Evaluation of HPV-16 and HPV-18 Genotyping for the Triage of Women With High-Risk HPV+ Cytology- Negative Results

Transcription

1 Anatomic Pathology / HPV DNA Testing in Women With Negative Cytology Evaluation of HPV-16 and HPV-18 Genotyping for the Triage of Women With High-Risk HPV+ Cytology- Negative Results Thomas C. Wright Jr, MD, 1 Mark H. Stoler, MD, 2 Abha Sharma, PhD, 3 Guili Zhang, PhD, 3 Catherine Behrens, MD, PhD, 3 Teresa L. Wright, MD 3 ; and the ATHENA (Addressing THE Need for Advanced HPV Diagnostics) Study Group Key Words: Adjunct testing; Cervical cancer screening; Cervical intraepithelial neoplasia; Cytology; Genotype; HPV DNA testing; Negative for intraepithelial lesions or malignancy; NILM DOI: /AJCPTUS5EXAS6DKZ Abstract The ATHENA (Addressing THE Need for Advanced HPV Diagnostics) HPV study evaluated the clinical usefulness of the cobas HPV Test (Roche Molecular Systems, Pleasanton, CA) for high-risk human papillomavirus (HR-HPV) testing (14 HR types) and individual HPV-16/HPV-18 genotyping in women undergoing routine cervical cytology screening in the United States. For the study, 47,208 women were recruited, including 32,260 women 30 years or older with negative cytology. All women with positive results for HR-HPV (n = 4,219) plus a subset of HR-HPV women (n = 886) were referred for colposcopy and biopsy. The overall prevalence of HR-HPV was 6.7% and of HPV-16/HPV-18 was 1.5%. Cervical intraepithelial neoplasia grade 2 (CIN 2) or worse was found in 1.2% of women examined. The estimated absolute risk of CIN 2 or worse in HPV-16+ and/or HPV-18+ women was 11.4% (95% confidence interval [CI], 8.4%-14.8%) compared with 6.1% (95% CI, 4.9%-7.2%) in HR-HPV+ and 0.8% (95% CI, 0.3%- 1.5%) in HR-HPV women. These analyses validate the 2006 American Society of Colposcopy and Cervical Pathology guidelines for HPV-16/HPV-18 genotyping, which recommend referral to colposcopy of HPV-16/ HPV-18+ women with negative cytology. Almost all high-grade cervical cancer precursors (cervical intraepithelial neoplasia [CIN] grades 2 and 3 [CIN 2 and 3]) and invasive cervical carcinomas are caused by persistent infection with 1 of 14 high-risk genotypes of human papillomavirus (HR-HPV). 1,2 This causal relationship has led to the introduction of sensitive molecular tests for HR-HPV into clinical practice, and testing for HR-HPV is now routinely used in the United States for managing women with mild cervical cytologic abnormalities (atypical squamous cells of undetermined significance [ASC-US]). HR-HPV testing is also used as an adjunct to cervical cytology when screening women 30 years or older. 3 However, when used for screening, the clinical usefulness of HR-HPV testing is limited by the fact that HR-HPV infections are relatively common among women without CIN 2, CIN 3, or invasive cervical cancer (CIN 2 or worse). Rather than referring all HR-HPV+ women 30 years or older with negative cytology (negative for intraepithelial lesions or malignancy [NILM]) for colposcopy, current management guidelines recommend repeating the cervical cytology and HR-HPV test in 12 months. 3 However, this conservative follow-up approach is somewhat problematic because it negates much of the benefit of the high clinical sensitivity of HR-HPV testing and can lead to a significant delay in the treatment of women with cervical cancer and a false-negative cervical cytologic result. Moreover, this strategy creates uncertainty for women who are HR-HPV+ and are deferred to follow-up rather than having information that guides immediate investigation for high-grade disease. These limitations may explain, in part, the relatively low utilization of HR-HPV testing by clinicians in the United States when screening women 30 years or older. 4,5 578 Am J Clin Pathol 2011;136: DOI: /AJCPTUS5EXAS6DKZ

2 Anatomic Pathology / Original Article One approach to limiting the number of HR-HPV+ referrals would be to stratify (ie, triage) women with positive results for HR-HPV so that only women at greatest risk for CIN 2 or worse would undergo colposcopy and intensive follow-up. It is well recognized that among the 14 HR-HPV genotypes, HPV-16 and HPV-18 confer the greatest risk for CIN 2 or worse because these 2 genotypes are associated with approximately two thirds of all invasive cervical carcinomas. 6,7 This suggests that genotyping for HPV-16 and/or HPV-18 could be used to identify a subset of HR-HPV+ women with a particularly elevated risk of having CIN 2 or worse. Although the clinical usefulness of using genotyping for HPV-16 and/ or HPV-18 to triage HR-HPV+ women with NILM cytologic results has been recently recognized in clinical management guidelines, the data supporting the use of HPV genotyping in this manner are relatively limited. 8 The ATHENA (Addressing THE Need for Advanced HPV Diagnostics) HPV study enrolled women 21 years or older undergoing routine cervical cytology screening in the United States. The trial was designed to evaluate the performance of a polymerase chain reaction based HPV assay (cobas HPV Test, Roche Molecular Systems, Pleasanton, CA) that detects 14 HR-HPV genotypes and provides 3 separate results: a pooled result for 12 HR-HPV genotypes (31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, and 68) and individual genotyping results for HPV-16 and HPV-18. Materials and Methods Study Design The ATHENA study is a prospective trial designed to evaluate the performance of a new HR-HPV test (cobas HPV Test) among women undergoing routine cervical cancer screening in the United States. The trial is being conducted in 2 phases: a baseline (cross-sectional) phase and a 3-year follow-up (longitudinal) phase that is due to be completed in December Only data from the completed crosssectional phase are reported here. The primary objective of the ATHENA study was to evaluate the performance of the cobas HPV Test in women 21 years or older with ASC-US cytology, and results are reported elsewhere. 