Human Papillomavirus Infection and Time to Progression and Regression of Cervical Intraepithelial Neoplasia

Nicolas F. Schlecht, Robert W. Platt, Eliane Duarte-Franco, Maria C. Costa, Joa˜o P. Sobrinho, Jose´ C. M. Prado, Alex Ferenczy, Thomas E. Rohan, Luisa L. Villa, Eduardo L. Franco

(1). This sequence forms the premise on which Background: Little is known about the duration of precan- cytologic screening for cervical cancer is based and corresponds cerous cervical lesions in relation to human papillomavirus to an underlying multistep carcinogenic process in the develop- (HPV) infection. We estimated rates of progression and re- ment of cervical intraepithelial neoplasia (CIN) (2). Low-grade Downloaded from https://academic.oup.com/jnci/article/95/17/1336/2520427 by guest on 29 September 2021 gression and sojourn times of cervical squamous intraepi- squamous intraepithelial lesions (LSILs) may progress to high- thelial lesions (SILs) according to HPV status. Methods: We grade SILs (HSILs) and invasive cervical cancer or may regress used data from a longitudinal study of HPV infection and to a normal state (3). However, few studies of cervical neoplasia cervical neoplasia in Sa˜o Paulo, Brazil. Cervical specimens have evaluated lesion recurrence (4,5) or disease progression (3) were taken from 2404 women for Pap cytology and polymer- over time. Likewise, lesion progression or regression has not ase chain reaction–based HPV testing every 4–6 months over been evaluated in relation to the presence of human - a period of 8 years. We used actuarial and non-actuarial viruses (HPV), the main etiologic agents in the initiation of analyses to measure time to and rates of lesion progression cervical neoplasia (6). The use of a biomarker that can predict and regression according to status and type of HPV infec- the rate of progression or regression and the duration of the tion. Results: During follow-up, 118 low-grade SIL (LSIL), preinvasive stages of cervical cancer could represent an attrac- 24 high-grade SIL (HSIL), and 173 atypical squamous cells tive means for targeting screening or chemoprevention. of undetermined significance (ASCUS) events were detected. Beginning in 1993, we initiated a cohort study involving Mean time to progression from ASCUS to LSIL or worse repeated measurements of HPV infection and cervical cytology and from LSIL to HSIL or worse was shorter in women with in women attending a comprehensive maternal and child health oncogenic HPV types than in women with no HPV infection program that serves low-income families living in neighbor- (mean times for ASCUS progression were 67.0 and 88.0 hoods located in the northern sector of the city of Sa˜o Paulo, months, respectively, in women with oncogenic HPV and no Brazil (7). In this population, early precursor lesions are gener- HPV, difference = 21.0 months, 95% confidence interval [CI] ally not treated, which enabled us to evaluate prospectively the = 11.3 to 30.7 months; mean times for LSIL progression occurrence of SIL events at regular intervals over time. In par- were 73.3 and 83.5 months, respectively, difference = 10.2 ticular, we sought to measure the frequency and rates of pro- gression and regression, as well as the durations (i.e., sojourn months, 95% CI = –0.15 to 20.6 months). Half of the LSILs time) of early cervical precursor lesions according to their HPV regressed to normal or ASCUS within 6 months. Mean times status. for regression from ASCUS to normal, from LSIL to ASCUS or normal, and from HSIL/cervical intraepithelial neoplasia SUBJECTS AND METHODS 2 to ASCUS or normal were longer for women with onco- genic HPV types (16.8 months, 95% CI = 7.5 to 26.2 months; Subject Recruitment 13.8 months, 95% CI = 8.8 to 18.7 months; and 17.1 months, Two study nurses approached 4990 women from daily lists of 95% CI = 4.1 to 30.1 months, respectively) than for women outpatients in the family medicine, gynecology, and family plan- with non-oncogenic HPV types (7.7 months, 95% CI = 5.2 to ning clinics at the Vila Nova Cachoeirinha municipal hospital in 10.2 months; 7.8 months, 95% CI = 5.3 to 10.2 months; 8.9 Sa˜o Paulo, Brazil, for an interview. Women who were poten- months, 95% CI = 3.3 to 14.6 months) or for women with no HPV infection (7.6 months, 95% CI = 6.9 to 8.4 months; 7.6 months, 95% CI = 6.4 to 8.7 months; and 7.0 months, 95% Affiliations of authors: N. F. Schlecht, E. L. Franco (Departments of On- CI = 5.0 to 8.9 months, respectively). Conclusion: Precursor cology and Epidemiology and Biostatistics), R. W. Platt (Departments of Epi- lesions of the persist longer and progress more demiology and Biostatistics and Pediatrics), E. Duarte-Franco (Department of quickly in women with oncogenic HPV infections than in Oncology), McGill University, Montreal, Quebec, Canada; M. C. Costa, J. P. Sobrinho, J. C. M. Prado, L. L. Villa, Ludwig Institute for Cancer Research, women with non-oncogenic infections or without HPV. Test- Sa˜o Paulo, Brazil; A. Ferenczy, Department of Pathology, McGill University; ing cervical lesions for oncogenic HPVs may help identify T. E. Rohan, Department of Epidemiology and Population Health, Albert Ein- those that are likely to progress rapidly. [J Natl Cancer Inst stein College of Medicine, New York, NY. 2003;95:1336–43] Correspondence to: Eduardo L. Franco, PhD, Department of Oncology, McGill University, 546 Pine Ave. West, Montreal, Quebec, Canada H2W 1S6 (e-mail: [email protected]). The natural history of cervical cancer involves reversible See “Notes” following “References.” changes in the cervical tissue from a normal state, in which no DOI: 10.1093/jnci/djg037 neoplastic changes are detected in the squamous epithelium, to Journal of the National Cancer Institute, Vol. 95, No. 17, © Oxford University varying states of cellular abnormalities that ultimately lead to Press 2003, all rights reserved.

