Inhibition of the CCR6-CCL20 axis prevents regulatory T cell recruitment and sensitizes head and neck squamous cell carcinoma to radiation therapy.

BACKGROUND:  Radioresistance of HNSCCs remains a major challenge for effective tumor control. Combined radiotherapy (RT) and immunotherapy (IT) treatment improved survival for a subset of patients with inflamed tumors or tumors susceptible to RT-induced inflammation. To overcome radioresistance and improve treatment outcomes, an understanding of factors that suppress anti-tumor immunity is necessary. In this regard, regulatory T cells (Tregs) are critical mediators of immune suppression in HNSCCs. In this study, we investigated how radiation modulates Treg infiltration in tumors through the chemokine CCL20. We hypothesized that radiation induces CCL20 secretion resulting in Treg infiltration and suppression of anti-tumor immunity. METHODS:  Human and mouse HNSCC cell lines with different immune phenotypes were irradiated at doses of 2 or 10 Gy. Conditioned media, RNA and protein were collected for assessment of CCL20. qPCR was used to determine CCL20 gene expression. In vivo, MOC2 cells were implanted into the buccal cavity of mice and the effect of neutralizing CCL20 antibody was determined alone and in combination with RT. Blood samples were collected before and after RT for analysis of CCL20. Tumor samples were analyzed by flow cytometry to determine immune infiltrates, including CD8 T cells and Tregs. Mass-spectrometry was performed to analyze proteomic changes in the tumor microenvironment after anti-CCL20 treatment. RESULTS:  Cal27 and MOC2 HNSCCs had a gene signature associated with Treg infiltration, whereas SCC9 and MOC1 tumors displayed a gene signature associated with an inflamed TME. In vitro, tumor irradiation at 10 Gy significantly induced CCL20 in Cal27 and MOC2 cells relative to control. The increase in CCL20 was associated with increased Treg migration. Neutralization of CCL20 reversed radiation-induced migration of Treg cells in vitro and decreased intratumoral Tregs in vivo. Furthermore, inhibition of CCL20 resulted in a significant decrease in tumor growth compared to control in MOC2 tumors. This effect was further enhanced after combination with RT compared to either treatment alone. CONCLUSION:  Our results suggest that radiation promotes CCL20 secretion by tumor cells which is responsible for the attraction of Tregs. Inhibition of the CCR6-CCL20 axis prevents infiltration of Tregs in tumors and suppresses tumor growth resulting in improved response to radiation.

Dichotomous effects of cellular expression of STAT3 on tumor growth of HNSCC.

STAT3 signaling has been shown to regulate cellular function and cytokine production in the tumor microenvironment (TME). Within the head and neck squamous cell carcinoma (HNSCC) TME, we previously showed that therapeutic targeting of STAT3 in combination with radiation resulted in improved tumor growth delay. However, given the independent regulatory effects STAT3 has on anti-tumor immunity, we aimed to decipher the effects of individually targeting STAT3 in the cancer cell, regulatory T cells (Tregs), and natural killer (NK) cell compartments in driving tumor growth and resistance to therapy in HNSCCs. We utilized a CRISPR knockout system for genetic deletion of STAT3 within the cancer cell as well as two genetic knockout mouse models, FoxP3-Cre/STAT3 fl and NKp46-Cre/STAT3 fl, for Tregs and NK cell targeting, respectively. Our data revealed differences in development of resistance to treatment with STAT3 CRISPR knockout in the cancer cell, driven by differential recruitment of immune cells. Knockout of STAT3 in Tregs overcomes this resistance and results in Treg reprogramming and recruitment and activation of antigen-presenting cells. In contrast, knockout of STAT3 in the NK cell compartment results in NK cell inactivation and acceleration of tumor growth. These data underscore the complex interplay between the cancer cell and the immune TME and carry significant implications for drug targeting and design of combination approaches in HNSCCs.

Loss of cancer cell STAT1 improves response to radiation therapy and promotes T cell activation in head and neck squamous cell carcinoma.

