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ORIGINAL RESEARCH |
From the Department of Gynecology, Reinier de Graaf Gasthuis, Delft, The Netherlands; Pathology Laboratory, SSZOG, Winschoten, The Netherlands; Department of Clinical Chemistry, Stichting Ziekenhuis De Heel, Zaandam, The Netherlands; and Department of Molecular Biology, SSDZ/Reinier de Graaf Gasthuis, Delft, The Netherlands.
Address reprint requests to: W. A. ter Harmsel, MD, PhD Department of Gynecology Reinier de Graaf Gasthuis Reinier de graafweg 11, PO Box 5012 2600 GA Delft The Netherlands
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Abstract |
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Methods: Fifty-four women with HPV 16 polymerase chain reaction (PCR)–positive tests were selected from the gynecologic outpatient clinic of the Reinier de Graaf Hospital, Delft, The Netherlands. At least three successive PCR tests were performed in each woman at intervals of 6 months. The PCR HPV 16 assay was performed in conjunction with cervical smear, and colposcopy and biopsy, if indicated. Women with at least three consecutive positive PCR tests were defined as having persistent HPV 16 infections. Women with one positive test followed by two negative tests were defined as having transient infections. Subdivided into two groups, 25 women had persistent infections and 29 had transient infections.
Results: In significantly more women in the persistent group compared with the transient group, CIN developed (11 of 25 versus six of 29, P = .036). Lesions in women with persistent HPV 16 infection were more severe (six of 11 were CIN III versus one of six P = .041).
Conclusion: Persistent infection with HPV 16 is associated with a higher risk of developing CIN, which is often high-grade.
A large number of epidemiologic and molecular cell biologic data offer strong evidence that specific human papilloma viruses (HPVs) are the main cause of cervical intraepithelial neoplasia (CIN) and cervical carcinoma.1 A distinction has been made between HPV types of low, intermediate, and high oncogenic potential.2 Clinically, the viruses have been subdivided into low-risk (HPV 6, 11, 42, and 44) and high-risk (HPV 16, 18, 31, and 33) groups. The low-risk group has not been associated with cervical carcinoma and high-grade CIN, but the high-risk group has. Both in vitro and in vivo studies strongly suggest that high-risk HPV genotypes influence the development of cervical cancer. The percentage of CIN lesions containing HPV viruses with oncogenic potential reportedly increases with lesion severity. Human papilloma virus has been detected in up to 93% of cervical smears from patients with CIN I lesions, up to 95% of CIN II lesions, and up to 96% of CIN III lesions.3 HPV is found in 84–100% of cervical carcinomas.4
In large population studies, HPV has been identified in up to 20% of women with normal cytologic smears5 in the 20- to 25-year age group. Up to 14% of these women harbor oncogenic HPV 16.6 These percentages decrease with increasing age. In women 35–50 years old with normal cervical cytology participating in a screening program in the region of Delft, The Netherlands, the prevalence of HPV-positive cervical smears was 5%, and the prevalence of HPV 16 was 2%.7 These observations suggest that the relationship between HPV infection and development of CIN and cervical carcinoma is not straightforward. CIN or cervical carcinoma will not develop in the majority of women infected with oncogenic HPV.1 Other factors, such as age at first sexual intercourse, number of sexual partners,8 smoking,9 immunologic factors, and viral load,10 also are thought to influence cervical carcinogenesis. Studies investigating the potential for progression of low-grade cervical disease associated with HPV 16 are limited. Results from studies investigating this relationship are mixed.11–15
For this reason, we undertook a prospective study of HPV 16 polymerase chain reaction (PCR)–positive women with normal cervical cytology. The objective of the study was to examine the influence of duration of cervical HPV 16 infection on the development of CIN. During the study, patients were treated according to current protocols, irrespective of their HPV status. Women were taken off study after any therapeutic intervention. The effect of HPV 16 infection on the development of CIN was compared with previously described risk factors.
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Materials and Methods |
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Both groups were invited to participate in a follow-up study consisting of a standardized interview asking age at first sexual intercourse, smoking habits, history of sexually transmitted diseases (STDs), number of sexual partners, and use of oral contraceptives. All women were offered further cervical smears and HPV PCR testing at intervals of 6 months. The endpoint of the study was therapeutic intervention.
Differences in quantitative variables were tested with Student’s t test and Fisher exact test. Qualitative variables were tested with the 
2 test. Differences between groups were considered significant when P < .05 (two-sided). Kaplan-Meier CIN-free survival curves were analyzed using the Statistical Package for Social Sciences (SPSS/PC+;SPSS Inc., Chicago, IL). The curves were compared by log-rank test.
Cervical smears were taken with a Cervex-Brush (Rovers BV, Oss, The Netherlands). If there was uncertainty regarding the visibility of the endocervix, a cytobrush was used to take an endocervical sample. After smear preparation, the Cervex-Brush and the endocervical brush were placed in 5 mL phosphate-buffered saline, pH 7.2, supplemented with merthiolate 1:10,000 v/v. Polymerase chain reaction was used to detect HPV using general primer GP5/6, which has been described extensively elsewhere16; PCR was performed as described by Claas et al.17 The amplified products were analyzed with a dot blot method. Filters were hybridized with a mixture of 
-32p 5'-end-labeled HPV type-specific oligonucleotide probes under conditions of high stringency. Positive and negative controls were included in each experiment. In a randomly selected group from every type of specimen, an amplification of a part of the ß-globin gene was performed using specific primers to check the efficiency of the amplification reaction.18 All specimens showed beta-globin–specific amplified products, which indicated reliable interpretation of PCR results.
