Development and Duration of Human Papillomavirus Lesions, after Initial Infection

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1 MAJOR ARTICLE Development and Duration of Human Papillomavirus Lesions, after Initial Infection Rachel L. Winer, 1 Nancy B. Kiviat, 2 James P. Hughes, 3 Diane E. Adam, 1 Shu-Kuang Lee, 3 Jane M. Kuypers, 2 and Laura A. Koutsky 1 Departments of 1 Epidemiology, 2 Pathology, and 3 Biostatistics, University of Washington, Seattle Background. To determine the potential value of human papillomavirus (HPV) vaccines, information concerning the incidence and duration of clinically important lesions is needed. Methods. A total of 603 female university students were followed for a mean duration of 38.8 months. Triannual gynecologic examinations included cervical and vulvovaginal specimen collection for Pap and HPV DNA testing. Women with cytologic evidence of a high-grade squamous intraepithelial lesion (SIL) were referred for colposcopically directed biopsy. Results. Among women with incident HPV infection, the 36-month cumulative incidence of cervical SILs found by cytologic testing (47.2%; 95% confidenc interval [CI], 38.9% 56.4%) was higher than that of vaginal SILs (28.8%; 95% CI, 21.3% 38.2%). The median time to clearance of cervical and vaginal SILs was 5.5 and 4.7 months, respectively. Among women with incident HPV-16 or HPV-18 infection, the 36-month cumulative incidence of cervical intraepithelial neoplasia (CIN) grade 2 was 20.0% (95% CI, 10.8% 35.1%), and that of CIN grade 3 was 6.7% (95% CI, 2.5% 17.0%). The 36-month cumulative incidence of clinically ascertained genital warts among women with incident HPV-6 or HPV-11 infection was 64.2% (95% CI, 50.7% 77.4%). Conclusions. Intraepithelial lesions are common early events among women with incident HPV infection, and the interval between incident HPV-16 or HPV-18 infection and biopsy-confi med CIN grade 2 3 appears to be relatively short. Genital human papillomavirus (HPV) infections are common among young women [1 4]. Although there are abundant data describing the persistence of HPV infection, few studies have examined the incidence and duration of clinically important endpoints, including squamous intraepithelial lesions (SILs) and genital warts. Furthermore, there is an impression that SILs are uncommon manifestations of HPV infection [5] and that high-grade SILs (HSILs) usually take years to develop after initial infection with HPV [6]. We hypothesized that this may be a function of the screening interval and that SILs would be detected more frequently and earlier Received 20 February 2004; accepted 8 September 2004; electronically published 21 January Presented in part: 36th Annual Meeting of the Society for Epidemiologic Research, Atlanta, June 2003 (abstract 396-S). Financial support: National Institutes of Health (grants A and 5T32AI ). Reprints and correspondence: Dr. Laura A. Koutsky, University of Washington HPV Research Group, Lake Union Place, Ste. 300, 1914 N 34th St., Seattle, WA (kouts@u.washington.edu). The Journal of Infectious Diseases 2005; 191: by the Infectious Diseases Society of America. All rights reserved /2005/ $15.00 (but that the duration of lesions would be relatively short) with more frequent screening. This prospective study was designed to estimate the incidences and induction periods of clinical endpoints among women with incident HPV infection. With prophylactic HPV vaccines showing promise [7], such information is needed to determine the potential population-level impact of these vaccines on rates of genital warts, abnormal Pap test results, colposcopic examinations, biopsies, and treatments for intraepithelial lesions. PATIENTS, MATERIALS, AND METHODS Study population. Female students years of age were recruited between July 1990 and September 1997 to participate in a longitudinal study of HPV infection. Methods of recruitment and data collection have been described elsewhere [1]. A total of 603 women ( 20% of eligible women receiving letters of invitation) were enrolled. Informed consent was obtained from all study participants, and the human-experimentation guidelines of the University of Washington Institutional Review Board were followed. Development and Duration of HPV Lesions JID 2005:191 (1 March) 731

