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Head and neck cancer is often diagnosed late and prognosis for most head and neck cancer patients remains poor. To aid early detection, we developed a risk prediction model based on demographic and lifestyle risk factors, human papillomavirus (HPV) serological markers, and genetic markers. A total of 10,126 head and neck cancer cases and 5,254 controls from 5 North American and European studies were included. HPV serostatus was determined by antibodies for HPV16 early oncoproteins (E6, E7) and regulatory early proteins (E1, E2, E4). The data were split into a training set (70%) for model development and a hold-out testing set (30%) for model performance evaluation, including discriminative ability and calibration. The risk models including demographic, lifestyle risk factors and polygenic risk score showed a reasonable predictive accuracy for head and neck cancer overall. A risk model that also included HPV serology showed substantially improved predictive accuracy for oropharyngeal cancer (AUC=0.94, 95%CI=0.92–0.95 in men and AUC=0.92, 95%CI=0.88–0.95 in women). The 5-year absolute risk estimates showed distinct trajectories by risk factor profiles. Based on the UK Biobank cohort, the risks of developing oropharyngeal cancer among 60 years old and HPV16 seropositive in the next 5 years ranged from 5.8% to 14.9% with an average of 8.1% for men, 1.3% to 4.4% with an average of 2.2% for women. Absolute risk was generally higher among individuals with heavy smoking, heavy drinking, HPV seropositivity, and those with higher polygenic risk score. These risk models may be helpful for identifying people at high risk of developing head and neck cancer.

TNM8 introduced a new staging system for HPV positive oropharyngeal squamous cell carcinoma (OPSCC). This study aimed to investigate whether the changes made in TNM8 offer the perceived benefit in prognostication when compared to TNM7 in a specific patient population in the North East of England. A retrospective cohort comparison study of all patients with HPV positive OPSCC (n = 106) through the Newcastle Head and Neck MDT between January 2012 to December 2014. Overall survival (OS) and Disease specific survival (DSS) data at 3 years was gathered for both TNM7 and TNM8. Log rank test was used to compare survival curves. Harrell’s C-index adjusted for age and smoking status was used to assess prognostic ability of the two staging methods. TNM8 downstages disease (TNM7 stage IV patients n = 74, TNM8 stage IV patients n = 2) but gives a more even distribution of patients across disease stages. Survival for TNM8 stage II and III is similar. In our small cohort, the log-rank test detected differences in OS between stages for both scoring methods (TNM7 p = 0.006, TNM8 p

This study reports oncological outcomes of transoral robotic surgery (TORS) and neck dissection (ND) alone for head and neck squamous cell carcinoma (HNSCC) and aims to analyse the influence of resection margins on local recurrence rates.Fifty-one patients treated with curative intent for HNSCC, with TORS and ND alone between 2013 and 2019 at two tertiary centres were included in this observational multi-centre prospective cohort study. Oncological outcomes are reported on 47 patients for whom the aim was to treat with TORS and ND alone; this excluded four patients who were recommended adjuvant radiotherapy based on resective pathology but did not receive treatment. Local control is the primary endpoint; disease specific, progression free and overall survival are secondary outcomes.With a median follow up of 43 months, estimated outcomes at 3 years (n = 47) were as follows: local control 92%, progression free survival 80%, disease specific survival 94%, and overall survival 84%. Presence of a positive margin on the main specimen was the only statistically significant predictor of local recurrence on univariate Cox regression analysis. Time dependent receiver operating characteristic curve identified margins of 1.1 mm as a threshold for local control, with area under the curve 0.788 (95% CI 0.616-0.960), indicating a good classifier.This is the first UK surgery alone series reporting mature oncological outcomes following TORS and ND. Positive margins on the resected specimen are the strongest predictor of local recurrence, with conventional definitions of "close margins" having no impact.

Single molecule biomarkers are used extensively in head and neck pathology for diagnosis and increasingly for prognosis. Companion markers for therapy such as PDL-1 and NTRK are now finding applications in head and neck cancer care. Immunohistochemistry is an attractive option because of its rapid turnaround time and convenience but molecular testing is often necessary for validation. This chapter will focus on some selected biomarkers being developed for translational purposes. Adoptive T cell therapies are being trialled for head and neck cancer and have limited efficacy currently. Identification of biomarkers as targets is an attractive option for development, and the use of molecular sequencing to identify individual neo-antigens is a promising way forward for precision medicine approaches including adoptive T cell therapies.

BACKGROUND: This study was undertaken to develop and validate a gene expression signature that characterises oral potentially malignant disorders (OPMD) with a high risk of undergoing malignant transformation. METHODS: Patients with oral epithelial dysplasia at one hospital were selected as the ‘training set’ (n = 56) whilst those at another hospital were selected for the ‘test set’ (n = 66). RNA was extracted from formalin-fixed paraffin-embedded (FFPE) diagnostic biopsies and analysed using the NanoString nCounter platform. A targeted panel of 42 genes selected on their association with oral carcinogenesis was used to develop a prognostic gene signature. Following data normalisation, uni- and multivariable analysis, as well as prognostic modelling, were employed to develop and validate the gene signature. RESULTS: A prognostic classifier composed of 11 genes was developed using the training set. The multivariable prognostic model was used to predict patient risk scores in the test set. The prognostic gene signature was an independent predictor of malignant transformation when assessed in the test set, with the high-risk group showing worse prognosis [Hazard ratio = 12.65, p = 0.0003]. CONCLUSIONS: This study demonstrates proof of principle that RNA extracted from FFPE diagnostic biopsies of OPMD, when analysed on the NanoString nCounter platform, can be used to generate a molecular classifier that stratifies the risk of malignant transformation with promising clinical utility.

