HPV-associated Oropharyngeal Squamous Cell Carcinomas Research Articles

Clinical outcomes following observation, post-operative radiation therapy, or post-operative chemoradiation for HPV-associated oropharyngeal squamous cell carcinomas

BackgroundIntermediate and high-risk features (IRFs/HRFs) for locoregional recurrence following initial surgery for oropharyngeal SCCs (OP-SCCs) were defined prior to the known association of HPV with OP-SCC. There are limited reports on practice patterns and outcomes associated with post-operative radiation therapy (PORT) or chemoradiation (POCRT) for HPV-associated OP-SCCs. Materials/Methods:The National Cancer Database was queried for patients with HPV-associated OP-SCCs managed initially with surgery with IRFs or HRFs. IRFs were defined as pT3/T4 disease, pN1-3 disease, and lymphovascular space invasion, and HRFs as positive margins and extranodal extension (ENE). Patients were stratified into no adjuvant therapy, PORT, or POCRT arms. Kaplan-Meier analysis was utilized for comparison of overall survival (OS) between treatment arms followed by a Cox multivariate (MVA) proportional-hazards model and propensity score analyses with inverse probability treatment weighting (IPTW). ResultsWe identified 6,301 patients; 51.2% had IRFs only and 48.8% had HRFs. Regarding treatment, 25.5%, 38.2%, and 36.3% of patients received no RT, PORT, and POCRT, respectively. Patients with IRFs who did not receive RT or CRT had inferior 8-year OS (81.1% vs. 87.8%; p < 0.001) that remained significant on IPTW MVA (hazard ratio (HR) = 1.69 (95% CI: 1.27–2.24; p < 0.001). Among patients with HRFs, 8-year OS was not significantly different between patients receiving PORT vs. POCRT (77.3% vs. 79.2%; p = 0.22) that remained insignificant on IPTW MVA (HR = 0.91(0.72–1.17); p = 0.48). ConclusionA significant proportion of HPV-associated OP-SCC patients with IRFs or HRFs did not receive PORT, which was associated with inferior OS. We did not demonstrate with statistical power that POCRT vs. PORT was associated with superior OS in patients with HRFs, though prospective studies are warranted.

CDKN2A/p16 evaluation in cytology specimens.

CDKN2A/p16 evaluation in cytology specimens.

Abstract 1212: Horizontal transfer of functional human papillomavirus genes in head and neck squamous cell carcinoma

Abstract Human papillomavirus (HPV) infections are currently associated with more than 70% of oropharyngeal squamous cell carcinomas (OPSCCs). HPV-positive OPSCCs display a distinct biologic behavior and treatment response and thus are now considered a distinct entity than HPV-negative OPSCCs. However, the underlying process of HPV-related OPSCC carcinogenesis is not entirely clear. HPV as a driver of cervical carcinogenesis is well-characterized with identification of a canonical mechanism dependent on HPV DNA integration, loss of HPV E2 gene expression, and increased expression of HPV E6 and E7 oncogenes with no active infection present in malignant lesions. However, whole genome sequencing found no HPV DNA integration in ~25% of HPV-associated OPSCC, suggesting an alternative mechanism of HPV carcinogenesis. Expression of all HPV genes, including self-assembling capsid proteins, in a substantial portion of HPV+ OPSCC, led us to hypothesize that tumors with episomal HPV are capable of transferring HPV genes to surrounding cells and that HPV gene transfer contributes to carcinogenesis in this subset of OPSCC. Our data support this hypothesis, showing that growth media from HPV+ OPSCC cell lines, as well as isolates from HPV+ tumors or nodal metastases, effectively transferred functional HPV genes to cells lacking HPV. The HPV DNA Transferring Agent (HDTA) derived from HPV+ OPSCC contained HPV L1 capsid protein and intact HPV genome protected from DNAase degradation. Initial characterization revealed that anti-L1 antibody blocked gene transfer and that exposure of cells to the HDTA initiated a robust innate immune response in recipient cells. Negative staining of HDTA isolate from metastatic lymph nodes revealed many pleomorphic, electron-dense particles with diameter ranging between 20 and 80nM, with approximately 40% of particles being between 50 and 60nM, consistent with the size of HPV. We propose that development and/or maintenance of OPSCCs depend on continuous production of HDTA capable of transferring HPV genes into neighboring cells. This paradigm of tumorigenesis/maintenance in OPSCC is most likely to apply to cancers lacking HPV integration, but may also apply to tumors with integration, since recent data suggested that integrated HPV may excise from the genome to recreate a transient episomal like structure, even containing cellular DNA. Our findings offer novel insights into oropharyngeal carcinogenesis and suggest targeting HDTA as a new therapeutic strategy for HPV-positive OPSCCs. Citation Format: Jason Tasoulas, Wendell Gray Yarbrough, Natalia Issaeva. Horizontal transfer of functional human papillomavirus genes in head and neck squamous cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1212.