2 Visits were scheduled at 4-month intervals. At every visit, demographic, medical, and sexual history information was obtained, and cervical and vulvovaginal Dacron-tipped swab specimens were collected for HPV DNA testing. Cervical cytologic specimens were obtained using a cytobrush to collect cellular material from the endocervix and a plastic spatula to collect cells from the squamocolumnar junction and ectocervix. Vaginal cytologic specimens were obtained using a Dacron-tipped swab to collect cells from the lateral vaginal walls. Colposcopic examination of the cervix was performed at every visit. All women with cytologic or colposcopic evidence of an HSIL were referred for colposcopically directed biopsy. Women with repeated cytologic test results showing a low-grade SIL (LSIL) or equivocal finding were also referred. HPV DNA testing. As described elsewhere, specimens were analyzed for the presence of HPV DNA by use of polymerase chain reaction (PCR) amplificatio and dot-blot hybridization methods [1]. One-fiftiet of each sample was amplifie in duplicate with the consensus primers MY09, MY11, and HMB01 and with human b-globin control primers. The products of these amplification were then probed with a biotin-labeled generic probe designed to detect most genital HPV types. Specimens found to be positive by generic probe were tested with individual and mixtures of biotin-labeled, type-specifi oligonucleotide probes, to determine the presence of HPV types 6, 11, 16, 18, 31, 45, and 56 and the following type mixtures: 31/ 33/35/39, 40/42/53/54, and 51/52/55/58. Samples hybridizing with the generic probe but not with one of the type-specifi probes were classifie as positive for uncharacterized genital HPV types. Samples that were found to be negative for both HPV and b-globin DNA by PCR were considered inadequate for analysis. Unless noted otherwise, cervical and vulvovaginal HPV DNA test results were combined. Cytologic and histologic testing. Pap smears were reviewed by a cytotechnologist; all smears showing abnormalities and all biopsies were reviewed by the study pathologist. The study pathologist has participated in blinded studies of interpathologist agreement, and her cytologic and histologic interpretations were found to be similar to those of other expert gynecologic pathologists [8]. Pap smear finding were classified according to the Bethesda system [9], as normal, atypical squamous cells of undetermined significanc (ASCUS), LSIL, or HSIL. Biopsy tissue was diagnosed as showing cervical intraepithelial neoplasia (CIN) grade 1, 2, or 3. None of the women developed cytologic or histologic evidence of invasive cervical cancer. Statistical analysis. An incident HPV infection was define as the firs positive HPV DNA test result among women who tested negative at enrollment. In analyses restricted to typespecifi HPV infections, an incident infection was define as the firs positive type-specifi result among women who tested negative for that type at enrollment. The cumulative probabilities of developing HPV-related lesions were estimated using the Kaplan-Meier method. In analyses restricted to women with incident HPV infection, at-risk time was calculated from the date of firs incident HPV DNA positivity to the date of detection of firs abnormality. To assess development of HPV-related lesions among women without HPV infection, at-risk time was calculated from enrollment to the date of lesion detection (including the enrollment visit), and women were censored at the date of