Diagnostic accuracy of p16INK4a immunohistochemistry in oropharyngeal squamous cell carcinomas: A systematic review and meta‐analysis

The accurate diagnosis of human papillomavirus (HPV) causality in oropharyngeal squamous cell carcinomas (OPSCC) is likely to influence therapeutic decisions in affected patients in the near future. We conducted a systematic review and meta‐analysis to determine the diagnostic accuracy of p16^INK4a immunohistochemistry (IHC) to identify HPV‐induced OPSCC. We identified all studies that performed p16^INK4a IHC (index test) and HPV E6/E7 mRNA detection using an amplification‐based method (gold standard to indicate a transforming relevance of HPV) in OPSCC. Testing with one or more comparator tests (HPV DNA PCR, HPV DNA in situ hybridization (ISH) and p16^INK4a IHC/HPV DNA PCR combined testing) was an optional criterion for inclusion. Among 1,636 retrieved studies 24 fulfilled the inclusion criteria. The pooled sensitivity of p16^INK4a IHC, HPV DNA PCR, HPV DNA ISH and p16^INK4a IHC/HPV DNA PCR combined testing was 94% (95%‐confidence interval (CI) 91–97%), 98% (CI 94–100%), 85% (CI 76–92%) and 93% (CI 87–97%), respectively. The pooled specificity was 83% (CI 78–88%), 84% (CI 74–92%), 88% (CI 78–96%) and 96% (CI 89–100%), respectively. p16^INK4a IHC/HPV DNA PCR combined testing was as sensitive as either p16^INK4a IHC or HPV DNA PCR alone but significantly more specific than either separate test. In conclusion, p16^INK4a IHC is highly sensitive but moderately specific to diagnose HPV‐transformed OPSCC when used as a single test. Combined p16^INK4a IHC and HPV DNA PCR testing significantly enhances specificity while maintaining high sensitivity. This diagnostic test combination thus represents an attractive testing strategy for the reliable diagnosis of HPV‐induced OPSCC in the clinical setting and may constitute an inclusion criterion for future therapeutic trials.     

Epidemiologic associations of HPV‐positive oropharyngeal cancer and (pre)cancerous cervical lesions

Human papillomavirus (HPV)‐induced oropharyngeal squamous cell carcinoma (OPSCC) remains increasing worldwide. We aimed to investigate if the HPV‐prevalence of OPSCC in the Netherlands is rising as well, also in female patients. In addition, we evaluated the association between HPV‐positive OPSCC and suspicious Pap results of the cervix in these female patients. Patients with OPSCC treated in the period 2000–2015 at the VU University Medical Center Amsterdam, were included (n = 926). The presence of an oncogenic HPV infection was determined by p16‐immunostaining, followed by a high‐risk HPV general primer 5+/6+ DNA PCR on the p16‐immunopositive cases. A review of pathology reports in all female patients (n = 305) was undertaken to identify cytological signs of HPV‐related (pre)cancer of the cervix. In total 281 of 926 (30.3%) OPSCCs were HPV‐positive. Moreover, a significant increase in the prevalence of HPV‐positive OPSCCs was observed from 14.0% in 2000 to 48.1% in 2015 (p < 0.001). Among the female patients with an HPV‐positive OPSSC (n = 70), the results of cervical smears were available in 56 of 70 patients (80.0%). Of the female patients with HPV‐positive OPSCC, 9 of 56 (16.1%) patients had a vaginal cuff Papanicolaou (Pap) test ≥3b in their medical history compared to 7 of 168 (4.2%) in the HPV‐negative group (p = 0.003). In conclusion, a continuous increase in the HPV‐attributable fraction of OPSCC was demonstrated in the period 2000–2015 in the Amsterdam region. HPV‐positive OPSCC has a significant association with a history of suspicious Pap results of the cervix in female patients.     