Quality assured pathology services are integral to provision of optimal management for patients with head and neck cancer. Pathology services vary globally and are dependent on resources in terms of both laboratory provision and availability of a highly trained and accredited workforce. Ensuring a high-quality pathology service depends largely on close working and effective communication between the clinical team providing treatment and the pathologists providing laboratory input. Laboratory services should be quality assured by achieving external accreditation, most often by conforming to International Organization for Standardization (ISO) standards such as ISO15189 sometimes with ISO17025 or alternatively ISO17020. Quality of diagnostic reporting can be assured by the ISO but clinical teams should endeavor to work with pathologists who engage in continuing professional development, external quality assurance and audit. Research also contributes to diagnostic reporting quality. A number of initiatives in the UK such as the EPSRC/MRC funded Molecular Pathology Nodes and the National Cancer Research Institute Cellular-Molecular Pathology initiative (C-M Path), for example, have linked pathologists, industry and researchers. This has resulted in centers leading in digital innovation, artificial intelligence, translational research and clinical trials supported by pathologists. For rare tumors and contemporary molecular diagnostics, biopsy material can increasingly be shared with expert specialist pathologists working in specialist centers, particularly by using digital pathology platforms with potentially global reach. High quality services for the majority of diagnostic processes required for head and neck cancer management is best provided by local pathologists where communication with the treating team is more effective than with pathologists working in remote centers. Quality assurance is an increasingly important aspect of pathology, assuring not only effective turnaround times and accuracy for the diagnostic service but also high quality consistent reporting for clinical trials where even small pathology errors can potentially produce a significant bias and in the worst case negate the value of a completed trial. Better outcomes have been associated with centers engaged in clinical trials than in non-participating centers. Provision of a quality assured pathology service should extend to both the research and diagnostic services.

Typically, HPV-related cancers are sexually transmitted, however, the natural history of HPV-related oropharyngeal squamous cell carcinoma (OPSCC) is unclear. HPV16 transmission has been reported previously between five couples with OPSCC. We report the clinico-pathological features of a further four couples with HPV-related OPSCC and compare them with the published cases.We identified four couples in long-term heterosexual relationships that all had HPV-related OPSCC. The couples were treated at three UK hospitals and presented between 2009 and 2015. HPV tests included p16 immunohistochemistry, high-risk HPV DNA in-situ hybridisation and Roche Cobas HPV test. DNA sequencing was used to determine the HPV variant.The four couples represented2% of patients with HPV-related OPSCC at the three contributing hospitals (8 of 457 consecutive patients). The couples' tumours all contained HPV16. The mean age was 63 years old (range 52-72 years). The interval between the index cancer and the partner's cancer was 16, 24, 26 and 64 months respectively. The majority of patients had Stage I disease (UICC TNM8). Six of eight patients are disease free, one patient is alive with disease and there was one death from loco-regional recurrence.This report highlights the occurrence of HPV-related OPSCC in heterosexual couples and raises the possibility of transmission of HPV16. Despite increasing prevalence of HPV-related OPSCC and increased awareness of the disease, there is a paucity of couples with the disease, suggesting either under-reporting or that the development of OPSCC following HPV transmission between couples is a rare event.

Head and neck cancer (HNC) is a preventable malignancy that continues to cause substantial morbidity and mortality worldwide. Using data from the ARCAGE and Rome studies, we investigated the main predictors of survival after larynx, hypopharynx and oral cavity (OC) cancers. We used the Kaplan-Meier method to estimate overall survival, and Cox proportional models to examine the relationship between survival and sociodemographic and clinical characteristics. 604 larynx, 146 hypopharynx and 460 OC cancer cases were included in this study. Over a median follow-up time of 4.6 years, nearly 50% (n = 586) of patients died. Five-year survival was 65% for larynx, 55% for OC and 35% for hypopharynx cancers. In a multivariable analysis, we observed an increased mortality risk among older (≥71 years) versus younger (≤50 years) patients with larynx/hypopharynx combined (LH) and OC cancers [HR = 1.61, 95% CI 1.09-2.38 (LH) and HR = 2.12, 95% CI 1.35-3.33 (OC)], current versus never smokers [HR = 2.67, 95% CI 1.40-5.08 (LH) and HR = 2.16, 95% CI 1.32-3.54 (OC)] and advanced versus early stage disease at diagnosis [IV versus I, HR = 2.60, 95% CI 1.78-3.79 (LH) and HR = 3.17, 95% CI 2.05-4.89 (OC)]. Survival was not associated with sex, alcohol consumption, education, oral health, p16 expression, presence of HPV infection or body mass index 2 years before cancer diagnosis. Despite advances in diagnosis and therapeutic modalities, survival after HNC remains low in Europe. In addition to the recognized prognostic effect of stage at diagnosis, smoking history and older age at diagnosis are important prognostic indicators for HNC.

Background: Determination of human papillomavirus (HPV) status has become clinically relevant for patient stratification under UICC TNM8 staging. Within the United Kingdom, a combination of p16 IHC and HPV DNA-ISH is recommended for classifying HPV status. This study will assess a series of clinically applicable second-line molecular tests to run in combination with p16 IHC to optimally determine HPV status. Methods: The ability of HPV RNA-ISH, HPV DNA-ISH, and HPV DNA-PCR to identify p16-positive/HPV-positive patients was investigated in a population-based oropharyngeal squamous cell carcinoma (OPSCC) cohort of patients diagnosed in Northern Ireland from 2000 to 2011. Results: Only 41% of the Northern Irish OPSCC patient population was associated with HPV-driven carcinogenesis. Both ISH assays were more specific than the DNA-PCR assay (100% and 95% vs. 67%) and were less likely to be affected by preanalytic factors such as increasing block age. A pooled HPV genotype probe for RNA-ISH was found to be the most accurate molecular assay assessed (95% accuracy) when compared with p16 positivity. Conclusions: Our study demonstrates the advantage of tissue-based molecular assays when determining HPV status in retrospective samples. Specifically, we demonstrate the enhanced sensitivity and specificity of ISH techniques compared with PCR-based methodology when working with formalin-fixed paraffin-embedded tissue, and found HPV RNA-ISH to be the most effective assay for determining HPV status. Impact: As p16 IHC is a relatively inexpensive, accessible, and sensitive test for stratifying patients by HPV status, this study finds that more patients would benefit from first-line p16 IHC followed by specific HPV testing using HPV RNA-ISH to confirm HPV status.

