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Human papillomavirus testing in primary screening for the detection of high-grade cervical lesions: a study of 7932 women

Human papillomavirus testing in primary screening for the detection of high-grade cervical... British Journal of Cancer (2001) 89(12), 1616–1623 © 2001 Cancer Research Campaign doi: 10.1054/ bjoc.2001.1845, available online at http://www.idealibrary.com on http://www.bjcancer.com Human papillomavirus testing in primary screening for the detection of high-grade cervical lesions: a study of 7932 women 1 1 2 1 1 1 3 2 2 C Clavel , M Masure , J-P Bory , I Putaud , C Mangeonjean , M Lorenzato , P Nazeyrollas , R Gabriel , C Quereux and P Birembaut 1 2 Laboratoire Pol Bouin, C.H.U. de Reims, 45, rue Cognacq-Jay, 51100 Reims, France; Department of Obstetrics and Gynecology, C.H.U. de Reims, 51100 Reims, France; Department of Cardiology and Department of Statistics, C.H.U. de Reims, 51100, France Summary High-risk human papillomaviruses (HR-HPV) are the necessary cause of cervical carcinomas. To determine whether HPR-HPV DNA detection in primary routine screening could represent a sensitive and reliable technique for the detection of high-grade squamous intraepithelial lesions (HGSIL), laboratory analysis using 2 cytologic techniques (conventional and liquid-based), HPV testing with Hybrid Capture II assay (HC-II), followed by colposcopic examination of women with abnormal cervical finding and/or persistent HR-HPV infection, was conducted in 7932 women who had routine cervical examination. The sensitivity of HPV testing for detecting a histologically proven HGSIL was 100%, higher than that of conventional (68.1%) and liquid-based (87.8%) cytology. The low specificities of 85.6% and 87.3% of HPV testing slightly increased to 88.4% and 90.1% if HPV testing was reserved for woman >30 years old. The quantitative approach provided by the HC-II assay for the assessment of the viral load was not reliable for predicting HGSIL in normal smears. HR-HPV testing could be proposed in primary screening in association with cytology. With conventional cytology it significantly improves the detection of HGSIL. With the use of the same cervical scrape for HPV testing and liquid-based cytology, HR-HPV testing would allow to select positive samples treated in a second time for cytology which gives a good specificity. © 2001 Cancer Research Campaign http://www.bjcancer.com Keywords: HPV; cervical cancer; screening It is now well established that oncogenic (high-risk) human cervical scrapes offers a diagnostic assay without the sampling papillomaviruses (HPV) are a causal factor in the development problems and subjectivity of cytology, but such an approach needs of cervical intraepithelial and invasive neoplasias (Lorincz et al, a specific, sensitive, reliable and easy to perform method. 1992; Zur Hausen, 1994; Bosch et al, 1995; Walboomers et al, 5 years ago, a commercial HPV detection test, Hybrid Capture-I 1999). Infections with high-risk HPV (HR-HPV) are associated (HC-I), followed by second-generation test Hybrid Capture II (HC- with a relative risk of between 8 and 11 for the development of II) was introduced (Lorincz, 1996). HC-II is a non-radioactive, squamous intraepithelial lesions (SIL) and essentially low-grade reproducible, relatively rapid, liquid hybridization assay in micro- SIL (LGSIL) containing HR-HPV progress to high-grade SIL titres designed to detect 18 HPV types divided into high-risk (types (HGSIL) (Koutsky et al, 1992; Gaarenstroom et al, 1994). Because 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, and 68) and low-risk of this, there is an increasing interest in using HPV DNA detection (types 6, 11, 42, 43 and 44) groups. The sensitivity of this assay is either alone or in addition to classic cytologic examination, essen- quite similar to that of Polymerase Chain Reaction (PCR) (Clavel tially as a method for triaging women with a cytologic diagnosis of et al, 1998b; Peyton et al, 1998; Riethmuller et al, 1999). atypical squamous cells of undetermined significance (ASCUS) in Nevertheless current screening remains based on the cytopatho- their cervical smears (Cox et al, 1995; Wright et al, 1998; Manos logic classification of cervical smears, according to the Bethesda et al, 1999) and now for primary cervical screening (Cuzick et al, system, leading to colposcopy and histological sampling. Women 1995, 1999; Clavel et al, 1998a, 1999; Meijer et al, 1998; Kuhn in wealthier countries are largely protected via the Papanicolaou et al, 2000; Ratnam et al, 2000; Schiffman et al, 2000). Never- test. However, this test is not perfect and false-negative rates of 5 theless, the low sensitivity of HPV detection in previous studies to 50% have been reported (Schneider et al, 1996; Cuzick et al, has led some authors to consider that HPV testing does not appear 1999). Thus, in the last few years, significant technical advances to be of value for identifying women with abnormal smears who have raised the possibility of improving conventional cytology. could be safely followed up with cytology alone (Kaufman and Particularly, liquid-based cytology has improved sampling, fixa- Adam, 1999). Moreover, the low positive predictive value and the tion, staining and background, ensuring a more representative cost-effectiveness of HPV DNA testing have to be taken in consid- sample with a dramatic improvement in sensitivity (Papillo et al, eration for primary screening. However, HPV DNA testing in 1998; Sherman et al, 1998; Weintraub and Morabia, 2000). Recently, Cuzick et al (2000) have emphasized the urgent need to Received 25 January 2001 undertake a large trial of HPV testing in conjunction with other new Revised 7 March 2001 technologies including liquid-based cytology. Here we report our Accepted 20 March 2001 experience on HPV testing in primary screening of 7932 women, for Correspondence to: C Clavel the detection of HGSIL. For that, we compared the results of HPV 1616 HPV testing for primary cervical screening 1617 testing to the results of conventional and liquid-based cytology. smears within normal limits but presenting a HR-HPV infection Because most LGSIL regresses spontaneously, our primary were also systematically recalled 6 months later for a new cytolog- endpoint was the histological diagnosis of HGSIL at the biopsy. ical examination and HR-HPV testing followed by colposcopy if a lesion and/or a persistent HR-HPV infection was detected. Punch biopsy specimens were taken from the areas colposcopically suspi- MATERIAL AND METHODS cious for CIN. The women with a second HR-HPV test positive without any detectable lesions were recalled 6 to 12 months later for Study population a third control with cytological examination and HR-HPV testing A total of 7932 women with a median age of 34 years (range 15 to and the same indications as above for colposcopy and biopsy. 76 years) were recruited for the study between August 1997 and Women with regressive HR-HPV infection were also recalled for February 2001. This population was restricted to women who colposcopy 12 months later. By contrast, women with initial normal underwent their biennal or triennial routine screening in the smears and without any HR-HPV infection were followed with a Department of Obstetrics and Gynecology of the CHU of Reims. classical biennal or triennal cervical screening with a new cytolog- We excluded subjects on the basis of a recent cytologic abnor- ical examination and HR-HPV testing at the second control. Some mality and/or an untreated cervical lesion in the past 2 years, preg- of these women had also randomly a colposcopic examination. nant women and patients with AIDS. All women were informed of There were not particular selection criteria for colposcopy. The the aim of the study and gave their consent. primary endpoint of our study was the detection of a histologically proven HGSIL at the biopsy and/or on the LEEP specimen. Cytologic diagnosis HPV testing At the first gynaecologic examination, in 2281 women, 2 samples were taken: first, a cytologic smear with an Ayre’s spatula, for When conventional cytology was performed, specimens for HPV classical cytology, then one scrape for the HC-II test with a DNA testing were suspended in 1 ml of ViraPap/Viratype transport Cervexbrush (Medscan, Uppsala, Sweden). These samples were medium (Digene, Silver Spring, MD) and stored at –20˚C until suspended in 1 ml of specimen transport medium for HPV testing further processing. When samples were used for liquid based (Digene, Silver Spring, MD). In another way, 5651 women had cytology, 4 ml of the sample were centrifuged and the cell pellet was only one cervical scrape with a Cervexbrush at the first entry. resuspended in 200 ml of phosphate-buffered saline for HPV testing. Samples were prepared for liquid-based cytology with the HPV DNA detection was performed by the commercially available ThinPrep technique (Cytyc