Prognostic implications of p16 and HPV discordance in oropharyngeal cancer (HNCIG-EPIC-OPC): a multicentre, multinational, individual patient data analysis.

BACKGROUND: p16INK4a (p16) immunohistochemistry is the most widely used biomarker assay for inferring HPV causation in oropharyngeal cancer in clinical and trial settings. However, discordance exists between p16 and HPV DNA or RNA status in some patients with oropharyngeal cancer. We aimed to clearly quantify the extent of discordance, and its prognostic implications. METHODS: In this multicentre, multinational individual patient data analysis, we did a literature search in PubMed and Cochrane database for systematic reviews and original studies published in English between Jan 1, 1970, and Sept 30, 2022. We included retrospective series and prospective cohorts of consecutively recruited patients previously analysed in individual studies with minimum cohort size of 100 patients with primary squamous cell carcinoma of the oropharynx. Patient inclusion criteria were diagnosis with a primary squamous cell carcinoma of oropharyngeal cancer; data on p16 immunohistochemistry and on HPV testing; information on age, sex, tobacco, and alcohol use; staging by TNM 7th edition; information on treatments received; and data on clinical outcomes and follow-up (date of last follow-up if alive, date of recurrence or metastasis, and date and cause of death). There were no limits on age or performance status. The primary outcomes were the proportion of patients of the overall cohort who showed the different p16 and HPV result combinations, as well as 5-year overall survival and 5-year disease-free survival. Patients with recurrent or metastatic disease or who were treated palliatively were excluded from overall survival and disease-free survival analyses. Multivariable analysis models were used to calculate adjusted hazard ratios (aHR) for different p16 and HPV testing methods for overall survival, adjusted for prespecified confounding factors. FINDINGS: Our search returned 13 eligible studies that provided individual data for 13 cohorts of patients with oropharyngeal cancer from the UK, Canada, Denmark, Sweden, France, Germany, the Netherlands, Switzerland, and Spain. 7895 patients with oropharyngeal cancer were assessed for eligibility. 241 were excluded before analysis, and 7654 were eligible for p16 and HPV analysis. 5714 (74·7%) of 7654 patients were male and 1940 (25·3%) were female. Ethnicity data were not reported. 3805 patients were p16-positive, 415 (10·9%) of whom were HPV-negative. This proportion differed significantly by geographical region and was highest in the areas with lowest HPV-attributable fractions (r=-0·744, p=0·0035). The proportion of patients with p16+/HPV- oropharyngeal cancer was highest in subsites outside the tonsil and base of tongue (29·7% vs 9·0%, p<0·0001). 5-year overall survival was 81·1% (95% CI 79·5-82·7) for p16+/HPV+, 40·4% (38·6-42·4) for p16-/HPV-, 53·2% (46·6-60·8) for p16-/HPV+, and 54·7% (49·2-60·9) for p16+/HPV-. 5-year disease-free survival was 84·3% (95% CI 82·9-85·7) for p16+/HPV+, 60·8% (58·8-62·9) for p16-/HPV-; 71·1% (64·7-78·2) for p16-/HPV+, and 67·9% (62·5-73·7) for p16+/HPV-. Results were similar across all European sub-regions, but there were insufficient numbers of discordant patients from North America to draw conclusions in this cohort. INTERPRETATION: Patients with discordant oropharyngeal cancer (p16-/HPV+ or p16+/HPV-) had a significantly worse prognosis than patients with p16+/HPV+ oropharyngeal cancer, and a significantly better prognosis than patients with p16-/HPV- oropharyngeal cancer. Along with routine p16 immunohistochemistry, HPV testing should be mandated for clinical trials for all patients (or at least following a positive p16 test), and is recommended where HPV status might influence patient care, especially in areas with low HPV-attributable fractions. FUNDING: European Regional Development Fund, Generalitat de Catalunya, National Institute for Health Research (NIHR) UK, Cancer Research UK, Medical Research Council UK, and The Swedish Cancer Foundation and the Stockholm Cancer Society.

Might Oral Human Papillomavirus (HPV) Infection in Healthy Individuals Explain Differences in HPV-Attributable Fractions in Oropharyngeal Cancer? A Systematic Review and Meta-analysis.

