PERSISTENCE AND LOAD OF HIGH-RISK HPV ARE PREDICTORS FOR DEVELOPMENT OF HIGH-GRADE CERVICAL LESIONS: A LONGITUDINAL FRENCH COHORT STUDY

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1 Int. J. Cancer: 106, (2003) 2003 Wiley-Liss, Inc. Publication of the International Union Against Cancer PERSISTENCE AND LOAD OF HIGH-RISK HPV ARE PREDICTORS FOR DEVELOPMENT OF HIGH-GRADE CERVICAL LESIONS: A LONGITUDINAL FRENCH COHORT STUDY Véronique DALSTEIN 1, Didier RIETHMULLER 1,2, Jean-Luc PRÉTET 1, Karine LE BAIL CARVAL 2, Jean-Loup SAUTIÈRE 2, Jean-Pierre CARBILLET 3, Bernadette KANTELIP 3, Jean-Patrick SCHAAL 1,2 and Christiane MOUGIN 1 * 1 Laboratoire de Biologie Cellulaire et Moléculaire, Institut d Etude et de Transfert de Gènes, Centre Hospitalier Universitaire, Besançon, France 2 Clinique Universitaire de Gynécologie, Obstétrique et de la Reproduction, Centre Hospitalier Universitaire, Besançon, France 3 Laboratoire d Anatomo-pathologie, Centre Hospitalier Universitaire, Besançon, France Oncogenic HPV types are the major cause of worldwide cervical cancer, but only a small proportion of infected women will develop high-grade cervical intraepithelial neoplasia or cancer (CIN2/3 ). We performed a prospective study including 781 women with normal, atypical squamous cells of undetermined significance (ASCUS) or low-grade squamous intraepithelial lesion (LGSIL) cytology, and infected or not by high-risk (HR) HPV tested by Hybrid Capture II. Women were followed up every 6 months for a median period of 22 months. Among the HR-HPV-positive women at entry, more than half cleared their virus in 7.5 months; the clearance rate was greater for low viral loads than for high loads and also was higher in women with an initial ASCUS/LGSIL smear than in women with normal cytology. The incidence of cytologic abnormalities strongly depended on baseline viral load and HR-HPV persistence. Maintenance of cytologic abnormalities was associated with the outcome of HR-HPV status (negative<transient<persistent) but not with baseline load. Progression to CIN2/3 was achieved only in women with persistent HR-HPV infection. The risk of CIN2/3 also was increased with initial high loads (>100 pg/ml). Conversely, women who were consistently HR-HPV negative or transiently HR-HPV positive, whatever the cytology at baseline was, did not develop CIN2/3 during follow-up. seemed to affect only the rate of incident HR-HPV infection. In conclusion, our data suggest that women repeatedly tested positive for HR-HPV are at risk of developing CIN2/3, even when initial cytology is normal. A high viral load could be used as a short-term marker of progression toward precancerous lesions, although lower load does not inevitably exclude progressive disease Wiley-Liss, Inc. Key words: human papillomavirus (HPV); cervical cancer; cervical intraepithelial neoplasia; high-grade squamous intraepithelial lesions (HGSIL); Hybrid Capture II High-risk human papillomavirus (HR-HPV) infection has been recognized as the necessary cause of cervical cancer since the 1990s. 1 3 However, HPV infection also is the most common sexually transmitted infection, its prevalence being especially high in young women. 4,5 Thus, the majority of HPV infections are transient, usually cleared in 8 10 months, 6,7 and only a few of them will actually lead to invasive cervical carcinoma During the course of cervical disease, high-grade squamous intraepithelial lesions (HGSIL), including cervical intraepithelial neoplasia grades 2 and 3 (CIN2-3), are considered as the cervical cancer precursors. Several longitudinal studies have consistently reported that persistence of HR-HPV in the genital tract was necessary for the development and progression of cervical dysplastic lesions. 4,6,11 13 Conversely, in women found to be HR-HPV negative, HGSIL seemed to be unlikely to develop at least during a follow-up (FU) of 2 years, and smears showing mild or borderline atypia returned to normal. 10,14 Since HR-HPV infection is the major risk factor for the appearance of cervical carcinomas and since sensitive and reproducible methods for HR-HPV DNA detection are available, HR-HPV status should be determined in screening and management of women at risk for progression to CIN2, CIN3 or worse (CIN2/ 3 ). 