1336 ARTICLES Journal of the National Cancer Institute, Vol. 95, No. 17, September 3, 2003 tially eligible were presented with a detailed overview of the HPV DNA Detection study and invited to participate. Recruitment began in November 1993 and continued until March 1997. Cervical specimens were tested for the presence of HPV Women were eligible to participate if they 1) were between DNA by using the MY09/11 polymerase chain reaction (PCR) 18 and 60 years of age; 2) were permanent residents of Sa˜o protocol (9,10). The amplified products were typed by hybrid- Paulo (city), Brazil; 3) were not currently pregnant and had no ization with individual oligonucleotide probes specific for 27 intention of becoming pregnant during the next 12 months; 4) genital HPV types (10). Amplified products that hybridized with had an intact uterus and no current referral for hysterectomy; 5) the generic probe but with none of the type-specific probes were reported no use of vaginal medication in the previous 2 days; and further tested by restriction fragment length polymorphism 6) had not received treatment for cervical disease in the previous analysis (11) to extend the range of identifiable HPV types. To 6 months. In addition to these criteria, women were considered verify the specificity of the hybridizations, we included more eligible only if they expressed willingness to comply with all than 30 type-specific positive controls in each HPV test run. To scheduled return visits, at least for the initial 2 years. check the integrity of the host DNA extracted from the speci- Subjects entered the study only after giving signed informed mens, assays also included an additional set of primers to am- ␤ consent. The study protocol was approved by institutional ethi- plify the -globin gene (9). HPV types were separated into two Downloaded from https://academic.oup.com/jnci/article/95/17/1336/2520427 by guest on 29 September 2021 cal and research review boards of the participating institutions in groups by level of oncogenicity. Oncogenic types included Canada (McGill University) and Brazil (Ludwig Institute for HPV16, -18, -31, -33, -35, -39, -45, -51, -52, -56, -58, -59, and Cancer Research). A detailed description of the design and -68; non-oncogenic types included 6/11, -26, -32, -34, -40, -42, methods of the study has been published previously (7). All -44, -53, -54, -55, -57, -62, -64, -66, -67, -69, -70, -71, -72, -73, participants were seen every 4 months in the first year (at 0, 4, -81, -82, -83, -84, CP6108, and other unknown types. All HPV 8, and 12 months) and every 6 months thereafter. Delays in assays were done on coded specimens, with no identification returning for a given follow-up appointment were allowed; the that could link specimens from the same woman. Appropriate visit numbering sequence was maintained, even when subjects precautions were taken to reduce the possibility of specimen returned for their follow-up visit after the scheduled date, with contamination. the information and specimens collected being assigned to the Statistical Analyses originally scheduled follow-up visit. As a result, the same num- ber of scheduled visits was retained, precluding missing interval For the analyses reported here, follow-up began in November visits. However, all time-to-event analyses were based on the 1993 and continued through mid-April 2002. Subjects with actual time of the visits. Cervical specimens were taken for atypical squamous cells of undetermined significance (ASCUS), conventional Pap smear and HPV testing at every visit. The LSIL, or HSIL on cytology were included in each risk set at the study nurses also performed a detailed interview at enrollment to time of their first detected result. Depending on the index lesion collect information on sociodemographic factors, reproductive of interest, prevalent cases of equivalent grade of abnormality at health, sexual activity, and smoking status. Information on sex- enrollment were excluded from the risk set at baseline. Subjects ual activity and reproductive health was also collected at each who were biopsied were censored at the time of their biopsy if return visit during the first 12 months and annually thereafter. no transition event had occurred before the biopsy date to reduce the potential for interference by the biopsy procedure on esti- Cervical Cell Specimens mates of time to regression. Women who dropped out of the study were censored at their last visit date. In time-to-event An Accelon biosampler (Medscand, Hollywood, FL) was analyses, time to event was measured from the date of the index used to collect ecto- and endocervical samples. After cells were visit (i.e., the first instance of an abnormal cytologic result) to smeared on a glass slide and fixed for Pap cytology, the sampler the date of the visit at which transition to a more or less severe containing the residual exfoliated cells was immersed in a tube cytologic category (for estimates of progression or regression, containing Tris–EDTA buffer (pH 7.4) (7). The exfoliated cell respectively) was first detected or, for censored subjects, to the samples were sent to the Ludwig Institute for Cancer Research last recorded return visit date. Such data represent interval- in Sa˜o Paulo for storage and HPV testing. The Pap smears were censored data because the exact dates of HPV infection and SIL shipped to McGill University for coding and classification by an incidence are not known. The time to regression from HSIL or expert cytopathologist (A. Ferenczy) who was blinded to previ- LSIL was defined as the time from the index visit until the first ous cytology outcomes and to HPV results for the same and follow-up visit at which a subject presented with LSIL, ASCUS, previous samples. The reports were based on the or a normal Pap smear (depending on the regression endpoint of 1991 for cytologic diagnoses (8). Referral to interest), whether or not a worse cytologic event had been de- was made by the Study Management Center at Mc- tected during intervening visits. Times to progression or regres- Gill University on the basis of either local or review cytology sion were evaluated for the overall group at risk by age and reports or the cervicography examination, which was performed ethnic group and for groups stratified by HPV status at the visit once every 2 years for all women. when the index lesion was first detected or at the visit closest to The progression and regression states were classified by le- the index event at which a valid HPV result was obtained. On- sion severity (LSIL or HSIL) and were subcategorized within cogenic status of HPV in the index specimen was coded accord- these lesional grades. LSILs were separated into those with koi- ing to the following hierarchic categories: 1) no HPV detected, locytosis (LSIL/HPV) and those without koilocytosis but with 2) only non-oncogenic types detected in the index specimen, and squamous abnormalities (LSIL/SQ). Similarly, HSILs were 3) any oncogenic HPV type detected. separated into those indicative of CIN2 (HSIL/CIN2) and those The cumulative probability of a lesion’s remaining in the indicative of CIN3 (HSIL/CIN3). same stage or progressing to the next stage was estimated by

