HNSCC Tumors Research Articles

Metformin Induces Pro-Tumorigenic Cytokines And Natural Killer Cells In Patients With Locally Advanced Head and Neck Squamous Cell Carcinoma

Concurrent chemotherapy and radiation (CRT) continues to be standard of care for unresectable locally advanced head and neck squamous cell carcinoma (LAHNSCC). In the HPV (human papillomavirus) positive non-smoker population the cure rate approaches greater than 90%. However, the cure rate can be as low as 50% in HPV negative smokers necessitating newer and more effective treatments. Interestingly, diabetic HNSCC patients taking the biguanide, metformin, have been shown to have superior survival outcomes. However, the mechanism by which metformin results in superior outcomes for these patients is unclear. In vivo, metformin was shown to prevent HNSCC tumor formation in mouse models via activation of AMPK and inhibition of the mTOR pathway. Metformin has also been shown to activate CD8+ T cells and activation of AMPK has been shown to activate NKT cells. Therefore, we hypothesized that the addition of metformin to CRT would be safe and would result in enhanced tumor responses through activation of AMPK and activation of the innate immune system. Here we report our findings on the effect of metformin on the immune system utilizing our phase 1 open label clinical trial (NCT02325401) entitled “Dose-finding Study of Metformin With Chemoradiation in Locally Advanced Head and Neck Squamous Cell Carcinoma”. Trial eligibility criteria included newly diagnosed, non-diabetic, LAHNSCC patients eligible for definitive CRT. Patients received a 14-day lead-in of metformin in escalating dose cohorts (1000, 2550, or 3000 mg daily in divided doses) followed by a combination of metformin and standard of care CRT (cisplatin 100mg/m2 days 1, 22, and 43, 70 Gy radiation in 35 fractions) using a modified toxicity probability interval design. Blood was collected before and after metformin treatment and was processed for serum and separation of peripheral blood mononuclear cells (PBMCs). PBMCs were analyzed by flow cytometry to determine immune cell subsets and serum was analyzed by Luminex (Human 10-plex) for cytokine release. Eighteen patients have been enrolled out of 18 planned patients for dose escalation. Median time to follow-up is 21 months (range 3-29 months). Patient serum ELISA examination revealed overall increases in the pro-tumor immune cytokines TNF-a, IL-2, and decrease in the anti-tumor immune cytokine IL-4 in response to metformin. Metformin treatment also resulted in increased populations of cytotoxic T cells, NK and NKT cells and overall activation of innate immunity. Clinically, median overall survival has not been reached, and in fact, no patients on study have relapsed. Metformin administration in LAHNSCC results in activation of pro-tumorigenic immunity which may result in enhanced tumor responses and decreased tumor relapse.

Pembrolizumab and its use in the treatment of recurrent or metastatic head and neck cancer.

Until recently, palliative options for the treatment of platinum-refractory recurrent or metastatic head and neck squamous cell carcinoma (R/M HNSCC) have been cytotoxic chemotherapy and EGFR inhibitors. These agents offer limited efficacy with substantial toxicity. The development of novel immune checkpoint inhibitors has challenged the standard treatment. Pembrolizumab is a potent and highly selective humanized monoclonal antibody that blocks the interaction between PD-1, an immune checkpoint receptor and its ligands PD-L1 and -2. In August 2016, the US FDA approved the use of pembrolizumab in R/M HNSCC following disease progression on or after platinum-containing chemotherapy. This review highlights the pharmacology, therapeutic efficacy and tolerability data relevant to the use of pembrolizumab for the treatment of R/M HNSCC. Readers will gain greater insight into the HNSCC tumor microenvironment, available biomarkers, and learn about important clinical considerations associated with the use of pembrolizumab and similar immune checkpoint inhibitors.

Sensitivity and specificity of oral HPV detection for HPV-positive head and neck cancer

BackgroundThe incidence of HPV-related head and neck squamous cell carcinoma (HPV-HNSCC) is increasing. Oral samples are easy and non-invasive to collect, but the diagnostic accuracy of oral HPV detection methods for classifying HPV-positive HNSCC tumors has not been well explored. MethodsIn a systematic review, we identified eight studies of HNSCC patients meeting our eligibility criteria of having: (1) HPV detection in oral rinse or oral swab samples, (2) tumor HPV or p16 testing, (3) a publication date within the last 10 years (January 2007–May 2017, as laboratory methods change), and (4) at least 15 HNSCC cases. Data were abstracted from each study and a meta-analysis performed to calculate sensitivity and specificity. ResultsEight articles meeting inclusion criteria were identified. Among people diagnosed with HNSCC, oral HPV detection has good specificity (92%, 95% CI = 82–97%) and moderate sensitivity (72%, 95% CI = 45–89%) for HPV-positive HNSCC tumor. Results were similar when restricted to studies with only oropharyngeal cancer cases, with oral rinse samples, or testing for HPV16 DNA (instead of any oncogenic HPV) in the oral samples. DiscussionAmong those who already have HNSCC, oral HPV detection has few false-positives but may miss one-half to one-quarter of HPV-related cases (false-negatives). Given these findings in cancer patients, the utility of oral rinses and swabs as screening tests for HPV-HNSCC among healthy populations is probably limited.