15,16 However, the use of HPV testing combined with cervical cytology to detect cervical cancer or its precursors is still controversial. 17,18 In addition, if it is well accepted that high-grade lesions must be treated to avoid invasive cancer, management of low-grade SIL (LGSIL) and atypical squamous cells of undetermined significance (ASCUS) smears is much more confusing, and now there is no clear consensus for the use of HPV testing in routine clinical practice. Recent data have also pointed out that viral load could be used as a sensitive indicator for progression to HGSIL, whereas others have shown that HPV load had no relation to risk of CIN3. 23,24 To address these questions, we enrolled in a prospective study a hospital-based cohort of women with normal, ASCUS or LGSIL smears infected or not by HR-HPV, and we investigated clinical outcome, especially progression to CIN2/3, according to HPV persistence and viral load. MATERIAL AND METHODS Among the 5,800 cohort women attending the Gynaecology Clinic of the Besançon s University Hospital between August 1997 and January 2002, 781 women with normal, ASCUS or LGSIL smears were available for long-term FU. Women with HGSIL were excluded. At enrollment and at each FU visit scheduled every 6 months, women underwent a pelvic examination, and 2 samples of exfoliated cells were obtained from the cervix: a first Abbreviations: ASCUS, atypical squamous cells of undetermined significance; CI, confidence interval; CIN, cervical intraepithelial neoplasia; CIN2/3 : cervical intraepithelial neoplasia grade 2, 3 or worse; FU, follow-up; HGSIL, high-grade squamous intraepithelial lesion; HR-HPV, high-risk human papillomavirus; LGSIL, low-grade squamous intraepithelial lesion; RLU, relative light units; RR, relative risk. Presented in part at the 19th International Papillomavirus Conference, Florianopolis, Brazil, September 2001, and selected for awards (Papillomavirus Report, vol. 12, no. 6, ). Grant sponsor: Ligue Contre le Cancer, Comité du Doubs. *Correspondence to: Laboratoire de Biologie Cellulaire et Moléculaire, Centre Hospitalier Universitaire, Hôpital Jean Minjoz, Bld Fleming, Besançon cedex, France. Fax: christiane.mougin@ufc-chu.univ-fcomte.fr Received 28 October 2002; Revised 11 March 2003; Accepted 19 March 2003 DOI /ijc.11222

2 HPV PERSISTENCE AND LOAD FOR HGSIL DEVELOPMENT 397 sample was collected with a Cytobrush Plus (Medscand Medical, Malmö, Sweden) for conventional cytology and a second sample was collected with the Digene Cervical Sampler (Digene, Gaithersburg, MD) for HPV testing, placed in a vial containing 1 ml Digene Specimen Transport Medium and stored at 20 C until processed. During the FU, women found to have HGSIL on cytology or 2 consecutive smears in favour of an ASCUS or a LGSIL and/or 2 consecutive positive HR-HPV tests were referred for a colposcopic examination and biopsy if indicated. Only women with histologically confirmed HGSIL or worse (CIN2/3 ) or with discordant cytology and histology diagnosis (HGSIL on smear and CIN1/ condyloma on biopsy) were treated by loop electro-excision procedure (LEEP). The main study endpoint was the diagnosis of a CIN2/3 on the biopsy and/or on the LEEP specimen. In addition, the following events were studied in different subgroups, according to baseline data: (i) incident HPV infection in HPV-negative women; (ii) HPV clearance in HPV-positive women; (iii) incidence of abnormal smears in cytologically normal women; and (iv) persistence of minor or mild cervical abnormalities in women with ASCUS or LGSIL. HPV DNA testing was performed with the Hybrid Capture II (HCII) system (Digene) according to the manufacturer s instructions, using the specific HPV RNA probe cocktail for carcinogenic high-risk HPV types (HR-HPV) 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59 and 68. Presence or absence of HPV DNA in the specimen was defined according to the strength in relative light units (RLU) compared to 1 pg/ml HPV16 DNA-positive control (PC). The sample was considered positive when the ratio of RLU/PC was 1. Values provided