Introduction The incidence of oropharyngeal squamous cell carcinoma (OPSCC) has been increasing worldwide. High-risk human papillomavirus (HPV) infection is now a well-recognised risk factor for oropharyngeal cancers. However, the information regarding the prevalence and outcome of HPV-related OPSCC is sparse in India. The study was conducted to identify the frequency of HPV infection in oropharyngeal cancer and also to study the treatment response and survival according to HPV positivity and p16INK4A expression. Materials and methods The study sample consists of 100 paraffin-embedded tissue blocks of histologically proven OPSCC patients who had undergone treatment at a tertiary cancer centre in Kerala, India, from January 2010 to December 2012. The patients' medical records were examined to obtain demographic data, information on habits, and clinical, histopathological, and treatment information. Follow-up information on disease status and vital status was collected until May 2023. Paraffin-embedded tissue blocks of these patients were collected from the archives of the Division of Pathology. Immunohistochemistry (IHC) was used to identify p16 expression. HPV DNA was isolated from the paraffin-embedded tissue blocks by polymerase chain reaction. Statistical analysis and results Survival curves were obtained using the Kaplan-Meier method and compared with the log-rank test. The influence of p16 status and HPV DNA positivity on survival and recurrence was assessed using Cox regression. A total of 100 patients diagnosed with oropharyngeal malignancy and their paraffin-embedded blocks were used for the present study. p16 IHC was invalid for three patients, and 16 patients had invalid HPV DNA. Two patients were excluded from survival analysis because they had both invalid HPV DNA and p16 expression. A total of 98 patients were included in the analysis. Out of 98 samples assessed, 47 tested positive for p16 expression, 48 were negative, and three showed invalid results. Among the 98 patients, HPV DNA results were available for 82 patients. HPV DNA positivity was reported in 25 patients, and 57 samples were HPV negative. There was no significant correlation between p16 expression and HPV status. The median follow-up was 134 months (1-160 months). The five-year overall survival (OS) probability was 42.6% (95% confidence interval (CI) 28.49-56.71) and 51.2% (95% CI 35.92-66.48), respectively, for p16-negative and p16-positive tumours (p=0.689). The corresponding figures for five-year disease-free survival (DFS) were 49.0% (95% CI 34.7-63.3) and 51.9% (95% CI 36.62-67.18), p=0.959. The five-year OS for HPV DNA-negative tumours was 45.5% (95% CI 32-59.02) compared to 49.1% (95% CI 28.72-69.48) in HPV DNA-positive tumours. There was an absolute difference of 20% in five-year OS between double-positive and double-negative tumours. Conclusion This study demonstrated a p16 positivity rate of 49.47% and an HPV DNA positivity rate of 30.37%. However, only 15.18% of cases showed double positivity. No significant correlation was observed between p16 expression and HPV status. Double positivity (p16 and HPV positive) was associated with better OS and DFS compared to double-negative (p16 and HPV negative) and single-positive (either p16 positive or HPV positive) cases. This subgroup of patients might benefit from potential de-escalation strategies and should be the target population for future studies.

BACKGROUND:Oral cancer is a major health problem in India. Recently an increase in the oral cancer incidence among young individuals without any habits was observed, which may be due to Human Papilloma Virus (HPV). However, information on the prevalence of HPV in oral cancer in India is sparse. This study aims to document the frequency of HPV16 infection in oral squamous cell carcinoma (SCC) and to study its correlation with treatment outcome. METHODS:Paraffin-embedded tissue blocks of 183 patients with SCC of the oral cavity were studied. HPV16 prevalence was detected by a polymerase chain reaction and expression of p16INK4a was analyzed by immunohistochemistry. This was correlated with clinical outcomes. RESULTS:The prevalence of HPV 16 was 6.6% and all were carcinoma tongue. All HPV-positive patients showed intense expression of p16INK4a. The five-year overall survival (OS) and disease-free survival (DFS) for HPV positive patients were higher compared to HPV negative patients (100% versus 74.5% and 100% versus 70.8% respectively). The five-year OS and DFS for patients with p16INK4a intense expression were also higher compared to p16INK4a weak expression (91.2% versus 57.4% and 82% versus 22.5% respectively). Five-year OS and DFS of patients with HPV16 positivity and p16INK4a intense expression were significantly higher than patients with HPV negative and p16INK4a weak expression (100% versus 54.7% and 100% versus 22.5% respectively). CONCLUSION:The frequency of HPV16 positivity in oral cancer was low in this study with a site predilection to the oral tongue. p16INK4a expression did not correlate with HPV status. A better treatment outcome was observed in HPV-positive cancers. We also observed better treatment outcomes in patients with p16INK4A overexpression irrespective of HPV status.

