Performance of the cobas HPV Test for the Triage of Atypical Squamous Cells of Undetermined Significance Cytology in Cervical Specimens Collected in SurePath.

OBJECTIVES: Determine performance of the cobas human papillomavirus (HPV) test for triage of atypical squamous cells of undetermined significance (ASC-US) in SurePath. METHODS: Women presenting for routine screening had cervical specimens collected in SurePath and specimen transport medium (STM); those with ASC-US cytology underwent colposcopy. Performance of cobas HPV in SurePath specimens that had undergone a preanalytic procedure to reverse possible cross-linking of HPV DNA was compared with Hybrid Capture 2 (hc2) specimens in STM. RESULTS: Among 856 women, HPV prevalence was 45.8%; HPV 16 and HPV 18 prevalences were lower than expected in the 21- to 29-year-old group in this highly vaccinated population. cobas HPV performance in SurePath was comparable to hc2 in STM. Sensitivity and specificity for detection of cervical intraepithelial neoplasia grade 3 or worse were 87.5% (95% confidence interval [CI], 71.9%-95.2%) and 55.5% (95% CI, 52.1%-58.9%) for cobas and 85.3% (95% CI, 69.9%-93.6%) and 54.7% (95% CI, 51.4%-57.9%) for hc2. Sensitivity was negatively affected by random biopsies performed at colposcopy; comparable sensitivities were achieved in the nonvaccinated and vaccinated populations with disease determined by directed biopsy only. CONCLUSIONS: Performance of cobas HPV for ASC-US triage in pretreated SurePath specimens meets criteria for validation. Preliminary data indicate reliable performance of HPV testing in a highly vaccinated population.

Triaging HPV-positive women with p16/Ki-67 dual-stained cytology: Results from a sub-study nested into the ATHENA trial.

OBJECTIVES: In addition to genotyping for HPV16/18, dual-immunostaining for p16/Ki-67 has shown promise as a triage of HPV-positive women. We assessed the performance of p16/Ki-67 dual-stained cytology for triaging HPV-positive women undergoing primary HPV screening. METHODS: All women ≥25years with valid cervical biopsy and cobas® HPV Test results from the cross-sectional phase of ATHENA who were referred to colposcopy (n=7727) were eligible for enrolment. p16/Ki-67 dual-stained cytology was retrospectively performed on residual cytologic material collected into a second liquid-based cytology vial during the ATHENA enrolment visit. The diagnostic performance of dual-stained cytology, with or without HPV16/18 genotyping, for the detection of biopsy-confirmed cervical intraepithelial neoplasia grade 3 or worse (CIN3+) was determined and compared to Pap cytology. Furthermore, the number of colposcopies required per CIN3+ detected was determined. RESULTS: Dual-stained cytology was significantly more sensitive than Pap cytology (74.9% vs. 51.9%; p<0.0001) for triaging HPV-positive women, whereas specificity was comparable (74.1% vs. 75.0%; p=0.3198). Referral of all HPV16/18 positive women combined with dual-stained cytology triage of women positive for 12 "other" HPV genotypes provided the highest sensitivity for CIN3+ (86.8%; 95% CI: 81.9-90.8). A similar strategy but using Pap cytology for the triage of women positive for 12 "other" HPV genotypes was less sensitive (78.2%; 95% CI: 72.5-83.2; p=0.0003), but required a similar number of colposcopies per CIN3+ detected. CONCLUSIONS: p16/Ki-67 dual-stained cytology, either alone or combined with HPV16/18 genotyping, represents a promising approach as a sensitive and efficient triage for colposcopy of HPV-positive women when primary HPV screening is utilized.

Development and Validation of a Preanalytic Procedure for Performing the cobas HPV Test in SurePath Preservative Fluid.

