The samples in PreservCyt media were sent to the University of California San Francisco laboratory, where 7 mL was removed for polymerase chain reaction under polymerase chain reaction sterile conditions and the remaining sent to the University of California San Francisco Anatomic Pathology laboratory for cytology processing. Testing for HPV types 6, 11, 16, 18, 26, 31, 33, 35, 39, 40, 42, 45, 51, 52, 53, 54, 55, 56, 58, 59, 61, 62, 64, 66, 67, 68, 69, 70, 71, 72, 73, 81, 82, 83, 84, IS39, and CP6108 was performed as previously described (Roche Molecular Diagnostics, Inc., ALmeda, CA).13,14 Polymerase chain reaction data were classified as negative or positive for the types identified. Samples without β-globin signal or positive for three or more HPV types were re-prepped and retested. Samples with two negative β-globin signals were considered inadequate and excluded from analysis. In addition, 5% of samples chosen at random were tested in duplicate.
Sample size estimates were based on HPV status. Comparison of CIN 3 or CIN 2 and CIN 1 or less by HPV status gave us an estimated power over 0.99 with α=0.05 and two-sided test. Comparison of CIN 3 and CIN 2 gave us a lower estimated power over 0.64. For purposes of the risk analysis, the following outcome categories were used: CIN 1 or less, CIN 2, and CIN 3. Since CIN 1 is considered benign, CIN 1 and benign (referred to CIN 1 or less in the text) diagnoses were combined for analysis. Statistical analyses were performed on each of the pairs of outcomes (ie, CIN 3 compared with CIN 1 or less, CIN 3 compared with CIN 2, CIN 2 compared with CIN 1 or less). Although CIN 3 was our primary outcome, we compared CIN 2 and CIN 1 or less and CIN 2 and CIN 3 to see if risk factors might differentiate CIN 3 compared with CIN 2. Predictor variables examined included those listed in Table 2. Reported history of sexually transmitted diseases and vaginal infections was used instead of chart review since the chart review likely underestimated the number of infections. We report both in Table 2. We collapsed HPV status into three categories: 1) high-risk HPV with type other than HPV 16 and/or 18; 2) HPV 16/18 only; and 3) low-risk only and HPV negative. Low-risk HPV-positive and HPV-negative results were pooled because they did not show any statistical significant differences between the CIN comparison groups (data not shown). High-risk HPV types included 16, 18, 26, 31, 33, 35, 39, 45, 51, 52, 53, 56, 58, 59, 66, 68, 73, and 82.15
In this analysis, we used t tests to evaluate the differences in average number of biopsies among CIN comparison groups. To compare the differences among included and excluded samples, we used t tests for continuous variables, and χ2 and Fisher exact tests for categorical variables. Because our outcome variable was polytomous, multinomial logistic regression method was used to investigate the associations between selected predictor variables and three CIN outcomes.16 Initial models included candidate predictors singly, and all those with associations significant at the 10% level were considered further in models with multiple predictors. To adjust for possible years of exposure to HPV and site differences, years of sexual activity and clinical site were also included in the final model. The model estimated the odds ratios of having the more serious diagnoses in all three comparisons simultaneously. All the analyses were performed with SAS 9.1.