Increasing prevalence rates of HPV attributable oropharyngeal squamous cell carcinomas in the Netherlands as assessed by a validated test algorithm

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 GP5+/6+ 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 = 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.     

No role for human papillomavirus in esophageal squamous cell carcinoma in China

Certain regions of China have high rates of esophageal squamous cell carcinoma (ESCC). Previous studies of human papillomavirus (HPV), a proposed causal factor, have produced highly variable results. We attempted to evaluate HPV and ESCC more definitively using extreme care to prevent DNA contamination. We collected tissue and serum in China from 272 histopathologically‐confirmed ESCC cases with rigorous attention to good molecular biology technique. We tested for HPV DNA in fresh‐frozen tumor tissue using PCR with PGMY L1 consensus primers and HPV16 and 18 type‐specific E6 and E7 primers, and in formalin‐fixed paraffin‐embedded tumor tissue using SPF₁₀ L1 primers. In HPV‐positive cases, we evaluated p16^INK4a overexpression and HPV E6/E7 seropositivity as evidence of carcinogenic HPV activity. β‐globin, and thus DNA, was adequate in 98.2% of the frozen tumor tissues (267/272). Of these, 99.6% (95% confidence interval (CI) = 97.9–100.0%) were negative for HPV DNA by PGMY, and 100% (95% CI = 98.6–100%) were negative by HPV16/18 E6/E7 PCR. In the corresponding formalin‐fixed paraffin‐embedded tumor specimens, 99.3% (95% CI = 97.3–99.9%) were HPV negative by SPF₁₀. By PGMY, 1 case tested weakly positive for HPV89, a noncancer causing HPV type. By SPF₁₀, 2 cases tested weakly positive: 1 for HPV16 and 1 for HPV31. No HPV DNA‐positive case had evidence of HPV oncogene activity as measured by p16^INK4a overexpression or E6/E7 seropositivity. This study provides the most definitive evidence to date that HPV is not involved in ESCC carcinogenesis in China. HPV DNA contamination cannot be ruled out as an explanation for high HPV prevalence in ESCC tissue studies with less stringent tissue procurement and processing protocols.     

Prognostic significance of human papillomavirus 16 viral load level in patients with oropharyngeal cancer

Human papillomavirus (HPV) infection in patients with oropharyngeal squamous cell carcinoma (OPSCC) is a major determinant for better prognosis. However, there remain HPV-positive patients who have poor outcomes. The stratification strategy for detecting high-risk patients among those with HPV-positive OPSCC has not been well delineated, especially for Asian patients. We undertook a retrospective cohort study on the survival rate of 89 Japanese patients diagnosed with primary OPSCC. The tumors were concurrently analyzed for the presence of HPV E6 DNA/mRNA, viral DNA load, p16 expression, viral physical status, and viral variant lineage. Human papillomavirus 16 viral DNA was found in 45 (51%) OPSCCs. Human papillomavirus 16 DNA-positive OPSCCs with higher viral load (classified as HPV16 DNA-medium/high OPSCCs) showed significantly favorable overall survival and progression-free survival compared with HPV16 DNA-positive OPSCCs with lower viral load (<10 copies/cell; HPV16 DNA-low OPSCCs) and HPV16 DNA-negative OPSCCs. E6 mRNA expression was observed in all HPV16 DNA-medium/high OPSCCs but not in HPV16 DNA-low OPSCCs. Notably, p16-positive and HPV16 DNA-negative/low OPSCCs showed significantly worse survival than p16-positive and HPV16 DNA-medium/high OPSCCs and resembled HPV-unrelated OPSCCs with regard to survival and risk factor profile. Although not significant, a trend toward shorter survival was observed for HPV16-integrated OPSCCs. Phylogenetic analysis revealed two major types of HPV16 variants termed Asian (A4) and European (A1/A2/A3) variants, but no difference in survival between these variants was observed. Altogether, these findings suggest that HPV viral load is a potentially informative factor for more accurate risk stratification of patients with OPSCC.     

