Oral Cavity Head And Neck Squamous Cell Carcinoma Research Articles

Abstract 1958: Inhibition of CDK4/6 in head and neck squamous cell carcinoma dismantles key DNA repair pathways in response to radiation treatment

Abstract Introduction: Conventional treatment for head and neck squamous cell carcinoma (HNSCC) relies on surgery and radiation for cure; locoregional failure is often related to recurrence after radiation. CDKN2A, coding for the CDK4/6 inhibitory protein, p16INK4a (p16), is among the most commonly lost tumor suppressors in HNSCC. Palbociclib, a chemotherapeutic agent that is FDA-approved for breast cancer, is a selective CDK4/6 inhibitor. FOXM1 is a key cell cycle regulatory protein whose activation is controlled by CDK4/6. FOXM1 controls cell proliferation, DNA damage repair and suppresses senescence. The current study examines the role of CDK4/6 and its targets in response to radiation in HNSCC. We demonstrate inhibiting CDK4/6 can synergize with radiation, partially via suppression of FOXM1 signaling and impact on senescence. Experimental Procedure: Our study focuses on utilizing palbociclib to increase radiation response in HPV(-) oral cavity HNSCC and understanding mechanisms of potential radiosensitization. We examined cell viability after concurrent palbociclib and radiation (P+RT) application using proliferation assays in 2-dimensional (2D) and 3D organoid models of immortalized cell lines (HN5 and Cal27), and calculated synergy by Compusyn analysis. After 3-days treatment, we performed western blot to evaluate DNA damage response (DDR) proteins and β-galactosidase (β -gal) staining to evaluate the senescent phenotype. Flow cytometry studies were used to measure cell cycle arrest and QPCR to measure expression of cell cycle regulators. Results: P+RT (1µM palbociclib + 2 or 4Gy RT) demonstrated consistent synergy in both 2D and 3D viability assays. Comparing P+RT to RT alone, flow cytometry data revealed a 2-fold increase in the G1 arrested population, and DNA damage (measured by γ-H2AX level) increased by 2.3 fold. Additionally, Rad51 and Ku80 showed a 2 and 10-fold decrease respectively, suggesting an impact on DDR repair via both the homologous recombination and non-homologous end joining pathways. On transient knockdown of CDK-4 or 6, or both, we observed that senescence was the prevailing response - concurrent inhibition of CDK4 and CDK6 was the most profound, and levels of senescence based on β -gal staining results matched that observed with palbociclib treatment. P+RT demonstrated a robust 3-fold decrease in FOXM1 mRNA level and a 10-fold decrease in protein expression. We observed a 5-fold decrease in FOXM1 levels with CDK6 inhibition compared to a 2-fold decrease with CDK4 inhibition alone. Independently, FOXM1 inhibition induced apoptosis in HNSCC to a level higher than staurosporine-control treated cells. Conclusions: Our results suggest that palbociclib may make HNSCC more susceptible to RT via the induction of senescence related to suppressed signaling through FOXM1.We propose P+RT as a rational and potentially effective treatment strategy in HNSCC Citation Format: Nitisha Shrivastava, Carlos Thomas, Daniel Li, Cory D. Fulcher, Michael B. Prystowsky, Indranil Basu, Chandan Guha, Thomas J. Ow. Inhibition of CDK4/6 in head and neck squamous cell carcinoma dismantles key DNA repair pathways in response to radiation treatment [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 1958.

Abstract IA08: Understanding programs of invasion and metastasis in head and neck cancer

