Ayman Oweida

Ionizing radiation sensitizes tumors to PD-L1 immune checkpoint blockade in orthotopic murine head and neck squamous cell carcinoma

ABSTRACT Immunotherapy clinical trials targeting the programmed-death ligand axis (PD-1/PD-L1) show that most head and neck squamous cell carcinoma (HNSCC) patients are resistant to PD-1/PD-L1 inhibition. We investigated whether local radiation to the tumor can transform the immune landscape and render poorly immunogenic HNSCC tumors sensitive to PD-L1 inhibition. We used the first novel orthotopic model of HNSCC with genetically distinct murine cell lines. Tumors were resistant to PD-L1 checkpoint blockade, harbored minimal PD-L1 expression and tumor infiltrating lymphocytes at baseline, and were resistant to radiotherapy. The combination of radiation and PD-L1 inhibition significantly enhanced tumor control and improved survival. This was mediated in part through upregulation of PD-L1 on tumor cells and increased T-cell infiltration after RT, resulting in a highly inflamed tumor. Depletion of both CD4 and CD8 T-cells completely abrogated the effect of anti PD-L1 with radiation on tumor growth. Our findings provide evidence that radiation to the tumor can induce sensitivity to PD-L1 checkpoint blockade in orthotopic models of HNSCC. These findings have direct relevance to high risk HNSCC patients with poorly immunogenic tumors and who may benefit from combined radiation and checkpoint blockade.

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.

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

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.

Enhancing radiosensitization in EphB4 receptor-expressing Head and Neck Squamous Cell Carcinomas

Members of the Eph family of receptor tyrosine kinases have been implicated in a wide array of human cancers. The EphB4 receptor is ubiquitously expressed in head and neck squamous cell carcinoma (HNSCC) and has been shown to impart tumorigenic and invasive characteristics to these cancers. In this study, we investigated whether EphB4 receptor targeting can enhance the radiosensitization of HNSCC. Our data show that EphB4 is expressed at high to moderate levels in HNSCC cell lines and patient-derived xenograft (PDX) tumors. We observed decreased survival fractions in HNSCC cells following EphB4 knockdown in clonogenic assays. An enhanced G2 cell cycle arrest with activation of DNA damage response pathway and increased apoptosis was evident in HNSCC cells following combined EphB4 downregulation and radiation compared to EphB4 knockdown and radiation alone. Data using HNSCC PDX models showed significant reduction in tumor volume and enhanced delay in tumor regrowth following sEphB4-HSA administration with radiation compared to single agent treatment. sEphB4-HSA is a protein known to block the interaction between the EphB4 receptor and its ephrin-B2 ligand. Overall, our findings emphasize the therapeutic relevance of EphB4 targeting as a radiosensitizer that can be exploited for the treatment of human head and neck carcinomas.

Impact of immunotherapy among patients with melanoma brain metastases managed with radiotherapy

BACKGROUND Patients with melanoma brain metastases (MBM) have been excluded from trials evaluating immunotherapy in melanoma. As such, immunotherapys role in MBM is poorly understood, particularly in combination with radiotherapy. METHODS The National Cancer Database was queried for patients with MBM receiving brain radiotherapy. They were classified according to immunotherapy receipt. Multivariate Cox regression was performed to identify factors associated with survival. RESULTS Among 1287 patients, 185 received immunotherapy. Factors associated with improved survival included younger age, academic facility, lower extracranial disease burden, stereotactic radiotherapy, chemotherapy, and immunotherapy. CONCLUSIONS Adding immunotherapy to radiotherapy for MBM is associated with improved survival.

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

BackgroundMedulloblastoma 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.MethodsThe 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.ResultsWe 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.ConclusionsOverall, 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.

Nomogram for preoperative prediction of nodal extracapsular extension or positive surgical margins in oropharyngeal squamous cell carcinoma