Abstract 07: An oncogenic transcriptional network anchored by ETS1, p63 and subtype specific drivers of HNSCC revealed by epigenomic and genomic interrogation

Abstract Head and neck squamous cell carcinoma (HNSCC) is a heterogeneous disease of significant mortality and morbidity, with a 5-year survival rate that has shown only modest improvement. This persistent patient mortality and morbidity can be attributed, in part, to an overall lack of novel targeted treatment options. Indeed, recent genome sequencing studies have highlighted the striking heterogeneity of HNSCC tumors and have failed to identify pervasive and broadly actionable driver mutations and/or other genetic alterations. Despite this genetic heterogeneity, HNSCC tumors can be classified into gene-expression defined subtypes, raising the possibility that such distinct gene expression programs and regulatory networks are closely interwoven with an intrinsic heritable and signature epigenetic state. We hypothesized that there exist key oncogenic transcriptional regulators of subtype dependency and that defining the epigenetic landscape of HNSCC tumors will reveal the identity of these crucial factors. This will be impactful since targeting of tumor addiction to key oncogenic Transcription Factors (TFs), although successful in a number of other tumor types, remains largely unexplored in HNSCC due to limited knowledge of the identity of these regulators in this cancer. To decipher the genomic and epigenomic drivers of HNSCC tumor subtypes, we examined a large cohort of cell lines that represent the Atypical, Basal and Mesenchymal subtypes. By using global H3K27 acetylation levels as a surrogate marker, we first generated a consensus genome-wide regulatory enhancer and Super-Enhancer map. Interestingly, we observed a differential enrichment pattern of H3K27Ac signals that recapitulated the segregation of cell-lines according to subtype based on differential gene expression profiles. We leveraged the differential enhancer map to identify enriched DNA-Binding Motifs to generate a Transcription Factor Combinatorial Regulatory Network, based on the co-occurrence frequency of TFs relative to genomic background. Our analysis highlighted a Cis-Regulatory Module (CRM) Network, specific to each molecular subtype of HNSCC. Furthermore, we identified key TF motifs that are embedded within these CRMs and enriched for each subtype-specific network, which we posit represent the pertinent TF drivers of the distinct gene-expression network found in various HNSCC tumors. Here, as proof of principle, we have examined the potential interaction between TP63 and ETS1, two oncogenic TFs revealed by our epigenomic and genomic analysis to be crucial mediators of HPV-negative HNSCC tumors. To better understand the TP63-ETS1 driven molecular processes in HPV-negative tumors, we performed transcriptomic analysis of the effects of knockdown of TP63 and ETS1 by RNA-Seq and identified their global targets by ChIP-Seq. Interestingly, our comprehensive analysis showed that TP63 and ETS1 share a significant number of genomic binding sites, exhibit a marked preference for binding to Super-Enhancers and together act in a concerted fashion to drive expression of unique set of pathways and processes. By integrating our findings with meta-analysis of HNSCC patient datasets, we have uncovered a core TP63-ETS1 driven signature network that serves as a regulatory anchor of common facets of cancer biology, such as proliferation and angiogenesis, as well as drug-targetable tumor-specific processes involving cancer stem cells. Our study has harnessed the power of an integrative genomic/epigenomic approach to characterize HNSCC subtypes and heterogeneity and to provide a robust framework for the discovery of novel tumor drivers that can be leveraged for targeted therapeutics. Citation Format: Christian Gluck, Isha Sethi, Maria Tsompana, Satrajit Sinha. An oncogenic transcriptional network anchored by ETS1, p63 and subtype specific drivers of HNSCC revealed by epigenomic and genomic interrogation [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 07.

Abstract 44: Synergistic antitumor activity of olaparib combined with AZD1775 in head and neck squamous cell carcinoma

Abstract Purpose: Although the treatment of locally advanced head and neck cancer (HNSCC) has evolved in recent years, the cure rate for patients with aggressive tumors and high-risk of failure remains poor due to resistance to standard chemoradiotherapy. Since TP53, the gene that encodes the protein p53, is by far the most commonly mutated gene found in HNSCC, and is in turn associated with treatment failure and poor survival outcomes. Although HPV+ HNSCC has a good prognosis, a group of HPV+ patients' tumors remain refractory to conventional treatments and unusual patterns of recurrence and metastasis are emerging. Therefore, more effective therapies are urgently needed. Our recent work has demonstrated that increased DNA damage response associated induction of replication stress and impaired Rad51-mediated homologous recombination (HR) sensitizes HNSCC tumor cells to WEE1 kinase inhibition with AZD1775. While the use of single molecularly targeted agents has demonstrated limited therapeutic benefits, there is growing interest in targeting multiple pathways or multiple steps within a single pathway to achieve even more effective cancer treatments. In this context, combinations of agents that target the DNA damage and HR responses are an exciting new area of investigation. Given the synthetic lethality of PARP inhibitor, olaparib in BRCA1/2-mutant or HR repair-defective cancers, combining PARP inhibitors with agents that inhibit HR is another interesting area of intensive investigation. Therefore, we hypothesize that simultaneous targeting of PARP and WEE1 will decrease the clonogenic survival in vitro and inhibit tumor growth and prolong animal survival in treated mice in an in vivo orthotopic nude mouse model of oral cancer. Experimental Design: Clonogenic survival assays and an orthotopic mouse model of oral cancer were used to examine the in vitro and in vivo sensitivity of high-risk mutant p53 and HPV+ HNSCC cell lines to olaparib in combination with AZD1775 respectively. Cell cycle analysis, DNA damage (γH2AX), HR, live cell imaging, and apoptosis were performed to dissect molecular mechanisms. Results: We found that olaparib synergized with AZD1775 in vitro to inhibit growth of HNSCC cells irrespective of p53 status or HPV positivity (median CI = 0.07-0.19). Mechanistically, these drugs interact synergistically to induce DNA damage, replication stress, and impaired Rad51-mediated HR through activation of CDK1 and increased Chk1 phosphorylation preceded by enhanced activity of ATR, culminating in aberrant mitosis associated with apoptotic cell death. Assessment of olaparib and AZD1775 combination therapy in vivo in an orthotopic mouse model of oral cancer is currently underway. Conclusions: Olaparib synergizes with AZD1775 in HNSCC cells in vitro through apoptosis. Our data provide an early-preclinical evidence for the rational combination of WEE1 and PARP inhibitors in the treatment of advanced HNSCC. Citation Format: Hideaki Takahashi, Antje Lindemann, Ameeta A. Patel, Weiwei Liu, Noriaki Tanaka, Lin Tang, Mei Zhao, Walter N. Hittelman, Jeffrey N. Myers, Abdullah A. Osman. Synergistic antitumor activity of olaparib combined with AZD1775 in head and neck squamous cell carcinoma [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 44.