109 Background: Trials in human papilloma virus associated oropharyngeal squamous cell carcinoma (HPVOPSCC), substituting cetuximab (CETUX) for cisplatin (CIS) with radiotherapy (RT), resulted in decreased efficacy without improved toxicity or symptom burden. We reported that high intratumoral immune cell (ITIC) CD103 expression (> 30%), a marker of tissue-resident memory T cells, is associated with better prognosis in unselected patients with HPVOPSCC treated with CIS/RT. In this study our aim was to determine whether low risk HPVOPSCC patients treated with CETUX/RT with high CD103 have a superior prognosis. Methods: TROG 12.01 and De-ESCALaTE are randomised multicentre trials that compared 70Gy RT/CETUX with 70Gy RT/CIS (weekly in TROG 12.01, 3-weekly in De-ESCALaTE) in patients with HPVOPSCC, low risk by Ang criteria: AJCC 7th Stage III (excluding T1-2N1) or stage IV (excluding N2b-c if smoking history > 10 pack years and/or distant metastases). In TROG 12.01 T4 and/or N3 patients were also excluded. Eligible patients required tumor samples available for immune cell quantification on immunohistochemistry. Data from the two trials were pooled, with analyses performed in eligible randomised patients who commenced treatment. The primary endpoint was failure-free survival (FFS) in patients receiving CETUX/ RT comparing CD103 ITIC > 30% (high) vs. < 30% (low). High/low CD103 were compared using Cox model adjusting for age, stage and trial. Results: Samples for CD103 testing were available in 159/182 patients on TROG 12.01 and 145/334 on De-ESCALaTE. ITIC CD103 expression was high in 26% of patients. The median follow-up was 3.2 years. The 3 -year failure-free survival rates in patients treated with CETUX/RT were 92% (95% CI: 78-97%) in high CD103 and 74% (95% CI: 64-82%) in low CD103, adjusted HR 0.25 (95% CI: 0.14-0.44); p < 0.001. The 3 -year overall survival (OS) in patients treated with CETUX/RT were 100% in high CD103 and 86% (95% CI: 76-92%) in low CD103, p < 0.001. Superior FFS in the high CD103 group was independent of stage. In patients treated with CIS/RT there was no significant difference in FFS (3-year 86% in high CD103 and 90% in low CD103; p = 0.55) or in OS (3-year 100% in high CD103 and 95% in low CD103; p = 0.14). The increase in failures in the low CD103 patients treated with CETUX/RT was evenly split between distant and locoregional failures. Conclusions: ITIC CD103 separates CETUX/RT treated low risk HPVOPSCC into excellent and poor prognosis subgroups. In a low risk population CIS/RT achieves excellent outcomes in both high and low ITIC CD103 groups. The high ITIC CD103 population is a rational target for future de-intensification trials.

Background Intraoperative analysis of sentinel lymph nodes would enhance the care of early-stage oral squamous cell carcinoma (OSCC). We determined the frequency and extent of cytokeratin 19 (CK19) expression in OSCC primary tumours and surrounding tissues to explore the feasibility of a “clinic-ready” intraoperative diagnostic test (one step nucleic acid amplification—OSNA, sysmex). Methods Two cohorts were assembled: cohort 1, OSCC with stage and site that closely match cases suitable for sentinel lymph node biopsy (SLNB); cohort 2, HNSCC with sufficient fresh tumour tissue available for the OSNA assay (>50 mg). CK19 assays included qRT-PCR, RNA in situ hybridisation (ISH), and immunohistochemistry (IHC), as well as OSNA. Results CK19 mRNA expression was detected with variable sensitivity, depending on method, in 60–80% of primary OSCC tumours, while protein expression was observed in only 50% of tumours. Discordance between different techniques indicated that OSNA was more sensitive than qRT-PCR or RNA-ISH, which in turn were more sensitive than IHC. OSNA results showed CK19 expression in 80% of primary cases, so if used for diagnosis of lymph node metastasis would lead to a false-negative result in 20% of patients with cervical lymph node metastases. Conclusions OSNA in its current form is not suitable for use in OSCC SLNB due to inadequate expression of the CK19 target in all case. However, the same assay technology would likely be very promising if applied using a more ubiquitous squamous epithelial target. Electronic supplementary material The online version of this article (doi:10.1245/s10434-016-5337-6) contains supplementary material, which is available to authorized users.

Background: Oral squamous cell carcinoma (OSCC) is a global healthcare problem associated with poor clinical outcomes. Early detection is key to improving patient survival. OSCC may be preceded by clinically recognizable lesions, termed oral potentially malignant disorders (OPMD). As histologic assessment of OPMD does not accurately predict their clinical behavior, biomarkers are required to detect cases at risk of malignant transformation. Epidermal growth factor receptor gene copy number (EGFR GCN) is a validated biomarker in lung non–small cell carcinoma. We examined EGFR GCN in OPMD and OSCC to determine its potential as a biomarker in oral carcinogenesis. Methods: EGFR GCN was examined by in situ hybridization (ISH) in biopsies from 78 patients with OPMD and 92 patients with early-stage (stages I and II) OSCC. EGFR ISH signals were scored by two pathologists and a category assigned by consensus. The data were correlated with patient demographics and clinical outcomes. Results: OPMD with abnormal EGFR GCN were more likely to undergo malignant transformation than diploid cases. EGFR genomic gain was detected in a quarter of early-stage OSCC, but did not correlate with clinical outcomes. Conclusion: These data suggest that abnormal EGFR GCN has clinical utility as a biomarker for the detection of OPMD destined to undergo malignant transformation. Prospective studies are required to verify this finding. It remains to be determined if EGFR GCN could be used to select patients for EGFR-targeted therapies. Impact: Abnormal EGFR GCN is a potential biomarker for identifying OPMD that are at risk of malignant transformation. Cancer Epidemiol Biomarkers Prev; 25(6); 927–35. ©2016 AACR.

Background The contemporary treatment of oropharyngeal squamous cell carcinoma (SCC) is an area of debate. We report outcomes of a minimally invasive approach involving transoral laser microsurgery (TLM). Methods A consecutive series of patients (n = 153) undergoing primary TLM for oropharyngeal SCC from 2006 to 2013 was studied. Human papillomavirus (HPV) status was determined by p16 immunohistochemistry and high-risk HPV DNA in situ hybridization. Survival analyses were evaluated using Kaplan–Meier statistics. Results Tumor subsites included tonsil (n = 94; 61.5%), tongue base (n = 38; 24.8%), and soft palate (n = 21; 13.7%), with the majority being American Joint Committee on Cancer (AJCC) stage III/IVa (n = 124; 81.0%) and HPV-positive (n = 101; 66.0%). Three-year overall survival (OS), disease-specific survival (DSS), and disease-free survival (DFS) were 84.5%, 91.7%, and 78.2%, respectively. HPV-positivity portended favorable oncologic outcomes. One-year gastrostomy tube (G-tube) dependency was 1.3%. Conclusion To the best of our knowledge, this is the largest single-center TLM oropharyngeal SCC series to date. Our data suggest that TLM +/− postoperative radiotherapy (PORT) results in at least as good oncologic outcomes as chemoradiotherapy (CRT), while conferring swallowing function advantages. © 2016 Wiley Periodicals, Inc. Head Neck , 2016 © 2016 Wiley Periodicals, Inc. Head Neck 38:1263–1270, 2016

