HPV-associated Head And Neck Squamous Cell Carcinoma Research Articles

Immune Landscape and Role of Immunotherapy in Treatment of HPV-Associated Head and Neck Squamous Cell Carcinoma (HNSCC)

Immune Landscape and Role of Immunotherapy in Treatment of HPV-Associated Head and Neck Squamous Cell Carcinoma (HNSCC)

Recurrent Human Papillomavirus-Related Head and Neck Cancer Undergoes Metabolic Reprogramming and Is Driven by Oxidative Phosphorylation.

Human papillomavirus (HPV) infection drives the development of some head and neck squamous cell carcinomas (HNSCC). This disease is rapidly increasing in incidence worldwide. Although these tumors are sensitive to treatment, approximately 10% of patients fail therapy. However, the mechanisms that underlie treatment failure remain unclear. We performed RNA sequencing (RNA-seq) on tissues from matched primary- (pHNSCC) and metachronous-recurrent cancers (rHNSCC) to identify transcriptional differences to gain mechanistic insight into the evolutionary adaptations of metachronous-recurrent tumors. We used HPV-related HNSCC cells lines to investigate the effect of (i) NRF2 overexpression on growth in vitro and in vivo, (ii) oxidative phosphorylation (OXPHOS) inhibition using IACS-010759 on NRF2-dependent cells, and (iii) combination of cisplatin and OXPHOS inhibition. The OXPHOS pathway is enriched in recurrent HPV-associated HNSCC and may contribute to treatment failure. NRF2-enriched HNSCC samples from The Cancer Genome Atlas (TCGA) with enrichment in OXPHOS, fatty-acid metabolism, Myc, Mtor, reactive oxygen species (ROS), and glycolytic signaling networks exhibited worse survival. HPV-positive HNSCC cells demonstrated sensitivity to the OXPHOS inhibitor, in a NRF2-dependent manner. Further, using murine xenograft models, we identified NRF2 as a driver of tumor growth. Mechanistically, NRF2 drives ROS and mitochondrial respiration, and NRF2 is a critical regulator of redox homeostasis that can be crippled by disruption of OXPHOS. NRF2 also mediated cisplatin sensitivity in endogenously overexpressing primary HPV-related HNSCC cells. These results unveil a paradigm-shifting translational target harnessing NRF2-mediated metabolic reprogramming in HPV-related HNSCC.

Abstract 1930: 4-nitroquinoline-1-oxide enhances induction of apoptosis preferentially in HPV positive head and neck squamous cell carcinoma cells

Abstract Background: Tobacco use and human papillomavirus (HPV) infection are both strong contributors to the oncogenesis of head and neck squamous cell carcinoma (HNSCC). Despite HPV's causative role in HNSCC, it confers no statistically significant difference in mutational profile between HPV-associated HNSCC smokers and non-smokers. Our earlier preliminary data suggested that HPV positive (+) HNSCC cells have better activation of DNA repair proteins compared to HPV negative (-) HNSCC cells when treated with 4NQO, a mutagen and carcinogen which serves as a surrogate for tobacco exposure. This may partially explain HPV+ HNSCC's higher mutational resistance. In addition to increased DNA repair, HPV+ cells may also eliminate mutations through higher rates of apoptosis compared to HPV- cells. Hence, we wanted to test this hypothesis by analyzing the effects of 4NQO on apoptosis in HPV+ and HPV- HNSCC cells. Methods: NOK, NOK E6.E7, SCC40 (HPV-), SCC104 and UMSCC47 (HPV+) cells were treated with different concentrations of 4NQO (0, 0.1, and 0.2 ug/ml) for 24 and 48 hours. Untreated (control) and 4NQO treated cells were assessed for cell viability using MTT assay. Apoptotic cells were determined using Annexin-V FITC flow cytometry analysis, and Western blot was performed to check the apoptotic proteins for the afore-mentioned control and 4NQO treatments. Results: MTT assay showed decreased cell viability of NOK E6.E7, SCC47 and SCC104 cells in response to 4NQO treatment compared to relatively higher cell viability of SCC40 cells. Flow cytometry confirmed that HPV+ HNSCC cells are relatively more apoptotic compared to HPV- cells following 4NQO treatment at 24 and 48 hours. This is further supported by activation of pro-apoptotic proteins including p53, cleaved caspase 3, cleaved PARP, cleaved caspase 9, and p21 to a greater extent in HPV+ cells at different time points of 4NQO treatment using Western blot analysis. Conclusion: Our in-vitro data suggest HPV may stimulate a protective measure against smoking-induced mutations by increasing apoptosis before cells can contribute to the HNSCC mutational profile. Taken together, these data explain why HPV associated HNSCC have a better prognosis than HPV negative irrespective of smoking history. Citation Format: Gauri Shishodia, Alice Lee, Rhodee Ric Toledo, Emily Zimmerman, Xiaohua Rong, Adam Xiao, Lynn Harrison, Cherie Ann Nathan. 4-nitroquinoline-1-oxide enhances induction of apoptosis preferentially in HPV positive head and neck squamous cell carcinoma cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1930.