The formation of chemical cross-links between nucleic acids and proteins in formalin-containing media presents challenges for human papillomavirus (HPV) testing of cervical samples collected in SurePath Preservative Fluid. A preanalytic process involving addition of a nucleophilic buffer and heating the sample to 120°C was developed to reverse the effects of cross-linking and improve nucleic acid accessibility for the cobas HPV Test in SurePath. Cycle threshold (CT) values for cobas HPV detection were evaluated over time and various temperatures, and mean CT differences between pretreated and both untreated SurePath samples and those collected in PreservCyt were assessed. Without pretreatment, low viral levels (1 × limit of detection) of HPV were no longer detectable by 7 days. For prospectively collected specimens, mean (95% CI) CT differences between pretreated and untreated samples indicated enhanced HPV DNA recovery in all categories of treated samples: -2.58 (-3.16 to -2.01), -2.63 (-3.62 to -1.64), and -3.39 (-4.95 to -1.82), respectively, for other 12 high-risk HPV types, HPV16, and HPV18. Furthermore, mean (95% CI) CT differences of pretreated SurePath samples were comparable to simultaneously collected PreservCyt samples: -0.48 (-0.98 to 0.02) and -0.23 (-0.93 to 0.46), respectively, for HPV16 and HPV18; a borderline significant difference [-0.35 (-0.57 to -0.13)] was observed for other 12 high-risk HPV types. This preanalytic procedure therefore ensures a validated, safe, and accurate method for cobas HPV testing in SurePath.

Knowledge of Patients' Human Papillomavirus Status at the Time of Cytologic Review Significantly Affects the Performance of Cervical Cytology in the ATHENA Study.

OBJECTIVES: With human papillomavirus (HPV) testing, patients' HPV status may be known when reviewing cytology specimens. METHODS: 41,955 women 25 years or older had cytology and HPV screening. Originally, cytology was reviewed blinded to HPV status. We re-reviewed unblinded to HPV status a subset of 428 cytology slides from women with cervical intraepithelial neoplasia grade 2 + (CIN2+) and 1,287 from women without CIN2+. RESULTS: Of the original interpretations of atypical squamous cells of undetermined significance (ASC-US), 33.7% were downgraded to negative after unblinded review, and 8.7% were upgraded to atypical squamous cells, cannot rule out a high-grade squamous intraepithelial lesion or high-grade squamous intraepithelial lesion. Of the original interpretations of ASC-US, 66.7% were downgraded on unblinded review in HPV-negative women and 30.2% were upgraded in HPV 16+/HPV 18+ women. Unblinding increases the sensitivity for cervical intraepithelial neoplasia grade 3+ of cotesting from 54.1% to 62.4% (P = .0015) and the sensitivity of HPV primary screening from 72.2% to 77.1% (P = .0029). With cotesting, specificity with unblinding is improved, whereas with HPV primary screening, there would be a small decrease in specificity. CONCLUSIONS: Unblinded cytology increases overall sensitivity with either cotesting or HPV primary screening; specificity is either slightly improved or is not affected by unblinding.

Evaluating HPV-negative CIN2+ in the ATHENA trial.

A post hoc analysis of the ATHENA study was performed to determine whether true HPV-negative cervical lesions occur and whether they have clinical relevance. The ATHENA database was searched for all CIN2 or worse (CIN2+) cases with cobas HPV-negative results and comparison was made with Linear Array (LA) and Amplicor to detect true false-negative HPV results. Immunostaining with p16 was performed on these cases to identify false-positive histology results. H&E slides were re-reviewed by the study pathologists with knowledge of patient age, HPV test results and p16 immunostaining. Those with positive p16 immunostaining and/or a positive histopathology review underwent whole tissue section HPV PCR by the SPF10/LiPA/RHA system. Among 46,887 eligible women, 497 cases of CIN2+ were detected, 55 of which tested negative by the cobas(®) HPV Test (32 CIN2, 23 CIN3/ACIS). By LA and/or Amplicor, 32 CIN2+ (20 CIN2, 12 CIN3/ACIS) were HPV positive and categorized as false-negatives by cobas HPV; nine of 12 false-negative CIN3/ACIS cases were p16+. There were 23 cases (12 CIN2, 11 CIN3/ACIS) negative by all HPV tests; seven of 11 CIN3/ACIS cases were p16+. H&E slides were available for six cases for re-review and all were confirmed as CIN3/ACIS. Tissue PCR was performed on the six confirmed CIN3/ACIS cases (and one without confirmation): four were positive for HPV types not considered oncogenic, two were positive for oncogenic genotypes and one was indeterminate. In summary, subanalysis of a large cervical cancer screening study did not identify any true CIN3/ACIS not attributable to HPV.