Objectives The incidence of human papillomavirus (HPV)-positive head and neck cancer, particularly oropharyngeal, has been increasing rapidly. Understanding of this disease, and modelling of suitable therapeutics, requires sustainable cell cultures, yet they remain limited in number and of variable origin. A comprehensive understanding of these resources is therefore of great importance. Materials and Methods Viral gene expression assays and pathological testing methods were used in the six currently available HPV-positive cell lines derived from head and neck (H&N) subsites, two HPV-negative H&N and two cervical carcinoma cell lines. A 2D migration assay monitored cell movement, speed and pattern of migration. Results All six H&N and two cervical cell lines were confirmed HPV-positive by gold standard testing, yet variability between tests was apparent. Although migration was not significantly different between cell lines, each demonstrated unique migration patterns. Conclusion Patient-derived cancer cells, arising as a consequence of natural oncogenic processes rather than in vitro manipulation, are essential for understanding cancer biology. We have characterised the available HPV-positive H&N cell lines and provided clear evidence of a persisting viral oncogenic driver in each, as such supporting their ongoing use as a model of HPV-positive H&N cancer. Importantly, we also highlight a need for caution to be exercised when translating future in vitro findings associated with these lines particularly in the context of oropharyngeal cancer given irregularities in tumour provenance (origin site and clinicopathological features).

Pre-trial radiotherapy quality assurance (RTQA) for the SPARC trial of preoperative pancreas SBRT highlighted overgenerous target contouring. We aimed to assess for improvement following a training workshop and appraise the effectiveness of a novel web-conferencing format for on-trial RTQA.

Background The incidence of oropharyngeal squamous cell carcinoma (OPSCC) has been rising in Western countries and this has been attributed to human papillomavirus (HPV) infection. p16 expression is a marker for HPV infection and p16 positive OPSCC is now recognized as a separate disease entity. There are only limited data available regarding HPV-related OPSCC in Asian countries and no data from Malaysia. Methods We identified 60 Malaysian patients with OPSCC over a 12-year period (2004–2015) from four different hospitals in two major cities, Kuala Lumpur and Penang. The detection of HPV was carried out using p16 immunohistochemistry and high risk HPV DNA in situ hybridisation. Results Overall, 15 (25%) tumours were p16 positive by immunohistochemistry, 10 of which were also positive for high risk HPV DNA by in situ hybridisation. By comparison, a matched cohort of UK patients had a p16 positive rate of 49%. However, between 2009 and 2015, where cases were available from all four hospitals, 13 of 37 (35%) cases were p16 positive. In our Malaysian cohort, 53% of patients were of Chinese ethnicity and 80% of the p16 positive cases were found in these patients; no Indian patients had p16 positive disease, despite representing 35% of the total cohort. Conclusion The proportion of OPSCCs associated with HPV in Malaysia appears to be lower than in European and American cohorts and could possibly be more prevalent amongst Malaysians of Chinese ethnicity. Further, our data suggests that the burden of HPV-related OPSCC could be increasing in Malaysia. Larger cross-sectional studies of Malaysian patients are required to determine the public health implications of these preliminary findings.

Head and neck squamous cell carcinomas (HNSCC) are treated with surgery, radiotherapy and cisplatin-based chemotherapy, but survival from locally-advanced disease remains poor, particularly in patients whose tumors are negative for Human papillomavirus (HPV). Type 1 IGF receptor (IGF-1R) is known to promote tumorigenesis and resistance to cancer therapeutics. Here, we assessed IGF-1R immunohistochemistry on tissue microarrays containing 852 cores from 346 HNSCC patients with primary tumors in the oropharynx (n = 231), larynx (85), hypopharynx (28), oral cavity (2). Of these, 236 (68%) were HPV-negative, 110 (32%) positive. IGF-1R was detected in the cell membrane of 36% and cytoplasm of 92% of HNSCCs; in 64 cases with matched normal tonsillar epithelium, IGF-1R was overexpressed in the HNSCCs (P < 0.001). Overall survival (OS) and disease-specific survival (DSS) were reduced in patients whose tumors contained high membrane IGF-1R [OS: hazard ratio (HR) = 1.63, P = 0.006; DSS: HR = 1.63, P = 0.016], cytoplasmic IGF-1R (OS: HR = 1.58, P = 0.009; DSS: HR = 1.58, P = 0.024) and total IGF-1R (OS: HR = 2.02, P < 0.001; DSS: HR = 2.2, P < 0.001). High tumor IGF-1R showed significant association with high-tumor T-stage (P < 0.001) and HPV-negativity (P < 0.001), and was associated with shorter OS when considering patients with HPV-positive (P = 0.01) and negative (P = 0.006) tumors separately. IGF-1R was independently associated with survival in multivariate analysis including HPV, but not when lymphovascular invasion, perineural spread and T-stage were included. Of these factors, only IGF-1R can be manipulated; the association of IGF-1R with aggressive disease supports experimental incorporation of anti-IGF-1R agents into multimodality treatment programs for HPV-negative and high IGF-1R HPV-positive HNSCC.

Assessment of human papillomavirus (HPV) status is a requirement for the diagnosis of HPV-associated oropharyngeal squamous cell carcinoma (OPSCC) and metastatic squamous cell carcinoma in cervical lymph nodes where the location of the primary neoplasm is unknown. Within the diagnostic histopathology laboratory, there should be a validated and reproducible HPV testing strategy that can provide HPV status within a reasonable timeframe to inform patient care. Although these requirements are recognized by the head and neck oncology community, there is no internationally accepted standard for HPV testing. A two-tiered approach incorporating p16 immunohistochemistry with specific HPV testing by DNA in situ hybridization is a pragmatic way of providing HPV testing in clinical practice. A novel RNA in situ hybridization methodology targeting E6 and E7 mRNA has been validated and is likely to be available as an in vitro diagnostic device soon. This review will outline the current concepts around the diagnosis of HPV-associated head and neck SCC and suggest a diagnostic algorithm that can be instituted in most diagnostic cellular pathology laboratories.