Survival of Head and Neck Cancer Cells Relies upon LZK Kinase-Mediated Stabilization of Mutant p53.

Head and neck squamous cell carcinoma (HNSCC) includes epithelial cancers of the oral and nasal cavity, larynx, and pharynx and accounts for ∼350,000 deaths per year worldwide. Smoking-related HNSCC is associated with few targetable mutations but is defined by frequent copy-number alteration, the most common of which is gain at 3q. Critical 3q target genes have not been conclusively determined for HNSCC. Here, we present data indicating that MAP3K13 (encoding LZK) is an amplified driver gene in HNSCC. Copy-number gain at 3q resulted in increased MAP3K13 mRNA in HNSCC tumor samples and cell lines. Silencing LZK reduced cell viability and proliferation of HNSCC cells with 3q gain but not control cell lines. Inducible silencing of LZK caused near-complete loss of colony-forming ability in cells harboring 3q gain. These results were validated in vivo by evidence that LZK silencing was sufficient to reduce tumor growth in a xenograft model of HNSCC. Our results establish LZK as critical for maintaining expression of mutant stabilized p53. Cancer Res; 77(18); 4961-72. ©2017 AACR.

γH2AX/53BP1 foci as a potential pre-treatment marker of HNSCC tumors radiosensitivity – preliminary methodological study and discussion

In order to improve patients’ post-treatment quality of life, a shift from surgery to non-surgical (chemo)radio-treatment is recognized in head and neck oncology. However, about half of HNSCC tumors are resistant to irradiation and an efficient marker of individual tumor radiosensitivity is still missing. We analyzed whether various parameters of DNA double strand break (DSB) repair determined in vitro can predict, prior to clinical treatment initiation, the radiosensitivity of tumors. We compared formation and decrease of γH2AX/53BP1 foci in 48 h after irradiating tumor cell primocultures with 2 Gy of γ-rays. To better understand complex tumor behavior, three different cell type primocultures – CD90−, CD90+, and a mixed culture of these cells – were isolated from 1 clinically radioresistant, 2 radiosensitive, and 4 undetermined HPV–HNSCC tumors and followed separately. While DSB repair was delayed and the number of persisting DSBs increased in the radiosensitive tumors, the results for the radioresistant tumor were similar to cultured normal human skin fibroblasts. Hence, DSB repair kinetics/efficiency may correlate with clinical response to radiotherapy for a subset of HNSCC tumors but the size (and therefore practical relevance) of this subset remains to be determined. The same is true for contribution of different cell type primocultures to tumor radioresistance.