Journal of the National Cancer Institute, Vol. 95, No. 17, September 3, 2003 ARTICLES 1337 actuarial analysis using Kaplan–Meier curves (12) as a function were 71% of HSIL smears (15 oncogenic types and two non- of the length of follow-up, stratified by HPV status at the index oncogenic types). visit. The life table method was also used to estimate the pro- The total follow-up time for subjects with no lesions at en- portion of women who remained positive for a precursor lesion rollment was 128 129 person-months, for a mean follow-up du- during follow-up without progressing to a higher stage, accord- ration of 53.3 months per subject. The average interval between ing to HPV status at the index visit (13). Ninety-five percent visits was 4.8 months for the first year and 6.8 months for confidence intervals (CIs) for the actuarial estimates were cal- subsequent years. Although visits were scheduled according to culated using the standard error of the cumulative probability at the study design to occur every 6 months after the first year, the the end of a particular interval in which an event occurred (14) actual intervals between visits in the second and subsequent or, for binomial proportions, via exact estimation. Statistical years ranged from 2.9 months to 81.3 months. comparisons (two-sided tests) of lesion sojourn times according The majority of ASCUS (147/173) and LSIL (104/118) to HPV status were performed by using the log-rank test. Pro- events detected by cytology regressed to a lower grade during gression and regression density estimates were also calculated follow-up (Table 1). Actuarial analyses showed that half of these by dividing the number of incident events (of progression or abnormalities regressed within 6 months of first detection. The regression, respectively) by the length of follow-up in person- overall mean time to regression of ASCUS abnormalities was Downloaded from https://academic.oup.com/jnci/article/95/17/1336/2520427 by guest on 29 September 2021 months of subjects at risk (i.e., those with the index lesion shorter than that of LSILs. The mean time to regression from event). Density estimates were likewise stratified by HPV status LSIL to ASCUS or normal was generally longer for lesions with of the index lesion. Density rates and mean durations were com- oncogenic HPV types (13.8 months) than for lesions with non- ,months 6.0 ס pared by pooled variance z tests and t tests, respectively. Statis- oncogenic HPV types (7.8 months) (difference to 12.7 months) or for HPV-negative lesions 0.7– ס tical analyses were performed by using SPSS version 11.0 95% CI to 11.4 1.0 ס months, 95% CI 6.2 ס SPSS, Chicago, IL) and PEPI version 4.0× (Sagebrush Press, (7.6 months) (difference) Salt Lake City, UT). months, respectively). Median durations, however (i.e., 6.1, 5.3, For non-actuarial estimates of mean lesion duration, we used and 6.0 months for LSILs with oncogenic HPV types, with a standard formula based on the epidemiologic tenet that, within non-oncogenic HPV types, and without HPV, respectively), dif- a stationary population and in the absence of migration, the fered less than the respective means. Similar differences in re- prevalence proportion (P) is a function of the incidence rate (I) gression times within levels of HPV status were observed for the and of the mean duration (D) of the condition. Therefore, the subcategories of LSIL. ס average duration can be estimated with the general formula D To evaluate mean sojourn times with respect to cumulative P/[I ×(1–P)], where P is calculated as a weighted average of HPV status during the first year of follow-up, we also calculated the point prevalence over time for each lesion grade. This for- mean lesion duration based on a non-actuarial formula (see the mula holds provided that the point prevalence within each stra- “Subjects and Methods” section) in which subjects were strati- tum is less than 10% (15), a condition that was met for all of the fied by cumulative HPV status during the first year of follow-up. individual lesion grades analyzed in this study. The non- Given the particular relevance of HPV16 in cervical cancer eti- actuarial estimates were stratified by the cumulative HPV status ology, subjects who tested positive for that type in any of the over the first year of follow-up, taking into account test results visits were evaluated separately from those with other oncogenic from the first four visits. HPV types. The mean ASCUS duration in women who were RESULTS HPV-negative at all four visits was 7.9 months, which was lower than that in women with only non-oncogenic HPV types (10.5 Of the 4990 women initially identified, 3589 women initially months), in women with any oncogenic HPV type (excluding met the eligibility criteria and were invited to participate in the HPV16 at any visit [15.4 months]), and in women positive for study. Between November 1993 and March 1997, 2528 women HPV16 at any visit (13.4 months). The equivalent mean LSIL were enrolled in the study, a response rate of 70.4%. A further durations were 8.9, 10.3, 12.2, and 13.4 months for HPV-negative 66 women who were found not to fit the eligibility criteria women and women with non-oncogenic HPV types, oncogenic were excluded after enrollment. Fifty-one women (2.1%) HPV types, and HPV16, respectively. The equivalent mean HSIL presented with prevalent lesions (LSIL or HSIL) at enrollment, durations were 7.6, 5.7, 15.6, and 57.0 months, respectively. and seven women had unsatisfactory baseline cytology results. With respect to progression to a higher preinvasive lesion Thus, 2404 women remained for analysis. Among these grade, we observed the reverse of the pattern described above for women, 131 (5.4%) developed incident SIL events: 118 de- regression by HPV infection status (Table 2). That is, lesions in veloped LSIL and 24 developed HSIL over the period of follow- subjects with no HPV detected in the index specimen took up, although these were not mutually exclusive events. Among longer to progress than those with oncogenic HPV. For example, women without any abnormal smears (i.e., excluding those mean time to progression from ASCUS to LSIL or worse and -there from LSIL to HSIL or worse was shorter for women with on ,(2361 ס with ASCUS or worse) at enrollment (N were 173 incident reports of ASCUS within the 8-year follow- cogenic HPV types than for women with no HPV infection (for -and 88.0 months, respec 67.0 ס up period. The prevalence of oncogenic and non-oncogenic ASCUS progression, means to 30.7 11.3 ס months [95% CI 21.0 ס HPV types was similar across visits and ranged from 7.8% to tively; difference ,and 83.5 months 73.3 ס for oncogenic types and from 6.2% to 7.6% for non- months]; for LSIL progression, means 9.2% to 0.15– ס months [95% CI 10.2 ס oncogenic types. With respect to HPV prevalence by level of respectively; difference incident abnormality detected by cytology, 32% of ASCUS 20.6 months]) or in women with non-oncogenic HPV types, with smears were HPV-positive (31 oncogenic types and 24 non- respect to LSIL- to HSIL progression. Among women with per- data not shown), 20% progressed to HSIL ,24 ס oncogenic types), whereas 61% of LSIL smears were HPV- sistent LSIL (n positive (45 oncogenic types and 28 non-oncogenic types), as or cervical cancer during follow-up.

