Catherine M. Behrens

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.

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

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.

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).

Prevalence of high-risk human papilloma virus genotypes and associated risk of cervical precancerous lesions in a large U.S. screening population: Data from the ATHENA trial

OBJECTIVE We assessed the age-related prevalence of high risk human papillomavirus (HR-HPV) genotypes and the genotype-associated risk for high-grade cervical intraepithelial neoplasia (CIN) in a large U.S. screening population. METHODS A total of 40,901 women aged ≥25 years were screened with liquid-based cytology and HPV testing in the ATHENA (Addressing the Need for Advanced HPV Diagnostics) trial. Genotyping was performed using the LINEAR ARRAY HPV Genotyping Test. RESULTS HPV16 was the most prevalent genotype in all age groups, ranging from 3.5% to 0.8% in women aged 25-29 and ≥50 years, respectively. The next most prevalent genotypes were HPV52, HPV31 and HPV18. In the overall population, HPV16 conferred the greatest absolute risk of ≥CIN3 both in women aged 25-29 and ≥30 years (14.2% and 15.1%, respectively) followed by HPV31 (8.0% and 7.9%), HPV52 (6.7% and 4.4%) and HPV18 (2.7% and 9.0%). Similar trends were seen in women with negative cytology. The percent positivity increased markedly with disease progression for HPV16 and HPV18 which were responsible for 45.6% and 8.4% of ≥CIN3, respectively. Of note, HPV 18 was responsible for 50% of adenocarcinoma in situ (AIS) and 50% of invasive cancer cases. CONCLUSIONS HPV16 played a major role in the development of ≥CIN3 irrespective of age, supporting the identification of HPV16 in primary screening for all women. Identification of HPV18 is also warranted, given its significant contribution to AIS and cancer. Identification of non-16/18 genotypes as a pool should provide sufficient information for screening.

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.

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

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.

Development and Characterization of the cobas Human Papillomavirus Test

ABSTRACT The cobas human papillomavirus (HPV) test, approved by the FDA in April 2011, is a fully automated assay for the detection of 14 high-risk (hr) HPV genotypes from cervical specimens collected in liquid-based cytology medium using real-time PCR amplification of the L1 gene and TaqMan probes. Results are simultaneously reported as positive or negative for the pooled 12 oncogenic HPV types (HPV31, -33, -35, -39, -45, -51, -52, -56, -58, -59, -66, and -68) from channel 1, with HPV16 and HPV18 genotypes read individually from channels 2 and 3. A fourth channel detects the human β-globin gene as a control for sample adequacy and assay inhibition. To optimize clinical sensitivity and specificity, cutoff values (cycle thresholds [CT ]) were established for each channel based on the detection of cervical intraepithelial neoplasia grade 2 (CIN2) or greater (≥CIN2). For women aged ≥21 years with cytology results indicating atypical squamous cells of undetermined significance (ASC-US), CT values provided a sensitivity of 90% (95% confidence interval [CI], 81.5% to 94.8%) for the detection of ≥CIN2 and a specificity of 70.5% (95% CI, 68.1% to 72.7%). The analytic sensitivity (limit of detection) ranged from 150 to 2,400 copies/ml, depending on genotype. The analytic specificity, evaluated by comparing the HPV result with a combined comparator of Sanger sequencing and the Qiagen digene HC2 high-risk HPV DNA test (hc2), demonstrated overall positive agreement of 96.3% for 14 hrHPV types in women with ASC-US cytology results who were aged ≥21 years and 86.1% in women with NLIM (negative for intraepithelial neoplasia or malignancy) cytology who were aged ≥30 years. These and other performance validation studies demonstrate that the cobas HPV test is a fully automated and clinically validated robust test.

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.

The low risk of precancer after a screening result of human papillomavirus-negative/atypical squamous cells of undetermined significance papanicolaou and implications for clinical management

Different US practice guidelines have conflicting recommendations for when women should return after a screening result of human papillomavirus (HPV)‐negative with an equivocal Papanicolaou (Pap) result of atypical squamous cells of undetermined significance (ASC‐US) (ie, return in either 3 or 5 years). One way to determine management is to compare the risk of precancer/cancer after an HPV‐negative/ASC‐US result with the risks after other negative screening results. For example, if the risk after an HPV‐negative/ASC‐US result was similar to the risk after a negative Pap test, a 3‐year return would be preferred because guidelines agree that women with negative Pap test results should return in 3 years. Alternatively, if the risk after an HPV‐negative/ASC‐US result is similar to that after a cotest‐negative result (HPV negative/Pap test negative), a 5‐year return would be preferred because guidelines agree that women testing cotest negative should return in 5 years.