Collectively our data show that EGFR mutations and HPV infection represent essential, alternative oncogenic mechanisms in ISP and ISP-associated SNSCC.

In this study, high-risk HPV (hrHPV) incidence, prognostic biomarkers, and outcome were assessed in HIV-positive (case) and HIV-negative (control) patients with head and neck squamous cell cancer (HNSCC). HIV-positive cases were matched to controls by tumor site, sex, and age at cancer diagnosis. A tissue microarray (TMA) was constructed and DNA isolated from tumor tissue. MultiPlex-PCR MassArray, L1-PCR and In Situ Hybridization were used to assess hrHPV. TMA sections were stained for p16ink4a, TP53, RB, CCND1, EGFR, and scored for intensity and proportion of positive tumor cells. The HNSCC cohort included 41 HIV-positive cases and 41 HIV-negative controls. Tumors from 11/40 (28%) cases, and 10/41 (24%) controls contained hrHPV. p16 expression, indicative of E7 oncogene activity, was present in 10/11 HPV-positive cases and 7/10 HPV-positive controls. Low p16 and high TP53 expression in some HPV-positive tumors suggested HPV-independent tumorigenesis. Survival did not differ in cases and controls. RB expression was significantly associated with poor survival (p=0.01). High TP53 expression exhibited a trend for poorer survival (p=0.12), but among cases, association with poor survival reached statistical significance (p=0.04). The proportion of HPV-positive tumors was similar, but the heterogeneity of HPV types was higher in the HIV-positive cases than in HIV-negative controls. High RB expression predicted poor survival, and high TP53 expression was associated with poorer survival in the HIV-positive cases but not HIV-negative controls. Implications HIV infection did not increase risk of death from HNSCC, and HPV-positive tumors continued to be associated with a significantly improved survival, independent of HIV status.

Background HPV-positive oropharyngeal cancer is generally associated with excellent response to therapy, but some HPV-positive tumors progress despite aggressive therapy. This study evaluates viral oncogene expression and viral integration sites in HPV16 and HPV18-positive squamous carcinoma cell lines. Methods E6-E7 alternate transcripts were assessed by RT-PCR. Detection of integrated papillomavirus sequences (DIPS-PCR) and sequencing identified viral insertion sites and affected host genes. Cellular gene expression was assessed across viral integration sites. Results All HPV-positive cell lines expressed alternate HPVE6/E7 splicing indicative of active viral oncogenesis. HPV integration occurred within cancer-related genes TP63, DCC, JAK1, TERT, ATR, ETV6, PGR, PTPRN2, and TMEM237 in 8 HNSCC lines but UM-SCC-105 and UM-GCC-1 had only intergenic integration. Conclusions HPV integration into cancer-related genes occurred in 7/9 HPV-positive cell lines and of these six were from tumors that progressed. HPV integration into cancer-related genes may be a secondary carcinogenic driver in HPV-driven tumors.

Background: The molecular drivers of human papillomavirus-related head and neck squamous cell carcinoma (HPV + HNSCC) are not entirely understood. This study evaluated the relationship between HPV integration, expression of E6/E7, and patient outcomes in p16+ HNSCCs. Methods: HPV type was determined by HPV PCR-MassArray, and integration was called using detection of integrated papillomavirus sequences polymerase chain reaction (PCR). We investigated whether fusion transcripts were produced by reverse transcriptase polymerase chain reaction (RT-PCR). E6/E7 expression was assessed by quantitative RT-PCR. We assessed if there was a relationship between integration and E6/E7 expression, clinical variables, or patient outcomes. Results: Most samples demonstrated HPV integration, which sometimes resulted in a fusion transcript. HPV integration was positively correlated with age at diagnosis and E6/E7 expression. There was a significant difference in survival between patients with vs without integration. Conclusions: Contrary to previous reports, HPV integration was associated with improved patient survival. Therefore, HPV integration may act as a molecular marker of good prognosis. K E Y W O R D S head and neck squamous cell carcinoma (HNSCC), human papillomavirus (HPV), integration, oropharynx, survival

