Human papillomavirus type 16 (HPV16) plays a role in the development of a subgroup of head and neck squamous cell carcinomas (HNSCC). However, uncertainty exists about the true impact of HPV in this tumor type as conflicting reports have been published with prevalence rates from 0 to 100%. We aimed to find a detection algorithm of a biologically and thus clinically meaningful infection, applicable for high-throughput screening of frozen and formalin-fixed paraffin embedded (FFPE) specimens. By considering detection of HPV E6 oncogene expression in frozen biopsies as gold standard for a meaningful HPV infection, the value of several assays was evaluated on FFPE tumor specimens and sera of 48 HNSCC patients. The following assays were evaluated on FFPE tissue samples: HPV DNA general primer (GP)5+/6+ PCR, viral load analysis, HPV16 DNA FISH detection, HPV16 E6 mRNA RT-PCR, p16 immunostaining, and on corresponding serum samples detection of antibodies against the HPV16 proteins L1, E6 and E7. Comparing single assays on FFPE tissue samples detection of E6 expression by RT-PCR was superior, but application remains at present limited to HPV16 detection. Most suitable algorithm with 100% sensitivity and specificity appeared p16 immunostaining followed by GP5+/6+ PCR on the p16-positive cases. We show that clinically meaningful viral HPV infections can be more reliably measured in FFPE HNSCC samples in a standard and high throughput manner, paving the way for prognostic and experimental vaccination studies, regarding not only HNSCC, but possibly also cancer types with HPV involvement in subgroups such as penile and anal cancer.

Given the strong etiologic link between high-risk HPV infection and cervical cancer high-risk HPV testing is now being considered as an alternative for cytology-based cervical cancer screening. Many test systems have been developed that can detect the broad spectrum of hrHPV types in one assay. However, for screening purposes the detection of high-risk HPV is not inherently useful unless it is informative for the presence of high-grade cervical intraepithelial neoplasia (CIN 2/3) or cancer. Candidate high-risk HPV tests to be used for screening should reach an optimal balance between clinical sensitivity and specificity for detection of high-grade CIN and cervical cancer to minimize redundant or excessive follow-up procedures for high-risk HPV positive women without cervical lesions. Data from various large screening studies have shown that high-risk HPV testing by hybrid capture 2 and GP5+/6+-PCR yields considerably better results in the detection of CIN 2/3 than cytology. The data from these studies can be used to guide the translation of high-risk HPV testing into clinical practice by setting standards of test performance and characteristics. On the basis of these data we have developed guidelines for highrisk HPV test requirements for primary cervical screening and validation guidelines for candidate HPV assays. ' 2008 Wiley-Liss, Inc.Key words: HPV-DNA testing; cervical screening; HPV test guidelines; HPV test requirements; HPV test statistics It is now well-established and widely, if not universally, accepted that virtually all cervical cancer and its immediate precancerous lesions arise from persisting cervical infections by $15 cancer-associated (high-risk or hr) human papillomavirus (hrHPV) genotypes.1,2 The most important of these HPV genotypes are HPV16 and HPV18, which account for $70% of all invasive cervical cancers with minor variations in this percentage between continents.3 A new paradigm of cervical carcinogenesis replaces an older model of stepwise progression from low-grade to highgrade morphological changes and can now be summarized as four reliably measured stages: (i) HPV acquisition, (ii) HPV persistence (vs. clearance), (iii) progression of a persisting infection to cervical precancer (with incidental co-occurrence of both conditions) and (iv) invasion. 4,5 On the basis of this nearly absolute etiologic link between carcinogenic HPV and cervical cancer, testing for hrHPV is now being considered as an alternative for cytology-based cervical cancer screening. However, before cost-effective implementation of population-based hrHPV testing in cervical cancer screening and prevention can be envisaged, any candidate HPV testing technologies must offer an optimal balance between clinical sensitivity and specificity for detection of cervical intraepithelial neoplasia grade 2 or 3 and treatable cancer (!CIN 2) to minimize redundant or excessive follow-up procedures. Reliable clinical performance needs to be established before any candidate screening test is widely disseminated and adopted into cl...