1338 ARTICLES Journal of the National Cancer Institute, Vol. 95, No. 17, September 3, 2003 Table 1. Actuarial estimates and 95% confidence intervals (CIs) of time to regression of first incident cervical abnormality events stratified by human papillomavirus (HPV) status in index lesions*

Index lesions and No. of regression event, events/ Person- Regression Median time to Mean time to Proportion¶ remaining with abnormality (95% CI) at HPV status in total No. months of density rate§ regression, mo࿣ regression, mo࿣ index lesion† of lesions‡ follow-up (95% CI) (95% CI) (95% CI) 6mo 12mo 18mo 24mo

ASCUS to normal Overall 147/173 1325.9 11.1 (9.4 to 13.0) 6.1 (5.9 to 6.2) 9.2 (7.4 to 11.0) 57.1 (49.5 to 64.7) 19.3 (12.8 to 25.8) 6.7 (2.6 to 10.8) 2.7 (0.0 to 5.4) Negative 105/117 827.5 12.7 (10.4 to 15.3) 6.1 (6.0 to 6.2) 7.6 (6.9 to 8.4) 58.0 (48.8 to 67.2) 14.8 (7.9 to 21.7) 4.2 (0.3 to 8.1) 0.0 (0.0 to 52.7) Non-oncogenic 19/24 151.3 12.6 (7.8 to 19.3) 5.8 (5.3 to 6.4) 7.7 (5.2 to 10.2) 45.5 (24.7 to 66.3) 15.2 (0.0 to 32.1) 7.6 (0.0 to 21.1) 7.6 (0.0 to 21.1) Oncogenicն 23/30 341.3 6.7 (4.4 to 9.9) 6.2 (5.9 to 6.5) 16.8 (7.5 to 26.2) 60.7 (42.7 to 78.7) 40.5 (21.7 to 59.3) 16.2 (1.7 to 30.7) 11.6 (0.0 to 24.5) LSIL (any) to ASCUS or normal Overall 104/118 1057.8 9.8 (8.1 to 11.9) 6.0 (5.9 to 6.1) 10.5 (8.1 to 12.9) 48.9 (39.7 to 58.1) 22.3 (14.5 to 30.1) 10.0 (3.9 to 16.1) 7.5 (2.0 to 13.0) Negative 37/44 304.5 12.2 (8.7 to 16.6) 6.0 (5.9 to 6.1) 7.6 (6.4 to 8.7) 50.6 (35.5 to 65.7) 16.0 (4.4 to 27.6) 4.0 (0.0 to 11.4) — Non-oncogenic 27/28 208.7 12.9 (8.7 to 18.5) 5.3 (3.7 to 7.0) 7.8 (5.3 to 10.2) 32.1 (14.9 to 49.3) 17.0 (2.9 to 31.1) 8.5 (0.0 to 19.5) 4.3 (0.0 to 12.3) Downloaded from https://academic.oup.com/jnci/article/95/17/1336/2520427 by guest on 29 September 2021 Oncogenicն 39/44 532.9 7.3 (5.3 to 9.9) 6.1 (4.8 to 7.3) 13.8 (8.8 to 18.7) 58.6 (43.9 to 73.3) 18.0 (3.9 to 32.1) 15.9 (4.5 to 27.3) 13.2 (2.6 to 23.8) LSIL/HPV to ASCUS or normal Overall 47/56 547.5 8.6 (6.4 to 11.3) 6.1 (5.7 to 6.6) 12.1 (8.2 to 15.9) 54.1 (40.8 to 67.4) 25.5 (13.5 to 37.5) 15.3 (4.7 to 25.9) 12.3 (2.3 to 22.3) Negative 15/20 136.0 11.0 (6.4 to 17.8) 6.0 (5.8 to 6.2) 7.8 (5.9 to 9.7) 52.6 (30.1 to 75.1) 15.5 (0.0 to 32.9) 15.5 (0.0 to 32.9) — Non-oncogenic 14/15 137.2 10.2 (5.8 to 16.7) 6.5 (3.1 to 9.8) 9.7 (5.3 to 14.2) 53.3 (28.0 to 78.6) 32.0 (7.9 to 56.1) 16.0 (0.0 to 35.8) 8.0 (0.0 to 22.9) Oncogenicն 17/20 268.3 6.3 (3.8 to 9.9) 11.7 (3.8 to 19.6) 15.3 (7.6 to 23.1) 59.0 (37.2 to 80.8) 30.9 (9.7 to 52.1) 18.5 (0.1 to 36.9) 18.5 (0.1 to 36.9) LSIL/SQ to ASCUS or normal Overall 63/71 598.5 10.5 (8.2 to 13.4) 6.0 (5.9 to 6.1) 9.9 (6.9 to 12.8) 49.4 (37.6 to 61.2) 19.1 (9.5 to 28.7) 8.7 (1.6 to 15.8) 5.2 (0.0 to 10.9) Negative 25/29 198.5 12.6 (8.4 to 18.3) 6.0 (5.9 to 6.1) 7.4 (6.2 to 8.6) 50.0 (31.4 to 68.6) 16.7 (2.0 to 31.4) 0.0 (0.0 to 63.2) — Non-oncogenic 15/15 86.6 17.3 (10.1 to 27.9) 5.8 (5.2 to 6.3) 5.8 (4.8 to 6.7) 26.7 (4.4 to 49.0) 0.0 (0.0 to 52.7) — — Oncogenicն 23/26 307.6 7.5 (4.9 to 11.0) 6.0 (5.9 to 6.1) 14.1 (7.2 to 20.9) 60.8 (41.8 to 79.8) 32.4 (14.0 to 50.8) 20.3 (4.4 to 33.6) 12.2 (0.0 to 25.1) HSIL/CIN2 to ASCUS or normal Overall 13/16 152.1 8.5 (4.8 to 14.2) 6.1 (6.0 to 6.1) 11.6 (5.7 to 17.5) 66.7 (42.8 to 90.6) 14.8 (0.0 to 33.6) 14.8 (0.0 to 33.6) 14.8 (0.0 to 33.6) Negative 6/6 41.8 14.3 (5.8 to 29.8) 6.0 (5.7 to 6.3) 7.0 (5.0 to 8.9) 66.7 (28.9 to 100.0) 0.0 (0.0 to 52.7) — — Non-oncogenic 2/2 17.9 11.2 (1.9 to 37.0) 6.1 (ND) 8.9 (3.3 to 14.6) 100 (22.4 to 100) 0.0 (0.0 to 77.6) — — Oncogenic** 5/8 92.4 5.4 (2.0 to 12.0) 6.0 (0.0 to 13.2) 17.1 (4.1 to 30.1) 57.1 (20.4 to 93.8) 38.1 (0.0 to 77.1) 38.1 (0.0 to 77.1) 38.1 (0.0 to 77.1) low-grade squamous intra-epithelial lesion (LSIL) with koilocytotic atypia induced by a productive ס atypical squamous cells of undetermined significance; LSIL/HPV ס ASCUS* (high-grade squamous intra-epithelial lesion (HSIL ס LSIL showing squamous effects equivalent to cervical intra-epithelial neoplasia grade 1; HSIL/CIN2 ס HPV infection; LSIL/SQ not estimable because of insufficient follow-up time for the group or because ס — ;not determined ס with moderate dysplasia equivalent to cervical intra-epithelial neoplasia grade 2. ND all regression events had already occurred at a previous time point. †Index lesion is defined as the first detected event of the stated cytologic abnormality. ‡Numbers of lesions regressed/total number of index lesions. Number of HPV stratum-specific samples may not add up to the overall number if valid HPV results were unavailable for some samples. §Regression density rate is equal to the number of incident events per 100 person-months. ࿣Estimates from actuarial analysis using the Kaplan–Meier method. ¶Proportion of subjects remaining with lesion at end of interval period (6, 12, 18, or 24 months) derived by life table analysis. նAny oncogenic HPV type in index sample. **No HPV16-positive specimens were identified.