Trends in Use and Clinical Outcomes Associated with Post-Operative Radiation Therapy for HPV-Associated Oropharyngeal Squamous Cell Carcinomas

<h3>Purpose/Objective(s)</h3> Intermediate and high-risk features (IRFs/HRFs) for locoregional recurrence following initial surgery for oropharyngeal SCCs (OP-SCCs) were defined prior to the known association of HPV with OP-SCC. There are limited reports on practice patterns and outcomes associated with post-operative radiation therapy (RT) or chemoradiation (CRT). We aimed to address these questions with the National Cancer Database (NCDB). <h3>Materials/Methods</h3> The NCDB was queried for patients with HPV-associated OP-SCCs managed initially with surgery with IRFs or HRFs. IRFs were defined as T3/T4 disease, node positive disease, and the presence of lymphovascular space invasion (LVSI), and HRFs as positive margins and extranodal extension (ENE). Patients were stratified into groups if they received no adjuvant therapy, RT, or CRT. Patients treated with RT or C-RT were required to have documentation of receipt of 50-70 Gy. Kaplan-Meier analysis was utilized for comparison of overall survival (OS) between treatment arms followed by a Cox multivariate (MVA) proportional-hazards model and propensity score analyses performed using inverse probability treatment weighting (IPTW). <h3>Results</h3> We identified 6,301 patients; 1,319 patients had positive margins, 2,266 patients had ENE, and 3,074 patients had one or two HRFs. The median number of RFs was 2 (range: 1-5). Regarding treatment, 1,606, 2,407, and 2288 patients received no RT, RT, and CRT, respectively. The median RT dose was 60 Gy (range: 50-70 Gy). Median age was 58 years (range: 24-90) and the median Charlson-Deyo comorbidity score was 0 (range: 0-3). Among patients with IRFs, 8-year OS with no RT (84.3%) was significantly lower vs. RT (90.1%) or CRT (88.8%; <i>p</i> < 0.0001) with no difference with RT vs. CRT (<i>p</i> = 0.13). On Cox MVA, RT was associated with significantly improved OS vs. no RT (hazard ratio (HR) = 0.57 (95% CI: 42.7-75.2%); <i>p</i> < 0.0001) but not CRT (HR = 1.37 (95% CI: 0.97-1.93). On IPTW analyses, receipt of RT or CRT was associated with significantly improved OS on Cox MVA (HR = 0.72 (95% CI: 0.57-0.91; <i>p</i> = 0.006). Among patients with HRFs, 8-year OS with no RT (69.1%) was significantly lower vs. RT (79.3%) or CRT (82.1%; <i>p</i> < 0.001) with no difference with RT vs. CRT (<i>p</i> = 0.22). On sub-group analysis of patients with positive margins, 8-year OS was not significantly different between RT (80.5%) and CRT (81.2%; <i>p</i> = 0.72). On Cox MVA comparing RT to CRT, CRT was not associated with significantly improved OS (HR = 0.94 (95% CI: 0.74-1.18)) for patients with HRFs. On IPTW analyses, CRT was not associated with improved 8-year OS (81.8%) vs. RT (81.5%) for patients with HRFs or on Cox MVA (HR = 0.97 (95% CI: 0.76-1.24); <i>p</i> = 0.81)). <h3>Conclusion</h3> A significant proportion of HPV-associated OP-SCC patients with IRFs or HRFs did not receive RT, which was associated with inferior OS. CRT did not result in superior outcomes compared to RT for patients with HRFs. Traditional HRFs as indications for CRT require re-evaluation for HPV-associated OP-SCC patients in prospective studies.

Impact of HIV infection on clinical outcomes among people diagnosed with head and neck cancer.