HPV16 increases the number of migratory cancer stem cells and modulates their miRNA expression profile in oropharyngeal cancer

Human papillomavirus type 16 (HPV16) is a major risk for development of oropharyngeal squamous‐cell‐carcinoma (OPSCC). Although HPV⁺ OPSCC metastasize faster than HPV^? tumors, they have a better prognosis. The molecular and cellular alterations underlying this pathobiology of HPV⁺ OPSCC remain elusive. In this study, we examined whether expression of HPV16‐E6E7 targets the number of migratory and stationary cancer stem cells (CSC). Furthermore, we wanted to elucidate if aberrantly expressed miRNAs in migratory CSC may be responsible for progression of OPSCCs and whether they may serve as potential novel biomarkers for increased potential of metastasis. Our studies revealed that HPV16‐E6E7 expression leads to an increase in the number of stationary (CD44^high/EpCAM^high) stem cells in primary keratinocyte cultures. Most importantly, expression of E6E7 in the cell line H357 increased the migratory (CD44^high/EpCAM^low) CSC pool. This increase in migratory CSCs could also be confirmed in HPV⁺ OPSCC. Differentially expressed miRNAs from HPV16‐E6E7 positive CD44^high/EpCAM^low CSCs were validated by RT‐qPCR and in situ hybridization on HPV16⁺ OPSCCs. These experiments led to the identification of miR‐3194‐5p, which is upregulated in primary HPV16⁺ OPSCC and matched metastasis. MiR‐1281 was also found to be highly expressed in HPV⁺ and HPV^? metastasis. As inhibition of this miRNA led to a markedly reduction of CD44^high/EpCAM^low cells, it may prove to be a promising drug target. Taken together, our findings highlight the capability of HPV16 to modify the phenotype of infected stem cells and that miR‐1281 and miR3194‐5p may represent promising targets to block metastatic spread of OPSCC.     

P16INK4A as a surrogate biomarker for human papillomavirus‐associated oropharyngeal carcinoma: Consideration of some aspects

Human papillomavirus (HPV)‐associated oropharyngeal squamous cell carcinomas (OPSCCs) frequently show different clinical and pathological features, which tend to be younger age, better performance status, less tobacco and alcohol consumption, more poorly differentiated histopathology, but usually with better treatment response and prognosis compared with HPV‐negative OPSCCs. In tumor tissue, HPV infection is closely correlated with p16^INK4A expression, which has been suggested to be a surrogate biomarker of HPV infection. However, there is diversity of sensitivity and specificity about p16^INK4A in surrogate detection of HPV status. Herein, we summarize the current knowledge and note some aspects for consideration concerning p16^INK4A as a surrogate biomarker for HPV‐associated OPSCC.     

P16INK4A immunostaining is a strong indicator for high‐risk‐HPV‐associated oropharyngeal carcinomas and dysplasias,but is unreliable to predict low‐risk‐HPV‐infection in head and neck papillomas and laryngeal dysplasias