Corporation, Marlborough, Mass) and HC-II System (Digene). All scrapes were analysed for the presence 4 ml of the sample were used for HPV testing. In total, considering of HR-HPV types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59 and 68. the first samples and the follow-up of women, we collected 10 101 This enzyme-linked immunosorbent assay is based on a sandwich cervical smears and scrapes. Smears were classified according to hybridization followed by a nonradioactive alkaline phosphatase the Bethesda system for reporting cervical or vaginal cytological reaction with chemoluminescence in microplates. The chosen posi- –1 diagnosis. We selected women with adequate smears including tive threshold of this test was 1.0 pg ml of HPV DNA. metaplastic and/or endocervical cells according to the criteria of Samples were classified as positive for HR-HPV DNA if the Bethesda, which represented 92% of our total smears. However, relative light unit (RLU) reading obtained from the luminometer even if the smear was not considered as adequate, we also included was equal to or greater than the mean of the 3 positive control women with smears evocative of lesions. In another way, 4.8% of values supplied by the HC-II kit. As some authors have reported the samples treated for liquid-based cytology could not be used for that increasing HPV DNA levels of HR-HPV types were the prin- HPV testing and have been excluded. The cytotechnicians and cipal predictors of CIN (Cox et al, 1995), we used as proposed, the pathologists involved in the study were not informed about the ratio RLU/positive controls values to quantify HR-HPV DNA in results of the HPV testing. All smears showing cytological abnor- our samples. Moreover, we added other positive controls such as malities and biopsy specimens were examined by the same 2 inde- SiHa cell lines (1 to 2 copies of HPV type 16 per cell) to check the pendent pathologists, without knowledge of cytology results for reproducibility of the HC-II sensitivity. the biopsies examination. The results were compared and if the first 2 diagnoses disagreed, a third pathologist reviewed the case Statistical methods with no knowledge of preceding diagnoses. Consensus diagnoses The statistical methods used were mostly descriptive. Sensitivity, were determined by two-thirds majority when possible and specificity, positive and negative predictive values were deter- remaining discrepancies resolved by conference review. Patients mined by comparing the results of each test to the gold standard of with HGSIL were systematically treated by loop electrosurgical histology. A few high-grade lesions may have been missed if they excision procedure (LEEP). Data from these LEEP specimens were negative on both tests and thus women were not referred were included in the disease definitions. for colposcopy. 95% confidence interval for these values were assessed using either binomial or normal distribution, according to Colposcopic referral the data. Moreover, differences between HR-HPV detection and cytologic diagnosis values were compared using Fisher exact test In our protocol (Table 1), all the women presenting cytological or Chi-2 statistics as adequate, with a P value set to 5%. abnormalities evocative of cervical lesions (from ASCUS to HGSIL) were systematically recalled for colposcopy in the next few RESULTS weeks, at an interval ranging from 14 to 160 days (mean 95 days) after entry examination. Punch biopsy specimens were taken from At their first examination, in our population of 7932 women, 1214 the areas colposcopically suspicious for CIN. All the women with (15.3%) presented a HR-HPV infection. © 2001 Cancer Research Campaign British Journal of Cancer (2001) 84(12), 1616–1623 1618 C Clavel et al Table 1 Protocol for the follow-up of the women Cytology FIRST SCRAPE HPV detection _/It I~ Cytology - Cytology - Cytology + HPV - HPV + HPV + or - c __ J c_J c=J ------- ______________ d_b __ _ IMMEDIATE Colposcopy - Colposcopy + RECALLING Biopsy ------- --------- _/ _____ D __ _ 6 MONTHS Cytology LATER HPV detection /I t I~ Cytology - Cytology - Cytology + HPV - HPV + HPV + or - --------- ----- (..________,___.) Colposcopy - Colposcopy + Biopsy ---- c ____ ) I I ------- --------- t------------------- 6 to 12 MONTHS LATER Cytology Colposcopy HPV detection .__________,___ I I etc ------- ------- --------------------------- ------------------- --------- 3 YEARS LATER Cytology HPV detection Table 2 represents the prevalence of HR-HPV infection HPV detection and cytological and histological observed in our 7932 women according to their age, at the first diagnoses examination. There was a peak of infection in the third These results are summarized in Table 3. decade (23.6% of women) with a progressive decrease after 30 The prevalence of HR-HPV infection was significantly related years. to the severity of the cytologically detected lesions (P < 0.001). British Journal of Cancer (2001) 84(12), 1616–1623 © 2001 Cancer Research Campaign HPV testing for primary cervical screening 1619 Table 2 Prevalence of high-risk HPV infections according to age at the first women with normal smears with liquid-based cytology, 542 examination (10.4%) had a HR-HPV infection. In this cohort of 542 women, at the present time, 237 women (43.7%) had a follow-up on a period Age Women High-risk HPV from 4 to 20 months (median = 12 months). A persistent HR-HPV < 20 418 (5.3%) 84 (20.1%) infection at the second and eventually third examination was 21–30 1843 (23.2%) 435 (23.6%) observed in 95 women (40.1%) and was associated with the detec- 31–40 2076 (26.2%) 289 (13.9%) tion of a HGSIL in 10 patients, 4 to 12 months after the first entry 41–50 1925 (24.3%) 235 (12.2%) (median = 6 months). No HGSIL was detected by cytology and 51–60 1014 (12.8%) 110 (10.8%) colposcopy in women with regressive HR-HPV infection what- > 60 656 (8.3%) 61 (9.3%) ever the initial technique of cytology used. In addition, none of the Total 7932 1214 (15.3%) 1225 women with normal smears and without any HR-HPV infec- tion at the first control (893 with conventional cytology and 332 with liquid-based cytology), developed HGSIL detectable at the 25 women had a smear evocative of HGSIL at the first exami- second systematic cytological control and at the colposcopic nation with conventional cytology with a HR-HPV infection in all examination performed in 172 of them, on a period of 24 to 36 of them. The diagnosis of HGSIL was histologically confirmed in months (median = 30 months). 21 of them. 82 women had a smear evocative of HGSIL with liquid-based cytology, with a HR-HPV positive test in 79 (96.3%) of them. The diagnosis of HGSIL was confirmed by the biopsy in Results according to the technique of cytology and the 58 patients who were all tested positive for HR-HPV DNA. Out of age of women 77 women who had a smear evocative of LGSIL at the first exam- Tables 4 and 5 represent the respective sensitivity, specificity, ination with conventional cytology, 56 (72.7%) had a HR-HPV positive and negative predictive values of the various methods infection. 65 of them had a follow-up and 6 had an underlying used for the detection of a HGSIL histologically proven in the HGSIL associated with a HR-HPV. 200 women had a smear global population and in women aged >30 years. In these tables, evocative of LGSIL with liquid-based cytology with a HR-HPV only results of cytology and HPV testing at the first examination infection in 168 (84%). In the cohort of 182 women with a follow- have been considered for the evaluation of these techniques, since up, 8 HGSIL were detected, all associated with HR-HPV. most women had a liquid-based cytology for their follow-up. As 34 women presented ASCUS with conventional cytology with expected, liquid-based cytology gave a significantly higher HR-HPV in 19 of them (55.9%). In 19 women with a follow-up, 5 sensitivity (87.8% in the general population and 84.4% in women HGSIL were detected all tested positive for HR-HPV DNA. 175 aged > 30 years) than conventional cytology (68.1% in the general women presented ASCUS with liquid-based cytology. HR-HPV population and 57.7% in women aged > 30 years) (P < 0.05). DNA was detected in 94 cases (53.7%). At the colposcopic control However, the sensitivity of HR-HPV testing remained higher, at obtained in 135 women, 6 HGSIL were diagnosed all associated 100% in all cases. Nevertheless, if the negative predictive value with a HR-HPV infection. was always of 100%, the specificity and the positive predictive In the remaining population of 2145 women with cervical value of the HR-HPV testing were clearly lower than those of the smears within normal limits with conventional cytology, HR-HPV cytology whatever the technique used. Limiting the screening to DNA was detected in 231 (10.8%). Out of these 231 women tested women aged > 30 years slightly increased the specificity of the HR-HPV positive, according to our protocol, 168 women (72.7%) HR-HPV testing to 