e18080 Background: Incidence of non-AIDS-defining cancers, including head and neck cancer (HNC), is rising among people with HIV (PWH) in the combination antiretroviral therapy (cART) era. The following study compares demographics, clinical outcomes, and the tumor microenvironment of HIV+ and Uninfected HNC patients at a single institution. Methods: Yale Tumor Registry query identified 3,356 HNC patients from 2002 to 2018 for analysis, including 50 PWH. In addition, quantitative immunofluorescence (QIF) was performed on tumor tissue from 22 PWH and 75 matched Uninfected patients. HPV status was based on p16 staining done universally among oropharyngeal squamous cell carcinomas (OPSCC) after 2010. Results: PWH were younger at cancer presentation compared to Uninfected patients (55.5 vs. 62.0, p &lt; 0.001), with differences in race/ethnicity and insurance status (p &lt; 0.001 for both). Biologic sex, tobacco use, alcohol consumption, anatomic site, stage at presentation, stage-specific treatment, and time to initiation of treatment were comparable between HIV+ and Uninfected cohorts. 89% of the PWH were on cART, with 79% achieving viral loads ≤200 copies/mL and a median CD4 count of 341 cells/mm³. Median survival among PWH was 39.1 months, compared to 100.8 months among Uninfected patients (p &lt; 0.001). In a multivariate (MV) analysis that included age, sex, race/ethnicity, tobacco use, anatomic site, stage, time to treatment initiation and insurance status, HIV was an independent predictor of poor outcome (HR 1.88 with 95% CI: 1.25-2.81). Difference in survival was noted particularly in early stage (stages 0, I, II) cancer, with a median survival of 73.8 months in PWH compared to 141.9 months in Uninfected patients (p = 0.001). Survival was comparable among late stage (stages III, IV) HNC patients. Among PWH, increased HIV viral load was associated with poor outcome in a MV analysis (p = 0.03). Among HPV-associated OPSCC, PWH had decreased survival compared to Uninfected patients (p &lt; 0.001). In our study of the tumor microenvironment by QIF, tumors of PWH had lower tumoral CD8 T cell infiltration (p = 0.04) and lower PD-L1 expression in tumor, stroma, and combined compartments (CPS) (p = 0.01, p = 0.03, and p = 0.01, respectively) compared to Uninfected patients. Conclusions: In our single institution study, HNC patients living with HIV experienced decreased overall survival, with HIV serving as an independent predictor of poor outcome in a multivariate analysis which included insurance status and treatment approaches. Difference in outcome was significant among early stage tumors and among HPV+ OPSCC. CD8 T cell infiltration and PD-L1 expression, both associated with improved outcomes in the general population, are decreased within the TME of PWH. Our data suggest that HIV-associated HNC is associated with poorer outcomes and highlight differences in tumor biology that require further detailed characterization in large cohorts.

Comparison of the Seventh and Eighth Edition of American Joint Committee on Cancer (AJCC) Staging for Selected and Nonselected Oropharyngeal Squamous Cell Carcinomas.

ObjectivesThe eighth edition American Joint Committee on Cancer (AJCC) Staging incorporates significant changes to the seventh edition in the staging of oropharyngeal squamous cell carcinomas (OPSCC). An important change was the inclusion of OPSCC associated with the human papilloma virus (HPV). Our goal is to compare the performance of both staging systems for patients with HPV-selected and unselected clinical characteristics for OPSCC.MethodsUsing the Surveillance, Epidemiology, and End Results (SEER) database, 2004-2016, we identified patients with likely HPV-associated OPSCC based on surrogate markers (white males aged <65 years old with squamous cell carcinomas of the tonsil and base of tongue), excluding those who underwent surgery. We re-classified these patients using seventh and eighth edition staging for HPV-selected OPSCC and compared the prediction performance of both staging editions for overall survival (OS) and disease-specific survival (DSS). We performed the same analysis for clinically unselected patients with OPSCC.ResultsOur analysis included 9554 patients with a median follow-up of 67 months. Comparing the eighth versus seventh edition for our HPV-selected cohort, clinical staging changed for 92.3% of patients and 10-year OS was 62.2%, 61.2%, 35.3%, and 15.5% for Stage I, II, III, and IV, versus 52.9%, 59.2%, 61.6%, 55.1%, 38.3%, and 15.5% for stage I, II, III, IVA, IVB, and IVC, respectively. A similar pattern was observed for 10-year DSS. The concordance statistics for our HPV-selected cohort were improved for both AJCC 7 (0.6260) and AJCC 8 (0.6846) compared with the unselected cohort, 0.5860 and 0.6457 for AJCC 7 and 8, respectively.ConclusionThe overall performance of discrimination improved from AJCC 7 to AJCC 8 for both clinically selected and unselected patients, but more notably for our HPV-selected cohort. Despite the lack of statistically significant differentiation between Stages I and II in AJCC 8 in either groups, markedly improved discrimination was observed between Stages I/II, III, and IV in the HPV-selected cohort.

De-Escalating Strategies in HPV-Associated Head and Neck Squamous Cell Carcinoma.

HPV-related head and neck squamous cell carcinoma (HNSCC) has emerged as a diverse clinical and biological disease entity, mainly in young patients with oropharyngeal tumors who are nonsmokers and nondrinkers. Indeed, during the past few years, the pendulum has shifted towards a new epidemiological reality, the “HPV pandemic”, where the majority of oropharyngeal squamous cell carcinomas (OPSCCs) are attributed to HPV. The oncogenic potential of the virus is associated to its capacity of integrating oncogenes E6 and E7 into the host cell, leading to the inactivation of several tumor suppressor genes, such as Rb. HPV status can affect prognosis in OPSCC, but its role as a predictive biomarker remains to be elucidated. Given the favorable prognosis associated with HPV-positive disease, the concept of de-escalation treatment strategies has been developed with the primary intent being the reduction of treatment-related long-term toxicities. In this review, we aim to depict current data regarding treatment de-escalation in HPV-associated OPSCC and discuss ongoing clinical trials.

