Human Papillomavirus (HPV) DNA Triage of Women with Atypical Squamous Cells of

Transcription

1 JCM Accepts, published online ahead of print on 1 February 2012 J. Clin. Microbiol. doi: /jcm Copyright 2012, American Society for Microbiology. All Rights Reserved Human Papillomavirus (HPV) DNA Triage of Women with Atypical Squamous Cells of Undertermined Significance (ASC-US) with Cobas 4800 HPV and Hybrid Capture 2 Tests for Detection of High-Grade Lesions of the Uterine Cervix Simon Grandjean Lapierre 1, Philippe Sauthier 1,2, Marie-Hélène Mayrand 1,2, Simon Dufresne 1, Patrick Petignat 3, Diane Provencher 1,2, Pierre Drouin 1,2, Philippe Gauthier 1,2, Marie-Josée Dupuis 1,2, Bertrand Michon 1,2, Stéphan Ouellet 1,2, Rachid Hadjeres 1,2, Alex Ferenczy 4, Eduardo LF Franco 5 and François Coutlée * 1,2,5 1- Département de Microbiologie et Infectiologie, de Gynécologie-Obstétrique et Pathologie, Centre Hospitalier de l Université de Montréal, Montréal, Québec, Canada. 2- Départements de Microbiologie-Immunologie, de Gynécologie-Obstétrique et de Pathologie, Université de Montréal, Montréal, Québec. 3- Unité d Oncogynécologie, Hôpitaux Universitaires de Genève, Genève, Suisse. 4- Department of Pathology and Obstetrics & Gynecology, Jewish General Hospital and McGill University, Québec, Canada 5- Division of Cancer Epidemiology, McGill University, Montreal, Québec. Address correspondence to: François Coutlée, Département de Microbiologie et Infectiologie, Hôpital Notre-Dame du Centre Hospitalier de l'université de Montréal, 1560 Sherbrooke est, Montréal (Québec), H2L 4M1, Canada. Tel , 25162; fax: francois.coutlee@ssss.gouv.qc.ca. Source of support: This work was supported by Roche Molecular Systems. Running Head: HPV DNA triage in ASCUS Word count:

2 ABSTRACT (word=200) The triage of women with high-risk HPV (HR HPV) positive atypical squamous cells of undetermined significance (ASC-US) smears to colposcopy is now an integrated option in clinical guidelines. The performance of cobas 4800 HPV and Hybrid Capture 2 (HC2) for HR HPV DNA detection in cervical samples in Preservcyt TM was compared in 396 women referred to colposcopy for ASC-US. Of these, 316 did not have cervical intraepithelial neoplasia (CIN), 47 had CIN1, 29 had CIN2 or CIN3 (CIN2+), and 4 had CIN of unknown grade. HR HPV was detected in 129 (32.6 %) and 149 (37.6 %) samples with HC2 and cobas 4800 HPV, respectively (p=0.15). The clinical sensitivity and specificity to detect CIN2+ was 89.7 % (95 % CI ) and 66.7 % (95% CI ), 93.1 % (95 % CI ) and 72.2 % (95 % CI ) with cobas 4800 HPV and HC2, respectively. The performance of cobas 4800 HPV was similar to HC2 to identify women with ASC-US who would benefit the most from colposcopy. Downloaded from on August 22, 2018 by guest 2