9 Preplanned secondary objectives included a comparison of the risks of CIN 2 or worse among women 30 years or older with NILM cytology and positive for HPV-16 and/or HPV-18 with the risks for women who were positive for the 12 other HR-HPV genotypes or were HR-HPV. The current analysis is restricted to the subset of women 30 years or older with NILM cytology. The protocol was approved by the institutional review board of all study sites, and all women provided written informed consent before undergoing any study procedures. Study Population In total, 47,208 nonpregnant women 21 years or older undergoing routine cervical cancer screening were enrolled between May 2008 and August 2009 at 61 clinical centers in 23 states across the United States. Of these women, 46,887 (99.3%) met the study inclusion/exclusion criteria: intact uterus, willing to undergo colposcopy and biopsy if required, no treatment for CIN in the preceding 12 months, and no current or planned participation in a clinical trial for HPV treatment. Protocol Study Visit 1: Enrollment Visit, All Participants After informed consent and demographic and medical history were obtained, 2 liquid-based cervical cytology samples (ThinPrep, Hologic, Bedford, MA) were obtained from the cervix of each participant using a spatula and an endocervical brush. One sample was used for liquid-based cytology and for HR-HPV testing. The second liquid-based cytology sample was reserved for additional testing. Study Visit 2: Colposcopy Visit, Selected Participants Based on the results of the cervical cytology and HR-HPV testing performed on cervical samples obtained at the enrollment visit, a subset of the enrollees was selected for colposcopy as follows. Before reporting screening test results to the clinical centers, results were entered into a subject selection and randomization database that generated a list of women selected for colposcopy. This subset (n = 5,726) included all women 25 years or older with NILM cervical cytology and a positive HR-HPV test by first-generation Roche HPV tests (ie, positive for HR-HPV by the AMPLICOR HPV Test and/or LINEAR ARRAY High Risk HPV Genotyping Test [LA], Roche Molecular Systems, Pleasanton, CA) and 1,041 randomly selected women 25 years or older with NILM cytology who were negative for HPV by the AMPLICOR and LA tests. Randomization was done in a block size of 35 using SAS software, version (SAS Institute, Cary, NC). Results of the second-generation Roche HPV test (cobas HPV Test) were not used to select women for colposcopy because the test cutoff value had not been finalized at the time that enrollment into the ATHENA study was initiated. Colposcopy with biopsy and/or endocervical curettage (ECC) was performed in nonpregnant women selected for colposcopy within 12 weeks of the enrollment visit according to a standardized protocol that included biopsy of all cervical lesions and a random biopsy sample at the squamocolumnar junction in women without cervical lesions and a satisfactory colposcopy. All women with an unsatisfactory colposcopy underwent ECC. Participants and colposcopists were blinded to enrollment visit test results until the colposcopy visit was completed. Biopsies and ECCs were reviewed in a Am J Clin Pathol 2011;136: DOI: /AJCPTUS5EXAS6DKZ 579

3 Wright et al / HPV DNA Testing in Women With Negative Cytology blinded manner by a consensus panel of 3 gynecologic pathologists and diagnosed using standard criteria and the CIN terminology, as previously described in detail. 9,10 The consensus panel diagnosis was used for all analyses. Women who underwent colposcopy and did not meet the primary study end point of histologically diagnosed CIN 2 or worse by consensus pathology were eligible for the follow-up phase (not described herein). Cytology and HR-HPV Testing Sample processing and testing were performed at 5 clinical laboratories in the United States. Two 2-mL aliquots were removed from the first liquid-based cervical cytology specimen (PreservCyt Solution, Hologic) before preparation of the cytology slide. One 2-mL aliquot was used for HR-HPV testing with first-generation HPV tests (both AMPLICOR HPV Test and LA test) according to the manufacturer s instructions. The AMPLICOR HPV Test detects 13 HR-HPV types (16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, and 68; cutoff, 0.2 optical density), and the LA test detects 16 HR-HPV types (16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, 68, 73, and 82). The other 2-mL aliquot was tested at 1 of 5 laboratories using the second-generation HPV test (cobas HPV Test) that detects 12 pooled HR-HPV genotypes (31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, and 68) and HPV-16 and HPV-18 individually and includes a separate β-globin control. Cycle threshold cutoff values for the cobas HPV Test were established by using samples from the first approximately 29,000 women enrolled in the ATHENA study; subsequent cross-validation of the test cutoff was achieved by using samples from the remaining approximately 18,000 participants. The remainder of the liquid-based cervical cytology specimen was used to prepare a slide for cytologic evaluation (ThinPrep Pap Test, Hologic) and processed and evaluated without computerized imaging, according to the standard procedures in 4 clinical laboratories and reported using the Bethesda System. 