Six hundred seventy-eight women were consented and completed a baseline questionnaire. Fifty-six women were excluded from the analysis based on either missing a cytology or biopsy diagnosis from the centralized laboratory (Fig. 1). There were 43 women who had normal colposcopy and no biopsy or ECC indicated. Of these, 19 had a negative cytology interpretation at the screening visit and were included for analysis as “benign.” Those with abnormal Pap test results (ASC-US or worse) from the screening visit were excluded because a histologic diagnosis was not available in the face of persistent abnormal cytology. Demographic characteristics and selected behaviors of the 622 women by CIN status are given in Table 2. Overall, the group was racially/ethnically diverse; 32.5% were white, 17.5% black, 15% Latino, 6.9% Asian, and the remaining mixed or other. The cohort appeared to have relative high-risk sexual behaviors compared with national data4,17 defined by a high number of previous pregnancies (37.9%, 95% confident interval [CI] 34.1–41.8%), C trachomatis infections (25.7%, 95% CI 22.3–29.1%), total number of lifetime sexual partners (mean 8 [standard deviation (SD) 8.6]), and history of anal intercourse (38.9%, 95% CI 34.1–41.7%). Comparison between the women who were included compared with excluded (n=56) for analysis showed that the excluded group had a fewer number of C trachomatis infections (12.5%; 95% CI 3.8–21.2%; P=.03) and were less likely to report a history of anal intercourse (25.0%; 95% CI 21.6–28.4%; P=.04) than those included. No other differences were found for any of the variables listed in Table 2.
Of the 622 women, 41 (6.6%; 95% CI 4.6–8.6%) had CIN 3, 81 (13%; 95% CI 10.4–15.6%) had CIN 2, 157 (25.2%; 95% CI 21.8–28.6%) had CIN 1, and 343 (55.1%; 95% CI 51.2–59.0%) were considered benign. Of the 622 women, 36 had missing referral diagnosis from Kaiser Permanente Northern California. Table 1 compares the cytologic referral diagnosis from Kaiser Permanente Northern California to the centralized histologic diagnosis. The majority of CIN 2 and 3 cases (95%; 95% CI 91.1–98.8%) were diagnosed from ASC-US or LSIL referral diagnosis. High-grade squamous intraepithelial lesions diagnoses were rare (1.9%; 95% CI 0.1–2.9%) and had a similar rate as reported by the cytology laboratories at Kaiser Permanente Northern California for the year 1999–2000, near the time the study was initiated. Only 4 of the 11 HSIL diagnoses were confirmed by biopsy. The number of biopsies taken influenced final diagnosis. From the 622 women, there were 1,651 biopsies. Those with benign diagnosis, including only those with biopsies, had the least average number of biopsies (mean 2.3 [SD 1.2] biopsies per woman). This was significantly lower than those with CIN 1, CIN 2, and CIN 3 (mean number of biopsies per women was 3.1 [SD 1.2], 2.9 [SD 1.1], and 3.5 [SD 1.1], respectively); P<.001. In addition, the average number of biopsies of those with CIN 3 was also significantly different from those with CIN 2 (P<.01), but not with CIN 1.
Twelve HPV tests were considered inadequate or missing. Figure 2 shows the number of HPV positive tests within each histologic category. Human papillomavirus 16/18 was strongly associated with grade of CIN, with the observed percentage of women with HPV 16/18 gradually increasing with increasing severity of lesion. Women with benign examinations had the lowest rate of HPV 16/18 detection (20.5%, 95% CI 16.2–24.8%), and those with CIN 3 had the highest (65.9%, 95% CI 51.2–80.1%). Rates of HPV 16/18 were significantly higher in women with CIN 3 than in those with either CIN 1 (24.4%; 95% CI 17.6–31.2%)or benign diagnoses (P<.001 for both), and marginally higher than in those with CIN 2 (48.2%; 95% CI 37.3–59.2%, P=.06). Rates of HPV 16/18 in women with CIN 2 were also higher than in those with benign or CIN 1 diagnoses (P<.001 for both). No differences for HPV 16/18 detection were seen among women with benign and CIN 1 outcomes. Only two women with CIN 3 were HPV negative, and none had low-risk HPV only.
Prevalence of any high-risk HPV was also higher in women with CIN 2 (87.7%, 95% CI 80.5–94.9%) or CIN 3 (95.1%, 95% CI 88.4–100%) than in those with CIN 1 (65.8%, 95% CI 58.3–73.3%) or benign diagnoses (54.5%, 95% CI 49.1–59.9%, P<.003 and P<.01, respectively).