DNA Methylation and HPV-Associated Head and Neck Cancer.

Head and neck squamous cell carcinoma (HNSCC), especially oropharyngeal squamous cell carcinoma (OPSCC), has recently been found to be significantly associated with human papillomavirus (HPV) infection. The incidence of OPSCC has been increasing and surpassed the number of cervical cancer cases in the United States. Although HPV-associated OPSCC has a relatively better prognosis than HPV-negative cancer, approximately 20% of HPV-associated HNSCC patients show a poor prognosis or therapeutic response, and the molecular mechanism behind this outcome in the intermediate-risk group is yet to be elucidated. These biological differences between HPV-associated HNSCC and HPV-negative HNSCC are partly explained by the differences in mutation patterns. However, recent reports have revealed that epigenetic dysregulation, such as dysregulated DNA methylation, is a strikingly common pathological feature of human malignancy. Notably, viral infections can induce aberrant DNA methylation, leading to carcinogenesis, and HPV-associated HNSCC cases tend to harbor a higher amount of aberrantly methylated DNA than HPV-negative HNSCC cases. Furthermore, recent comprehensive genome-wide DNA-methylation analyses with large cohorts have revealed that a sub-group of HPV-associated HNSCC correlates with increased DNA methylation. Accordingly, in this review, we provide an overview of the relationship between DNA methylation and HPV-associated HNSCC.

Determining the molecular landscape and impact on prognosis in HPV-associated head and neck cancer

BackgroundHuman papillomavirus (HPV) associated head and neck squamous cell carcinoma (HNSCC) has a better prognosis than HNSCC due to other risk factors. However, there is significant heterogeneity within HPV-associated HNSCC and 25% of these patients still do poorly despite receiving aggressive therapy. We currently have no good molecular tools to differentiate and exclude this “high-risk” sub-population and focus on “low-risk” patients for clinical trials. This has been a potential barrier to identifying successful de-escalation treatment strategies in HPV-associated HNSCC. We conducted an analysis of molecular markers with a well-known role in the pathogenesis of HPV-associated HNSCC and hypothesized that these markers could help independently predict recurrence and prognosis in these patients and therefore help identify at the molecular level “low-risk” patients suitable for de-escalation trials.MethodsWe analyzed 24 tumor specimens of patients with p16+ HNSCC who underwent definitive resection as primary treatment. Tissue microarray (TMA) was generated from the 24 pathology blocks and immunohistochemistry (IHC) was performed using highly specific antibodies for our chosen biomarkers (PI3K-PTEN, AKT pathway, mTOR, 4EBP1, S6, and pAMPK, ERCC-1). Transcriptome data was also obtained for 7 p16+ HNSCC patients from The Cancer Genome Atlas (TCGA). Data from the TMA and TCGA were analyzed for association of relapse-free survival (RFS) and overall survival (OS) with protein and gene expression of the chosen biomarkers.ResultsIncreased pAMPK protein activity by IHC and AMPK gene expression by TCGA gene expression data was correlated with improved RFS with a trend towards statistical significance.ConclusionsThis data suggests that increased pAMPK activity and expression may portend a better prognosis in HPV-associated HNSCC undergoing primary definitive resection. However, these findings require validation in larger studies.

Characteristics and Impact of HPV-Associated p16 Expression on Head and Neck Squamous Cell Carcinoma in Thai Patients.