3 INTRODUCTION The primary screening of cancer of the uterine cervix is based in most countries on Pap cytology testing. Women with an abnormal cytology are referred for colposcopy-directed biopsy to assess the presence and grade of cervical disease. In North America, for each new case of cervical cancer, there are between 50 to 100 cases of abnormal smears consistent with low-grade (LSIL) or high-grade squamous intraepithelial lesions (HSIL) 10. Additionally, there are at least twice as many cases of atypical squamous cells of undetermined significance (ASC-US). Up to 15% of women with ASC-US have underlying high-grade cervical intraepithelial neoplasia (CIN 2 or CIN3) 19. Three clinical options have been proposed by the American Society for Colposcopy and Cervical Pathology to refer women with ASC-US to colposcopy, namely, repeat cytology, immediate referral to colposcopy, and reflex testing with generic assays for detection of high-risk human papillomavirus (HR HPV) genotypes 6,21. When liquid-based cytology is used or when co-collection for HR HPV DNA testing can be obtained, HR HPV testing is the preferred triage approach 21. Since the introduction of the Hybrid Capture 2 assay (HC2 ; Qiagen, Inc., Mississauga, Ontario, Canada), several new HPV generic assays have been developed for the detection of HR HPV for ASC-US triage 16. The cobas 4800 HPV test (Roche Diagnostics, Laval, Québec, Canada) is a novel assay that detects HPV66 in addition to the 13 HR HPV genotypes detected by HC2. The cobas 4800 HPV detects HPV66 in addition to the panel of 13 types detected with HC2. Nevertheless, HPV66 is detected by HC2 due to cross-hybridization 9. One report found that cross-reactivity with low-risk genotypes was more frequently encountered with HC2 than the cobas 4800 HPV test 13. Few studies have evaluated the screening performance of the cobas 4800 HPV test 3,5,11,13,14,18,22. 3

4 In this work, we compared the clinical performance of cobas 4800 HPV to that of HC2 on clinical specimens collected from women referred for colposcopy because of ASC-US. HPV genotypic analysis was also performed with the Linear Array HPV genotyping (LA) assay (Roche Diagnostics, Laval, Canada) on all samples to assess cross-reactivity of the HPV generic assays. MATERIALS AND METHODS Study design and population. Participants were recruited consecutively from December 2005 to December 2007 if they were referred for colposcopy because of at least one ASC-US cytology, were 24 years old, and had not received treatment for CIN in the last 2 years 9. Cervical cells were first collected with a cytobrush for a conventional cytology. A second cervical specimen was collected with a cytobrush prior to colposcopic examination. The cytobrush was washed into the PreservCyt collection medium (Hologic, Inc., Marlborough, MA) and kept at room temperature for one week, and at -20 C thereafter. Samples in Preservcyt were processed for HPV DNA testing within 1 week for HC2 testing and LA, and 3 to 5 years later depending on patients inclusion dates with cobas 4800 HPV (Roche Diagnostics). Colposcopy-guided biopsies of lesions were obtained and histological diagnosis was established without knowledge of HPV test results. Biopsy samples with CIN2 or CIN3 (CIN2+) were confirmed by a second pathologist. This study was approved by the ethics committee of the Centre Hospitalier de l Université de Montréal. All participants provided written informed consent. HPV DNA testing. Technologists performing each HPV DNA test were blinded to other HPV test results as well as to cytology, colposcopy, and histology results. Four ml of PreservCyt samples were processed in Qiagen sample conversion kit and tested with HC2 according to the manufacturer's instructions with probe set B, a pool of full length HPV RNA 4

5 probes against 13 HR genotypes including 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59 and 68. Sample reactivity was measured in relative light units (RLU). A specimen was considered positive for HR HPV DNA if the ratio of the specimen RLU to the mean RLU of triplicates of a positive control at 1 pg per ml was >3.00. Samples with ratios between 1.00 and 3.00 were retested twice and were considered positive if 2 out of 3 results had a ratio >1.00. Four hundred µl of samples in Preservcyt were tested with cobas 4800 HPV according to manufacturer s recommendations. This assay detects simultaneously 14 HR HPV genotypes. HPV16 and HPV18 are detected individually while 12 HR genotypes (31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66 and 68) are detected as a group. The assay also evaluates the quality of samples by testing simultaneously for the presence of β-globin DNA. DNA was also extracted from Preservcyt samples using the AmpliLute Liquid Media Extraction Kit on the Qiagen MDx platform, as described in the manufacturer s instructions. Fifty µl of DNA extracted with the AmpliLute Liquid Media Extraction Kit was analyzed for HPV genotyping with LA according to a standard protocol 8. Samples positive with the crossreactive probe for HPV-52 and containing one of the cross-reactive types other than 52 (HPV- 33, -35, or -58) were further tested with a real-time PCR assay specific for HPV Thirtysix mucosal HPV genotypes are detected with LA (HPV types 6, 11, 16, 18, 26, 31, 33, 34 (formerly known as type 64), 35, 39, 40, 42, 44 (formerly known as type 55), 45, 51, 52, 53, 54, 56, 58, 59, 61, 62, 66, 67, 68, 69, 70, 71, 72, 73, 81, 82 (including subtype IS39), 83, 84, and 89 (formerly known as CP6108)). Statistical Analysis. Twelve genotypes are currently considered to be HR (HPV16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59)and 13 to be probable/possible HR types (HPV26, 30, 34, 53, 66, 67, 68, 69, 70, 73, 82, 85, 97) 2. The crude percent agreement between tests was the percentage of paired tests with identical results. The modified Wald method was used to calculate 95% confidence intervals (CI) around binomial proportions. The kappa statistic was 5