Validation of the TNM-8 AJCC Classification for HPV-Positive Oropharyngeal Cancers in Patients Undergoing Trans-Oral Robotic Surgery (TORS)

High-risk human papillomaviruses (HPV) are associated with some oropharyngeal squamous cell carcinomas (OPSCC) and confer a significant prognostic factor. HPV-OPSCC have better survival outcomes compared to HPV negative tumours. The new TNM-8 AJCC staging (2018) is specific to HPV-associated OPSCC. In the re-classification study by the ‘International Collaboration on Oropharyngeal Cancer Network for Staging’, 98% of HPV-OPSCC patients were treated with primary chemo-radiation. The aim of our study was to validate the TNM-8 AJCC classification in HPV-OPSCC treated primarily with surgery (trans-oral robotic surgery or open). There were 102 patients with HPV-OPSCC treated between July 2009 and December 2014 at the Royal Adelaide Hospital. The median age was 57 years (range: 38-83) and mostly males (84.5%). 27.2% were active smokers and 50.5% reformed smokers. Early T stage cancer accounted for 72.8% (T1/2). Primary treatment was with surgery (TORS or open) and adjuvant therapy (70%) while primary chemo-radiation was the treatment for 30% of patients. Kaplan-Meier and Cox regression analyses were performed for both the 7th and 8th AJCC staging systems. The re-classification of this cohort to the AJCC 8th edition staging system resulted in a change of 54 patients from stage 4 to stages 1 and 2. This was mainly an effect of changes with N2a and N2b nodal disease being reclassified to N1 and demonstrates the prognostic effect of HPV in OPSCC. Survival outcomes were comparable with the ICON-S data. The new TNM-8 classification is therefore validated in a cohort treated, predominantly with primary surgery and adjuvant therapy. Funding Statement: None. Declaration of Interests: All authors declare no conflicts of interest. Ethics Approval Statement: Ethical approval was obtained from the CALHN REC, South Australia (Ref 13,407) to analyse clinical data on TORS.

Human Papillomavirus DNA Detection by Droplet Digital PCR in Formalin-Fixed Paraffin-Embedded Tumor Tissue from Oropharyngeal Squamous Cell Carcinoma Patients.

High-risk human papillomavirus infection impacts staging and prognosis of oropharyngeal squamous cell carcinomas (OPSCCs). Determination of HPV status in tumor tissue by p16-immunohistochemistry (p16-IHC) can be challenging; therefore, complementary methodologies could be useful in a clinical setting. To test for accuracy and clinical relevance of HPV-DNA detection in formalin-fixed and paraffin-embedded (FFPE) tumor samples by droplet digital PCR (ddPCR). Fifty OPSCCs were tested for p16-IHC status followed by HPV-16/18 DNA detection/quantification in FFPE-recovered DNA using ddPCR. Accuracy for HPV status determination and association with patient information were also evaluated. 32.0% (16/50) of the cases were p16-IHC positive (p16 +), 42.0% (21/50) had detectable levels of HPV-16 DNA, and none were positive for HPV-18 DNA. A higher median viral load of HPV-16 DNA was observed in p16 + cases (p <0.0001). Concordance between p16-IHC and HPV-16 DNA ranged from 78.0 to 86.0% and accuracy rates were between 78.0 and 86.0%. P16-IHC and HPV-16 DNA detection was associated with gender, smoking status, and tumor subsite, while only HPV-16 DNA was associated with cT stage. The combination of HPV positivity by p16-IHC and ddPCR showed higher overall survival rates in comparison with p16 + /HPV-DNA- and p16 - /HPV-DNA- results. Type-specific HPV-DNA detection by ddPCR is highly specific but moderately sensitive for the determination of HPV status and showed clinical relevance, mainly when associated with p16-IHC status. Results highlight the importance of performing HPV-DNA testing in combination with p16-IHC for proper identification of HPV-associated OPSCC and to improve clinical management of OPSCC patients.

Loss of HPV type 16 E7 restores cGAS-STING responses in human papilloma virus-positive oropharyngeal squamous cell carcinomas cells.

Human papilloma viruses (HPV) are the main culprit in cervical and oropharyngeal cancers. HPV positive (+) cancers are regarded as 'oncogene addicted', displaying an absolute requirement for the continued expression of the oncogenes for their viability owing their survival, and thus making these genes salient targets for developing specific therapeutic agents. There is a strong association between HPV and oropharyngeal squamous cell carcinomas (OPSCC), a subset of head and neck cancers (HNCs). Alarmingly, HPV-associated OPSCC are on the rise globally, and the number of cases of HPV+OPSCCs surpasses that of cervical cancer in the USA. Here, we show that major HPV oncogenes, E6 and E7, are essential for the survival of HPV positive (+) OPSCCs, making these oncogenes salient targets for HPV-driven OPSCCs. HPV E7 is known to interact with STING, a component of the viral DNA-sensing cGAS-STING machinery which activates a pro-typical anti-viral type I interferon (IFN) response. Our recent work showed that E7 from HPV type 16 is responsible for the blockade of cGAS-STING responses in HPV+OPSCC cells. In this study, we show that CRISPR/Cas9-mediated loss of E7 from HPV+OPSCC cells, SCC2 and SCC104, restored cGAS-STING responses. Future work could involve HPV oncogene targeting leading to HPV+OPSCC tumour regression and that the combined use of STING agonists would induce favourable tumour clearance by activating appropriate anti-tumour responses.