Highlights of This Issue

Defects in homologous recombination repair genes (e.g., BRCA1 and BRCA2 mutations) can sensitize tumors to poly(ADP-ribose) polymerase (PARP) inhibition through synthetic lethality. This phase I–II study was the first to evaluate single-agent oral rucaparib, a potent PARP inhibitor, at multiple doses in patients with advanced solid tumors (Part 1; phase I), establish the recommended phase II dose (RP2D) of 600 mg twice daily, and evaluate the RP2D in patients with platinum-sensitive, high-grade ovarian carcinoma (HGOC) associated with a germline BRCA1/2 mutation (Part 2A; phase II). Rucaparib was tolerable and had robust antitumor activity in patients with platinum-sensitive germline BRCA1/2-mutated HGOC.Germinal center B-cell (GCB) has a better prognosis than non-GCB diffuse large B-cell lymphoma (DLBCL) to upfront therapy. Cell-of-origin (COO) determination using gene expression profiling (GEP) is more sensitive, reproducible, and robust than immunohistochemistry. Czuczman and colleagues designed a randomized study using COO-based subtyping to elucidate differences in activity of lenalidomide over salvage monotherapy in relapsed/refractory DLBCL. Lenalidomide elicited higher ORR and longer PFS in non-GCB patients (most pronounced in ABC-designated patients). These results provide an additional rationale for the ongoing phase 3 trial, ROBUST (NCT02285062), of lenalidomide plus immunochemotherapy versus immunochemotherapy alone in patients with treatment-naive GEP-selected ABC-subtype DLBCL.The improvement of imaging and therapeutic targeting of tumors requires the identification of novel target-specific tracers. To address HNSCC tumors, Altmann and colleagues performed alternating biopanning on membrane proteome of HNO97 tumor cells fractionated by the ProteomeLab PF2D system and corresponding HNO97 cells using a sunflower trypsin inhibitor1-based phage display (SFTI8Ph) library. A novel ITGαvβ6-binding peptide (SFITGv6) was identified providing high stability and demonstrating affinity for ITGαvβ6-positive HNSCC and other carcinomas. SFITGv6 accumulated in tumor tissues but not in normal tissues or inflammatory lesions, and thus represents a promising tracer for imaging and endoradiotherapy.Deubiquitylating (DUB) enzymes have emerged as potential therapeutic targets. Das and colleagues examined the functional significance of DUB enzyme USP1 in multiple myeloma (MM) using an irreversible USP1 inhibitor SJB3-019A. USP1 is highly expressed in patient multiple myeloma (MM) cells. USP1 blockade with SJB3-019A decreases the viability of MM cells, inhibits DNA repair and USP1/ID/Notch/Sox pathways, and overcomes bortezomib-resistance. SJB induced generation of more mature and differentiated plasma cells. Combination of SJB with bortezomib, ACY1215, lenalidomide, or pomalidomide triggers synergistic anti-MM activity. These preclinical data provide basis for clinical evaluation of USP1 inhibitors in MM to overcome proteasome inhibitor resistance.

Abstract 2970: Mitigating tumor-stroma metabolic symbiosis for cancer therapy

Abstract Head and neck squamous cell carcinoma (HNSCC) affects 40,000 patients annually and is associated with <50% 5-year survival. There is an urgent need to better understand the biology of the disease in order to develop more effective therapeutic approaches. HNSCC tumors are dysplastic with up to 80% fibroblasts. We recently reported that tumor-associated fibroblasts (TAFs) make HNSCC more aggressive. Furthermore, we reported that TAFs produce hepatocyte growth factor (HGF), which binds to the c-Met receptor expressed on HNSCC to drive aggressiveness. Although we did not detect HGF secretion from HNSCC cell lines, we reported that paracrine activation of c-Met by TAF-secreted HGF facilitates HNSCC progression. Reciprocal signaling between the tumor and stroma has been reported in several cancers to facilitate tumor growth, invasion and resistance to therapy. Recent studies have shown that c-Met activation promotes glycolysis. Although highly glycolytic, the mechanisms regulating HNSCC glycolysis remain unknown. We show that TAF-secreted HGF through c-Met activation on HNSCC (a) induces aerobic glycolysis accompanied by lactate production, and b) regulates expression of basic fibroblast growth factor (bFGF). Studies have shown that HNSCC tumors have high lactate levels resulting from increased glycolysis, and this correlates with reduced survival. Our data demonstrate that TAF-secreted HGF increases key glycolytic enzymes including hexokinase II. Furthermore, HGF increases glycolysis and lactate production from HNSCC. We demonstrate that HGF also increases levels of the bidirectional lactate transporter, monocarboxylate transporter 1 (MCT1). We demonstrate that MCT1 levels are increased in both HNSCC and TAFs under co-culture conditions. In addition, HGF stimulation increases levels of MCT1 in HNSCC indicating a possible mechanism whereby HNSCC remove the excess lactate generated during glycolysis. The mechanisms whereby HNSCC tumors survive highly acidic conditions remain unknown. Since MCT1 levels are increased in TAFs as well, we sought to determine if TAFs utilize the lactate as a carbon source to generate energy. Indeed, we found that bFGF secreted by HNSCC, binds to its cognate FGF receptor (FGFR) on TAFs to facilitate latate utilization through mitochondrial oxidative phosphorylation (OXPHOS). Thus there exists a metabolic symbiosis between HNSCC and TAFs that contribute to tumor growth. Through these studies, we delineate the mechanisms of glycolysis regulation in HNSCC and demonstrate that inhibition of the cross-talk between HNSCC and TAFs can be used as a novel therapeutic approach. Citation Format: Dhruv Kumar, Jacob New, Vikalp Vishwakarma, Hemant Chavan, Partha Kasturi, Sufi M. Thomas. Mitigating tumor-stroma metabolic symbiosis for cancer therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2970. doi:10.1158/1538-7445.AM2017-2970

Abstract 4911: Allosteric inhibition of the Receptor Tyrosine Kinases c-MET, RON and VEGFR-2 via the co-receptor CD44v6 by the novel compound AMC303