BACKGROUND: Differences in oral human papillomavirus (HPV) prevalence and contrasts in HPV-attributable fractions (AFs) in oropharyngeal cancer (OPC) have not been evaluated in depth. METHODS: A systematic review was performed to identify studies in which at least 50 healthy individuals were tested for oral HPV infection. Information on sex, age, tobacco/alcohol consumption, sex practices, specimen collection, HPV detection, and population type was extracted. Prevalences were pooled using random-effects models for meta-analyses of binomial data. Correlations were assessed by the Spearman test. RESULTS: Forty-eight reports comprising 28 544 individuals fulfilled inclusion criteria. Global oral HPV prevalence was 4.9%. Estimates were highest in Europe, although regional differences were not statistically significant. HPV16 prevalence was 1.0% globally, and regional differences became statistically significant. A lifetime history of >6 sex partners showed a higher risk of oral HPV infection. The age-specific HPV distribution revealed a prevalence of ≥5% over 40 years of age and a lower prevalence at younger ages. There was no association between oral HPV prevalence and HPV-AFs or age-standardized rates (ASRs) of OPC, genital HPV in healthy women, or tobacco use. CONCLUSIONS: Differences in HPV-AFs or ASRs of OPC cannot be explained by differences in the prevalence of oral HPV infection across healthy populations. Consistent research on determinants of oral HPV prevalence, acquisition, clearance, and persistence is warranted.

Epidemiology of HPV-Positive Tumors in Europe and in the World.

Strong evidence has accumulated in the last 15 years showing that infection by certain human papillomaviruses (HPVs) is etiologically involved in a subset of head and neck cancers (HNCs). In this chapter, epidemiologic-related topics on HNCs are reviewed: (i) HPV-attributable fractions and HPV-type distributions by different anatomical HNC sites, using not only HPV DNA but other more specific markers of causality; (ii) an update of the HPV-related HNCs burden worldwide and by regions; and finally, (iii) the determinants for HPV positivity in HNCs, focussing on gender, age, smoking habits, sexual behavior, and other related factors such as tonsillectomy performance. This information is essential in order to understand the burden of the disease and its dynamics and changing patterns, as well as for planning and assessment of the potential impact of HPV-based preventive strategies for HNCs.

HPV DNA, E6/E7 mRNA, and p16INK4a detection in head and neck cancers: a systematic review and meta-analysis.

BACKGROUND: We aimed to provide updated information about the global estimates of attributable fraction and type distribution of human papillomavirus (HPV) in head and neck squamous cell carcinomas by doing a systematic review and meta-analysis. METHODS: We did a literature search on PubMed to identify studies that used PCR for detection of HPV DNA in head and neck squamous cell carcinomas with information about HPV genotype distribution. We included studies that tested 20 or more biopsies per cancer site and were published between July 15, 1990, and Feb 29, 2012. We collected information about sex, risk factors, HPV detection methods, and biomarkers of potentially HPV-induced carcinogenesis (E6/E7 mRNA and p16(INK4a)). If it was not possible to abstract the required information directly from the paper, we contacted the authors. We did a meta-analysis to produce pooled prevalence estimates including a meta-regression to explore sources of heterogeneity. FINDINGS: 148 studies were included, contributing data for 12 163 cases of head and neck squamous cell carcinoma from 44 countries. HPV DNA was detected in 3837 cases. HPV16 accounted for 82·2% (95% CI 77·7-86·4) of all HPV DNA positive cases. By cancer site, pooled HPV DNA prevalence estimates were 45·8% (95% CI 38·9-52·9) for oropharynx, 22·1% (16·4-28·3) for larynx (including hypopharynx), and 24·2% (18·7-30·2) for oral cavity. The percent positivity of p16(INK4a) positive cases in HPV-positive oropharyngeal cancer cases was 86·7% (95% CI 79·2-92·9) and of E6/E7 mRNA positive cases was 86·9% (73·2-96·8). The estimate of HPV attributable fraction in oropharyngeal cancer defined by expression of positive cases of E6/E7 mRNA was 39·8% and of p16(INK4a) was 39·7%. Of subsites, tonsils (53·9%, 95% CI 46·4-61·3) had the highest HPV DNA prevalence. HPV DNA prevalence varied significantly by anatomical site, geographic region, but not by sex or tobacco or alcohol consumption. INTERPRETATION: The contribution of HPV prevalence in head and neck squamous cell carcinoma and in particular that of HPV16 in the oropharynx shows the potential benefit of prophylactic vaccines. FUNDING: European Commission.