an approximate determination of the global HR-HPV DNA load. Data analysis Baseline viral load was arbitrarily stratified in 3 levels: low (1 9 RLU/PC), intermediate (10 99 RLU/PC) and high ( 100 RLU/ PC), and then combined in 2 levels in some analyses. HR-HPV infection course was stratified in 3 groups: (i) transient infection, defined as having negative HPV test(s) at the end of FU after a positive HPV test at enrollment; (ii) persistent infection, defined as having positive HPV tests all along FU; and (iii) incident infection defined as having 1 or several positive HPV tests during FU, after an initial negative HPV test. For the analysis of incident cytologic abnormalities, any first abnormal smear (ASCUS or any SIL) was taken into account. For the analysis of persistent cytologic abnormalities, we focused on females who, at baseline and during the entire period of FU, had abnormal smears whatever the diagnosis was (ASCUS or any SIL). Comparison of means was assessed with Student s t-test. Incidence rates were given in rate per 100 women-months and their 95% confidence intervals. Cumulative probabilities and their 95% CI were estimated using the Kaplan-Meier method. In univariate analysis, the different categories of variables were compared by using a log-rank test. Depending on the estimated effect in this univariate analysis, the categories of several variables were combined for the multivariate analysis, and estimates of the longitudinal relative risks (RR) and respective 95% CI of the different events were calculated from the Cox proportional hazard model. For incident CIN2/3, analysis by Cox regression was restricted to the women who were HPV positive at entry to be able to consider the viral load in the model. All s presented were 2-sided and considered as significant when Statistical analysis was performed using the STATA Statistical Software (Release 5.0, 1997, Stata, College Station, TX). RESULTS Characteristics of the study population At enrollment, the median age of the 781 patients eligible for analysis was 35.7 years (range 16 76). repartition of the study population is presented in Table I. The mean number of visits was TABLE I AGE REPARTITION OF THE 781 WOMEN INCLUDED IN THE STUDY, PREVALENCE OF HR-HPV INFECTIONS AND PREVALENCE OF CYTOLOGIC ABNORMALITIES (ASCUS OR LGSIL) group No. subjects (% by column) No. HR-HPV positive (% by row) No. ASCUS/LGSIL smears (% by row) (2.82) 14 (63.64) 11 (50.00) (24.20) 98 (51.85) 42 (22.22) (38.54) 84 (27.91) 31 (10.30) (24.58) 49 (25.52) 26 (13.54) (9.86) 13 (16.88) 11 (14.29) Total 781 (100) 258 (33.03) 121 (15.49) 2.5 and the median FU period was 22 months (range 3 52). The baseline cytology included 652 normal smears (83.5%), 24 AS- CUS (3.1%), 97 LGSIL (12.4%) and 8 unsatisfactory smears (1%). The 8 women with inadequate baseline smears were excluded from further analysis. Among them, 1 was HPV positive and 7 HPV negative. Their mean age was 33.0 years, and the mean duration of FU was 18.6 months. All of them tested cytologically normal at the second visit. None of them developed a CIN2/3 during FU. The overall prevalence of HR-HPV was 33% at entrance in the study (Table I). The rate of HPV infection was 27% (176/652) in normal smears, 54% (13/24) in ASCUS smears and 70% (68/97) in LGSIL smears. The median age of HPV-positive women was significantly lower than that of HPV-negative women: 32.2 years (range 16 65) and 37.7 years (range 16 76), respectively (p ). Among HPV-positive women, median age was 33.0 years in those with normal smears and 27.9 years in those with abnormal smears (p 0.036). Incidence of HR-HPV infection Among the 516 women who were HPV negative at entrance in our study, the rate of cumulative incident-positive HR-HPV testing during FU was 1.62% at 12 months (95% CI ), 5.50% at 24 months (95% CI ) and 13.25% at 36 months (95% CI ). Among the 41 women with incident infection, 33 (80.5%) were cytologically normal at baseline, 7 (17.1%) demonstrated an LGSIL and 1 (2.4%) an ASCUS smear. Obviously, HPV infection acquisition significantly depended on age; the cumulative 36-month incidence was 27% in women aged 30 years vs. 10% in women 30 years old (log-rank test: p ). So in our cohort, women 30 years old were 4-fold less likely to have an incident HPV infection compared to younger women (RR 0.25; 95% CI ; p ). Also there was an increased risk of incident infection associated with an abnormal cytology at baseline (RR 6.7, 95% CI ). Viral clearance in HR-HPV-positive women Among the 257 women who tested HR-HPV positive at study entry, 130 (50.6%) cleared their viral infections in a median period of 7.5 months (range 3 42). Incidence rates of clearance according to age, baseline cytology and baseline HR-HPV load are presented in Table II. The survival analysis revealed that women with AS- CUS or LGSIL smears were more likely to clear their HPV infections than women with normal smears, whereas women with loads 10 pg/ml were less likely to get free of the virus than women with lower viral loads (Table II). did not influence the rate of definite viral clearance. These findings were confirmed in the multivariate analysis; we observed an RR of 1.67 for clearance among women with ASCUS or LGSIL and an RR of 0.65 among women with baseline viral loads 10 pg/ml. Incidence of cytologic lesions in women with baseline normal cytology Among the 652 women with normal baseline Pap smears, 5 were excluded for further analysis because of unsatisfactory smears at the end of FU. Thus, among the 647 eligible women, the rate of incident abnormal cytology was 0.33 per 100 womenmonths, and the cumulative incidence was 4.4% at 12 months and

3 398 DALSTEIN ET AL. TABLE II HPV CLEARANCE IN WOMEN WITH POSITIVE HPV TESTING AT ENTRANCE IN THE STUDY, ACCORDING TO INITIAL AGE, BASELINE CYTOLOGY AND BASELINE HR-HPV LOAD subjects events (%) Womenmonths Rate/100 women-months % of 12-month cumulative incidence (log-rank test) RR 1 of HPV clearance (Cox model) Total (50.6) 3, ( ) ( ) (45.5) 1, ( ) ( ) (referent) (54.5) 1, ( ) ( ) 1.08 ( ) Baseline cytology Normal (51.1) 2, ( ) ( ) (referent) ASCUS/LGSIL (49.4) ( ) ( ) 1.67 ( ) Baseline viral load 1 9 pg/ml (64.7) ( ) ( ) (referent) pg/ml (43.6) 2, ( ) ( ) 0.65 ( ) 1 Calculated from a Cox proportional hazard model. TABLE III INCIDENT ABNORMAL CYTOLOGY (ASCUS OR ANY SIL) IN WOMEN WITH INITIAL NORMAL SMEAR, ACCORDING TO AGE, BASELINE HR-HPV LOAD AND VIRAL INFECTION OUTCOME subjects events (%) Womenmonths Rate/100 womenmonths % of 12-month cumulative incidence (log-rank test) RR 1 of abnormal cytology (Cox model) Total (7.4) 14, ( ) 4.44 ( ) (7.8) 3, ( ) 6.28 ( ) Not considered - in the model (7.3) 11, ( ) 3.93 ( ) Baseline viral load Negative (2.3) 1, ( ) 0.66 ( ) 1 (referent) 100 pg/ml (18.3) 2, ( ) ( ) ( ) pg/ml (29.8) ( ) ( ) 8.66 ( ) HR-HPV outcome Negative (2.3) 12, ( ) 0.66 ( ) 1 (referent) Transient (12.2) 1, ( ) ( ) ( ) Persistent (31.3) 1, ( ) ( ) 9.13 ( ) *Calculated from a Cox proportional hazard model. 7.3% at 24 months (95% CI ) (Table III). The appearance of an abnormal smear was highly associated with initial HR-HPV load and viral persistence but not with age (Table III). These associations were still observed in the multivariate model. Compared to HPV-negative women at baseline, women with low and intermediate viral loads ( 100 pg/ml) were more likely to develop cytologic abnormalities (RR 1.65) as well as women with high viral loads 100 (RR 8.66). Moreover, women with transient and persistent infections were more likely (RR 2.38 and 9.13, respectively) to reveal ASCUS or any SIL during FU than HPVnegative women. Outcome of cytologic abnormalities in women with baseline ASCUS or LGSIL cytology Among the 121 women with a cytology of ASCUS or LGSIL at baseline, further analysis was not available for 3 of them because of consistently inadequate smears. For the 118 eligible patients, the cumulative rate of persistent abnormal cytology was 35.5% at 12 months and 47.4% at 24 months (95% CI ) (Table IV). Again, the outcome of cervical lesions was influenced by the initial HPV status and viral persistence and not by age. The baseline viral load was not specified here because there were no significant differences for persistent lesions when considering low and high viral loads. Comparatively, in the multivariate analysis, only the repeated presence