Neck Squamous Cancer Research Articles

Head and neck squamous cancer progression is marked by CLIC4 attenuation in tumor epithelium and reciprocal stromal upregulation of miR-142-3p, a novel post-transcriptional regulator of CLIC4.

Chloride intracellular channel 4 (CLIC4) is a tumor suppressor implicated in processes including growth arrest, differentiation, and apoptosis. CLIC4 protein expression is diminished in the tumor parenchyma during progression in squamous cell carcinoma (SCC) and other neoplasms, but the underlying mechanisms have not been identified. Data from The Cancer Genome Atlas suggest this is not driven by genomic alterations. However, screening and functional assays identified miR-142-3p as a regulator of CLIC4. CLIC4 and miR-142-3p expression are inversely correlated in head and neck (HN) SCC and cervical SCC, particularly in advanced stage cancers. In situ localization revealed that stromal immune cells, not tumor cells, are the predominant source of miR-142-3p in HNSCC. Furthermore, HNSCC single-cell expression data demonstrated that CLIC4 is lower in tumor epithelial cells than in stromal fibroblasts and endothelial cells. Tumor-specific downregulation of CLIC4 was confirmed in an SCC xenograft model concurrent with immune cell infiltration and miR-142-3p upregulation. These findings provide the first evidence of CLIC4 regulation by miRNA. Furthermore, the distinct localization of CLIC4 and miR-142-3p within the HNSCC tumor milieu highlight the limitations of bulk tumor analysis and provide critical considerations for both future mechanistic studies and use of miR-142-3p as a HNSCC biomarker.

Abstract B130: Elevated cancer-intrinsic type I interferon signaling confers a dependency on the RNA editor ADAR1

Abstract The deamination of adenosine to inosine (known as A-to-I editing) is one of the most prevalent RNA modifications that occurs in metazoans and is mediated by the Adenosine Deaminase Acting on RNA (ADAR) family of enzymes. The enzyme ADAR/ADAR1 catalyzes the majority of A-to-I editing where it has been demonstrated to effect coding sequence, miRNA function and silencing of Alu repetitive elements1. A critical function of ADAR1 is to edit double stranded RNA (dsRNA) structures that can activate the cytoplasmic nucleoside sensors MDA5 and PKR, preventing aberrant activation of an innate immune type I interferon (IFN) response2,3. Consistent with this hypothesis, mutations in ADAR1 and other enzymes involved in nucleoside metabolism and/or sensing are found in Aicardi-Goutiéres Syndrome (AGS), an interferonopathy associated with spontaneous interferon production4. Through analysis of pooled CRISPR and shRNA screening data, we have identified that cancer cell lines with elevated expression of specific type I interferon stimulated genes (ISGs) are dependent on ADAR1 for their survival. Tumor cells can display elevated intrinsic type I interferon signaling and associated dsRNA burden due to multiple factors, including chronic cytoplasmic DNA activation of STING, oncovirus infection and other proinflammatory signals. We have identified a core subset of 26 ISGs that predict a cellular dependency on ADAR1 and demonstrate elevated expression of these ISGs in a diverse range of tumor types including head and neck squamous cell carcinoma, breast cancer, esophageal cancer, and lung cancer. Utilizing an isogenic cell line with low basal type I interferon signaling and knockout of ADAR1, we demonstrate that we can induce cell death through exogenous treatment with the dsRNA mimetic poly-(I:C) or type I interferons (IFNα or IFNβ) but not the type II interferon IFNγ. We believe that chronic ISG expression and dsRNA burden creates a dependency on ADAR1 to prevent activation of MDA5 and PKR. These data suggest an ADAR1 inhibitor could be beneficial for the treatment of tumors with elevated ISG expression. Eisenberg et al., Nature Review Genetics (2018) Ahmad et al., Cell (2018) Chung et al., Cell (2018) Crow et al., Nature Review Immunology (2015) Citation Format: Alexandra K. Gardino, Cindy Collins, Maureen S. Lynes, P. Ann Boriack-Sjodin, Robert A. Copeland, Scott A. Ribich. Elevated cancer-intrinsic type I interferon signaling confers a dependency on the RNA editor ADAR1 [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr B130. doi:10.1158/1535-7163.TARG-19-B130

Dissecting multi drug resistance in head and neck cancer cells using multicellular tumor spheroids

One of the hallmarks of cancers is their ability to develop resistance against therapeutic agents. Therefore, developing effective in vitro strategies to identify drug resistance remains of paramount importance for successful treatment. One of the ways cancer cells achieve drug resistance is through the expression of efflux pumps that actively pump drugs out of the cells. To date, several studies have investigated the potential of using 3-dimensional (3D) multicellular tumor spheroids (MCSs) to assess drug resistance; however, a unified system that uses MCSs to differentiate between multi drug resistance (MDR) and non-MDR cells does not yet exist. In the present report we describe MCSs obtained from post-diagnosed, pre-treated patient-derived (PTPD) cell lines from head and neck squamous cancer cells (HNSCC) that often develop resistance to therapy. We employed an integrated approach combining response to clinical drugs and screening cytotoxicity, monitoring real-time drug uptake, and assessing transporter activity using flow cytometry in the presence and absence of their respective specific inhibitors. The report shows a comparative response to MDR, drug efflux capability and reactive oxygen species (ROS) activity to assess the resistance profile of PTPD MCSs and two-dimensional (2D) monolayer cultures of the same set of cell lines. We show that MCSs provide a robust and reliable in vitro model to evaluate clinical relevance. Our proposed strategy can also be clinically applicable for profiling drug resistance in cancers with unknown resistance profiles, which consequently can indicate benefit from downstream therapy.