Evaluation of the impact of a breast cancer awareness program in rural Ghana: a cross-sectional survey.

Community awareness is crucial to early detection of breast cancer in low- and middle-income countries. In Ghana 60% of the cases are detected at late stages. Breast Care International (BCI) is a Ghanaian non-governmental organization dedicated to raising breast cancer awareness. A cross-sectional survey was designed to assess the impact of BCI program on knowledge, attitudes and practices (KAP) toward breast cancer among women from rural communities of Ghana. A total of 232 women were interviewed in June 2011 in the Ashanti region; of these 131 participants were from a community that received the BCI program in August 2010 (intervention group) and 101 from another community that received the program post-survey (referent group). Data analysis was performed using Epi-Info version 3.5.3. Knowledge about breast cancer among participants who received the program was better than among those who did not. Only 53.5% of participants from the referent group knew that breast cancer usually appears as painless breast lump when compared to 82.3% from the intervention group. Participants who attended the program were significantly more likely to obtain higher knowledge scores (odds ratio (OR) = 2.10, 95% confidence interval (CI) = 1.14-3.86) and to state practicing breast self-examination (OR = 12.29, 95% CI = 5.31-28.48). The BCI program improved KAP toward breast cancer. Further research is warranted to provide stronger evidence that the program improves breast cancer early detection.

TNM 8 staging system beyond p16: Double HPV/p16 status is superior to p16 alone in predicting outcome in oropharyngeal squamous cell carcinoma.

PURPOSE: The assessment of p16INK4a (p16) in oropharyngeal squamous cell carcinoma (OPSCC) has been incorporated into tumor classification, as p16 has been shown to impact survival probability. However, a recent study demonstrated that human papillomavirus (HPV) status in addition to p16 may have a better discriminatory effect on survival probability. This study aims to determine the impact of combined evaluation of p16 and HPV on prognosis. METHODS: This was a multicenter, multinational analysis including retrospective and prospective cohorts of patients treated for primary OPSCC with curative intent, based on the data of the HNCIG-EPIC study. The primary outcome was to determine how the combined assessment of HPV and p16 status predicts prognosis of patients with OPSCC compared to p16 assessment alone. We employed multivariable analyses models to compute hazard ratios regarding survival. Analyses were stratified by stage, smoking status, and sub-anatomical region. RESULTS: The study included 7654 patients, with approximately half of the tumors being p16-negative (50.3 %, n = 3849). A total of 9.2 % of patients had discordant p16 and HPV status (n = 704). HPV status significantly impacted overall survival and disease-free survival regardless of p16 status and across both UICC 8th stage I-II and III-IVb cancers. p16-positive/HPV-positive OPSCC patients exhibited the best survival probability. CONCLUSION: The detection of HPV had a significant impact on survival probability for OPSCC patients with both p16-positive and p16-negative tumors. HPV testing should be integrated in cancer staging, especially in regions of low attributable fraction, alongside p16 evaluation to ensure a comprehensive assessment of prognosis.

The Isothermal Amplification AmpFire Assay for Human Papillomavirus (HPV) Detection and Genotyping in Formalin-Fixed, Paraffin-Embedded Oropharyngeal Cancer Samples.

Human papillomavirus (HPV)-related oropharyngeal squamous cell carcinomas (OPSCCs) represent a distinct clinical entity compared with HPV-negative tumors with particular regard to treatment response and survival outcome. The aim of this study was to assess the AmpFire Multiplex HR-HPV tests, for the detection and genotyping of 15 high-risk (HR) HPV types in formalin-fixed, paraffin-embedded (FFPE) samples and identify HPV-driven OPSCC. DNA from 160 OPSCC FFPE specimens plus 23 samples from other head and neck primary sites was tested. Results were compared with those obtained using Linear Array HPV-DNA Genotyping Test. Linear Array and AmpFire Multiplex HR-HPV tests showed, for all samples and specifically for OPSCCs, an overall concordance agreement of 98.9% and 99.4% and a Cohen κ coefficient of 0.972 and 0.984, respectively. A concordance of 100% for HPV16 and HPV18 was observed. The overall agreement between p16INK4a overexpression and HPV detection by the AmpFire Multiplex HR-HPV assay in 145 OPSCC samples was 93.8%, with a Cohen κ coefficient of 0.848. The AmpFire HPV Tests are simple assays for detection and genotyping of HPV-DNA in OPSCC FFPE samples and can be easily implemented in the clinical practice setting for HPV-DNA detection.