88.4% in scrapes treated for liquid-based had a follow-up on a period from 4 to 36 months (median = 24 cytology and to 90.1% in smears examined with conventional months). A persistent HR-HPV infection at the second and eventu- cytology, but did not modify the positive predictive value for ally third examination was observed in 66 women (39.3%) and HGSIL detection. However, we have to emphasize that in our was associated with the detection of a HGSIL in 15 patients, 4 to same referral population, with the same follow-up protocol, 58 out 20 months after the first entry (median = 10 months). Out of 5194 Table 3 Follow-up from the first smear to the final diagnosis First smear HG detected First smear HG detected Conventional HR-HPV Follow-up at the Thin-prep HR-HPV Follow-up at the cytology detection histology cytology detection histology 2145 WL 231 168 HPV+ 15 HG 5194 WL 542 237 HPV + 10 HG (94.0%) (10.8%) 893 HPV – (91.9%) (10.4%) 332 HPV– 34 ASCUS 19 19 HPV + 5 HG 175 ASCUS 94 89 HPV + 6 HG (1.5%) (55.9%) 7 HPV – (3.1%) (53.7%) 46 HPV – 77 LG 56 54 HPV + 6 HG 200 LG 168 160 HPV + 8 HG (3.5%) (72.7%) 11 HPV – (3.5%) (84.0%) 22 HPV – 25 HG 25 25 HPV + 21 HG 82 HG 79 79 HPV + 58 HG (1.1%) (100.0%) (1.4%) (96.3%) 3 HPV – 2281 331 1177 47 HG 5651 883 968 82 HG WL: without lesion; LG: low-grade lesion; HG: high-grade lesion; HR-HPV: high-risk HPV. © 2001 Cancer Research Campaign British Journal of Cancer (2001) 84(12), 1616–1623 1620 C Clavel et al Table 4 Evaluation of cytology and high-risk HPV testing for the detection of histologically proven high-grade lesion Methods Sensitivity Specificity PPV NPV (95% C.I.) (95% C.I.) (95% C.I.) (95% C.I.) HPV detection 47/47 (100%) 1950/2234 (87.3%) 47/331 (14.2%) 1950/1950 (100%) 93.8–100% 85.9–88.7% 10.4–18.0% 99.8–100% Conventional cytology 32/47 (68.1%) 2130/2234 (95.3%) 32/136 (23.5%) 2130/2145 (99.3%) 55.4–79.2% 94.5–96.2% 16.4–30.7% 99.0–99.6% HPV detection 82/82 (100%) 4768/5569 (85.6%) 82/883 (9.3%) 4768/4768 (100%) 96.4–100% 84.7–86.5% 7.4–11.2% 99.9–100% Thin prep cytology 72/82 (87.8%) 5184/5569 (93.1%) 72/457 (15.7%) 5184/5194 (99.8%) 80.3–93.3% 92.4–93.8% 12.4–19.1% 99.7–99.9% PPV: Positive predictive value, NPV: Negative predictive value, C.I.: Confidence interval. Table 5 Evaluation of cytology and high-risk HPV testing for the detection of a histologically proven high-grade lesion in women aged > 30 years Methods Sensitivity Specificity PPV NPV (95% C.I.) (95% C.I.) (95% C.I.) (95% C.I.) HPV detection 26/26 (100%) 1373/1524 (90.1%) 26/177 (14.7%) 1373/1373 (100%) 89.2–100% 88.6–91.6% 9.5–19.9% 99.8–100% Conventional cytology 15/26 (57.7%) 1457/1524 (95.6%) 15/82 (18.3%) 1457/1468 (99.2%) 39.1–75.0% 94.6–96.6% 9.9–26.6% 98.5–99.4% HPV detection 45/45 (100%) 3603/4076 (88.4%) 45/518 (8.7%) 3603/3603 (100%) 93.6–100% 87.4–89.4% 6.3–11.1% 99.9–100% Thin prep cytology 38/45 (84.4%) 3863/4076 (94.8%) 38/251 (15.1%) 3863/3870 (99.8%) 73.2–92.8% 94.1–95.5% 10.7–19.6% 99.5–99.8% PPV: Positive predictive value, NPV: Negative predictive value, C.I.: Confidence Interval. Table 6 Evaluation of the viral load (VL) estimated by the HC-II assay for the detection of a high-grade lesion in cytologically normal smears Methods VL Sensitivity Specificity PPV NPV HPV testing associated >3 15/15 (100%) 53/216 (24.5%) 15/178 (8.4%) 53/53 (100%) with conventional cytology >10 15/15 (100%) 88/216 (40.7%) 15/143 (10.5%) 88/88 (100%) HPV testing associated >3 10/10 (100%) 203/532 (38.1%) 10/329 (2.9%) 203/203 (100%) with thin prep cytology >10 7/10 (70%) 313/532 (58.8%) 7/226 (3.1%) 313/316 (99.0%) PPV: Positive predictive value; NPV: Negative predictive value. Table 7 Evaluation of cytology and high-risk HPV testing for the detection of a histologically proven high-grade lesion in function of the viral load (VL) Methods Sensitivity Specificity PPV NPV (95% C.I.) (95% C.I.) (95% C.I.) (95% C.I.) HPV detection. VL>3 44/47 (93.6%) 2008/2234 (89.9%) 44/270 (16.3%) 2008/2011 (99.8%) 83.5–98.6% 88.6–91.1% 11.2–20.7% 99.6–99.7% HPV detection. VL>10 42/47 (89.4%) 2049/2234 (91.7%) 42/227 (18.5%) 2049/2054 (99.7%) 78.4–96.1% 90.6–92.9% 13.5–23.6% 99.5–99.9% Conventional cytology 32/47 (68.1%) 2130/2234 (95.3%) 32/136 (23.5%) 2130/2145 (99.3%) 55.4–79.2% 94.5–96.2% 16.4–30.7% 99.0–99.6% HPV detection. VL>3 80/82 (97.6%) 5007/5569 (89.9%) 80/642 (12.5%) 5007/5009 (99.9%) 91.9–99.7% 89.1–90.7% 9.9–15.0% 99.8–99.9% HPV detection. VL>10 72/82 (87.8%) 5137/5569 (92.2%) 72/504 (14.3%) 5137/5147 (99.8%) 80.3–93.3% 91.6–92.9% 11.2–17.3% 99.7–99.9% Thin prep cytology 72/82 (87.8%) 5184/5569 (93.1%) 72/457 (15.7%) 5184/5194 (99.8%) 80.3–93.3% 92.4–93.8% 12.4–19.1% 99.7–99.9% PPV: Positive predictive value; NPV: Negative predictive value; C.I.: Confidence interval. British Journal of Cancer (2001) 84(12), 1616–1623 © 2001 Cancer Research Campaign HPV testing for primary cervical screening 1621 of 129 HGSIL (45.0%) were detected in women aged < 30 years. management of these women. Indeed in our series, HR-HPV This finding explains the absence of modification of the positive infection is frequent in this population of women (80.9%). In predictive value after 30 years. consequence, its use as a discriminating test for colposcopic referral seems of limited interest, even though all the HGSIL detected at the histological control of cytological LGSIL, were Value of the semi-quantitative viral load estimated by positive for HR-HPV DNA. In another way, in the literature, the HC-II assay for predicting the persistence of about 50% of smears with ASCUS are tested positive for HR- HR-HPV infection and the apparition of HGSIL HPV DNA and 5 to 10% of ASCUS correspond to an underlying HGSIL associated with HR-HPV infection. Thus, Manos et al Table 6 represents the evaluation of different viral load estimated (1999) and the ALTS group (Solomon et al, 2001) have proposed in RLU (3 and 10 RLU) by the HC-II assay for predicting the that, for women with ASCUS, HPV DNA testing can help to detection of HGSIL in smears within normal limits at the first identify those who have underlying HGSIL. Our results are in cytological examination with conventional and liquid-based agreement with this proposition since 5 HGSIL have been cytology. The sensitivities remained high but the specificities were detected at the colpo-histological control of ASCUS obtained in very low in any case. Table 7 evaluates the efficiency of different 26 women with initial conventional cytology and 6 HGSIL in 135 viral load values (1,3 and 10 RLU) for general screening. The use women with liquid-based cytology, all associated with high-risk of a viral load > 10 increased specificities and predictive positive HPV infection. If we consider the negative predictive value of the values and the sensitivity of the HPV testing remained very high, HC-II assay of 100%, HPV testing is a reliable test to select equivalent or superior to that of cytology. women with ASCUS and HR-HPV infection who had to be referred for colposcopy. DISCUSSION In our series, we have observed that in total, 773 out of 7339 This work using the HC-II assay on a series of 7932 French women with smears within normal limits presented a HR-HPV women attending routine cytologic screening clearly confirms the infection. These numerous positive results, particularly in women high prevalence of HR-HPV infection with a peak at 23.6% in the < 30 years old, are largely responsible for the low specificity and third decade and a progressive decrease after 30 years. In the same positive predictive value of the HPV testing. Numerous HPV way, Herrero et al (2000) using PCR have reported such a high infections are known to regress spontaneously, especially in young prevalence of HPV infection in women under 25 years in rural women and the mean HPV infection duration is between 8 to 14 Costa Rica. The high percentage of women tested positive in our months (Ho et al, 1998; Franco et al, 1999). However, Rozendaal cohort may partly be due to the technique used with the possibility et al (1996) have emphasized that the women with normal smears of cross-hybridization with unknown and/or additional HPV types and HR-HPV genotypes are 116 times more at risk of developing not included in the HC-II assay probe cocktails (Vernon et al, HGSIL, in contrast of women without HR-HPV. Moreover, the 2000), which may increase the number of positive results. There is persistence of HR-HPV infection is significantly associated with also a significant prevalence and/or persistence of HR-HPV infec- progressive disease (Ho et al, 1995; Remmink et al, 1995). Indeed, tion in women with HGSIL. HR-HPV DNA has been detected in in our follow-up of 168 (72.7%) of 231 women with smears within all the 129 women with a HGSIL histologically confirmed. Thus, normal limits with conventional cytology and with HR-HPV in our experience at the present time, whatever the collection infection, 15 (22.7%) out of 66 women with persistent HR-HPV protocol used for HPV testing, the