6 calculated to ascertain agreement between HPV detection assays that was in excess of that due to chance. In general, a kappa value above 0.75 indicates excellent agreement, between 0.40 and 0.75 indicates fair to good agreement, and below 0.40 represents poor agreement beyond chance. Differences in the rate of detection of HR HPV types were tested for statistical significance using an exact Chi-square McNemar s test. CIN2+ served as the clinical endpoint for the evaluation of the clinical performance of HPV tests. Sensitivity, specificity, negative predictive value (NPV) and positive predictive value were calculated from contingency tables. All statistical tests were two-sided. P values <0.05 were considered statistically significant. Statistical analyses were performed with STATISTICA version 6 software (StatSoft, Tulsa, OK). RESULTS HPV prevalence with LA in the study population. Of the 408 women who agreed to participate, 396 were eligible to the study 9. The characteristics of recruited women were described recently 9. Briefly, the age of participants ranged from 24 to 75 years (median, 38 years). Combining the 182 women who had a normal colposcopic exam without biopsy with those having a normal biopsy on histology, 316 women did not have CIN, 47 had CIN1, 29 had CIN2+, and 4 had CIN that could not be graded. There was no case with invasive cancer. HR HPV detection with cobas 4800 HPV and HC2. Three samples were tested a second time with cobas 4800 HPV (2 were β-globin negative, one contained a clot) and all were adequate upon retesting. The prevalence stratified by CIN grades of HR HPV genotypes detected with HC2 and with cobas 4800 HPV is provided in Table 1. Overall, the detection rate of HR HPV DNA was higher with cobas 4800 HPV (37.6%, ) than with HC2 (32.6%, ), but the difference was not statistically significant (p=0.15). The detection rate of HR HPV increased with CIN grade for both generic HPV assays (Table 1). Results 6

7 obtained with LA and Amplicor HPV test on these samples have been reported in a previous publication 9. Agreement between cobas 4800 HPV and HC2 for detection of HR HPV. We calculated a 87.9% agreement between HC2 and cobas 4800 HPV (Table 2). After stratifying by CIN grade, kappa values indicated good to excellent agreement, except for women with CIN 2+. Of the 14 HC2-positive and cobas 4800 HPV-negative samples, 3 contained a HR HPV genotype (2 HPV58, 1 HPV16) and 6 contained a possible/probable HR genotype (4 HPV53, 2 HPV70) with LA. Eleven of these 14 women had a normal cervix, 1 had CIN1 and 2 had CIN2+ (1 HPV16, 1 HPV53). Of these 14 samples, 9 were retested with HC2 to assess if there was a loss of HPV DNA over time explaining the false-negative cobas 4800 HPV results. Five specimens could not be retested because of insufficient remaining quantities of sample. The 9 samples tested positive for HR HPV with HC2, including the two participants with CIN2+. Of the 34 HC2-negative but cobas 4800 HPV test-positive samples, 31 contained at least one HR HPV genotype (10 HPV16; 6 HPV31; 4 HPV56; 2 each for HPV55, 39, 51, and 52; 1 each for HPV 18, 35, and 45) and 2 contained one probable/possible HR genotype (1 HPV82, 1 HPV73). Most of the latter samples were collected from women without CIN (29 were CIN-free, 3 had CIN1, 1 had CIN2+ and 1 had CIN of unknown grade). Agreement between cobas 4800 HPV and LA for HR HPV genotyping. The agreement between cobas 4800 HPV and LA for the detection of HR HPV DNA (Table 3) was excellent for all CIN grades, with good to excellent kappa values >0.78 for each comparison. Even though LA was more frequently positive than cobas 4800 HPV for the 12 other HR HPV genotype group, the agreement between these assays was uniformly >92%. Cross-reactivity of the cobas 4800 HPV for types not included in the panel of 14 HR HPV genotypes was evaluated based on genotyping data obtained with LA. Seven samples (5 from women without CIN, 1 with CIN1, 1 with CIN2+) that were positive for HR HPV by 7