Resistance to radiation therapy (RT) remains an obstacle in HPV-negative head and neck squamous cell carcinomas (HNSCCs)-even with a combined RT-immunotherapy approach. Jak-Stat proteins have long been studied for both their immune regulatory role in the host immune response as well as their cancer cell signaling role in shaping the tumor microenvironment (TME). Here, we identify STAT1 as a mediator of radioresistance in HPV-negative preclinical mouse models of HNSCC, by which knockout of STAT1 in the cancer cell (STAT1 KO)-but not in the host-resulted in decreased tumor growth alongside increased immune activation. We show that RT increases STAT1/pSTAT1 expression, which may act as a marker of radioresistance. Whereas RT increased JAK-STAT and interferon (IFN) signaling, transcriptomic analysis revealed that STAT1 KO in the cancer cell resulted in decreased expression of IFN-associated genes of resistance. In vitro experiments showed that STAT1 KO increased T cell chemoattraction and decreased baseline growth. These results indicate that STAT1 may serve a tumor-promoting role in the cancer cell and will inform biomarker development and treatment regimens for HNSCC incorporating RT.

Response to radiotherapy in pancreatic ductal adenocarcinoma is enhanced by inhibition of myeloid-derived suppressor cells using STAT3 anti-sense oligonucleotide.

Pancreatic ductal adenocarcinoma (PDAC) has a heterogeneous tumor microenvironment (TME) comprised of myeloid-derived suppressor cells (MDSCs), tumor-associated macrophages, neutrophils, regulatory T cells, and myofibroblasts. The precise mechanisms that regulate the composition of the TME and how they contribute to radiotherapy (RT) response remain poorly understood. In this study, we analyze changes in immune cell populations and circulating chemokines in patient samples and animal models of pancreatic cancer to characterize the immune response to radiotherapy. Further, we identify STAT3 as a key mediator of immunosuppression post-RT. We found granulocytic MDSCs (G-MDSCs) and neutrophils to be increased in response to RT in murine and human PDAC samples. We also found that RT-induced STAT3 phosphorylation correlated with increased MDSC infiltration and proliferation. Targeting STAT3 using an anti-sense oligonucleotide in combination with RT circumvented RT-induced MDSC infiltration, enhanced the proportion of effector T cells, and improved response to RT. In addition, STAT3 inhibition contributed to the remodeling of the PDAC extracellular matrix when combined with RT, resulting in decreased collagen deposition and fibrotic tissue formation. Collectively, our data provide evidence that targeting STAT3 in combination with RT can mitigate the pro-tumorigenic effects of RT and improve tumor response.

Role of epidermal growth factor receptor inhibitor-induced interferon pathway signaling in the head and neck squamous cell carcinoma therapeutic response.

BACKGROUND: Epidermal growth factor receptor (EGFR) is frequently amplified or overexpressed in head and neck squamous cell carcinoma (HNSCC) and is a clinically validated target for the therapeutic antibody, cetuximab, in the management of this cancer. The degree of response to EGFR inhibitors measured by tumor shrinkage varies widely among HNSCC patients, and the biological mechanisms that underlie therapeutic heterogeneity amongst HNSCC patients remain ill-defined. METHODS: EGFR-dependent human and murine HNSCC cell lines were treated with the EGFR/ERBB inhibitors, gefitinib and AZD8931, and submitted to RNAseq, GSEA, and qRT-PCR. Conditioned media was analyzed by ELISA and Luminex assays. Murine HNSCC tumors were stained for T cell markers by immunofluorescence. Primary HSNCC patient specimens treated with single agent cetuximab were stained with Vectra multispectral immunofluorescence. RESULTS: The transcriptional reprogramming response to EGFR/ERBB-specific TKIs was measured in a panel of EGFR-dependent human HNSCC cell lines and interferon (IFN) α and γ responses identified as top-ranked TKI-induced pathways. Despite similar drug sensitivity, responses among 7 cell lines varied quantitatively and qualitatively, especially regarding the induced chemokine and cytokine profiles. Of note, the anti-tumorigenic chemokine, CXCL10, and the pro-tumorigenic factor, IL6, exhibited wide-ranging and non-overlapping induction. Similarly, AZD8931 exerted potent growth inhibition, IFNα/IFNγ pathway activation, and CXCL10 induction in murine B4B8 HNSCC cells. AZD8931 treatment of immune-competent mice bearing orthotopic B4B8 tumors increased CD8 + T cell content and the therapeutic response was abrogated in nu/nu mice relative to BALB/c mice. Finally, Vectra 3.0 analysis of HNSCC patient tumors prior to and after 3-4 weeks of single agent cetuximab treatment revealed increased CD8 + T cell content in specimens from patients exhibiting a therapeutic response relative to non-responders. CONCLUSIONS: The findings reveal heterogeneous, tumor cell-intrinsic, EGFR/ERBB inhibitor-induced IFN pathway activation in HNSCC and suggest that individual tumor responses to oncogene-targeted agents are a sum of direct growth inhibitory effects and variably-induced participation of host immune cells.