sensitivity of HC-II for infection have presented a HGSIL within 4 to 20 months after the detecting HGSIL is 100%, but the specificity remains low, at first entry. At the present time, we have a shorter follow-up of only 87.6% when the initial sample has been associated with conven- 237 (43.7%) out of 542 women with normal smears with liquid- tional cytology, and at 85.6% when the initial sample has been based cytology and HR-HPV infection and the number of 10 treated for liquid-based cytology, with positive predictive values HGSIL detected may not exactly reflect the total HGSIL present in of 14.2% and 9.3% respectively. These results are quite similar to this population, but these preliminary data are also very promising. those of previous studies using HPV testing with HC-II for In another way, our more frequent follow-up of women with primary screening (Cuzick et al, 1999; Schiffman et al, 2000). normal smears and HR-HPV positive, induces a bias of selection Moreover, in our work, the sensitivity of liquid-based cytology is which logically results in more numerous diagnoses of HGSIL. also significantly higher than that of conventional cytology However, practically, a systematic intensive follow-up in parallel (87.8% vs 68.1%) (P < 0.05) as previously reported in the litera- of women with normal smears and without any HR-HPV infec- ture when the ThinPrep method has been compared to conventional tion, is difficult to propose and to obtain. Nevertheless, we cytology (Papillo et al, 1998; Sherman et al, 1998; Weintraub and underline that no HGSIL has been detected in our cohort of 1225 Morabia, 2000). However, if liquid-based cytology alone is a very women with initial HR-HPV negative and normal smears, efficient method, HR-HPV testing combined with cytology signif- followed with classic biennal or triennal screening. Thus, icantly increases the detection of HGSIL. according to our protocol of follow-up, the detection of a persis- The use of HPV testing for triage test in smears evocative of tent HR-HPV infection selects a population of women with normal ASCUS and LGSIL has been recently discussed in large series. smears at risk for developing a HGSIL. Adam et al (1998) have found that screening for HPV DNA with Considering that HPV infection in younger women often PCR, does not have prognostic value in women reported as spontaneously regress and that the incidence of cervical cancer having ASCUS or smears evocative of low-grade lesions. The in women younger than 30 years is very low, numerous authors ALTS group (2000) has considered that, because a very high (Wright et al, 1998; Cuzick et al, 1999; Stoler, 2000) have percentage of women with cytological evidence of LGSIL are recommended the use of HPV testing for older women (>30–35 positive for HPV DNA with the HC-II assay, there is limited years old). This could improve the specificity and the positive potential for HPV testing to direct decisions about the clinical predictive value of the test. Nevertheless, these propositions do © 2001 Cancer Research Campaign British Journal of Cancer (2001) 84(12), 1616–1623 1622 C Clavel et al not consider the frequent early age of the first sexual intercourse a good technical approach and a high professional skill to be as a source of HPV infection with a higher risk for developing a highly sensitive. Considering that the mean time from detectable HGSIL before 30 years. In our experience, if we reserve HPV LGSIL to preclinical invasive cancer is 12–13 years (Gustafson testing in women > 30 years old, the positive predictive value of and Adami, 1989), Meijer et al (1998) have proposed that women HPV testing is not modified, but the specificity slightly with cytologically normal smears and a negative HR-HPV test increases from 87.3% to 90.1% in samples associated with could be rescreened every 8 years. If we subscribe to this conventional cytology and from 85.6% to 88.4% in samples screening policy, HPV testing combined with cytology would treated for liquid-based cytology. Again, we have to emphasize significantly lower the number of cervical smears and the inci- that 58 out of our 129 patients (45.0%) with HGSIL were women dence of colposcopy with a more efficient screening. Another < 30 years old. This clearly explains the absence of modification proposition is to begin the primary cervical screening with HPV of the predictive positive values after 30 years. Consequently, detection. This policy has been proposed in low-resource settings limiting HPV testing in women > 30 years in primary screening where HPV DNA testing programmes may be easier to implement will miss the increasing number of HGSIL present under this than cytologic screening (Kuhn et al, 2000; Schiffman et al, age. 2000). In our wealthier countries, we can use the same cervical There may be utility in semiquantitative measurement of high scrape for HPV testing and liquid-based cytology. HR-HPV viral load to increase the specificity of HPV testing. Indeed, testing would allow to select positive samples treated in a second Cuzick et al (1994) have shown that in women with cytologic time for liquid-based cytology which gives a better sensitivity abnormalities, HPV type 16 positivity at a high-level detected by a than conventional cytology and a good specificity. Women with semi-quantitative PCR is strongly related to HGSIL. Ho et al cervical abnormalities in their smears will be immediately (1995) have also suggested that SIL with a high viral load are more recalled for colposcopy. A more intensive cytological screening likely to persist than those with a low level of HPV DNA. A recent with HPV positive women with normal smears, with an algorithm study using a sensitive real time quantitative PCR assay has clearly of 6 to 12 months, to confirm the persistence of HPV infection demonstrated that cervical carcinoma in situ associated with HPV and/or the occurrence of SIL. All these propositions of new type 16 occurs mainly in HPV type 16 positive women who had managements for the cervical screening require extensive and consistently high viral loads long term (Ylitalo et al, 2000). Thus, multicentric studies in different countries to be validated in terms a high viral load may be considered as a risk factor and preferen- of efficiency and costs–benefits. In any case, the introduction of tially observed in potentially evolutive lesions and in HGSIL. This the HPV testing represents a new promising technology for parameter could be semi-quantitatively evaluated by the relative primary cervical screening. light unit values (RLU) provided by the HC-II assay. Our present results with HC-II show that when this parameter is basically ACKNOWLEDGEMENTS applied for predicting the apparition of a HGSIL in women without any detectable lesions, the specificity is very low <60% This work was supported by a grant from the European but the sensitivity remains very good. Indeed for a cervical Community (L’Europe contre le Cancer), the ARC (Association de sample/positive control ratio >10, the best results are obtained Recherche sur le Cancer), the ARERS, the Ligue Contre le Cancer when HPV testing is performed with conventional cytology, with a (Comités de la Marne, de l’Aisne, de la Haute-Marne et de sensitivity for predicing a HGSIL of 100%, but with a specificity l’Aube), the CHU of REIMS and by the LIONS Club of SOIS- of 40.7% and a predictive positive value of 10.5%. In another way, SONS. We thank all the gynaecologists and women who partici- when a viral load >10 is considered for general screening, the pated in this study. specificities and predicitive positive values increase and the sensi- tivity is equivalent or better to that of liquid-based cytology for detecting a HGSIL. However we have to underline that the values of viral load are semi-quantitative global measurements for detec- REFERENCES tion of one or multiple HPV types among the 13 in the kit. Adam E, Kaufman RH, Berkova Z et al (1998) Is human papillomavirus testing an Recently, Liaw et al (2001) have reported that HPV type 16 infec- effective triage method for detection of high-grade (grade 2 or 3) cervical tion is generally associated with an increased risk of subsequent intraepithelial neoplasia? 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Obstet Weintraub J and Morabia A (2000) Efficacy of a liquid-based thin layer method for Gynecol 79: 328–337 cervical cancer screening in a population with low incidence of cervical cancer. Manos MM, Kinney WK, Hurley LB et al (1999) Identifying women with cervical Diagn Cytopathol 22: 52–59 neoplasia using human papillomavirus testing for equivocal Papanicolaou Wright TC Jr, Lorincz AT, Ferris DG et al (1998) Reflex human papillomavirus results. JAMA 281: 1605–1610 deoxyribonucleic acid testing in women with abnormal Papanicolaou smears. Meijer CJLM, Helmerhorst TJM, Rozendaal L et al (1998) HPV typing and testing Am J Obstet Gynecol 178: 962–966 in gynaecological pathology; has the time come? Histopathology 33: 83–86 Ylitalo N, Sorensen P, Josepfsson AM et al (2000) Consistent high viral load of Papillo, JL, Zarka MA, St-John TL et al (1998) Evaluation of the ThinPrep Pap test human papillomavirus 16 and risk of cervical carcinoma in situ: a nested-case in clinical practice. A seven-month 16,314-case experience in Northern control study. Lancet 355: 2194–2198 Vermont. Acta Cytologica 42: 203–208 Zur Hausen H (1994) Molecular pathogenesis of cancer of the cervix and its Peyton CL, Schiffman MH, Lorincz AT et al (1998) Comparison of PCR and Hybrid causation by specific human papillomavirus types. Curr Top Microbiol Capture-based human papillomavirus detection systems using multiple cervical Immunol 186: 131–156 specimen collection strategies. J Clin Microbiol 36: 3248–3254 © 2001 Cancer Research Campaign British Journal of Cancer (2001) 84(12), 1616–1623 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png British Journal of Cancer Springer Journals