Concordance of p16INK4a and E6*I mRNA among HPV-DNA-Positive Oropharyngeal, Laryngeal, and Oral Cavity Carcinomas from the ICO International Study.

Background: Tests or test algorithms for diagnosing HPV-driven oral cavity and laryngeal head and neck carcinomas (HNC) have not been yet validated, and the differences among oral cavity and laryngeal sites have not been comprehensively evaluated. We aimed to assess the utility of a diagnostic algorithm for the detection of HPV-driven oral cavity (OCC), oropharyngeal (OPC) and laryngeal (LC) carcinomas using HPV-DNA testing followed by p16INK4a immunohistochemistry, taking E6*I mRNA detection as the reference standard. Methods: Formalin-fixed paraffin-embedded OCC, OPC, and LC carcinomas were collected from pathology archives in 29 countries. All samples were subjected to histopathological evaluation, DNA quality control, and HPV-DNA detection. All HPV-DNA-positive samples (including 78 OCC, 257 OPC, and 51 LC out of 3680 HNC with valid HPV-DNA results) were also tested for p16INK4a immunohistochemistry and E6*I mRNA. Three different cutoffs of nuclear and cytoplasmic staining were evaluated for p16INK4a: (a) >25%, (b) >50%, and (c) ≥70%. The concordance of p16INK4a and E6*I mRNA among HPV-DNA-positive OCC, OPC, and LC cases was assessed. Results: A total of 78 OCC, 257 OPC, and 51 LC were HPV-DNA-positive and further tested for p16INK4a and E6*I mRNA. The percentage of concordance between p16INK4a (cutoff ≥ 70%) and E6*I mRNA among HPV-DNA-positive OCC, OPC, and LC cases was 79.5% (95% CI 69.9−89.1%), 82.1% (95% CI 77.2−87.0%), and 56.9% (95% CI 42.3−71.4%), respectively. A p16INK4a cutoff of >50% improved the concordance although the improvement was not statistically significant. For most anatomical locations and p16INK4a cutoffs, the percentage of discordant cases was higher for HPV16- than HPV-non16-positive cases. Conclusions: The diagnostic algorithm of HPV-DNA testing followed by p16INK4a immunohistochemistry might be helpful in the diagnosis of HPV-driven OCC and OPC, but not LC. A different p16INK4a expression pattern was observed in those cases HPV-DNA-positive for types other than HPV16, as compared to HPV16-positive cases. Our study provides new insights into the use HPV-DNA, p16INK4a, and HPV-E6*I mRNA for diagnosing an HPV-driven HNC, including the optimal HPV test or p16INK4a cutoffs to be used. More studies are warranted to clarify the role of p16INK4a and HPV status in both OPC and non-OPC HNC.

Role of Human Papillomavirus Infection in Head and Neck Cancer in Italy: The HPV-AHEAD Study.

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.

Competing mortality in oropharyngeal carcinoma according to human papillomavirus status.

BACKGROUND: The objective of the present study is to assess differences in the competing causes of death in patients with oropharyngeal carcinoma (OPC) as a function of the human papillomavirus (HPV) status. METHODS: We studied retrospectively 423 patients with OPC with known HPV status. Among the patients included in the study, 53 (12.5%) were HPV-positive. We analyzed overall survival and competing causes of mortality according to the HPV status of the patients. RESULTS: Patients with HPV-negative tumors had lower OPC cancer-specific survival (P = .0001), second primary neoplasm survival (P = .0001), and noncancer-related causes survival (P = .13) than patients with HPV-positive tumors. This resulted in significant differences in overall survival depending on HPV status (P = .0001). CONCLUSION: Conclusion: HPV-positive OPC has a better overall survival than HPV-negative OPC. Patients with HPV-positive tumors presented a significant lower OPC cancer-specific and second primary neoplasm mortality and a marginally nonsignificant lower noncancer mortality as compared to HPV-negative tumors.

Human papillomavirus in premalignant oral lesions: No evidence of association in a Spanish cohort.

