1 Department of Pathology, Vrije Universiteit Medical Centre, Amsterdam, the Netherlands.
2 Division de Investigacion, Instituto Nacional de Cancerologia, Bogota, Colombia.
3 International Agency for Research on Cancer, Lyon, France.
Received for publication November 22, 2002; accepted for publication February 26, 2003.
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ABSTRACT |
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cervix neoplasms; cohort studies; cytology; oral contraceptives; papillomavirus
Abbreviations: Abbreviations: CIN III, cervical intraepithelial neoplasia, grade III; EIA, enzyme immunoassay; HPV, human papillomavirus; PCR, polymerase chain reaction.
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INTRODUCTION |
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HPV infection is mostly a transient phenomenon resulting in no cervical lesions or leading to low-grade lesions that often regress spontaneously (8, 9). HPV presence is thus a necessary, but not sufficient cause of cervical neoplasia development (1). Persistence of an HPV infection appears to be a prerequisite for the development of cervical intraepithelial neoplasia, grade III (CIN III), and cervical cancer (1013). Viral, host, and environmental factors may influence the course of HPV infection (2, 9, 14).
In 1993, the National Cancer Institute, Colombia, in collaboration with the International Agency for Research on Cancer, started a population-based cohort study on the natural history of HPV infections and cervical neoplasia in a group of low-income women from Bogota, Colombia. The country has one of the highest incidences of cervical cancer in the world (age-standardized rate, 34.4/100,000) (15), and the present study cohort showed a high prevalence of HPV at enrollment (15 percent among cytologically normal women, 26 percent among women aged <20 years) (3).
We present here results from a 5-year follow-up analysis of HPV-positive women who had normal cervical cytology at baseline. Analysis focuses on the possible role of HPV types, viral load, and various womens characteristics on the clearance rate of HPV infection.
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MATERIALS AND METHODS |
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A total of 1,995 (91 percent) women who had a valid questionnaire, a valid HPV test, and adequate cervical cytology were thus included in the cohort study (3). Informed consent was obtained from all study participants. The ethical committees at the National Cancer Institute, Colombia, and the International Agency for Research on Cancer approved the study protocol and the manner in which informed consent was obtained from subjects.
Follow-up consisted of a visit every 69 months. At each visit, a short follow-up questionnaire was administered, and a cervical scrape was obtained for cytologic evaluation and HPV testing. Follow-up ended in December 2000 or at the diagnosis of CIN III, whichever occurred first. Women who had a CIN III diagnosis underwent confirmatory biopsies and treatment. Womens HPV status was not known during the follow-up and did not influence clinical management.
The analysis described here was carried out on a subset of the study cohort. We selected 330 women who were positive for one or more HPV types at baseline. We then restricted the analysis to women whose cytology was normal (i.e., we excluded three women with inadequate cytology and 51 women with abnormal cytology; the majority (39/51) evidenced low-grade lesions). Finally, we excluded 49 women who did not have at least one follow-up visit, thus yielding a total of 227 women. Since each woman could have one or more infections with different types of HPV, we based the analyses on type-specific HPV infections rather than on individual women. Clearance of a given HPV type was defined as disappearance of the HPV type detected at enrollment. At any visit, if the HPV test was inadequate, clearance status was considered unknown and follow-up was continued.
HPV detection by polymerase chain reaction
Testing for HPV was conducted by using a standard GP5+/GP6+ polymerase chain reaction (PCR) enzyme immunoassay (EIA) (3). Briefly, HPV-positive samples were subjected to EIA-HPV group-specific analysis by using cocktail probes for high-risk and low-risk HPVs (16). The high-risk HPV cocktail probe consisted of oligoprobes for HPV 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, and 68; the low-risk HPV probe consisted of oligoprobes for HPV 6, 11, 26, 34, 40, 42, 43, 44, 53, 54, 55, 57, 61, 70, 71 (CP8061), 72, 73, 81 (CP8304), 82 (MM4), 83 (MM7), 84 (MM8), IS39, and CP6108. Additionally, HPV positivity was assessed by Southern blot hybridization of GP5+/GP6+ PCR products with the general probe of specific DNA fragments from cloned DNA of HPV 6, 11, 16, 18, 31, and 33 (17). Samples positive by Southern blot analyses and negative by high-risk/low-risk EIA were considered HPV X or undetermined type and were classified as low risk. The low-risk cocktail probe contained some HPV typesnamely, 26, 34, 53, 73, and IS39of unknown oncogenic potential. These types were classified as "high-risk" for analysis purposes.