Separate multinomial logistic regression models were fitted for each candidate predictor variable, with results summarized using P values from tests of association for each of the following outcome comparisons: CIN 3 compared with CIN 1 or less, CIN 3 compared with CIN 2, and CIN 2 compared with CIN 1 or less. These results are summarized in Table 2. The first comparison (CIN 3 compared with CIN 1 and less) showed that the following variables associated with risk for CIN 3 at P<.1 level: total months of oral contraceptive pill (OCP) use, total months on medroxyprogesterone, number of sex partners in last 2 months, and number of cigarettes smoked in the last 24 hours. The second comparison (CIN 3 compared with CIN 2) found only number of sex partners in last 2 months significant at P<.1 level. The third comparison (CIN 2 compared with CIN 1 or less) showed that the risk of CIN 2 is associated with younger age, history of chlamydia, and total months on medroxyprogesterone at the P<.1 level.
Predictor variables with associations significant at P<.1 level in Table 2 were included together in a final multinomial logistic model, with results summarized as odds ratios (and 95% CIs) for the outcome comparisons introduced above. The model also adjusted for years of sexual activity and clinic site. The first comparison for CIN 3 compared with CIN 1 or less found total years of OCP use increased risk of CIN 3. For each one additional year on OCPs, the odds of having CIN 3 increases by 36% on average. Although not statistically significant, having two or more recent sex partners had an increased odds of CIN 3 (P=.08).
The second comparison for CIN 2 and CIN 1 or less found that for each additional year on medroxyprogesterone increases the odds of CIN 2 by 46%. Both analyses found HPV 16 and/or 18 and other high-risk HPV detection significant: the former increases the odds of CIN 3 by almost 3,000%, and CIN 2 by 600%; the latter increases the odds of CIN 3 by 500%, and CIN 2 by 260%, comparing with those negative for HPV or low-risk HPV. No statistically significant differences were found for the comparison between CIN 3 and 2. The results are presented in Table 3.
Little is known about adolescents and young women who develop CIN 3 because most studies focus on older women. In this study of adolescents and young women attending a large health maintenance organization, less than 7% of the population referred for abnormal cytology was found to have CIN 3. Similar to older adults, most of the study participants were referred because of ASC-US and LSIL.18 In contrast, the rate of CIN 3 among study participants referred for ASC-US or LSIL was less than half of that reported for older adults. It is estimated that 10–16% of adult women with HPV-positive ASC-US/LSIL will have underlying CIN 3.19–21 We found CIN 3 in 6.3% (95% CI 4.3–8.3%) of adolescents and young women with ASC-US or LSIL on referral Pap. This lower rate of CIN 3, along with data showing high rates of LSIL regression,13 support the new 2006 American Society for Colposcopy and Cervical Pathology Consensus Guidelines, which recommend following adolescents with ASC-US/LSIL by cytology rather than immediate referral to colposcopy.18 Our data suggest that extending this conservative management to young women under the age of 25 years may be reasonable and that further analysis by guideline groups is warranted. The consensus guidelines also recommend continued follow-up of these young women with ASC-US/LSIL because the possibility of CIN 3 is not nil, as demonstrated in our study. However, the likelihood of progression to cancer is extremely low during this young age period.2 This assumption was supported by the lack of finding a single cancer case in our study.
Although the rate of CIN 3 was relatively low, the risks for having CIN 3 were similar to those found in previous studies of adults. The strongest association with CIN 3 was that of having HPV 16/18 or other high-risk HPV infections.22 Because ours was a sexually active group of adolescents and young women, it is not surprising that HPV DNA detection was high in all histology groups, including those with benign histology. However, we noted that the rate of HPV 16 and 18 among those with CIN 3 was threefold that of those with benign/CIN 1 histology. The high rate found in CIN 3 (65%) is identical to the rates attributed to cervical cancer and CIN 3 by HPV 16 and 18 worldwide.15,23 Our findings suggest there may be clinical utility in separating these types for clinical testing in young women as well as older adults.