1288P - Safety and efficacy of anti-PD-1 inhibitor ABBV-181 in lung and head and neck carcinoma

BackgroundABBV-181 is a humanized anti-PD1 monoclonal antibody; dose finding and early safety, PK and pharmacodynamic data have been reported (ESMO18). This report summarizes data from the head and neck squamous cell carcinoma (HNSCC) and non-small cell lung cancer (NSCLC) expansion cohorts in the Ph1 FIH study (NCT03000257). MethodsPatients (pts) with previously treated, advanced HNSCC and NSCLC received ABBV-181 IV, 250mg Q2W or 500mg Q4W to progression. Response was assessed by RECIST v1.1 and iRECIST. ResultsAs of April 2019, 81 pts were dosed.Table1288PTablen (%)HNSCC n=41NSCLC n=40Median days on treatment, range72, 1–40771, 1–421Dose: 250mg/500mg31/1019/21AE any grade40 (98)40 (100)Grade (G) ≥3 AE23 (56)27 (67)ABBV-181 related G≥3 AE4 (10)3 (7)Immune-mediated AE15 (37)15 (37)AE resulting in discontinuation of ABBV-181 Malignant neoplasm progresion General physical health Confusion Pneumonitisa Acute kidney injurya Hypothyroidisma Immune-mediated hepatitisa Disturbance in attention Dysponea Intracranial hemorrhage Neoplasm progression Acute respiratory distress syndrome Upper respiratory tract infection5 (12) 2 (5) 1 (2) 0 0 0 1 (2) 0 0 0 0 1 (2) 1 (2) 09 (22) 3 (7) 0 1 (2) 1 (2) 1 (2) 1 (2) 1 (2) 1 (2) 1 (2) 1 (2) 0 0 1(2)G 5 AEsb Malignant neoplasm progression Acute respiratory distress syndrome Dyspnea Neoplasm progression Upper respiratory tract infection General physical condition abnormal7 (17) 4 (10) 1 (2) 0 1 (2) 0 1 (2)7 (18) 6(15) 0 1 (2) 0 1 (2) 0aABBV-181 related.bnone considered related to ABBV-181. Most frequent G≥1 AE (n): anemia (20), asthenia (20) and fatigue (16). ABBV-181PK is approximately dose proportional, with comparable dose-normalized exposures at both doses; trough levels resulted in sustained PD-1 saturation on circulating CD4 T cells. Transient decreases in circulating T cells and increased Ki67+ CD8 T cells and serum CXCL9/CXCL10 were observed at both doses. In pts with ≥ 1 post baseline assessment, responses by investigator: 7/40 HNSCC pts (7 partial response, 6 by RECISTv.1.1, 1 by iRECIST), 6/36 NSCLC (5 PR, 1 complete response, all by RECIST v.1.1). 3 HNSCC and 2 NSCLC responders had PD-L1+ tumors. ConclusionsSafety, PK and PD data support dosing ABBV-181 at 250mg Q2W or 500mg Q4W with expected biological and pharmacodynamic activity for an anti-PD-1 agent. Efficacy data demonstrate clinical activity per RECISTv.1.1 and iRECIST supporting continued development of ABBV-181. Clinical trial identificationNCT03000257. Editorial acknowledgementFatemeh Atashi (AbbVie employee). Legal entity responsible for the studyAbbVie Inc. FundingAbbVie Inc. DisclosureA. Italiano: Advisory / Consultancy: Roche, Daiichi Sankyo, ImmuneDesign, Epizyme, Bayer, Lilly; Honoraria (self): Bayer, Daiichi Sankyo, Lilly, Epizyme, Novartis, Roche; Research grant / Funding (self): Roche, Bayer, AstraZeneca/MedImmune, PharmaMar, MSD Oncology, Merck Serono. P. Cassier: Honoraria (self): Novartis, Roche/Genentech, Blueprint Medicines, Amgen; Research grant / Funding (institution): Novartis, Roche/Genentech, Lilly, Blueprint Medicines, Bayer, AstraZeneca, Celgene, Plexxikon, AbbVie, Bristol-Myers Squibb, Merck Serono, Merck, Sharp and Dohme. C. Lin: Advisory / Consultancy: Novartis, Boehringer Ingelheim, Blueprint Medicines; Travel / Accommodation / Expenses: Lilly, Daiichi Sankyo, BeiGene, Novartis; Honoraria (self): Novartis, Roche, Daiichi Sankyo. K. Peltola: Advisory / Consultancy: Orion Pharma, BMS, MSD, Pfizer, Ipsen and Roche; Shareholder / Stockholder / Stock options: Faron Pharmaceuticals; Speaker Bureau / Expert testimony: BMS, Pfizer, MSD; Speaker Bureau / Expert testimony: Ipsen; Travel / Accommodation / Expenses: Roche and BMS. A. Gazzah: Travel / Accommodation / Expenses: Boehringer, Novartis, Pfizer, Roche. E. Calvo: Advisory / Consultancy: Novartis, Nanobiotix, Janssen-Cilag, PsiOxus, Seattle Genetics, EUSA Pharma, AbbVie, Celgene, AstraZeneca, Guidepoint Global, Roche/Genentech, GLG, Pfizer, Servier, Amcure; Speaker Bureau / Expert testimony: Novartis; Research grant / Funding (self): AstraZeneca, BeiGene, Novartis, START, Oncoart Associated, ; Leadership role: President and Founder of Foundation Intheos. D. Tosi: Advisory / Consultancy: Biomarin ; Research grant / Funding (self): Novartis, Astellas, Janssen; Travel / Accommodation / Expenses: Janssen, Pfizer, and Astellas Pharma, Nutricia and Amicus. B. Gao: Advisory / Consultancy: MSD. L. warburton: Travel / Accommodation / Expenses: MSD, Merck. M. Tanner: Advisory / Consultancy: Roche, Novartis and Pfizer; Speaker Bureau / Expert testimony: Roche, Novartis, Pfizer and Amgen. S. Englert: Full / Part-time employment: AbbVie Inc.; Shareholder / Stockholder / Stock options: AbbVie Inc. S. Lambert: Full / Part-time employment: AbbVie Inc.; Shareholder / Stockholder / Stock options: AbbVie Inc. A. Parikh: Shareholder / Stockholder / Stock options: AbbVie Inc.; Full / Part-time employment: AbbVie Inc. D. Afar: Shareholder / Stockholder / Stock options: AbbVie Inc.; Full / Part-time employment: AbbVie Inc. G. Vosganian: Shareholder / Stockholder / Stock options: AbbVie Inc.; Full / Part-time employment: AbbVie Inc. V. Moreno: Advisory / Consultancy: Merck; Travel / Accommodation / Expenses: Regeneron/Sanofi; Research grant / Funding (self): Medscape/Bayer. All other authors have declared no conflicts of interest.