Human papillomavirus (HPV) is a risk factor for the development of benign and malignant mucosal head and neck lesions. P16^INK4A is often used as a surrogate marker for HPV‐infection, although there is still controversy with respect its reliability. Our aim was to determine if p16^INK4A overexpression can accurately predict both high‐risk and low‐risk‐HPV‐presence in (pre)malignant and benign head and neck lesions. P16^INK4A immunohistochemistry was performed on paraffin‐embedded tissue sections of 162 oropharyngeal squamous cell carcinomas (OPSCC), 14 tonsillar and 23 laryngeal dysplasias, and 20 tonsillar and 27 laryngeal papillomas. PCR, enzyme‐immunoassay and FISH analysis were used to assess HPV‐presence and type. Of the 162 OPSCC and 14 tonsillar dysplasias, 51 (31%) and 10 (71%) were HPV16‐positive, respectively. All tonsillar papillomas were HPV‐negative and four laryngeal dysplasias and 26 laryngeal papillomas were positive for HPV6 or ?11. P16^INK4A immunohistochemistry revealed a strong nuclear and cytoplasmic staining in 50 out of 51 HPV16‐positive and 5 out of 111 HPV‐negative OPSCC (p < 0.0001) and in all HPV16‐positive tonsillar dysplasias, whereas highly variable staining patterns were detected in the papillomas and laryngeal dysplasias, irrespective of the HPV‐status. In addition, the latter lesions generally showed a higher nuclear than cytoplasmic p16^INK4A immunostaining intensity. In conclusion, our data show that strong nuclear and cytoplasmic p16^INK4A overexpression is a reliable surrogate indicator for HPV16 in OPSCC and (adjacent) dysplasias. For HPV6 or ?11‐positive and HPV‐negative benign and premalignant lesions of the tonsil and larynx, however, p16^INK4A immunostaining is highly variable and cannot be recommended to predict HPV‐presence.     

Is the improved prognosis of p16 positive oropharyngeal squamous cell carcinoma dependent of the treatment modality?

The incidence of human papilloma virus (HPV) induced oropharyngeal squamous cell carcinoma (OPSCC) increases in the western countries. These OPSCC show distinct molecular characteristics and are characterized by an overexpression of p16, considered a surrogate marker for HPV infection. When compared to patients with p16 negative OPSCC, patients with HPV induced p16 positive OPSCC show a significantly better prognosis, which is reported to be caused by increased radiosensitivity. The objective of the present study was to analyze the impact of p16 expression status on the prognosis of OPSCC treated by either radiotherapy (RT) or primary surgery. Results are based upon a tissue microarray (TMA) of 365 head neck squamous cell carcinomas (HNSCC) including 85 OPSCC with clinico‐pathological and follow‐up data. p16 positivity correlated significantly with oropharyngeal tumor localization (p < 0.001). Patients with p16 positive OPSCC exhibited a significantly better overall survival than those with p16 negative tumors (p = 0.007). In a multivariate analysis, survival benefit of patients with p16 positive OPSCC was independent of clinico‐pathological parameters such as cT and cN classification and treatment modality. The improved prognosis of p16 positive OPSCC is found after RT as well as after surgery.     

Combined P16 and human papillomavirus testing predicts head and neck cancer survival

While its prognostic significance remains unclear, p16^INK4a protein expression is increasingly being used as a surrogate marker for oncogenic human papillomavirus (HPV) infection in head and neck squamous cell carcinomas (HNSCC). To evaluate the prognostic utility of p16 expression in HNSCC, we prospectively collected 163 primary tumor specimens from histologically confirmed HNSCC patients who were followed for up to 9.4 years. Formalin fixed tumor specimens were tested for p16 protein expression by immunohistochemistry (IHC). HPV type‐16 DNA and RNA was detected by MY09/11‐PCR and E6/E7 RT‐PCR on matched frozen tissue, respectively. P16 protein expression was detected more often in oropharyngeal tumors (53%) as compared with laryngeal (24%), hypopharyngeal (8%) or oral cavity tumors (4%; p < 0.0001). With respect to prognosis, p16‐positive oropharyngeal tumors exhibited significantly better overall survival than p16‐negative tumors (log‐rank test p = 0.04), whereas no survival benefit was observed for nonoropharyngeal tumors. However, when both p16 and HPV DNA test results were considered, concordantly positive nonoropharyngeal tumors had significantly better disease‐specific survival than concordantly negative nonoropharyngeal tumors after controlling for sex, nodal stage, tumor size, tumor subsite, primary tumor site number, smoking and drinking [adjusted hazard ratio (HR) = 0.04, 0.01–0.54]. Compared with concordantly negative nonoropharyngeal HNSCC, p16(+)/HPV16(?) nonoropharyngeal HNSCC (n = 13, 7%) demonstrated no significant improvement in disease‐specific survival when HPV16 was detected by RNA (adjusted HR = 0.83, 0.22–3.17). Our findings show that p16 IHC alone has potential as a prognostic test for oropharyngeal cancer survival, but combined p16/HPV testing is necessary to identify HPV‐associated nonoropharyngeal HNSCC with better prognosis.     