Abstract Despite advances in our understanding of tumor biology as well as radiation, chemotherapy, and surgical treatments, head and neck squamous cell carcinoma (HNSCC) remains the sixth leading cause of cancer-related mortality and a major cause of morbidity, with over 550,000 new cases per year. Although HNSCC mortality has slightly declined with improved tolerance of therapy, the majority of deaths in HNSCC are related to advanced disease with locoregional lymph node (LN) or distant metastasis. Indeed, the presence of LN metastases reduces survival by approximately 50% in all HNSCC patients. Unfortunately, a firm biologic understanding of pathways that underlie metastasis in HNSCC (and many other solid tumors) remains elusive. Tumor heterogeneity in HNSCC is a major challenge in clinical management and can manifest as variability among tumors (intertumoral heterogeneity) or among cells harboring distinct mutations or expressing unique transcriptional programs from the same tumor (intratumoral heterogeneity). Prior studies have suggested that intratumoral heterogeneity is present in HNSCC based on comparative genomic hybridization assays and DNA ploidy analyses, with recent studies validating intratumoral heterogeneity using whole-genome sequencing. In addition, there is growing interest in subpopulations of HNSCC cells, which may have unique properties such as the ability to self-renew or invade, contributing to recurrence or metastasis, respectively. The clinical significance of intratumoral heterogeneity has recently been demonstrated with a quantitative measure of genetic heterogeneity based on whole-exome sequencing (WES). This measure, termed mutant-allele tumor heterogeneity (MATH), suggests that intratumoral heterogeneity is associated with tumor progression and poor responses to treatment, supporting the idea that greater genetic variability predicts a worse clinical outcome in HNSCC. However, whether intratumoral transcriptional heterogeneity also contributes to clinical outcomes remained an open question. We recently utilized single cell RNA-sequencing (scRNA-seq), a powerful new approach to characterize expression heterogeneity present within oral cavity HNSCC (Puram et al., Cell 2017). The strength of these analyses is the ability to capture the transcriptional profile of each individual cell without contamination from other cells that may be present within the sample. As a result, single-cell sequencing offers 1) more detailed, nuanced expression data than analyses of tumor fragments and 2) captures the transcriptional profile of every cell, even rare populations, that might be overlooked in pooled RNA. We sequenced ~6000 single cells from 18 samples, including five matched primary/lymph node pairs. Our analyses reveal that stromal and immune populations have well-defined and consistent expression programs across tumors. In contrast, malignant cells are highly heterogeneous with variable gene signatures related to cell cycle, stress, hypoxia, epithelial structures, and partial epithelial-to-mesenchymal transition (p-EMT). Cells expressing the p-EMT program spatially localized to the leading edge of primary tumors and in further in vitro analyses, we found that p-EMT cells were more invasive. Integration of single-cell transcriptomes with bulk expression profiles for >500 tumors from The Cancer Genome Atlas using a deconvolution approach identified a malignant cell-specific signature for each of these samples. Remarkably, this analysis identified the p-EMT program as the primary source of variability in HNSCC, while enabling us to redefine HNSCC subtypes by their malignant and stromal composition. Further integration with clinical annotations uncovered a significant association between the p-EMT program and nodal metastasis, grade, and adverse pathologic features including lymphovascular invasion and extracapsular extension. Consistent with these findings, the p-EMT score was a significant predictor of occult nodal metastases in clinically N0 patients, with predictive power equivalent to tumor thickness/size. Our studies provide a detailed map of expression heterogeneity in the HNSCC ecosystem and define an in vivo p-EMT program associated with metastasis and disease progression. Citation Format: Sidharth V. Puram. Understanding programs of invasion and metastasis in head and neck cancer [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 IA08.

Development and Validation of a Novel and Rapid Molecular Detection Method for High-Risk Human Papillomavirus in Formalin-Fixed, Paraffin-Embedded Tumor Tissue

The most widely applied algorithm for human papillomavirus (HPV) detection in formalin-fixed, paraffin-embedded (FFPE) specimens of oropharyngeal head and neck squamous cell carcinoma (HNSCC) consists of p16INK4A immunostaining followed by PCR-based detection of high-risk HPV DNA on p16INK4A-immunopositive samples. However, in nonoropharyngeal HNSCC this algorithm fails, hampering correct interpretation of the prevalence and prognosis of HPV in these cases. In this study, we developed and validated a molecular HPV detection method for FFPE specimens of oropharyngeal and nonoropharyngeal HNSCC. Sectioning of FFPE blocks was circumvented by using punch biopsies from tumor-enriched regions of FFPE tissue blocks, and combined extraction was applied to obtain high-quality DNA and RNA from the punch biopsy. Next, PCR-based detection of HPV DNA was performed for 15 high-risk HPV types with subsequent detection of E6 mRNA for validation. The combined DNA/RNA FFPE test of tissue cores was assessed in well-characterized cohorts with known HPV status based on earlier work, that is, a cohort of oropharyngeal HNSCC (n = 80) and oral cavity HNSCC (n = 25), and reached an accuracy of 97% and 100%, respectively. In conclusion, our method is rapid, simple, and shows an excellent diagnostic performance for detection of HPV type 16. Ultimately, it can be applied for large cohort studies to determine the etiologic fraction and prognostic implication of HPV in nonoropharyngeal HNSCC.