BACKGROUND: Human papillomavirus (HPV) is the cause of a fraction of head and neck squamous cell carcinoma. Although this relation is well-known, it is still not clear the role of HPV in premalignant oral lesions such as oral lichen planus (OLP) and dysplasia. We aimed to evaluate the HPV-DNA prevalence and type distribution in a set of oral biopsies obtained from patients diagnosed with OLP and dysplasia, as well as the role of HPV in these lesions. METHODS: A retrospective cohort of all premalignant oral lesions consecutively diagnosed from March 30th 1995 to May 21st 2014 at Hospital of Bellvitge and Odontological University Hospital of Bellvitge was identified and classified in four groups: OLP (groups 1 and 2) and dysplasias (groups 3 and 4) that progressed or not to invasive cancer during follow-up. A random selection targeting 25 cases was aimed to be performed for each group. All selected cases were subjected to pathological evaluation, DNA quality control and HPV-DNA detection. HPV-DNA positive samples were further subject to p16INK4a analysis. RESULTS: A total of 83 cases yielded a valid HPV-DNA result. From those, 7 and 34 cases were OLP that progressed or not to invasive cancer during follow-up, whereas 24 and 18 cases were displasias that progressed or not to invasive cancer during follow-up, respectively. HPV-DNA was detected in 4 samples (3 dysplastic lesions and 1 OLP). Two samples were HPV16 positive (2%), 1 sample HPV18 positive (1%) and 1 sample (1%) was HPV indeterminate. Two out of four HPV-DNA positive cases had high p16INK4a expression and none of the HPV positive cases progressed to invasive cancer during long-term follow-up. CONCLUSIONS: We found a low HPV-DNA attributable fraction in premalignant lesions of the oral cavity, suggesting that HPV is unlikely to play a significant role in oral carcinogenesis in our setting.

Identifying populations most susceptible to get benefit from broadening the scope for prevention of cervical cancer: Example from Uruguay.

OBJECTIVES: To identify factors associated with high-risk human papillomavirus (HPV) infection and high grade squamous intraepithelial lesion (HSIL) among a high-risk group of HPV-unvaccinated women in Montevideo. METHODS: Participants completed a questionnaire on socio-demographics, sexual behavior and gynecological history and received a gynecological examination. HPV DNA was detected by PCR using MY09/11 primers. Logistic regression analyses were performed to identify factors associated with high-risk HPV infection and HSIL. RESULTS: A total of 469 women with HPV DNA and cytological results completed the questionnaire. Among women older than 30 years, those with high number of sexual partners and regular housing conditions were more likely to be positive for high-risk HPV infection (adjusted OR: 2.94, 95%CI: 1.01-8.51 and 2.68, 95%CI: 1.01-7.21, respectively). A marginally non-statistically significant association between getting a HSIL and having a high number of sexual partners was also observed (adjusted OR: 3.22, 95%CI: 0.97-10.75). CONCLUSIONS: In an era of development of new strategies for accelerating the reduction of cervical cancer incidence and mortality, our results may contribute to identify populations most susceptible to get benefit from broadening the scope for prevention of cervical cancer and could be used with other triage strategies.

HPV-relatedness definitions for classifying HPV-related oropharyngeal cancer patient do impact on TNM classification and patients' survival.

BACKGROUND: Given the different nature and better outcomes of oropharyngeal carcinoma (OPC) associated with human papillomavirus (HPV) infection, a novel clinical stage classification for HPV-related OPC has been accepted for the 8th edition AJCC TNM (ICON-S model). However, it is still unclear the HPV-relatedness definition with best diagnostic accuracy and prognostic value. MATERIAL AND METHODS: The aim of this study was to compare different staging system models proposed for HPV-related OPC patients: 7th edition AJCC TNM, RPA stage with non-anatomic factors (Princess Margaret), RPA with N categories for nasopharyngeal cancer (MD-Anderson) and AHR-new (ICON-S), according to different HPV-relatedness definitions: HPV-DNA detection plus an additional positive marker (p16INK4a or HPV-mRNA), p16INK4a positivity alone or the combination of HPV-DNA/p16INK4a positivity as diagnostic tests. RESULTS: A total of 788 consecutive OPC cases diagnosed from 1991 to 2013 were considered eligible for the analysis. Of these samples, 66 (8.4%) were positive for HPV-DNA and (p16INK4a or HPV-mRNA), 83 (10.5%) were p16INK4a positive and 58 (7.4%) were double positive for HPV-DNA/p16INK4a. ICON-S model was the staging system, which performed better in our series when using at least two biomarkers to define HPV-causality. When the same analysis was performed considering only p16INK4a-positivity, RPA stage with non-anatomic factors (Princess Margaret) has the best classification based on AIC criteria. CONCLUSION: HPV-relatedness definition for classifying HPV-related OPC patient do impact on TNM classification and patients' survival. Further studies assessing HPV-relatedness definitions are warranted to better classify HPV-related OPC patients in the era of de-escalation clinical trials.