BACKGROUND:While recommending a human papillomavirus (HPV) single-dose vaccination schedule in 2022, the World Health Organization highlighted the need for long-term follow-up studies to monitor waning of protection. We report on vaccine efficacy against HPV infections in 1-, 2-, and 3-dose schedules and protection against cervical precancers at a median follow-up of 12 years postvaccination. METHODS:This randomized multicenter study in India was originally designed to vaccinate unmarried girls aged 10-18 years with either 2 or 3 doses of quadrivalent HPV vaccine. A ministerial decree to halt vaccination in trials resulted in the creation of cohorts receiving different doses, including just a single dose. Cohorts were assessed for incident and persistent infections by genotyping cervical samples collected yearly for 4 consecutive years after participants were married. Cervical screening with an HPV test was initiated at age 25 years for married participants. Age- and site-matched unvaccinated married women were recruited to be compared with vaccinated cohorts. Vaccine efficacy was assessed using proportional incidence ratios. RESULTS:The number of participants in the 1-, 2- (at 0 and 6 months), and 3-dose cohorts was 4949, 4980, and 4348, respectively. Of the recipients, 71%-82% in the different cohorts were eligible to provide samples for genotyping. Vaccine efficacy against persistent HPV 16 and 18 infection was 92.0% (95% confidence interval [CI] = 87.0% to 95.0%) in 3022 recipients of the single dose; and comparable with that observed in the 2-dose arm (94.8%, 95% CI = 90.0% to 97.3%) and the 3-dose arm (95.3%, 95% CI = 90.9% to 97.5%). No high-grade precancer associated with HPV 16 and 18 was detected among vaccinated participants compared with 8 precancers detected among the unvaccinated women. CONCLUSION:This observational cohort study has established that a single dose of HPV vaccine provides high protective efficacy against persistent HPV 16 and 18 infections and associated neoplasia 15 years postvaccination.

Background: The recent World Health Organization recommendation supporting single-dose of HPV vaccine will significantly reduce programmatic cost, mitigate the supply shortage, and simplify logistics, thus allowing more low- and middle-income countries to introduce the vaccine. From a programmatic perspective the durability of protection offered by a single-dose will be a key consideration. The primary objectives of the present study were to determine whether recipients of a single-dose of quadrivalent HPV vaccine had sustained immune response against targeted HPV types (HPV 6,11,16,18) at 10 years post-vaccination and whether this response was superior to the natural antibody titres observed in unvaccinated women.Methods: Participants received at age 10-18 years either one, two or three doses of the quadrivalent HPV vaccine. Serology samples were obtained at different timepoints up to 10 years after vaccination from a convenience sample of vaccinated participants and from age-matched unvaccinated women at one timepoint. The evolution of the binding and neutralizing antibody response was presented by dose received. 10-year durability of immune responses induced by a single-dose was compared to that after three doses of the vaccine and in unvaccinated married women.Results: The dynamics of antibody response among the single-dose recipients observed over 120 months show stabilized levels 18 months after vaccination for all four HPV types. Although the HPV type-specific (binding or neutralizing) antibody titres after a single-dose were significantly inferior to those after three doses of the vaccine (lower bounds of GMT ratios < 0.5), they were all significantly higher than those observed in unvaccinated women following natural infections (GMT ratios: 2.05 to 4.04-fold higher). The results correlate well with the high vaccine efficacy of single-dose against persistent HPV 16/18 infections reported by us earlier at 10-years post-vaccination.Conclusion: Our study demonstrates the high and durable immune response in single-dose recipients of HPV vaccine at 10-years post vaccination.(c) 2022 The Author(s). Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