11 Statistical Analysis Only the data for women 30 years or older with a valid NILM cytology result and valid results for all 3 HPV assays (AMPLICOR, LA, and cobas HPV Test) are included in this analysis. The absolute risks and relative risks of highgrade cervical disease with the respective 95% confidence intervals (CIs) were determined for different categories of HPV DNA result (by cobas HPV Test) in women with NILM cytology. HPV results of genotypes 16 and 18 were analyzed as individual results and as combined 16/18 results (genotype 16 and/or 18). Genotype 16+ results included cases positive for genotype 16 alone, with or without genotype 18, and with or without the 12 other HR-HPV types present. Genotype 18+ results included cases positive for genotype 18 alone, with or without a positive result for the 12 other HR-HPV genotypes, and cases negative for genotype 16. The 12 other HR-HPV+ samples were positive only for these 12 HR-HPV types. Verification bias adjusted estimates for absolute risk and relative risk in women with NILM cytology were obtained by calculating the projected number of women with and without disease who would have been found if all women had undergone colposcopy. CIs for absolute and relative risks were estimated by bootstrapping (1,000 times). 12 Results Enrollment and Demographics Among the 47,208 subjects enrolled into the trial, a total of 32,260 were 30 years or older and had valid HPV test results and an NILM cytology result at their enrollment visit. Of these women, 4,219 were classified as HR-HPV+ using the first-generation HPV tests and referred for colposcopy on that basis. An additional 886 women with NILM cytology who were HR-HPV were randomized to undergo colposcopy to adjust for verification bias Figure 1. Of the 5,105 women referred for colposcopy, 4,422 (86.6%) underwent the procedure and 4,258 (83.4%) had valid results on their cervical biopsies/ecc. Among all women referred for colposcopy, the same percentage (86.6%) from each HPV group (HPV+ and women randomized from the HPV group) completed the colposcopy visit. The mean age of the women in the NILM population included in the analysis was 44.9 years Table 1. Approximately one third were 30 to 39 years old, and 37.5% were postmenopausal. Most women (90.8%) had undergone cervical cytologic screening within the past 5 years. Prevalence of HR-HPV Among the 32,260 women 30 years or older with NILM cytology, the overall prevalence of HR-HPV (14 types) detected using the cobas HPV Test was 6.7% and the overall prevalence rates for HPV-16, HPV-18, and 12 other HR-HPV genotypes were 1.0%, 0.5%, and 5.2%, respectively Table 2. The overall prevalence of HR-HPV (14 types), as well as HPV-16 and HPV-18 individually, decreased with increasing age. Among women 50 to 59 years old, the prevalence of HR-HPV positivity was 5.3% and the prevalence of HPV-16 and HPV-18 combined was only approximately 1% (Table 2). Absolute Risks of CIN by HR-HPV Test Result Referral of HR-HPV+ women with NILM cytology was based on HPV status determined by using the first-generation HPV tests (AMPLICOR and LA tests), whereas the absolute risk of cervical disease was estimated based on the results 580 Am J Clin Pathol 2011;136: DOI: /AJCPTUS5EXAS6DKZ

4 Anatomic Pathology / Original Article Evaluable women with NILM cytology at visit 1 (n = 32,260) Selected/randomized to visit 2 (n = 5,105) HR-HPV+* (n = 4,219) HR-HPV * (n = 886) Visit 2 colposcopy result <CIN 2 (n = 4,127) CIN 2 (n = 131) Invalid result (n = 113) No biopsy taken (n = 51) Not selected/randomized to visit 2 (n = 27,155) HR-HPV * and not randomized (n = 27,083) HR-HPV+* and error in randomization (n = 72) Exited due to loss to follow-up or other reasons (n = 683) Figure 1 Flow of evaluable women 30 years or older with NILM cytology through the study. * By Roche first-generation HPV tests. Biopsy obtained according to a standardized protocol and reviewed by central pathology review panel. Invalid biopsy result includes biopsy sample inadequate for analysis and biopsy sample obtained outside of 12-wk window. CIN, cervical intraepithelial neoplasia; HPV, human papillomavirus; HR, high-risk; NILM, negative for intraepithelial lesions or malignancy. obtained with the second-generation cobas HPV Test. It should be noted that the AMPLICOR test is more sensitive than the cobas HPV Test from an analytic perspective, as indicated by limit of detection values expressed as cells per milliliter. 