Human papillomavirus testing in primary screening for the detection of high-grade cervical lesions: a study of 7932 women

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Springer Journals
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Copyright © 2001 by The Author(s)
Subject
Biomedicine; Biomedicine, general; Cancer Research; Epidemiology; Molecular Medicine; Oncology; Drug Resistance
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0007-0920
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1532-1827
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10.1054/bjoc.2001.1845
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Abstract

British Journal of Cancer (2001) 89(12), 1616–1623 © 2001 Cancer Research Campaign doi: 10.1054/ bjoc.2001.1845, available online at http://www.idealibrary.com on http://www.bjcancer.com Human papillomavirus testing in primary screening for the detection of high-grade cervical lesions: a study of 7932 women 1 1 2 1 1 1 3 2 2 C Clavel , M Masure , J-P Bory , I Putaud , C Mangeonjean , M Lorenzato , P Nazeyrollas , R Gabriel , C Quereux and P Birembaut 1 2 Laboratoire Pol Bouin, C.H.U. de Reims, 45, rue Cognacq-Jay, 51100 Reims, France; Department of Obstetrics and Gynecology, C.H.U. de Reims, 51100 Reims, France; Department of Cardiology and Department of Statistics, C.H.U. de Reims, 51100, France Summary High-risk human papillomaviruses (HR-HPV) are the necessary cause of cervical carcinomas. To determine whether HPR-HPV DNA detection in primary routine screening could represent a sensitive and reliable technique for the detection of high-grade squamous intraepithelial lesions (HGSIL), laboratory analysis using 2 cytologic techniques (conventional and liquid-based), HPV testing with Hybrid Capture II assay (HC-II), followed by colposcopic examination of women with abnormal cervical finding and/or persistent HR-HPV infection, was conducted in 7932 women who had routine cervical examination. The sensitivity of HPV testing for detecting a histologically proven HGSIL was 100%, higher than that of conventional (68.1%) and liquid-based (87.8%) cytology. The low specificities of 85.6% and 87.3% of HPV testing slightly increased to 88.4% and 90.1% if HPV testing was reserved for woman >30 years old. The quantitative approach provided by the HC-II assay for the assessment of the viral load was not reliable for predicting HGSIL in normal smears. HR-HPV testing could be proposed in primary screening in association with cytology. With conventional cytology it significantly improves the detection of HGSIL. With the use of the same cervical scrape for HPV testing and liquid-based cytology, HR-HPV testing would allow to select positive samples treated in a second time for cytology which gives a good specificity. © 2001 Cancer Research Campaign http://www.bjcancer.com Keywords: HPV; cervical cancer; screening It is now well established that oncogenic (high-risk) human cervical scrapes offers a diagnostic assay without the sampling papillomaviruses (HPV) are a causal factor in the development problems and subjectivity of cytology, but such an approach needs of cervical intraepithelial and invasive neoplasias (Lorincz et al, a specific, sensitive, reliable and easy to perform method. 1992; Zur Hausen, 1994; Bosch et al, 1995; Walboomers et al, 5 years ago, a commercial HPV detection test, Hybrid Capture-I 1999). Infections with high-risk HPV (HR-HPV) are associated (HC-I), followed by second-generation test Hybrid Capture II (HC- with a relative risk of between 8 and 11 for the development of II) was introduced (Lorincz, 1996). HC-II is a non-radioactive, squamous intraepithelial lesions (SIL) and essentially low-grade reproducible, relatively rapid, liquid hybridization assay in micro- SIL (LGSIL) containing HR-HPV progress to high-grade SIL titres designed to detect 18 HPV types divided into high-risk (types (HGSIL) (Koutsky et al, 1992; Gaarenstroom et al, 1994). Because 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, and 68) and low-risk of this, there is an increasing interest in using HPV DNA detection (types 6, 11, 42, 43 and 44) groups. The sensitivity of this assay is either alone or in addition to classic cytologic examination, essen- quite similar to that of Polymerase Chain Reaction (PCR) (Clavel tially as a method for triaging women with a cytologic diagnosis of et al, 1998b; Peyton et al, 1998; Riethmuller et al, 1999). atypical squamous cells of undetermined significance (ASCUS) in Nevertheless current screening remains based on the cytopatho- their cervical smears (Cox et al, 1995; Wright et al, 1998; Manos logic classification of cervical smears, according to the Bethesda et al, 1999) and now for primary cervical screening (Cuzick et al, system, leading to colposcopy and histological sampling. Women 1995, 1999; Clavel et al, 1998a, 1999; Meijer et al, 1998; Kuhn in wealthier countries are largely protected via the Papanicolaou et al, 2000; Ratnam et al, 2000; Schiffman et al, 2000). Never- test. However, this test is not perfect and false-negative rates of 5 theless, the low sensitivity of HPV detection in previous studies to 50% have been reported (Schneider et al, 1996; Cuzick et al, has led some authors to consider that HPV testing does not appear 1999). Thus, in the last few years, significant technical advances to be of value for identifying women with abnormal smears who have raised the possibility of improving conventional cytology. could be safely followed up with cytology alone (Kaufman and Particularly, liquid-based cytology has improved sampling, fixa- Adam, 1999). Moreover, the low positive predictive value and the tion, staining and background, ensuring a more representative cost-effectiveness of HPV DNA testing have to be taken in consid- sample with a dramatic improvement in sensitivity (Papillo et al, eration for primary screening. However, HPV DNA testing in 1998; Sherman et al, 1998; Weintraub and Morabia, 2000). Recently, Cuzick et al (2000) have emphasized the urgent need to Received 25 January 2001 undertake a large trial of HPV testing in conjunction with other new Revised 7 March 2001 technologies including liquid-based cytology. Here we report our Accepted 20 March 2001 experience on HPV testing in primary screening of 7932 women, for Correspondence to: C Clavel the detection of HGSIL. For that, we compared the results of HPV 1616 HPV testing for primary cervical screening 1617 testing to the results of conventional and liquid-based cytology. smears within normal limits but presenting a HR-HPV infection Because most LGSIL regresses spontaneously, our primary were also systematically recalled 6 months later for a new cytolog- endpoint was the histological diagnosis of HGSIL at the biopsy. ical examination and HR-HPV testing followed by colposcopy if a lesion and/or a persistent HR-HPV infection was detected. Punch biopsy specimens were taken from the areas colposcopically suspi- MATERIAL AND METHODS cious for CIN. The women with a second HR-HPV test positive without any detectable lesions were recalled 6 to 12 months later for Study population a third control with cytological examination and HR-HPV testing A total of 7932 women with a median age of 34 years (range 15 to and the same indications as above for colposcopy and biopsy. 