of HR-HPV was a key risk factor for the persistence of cytologic lesions (RR 9.9), whereas a single HPV positivity at enrollment did not appear to be associated with persistence of the lesions. When we focused especially on the 79 women who tested positive for HR-HPV at baseline, 38 became HPV negative during the study period. Among them, 34 were diagnosed as having normal cytology during FU. In the majority of cases (31/34), HPV clearance and cytologic normalization were observed at the same visit. In 1 case, HPV clearance occurred 6 months before cytologic normalization, whereas in 2 cases, HPV clearance occurred 7 months after cytologic normalization. In the remaining 4 cases, cytology still demonstrated LGSIL or ASCUS, whereas HPV DNA became undetectable. Risks for incident CIN2/3 in the overall population Further analysis was dedicated to incident CIN2/3, according to several baseline and follow-up factors. In the overall study population, 31 patients developed a CIN2/3, diagnosed as CIN2 for 13 subjects, CIN3 for 17 and adenocarcinoma for 1. Moderate and severe dysplasia did not occur significantly more frequently in women aged 30 years than in women aged 30 years (Table V). However, a higher rate of incident CIN2/3 has been observed among women who presented cervical abnormalities at baseline (0.67 per 100 women-months, 13.7% of cumulative incidence at 18 months) compared to women having an initial normal Pap smear (0.13 per 100 women-months, 2.9% of cumulative incidence at 18 months). Not surprisingly, there was evidence of an association between development of CIN2/3 and HPV exposure. The rate of CIN2/3 was only 0.19 per 100 women-months when taking into account the global study population, 0.81 per 100 women-months when considering only baseline HPV-positive women and 1.75 per 100 women-months when considering only women who have been persistently exposed to HR-HPV. By contrast, no women who tested HR-HPV negative at baseline progressed to CIN2/3, nor women who cleared their virus during FU. Figure 1 illustrates the cumulative incidence of CIN2/3 according to HPV load at entry in the study and to HPV infection outcome.

4 HPV PERSISTENCE AND LOAD FOR HGSIL DEVELOPMENT 399 TABLE IV PERSISTENCE OF CYTOLOGIC ABNORMALITIES IN WOMEN WITH INITIAL ASCUS OR LGSIL SMEAR, ACCORDING TO AGE, BASELINE HR-HPV TESTING AND VIRAL INFECTION OUTCOME subjects Womenmonths events (%) Rate/100 women-months % of 12-month cumulative incidence (Log-Rank test) RR 1 Total 118 1, (26.7) 2.71 ( ) ( ) (25.0) 2.91 ( ) ( ) Not considered (27.3) 2.60 ( ) ( ) in the model Baseline HR-HPV test Negative (12.8) 1.01 ( ) ( ) (referent) Positive (32.9) 4.02 ( ) ( ) 0.91 ( ) HR-HPV outcome Negative or transient (9.1) 0.89 ( ) ( ) (referent) Persistent (58.5) 6.81 ( ) ( ) 9.94 ( ) 1 Calculated from a Cox proportional hazard model. (Cox model) Since there was no event recorded in the baseline HPV-negative group, the multivariate analysis was restricted to the baseline HPV-positive population (Table VI). Thus, only the viral load at enrollment was demonstrated to play a significant role in CIN2/3 development: a load of pg/ml was associated with a slightly increased risk (RR 1.4) for CIN2/3, and a load 100 pg/ml with an almost 3-fold increased risk (RR 2.7) compared to women harboring 1 9 pg/ml HPV DNA. Neither initial smear, nor age, was associated with an increased risk of developing a CIN2/3. Finally, we focused on the 31 women who developed a highgrade lesion during FU. At time of CIN2/3 diagnosis, every woman was still HR-HPV positive. Three of them (9.7%) had viral loads 10 pg/ml, 3 (9.7%) from pg/ml and 25 (80.6%) 100 pg/ml. Cytology performed a few weeks before CIN2/3 diagnosis revealed 18 (58.1%) HGSIL, 5 (16.1%) ASCUS, 3 (9.7%) LGSIL and 5 (16.1%) normal smears. In other words, we found 58.1% of concordant results and 41.8% of discordant results between cytology and histology. The median delay between entrance in the study and CIN2/3 diagnosis was 11 months. This delay depended on baseline cytology: 12 months for the 20 women with normal smear at entrance in the study vs. 7 months for the 11 women with minor or mild cytologic abnormalities (p 0.016). The mean delay also depended on baseline viral load: 15.4 months for viral load 10 pg/ml, 12.7 months for a load comprised of pg/ml and 10 months for a viral load 100 pg/ml (p 0.034). Although the delay seemed to be shorter in women 30 years old (10.7 months) than in women aged 30 years (13.5 months), this difference was not significant. DISCUSSION As far as we know, this is one of the 2 prospective longitudinal studies on HPV that were carried out in France, the other being published recently by Bory et al. in Our analysis concerned a subset of a large cohort of women with or without HPV infection at entry in the study and with normal or abnormal cytology with the exception of HGSIL or worse. The main endpoint was clinical progression, defined as having histologic CIN2/3 diagnosis. We also focused on specific events linked with natural history of HPV infection and cervical lesions. In our present study, for cytologic analysis we used conventional cytology. The sensitivity of conventional cytology to detect a CIN2/3 reached 74% (data not shown). Concerning the choice of HPV-testing method, HCII enabled us to screen easily and accurately a large number of samples. The amount of light generated by the HCII assay is proportional to the target DNA in the specimen and a linear range of quantification is achievable over approximately 4 logs, which allows reliable measurement of viral load. 26 Although this test does not permit us to quantify a specific HR-HPV type, it appeared of good utility in our clinical practice. Indeed, each type of HR-HPV present in the cervix can contribute to the early steps of neoplasia. 20 Moreover, co-infection with 1 HR-HPV type seems to be relatively common, about 15% of all HPV infections. 27 Thus, with the use of a specific sampler and a standardized sampling procedure, we drew the hypothesis that HCII values could provide a suitable estimation of the global HR-HPV DNA load. The use of HPV persistence as one of the criteria to refer women for colposcopy might lead to a bias: the more extensive surveillance of repeatedly HPV-positive women could result in higher rates of CIN2/3 detection compared to HPV-negative and transiently HPV-positive women. On the one hand, from an ethical point of view, it was not conceivable for the clinicians to ignore HPV data, knowing that persistence of HR- HPV can lead to CIN2/3 development. 4,9,12,28 On the other hand and for practical reasons, it was not possible to refer all eligible women for colposcopy. An alternative could have been to randomly select some women in the HPV-negative and transient groups for colposcopy. For instance, we cannot control this bias and have to keep in mind that the rate of CIN2/3 development in the HPV-negative and transient groups may be slightly underestimated in our present work. In agreement with previous data, an inverse relation was seen between age and prevalent oncogenic HPVs. 29 Moreover, the prevalence of HPV DNA detected at baseline for ASCUS and LGSIL smears was in the range found by most reports As already noted, the mean age of HPV-positive women with minor or mild cytologic lesions was lower than that of women with smears within normal limits, suggesting that in older women, HPV infections are less likely to manifest themselves cytologically. 33 Here we reported a lower rate of cumulative incident HPV infection (13.25% at 36 months) compared to Ho et al. s study on the HPV infection dynamics (43% at 36 months). 6 The risk of incident HPV infection is defined by age, and in our cohort, women were relatively older (mean age 35.7 years) than in the cohort of Ho et al. (mean age 20 years). 6 These incident HR-HPV infections might correspond to true new sexually acquired HPV infections, rather than a reactivation of latent infections, 34 since women 30 years were 4-fold more likely to get such an infection than older women. Moreover, we cannot exclude that some HPV findings were false-negative related either to fluctuant infection 35 or to very low amounts of virus. These hypotheses can be applied particularly to the 8 HPV-negative women who were diagnosed with an abnormal smear at enrollment. In the subgroup of women who were HPV positive at entrance in our study, the median duration of viral infection was 7.5 months, which is consistent with previous studies. 6 However, this duration may still suffer from imprecision in the estimates of time at first exposure. The baseline cytologic diagnosis and the level of viral load given by the HCII assay may influence the course of viral clearance. We observed that clearance occurred earlier in women with baseline ASCUS or LGSIL cytology than in women with