13,14 Thus, among the 4,258 women who underwent Table 1 Baseline Characteristics of Women 30 Years or Older With NILM Cytology * Evaluable Women Characteristic (N = 32,260) Age, y Mean ± SD 44.9 ± ,398 (35.3) 40 20,862 (64.7) Race White 27,197 (84.3) Black or African American 4,199 (13.0) Asian 466 (1.4) American Indian or Alaskan Native 173 (0.5) Native Hawaiian or other Pacific Islander 72 (0.2) Any combination or missing data 153 (0.5) Ethnicity Hispanic or Latino 5,736 (17.8) Postmenopausal 12,100 (37.5) HPV vaccine 45 (0.1) Immunocompromised or immunosuppressed 208 (0.6) Family history of cervical disease related to cervical cancer Yes 1,804 (5.6) No 30,133 (93.4) Unknown 323 (1.0) Previous Papanicolaou cytology test 29,301 (90.8) (within past 5 y) HPV, human papillomavirus; NILM, negative for intraepithelial lesions or malignancy. * Data are given as number (percentage) unless otherwise indicated. Evaluable women had valid HPV test results. colposcopy and had a valid biopsy result, only 1,744 women were HR-HPV+ using the cobas HPV Test and the remaining 2,514 women were HR-HPV. Of note, among women with negative results by the cobas HPV Test and who had combined AMPLICOR and LA test positive results, the crude prevalence of CIN 2 or worse was comparable with the prevalence of CIN 2 or worse for women with negative results on the AMPLICOR and LA tests (0.9% and 0.8%, respectively), thereby confirming the clinical validation of the appropriate cutoff for the cobas HPV Test. Table 2 Prevalence of HR-HPV in Women 30 Years or Older With NILM Cytology * HPV Test Result Age Group, y Total HR-HPV+ HPV-16+ HPV Other HR-HPV , , , , Overall 32, HPV, human papillomavirus; HR, high-risk; NILM, negative for intraepithelial lesions or malignancy. * HR-HPV genotypes were detected using the cobas HPV Test, and data are given as percentages. HR-HPV+ includes HPV-16+ and/or HPV-18+ and/or 12 other HR-HPV+ types; HPV-16+ includes HPV-16+, with or without HPV-18+, and with or without 12 other HR-HPV+ types; HPV-18+ includes HPV-16, HPV-18+, with or without 12 other HR-HPV+ types; 12 other HR-HPV+ include HPV-16, HPV-18, 12 other HR-HPV+ types. Evaluable women with valid HPV results and valid biopsy results. Am J Clin Pathol 2011;136: DOI: /AJCPTUS5EXAS6DKZ 581

5 Wright et al / HPV DNA Testing in Women With Negative Cytology Based on the consensus pathology review diagnosis, the samples for 3,898 of 4,258 evaluable women who underwent colposcopy were classified as within normal limits, while 229 had biopsy-confirmed CIN 1, 51 had biopsy-confirmed CIN 2, and 80 had biopsy-confirmed CIN 3 or worse. Cases classified as CIN 3 or worse included 5 cases of adenocarcinoma in situ. Verification bias adjusted estimates of the overall absolute risk among women 30 years or older with NILM cytology at ATHENA enrollment were 1.2% (95% CI, 0.6%-1.8%) for CIN 2 or worse and 0.5% (95% CI, 0.3%-0.9%) for CIN 3 or worse Table 3. The estimated absolute risk was dependent on HR-HPV status, and HPV-16+ women had the highest absolute risks for CIN 2 or worse and CIN 3 or worse. The estimated absolute risk for CIN 3 or worse ranged from 0.3% (95% CI, 0.02%-0.7%) in women who were negative for HR-HPV to 11.7% (95% CI, 7.9%-15.8%) in women who were positive for HPV-16. Estimated absolute risks for all cobas HPV Test results stratified by age are shown in Table 3. Absolute risk in women who were HR-HPV+ showed a modest decrease with age across all combinations of results. However, the decrease in risk was more marked among women who were HPV-16+ and/or HPV-18+ compared with women who were not HPV- 16+/HPV-18+ (ie, positive for 12 other HR-HPV types). Relative Risks of CIN by HR-HPV Test Result Verification bias adjusted estimates for relative risk of CIN 2 or worse and CIN 3 or worse by HR-HPV status in women 30 years or older with NILM cytology are provided in Table 4. The relative risks for CIN 2 or worse and CIN 3 or worse were highest in women found to be HPV-16+ using the cobas HPV Test; the relative risk for CIN 2 or worse was 16.3 (95% CI, ) and for CIN 3 or worse was 42.0 (95% CI, ), compared with HR-HPV women. Furthermore, compared with HR-HPV women, the relative risks for CIN 2 or worse and CIN 3 or worse were intermediate in HPV-18+ women: 8.4 ( 95% CI, ) and 20.5 (95% CI, ), respectively. The relative risks were also intermediate in Table 4 Estimated Relative Risk of High-Grade Disease According to the HPV Test Result in Women 30 Years or Older With NILM Cytology * Table 3 Impact of HR-HPV Status on Estimated Absolute Risk of High-Grade Cervical Disease in Women With NILM Cytology * Age Group, y Estimated Relative Risk HPV Test Result CIN 2 or Worse CIN 3 or Worse HPV-16+ vs HPV 16.3 ( ) 42.0 ( ) HPV-16+/HPV-18+ vs HPV 13.7 ( ) 35.0 ( ) HR-HPV+ vs HPV 7.3 ( ) 14.4 ( ) 12 other HPV+ vs HPV 5.5 ( ) 8.7 ( ) HPV-18+ vs HPV 8.4 ( ) 20.5 ( ) HPV-16+ vs 12 other HPV+ 3.0 ( ) 4.8 ( ) CI, confidence interval; CIN, cervical intraepithelial neoplasia; HPV, human papillomavirus; HR, high-risk; NILM, negative for intraepithelial lesion or malignancy. * Relative risk was calculated using absolute risk values to 3 decimal places. HR-HPV was detected using the cobas HPV Test. Data are given as the estimated relative risk (95% CI). HR-HPV+ includes HPV-16+ and/or HPV-18+ and/or 12 other HR-HPV+ types; HPV-16+ includes HPV-16+, with or without HPV-18+, and with or without 12 other HR-HPV+ types; HPV-18+ includes HPV-16, HPV-18+, with or without 12 other HR-HPV+ types; 12 other HR-HPV+ types include HPV-16, HPV-18, 12 other HR-HPV+ types. HPV Test Result Overall CIN 2 or worse HR-HPV+ 8.4 ( ) 5.4 ( ) 1.3 ( ) 5.4 ( ) 6.1 ( ) HPV-16+/HPV ( ) 7.4 ( ) 4.4 ( ) 0.0 ( ) 11.4 ( ) HPV ( ) 10.0 ( ) 2.4 ( ) 0.0 ( ) 13.6 ( ) HPV ( ) 2.4 ( ) 3.7 ( ) 0.0 ( ) 7.0 ( ) 12 other HR-HPV+ 5.6 ( ) 4.9 ( ) 0.6 ( ) 6.6 ( ) 4.6 ( ) HR-HPV 0.1 ( ) 0.8 ( ) 1.2 ( ) 3.1 ( ) 0.8 ( ) Overall 1.2 ( ) CIN 3 