Primary cervical cancer screening with human papillomavirus: end of study results from the ATHENA study using HPV as the first-line screening test.

OBJECTIVES: ATHENA evaluated the cobas HPV Test as the primary screen for cervical cancer in women ≥25years. This reports the 3-year end-of-study results comparing the performance of HPV primary screening to different screening and triage combinations. METHODS: 42,209 women ≥25years were enrolled and had cytology and hrHPV testing. Women with abnormal cytology (≥atypical squamous cells of undetermined significance) and those HPV positive were referred to colposcopy. Women not reaching the study endpoint of CIN2+ entered the 3-year follow-up phase. RESULTS: 3-year CIR of CIN3+ in cytology-negative women was 0.8% (95% CI; 0.5-1.1%), 0.3% (95% CI 0.1-0.7%) in HPV-negative women, and 0.3% (95% CI; 0.1-0.6%) in cytology and HPV negative women. The sensitivity for CIN3+ of cytology was 47.8% (95% CI; 41.6-54.1%) compared to 61.7% (95% CI; 56.0-67.5%) for the hybrid strategy (cytology if 25-29years and cotesting with cytology and HPV if ≥30years) and 76.1% (95% CI; 70.3-81.8%) for HPV primary. The specificity for CIN3+ was 97.1% (95% CI; 96.9-97.2%), 94.6% (95% CI; 94.4-94.8%), and 93.5% (95% CI; 93.3-93.8%) for cytology, hybrid strategy, and HPV primary, respectively. Although HPV primary detects significantly more cases of CIN3+ in women ≥25years than either cytology or hybrid strategy, it requires significantly more colposcopies. However, the number of colposcopies required to detect a single CIN3+ is the same as for the hybrid strategy. CONCLUSIONS: HPV primary screening in women ≥25years is as effective as a hybrid screening strategy that uses cytology if 25-29years and cotesting if ≥30years. However, HPV primary screening requires less screening tests.

Relevance of random biopsy at the transformation zone when colposcopy is negative.

OBJECTIVE: A post hoc analysis to determine the diagnostic yield of random biopsy in detecting high-grade cervical disease in women with negative colposcopy. METHODS: The ATHENA (Addressing the Need for Advanced HPV Diagnostics) trial screened more than 47,000 women with cytology and high-risk human papillomavirus (HPV) DNA genotyping. Colposcopy was performed in all women with abnormal cytology or positive HPV results. A single random biopsy was taken at the squamocolumnar junction if colposcopy was adequate and no lesions were identified. RESULTS: The random biopsy diagnosed 20.9% (81/388, 95% confidence interval [CI] 16.9-25.3%) and 18.9% (45/238, 95% CI 14.1-24.5%) of the total cervical intraepithelial neoplasia (CIN) grade 2 or worse and grade 3 or worse, respectively. This additional disease was detected in both HPV 16 or 18+ and for 12 other high-risk HPV+ women. For HPV 16 or 18, the absolute risk for detection of CIN 2 or worse on random biopsy in the overall population was 13.1% (40/305, 95% CI 9.8-17.4%) and 8.2% (25/305, 95% CI 5.6-11.8%) for CIN 3 or worse. By contrast, the absolute risk for 12 other high-risk HPV+ women was 3.5% (29/820, 95% CI 2.5-5%) and 1.7% (14/820, 95% CI 1.0-2.8%) for CIN 2 or worse and CIN 3 or worse, respectively. CONCLUSION: A single random biopsy increased the detection of high-grade disease when no lesions were visualized at colposcopy. The absolute risks of disease associated with the random biopsy were highest for women positive for genotype 16 or 18. Our study supports performing a random biopsy in women undergoing colposcopy without visible lesions, particularly in those positive for HPV 16 or 18. LEVEL OF EVIDENCE: : II.