High-risk human papillomavirus (hrHPV) testing has a higher sensitivity but lower specificity than cytology for detection of high-grade intraepithelial neoplasia (CIN). To avoid over-referral to colposcopy and overtreatment, hrHPV-positive women require triage testing and/or followup. A total of 25,658 women (30-60 years) enrolled in a population-based cohort study had an adequate baseline Pap smear and hrHPV test. The end-point was cumulative two-year risk of CIN grade 3 or worse (CIN31). In a post-hoc analysis, fourteen triage/followup strategies for hrHPV-positive women (n 5 1,303) were evaluated for colposcopy referral rate, positive (PPV) and negative predictive value (NPV). Five strategies involved triage testing without a repeat test and nine strategies involved triage testing followed by one repeat testing. The tests were cytology, hrHPV, HPV16/ 18 genotyping and HPV16/18/31/33/45 genotyping. Results were adjusted for women in the cohort study who did not attend repeat testing. Of the strategies without repeat testing, combined cytology and HPV16/18/31/33/45 genotyping gave the highest NPV of 98.9% (95%CI 97.6-99.5%). The corresponding colposcopy referral rate was 58.1% (95%CI 55.4-60.8%). Eight of the nine strategies with retesting had an estimated NPV of at least 98%. Of those, cytology triage followed by cytology at 12 months had a markedly lower colposcopy referral rate of 33.4% (95%CI 30.2-36.7%) than the other strategies. The NPV of the latter strategy was 99.3% (95%CI 98.1-99.8%). Triage hrHPV-positive women with cytology, followed by repeat cytology testing yielded a high NPV and modest colposcopy referral rate and appear to be the most feasible management strategy.Strong evidence is now available that testing for high-risk human papillomavirus (hrHPV) infection is more sensitive than cytology in detecting high-grade cervical intraepithelial neoplasia (CIN). 1-7 However, hrHPV testing also detects more transient hrHPV infections than cytology, 1,8 which may lead to over-referral for colposcopy and thus overtreatment. 5 Management of hrHPV-positive women is, therefore, of major concern. In particular, in countries with an efficient cytology-based screening program and a moderate colposcopy referral rate such as The Netherlands and the United Kingdom, the increased burden on healthcare resources upon introduction of a less-specific screening test may be Key words: human papillomavirus, uterine cervical neoplasms, cervical intraepithelial neoplasia, colposcopy, early detection of cancer CJLM was the project leader, designed the study with SB, LR and PJFS and had access to all data. DCR, together with JB, CJLM, FJvK, VC and PJFS, drafted the manuscript. DCR, JB and VC were responsible for data analysis. ATH, DAMH CJLM and PJFS were responsible for HPV testing and HPV genotyping. LR was responsible for database management. DCR and WMV were responsible for the logistics. RHMV was responsible for communication with gynaecologists. FJvK and LR were responsible for cytology testing. All authors critically...

Human papillomavirus (HPV) infection has been etiologically linked to oropharyngeal squamous cell carcinoma (OPSCC). The prevalence of HPV-positive OPSCC varies between studies, ranging from 20 to 90%. This may be related to the lack of a standardized HPV detection assay as well as to the time period in which HPV prevalence is investigated, as rising incidence rates are reported over the last decades. Here, we validated our previously defined test algorithm for HPV detection in formalin-fixed paraffin-embedded (FFPE) tumor specimen consisting of p16 INK4A immunostaining followed by high-risk HPV DNA detection by GP51/61 PCR on the positive cases (Smeets et al., Int J Cancer 2007;121:2465-72). In addition, we analyzed HPV prevalence rates in OPSCCs in the years 1990-2010. The test algorithm was validated on a consecutive series of 86 OPSCCs collected during 2008-2011, of which both fresh frozen and FFPE samples were available. We performed HPV-E6 RT-PCR on the frozen samples as gold standard and applied the algorithm to the corresponding FFPE samples. The test algorithm showed an accuracy of 98%. Using the validated algorithm, we determined the presence of an oncogenic HPV infection in 240 OPSCCs of patients diagnosed in the years 1990-2010 at our center. A significant increase in the proportion of HPV-positive samples was observed, from 5.1% in 1990 to 29.0% in 2010 (p 5 0.001). In conclusion, we confirmed the accuracy of the test algorithm for HPV detection in FFPE tumor specimen and we found a significant increase in the prevalence of HPV in OPSCC over the last two decades at our center.Head and neck squamous cell carcinoma (HNSCC) is the sixth most common cancer worldwide, accounting for $4% of all tumors. 1 Tobacco smoking and alcohol consumption are the main risk factors in the etiology of HNSCC. Over the last three decades, it has become clear that infection with high-risk human papillomavirus (HPV) is also etiologically linked to the development of HNSCCs, particularly those carcinomas that arise in the oropharyngeal region. HPV-positive oropharyngeal squamous cell carcinomas (OPSCCs) are considered to be a different tumor entity, based on prominent biological and epidemiological differences, when compared to the HPV-negative OPSCCs. 2 The biological characteristics of HPV-positive OPSCCs are distinct with respect to gene expression profiles, patterns of genetic changes, frequency of mutations in TP53 and expression of p16 INK4A (p16), encoded by the CDKN2A gene. 2,3 Epidemiologically, it has been reported that most patients with HPV-positive OPSCC are younger, are less likely to have a history of tobacco and alcohol use and have a higher number of sexual partners, in particular oral sex partners, than patients with HPV-negative OPSCC. 4-7 Furthermore, survival is markedly better for patients with an HPV-positive OPSCC, despite the fact that HPV-positive OPSCCs often present as poorly differentiated, aggressively growing tumors, that have already metastasized to the lymph nodes in the neck. [8][9][10][11]...

