Type specific persistence of high risk human papillomavirus (HPV) as indicator of high grade cervical squamous intraepithelial lesions in young women: population based prospective follow up study

Examination at enrolment

At enrolment all 11 088 women were interviewed personally by specially trained female nurses. The nurses collected data on demographic variables, smoking, reproductive background, contraception, sexual habits, previous sexually transmitted diseases, and history of cervical smear tests. The participants also had a gynaecological examination, in which we carried out a smear test and obtained endo-ectocervical cells for detection of HPV DNA. All swabs were placed in a tube with TE-buffer (10 mM Tris-HCl and 1 mM EDTA, pH=8.0). In addition, all participants gave two blood samples. All biological material was kept at −80½C until tested.

Examination at follow up

In October 1993 we invited the entire cohort for a second examination. Initially, the cohort was linked to the central personal register using the CPR number as key identifier. We traced all the women in the cohort using this register and retrieved information on vital status and current address. We invited the women to participate in the second phase in the same order as they were originally enrolled in the study. During the following 18 months (that is, until January 1995) 8656 women (78%) underwent this second examination. Women were interviewed about suspected risk factors for cervical cancer, focusing on the time between enrolment (first examination) and follow up (second examination). We also did a smear test and took cervical swabs for HPV testing (placed in phosphate buffered saline with 0.05% methiolate) and two blood samples from each woman using the same procedure as at the initial examination, all biological material being stored at −80½C.

Passive follow up

We also had the cohort under passive surveillance for occurrence of abnormal cytology. In a high proportion of Danish counties, all cytological and histological diagnoses are registered in a computerised pathology register (the smears taken in the present study were also registered in the pathology register). In November 1995 we linked the original cohort of 11 088 women to the pathology register files, and all women were traced in the register. Although the Danish Board of Health recommends cervical smear testing every three years, many women tend to get screened more often.8 By means of the pathology register we were able to get information about all such examinations on every woman in our study since their first smear test and up to the date of the register linkage.

Study population

We excluded women with a history or current evidence of cervical neoplasia. The figure shows the different exclusions for the entire cohort. We excluded 11 women in whom cervical neoplasia had been diagnosed in the first nine months (the time was chosen to ensure comparability with another study4) to avoid inclusion of potentially prevalent cases in the study (four had atypical squamous cells of undetermined significance, four had low grade lesions, three had high grade lesions). After all exclusions 10 177 women remained in the follow up study.

Schematic overview of overall study design. Final study population comprised 653 women in the control group had at least one normal smear test result at or after the second examination and 25, 115, and 112 women who were diagnosed within three months after the second examination

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Identification of potential cases

A total of 428 potential cases developed in the cohort. Of these, we identified 329 at the second examination. The linkage with the pathology register resulted in 99 more women with an incident diagnosis of lesions on the uterine cervix that qualified them as potential cases in the time period between nine months after the first examination (that is, enrolment in the cohort) and November 1995. The cytological diagnoses covered a spectrum from “non-specific viral changes, not further specified,” “koilocytosis,” and “atypia” to dysplasia (mild, moderate, severe) and carcinoma in situ.

Review and confirmation of case diagnoses

From the files of the pathology register we identified the microscopy number on all abnormal smear results (and biopsies if taken) as well as on the enrolment smear and smears taken and diagnosed as negative during follow up from every potential case. The smear samples and biopsy slides were subsequently located and retrieved from the respective pathology departments. This material was reviewed in a masked fashion by one pathologist (GP) using the Bethesda nomenclature system.9

In cases of discrepancy between the original diagnosis and the review diagnosis, another pathologist (MES), who was unaware of any of the two previous diagnoses, reviewed the material. In most cases there was agreement at the first review, and in the remaining cases there was agreement between two of the three pathologists (GP, MES, PAP).