NMR-based metabolomics in real-time monitoring of treatment induced toxicity and cachexia in head and neck cancer: a method for early detection of high risk patients

IntroductionNutritional treatment in head and neck squamous cell carcinoma cancer (HNSCC) patients undergoing radio-/chemo-radiotherapy (RT/CHRT) is complex and requires a multidisciplinary approach. In this study the real-time dynamic changes in serum metabolome during RT/CHRT in HNSCC patients were monitored using NMR-based metabolomics.ObjectivesThe main goal was to find the metabolic markers that could help prevent of acute radiation sequelae (ARS) escalation.Methods170 HNSCC patients were treated radically with RT/CHRT. Blood samples were collected weekly, starting from the day before the treatment and stopping within the week after the RT/CHRT completion, resulting in a total number of 1328 samples. 1H NMR spectra were acquired on Bruker 400 MHz spectrometer at 310 K and analyzed using principal component analysis (PCA) and orthogonal partial least squares discriminant analysis (OPLS-DA). Additional statistical analyses were performed on the quantified metabolites.ResultsPCA has detected a group of distinct outliers corresponding to ketone bodies (3HB, Ace, AceAce). These outliers were found to identify the individuals at high risk of weight loss, mainly by the 3HB changes, which was confirmed by the patients’ medical data. In the OPLS-DA models a transition from the lowest to the highest weight loss is seen, defining the metabolic time trajectories for the patients from the studied groups during RT/CHRT. 3HB is a relatively sensitive marker that allows earlier identification of the patients at higher risk of > 10% weight loss.ConclusionOur findings indicate that metabolic alterations, characteristic for malnutrition or cachexia, can be detected already at the beginning of the treatment, making it possible to monitor the patients with a higher risk of weight loss.

Prognostic Value of Pre-Treatment Neutrophil-to-Lymphocyte Ratio and Platelet in Patients with Operable Head and Neck Squamous Cell Carcinoma

Background: Peripheral blood inflammation factor neutrophil-lymphocyte Ratio (NLR), platelet and nutritional factor albumin (ALB) have recently been proposed as prognostic markers in solid tumors. The current study the impact of those hematological parameters and oncologic outcomes in patients with operable head and neck squamous carcinoma cancer (HNSCC) using database approach were evaluated. Methods: 198 patients with oropharyngeal, hypopharyngeal and laryngeal cancers receiving multimodality protocols between 2012 and 2014 were included. Neutrophil, lymphocyte, platelet and albumin from the last phlebotomy before treatment were identified. Patients were risk-stratified and were tested for association with survival and disease-control outcomes. Results: 5-year LRC were decreased (p=0.004, respectively) for 140 patients with NLRpre-treatment 248×109/L were better among tested models. On Bayesian information criteria (BIC) analysis, the optimal prognostic model was then used to develop nomograms predicting 3- and 5-year locoregional recurrence (LRC). External validity evaluated by studying another 57 patients from other hospital confirmed the reliability of prediction model. Conclusion: Pre-treatment NLR elevation and platelet >248×109/L are promising predictors of prognosis in patients with operable HNSCC. The analyses performed show that pre-treatment hematological inflammatory markers and platelet-based Nomograms provide a distinct risk stratification. Further works should be complete to elucidate the utility of anti-inflammatory and anti- platelet treatments in modifying risk in peri-operation HNSCC patients. Funding Statement: This study is sponsored by grants from Medical Health Science and Technology Project of Zhejiang Provincial Health Commission (Grant No.2019336033). Medical Health Science Project of Hangzhou (Grant No. OO20190775). Declaration of Interests: The authors declare no conflict of interest. Ethics Approval Statement: This study was in accordance with Declaration of Helsinki and approved by the Research Ethic Committees of Affiliated Sir Run Run Shaw Hospital, Zhejiang University medical college and The Second Affiliated Hospital of Nanchang University Medical College. Both the institutional review board approved data extraction was performed from an existing HNSCC.

