This study presents the results of a 3-year longitudinal evaluation of the AHPV assay as an adjunctive method for screening women 30 years and older who have NILM Pap cytology results. Consistent with previously published data,[28,29] these results demonstrate that HR-HPV oncogenic E6/E7 mRNA testing has a sensitivity similar to an HR-HPV DNA-based test for detection of CIN2+ and CIN3+ and slightly, but significantly, improved specificity compared with HR-HPV DNA testing for both end points. We found that use of AHPV as an adjunctive method for HPV-induced cervical disease screening provided disease detection capability similar to HC2 while reducing the false-positive rate (from 5.2% to 3.7%) relative to the HPV DNA-based test. Reduction of HPV detection in women without cervical disease minimizes the anxiety and burden associated with spurious positive HPV molecular test results in women with NILM cytology, decreases health care costs, and reduces unnecessary follow-up procedures, thereby improving the safety of cervical cancer screening (unnecessary colposcopy is considered a significant "harm" in the recent American Cancer Society guidelines).
Importantly, we show that women with a NILM cytology result who also had a positive AHPV result are approximately 24 times more likely to have CIN2+ disease after 3 years than women with a negative AHPV result. This risk increased to approximately 68-fold for detection of CIN3+ disease. Similar but slightly lower risk estimates were obtained with HC2, demonstrating comparable accuracy of the AHPV and HC2 for identifying participants with CIN2+ and CIN3+ in this respect.
After 3 years of follow-up, women in this study who were HPV negative at baseline using any test method had very low risk for CIN2+ (<0.3%), a result similar to previously published studies with HC2.[42,43] These findings reinforce evidence from previous studies showing that HR-HPV nucleic acid testing should be performed as an adjunctive test to routine Pap for cervical cancer screening of women 30 years or older to increase sensitivity of disease detection. Correspondingly, compared with annual cytology-only screening, this study supports longer screening intervals for women negative for both abnormal cytology and HPV E6/E7 mRNA, due to the high NPV and low risk of disease afforded by this screening algorithm for 3 years following a test-negative baseline visit. Extension of cervical cancer screening intervals following negative HPV and cytology test results in women 30 years or older is a key recommendation of current US screening guidelines from both the American Cancer Society and the US Preventive Services Task Force.
Conversely, since the positive predictive value of any HPV test in women with NILM cytology is low, additional AHPV testing to detect persistent HR-HPV infection during follow-up care in women with an initial AHPV-positive result is likely a better option than direct referral to colposcopy. Alternatively, genotyping with referral for HPV 16– or HPV 18–positive women can optimize referral and minimize loss to follow-up.
Several design features were employed in the CLEAR study to achieve accurate determination of the performance characteristics for both AHPV and HC2 assays. First, all biopsy samples were subjected to adjudicated review by three independent expert pathologists. Second, molecular test performance was compared with a consensus histology diagnosis, the gold standard for determining cervical disease status. Third, AHPV performance was compared directly with HC2 performance, the most broadly used and characterized HPV DNA test. Fourth, performance characteristics of both assays obtained from baseline results were adjusted for verification bias by conducting colposcopy and biopsy in 3.4% of HPV-negative women. This process is recommended in low-prevalence populations to avoid overestimating assay sensitivity and underestimating assay specificity.[45–47] Finally, women were followed for 3 years with annual cytology testing and referral to colposcopy for abnormal results.
A limitation of this study was that a portion of HPV-negative women with normal cytology were subjected to colposcopy and biopsy at the baseline visit but not at the subsequent follow-up visits. Thus, the relative risk estimates reported here for disease in HPV-positive vs HPV-negative women evaluated during years 1, 2, and 3 of the follow-up period may be overstated. This potential bias is present in previously reported longitudinal cotesting studies[17,19,48] and is unavoidable, since implementation of invasive procedures on thousands of women with normal cytology and negative HPV test results presents a burden to study participants and is not supported by current US and European practice guidelines. However, as in previous longitudinal cotesting studies, women enrolled in CLEAR who exited at the final (third) year of follow-up had yielded negative cytology and/or negative HC2 and AHPV results from four consecutive examinations. Thus, their risk of harboring an occult CIN lesion is likely to be exceedingly small,[42,43] such that any potential error encountered here most likely constitutes a very small fraction of the overall magnitude of the risks reported.
Another limitation of this study was that colposcopists were aware of the women's HPV test status during the first half of the baseline portion of the study, because during that period, only women who tested positive in the AHPV or HC2 were referred to colposcopy. When the colposcopists were unmasked, they may have been more diligent to find cervical disease with prior knowledge of current HPV infection status. However, after randomly selected HPV-negative women were referred to colposcopy, the colposcopists were masked to HPV status, and throughout the entire study, colposcopists were masked as to which HPV assay caused the referral. Thus, any potential "colposcopy bias" would be identical for both molecular tests.
In summary, these results demonstrate that the clinical performance of HR-HPV E6/E7 mRNA testing using the AHPV is consistent with current US cervical cancer screening guidelines for women with a NILM cytology result who are 30 years of age or older. There was a significantly greater risk of CIN2+ in AHPV-positive vs AHPV-negative participants, as well as a statistically and clinically significant improvement in specificity for detection of CIN2+ by the AHPV compared with HPV DNA testing with the HC2 assay. Thus, these data confirm the clinical utility of AHPV testing in an adjunct cervical cancer screening setting.
CLEAR Study Central Pathology Review Panel: Thomas C. Wright Jr., MD, Department of Pathology, Columbia University School of Medicine, New York, NY; Mark Stoler, MD, Department of Pathology, University of Virginia Health System, Charlottesville, VA; and Alex Ferenczy MD, BioVision, Incorporated, Outremont, Canada.
The CLEAR study testing sites and participants are as follows: Laboratory testing sites: B. A. Body, Laboratory Corporation of America, Burlington, NC; B. Yen-Lieberman, Cleveland Clinic Foundation, Cleveland, OH; C. Ginocchio, North Shore Long Island Jewish Health System Laboratory, Lake Success, NY; and T. Davis, Eskenazi Health Services, Indianapolis, IN. Collection sites (principal investigators and institutions): M. Rabinowitz, Community Medical Research, Miami Beach, FL; A. T. Hanna, Diverse Research Solutions, Oxnard, CA; K. Ault, Emory University School of Medicine, Atlanta, GA; G. M. Grossman, HealthCare Partners, Los Angeles, CA; W. Campbell, HealthCare Partners, Torrance, CA; M. Stripling, NEA Baptist Clinic Women's Clinic, Jonesboro, AR; D. Ronk, Planned Parenthood of Eastern Arkansas and Oklahoma, Tulsa, OK; R. Muckerman, PPS Clinical Research, Chesterfield, MO; B. Naghi, Peninsula Research Associates, Rolling Hills Estates, CA; T. Easter, REMEK Research, Montclair, CA; E. Guzman, Guzman MD Research, Downey, CA; A. Wagner, Saginaw Valley Medical Research, Saginaw, MI; C. Saffer MD, West Coast OB/GYN, San Diego, CA; R. Healy, Adams Patterson OB-GYN, Memphis, TN; and W. Wilkerson, Insignia Clinical Research, Tampa, FL.
This study was funded by Hologic.
Am J Clin Pathol. 2015;144(3):473-483. © 2015 American Society for Clinical Pathology