Abstract 1132: Multiplexed immunofluorescence and multispectral imaging-based quantification of tumor and immune cell populations reveals spatial relationships in oral cavity squamous cell carcinoma

Abstract Recent single cell expression profiling of head and neck squamous cell carcinoma (HNSCC) identified a partial epithelial-to-mesenchymal transition (p-EMT) subpopulation of cancer cells. We have previously demonstrated an association of p-EMT subpopulations with adverse clinical and pathologic features, in both TCGA expression data and an independent cohort of single institutional patients. Here, we aim to understand cellular co-expression of EMT markers and spatial relationships with immune infiltrates in oral cavity HNSCC. Paraffin blocks of 10 oral cavity HNSCC patients whose tumors had previously undergone single cell expression profiling were characterized by a 13-marker panel of tumor cell, p-EMT, epithelial differentiation, and immune markers. Multiplexed immunofluorescence was used to perform simultaneous staining within two serial tissue sections. Multispectral imaging was performed using wide field microscopy (Vectra 3), and algorithms were used for spectral unmixing, tissue and cell segmentation, and cell phenotyping. Following user training, tissue and cell segmentation and cell phenotyping were successfully performed in an automated fashion. Whole slides containing entire tissue sections were analyzed, enabling high throughput analysis of hundreds of thousands of individual cells per tumor. p-EMT quantification by tumor, as measured by simultaneous signal for p-EMT markers, showed consistent co-localization, in accordance with prior single cell expression profiling data. We found that individual cells defined as p-EMT positive, based on co-expression of two or more p-EMT markers, co-localized to the leading edge of tumor nests, in close apposition to the stroma. Co-localization of LAMB3 and LAMC2 was the most robust marker combination defining this subpopulation. In addition, helper and cytotoxic T cells were identified across tumors, including activated and exhausted T-cell subsets. The relationships of these cellular subpopulations to other immune cells, which may drive immune cell exhaustion, were explored by radial measurements associated with cell densities and intercellular distances. Tumor and immune cell profiling using 13 markers across serial sections enabled high throughput characterization of individual cells with spatial information in a manner not previously possible. Distinct spatial relationships among p-EMT and epithelial tumor cells were identified, including a potential correlation with immune cells. This technology may have clinical utility in HNSCC, which includes predicting the need for therapy and response to immunotherapy. Citation Format: Joao Paulo Oliveira-Costa, Anuraag S. Parikh, Linda T. Neiman, Derin Sevenler, Doyeon Koo, Chenyue Lu, William C. Faquin, Itay Tirosh, Jeremy D. Richmon, Kevin S. Emerick, Daniel G. Deschler, Mark A. Varvares, Derrick T. Lin, Sidarth V. Puram, Bradley E. Bernstein, Shannon L. Stott. Multiplexed immunofluorescence and multispectral imaging-based quantification of tumor and immune cell populations reveals spatial relationships in oral cavity squamous cell carcinoma [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 1132.

Abstract 31: The INSPIRE trial: A randomized trial of neoadjuvant and adjuvant therapy with the IRX-2 regimen in patients with newly diagnosed stage II, III, or IVa squamous cell carcinoma of the oral cavity