Abstract A06: Interrogation of HPV-positive oropharyngeal squamous cell carcinomas with RNA-based Nanostring assay demonstrates expression profile associated with treatment resistance

Abstract Background: Human papilloma virus (HPV)-associated oropharyngeal squamous cell carcinoma (OPSCC) is an increasingly common malignancy and public health challenge. HPV-associated OPSCCs typically respond to treatment, and there is an increasing focus on deintensification trials to minimize treatment related morbidity. However, treatment failure rates in locally advanced HPV-associated OPSCC range from 14%-32%, and there is a need to interrogate potential biomarkers of treatment response and resistance to guide patient stratification for deintensification trials and to identify novel treatment targets for high-risk patients. Methods: We prospectively identified a cohort of 8 patients with HPV-associated OPSCC who subsequently developed a biopsy-proven tumor recurrence. We also identified 8 patients with HPV-associated OPSCC matched to the above cohort by gender, smoking history, TNM stage, and treatment modality who developed a complete response to treatment. The primary samples of each cohort were interrogated with an RNA-based Nanostring immuno-oncology assay. Results: We identified that treatment-responsive tumors had significantly higher expression of NK cell markers than nonresponders (KIR3DL2, q=0.003, KIR2DL3, q=0.04). These data may indicate a potential role of the innate immune system in tumor eradication. We also identified increased expression of CCL21 in treatment-resistant tumors (q=0.04), a biomarker that has been explored in colorectal cancer due to its association with cancer stem cell-like properties. Interrogation of the tumors by smoking status demonstrated that smokers had significantly higher expression of FGF18 (q=0.003), a fibroblast growth factor linked to epithelial-to-mesenchymal transition and cisplatin resistance. We also looked at the immune checkpoint B7-H3 (CD276), a novel immunotherapeutic target associated with worse outcomes in head and neck cancer. We demonstrate that B7-H3 expression is associated with expression of epithelial-to-mesenchymal transition markers such as TWIST1 (q= 0.01), SOX11 (q= 0.006), COL5A1 (q=0.0001), and CXCL8 (q=0.02). Interestingly, low expression of B7-H3 is associated with increased CD8B expression. Conclusion: In this genomic analysis of HPV-associated oropharyngeal squamous cell carcinomas, we identify an association between presence of NK cells in the tumor microenvironment and treatment responsiveness. We also identify CCL21 as a potential biomarker of treatment resistance. Further studies are needed to confirm these biomarkers and examine the link between smoking status and immune microenvironment. Finally, we demonstrate that the immune checkpoint B7-H3 is associated with markers of epithelial-to-mesenchymal transition and warrants further exploration as a novel therapeutic target. Citation Format: Zixing Liu, Angela Mazul, Malachi Griffith, Paul Zolkind. Interrogation of HPV-positive oropharyngeal squamous cell carcinomas with RNA-based Nanostring assay demonstrates expression profile associated with treatment resistance [abstract]. In: Proceedings of the AACR-AHNS Head and Neck Cancer Conference: Optimizing Survival and Quality of Life through Basic, Clinical, and Translational Research; 2019 Apr 29-30; Austin, TX. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(12_Suppl_2):Abstract nr A06.

Inclusion of an E7 DNA Amplification Test Improves the Robustness of Human Papillomavirus-Associated Oropharyngeal Squamous Cell Carcinoma Diagnosis.