Discrimination of Head and Neck Squamous Cell Carcinoma Patients and Healthy Adults by 10-Color Flow Cytometry: Development of a Score Based on Leukocyte Subsets.

Background: Leukocytes in peripheral blood (PB) are prognostic biomarkers in head and neck squamous cell carcinoma cancer patients (HNSCC-CPs), but differences between HNSCC-CPs and healthy adults (HAs) are insufficiently described. Methods: 10-color flow cytometry (FCM) was used for in-depth immunophenotyping of PB samples of 963 HAs and 101 therapy-naïve HNSCC-CPs. Absolute (AbsCC) and relative cell counts (RelCC) of leukocyte subsets were determined. A training cohort (TC) of 43 HNSCC-CPs and 43 HAs, propensity score (PS)-matched according to age, sex, alcohol, and smoking, was used to develop a score consecutively approved in a validation cohort (VC). Results: Differences in AbsCC were detected in leukocyte subsets (p < 0.001), but had low power in discriminating HNSCC-CPs and HAs. Consequently, RelCC of nine leukocyte subsets in the TC were used to calculate 36 ratios; receiver operating characteristic (ROC) curves defined optimum cut-off values. Binary classified data were combined in a score based on four ratios: monocytes-to-granulocytes (MGR), classical monocytes-to-monocytes (clMMR), monocytes-to-lymphocytes (MLR), and monocytes-to-T-lymphocytes (MTLR); ≥3 points accurately discriminate HNSCC-CPs and HAs in the PS-matched TC (p = 2.97 × 10−17), the VC (p = 4.404 × 10−178), and both combined (p = 7.74 × 10−199). Conclusions: RelCC of leukocyte subsets in PB of HNSCC-CPs differ significantly from those of HAs. A score based on MGR, clMMR, MLR, and MTLR allows for accurate discrimination.

Initial biological evaluations of 18F-KS1, a novel ascorbate derivative to image oxidative stress in cancer

BackgroundReactive oxygen species (ROS)-induced oxidative stress damages many cellular components such as fatty acids, DNA, and proteins. This damage is implicated in many disease pathologies including cancer and neurodegenerative and cardiovascular diseases. Antioxidants like ascorbate (vitamin C, ascorbic acid) have been shown to protect against the deleterious effects of oxidative stress in patients with cancer. In contrast, other data indicate potential tumor-promoting activity of antioxidants, demonstrating a potential temporal benefit of ROS. However, quantifying real-time tumor ROS is currently not feasible, since there is no way to directly probe global tumor ROS. In order to study this ROS-induced damage and design novel therapeutics to prevent its sequelae, the quantitative nature of positron emission tomography (PET) can be harnessed to measure in vivo concentrations of ROS. Therefore, our goal is to develop a novel translational ascorbate-based probe to image ROS in cancer in vivo using noninvasive PET imaging of tumor tissue. The real-time evaluations of ROS state can prove critical in developing new therapies and stratifying patients to therapies that are affected by tumor ROS.MethodsWe designed, synthesized, and characterized a novel ascorbate derivative (E)-5-(2-chloroethylidene)-3-((4-(2-fluoroethoxy)benzyl)oxy)-4-hydroxyfuran-2(5H)-one (KS1). We used KS1 in an in vitro ROS MitoSOX-based assay in two different head and neck squamous cancer cells (HNSCC) that express different ROS levels, with ascorbate as reference standard. We radiolabeled 18F-KS1 following 18F-based nucleophilic substitution reactions and determined in vitro reactivity and specificity of 18F-KS1 in HNSCC and prostate cancer (PCa) cells. MicroPET imaging and standard biodistribution studies of 18F-KS1 were performed in mice bearing PCa cells. To further demonstrate specificity, we performed microPET blocking experiments using nonradioactive KS1 as a blocker.ResultsKS1 was synthesized and characterized using 1H NMR spectra. MitoSOX assay demonstrated good correlations between increasing concentrations of KS1 and ascorbate and increased reactivity in SCC-61 cells (with high ROS levels) versus rSCC-61cells (with low ROS levels). 18F-KS1 was radiolabeled with high radiochemical purity (> 94%) and specific activity (~ 100 GBq/μmol) at end of synthesis (EOS). Cell uptake of 18F-KS1 was high in both types of cancer cells, and the uptake was significantly blocked by nonradioactive KS1, and the ROS blocker, superoxide dismutase (SOD) demonstrating specificity. Furthermore, 18F-KS1 uptake was increased in PCa cells under hypoxic conditions, which have been shown to generate high ROS. Initial in vivo tumor uptake studies in PCa tumor-bearing mice demonstrated that 18F-KS1 specifically bound to tumor, which was significantly blocked (threefold) by pre-injecting unlabeled KS1. Furthermore, biodistribution studies in the same tumor-bearing mice showed high tumor to muscle (target to nontarget) ratios.ConclusionThis work demonstrates the strong preliminary support of 18F-KS1, both in vitro and in vivo for imaging ROS in cancer. If successful, this work will provide a new paradigm to directly probe real-time oxidative stress levels in vivo. Our work could enhance precision medicine approaches to treat cancer, as well as neurodegenerative and cardiovascular diseases affected by ROS.