We found the risk for CIN 3 associated with time on hormonal contraceptives most interesting because most of the women had been on OCPs for a limited time. Although OCP use has been identified as a potential risk factor for both cervical cancer and CIN 3, the absolute attributable risk remains highly controversial, and when associated, the risk becomes appreciable only after some time period on OCPs ranging from 3 to 5 years.10,11,24,25 The significant association between CIN 3 and OCP use in our study, after controlling for HPV status, suggests that OCP use does contribute a small risk in some women. Given that these women had limited sexual exposure to HPV and OCPs, there may be some epigenetic event that occurs with exogenous hormones and HPV exposure explaining the occurrence of these CIN 3 lesions at this young age.26 Several in vitro studies demonstrate the biologic plausibility for the association between estrogen and invasive cervical cancer.27–30
The inability of our analysis to discriminate between CIN 2 and 3, suggest that CIN 2 and 3 share some risks factors. On the other hand, risks that discriminated CIN 3 compared with CIN 1 or less and CIN 2 compared with CIN 1 or less were different, underscoring the likely difference between these two lesions. It is worth noting that the prevalence of HPV 16 and 18 was greater in the CIN 3 than CIN 2 group; 66% compared with 48%, respectively, and the multinomial models showed a much greater risk for CIN 3 if positive for HPV 16/18 than for CIN 2. Only one risk factor was found that distinguished the CIN 2 and the CIN 1/benign group, and this was the use of medroxyprogesterone. Some epidemiology studies have shown medroxyprogesterone use to be associated with cervical cancer.31 The association with CIN 2, but not CIN 3, suggests to us that progesterone-only contraceptives may have other attributes which enhance the expression of CIN 2–type lesions but lack the ability to cause sentinel events that lead to CIN 3 in young women. On the other hand, the atrophy induced by medroxyprogesterone may have led to the misdiagnosis of CIN 2 in this group.32 Cervical intraepithelial neoplasia 2 remains a controversial diagnosis with some questioning its existence and reproducibility. We believe our results suggest that CIN 2 and 3 are likely different biologically.33 The lack of finding differences between CIN 2 and 3 may have been limited by our sample size for these groups. Examination of biologic markers is likely to be better suited to distinguish these lesions, if there is a true biologic distinction.
The main limitation of the study was lack of information on those not participating in the study. Although we examined differences between those included and excluded by our criteria, we were unable to interview the young women who we were not able to contact or get permission to contact, limiting our ability to generalize. However, this remains one of the largest studies to examine risk factors for CIN 3 in young women. Second, we did not verify all pathologic diagnosis using two reviewers; only CIN 3 diagnoses were confirmed by a second pathologist. The centralized readings for all cytology and histology allowed consistency among diagnoses. The tight association between HPV 16 and 18 infections and diagnosis suggests that the readings were consistent with other publications using two or more reviewers.23 The lack of association with smoking was possibly due to the relative low nicotine exposure of smokers; few smoked more than five cigarettes a day.
In summary, our data show that CIN 3 is relatively rare in adolescents and young women referred for abnormal cytology. Those with CIN 3 appear to have an extremely low likelihood of progression to cancer because none was found in the time frame of this study. The primary risk for CIN 2 and 3 in this age group remains HPV 16 and 18 infections. Studies are warranted to examine the utility of using type-specific HPV DNA 16 and 18 assays for screening and evaluation in these young women. Our data also found that time on hormonal contraceptives contribute a small increased risk for the development of CIN 3 in young women, even with limited exposure to these exogenous hormones. Different associations were found with CIN 2 lesions than CIN 3, suggesting that CIN 2 is a different biologic lesion than CIN 3.
1. Dunne EF, Unger ER, Sternberg M, McQuillan G, Swan DC, Patel SS, et al. Prevalence of HPV infection among females in the United States. JAMA 2007;297:813–9.