During follow-up, a new GP5+/GP6+ PCR reverse line blot analysis was developed and was used to type the same 37 different HPV types detected by EIA. The findings from the GP5+/GP6+ PCR reverse line blot analysis were compared with those from the PCR-EIA assay and were found to agree in 96 percent of cases (kappa = 0.77) (18).
Viral load analysis
PCR-EIA can be used as a semiquantitative method to assess the relative amount of HPV DNA in cervical scrapes because of the linear relation between the amount of DNA and the optical density in the range of 10106 genome equivalents. A semiquantitative, noncompetitive GP5+/GP6+ PCR-EIA was carried out according to the method described by Jacobs et al. (19). Viral load of the samples was analyzed in two ways: 1) by using an a priori classification of low (optical density, <0.5), medium (optical density, 0.5<1.5), and high (optical density,
1.5); and 2) by classifying the optical density into quintile groups to obtain a clearer picture of the dose-response relation.
Statistical methods
The time of clearance of an HPV infection was modeled by using methods for interval-censored survival time data. Doing so is necessary since clearance times are never observed precisely but are known only to have occurred between two visits. The survival function, which describes the probability that an HPV infection has cleared as a function of time, was estimated by using the nonparametric maximum likelihood estimator (20, 21), which is the natural generalization of the Kaplan-Meier estimator to interval-censored data. Rate ratio estimates were derived by applying Cox regression to imputed clearance times, as described by Pan (22). The analyses were implemented in R language (23) by using the survival package. Custom software was written to calculate the survival function and carry out the multiple imputation.
The following factors potentially associated with HPV clearance were considered: HPV type in which a classification (2) based on phylogenetic groups was used (low-risk types, HPV 16, types linked to HPV 16 (31, 33, 35, 52, 58), HPV 18 and types linked to HPV 18 (18, 39, 45, 59, 68), other high-risk types), multiplicity of infection, viral load (in quintile groups), age (<18, 1824, 2534, 35 years or older), educational level (none or primary, secondary, higher), number of regular sexual partners (1,
2), parity (0, 12,
3 children), use of oral contraceptives (ever, never), intrauterine device use (ever, never), and smoking (ever, never). For each factor, the value at baseline was used. The basic unit of analysis when modeling clearance times was an HPV infection. To account for possible correlations in the clearance of times of multiple infections in the same woman, a cluster term was included in the model, and standard errors were calculated that are robust to correlations within clusters (24).
HPV infection may clear as a consequence of treatment of cervical lesions. Follow-up time was therefore censored at the date of any biopsy or treatment and, in any case, when a cytologic diagnosis of CIN III or worse was observed. This choice led to the censoring of five women. Three were infected with HPV 16 only; two developed CIN III (at months 17 and 54 after enrollment), and one developed invasive cervical carcinoma (at month 58). The other two women had single infections with HPV 52 and HPV 58 and developed one CIN III at month 5 and one CIN III at month 10 after enrollment, respectively.
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RESULTS |
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Table 1 shows the characteristics of the study population subdivided into three age groups. Median age of the participants was 29 years. Twenty-eight percent of the women reported a primary education only, 76 percent had one lifelong sexual partner, and 20 percent had not given birth to any children. Ever use of oral contraceptives was reported by 54 percent and ever use of an intrauterine device by 57 percent of the women. Twenty-nine percent of the women reported ever smoking.