To evaluate whether lesions progressed more quickly with from ASCUS to HSIL or cancer for women with oncogenic HPV age, we estimated rates of progression separately for younger infections were 73.4 months and 80.4 months in older and months, 95% CI 7.0 ס and older women. On average, women aged 31–65 years pro- younger women, respectively (difference .(to 24.2 months 10.2– ס gressed to HSIL from an incident LSIL more rapidly (mean time months) than women aged 16–30 years We also evaluated rates of progression and regression for 77.9 ס to progression subjects with respect to ethnic origin (data not shown). The 10.5 ס months, difference 88.4 ס mean time to progression) to 19.5 months]). By contrast, the mean regression density rates of incident ASCUS, LSIL, and HSIL to 1.5 ס months [95% CI were (856 ס and nonwhites (n (1542 ס time to progression from ASCUS to HSIL was shorter in normal for whites (n younger women (81.8 months) than in older women (90.4 not statistically significantly different (differences in lesion re- -regressed abnormalities per 100 person 0.61 ס to 13.2 gression rates 4.0 ס months [95% CI 8.6 ס months, difference to 11.2]; 2.4 per 100 person-months 10.0– ס months]). After stratification by HPV status, older women with months [95% CI to 14.6]; and 4.7 per 100 person-months [95% 9.8– ס oncogenic HPV types, including HPV16, had a higher cumula- [95% CI -to 76.1], respectively). Similarly, we saw little dif 66.7– ס tive risk of progression to HSIL than younger women, regardless CI of baseline abnormality (Fig. 1), although the differences were ference in rates of progression to HSIL or worse between ethnic not statistically significant. Mean times to progression from groups. LSIL to HSIL or cancer for women with oncogenic HPV infec- Given the potential for biopsy interventions to interfere with tions were 68.4 months in women aged 31–65 years and 75.6 the natural history of HSIL, we estimated the time to regression months, for HSILs for women based on whether their biopsies were 7.2 ס months in women aged 16–30 years (difference -to 24.2 months). Mean times to progression performed before or after a regression event to ASCUS or nor 9.8– ס CI 95%

Journal of the National Cancer Institute, Vol. 95, No. 17, September 3, 2003 ARTICLES 1339 Table 2. Actuarial estimates and 95% confidence intervals (CIs) of time to progression of first incident cervical abnormality events stratified by human papillomavirus (HPV) status in index lesions*