76 years) were recruited for the study between August 1997 and Women with regressive HR-HPV infection were also recalled for February 2001. This population was restricted to women who colposcopy 12 months later. By contrast, women with initial normal underwent their biennal or triennial routine screening in the smears and without any HR-HPV infection were followed with a Department of Obstetrics and Gynecology of the CHU of Reims. classical biennal or triennal cervical screening with a new cytolog- We excluded subjects on the basis of a recent cytologic abnor- ical examination and HR-HPV testing at the second control. Some mality and/or an untreated cervical lesion in the past 2 years, preg- of these women had also randomly a colposcopic examination. nant women and patients with AIDS. All women were informed of There were not particular selection criteria for colposcopy. The the aim of the study and gave their consent. primary endpoint of our study was the detection of a histologically proven HGSIL at the biopsy and/or on the LEEP specimen. Cytologic diagnosis HPV testing At the first gynaecologic examination, in 2281 women, 2 samples were taken: first, a cytologic smear with an Ayre’s spatula, for When conventional cytology was performed, specimens for HPV classical cytology, then one scrape for the HC-II test with a DNA testing were suspended in 1 ml of ViraPap/Viratype transport Cervexbrush (Medscan, Uppsala, Sweden). These samples were medium (Digene, Silver Spring, MD) and stored at –20˚C until suspended in 1 ml of specimen transport medium for HPV testing further processing. When samples were used for liquid based (Digene, Silver Spring, MD). In another way, 5651 women had cytology, 4 ml of the sample were centrifuged and the cell pellet was only one cervical scrape with a Cervexbrush at the first entry. resuspended in 200 ml of phosphate-buffered saline for HPV testing. Samples were prepared for liquid-based cytology with the HPV DNA detection was performed by the commercially available ThinPrep technique (Cytyc Corporation, Marlborough, Mass) and HC-II System (Digene). All scrapes were analysed for the presence 4 ml of the sample were used for HPV testing. In total, considering of HR-HPV types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59 and 68. the first samples and the follow-up of women, we collected 10 101 This enzyme-linked immunosorbent assay is based on a sandwich cervical smears and scrapes. Smears were classified according to hybridization followed by a nonradioactive alkaline phosphatase the Bethesda system for reporting cervical or vaginal cytological reaction with chemoluminescence in microplates. The chosen posi- –1 diagnosis. We selected women with adequate smears including tive threshold of this test was 1.0 pg ml of HPV DNA. metaplastic and/or endocervical cells according to the criteria of Samples were classified as positive for HR-HPV DNA if the Bethesda, which represented 92% of our total smears. However, relative light unit (RLU) reading obtained from the luminometer even if the smear was not considered as adequate, we also included was equal to or greater than the mean of the 3 positive control women with smears evocative of lesions. In another way, 4.8% of values supplied by the HC-II kit. As some authors have reported the samples treated for liquid-based cytology could not be used for that increasing HPV DNA levels of HR-HPV types were the prin- HPV testing and have been excluded. The cytotechnicians and cipal predictors of CIN (Cox et al, 1995), we used as proposed, the pathologists involved in the study were not informed about the ratio RLU/positive controls values to quantify HR-HPV DNA in results of the HPV testing. All smears showing cytological abnor- our samples. Moreover, we added other positive controls such as malities and biopsy specimens were examined by the same 2 inde- SiHa cell lines (1 to 2 copies of HPV type 16 per cell) to check the pendent pathologists, without knowledge of cytology results for reproducibility of the HC-II sensitivity. the biopsies examination. The results were compared and if the first 2 diagnoses disagreed, a third pathologist reviewed the case Statistical methods with no knowledge of preceding diagnoses. Consensus diagnoses The statistical methods used were mostly descriptive. Sensitivity, were determined by two-thirds majority when possible and specificity, positive and negative predictive values were deter- remaining discrepancies resolved by conference review. Patients mined by comparing the results of each test to the gold standard of with HGSIL were systematically treated by loop electrosurgical histology. A few high-grade lesions may have been missed if they excision procedure (LEEP). Data from these LEEP specimens were negative on both tests and thus women were not referred were included in the disease definitions. for colposcopy. 95% confidence interval for these values were assessed using either binomial or normal distribution, according to Colposcopic referral the data. Moreover, differences between HR-HPV detection and cytologic diagnosis values were compared using Fisher exact test In our protocol (Table 1), all the women presenting cytological or Chi-2 statistics as adequate, with a P value set to 5%. abnormalities evocative of cervical lesions (from ASCUS to HGSIL) were systematically recalled for colposcopy in the next few RESULTS weeks, at an interval ranging from 14 to 160 days (mean 95 days) after entry examination. Punch biopsy specimens were taken from At their first examination, in our population of 7932 women, 1214 the areas colposcopically suspicious for CIN. All the women with (15.3%) presented a HR-HPV infection. © 2001 Cancer Research Campaign British Journal of Cancer (2001) 84(12), 1616–1623 1618 C Clavel et al Table 1 Protocol for the follow-up of the women Cytology FIRST SCRAPE HPV detection _/It I~ Cytology - Cytology - Cytology + HPV - HPV + HPV + or - c __ J c_J c=J ------- ______________ d_b __ _ IMMEDIATE Colposcopy - Colposcopy + RECALLING Biopsy ------- --------- _/ _____ D __ _ 6 MONTHS Cytology LATER HPV detection /I t I~ Cytology - Cytology - Cytology + HPV - HPV + HPV + or - --------- ----- (..________,___.) Colposcopy - Colposcopy + Biopsy ---- c ____ ) I I ------- --------- t------------------- 6 to 12 MONTHS LATER Cytology Colposcopy HPV detection .__________,___ I I etc ------- ------- --------------------------- ------------------- --------- 3 YEARS LATER Cytology HPV detection Table 2 represents the prevalence of HR-HPV infection HPV detection and cytological and histological observed in our 7932 women according to their age, at the first diagnoses examination. There was a peak of infection in the third These results are summarized in Table 3. decade (23.6% of women) with a progressive decrease after 30 The prevalence of HR-HPV infection was significantly related years. to the severity of the cytologically detected lesions (P < 0.001). British Journal of Cancer (2001) 84(12), 1616–1623 © 2001 Cancer Research Campaign HPV testing for primary cervical screening 1619 Table 2 Prevalence of high-risk HPV infections according to age at the first women with normal smears with liquid-based cytology, 542 examination (10.4%) had a HR-HPV infection. In this cohort of 542 women, at the present time, 237 women (43.7%) had a follow-up on a period Age Women High-risk HPV from 4 to 20 months (median = 12 months). A persistent HR-HPV < 20 418 (5.3%) 84 (20.1%) infection at the second and eventually third examination was 21–30 1843 (23.2%) 435 (23.6%) observed in 95 women (40.1%) and was associated with the detec- 31–40 2076 (26.2%) 289 (13.9%) tion of a HGSIL in 10 patients, 4 to 12 months after the first entry 41–50 1925 (24.3%) 235 (12.2%) (median = 6 months). No HGSIL was detected by cytology and 51–60 1014 (12.8%) 110 (10.8%) colposcopy in women with regressive HR-HPV infection what- > 60 656 (8.3%) 61 (9.3%) ever the initial technique of cytology used. In addition, none of the Total 7932 1214 (15.3%) 1225 women with normal smears and without any HR-HPV infec- tion at the first control (893 with conventional cytology and 332 with liquid-based cytology), developed HGSIL detectable at the 25 women had a smear evocative of HGSIL at the first exami- second systematic cytological control and at the colposcopic nation with conventional cytology with a HR-HPV infection in all examination performed in 172 of them, on a period of 24 to 36 of them. The diagnosis of HGSIL was histologically confirmed in months (median = 30 months). 21 of them. 82 women had a smear evocative of HGSIL with liquid-based cytology, with a HR-HPV positive test in 79 (96.3%) of them. The diagnosis of HGSIL was confirmed by the biopsy in Results according to the technique of cytology and the 58 patients who were all tested positive for HR-HPV DNA. Out of age of women 77 women who had a smear evocative of LGSIL at the first exam- Tables 4 and 5 represent the respective sensitivity, specificity, ination with conventional cytology, 56 (72.7%) had a HR-HPV positive and negative predictive values of the various methods infection. 