Abstract Introduction: The receptor tyrosine kinases (RTKs) c-MET and RON and more importantly their crosstalk, play a crucial role in mediating local invasion, systemic dissemination and resistance in different types of cancer. Both RTKs are activated by the ligands HGF and MSP respectively and homo- or heterodimerize with each other. The RTK VEGFR-2 is the most important receptor for angiogenesis in solid epithelial tumors. CD44v6, a member of the CD44 family of transmembrane glycoproteins, was identified as an essential co-receptor for activation of c-MET, RON and VEGFR-2. AMC303, a peptide based allosteric and selective inhibitor of CD44v6, was investigated for its inhibitory effects on c-MET, RON and VEGFR-2 pathways in vitro and its effect on tumor growth and metastases in vivo. Methods: Affinity was determined using Microscale Thermophoresis. For in vitro blocking assays AMC303 was added 30 min prior to induction with the ligands. Analysis of RTK activation was carried out by western blot. Analysis of cell migration and invasion was performed with Boyden Chamber assays. VEGF-A induced tube formation was microscopically analyzed. For in vivo studies nude mice were orthotopically implanted with human tumor cells (L3.6pl). Animals were treated with AMC303 i.v. at 0.1, 1, 10 mg/kg QOD or QWK for 3 weeks. Regression of metastases was investigated at 1 mg/kg QOD. HPLC was used to detect the amount of AMC303 in the primary tumor lysates. Results: AMC303 binds to the ectodomain of CD44v6 with high affinity. In various pancreatic, breast, colon, lung and HNSCC tumor cell lines activation of c-MET and RON by their ligands was inhibited by AMC303 in vitro and consequently ligand induced cell scattering, migration and invasion was significantly reduced. In endothelial cells, activation of VEGFR-2 and VEGF-A induced formation of a tubular network was blocked by AMC303 treatment. In vivo treatment with AMC303 inhibited tumor growth in a dose dependent manner. The metastatic spreading of the primary tumor was prevented when animals were treated at early disease stage. Most strikingly, a marked regression of established liver metastases was observed at progressed disease state when animals were treated with AMC303 at 1 mg/kg QOD for 3 weeks. Conclusions: AMC303 inhibits activation of the RTKs c-MET and RON allosterically in different epithelial tumor cells and VEGFR-2 in endothelial cells by extracellular binding to CD44v6. This unique and novel mode of action results in a strong anti-tumor and anti-metastatic effect in vivo which together with its wide safety and tolerability window in preclinical toxicity studies is strongly supporting the clinical investigation. AMC303 is currently tested in a Phase I study in patients with solid epithelial tumors. Citation Format: Vanessa Al-Rawi, Thorsten Laeufer, Katrin Glocker, Yvonne Heneka, Alexandra Matzke-Ogi. Allosteric inhibition of the Receptor Tyrosine Kinases c-MET, RON and VEGFR-2 via the co-receptor CD44v6 by the novel compound AMC303 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4911. doi:10.1158/1538-7445.AM2017-4911

Abstract 4108: Targeting IL-6 signaling overcomes cetuximab resistance in head and neck squamous cell carcinoma

Abstract Head and neck squamous cell carcinoma (HNSCC), the sixth most common cancer worldwide, has a five-year survival rate of only 50%. The first targeted agent FDA approved for treatment of HNSCC was cetuximab, a monoclonal antibody targeting the epidermal growth factor receptor (EGFR). Despite EGFR overexpression in up to 90% of HNSCC tumors and ample evidence supporting EGFR as a therapeutic target in HNSCC, the response rate for single-agent cetuximab is below 20%, and resistance to cetuximab-containing therapy remains a major obstacle in the effective treatment of HNSCC. Identification and targeting of mediators of cetuximab resistance is needed to improve patient outcomes. Secretion of the cytokine interleukin 6 (IL-6) has been proposed as a mechanism of resistance to cetuximab in HNSCC, and inhibition of signal transducer and activator of transcription 3 (STAT3), a downstream mediator of IL-6 signaling, has been shown to overcome cetuximab resistance in preclinical HNSCC models. Thus, we hypothesize that IL-6 signaling mediates resistance to cetuximab and that co-treatment with agents targeting the IL-6 pathway will enhance the therapeutic efficacy of cetuximab in HNSCC. We have generated cetuximab-resistant variants of the cetuximab-sensitive HNSCC cell lines Cal33 and PE/CA-PJ49. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) analysis indicated that IL6 expression is increased 3.5- to 8-fold in cetuximab-resistant variants compared to the parental cells from which they were derived. In addition, a colony formation assay revealed that, as expected, cetuximab-treated parental cells formed fewer colonies than vehicle-treated cells, while cetuximab treatment had no effect on the ability of cetuximab-resistant PE/CA-PJ49 variants to form colonies. In contrast, treatment with tocilizumab, an IL-6 receptor-neutralizing monoclonal antibody, markedly reduced the colony-forming ability of the cetuximab-resistant variants, but not parental cells. These results suggest that targeted inhibition of IL-6 signaling may effectively inhibit proliferation in cetuximab-refractory HNSCC cells. Citation Format: Rachel A. O'Keefe, Neil Bhola, Toni M. Brand, Yan Zeng, Daniel E. Johnson, Jennifer R. Grandis. Targeting IL-6 signaling overcomes cetuximab resistance in head and neck squamous cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4108. doi:10.1158/1538-7445.AM2017-4108

Abstract 4667: mtDNA sequencing in HNSCC as a measurer of intratumoral heterogeneity and lymph node metastasis