Tumor infiltrating B cells co-localize with CD4 T effector cells within organized tertiary lymphoid structures to present antigen and educate the anti-tumor immune response in human primary tumors

Abstract Only 20% of cancer patients produce a durable response to current immunotherapies. Thus, a need exists to develop additional therapeutic strategies to treat these patients, which includes evaluation of other tumor infiltrating immune cells that could further augment the T cell response. Tumor infiltrating B cells (TIL-B) represent a possible target for immunotherapy due to their crucial role in the immune response. In fact, current data demonstrate that detection of TIL-Bs within tertiary lymphoid structures (TLS) correlate with increased survival in patients with solid tumors and enhanced response to anti-PD1 immunotherapy. We hypothesize that TIL-Bs help generate potent, long-term immune responses against cancer by presenting tumor antigens to CD4 TILs within TLS. We observed increased numbers of activated TIL-Bs in the primary tumors of head and neck squamous cell carcinoma and non-small cell lung cancer patients. We further assessed the transcriptional signature and location of TIL-Bs within the TME and demonstrated that they are adjacent to Th1 and Tfh CD4+ tumor infiltrating lymphocytes (TILs) within organized, germinal center containing TLS. Further, we generated an antigen presentation assay in vitro, and we observed increased CD4+ TIL responses when TIL-Bs presented autologous tumor antigens. These data suggest that TIL-Bs influence the function of CD4+ TILs in patient tumors within TLS. Ultimately, results from this study will increase effective targeting of TIL-Bs within patient primary tumors.

Incidence and risk factors of interstitial lung disease of patients with head and neck cancer treated with cetuximab.

Interstitial lung disease (ILD) is known as a potentially severe adverse event associated with epidermal growth factor receptor (EGFR)-targeted therapy. The incidence and risk factors of ILD in patients with head and neck squamous cancer (HNSCC) treated with cetuximab, an anti-EGFR monoclonal antibody, have not been established. We retrospectively reviewed patients with HNSCC who received cetuximab from December 2012 to December 2016 at our institute and evaluated the incidence and risk factors of ILD. Of the 201 patients with HNSCC, ILD was observed in 9 patients (4.5%), 8 of whom had grade 3 or higher. High Krebs von den Lungen-6 (KL-6) and ≥50 pack-years of smoking were significantly predictive of associated with ILD (P = 0.00011 and 0.05, respectively). The incidence of ILD in patients with HNSCC treated with cetuximab was <5%, but most of the ILD cases were severe. High KL-6 and smoking histories might be predictive for ILD among patients with HNSCC.

Primary Thyroid Squamous Cell Carcinoma With Complete Response to Radical Surgery and Concurrent Chemoradiotherapy: Diagnostic Pathologic Workup and Clinical Management

Abstract Primary squamous cell carcinoma (SqCC) of the thyroid gland is an extremely rare, highly aggressive malignancy associated with locally advanced disease and frequent metastases to regional lymph nodes and distant sites. There are no standardized guidelines for treating primary thyroid SqCC because of its rarity, but some investigators have recommended staging and treating it as a variant of anaplastic thyroid carcinoma. Prognosis, however, remains poor with minimal response to treatment and an estimated median survival of 6 to 14 months. We report a case of primary SqCC of the thyroid that was successfully treated with aggressive locoregional resection followed by concurrent radical dose radiotherapy and cisplatin-based chemotherapy. The patient remains free of disease more than 4 years after surgery and adjuvant chemoradiation. Because of potential prognostic and management implications, it is crucial to make an accurate pathologic diagnosis of primary SqCC of the thyroid and distinguish it from metastatic squamous carcinoma and other primary thyroid malignancies with squamous features. A thorough clinicopathologic diagnostic workup including immunohistochemistry, computed tomography scan, and endoscopy is mandatory. In this case, treatment similar to the standard management used for patients with locally advanced mucosal head and neck squamous cancer led to a successful outcome.

Abstract A053: Activated B cells in human primary tumors present antigen and increase antitumor function of CD4 T-cells in tertiary lymphoid structures