firs positive HPV DNA test result. Women could contribute at-risk time to both HPV-negative and incident HPV analyses, provided they did not develop an abnormality prior to detection of incident HPV infection. For the genital-warts analysis, separate cumulative incidences were estimated among women with incident HPV-6 or HPV-11 infection and among women with incident infections other than HPV-6 or HPV-11. The same method was used to estimate the cumulative incidence of CIN grade 2 3 among women with and without incident HPV-16 or HPV-18 infection. In analyses distinguishing between cervical and vaginal SILs, date of firs detection was define as the firs date at which a cervical or vaginal lesion was detected, regardless of whether a lesion had been detected at the other site at an earlier date. Date of CIN grade 2 3 was define as the study visit date immediately preceding the date at which a biopsy-confi med diagnosis was made. The Kaplan-Meier method was also used to estimate median time to clearance of LSILs (based on cytologic testing) and genital warts. Date of development was define as the date at which the abnormality was firs detected, and date of clearance was define as the firs date at which the abnormality was not detected. Cox proportional-hazards methods were used to compare durations of firs and second episodes of SILs and to make comparisons between cervical and vaginal SILs with respect to both duration and time to development. Robust variances [10] were used when multiple observations from the same women were included in the analysis. To determine what the cumulative probabilities of incident cervical SILs would be if women were screened yearly or every 2 years rather than every 4 months, analyses were restricted to women who were sexually experienced at the time of enrollment. At-risk time was calculated from the enrollment date, and follow-up data were restricted to visits closest to 12, 24, 36, and 48 months after enrollment for yearly screening and 24 and 48 months after enrollment for biennial screening. RESULTS A total of 602 women provided at least 1 set of adequate samples for HPV DNA testing and cytologic evaluation. Altogether, these women completed 5500 visits. Their mean follow-up time was 38.8 months (SD, 19.5 months), the mean number of visits per person was 9.1 (SD, 4.1), and the median time between 732 JID 2005:191 (1 March) Winer et al.

3 Figure 1. Cumulative incidence of developing cervical (A) or vaginal (B) squamous intraepithelial lesions (SILs), cervical intraepithelial neoplasia (CIN) grade 2 3 (C), and cervical atypical squamous cells of undetermined significance (ASCUS) or a more severe abnormality (D) among women with incident human papillomavirus (HPV) infection (thin black line; n p 195 ), women with prevalent HPV infection (thick black line; n p 119 ), and women with no HPV infection (gray line; n p 289 ). The X-axes show the no. of months from time of detection of first incident HPV infection or from enrollment date, for women with prevalent HPV infection or no HPV infection, respectively.

4 Figure 2. Cumulative incidence of developing cervical intraepithelial neoplasia (CIN) grade 2 3 among women with incident human papillomavirus (HPV) 16 or HPV-18 infection (thick black line; n p 60) or with any incident HPV infection other than HPV-16 or HPV-18 (thin black line; n p 137). The X-axis shows the no. of months from time of detection of first incident HPV-16 or HPV-18 infection or first infection with any type other than HPV-16 or HPV-18. visits was 4.3 months. The mean age of these women at enrollment was 19.2 years (SD, 0.5 years). At enrollment, 170 women were virgins, and the mean lifetime number of partners of the 432 women who were sexually active was 2.5 (SD, 2.8). HPV DNA was detected in the genital-tract specimens from 119 (20.3%) of 585 women providing adequate samples for testing at enrollment, and