or worse HR-HPV+ 5.7 ( ) 3.5 ( ) 1.1 ( ) 2.7 ( ) 4.1 ( ) HPV-16+/HPV ( ) 6.6 ( ) 4.4 ( ) 0.0 ( ) 9.8 ( ) HPV ( ) 7.5 ( ) 2.4 ( ) 0.0 ( ) 11.7 ( ) HPV ( ) 2.4 ( ) 3.7 ( ) 0.0 ( ) 5.7 ( ) 12 other HR-HPV+ 3.0 ( ) 2.8 ( ) 0.3 ( ) 3.3 ( ) 2.4 ( ) HR-HPV 0.0 ( ) 0.4 ( ) 0.0 ( ) 1.6 ( ) 0.3 ( ) Overall 0.5 ( ) CI, confidence interval; CIN, cervical intraepithelial neoplasia; HPV, human papillomavirus; HR, high-risk; NILM, negative for intraepithelial lesion or malignancy. * Estimated absolute risk is the number of subjects with disease/number of subjects with positive test results adjusted for verification bias. HR-HPV was detected using the cobas HPV Test, and data are given as the estimated absolute risk (95% CI) in percentages. HR-HPV+ includes HPV-16+ and/or HPV-18+ and/or 12 other HR-HPV+ types; HPV-16+ includes HPV-16+, with or without HPV-18+, and with or without 12 other HR-HPV+ types; HPV-18+ includes HPV-16, HPV-18+, with or without 12 other HR-HPV+ types; 12 other HR-HPV+ types include HPV-16, HPV-18, 12 other HR-HPV+ types. Data for subjects 70 years or older with NILM cytology are not included in this table because no CIN 2 or worse was diagnosed in this age group. 582 Am J Clin Pathol 2011;136: DOI: /AJCPTUS5EXAS6DKZ

6 Anatomic Pathology / Original Article women with positive results for 12 other HR-HPV types: 5.5 (95% CI, ) and 8.7 (95% CI, ), respectively. Discussion The ATHENA study is the first trial to evaluate the clinical usefulness of HR-HPV DNA testing and HPV- 16/HPV-18 genotyping among a large cohort of women undergoing routine cervical cytologic screening in the United States. The current analysis focused on women 30 years or older with NILM cytology results. The main findings are that the absolute and relative risks of CIN 2 or worse and CIN 3 or worse in women with NILM cytology are impacted not only by HR-HPV (14 types) status, but even more so by whether HPV-16 or HPV-18 is detected. Prior cross-sectional and prospective studies have clearly demonstrated that HR-HPV status is an important predictor of the current and future detection of CIN 2 or worse in women with NILM cytology. Based on this finding, current US cervical cancer screening guidelines recommend extending the screening interval to at least 3 years in women 30 years or older with NILM cytology who are HR-HPV and retesting women who are HR-HPV+ at 12 months using cervical cytology and HPV tests. Women who are HPV with low-grade squamous intraepithelial lesions or greater on the repeated cytology and women who are persistently HR-HPV+ should be referred for colposcopy. 3 Therefore, it is not unexpected that in the current analysis, HR-HPV status had a marked impact on the risks of CIN 2 or worse and CIN 3 or worse in women 30 years or older with NILM cytology; however, data have not been previously reported for HPV-16/HPV-18. In women with NILM cytology who were also HR-HPV+ (14 types), the estimated absolute risk of CIN 3 or worse at baseline was 4.1% (95% CI, 3.1%-5.0%), and the relative risk of CIN 3 or worse was 14.4 (95% CI, ) compared with women who were HR-HPV. The presence of HPV-16 or HPV-18 in women 30 years or older with NILM cytology was found to greatly magnify the risk of CIN 3 or worse, as did the presence of HPV-16 alone. The absolute risk for CIN 3 or worse in women with NILM cytology who were HPV-16+ was 11.7% (95% CI, 7.9%-15.8%); the relative risk of CIN 3 or worse compared with women who were HR-HPV (14 types) was 42.0 (95% CI, ). The absolute risk in women who were HR-HPV+ showed a modest decrease with age across all combinations of results, and the decrease was more marked among women who were HPV-16+ and/or HPV-18+ compared with women who were not HPV-16+/HPV-18+ (ie, positive for 12 other HR-HPV types). The decrease in risk of high-grade disease is consistent with the reported peak age of CIN 3 in the United States (25-29 years) 21 and was also observed in the overall population of the ATHENA trial. 22 Two prospective follow-up studies have shown the importance of HPV-16 in predicting the women with NILM cytology in whom high-grade CIN will develop. In the study by Khan et al, 23 in women enrolled in the Kaiser Permanente health management organization (Portland, OR), the 10-year cumulative incidence rate (CIR) of CIN 3 or worse in women 30 years or older with NILM cytology at enrollment was 20.7% (95% CI, 8.6%-32.8%) among HPV-16+ women and 17.7% (95% CI, 0.0%-36.0%) among HPV-18+ women. In contrast, the 10-year CIR of CIN 3 or worse was only 1.5% (95% CI, 0.3%-2.7%) among HR-HPV+ women who were negative for both HPV-16 and HPV In the Kaiser study, the increased detection of CIN 3 or worse in women who were HPV-18+ was delayed by approximately 2 years compared with the increase seen in women who were HPV A 2-year delay in the oncogenic effects of HPV-18 compared with HPV-16 could explain the somewhat lower impact of HPV-18 compared with HPV-16 observed in the current cross-sectional analysis of the ATHENA trial. Similar data have recently been reported from a Danish prospective follow-up study. After a 12-year follow-up, the estimated probability of developing CIN 3 or worse in HPV-16+ women with NILM cytology was 26.7% (95% CI, 21.1%-31.8%), and for HPV-18+ women, it was 19.1% (95% CI, 10.4%-27.3%). In contrast with the Kaiser study, an increased risk similar to that associated with HPV-18 was also found in women who were positive for HPV-31 and HPV-33. Women with an HR-HPV other than types 16, 18, 31, or 33 had a relatively lower absolute risk of CIN 3 (6.0% [95% CI, 3.8%-8.3%]). 