65 of them had a follow-up and 6 had an underlying used for the detection of a HGSIL histologically proven in the HGSIL associated with a HR-HPV. 200 women had a smear global population and in women aged >30 years. In these tables, evocative of LGSIL with liquid-based cytology with a HR-HPV only results of cytology and HPV testing at the first examination infection in 168 (84%). In the cohort of 182 women with a follow- have been considered for the evaluation of these techniques, since up, 8 HGSIL were detected, all associated with HR-HPV. most women had a liquid-based cytology for their follow-up. As 34 women presented ASCUS with conventional cytology with expected, liquid-based cytology gave a significantly higher HR-HPV in 19 of them (55.9%). In 19 women with a follow-up, 5 sensitivity (87.8% in the general population and 84.4% in women HGSIL were detected all tested positive for HR-HPV DNA. 175 aged > 30 years) than conventional cytology (68.1% in the general women presented ASCUS with liquid-based cytology. HR-HPV population and 57.7% in women aged > 30 years) (P < 0.05). DNA was detected in 94 cases (53.7%). At the colposcopic control However, the sensitivity of HR-HPV testing remained higher, at obtained in 135 women, 6 HGSIL were diagnosed all associated 100% in all cases. Nevertheless, if the negative predictive value with a HR-HPV infection. was always of 100%, the specificity and the positive predictive In the remaining population of 2145 women with cervical value of the HR-HPV testing were clearly lower than those of the smears within normal limits with conventional cytology, HR-HPV cytology whatever the technique used. Limiting the screening to DNA was detected in 231 (10.8%). Out of these 231 women tested women aged > 30 years slightly increased the specificity of the HR-HPV positive, according to our protocol, 168 women (72.7%) HR-HPV testing to 88.4% in scrapes treated for liquid-based had a follow-up on a period from 4 to 36 months (median = 24 cytology and to 90.1% in smears examined with conventional months). A persistent HR-HPV infection at the second and eventu- cytology, but did not modify the positive predictive value for ally third examination was observed in 66 women (39.3%) and HGSIL detection. However, we have to emphasize that in our was associated with the detection of a HGSIL in 15 patients, 4 to same referral population, with the same follow-up protocol, 58 out 20 months after the first entry (median = 10 months). Out of 5194 Table 3 Follow-up from the first smear to the final diagnosis First smear HG detected First smear HG detected Conventional HR-HPV Follow-up at the Thin-prep HR-HPV Follow-up at the cytology detection histology cytology detection histology 2145 WL 231 168 HPV+ 15 HG 5194 WL 542 237 HPV + 10 HG (94.0%) (10.8%) 893 HPV – (91.9%) (10.4%) 332 HPV– 34 ASCUS 19 19 HPV + 5 HG 175 ASCUS 94 89 HPV + 6 HG (1.5%) (55.9%) 7 HPV – (3.1%) (53.7%) 46 HPV – 77 LG 56 54 HPV + 6 HG 200 LG 168 160 HPV + 8 HG (3.5%) (72.7%) 11 HPV – (3.5%) (84.0%) 22 HPV – 25 HG 25 25 HPV + 21 HG 82 HG 79 79 HPV + 58 HG (1.1%) (100.0%) (1.4%) (96.3%) 3 HPV – 2281 331 1177 47 HG 5651 883 968 82 HG WL: without lesion; LG: low-grade lesion; HG: high-grade lesion; HR-HPV: high-risk HPV. © 2001 Cancer Research Campaign British Journal of Cancer (2001) 84(12), 1616–1623 1620 C Clavel et al Table 4 Evaluation of cytology and high-risk HPV testing for the detection of histologically proven high-grade lesion Methods Sensitivity Specificity PPV NPV (95% C.I.) (95% C.I.) (95% C.I.) (95% C.I.) HPV detection 47/47 (100%) 1950/2234 (87.3%) 47/331 (14.2%) 1950/1950 (100%) 93.8–100% 85.9–88.7% 10.4–18.0% 99.8–100% Conventional cytology 32/47 (68.1%) 2130/2234 (95.3%) 32/136 (23.5%) 2130/2145 (99.3%) 55.4–79.2% 94.5–96.2% 16.4–30.7% 99.0–99.6% HPV detection 82/82 (100%) 4768/5569 (85.6%) 82/883 (9.3%) 4768/4768 (100%) 96.4–100% 84.7–86.5% 7.4–11.2% 99.9–100% Thin prep cytology 72/82 (87.8%) 5184/5569 (93.1%) 72/457 (15.7%) 5184/5194 (99.8%) 80.3–93.3% 92.4–93.8% 12.4–19.1% 99.7–99.9% PPV: Positive predictive value, NPV: Negative predictive value, C.I.: Confidence interval. Table 5 Evaluation of cytology and high-risk HPV testing for the detection of a histologically proven high-grade lesion in women aged > 30 years Methods Sensitivity Specificity PPV NPV (95% C.I.) (95% C.I.) (95% C.I.) (95% C.I.) HPV detection 26/26 (100%) 1373/1524 (90.1%) 26/177 (14.7%) 1373/1373 (100%) 89.2–100% 88.6–91.6% 9.5–19.9% 99.8–100% Conventional cytology 15/26 (57.7%) 1457/1524 (95.6%) 15/82 (18.3%) 1457/1468 (99.2%) 39.1–75.0% 94.6–96.6% 9.9–26.6% 98.5–99.4% HPV detection 45/45 (100%) 3603/4076 (88.4%) 45/518 (8.7%) 3603/3603 (100%) 93.6–100% 87.4–89.4% 6.3–11.1% 99.9–100% Thin prep cytology 38/45 (84.4%) 3863/4076 (94.8%) 38/251 (15.1%) 3863/3870 (99.8%) 73.2–92.8% 94.1–95.5% 10.7–19.6% 99.5–99.8% PPV: Positive predictive value, NPV: Negative predictive value, C.I.: Confidence Interval. Table 6 Evaluation of the viral load (VL) estimated by the HC-II assay for the detection of a high-grade lesion in cytologically normal smears Methods VL Sensitivity Specificity PPV NPV HPV testing associated >3 15/15 (100%) 53/216 (24.5%) 15/178 (8.4%) 53/53 (100%) with conventional cytology >10 15/15 (100%) 88/216 (40.7%) 15/143 (10.5%) 88/88 (100%) HPV testing associated >3 10/10 (100%) 203/532 (38.1%) 10/329 (2.9%) 203/203 (100%) with thin prep cytology >10 7/10 (70%) 313/532 (58.8%) 7/226 (3.1%) 313/316 (99.0%) PPV: Positive predictive value; NPV: Negative predictive value. Table 7 Evaluation of cytology and high-risk HPV testing for the detection of a histologically proven high-grade lesion in function of the viral load (VL) Methods Sensitivity Specificity PPV NPV (95% C.I.) (95% C.I.) (95% C.I.) (95% C.I.) HPV detection. VL>3 44/47 (93.6%) 2008/2234 (89.9%) 44/270 (16.3%) 2008/2011 (99.8%) 83.5–98.6% 88.6–91.1% 11.2–20.7% 99.6–99.7% HPV detection. VL>10 42/47 (89.4%) 2049/2234 (91.7%) 42/227 (18.5%) 2049/2054 (99.7%) 78.4–96.1% 90.6–92.9% 13.5–23.6% 99.5–99.9% Conventional cytology 32/47 (68.1%) 2130/2234 (95.3%) 32/136 (23.5%) 2130/2145 (99.3%) 55.4–79.2% 94.5–96.2% 16.4–30.7% 99.0–99.6% HPV detection. VL>3 80/82 (97.6%) 5007/5569 (89.9%) 80/642 (12.5%) 5007/5009 (99.9%) 91.9–99.7% 89.1–90.7% 9.9–15.0% 99.8–99.9% HPV detection. VL>10 72/82 (87.8%) 5137/5569 (92.2%) 72/504 (14.3%) 5137/5147 (99.8%) 80.3–93.3% 91.6–92.9% 11.2–17.3% 99.7–99.9% Thin prep cytology 72/82 (87.8%) 5184/5569 (93.1%) 72/457 (15.7%) 5184/5194 (99.8%) 80.3–93.3% 92.4–93.8% 12.4–19.1% 99.7–99.9% PPV: Positive predictive value; NPV: Negative predictive value; C.I.: Confidence interval. British Journal of Cancer (2001) 84(12), 1616–1623 © 2001 Cancer Research Campaign HPV testing for primary cervical screening 1621 of 129 HGSIL (45.0%) were detected in women aged < 30 years. management of these women. Indeed in our series, HR-HPV This finding explains the absence of modification of the positive infection is frequent in this population of women (80.9%). In predictive value after 30 years. consequence, its use as a discriminating test for colposcopic referral seems of limited interest, even though all the HGSIL detected at the histological control of cytological LGSIL, were Value of the semi-quantitative viral load estimated by positive for HR-HPV DNA. In another way, in the literature, the HC-II assay for predicting the persistence of about 50% of smears with ASCUS are tested positive for HR- HR-HPV infection and the apparition of HGSIL HPV DNA and 5 to 10% of ASCUS correspond to an underlying HGSIL associated with HR-HPV infection. Thus, Manos et al Table 6 represents the evaluation of different viral load estimated (1999) and the ALTS group (Solomon et al, 2001) have proposed in RLU (3 and 10 RLU) by the HC-II assay for predicting the that, for women with ASCUS, HPV DNA testing can help to detection of HGSIL in smears within normal limits at the first identify those who have underlying HGSIL. Our results are in cytological examination with conventional and liquid-based agreement with this proposition since 5 HGSIL have been cytology. The sensitivities remained high but the specificities were detected at the colpo-histological control of ASCUS obtained in very low in any case. Table 7 evaluates the efficiency of different 26 women with initial conventional cytology and 6 HGSIL in 135 viral load values (1,3 and 10 RLU) for general screening. The use women with liquid-based cytology, all associated with high-risk of a viral load > 10 increased specificities and predictive positive HPV infection. If we consider the negative predictive value of the values and the sensitivity of the HPV testing remained very high, HC-II assay of 100%, HPV testing is a reliable test to select equivalent or superior to that of cytology. women with ASCUS and HR-HPV infection who had to be referred for colposcopy. DISCUSSION In our series, we have observed that in total, 773 out of 7339 This work using the HC-II assay on a series of 7932 French women with smears within normal limits presented a HR-HPV women attending routine cytologic screening clearly confirms the infection. These numerous positive results, particularly in women high prevalence of HR-HPV infection with a peak at 23.6% in the < 30 years old, are largely responsible for the low specificity and third decade and a progressive decrease after 30 years. In the same positive predictive value of the HPV testing. Numerous HPV way, Herrero et al (2000) using PCR have reported such