Abstract Introduction: IRX-2 is a primary-cell-derived immune-restorative biologic consisting of a well-defined set of human cytokines that act on multiple immune system cell types to overcome tumor-mediated immunosuppression. The IRX-2 biologic stimulates T cells and natural killer cells and turns immature or defective dendritic cells into mature antigen-presenting cells. The IRX-2 biologic is provided as part of the IRX-2 regimen, which contains cyclophosphamide, indomethacin, and zinc to support an anticancer immune response. In a phase 2a clinical trial in 27 patients with therapy-naïve head and neck squamous cell carcinoma (HNSCC), treatment with the IRX-2 regimen was well tolerated and associated with immunologically mediated antitumor effects. The new multicenter randomized IRX-2 Neoadjuvant Therapy in Head and Neck SCC to Provide Immune Response Enhancement (INSPIRE) trial (NCT02609386) is designed to evaluate the safety and effectiveness of combined neoadjuvant and adjuvant therapy with the IRX-2 regimen in patients with oral cavity HNSCC, a disease known to have a disordered immune function. Study Design: Patients enrolled in INSPIRE must be at least 18 years of age; have previously untreated Stage II, III, or IVa SCC of the oral cavity that is surgically resectable with curative intent; and have a Karnofsky Performance status ≥70%. Up to 200 patients will be randomized 2:1 to either the IRX-2 biologic regimen arm or the IRX-2 regimen control arm (the IRX regimen minus the IRX-2 biologic). The neoadjuvant stage of INSPIRE begins 21 days before resection, when patients in both treatment arms will receive the IRX-2 regimen each day until resection. This regimen consists of the immunomodulatory chemotherapeutic agent cyclophosphamide, provided as a low-dose intravenous infusion, as well as two oral drugs, a zinc-containing multivitamin and indomethacin, a nonselective COX-1/COX-2 inhibitor. During this period, patients in the IRX-2 biologic regimen arm will also receive 10 days of the IRX-2 biologic subcutaneously injected into their bilateral sternocleidomastoid insertion regions. After resection, patients receive standard adjuvant radiation or chemoradiation therapy followed by adjuvant IRX-2 booster regimens at 3, 6, 9, and 12 months. During each 10-day booster period, patients in the IRX-2 biologic regimen arm also receive, for 5 consecutive days, the IRX-2 biologic subcutaneously injected into their bilateral deltoid regions. Study Endpoints: INSPIRE is now enrolling patients at 12 selected institutions in the United States. The primary endpoint of INSPIRE is event-free survival (EFS). Secondary endpoints include overall survival (OS) and safety as assessed by the incidence and severity of adverse events. Exploratory endpoints include changes in tumor size and histologic differences between pre- and post-treatment specimens in lymphocytic infiltration, assessed by both cell-surface marker expression (Perkin Elmer multiplex IHC) and immune cell gene signatures (NanoString). Conclusion: With enrollment of up to 200 patients, the randomized INSPIRE trial provides the opportunity to assess the ability of the IRX-2 regimen to improve EFS and OS by inhibiting tumor-mediated immunosuppression. Analysis of the exploratory endpoints will generate data to better describe the mechanism of action of the IRX-2 regimen and provide insight into the differences between inflammatory and non-inflammatory responses to SCCs of the oral cavity. Citation Format: Gregory T. Wolf, Mihir Patel, Audrey Erman, Jason G. Newman, Greg Krempl, Jorge Nieva, R. Bryan Bell, Michael Kaplan, Dennis Kraus, Jeffrey Moyer, Aru Panwar, Joseph Valentino. The INSPIRE trial: A randomized trial of neoadjuvant and adjuvant therapy with the IRX-2 regimen in patients with newly diagnosed stage II, III, or IVa squamous cell carcinoma of the oral cavity [abstract]. In: Proceedings of the AACR-AHNS Head and Neck Cancer Conference: Optimizing Survival and Quality of Life through Basic, Clinical, and Translational Research; April 23-25, 2017; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(23_Suppl):Abstract nr 31.

Tumor Metabolism in the Microenvironment of Nodal Metastasis in Oral Squamous Cell Carcinoma.

Objective In many cancers, including head and neck squamous cell carcinoma (HNSCC), different regions within a tumor have different metabolic phenotypes. Transfer of metabolites between compartments promotes tumor growth and aggressive behavior. Metabolic compartmentalization in HNSCC nodal metastases has not been studied, nor has its impact on extracapsular extension or clinical outcomes been determined. Study Design Retrospective analysis based on immunohistochemistry staining. Setting Tertiary care center. Subjects and Methods Primary tumors and nodal metastases from 34 surgically treated oral cavity HNSCC patients with extracapsular extension (ECE) were stained for monocarboyxlate transporter (MCT) 4, MCT1, translocase of outer mitochondrial membrane 20, and Ki-67. Strength of staining was assessed using a computer-assisted pathology algorithm. Immunohistochemistry (IHC) scores along with clinical factors were used to predict disease-free survival (DFS). Results Patterns of IHC staining showed metabolic compartmentalization both at the primary tumor sites and in nodal metastases. MCT4 staining in the perinodal stroma was significantly higher in specimens with ECE greater than 1 mm (macro-ECE, P = .01). Patients with high perinodal MCT4 staining were compared with those with low perinodal MCT4 staining. On multivariate analysis, only high perinodal MCT4 staining had a significant impact on DFS ( P = .02); patients with high perinodal MCT4 had worse survival. DFS was not significantly worsened by advancing T stage, N stage, ECE extent, or perineural invasion. Conclusion Oral HNSCC displays compartmentalized tumor metabolism at both primary and metastases. Greater cancer-associated stromal conversion around ECE, denoted by high stromal MCT4, may be a biomarker for aggressive disease and worsened DFS.