Evaluation of the eighth TNM classification on p16-positive oropharyngeal squamous cell carcinomas in the Netherlands and the importance of additional HPV DNA testing

BackgroundOropharyngeal squamous cell carcinomas (OPSCCs) are traditionally caused by smoking and excessive alcohol consumption. However, in the last decades high-risk human papillomavirus (HPV) infections play an increasingly important role in tumorigenesis. HPV-driven OPSCCs are known to have a more favorable prognosis, which has led to important and marked changes in the recently released TNM-8. In this 8th edition, OPSCCs are divided based on p16 immunostaining, with p16 overexpression as surrogate marker for the presence of HPV. The aims of this study are to evaluate TNM-8 on a Dutch consecutive cohort of patients with p16-positive OPSCC and to determine the relevance of additional HPV DNA testing.Patients and methodsAll OPSCC patients without distant metastases at diagnosis and treated with curative intent at VU University Medical Center (2000–2015) and Erasmus Medical Center (2000–2006) were included (N = 1204). HPV status was determined by p16 immunostaining followed by HPV DNA PCR on the p16-immunopositive cases. We compared TNM-7 and TNM-8 using the Harrell’s C index.ResultsIn total, 388 of 1204 (32.2%) patients were p16-immunopositive. In these patients, TNM-8 had a markedly better predictive prognostic power than TNM-7 (Harrell’s C index 0.63 versus 0.53). Of the 388 p16-positive OPSCCs, 48 tumors (12.4%) were HPV DNA-negative. This subgroup had distinct demographic, clinical and morphologic characteristics and showed a significantly worse five-year overall survival compared with the HPV DNA-positive tumors (P < 0.001).ConclusionsTNM-8 has a better predictive prognostic power than TNM-7 in patients with p16-positive OPSCC. However, within p16-positive OPSCCs, there is an HPV DNA-negative subgroup with distinct features and a worse overall survival, indicating the importance to perform additional HPV DNA testing when predicting prognosis and particularly for selecting patients for de-intensified treatment regimens.     

Mucosal alpha‐papillomaviruses are not associated with esophageal squamous cell carcinomas: Lack of mechanistic evidence from South Africa,China and Iran and from a world‐wide meta‐analysis

Epidemiological and mechanistic evidence on the causative role of human papillomaviruses (HPV) in esophageal squamous cell carcinoma (ESCC) is unclear. We retrieved alcohol‐ and formalin‐fixed paraffin‐embedded ESCC tissues from 133 patients seropositive for antibodies against HPV early proteins, from high‐incidence ESCC regions: South Africa, China and Iran. With rigorous care to prevent nucleic acid contamination, we analyzed these tissues for the presence of 51 mucosotropic human alpha‐papillomaviruses by two sensitive, broad‐spectrum genotyping methods, and for the markers of HPV‐transformed phenotype: (i) HPV16/18 viral loads by quantitative real‐time PCR, (ii) type‐specific viral mRNA by E6*I/E6 full‐length RT‐PCR assays and (iii) expression of cellular protein p16^INK4a. Of 118 analyzable ESCC tissues, 10 (8%) were positive for DNA of HPV types: 16 (4 tumors); 33, 35, 45 (1 tumor each); 11 (2 tumors) and 16, 70 double infection (1 tumor). Inconsistent HPV DNA+ findings by two genotyping methods and negativity in qPCR indicated very low viral loads. A single HPV16 DNA+ tumor additionally harbored HPV16 E6*I mRNA but was p16^INK4a negative (HPV16 E1 seropositive patient). Another HPV16 DNA+ tumor from an HPV16 E6 seropositive patient showed p16^INK4a upregulation but no HPV16 mRNA. In the tumor tissues of these serologically preselected ESCC patients, we did not find consistent presence of HPV DNA, HPV mRNA or p16^INK4a upregulation. These results were supported by a meta‐analysis of 14 other similar studies regarding HPV‐transformation of ESCC. Our study does not support the etiological role of the 51 analyzed mucosotropic HPV types in the ESCC carcinogenesis.     