The effects of ephrinB2 signaling on proliferation and invasion in glioblastoma multiforme.

The aggressive nature of glioblastoma multiforme (GBM) may be attributed to the dysregulation of pathways driving both proliferation and invasion. EphrinB2, a membrane-bound ligand for some of the Eph receptors, has emerged as a critical target regulating these pathways. In this study, we investigated the role of ephrinB2 in regulating proliferation and invasion in GBM using intracranial and subcutaneous xenograft models. The Cancer Genome Atlas analysis suggested high transcript and low methylation levels of ephrinB2 as poor prognostic indicators in GBM, consistent with its role as an oncogene. EphrinB2 knockdown, however, increased tumor growth, an effect that was reversed by ephrinB2 Fc protein. This was associated with EphB4 receptor activation, consistent with the data showing a significant decrease in tumor growth with ephrinB2 overexpression. Mechanistic analyses showed that ephrinB2 knockdown has anti-invasive but pro-proliferative effects in GBM. EphB4 stimulation following ephrinB2 Fc treatment in ephrinB2 knockdown tumors was shown to impart strong anti-proliferative and anti-invasive effects, which correlated with decrease in PCNA, p-ERK, vimentin, Snail, Fak, and increase in the E-cadherin levels. Overall, our study suggests that ephrinB2 cannot be used as a sole therapeutic target. Concomitant inhibition of ephrinB2 signaling with EphB4 activation is required to achieve maximal therapeutic benefit in GBM.

A comparison of overall survival for patients with T4 larynx cancer treated with surgical versus organ-preservation approaches: A National Cancer Data Base analysis.

BACKGROUND: Although laryngectomy is the treatment of choice for patients with T4 larynx cancer, many patients are unable or unwilling to undergo laryngectomy and instead pursue larynx-preservation strategies combining radiotherapy (RT) and chemotherapy. Herein, the authors analyzed the National Cancer Data Base to evaluate overall survival (OS) between patients treated with surgical and organ-preserving modalities. METHODS: The National Cancer Data Base was queried for patients diagnosed from 2004 through 2012 with T4M0 laryngeal cancer who underwent either laryngectomy (surgery) with adjuvant RT (SRT), chemotherapy starting concurrently within 7 days of RT (CCRT), or multiagent induction chemotherapy starting 43 to 98 days before RT (ICRT). Multivariate analysis and propensity score matching were used to explore the association between the intervention and OS. Recursive partitioning analysis was performed to identify groups benefiting from particular modalities. RESULTS: A total of 1559 patients who underwent SRT, 1597 patients who underwent CCRT, and 386 patients who underwent ICRT were included. Adjusting for covariates, CCRT was found to be associated with inferior OS compared with SRT (hazard ratio [HR], 1.55; 95% confidence interval [95% CI], 1.41-1.70 [P<.01]) and with ICRT (HR, 1.25; 95% CI, 1.07-1.45 [P<.01]). OS among the patients treated with SRT did not appear to differ significantly from that of the ICRT cohort (HR, 0.87; 95% CI, 0.73-1.03 [P = 0.10]), a finding confirmed with propensity score matching. Recursive partitioning analysis identified no subset of patients that derived an OS benefit from either approach over the other. CONCLUSIONS: OS among patients undergoing SRT was found to be superior to that of patients treated with CCRT but did not significantly differ from that of those undergoing ICRT. Because these intriguing findings require validation, SRT should remain the standard of care for patients with this disease. However, organ preservation with ICRT may be a reasonable alternative in certain patients. Cancer 2017;123:600-608. © 2016 American Cancer Society.