377P Induction chemotherapy with APF (bevacizumab + cisplatin + 5-fluorouracil) for betel-nut-chewing associated head and neck squamous cell carcinoma - a retrospective analysis

Background Head and neck squamous cell carcinoma (HNSCC) in Taiwan is associated with betel-nut chewing. Indcution chemotherapy with TPF is standard after theTAX324 trial. But the side effects of TPF regimen is of concern in Asia. 12% neutropenic fever and 83% grade ¾ neutropenia were found in the TAX 324 study. We hypothesise the APF regimen may avoid taxotere-related toxicity and retain treatment efficacy. This retrospective study was aimed at evaluating the efficacy of APF for this population. Methods Patients with locally HNSCC were retrospectively reviewed in a university hospital in Taiwan from Oct 2014 to Dec 2015. Patients receiving induction chemotherapy wtih APF for curative intent were recorded for analysis. The history of alcohol drinking, betel nut chewing, and cigarette smoking were coded according to medical records. The clinical response described in medical records and image reports were retrieved. Results Twenty-seven HNSCC patients receiving induction chemotherapy with APF for curative intent were recorded. In this population, 26 (96%) were male, 9(33.3%) were oral cavity HNSCC, 6 (22.2%) were oropharynx HNSCC, 11 (40.7%) were hypopharynx HNSCC. 18(66.7%) had history of betel-nut chewing, 24 (88.9%) had history of smoking, 20 (74.1%) had history of alcohol drinking. 24 (88.9%) were clinical stage III, IVa and IVb. The response rate after indcution chemotherapy with APF(1~3 cycles, median 2 cycles) were 92.6%. 4 (14.8%) patients were in complete remission, 3 (11.1%) patients were in pathologic complete remission. 4 (14.8%) patients had grade 3/ 4 neutropenia, 1 (3.7%) patient had neutropenic fever, 1 (3.7%) had grade 3 acute kidney injury. No tumor bleeding, nor treatment related death was recorded. Conclusions The effect of induction chemotherapy with APF for betel-nut-chewing- associated HNSCC is comparative to other studies with TPF regimen. And the side effect profile of APF was acceptable. Further confirmatory studies of induction chemtherapy with APF for this population are mandatory. Legal entity responsible for the study Huai-Cheng Huang Funding National Taiwan University Hospital Disclosure All authors have declared no conflicts of interest.

384P Afatinib for betel-nut-chewing associated head and neck squamous cell carcinoma - a retrospective analysis

Background Head and neck squamous cell carcinoma (HNSCC) in Asia and South Pacific is associated with betel-nut chewing. In Lux-Head & Neck1, the impact of afatinib for betel-nut-chewing associated HNSCC is not well characterized. The biology of betel-nut-chewing associated HNSCC is not similar to other types of HNSCC. This retrospective study is aimed to evaluate the efficacy of afatinib for this population. Methods Patients with locally advanced or metastatic HNSCC were retrospectively reviewed in a university hospital in Taiwan from Apr 2014 to Feb 2015. Patients taking afatinib for palliative intent were recorded for analysis. The history of alcohol drinking, betel nut chewing, and cigarette smoking were coded according to medical records. The clinical response described in medical records were retrieved. Results Eighty HNSCC patients taking afatinib for palliative intent were recorded. In this population, 72 (90%) were male, 58 (72.5%) were oral cavity HNSCC, 53(66%) had history of betel-nut chewing, 46 (57.5%) had history of smoking, 67 (84%) had local recurrence, and 26 (32%) had distant metastases. The progression free survival between betel-nut-chewing and non-betel-nut-chewing were: median (95% confidence interval): 2.6 (2.0-3.2) vs 3.0 (2.2-3.9) months (hazard ratio:1.23 (95% CI: 0.732-2.072), p = 0.433). Patients without distant metastases had a better survival (HR: 0.31, 95% CI: 0.15-0.62), p = 0.001). Patients with poorly differentiated squamous cell carcinoma may predict worse survival (HR: 5.51, 95% CI: 0.87-34. 82, p = 0.069). An earlier treatment with a fatinib may predict better survival (palliative chemotherapy lines before afatinib: 0 or 1 line vs 2 lines or more: HR: 0.5, 95% CI: 0.29-0.86, p = 0.013). Conclusions The effect of afatinib for betel-nut-chewing associated HNSCC is comparative to the result of LUX-Head & Neck 1. Further confirmatory study of afatinib for this population is mandatory. Legal entity responsible for the study Hsiang-Fong Kao Funding National Taiwan University Hospital Disclosure All authors have declared no conflicts of interest.