BackgroundThe rise in human papillomavirus (HPV) infection rates over the last few decades in the USA has contributed to a significant increase in the overall incidence of patients diagnosed with squamous cell carcinoma of the head and neck. These head and neck carcinomas develop in the oropharynx, with more than 90% of them caused by infection with high-risk HPV type 16. Patients diagnosed with HPV-induced oropharyngeal squamous cell carcinomas (OPSCCs) have a better prognosis and treatment response than those diagnosed with head and neck cancers caused by alcohol consumption and tobacco use. To identify patients with HPV-positive OPSCC, new guidelines recommend positive staining of oropharyngeal tissues for p16 INK4a (p16) by immunohistochemistry (IHC). Herein we discuss the testing algorithm that was adopted to address discrepant results between p16 IHC and a DNA in situ hybridization (ISH) test used routinely to diagnose HPV-positive OPSCC patients.MethodsA DNA polymerase chain reaction (PCR) test that amplifies HPV16 and HPV18 E7 was developed to aid in the diagnosis of HPV-positive OPSCC in a subset of patients. Specimens from these patients stained positive for p16 by an IHC test, but negative for high-risk HPV by a commercial DNA ISH test. Moreover, these results did not match the histopathological characteristics of the specimens, nor the clinical presentations of the patients.ResultsOf 21 patients’ specimens that were tested for p16 by IHC, 11 specimens showed concordant results with the high-risk HPV 16/18 DNA ISH test. Whereas, in eight p16 IHC positive specimens, HPV viral DNA was not detected by HPV16/18 DNA ISH, and two specimens were not tested by DNA ISH. When these eight p16 IHC positive specimens with discrepant p16 IHC and DNA ISH results were further tested by DNA PCR, six specimens showed concordance with p16 IHC with positive results for HPV16 E7, while two specimens were negative for HPV16 E7 by DNA PCR. All tested specimens were negative for HPV18 E7 by DNA PCR. Thus, the addition of the HPV16 and HPV18 E7 DNA PCR test identified a significant number of false negative test results by the HPV16/18 DNA ISH test and likely several false positive results by p16 IHC.ConclusionsInclusion of an HPV16 E7 DNA PCR test improved the robustness of HPV-associated OPSCC diagnosis in patients with discrepant results from p16 IHC staining and a DNA ISH test, and identified patients for proper management with less misclassification.

Pretreatment metabolic tumor volume as a prognostic factor in HPV-associated oropharyngeal cancer in the context of AJCC 8th edition staging.

This study evaluates the prognostic significance of 18 F-fluorodeoxyglucose-positron emission tomography ([F-18]FDG-PET)-derived metabolic tumor volume (MTV) in human papillomavirus (HPV)-associated oropharyngeal squamous cell carcinomas (OPSCCs) in the context of AJCC 8th edition staging. We performed a retrospective study of HPV-associated OPSCCs treated with postoperative or definitive radiation. The prognostic significance of pretreatment MTV for freedom from recurrence (FFR), freedom from distant metastasis (FFDM), and overall survival (OS) was determined using Kaplan-Meier analysis. Multivariate analysis (MVA) was performed using Cox regression. In this 153-patient cohort, stratifying by the optimum MTV (24 cm3 ) was prognostic for FFR (P = .0002), FFDM (P = .001), and OS (P < .0001). Metabolic tumor volume (MTV) was prognostic of FFR in AJCC 8th edition stage I/II (P = .03), and stage III patients (P = .04). On multivariate analysis only MTV was a significant factor for OS. Metabolic tumor volume (MTV) is a significant prognostic factor in HPV-associated OPSCCs, independent of AJCC 8th edition stage.

Sex differences in patients with high risk HPV-associated and HPV negative oropharyngeal and oral cavity squamous cell carcinomas

Background Human papilloma virus (HPV)-associated head and neck cancer is now recognized as a distinct clinical entity from HPV-negative tumors, which are primarily associated with tobacco and alcohol exposure.Little is known, however, about the behavior of HPV-associated oropharynx (OP) and oral cavity (OC) SCCs as two distinct cancers and how sex affects the overall survival (OS) in these two cancers. The objective of our study is to determine if sex is associated with overall survival (OS) in patients with high-risk human papillomavirus (HPV)-positive and HPV-negative squamous cell carcinomas (SCC) in the oropharynx and oral cavity sites.MethodsThis is a retrospective cohort study using a national database. Data were extracted from the National Cancer Database (NCDB) of patients diagnosed with OP or OC SCC from 2010 to 2014. Univariate and multivariate survival analyses were conducted with chi-square tests, Kaplan-Meier estimates, log-rank tests, and Cox proportional hazards multivariable modeling.ResultsA total of 30,707 patients (13,694 OP HPV-associated, 7933 OP HPV-, 1220 OC HPV-associated, 7860 OC HPV-) were identified. In all four groups, women tended to be older and have lower T and N clinical classification than men. Though there were no significant differences in OS between the sexes in OP HPV-associated cancers, female sex was associated with worse OS in OP HPV- cancers (HR: 1.15; 95% CI 1.04–1.28, p = 0.004), whereas it was associated with improved OS in OC HPV-associated and HPV- cancers (HPV-associated: HR: 0.71; 95% CI 0.50–0.99, p = 0.048; HPV-: HR: 0.87; 95% CI 0.78–0.95, p = 0.004).ConclusionThe effect of sex on OS in OC and OP SCC appears to vary based on tumor location and HPV status. While the source of this difference in prognostic association is unclear, it may be related to an emerging difference in the biology of HPV carcinogenesis in these locations.

The epidemiology of the human papillomavirus related to oropharyngeal head and neck cancer.