Long-term follow-up of a cohort of unmarried girls who received one, two, or three doses of quadrivalent HPV vaccine, between 10 and 18 years of age, in an Indian multi-centric study allowed us to compare antibody responses between the younger and older age cohorts at 10-years post-vaccination, and study the impact of initiation of sexual activity and cervical HPV infections on antibody levels. Among the younger (10-14 years) recipients of a single dose, 97.7% and 98.2% had detectable binding antibody titers against HPV 16 and HPV 18 respectively at ten years post-vaccination. The proportions among those receiving a single dose at age 15-18 years were 92.3% and 94.2% against HPV 16 and HPV 18 respectively. Mean HPV 16 binding antibody titers were 2.1 folds (95%CI 1.4 to 3.3) higher in those vaccinated at ages 10-14 years, and 1.9 folds (95%CI 1.2 to 3.0) higher in those vaccinated at 15-18 years compared to mean titers seen in the unvaccinated women. Compared to previous timepoints of 36 or 48 months, binding antibodies against HPV 16 and neutralizing antibodies against both HPV 16 and HPV 18 were significantly higher at 10 years. This rise was more pronounced in participants vaccinated at 15-18 years. No association of marital status or cervical HPV infections was observed with the rise in titer. Durability of antibody response in single dose recipients correlated well with the high efficacy of a single dose against persistent HPV 16/18 infections irrespective of age at vaccination, as we reported earlier.

PDF file - 52KB, Comparison of HPV16 DNA positivity by sex, smoking status and lung histology.

Abstract The clinical presentation of patients with Takayasu arteritis (TAK), a large vessel vasculitis, is heterogenous and absence on an ideal biomarker renders assessment of disease activity difficult. In the present study we determined the biomarker profile of patients with TAK and subclassified them based on the same. Methods: Serum of consecutive patients with TAK and 36 controls were subjected to bead-based-multiplex assay or ELISA to quantify cytokines or chemokines or growth factors representing various pathophysiological pathways in TAK. The concentration of analytes between patients and controls and among various subsets of patients were compared. Unbiased clustering of patients was performed by dimensionality reduction methods and correlation networks were constructed. The surgical arterial biopsies of a subset of patients were examined for the extent of inflammation, fibrosis and myxomatous changes in arterial wall and the changes were correlated with cytokine concentration. Results: 85 patients with TAK [66 females, mean age: 28.8 ± 8.9 years, symptom duration of 24 months (IQR: 8–48 months)] and 36 controls were recruited. Levels of B cell antigen (BCA-1) and Pentraxin-3 were higher in patients than controls after applying correction for multiple testing (p < 0.02 for both). IL18 levels were highest in type 5 subtype (p = 0.015) while angiopoietin-2 and BCA-1 levels were highest in type 4 disease (p = 0.015, 0.003 and 0.009 respectively). The level of angiopoietin-2 was higher in patients with active disease (n = 52) than stable disease at baseline (p = 0.007) while IL6 and MMP-2 were paradoxically lower in patients with active disease (p = 0.029 and 0.021 respectively). Serum IL18 and IL2Rα levels trended to correlate positively with intensity of inflammation in the studied samples of aortic tissues. Among treatment naïve patients, those with lower concentration serum IL15, IFNγ and IL12p70 separated into one cluster. The angiogenic factors, MMP-9 and pentraxin-3 demonstrated interconnectedness in patients with active disease and not in those with stable disease. Conclusions: Patients with TAK have varied biomarker profile that depends on angiographic type and intensity of inflammation. While BCA-1 and Pentraxin levels were higher in TAK as compared with controls, angiopoietin-2, Il-6 and MMP-2 levels differentiated active from stable TAK. The biomarkers interconnected among each other differently in active and stable patients.

Background: Human papillomavirus (HPV) is one of the most common sexually transmitted infections worldwide. Although the efficacy of the HPV vaccine in preventing the development of cervical pre-malignant lesions has been well demonstrated, the efficacy of the HPV vaccine in preventing HPV infection in the upper respiratory tract has been poorly studied. Methods: In the context of the IARC cohort study of two versus three doses of HPV vaccine in India, we compared the HPV type prevalence in the oral cavity of women vaccinated with three doses, two doses, or a single dose of quadrivalent HPV vaccine with that of unvaccinated women. A total of 997 oral samples, from 818 vaccinated women and 179 unvaccinated women, were collected at three study sites. All the participants were sexually active at the time of sample collection. Results: The age-standardized proportion (ASP) of HPV16/18 infections was 2.0 % (95 % CI, 1.0-3.0 %) in vaccinated women and 4.2 % (95 % CI, 1.2-7.2 %) in unvaccinated women. HPV16 was detected in 3.5 % of single-dose recipients, 1.2 % of two-dose recipients (days 1 and 180), and 1.5 % of three-dose recipients (days 1, 60, and 180), whereas 3.3 % of the unvaccinated women tested positive for HPV16. The same trend was observed for HPV18. Discussion: Our findings agree with those of previous studies on the efficacy of HPV vaccination in reducing oral HPV infections and provide indications that a single vaccine dose may be less efficient than two or three doses in preventing oral HPV infection.