Interlaboratory variation in the performance of liquid-based cytology: insights from the ATHENA trial.

Although it is recognized that cervical cytology is highly subjective, and that there is considerable interlaboratory variation in how slides are evaluated, little is known as to how this impacts the performance of cytology. In the ATHENA trial, liquid-based cytology specimens from 46,887 eligible women ≥21 years of age were evaluated at four large regional US laboratories, providing a unique opportunity to evaluate the impact of interlaboratory variations on the performance of cervical cytology. All women with abnormal cytology (atypical squamous cells of undetermined significance or higher) were referred to colposcopy, as were all high-risk human papillomavirus (hrHPV)-positive women ≥25 years of age and a random subset of those ≥25 years of age who were negative by both hrHPV testing and cytology. Sociodemographics, risk factors for cervical disease, and prevalence of cervical intraepithelial neoplasia (CIN) were similar across the laboratories. There were considerable differences among the laboratories both in overall cytological abnormal rates, ranging from 3.8 to 9.9%, and in sensitivity of cytology to detect CIN grade 2 or worse (CIN2+), from 42.0 to 73.0%. In contrast, the hrHPV positivity rate varied only from 10.9 to 13.4%, and the sensitivity of hrHPV testing from 88.2 to 90.1%. These observations suggest that hrHPV testing without cytology should be considered as the initial method for cervical cancer screening.

Human papillomavirus testing for triage of women with low-grade squamous intraepithelial lesions.

Low-grade squamous intraepithelial lesion (LSIL) is a common cytologic finding in cervical screening, yet only about 10-20% have significant histologic abnormalities and these are almost always positive for high-risk human papillomavirus (hrHPV). This analysis aims to clarify the role of hrHPV DNA testing in the triage of women with LSIL cytology. In the ATHENA screening trial, we examined 1,084 cases of LSIL, of which 925 had an evaluable biopsy, to determine the extent to which hrHPV testing can identify those patients who have precursor lesions in need of immediate clinical referral and those who have changes more likely to regress spontaneously. Overall, 71.2% of LSIL cases were hrHPV positive, but the prevalence was age dependent, with only 56.1% in women ≥ 40 years. Among women with LSIL, 11.6% (107/925) had a cervical intraepithelial neoplasia grade 2 or worse (CIN2+) histologic diagnosis and, of these, only nine were hrHPV negative. For CIN3+, 91.7% (44/48) of women with LSIL were hrHPV positive. The negative predictive value of hrHPV testing for CIN3+ in LSIL was 100% for women aged ≥ 40 years. Women who were HPV16 positive had a higher positive predictive value for CIN2+ (25.4%) than those who were positive for 12 other pooled hrHPV types (11.5%). Testing for hrHPV in women with LSIL is effective in identifying high-grade cervical lesions, thereby avoiding unnecessary referrals to colposcopy and potential over-treatment of non-progressive lesions, especially for women aged ≥ 40 years.

Comparison of cervical cancer screening strategies incorporating different combinations of cytology, HPV testing, and genotyping for HPV 16/18: results from the ATHENA HPV study.

OBJECTIVE: The objective of the study was to compare 9 cervical cancer screening strategies to the current screening standard (cytology with human papillomavirus [HPV] triage of atypical squamous cells of undetermined significance) for the detection of high-grade cervical disease. STUDY DESIGN: Women (n = 34,254) aged 30 years or older from the Addressing the Need for Advanced HPV Diagnostics (ATHENA) study underwent screening with cytology and HPV testing with simultaneous HPV16/18 genotyping; those with atypical squamous cells of undetermined significance cytology or greater or HPV-positive status were referred for colposcopy. RESULTS: In general, screening strategies that offered greater sensitivity also required more referral to colposcopy. HPV testing was more sensitive than cytology for detection of cervical intraepithelial neoplasia grade 2 or greater, but strategies that depended on cytology for triage of HPV-positive women decreased this sensitivity. Various strategies of cotesting with cytology increased sensitivity but did so by increasing testing. Strategies that included integrated HPV16/18 testing provided more efficient referral to colposcopy. CONCLUSION: Strategies that maximize detection of women at greatest risk of cervical intraepithelial neoplasia grade 3 or greater by immediate referral to colposcopy, with follow-up testing of women at intermediate risk, maximize the benefits of cervical cancer screening while decreasing the potential harm. Incorporating screening with HPV and triage of HPV-positive women by a combination of genotyping for HPV16/18 and cytology provided a good balance between maximizing sensitivity (benefit) and specificity by limiting the number of colposcopies (potential harm).