Background:Head and neck squamous cell carcinoma (HNSCC) is a common malignancy in Asia. Infection by human papilloma virus (HPV) has been recognized as an etiological risk for HNSCC, especially oropharyngeal region. While the association between HPV and HNSCC has been well evaluated in Western countries, only a few investigated the HPV-associated HNSCC in Southeast Asia. This study evaluated the prevalence, the characteristics, and the impact of HPV on the treatment outcomes in Thai HNSCC patients. Methods:Non-nasopharyngeal HNSCC patients treated at Ramathibodi Hospital during 2007-2013 were identified through the cancer registry database. Baseline patient, treatment data and survivals were retrospectively reviewed. The formalin-fixed paraffin-embedded (FFPE) tissue sections were retrieved for p16 analysis. The HPV status was determined by p16 immunohistochemistry. The survival outcomes were analyzed in cases which p16 status was confirmed. Results:Total of 200 FFPE tissues of HNSCC patients was evaluated for p16 expression. Positive p16 status was observed in 24 cases (12%); majority of p16-positive were men (20:4 cases). The oropharynx (37.9%) was the most common site found in p16-positive while oral cavity (3.2%) was the least common site. Interestingly, 66.7% of p16-positive were former/current smokers, and 70.8% of this subgroup was categorized as clinical AJCC stage III-IV. The p16-positive HNSCC was significantly superior in 5-year overall survival [5-yrs OS 63% vs. 40%, p=0.03], 5-year disease-free survival [5-yrs DFS 61% vs. 36%, p=0.03] and in 5-year locoregional relapse-free survival [5-yrs LRFS 93% vs. 68%, p=0.018] when compared with p16-negative. Conclusions:In comparison to the results from the Western countries, the prevalence of HPV-related HNSCC in Thai patients was less, and differences in some characteristics were observed. Nevertheless, improvement in OS, DFS and LRFS were observed in p16-positive patients. Our analyses suggested that p16 status is also a strong prognostic marker for HNSCC patients in Thailand.

Endogenously-Activated Ultrasmall-in-Nano Therapeutics: Assessment on 3D Head and Neck Squamous Cell Carcinomas.

Negative or positive HPV-associated Head and Neck Squamous Cell Carcinomas (HNSCCs) are high recurrence neoplasms usually resulting in a poor prognosis, mainly due to metastasis formation. Despite the low overall patient survival rate and the severe side effects, the treatment of choice is still cisplatin-based chemotherapy. Here, we report a straightforward protocol for the production of high throughput 3D models of negative or positive HPV-associated HNSCCs, together with their employment in the therapeutic evaluation of gold ultrasmall-in-nano architectures comprising an endogenously-activatable cisplatin prodrug. Beyond enhancing the biosafety of cisplatin, our approach paves the way for the establishment of synergistic co-therapies for HNSCCs based on excretable noble metals.

Oropharyngeal HPV Detection Techniques in HPV-associated Head and Neck Cancer Patients.

The incidence of human papilloma virus (HPV)-related head and neck squamous cell carcinoma (HNSCC) has been increasing in the last decades. Analysis of oral brushing or rinsing samples for screening or stratification could potentially improve screening and prevention. Oral brushes and mouthwashes were taken from 20 patients with HPV-associated HNSCC before definite therapy. HPV genotyping was performed for the detection of 14 high-risk HPV subtypes and correlated to DNA isolated from tumor tissue. Ten of 20 patients were tested HPV positive by using either method. There was a significant correlation between macroscopic visibility of tumor and positive HPV detection (p<0.001) and HPV detection and tumor size (p<0.001). HPV was detected in all macroscopically visible tumors. Half of the HPV cases who had macroscopically invisible tumors were missed by both methods. Both techniques are limited in the detection of macroscopically non-visible and small tumors. Therefore, the application of these techniques for screening or diagnosis of HNSCC is not recommended.

STOP HPV study protocol: a nationwide case–control study of the association between oropharyngeal cancer and human papillomavirus (HPV) infection in Brazil

IntroductionHuman papillomavirus (HPV) is the most common sexually transmitted infection and is associated with several types of cancer. The number of cases of HPV-associated head and neck squamous cell carcinomas...

Immunotherapy for head and neck cancers

Head and neck squamous cell carcinoma (HNSCC) is a frequent tumour which arises from various anatomical areas in the head and neck region. HNSCC has multiple resistance mechanisms through which it evades the immune responses. It is particularly characterized by an immunosuppressive environment which includes the release of immunosuppressive factors, expansion, and expansion of immune cells which have inhibitory activity reduction of tumour immunogenicity. Human papillomavirus positive (HPV+) HNSCC tumours have one of the higher levels of T cells infiltration. Studies which explore this relationship to the prognosis of patients vary, with some showing benefit only with high CD8/Treg ratio as seen with HPV+ diseaseand others showing improved prognosis with a higher number of TIL Treg. High CD8+ TIL seen in HPV + disease has been shown in several studies to confer improved disease-free survival. The most successful vaccination strategy is preventive vaccination for HPV. Investigations using different approaches have been carried out on therapeutic vaccines for HPV-associated HNSCC. Despite immune responses being seen in a number of studies, these vaccines are still not effective for clinical use as of yet.