In cases where both cervical smear result and biopsy contributed to the diagnosis, the more severe diagnosis formed the basis of the final diagnosis. Among 428 potential cases, 58 were downgraded to normal in the review procedure, and 370 (86%) had a confirmed diagnosis of incident atypical squamous cells of undetermined significance or cervical neoplasia. This covered 40 with atypical squamous cells, 165 with low grade squamous intraepithelial lesions, and 165 with high grade squamous intraepithelial lesions (figure). Histological examination results to confirm the diagnosis were available in 136 (83%) high grade cases and 60 (35%) low grade cases. None of the smear results that were originally negative at enrolment were upgraded at the review procedure.

Subcohort selection

We randomly selected a sample of 1000 from the 10 758 women in the entire cohort who had cytologically normal results at enrolment. We retrieved the smear samples taken at enrolment and during follow up from the files of the pathology departments, and they were reviewed by one pathologist (PAP). In cases of discrepancy between the original diagnosis and the reviewed diagnosis, another pathologist blindly reviewed the smear (MES).

We excluded 39 women from the subcohort because of previous cervical neoplasia (n=38) or abnormal cytology detected within nine months of enrolment (n=1). During follow up, 40 women had an abnormal smear test result, and we included them in the group of potential cases. This left 921 women without any history of cervical neoplasia (that is, no history of cervical neoplasia before enrolment and no abnormal cervical cytology during follow up). At the review procedure none of the enrolment or follow up smears was upgraded.

Final study population

We excluded cases diagnosed later than three months after the follow up examination. This time limit was chosen so the HPV status at the follow up visit would still reflect the status at diagnosis. In the analyses including HPV status at follow up, we excluded cases that were diagnosed before the follow up examination and in which cervical biopsies or surgical treatment (cone) had been carried out (two with atypical squamous cells, 13 with low grade lesions, 19 with high grade lesions). For us to define women in the subcohort as “cytologically normal” we considered that they had to have a normal cervical smear result at or after the follow up examination. On the basis of these restriction criteria, we excluded 15 women with atypical squamous cells, 46 with low grade lesions, 51 with high grade lesions, and 265 controls from the analyses. We excluded four other women with low grade lesions, two with high grade lesions, and three controls because their cervical swabs were inadequate for HPV analysis. Thus, the final study population comprised 252 incident cases (25 with atypical squamous cells, 115 with low grade lesions, and 112 with high grade lesions) and 653 cytologically normal women (see figure). Among the cases, 191 (76%) women were identified at the second examination and 61 women with an incident diagnosis of cervical neoplasia were identified from the pathology register linkage.

HPV DNA detection

The cervical samples were analysed by the general primer GP5+/6+ mediated polymerase chain reaction-enzyme immunoassay method.10 Briefly, we added 10 μl of the crude cervical cell suspension to the polymerase chain reaction mixture (10 mM TRIS HCl, pH 8.3; 50 mM KCl; 3.5 mM MgCl2; 1 unit of thermostable DNA polymerase (Amplitaq, Perkin Elmer Cetus, Norwalk, CT); 200 μmol of each dNTP; and 25 pmol of each primer (GP5+ and biotinylated GP6+)). We incubated the mixture for five minutes at 94½C for DNA denaturation, followed by 40 cycles of amplification with a polymerase chain reaction processor (Biomed, Theres, Germany). Each cycle included a denaturation step to 94½C for one minute, an annealing step to 40½C for two minutes, and a chain elongation step to 72½C for 90 seconds. To ensure a complete extension of the amplified DNA we prolonged the final elongation step by four minutes.

We analysed the biotinylated GP5+/6+ polymerase chain reaction products by enzyme immunoassay using HPV high risk (HR) and HPV low risk (LR) oligococktail probes to identify 14 high risk HPV types (HPV 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, 68) and six low risk types (HPV 6, 11, 40, 42, 43, 44). We also typed the high risk and low risk positive swabs individually usin specific enzyme immunoassays. In addition, we analysed GP5+/6+ polymerase chain reaction products for the presence of other HPV types not identified by the high risk and low risk enzyme immunoassays; this was done with gel electrophoresis, followed by Southern blot analysis under low stringent conditions with a cocktail probe of different HPV types.11 We classified samples that were positive by this Southern blot analysis but negative by both high risk and low risk enzyme immunoassay as HPV X positive.