Comparison of cobas human papillomavirus test results from primary versus secondary vials of PreservCyt specimens and evaluation of potential cross-contamination.

BACKGROUND: The preferred workflow for high-risk human papillomavirus (hrHPV) testing in the majority of laboratories involves cytology processing of samples collected in liquid-based cytology medium followed by hrHPV testing. The cobas HPV Test received approval from the US Food and Drug Administration in April 2011, and the supporting clinical trial design necessitated prealiquoting the sample used for hrHPV testing from the PreservCyt primary vial into a secondary vial that was placed on the cobas 4800 System. METHODS: To validate use of the postcytology residual sample in the primary vial, the authors presented the results of cross-contamination studies and a comparison of the cobas HPV Test results from the prealiquot in the secondary vial with results obtained from the postcytology primary vial on samples processed on either the Hologic ThinPrep 2000 System (T2000) or ThinPrep 3000 System (T3000). RESULTS: Cross-contamination checkerboard studies with 100 samples processed on the T2000 system and 120 samples processed on the T3000 system indicated no conversion of primary vial results from positive to negative or from negative to positive. For clinical samples, approximately 1100 archived specimens from the ATHENA (Addressing the Need for Advanced HPV Diagnostics) study and 1100 combined archived and fresh primary vial specimens that had been processed on the T2000 and T3000 processors, respectively, were compared. The overall percentage agreement between the primary and secondary vials was at least 93.5%. CONCLUSIONS: The postcytology residual of samples collected in PreservCyt primary vials and run on the T2000 and T3000 processors provided reliable cobas HPV Test results that were comparable to results from the precytology secondary vial, without any evidence of contamination.

The interplay of age stratification and HPV testing on the predictive value of ASC-US cytology. Results from the ATHENA HPV study.

We have previously shown that human papillomavirus (HPV) genotyping, using the cobas HPV Test (Roche Molecular Systems, Pleasanton, CA), can be used to identify women with atypical squamous cells of undetermined significance (ASC-US) at the highest risk for cervical intraepithelial neoplasia (CIN) grade 2 or worse. We investigated the impact of age stratification on the risk of CIN 2 or worse in women with ASC-US and the performance of HPV genotyping in different age strata. The sensitivity of the cobas HPV Test was 93.3% in the 21- to 29-year-old age group and 67.7% in the 40 years or older group, most likely owing to pathologic misclassification of CIN 2 or worse in older women. The prevalence of CIN 2 or worse in younger women was nearly 4-fold that detected in older women and was predominantly HPV-16-related. Age-specific evaluation of ASC-US cytology in conjunction with HPV genotype status enables more effective risk assessment and could be used in clinical management.

The ATHENA human papillomavirus study: design, methods, and baseline results.

OBJECTIVE: The objective of the study was to describe baseline data from Addressing the Need for Advanced HPV Diagnostics, a prospective, multicenter US cervical cancer screening trial. STUDY DESIGN: A total of 47,208 women aged 21 years or older undergoing routine screening were enrolled; liquid-based cytology and human papillomavirus (HPV) testing were performed. Women with abnormal cytology underwent colposcopy, as did high-risk HPV (hrHPV)-positive women and a random subset of women negative by both tests aged 25 years or older. Verification bias adjustment was applied; 95% confidence intervals were computed by the bootstrap method. RESULTS: The prevalence of cytologic abnormalities was 7.1%. hrHPV, HPV 16, and HPV 18 were detected using the cobas HPV Test in 12.6%, 2.8%, and 1.0% of women, respectively. Both cytologic abnormalities and hrHPV positivity declined with increasing age. The adjusted prevalence of cervical intraepithelial neoplasia grade 2 (CIN2) or greater in women aged 25-34 years was 2.3%, decreasing to 1.5% among older women. CONCLUSION: The Addressing the Need for Advanced HPV Diagnostics study provides important estimates of the prevalence of cytologic abnormalities, hrHPV positivity, and CIN2 or greater in a US screening population.