Background and objectives Human papillomavirus (HPV)-driven oropharyngeal squamous cell carcinoma (OPSCC) is increasing globally. In Taiwan, HPV-positive OPSCC is obscured by tobacco, alcohol, and betel quid use. We investigated the role of high-risk HPV (hrHPV) in a large retrospective Taiwan OPSCC cohort. Methods and results The cohort of 541 OPSCCs treated at Chang Gung Memorial Hospital from 1998–2016 consisted of 507 men (94%) and 34 women (6%). Most used tobacco (81%), alcohol (51%), and betel quid (65%). Formalin-fixed, paraffin-embedded tissue was used for p16 staining (a surrogate marker for HPV) and testing for HPV DNA presence and type by Multiplex HPV PCR-MassArray. HPV DNA and/or p16 staining (HPV-positive) was found in 28.4% (150/528) tumors. p16 and HPV DNA were strongly correlated (F < 0.0001). HPV16 was present in 82.8%, and HPV58 in 7.5% of HPV-positive tumors. HPV was associated with higher age (55.5 vs. 52.7 years, p = 0.004), lower T-stage (p = 0.008) better overall survival (OS) (hazard ratio [HR] 0.58 [95% CI 0.42–0.81], p = 0.001), and disease-free survival (DFS) (HR 0.54 [95% CI 0.40–0.73], p < 0.0001). Alcohol was strongly associated with recurrence and death (OS: HR 2.06 [95% CI 1.54–2.74], p < 0.0001; DFS: HR 1.72 [95% CI 1.33–2.24], p < 0.0001). OS and DFS in HPV-positive cases decreased for alcohol users (p < 0.0001). Obscured by the strong alcohol effect, predictive associations were not found for tobacco or betel quid. Conclusions As with HPV-positive OPSCC globally, HPV is an increasingly important etiological factor in Taiwanese OPSCC. HPV-positive OPSCC has considerable survival benefit, but this is reduced by alcohol, tobacco, and betel quid use. hrHPV is a cancer risk factor in males and females. Vaccinating both sexes with a multivalent vaccine including HPV58, combined with alcohol and tobacco cessation policies will be effective cancer-prevention public health strategies in Taiwan.

IntroductionHuman papillomavirus (HPV) is the primary cause of cervical and other anogenital cancers and is also associated with head and neck cancers. Incidence of HPV-related oropharyngeal squamous cell cancers (OPSCCs) is increasing, and HPV-related OPSCCs have surpassed cervical cancer as the most common HPV-related cancer in the USA. Given the multisite nature of HPV, there is strong interest in collecting data from both genital and oral sites, as well as associated data on social and sexual behaviours. The overarching goal of this study is to evaluate patterns of oral HPV infection incidence, clearance and persistence and their relationship to sexual behaviour history.Methods and analysisParticipants are recruited from two populations: college students at a large public university and general population from the surrounding area. At the first study visit, participants complete a detailed sexual history, health and behaviour questionnaire. Follow-up visits occur every 3–4 months over 3 years, when participants complete an abbreviated questionnaire. All participants provide a saliva sample at each visit, and eligible participants may provide a cervicovaginal self-swab. Genetic material isolated from specimens is tested for 15 high-risk and 3 low-risk HPV types. Statistical analyses will examine outcome variables including HPV prevalence, incidence, persistence and clearance. Logistic regression models will be used to estimate odds ratios and 95% confidence intervals for associations between the outcomes of interest and demographic/behavioural variables collected in the questionnaires. The longitudinal HPV infection data and detailed sexual history data collected in the questionnaires will allow us to develop individual-based network models of HPV transmission and will be used to parameterise multiscale models of HPV-related OPSC carcinogenesis.Ethics and disseminationThis study has been approved by the University of Michigan Institutional Review Board. All participants are consented in person by trained study staff. Study results will be disseminated through peer-reviewed publications.

