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BackgroundDiffuse 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.MethodsWe 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.ResultsMice 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.ConclusionIn 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.

Abstract 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.

Abstract 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.

Abstract 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.

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.