Abstract Head and neck squamous cell carcinoma (HNSCC), accounts for 650,000 new cases worldwide. It is notorious for poor prognosis, which reflects its propensity to present as metastatic disease upon diagnosis. The prognosis and treatment regiments vary dramatically upon the manifestation of lymph node (LN) metastasis. Detection of regional LN metastasis is usually based on the clinical examination and imaging, followed by post-operational histological analysis. Unfortunately, current tools for detection of tumor cells in surgically resected LN may, in some cases, miss the presence of micrometastasis. Novel strategies for detection of micrometastasis based on the analysis of tumor-derived genetic aberrations by next generation sequencing (NGS) offer new hope for improved risk assessment and better selection of the treatment regimen. However, detection of mutations in minuscule metastatic lesions comprised of only few neoplastic cells may still fall below the detection threshold of these highly sensitive techniques. By virtue of their clonal nature, higher mutation rate and copy number, assessing tumor-specific mitochondrial DNA (mtDNA) mutations in histologically clean LN may provide a more sensitive diagnostic tool and eventually reduce the false negative rate in patients. Additionally, due to its circular configuration, mtDNA thought to be more stable than genomic DNA, and may also be suitable for sequencing formalin-fixed paraffin-embedded (FFPE) -derived genomic material, an invaluable resource for clinical research. However, current methods for library preparation are still imperfect for detection of low prevalence variants and heteroplasmy with high depth of coverage, especially in highly degraded samples. To this end, we aimed to set up a novel NGS assay for mtDNA analysis in primary and metastatic samples. Amplicon-based NGS library preparation approach, consisting of 148 primers pairs that cover the entire mitochondrial genome (with a dual coverage of 86.18%) was designed in collaboration with Fluidigm to be used on Access Array™ platform. To enhance the coverage of degraded material the amplicon size was limited to ~170 base pairs. Primers for three nuclear housekeeping genes were added to the panel to allow quantitative copy number analysis. To address mitochondrial mutational patterns, heteroplasmy and heterogeneity, we have used this novel high-throughput enrichment technology to sequence mtDNA from different areas of 16 primary HNSCC tumors (fresh frozen and FFPE), and multiple matched metastatic or histologically clean lymph-nodes. We obtained over 99% coverage with a median average depth of 3000X and no difference between fresh frozen and FFPE samples. This method allows obtaining a snapshot on the extent of mitochondrial heterogeneity of these lesions and may be used for detection of low frequency tumor-associated mtDNA mutations as a measurer of possible metastatic processes in histologically clean LN. Citation Format: Adrian D. Schubert, Evgeny Izumchenko, William H. Westra, Aditi Chatterjee, Wayne M. Koch, Mohammad O. Hoque, David Sidransky. mtDNA sequencing in HNSCC as a measurer of intratumoral heterogeneity and lymph node metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4667. doi:10.1158/1538-7445.AM2017-4667

Abstract 1850: Development of a NOTCH1 loss of function and PIK3CA mutant transgenic model of HNSCC

Abstract Head and neck squamous cell carcinoma (HSNCC) is a debilitating disease with a poor 5-year survival rate. The use of in vivo models to study tumorigenesis and to identify potential targeted therapies, especially those that work in combination with immune checkpoint inhibitors, is essential in improving overall patient care. However, the overall number of HNSCC transgenic models with recurrent genetic lesions identified by the HNSCC TCGA project has been limited. For example, TCGA data has shown both Notch1 loss of function mutations and PIK3CA activating mutations as some of the most common in HNSCC, but bi-genic models of these lesions do not yet exist. Here, we have designed a model which utilizes K14-CreERT to drive a bi-genic model with Notch1flox and PIK3CAH1047R mutations in epithelial compartments. Characterization of this line both in the context of spontaneous and carcinogen induced tumor formation is a tool which can be utilized to better understand common mutations in HNSCC. Immunohistochemistry confirms the overexpression of PIK3CA in tamoxifen induced mice. Furthermore, early characterization of this model with chronic treatment of the smoking analogue 4-Nitroquinoline N-oxide (4NQO) in drinking water shows tumor formation on the tongue and surrounding head and neck regions in as little as 12-16 weeks. Hematoxylin and Eosin staining confirmed the squamous tumor formation, while Ki67 staining showed enhanced proliferation of the transformed cells. In the future, this model can be used to study tumor formation over time and in response to targeted inhibitors and common therapies that are being advanced as companion diagnostic strategies for either NOTCH1 deficient or PIK3CA aberrant HNSCC tumors. Further detailed characterization of this model will allow for a deeper understanding of the mechanisms which drive HNSCC and will translate to improved patient care. Citation Format: Samantha N. Devenport, Nicole L. Michmerhuizen, Elizabeth Leonard, Brittany Jewell, Jacob Swanson, Sunny Wong, Thomas E. Carey, J Chad Brenner. Development of a NOTCH1 loss of function and PIK3CA mutant transgenic model of HNSCC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1850. doi:10.1158/1538-7445.AM2017-1850

Abstract 5476: CIP2A can be a therapeutic target of rapamycin in radioresistant head and neck cancer with P53 mutation