5 400 DALSTEIN ET AL. TABLE V CIN2/3 DEVELOPMENT IN THE OVERALL STUDY POPULATION, ACCORDING TO AGE, BASELINE CYTOLOGY, BASELINE HR-HPV LOAD AND VIRAL INFECTION OUTCOME subjects No. (%) of events Womenmonths Rate/100 women-months % of 18-month cumulative incidence (log-rank test) Total (4.0) 16, ( ) 4.27 ( ) - 30 years (4.8) 3, ( ) 5.49 ( ) p years (3.7) 12, ( ) 3.87 ( ) Baseline cytology Normal (3.1) 15, ( ) 2.92 ( ) p ASCUS/LGSIL (9.1) 1, ( ) 13.7 ( ) Baseline viral load Negative , pg/ml 85 6 (7.0) 1, ( ) 3.98 ( ) p pg/ml 74 6 (8.1) 1, ( ) ( ) 100 pg/ml (19.4) 1, ( ) ( ) HR-HPV outcome Negative , p Transient , Persistent (25) 1, ( ) ( ) normal smear. Nobbenhuis et al. described that clearance rates of HR-HPV decreased with increased severity of the lesion. 14 But when looking more precisely at their findings, it appeared that the 18-month cumulative rates of HPV clearance were less (about 50%) in women with normal cytology than in women with very mild dyskariosis for example (about 65%), which is consistent with our findings (data not shown). However, we have to be careful in comparing the data of the 2 studies, since cytologic diagnoses were not based on the same classification. As previously reported, 36 we showed that women with low levels of HPV DNA ( 10 pg/ml) were more likely to clear their HPV infections than those with high-level infections. Indeed, infections with high levels of oncogenic HPV DNA were reported to be more likely to persist, in particular if HPV16 is present. 22 In agreement with Nobbenhuis et al., 14 we did not find any role for age in HPV clearance. Our data also confirmed the clear association between the outcome of cytology and HPV exposure, as already reported by others. 28,37 Not surprisingly, baseline HPV positivity was associated with an increased risk of incident cytologic abnormalities, especially when the load was high ( 100 pg/ml, RR 8.66), when compared to HR-HPV negativity. We also were able to confirm that repeated detection of HR-HPV DNA was highly associated (RR 9.13) to incidence of abnormal smears. Fewer incident AS- CUS/LGSIL but no HGSIL were found in women with transient HR-HPV infections, which was already mentioned. 28 Likewise, when we looked at the risk of having a persistent cervical lesion, we noticed that only repeated positive HR-HPV testing was demonstrated to have a significant impact (RR 9.94). Moreover, it has been claimed that HPV clearance precedes cytologic regression. 14 Unfortunately, the design of our study did not permit us to confirm whether HPV clearance really took place before the disappearance of cytologic abnormalities. Surprisingly, 2 women became HPV negative after smears were normalized and 4 women became HPV negative without cytologic normalization. In these former patients, we cannot exclude that HR-HPV are still present but under the detection cutoff. No effect for age was assessed in our several analyses concerning the outcome of cytology. As previously described, we confirmed that HPV-negative women have a very low risk of developing a CIN2/3. 10,14 No high-grade lesion was detected among HPV-negative women for a median follow-up period of 24 months. Considering their very low risk, these women could be rescreened some years later (5 8 years), 15 but this needs to be further evaluated. Furthermore, none of the women who cleared their infections developed a CIN2/3, which confirms previous data. 28 Again, those women do not appear to be at risk for progression of disease. Thus, a single positive HPV test does not mean that there is an underlying cervical lesion and is not sufficient to evaluate the real subsequent risk for CIN2/3. 