Background In North America and Western Europe, human papillomavirus (HPV)-driven oropharyngeal squamous cell carcinoma (OPSCC) has increased dramatically over the past 40 years, whereas HPV-negative OPSCC, typically associated with alcohol and tobacco as etiological factors, has declined. In Taiwan, the OPSCC rate is increasing; however, there is limited understanding of the role of HPV, as tobacco, alcohol, and betel quid use are still very prominent. Here we investigated the involvement of HPV and its prognostic implications for OPSCC in Taiwan. Methods and findings We studied a retrospective cohort of 541 OPSCCs undergoing care between 1998 and 2016 at the Chang Gung Memorial Hospital-Linkou, Taiwan. Clinical and risk exposure data were retrieved from hospital charts. Most cases were males (94%) and had concomitant (87%) exposure to alcohol (51%), tobacco (83%), and betel quid (65%). Formalin-fixed, paraffin-embedded tissue sections were immunostained for p16, a surrogate for active HPV, and DNA was tested for HPV detection and genotyping by Multiplex PCR-MassArray. Tumors with p16 and/or HPV DNA positivity were identified as HPV-positive. HPV status was assigned to 528 tumors. The prevalence of HPV-positive OPSCC was 28.4% (150/528), with a strong correlation between p16 and HPV DNA results (F < 0.0001). We observed an incremental trend for HPV-positive OPSCC over 18 years. HPV16 alone was found in 76.9%, and HPV58 in 7.5% tumors. Among males more tumors were HPV-negative than positive, but among the 34 females more tumors were HPV-positive (62% vs. 38%, p < 0.0001), more commonly from the tonsils (p < 0.01), and less prone to use tobacco, alcohol, and betel quid (p < 0.0001). For all patients, HPV-positive OPSCC was associated with higher age at diagnosis (55.5 vs. 52.7 years, p = 0.004), and lower T-stage (p = 0.02). HPV-positivity in OPSCC tumors was an independent predictor of better overall survival (OS) (hazard ratio [HR] 0.57 [95% CI 0.41-0.80], p = 0.0009), and disease-free survival (DFS) (HR 0.53 [95% CI 0.40-0.72], p < 0.0001) in multivariable estimations for up to 5-year post-diagnosis. Alcohol consumption was strongly associated with higher risk of recurrence and death (OS: HR 2.02 [95% CI 1.52-2.68], p < 0.0001; DFS: HR 1.70 [95% CI 1.31-2.21], p < 0.0001). No predictive associations were found for smoke or betel quid. Kaplan-Meier's OS and DFS were longer in HPV-positive cases (p < 0.0001), but this advantage decreased for alcohol users (p < 0.0001). Lastly, HPV-positive OPSCC had the best outcomes in non-drinkers, non-smokers, and non-betel quid chewers, but the prognostic benefit of HPV persisted in the presence of these risk factors. Conclusions Consistent with the increasing role of HPV on OPSCC globally, HPV is an important etiological factor in more than one-fourth of OPSCC cases from Chang Gung Hospital. Like in Western countries, HPV provides considerable independent survival benefits to OPSCC, but the added prognostic value is reduced by exposure to the risk factors, alcohol, smoking, and betel quid. There is a need to consider HPV as an etiologic factor in treatment and in cancer-prevention policies in Taiwan.

ObjectivesThe Michigan HPV and Oropharyngeal Cancer study aimed to evaluate patterns of oral and cervicogenital human papillomavirus (HPV) infection prevalence, incidence, and clearance as well as their relationship to sexual behaviours.DesignCohortSettingGeneral public in and around Ann Arbor, Michigan.Participants394 college-age and older-adult participants of both sexes provided oral samples, and 325 completed at least 2 visits. 130 who provided a cervicogenital samples, and 127 completed at least 2 visits.OutcomesIncidence and clearance rates as well as HRs for oral and cervicogenital HPV.ResultsOral HPV infections were transient, with only 16% of genotypes persisting to the next visit. The mean time to clearance of a genotype was 46 days (95% CI 37 to 58). In contrast, cervicogenital infections were more persistent, with 56% of genotypes persisting to the next visit. The mean time to clearance of a genotype was 87 days (95% CI 74 to 102). HPV vaccination was associated with reduced incidence of cervicogenital HPV infection (HR 0.63; 95% CI 0.47 to 0.83) but not oral HPV infection. Incidence of oral HPV infection was associated with 2+ recent deep kissing partners (HR 2.00; 95% CI 1.13 to 3.56). Incidence of both oral (HR: 1.70; 95% CI 1.08 to 2.68) and cervicogenital (HR 2.46; 95% CI 1.69 to 3.59) was associated with 2+ recent sexual partners.ConclusionsDetection of oral HPV was highly transient, but incidence was associated with recent deep kissing and sexual partners. Detection of cervicogenital HPV was more persistent, and incidence was positively associated with recent sexual partners and negatively associated with HPV vaccination.