1 Julius Center for General Practice and Patient Oriented Research, University Medical Center Utrecht, Utrecht, the Netherlands.
2 Delft Diagnostic Laboratory, Delft, the Netherlands.
3 Department of Pathology, University Medical Center Utrecht, Utrecht, the Netherland.
Received for publication September 25, 2001; accepted for publication February 24, 2002.
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ABSTRACT |
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cervix neoplasms; papillomavirus, human; vaginal smears
Abbreviations: Abbreviations: CI, confidence interval; CIN, cervical intraepithelial neoplasia; HPV, human papillomavirus; PCR, polymerase chain reaction; Pap, Papanicolaou.
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INTRODUCTION |
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In the Netherlands (year 2000 population: 16 million), nearly 1 million preventive cervical Papanicolaou (Pap) smears are obtained annually, while the absolute number of cervical cancers is less than 1,000. Approximately 0.4 percent of the women who undergo a preventive smear are diagnosed as having high-grade squamous intraepithelial lesions and are referred to a gynecologist for a biopsy; for approximately 4 percent of the women, the smears are classified as low-grade squamous intraepithelial lesions, and the Pap smear has to be repeated within 6 months (observations from our own laboratory).
It has been hypothesized that molecular diagnosis of HPV infection could have considerable implications for population-based screening. Recently, Wallin et al. reported a prospective, nested case-control study showing that women whose cytology was normal at baseline but who were positive for HPV DNA had a 16.4 (95 percent confidence interval (CI): 4.4, 75.1) times higher risk of developing invasive cancer compared with noninfected women (6) during a mean follow-up period of 5.6 years. To estimate the risk of cervical neoplasia for HPV-positive and -negative women, we used data from the Dutch mass screening program to perform a nested case-control study. The percentage of women with HPV DNA-positive smears and a normal Pap smear prior to the occurrence of in situ and invasive cancer was compared with the percentage of women with normal Pap smears who did not develop cervical neoplasia. We calculated the risk for HPV-positive and -negative women of developing cervical cancer and the role of different HPV types in this risk. If available, follow-up Pap smears were also analyzed to assess the persistence of HPV infection.
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MATERIALS AND METHODS |
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Study subjects
All women were participants in the national screening program. Data from the Utrecht region were used in this study. The entire screening program comprised 9 years (19761984) and was subdivided into three periods of 3 years each. Every 3 years, all women aged 3554 years were invited to have a Pap smear, which was obtained by experienced laboratory assistants in a mobile unit in the municipalities in which the women were living. All smears were processed at the Cyt-U-University Laboratory of Utrecht University and were screened by experienced cytotechnologists. Cytologic findings were recorded according to the Pap classification. If a cytologic diagnosis was consistent with severe dysplasia, carcinoma in situ, or invasive cancer, the general practitioner was asked to refer the woman to a gynecologist. If a cytologic diagnosis was consistent with mild or moderate dysplasia, the general practitioner was requested to obtain a repeat smear after 6 or 3 months, respectively. All women with a diagnosis of "within normal limits" or "reactive change" were considered cytologically normal. Women whose former smears had been positive were not included in the analysis. The national pathology search system (Pathologisch Anatomisch Landelijk Geautomatiseerd Archief (PALGA)) was used to evaluate and take into account all subsequent cytologic and histologic diagnoses.
Women were eligible as cases if 1) they had received a histopathologic diagnosis of CIN 3 or microinvasive or invasive squamous cervical cancer in 19801986; 2) they had participated at least once in the cervical cancer screening program in the Netherlands during 19761984, and the smears had been obtained at least 2 years prior to the diagnosis of CIN 3 or microinvasive or invasive squamous cervical cancer; and 3) the Pap smear was classified origi-nally, as well as after reclassification by a pathologist (H. D.), as normal. Subtle koilocytic change was considered reactive change if no sign of an intraepithelial lesion was present. Reclassification of the Pap smears was independent of the results of HPV-DNA detection.
All smears were stored in the central cytology archives of Utrecht University. A total of 92 cases fulfilled the inclusion criteria, and we could retrieve Pap smears from 72. Sixty-two (80.5 percent) of the cases were diagnosed as having CIN 3, three (3.9 percent) cases as having microinvasive cancer, and 12 (15.6 percent) cases as having invasive cancer. If available, second and third smears were retrieved and tested as well.