8 cobas 4800 HPV tested negative with LA for any of the 14 HR genotypes targeted by the generic assay: 3 contained one of the probable/possible HR genotypes 73 or 82, 1 contained low-risk genotype 61 and 3 were negative for HPV DNA (Table 3). Four of these samples tested positive for HR HPV with HC2, including two out of the three LA-negative samples 9. Clinical performance of cobas 4800 HPV. This was evaluated by comparing the ability of cobas 4800 HPV and HC2 to detect CIN2+ (Table 4). The sensitivity of 89.7% of cobas 4800 HPV was similar to that of HC2. Although the specificity of cobas 4800 HPV was lower than that of HC2, the difference was not significant (p=0.13) and the 95% CI overlapped. When the analysis was restricted to women 30 years of age, the specificity of cobas 4800 HPV increased to 74.1% (95% CI ). Only one sample from a woman with CIN 2+ was negative with both tests and with LA. A follow-up colposcopy and biopsy for this participant did not reveal CIN. Of the 2 samples from women with CIN 2+ that tested negative with HC2, one tested positive for 12 other HR HPV genotypes with cobas 4800 HPV and was positive for HPV51 with LA. These results were confirmed on a follow-up visit. Of the 3 samples from women with CIN 2+ that were negative with cobas 4800 HPV, 2 tested positive with HC2. One contained HPV16 and 53 DNA whereas the other specimen tested positive for HPV53 DNA. A specimen obtained at a follow-up visit for the latter participant tested negative for HR HPV DNA with HC2. Of the 316 women with a normal colposcopy at accrual, 99 were positive for HR HPV DNA with cobas 4800 HPV. A follow-up colposcopy was performed from 61 to 522 days (median of 190 days) after the first colposcopy for 63 of these 99 HR HPV-positive women. Eight participants were found to have CIN2+ despite an initial colposcopy without CIN. Only 3 of these 8 women had a repeat testing with cobas 4800 HPV and all were HR HPV-positive (HPV16 at both visits for 2, 12 other HR HPV at both visits for 1). Results for HC2 with a shorter follow-up period have been presented elsewhere 9. 8

9 DISCUSSION Reflex HR HPV DNA testing is now recognized as a cost-effective strategy to refer women with an ASC-US cytology for colposcopy 12,17. The immediate colposcopy option for ASC-US would have subjected the majority of women to unnecessary procedures, as only 7.3% of women in our experience had CIN2+. The objective of our work was to compare for triaging women with ASC-US for colposcopy the clinical performance of a novel real-time PCR assay, the cobas 4800 HPV test, with a worldwide validated standard HPV DNA assay for the evaluation of new generic HPV tests 15. The assay has been evaluated in a few reports, one using a prototype assay, one focusing on ASCUS triage, one on cervical cancer screening, one on the clinical validation of the assay and two others on samples from women with various grades of disease 3,5,11,13,14,18. The prevalence of HR HPV infection of 37.6% in our population of women with ASC- US was lower than the 48% rate reported in the ASC-US-Low-Grade Squamous intraepithelial Lesion Triage Study (ALTS) or the 44.7% rate calculated in a meta-analysis of published studies on ASC-US and HR HPV, but was higher than the rate of 32.6% reported in the Addressing THE Need for Advanced HPV Diagnostics (ATHENA) study 1,4,18. These discrepancies most likely reflect the difference in the age structure of each study population, as HPV prevalence decreases as age increases 20. We specifically recruited women at least 24 years of age and the median age of our participants (38 years old) was higher than those of the ALTS study (29 years old). An excellent agreement was obtained between cobas 4800 HPV and HC2 or LA for the detection of HR HPV DNA in CIN. We calculated an agreement >93% between cobas 4800 HPV and LA for detection of HPV16 and the 12 other HR HPV genotypes. The highest agreement was found for HPV18. The agreement between both assays for HPV16 detection 9