2. Ries LA, Melbert D, Krapcho M, Mariotto A, Miller BA, Feuer EJ, et al. SEER Cancer Statistics Review, 1975–2004. Bethesda (MD): National Cancer Institute; 2007.
3. Moscicki AB, Hills N, Shiboski S, Powell K, Jay N, Hanson E, et al. Risks for incident human papillomavirus infection and low-grade squamous intraepithelial lesion development in young females. JAMA 2001;285:2995–3002.
4. Mosher WD, Chandra A, Jones J. Sexual behavior and selected health measures: men and women 15-44 years of age, United States, 2002. Adv Data 2005;1–55.
5. Moscicki AB, Shiboski S, Broering J, Powell K, Clayton L, Jay N, et al. The natural history of human papillomavirus infection as measured by repeated DNA testing in adolescent and young women. J Pediatr 1998;132:277–84.
6. Munoz N, Mendez F, Posso H, Molano M, van den Brule AJ, Ronderos M, et al. Incidence, duration, and determinants of cervical human papillomavirus infection in a cohort of Colombian women with normal cytological results. J Infect Dis 2004;190:2077–87.
7. Moscicki AB, Schiffman M, Kjaer S, Villa LL. Chapter 5: Updating the natural history of HPV and anogenital cancer. Vaccine 2006;24 suppl:S3/42–51.
8. Winer RL, Kiviat NB, Hughes JP, Adam DE, Lee SK, Kuypers JM, et al. Development and duration of human papillomavirus lesions, after initial infection. J Infect Dis 2005;191:731–8.
9. International Collaboration of Epidemiological Studies of Cervical Cancer, Appleby P, Beral V, Berrington de Gonzalez A, Colin D, Franceschi S, et al. Cervical cancer and hormonal contraceptives: collaborative reanalysis of individual data for 16,573 women with cervical cancer and 35,509 women without cervical cancer from 24 epidemiological studies. Lancet 2007;370:1609–21.
10. McIntyre-Seltman K, Castle PE, Guido R, Schiffman M, Wheeler CM, ALTS Group. Smoking is a risk factor for cervical intraepithelial neoplasia grade 3 among oncogenic human papillomavirus DNA-positive women with equivocal or mildly abnormal cytology. Cancer Epidemiol Biomarkers Prev 2005;14:1165–70.
11. Richardson H, Abrahamowicz M, Tellier PP, Kelsall G, du Berger R, Ferenczy A, et al. Modifiable risk factors associated with clearence of type-specific cervical papillomavirus infections in a cohort of university students. Cancer Epidemiol Biomarkers Prev 2005;14:1149–56.
12. Nugent RP, Krohn MA, Hillier SL. Reliability of diagnosing bacterial vaginosis is improved by a standardized method of gram stain interpretation. J Clin Microbiol 1991;29:297–301.
13. Moscicki AB, Shiboski S, Hills NK, Powell KJ, Jay N, Hanson EN, et al. Regression of low-grade squamous intra-epithelial lesions in young women. Lancet 2004;364:1678–83.
14. Gravitt P, Peyton CL, Alessi TQ, Wheeler C, Coutlée F, Hildesheim A, et al. Improved amplification of genital human papillomaviruses. J Clin Microbiol 2000;38:357–61.
15. Muñoz N, Bosch FX, de Sanjose S, Herrero R, Castellsague X, Shah KV, et al. Epidemiological classification of human papillomavirus types associated with cervical cancer. N Engl J Med 2003;348:518–27.
16. Stokes ME, Davis CS, Koch GG. Categorical data analysis using the SAS system. 2nd ed. Cary (NC): SAS Institute Inc; 2000.
17. Centers for Disease Control. National and state-specific pregnancy rates among adolescents - United States, 1995–1997 [Published erratum appears in MMWR Morb Mortal Wkly Rep 2000;49:672]. MMWR Morb Mortal Wkly Rep 2000;49:605–11.