All cervical smears were Papanicolaou-stained, screened routinely, and reviewed without knowledge of the HPV PCR results by a cytologic technician and a pathologist (FS). Initial classifications were made according to the current Dutch cytologic classification system (KOPAC) and the Papanicolaou system, as adopted in the Netherlands.19 These were converted to the Bethesda system for this paper. Histologic tissues were classified according to CIN classification.
Colposcopy with or without directed biopsies was performed subsequent to a cytologic diagnosis of low-or high-grade squamous intraepithelial lesion (SIL), as recommended by the Dutch Society of Colposcopy. Human papillomavirus PCR results did not influence the decision for biopsy, because the treating gynecologists were blinded to the HPV PCR test results. Any further treatment was based on histologic diagnosis only, irrespective of HPV status. Women were discharged from the study after therapy.
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Results |
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CIN developed in 11 of 25 women with persistent infections, follow-up average 23.5 months (range 8–60 months). Two CIN I lesions, three CIN II lesions, and six CIN III lesions developed. CIN III lesions reached statistical significance when P = .041. Sexually transmitted disease was a significant independent risk factor for persistent HPV 16 infection (P = .001). No other characteristics were statistically significant risk factors (Table 1
).
Women with persistent cervical HPV 16 infections have a significantly increased chance of CIN developing. Figure 1 shows Kaplan-Meier curves for the development of CIN in persistent and transient groups. The difference is statistically significant (P = .036).
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Discussion |
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On the basis of both clinical and ethical consideration, we chose to intervene in cases of cytologically or colposcopically proven CIN lesions. In contrast to other studies in which low-or high-grade CIN was left untreated,15 we removed these lesions, and these patients were excluded from the study. Our results are comparable to those of Koutsky et al14 and Remmink et al.15 Even though our approach was different, we also found a highly significant association between development of CIN and persistence of HPV 16 infection (Figure 1). During our study period of 2 years, the probability of CIN developing in women with persistent HPV 16 infection and normal cytology was 44%. This percentage is higher than the 28% cumulative incidence of developing CIN in 2 years, as reported by Koutsky et al.14 However, both studies showed that high-grade CIN develops quickly in women with persistent HPV 16 infection.
CIN developed in 22% of women with transient HPV 16 infections when HPV could no longer be detected. We believe that even transient infection with HPV 16 exposes women to a significant risk of development of CIN. A relatively high percentage of low-grade SIL in our study turned out to be CIN III on histologic examination. The margin of error in diagnosing CIN based on cytology and colposcopy alone is well known. This throws some doubt on the reported high rates of progression observed in the study of Remmink et al,15 in which the initial diagnosis is based only on cytology and colposcopy, and the final diagnosis is based on histology. Many of the baseline lesions in this study could have been high-grade CIN.
In this study, we were concerned only with HPV 16. This choice was based on the particularly high prevalence of HPV 16 reported in previous studies. If we had investigated other oncogenic HPVs such as 18, 31, and 33, which also are found in high-grade CIN, an even higher correlation between HPV positivity and development of CIN probably would have been found. The use of more sensitive primers in HPV detection could also improve the results. Another important factor not taken into account in this study was viral load. According to Cox et al10 and Cuzick et al25 this has enormous predictive value for the development of CIN. In cases of persistent HPV infection, the viral load could be higher than in cases of transient infection.
We also examined other known risk factors for CIN and their relationship to persistent or transient HPV 16 infection. The only significant risk factor was the number of sexually transmitted diseases in the medical history. This can be explained by the fact that repeated contact with HPV carriers helps to maintain cervical infection. Schiffman and Brinton26 also reported this association.
This study provides evidence for the influence of HPV 16 cervical infection on the development of CIN. Persistent HPV 16 infection correlated with a higher risk of development of CIN than transient infection. However, even a single positive HPV 16 test predisposes women to considerable risk of CIN developing. CIN that develops in persistent HPV 16 infection is more severe than that in women infected for a short period only.
On the basis of our observations, we propose that patients with positive HPV 16 tests, regardless of the cytologic results, should be monitored more closely because of the high probability of development of CIN. Routine implementation of HPV testing in cervical screening programs is under discussion. On the basis of the carcinogenetic model for cervical carcinoma in which HPV is central, it would be logical if all women were tested for HPV at least once. Using this study as an example, costs for this approach are very high: 5764 tests, at $25 per test, to identify 25 persistently and 29 transiently HPV 16–infected women. Women with normal cervical cytology have a very low prevalence of HPV. Therefore, HPV testing in this group would incur high costs to identify a few HPV-positive women. On the other hand, nearly all women with cytologically proven high-grade SIL harbor HPV. On the basis of their cytology results, colposcopy will be performed anyway. Between these two extremes are some smears, approximately 10%, depending on the cytology laboratory, that are classified as atypical squamous cells of uncertain significance. These smears carry an estimated 10% risk of underlying CIN. This volume entails considerable financial and psychologic costs, because of necessary repeat smears and referrals for colposcopy and treatment. The problems with cytology in this group of women might be averted by HPV testing. The cost-effectiveness of HPV screening in this group of women would be very much higher than that of screening the entire population.
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Footnotes |
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Received November 17, 1997. Received in revised form May 28, 1998. Accepted June 19, 1998.
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