Incidence and Outcomes of HPV-Associated Head and Neck Cancers in the United States: A Large Population-Based Analysis

Human papillomavirus (HPV)-associated oropharyngeal cancers are increasing in the United States (US); yet there are no large epidemiologic studies to define the national burden of HPV-associated head and neck squamous cell carcinomas (HNSCC) of oropharynx and non-oropharynx subsites. A custom Surveillance, Epidemiology, and End Results (SEER) database identified 5,936 patients with HNSCC (oropharynx, nasopharynx, hypopharynx, or other pharynx) and known HPV status, diagnosed from 2013-2014. Age-adjusted HPV-positive HNSCC incidence rates per 100,000 persons were calculated using SEER*Stat. Associations between HPV status and cancer-specific mortality (CSM) were determined using multivariable Fine-Gray competing-risks regression. From 2013-2014, the US incidence of HPV-positive versus negative HNSCC was 2.43 (95% Confidence Interval [CI] 2.35-2.50) versus 1.28 (1.22-1.33) per 100,000 persons, respectively. HPV-positive incidence was 2.26 (2.19-2.34), 0.06 (0.05-0.07), 0.06 (0.05-0.07), and 0.04 (0.01-0.03) for oropharynx, nasopharynx, hypopharynx, and other pharynx HNSCC, respectively. White (2.80, 2.71-2.90), non-Hispanic (2.68, 2.59-2.77) and male patients (4.38, 4.23-4.53) had the highest incidences of HPV-positive disease, with a unimodal age incidence distribution peaking at ages 60-64 (8.04, 7.49-8.62); notably, non-Hispanic white men ages 60-64 had an incidence of 18.56 (17.09-20.13). HPV-positive disease was associated with lower CSM in oropharynx HNSCC (Adjusted Hazard Ratio [AHR] 0.40, P<0.001; 23-month CSM 4.5% versus 11.2%), but not in non-oropharynx HNSCC (AHR 1.08, P=0.81; 23-month CSM 12.2% versus 11.4%), Poropharynx*HPV interaction = 0.002. This large epidemiological study defines the US burden of HPV-associated HNSCC. The highest incidence of US incidence of HPV-positive HNSCC was in non-Hispanic white men, where it represents the sixth most common non-skin solid malignancy in patients 64 and younger. The favorable prognosis associated with HPV appears to be limited to the oropharynx.

Invasion and metastasis mechanism of human papillomavirus in head and neck squamous cell carcinomas

Human papillomavirus (HPV) is a major causative agent of head and neck squamous cell carcinomas (HNSCC). Over the past several decades, an increasing number of studies established the strong association of HPV with the invasion and metastasis of HNSCC. In the present study, we reviewed the gene mutations in HPV-associated HNSCC and the unique mechanism of E6- and E7-mediated carcinogenesis via interactions with an array of cellular elements. We further discussed the progress in the mechanisms of invasion and metastasis; these mechanisms include non-coding RNAs, deregulating cellular energetics, tumor microenvironment, cancer stem cells, angiogenesis, and lymphangiogenesis.

Incidence and Risk of Second Primary Malignant Neoplasm After a First Head and Neck Squamous Cell Carcinoma

Second primary malignant neoplasms (SPMNs) are the leading cause of death in survivors of head and neck squamous cell carcinoma (HNSCC). Recently, human papillomavirus (HPV) has emerged as a risk factor for oropharyngeal squamous cell carcinoma and has different prognosis from classic tobacco/alcohol-associated HNSCC. This suggests that there also may be different risks and burden of SPMNs among patients who's HNSCC were from HPV or tobacco and/or alcohol. To assess SPMN risks and burden in a large US cohort of patients with a first potentially HPV-associated HNSCC vs non-HPV-associated HNSCC. In this population-based retrospective cohort study, 109 512 adult patients diagnosed with HNSCC between 2000 and 2014 were identified from the Surveillance, Epidemiology, and End Results registry. HPV-relatedness based on whether patients' first HNSCC was potentially associated with HPV. Patients were grouped into 2 cohorts: potentially HPV-associated HNSCC, and non-HPV-associated HNSCC. The primary outcome was incidence of SPMN (defined as the first subsequent primary cancer occurring at least 2 months after first cancer diagnosis). Excess SPMN risk was calculated using relative (standardized incidence ratios [SIRs]) and absolute (excess absolute risk [EAR] per 10 000 person-years at risk [PYR]). A total of 109 512 patients with HNSCC (mean [SD] age, 61.9 [12.1] years; 83 305 [76.1%] men) were identified. The overall SIR was 2.18 (95% CI, 2.14-2.22) corresponding to 160 excess cases per 10 000 PYR. The risk among patients with first potentially HPV-associated HNSCC (SIR, 1.98; EAR, 114 excess cases per 10 000 PYR) was lower than those with first non-HPV-associated HNSCC (SIR, 2.28; EAR, 188 excess cases per 10 000 PYR). Overall, the largest SIRs and EARs were observed for cancers of the head and neck, lung, and esophagus. However, the risks of SPMN were lower among potentially HPV-associated HNSCC patients. Patients diagnosed with HNSCC experience excess risk of SPMN, which was higher among those with non-HPV-associated HNSCC than from potentially HPV-associated HNSCC. Clinicians should implement strategies that prevent or detect SPMN early in patients with HNSCC.