Abstract Immunotherapy, specifically anti-PD1, has improved patient survival in a range of tumor types including head and neck squamous cell carcinoma (HNSCC) and non-small cell lung cancer (NSCLC). Despite the success of anti-PD1 therapy, only 20% of patients produce a durable response to this treatment. Thus, a need exists to develop additional therapeutic strategies to treat these patients, which includes evaluation of other tumor-infiltrating immune cells that could further augment the CD8+ and CD4+ T-cell response. Tumor-infiltrating B cells (TIL-B) represent a possible target for immunotherapy due to their predominance in the tumor microenvironment (TME) and crucial role in the immune response. However, TIL-B function in cancer and in the context of immunotherapy has been understudied. In fact, conclusions on an anti- or protumor role for TIL-Bs in the TME is dependent on the study. However, in HNSCC and NSCLC patients, current evidence suggests an antitumor role for TIL-Bs. Specifically, detection of TIL-Bs within tertiary lymphoid structures (TLS) correlates with better prognosis. While TIL-Bs have been identified in HNSCC and NSCLC patients, their complete phenotypic signature and function in the TME has been understudied with no focus on their role as antigen presenting cells (APCs) and their influence on CD8+ and CD4+ tumor infiltrating lymphocytes (TILs). We hypothesize that TIL-Bs help generate potent, long-term immune responses against cancer by presenting tumor antigens to CD4 TILs within TLS.Using unmanipulated, primary human B cells from fresh tumor, we quantified and further characterized TIL-Bs in HNSCC and NSCLC utilizing single-cell RNAseq and multiparameter flow cytometry. We observed increased numbers of activated TIL-Bs in these primary tumors compared to other immune subsets, specifically CD27+ TIL-Bs. We further assessed the TIL-Bs by correlating phenotype of the TIL-B with its location in the TME, predominantly separating out differences between TIL-Bs within TLS and outside TLS. In addition, we generated a specific antigen presentation assay in vitro, and we observed three types of CD4+ TIL responses when TIL-Bs presented autologous tumor antigens. There were activated responder CD4+ TILs that proliferated when combined with TIL-Bs alone, which indicates stimulation with endogenous tumor antigens. There were antigen-associated responders that required exogenous autologous tumor lysate to elicit a CD4+ TIL response, and there were patient CD4 TILs that did not respond to antigen presentation by TIL-Bs. Within the activated and antigen-associated responders, the TIL-B phenotype influenced the CD4+ TIL phenotype; if the TIL-Bs were activated (CD27+), the CD4+ TILs were T helper (antitumor) CD4+ T-cells and if the TIL-Bs were non-activated (CD27-), the CD4+ TILs were T regulatory cells (protumor). These data suggest that TIL-Bs influence the phenotype and function of CD4+ TILs in patient tumors. In conclusion, activated TIL-Bs are increased in human primary tumors, they can present antigen to CD4+ TILs and influence their overall phenotype. Determining the complete activation signature of TIL-Bs in HNSCC and NSCLC patients will determine the extent of their antitumor function in these cancers. Comparison of TIL-Bs in HNSCC and NSCLC is important as there are not many unified studies on TIL-B function across tumor types. Further, because HNSCC has two etiologies (viral vs. carcinogen induced), we are able to better study the differential function of activated and nonactivated TIL-Bs in solid tumors. Ultimately, results from this study will help predict how to target TIL-B functions in future TIL-B-specific immunotherapies or in combination with current immunotherapies for HNSCC and NSCLC patients like blockade of the inhibitory receptor, PD-1. Citation Format: Tullia C. Bruno, Ayana T. Ruffin, Anthony R. Cillo, Robert L. Ferris, Dario A.A. Vignali. Activated B cells in human primary tumors present antigen and increase antitumor function of CD4 T-cells in tertiary lymphoid structures [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr A053.

AdoMet triggers apoptosis in head and neck squamous cancer by inducing ER stress and potentiates cell sensitivity to cisplatin.

S-Adenosyl-l-methionine (AdoMet) is a naturally and widely occurring sulfonium compound that plays a primary role in cell metabolism and acts as the principal methyl donor in many methylation reactions. AdoMet also exhibits antiproliferative and proapoptotic activities in different cancer cells. However, the molecular mechanisms underlying the effects exerted by AdoMet have only been partially studied. Inthe current study, we evaluated the antiproliferative effect of AdoMet on Cal-33 oral and JHU-SCC-011 laryngeal squamous cancer cells to define the underlying mechanisms. We demonstrated that AdoMet induced apoptosis in Cal-33 and JHU-SCC-011 cells, involving a caspase-dependent mechanism paralleled by an increased Bax/Bcl-2 ratio. Moreover, we showed, for the first time, that AdoMet induced ER-stress in Cal-33 cells and activated the unfolded protein response, which can be responsible for apoptosis induction through the activation of CHOP and JNK. In addition, AdoMet-induced ER-stress was followed by autophagy with a consistent increase in the levels of the autophagic marker LC3B-II, which was indeed potentiated by the autophago-lysosome inhibitor chloroquine. As both escape from apoptosis and decreased activation of JNK are mechanisms of resistance to cisplatin (cDPP), an agent usually used in cancer therapy, we have evaluated the effects of AdoMet in combination with cDPP on Cal-33 cells. Our data showed that the combined treatment resulted in a strong synergism in inhibiting cell proliferation and in enhancing apoptosis via intrinsic mechanism. These results demonstrate that AdoMet has ER-stress-mediated antiproliferative activity and synergizes with cDDP on cell growth inhibition, thus providing the basis for its use in new anticancer strategies.

Fibroblast growth factor receptor 3‐mediated reactivation of ERK signaling promotes head and neck squamous cancer cell insensitivity to MEK inhibition

Recurrent or metastatic head and neck squamous cell carcinoma (HNSCC) has been a longstanding challenge for head and neck oncologists, and current treatments still have limited efficacy. ERK is aberrantly overexpressed and activated in HNSCC. Herein, we aimed to investigate the cause of the limited therapeutic effect of selumetinib, a selective inhibitor of MEK in HNSCC, as MEK/ERK reactivation inevitably occurs. We assessed the effects of combining selumetinib with fibroblast growth factor receptor 3 (FGFR3) inhibitor (PD173074) on tumor growth. Selumetinib transiently inhibited MAPK signaling and reactivated ERK signaling in HNSCC cells. Rebound in the ERK and Akt pathways in HNSCC cells was accompanied by increased FGFR3 signaling after selumetinib treatment. Feedback activation of FGFR3 was a result of autocrine secretion of the FGF2 ligand. The FGFR3 inhibitor PD173074 prevented MAPK rebound and sensitized the response of HNSCC cells to selumetinib. These results provided rational therapeutic strategies for clinical studies of this subtype of patients that show a poor prognosis with selumetinib. Our data provide a rationale for combining a MEK inhibitor with inhibitors of feedback activation of FGFR3 signaling in HNSCC cells. ERK rebound as a result of the upregulation of FGFR3 and the ligand FGF2 diminished the antitumor effects of selumetinib, which was overcome by combination treatment with the FGFR3 inhibitor.

In vitro and in vivo superior radiosensitizing effect of berbamine for head and neck squamous cell carcinoma.