Ephrin-B2 overexpression predicts for poor prognosis and response to therapy in solid tumors.

Ephrin B2 is variably expressed on tumor cells and its blockade has been shown to inhibit angiogenesis in animal models of pancreatic, colorectal, lung and head, and neck squamous cell carcinomas. However, the implications of ephrinB2 expression in cancer patients have remained elusive. In this study, we analyzed the cancer genome atlas (TCGA) for ephrinB2 expression. We report significant correlations between EFNB2 expression, overall survival and disease-free survival in head and neck squamous cell carcinoma (HNSCC, n = 519), pancreatic adenocarcinoma (n = 186), and bladder urothelial carcinoma (n = 410). In HNSCC patients, high-EFNB2 mRNA expression was associated with tumor HPV negativity, oral cavity location, alcohol intake, higher TP53 mutation, and EGFR amplification. EphrinB2 overexpression also correlated with worse response to chemotherapy and radiotherapy. The therapeutic potential of blocking ephrinB2 was validated in HNSCC patient-derived tumor xenografts and showed significant improvement in survival and tumor growth delay. Our data shows that ephrinB2 overexpression can serve as a critical biomarker for patient prognosis and response to therapy. These results should guide design of future clinical trials exploring EphrinB2 inhibition in cancer patients. © 2016 Wiley Periodicals, Inc.

Survival impact of pre-treatment neutrophils on oropharyngeal and laryngeal cancer patients undergoing definitive radiotherapy.

BACKGROUND: Squamous cell carcinoma of the head and neck (HNSCC) represents an array of disease processes with a generally unfavorable prognosis. Inflammation plays an important role in tumor development and response to therapy. We performed a retrospective analysis of HNSCC patients to explore the relationship of the lymphocyte and neutrophil counts, the neutrophil-to-lymphocyte ratio (NLR) overall survival (OS), cancer-specific survival (CSS), local control (LC) and distant control (DC). MATERIALS/METHODS: All patients received definitive treatment for cancers of the oropharynx or larynx between 2006-2015. Neutrophil and lymphocyte counts were collected pre-, during-, and post-treatment. The correlations of patient, tumor, and biological factors to OS, CSS, LC and DC were assessed. RESULTS: 196 patients met our inclusion criteria; 171 patients were Stage III or IV. Median follow-up was 2.7 years. A higher neutrophil count at all treatment time points was predictive of poor OS with the pre-treatment neutrophil count and overall neutrophil nadir additionally predictive of DC. Higher pre-treatment and overall NLR correlated to worse OS and DC, respectively. CONCLUSION: A higher pre-treatment neutrophil count correlates to poor OS, CSS and DC. Lymphocyte counts were not found to impact survival or tumor control. Higher pre-treatment NLR is prognostic of poor OS.

Combined EphB2 receptor knockdown with radiation decreases cell viability and invasion in medulloblastoma.

BACKGROUND: Medulloblastoma is one of the most common types of pediatric brain tumor characterized by the subpopulation of cells that exhibit high invasive potential and radioresistant properties. In addition, dysregulated function and signaling by Eph family of receptors have been shown to impart pro-tumorigenic characteristics in this brain malignancy. In the current study, we investigated whether EphB2 knockdown in combination with radiation can alter invasiveness and decrease medulloblastoma tumor growth or viability in vitro. METHODS: The expression of EphB2 receptor was analyzed by immunohistochemistry and Western blotting. Microarray analysis and mRNA analysis was performed on medulloblastoma patient datasets and compared to the normal cerebellum. The radiosensitization effect following EphB2 knockdown was determined by clonogenic assay in human medulloblastoma cells. Effects of EphB2-siRNA in absence or presence of radiation on cell cycle distribution, cell viability, and invasion were analyzed by flow cytometry, MTT assay, trypan blue exclusion assay, xcelligence system, and Western blotting. RESULTS: We observed that EphB2 is expressed in both medulloblastoma cell lines and patient samples and its downregulation sensitized these cells to radiation as evident by decreased clonogenic survival fractions. EphB2 expression was also high across different medulloblastoma subgroups compared to normal cerebellum. The radiosensitization effect observed following EphB2 knockdown was in part mediated by enhanced G2/M cell cycle arrest. We also found that the combined approach of EphB2 knockdown and radiation exposure significantly reduced overall cell viability in medulloblastoma cells compared to control groups. Similar results were obtained in the xcelligence-based invasion assay. Western blot analysis also demonstrated changes in the protein expression of cell proliferation, cell survival, and invasion molecules in the combination group versus others. CONCLUSIONS: Overall, our findings indicate that specific targeting of EphB2 receptor in combination with radiation may serve as an effective therapeutic strategy in medulloblastoma. Future studies are warranted to test the efficacy of this approach in in vivo preclinical models.