a high infections are known to regress spontaneously, especially in young prevalence of HPV infection in women under 25 years in rural women and the mean HPV infection duration is between 8 to 14 Costa Rica. The high percentage of women tested positive in our months (Ho et al, 1998; Franco et al, 1999). However, Rozendaal cohort may partly be due to the technique used with the possibility et al (1996) have emphasized that the women with normal smears of cross-hybridization with unknown and/or additional HPV types and HR-HPV genotypes are 116 times more at risk of developing not included in the HC-II assay probe cocktails (Vernon et al, HGSIL, in contrast of women without HR-HPV. Moreover, the 2000), which may increase the number of positive results. There is persistence of HR-HPV infection is significantly associated with also a significant prevalence and/or persistence of HR-HPV infec- progressive disease (Ho et al, 1995; Remmink et al, 1995). Indeed, tion in women with HGSIL. HR-HPV DNA has been detected in in our follow-up of 168 (72.7%) of 231 women with smears within all the 129 women with a HGSIL histologically confirmed. Thus, normal limits with conventional cytology and with HR-HPV in our experience at the present time, whatever the collection infection, 15 (22.7%) out of 66 women with persistent HR-HPV protocol used for HPV testing, the sensitivity of HC-II for infection have presented a HGSIL within 4 to 20 months after the detecting HGSIL is 100%, but the specificity remains low, at first entry. At the present time, we have a shorter follow-up of only 87.6% when the initial sample has been associated with conven- 237 (43.7%) out of 542 women with normal smears with liquid- tional cytology, and at 85.6% when the initial sample has been based cytology and HR-HPV infection and the number of 10 treated for liquid-based cytology, with positive predictive values HGSIL detected may not exactly reflect the total HGSIL present in of 14.2% and 9.3% respectively. These results are quite similar to this population, but these preliminary data are also very promising. those of previous studies using HPV testing with HC-II for In another way, our more frequent follow-up of women with primary screening (Cuzick et al, 1999; Schiffman et al, 2000). normal smears and HR-HPV positive, induces a bias of selection Moreover, in our work, the sensitivity of liquid-based cytology is which logically results in more numerous diagnoses of HGSIL. also significantly higher than that of conventional cytology However, practically, a systematic intensive follow-up in parallel (87.8% vs 68.1%) (P < 0.05) as previously reported in the litera- of women with normal smears and without any HR-HPV infec- ture when the ThinPrep method has been compared to conventional tion, is difficult to propose and to obtain. Nevertheless, we cytology (Papillo et al, 1998; Sherman et al, 1998; Weintraub and underline that no HGSIL has been detected in our cohort of 1225 Morabia, 2000). However, if liquid-based cytology alone is a very women with initial HR-HPV negative and normal smears, efficient method, HR-HPV testing combined with cytology signif- followed with classic biennal or triennal screening. Thus, icantly increases the detection of HGSIL. according to our protocol of follow-up, the detection of a persis- The use of HPV testing for triage test in smears evocative of tent HR-HPV infection selects a population of women with normal ASCUS and LGSIL has been recently discussed in large series. smears at risk for developing a HGSIL. Adam et al (1998) have found that screening for HPV DNA with Considering that HPV infection in younger women often PCR, does not have prognostic value in women reported as spontaneously regress and that the incidence of cervical cancer having ASCUS or smears evocative of low-grade lesions. The in women younger than 30 years is very low, numerous authors ALTS group (2000) has considered that, because a very high (Wright et al, 1998; Cuzick et al, 1999; Stoler, 2000) have percentage of women with cytological evidence of LGSIL are recommended the use of HPV testing for older women (>30–35 positive for HPV DNA with the HC-II assay, there is limited years old). This could improve the specificity and the positive potential for HPV testing to direct decisions about the clinical predictive value of the test. Nevertheless, these propositions do © 2001 Cancer Research Campaign British Journal of Cancer (2001) 84(12), 1616–1623 1622 C Clavel et al not consider the frequent early age of the first sexual intercourse a good technical approach and a high professional skill to be as a source of HPV infection with a higher risk for developing a highly sensitive. Considering that the mean time from detectable HGSIL before 30 years. In our experience, if we reserve HPV LGSIL to preclinical invasive cancer is 12–13 years (Gustafson testing in women > 30 years old, the positive predictive value of and Adami, 1989), Meijer et al (1998) have proposed that women HPV testing is not modified, but the specificity slightly with cytologically normal smears and a negative HR-HPV test increases from 87.3% to 90.1% in samples associated with could be rescreened every 8 years. If we subscribe to this conventional cytology and from 85.6% to 88.4% in samples screening policy, HPV testing combined with cytology would treated for liquid-based cytology. Again, we have to emphasize significantly lower the number of cervical smears and the inci- that 58 out of our 129 patients (45.0%) with HGSIL were women dence of colposcopy with a more efficient screening. Another < 30 years old. This clearly explains the absence of modification proposition is to begin the primary cervical screening with HPV of the predictive positive values after 30 years. Consequently, detection. This policy has been proposed in low-resource settings limiting HPV testing in women > 30 years in primary screening where HPV DNA testing programmes may be easier to implement will miss the increasing number of HGSIL present under this than cytologic screening (Kuhn et al, 2000; Schiffman et al, age. 2000). In our wealthier countries, we can use the same cervical There may be utility in semiquantitative measurement of high scrape for HPV testing and liquid-based cytology. HR-HPV viral load to increase the specificity of HPV testing. Indeed, testing would allow to select positive samples treated in a second Cuzick et al (1994) have shown that in women with cytologic time for liquid-based cytology which gives a better sensitivity abnormalities, HPV type 16 positivity at a high-level detected by a than conventional cytology and a good specificity. Women with semi-quantitative PCR is strongly related to HGSIL. Ho et al cervical abnormalities in their smears will be immediately (1995) have also suggested that SIL with a high viral load are more recalled for colposcopy. A more intensive cytological screening likely to persist than those with a low level of HPV DNA. A recent with HPV positive women with normal smears, with an algorithm study using a sensitive real time quantitative PCR assay has clearly of 6 to 12 months, to confirm the persistence of HPV infection demonstrated that cervical carcinoma in situ associated with HPV and/or the occurrence of SIL. All these propositions of new type 16 occurs mainly in HPV type 16 positive women who had managements for the cervical screening require extensive and consistently high viral loads long term (Ylitalo et al, 2000). Thus, multicentric studies in different countries to be validated in terms a high viral load may be considered as a risk factor and preferen- of efficiency and costs–benefits. In any case, the introduction of tially observed in potentially evolutive lesions and in HGSIL. This the HPV testing represents a new promising technology for parameter could be semi-quantitatively evaluated by the relative primary cervical screening. light unit values (RLU) provided by the HC-II assay. Our present results with HC-II show that when this parameter is basically ACKNOWLEDGEMENTS applied for predicting the apparition of a HGSIL in women without any detectable lesions, the specificity is very low <60% This work was supported by a grant from the European but the sensitivity remains very good. Indeed for a cervical Community (L’Europe contre le Cancer), the ARC (Association de sample/positive control ratio >10, the best results are obtained Recherche sur le Cancer), the ARERS, the Ligue Contre le Cancer when HPV testing is performed with conventional cytology, with a (Comités de la Marne, de l’Aisne, de la Haute-Marne et de sensitivity for predicing a HGSIL of 100%, but with a specificity l’Aube), the CHU of REIMS and by the LIONS Club of SOIS- of 40.7% and a predictive positive value of 10.5%. In another way, SONS. 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British Journal of CancerSpringer Journals

Published: Jun 12, 2001

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