To select the controls, we considered all women 1) who participated at least once in the cervical cancer screening program during 19761984; 2) whose Pap smears were classified originally, as well as after reclassification by the study pathologist, as normal; 3) who had no histopathologic diagnosis of CIN 3 or microinvasive or invasive squamous cervical cancer in 19801986; and 4) whose follow-up period was similar to that of the case. For each case, three controls were randomly selected by computer. Controls were required to fulfill these four criteria and were allowed to differ in age by a maximum of 5 years. From 270 of the 276 controls selected, smears could be retrieved.
HPV testing
DNA isolation
DNA was isolated from Pap-stained Pap smears. Glass slides were soaked in xylene for 35 days, and the coverslips were then removed. Cells were scraped from the slides by using a clean scalpel for each slide; the cells were then transferred to a guanidinium isothiocyanate-containing lysis buffer (L6). DNA was extracted by using the method described by Boom et al., with the following modifications (7). Before the silica suspension was added, the cellular material was incubated in lysis buffer L6 at 37°C for 10 minutes. Washing with buffer L2 and 70 percent ethanol was performed only once. DNA was eluted in 100 µl 10 mM Tris hydrochloric acid (pH 8.2) and was diluted 1:10 in 10 mM Tris (hydroxymethyl)-amino-methane (pH 8.2) before polymerase chain reaction (PCR) analysis.
PCR
HPV DNA was amplified by using the broad-spectrum, short-fragment PCR 10 primer set, which amplifies a fragment of 65 base pairs in the L1 region of the HPV genome (3). This general HPV primer set amplifies DNA from a broad range of anogenital HPV genotypes. To identify HPV DNA-positive cases, the short-fragment PCR 10 amplified fragments were first analyzed by hybridization to a mixture of general HPV-specific probes in a microtiterplate assay. HPV DNA-positive PCR products were further characterized by using a reverse hybridization line probe assay to identify the HPV genotypes, as described in detail elsewhere (8). Briefly, short-fragment PCR 10 products were denatured and incubated with a reverse hybridization strip by using stringent hybridization conditions. After a stringent wash, hybrids were detected by incubation with conjugate and substrate, generating a purple precipitate at the probe line. All incubations and washing steps were performed automatically by using an Auto-LiPA (Innogenetics NV, Ghent, Belgium). Results were interpreted visually. The HPV line probe assay identifies 25 genotypes (HPV 6, 11, 16, 18, 31, 33, 34, 35, 39, 40, 42, 43, 44, 45, 51, 52, 53, 54, 56, 58, 59, 66, 68/73, 70, and 74). HPV 6, 11, 34, 40, 42, 43, 44, 53, 54, 70, and 74 were considered low-risk types, whereas HPV 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, and 68 were considered high-risk types. This HPV detection and genotyping system has been evaluated extensively (9).
In each experiment, separate positive (limited quantity of cells containing HPV 18) and negative PCR controls were processed. The quality of the isolated DNA was checked by PCR amplification of a 110-base-pair fragment of the ß-globin gene (10). If the ß-globin PCR was negative, HPV PCR was not performed and the sample was excluded for analysis. HPV detection was performed blinded to case-control status.
Data analysis
We calculated odds ratios with corresponding 95 percent confidence intervals to estimate the risk of developing CIN 3 or microinvasive or invasive cervical cancer for women with a cytologically normal smear at baseline who were HPV infected, as compared with noninfected women. All odds ratios were determined by using logistic regression analysis and were adjusted for age at first smear. HPV detection was treated as a binary variable (positive vs. negative) to calculate the risk estimates. Specific HPV types were grouped according to their association with invasive cervical cancer, as described in the following paragraph (11).
The most prevalent genotype (HPV 16) was maintained as a separate category. For grouped analyses, HPV types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, and 68 were categorized as a single "high-risk" group, as defined earlier (12). The remaining HPV genotypes (6, 11, 34, 40, 42, 43, 44, 53, 54, 66, 70, and 74) diagnosed in this study were categorized as "low-risk" types. Subjects infected with multiple types of HPV were classified as "type 16" if only HPV 16 was present, as "high risk" if any high-risk HPV genotype (including HPV 16) was present, and as "low risk" if only low-risk genotypes were found. A separate analysis was conducted for women with HPV types 18, 31, and 33.