Reconciling two opposing effects of radiation therapy: stimulation of cancer cell invasion and activation of anti-cancer immunity.

PURPOSE: The damage caused by radiation therapy to cancerous and normal cells inevitably leads to changes in the secretome profile of pro and anti-inflammatory mediators. The inflammatory response depends on the dose of radiation and its fractionation, while the inherent radiosensitivity of each patient dictates the intensity and types of adverse reactions. This review will present an overview of two apparently opposite reactions that may occur after radiation treatment: induction of an antitumor immune response and a protumoral response. Emphasis is placed on the molecular and cellular mechanisms involved. CONCLUSIONS: By understanding how radiation changes the balance between anti- and protumoral effects, these forces can be manipulated to optimize radiation oncology treatments.

Toll-Like Receptors and the Response to Radiotherapy in Solid Tumors: Challenges and Opportunities.

Toll-like receptors (TLRs) are indispensable for the activation, maintenance and halting of immune responses. TLRs can mediate inflammation by recognizing molecular patterns in microbes (pathogen-associated molecular patterns: PAMPs) and endogenous ligands (danger-associated molecular patterns: DAMPs) released by injured or dead cells. For this reason, TLR ligands have attracted much attention in recent years in many cancer vaccines, alone or in combination with immunotherapy, chemotherapy and radiotherapy (RT). TLRs have been shown to play controversial roles in cancer, depending on various factors that can mediate tumor progression or apoptosis. Several TLR agonists have reached clinical trials and are being evaluated in combination with standard of care therapies, including RT. Despite their prolific and central role in mediating immune responses, the role of TLRs in cancer, particularly in response to radiation, remains poorly understood. Radiation is recognized as either a direct stimulant of TLR pathways, or indirectly through the damage it causes to target cells that subsequently activate TLRs. These effects can mediate pro-tumoral and anti-tumoral effects depending on various factors such as radiation dose and fractionation, as well as host genomic features. In this review, we examine how TLR signaling affects tumor response to RT, and we provide a framework for the design of TLR-based therapies with RT.

A novel preclinical model of craniospinal irradiation in pediatric diffuse midline glioma demonstrates decreased metastatic disease.

Background: Diffuse midline glioma (DMG) is an aggressive pediatric central nervous system tumor with strong metastatic potential. As localized treatment of the primary tumor improves, metastatic disease is becoming a more important factor in treatment. We hypothesized that we could model craniospinal irradiation (CSI) through a DMG patient-derived xenograft (PDX) model and that CSI would limit metastatic tumor. Methods: We used a BT245 murine orthotopic DMG PDX model for this work. We developed a protocol and specialized platform to deliver craniospinal irradiation (CSI) (4 Gy x2 days) with a pontine boost (4 Gy x2 days) and compared metastatic disease by pathology, bioluminescence, and MRI to mice treated with focal radiation only (4 Gy x4 days) or no radiation. Results: Mice receiving CSI plus boost showed minimal spinal and brain leptomeningeal metastatic disease by bioluminescence, MRI, and pathology compared to mice receiving radiation to the pons only or no radiation. Conclusion: In a DMG PDX model, CSI+boost minimizes tumor dissemination compared to focal radiation. By expanding effective DMG treatment to the entire neuraxis, CSI has potential as a key component to combination, multimodality treatment for DMG designed to achieve long-term survival once novel therapies definitively demonstrate improved local control.

Targeting resistance to radiation-immunotherapy in cold HNSCCs by modulating the Treg-dendritic cell axis.