The Use of HPV16-E5, EGFR, and pEGFR as Prognostic Biomarkers for Oropharyngeal Cancer Patients.

Background: Anti-epidermal-growth-factor-receptor (EGFR) therapies in combination with radiotherapy are being studied on de-escalation clinical trials for HPV-related oropharyngeal cancer (OPC) patients. The HPV16-E5 oncoprotein increases recycling of activated EGFR to the cell surface, enhancing factor signal transduction. Our aim was to evaluate viral HPV16-E5 oncogene expression as well as EGFR and phosphorylated-EGFR (pEGFR), protein levels as biomarkers for clinical outcome in a retrospective cohort of OPC patients. Methods: Formalin-fixed-paraffin-embedded OPCs were collected from 1990 to 2013. OPC samples containing HPV-DNA were subject to viral E6 * I mRNA detection and p16INK4a immunohistochemistry (IHC). HPV16-positive cases were evaluated for HPV16-E5 (RT-PCR) and EGFR/pEGFR (IHC). A stratified and matched random sample of HPV-negative samples was used as control and evaluated for EGFR/pEGFR. Overall survival (OS) and disease free survival (DFS) estimates were assessed for locally advanced OPC patients (stage III, IVa,b 7th edition). Results: Among 788 OPC patient samples, 53 were double positive for HPV16-DNA/p16INK4a. HPV16-E5 expression was found in 41 of 53 samples (77.4%). EGFR expression was observed in 37.7 vs 70.8% of HPV16-positive vs HPV-negative samples, respectively; (adjusted OR = 0.15) 5% CI = 0.04-0.56]). Expression of pEGFR followed an inverse pattern with 39.6 and 24.9% detection in HPV16-positive and HPV-negative samples; (adjusted OR = 1.58 [95% CI = 0.48-5.17]). Within HPV16-positive cases, no association between HPV16-E5/EGFR nor pEGFR was observed. With a median follow-up of 39.36 months (min = 0.03 - max = 272.07), the combination of HPV status and EGFR or pEGFR expression were predictors of better OS (p < 0.001, for both) and DFS (p < 0.001 for EGFR and p = 0.003 for pEGFR). Conclusions: HPV16-E5 is highly expressed on HPV16-positive OPCs. Interestingly, HPV16-positive cases expressed significantly more pEGFR while HPV-negative cases expressed more EGFR. The combinations of HPV status and EGFR or pEGFR may be useful biomarkers for evaluating prognosis outcome in OPC patients.

Distinctive Expression and Amplification of Genes at 11q13 in Relation to HPV Status with Impact on Survival in Head and Neck Cancer Patients.

Clear differences have been established between head and neck squamous cell carcinomas (HNSCC) depending on human papillomavirus (HPV) infection status. This study specifically investigated the status of the CTTN, CCND1 and ANO1 genes mapping at the 11q13 amplicon in relation to the HPV status in HNSCC patients. CTTN, CCND1 and ANO1 protein expression and gene amplification were respectively analyzed by immunohistochemistry and real-time PCR in a homogeneous cohort of 392 surgically treated HNSCC patients. The results were further confirmed using an independent cohort of 279 HNSCC patients from The Cancer Genome Atlas (TCGA). The impact on patient survival was also evaluated. CTTN, CCND1 and ANO1 gene amplification and protein expression were frequent in HPV-negative tumors, while absent or rare in HPV-positive tumors. Using an independent validation cohort of 279 HNSCC patients, we consistently found that these three genes were frequently co-amplified (28%) and overexpressed (39⁻46%) in HPV-negative tumors, whereas almost absent in HPV-positive tumors. Remarkably, these alterations (in particular CTTN and ANO1 overexpression) were associated with poor prognosis. Taken together, the distinctive expression and amplification of these genes could cooperatively contribute to the differences in prognosis and clinical outcome between HPV-positive and HPV-negative tumors. These findings could serve as the basis to design more personalized therapeutic strategies for HNSCC patients.