10 was slightly lower in our study in the presence of CIN2+, a result that may reflect the small number of women with this grade of CIN. A very good agreement between cobas 4800 HPV and LA had already been reported in one study 3. These investigators also found that a greater number of women without CIN tested positive for the 12 other HR HPV by LA only. We also found that agreement was stronger between these assays for detection of the 12 other HR HPV genotypes in the presence of CIN than in women without CIN. When results obtained with cobas 4800 HPV were compared to those obtained with LA for women with consecutive samples positive for HR HPV with cobas 4800 HPV, agreement between tests was obtained for 38 of the 40 pairs of visits. The clinical performance of cobas HPV 4800 to refer women with ASC-US for colposcopy was similar to HC2. An international panel of experts has recently published guidelines on requirements for HPV detection assays in primary screening 15. The candidate test should have a clinical sensitivity for CIN2+ not less than 90% of the clinical sensitivity of HC2 in women of at least 30 years of age and a specificity no less than 98% of that measured for HC2. This recommendation was based on a pooled sensitivity for HC2 of 97.9% in primary screening in several studies. The cobas 4800 HPV test fulfilled these requirements for clinical use in a recent validation of the cobas 4800 HPV test 11. However, that work included only 11 women with ASCUS. One study also evaluated the clinical performance of cobas 4800 HPV for detection of CIN 2+ among women with ASC-US 18. In ATHENA, the sensitivity and specificity of cobas 4800 HPV on samples from 1578 women were respectively 90% (95% CI 81.5 to 94.8) and 70.5% (95% CI 68.1 to 72.7), values that are very similar to those obtained in the current study. In another study, the cobas 4800 HPV test demonstrated comparable analytical performance to HC2 for the detection of HR HPV infection, but had lower cross-reactivity with low risk types 13. A last evaluation demonstrated that cobas 4800 HPV test had a similar performance to that of HC2 outside of the context of 10

11 ASCUS triage 14. In agreement with current guidelines, we found that repeating HR HPV testing in women initially positive for HR HPV but with a normal colposcopy allows detection of CIN 2+ with cobas 4800 HPV that was undetected at the first colposcopy 21. Our study has several strengths. We consecutively recruited women with well-defined inclusion criteria. All patients were referred for colposcopy because of at least one previous ASC-US smear. Colposcopists were masked to HPV results. HPV assays were applied on the same cytobrush sample and performed in parallel in a diagnostic setting. HPV DNA detection tests were standardized. Discrepancies between HPV generic assays were evaluated with a standardized genotyping assay applied prospectively on all samples. A prospective follow-up was included for several patients to further ascertain cervical status. Our study was, however, limited by the absence of standardized management of participants for repeat HPV testing and colposcopic evaluation of women without lesion at the initial colposcopy and by loss to follow-up. Also, the cobas 4800 HPV test was performed several years after testing was done with HC2 on samples in Preservcyt. Most of the samples testing negative with the novel assay but positive initially with HC2 could be retested and were all positive with HC2. Our study included a small number of participants with CIN2+. The cobas 4800 HPV test will refer fewer women to colposcopy than automatic referral of all women with ASC-US. It will also distinguish women infected with HPV 16 or HPV 18 from other HR HPV genotypes, although this feature is not yet included in clinical guidelines for the management of women with ASC-US. When utilized by a diagnostic laboratory, the performance of cobas 4800 HPV was equivalent to that of HC2. ACKNOWLEDGMENT This work was supported by Roche Molecular Systems. 11