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.

BACKGROUND: The ATHENA study was designed to assess the performance of carcinogenic human papillomavirus (HPV) testing and HPV16 or HPV18 genotyping compared with liquid-based cytology for cervical cancer screening in a large US population aged 21 years and older. We did a subanalysis of this population to compare the screening performance of the cobas HPV test versus liquid-based cytology in women aged 25 years and older, and assess management strategies for HPV-positive women. METHODS: Women aged 25 years or older who were attending routine cervical screening were enrolled from 61 clinical centres in 23 US states. Cervical specimens were obtained for liquid-based cytology and HPV DNA testing with two first-generation assays (Amplicor HPV test and Linear Array HPV genotyping test) and the second-generation cobas HPV test (with individual HPV16 and HPV18 detection). Colposcopy and diagnostic biopsies were done on women with atypical squamous cells of undetermined significance (ASC-US) or worse cytology, those who tested positive with either first-generation HPV test, and a random sample of women who tested negative for HPV and cytology. All women not selected for colposcopy received their results and exited the study. Participants and colposcopists were masked to cytology and HPV test results until the colposcopy visit was completed. The primary endpoint for this substudy was histologically confirmed cervical intraepithelial neoplasia grade 3 (CIN3) or worse. This study is registered with ClinicalTrials.gov, number NCT00709891; the study is in the follow-up phase, which is due to be completed in December, 2012. FINDINGS: From May 27, 2008, to Aug 27, 2009, 47,208 women were enrolled, of whom 41,955 met our eligibility criteria. Valid cobas HPV and liquid-based cytology test results were available for 40,901 women (97%), who were included in this analysis. Of these, 4275 women (10%) tested cobas HPV positive and 2617 (6%) had abnormal cytology. 431 women were diagnosed with CIN2 or worse and 274 with CIN3 or worse. In women who had colposcopy, the cobas HPV test was more sensitive than liquid-based cytology for detection of CIN3 or worse (252/274 [92·0%, 95% CI 88·1-94·6] vs 146/274 [53·3%, 95% CI 47·4-59·1]; difference 38·7%, 95% CI 31·9-45·5; p<0·0001). Addition of liquid-based cytology to HPV testing increased sensitivity for CIN3 or worse to 96·7% (265/274, 95% CI 93·9-98·3), but increased the number of screen positives by 35·2% (5783/40,901 vs 4275/40,901) compared with HPV testing alone. As a triage test to identify CIN3 or worse in HPV-positive women, detection of HPV16, HPV18, or both alone was equivalent to detection of ASC-US or worse alone in terms of sensitivity (150/252 [59·5%] vs 133/252 [52·8%]; p=0·11) and positive predictive value (PPV) (150/966 [15·5%] vs 133/940 [14·1%]; p=0·20). Among HPV-positive women, detection of HPV16, HPV18, or both or low-grade squamous intraepithelial lesion or worse cytology had better sensitivity (182/252 [72·2%]; p<0·0001) and similar PPV (182/1314 [13·9%]; p=0·70) for detection of CIN3 or worse than ASC-US or worse cytology alone. Furthermore, detection of HPV16, HPV18, or both or high-grade squamous intraepithelial lesion or worse cytology had higher sensitivity (165/252 [65·5%]; p=0·0011) and PPV (165/1013 [16·3%]; p=0·031) for detection of CIN3 or worse than ASC-US or worse cytology alone. INTERPRETATION: HPV testing with separate HPV16 and HPV18 detection could provide an alternative, more sensitive, and efficient strategy for cervical cancer screening than do methods based solely on cytology. FUNDING: Roche Molecular Systems.