PDF file - 67KB, Antibodies to type-specific HPV proteins and the risk of lung cancer.

PDF file - 55KB, Association between type-specific HPV antibodies and risk of lung cancer by seropositivity threshold.

Supplemental Table 1. Study-specific definitions for categorization of tobacco and alcohol use.

Background The incidence of high-risk human papillomavirus (hrHPV)-driven head and neck squamous cell carcinoma, in particular oropharyngeal cancers (OPC), is increasing in high-resource countries. Patients with HPV-induced cancer respond better to treatment and consequently have lower case-fatality rates than patients with HPV-unrelated OPC. These considerations highlight the importance of reliable and accurate markers to diagnose truly HPV-induced OPC. Methods The accuracy of three possible test strategies, i.e. (a) hrHPV DNA PCR (DNA), (b) p16 (INK4a) immunohistochemistry (IHC) (p16), and (c) the combination of both tests (considering joint DNA and p16 positivity as positivity criterion), was analysed in tissue samples from 99 Belgian OPC patients enrolled in the HPV-AHEAD study. Presence of HPV E6*I mRNA (mRNA) was considered as the reference, indicating HPV etiology. Results Ninety-nine OPC patients were included, for which the positivity rates were 36.4%, 34.0% and 28.9% for DNA, p16 and mRNA, respectively. Ninety-five OPC patients had valid test results for all three tests (DNA, p16 and mRNA). Using mRNA status as the reference, DNA testing showed 100% (28/28) sensitivity, and 92.5% (62/67) specificity for the detection of HPV-driven cancer. p16 was 96.4% (27/28) sensitive and equally specific (92.5%; 62/67). The sensitivity and specificity of combined p16 + DNA testing was 96.4% (27/28) and 97.0% (65/67), respectively. In this series, p16 alone and combined p16 + DNA missed 1 in 28 HPV driven cancers, but p16 alone misclassified 5 in 67 non-HPV driven as positive, whereas combined testing would misclassify only 2 in 67. Conclusions Single hrHPV DNA PCR and p16 (INK4a) IHC are highly sensitive but less specific than using combined testing to diagnose HPV-driven OPC patients. Disease prognostication can be encouraged based on this combined test result.

IARC Perspective on Oral Cancer PreventionAn expert panel review of strategies for preventing oral cancer report that the use of tobacco (both smoking and smokeless), areca nut exposure, and heavy alcohol consumption are major contributors to this illness.

Background A randomised trial designed to compare three and two doses of quadrivalent human papillomavirus (HPV) vaccine in adolescent girls in India was converted to a cohort study after suspension of HPV vaccination in trials by the Indian Government. In this Article, the revised aim of the cohort study was to compare vaccine efficacy of single dose to that of three and two doses in protecting against persistent HPV 16 and 18 infection at 10 years post vaccination. Methods In the randomised trial, unmarried girls aged 10-18 years were recruited from nine centres across India and randomly assigned to either two doses or three doses of the quadrivalent HPV vaccine (Gardasil [Merck Sharp & Dohme, Whitehouse Station, NJ, USA]; 0middot5 mL administered intramuscularly). After suspension of recruitment and vaccination, the study became a longitudinal, prospective cohort study by default, and participants were allocated to four cohorts on the basis of the number vaccine doses received per protocol: the two-dose cohort (received vaccine on days 1 and 180 or later), three-dose cohort (days 1, 60, and 180 or later), two-dose default cohort (days 1 and 60 or later), and the single-dose default cohort. Participants were followed up yearly. Cervical specimens were collected from participants 18 months after marriage or 6 months after first childbirth, whichever was earlier, to assess incident and persistent HPV infections. Married participants were screened for cervical cancer as they reached 25 years of age. Unvaccinated women age-matched to the married vaccinated participants were recruited to serve as controls. Vaccine efficacy against persistent HPV 16 and 18 infections (the primary endpoint) was analysed for single-dose recipients and compared with that in two-dose and three-dose recipients after adjusting for imbalance in the distribution of potential confounders between the unvaccinated and vaccinated cohorts. This trial is registered with ISRCTN, ISRCTN98283094, and ClinicalTrials.gov, NCT00923702. Findings Vaccinated participants were recruited between Sept 1, 2009, and April 8, 2010 (date of vaccination suspension), and followed up over a median duration of 9middot0 years (IQR 8middot2-9middot6). 4348 participants had three doses, 4980 had two doses (0 and 6 months), and 4949 had a single dose. Vaccine efficacy against persistent HPV 16 and 18 infection among participants evaluable for the endpoint was 95middot4% (95% CI 85middot0-99middot9) in the single-dose default cohort (2135 women assessed), 93middot1% (77middot3-99middot8) in the two-dose cohort (1452 women assessed), and 93middot3% (77middot5-99middot7) in three-dose recipients (1460 women assessed). Interpretation A single dose of HPV vaccine provides similar protection against persistent infection from HPV 16 and 18, the genotypes responsible for nearly 70% of cervical cancers, to that provided by two or three doses. Funding Bill & Melinda Gates Foundation. Copyright (c) 2021 World Health Organization; licensee Elsevier. This is an Open Access article published under the CC BY 3.0 IGO license which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. In any use of this article, there should be no suggestion that WHO endorses any specific organisation, products or services. The use of the WHO logo is not permitted. This notice should be preserved along with the article's original URL.