If two or more smears from the cases were available, they were classified as follows: 1) all smears HPV negative, 2) all HPV DNA positive and HPV genotypes identical, 3) all smears HPV DNA positive but HPV genotypes different, 4) all smears HPV DNA positive and HPV genotypes compatible (at least one type persistent), 5) baseline smear HPV negative but subsequent smear(s) HPV positive, and 6) baseline smear HPV positive but subsequent smear(s) HPV negative.
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RESULTS |
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Analysis of multiple smears and persistence of HPV
The HPV status of the cases for whom a second or a second and third smear was available is presented in table 3. Follow-up smears were available from 49 cases, with an average sampling interval of 3 years. Five women were HPV negative according to both the baseline and second smears, and 37 cases were HPV positive on both smears. In six women, HPV was not detected in the baseline smear, whereas the second smear was HPV positive, and, in three of these women, HPV 16 was found. For one case, the baseline smear was HPV positive but the second smear negative.
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For 11 of the 22 cases whose baseline smears were found to be negative for HPV, a second smear was available. HPV was not detected in five of these smears, three contained HPV 16, and three contained other high-risk HPV genotypes. For the five cases whose baseline and second smears were HPV negative, a third smear was available for three women, but HPV also was not detected in those smears.
Of the 38 cases whose baseline smears were HPV positive, 37 (97.4 percent) cases were still HPV-DNA positive after an average follow-up period of 3 years, and only one case became HPV negative. Among the 37 persistently HPV-positive cases, the HPV genotypes of the two smears were identical in 24 (64.9 percent), and 17 (45.9 percent) cases showed evidence of HPV 16. Genotypes were compatible in five (13.5 percent) and different in eight (21.6 percent) cases. Of the 17 cases for whom three smears were available, 10 (58.8 percent) had at least two consecutive smears that were positive for HPV 16.
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DISCUSSION |
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In this study, women with cytologically normal smears at baseline who developed CIN 3 or microinvasive or invasive cancer during an average follow-up period of 5.6 years were compared with women whose smears were normal and who did not develop disease. HPV DNA was detected in 71 percent of the cytologically normal smears at baseline (212 years earlier) from women who developed CIN 3 or microinvasive or invasive cancer, whereas only 12 percent of the smears from controls were HPV positive. This finding resulted in an odds ratio of 19.2 (95 percent CI: 10.3, 35.7) for development of cervical neoplasia in women whose Pap smears were HPV positive but whose cytology was normal compared with noninfected women whose cytology was normal.
Besides the presence of HPV in general, specific HPV genotypes also play an important role. In this study, a very high odds ratio of 104.8 (95 percent CI: 29.5, 372.7) was observed for HPV 16 infection, whereas infection with the high-risk HPV genotypes 18, 31, or 33 resulted in an odds ratio of 50.6 (95 percent CI: 13.5, 190.0). Cases, more often than controls, had multiple HPV types in their smears (19 percent and 1 percent, respectively; odds ratio = 50.6 (95 percent CI: 13.5, 190.0)).
Increasing and overwhelming evidence of a causal link between certain types of HPV and the development of cervical cancer has led to discussion of the role of HPV testing in cervical screening. Recently, Wallin et al. reported a similar nested case-control study in Sweden; 30 percent of the women with cancer were HPV positive at baseline, whereas only 3 percent of the controls had HPV DNA (6). The mean interval between the baseline Pap smear and histologic diagnosis of in situ cancer or invasive cancer was similar, as reported here (5.3 years and 5.6 years, respectively). However, in our study, the HPV-DNA detection rate was higher for cases (72 percent vs. 30 percent) as well as controls (12 percent vs. 3 percent). This finding may be related to differences between the patient populations in Sweden and the Netherlands. It is more likely that this phenomenon is due to the different methods used to detect HPV DNA. The short-fragment PCR 10 amplifies a fragment of only 65 base pairs, whereas the My09/11 primers (as used by Wallin et al.) generate a fragment of 450 base pairs. Long-term storage of Pap smears at room temperature may lead to gradual degradation of target DNA; consequently, amplification of a small target by the short-fragment PCR 10 primers may be more sensitive compared with the bigger My09/11 target. As shown earlier, the short-fragment PCR 10 primers also permit adequate HPV-DNA detection in formalin-fixed, paraffin-embedded tissues (3).
