). However, the specificity in the 50- to 65-year group was significantly lower than that in the 25- to 49-year group (89.3% vs. 92.4%), and the PPV in the 50- to 65-year group was significantly lower than that in the 25- to 49-year group (9.0% vs. 15.1%). The differences in effectiveness between the 25- to 49- and 50- to 65-year groups were similar when CIN3+ was the identifying target, as shown in Table 2.
|Sensitivity||89/102 (87.3%, 79.2–93.0)||37/38 (97.4%, 86.2–99.9)||P = 0.112||95/109 (87.2%, 79.4–92.8)||40/41 (97.6%, 87.1–99.9)||P = 0.070|
|Specificity||511/1017 (50.3%, 47.1–53.4)||203/586 (34.6%, 30.8–38.7)||P = 0.000||6511/7044 (92.4%, 91.8–93.0)||3411/3818 (89.3%, 88.3–90.3)||P = 0.000|
|PPV||89/595 (15.0%, 12.2–18.1)||37/420 (8.8%,6.3–11.9)||P = 0.003||95/628 (15.1%,12.4–18.2)||40/447 (9.0%,6.5-12.0)||P = 0.003|
|NPV||511/524 (97.5%, 95.8–98.7)||203/204 (99.5%, 97.3–99.99)||P = 0.128||6511/6525 (99.8%, 99.6–99.9)||3411/3412 (99.97%, 99.84–100.0)||P = 0.024|
|Sensitivity||52/59 (88.1%, 77.1–95.1)||27/28 (96.4%, 81.7–99.9)||P = 0.428||55/62 (88.7%, 78.1–95.3)||29/30 (96.7%, 82.8–99.9)||P = 0.266|
|Specificity||517/1060 (48.8%, 45.7–51.8)||203/596 (34.1%, 30.3–38.0)||P = 0.000||6518/7091 (91.9%, 91.3–92.5)||3411/3829 (89.1%, 88.1–90.1)||P = 0.000|
|PPV||52/595 (8.7%, 6.6–11.3)||27/420 (6.4%, 4.3–9.2)||P = 0.176||55/628 (8.8%, 6.7–11.3)||29/447 (6.5%, 4.4–9.2)||P = 0.172|
|NPV||517/524 (98.7%, 97.3–99.5)||203/204 (99.5%, 97.3–99.99)||P = 0.454||6518/6525 (99.9%, 99.8–99.96)||3411/3412 (99.97%, 99.84–100.0)||P = 0.278|
Comparison among four screening strategies to identify high-grade CIN
As shown in Table 3 and Figure 4, screening Strategy 1 as co-testing demonstrated an optimal effectiveness, with a sensitivity of 72.7% and specificity of 96.9%, for identifying high-grade CIN, but it consumed maximum tests (220.2) and colposcopies (4.4) to detect one case of high-grade CIN. Compared with Strategy 1, Strategy 2 had the same sensitivity, specificity and number of performed colposcopies to detect one case of high-grade CIN, but it required nearly half of the tests to detect one case of high-grade CIN. Strategy 3 had the highest specificity (98.0%) and PPV (29.2%) with the lowest number of performed colposcopies (3.4) to detect one case of high-grade CIN. But it had the lowest sensitivity (63.6%) and NPV (99.5%) of the four strategies and required more performed tests (134.2) than Strategy 2 to detect one case of high-grade CIN. Strategy 4 using primary HPV testing with type 16/18 genotyping demonstrated the highest sensitivity (78.6%) and NPV (99.7%) of the four strategies, and it had a similar specificity (96.8%), PPV (23.9%) and number of performed colposcopies (4.2) as strategies 1 and 2 to detect one case of high-grade CIN.
|Strategy1||72.7||96.9||22.7||99.6||4.4 (423/96)||220.2 (110.1/110.1)|
|Strategy2||72.7||96.9||22.7||99.6||4.4 (423/96)||112.7 (110.1/2.6)|
|Strategy3||63.6||98.0||29.2||99.5||3.4 (305/89)||134.2 (11.4/122.8)|
|Strategy4||78.6||96.8||23.9||99.7||4.2 (461/110)||106.3 (7.0/99.4)|
|Strategy1||81.2||96.6||16.3||99.8||6.1 (423/69)||306.4 (153.2/153.2)|
|Strategy2||81.2||96.6||16.3||99.8||6.1 (423/69)||156.7 (153.2/3.6)|
|Strategy3||72.4||97.8||20.7||99.8||4.8 (305/63)||189.6 (16.1/173.5)|
|Strategy4||85.1||96.4||16.1||99.9||6.2 (461/74)||158.1 (10.4/147.7)|
This was the first large-scale cross-sectional study on the application of HPV testing with type 16/18 genotyping for cervical cancer screening in Chinese women. Using pathologic diagnosis of colposcopic biopsy as a standard, we assessed the effectiveness of HPV testing, with cytology as a control, to identify high-grade CIN. Considering that cytological diagnosis significantly relies on cytologists, we employed at least two cytologists to blindly read the same slide to ensure the accuracy of cytological diagnosis. Our results revealed that Youden's index (YI) of HPV detecting CIN2+ and CIN3+ was higher than that of cytology after bias correction (0.81 vs. 0.64, 0.82 vs. 0.71) with sensitivities of 90.0% and 91.3%, respectively, which were significantly higher than that of cytology (66.7% and 73.9%), and specificities of 91.3% and 90.9%, respectively, which were slightly lower, yet still acceptable, than cytology (97.2% and 97.0%). Our results are consistent with previous studies on HPV testing in areas other than China [5, 14, 15], suggesting that HPV testing demonstrates effective performance in detecting high-grade CIN and provides more assuring negative results than cytology. Because the results of HPV testing do not depend on cytologists, HPV testing is suitable for cervical cancer screening in China and other developing countries where there is a shortage of cytologists.
