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.     

Long‐term HPV type‐specific risks of high‐grade cervical intraepithelial lesions: A 14‐year follow‐up of a randomized primary HPV screening trial

Quantitative knowledge of the long‐term human papillomavirus (HPV) type‐specific risks for high‐grade cervical intraepithelial neoplasias Grades 2 and 3 (CIN2 and CIN3) is useful for estimating the effect of elimination of specific HPV types and clinical benefits of screening for specific HPV types. We estimated HPV type‐specific risks for CIN2 and CIN3 using a randomized primary HPV screening trial followed up for 14.6 years using comprehensive, nationwide registers. Poisson regression estimated cumulative incidences, population attributable proportions (PAR) and incidence rate ratios (IRRs) of high‐grade lesions by baseline HPV type, with censoring at date of first CIN2/3 or last registered cytology. Multivariate analysis adjusted for coinfections. IRRs were highest during the first screening round, but continued to be high throughout follow‐up (IRRs for CIN3 associated with high‐risk (HR) HPV positivity were 226.9, 49.3, 17.7 and 10.3 during the first, second and third screening round and for >9 years of follow‐up, respectively). Increased long‐term risks were found particularly for HPV Types 16, 18 and 31 and for CIN3+ risks. HPV16/18/31/33 had 14‐year cumulative incidences for CIN3+ above 28%, HPV35/45/52/58 had 14 year risks between 14% and 18% and HPV39/51/56/59/66/68 had risks <10%. HPV16 contributed to the greatest proportion of CIN2+ (first round PAR 36%), followed by Types 31, 52, 45 and 58 (7–11%). HPV16/18/31/33/45/52/58 together contributed 73.9% of CIN2+ lesions and all HR types contributed 86.9%. In summary, we found substantial differences in risks for CIN2 and CIN3 between different oncogenic HPV types. These differences may be relevant for both clinical management and design of preventive strategies.     

Long‐term HPV type‐specific risks for ASCUS and LSIL: A 14‐year follow‐up of a randomized primary HPV screening trial

Human papillomavirus (HPV) infections result in a significant burden of low‐grade cervical lesions. Between 1997 and 2000, our randomized trial of primary HPV screening enrolled 12,527 women participating in population‐based screening. Women between 32 and 38 years of age (median: 34, interquartile range: 33–37) were randomized to HPV and cytology double testing (intervention arm, n = 6,257 enrolled, n = 5,888 followed‐up) or to cytology, with samples frozen for future HPV testing (control arm, n = 6,270 enrolled, n = 5,795 followed‐up). We estimated the HPV type‐specific, long‐term absolute risks (AR), and population attributable proportions (PAR) for cytological diagnoses of atypical squamous cells of undetermined significance (ASCUS) or low‐grade squamous intraepithelial lesion (LSIL) and for histopathologically diagnosed cervical intraepithelial neoplasia grade 1 (CIN1). The women were followed using comprehensive, nationwide register‐based follow‐up. During a mean follow‐up time of 11.07 years, 886 ASCUS and LSIL lesions were detected, 448 in the intervention arm and 438 in the control arm. Poisson regression estimated the incidence rate ratios (IRRs) of low‐grade lesions by HPV type. The IRRs were strongly dependent on follow‐up time. The IRRs for ASCUS/LSIL associated with high‐risk HPV positivity were 18.6 (95% CI: 14.9–23.4) during the first screening round, 4.1 (95% CI: 2.8–6.2) during the second, 2.6 (95% CI: 1.7–4.1) during the third, and 1.1 (95% CI: 0.7–1.8) for >9 years of follow‐up, with similar declines seen for the individual types. Type 16 contributed consistently to the greatest proportion of ASCUS, LSIL, and CIN1 risk in the population (first screening round PAR: ASCUS: 15.5% (95% CI: 9.7–21.9), LSIL: 14.7% (95% CI: 8.0–20.9), and CIN1: 13.4% (95% CI: 3.2–22.5)), followed by type 31 [8.4% (95% CI: 4.2–12.5) for ASCUS to 17.3% (95% CI: 6.8–26.6) for CIN1]. In summary, most ASCUS/LSIL lesions associated with HPV infection are caused by new HPV infections and most lesions are found during the first screening round.     

Cervical cancer risk in HPV‐positive women after a negative FAM19A4/mir124‐2 methylation test: A post hoc analysis in the POBASCAM trial with 14 year follow‐up

DNA methylation analysis of cervical scrapes using FAM19A4 and mir124‐2 genes has shown a good clinical performance in detecting cervical cancer and advanced CIN lesions in need of treatment in HPV‐positive women. To date, longitudinal data on the cancer risk of methylation test‐negative women are lacking. In our study, we assessed the longitudinal outcome of FAM19A4/mir124‐2 methylation analysis in an HPV‐positive screening cohort with 14 years of follow‐up. Archived HPV‐positive cervical scrapes of 1,040 women (age 29–61 years), who were enrolled in the POBASCAM screening trial (ISRCTN20781131) were tested for FAM19A4/mir124‐2 methylation. By linkage with the nationwide network and registry of histo‐ and cytopathology in the Netherlands (PALGA), 35 cervical cancers were identified during 14 years of follow‐up comprising three screens (baseline, and after 5 and 10 years). The baseline scrape of 36.1% (n = 375) women tested positive for FAM19A4/mir124‐2 methylation, including 24 women with cervical cancer in follow‐up, and 30.6% (n = 318) had abnormal cytology (threshold borderline dyskaryosis or ASCUS), including 14 women with cervical cancer in follow‐up. Within screening round capability of FAM19A4/mir124‐2 methylation to detect cervical cancer was 100% (11/11, 95% CI: 71.5–100). Kaplan–Meier estimate of 14‐year cumulative cervical cancer incidence was 1.7% (95% CI: 0.66–3.0) among baseline methylation‐negative and 2.4% (95% CI: 1.4–3.6) among baseline cytology‐negative women (risk difference: 0.71% [95% CI: 0.16–1.4]). In conclusion, a negative FAM19A4/mir124‐2 methylation test provides a low cervical cancer risk in HPV‐positive women of 30 years and older. FAM19A4/mir124‐2 methylation testing merits consideration as an objective triage test in HPV‐based cervical screening programs.     