Background: The main risk factors for head and neck cancer (HNC) are tobacco and alcohol use. However, an important fraction of oropharyngeal cancer (OPC) is caused by human papillomaviruses (HPV), a subgroup with increasing incidence in several western countries. Methods: As part of the HPV-AHEAD study, we assessed the role of HPV infection in 772 archived tissue specimens of Belgian HNC patients: 455 laryngeal (LC), 106 oral cavity (OCC), 99 OPC, 76 hypopharyngeal (HC), and 36 unspecified parts of the head and neck. All specimens were tested for HPV DNA (21 genotypes); whereof all HPV DNA-positives, all HPV DNA-negative OPCs and a random subset of HPV DNA-negatives of the other HNC sites were tested for the presence of type-specific HPV RNA and p16(INK4a) over-expression. Results: The highest HPV DNA prevalence was observed in OPC (36.4 %), and was significantly lower (p < 0.001) in the other HNCs (OCC:7.5 %, LC:6.6 %). HPV16 was the most common HPV-genotype in all HNCs. Approximately 83.0 % of the HPV DNA-positive OPCs tested HPV RNA or p16-positive, compared to about 37.5 % and 44.0 % in OCC and LC, respectively. Estimation of the attributable fraction of an HPV infection in HNC was very similar for HPV RNA or p16 in addition to DNA-positivity; with 30 % for OPC, and 3 % for OCC and LC. Conclusion: Our study confirms the heterogeneity of HPV DNA prevalence across anatomical sites in HNC, with a predominance of HPV16 in all sites. The estimated proportion of HPV-driven HNC in Belgium, during the period 1980-2014, was 10 times higher in OPC compared to OCC and LC.

In context of the ongoing multi-centric HPV vaccine study in India, unvaccinated married women (N = 1484) aged 18-23 years were recruited in 2012-2015 as age-matched controls to the vaccinated women and followed up yearly. We assess type-specific prevalence, natural history and potential determinants of human papillomavirus (HPV) infection in these unvaccinated women. Cervical samples were collected yearly for at least four consecutive years. A Multiplex Type-Specific E7-Based polymerase chain reaction assay was used to detect 21 HPV types. HPV prevalence was 36.4% during 6 years. Most common HPV types were 16 (6.5%) and 31 (6.1%). Highest persistence were observed for HPV 35 (62.5%) and 52 (25%). New HPV acquisition rate was 5.6/1000 person-months of observation (PMO), highest for HPV 16 (1.1/1000 PMO). Type-specific clearance rates ranged between 2.9-5.5/100 PMO. HPV 16 and/or 18 infections were 41% (95% CI 4-63%) lower among women with 2-<3 years between marriage and first cervical sample collection compared to those with <2 years. HPV prevalence and acquisition rates in young Indian women were lower than their Western counterparts. HPV 16 infections being most common shows the importance and potential impact of HPV vaccination in India. Women with 2-3 years exposure had reduced risk possibly due to higher infections clearance.