Double positivity for HPV-DNA/p16ink4a is the biomarker with strongest diagnostic accuracy and prognostic value for human papillomavirus related oropharyngeal cancer patients.

BACKGROUND: The etiologic role of human papillomaviruses (HPV) in oropharyngeal cancer (OPC) is well established. Nevertheless, information on survival differences by anatomic sub-site or treatment remains scarce, and it is still unclear the HPV-relatedness definition with best diagnostic accuracy and prognostic value. METHODS: We conducted a retrospective cohort study of all patients diagnosed with a primary OPC in four Catalonian hospitals from 1990 to 2013. Formalin-fixed, paraffin-embedded cancer tissues were subjected to histopathological evaluation, DNA quality control, HPV-DNA detection, and p16INK4a/pRb/p53/Cyclin-D1 immunohistochemistry. HPV-DNA positive and a random sample of HPV-DNA negative cases were subjected to HPV-E6*I mRNA detection. Demographic, tobacco/alcohol use, clinical and follow-up data were collected. Multivariate models were used to evaluate factors associated with HPV positivity as defined by four different HPV-relatedness definitions. Proportional-hazards models were used to compare the risk of death and recurrence among HPV-related and non-related OPC. RESULTS: 788 patients yielded a valid HPV-DNA result. The percentage of positive cases was 10.9%, 10.2%, 8.5% and 7.4% for p16INK4a, HPV-DNA, HPV-DNA/HPV-E6*I mRNA, and HPV-DNA/p16INK4a, respectively. Being non-smoker or non-drinker was consistently associated across HPV-relatedness definitions with HPV positivity. A suggestion of survival differences between anatomic sub-sites and treatments was observed. Double positivity for HPV-DNA/p16INK4a showed strongest diagnostic accuracy and prognostic value. CONCLUSIONS: Double positivity for HPV-DNA/p16INK4a, a test that can be easily implemented in the clinical practice, has optimal diagnostic accuracy and prognostic value. Our results have strong clinical implications for patients' classification and handling and also suggest that not all the HPV-related OPC behave similarly.

The role of HPV on the risk of second primary neoplasia in patients with oropharyngeal carcinoma.

OBJECTIVES: It has been reported that patients with HPV-positive oropharyngeal cancer (OPC) have a lower risk of appearance of second primary neoplasm (SPN) than HPV-negative OPC patients. The aim of our study was to analyze the risk of developing SPN in a large group of patients with OPC according to HPV status in the primary tumor. MATERIALS AND METHODS: We included 412 OPC patients treated at our center from 1991 to 2014 for which the HPV DNA positivity was evaluated by PCR in available tumor specimens. HPV DNA positive samples were further tested for HPV E6∗I mRNA detection and/or p16INK4a immunohistochemistry. We estimated the incidence of SPN in all cancer sites and in cancer sites related to tobacco and alcohol consumption according to the HPV status in the primary tumor. RESULTS: Fifty-one (12.4%) out of 412 OPCs included in the study were HPV-related. Five-year SPN-free survival for HPV-negative versus HPV-positive OPC patients was 57.0% and 89.0% (P<0.001), respectively. Corresponding estimates for 10-year SPN-free survival were 35.2% versus 78.5% (P<0.001). When restricting the analyses to tobacco/alcohol-related SPNs, the corresponding survival rates where 62.0% versus 97.6% (P<0.001) and 42.2% versus 97.6%, (P<0.001), for 5-year and 10-year survival rates, respectively. HPV status and previous toxic habits might allow classifying patients regarding the risk of tobacco/alcohol-related SPNs. CONCLUSION: HPV-related OPC patients have a significant lower risk of SPN development, particularly in those locations related to tobacco use or alcohol consumption.

Development and validation of a protocol for optimizing the use of paraffin blocks in molecular epidemiological studies: The example from the HPV-AHEAD study.