Our HPV detection and typing system has been extensively evaluated and compared with other systems. Two important conclusions from our previous work (8, 9, 15, 16) are that 1) the short-fragment PCR 10 is more sensitive than other primer sets because of the very small amplified fragment of only 65 base pairs, and 2) the reliability of the genotyping method is very high compared with sequence analysis and alternative hybridization systems.
Any general PCR primer set for broad-spectrum HPV detection will have some degree of differential activity. However, so far, the short-fragment PCR 10 line probe assay system has shown a very high degree of concordance with alternative typing systems (3, 8, 9, 15, 16). Despite the different HPV-DNA detection rates, the odds ratio for the presence of HPV reported by Wallin et al. (6) is comparable to our results.
In the present study, as well as in recent reports, the odds ratios were calculated on the basis of a point prevalence of HPV at baseline (6, 13). However, the present study showed that HPV infection is a dynamic process. Of the 49 cases for whom follow-up smears were available for analysis, 11 were found to be HPV negative at baseline, and five remained negative. However, in six (54 percent) of the 11 cases, HPV DNA was detected in the second smear. This finding may be due to sampling errors or a low concentration of HPV DNA, resulting in a false-negative result at baseline. Alternatively, HPV positivity in the second smear may indicate a recent HPV infection that progressed rapidly to CIN 3 or invasive carcinoma. Biopsy specimens from the cases in this study were not available for analysis, but it has been clearly demonstrated that cervical biopsies from patients with CIN 3 or invasive carcinoma are virtually all positive for HPV DNA, especially when a highly sensitive assay is used (1, 3). Besides detecting HPV DNA and identifying high-risk genotypes, quantitative analyses may also play a role in predicting cancer risk. Josefsson et al. performed a case-control study of HPV 16 (by using type-specific PCR with an amplified fragment size of 180 base pairs) and reported that, at baseline, 42 percent of the cases were positive for HPV 16 compared with 7 percent of the controls (13). This HPV 16 detection rate was similar to the results from the present study (38 percent in cases and 1 percent in controls). Moreover, Josefsson et al. found that the odds ratio for development of cervical neoplasia increased proportionally with the concentration of HPV 16 DNA in the Pap smear, suggesting that the viral load of high-risk HPV genotypes is associated with cancer risk.
Wallin et al. reported that, in all HPV-positive cases, the HPV genotypes in the baseline smear and the biopsy specimen were similar, whereas none of the controls had the same HPV type in both smears (6). Unfortunately, the diagnostic biopsy specimens from the cases in our study were not available for analysis. However, for a considerable number of cases, multiple Pap smears were available, and the majority (78.4 percent) showed persisting HPV (identical or compatible) genotypes. Importantly, HPV 16 appeared to be highly persistent in the cases. These findings are consistent with the recent study of Nobbenhuis et al., which showed the importance of persisting HPV infections, especially of the high-risk genotypes (17). The finding that cases, more often than controls, had multiple HPV types in their smears was also reported by Morrison et al. (18). PCR detection of simultaneous infection with more than one HPV type was associated with a very high risk of squamous intraepithelial lesions (odds ratio for one type = 7.2, 95 percent CI: 2.4, 21.9; odds ratio for more than one type = 43.0, 95 percent CI: 6.9, 266.6).
We were not able to retrieve all smears that were selected, especially for the cases; 15 (16.3 percent) of the 92 smears could not be retrieved. Most probably, those smears were more often revised by the pathologist and gynecologist after the women seemed to have CIN 3 or invasive cervical cancer. Because HPV was not an issue at the baseline evaluation, it is very unlikely that smears were lost because of HPV status and may have caused a selection bias.
In conclusion, the present study showed that the presence of HPV in cytologically normal Pap smears is associated with a significantly increased risk of developing invasive cervical cancer. In particular, in our study, infection with high-risk or multiple HPV genotypes resulted in a strongly increased risk of developing cervical neoplasia. If additional markers are used, such as identifying viral genotypes and measuring viral load, the positive-predictive value of HPV DNA as a primary screening test may increase.
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ACKNOWLEDGMENTS |
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NOTES |
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REFERENCES |
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