BACKGROUND: Numerous trials combining radiation therapy (RT) and immunotherapy in head and neck squamous cell carcinoma (HNSCC) are failing. Using preclinical immune cold models of HNSCC resistant to RT-immune checkpoint inhibitors, we investigate therapeutic approaches of overcoming such resistance by examining the differential microenvironmental response to RT. METHODS: We subjected two HPV-negative orthotopic mouse models of HNSCC to combination RT, regulatory T cells (Treg) depletion, and/or CD137 agonism. Tumor growth was measured and intratumorous and lymph node immune populations were compared among treatment groups. Human gene sets, genetically engineered mouse models DEREG and BATF3-/-, flow and time-of-flight cytometry, RNA-Seq, Treg adoptive transfer studies, and in vitro experiments were used to further evaluate the role of dendritic cells (DCs) and Tregs in these treatments. RESULTS: In MOC2 orthotopic tumors, we find no therapeutic benefit to targeting classically defined immunosuppressive myeloids, which increase with RT. In these radioresistant tumors, supplementing combination RT and Treg depletion with anti-CD137 agonism stimulates CD103+ DC activation in tumor-draining lymph nodes as characterized by increases in CD80+ and CCR7+ DCs, resulting in a CD8 T cell-dependent response. Simultaneously, Tregs are reprogrammed to an effector phenotype demonstrated by increases in interferonγ+, tumor necrosis factorα+, PI3K+, pAKT+ and Eomes+ populations as well as decreases in CTLA4+ and NRP-1+ populations. Tumor eradication is observed when RT is increased to an 8 Gy x 5 hypofractionated regimen and combined with anti-CD25+ anti-CD137 treatment. In a human gene set from oral squamous cell carcinoma tumors, high Treg number is associated with earlier recurrence. CONCLUSIONS: Regulating Treg functionality and DC activation status within the lymph node is critical for generating a T cell effector response in these highly radioresistant tumors. These findings underscore the plasticity of Tregs and represent a new therapeutic opportunity for reprogramming the tumor microenvironment in HNSCCs resistant to conventional radioimmunotherapy approaches.

Induction of ADAM10 by Radiation Therapy Drives Fibrosis, Resistance, and Epithelial-to-Mesenchyal Transition in Pancreatic Cancer.

Stromal fibrosis activates prosurvival and proepithelial-to-mesenchymal transition (EMT) pathways in pancreatic ductal adenocarcinoma (PDAC). In patient tumors treated with neoadjuvant stereotactic body radiation therapy (SBRT), we found upregulation of fibrosis, extracellular matrix (ECM), and EMT gene signatures, which can drive therapeutic resistance and tumor invasion. Molecular, functional, and translational analysis identified two cell-surface proteins, a disintegrin and metalloprotease 10 (ADAM10) and ephrinB2, as drivers of fibrosis and tumor progression after radiation therapy (RT). RT resulted in increased ADAM10 expression in tumor cells, leading to cleavage of ephrinB2, which was also detected in plasma. Pharmacologic or genetic targeting of ADAM10 decreased RT-induced fibrosis and tissue tension, tumor cell migration, and invasion, sensitizing orthotopic tumors to radiation killing and prolonging mouse survival. Inhibition of ADAM10 and genetic ablation of ephrinB2 in fibroblasts reduced the metastatic potential of tumor cells after RT. Stimulation of tumor cells with ephrinB2 FC protein reversed the reduction in tumor cell invasion with ADAM10 ablation. These findings represent a model of PDAC adaptation that explains resistance and metastasis after RT and identifies a targetable pathway to enhance RT efficacy. SIGNIFICANCE: Targeting a previously unidentified adaptive resistance mechanism to radiation therapy in PDAC tumors in combination with radiation therapy could increase survival of the 40% of PDAC patients with locally advanced disease.See related commentary by Garcia Garcia et al., p. 3158 GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/81/12/3255/F1.large.jpg.

Epidemiology and treatment trends for primary tracheal squamous cell carcinoma.