Viral DNA integration and methylation of human papillomavirus type 16 in high-grade oral epithelial dysplasia and head and neck squamous cell carcinoma.

This study evaluated the integration and methlyation of human papillomavirus type 16 (HPV16) in head and neck squamous cell carcinoma (HNSCC) and its oral precursor, high-grade oral epithelial dysplasia (hgOED). Archival samples of HPV16-positive hgOED (N = 19) and HNSCC (N = 15) were evaluated, along with three HNSCC (UMSCC-1, -47 and -104) and two cervical cancer (SiHa and CaSki) cell lines. HgOED cases were stratified into three groups with increasing degrees of cytologic changes (mitosis, karyorrhexis and apoptosis). The viral load was higher and the E2/E6 ratio lower (indicating a greater tendency toward viral integration) in group 3 than in groups 1 or 2 (p = 0.002, 0.03). Methylation was not observed in hgOED cases and occurred variably in only three HNSCC cases (26.67%, 60.0% and 93.3%). In HNSCC cell lines, lower E7 expression correlated with higher levels of methylation. HgOED with increased cytologic change, now termed HPV-associated oral epithelial dysplasia (HPV-OED), exhibited an increased viral load and a tendency toward DNA integration, suggesting a potentially increased risk for malignant transformation. More detailed characterization and clinical follow-up of HPV-OED patients is needed to determine whether HPV-OED is a true precursor to HPV-associated HNSCC and to clarify the involvement of HPV in HNSCC carcinogenesis.

Abstract 220: Intra-tumor immune heterogeneity is associated with prognosis of oropharyngeal head and neck squamous cell carcinoma

Abstract As cell-cell interactions among tumor and immune cells are known to contribute to tumor progression, in depth evaluation of tumor-immune heterogeneity will enable biomarker-guided patient stratification and improvement of treatment response. Here we examined head and neck squamous cell carcinomas (HNSCC) to reveal the prognostic significance of leukocytes in tumors with regards to their complexity, effector status and spatial characteristics via multiplex immunohistochemistry and image cytometry. To accomplish this, we examined oropharyngeal HNSCC (N = 38), where lymphoid, myeloid, and hypo-inflamed leukocyte complexity correlated with HPV-status (Tsujikawa et al. Cell Reports, 2017). In the present study, quantitative analysis of cell density and distribution of 14-distinct immune cell lineages (e.g., CD8+ T cells, regulatory T cells, Th1, Th2, Th17, Th0 lymphocytes, B cells, NK cells, CD163+ and CD163- macrophages, mature and immature dendritic cells, mast cells, granulocytes) was analyzed via immune cell density mapping and tissue segmentation algorithms. We revealed tropism of polarized Th1-type cells within tumor nests versus stroma in HPV-associated HNSCC. In addition, Cox regression analysis of cell density and distribution of the 14 immune populations revealed that CD66b+ granulocyte infiltration within tumor nests reflected a negative prognostic indicator for HNSCC outcome. Furthermore, cell-cell proximity analysis in HPV-associated HNSCC further revealed a correlation between PD-L1 positive immune cells, and micro-regionally polarized immune characteristics biased towards Th1, coincident with high density of CD8+ T cells. These results reveal intra-tumor immune heterogeneity is associated with micro-regional immune complexity profiles, and provide insight into in situ immune characteristics that may aid patient stratification for immune therapy going forward. Acknowledgement: This project was supported by the Japan Society for the Promotion of Science Grant-in-Aid for Young Scientists (Start-up, 17H07016), Oregon Clinical and Translational Research Institute (OCTRI), grant number (UL1TR000128) from the National Center for Advancing Translational Sciences (NCATS) at the National Institutes of Health (NIH), and P30 CA069533-17 OHSU Knight Cancer Institute. LMC acknowledges support from the NIH/NCI, DOD BCRP Era of Hope Scholar Expansion Award, Susan G. Komen Foundation, Stand Up To Cancer - Lustgarten Foundation Pancreatic Cancer Convergence Dream Team Translational Research Grant, Breast Cancer Research Foundation, and the Brenden-Colson Center for Pancreatic Health. Citation Format: Takahiro Tsujikawa, Guillaume Thibault, Young Hwan Chang, Edward El Rassi, Daniel R. Clayburgh, David Sauer, Akihito Arai, Molly F. Kulesz-Martin, Motomi Mori, Shigeru Hirano, Paul W. Flint, Lisa M. Coussens. Intra-tumor immune heterogeneity is associated with prognosis of oropharyngeal head and neck squamous cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 220.