The HPV prevalence and its distribution in different ages may affect the aptness of HPV testing in cervical cancer screening. HPV testing is not yet recommended for young women less than 25 years old around the world due to the high HPV prevalence in this age period. In our study, the positivity rate of 14 genotypes of high-risk HPVs was 9.8% in 11,064 women, which was consistent with 10.4% of the worldwide prevalence . After age stratification, the positivity rate in the 21- to 24-year group was as high as 15.4%, which dropped to 8.5% in the 30- to 39-year group, and then gradually climbed to the second peak of 14.4% in the 60- to 65-year group, which equaled that in the 21- to 24-year group and seemed to be higher than that in European and American women of that age in some reports [4, 17]. De SanJose  reported the worldwide HPV prevalence via a meta-analysis and found that the second peak of HPV prevalence in women after 44 years old presented with an area difference. For instance, in European and North American women, the HPV prevalence commonly declines after the age of 25 [4, 15, 18, 19], while in Asian and Latin American women, the second rise in the HPV prevalence may emerge in some areas, such as China [20, 21], Japan [22, 23], Chile , Colombia , and Mexico . Some factors may be related to the second peak in the HPV prevalence, such as increased extramarital sexual behaviors of their husbands or themselves, or decreased immunity due to a female hormone decline during peri- or post-menopause, which could lead to potential activation of HPV with a low replication status [16, 27, 28]. To explore whether HPV testing was suitable for Chinese women aged 50 or above with a high prevalence of HPV, we divided the population into 21- to 24-, 25- to 49-, and 50- to 65-year groups. Since there were too few samples in the 21- to 24-year group because many women were going out for work or study, or were unmarried, we excluded them from the analysis. Our results showed that the sensitivity of HPV testing in the 50- to 65-year group was higher than that in the 25- to 49-year group, but the difference was not significant, while the specificity was significantly lower than that in the 25- to 49-year group. The NPV and PPV of HPV detecting high-grade CIN also revealed similar differences between the 50–65- and 25- to 49-year groups. Our findings suggest that primary HPV testing could have a higher false-positive proportion for detecting high-grade CIN in peri- or post-menopausal Chinese women, which probably results in more unnecessary colposcopies. It may be valuable to further study whether HPV-based screening is suitable in peri- or post-menopausal women who live in the area where the second peak of HPV prevalence emerges.
HPV or cytology testing alone has insufficient sensitivity or specificity, but the combination of two techniques can strengthen the detection advantage and overcome each test's individual weaknesses, elevating the accuracy of screening [29, 30]. With HPV and cytology co-testing screening as a control strategy, we found that the strategy of primary cytology with HPV testing triage, because its standard for referring to colposcopy was the same as co-testing (cytology LSIL or worse, or HPV+/cytology ASC-US), showed equal effectiveness and number of colposcopies while consuming only half of the tests to detect one case of high-grade CIN compared to co-testing. The strategy of primary HPV testing with cytology triage revealed the highest specificity and PPV, resulting in fewer performed cytology tests and colposcopies to detect one case of high-risk CIN, but it showed the lowest sensitivity and NPV in the first round of screening, which may be associated with poor effectiveness of triage by cytology due to its low sensitivity. It has been reported that HPV 16/18 genotyping demonstrates optimal triage performance in American women who are HPV positive [5, 7, 31, 32]. In the study, we also found that primary HPV testing with type 16/18 genotyping showed the highest sensitivity and NPV and promised similar specificity and PPV for detecting high-grade CIN compared with co-testing screening. Importantly, this strategy further reduced the number of necessary cytology tests compared to other HPV testing techniques, requiring only 7.0 or 10.4 tests to detect one case of CIN 2+ or CIN3+, respectively. Therefore, the strategy of primary HPV testing with type 16/18 genotyping may be more feasible than other strategies in China or other developing countries where screening is usually unruled and nonstandard and cytologists are lacking.
Since our study is cross-sectional, our conclusions are preliminary, and there may be bias. For instance, Strategy 4 sends HPV 16/18 cases with NILM cytology directly to colposcopy, whereas these women in Strategy 3 will be sent to colposcopy one year later, and some of them will not need colposcopy in the second round of screening because of transient HPV16/18 infection. Therefore, the sensitivity of Strategy 3 will be considerably increased, while the PPV of Strategy 4 will be considerably decreased by follow-up performed on HPV positive/NILM women. It has been observed that direct referral of HPV 16/18 positive women to colposcopy is possibly an excessive treatment.
Considering our results together, HPV testing has optimal effectiveness for detecting high-grade CIN and is suitable for cervical cancer screening in Chinese women who are older than 25 years of age. The strategy of primary HPV testing with type 16/18 genotyping shows the highest sensitivity and NPV with similar specificity and PPV among different screening strategies, and it requires fewer performed cytology tests and colposcopies to detect one case of high-grade CIN, suggesting that this strategy possesses optimal cost/effectiveness in the first round of screening and is a feasible strategy of cervical cancer screening for Chinese women.
This work was supported by the research project of Ministry of Public Health, China (Grant number 201402010) and National key research and development program, China (Grant number 2016YFC0902900). We thank doctor Yun Liang and other cytologists and pathologists of the Department of Pathology in our hospital for their cytological and histological diagnosis for all participants; Doctor Yifan Cheng, Xiaonan Lu, Qifang Tian, and Xueqian Qian in our hospital for their help in sample collection and colposcopy/biopsy; Professor Kun Chen, a statistician in Public Health School, Zhejiang University, for his help in study design and statistical analysis; and local doctors for their help in the recruitment of eligible women and other works.
Conflict of Interest