Purpose: Screening women for high-grade cervical intraepithelial neoplasia or cervical cancer (CIN3 þ ) by high-risk human papillomavirus (hrHPV) testing has as side-effect the detection of hrHPV-positive women without clinically relevant lesions. Here, we developed an objective assay assessing the methylation status of the promoter regions of CADM1 and MAL to triage hrHPV-positive women for CIN3 þ .Experimental Design: In a training set (51 women with CIN3 þ and 224 without CIN2 þ ), panels consisting of one to four quantitative methylation-specific PCR (qMSP) assays (CADM1-m12,CADM1-m18,MAL-m1,MAL-m2) were analyzed. Cross-validated receiver-operating characteristics (ROC) curves were constructed and the panel with highest partial cross-validated area under the curve (AUC) was used for validation in an independent set of 236 consecutive hrHPV-positive women from a screening cohort. In the validation set, the ROC curve of the panel was compared with CIN3 þ sensitivity and specificity of cytology and of cytology combined with HPV16/18 genotyping. Results: In the training set, CADM1-m18 combined with MAL-m1 was the best panel (cross-validated partial AUC ¼ 0.719). In the validation set, this panel revealed CIN3 þ sensitivities ranging from 100%

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Primary testing for human papillomavirus (HPV) in cervical screening requires triage to differentiate women with transient infection from those with persistent infection who require more intensive management given their risk for cervical (pre)cancer. In this study, the clinical performance of a novel methylation marker FAM19A4 for the triage of high-risk (hr)HPV-positive women was evaluated. Using a trainingvalidation set approach, we analyzed a FAM19A4 quantitative methylation-specific PCR (qMSP). The training set comprised hrHPV-positive cervical scrapes of 43 women with cervical intraepithelial neoplasia grade 3 or worse (CIN3þ) and 135 women with CIN1. The validation set comprised hrHPV-positive cervical scrapes of 52 women with CIN2þ, including 33 CIN3þ, 19 CIN2, and 166 women with CIN1. The methylation threshold of FAM19A4 qMSP that gave rise to CIN3þ specificity of 70% in the training set was applied in the validation set. This resulted in CIN3þ sensitivity of 75.8% [95% confidence interval (CI), 61.1-90.4] at 67.0% (95% CI, 60.3-73.8) specificity. Next, the validated qMSP was applied to an independent series of hrHPV-positive cervical scrapes of 22 women with cervical cancer, 29 with advanced CIN2/3 [i.e., women with a known preceding hrHPV infection (PHI) lasting !5 years as proxy of longer duration of lesion existence], and 19 with early CIN2/3 (i.e., PHI <5 years). All carcinomas (22/22) and advanced CIN2/3 lesions (29/29) were FAM19A4 methylation-positive, compared with 42.1% (8/19; 95% CI, 19.9-64.3) of early CIN2/3 lesions. In conclusion, FAM19A4 is an attractive triage marker for hrHPVpositive women, with a high reassurance for the detection of cervical carcinoma and advanced CIN2/3 lesions. Cancer Prev Res; 7(12); 1251-7. Ó2014 AACR.

Combined detection of cell adhesion molecule 1 (CADM1) and T-lymphocyte maturation-associated protein (MAL) promoter methylation in cervical scrapes is a promising triage strategy for high-risk human papillomavirus (hrHPV)-positive women. Here, CADM1 and MAL DNA methylation levels were analysed in cervical scrapes of hrHPV-positive women with no underlying high-grade disease, high-grade cervical intraepithelial neoplasia (CIN) and cervical cancer. CADM1 and MAL methylation levels in scrapes were first related to CIN-grade of the corresponding biopsy and second to CIN-grade stratified by the presence of 'normal' or 'abnormal' cytology as present in the accompanying scrape preceding the cervical biopsy. The scrapes included 167 women with CIN1, 54 with CIN2/3 and 44 with carcinoma. In a separate series of hrHPV-positive scrapes of women with CIN2/3 (n 5 48), methylation levels were related to duration of preceding hrHPV infection (PHI; <5 and 5 years). Methylation levels were determined by quantitative methylation-specific PCR and normal cytology scrapes of hrHPV-positive women with histologically CIN1 served as reference. CADM1 and MAL methylation levels increased proportional to severity of the underlying lesion, showing an increase of 5.3-and 6.2-fold in CIN2/3, respectively, and 143.5-and 454.9-fold in carcinomas, respectively, compared to the reference. Methylation levels were also elevated in CIN2/3 with a longer duration of PHI (i.e. 11.5-and 13.6-fold, respectively). Moreover, per histological category, methylation levels were higher in accompanying scrapes with abnormal cytology than in scrapes with normal cytology. Concluding, CADM1 and MAL promoter methylation levels in hrHPV-positive cervical scrapes are related to the degree and duration of underlying cervical disease and markedly increased in cervical cancer.