Objectives: To WA the performance of an oral cancer prognostic 13-gene signature for the prediction of survival of patients diagnosed with HPV-negative and p16-negative oral cavity cancer. Materials and Methods: Diagnostic formalin-fixed paraffin-embedded oral cavity cancer tumor samples were obtained from the Fred Hutchinson Cancer Research Center/University of Washington, University of Calgary, University of Michigan, University of Utah, and seven ARCAGE study centers coordinated by the International Agency of Research on Cancer. RNA from 638 Human Papillomavirus (HPV)-negative and p16-negative samples was analyzed for the 13 genes using a NanoString assay. Ridge-penalized Cox regressions were applied to samples randomly split into discovery and validation sets to build models and evaluate the performance of the 13-gene signature in predicting 2-year oral cavity cancer-specific survival overall and separately for patients with early and late stage disease. Results: Among AJCC stage I/II patients, including the 13-gene signature in the model resulted in substantial improvement in the prediction of 2-year oral cavity cancer-specific survival. For models containing age and sex with and without the 13-gene signature score, the areas under the Receiver Operating Characteristic Curve (AUC) and partial AUC were 0.700 vs. 0.537 (p < 0.001), and 0.046 vs. 0.018 (p < 0.001), respectively. Improvement in predicting prognosis for AJCC stage III/IV disease also was observed, but to a lesser extent. Conclusions: If confirmed using tumor samples from a larger number of early stage oral cavity cancer patients, the 13-gene signature may inform personalized treatment of early stage HPV-negative and p16-negative oral cavity cancer patients.

OBJECTIVES:The 8th edition AJCC staging guidelines for head and neck squamous cell carcinoma (HNSCC) recently introduced pathologic staging criteria for nodal disease among p16-positive patients. In this study we evaluate pathologic staging in p16-negative HNSCC. MATERIALS AND METHODS:We compared pathologic staging to the 7th and 8th edition AJCC staging systems using a statewide population-based cohort. All M0 p16-negative surgical patients were included. The outcome was five-year overall survival. RESULTS:Of 304 patients identified, 113 were N0, 157 had 1-4 positive nodes, and 34 had ≥4 nodes. Survival was 71% (95% CI 61-78%) with no nodes, 48% (36%-60%) for 1-4 nodes, and 24% (11 - 39%) for > 4 nodes. When compared to the AJCC systems, the pathologic staging yielded a larger total survival gradient, more montonic survival, better consistency across primary sites, and a slightly lower Bayesian information criterion (1510 vs 1538). After adjusting for disease characteristics, demographics, and tobacco use, hazard ratios for survival were similar using pathologic and AJCC criteria. CONCLUSION:In this cohort, pathological staging was more prognostic than AJCC staging. This is the first study to evaluate pathologic staging in p16-negative cancer; if these findings are verified, a universal nodal staging system could be introduced.

Cancer of the oropharynx has attracted considerable attention in recent years given: (1) an increasing incidence in selected populations over the past three decades; (2) the discovery of human papillomavirus (HPV) infection as the driver of the increase, as opposed to the traditional risk factors such as tobacco (smoking and chewing) and alcohol; and (3) the promise of new prevention and treatment strategies. As a result of such developments, the International Agency for Research on Cancer (IARC) and the US National Cancer Institute (NCI), convened the fourth Cancer Seminar meeting in November 2018 to focus on this topic. This report summarizes the proceedings: a review of recent science on the descriptive epidemiology, etiology, biology, genetics, early detection, pathology and treatment of HPV-positive oropharyngeal cancer, and the formulation of key research questions to be addressed.

Literature on the role of human papillomavirus (HPV) in head and neck cancer (HNC) in Italy is limited, especially for non-oropharyngeal tumours. Within the context of the HPV-AHEAD study, we aimed to assess the prognostic value of different tests or test algorithms judging HPV carcinogenicity in HNC and factors related to HPV positivity at the European Institute of Oncology. We conducted a retrospective cohort study (2000–2010) on a total of 696 primary HNC patients. Formalin-fixed, paraffin-embedded cancer tissues were studied. All HPV-DNA-positive and a random sample of HPV-DNA-negative cases were subjected to HPV-E6*I mRNA detection and p16INK4a staining. Multivariate models were used to assess for factors associated with HPV positivity and proportional hazards for survival and recurrence. The percentage of HPV-driven cases (considering HPV-E6*I mRNA positivity) was 1.8, 2.2, and 40.4% for oral cavity (OC), laryngeal (LC), and oropharyngeal (OPC) cases, respectively. The estimates were similar for HPV-DNA/p16INK4a double positivity. Being a non-smoker or former smoker or diagnosed at more recent calendar periods were associated with HPV-E6*I mRNA positivity only in OPC. Being younger was associated with HPV-E6*I mRNA positivity in LC. HPV-driven OPC, but not HPV-driven OC and LC, showed better 5 year overall and disease-free survival. Our data show that HPV prevalence in OPC was much higher than in OC and LC and observed to increase in most recent years. Moreover, HPV positivity conferred better prognosis only in OPC. Novel insights on the role of HPV in HNC in Italy are provided, with possible implications in the clinical management of these patients.