To summarize the epidemiology of human papillomavirus (HPV)-related oropharyngeal squamous cell carcinomas (OSCC). Articles in the English language referenced in MEDLINE/PubMed from the year 2000 onward. Relevant articles pertaining to the epidemiology of HPV-related OSCC were selected for review, with a particular preference for articles from 2008 onward. The incidence of HPV-related OSCC continues to increase in North America and Western Europe, with up to 70% of new oropharyngeal cancer cases being attributed to HPV, whereas data from the developing world remain lacking. There is evidence to support distinct risk factors for HPV-related OSCC as compared with HPV-unrelated OSCC. The long-term natural history of HPV infections remains unknown. HPV-related OSCC are associated with an improved prognosis both at the time of primary diagnosis and disease progression. There is preliminary evidence to show that HPV vaccination initiatives are effective in preventing HPV cervical infections, although data related to oral HPV infections are lacking. The epidemiology of HPV-related OSCC is evolving. Further efforts are needed to characterize the natural history of oral HPV infection and its relationship with the eventual development of HPV-associated OSCC to develop effective tools for secondary prevention. NA.

Abstract A1-63: Characterizing the DNA damage repair defect in HPV-positive oropharyngeal squamous cell carcinoma

Abstract Although tobacco-associated head and neck cancers (HNCs) are declining in incidence, overall HNC rates are escalating due to increasing prevalence of human papillomavirus (HPV)-associated tumors, especially oropharyngeal squamous cell carcinomas (OPSCCs). Clinically, patients with HPV-associated OPSCCs have improved overall survival and increased response to therapy, especially agents which act by damaging DNA. Based on these observations, we hypothesized that HPV-positive OPSCCs harbor a defect in DNA repair activity and are sensitive to DNA repair-targeted therapy. We evaluated DNA repair activity by immunofluorescent staining for DNA damage-induced formation of DNA repair protein foci. Baseline expression of DNA repair proteins was determined by NanoString nCounter analysis of transcript levels and immunoblotting for protein levels. Therapeutic sensitivity was assessed in vitro using the colony formation assay and in vivo using both cell line-derived xenografts and a patient-derived xenograft. Consistent with our hypothesis, in vitro disease models demonstrated delayed resolution of radiation-induced DNA double strand breaks (DSBs) as assessed by γH2AX foci staining and neutral comet assay. Investigation of the two main DSB repair mechanisms, non-homologous end joining (NHEJ) and homologous recombination (HR), indicated intact activation of both pathways but strikingly diminished recruitment of downstream repair factors DNA-Pk (NHEJ) and BRCA2 (HR) to sites of damage. In addition, protein expression of both DNA-PK and BRCA2 was decreased in HPV-positive compared to HPV-negative HNC cell lines. We next studied susceptibility of HPV-positive OPSCCs to PARP inhibition, a class of anticancer agents which block DNA repair signaling pathways and have proven effective clinically in DNA repair-deficient cancers. In vitro colony formation assays revealed a negative effect on cell survival in HPV-positive but not HPV-negative HNCs treated with the PARP inhibitor veliparib. Importantly, these results were confirmed in vivo in both cell line-derived xenografts and a patient-derived xenograft. In summary, our findings demonstrate for the first time the presence of a significant DNA DSB repair defect in HPV-positive OPSCCs encompassing both NHEJ and HR repair, and suggest therapies targeting DNA repair pathways may help improve therapeutic ratio in this disease. Citation Format: Alice N. Weaver, Tiffiny S. Cooper, Hoa Q. Trummell, James A. Bonner, Eben L. Rosenthal, Eddy S. Yang. Characterizing the DNA damage repair defect in HPV-positive oropharyngeal squamous cell carcinoma. [abstract]. In: Proceedings of the AACR Special Conference on Translation of the Cancer Genome; Feb 7-9, 2015; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(22 Suppl 1):Abstract nr A1-63.

Abstract 828: Methylation status of HPV16 E2-binding sites identifies subtypes of HPV-associated oropharyngeal squamous cell carcinomas

Abstract Background: The viral E2 protein is a transcriptional repressor of the HPV oncogenes E6/E7 and loss of E2 function is considered a key step in carcinogenesis. Integration of HPV into the host genome may disrupt the E2 gene. Furthermore, methylation of CpG dinucleotides in E2 binding sites (E2BSs) in the HPV upstream regulatory region (URR) may interfere with transcriptional repression of E6 and E7 by the E2 protein. We hypothesized that CpG methylation of the E2BS is found in a proportion of HPV16-associated oropharyngeal squamous cell cancers (OPSCC) in association with E2 gene integrity and viral integration. Methods: Methylation of 10 CpGs within the URR, encompassing E2BSs 1, 2, 3 and 4, was quantitatively analyzed by bisulfite pyrosequencing in 57 HPV16-associated OPSCC. E2 status was analyzed by gene amplification and quantitative real-time RT-PCR. Viral integration was determined by integration-specific PCR methods. Results: Three subgroups with differential E2BSs3 and 4 methylation were identified: a) complete methylation (&amp;gt;80%) associated with presence of integrated HPV genomes with intact E2 gene, b) intermediate methylation levels (20-80%) with predominantly episomal HPV genomes with intact E2, and c) no methylation (&amp;lt;20%) with integrated HPV with disrupted E2. Patients with complete and without methylation had poor 5-year overall survival compared to patients with intermediate methylation (stage-adjusted hazard ratio 3.8 (95%CI 1.3-11.2) and 3.1 (95%CI 1.14-8.28); p = 0.03). Conclusions: E2BSs3 and 4 methylation in oropharyngeal cancers with episomal HPV and integrated HPV with intact E2 gene might explain deregulated viral oncogene expression in the presence of E2. The methylation level appears to be of prognostic significance for patients with HPV-associated OPSCC. Citation Format: Ernst-Jan M. Speel, Miriam Reuschenbach, Christian U. Huebbers, Elena-Sophie Prigge, Justo L. Bermejo, Simon Kalteis, Simon Preuss, Jutta Kolligs, Nadine C. Olthof, Bernd Kremer, Steffen Wagner, Jens P. Klussmann, Svetlana Vinokurova, Magnus von Knebel Doeberitz. Methylation status of HPV16 E2-binding sites identifies subtypes of HPV-associated oropharyngeal squamous cell carcinomas. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 828. doi:10.1158/1538-7445.AM2015-828