SILs were detected in 24 (3.5%) of 589 women. Of these 24 women, 23 (95.8%) also tested positive for HPV DNA at the enrollment visit. Four virgins did not provide samples for cytologic evaluation at enrollment. During the course of the study, there were 129 incident cases of SIL and 23 incident cases of biopsy-confi med CIN grade 2 3. Of the 129 incident cases of SIL, 26 (20.2%) were detected on the same day that the woman was firs found to be positive for HPV DNA. Eighty-fiv cases of SIL were firs detected in the cervix, 17 in the vagina, and 27 in both sites. Vaginal SILs were detected at subsequent visits in 12 (14.1%) of the 85 women whose firs SIL was detected in the cervix, and cervical SILs were detected at subsequent visits in 6 (35.3%) of the 17 women whose firs SIL was detected in the vagina. Seventeen of the 23 cases of histologically confi med CIN grade 2 3 were diagnosed among women with incident HPV infection. Sixteen cases were referred for biopsy on the basis of abnormal cytologic test results, and 1 case had colposcopic signs of an HSIL. Cumulative incidence of lesions. Among women with incident HPV infection, the cumulative incidence of cervical SILs (47.2% at 36 months; 95% confidenc interval [CI], 38.9% 56.4%) was higher than that of vaginal SILs (28.8% at 36 months; 95% CI, 21.3% 38.2%; P!.01) (figu e 1A and 1B). Among women who developed lesions within 36 months of incident infection (95%), the median time from firs incident HPV infection to detection of lesions was 4.0 months (interquartile range [IQR], months) for cervical SILs and 8.0 months (IQR, months) for vaginal SILs. The distribution of HPV types detected at the same visit that SILs were detected was similar for cervical and vaginal SILs. Among women with incident HPV infection of any type, the 36-month cumulative incidence of CIN grade 2 3 was 11.1% (95% CI, 6.5% 18.5%) (figu e 1C), and, among women with incident HPV-16 or HPV-18 infection, it was 27.2% (95% CI, 16.3% 43.3%) (figu e 2). Among women who received diagnoses within 40 months of incident infection (94%), the median time from firs HPV detection to CIN grade 2 3 was 14.1 months (IQR, months). The median time from detection of incident HPV infection to diagnosis was similar for CIN grade 2 and CIN grade 3. Among women with CIN grade 1 ( n p 16), CIN grade 2 ( n p 13), and CIN grade 3 ( n p 4), the median time from detection of incident HPV infection to detection of firs cervical cytologic abnormality was 4 months and did not differ by lesion grade. Nine (52.9%) of 17 cases of CIN grade 2 3 (including all 4 cases of CIN grade 3) among women with incident HPV infection were positive for HPV-16 or HPV-18 DNA at the visit prior to biopsy (figu e 3). Six of the other 8 cases of CIN grade 2 were found to be positive for other high-risk types. There were no cases of CIN grade 2 3 among HPV-negative women within 1048 person-years of observation time. Although the 36-month cumulative incidence of cervical SILs among HPV-negative women was only 1.6% (95% CI, 734 JID 2005:191 (1 March) Winer et al.

5 Figure 3. Case plot of cervical intraepithelial neoplasia (CIN) grade 2 3 among women with incident human papillomavirus (HPV) infection, from date of enrollment. 0.7% 4.0%), the 36-month cumulative incidence of ASCUS or a more severe abnormality was 25.2% (95% CI, 20.3% 31.1%) (figu e 1D). By modeling the screening interval (that is, by assuming screening every year, every 2 years, or every 4 months), we showed that the frequency of screening greatly impacted estimates of the frequency of detecting cervical cytologic abnormalities (table 1). Whether a Pap test findin of ASCUS or a more severe abnormality or of SIL was used as the threshold, more-frequent screening yielded higher cumulative incidence estimates at both 24 and 48 months. Lesion duration. Of 112 women who developed incident LSILs before their last follow-up visit, 