Index lesion and progression event, No. of Person- Progression Mean time to Proportion¶ progressing to worse abnormality (95% CI) at HPV status in events/total months of density rate§ progression, mo࿣ index lesion† No. of lesions‡ follow-up (95% CI) (95% CI) 6mo 12mo 18mo

ASCUS to LSIL or worse Overall 18/174 6867.3 0.3 (0.2 to 0.4) 82.7 (78.4 to 87.1) 3.7 (0.8 to 6.6) 7.9 (3.6 to 12.2) 8.6 (4.1 to 13.1) Negative 6/117 4992.2 0.12 (0.05 to 0.3) 88.0 (84.3 to 91.7) 0.9 (0.0 to 2.7) 3.9 (0.2 to 7.6) 3.9 (0.2 to 7.6) Non-oncogenic 5/24 756.5 0.7 (0.2 to 1.5) 67.9 (51.9 to 83.9) 9.8 (0.0 to 22.7) 21.5 (2.7 to 40.3) 21.5 (2.7 to 40.3) Oncogenicն 7/31 1112.8 0.63 (0.3 to 1.2) 67.0 (54.6 to 79.5) 10.3 (0.0 to 21.5) 14.2 (1.3 to 27.1) 18.2 (3.7 to 32.7) LSIL (any) to HSIL or worse Overall 11/119 5365.5 0.2 (0.1 to 0.4) 85.7 (80.8 to 90.6) 1.7 (0.0 to 4.1) 3.6 (0.1 to 7.1) 6.5 (1.8 to 11.2) Negative 2/44 1676.3 0.12 (0.02 to 0.4) 83.5 (78.0 to 89.1) 0.0 (0.0 to 6.6) 0.0 (0.0 to 7.0) 2.8 (0.0 to 8.3) Non-oncogenic 1/28 1563.8 0.06 (0.0 to 0.3) 91.3 (85.1 to 97.4) 0.0 (0.0 to 10.1) 3.6 (0.0 to 10.7) 3.6 (0.0 to 10.7) Oncogenicն 8/45 2107.7 0.38 (0.2 to 0.7) 73.3 (64.8 to 81.8) 4.6 (0.0 to 10.7) 6.9 (0.0 to 14.5) 11.9 (2.1 to 21.7) Downloaded from https://academic.oup.com/jnci/article/95/17/1336/2520427 by guest on 29 September 2021 LSIL/SQ to HSIL/CIN3 Overall** 4/72 3482.6 0.11 (0.04 to 0.3) 89.2 (84.2 to 94.2) 1.0 (0.0 to 3.0) 1.4 (0.0 to 4.1) 3.0 (0.0 to 7.1) LSIL/HPV to HSIL/CIN3 Overall** 2/56 2266.5 0.09 (0.01 to 0.3) 86.4 (81.9 to 90.9) 1.8 (0.0 to 5.3) 1.8 (0.0 to 5.3) 1.8 (0.0 to 5.3) low-grade squamous intra-epithelial lesion (LSIL) showing squamous effects ס atypical squamous cells of undetermined significance; LSIL/SQ ס ASCUS* ס LSIL with koilocytotic atypia induced by a productive HPV infection; HSIL/CIN3 ס equivalent to cervical intra-epithelial neoplasia grade 1; LSIL/HPV high-grade squamous intra-epithelial lesion (HSIL) with severe dysplasia equivalent to cervical intra-epithelial neoplasia grade 3. †Index lesion is defined as the first detected event of the stated cytologic abnormality. ‡Number of lesions progressed/total number of index lesions. Number of HPV stratum-specific samples may not add up to the overall number if valid HPV results were unavailable for some samples. §Progression density rate is equal to the number of incident events per 100 person-months. ࿣Estimates from actuarial analysis using the Kaplan–Meier method. Median time to progression was not estimated as less than 50% of the index lesions progressed. Mean times were restricted to the longest follow-up time, regardless of event status. ¶Proportion of subjects progressing to worse abnormality at end of interval period (6, 12, or 18 months) derived by life table analysis. նAny oncogenic HPV types in baseline smear. **All baseline (index) LSIL events were oncogenic HPV-positive. mal was detected by cytology during study follow-up (data not the natural history of HPV and cervical neoplasia suggests that shown). As the study progressed, recommendations for biopsy a relationship between the likelihood of precursor lesions per- became more aggressive. Biopsies were done at the discretion of sisting and progressing to cancer is dependent on the character- the attending colposcopists if lesional tissue could be visualized istics of the HPV infection (4,6,19,20). In the current longitudi- under magnification. Of the 24 women with incident HSIL in- nal study, we found that precursor lesions of the cervix detected cluded in the analyses of regression, 23 had a biopsy, at an by cytology persisted longer and were more likely to progress in average of 27.9 months after the first cytologic detection of women with oncogenic HPV infections than in women with HSIL. Some lesions appeared to regress as determined by cy- non-oncogenic HPV infections or in uninfected women. Using tology before the biopsy was performed. We therefore evaluated repeated screening by cytology, we were able to evaluate the rates of regression separately among women whose biopsy was progression and regression of cervical lesions over time in a taken before regression was determined by cytology and among more systematic fashion than that in previous studies, which women whose biopsy was taken after (including the subject who involved registry data and screening programs based on passive did not have a biopsy). Irrespective of biopsy status, the mean data collection. time to regression of all incident HSILs was 12.8 months (95% Our study does have several limitations. First, given that our ס CI 7.6 to 18.0 months). Although the number of HSILs with outcome ascertainment was based on cytologic analysis, one ס ס (n 12) and without (n 12) possible biopsy interference was potential limitation is misclassification of lesion outcome his- small, it appeared that women who had a biopsy before a re- tory, even though the cytologic assessments were carefully con- gression event (to ASCUS or normal) was detected by cytology ducted in a reference laboratory following a strict quality-control ס retained their lesions longer (mean time to regression 16.9 protocol. We opted for an intensive, expert cytologic review of months) than those whose biopsy was performed after regression all subjects in the study every 4–6 months and referred all in- ס ס ס (mean 8.3 months; difference 8.6 months, 95% CI –1.0 stances of HSILs for colposcopy. This approach reduced the to 18.1 months). The mean duration of HSIL persistence after likelihood of unnecessary biopsies, which can interfere with the having the biopsy (calculated by subtracting time to biopsy from natural history of early lesions (21). Nevertheless, the occur- time to regression) was 5.6 months. rence of false-negative Pap tests could have resulted in under- DISCUSSION estimates (or shorter estimates) of regression time and in either overestimates or underestimates of progression time, depending To date, few studies have investigated rates of progression on whether these test results occurred at lesion outset or during and regression of preinvasive cervical lesions in relation to cer- the sojourn period. In a prospective study of histologically di- vical cancer risk factors (2,3,16–18). The available evidence on agnosed carcinoma in situ (CIS), McIndoe et al. (16) found that