OBJECTIVE: Management of tracheal squamous cell carcinoma (TSCC) has been complicated by the lack of prognostic data and staging. We describe the epidemiology of TSCC and current treatment approaches. METHODS: Five hundred thirty-two adult patients with primary TSCC from 2004 to 2012 in the National Cancer Database were identified. Demographic, clinical factors, and 5-year overall survival were analyzed. Staging was classified as localized, regional extension, and distant spread. Treatment modality was defined as "no treatment (NT)," "limited surgery (LS)," "curative surgery (CS)," "LS with any adjuvant therapy (AT) (LS+AT)," "CS with AT (CS+AT)," "radiation therapy (RT)," or "chemoradiation (CRT)." RESULTS: Overall survival was 25%. Majority of cases were males, white, and occurred in sixth/seventh decades. Twenty-six percent of cases received CRT, 20% underwent LS+AT or CS+AT, 20% underwent LS or CS only, and 17% underwent RT alone. On multivariate analysis, CS (HR 0.42, 95% CI: 0.26-0.69), CS+AT (HR 0.44, 95% CI: 0.36-0.77), CRT (HR 0.48, 95% CI: 0.35-0.67), and RT (HR, 0.66 95% CI: 0.46-0.94) were associated with decreased likelihood of death compared to NT. Elderly patients and those with poor performance status had worse outcomes even on multivariate analysis. CONCLUSIONS: TSCC is increasingly treated with surgery and systemic therapy in addition to RT, with improved survival outcomes. CS, CS+AT, CRT, or RT provided improved survival advantage in patients with variable levels of improvement based on the extent of the disease. Prospective trials would help differentiate survival advantages between treatment modalities. Patients' goals of care, comorbidities, and age should be considered when deciding appropriate treatment recommendations. LEVEL OF EVIDENCE: NA Laryngoscope, 130:405-412, 2020.

Comprehensive Structural and Molecular Comparison of Spike Proteins of SARS-CoV-2, SARS-CoV and MERS-CoV, and Their Interactions with ACE2.

The severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) has recently emerged in China and caused a disease called coronavirus disease 2019 (COVID-19). The virus quickly spread around the world, causing a sustained global outbreak. Although SARS-CoV-2, and other coronaviruses, SARS-CoV and Middle East respiratory syndrome CoV (MERS-CoV) are highly similar genetically and at the protein production level, there are significant differences between them. Research has shown that the structural spike (S) protein plays an important role in the evolution and transmission of SARS-CoV-2. So far, studies have shown that various genes encoding primarily for elements of S protein undergo frequent mutation. We have performed an in-depth review of the literature covering the structural and mutational aspects of S protein in the context of SARS-CoV-2, and compared them with those of SARS-CoV and MERS-CoV. Our analytical approach consisted in an initial genome and transcriptome analysis, followed by primary, secondary and tertiary protein structure analysis. Additionally, we investigated the potential effects of these differences on the S protein binding and interactions to angiotensin-converting enzyme 2 (ACE2), and we established, after extensive analysis of previous research articles, that SARS-CoV-2 and SARS-CoV use different ends/regions in S protein receptor-binding motif (RBM) and different types of interactions for their chief binding with ACE2. These differences may have significant implications on pathogenesis, entry and ability to infect intermediate hosts for these coronaviruses. This review comprehensively addresses in detail the variations in S protein, its receptor-binding characteristics and detailed structural interactions, the process of cleavage involved in priming, as well as other differences between coronaviruses.

PARP Inhibition Enhances Radiotherapy of SMAD4-Deficient Human Head and Neck Squamous Cell Carcinomas in Experimental Models.

PURPOSE: SMAD4 loss causes genomic instability and the initiation/progression of head and neck squamous cell carcinoma (HNSCC). Here, we study whether SMAD4 loss sensitizes HNSCCs to olaparib (PARP inhibitor) in combination with radiotherapy (RT). EXPERIMENTAL DESIGN: We analyzed HNSCC The Cancer Genome Atlas data for SMAD4 expression in association with FANC/BRCA family gene expression. Human HNSCC cell lines were screened for sensitivity to olaparib. Isogenic HNSCC cell lines were generated to restore or reduce SMAD4 expression and treated with olaparib, radiation, or the combination. HNSCC pretreatment specimens from a phase I trial investigating olaparib were analyzed. RESULTS: SMAD4 levels correlated with levels of FANC/BRCA genes in HNSCC. HNSCC cell lines with SMAD4 homozygous deletion were sensitive to olaparib. In vivo, olaparib or RT monotherapy reduced tumor volumes in SMAD4-mutant but not SMAD4-positive tumors. Olaparib with RT dual therapy sustained tumor volume reduction in SMAD4-deficient (mutant or knockdown) xenografts, which exhibited increased DNA damage and cell death compared with vehicle-treated tumors. In vitro, olaparib alone or in combination with radiation caused lower clonogenic survival, more DNA damage-associated cell death, and less proliferation in SMAD4-deficient cells than in SMAD4-positive (endogenous SMAD4 or transduced SMAD4) cells. Applicable to clinic, 5 out of 6 SMAD4-negative HNSCCs and 4 out of 8 SMAD4-positive HNSCCs responded to a standard treatment plus olaparib in a phase I clinical trial, and SMAD4 protein levels inversely correlated with DNA damage. CONCLUSIONS: SMAD4 levels are causal in determining sensitivity to PARP inhibition in combination with RT in HNSCCs.