18. Wright TC, Massad LS, Dunton CJ, Spitzer M, Wilkinson EJ, Solomon D, et al. 2006 consensus guidelines for the management of women with abnormal cervical cancer screening tests. Am J Obstet Gynecol 2007;197:346–55.
19. Cox JT, Schiffman M, Solomon D, ASCUS-LSIL Triage Study (ALTS) Group. Prospective follow-up suggests similar risk of subsequent cervical intraepithelial neoplasia grade 2 or 3 among women with cervical intraepithelial neoplasia grade 1 or negative colposcopy and directed biopsy. Am J Obstet Gynecol 2003;188:1406–12.
20. ASCUS-LSIL Triage Study (ALTS) Group. Results of a randomized trial on the management of cytology interpretations of atypical squamous cells of undetermined significance. Am J Obstet Gynecol 2003;188:1383–92.
21. ASCUS-LSIL Triage Study (ALTS) Group. A randomized trial on the management of low-grade squamous intraepithelial lesion cytology interpretations. Am J Obstet Gynecol 2003;188:1393–400.
22. Castle PE, Solomon D, Schiffman M, Wheeler CM. Human papillomavirus type 16 infections and 2-year absolute risk of cervical precancer in women with equivocal or mild cytologic abnormalities. J Natl Cancer Inst 2005;97:1066–71.
23. Franceschi S, Clifford GM. Fraction of cervical neoplasias due to human papillomavirus 16 and 18 in vaccine trials. Int J Cancer 2008;122:719–20.
24. Shields TS, Brinton LA, Burk RD, Wang SS, Weinstein SJ, Ziegler RG, et al. A case-control study of risk factors for invasive cervical cancer among U.S. women exposed to oncogenic types of human papillomavirus. Cancer Epidemiol Biomarkers Prev 2004;13:1574–82.
25. Miller K, Blumenthal P, Blanchard K. Oral contraceptives and cervical cancer: critique of a recent review. Contraception 2004;69:347–51.
26. Couto E, Hemminki K. Heritable and environmental components in cervical tumors. Int J Cancer 2006;119:2699–701.
27. Ruutu M, Wahlroos N, Syrjanen K, Johansson B, Syrjanen S. Effects of 17beta-estradiol and progesterone on transcription of human papillomavirus 16 E6/E7 oncogenes in CaSki and SiHa cell lines. Int J Gynecol Cancer 2006;16:1261–8.
28. Krusekopf S, Chauchereau A, Milgrom E, Henderson D, Cato AC. Co-operation of progestational steroids with epidermal growth factor in activation of gene expression in mammary tumor cells. J Steroid Biochem Mol Biol 1991;40:239–45.
29. Webster K, Taylor A, Gaston K. Oestrogen and progesterone increase the levels of apoptosis induced by the human papillomavirus type 16 E2 and E7 proteins. J Gen Virol 2001;82:201–13.
30. Scott ME, Ma Y, Farhat S, Shiboski S, Moscicki AB. Covariates of cervical cytokine mRNA expression by real-time PCR in adolescents and young women: effects of Chlamydia trachomatis infection, hormonal contraception, and smoking. J Clin Immunol 2006;26:222–32.
31. Depot-medroxyprogesterone acetate (DMPA) and risk of invasive squamous cell cervical cancer. The WHO Collaborative Study of Neoplasia and Steroid Contraceptives. Contraception 1992;45:299–312.
32. Valente PT, Schantz HD, Trabal JF. Cytologic changes in cervical smears associated with prolonged use of depot-medroxyprogesterone acetate. Cancer 1998;84:328–34.
© 2008 by The American College of Obstetricians and Gynecologists. Published by Wolters Kluwer Health, Inc. All rights reserved.
33. Syrjanen K, Kataja V, Yliskoski M, Chang F, Syrjanen S, Saarikoski S. Natural history of cervical human papillomavirus lesions does not substantiate the biologic relevance of the Bethesda System. Obstet Gynecol 1992;79:675–82.