BACKGROUND: The proxy marker for human papillomavirus (HPV), p16, is included in the new AJCC 8th/UICC 8th staging system, but due to incongruence between p16 status and HPV infection, single biomarker evaluation could lead to misallocation of patients. We established nomograms for overall survival (OS) and progression-free survival (PFS) in patients with oropharyngeal squamous cell carcinoma (OPSCC) and known HPV-DNA and p16 status, and validated the models in cohorts from high- and low-prevalent HPV countries.METHODS: Consecutive OPSCC patients treated in Denmark, 2000-2014 formed the development cohort. The validation cohorts were from Sweden, Germany, and the United Kingdom. We developed nomograms by applying a backward-selection procedure for selection of variables, and assessed model performance.RESULTS: In the development cohort, 1313 patients, and in the validation cohorts, 344 German, 503 Swedish and 463 British patients were included. For the OS nomogram, age, gender, combined HPV-DNA and p16 status, smoking, T-, N-, and M-status and UICC-8 staging were selected, and for the PFS nomogram the same variables except UICC-8 staging. The nomograms performed well in discrimination and calibration.CONCLUSIONS: Our nomograms are reliable prognostic methods in patients with OPSCC. Combining HPV DNA and p16 is essential for correct prognostication. The nomograms are available at www.orograms.org .

Objectives HPV16-positive oropharyngeal cancer (OPC) patients experience better outcomes compared to HPV16-negative patients. Currently, strategies for treatment de-escalation are based on HPV status, smoking history and disease stage. However, the appropriate cut-point for smoking and the role of other non-clinical factors in OPC survival remains uncertain. Materials and Methods We examined factors associated with OPC outcome in 321 patients recruited in a large European multi-center study. Seropositivity for HPV16 E6 was used as a marker of HPV16 positive cancer. Hazard ratios (HR) and confidence intervals (CI) were estimated using Cox proportional models adjusted for potential confounders. Results Overall 5-year survival following OPC diagnosis was 50%. HPV16-positive OPC cases were at significantly lower risk of death (aHR = 0.51, 95% CI: 0.32–0.80). A significant effect on OPC survival was apparent for female sex (aHR 0.50: 95% CI: 0.29–0.85) and being underweight at diagnosis (aHR: 2.41, 95% CI: 1.38–4.21). A 10 pack year smoking history was not associated with overall survival. Higher stage at diagnosis appeared as the only factor significantly associated with OPC recurrence (aHR: 4.88, 95% CI: 2.12–11.21). Conclusion This study confirms that HPV16 status is an independent prognostic factor for OPC survival while female sex lowers risk of death and being underweight at diagnosis increases the risk of death. Smoking was not an independent predictor of OPC survival.

Aims Oropharyngeal squamous cell carcinoma (OPSCC) can be divided into favourable and poor prognostic groups by association with human papilloma virus (HPV) and smoking. This study prospectively investigated a dose-intensified schedule in poor/intermediate prognosis OPSCC. Materials and methods Patients with p16/HPV-negative or p16-positive N2b OPSCC with a greater than 10 pack-year smoking history were eligible. Patients were planned to receive 64 Gy in 25 fractions with cisplatin. The primary end point was absence of grade 3 mucositis at 3 months. Results Fifteen patients were recruited over 14 months. All patients completed a minimum of 2 years of follow-up. All patients completed full-dose radiotherapy within a median treatment time of 32 days (31–35). Grade 3 mucositis was absent in all patients at 3 months. There was one grade 4 toxicity event due to cisplatin (hypokalaemia). Complete response rates at 3 months were 100% and 93% for local disease and lymph nodes, respectively. One patient developed metastatic disease and subsequently died. Overall survival at 2 years was 93% (95% confidence interval 61–99%). Conclusions The schedule of 64 Gy in 25 fractions with concomitant chemotherapy is tolerable in patients with poor and intermediate prognosis OPSCC.

Summary Objectives Patients with human papillomavirus (HPV)-related oropharyngeal squamous cell carcinoma (OPSCC) have a reduced risk of developing second primary upper aerodigestive tract (UADT) tumours compared to patients with HPV-negative primary tumours at the same site. To determine whether this finding might be explained by a lack of viral-induced field cancerisation or multifocal infection, we investigated whether there was epithelial dysplasia and/or evidence of HPV infection at other pharyngeal mucosal sites in patients presenting with the disease. Materials and methods Sixty-three patients with primary tonsil SCC and 108 pharyngeal endoscopic biopsies, representing at least one pharyngeal subsite from each patient, were included in this study. Tissue samples were tested using HPV PCR (GP5+/6+), p16 immunohistochemistry (IHC) and high risk HPV DNA in situ hybridisation (ISH). Results There were 46 patients with HPV-related SCC and 17 patients with HPV-negative disease. PCR detected HPV DNA in a fifth of pharyngeal endoscopic biopsies and was equally likely to be from a patient with HPV-related SCC as from a patient with HPV negative disease. All PCR positive cases were tested using p16 IHC and high risk HPV ISH and only three biopsies were positive. Significantly, these three biopsies all showed evidence of epithelial dysplasia and were from patients with an HPV positive index tumour. Conclusion Our data suggest that virus-induced field cancerisation and/or multifocal oncogenic HPV infection of the pharynx is uncommon in OPSCC and supports the concept that these patients have a lower risk of developing second primary tumours of the UADT.

Background The purpose of this study was to present the clinicopathological features of a series of patients with human papillomavirus (HPV)-associated head and neck second primary tumors. Methods Patients with HPV-associated head and neck second primary tumors from 3 centers were identified. HPV infection was evaluated using p16 by immunohistochemistry (IHC), high-risk HPV DNA by in situ hybridization (ISH), and HPV genotyping by DNA polymerase chain reaction (PCR) enzyme immunoassay (EIA). Results Eleven patients were identified: 5 with synchronous and 6 with metachronous HPV-positive second primary tumors, the latter demonstrating a mean time interval of 5 years. There were 13 second primary tumors: 11 oropharyngeal, 1 nasopharyngeal, and 1 floor of the mouth. Nine of 10 genotyped patients harbored HPV-16, and 1 patient had HPV-33 in 3 synchronous tumors. Conclusion HPV-associated second primary tumors may present as synchronous and/or metachronous lesions and can arise outside the oropharynx after prolonged intervals. Further work is necessary to identify patients at risk and to elucidate the mechanisms of HPV-associated head and neck second primary tumors.

Oropharyngeal 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.All 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.In 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).TNM-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.