BackgroundBerbamine (BBM), one of the bis-benzylisoquinoline products isolated from Berberis amurensis, has been demonstrated for its anticancer effect against leukemia, breast cancer, liver cancer, etc. There are some studies focusing on the chemosensitization effect of BBM. However, there is no report about whether BBM could enhance the anticancer effect of radiation, which made us to explore the possible radiosensitization effect of BBM.Materials and methodsHere, in vitro cytotoxicity of BBM was evaluated on two kinds of head and neck squamous cancer cell lines. Clonogenic assay was performed to study the radiosensitization effect of BBM. Western blot was utilized to elucidate the possible mechanism underlying the radiosensitization effect.ResultsBBM effectively inhibited the growth of two kinds of cancer cells in a time- and dose-dependent manner. Radiation plus BBM led to significantly more reduction of the colony-forming ability of cancer cells when compared with radiation alone. BBM plus radiation led to the most reduction of STAT3 phosphorylation, followed by the significant decrease of the ratio of Bax/Bcl-2. In vivo study demonstrated that the combinational administration of BBM and radiation generated the most significant tumor-delaying effect among all of the treatment regimens.ConclusionWe reported, in the current study, the potential role of BBM in not only treating cancer by itself but also offering a promising way to improve the efficacy of radiotherapy by inhibiting the activation of STAT3 and subsequently inducing the apoptosis of cancer.

Abstract PR10: Multimodality imaging of human lung squamous cell carcinoma reveals unique metabolic dependencies that are effectively targeted with metabolic-based therapies

Abstract Altered metabolism is known to commonly contribute to cancer growth, forming the conceptual basis for the development of metabolic therapies as cancer treatments. However, the specific metabolic characteristics of individual cancer types in vivo are still largely unknown, limiting the translatability of metabolic therapies in the clinic. In this study we used a multimodality imaging approach to identify metabolic dependencies in lung squamous cell carcinomas (SCC) in order to guide precise treatments with metabolic therapies. We performed in vivo metabolic profiling and molecular analysis of lung SCC using positron emission tomography (PET) imaging, liquid chromatography mass spectrometry (LC-MS), and immunohistochemistry (IHC). PET imaging lung SCC tumors with 18F-fluoro-2-deoxyglucose (18F-FDG) and 11C-Glutamine PET tracers identifed a conserved metabolic reliance on both glucose and glutamine that we confirmed with LC-MS based metabolomics and quantitative IHC. The mTOR kinase is a central regulator of growth and metabolism and is readily inhibited using allosteric and catalytic kinase inhibitors. Using the mTOR catalytic kinase inhibitor MLN128, we successfully inhibited glycolysis in lung SCC. However, lung SCC tumors were able to metabolically adapt to chronic mTOR inhibition through upregulation of glutamine metabolism. We discovered that chronic mTOR inhibition induced phosphorylation and inactivation of GSK3a/b resulting in upregulation and activation of cMYC and cJUN—both central regulators of the glutaminase (GLS) enzyme and glutaminolysis. Importantly, phospho-GSK3α/β and phospho-cJUN proteins serve as functional biomarkers that predict MLN128 resistance and upregulation of glutaminolysis. To overcome MLN128 resistance we treated lung SCC tumors with the glutaminase inhibitor CB-839. The combination of MLN128 and CB-839 effectively overcame therapy resistance in genetically engineered mouse models (GEMMs) and patient-derived xenografts (PDXs) of lung SCC. Lastly, data mining from The Lung Cancer Genome Atlas coupled to our PET imaging and IHC biomarkers analysis allowed us to construct a metabolic signature that is conserved in human lung SCC as well as a broad spectrum of hypermetabolic human tumors. These tumors include lung SCC, head and neck squamous cell carcinoma (HNSCC), osteosarcomas (OS), and triple-negative breast cancer (TNBC). Importantly, this metabolic signature is predictive of patient outcome and response to combined metabolic therapies targeting mTOR and glutaminase. We therefore propose a clinically translatable treatment strategy for lung SCC patients that is driven by PET imaging and IHC to first stratify patients by their metabolic signature, which is then used to guide the delivery of precise metabolic-based therapies that target key metabolic nodes such as mTOR and GLS. This abstract is also being presented as Poster B11. Citation Format: Milica Momcilovic, David B. Shackelford. Multimodality imaging of human lung squamous cell carcinoma reveals unique metabolic dependencies that are effectively targeted with metabolic-based therapies [abstract]. In: Proceedings of the Fifth AACR-IASLC International Joint Conference: Lung Cancer Translational Science from the Bench to the Clinic; Jan 8-11, 2018; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(17_Suppl):Abstract nr PR10.