Abstract IA18: Tumor suppressor gene silencing by somatic mutations and promoter methylation leads to genomic instability in head and neck squamous cell carcinoma

Abstract IA18: Tumor suppressor gene silencing by somatic mutations and promoter methylation leads to genomic instability in head and neck squamous cell carcinoma

OP046: Targeting cancer stem cells (CSC) in oral cavity head and neck squamous cell carcinoma (HNSCC) with dendritic cell (DC) vaccines

Targeting CSCs with a DC vaccine is an innovative and rationale approach that could specifically address the most replicative and therapy-resistant subpopulation of cancer cells within a solid tumor malignancy. Patients with oral cavity squamous cell carcinoma had tumors harvested and the cells sorted for ALDH high and ALDH low cells. ALDH high cells have been shown to possess the properties of cancer stem cells. DCs were generated from the peripheral blood of patients with HNSCC and pulsed in vitro with CSC lysate. DCs generated from the peripheral blood of patients with HNSCC and pulsed with CSC lysate confer greater specific antitumor efficacy by inducing anti-CSC immunity in vitro than DCs pulsed with whole tumor or non-CSC lysate. There was greater specific lysis of ALDH high target cells (vs. ALDH low targets) by CTL co-stimulated with DCs pulsed with ALDH high tumor cells suggesting a specific anti-CSC effect with this stimulation approach. Targeting CSCs in oral cavity HNSCC tumors with a DC vaccine is feasible and demonstrates an antitumor immune response.

Patterns of care in elderly patients with squamous cell carcinoma of the head and neck: A SEER-Medicare analysis.

5539 Background: Head and neck squamous cell carcinoma (HNSCC) is predominantly a disease of the elderly, however age and co-morbidity may affect therapy decisions. The purpose of this study was to assess the patterns of care of elderly HNSCC patients and identify factors associated with non-receipt of surgery and chemoradiation (CRT). Methods: Using the Surveillance, Epidemiology, and End Results (SEER)-Medicare linked database (1992-2007) we identified a retrospective cohort of non-metastatic HNSCC patients and categorized them into treatment cohorts. Comparisons were made between surgery vs. non-surgery, and CRT vs. non-CRT cohorts. Multivariate logistic regression models examined factors associated with non-receipt of treatment. Variables included tumor location, stage, year of diagnosis, age, gender, marital status, race, comorbidity, race-comorbidity interaction, SEER region, socioeconomic variables, and hospital affiliations. Results: The final cohort of 11,336 were 63% male and 83% white. 52% had no co-morbidities, 44% had oral cavity HNSCC, 52% were diagnosed between 2002 and 2007, 58% had surgery, and 21% received CRT. For the CRT vs. non-CRT model, increasing age (OR = 0.93; 95% CI 0.92 -0.94) and non-whites with co-morbidity (OR = 0.71; 95% CI 0.55 – 0.92) were associated with decreased likelihood of receiving CRT. More advanced staged disease, oropharyngeal tumor location, and diagnosis after 2002 were associated with increased likelihood of receiving CRT. For the surgery versus non-surgery model, age was not associated with receipt of surgery (OR 0.99; 95% CI 0.99-1.00), nor was existence of comorbidity in white patients (OR 0.97; 95% CI 0.88-1.05). However, recent year of diagnosis, non-oral cavity tumor location, and advanced stage were all associated with reduced likelihood of receiving surgery. During the 15 year surveillance period, patients were less likely to receive surgery, and CRT was used more often. Conclusions: Age may influence the non-receipt of CRT in this elderly cohort but does not appear to be significantly associated with the non-receipt of surgery. There has been an increasing trend in the receipt of CRT with a decrease in surgery over the past 15 years.