Worldwide use of formalin-fixed paraffin-embedded blocks (FFPE) is extensive in diagnosis and research. Yet, there is a lack of optimized/standardized protocols to process the blocks and verify the quality and presence of the targeted tissue. In the context of an international study on head and neck cancer (HNC)-HPV-AHEAD, a standardized protocol for optimizing the use of FFPEs in molecular epidemiology was developed and validated. First, a protocol for sectioning the FFPE was developed to prevent cross-contamination and distributed between participating centers. Before processing blocks, all sectioning centers underwent a quality control to guarantee a satisfactory training process. The first and last sections of the FFPEs were used for histopathological assessment. A consensus histopathology evaluation form was developed by an international panel of pathologists and evaluated for four indicators in a pilot analysis in order to validate it: 1) presence/type of tumor tissue, 2) identification of other tissue components that could affect the molecular diagnosis and 3) quality of the tissue. No HPV DNA was found in sections from empty FFPE generated in any histology laboratories of HPV-AHEAD consortium and all centers passed quality assurance for processing after quality control. The pilot analysis to validate the histopathology form included 355 HNC cases. The form was filled by six pathologists and each case was randomly assigned to two of them. Most samples (86%) were considered satisfactory. Presence of >50% of invasive carcinoma was observed in all sections of 66% of cases. Substantial necrosis (>50%) was present in <2% of samples. The concordance for the indicators targeted to validate the histopathology form was very high (kappa > 0.85) between first and last sections and fair to high between pathologists (kappa/pabak 0.21-0.72). The protocol allowed to correctly process without signs of contamination all FFPE of the study. The histopathology evaluation of the cases assured the presence of the targeted tissue, identified the presence of other tissues that could disturb the molecular diagnosis and allowed the assessment of tissue quality.

HPV Involvement in Head and Neck Cancers: Comprehensive Assessment of Biomarkers in 3680 Patients.

BACKGROUND: We conducted a large international study to estimate fractions of head and neck cancers (HNCs) attributable to human papillomavirus (HPV-AFs) using six HPV-related biomarkers of viral detection, transcription, and cellular transformation. METHODS: Formalin-fixed, paraffin-embedded cancer tissues of the oral cavity (OC), pharynx, and larynx were collected from pathology archives in 29 countries. All samples were subject to histopathological evaluation, DNA quality control, and HPV-DNA detection. Samples containing HPV-DNA were further subject to HPV E6*I mRNA detection and to p16(INK4a), pRb, p53, and Cyclin D1 immunohistochemistry. Final estimates of HPV-AFs were based on HPV-DNA, HPV E6*I mRNA, and/or p16(INK4a) results. RESULTS: A total of 3680 samples yielded valid results: 1374 pharyngeal, 1264 OC, and 1042 laryngeal cancers. HPV-AF estimates based on positivity for HPV-DNA, and for either HPV E6*I mRNA or p16(INK4a), were 22.4%, 4.4%, and 3.5% for cancers of the oropharynx, OC, and larynx, respectively, and 18.5%, 3.0%, and 1.5% when requiring simultaneous positivity for all three markers. HPV16 was largely the most common type. Estimates of HPV-AF in the oropharynx were highest in South America, Central and Eastern Europe, and Northern Europe, and lowest in Southern Europe. Women showed higher HPV-AFs than men for cancers of the oropharynx in Europe and for the larynx in Central-South America. CONCLUSIONS: HPV contribution to HNCs is substantial but highly heterogeneous by cancer site, region, and sex. This study, the largest exploring HPV attribution in HNCs, confirms the important role of HPVs in oropharyngeal cancer and drastically downplays the previously reported involvement of HPVs in the other HNCs.

Human papillomavirus and breast cancer: no evidence of association in a Spanish set of cases.

BACKGROUND/AIM: Great controversy exists about the association between Human Papillomavirus (HPV) and breast tumors. The aim of this study was to explore the presence of HPV DNA in a large set of breast cancer cases. MATERIALS AND METHODS: Techniques used followed the standards for an international retrospective survey of HPV-DNA genotyping, coordinated by our own group and the DDL Laboratories in Rijswijk, the Netherlands. Paraffin-embedded samples were used. SPF-10 broad-spectrum primers were applied, followed by deoxyribonucleic acid enzyme immunoassay and genotyping by reverse-line probe assay. RESULTS: A total of 78 samples were included in the study, 2 of benign conditions and 76 carcinomas, including different histological subtypes. HPV was not present in any of the specimens studied irrespective of histology, hormonal status and stage of disease. CONCLUSION: Our data do not support the involvement of HPV in breast carcinogenesis as no evidence of its presence was found.