20 In the current analysis, the verification bias adjusted absolute risks of CIN 2 or worse and of CIN 3 or worse in women 30 years or older with NILM cytology were 1.2% (95% CI, 0.6%-1.8%) and 0.5% (95% CI, 0.3%- 0.9%), respectively. Based on older data, this finding seems somewhat high, especially given that liquid-based cytology was used for screening in the ATHENA study and that only women 30 years or older are included in the current analysis. For example, a retrospective study of 128,805 women with NILM cytology enrolled in the National Breast and Cervical Cancer Early Detection Program found that the age-adjusted incidence rate of having a cervical cytology result of highgrade squamous intraepithelial lesions or worse within 3 years of the initial negative cytology was only approximately 0.2%. 24 However, more recent studies that have performed colposcopy in women with abnormal cytology and women who are HR-HPV+ have found a higher prevalence of highgrade CIN in women with NILM cytology. 18,25,26 A comprehensive review of 7 prospective European screening trials found that after 3 years of follow-up, the CIR of CIN 2 or worse in women with NILM cytology was 0.8% (95% CI, 0.4%-1.2%) and the CIR of CIN 3 or Am J Clin Pathol 2011;136: DOI: /AJCPTUS5EXAS6DKZ 583

7 Wright et al / HPV DNA Testing in Women With Negative Cytology worse was 0.5% (95% CI, 0.2%-0.8%). 18 North American screening studies that have not only performed colposcopy on HR-HPV+ women, but also adjusted for verification bias by performing colposcopy in a subset of women with negative screening test results, report absolute risks of high-grade CIN in women with NILM cytology that are both higher and lower than found in the ATHENA study. Kulasingam et al 25 used 2 different HR-HPV tests and liquid-based cytology to screen more than 4,000 women visiting Planned Parenthood clinics in Washington State. After adjusting for verification bias, the absolute risk of CIN 3 or worse was approximately 1.5% (calculated from data in Figure 2 in their article) in women of all ages with an NILM liquid-based cytology result. 25 Another study of similar design that used conventional cytology screened more than 10,000 Canadian women 30 years or older. In this study, the verification bias adjusted prevalence of CIN 2 or worse was 0.8% (calculated from data in Figure 1 in their article) in women 30 years or older with an NILM cytology result, which is similar to the 1.2% found in the current analysis. 26 There is considerable controversy as to the merits of adjusting for verification bias. In general, the proportion of women with negative results by both HPV testing and cervical cytology who undergo colposcopy in studies that adjust for verification bias is low, and, therefore, attempts to adjust for missed disease in this group tend to lead to magnification of these few cases. 27 Moreover, relatively few cases of highgrade CIN are identified in women who are both HPV and cytology negative, and this can also lead to unstable results. 16 Adjustment for verification bias may explain why the absolute risk of CIN 2 or worse in women who had NILM cytology and tested HR-HPV (14 types) was 0.8% (95% CI, 0.3%- 1.5%) in the current analysis. This rate is considerably higher than the expected rate of 1 in 1, The present study has a number of considerable strengths. These strengths include the large number of women who were evaluated, the fact that all women enrolled had both cytology and HPV testing performed, that liquid-based cytology was used, and that all cervical cytology was evaluated in 4 different laboratories under routine practice conditions. In addition, all colposcopy examinations and evaluations of samples were performed without knowledge of clinical information or the results of other laboratory tests, a standardized approach was taken to colposcopy with a biopsy of normal-appearing cervix obtained in women without visible cervical lesions at the time of colposcopy, and adjudicated pathology was used to determine the gold standard. This study clearly defines risk for disease at baseline in women 30 years or older undergoing routine cervical cancer screening in the United States who are found to have an NILM cytology result. Risk for high-grade neoplasia is greatly elevated in HPV-16+/HPV-18+ women and is further increased when restricted to HPV-16 alone. Risk associated with HPV-16+/HPV-18 is considerably greater than the risk associated with the 12 other HR-HPV genotypes. Moreover, the absolute risk for CIN 3 or worse in HPV-16+/HPV-18+ women 30 years or older with NILM in the ATHENA study (9.8%) is approximately the same as for women 21 years or older with ASC-US who are HR-HPV+ (14 pooled types) in the ATHENA study (8.4%). 9 Based on rather limited data, predominantly from the study by Khan et al, 23 the 2006 American Society of Colposcopy and Cervical Pathology Consensus Conference approved a strategy incorporating HPV-16/HPV-18 genotyping when managing women 30 years or older undergoing routine cervical cancer screening and found to be HR-HPV+ with NILM cytology. With this strategy, HPV- 16+/HPV-18+ women with NILM cytology are referred for immediate colposcopy, whereas women who are positive for the 12 other HR-HPV genotypes undergo repeated testing with both cytology and HR-HPV testing at 12 months. 