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HPV-DNA clearance
Figure 1 shows the proportion of persistent HPV infection as a function of time, overall and according to HPV phylogenetic groups. The clearance rate was not constant, but it was highest in the first 6 months of follow-up. Globally, 23 percent of HPV infections were still present at 1 year and 7 percent at 5 years. Clearance rates were lower for HPV 16 than for low-risk HPV types.
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DISCUSSION |
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In some previous studies (25), two or more consecutive HPV-negative tests were required before HPV infection was considered cleared. We considered an infection cleared when it was absent during a single visit; in our study, the time interval between visits was relatively long (69 months) and thus probably sufficient to clear the infection. Indeed, only five of 223 HPV infections that had cleared and could be reevaluated during a subsequent visit were positive again for infection with the same HPV type. Defining HPV clearance as two consecutive HPV-negative tests, rather than one only, therefore would not have materially altered our results.
In our analysis, the clearance rate of HPV 16 was lower than that of low-risk HPV types. High-risk HPV types phylogenetically linked with HPV 16 showed clearance rates intermediate between those of HPV 16 and low-risk types, whereas HPV types linked with HPV 18 and other high-risk types had clearance rates similar to those of low-risk types. Franco et al. (9), in a follow-up study from Brazil (Ludwig-McGill cohort), showed that 12-month clearance was higher for low-risk HPV types (12.2 percent, 95 percent confidence interval: 9.6, 15.4) than for high-risk HPV types (9.5 percent, 95 percent confidence interval: 7.5, 11.9), but no clear difference was found between infections with HPV 16 and those with high-risk types other than HPV 16 (8.9 percent, 95 percent confidence interval: 5.8, 13.1) (9). A tendency for HPV 16, but not HPV 18, to persist longer than other HPV types has been noted in previous follow-up studies (7, 8, 26, 27), but the reasons for this phenomenon remain unclear. HPV 16 is by far the predominant type in invasive cervical cancer (28), although HPV 18 is suspected to induce a more rapid transition to malignancy than HPV 16 does (29).
Studies using DNA sequencing have shown a considerable intratypic diversity for HPV 16 compared with other high-risk types, which may be critical to understanding the greater ability of HPV 16 to escape immunologic surveillance (30). Several studies have suggested that non-European variants of HPV 16 could be associated with longer persistence than European variants (31, 32) and more frequent progression into clinically relevant cervical lesions (3335). Unfortunately, we did not have information on HPV 16 variants.
Numerous authors have suggested an association between viral load and persistence of HPV infection (11, 3638). We used a semiquantitative PCR-EIA method to distinguish between high- and low-viral-load infections but found no evidence of a trend in persistence or a threshold effect. Other studies showed associations between high viral load and persistent cytologic abnormalities (39, 40). van Duin et al. (38) observed that high-viral-load infections in women with normal cytology conferred an increased risk of developing a CIN, most notably a high-grade CIN. Conversely, Lorincz et al. (41) reported that high viral load for 13 high-risk types of HPV, evaluated by using the Hybrid Capture 2 test, did not predict risk of CIN III.
It is worth noting that most previous studies (39) focused on HPV 16 only or measured overall viral load, whereas we had information on HPV type-specific viral load. Unfortunately, it is as yet unclear whether high viral load is the result of a few cells with a large number of virions or a large number of cells with a few virions.
Another interesting finding of our study was the similar clearance rate of single and multiple HPV infections. Some studies have shown an association between persistent HPV infection and the presence of multiple types (8). These studies have been interpreted as if women who have multiple types (e.g., women infected with human immunodeficiency virus) (42) had certain characteristics, such as a deficient immune response to HPV, that could predispose to persistent infection. Other studies (27), like ours, have shown that clearance of a type-specific HPV infection seems to be independent of the presence of a coinfection with other types, at least in immunocompetent women.