Statistical analysis

We investigated the associations between squamous intraepithelial lesions and HPV DNA detected at the two examinations by multiple logistic regression analyses performed separately for each type of lesion compared with the controls (subcohort). This corresponds to being either cytologically normal or having a specific case type in the full generalised logistic regression model considering all four outcome categories (normal, atypical squamous cells, low grade lesions, and high grade lesions) simultaneously, and makes the estimates directly comparable with case-control studies of any of the single adverse outcomes. We corrected all analyses for age at enrolment as a categorical variable, grouped in yearly intervals. The 95% confidence intervals were based on Wald's test performed on the log transformed odds ratios and back transformed.

We classified HPV types in relation to their association with cervical cancer. HPV types 16, 18, 31, 33, 35, 39, 45, 51, 52,56, 58, 59, 66, and 68 were grouped together in a high risk (“oncogenic”) group, and HPV types 6, 11, 40, 42, 43 and 44 were placed in the low risk (“non-oncogenic”) group. The uncharacterised HPV types (HPV X) were grouped together with the low risk types. Women with multiple types were grouped according to the highest risk group.

We performed the statistical analyses both on the unrestricted study population (that is, 40 with atypical cells, 165 with low grade lesions, 165 with high grade lesions, and 921 without any history of cervical neoplasia), on the final study population (25, 115, 112, and 653 respectively) (figure), and with intermediate restriction criteria for cases and the subcohort (data not shown). The overall pattern of the results was the same irrespective of the restriction criteria; however, the strength of association in the analyses including HPV status at follow up increased with the severity of the inclusion criteria for the subcohort and decreased with the length of time between the second examination and the case diagnosis (data not shown). We have presented only those results concerning the final (restricted) study population.

Finally, we investigated whether the restrictions leading to the final study population were associated with different distributions of some potential confounders registered at enrolment (age, number of sexual partners, age at first intercourse, and use of oral contraceptives). Thus, we compared the women in the final study population with those who were excluded from the unrestricted study population within the control group and within the case groups, and the respective distributions were nearly the same (data not shown).

Prevalence of HPV

In both cytologically normal women and all three case groups the most common type of HPV at enrolment was HPV 16 (table 1). Among women positive for HPV, the prevalence was similar among cytologically normal women (30%), women with atypical cells (25%), and women with low grade lesions (30%), but significantly higher in those with high grade lesions (48%). Uncharacterised HPV types were found in about 8% of women with low grade lesions and 7% of women with high grade lesions, whereas nearly a quarter of the cytologically normal women harboured these unknown types. At enrolment, among women positive for HPV, 12% of cytologically normal women, 36% of women with low grade lesions, and 25% of women with high grade lesions had multiple types of HPV.

The prevalence of HPV detected at enrolment in women who were cytologically normal at enrolment and during follow up was 14% (89/653). Among women in whom high grade lesions were subsequently detected, 80% (89/112) were HPV positive at enrolment. The corresponding figures for low grade lesions and atypical cells were 63 (55%) and 8 (32%). At the follow up examination, 11 (50%) women with atypical cells, 92 (89%) women with low grade lesions, and 84 (93%) women with high grade lesions were positive for HPV.

Clearance of HPV

Among cytologically normal women who were positive for HPV at the first examination, 62/87 (71%) cleared that specific HPV infection: 45 became HPV negative and 17 acquired another HPV type. In contrast with this, 21/53 (40%) women who were positive for HPV and had low grade lesions lost the HPV (two became HPV negative and 19 got a new type), and only 17/72 (26%) women who were positive for HPV and had high grade lesions cleared the specific infection (four (6%) became HPV negative and 13 (20%) acquired a new HPV type), such that in 72% of HPV positive women with high grade lesions, the same type(s) was found at both examinations (2% still had uncharacterised HPV types at both examinations).

