Research Proposal Specific Aims: PD-1 is expressed on T cells, B cells and macrophages, and binding of its ligand PD-L1 negatively regulates immune responses. Several cancers including neuroblastoma express PD-L1, which has been hypothesized to underlie their resistance to radiation therapy. The overall goal of this study is to characterize the host immune response in a mouse model of neuroblastoma following stereotactic radiation, and determine whether disruption of PD-1 binding is a feasible therapeutic avenue in neuroblastoma. Our hypothesis is that the immune response to neuroblastoma tumor following high-dose radiation can be augmented by treatment with an anti-PD-1 reagent. This hypothesis contains two component aims: Aim 1) To determine the nature of the immune response to radiation of neuroblastoma tumor by quantifying the immune cell types, both circulating and infiltrating tumor parenchyma, following radiation treatment. Aim 2) To determine whether anti-PD-1 antibody augments the host immune response by administering combined radiation and anti-PD-1 antibody treatment and quantifying immune cell types as well as tumor volume. Background and Significance: Neuroblastoma is the most common extracranial pediatric solid tumor, responsible for 15% of all childhood cancer deaths [1]. The majority of children diagnosed with advanced stage disease die from progression despite intensive multimodality therapy consisting of surgical resection, high-dose chemotherapy and external beam radiation [2]. Previous studies have suggested that increasing radiation doses to primary sites of disease can improve local recurrence rates and increase overall survival [3-7]; however, doses are limited in the pediatric population by the low radiation tolerance of surrounding normal tissue. Stereotactic body radiation therapy (SBRT), a technique by which precise tumor localization and multiple convergent beams increase the therapeutic ratio of radiation delivery, has yielded promising results in a number of cancers [8]. Case reports exist of successful treatment of neuroblastoma with SBRT [9-11], but other cases have resulted in recurrence [12]. A need exists for a complementary treatment that would enhance the effectiveness of radiation therapy for neuroblastoma by reducing required radiation dose while increasing tumor cell killing. Immunotherapy has arisen as the “fifth pillar” in cancer treatment, complementing surgery, chemotherapy, radiation, and molecularly targeted therapies. Radiation kills tumor cells not only by generation of double-strand breaks in DNA, but also by recruitment of the host immune response [13]. Radiation exposure provides a source of antigen that is well-suited for cross presentation by host antigen-presenting cells, leading to the induction of positive immunomodulatory pathways and trafficking of lymphocytes to the tumor microenvironment [14]. In addition, radiation changes the immunophenotype of cancer cells by enhancing MHC Class I expression and increasing presentation of antigenic peptides [15]. PD-L1 is expressed by many tumor cell types [16] including neuroblastoma [17] and acts to shield the tumor from the host immune system by binding the PD-1 receptor and blunting the cytotoxic effect of CD8+ T lymphocytes. Anti-PD-1 reagents improve survival for patients with advanced melanoma [18, 19] and have recently been shown to synergize with stereotactic radiation therapy to augment the host immune response and improve local tumor control in mouse models of melanoma and breast carcinoma. Overall, few publications exist on the topic of the host immune response in the setting of SBRT, and none with regards to neuroblastoma. Previous work in this laboratory has included the creation of a nude mouse model of SBRT for neuroblastoma, as well as determination of the effect of radiation on the volume and integrity of tumor vasculature in this model. Current projects include investigation of the potential for Notch blockade to enhance radiation-mediated destruction of tumor vasculature. In contrast to previous studies in nude mice, we will use the TH-MYCN immune competent neuroblastoma mouse model in order to gain a

more clinically relevant understanding of the host immune response to stereotactic radiation. A positive result from this preclinical study of anti-PD-1 antibody would provide a rationale for expanding clinical studies of anti-PD-1 reagents to neuroblastoma. Experimental Design: Aim #1 will require one treatment arm and one control arm with 12 mice in each group. Xenografts will be established in all 24 mice. Once tumors have reached 1 gram in size, the treatment arm will receive a single 12 Gray fraction of stereotactically guided radiation. Mice will be sacrificed at 72 hours following irradiation to quantify infiltrating immune cells via flow cytometry. Aim #2 will require one treatment arm and one control arm with 12 mice in each group. The treatment arm will receive two injections of 200µg anti-PD-1 antibody separated by three days. Subsequently all mice will receive a single 12 Gray fraction of radiation. Tumor volume will be assessed at 6, 24 and 72 hour time points by microbubble contrast-enhanced three-dimensional ultrasound (3D-CEUS), following which mice will be sacrificed for flow cytometry. A third cohort of 24 mice will incorporate methodological refinements based on earlier studies and ensure robust results. Neuroblastoma Xenograft Tumors: We will utilize the NXS2 murine neuroblastoma cell line [20] and the TH-MYCN transgenic model of neuroblastoma [21]. NXS2 is a moderately immunogenic, highly metastatic hybrid of the C1300 murine neuroblastoma cell line (A/J background) with murine dorsal root ganglional cells from C57BL/6J mice. The A/J mice will be anesthetized, the flank incised in a sterile manner, an inoculum of NXS2 cells will be injected into kidney, and the flank muscles and skin will be sutured closed [22]. TH-MYCN tumors arise spontaneously and to high penetrance within their native tissue of origin (sympathetic paraspinal, celiac and periadrenal sympathetic ganglia) and replicate many major genetic changes of human MYCN-amplified disease. Tumor sizes are monitored weekly by palpation and ultrasound imaging until they reach a size corresponding to 1 gm, typically after 4 weeks. All animal experiments will be approved by the Columbia University Institutional Animal Care and Use Committee. In Vivo Radiation Treatment: Radiation will be delivered using an image guided SARRP 200 (Small Animal Radiation Research Platform) from Xstrahl Limited with a dose-rate of 1Gy/min. This system allows us to utilize CT imaging within the machine to determine a three-dimensional, conformal plan that specifically targets tumor as seen on CT and minimizes dose to nontarget tissue. Flow Cytometry: Single-cell suspensions will be prepared from spleens, inguinal lymph nodes and tumors. Cells will be stained with fluorescent-labeled antibodies and analyzed by FACSCalibur flow cytometer. The following cell types will be quantified: CD8 T lymphocytes, CD4 T lymphocytes, regulatory T lymphocytes, effector memory T lymphocytes, plasma B lymphocytes, macrophages and natural killer cells. Microbubble Contrast Enhanced Ultrasonography (CEUS): We will use microbubble contrast-enhanced ultrasound, which provides rapid, reproducible, in vivo imaging of tumor vasculature. Microbubbles are gas-filled spheres which circulate in the blood stream when injected systemically. Studies in Dr. Yamashiro’s laboratory have previously demonstrated that CEUS utilizing targeted microbubble adhesion is effective in determining tumor response to anti-angiogenic therapy, and is likely to prove similarly useful in investigation of immune modulatory agents. Statistical Analysis: Statistical testing will be performed using Stata software, with normal data analyzed by unpaired t-test or ANOVA and non-normal data analyzed non-parametrically with Mann-Whitney or Kruskal-Wallis test. Using quantity of tumor-infiltrating CD8+ T lymphocytes as our primary outcome, with twelve animals per group we can detect an effect size of 1.0 between the two treatment groups, assuming a standard deviation of 1.0 in each group based on a 2-sample t-test with α=0.05 and power of 80%.

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