12 Reference List Arbyn, M., E. Paraskevaidis, P. Martin Hirsch, W. Prendiville, and J. Dillner Clinical utility of HPV-DNA detection: triage of minor cervical lesions, follow-up of women treated for high-grade CIN: an update of pooled evidence. Gynecol. Oncol. 99:S Bouvard, V., R. Baan, K. Straif, Y. Grosse, B. Secretan, F. El Ghissassi, L. Benbrahim Tallaa, N. Guha, C. Freeman, L. Galichet, and V. Cogliano A review of human carcinogens--part B: biological agents. Lancet. Oncol. 10: Castle, P. E., M. Sadorra, T. Lau, C. Aldrich, F. A. Garcia, and J. Kornegay Evaluation of a prototype real-time PCR assay for carcinogenic human papillomavirus (HPV) detection and simultaneous HPV genotype 16 (HPV16) and HPV18 genotyping. Jm Clin Microbiol 47: Castle, P. E., D. Solomon, M. Schiffman, and C. M. Wheeler Human papillomavirus type 16 infections and 2-year absolute risk of cervical precancer in women with equivocal or mild cytologic abnormalities. J Natl. Cancer Inst. 97: Castle, P. E., M. H. Stoler, T. C. J. Wright, A. Sharma, T. L. Wright, and C. M. Behrens Performance of carcinogenic human papillomavirus (HPV) testing and HPV16 or HPV18 genotyping for cervical cancer screening of women aged 25 years and older: a subanalysis of the ATHENA study. Lancet Oncol 12: Coutlee, F., M. H. Mayrand, M. Roger, and E. L. Franco Detection and typing of human papillomavirus nucleic acids in biological fluids. Public Health Genomics 12: Coutlee, F., D. Rouleau, G. Ghattas, C. Hankins, S. Vezina, P. Cote, J. Macleod, A. de Pokomandy, D. Money, S. Walmsley, H. Voyer, P. Brassard, and E. Franco Confirmatory real-time PCR assay for human papillomavirus (HPV) type 52 infection in anogenital specimens screened for HPV infection with the linear array HPV genotyping test. J. Clin. Microbiol. 45: Coutlee, F., D. Rouleau, P. Petignat, G. Ghattas, J. Kornegay, P. Schlagg, S. Boyle, C. Hankins, D. Vézina, P. Cote, J. Macleod, H. Voyer, P. Forest, S. Walmsley, and E. L. Franco Enhanced Detection and typing of Human Papillomavirus DNA in Anogenital Samples with PGMY primers and the LINEAR ARRAY HPV Genotyping Test. J. Clin. Microbiol. 44: Dufresne, S., P. Sauthier, M. H. Mayrand, P. Petignat, D. Provencher, P. Drouin, P. Gauthier, M. J. Dupuis, R. Hadjeres, E. L. Franco, and F. Coutlee HPV DNA triage of women with ASCUS living in Montreal: demonstration of differences between Amplicor HPV and Hybrid capture 2. J Clin Microbiol 49:

13 Franco, E. L. and D. M. Harper Vaccination against human papillomavirus infection: a new paradigm in cervical cancer control. Vaccine 23: Heideman, D. A., A. T. Hesselink, J. Berkhof, F. van Kemenade, W. J. Melchers, N. F. Daalmeijer, M. Verkuijten, C. J. Meijer, and P. J. Snijders Clinical validation of the cobas 4800 HPV test for cervical screening purposes. J. Clin. Microbiol. 49: Kim, J. J., T. C. Wright, and S. J. Goldie Cost-effectiveness of alternative triage strategies for atypical squamous cells of undetermined significance. JAMA 287: Mateos Lindemann, M. L., M. J. Rodriguez Dominguez, Chac&#x00f3, J. de Antonio, M. T. Sandri, A. Tricca, M. Sideri, H. Khiri, S. Ravet, S. Boyle, C. Aldrich, and P. Halfon Analytical Comparison of the cobas HPV Test with Hybrid Capture 2 for the Detection of High-Risk HPV Genotypes. J Mol Diagn 14: Mateos, M. L., Chac&#x00f3, J. de Antonio, Rodr&#x00ed, D. guez, M. nguez, I. Sanz, and M. D. Rubio Evaluación de un sistema de PCR a tiempo real (cobas 4800) para la detección separada de los genotipos 16 y 18 y otros genotipos de alto riesgo del virus del papiloma humano en la prevención del cáncer cervical. [Evaluation of a prototype real-time PCR assay for the separate detection of human papilloma virus genotypes 16 and 18 and other high risk human papillomavirus in cervical cancer screening]. Enferm. Infecc. Microbiol. Clin 29: Meijer, C. J., J. Berkhof, P. E. Castle, A. T. Hesselink, E. L. Franco, G. Ronco, M. Arbyn, F. X. Bosch, J. Cuzick, J. Dillner, D. A. Heideman, and P. J. Snijders Guidelines for human papillomavirus DNA test requirements for primary cervical cancer screening in women 30 years and older. Int J Cancer 124: Schiffman, M., N. Wentzensen, S. Wacholder, W. Kinney, J. C. Gage, and P. E. Castle Human papillomavirus testing in the prevention of cervical cancer. J Natl Cancer Inst 103: Solomon, D., M. Schiffman, R. Tarone, and S. g. ALTS Comparison of three management strategies for patients with atypical squamous cells of undetermined significance: baseline results from a randomized trial. J Natl Cancer Inst 93: Stoler, M. H., T. C. J. Wright, A. Sharma, R. Apple, K. Gutekunst, and T. L. Wright High-risk human papillomavirus testing in women with ASC-US cytology: results from the ATHENA HPV study. Am J Clin Pathol 135: The Atypical Squamous Cells of Undetermined significance/low Grade Squamous Lesions Triage Study (ALTS) Group Results of a randomized trial on the management of cytology interpretations of atypical squamous cells of undetermined significance. Am J Obstet Gynecol 188: Trottier, H. and E. L. Franco The epidemiology of genital human papillomavirus infection. Vaccine 24:S