Abstract LB-333: PARP-1 supports HPV infection and HPV-associated head and neck cancer

Abstract Background: As the incidence of human papillomavirus (HPV)-related head and neck squamous cell carcinoma (HNSCC) continues to rise, mechanistic knowledge of HPV16-driven HNSCC remains incomplete. HPV(+) and HPV(-) HNSCCs are molecularly distinct, with altered signaling pathways that have been characterized by proteomic technologies. We recently reported that poly(ADP-ribose)polymerase-1 (PARP-1) is overexpressed in HPV(+) compared to HPV(-) HNSCC. PARP-1 is a multi-functional protein with the ability to produce polymers of ADP-ribose attached to proteins and with major known roles in maintaining genomic integrity, DNA damage repair, transcription, DNA replication, and cell cycle regulation. However, PARP-1 functions in HPV infection and in head and neck cancer have not been fully uncovered. Here, we examine the role of PARP-1 in HPV infection, the interactions of HPV with cellular proteins, and the therapeutic implications of PARP-1 inhibition in HPV(+) HNSCC. Methods: PARP activity was assessed by immunoblotting for levels of poly(ADP-ribose) and by enzymatic PARP-1 ELISA. Expression of HPV genes was measured by RT-PCR. Murine kidney epithelial cells were established from PARP-1 wild-type and knockout mice, and the absence of PARP-1 expression was confirmed by RT-PCR and immunoblotting. Infection with HPV pseudovirus (PsV) containing a GFP expression plasmid was detected by L1 immunofluorescence staining and GFP detection. Survival studies were done in vitro and in vivo with PARP inhibitors veliparib and olaparib. Results: In addition to being overexpressed, we found that PARP-1 was highly enzymatically active in HPV(+) HNSCC as compared to HPV(-) tumors. HPV16 PsV and major capsid protein L1 activated PARP-1 in vitro and in cells. Depletion of PARP-1 and chemical PARP inhibition repressed HPV16 cellular entry, and infection of cells with HPV PsV increased sensitivity of HPV(-) cells to PARP inhibition. Conclusions: We identified a functional role of PARP-1 in initial HPV infection and in HPV-associated cancer. PARP-1 is activated by HPV major capsid protein L1, and HPV(+) HNSCCs maintain PARP-1 enzymatic activity. On top of its role in initial HPV infection, PARP-1 also appears to be important for survival of HPV-infected cells, as inhibition of PARP-1 sensitized cells to HPV PsV infection. These findings suggest that PARP inhibition may hold significant therapeutic potential in the treatment of HPV-associated HNSCC, with our work resulting in the initiation of a window clinical trial ongoing at the Yale Cancer Center. Citation Format: Cassie Pan, Asel Biktasova, Michael Hajek, Andrew Sewell, Tejas Sathe, Gary Bellinger, Wendell Yarbrough, Natalia Issaeva. PARP-1 supports HPV infection and HPV-associated head and neck cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr LB-333.

Abstract A33: Head and neck squamous cell carcinoma in adolescents and young adults: Survivorship patterns and disparities