Abstract The presence of p53 mutation was associated with poor survival in various tumors. The involvement of p53 in apoptosis and cell-cycle control makes it a plausible biomarker of prognosis . Classifying TP53 mutations in HNSCC described disruptive TP53 mutation in either the L2 or L3 loop of the DNA binding domain, resulting in polarity change within the protein, or stop cordon. Disruptive TP53 mutation in HNSCC tumors predicts for locoregional recurrence, due to increased radioresistance via the inhibition of senescence. Senescence induction contributes to cancer therapy responses and is crucial for p53-mediated tumor suppression. However, whether p53 inactivation actively suppresses senescence induction has been unclear. Here, we found that overexpression CIP2A (cancerous inhibitor of protein phosphatase 2A) positively correlated with presence of p53 mutation in head and neck cancer and mediates radioresistance through suppression of radiation induced senescence. And we demonstrated that rapamycin could induce senescence via CIP2A downregulation and increase radiosensitivity in p53 disruptive mutation cell line. This is the first investigation to analyze the role of CI2A in resistance to the radiotherapy of head and neck cancer. As a consequence, a greater understanding of radioresistance mechanisms through our results in head and neck cancer with p53 mutation will enable the rational design of combination regimens and sequential treatment algorithms to improve clinical outcomes and points to the usefulness of CIP2A as a biomarker to predict clinical response to rapamycin in head and neck cancer. Citation Format: Song Hee Kim, Won hyeok Lee, Myung Jin Lee, Hyo Won Chang, Ji Won Kim, Mi Ra Kim, Jung Je Park, Myung Woul Han, Sang Yoon Kim. CIP2A can be a therapeutic target of rapamycin in radioresistant head and neck cancer with P53 mutation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5476. doi:10.1158/1538-7445.AM2017-5476

Abstract 1332: NOTCH - HEY1 pathway induces EMT in head and neck squamous cell carcinoma

Abstract Background Head and neck squamous cell carcinoma (HNSCC) is noted to have a NOTCH mutation rate of 20% within the Cancer Genome Atlas (TCGA) dataset consistent with tumor suppressor gene status. However, NOTCH genes are also upregulated in wild type HNSCC and show signs of downstream activation in a substantial proportion of HNSCC. In this study, we aimed to further explore the potential for NOTCH pathways to be activated in HNSCC. Material and Methods mRNA sequence of TCGA data includes 520 HNSCC tumors and 46 normal tissues. We used 447 HNSCC excluding 73 tumors with NOTCH mutations. The activated groups of NOTCH downstream genes were defined as tumors with expression 1 standard deviation above the mean of normal tissue. We used four HNSCC cell lines (SKN3, Cal27, SCC61, SCC090) to ascertain the results of the TCGA analysis in vitro. Result In TCGA analysis, HEY1 activated HNSCC had significant more expression of NOTCH pathway genes. In particular NOTCH4 had the most significant correlation with HEY1 activation among NOTCH receptors. To confirm this result in vitro, HEY1 expression was compared between si-control and si-NOTCH4 of SKN3, Cal27, SCC61 and SCC090. As expected, HEY1 expression in all si-NOTCH4 treated cell lines were significant decreased. Next, we examined the relationship about NOTCH4 and an epithelial mesenchymal transition phenotype (EMT). Using TCGA data, we found that mesenchymal marker genes were significantly increased in high NOTCH4 group. In vitro experiments, si-NOTCH4 cells decreased mesenchymal marker gene expression such as Fibronectin and TWIST1. We also examined HEY1 function. Similar to NOTCH4, it was found that HEY1 is significantly related to EMT in silico and in vitro. Conclusion The NOTCH4-HEY1 pathway is significantly upregulated in HNSCC and induces EMT. Citation Format: Takahito Fukusumi, Theresa W. Guo, Shuling Ren, Akihiro Sakai, Mizuo Ando, Sunny Haft, Chao Liu, Joseph A. Califano. NOTCH - HEY1 pathway induces EMT in head and neck squamous cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1332. doi:10.1158/1538-7445.AM2017-1332

Abstract 2684: Multiplex immunofluorescence profiling of tumor infiltrating immune subsets in HNSCC biopsies provides a powerful tool when combined with patient outcome data

Abstract Head and neck squamous cell carcinoma (HNSCC) is one of the growing number of tumors for which an anti-tumor immune response is implicated to play a role in the course of the disease. Profiling of the tumor microenvironment including specific infiltrating lymphocyte subsets, antigen presenting cells, expression of regulatory molecules on both tumor and immune cells and spatial relationships between cells could lead to predictive signatures for responsiveness to both conventional and immune modulatory therapies. While flow cytometry results in multi-parametric analysis of immune populations within the tumor, their context i.e. the spatial relationship of these cells to tumor and to other immune cells, is lost. In this current study we use Cell IDx’s novel UltraPlex technology, which is based on a simple two-step, autostainer compatible protocol using hapten labeled primary antibody cocktails followed by fluor labeled anti-hapten secondary cocktails to simultaneously detect 4 cellular markers on the same cell in tumor biopsies. Image registration of serial sections increases multiplexing to 12-16 markers. This technology, which allows generation of full immune subset profiling, quantitative marker expression and relative cellular localization, was applied to a HNSCC tumor microarray as a test system. Data are presented showing the potential of this technology for correlation of tumor “immune signature” with patient outcome. Citation Format: Matt Levin, Mark Lingen, David Schwartz, Helen Snyder. Multiplex immunofluorescence profiling of tumor infiltrating immune subsets in HNSCC biopsies provides a powerful tool when combined with patient outcome data [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2684. doi:10.1158/1538-7445.AM2017-2684

Abstract 1797: EGFR as a marker for head and neck squamous cell carcinoma-derived small extracellular vesicles