28 In contrast, a persistent infection with HR-HPV types was found to be a key prognostic variable for risk of CIN2/3, which confirmed most studies. 13,28,29,38 Here we reported a cumulative 24-month incidence of CIN2/3 of 36% in women with persistent HPV infection. Moreover, HR-HPV load was demonstrated to have a significant influence on CIN2/3 development; the risk increases with high baseline amounts of HPV DNA. Thus, high viral load could be used as a short-term marker of progression toward precancerous lesions, although lower load does not inevitably exclude progressive disease. Nevertheless, a high viral load could be the result of an infection with multiple HPV types. With the HCII test, it is not possible to discriminate between an increased risk linked to a high viral load per se or to a multiple HPV infection. Recently, genotyping of HPV was performed as previously described. 5 Eleven of the 31 women who developed a CIN2/3 were genotyped, and 9 of them (82%) demonstrated a single HR-HPV infection, which could be in favour of a role for HPV load rather than multiple HR-HPV infections (data not shown). Viral load determined by quantitative methods has already been considered by several authors as highly predictive of future development of high-grade cervical lesions. 19,22,39 41 Some studies also demonstrated that HCII results can be considered as a reflection of HPV DNA load, 20,21,42,43 whereas others reported that the HCII load cannot be considered as predictive for high-grade cervical lesion development. 24,25 These discrepancies can be partly explained by the type of specimen collection. Indeed, in our study, we used a specific device for specimen collection dedicated only to HPV testing. Moreover, the Digene Cervical Sampler provides a collection of cervical cells, which is standardized and targeted to the transformation zone and which has been shown to be reproducible. 44 Another important point of our design was the use of prospectively collected cervical material and not archival cervicovaginal lavages, as Lorincz et al. did. 24 The material was dedicated especially for HPV testing and not for both liquid-based cytology and HCII assay, as Bory et al. did. 25 Whether it was claimed that older ages were risk factors for persistent HPV infection, 4,36 in our present study, age would not be a factor in determining persistence of oncogenic HPV types and subsequently would not have a significant influence on the course of preneoplastic cervical lesions. 29 Our data also indicate that the development of CIN2/3 is a very common event in HPV-infected women, as already mentioned by Koutsky et al. 11 Nevertheless, a number of studies strongly suggest that an important proportion of these lesions are more likely to regress rather than to progress Meanwhile, more accurate and relevant data in the

6 HPV PERSISTENCE AND LOAD FOR HGSIL DEVELOPMENT 401 FIGURE 1 Kaplan-Meier estimates of cumulative incidence of CIN2/3 among 773 women, according to HPV infection characteristics. TABLE VI RELATIVE RISKS (RR) OF CIN2/3 AMONG THE 257 HR-HPV-POSITIVE WOMEN FOR AGE, BASELINE CYTOLOGY AND BASELINE VIRAL LOAD; MULTIVARIATE ANALYSIS WITH COX PROPORTIONAL HAZARDS REGRESSION RR of CIN2/3 95% CI 30 1 (referent) Baseline cytology Normal 1 (referent) ASCUS/LGSIL Baseline viral load (referent) future may provide information to properly recommend improved screening programs and clinical management of cervical lesions. In conclusion, our data strongly confirm the key role of persistent HR-HPV infection in cervical disease course. The data also suggest that HR-HPV DNA measured with HCII is very likely to reflect the global viral load and to constitute a useful and accurate predictor to identify women at high risk for HGSIL development, which has to be confirmed by real-time quantitative methods. ACKNOWLEDGEMENTS We are grateful to Ms. S. Coumes-Marquet for technical assistance with HPV testing, to Dr. P. Arveux and Dr. C. Menat for helpful discussion on statistical analysis and to Dr. F.X. Bosch for lecture and encouragement. V.D. is the recipient of a predoctoral scholarship from the Fondation de France.

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