Background: Novel cancer immunotherapy includes application of immune checkpoint inhibitors to the treatment of solid tumors. The body’s own immune system is thereby harnessed to tip the balance in favor of antitumor activity. The intracellular enzyme indoleamine 2,3-dioxygenase (IDO) is a critical regulator of the tumor microenvironment (TME) via tryptophan metabolism. Programmed death protein 1 (PD-1) and programmed death-ligand 1 (PD-L1) are immune checkpoints involved in T-cell inhibition and immunosuppression. Another immune checkpoint, IDO1, breaks down tryptophan into its metabolites which results in immunosuppression in the TME. Aim: To assess the evidence on IDO in head and neck squamous cell carcinoma (HNSCC). Methods: Medline, EMBASE using Ovid, Scopus, Web of Science, Cochrane Library databases, and ClinicalTrials.gov were searched from inception until present day. Results: We included 32 studies. Of those, 5 involved cell lines, 7 assessed tumor immunohistochemistry, 6 measured IDO gene transcription, and 14 reported on clinical trials. The human cell lines studied commonly were SCC4, SCC15, and SCC25 in which IDO expression and activation by the Stimulator of Interferon Genes (STING) pathway played a central role. Retrospective immunohistochemistry studies of lower lip, oral cavity, tongue, tonsil, and larynx found that relatively high IDO expression correlated with worse survival. Gene transcription studies showed increased IDO in tumors that expressed PD-L1 and harbored human papillomavirus (HPV). Phase I/II clinical trials showed 1) objective responses (34%) and disease control rates (62%) for IDO1 inhibitor in combination with a PD-1 inhibitor, 2) consistent safety profile in combination versus monotherapy, and 3) IDO gene expression as a predictive biomarker for response to PD-L1 therapy. Conclusions: IDO is integral to TME immunity in HNSCC particularly in HPV positive tumors, modulating existing therapies and application in combinatorial immunotherapy. Retrospective studies have shown the presence of IDO in the TME and suggest a link to prognosis and prediction of HNSCC treatment outcome. However, the exact mechanism of IDO-driven immune modulation in the HNSCC TME remains unclear. We now require prospective longitudinal studies to track IDO activity and expression throughout HNSCC treatment, thence optimise IDO-based immunotherapy. Citation Format: Daniel J. Lin, James C. Ng, Lei Huang, Max Robinson, James O'Hara, Janet A. Wilson, Andrew L. Mellor. The immunotherapeutic role of indoleamine 2,3-dioxygenase (IDO) in head and neck squamous cell carcinoma: a systematic review [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr LB-184.

Objectives Oral squamous cell carcinoma (OSCC) is associated with a high degree of morbidity and mortality. OS-CCs are often preceded by oral potentially malignant disorders (OPMD) which have a higher propensity to undergo malignant transformation (MT) compared to clinically normal oral mucosa. Currently there is no reliable method to determine which OPMD cases will undergo MT. This study was performed to construct a prognostic classifier for patients with OPMD by integrating clinical, histopathological and molecular factors and to discover a gene expression signature that characterises OPMD with a high risk of undergoing MT. Findings Statistical analysis of an OPMD patient cohort (23 MT vs. 25 with no MT) showed that site of initial OPMD (p = 0.043), binary oral epithelial dysplasia (OED) grading (p = 0.009) and loss of heterozygosity at 3p/9p/17p (p = 0.026) were statistically significant. Other demographic factors, clinical features and the WHO 3-tiered OED grading system were not statistically significant. Gene expression experiments revealed several genes that were differentially expressed between OPMD that underwent MT and those that did not [false discovery rate of Conclusions Our findings show that a classifier combining histopathological and molecular factors outperforms conventional methods for prognosticating clinical outcome in patients with OPMD. We have also shown that formalin-fixed paraffin-embedded tissue can be used to generate a molecular classification with clinical utility.

The rapid worldwide rise in incidence of human papillomavirus (HPV)-positive oropharyngeal squamous cell carcinoma (OPSCC) has generated studies confirming this disease as an entity distinct from traditional OPSCC. Based on pathology, surgical studies have revealed prognosticators specific to HPV-positive OPSCC. The current AJCC/UICC staging and pathologic nodal (pN)-classification do not differentiate for survival, demonstrating the need for new, HPV-specific OPSCC staging. The objective of this study was to define a pathologic staging system specific to HPV-positive OPSCC.Data were assembled from a surgically-managed, p16-positive OPSCC cohort (any T, any N, M0) of 704 patients from five cancer centers. Analysis was performed for (a) the AJCC/UICC pathologic staging, (b) newly published clinical staging for non-surgically managed HPV-positive OPSCC, and (c) a novel, pathology-based, "HPVpath" staging system that combines features of the primary tumor and nodal metastases.A combination of AJCC/UICC pT-classification and pathology-confirmed metastatic node count (⩽4 versus ⩾5) yielded three groups: stages I (pT1-T2, ⩽4 nodes), II (pT1-T2, ⩾5 nodes; pT3-T4, ⩽4 nodes), and III (pT3-T4, ⩾5 nodes), with incrementally worse prognosis (Kaplan-Meier overall survival of 90%, 84% and 48% respectively). Existing AJCC/UICC pathologic staging lacked prognostic definition. Newly published HPV-specific clinical stagings from non-surgically managed patients, although prognostic, showed lower precision for this surgically managed cohort.Three loco-regional "HPVpath" stages are identifiable for HPV-positive OPSCC, based on a combination of AJCC/UICC primary tumor pT-classification and metastatic node count. A workable, pathologic staging system is feasible to establish prognosis and guide adjuvant therapy decisions in surgically-managed HPV-positive OPSCC.