1340 ARTICLES Journal of the National Cancer Institute, Vol. 95, No. 17, September 3, 2003 Fig. 1. Kaplan–Meier graphs for time to progression to high-grade squa- mous intraepithelial lesions (HSILs) from incident low-grade squamous intraepithelial lesions (LSILs) and atypical squamous cells of undeter- mined significance (ASCUS) de- tected by cytology according to age group and human papillomavirus (HPV) infection status in the index sample. A) progression of LSIL to HSIL in women aged 16–30 years, B) progression of LSIL to HSIL in women aged 31–65 years, C) pro- gression of ASCUS to HSIL in women aged 16–30 years, and D)

progression from ASCUS to HSIL in Downloaded from https://academic.oup.com/jnci/article/95/17/1336/2520427 by guest on 29 September 2021 women aged 31–65 years. Mutually exclusive categories for HPV infec- tion status in the index sample: nega- tive (dotted line), only non- oncogenic HPV types (dashed line), and any oncogenic HPV types (solid line). The ratios of number of events during follow-up to number at risk for each HPV category at the index visit are A) HPV-negative (1/19), non-oncogenic HPV (0/13), onco- genic HPV (3/25); B) HPV-negative (1/15), non-oncogenic HPV (1/25), oncogenic HPV (5/20); C) HPV- negative (2/42), non-oncogenic HPV (0/12), oncogenic HPV (2/26); and D) HPV-negative (1/93), non-oncogenic HPV (0/19), oncogenic HPV (4/18). The cumulative risk (95% confidence intervals [CIs]) at 48 months accordingtoHPV (0.0to0.29);B)0.14 ס to 0.22), oncogenic HPV 0.0) 0.0 ס to 0.23), non-oncogenic HPV 0.0) 0.08 ס status in the index sample are A) HPV-negative ,(to 0.21 0.0) 0.09 ס to 0.39); C) HPV-negative 0.03) 0.21 ס to 0.20), oncogenic HPV 0.0) 0.07 ס to 0.15), non-oncogenic HPV 0.0) 0.05 ס HPV-negative ,(to 0.26 0.0) 0.0 ס to 0.05), non-oncogenic HPV 0.0) 0.02 ס to 0.22); and D) HPV-negative 0.0) 0.09 ס to 0.31), oncogenic HPV 0.0) 0.0 ס non-oncogenic HPV .(to 0.37 0.0) 0.18 ס oncogenic HPV

54% of the cases had normal cytology during screening after a tivity by cytologic category in the present study are not substan- punch biopsy, with only one case of six developing invasive tially different from other published estimates for ASCUS and carcinoma within 4 years. We therefore censored subjects at the LSIL patients (23), which reflects positively on the quality of the time of their biopsy, anticipating that the procedure could influ- cervical specimens taken, as well as on the sensitivity of the ence the rates of disease determined by cytology. PCR-based method we used for HPV DNA detection. In this Studies using both histology and cytology to follow the natu- analysis, we chose to use the HPV DNA test results obtained ral history of cervical neoplasia have shown no effect of limited from the same specimen that was used for Pap cytology. This sampling by punch biopsy on the short-term course of dysplasia decision was based on the assumption that finding HPV in in- (22). In a review of 27 studies of CIN, Mitchell et al. (2) ob- cident lesions is a proxy for prior HPV infection states that led served similar probabilities of regression, persistence, and pro- to the lesion. Cross-sectional assessment of HPV and lesion gression based on biopsy evaluation and cytology. We found status precludes the determination of directionality in the asso- that HSIL persisted for an average of 5.6 months following a ciation between infection and lesion development. However, this biopsy. Although persistent lesions are more likely to be biop- approach was chosen to generate data similar to those obtained sied before regression than lesions of short duration, we found in screening and on which triage decisions are usually based. no evidence that biopsy affected the persistence of HSILs, at We used two methods to calculate the mean duration or so- least in the short term. Although more aggressive standards for journ time of incident cytologic lesions: an actuarial method and biopsy were adopted after a number of years into the study by a non-actuarial formula—the prevalence–incidence relation. Es- the local colposcopists (all women referred for colposcopy are timates of mean duration based on actuarial probability esti- currently obtaining biopsies following our recommendations), mates indicated that lesions with oncogenic HPV infections per- we cannot exclude the possibility that the biopsy procedures sisted longer than lesions with non-oncogenic HPV infections or performed earlier in the study were preferentially done for le- than lesions without HPV infection. In general, the average du- sions that appeared more severe on colposcopy. Such lesions ration estimates based on the non-actuarial formula were similar may have taken longer to regress, regardless of whether a biopsy to those obtained with the actuarial method. However, the mean had been done. duration of HSIL estimated by the non-actuarial formula was Another possible limitation was misclassification of HPV sta- longer than the equivalent estimate by actuarial analysis for tus. Misclassification was unlikely because we used a highly subjects with HPV16. The non-actuarial prevalence–incidence sensitive PCR-based testing method. The estimates of HPV posi- relation method is appropriate for estimating average duration of