Abstract Head and neck cancer will account for an estimated 61,760 new cancer cases and 13,190 cancer-related deaths in the United States in 2016, the majority of which (> 90%) will be squamous cell carcinoma (HNSCC). The prognosis for HNSCC is relatively poor, with an overall 5-year survival around 60%, and the outlook worsens with increasing stage at diagnosis, which is problematic since about two-thirds of patients present with advanced stage disease. This underscores the urgent need for discovery, development and translation of novel biomarkers to facilitate early detection of HNSCC and bolster the chance for positive patient outcomes. Exosomes are nano-sized (40-150 nm) membrane-encapsulated vesicles that may offer a new avenue for discovery and development of novel HNSCC biomarkers. However, they are released by essentially all cells, both normal and malignant, into the extracellular space, resulting in a noisy environment in biofluids from the perspective of biomarker measurement. As such, a membrane-bound surface marker that allows for enrichment for cancer-derived exosomes in patient samples would provide a clear benefit. Epidermal growth factor receptor (EGFR), which belongs to the ErbB/HER family of receptor tyrosine kinases, localizes to the plasma membrane and is over-expressed in as much as 90% of HNSCC tumors. Therefore, we hypothesized that EGFR will be over-represented on membranes of HNSCC-derived exosomes and thus may offer utility as a surface marker for detection and/or enrichment. To test this, we cultured an HNSCC cell line that over-expresses EGFR (FaDu), EGFR-deficient SW620 colorectal adenocarcinoma cells, and primary human oral epithelial cells pooled from 3 healthy donors. Cells were cultivated in supplier-recommended media with 10% fetal bovine serum (FBS) that was super-depleted of exosomes via 18 hour ultracentrifugation at 100,000g and 1% penicillin/streptomycin at 37°C with 5% CO2 in 150 cm2 flasks with 25mL media. After reaching 80-90% confluence, media was harvested, and exosomes were isolated and purified via differential ultracentrifugation. Exosome isolates were whole-mounted on carbon grids, Immuno-Gold labeled with anti-EGFR, and imaged on a JEOL JEM-1230 transmission electron microscope (Immuno-EM); anti-nucleoporin 153 (NUP-153), which localizes to the nucleus, was used as a negative control. Immuno-EM images indicate abundant EGFR expression on exosomes secreted by FaDu HNSCC cells, but EGFR is not detectable on the exosomes secreted by the oral epithelial cells from healthy donors. These results indicate that EGFR may offer utility for enrichment for HNSCC-derived exosomes in human biofluid isolates using magnetic beads or flow cytometry. Citation Format: Jenna A. Dombroski, Damaris Kuhnell, Susan Kasper, Scott M. Langevin. EGFR as a marker for head and neck squamous cell carcinoma-derived small extracellular vesicles [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1797. doi:10.1158/1538-7445.AM2017-1797

PO-0887: Experimental validation of a 3D model to simulate FMISO spatial retention in HNSCC tumor xenografts

PO-0887: Experimental validation of a 3D model to simulate FMISO spatial retention in HNSCC tumor xenografts

Natural Killer cells from primary human head and neck squamous cell carcinomas upregulate NKG2A

Abstract Immunotherapy has revolutionized cancer therapy primarily by targeting checkpoint inhibitors found on endogenous immune cells. We analyzed and compared NK cells from the blood and tumor of HNSCC patients by flow cytometry for the expression of NK cell activating and inhibitory receptors. We found that NK cells from the primary tumor, but not the blood of HNSCC patients, significantly downregulated activating receptors NKG2D, DNAM-1, NKp30, CD16 and 2B4. Surprisingly, the majority of inhibitory KIR receptors on tumor-infiltrating NK cells were decreased compared to NK cells from the blood. However, the inhibitory receptors NKG2A and PD-1 were found to be highly overexpressed in tumor-associated NK cells. We believe that this study provides the first in-depth look at NK cells from primary HNSCC tumors. We found that tumor-associated NK cells express a profoundly different phenotype than NK cells from the blood of the same patients, with multiple activating receptors downregulated and the inhibitory receptor NKG2A over-expressed. These results support the therapeutic strategy of targeting NKG2A.

Phenotypic heterogeneity of disseminated tumour cells is preset by primary tumour hypoxic microenvironments.

Hypoxia is a poor-prognosis microenvironmental hallmark of solid tumours, but it is unclear how it influences the fate of disseminated tumour cells (DTCs) in target organs. Here we report that hypoxic HNSCC and breast primary tumour microenvironments displayed upregulation of key dormancy (NR2F1, DEC2, p27) and hypoxia (GLUT1, HIF1α) genes. Analysis ofsolitary DTCs in PDX and transgenic mice revealed that post-hypoxic DTCs were frequently NR2F1hi/DEC2hi/p27hi/TGFβ2hi and dormant. NR2F1 and HIF1α were required for p27 induction in post-hypoxic dormant DTCs, but these DTCs did not display GLUT1hi expression. Post-hypoxic DTCs evaded chemotherapy and, unlike ER- breast cancer cells, post-hypoxic ER+ breast cancer cells were more prone to enter NR2F1-dependent dormancy. We propose that primary tumour hypoxic microenvironments give rise to a subpopulation of dormant DTCs that evade therapy. These post-hypoxic dormant DTCs may be the source of disease relapse and poor prognosis associated with hypoxia.