Several cross-sectional studies have shown that a womans age is the strongest determinant of HPV prevalence, with a peak in HPV infections generally observed in women younger than age 25 years (35, 43). Some studies have shown that HPV infections in older women may be more persistent than those in younger women (8, 44). Possible explanations for this difference are selection of persistent infections, a decrease in the immune response (14), hormonal changes, and specific lifestyle characteristics of older women (or of their sexual partners). However, we did not confirm any unfavorable effect of age on clearance, at least among women with a normal cytologic smear. We did find a steady increase in the proportion of HPV 16 infections, but not of high-risk types other than HPV 16, across the three age groups considered.
Womens characteristics associated with persistence and/or clearance of HPV infections are not totally consistent across studies (8, 9, 11, 44) and do not always coincide with characteristics found to increase cervical cancer risk. Use of oral contraceptives, for instance, is a risk factor for CIN III and cervical cancer (45). In our study, however, HPV infections were less persistent in women who had ever used oral contraceptives than in never users. A consistent association is lacking between oral contraceptive use and the prevalence of HPV infection in cross-sectional studies (6, 46) and among controls in case-control studies (45). In prospective studies, Moscicki et al. (25) found no association between oral contraceptive use and clearance of HPV infection, and Castle et al. (47) found no association between oral contraceptive use by HPV-positive women and subsequent incidence of CIN III or cervical cancer. High parity is associated with an increased risk of CIN III and cervical cancer (48). Our findings suggest that HPV infection in parous women may persist longer than among nulliparae.
Our follow-up study has several strengths, including the large number of women involved, the low proportion of those refusing to participate, and the presence of information on a broad range of viral and lifestyle factors. HPV testing and typing were conducted in a central laboratory by means of well-validated, sensitive methods. Some misclassification in our semiquantitative evaluation of viral load is possible, which would attenuate any underlying dose-response relation between viral load and HPV clearance. In the present study, follow-up visits were less frequent than those in some other investigations. Although this difference may have hampered to some extent our ability to capture rapid variations in HPV status, it enabled us to reduce the number of unnecessary treatments resulting from the cytologic manifestations of transient HPV infections. Finally, the information on HPV status became available only at the end of the present follow-up, ruling out any influence of this knowledge on the clinical management of study participants.
The decision to analyze prevalent rather than incident HPV infections has both advantages and disadvantages. An advantage is that we were able to include infections that had persisted for many years prior to the start of the study, especially in older women. A disadvantage is that we did not have the complete natural history of the infections studied. For this reason, the clearance curve in figure 1 should not be interpreted as the clearance of incident infections. Note that the clearance rate was high in the first 6 months of follow-up and diminished thereafter. This finding may be taken as evidence of some heterogeneity in the potential for persistence among prevalent infections. Our estimate of the median clearance time (6 months) is lower than in previously reported studies. This estimate correctly accounts for the uncertainty in the clearance time, which is not known exactly, but is known to only have occurred between two visits. An analysis that equates the clearance date with the visit at which the subject was HPV negative is incorrect and leads to an overestimation of persistence. In our study, such an analysis yields an (incorrect) estimate of median clearance time of 19.5 months.
In conclusion, we found that less than half of prevalent HPV infections persist after 6 months and only 7 percent after 5 years. The strongest risk factor for persistence of infection was the presence of HPV 16.
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ACKNOWLEDGMENTS |
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Dr. Molano obtained a fellowship from Colciencias of the Colombian government.
The authors are indebted to all of the gynecologists, nurses, and social workers who collaborated on the fieldwork. They also thank Dr. L. Rozendaal for comments and Drs. R. Pol, N. Fransen-Daalmeijer, R. Van Andel, and H. Schrijnemakers for technical support.
HPV Study Group: Drs. Mauricio Gonzalez, Joaquin Luna, Gilberto Martinez, Edmundo Mora, Gonzalo Perez, Jose Maria Fuentes, Constanza Gomez, Eva Klaus, Constanza Camargo, Cecilia Tobon, Teodolinda Palacio, Carolina Suarez, and Claudia Molina.
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NOTES |
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REFERENCES |
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