A rising incidence of oropharyngeal squamous cell carcinoma (OPSCC) incidence has occurred throughout the developed world, where it has been attributed to an increasing impact of human papillomavirus (HPV) on disease etiology. This report presents the findings of a multicenter cross-sectional retrospective study aimed at determining the proportion of HPV-positive and HPV-negative OPSCC within the United Kingdom. Archival tumor tissue blocks from 1,602 patients previously diagnosed with OPSCC (2002–2011) were collated from 11 centers. HPV status was determined with three validated commercial tests to provide valid data for 1,474 cases in total. Corresponding national incidence data from the same decade were obtained from UK Cancer registries. The overall proportion of HPV+ OPSCC between 2002 and 2011 was 51.8% [95% confidence interval (CI), 49.3–54.4], and this remained unchanged throughout the decade [unadjusted RR = 1.00 (95% CI, 0.99–1.02)]. However, over the same period, the incidence of OPSCC in the broader UK population underwent a 2-fold increase [age-standardized rate 2002: 2.1 (95% CI, 1.9–2.2); 2011: 4.1 (95% CI, 4.0–4.3)]. Although the number of OPSCCs diagnosed within the United Kingdom from 2002 to 2011 nearly doubled, the proportion of HPV+ cases remained static at approximately 50%. Our results argue that the rapidly increasing incidence of OPSCC in the United Kingdom cannot be solely attributable to the influence of HPV. The parallel increase in HPV+ and HPV− cases we documented warrants further investigation, so that appropriate future prevention strategies for both types of disease can be implemented. Cancer Res; 76(22); 6598–606. ©2016 AACR.

Human papillomavirus (HPV) testing is now recommended as part of the work up for patients with oropharyngeal squamous cell carcinoma (OPSCC) and those patients with cervical lymph node metastasis of unknown origin. The laboratory testing strategy should accurately assess the presence or absence of oncogenic HPV infection in routinely collected tumour samples that are subject to standard fixation protocols, alcohol-fixed cytological preparations and formalin-fixed tissue samples. The HPV status should correlate with biologically relevant outcome measures such as overall, disease-specific and disease-free survival. Whilst increased expression of p16 by immunohistochemistry is considered to be a surrogate marker of oncogenic HPV infection and is a validated independent prognostic biomarker, only HPV specific tests provide definitive evidence of the aetiological agent. We provide an overview of HPV testing in OPSCC, justifying the use of HPV specific tests. We examine the analytical accuracy of HPV specific tests against the 'reference' test--high risk HPV mRNA in fresh tissue--and contrast this with the performance of p16 immunohistochemistry as a stand alone test. We highlight the added value of HPV specific tests in prognostication, clinical trial design, and population-based disease surveillance. We consider that HPV specific testing is the starting point for developing increasingly informative biomarker panels in the context of 'stratified medicine'. We briefly frame test information in the context of disclosure of HPV status to patients. We conclude that only a testing strategy that includes HPV specific tests can deliver more effective care for patients with OPSCC. The international head and neck oncology community should work together to clearly define the minimum requirements for assigning a diagnosis of HPV-related OPSCC in order to ensure consistent reporting of this emerging and increasingly prevalent disease.

Ganglioneuroblastic transformation in olfactory neuroblastoma (ONB) is an exceptionally rare phenomenon. We document the case of a patient with a poorly differentiated sinonasal malignancy that recurred following treatment with chemoradiotherapy and showed ganglioneuroblastic transformation. Although the index tumour showed neuroendocrine differentiation, it did not have the typical clinico-pathological features associated with ONB. We highlight the diagnostic difficulties in establishing an accurate diagnosis for undifferentiated sinonasal tumours and present evidence that the index tumour was an ONB. The current report is only the third case of ONB showing complete ganglioneuroblastic transformation.

Human papillomavirus type 16 (HPV-16) has been established beyond doubt as a causative agent in oropharyngeal squamous cell carcinoma (SCC). The incidence of oropharyngeal cancer has risen in recent decades, as has the proportion of patients who have a biologically relevant HPV-16 infection. Combined data from 14 recently published studies (2006-2010) show that 57% of 1316 reported cases of oropharyngeal SCC were HPV-16 positive. They had significantly better prognosis (hazard ratio (HR) for 5-year overall survival range 0.05-0.64), although smoking and higher T stage often appear as confounding factors to this favourable prognostic benefit. HPV-16 therefore has increasing importance as a clinically useful prognostic biomarker, but a benefit in survival has been seen in the use of surgery, radiotherapy, and chemotherapy, so specific changes in the preferred methods of treatment are hard to justify. Future trials that include oropharyngeal SCC will consider HPV-16 routinely as a stratification factor, and its use as a predictive biomarker awaits the development of effective targeted treatments. The undeniable and impressive prognostic significance of HPV-16 should hasten its addition to standard pathological reporting of oropharyngeal SCC, and ultimately to its inclusion in TNM staging systems of the American Joint Committee on Cancer (AJCC) and the International Union against Cancer (UICC).

Although it is now well established that a significant proportion of oropharyngeal squamous cell carcinomas (SCC) harbour oncogenic human papillomavirus (HPV) sequences, the frequency with which these sequences are detected in oral SCC (excluding oropharyngeal subsites) is highly variable. In an attempt to establish the true prevalence of HPV-16 and HPV-18 subtypes in oral SCC, we screened 142 consecutive cases from a UK cohort using both conventional PCR with consensus primers and type-specific quantitative PCR (Q-PCR), while at the same time employing a rigorous protocol to avoid sample contamination. Q-PCR revealed HPV sequences in five cases; two contained HPV-16 alone, two HPV-18 alone, and one sample carried both genotypes. However, only two of these cases (both HPV-16-positive) had moderate viral loads (51 and 91 viral copies per 100 cells respectively) and were positive for HPV DNA by conventional PCR. Both cases contained HPV DNA in tumour cells as shown by Q-PCR analysis of micro-dissected tissue and by in situ hybridisation. The remaining three cases had only very low viral loads (between 3 and 7 viral copies per 100 cells), were negative by conventional PCR and lacked HPV DNA in tumour cells. Our data provide strong evidence that oncogenic HPV is uncommon in oral SCC and that routine HPV testing of these tumours cannot be advocated.

Background: Mitochondrial DNA (mtDNA) mutations occur in head and neck squamous cell carcinoma (HNSCC) and are most frequently detected in the displacement-loop (D-loop) region. The D-loop is considered to be important because it controls mitochondrial gene expression and mtDNA replication. There is currently no evidence that mtDNA mutations can be used as prognostic or predictive biomarkers in HNSCC. Methods: We used denaturing high performance liquid chromatography to screen the entire mitochondrial genome of six oral squamous cell carcinoma-derived cell lines and then focused on detecting D-loop abnormalities in 34 HNSCC tissue samples. Results: Mitochondrial DNA mutations are not ubiquitous in HNSCC because only half of the cell lines had detectable mtDNA abnormalities following screening of the entire mitochondrial genome and only 18% (6 of 34) of tissue samples had D-loop mutations. There was no correlation between D-loop mutations and determinates of clinical outcome; specifically, tumour stage and the expression of hypoxia-inducible genes included in a highly prognostic hypoxia metagene. Conclusions: Taken together, these data suggest that mtDNA D-loop mutations are stochastic events that may not significantly influence the biology of HNSCC and supports the hypothesis that mtDNA mutations in cancer represent bystander genotoxic damage as a consequence of tumour development and progression.