96 (85.7%) cleared their firs episode of SILs while enrolled in the study, and 10 (8.9%) subsequently developed CIN grade 2 3, were referred for treatment, and were removed from the duration analysis of cervical SILs. The median time to clearance was slightly longer for cervical SILs (5.5 months; IQR, months) than for vaginal SILs (4.7 months; IQR, months) ( P p.08). The duration of histologically confi med HSILs could not be determined, because all women were referred for treatment. Fourteen (15.7%) of 89 women had a recurrent episode of cervical LSILs. Durations were similar for firs (median, 5.5 months; IQR, months) and second (median, 5.9 months; IQR, months) episodes ( P p.55). Genital warts. The cumulative incidence of genital warts was highest among women with incident HPV-6 or HPV-11 infection (66.2% at 36 months; 95% CI, 52.8% 79.2%) (figu e 4). Given the rarity of incident HPV-11 infections, compared with incident HPV-6 infections, we did not have sufficien power to determine whether the cumulative incidences of genital warts were similar for each type. It is worth noting, however, that during the course of the study period, clinically visible genital warts were detected in 2 of 4 women with incident HPV-11 infection (and no HPV-6 infection) and in 28 of 41 women with incident HPV-6 infection (and no HPV-11 infec- Development and Duration of HPV Lesions JID 2005:191 (1 March) 735

6 Table 1. Cumulative incidence of cervical abnormalities among sexually active women ( n p 432), under the assumption of 4-, 12-, or 24-month screening intervals. Cumulative incidence (95% CI), %, at Abnormality, screening interval 12 months 24 months 36 months 48 months ASCUS or a more severe abnormality a 4 months 18.2 ( ) 33.0 ( ) 43.7 ( ) 62.2 ( ) 12 months 13.2 ( ) 21.6 ( ) 28.2 ( ) 33.1 ( ) 24 months 12.5 ( ) 21.1 ( ) SIL a 4 months 9.1 ( ) 16.7 ( ) 23.3 ( ) 29.0 ( ) 12 months 4.7 ( ) 10.2 ( ) 15.0 ( ) 18.3 ( ) 24 months 7.5 ( ) 11.7 ( ) NOTE. ASCUS, atypical squamous cells of undetermined significance; CI, confidence interval; SIL, squamous intraepithelial lesion. a Sample includes women who tested negative for this abnormality at enrollment. tion). Of 31 women developing clinically ascertained genital warts after incident HPV infection (2 women had cervical warts, 1 woman had vaginal warts, and 28 women had vulvar, perineal, or perianal warts), 83.9% were positive for HPV-6 or HPV-11 DNA prior to or during the time that warts were present. The median time between detection of incident HPV-6 or HPV-11 infection and detection of genital warts was 2.9 months (IQR, months). All women with probable or definit genital warts were referred for treatment; the median time to clearance with treatment was 5.9 months (IQR, months). DISCUSSION Contrary to the theory that prolonged infection is necessary for progression to high-grade neoplasia [11 13], our results support the hypothesis that HSILs are often an early manifestation of HPV infection in young women. Half of the incident cases of biopsy-confi med CIN grade 2 3 in our study occurred within 14 months of an incident HPV infection. This findin is consistent with previous studies that reported HSILs within 2 years of detection of HPV infection [2, 14]. Our data also Figure 4. Cumulative incidence of developing genital warts among women with incident human papillomavirus (HPV) 6 or HPV-11 infection (thick black line; n p 54, including 41 with HPV-6, 4 with HPV-11, 2 with concurrent HPV-6 and HPV-11, and 7 with HPV-6/11 who were not tested for HPV-6 and HPV-11 separately), any incident HPV infection other than HPV-6/11 (thin black line; n p 156 ), and no HPV infection (gray line; n p 289). The X-axis shows the no. of months from time of first incident HPV-6/11 infection or first infection with any type not including HPV-6/11 or the date of enrollment for women with no infection. 736 JID 2005:191 (1 March) Winer et al.