The complex relationship between human papillomavirus and cervical adenocarcinoma

Human Papillomavirus (HPV) is reported in 60–100% of cervical adenocarcinoma (CADC) globally. We investigated this relationship in a hospital‐based survey in China. 718 CADC samples from nine Chinese regions were analysed. Expert pathologists reviewed cases with p16 and progesterone receptor immunostaining. Cases were tested for HPV using whole‐tissue sections (WTS) and laser‐capture microdissection. All cases were HPV‐tested by L1 based broad‐spectrum SPF₁₀‐DEIA‐LiPA₂₅ PCR. Negative cases were tested for DNA adequacy and with E6 oncogene, type‐specific HPV PCRs. Using WTS‐PCR CADC showed overall 75% HPV‐positivity (33–100% for different histological types). LCM‐PCR showed that none of minimal deviation or serous CADC, and <10% of all clear cell and endometrioid CADC were HPV‐positive in tumour cells. Usual and adenosquamous CADC showed a single HPV genotype in 60 and 78% cases. In some cases, HPV was found in adjacent cervix but not in tumour. HPV 16, 18 and 45 accounted for 90% of HPV in tumour cells. Patients with HPV‐positive tumours were on average 6 years younger and presented at a lower clinicopathological stage as compared to patients with HPV‐negative cancers. CADC is diverse pathologically and in HPV status. Special histopathological tumor subtypes may develop through different cellular and molecular pathways. Between 20 and 40% usual and adenosquamous types, in particular these diagnosed in older women and at advanced FIGO stages, are not driven by oncogenic HPV. In these cases HPV may not be involved in carcinogenisis or maybe lost during tumour progression.     

Time trends in the prevalence of HPV in oropharyngeal squamous cell carcinomas in northern Spain (1990–2009)

Recent studies support an important role for human papillomavirus (HPV) in oropharyngeal squamous cell carcinomas (OPSCC), although the incidence varies widely depending on the geographic location and time period studied. The aim of this study was to determine the proportion of HPV in a large cohort of OPSCC in northern Spain in the years 1990–2009. Clinical records and paraffin embedded tumor specimens of 248 consecutive patients surgically treated for OPSCC (140 tonsillar and 108 base of tongue) between 1990 and 2009 were retrieved. OPSCC cases were histomorphologically evaluated, and protein expression of p16 and p53 was analyzed by immunohistochemistry. Detection of high‐risk HPV DNA was performed by GP5+/6+‐PCR and in situ hybridization (ISH). Thirty cases (12%) were positive for p16 immunostaining, of which eight (3.2% of the total series) were found positive for HPV type 16 by genotyping of GP5+6+‐PCR products. All HPV GP5+/6+‐PCR‐positive tumors were p53‐immunonegative, seven had a basaloid morphology and seven were also positive by HPV ISH. Presence of HPV correlated inversely with tobacco and alcohol consumption (p < 0.001), but not with age of onset of OPSCC. Overall survival was better in the HPV‐positive group, although not statistically significant (p = 0.175). OPSCC patients in northern Spain demonstrated a low involvement of HPV, increasing (although not significantly, p = 0.120) from 1.8% in 1990–1999 to 6.1% of cases in 2000–2009.     