Abstract Introduction: Head and neck squamous cell carcinoma (HNSCC) incidence and survivorship has been thoroughly described in the literature. Most of the literature has mainly focused on older adults. However, there are documented cases of early-onset HNSCC, typically in adolescents and young adults (AYA) aged 15-39 years. In fact, 12% of all pediatric cancers are in the head and neck region. As individuals live longer in the general population, it will become more important to track AYA cancer cases due to the potential for treatment effects, late toxicities, and comorbidities after cancer survivorship. Additionally, the emergence of the human papillomavirus (HPV)-associated oropharyngeal cancer as a dominant head and neck cancer means that more HNSCC patients are diagnosed at a younger age than previously known. These patients with HPV-associated oropharyngeal cancer are likely to live longer since HPV-associated HNSCC typically has better prognosis than other HNSCC. While numerous studies have investigated HNSCC survivorship in older adults and the elderly, limited research exists describing the incidence and survivorship in adolescents and young adults. The aim of this study is to characterize survivorship of HNSCC in the AYA population. Methods: In this retrospective study, we utilized a patient cohort of 3,366 first primary HNSCC cases from the Surveillance, Epidemiology, and End Results (SEER) 18 database diagnosed between ages 15-39 and the years 2000-2014. Actuarial survival curves stratified by age group (15-29, 30-34, 35-39) indicated differences in HNSCC survival among groups with a log-rank test. Patient characteristics including age, sex, race/ethnicity, HNSCC site, stage, and treatment modality were utilized in a Fine and Gray competing risk proportional hazard model to examine their impact on HNSCC death in this cohort. Results: The cohort was mostly male (65.1%) with an average age of 33.6. There was no significant HNSCC survival difference between the age-stratified survival curves (log-rank p=0.83). The Fine and Gray model also did not find a significant effect for age. All race/ethnicity groups had a significantly increased hazard of HNSCC death compared with non-Hispanic whites, with non-Hispanic American Indians/Alaska Natives having the highest increased hazard (aHR=4.01, 95% CI: 2.18, 7.38). Each increasing year of diagnosis was associated with a 5% decrease in hazard of death from HNSCC (aHR=0.95, 95% CI: 0.93, 0.97). Regional (aHR=3.90, 95% CI: 3.13, 4.86) and distant (aHR=6.77, 95% CI: 5.27, 8.70) stage had a higher HNSCC death hazard compared with localized stage. Patients who did not receive surgery had a 93% (aHR=1.93, 95% CI: 1.60, 2.33) increased hazard of HNSCC death compared with those who received surgery. Compared with oropharyngeal cancer, hypopharyngeal, oral cavity, and sinonasal cancers were associated with significantly increased hazard of HNSCC death, while nasopharynx was associated with a decreased hazard (aHR=0.68, 95% CI 0.52, 0.90). Conclusions: We found no survival differences between adolescents and young adults based on age at diagnosis; however, minorities, especially American Indians/Alaskan Natives, had the worst survival outcomes. Also, patients who did not have the definite treatment of surgery and had regional or distant stage faced increased death from HNSCC. More research needs to be done to understand the mechanisms underlying the survival disparities noted among minorities, especially American Indians/Alaskan Natives. Efforts should also focus on establishing risk factor awareness and educational interventions, earlier screening measures, and patient navigator programs to improve prognosis of HNSCC in the American Indian/Alaskan Natives and other high-risk populations. Citation Format: Sai D. Challapalli, Eric Adjei Boakye, Matthew C. Simpson, Nosayaba Osazuwa-Peters. Head and neck squamous cell carcinoma in adolescents and young adults: Survivorship patterns and disparities [abstract]. In: Proceedings of the Tenth AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2017 Sep 25-28; Atlanta, GA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2018;27(7 Suppl):Abstract nr A33.

Demethylation Therapy as a Targeted Treatment for Human Papillomavirus-Associated Head and Neck Cancer.

Purpose: DNA methylation in human papillomavirus-associated (HPV+) head and neck squamous cell carcinoma (HNSCC) may have importance for continuous expression of HPV oncogenes, tumor cell proliferation, and survival. Here, we determined activity of a global DNA-demethylating agent, 5-azacytidine (5-aza), against HPV+ HNSCC in preclinical models and explored it as a targeted therapy in a window trial enrolling patients with HPV+ HNSCC.Experimental Design: Sensitivity of HNSCC cells to 5-aza treatment was determined, and then 5-aza activity was tested in vivo using xenografted tumors in a mouse model. Finally, tumor samples from patients enrolled in a window clinical trial were analyzed to identify activity of 5-aza therapy in patients with HPV+ HNSCC.Results: Clinical trial and experimental data show that 5-aza induced growth inhibition and cell death in HPV+ HNSCC. 5-aza reduced expression of HPV genes, stabilized p53, and induced p53-dependent apoptosis in HNSCC cells and tumors. 5-aza repressed expression and activity of matrix metalloproteinases (MMP) in HPV+ HNSCC, activated IFN response in some HPV+ head and neck cancer cells, and inhibited the ability of HPV+ xenografted tumors to invade mouse blood vessels.Conclusions: 5-aza may provide effective therapy for HPV-associated HNSCC as an alternative or complement to standard cytotoxic therapy. Clin Cancer Res; 23(23); 7276-87. ©2017 AACR.