Diffusion tensor imaging in differentiation of residual head and neck squamous cell carcinoma from post-radiation changes

BackgroundDifferentiation between residual head and neck squamous cell carcinoma and post-radiation changes is difficult with routine computed tomography and magnetic resonance imaging. PurposeTo assess the reliability and reproducibility of mean diffusivity (MD) and fractional anisotropy (FA) parameters of diffusion tensor imaging in differentiation residual head and neck squamous cell carcinoma from post-radiation changes. Material and methodsA retrospective analysis of diffusion tensor imaging of 43 patients with head and neck squamous cell carcinoma cancer after radiotherapy. The MD and FA of the lesion were calculated by the same reader at two-time points. ResultsThere was a significantly lower difference (P = 0.001) in MD of both readings between residual head and neck squamous cell carcinoma (1.48 ± 0.06 and 1.47 ± 0.07 × 10−3 mm2/s) and post-radiation changes (1.72 ± 0.08 and 1.71 ± 0.11 × 10−3 mm2/s). The FA of residual head and neck squamous cell carcinoma of both readings (0.41 ± 0.09 and 0.42 ± 0.09) shows a significantly higher difference (P = 0.001) than post-radiation changes (0.17 ± 0.04 and 0.16 ± 0.03). There was excellent intra-reader agreement between both readings using MD (K = 0.958) and FA (K = 0.987). The threshold MD and FA used for differentiating the residual from post-radiation changes of both readings was 1.61, 1.65 × 10−3 mm2/s and 0.27, 0.25 with an area under the curve of 0.991, 0.934, 0.993 and 0.990 respectively. ConclusionMD and FA of diffusion tensor imaging are non-invasive reliable and reproducible parameters that can help in differentiation residual head and neck squamous cell carcinoma from post-radiation changes.

Abstract 5047: Exosomes derived from HIV-1-infected T cells activate ERK 1/2 through EGFR and TLR3 in cancer cells

Abstract Exosomes secreted from HIV-1-infected T cells play important roles in coordinating the immune responses and disease processes in HIV-infected people who are treated with antiretroviral therapy. We hypothesize that exosomes from HIV-infected T cells can promote growth and progression of non-AIDS-defining cancer cells. Activation of the MAPK ERK 1/2 is important for cancer cell survival, proliferation and invasion. We investigated the ability of exosomes from HIV-1-infected T cells to activate ERK 1/2 in head neck squamous cell carcinoma (HNSCC) and lung cancer cells for cancer cell proliferation and progression. Exosomes were isolated from Jurkat and J1.1, a latently HIV-1-infected Jurkat cells, using differential ultracentrifugation and quantified by acetylcholinesterase (AchE) activity. HNSCC cell line HSC3 and lung cancer cell lines NCI-H1299 and NCI-H1437 cells were treated with exosomes. To test the involvement of EGFR and TLR3 in ERK 1/2 activation by exosomes, EGFR neutralizing antibody Cetuximab or the TLR3/dsRNA inhibitor complex was added to cancer cells 30 min prior to adding exosomes. To test the entry of exosomes into cells, HEK293 cells were transiently transfected with EGFR-GFP, and seeded into glass bottom 35 mm dishes, time lapse Z-stacking imaging was performed and deconvolution was used to remove out of focus signals. By using Western blotting, we found that exosomes from J1.1 cells (HIV-1-infected), but not those from HIV-negative Jurkat cells, constantly induced ERK 1/2 phosphorylation in HNSCC and lung cancer cells. Cetuximab dramatically decreased ERK activation by exosomes from J1.1 cells, suggesting an EGFR-dependent ERK activation. While EGF induced a whole series of EGFR phosphorylation, exosomes from J1.1 cells failed to do so, suggesting that exosomes from J1.1 cells trigger ERK activation through a non-canonical EGFR pathway. Furthermore, ERK activation was significantly reduced in TLR3/dsRNA inhibitor pretreated cancer cells in response to exosomes from J1.1 cells, indicating exosomes triggered ERK activation is also TLR3 dependent. Time lapse imaging showed that exosomes quickly entered into cells within two minutes; however, Cetuximab greatly delayed exosome entry. To conclude, we found that exosomes from HIV-infected T cells can induce ERK activation in HNSCC and lung cancer cells through interaction with EGFR and TLR3. Citation Format: Zhimin Feng, Lechuang Chen, Hong Yue, Bingcheng Wang, Ge Jin. Exosomes derived from HIV-1-infected T cells activate ERK 1/2 through EGFR and TLR3 in cancer cells [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 5047.

Reactive oxygen species in cancer stem cells of head and neck squamous cancer.

One of the greatest challenges in systemic treatment of head and neck squamous cell carcinoma (HNSCC) is a small tumor cell population, namely, cancer stem-like cells (CSC). CSC can regenerate and maintain a heterogenic tumor by their self-renewal capacity. Their potential ability to be more resistant to and survival after chemo- and radiation therapy was also identified. Further studies have shown that reactive oxygen species (ROS) contribute to this CSC-associated resistance. In this review, we focus on the current knowledge of HNSCC-CSC, with regard to ROS as a possible and novel therapeutic approach in targeting CSC.

PD-L1 is expressed on human platelets and is affected by immune checkpoint therapy.

Cancer immunotherapy has been revolutionised by drugs that enhance the ability of the immune system to detect and fight tumors. Immune checkpoint therapies that target the programmed death-1 receptor (PD-1), or its ligand (PD-L1) have shown unprecedented rates of durable clinical responses in patients with various cancer types. However, there is still a large fraction of patients that do not respond to checkpoint inhibitors, and the challenge remains to find cellular and molecular cues that could predict which patients would benefit from these therapies. Using a series of qualitative and quantitative methods we show here that PBMCs and platelets from smokers and patients with head and neck squamous cell carcinoma (HNSCC) or lung cancer express and up-regulate PD-L1 independently of tumor stage. Furthermore, treatment with Atezolizumab, a fully humanised monoclonal antibody against PD-L1, in 4 patients with lung cancer caused a decrease in PD-L1 expression in platelets, which was restored over 20 days. Altogether, our findings reveal the expression of the main therapeutic target in current checkpoint therapies in human platelets and highlight their potential as biomarkers to predict successful therapeutic outcomes.