7 suggest that the time between firs detection of HPV and firs detection of cytologic abnormalities is similar for all grades of CIN. Many of the HSILs we detected were CIN grade 2. Although CIN grade 2 is less reproducible than CIN grade 3 [15], it is reassuring that 100% of women with diagnoses of CIN grade 2 were positive for HPV DNA at the visit prior to biopsy and that 88.2% were positive for high-risk types. Approximately 50% of women in our cohort developed cervical LSILs within 3 years of an incident HPV infection. Two other cohort studies reported significantl lower proportions of cervical abnormalities in women with incident HPV infection. Moscicki et al. [3] followed a cohort of young women in San Francisco every 6 months until firs HPV detection and every 4 months thereafter and found that only 15% developed LSILs within 3 years of initial HPV infection. Woodman et al. [2] followed young women in the United Kingdom at 6-month intervals and reported that 33% developed any type of cervical cytologic abnormality within 3 years of infection. More frequent screening and the fact that the median duration of lesions was!6 months could explain the higher cumulative incidence of lesions in our cohort. It is possible that Woodman et al. [2] and Moscicki et al. [3] missed detection of lesions that developed and cleared within 6 months. Irrespective of HPV status, detection rates tend to increase with more frequent screening, as is demonstrated in our projections of the number of cervical abnormalities that would have been detected if the sexually active women in our cohort had been tested yearly or every other year (intervals consistent with current screening guidelines [16]) rather than every 4 months. Furthermore, by testing every 4 months, we were able to more finel estimate the median duration of LSILs. Our estimate of!6 months was lower than Woodman et al. s [2] estimate of 9 months for the median duration of any type of cervical abnormality and was lower than estimates in earlier studies reporting that half of lesions regress within several years [2, 17, 18]. None of these earlier studies included active follow-up; rather, they relied on women attending screening visits according to their own schedules. Although less common than cervical SILs, vaginal SILs were not rare among women with HPV infection (almost 30% of women developed vaginal SILs within 3 years of incident HPV infection). The present study is, to our knowledge, the firs study to assess the incidence of vaginal SILs. Although the high incidence of vaginal SILs is somewhat surprising, given the rarity of vaginal cancers [19], all lesions cleared within 16 months, suggesting that early vaginal lesions are associated with a low risk for progression. Vaginal SILs were more likely to precede cervical SILs than vice versa, an observation that is consistent with the findin that HPV DNA is more frequently detected in the vulva or vagina before it is detected in the cervix [1]. One limitation of our study is that we did not test for individual HPV types other than types 6, 11, 16, 18, 31, 45, and 56. Although this decreased our ability to assess the associations between certain HPV types and risk of lesions, we were able to evaluate risk associated with the 2 types most frequently detected in cervical cancers (HPV-16 and HPV-18) and the 2 types most often associated with genital warts (HPV-6 and HPV-11). However, without tissue dissection, we cannot know with certainty which HPV types were present in the cells of all lesions. Furthermore, our projections of the 1- and 2-year cumulative incidences of abnormal Pap smear results are conservative, since women with confi med HSILs were treated and removed from the analysis. Also, although specimens for cervical and vaginal cytologic testing were obtained by independent sampling of the endo-/ectocervix and lateral vaginal walls, respectively, it is possible that cervical cells could have been picked up in vaginal cytologic specimens, or vice versa. Finally, as with any test, there is always the inherent possibility of falsepositive or false-negative results in HPV, cytologic, colposcopic, and histologic testing. The present study is one of the firs to examine the incidence and duration of clinically important endpoints of incident HPV infection. Although the incidence of LSILs in our study was higher than those previously reported, these lesions tended to regress quickly. Although the recommendations to refer women directly to colposcopically directed biopsy after detection of LSILs [20] may be appropriate for older women, our results suggest that incident LSILs detected in young women (i.e., those years of age) behave differently from prevalent LSILs detected in older women with persistent HPV infection. Applying such recommendations to young women would result in a largely unnecessary expense. When HSILs developed, they did so relatively early after incident HPV infection, primarily HPV-16 infection. Genital warts also developed quickly after infection with HPV-6 or HPV-11. These results have important implications for the implementation of a prophylactic HPV vaccine. Our results suggest that, within 4 years of achieving widespread vaccine coverage, a quadrivalent vaccine for HPV types 6, 11, 16, and 18 could reduce a substantial proportion of cases of CIN grade 2 3 and genital warts in young women. Acknowledgments We thank Sandra O Reilly for her contributions as research coordinator and Connie Nelson for her assistance in creating the figu es for the article. References 1. Winer RL, Lee SK, Hughes JP, Adam DE, Kiviat NB, Koutsky LA. Incident infection with genital human papillomavirus: rates and risk factors among a cohort of female university students. Am J Epidemiol 2003; 157: Development and Duration of HPV Lesions JID 2005:191 (1 March) 737

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