14 Wright, T. C. J., L. S. Massad, C. J. Dunton, M. Spitzer, E. J. Wilkinson, and D. Solomon consensus guidelines for the management of women with abnormal cervical cancer screening tests. Am. J. Obstet. Gynecol. 197: Wright, T. C. J., M. H. Stoler, A. Sharma, G. Zhang, C. Behrens, and T. L. Wright Evaluation of HPV-16 and HPV-18 genotyping for the triage of women with high-risk HPV+ cytology-negative results. Am J Clin Pathol 136: Downloaded from on August 22, 2018 by guest 14

15 Table 1 High risk HPV DNA detection with the cobas 4800 HPV and HC2 tests by grade of CIN for women referred to colposcopy because of an ASC-US cytology cobas 4800 HPV test results (%, 95% CI) HC2 test results (%, 95% CI) Diagnosis group n 12 other HR HPV + HPV18 + HPV16 + HR HPV + HR HPV + Total (29.8%, ) 7 (1.8%, ) 43 (10.9%, ) 149 (37.6%, ) 129 (32.6%, ) No CIN (24.1%, ) 5 (1.6%, ) 24 (7.6%, ) 99 (31.3%, ) 81 (25.6%, ) CIN (40.4%, ) 2 (4.3%, ) 7 (14.9%, ) 22 (46.8%, ) 20 (42.6%, ) CIN (75.9%, ) 0 (0.0%, ) 11 (37.9%, ) 26 (89.7%, ) 27 (93.1%, ) Diagnoses were obtained at colposcopy with biopsy in the presence of cervical lesions. Women without lesion visible at colposcopy were considered to be CIN-free. CIN grade could not be established for 4 women who were excluded from the table. The group of 12 other HR HPV genotypes includes HPV31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66 and 68. HR HPV combines results obtained for the 12 other HR HPV, HPV16 and HPV18. HC2 is the Hybrid Capture 2 assay that detects 13 HR HPV genotypes (HPV16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, and 68.); CIN, cervical intraepithelial neoplasia; CI, confidence interval. 15

16 Table 2 Agreement between cobas 4800 HPV and HC2 tests for the detection of HR HPV genotypes in endocervical specimens collected from women having an ASC-US cytology Number of samples (%) Diagnosis n Cobas + / HC2 + Cobas - / HC2 + Cobas + / HC2 - Cobas - / HC2 - % Agreement (95% CI) Kappa (95% IC) Total (29.0) 14 (3.5) 34 (8.6) 233 (58.8) 87.9 ( ) 0.74 ( ) No CIN (22.2) 11 (3.5) 29 (9.2) 206 (65.2) 87.3 ( ) 0.69 ( ) CIN (40.4) 1 (2.1) 3 (6.4) 24 (51.1) 91.5 ( ) 0.83 ( ) CIN (86.2) 2 (6.9) 1 (3.4) 1 (3.4) 89.7 ( ) 0.35 ( ) Four women had a diagnosis of CIN that could not be graded and were excluded from the table: one was positive for HPV16 in cobas 4800 HPV and LA, one was positive for 12 other HR HPV in cobas 4800 HPV and was HPV56-positive with LA. See footnote of Table 1 for abbreviations. 16