Epigenetic Mechanisms of Human Papillomavirus-Associated Head and Neck Cancer.

Growing evidence suggests that as many as half of all oropharyngeal squamous cell carcinomas (OPSCCs) harbor human papillomavirus (HPV) infections. Despite being more advanced at diagnosis, HPV-positive OPSCCs are associated with a better response to therapy and longer patient survival than HPV-negative OPSCCs. Human papillomavirus-positive OPSCC has also been shown to have distinct host gene expression profiles compared with HPV-negative OPSCC. Recently, this distinction has been shown to include the epigenome. It is well supported that cancers are epigenetically deregulated. This review highlights epigenetic differences between HPV-positive and HPV-negative OPSCCs. The epigenetic mechanisms highlighted include methylation changes to host and viral DNA, and host chromatin modification. We also review the current evidence regarding host DNA methylation changes associated with smoking, and deregulation of microRNA expression in HPV-positive OPSCC. To provide an overview of epigenetic mechanisms reported in HPV-positive OPSCC, with analogies to cervical cancer, and discussion of the challenges involved in studying epigenetic changes in HPV-associated OPSCC in combination with changes associated with smoking. Sources were a literature review of peer-reviewed articles in PubMed on HPV and either OPSCC or head and neck squamous cell carcinoma, and related epigenetic mechanisms. Epigenetic changes are reported to be a contributing factor to maintaining a malignant phenotype in HPV-positive OPSCC. The epigenetic mechanisms highlighted in this review can be studied for potential as biomarkers or as drug targets. Furthermore, continued research on the deregulation of epigenetic mechanisms in HPV-positive OPSCC (compared with HPV-negative OPSCC) may contribute to our understanding of the clinical and biologic differences between HPV-positive and HPV-negative OPSCC.

Treatment De-intensification in HPV-Associated Oropharyngeal Cancer: Evidence, Controversies, and Strategies

A subset of oropharyngeal squamous cell carcinomas (OPSCC) has been shown to be related to human papillomavirus (HPV). These cancers have improved prognosis compared to non-HPV-associated OPSCC. The recognition of this favorable prognosis cohort of patients has occurred in the midst of decades of investigations seeking to intensify local-regional therapy to improve tumor control and survival rates. With long-term follow-up, it is now clear that these intensification strategies are associated with significant late toxicities prompting a re-evaluation of the role of intensified local-regional therapy for HPV-associated OPSCC. The de-intensification strategies that have been evaluated or currently under evaluation are reviewed in this article.

Identification of a Human Papillomavirus–Associated Oncogenic miRNA Panel in Human Oropharyngeal Squamous Cell Carcinoma Validated by Bioinformatics Analysis of The Cancer Genome Atlas

High-risk human papillomavirus (HPV) is a causative agent for an increasing subset of oropharyngeal squamous cell carcinomas (OPSCCs), and current evidence supports these tumors as having identifiable risk factors and improved response to therapy. However, the biochemical and molecular alterations underlying the pathobiology of HPV-associated OPSCC (designated HPV(+) OPSCC) remain unclear. Herein, we profile miRNA expression patterns in HPV(+) OPSCC to provide a more detailed understanding of pathologic molecular events and to identify biomarkers that may have applicability for early diagnosis, improved staging, and prognostic stratification. Differentially expressed miRNAs were identified in RNA isolated froman initial clinical cohort of HPV(+/-) OPSCC tumors by quantitative PCR-based miRNA profiling. This oncogenic miRNA panel was validated using miRNA sequencing and clinical data from The Cancer Genome Atlas and miRNA in situ hybridization. The HPV-associated oncogenic miRNA panel has potential utility in diagnosis and disease stratification and in mechanistic elucidation of molecular factors that contribute to OPSCC development, progression, and differential response to therapy.