HPV and cervical lesions

Table 2 shows the odds ratios for incident atypical cells and squamous intraepithelial lesions according to HPV status at enrolment. Compared with women who were HPV negative at enrolment, HPV positive women had a significantly increased risk of developing atypical cells (odds ratio 3.2, 95% confidence interval 1.3 to 7.9), low grade lesions (7.5, 4.8 to 11.7), and high grade lesions (25.8, 15.3 to 43.6). When we divided the women who were HPV positive into a group of low risk or unknown HPV types and a group of high risk or oncogenic types, it was evident that the oncogenic HPV types were associated with the highest risk, especially for high grade lesions, when compared with HPV negative (7.8, 3.1 to 9.4 for low risk or unknown types v HPV negative) and (34.5, 19.7 to 60.2 for high risk or unknown types v HPV negative).

A similar picture emerged regarding the risk of cervical neoplasia in relation to HPV status at follow up (table 2), with HPV positive women having a strongly increased risk, especially of low grade lesions (34.3, 17.6 to 67.0) and high grade lesions (60.7, 25.5 to 144.0). Also here the group of high risk HPV types was responsible for the highest increase in risk.

We also examined the risk of incident cervical neoplasia taking into account the HPV status both at enrolment and at follow up (table 2). We found a similar overall pattern of risk for all three disease categories. However, the most substantial effect was seen for high grade lesions, where the odds ratio was 413.9(96.3 to 1779.5) when we compared women who were HPV positive at both examinations with women who were negative at both examinations.

When we compared women who were positive at both examinations with those who were positive only at enrolment, the odds ratio for low and high grade lesions increased (low grade 31.6, 7.1 to 140.5; high grade 20.4, 6.6 to 62.9) (data not shown). When we compared the same women with women who had HPV detected only at follow up we observed significantly increased odds ratios for both low grade (2.7, 1.5 to 5.0) and high grade lesions (10.6, 5.1 to 21.8) (data not shown).

Table 3 shows the results for women who stayed HPV negative compared with women who stayed HPV positive. For both low and high grade lesions the highest risk was associated with having a high risk HPV type detected at both visits, though the odds ratio for high grade lesions was the highest (low grade 117.7,45.2 to 306.8; high grade 691.6, 145.3 to 3292.7).

In addition, we found that for high grade lesions the risk was strongly increased if at least one identical HPV type was present at both examinations (813.0, 168.2 to 3229.2 for being positive for identical types at both examinations v being negative for HPV at both examinations, table 3). Even when we carried out an internal comparison of women with identical types at the two visits versus different types at the two visits, we observed a significantly increased risk of high grade lesions (4.2, 1.5 to 12.3) (data not shown). In contrast, we found no significant difference between having identical HPV types or different HPV types at the two examinations in relation to the risk of low grade lesions (1.6, 0.6 to 4.2). No women with atypical cells had identical types at the two visits.

Finally, we estimated the odds ratio for the association between being repeatedly postitive for HPV and high grade lesions in relation to age. The risk of high grade lesions in women positive for identical types at the two visits compared with women negative at both examinations tended to be stronger in women aged 25-29 years (810, 97 to 6754) than in women aged 20-24 years (567, 63 to 5688) (data not shown).

Conclusion

In conclusion, we can confirm previous reports that stated that HPV infection is common in young women and that most infections are transient with high rates of acquisition and clearance. More importantly, we have shown that HPV infection precedes the development of low and high grade squamous intraepithelial lesions and that high risk HPV infection is a good predictor of subsequent high grade lesions in young women. Our data also indicate that HPV is an even better predictor in older women with a lower background HPV prevalence. The outstanding predictor of high grade lesions, however, was being repeatedly positive for HPV with the same HPV type, in line with the previously suggested hypothesis that persistence of high risk HPV types is strongly associated with the development of high grade lesions.