Prevalence and types of high-risk human papillomaviruses in head and neck cancers from Bangladesh

BackgroundThere is a dramatic rise in the incidence of Human papillomavirus (HPV) – associated head and neck squamous cell carcinoma (HNSCC) in the world, with considerable variation by geography, gender and ethnicity. Little is known about the situation in Bangladesh, where tobacco- and areca nut-related head and neck cancers (HNCs) are the most common cancers in men. We aimed to determine the prevalence of HPV in HNSCC in Bangladesh and to explore the possible value of cell cycle markers in clinical diagnostic settings.MethodsOne hundred and ninety six archival HNSCC tissue samples were analysed for the presence of HPV DNA. The DNA quality was assured, and then amplified using a nested PCR approach. The typing of HPV was performed by automated DNA sequencing. Cellular markers p53, Cyclin D1 and pRb were tested on all samples by immunohistochemistry (IHC), as well as p16 as a putative surrogate for the detection of HPV.ResultsHPV DNA was detected in 36/174 (~21%) samples: 36% of cancers from the oropharynx; 31% of oral cancers, and 22% from the larynx. HPV-16 was most common, being present in 33 samples, followed by HPV-33 (2 samples) and HPV-31 (1 sample). Twenty-eight out of 174 samples were positive for p16, predominantly in HPV-positive tissues (p < 0.001). No statistically significant association was observed between the cellular markers and HPV DNA positive cases. However, p16 positivity had excellent predictive value for the presence of HPV by PCR.ConclusionThere is a significant burden of HPV-associated HNSCC in Bangladesh, particularly in the oropharynx but also in oral and laryngeal cancers. Whilst a combination of PCR-based DNA detection and p16 IHC is useful, the latter has excellent specificity, acceptable sensitivity and good predictive value for carriage of HPV in this population and should be used for prognostic evaluation and treatment planning of all HNSCC patients in South Asia, as in the Western world.

Abstract A26: Network integration of epigenomic data in HPV-associated head and neck cancer

Abstract The majority of published studies investigating driver genes have focused primarily on genomic mutations which have led to novel study designs (basket trials) where patients with a rare mutation, regardless of tumor histology, are matched to a drug expected to work through the mutated pathway. This dominant focus on genomic mutations has overshadowed consideration of inclusion of epigenetic information. Epigenetic silencing of driver genes leads to various genomic alterations, including mismatch repair deficiency, altered DNA repair and loss of chromosomal stability. In human papilloma virus (HPV) positive head and neck squamous cell carcinoma (HNSCC), recent studies are beginning to establish a mechanistic role for DNA methylation with potential to impact improved survival outcomes. The purpose of this study was to illustrate network integration of epigenomic data in head and neck cancer to prioritize elements that drive biological states. Causal Networks are small hierarchical networks of regulators whose activity can be modulated by the expression of downstream target genes to enhance understanding of the effect of upstream master regulators on disease or function. To further establish the driver potential of 11 genes: C14orf162/ CCDC177, CDH8, CRMP1, ELMO1, HTR1E, MEI1, MSX2, PCDH10, PCDHB11, PITX2, SYN2 previously reported as significantly differentially methylated between HPV positive and HPV negative HNSCC tumor samples, and subsequently validated by our group in two independent sample sets, their master regulatory networks were identified utilizing Causal Network Analysis (CNA) software from Ingenuity Pathway Analysis. To reflect expected gene expression direction implied by methylation changes, the inverse of the methylation ratio from HPV positive vs. HPV negative HNSCC was used for CNA. CNA identified 23 top hierarchical networks (significant z score of absolute 2) associated with HNSCC and their corresponding master regulatory molecules characterized as transcription regulators and kinase inhibitors among others. Of the 11 target genes, 7 had representation in multiple networks; CRMP1 in 21/23, MSX2 and SYN2 in 20/23, CDH8 and PITX2 in 17/23, PCDH10 in 10/23, and ELMO1 in 5/23 networks. Of the 23 networks, 12 indicated activation and 11 inhibition by at least 4 or more of the 7 target genes. Master regulators were within 2 or 3 hops (intermediate regulators) to the target genes. CNA raised the profile of CDH8, CRMP1, ELMO1, MSX2, PCDH10, SYN2, and PITX2 (7/11 target genes) for further consideration as epigenetic drivers of HPV-associated HNSCC. Support: Komen Foundation: KG110218 Citation Format: Maria J. Worsham, Kang Mei Chen, Indrani Datta, Josena K. Stephen, Dhananjay Chitale, Tamer Ghanem, Lamont Jones, Laura Garcia-Rodriquez, George Divine. Network integration of epigenomic data in HPV-associated head and neck cancer [abstract]. In: Proceedings of the AACR International Conference: New Frontiers in Cancer Research; 2017 Jan 18-22; Cape Town, South Africa. Philadelphia (PA): AACR; Cancer Res 2017;77(22 Suppl):Abstract nr A26.