8 While the risk threshold for performing colposcopy in response to specific cytology and HPV test results is still being defined, Castle et al 29 suggested that women with an absolute risk for CIN 3 or worse of 10% or more over 2 years should undergo colposcopy. Because the present study indicates that the risk of an HPV-16+ and/or HPV-18+ woman with NILM cytology having CIN 3 or worse is 9.8% at baseline, these data clearly support the recommendation for immediate referral to colposcopy in accordance with the 2006 American Society of Colposcopy and Cervical Pathology guidelines for HPV-16/HPV-18 genotyping of women with NILM cytology. From the 1 Department of Pathology, Columbia University School of Medicine, New York, NY; 2 University of Virginia Health System, Charlottesville; and 3 Roche Molecular Systems, Pleasanton, CA. Funded by Roche Molecular Systems, Pleasanton, CA. Address reprint requests to Dr Behrens: Roche Molecular Systems, 4300 Hacienda Dr, Pleasanton, CA Disclosure: Dr T.C. Wright has been a consultant and speaker for Merck, GlaxoSmithKline, and Roche Diagnostics and a consultant for Gen-Probe. Dr Stoler has been a consultant in clinical trial and HPV DNA test development for Third Wave, Hologic, QIAGEN, Roche Molecular Systems, and Gen-Probe. Drs Sharma, Zhang, Behrens, and T.L. Wright are employed by Roche Molecular Systems, the sponsor of the study. Acknowledgment: Editorial assistance was provided by Health Interactions, London, England. The ATHENA Central Pathology Review Panel includes Thomas C. Wright Jr, MD, Department of Pathology, Columbia University School of Medicine, NY; Mark H. Stoler, MD, University of Virginia Health System, Charlottesville; and Alexander Ferenczy, MD, Department of Pathology, McGill University, Montreal, Canada. The ATHENA study testing sites and participants are as follows: 584 Am J Clin Pathol 2011;136: DOI: /AJCPTUS5EXAS6DKZ

8 Anatomic Pathology / Original Article Laboratory testing sites: B.A. Body, LabCorp, Burlington, NC; A. Butcher, Roche Molecular Systems, Pleasanton, CA; C. Eisenhut, DCL Medical Laboratories, Indianapolis, IN; A. Rao, Scott & White Memorial, Temple, TX; and S. Young, TriCore Reference Laboratories, Albuquerque, NM. Collection sites (principal investigators and institutions): R. Ackerman, Comprehensive Clinical Trials, West Palm Beach, FL; R. Anders, Green Clinic, Ruston, LA; E. Andruczyk, Philadelphia Clinical Research, Philadelphia, PA; K. Aqua, Visions Clinical Research, Boynton Beach, FL; R. Black, Women s Health Specialist, Costa Mesa, CA; S. Blank, Mount Vernon Clinical Research, Atlanta, GA; P. Bressman, Tennessee Women s Care, Nashville, TN; K. Brody, Chattanooga Medical Research, Chattanooga, TN; J. Burigo, OB/GYN Specialists of the Palm Beaches, West Palm Beach, FL; S. Chavoustie, Segal Institute for Clinical Research, North Miami, FL; M. Davis, SC Clinical Research Center, Columbia, SC; A. Donovan, Bluegrass Clinical Research, Louisville, KY; S. Eder, Delaware Valley OB-GYN and Infertility Group, Plainsboro, NJ; C. Eubank, Advanced Research Associates, Corpus Christi, TX; S. Fehnel, Advanced Clinical Concepts, West Reading, PA; R. Feldman, Miami Research Associates, Miami, FL; R. Filosa, Center for Women s Health of Lansdale, Lansdale, PA; S. Fowler, Blue Skies Center for Women, Colorado Springs, CO; C. Goldberg, Visions Clinical Research, Tucson, AZ; R. Groom, Impact Clinical Trials, Las Vegas, NV; J. Grube, Physicians Research Options, Lakewood, CO; P. Grumley, Four Rivers Clinical Research, Paducah, KY; P. Hadley, Medical Network for Education and Research, Decatur, GA; M. Harris, Women s Health Research, Phoenix, AZ; L. Hazan, Impact Clinical Trials, Los Angeles, CA; J. Huey, HWC Women s Research Center, Englewood, OH; M. Jacobs, Texas Medical Center, Houston; S. Kleinpeter, Mobile OB/GYN, Mobile, AL; S. Lederman, Altus Research, Lake Worth, FL; J. Lenihan Jr, Tacoma Women s Specialist, Tacoma, WA; B. Levine, Phoenix OB-GYN Association, Moorestown, NJ; K. Lowder, The Woman s Clinic, Boise, ID; N. Lurvey, Impact Clinical Trials, Los Angeles, CA; J. Martin Jr, ecast Corporation, North Charleston, SC; R. McDavid, State of Franklin Healthcare Associates Research, Johnson City, TN; J. McGettigan, Quality of Life Medical & Research Center, Tucson, AZ; J. Michelson, Eastern Carolina Women s Center, New Bern, NC; F. Morgan, Tidewater Clinical Research, Virginia Beach, VA; R. Myers, St John s Center for Clinical Research, Jacksonville, FL; K. Osman, M & O Clinical Research, Ft Lauderdale, FL; R. Parker Jr, Lyndhurst Gynecologic Associates, PA, Winston- Salem, NC; J. Pollard, Enterprise Women s Center, Enterprise, AL; A. Rappleye, Salt Lake Research, Salt Lake City, UT; R. Reagan, Women s Health Care at Frost Street, San Diego, CA; H. Reisman, Atlanta North Gynecology Center for Research, Roswell, GA; L. Rogers, Women s Clinical Research, Newburgh, IN; N. Sager, Jacksonville Center for Clinical Research, Jacksonville, FL; G. Sieggreen, Women s OB-GYN, Saginaw, MI; W. Somerall Jr, Clinical Research Consultants, Hoover, AL; M. Sperling, Edinger Medical Group Research Center, Fountain Valley, CA; R. Surowitz, Health Awareness, Jupiter, FL; M. Swor, Physician Care Clinical Research, Sarasota, FL; S. Trupin, Woman s Health Practice, Champaign, IL; A. Tydings, Clinical Trials Management, Covington, LA; K. Waldrep, Advanced Research Associates, Dallas, TX; D. Walland, Fellows Research Alliance, Savannah, GA; D. Walland, Fellows Research Alliance, Hilton Head, SC; W. Wilkerson, Women s Care Florida, Tampa; W. Wilson, Advanced Research Associates, McAllen, TX; S. Wininger, Precision Trials, Phoenix, AZ; and S. Yassear, Yassear Clinical Research, Carmichael, CA. References 1. Walboomers JM, Jacobs MV, Manos MM, et al. Human papillomavirus is a necessary cause of invasive cervical cancer worldwide. J Pathol. 