Abstract 3944: In-vitro evaluation of the Hypoxia Inducible Factor (HIF) pathway in head and neck squamous cell carcinoma (HNSCC)

Abstract Objective: To evaluate the effect of hypoxia on the expression of genes regulated by oxygen (HIF1-α, HIF2-α, VEGF, cMET, OCT4 and EGFR) and on cell migration in UM-SCC-103 cells while controlling for mutations in the von Hippel-Lindau (VHL) gene, a regulator of HIF1. Methods: UM-SCC-103, a cell line derived from an oral cavity HNSCC, was used in all experiments. DNA and cDNA was sequenced to analyze UM-SCC-103 for mutations of VHL. Cells were cultured in normoxic (20% O2) and hypoxic (1% O2) conditions. RNA was isolated from each group at specific time points after onset of hypoxia (0, 6, 12 and 24 hrs), converted to cDNA and assessed by real-time qPCR for β-actin, HIF1-α, HIF2-α, VEGF, cMET, OCT4 and EGFR expression. A wound-healing assay was performed on cells grown to 90-95% confluence, then scratched with a plastic pipette tip, and incubated under normoxic or hypoxic conditions; measurements were taken at 0, 12, 16, and 20 hrs. Wound closure at each time point was quantified using ImageJ software by measuring the area of the wound. Mean percent change from baseline was calculated and analyzed with a paired t-test statistic. Results: Sequence analysis of VHL genomic DNA and cDNA revealed no mutations, indicating the presence of wild-type (wt) VHL. The HIF1-α transcript decreased to 35% below the normoxic control at 24 hours, while HIF2-α showed a transient 20% increase at 6 hours before returning to normoxic levels. Under hypoxic conditions, transcripts of VEGF steadily increased over time, reaching 61% over normoxic at 24 hrs. OCT4 was increased by 65% at 12 hrs and 36% at 24 hrs, relative to normoxic conditions. cMET increased by 10% at 24 hrs while EGFR expression level remained unchanged. The cell migration/wound-healing assay showed that cells incubated under hypoxic conditions had significantly increased migration at 12, 16 and 20 hrs compared to cells incubated under normoxic conditions (62% v. 20% wound closure p=0.0001, 86% v. 46% p=0.0001 and 97% v. 71% p=0.0015 respectively). Conclusions: UM-SCC-103 expresses wt-VHL, suggesting physiologic regulation of HIF under normoxic conditions. Under hypoxic conditions, decreased expression of HIF1-α message over time is consistent with a potential negative feedback loop inhibiting HIF1-α transcription. HIF2-α, thought to be associated with chronic hypoxia, is likely regulated differently, and showed no evidence of transcriptional down-regulation over 24 hrs. HIF downstream targets cMET, VEGF and OCT4 increased in hypoxia. Given the known association of cMET and VEGF with tumor cell migration and survival, respectively, and of OCT4 with the stem cell phenotype, our results are consistent with a role for hypoxia as a driver of a biologically aggressive phenotype in HNSCC. Wound-healing assays support this assertion, demonstrating increased cell migration in hypoxia. Hypoxia may also shift cells into the stem cell niche. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3944. doi:1538-7445.AM2012-3944

HLA and MICA associations with head and neck squamous cell carcinoma

Head and neck squamous cell carcinoma (HNSCC) is a very aggressive tumour arising from the epithelial lining of the upper aerodigestive tract. The precise mechanisms involved in the pathogenesis of HNSCC have not been elucidated. Previous studies observed aberrant HLA expression patterns on HNSCC tumour cells and this study focused on the allelic polymorphism of HLA genes and the MHC class I chain related gene A (MICA) and HNSCC. We investigated whether associations with HLA and/or MIC alleles or haplotypes are involved in the pathogenesis of HNSCC and could explain the observed HLA expression patterns. Patients and controls were typed for HLA-A, HLA-B, HLA-C, HLA-DRB1 and HLA-DQB1 with sequence specific priming (SSP), supplemented with sequencing based typing (SBT). MICA allelic polymorphism was included and MICA allele assignment was based upon the combination of high resolution SBT of exons 2-4 in combination with repeat analysis and nucleotide polymorphism of exon 5. HLA-B *35 (p=0.014, OR=0.31) and HLA-B *40 (p=0.013, OR=2.9) were significantly associated in respectively the metastasized patients and the oral cavity patients. In addition, the HLA-B *40-DRB1 *13 haplotype (p=0.016, OR=4.1) was more often observed in the oral cavity patient group. The biological significance of the prevalence of specific HLA haplotypes in patients with oral cavity HNSCC and metastasizing HNSCC requires further investigation.