Molecular characterization of p16‐immunopositive but HPV DNA‐negative oropharyngeal carcinomas

Recent studies have reported that p16 protein overexpression qualifies as a surrogate marker identifying an oncogenic human papillomavirus (HPV) infection in oropharyngeal squamous cell carcinoma (OPSCC). However, there is still a percentage of OPSCCs that are positive for p16 immunohistochemistry (p16 IHC) but lack HPV DNA. The objective of this study was to characterize this group at the molecular level by performing sensitive HPV DNA‐ and RNA‐based PCR methods and genetic profiling. All patients diagnosed with an OPSCC in the period 2000–2006 in two Dutch university medical centers were included (n = 841). The presence of HPV in a tumor sample was tested by p16 IHC followed by an HPV DNA GP5+/6+ PCR. p16 IHC scored positive in 195 samples, of which 161 were HPV DNA‐positive and 34 (17%) HPV DNA‐negative. In the latter group, a SPF₁₀‐LiPA25 assay, an HPV16 type‐specific E7 PCR and an E6 mRNA RT‐PCR were performed. Next, ten of these cases were further analyzed for loss of heterozygosity (LOH) of 15 microsatellite markers at chromosome arms 3p, 9p and 17p. Of the 34 p16‐positive but PCR‐negative OPSCCs, two samples tested positive by SPF₁₀ assay, HPV16 E7 PCR and HPV16 E6 mRNA RT‐PCR. Three samples tested positive by SPF₁₀ assay but negative by the HPV16‐specific assays. Nine of ten cases that were tested for LOH showed a genetic pattern comparable to that of HPV‐negative tumors. This study categorizes p16‐positive but HPV DNA‐negative OPSCCs as HPV‐negative tumors based on genetic profiling. This study highlights the importance of performing HPV testing in addition to p16 IHC for proper identification of HPV‐associated OPSCCs.     

The comparative and cost‐effectiveness of HPV‐based cervical cancer screening algorithms in El Salvador

Cervical cancer is the leading cause of cancer death among women in El Salvador. Utilizing data from the Cervical Cancer Prevention in El Salvador (CAPE) demonstration project, we assessed the health and economic impact of HPV‐based screening and two different algorithms for the management of women who test HPV‐positive, relative to existing Pap‐based screening. We calibrated a mathematical model of cervical cancer to epidemiologic data from El Salvador and compared three screening algorithms for women aged 30–65 years: (i) HPV screening every 5 years followed by referral to colposcopy for HPV‐positive women (Colposcopy Management [CM]); (ii) HPV screening every 5 years followed by treatment with cryotherapy for eligible HPV‐positive women (Screen and Treat [ST]); and (iii) Pap screening every 2 years followed by referral to colposcopy for Pap‐positive women (Pap). Potential harms and complications associated with overtreatment were not assessed. Under base case assumptions of 65% screening coverage, HPV‐based screening was more effective than Pap, reducing cancer risk by ～60% (Pap: 50%). ST was the least costly strategy, and cost $2,040 per year of life saved. ST remained the most attractive strategy as visit compliance, costs, coverage, and test performance were varied. We conclude that a screen‐and‐treat algorithm within an HPV‐based screening program is very cost‐effective in El Salvador, with a cost‐effectiveness ratio below per capita GDP.     

Proof‐of‐principle study of a novel cervical screening and triage strategy: Computer‐analyzed cytology to decide which HPV‐positive women are likely to have ≥CIN2

A challenge in implementation of sensitive HPV‐based screening is limiting unnecessary referrals to colposcopic biopsy. We combined two commonly recommended triage methods: partial HPV typing and “reflex” cytology, evaluating the possibility of automated cytology. This investigation was based on 1,178 exfoliated cervical specimens collected during the enrollment phase of The Study to Understand Cervical Cancer Early Endpoints and Determinants (SUCCEED, Oklahoma City, OK). We chose a colposcopy clinic population to maximize number of outcomes, for this proof‐of‐principle cross‐sectional study. Residual aliquots of PreservCyt were HPV‐typed using Linear Array (LA, Roche Molecular Systems, Pleasanton, CA). High‐risk HPV typing data and cytologic results (conventional and automated) were used jointly to predict risk of histologically defined ≥CIN2. We developed a novel computer algorithm that uses the same optical scanning features that are generated by the FocalPoint Slide Profiler (BD, Burlington, NC). We used the Least Absolute Shrinkage and Selection Operator (LASSO) method to build the prediction model based on a training dataset (n = 600). In the validation set (n = 578), for triage of all HPV‐positive women, a cytologic threshold of ≥ASC‐US had a sensitivity of 0.94, and specificity of 0.30, in this colposcopy clinic setting. When we chose a threshold for the severity score (generated by the computer algorithm) that had an equal specificity of 0.30, the sensitivity was 0.91. Automated cytology also matched ≥ASC‐US when partial HPV typing was added to the triage strategy, and when we re‐defined cases as ≥CIN3. If this strategy works in a prospective screening setting, a totally automated screening and triage technology might be possible.