17 Table 3 - Agreement between cobas 4800 HPV and LA tests for the detection of HPV16, HPV18, 12 other HR HPV genotypes in endocervical specimens collected from women having an ASC-US cytology Number of samples (%) Diagnosis n Cobas + / LA + Cobas - / LA + Cobas + / LA - Cobas - / LA - % Agreement (95% CI ) Kappa (95% CI) HPV18 Total (1.5) 0 (0.0) 1 (0.3) 389 (98.2) 99.7 ( ) 0.92 ( ) No CIN (1.3) 0 (0.0) 1 (0.3) 311 (98.4) 99.7 ( ) 0.89 ( ) CIN (4.3) 0 (0.0) 0 (0.0) 45 (95.7) 100 ( ) 1.00 ( ) CIN (0.0) 0 (0.0) 0 (0.0) 29 (100.0) ( ) not calculated HPV16 Total (9.3) 7 (1.8) 6 (1.5) 346 (87.4) 96.7 ( ) 0.83 ( ) No CIN (6.3) 6 (1.9) 4 (1.3) 286 (90.5) 96.8 ( ) 0.78 ( ) CIN (12.8) 0 (0.0) 1 (2.1) 40 (85.1) 97.9 ( ) 0.91 ( ) CIN (34.5) 1 (3.4) 1 (3.4) 17 (58.6) 93.1 ( ) 0.85 ( ) 12 other HR HPV Total (28.5) 21 (5.3) 5 (1.3) 257 (64.9) 93.4 ( ) 0.85 ( ) No CIN (23.1) ) 3 (0.9) 220 (69.6) 92.7 ( ) 0.82 ( ) CIN (36.2) 1 (2.1) 2 (4.3) 27 (57.4) 93.6 ( ) 0.87 ( ) CIN (75.9) 0 (0.0) 0 (0.0) 7 (24.1) 100 ( ) 1.00 ( ) 14 HR HPV types Total (35.9) 20 (5.1) 7 (1.8) 227 (57.3) 93.2 ( ) 0.86 ( ) No CIN (29.7) 18 (5.7) 5 (1.6) 199 (63.0) 92.7 ( ) 0.84 ( ) CIN (44.7) 1 (2.1) 1 (2.1) 24 (51.1) 95.7 ( ) 0.92 ( ) CIN (86.2) 1 (3.4) 1 (3.4) 2 (6.9) 93.1 ( ) 0.63 ( ) 17

18 See legend for tables 1 and HR HPV genotypes with the cobas 4800 HPV test combines results obtained for HPV 16, 18 and 12 other HR HPV genotypes. 18

19 Table 4 Clinical performance of cobas 4800 HPV and HC2 tests to detect CIN2+ in women with ASC-US HR HPV test Sensitivity (95% CI) Specificity (95% CI) PPV (95% CI) NPV (95% CI) Referral rate All women (n=29) (n=363) (n=396) Cobas 89.7% ( ) 66.7% ( ) 17.7% ( ) 98.8% ( ) 37.6% ( ) HC2 93.1% ( ) 72.2% ( ) 21.1% ( ) 99.2% ( ) 32.6% ( ) Downloaded from Women > 30 yrs (n=23) (n=278) (n=304) Cobas 87% ( ) 74.1% ( ) 21.3 ( ) 98.6 ( ) 31.3 ( ) HC ( ) 75.2% ( ) 23.3 ( ) 99.1 ( ) 29.9 ( ) 19 on August 22, 2018 by guest

20 Four women (three over 30 years of age) had a diagnosis of CIN that could not be graded : one was positive with HC-2 and with HPV 16 specific assay on cobas 4800 and one was positive for HR HPV excluding HPV 16 and 18 on cobas 4800 HPV test. The denominators (n) for the calculation of sensitivity, specificity and referral rate are indicated in the table). See footnote 1 of Table 1 for abbreviations. 20