Multiple imputation and clinico-serological models to predict human papillomavirus status in oropharyngeal carcinoma: An alternative when tissue is unavailable

In cancer epidemiological studies, determination of human papillomavirus (HPV) in oropharyngeal squamous cell carcinoma (OPSCC) typically depends on the availability of tumor tissue testing, and/or tumor tissue access. Identifying alternative methods for estimating HPV status can improve the quality of such studies when tissue is unavailable. We developed multiple predictive models for tumor HPV status and prognosis by combining both clinico-epidemiological variables and either serological multiplex assays of HPV or multiple imputation of HPV status (HPV_mi). Sensitivity, specificity and accuracy of these methods compared to either p16 immunostaining (p16 IHC) or survival were assessed. When compared to a reference of tumor tissue p16 IHC in 783 OPSCC patients, the clinic-HPV_sero model incorporating a composite of 20 HPV serological antibodies (HPV_sero) and 4 clinical factors (c-index: 0.96) performed better than using HPV_sero (c-index: 0.92) or HPV_mi (c-index: 0.76) alone. However, the model that contained a single HPV16 E6 antibody combined with four clinical variables, performed extremely well (clinic-s1-16E6; c-index: 0.95). When defining HPV status by HPV_sero, s1-16E6, HPV_mi or through p16 IHC, each of these definitions demonstrated improved overall and disease-free survival in HPV-positive OPSCC patients, when compared to HPV-negative patients (adjusted hazard ratios between 0.25 and 0.63). Our study demonstrates that when blood samples are available, a model that utilizes a single s1-16E6 antibody combined with several clinical features has excellent test performance characteristics to estimate HPV status and prognosis. When neither blood nor tumor tissue is available, multiple imputation, calibrated on local population characteristics, remains a viable, but suboptimal option.     

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.     

Baseline prevalence and type distribution of human papillomavirus in healthy Chinese women aged 18–25 years enrolled in a clinical trial

Baseline human papillomavirus (HPV) prevalence and type distribution were evaluated in young Chinese women enrolled in a clinical trial of an HPV vaccine ( ClinicalTrials.gov registration NCT00779766). Cervical specimens and blood samples were collected at baseline from women aged 18–25 years (n = 6,051) from four sites across Jiangsu province. Cervical specimens were tested for HPV DNA by SPF₁₀ PCR‐DEIA‐LiPA₂₅ version 1, and HPV‐16/18 type‐specific polymerase chain reaction. Anti‐HPV‐16 and anti‐HPV‐18 antibody titres were quantified by enzyme‐linked immunosorbent assay. At baseline, 15.3% of women were DNA positive for any of 14 HPV high‐risk (hr) types (HPV‐16/18/31/33/35/39/45/51/52/56/58/59/66/68). The most commonly detected hrHPV types in cervical specimens were HPV‐52 (4.0%) and HPV‐16 (3.7%). High‐risk HPV DNA‐positivity increased with severity of cytological abnormalities: 39.3% in atypical squamous cells of undetermined significance, 85.0% in low‐grade squamous intraepithelial lesions and 97.8% in high‐grade squamous intraepithelial lesions (HSIL). The hrHPV types most frequently detected in HSIL were HPV‐16 (63.0%), HPV‐18 (17.4%), HPV‐52 (17.4%), HPV‐58 (15.2%) and HPV‐33 (15.2%). The hrHPV types most frequently detected in cervical intraepithelial neoplasia 2+ were HPV‐16 (66.1%), HPV‐33 (16.1%), HPV‐52 (16.1%), HPV‐58 (14.5%) and HPV‐51 (11.3%). Multiple hrHPV infections were reported for 24.4% of hrHPV DNA positive women. Regardless of baseline HPV DNA status, 30.5% and 16.0% of subjects were initially seropositive for anti‐HPV‐16 and anti‐HPV‐18, respectively. In conclusion, the high baseline seropositivity rate and intermediate prevalence of cervical hrHPV types in Chinese women aged 18–25 years underlines the importance of early HPV vaccination in this population.