1999;189: Schiffman M, Castle PE, Jeronimo J, et al. Human papillomavirus and cervical cancer. Lancet. 2007;370: Wright TC Jr, Massad LS, Dunton CJ, et al consensus guidelines for the management of women with abnormal cervical cancer screening tests. Am J Obstet Gynecol. 2007;197: Saraiya M, Berkowitz Z, Yabroff KR, et al. Cervical cancer screening with both human papillomavirus and Papanicolaou testing vs Papanicolaou testing alone: what screening intervals are physicians recommending? Arch Intern Med. 2010;170: Saraiya M, Irwin KL, Carlin L, et al. Cervical cancer screening and management practices among providers in the National Breast and Cervical Cancer Early Detection Program (NBCCEDP). Cancer. 2007;110: Bosch FX, de Sanjose S. Chapter 1: Human papillomavirus and cervical cancer: burden and assessment of causality. J Natl Cancer Inst Monogr. 2003: de Sanjose S, Diaz M, Castellsague X, et al. Worldwide prevalence and genotype distribution of cervical human papillomavirus DNA in women with normal cytology: a meta-analysis. Lancet Infect Dis. 2007;7: American Society for Colposcopy and Cervical Pathology HPV Genotyping Clinical Update org/consensus.shtml. Accessed February Stoler MH, Wright TC Jr, Sharma A, et al. High-risk human papillomavirus testing in women with ASC-US cytology: results from the ATHENA HPV study. Am J Clin Pathol. 2011;135: Kurman RJ, Ellenson LH, Bonnett BM. Blaustein s Pathology of the Female Genital Tract. New York, NY: Springer Verlag; Solomon D, Davey D, Kurman R, et al. The 2001 Bethesda System: terminology for reporting results of cervical cytology. JAMA. 2002;287: Chernick M. Bootstrapping Methods: A Practitioner s Guide. New York, NY: Wiley Interscience; 1999:8, cobas HPV Test [package insert]. Pleasanton, CA: Roche Molecular Systems; The AMPLICOR Human Papillomavirus (HPV) test [package insert]. Pleasanton, CA: Roche Molecular Systems; Bigras G, de Marval F. The probability for a Pap test to be abnormal is directly proportional to HPV viral load: results from a Swiss study comparing HPV testing and liquid-based cytology to detect cervical cancer precursors in 13,842 women. Br J Cancer. 2005;93: Cuzick J, Clavel C, Petry KU, et al. Overview of the European and North American studies on HPV testing in primary cervical cancer screening. Int J Cancer. 2006;119: Am J Clin Pathol 2011;136: DOI: /AJCPTUS5EXAS6DKZ 585

9 Wright et al / HPV DNA Testing in Women With Negative Cytology 17. Ronco G, Segnan N, Giorgi-Rossi P, et al. Human papillomavirus testing and liquid-based cytology: results at recruitment from the New Technologies for Cervical Cancer randomized controlled trial. J Natl Cancer Inst. 2006;98: Dillner J, Rebolj M, Birembaut P, et al. Long term predictive values of cytology and human papillomavirus testing in cervical cancer screening: joint European cohort study. BMJ. 2008;337:a1754. doi: /bmj.a Kitchener HC, Gilham C, Sargent A, et al. A comparison of HPV DNA testing and liquid based cytology over three rounds of primary cervical screening: extended follow up in the ARTISTIC trial [published online ahead of print February 18, 2011]. Eur J Cancer. doi: /j. ejca Kjaer SK, Frederiksen K, Munk C, et al. Long-term absolute risk of cervical intraepithelial neoplasia grade 3 or worse following human papillomavirus infection: role of persistence. J Natl Cancer Inst. 2010;102: Moscicki AM, Schiffman M, Kjaer S, et al. Chapter 5: Updating the natural history of HPV and anogenital cancer. Vaccine. 2006;24(suppl 3):S3/42-S3/ Wright TC Jr, Stoler MH, Behrens CM, et al. The ATHENA human papillomavirus study: design, methods, and baseline results. Am J Obstet Gynecol. In press. 23. Khan MJ, Castle PE, Lorincz AT, et al. The elevated 10-year risk of cervical precancer and cancer in women with human papillomavirus (HPV) type 16 or 18 and the possible utility of type-specific HPV testing in clinical practice. J Natl Cancer Inst. 2005;97: Sawaya GF, Kerlikowske K, Lee NC, et al. Frequency of cervical smear abnormalities within 3 years of normal cytology. Obstet Gynecol. 2000;96: Kulasingam SL, Hughes JP, Kiviat NB, et al. Evaluation of human papillomavirus testing in primary screening for cervical abnormalities: comparison of sensitivity, specificity, and frequency of referral. JAMA. 2002;288: Mayrand MH, Duarte-Franco E, Rodrigues I, et al. Human papillomavirus DNA versus Papanicolaou screening tests for cervical cancer. N Engl J Med. 2007;357: Sasieni P. Estimating prevalence when the true disease status is incompletely ascertained. Stat Med. 2001;20: Wright TC Jr, Schiffman M, Solomon D, et al. Interim guidance for the use of human papillomavirus DNA testing as an adjunct to cervical cytology for screening. Obstet Gynecol. 2004;103: Castle PE, Sideri M, Jeronimo J, et al. Risk assessment to guide the prevention of cervical cancer. Am J Obstet Gynecol. 2007;197:356.e1-356.e6. doi: /j.ajog Am J Clin Pathol 2011;136: DOI: /AJCPTUS5EXAS6DKZ