Journal of the National Cancer Institute, Vol. 95, No. 17, September 3, 2003 ARTICLES 1341 incident conditions, such as cytologic abnormalities consistent Of the reviews indicated above, few evaluated progression or with ASCUS, LSIL, or HSIL that rarely exceed a prevalence of regression by risk stratifiers. Holowaty et al. (3) examined the 10% in most clinical settings (15). However, the non-actuarial influence of parity, age, oral contraceptive use, and number of formula does not account for censored data (i.e., incomplete positive smears on the relative risk of progression of LSILs. observations due to lesions that have not cleared at the study They found no relationship between these factors and the rela- closing date or losses to follow-up). By contrast, actuarial esti- tive risk of progression. Similarly, in our study, the mean time to mates of time to progression were restricted to the longest fol- progression was not statistically significantly different between low-up time available. As a result, mean times were underesti- age groups, although we found that, among women with high- mated when the largest observed analysis time occurred for a risk oncogenic HPV types, progression rates were higher in censored subject. This effect was greater for strata with smaller women 31 years and older than in younger women. These find- numbers of observations. Therefore, we did not separate out the ings support the World Health Organization’s recommendation subjects with HPV16 in the actuarial analyses. This difference in for focusing screening on older women at risk for cervical cancer estimates from the actuarial and non-actuarial analyses under- (24,25). We also observed shorter progression transit times from scores the fact that, due to the heterogeneity of follow-up times ASCUS to LSIL and worse in women with non-oncogenic (low- and censoring, no single statistical summary measure is appro- risk) and oncogenic (high-risk) HPV types compared with Downloaded from https://academic.oup.com/jnci/article/95/17/1336/2520427 by guest on 29 September 2021 priate to capture the average progression or regression times of women who had HPV-negative abnormalities. This observation lesions in a repeated-measurements cohort study. Whenever ap- provides further evidence of the importance of colposcopic propriate, therefore, we reported all three summary statistics evaluation of women with HPV-positive ASCUS smears, par- (non-actuarial means, actuarial means, and actuarial medians) to ticularly those with high-risk oncogenic HPV types (23). provide a more complete picture of transit times. In conclusion, using screening tests for oncogenic HPVs may help identify those lesions that are likely to progress quickly to It is conceivable that using shorter cytologic screening inter- more advanced stages. HPV testing of women with abnormal vals could have resulted in more precise estimates of the Pap smears may therefore help identify women who might ben- duration of lesion sojourn times. Indeed, the reduction in follow- efit from colposcopic evaluation and, if appropriate, chemopre- up frequency to every 6 months after the first year (when return ventive treatment. The ability to identify subjects whose lesions visits were scheduled at 4 months) may have increased the are likely to take longer to progress could also be cost-saving by observed sojourn time. However, this effect would have been increasing the follow-up intervals and reducing the morbidity equal across HPV groups because all cytology and HPV evalu- which may result from potentially unnecessary invasive diag- ations were carried out blindly with respect to previous results nostic and therapeutic procedures. for the same subject, making comparisons valid on a relative scale. REFERENCES Several reviews and meta-analyses have attempted to sum- marize rates of progression and regression along the continuum (1) Meijer CJ, van den Brule AJ, Snijders PJ, Helmerhorst T, Kenemans P, of cervical neoplastic changes. Östor (17) reported decreasing Walboomers JM. Detection of human papillomavirus in cervical scrapes by probabilities of regression for different CIN grades of increasing the polymerase chain reaction in relation to cytology: possible implications severity detected by histology. Mitchell et al. (2) observed prob- for cervical cancer screening. 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(18) Melnikow J, Nuovo J, Willan AR, Chan BK, Howell LP. Natural history of search, by Public Health Service grant CA70269 (to E. L. Franco, L. L. Villa,A. Downloaded from https://academic.oup.com/jnci/article/95/17/1336/2520427 by guest on 29 September 2021 cervical squamous intraepithelial lesions: a meta-analysis. Obstet Gynecol Ferenczy, and T. E. Rohan) from the National Cancer Institute, National Insti- 1998;92:727–35. tutes of Health, Department of Health and Human Services, and by grant (19) Londesborough P, Ho L, Terry G, Cuzick J, Wheeler C, Singer A. Human MA13647 (to E. L. Franco and A. Ferenczy) from the Canadian Institutes of papillomavirus genotype as a predictor of persistence and development of Health Research. N. F. Schlecht is a recipient of a pre-doctoral scholarship, R. high-grade lesions in women with minor cervical abnormalities. Int J Can- W. Platt is the recipient of a scientist award, and E. L. Franco is the recipient of cer 1996;69:364–8. a distinguished scientist award, all from the Canadian Institutes of Health Re- (20) Schlecht NF, Kulaga S, Robitaille J, Ferreira S, Santos M, Miyamura RA, search. et al. Persistent human papillomavirus infection as a predictor of cervical We thank Maria L. Baggio and Lenice Galan for management of the patients intraepithelial neoplasia. JAMA 2001;286:3106–14. and specimen collection, Silvaneide Ferreira and Romulo Mayamura for HPV (21) Richart RM. Influence of diagnostic and therapeutic procedures on the testing, and Juliette Robitaille for cytology reviews. distribution of cervical intraepithelial neoplasia. Cancer 1966;19:1635–8. Manuscript received January 21, 2003; revised June 18, 2003; accepted July (22) Youkeles L, Forsythe AB, Stern E. Evaluation of Papanicolaou smear and 3, 2003.

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