INTRODUCTION Extracapsular extension (ECE) in regional lymph nodes and positive surgical margins (PSM) are considered high-risk adverse pathologic features in patients with oropharyngeal squamous cell carcinoma (OPSCC) that each constitute an indication for postoperative adjuvant chemoradiation. We identify pre-operative clinical factors that can predict post-operative ECE and/or PSM and create a nomogram to help clinical decision making. METHODS Adult patients with non-metastatic OPSCC with initial surgical treatment and confirmed HPV status diagnosed between 2010 and 2014 were selected from the National Cancer Database. Clinical staging was modified to American Joint Committee on Cancer 8th edition parameters. Logistic regression was used for multivariate analysis to identify predictors of pathologic ECE and/or PSM. RESULTS 5065 patients were included. 47.5% of the 3336 HPV-positive (HPV+) patients had ECE/PSM. 40.4% of the 1729 HPV-negative (HPV-) patients with had ECE/PSM. A model was built that included age, clinical ECE, tumor grade, and clinical T and N staging for HPV+ patients. Increasing N-classification was highly predictive of pathologic ECE and/or PSM (N1 OR = 3.6, N2 OR = 7.0, N3 OR = 11.2, p < 0.01). Clinical ECE (OR = 4.1, p < 0.01), tumor grade (ORs 2.2-4.4 with p < 0.05), and increasing clinical T-classification (ORs 1.2-1.8, p < 0.05) were also associated with ECE and/or PSM. A similar model was built for HPV- with similar predictive capability. Two internally validated nomograms were designed that demonstrated good discrimination (HPV+ AUC = 0.66, 95% CI: 0.64-0.68, and HPV- AUC = 0.70, 95% CI: 0.67-0.72) and good calibration (goodness-of-fit statistic of HPV+ 6.32, p = 0.61 and HPV- 11.66, p = 0.17). CONCLUSIONS These are the first nomograms designed to help predict ECE or PSM for both HPV+ and HPV- OPSCC. The nomograms can facilitate shared decision-making between clinicians and patients as they consider upfront treatment selection for OPSCC.

Role of EphB3 receptor in mediating head and neck tumor growth, cell migration, and response to PI3K inhibitor

Eph proteins have emerged as critical drivers affecting tumor growth and progression in human malignancies. Our The Cancer Genome Atlas (TCGA) data analysis showed that EphB3, a receptor tyrosine kinase, is frequently coamplified with PIK3CA in head and neck squamous cell carcinoma (HNSCC). We therefore hypothesized that EphB3 amplification plays a protumorigenic role in HNSCC and that EphB3 and PIK3CA are cooperating oncogenes that contribute toward its pathogenesis. This hypothesis was not experimentally supported, because EphB3 knockdown failed to alter HNSCC tumor cell growth in vitro or in vivo with an orthotopic model. However, responsiveness of EphB3 knockdown tumors to the PI3K inhibitor, BKM120, was significantly decreased in terms of both tumor growth delay and survival. This is correlated with an increase in prosurvival proteins, S6 and BcL-XL, in the EphB3 shRNA tumors treated with BKM120 compared with controls. We further observed that EphB3 knockdown resulted in increased migration in vitro and increased EMT gene signature in vivo. To explain these results, we examined EphB3 phosphorylation levels in HNSCC at baseline. Although total EphB3 levels were high, we found low phospho-EphB3 levels in HNSCCs. Forced EphB3 phosphorylation with an ephrin-B2–Fc fusion protein resulted in decreased HNSCC migration and cell growth, and enhanced response to BKM120 in vitro. These data collectively indicate that progression of HNSCC selects for low/inhibited EphB3 activity to enhance their survival and migratory abilities and decrease response to PI3K signaling. Therefore, strategies focused on activating EphB3 might be helpful to inhibit tumor growth and enhance sensitivity to PI3K inhibitors in HNSCC. Mol Cancer Ther; 17(9); 2049–59. ©2018 AACR.

Inhibition of EphB4-ephrin-B2 signaling enhances response to Cetuximab-radiation therapy in head and neck cancers

Purpose: The clinical success of targeted therapies such as cetuximab and radiotherapy (RT) is hampered by the low response rates and development of therapeutic resistance. In the current study, we investigated the involvement of EphB4–ephrin-B2 protumorigenic signaling in mediating resistance to EGFR inhibition and RT in head and neck cancers. Experimental Design: We used patient-derived xenograft (PDX) models of head and neck squamous cell carcinoma (HNSCC) and HNSCC cell lines to test our hypothesis. Tumor tissues were subjected to PhosphoRTK array, and Western blotting to detect changes in EphB4–ephrin-B2 targets. mRNA sequencing and microarray data analysis were performed on PDX tumors and HNSCC cell lines, respectively, to determine differences in gene expression of molecules involved in tumor cell growth, proliferation, and survival pathways. Effects on cell growth were determined by MTT assay on HNSCC cells downregulated for EphB4/ephrin-B2 expression, with and without EGFR inhibitor and radiation. Results: Our data from locally advanced HNSCC patients treated with standard-of-care definitive chemo-RT show elevated EphB4 and ephrin-B2 levels after failure of treatment. We observed significant response toward cetuximab and RT following EphB4–ephrin-B2 inhibition, resulting in improved survival in tumor-bearing mice. Tumor growth inhibition was accompanied by a decrease in the levels of proliferation and prosurvival molecules and increased apoptosis. Conclusions: Our findings underscore the importance of adopting rational drug combinations to enhance therapeutic effect. Our study documenting enhanced response of HNSCC to cetuximab-RT with EphB4–ephrin-B2 blockade has the potential to translate into the clinic to benefit this patient population. Clin Cancer Res; 24(18); 4539–50. ©2018 AACR.