There is accumulating evidence that oropharyngeal squamous cell carcinomas (SCCs) that harbour oncogenic human papillomavirus (HPV) are biologically distinct and have a better prognosis. This information is a persuasive argument for the identification of these cancers in clinical practice. The concept of 'HPV related' SCC should be underpinned by knowledge that the malignant cells harbour a high risk HPV genotype and there is evidence of oncogenic viral protein expression with effects on cell signalling pathways. For satisfactory classification in clinical practice HPV tests should reliably work on fixed cells and tissue. There is evidence that detection of high risk HPV by consensus polymerase chain reaction (PCR) alone is insufficient to accurately classify tumours. However, there is convincing evidence that the detection of p16 protein by immunohistochemistry can be used as a surrogate marker for the elaboration of oncogenic HPV proteins. Recently, there have been calls for standardisation of HPV testing in head and neck cancers and two diagnostic algorithms have emerged: the first advocates screening for p16 by immunohistochemistry followed by detection of HPV DNA by in situ hybridisation: the second recommends detection of p16 followed by consensus PCR. The majority of pathology laboratories have the capability of delivering the first algorithm. Furthermore, the techniques employed are automated and are subject to stringent quality assurance measures; features that can deliver routine, accurate and cost effective HPV testing for oropharyngeal cancers.

TPS6097Background: Incidence of Oropharyngeal squamous cell carcinoma (OPSCC) is rapidly increasing as a result of Human Papillomavirus (HPV), genotype 16 infection. Existing treatments for HPV+ OP...

Like many solid tumours, oral squamous cell carcinomas (OSCC) invariably exhibit chromosomal instability (CIN) leading to aneuploidy. The mechanisms responsible for CIN in OSCC, however, are largely unknown. This study examined the fidelity of the spindle checkpoint, together with the number, structure and function of centrosomes in a series of well-characterised aneuploid immortal OSCC-derived cell lines that harbour p53 and p16(INK4A) defects. The spindle checkpoints were fully functional in 2 of 7 cell lines and attenuated in the remaining 5 cell lines. Overexpression of the spindle checkpoint protein, Cdc20, was observed in 2 of the cell lines with attenuated checkpoints. Defects in centrosome number, size and localisation were detected in 5 of the cell lines. Clonal cell populations contained cells with both normal and abnormal numbers of centrosomes, suggesting that the control of centrosome number may be inherently unstable in OSCC-derived cell lines. Centrosomal abnormalities were then examined in tissue samples of oral epithelial dysplasias and carcinomas. Abnormal centrosomes were detected in all the tissues examined albeit in a low percentage of cells (1% to5%). The percentage of cells containing centrosome abnormalities was significantly higher in the carcinomas than in the dysplasias (p0.02) and in the poorly differentiated SCCs relative to their moderately differentiated (p0.04) and well-differentiated (p0.01) counterparts. We suggest that the genetic alterations associated with the development of the immortal phenotype, together with spindle checkpoint and centrosome defects, are responsible, albeit in part, for the complex karyotypes observed in OSCC. The presence of centrosome abnormalities in oral dysplasias raises the possibility that such defects might contribute to malignant progression.

Radial forearm free flaps are used routinely to reconstruct oro-facial tissues following resection of oral squamous cell carcinoma. Surprisingly, there is little information regarding their behaviour following engraftment. The present report is a clinico-pathological study of 10 patients who had incisional biopsies of cutaneous free flaps after the presence of a white patch or erythema raised clinical suspicion. Tissues were stained with haematoxylin and eosin, diastase-periodic acid-Schiff reagent and labelled immunohistochemically for Ki-67 and p53. Four of 10 specimens showed severe epithelial dysplasia within the graft, which was contiguous with dysplasia in the adjacent oral mucosa; the remaining grafts had features typical of candidosis (n=4) or hyperkeratosis (n=2). Grafts with dysplasia had a significantly higher Ki-67 labelling index than lesions in the 'non-dysplastic' group. There were no significant differences in the Ki-67 labelling index between areas of dysplasia in the graft and areas of dysplasia in the adjacent oral epithelium. p53 staining was present in all strata of the epithelium in the dysplastic grafts and adjacent dysplastic mucosa, but was absent or weakly expressed in the stratum basale of grafts showing reactive changes only. None of the dysplastic lesions progressed to carcinoma despite a mean follow-up period of 32 months; one patient developed a recurrent mucosal tumour at the resection margin. These observations indicate that cutaneous free flaps grafted to a site of field cancerisation can develop severe epithelial dysplasia with concomitant deregulation of proliferation and increased p53 expression. Such changes raise the possibility that these lesions have the potential for malignant transformation.

BACKGROUND: Although smoking and HPV infection are recognized as important risk factors for oropharyngeal cancer, how their joint exposure impacts on oropharyngeal cancer risk is unclear. Specifically, whether smoking confers any additional risk to HPV-positive oropharyngeal cancer is not understood.METHODS: Using HPV serology as a marker of HPV-related cancer, we examined the interaction between smoking and HPV16 in 459 oropharyngeal (and 1445 oral cavity and laryngeal) cancer patients and 3024 control participants from two large European multi-centre studies. Odds ratios and credible intervals [CrI], adjusted for potential confounders, were estimated using Bayesian logistic regression.RESULTS: Both smoking [odds ratio (OR [CrI]: 6.82 [4.52, 10.29]) and HPV seropositivity (OR [CrI]: 235.69 [99.95, 555.74]) were independently associated with oropharyngeal cancer. The joint association of smoking and HPV seropositivity was consistent with that expected on the additive scale (synergy index [CrI]: 1.32 [0.51, 3.45]), suggesting they act as independent risk factors for oropharyngeal cancer.CONCLUSIONS: Smoking was consistently associated with increase in oropharyngeal cancer risk in models stratified by HPV16 seropositivity. In addition, we report that the prevalence of oropharyngeal cancer increases with smoking for both HPV16-positive and HPV16-negative persons. The impact of smoking on HPV16-positive oropharyngeal cancer highlights the continued need for smoking cessation programmes for primary prevention of head and neck cancer.