Intramucosal Inoculation of Squamous Cell Carcinoma Cells in Mice for Tumor Immune Profiling and Treatment Response Assessment.

Head and neck squamous cell carcinoma (HNSCC) is a debilitating and deadly disease with a high prevalence of recurrence and treatment failure. To develop better therapeutic strategies, understanding tumor microenvironmental factors that contribute to the treatment resistance is important. A major impediment to understanding disease mechanisms and improving therapy has been a lack of murine cell lines that resemble the aggressive and metastatic nature of human HNSCCs. Furthermore, a majority of murine models employ subcutaneous implantations of tumors which lack important physiological features of the head and neck region, including high vascular density, extensive lymphatic vasculature, and resident mucosal flora. The purpose of this study is to develop and characterize an orthotopic model of HNSCC. We employ two genetically distinct murine cell lines and established tumors in the buccal mucosa of mice. We optimize collagenase-based tumor digestion methods for the optimal recovery of single cells from established tumors. The data presented here show that mice develop highly vascularized tumors that metastasize to regional lymph nodes. Single-cell multiparametric mass cytometry analysis shows the presence of diverse immune populations with myeloid cells representing the majority of all immune cells. The model proposed in this study has applications in cancer biology, tumor immunology, and preclinical development of novel therapeutics. The resemblance of the orthotopic model to clinical features of human disease will provide a tool for enhanced translation and improved patient outcomes.

Effects of ranibizumab and amfenac on the functional abilities and radiosensitivity of uveal melanoma cells.

PURPOSE: To evaluate the effects of ranibizumab and amfenac in human uveal melanoma cell lines and to explore the ability of these compounds to sensitize uveal melanoma cells to radiation therapy. METHODS: The 92.1 human uveal melanoma cell line was cultured and subjected to the proposed treatment (ranibizumab, amfenac, and a combination of both). Proliferation, migration, and invasion assays of the 92.1 uveal melanoma cell line were assessed after pretreatment with ranibizumab (125 mg/mL), amfenac (150 nM), or a combination of both. In addition, proliferation rates were assessed after treatment with ranibizumab and amfenac, and the cells were subsequently exposed to various radiation doses (0, 4, and 8 Gy). RESULTS: Proliferation assay: cells treated with a combination of ranibizumab and amfenac had lower proliferation rates than controls (p=0.016) and than those treated with only ranibizumab (p=0.033). Migration assay: a significantly lower migration rate was observed in cells treated with amfenac than the control (p=0.014) and than those treated with ranibizumab (p=0.044). Invasion assay: there were no significant differences among the studied groups. Irradiation exposure: in the 4 Gy dose group, there were no significant differences among any groups. In the 8 Gy dose group, treatment with ranibizumab, amfenac, and their combination prior to application of the 8 Gy radiation led to a marked reduction in proliferation rates (p=0.009, p=0.01, and p=0.034, respectively) compared with controls. CONCLUSION: Combination of ranibizumab and amfenac reduced the proliferation rate of uveal melanoma cells; however, only amfenac monotherapy significantly decreased cell migration. The radiosensitivity of the 92.1 uveal melanoma cell line increased following the administration of ranibizumab, amfenac, and their combination. Further investigation is warranted to determine if this is a viable pretreatment strategy to render large tumors amenable to radiotherapy.