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Introduction: Pediatric high-grade gliomas (pHGGs) are routinely fatal with a median overall survival (OS) at recurrence of 5.6 months (mos). A safe, effective immunotherapy for pHGG has eluded investigators. Oncolytic HSV-1 G207 contains mutations that prevent a productive infection of normal cells but permit replication in tumor cells. In addition to direct tumor cell lysis, G207 activates innate/adaptive immune cells and promotes cross-presentation of tumor antigens to generate an anti-tumor immune response. A 5 Gy radiation dose increases viral replication and spread. We evaluated the safety and efficacy of G207 alone and combined with radiation in children with progressive supratentorial HGG (NCT02457845). Methods: We employed a 3 + 3 design with 4 cohorts. Children 3-18 years old with biopsy-confirmed HGG underwent stereotactic placement of up to four intratumoral catheters. The next day, we administered 107 or 108 plaque-forming units (pfu) of G207 by controlled rate infusion over 6 hours. Within 24 hours of G207, patients in dose level 3 and 4 received 5 Gy to the gross tumor volume. The primary objective was safety/tolerability. We assessed secondary objectives of virus shedding in blood, saliva and conjunctiva by PCR, response by MRI and evaluation of matched pre- and post-G207 tissue for tumor-infiltrating lymphocytes (TILs), and seroconversion by immunofluorescence assay. Results: 12 patients (age range 7-18) with progressive, IDH wild-type pHGG received G207. At screening, 10 patients had tumors with a bi-perpendicular sum ≥ 5.5 cm, 3 had multi-focal disease, 8 had failed ≥ 2 prior treatment regimens, and 4 had failed ≥ 3 regimens. 3-4 catheters (44 total) were placed safely throughout the cerebrum and resulted in no neurologic sequelae. G207 alone or with radiation was safe and tolerable in all patients with no dose-limiting toxicities, attributable grade 3 or 4 toxicities/serious adverse events, or evidence of virus shedding. 11 participants had radiographic, neuropathologic, and/or clinical responses. Median OS was 12.2 mos (95% CI 8.0, 16.4). Thus far, 36% of patients have lived >18 mos, the median OS for newly diagnosed pHGG. Compared to patients who seroconverted post-G207 (n=3), patients with baseline HSV-1 antibodies (n=3) had a shorter median survival: 5.1 mos (3.0, 7.2) vs 18.3 mos (9.2, 27.4). G207 significantly increased CD4+ and CD8+ TILs. Conclusions: G207 alone and combined with radiation was tolerable and safe with evidence of responses in children with pHGG. The promising median OS (12.2 mos) compares favorably with historical data (5.6 mos). Baseline HSV-1 seropositivity and seroconversion are potential biomarkers of treatment response that require further investigation. Importantly, G207 converted ‘cold' tumors to ‘hot' with a dramatic increase in TILs. A multi-institutional Phase II clinical trial of G207 in pHGG is forthcoming (NCT04482933). Citation Format: Gregory K. Friedman, James M. Johnston, Asim K. Bag, Joshua D. Bernstock, Rong Li, Inmaculada Aban, Kara Kachurak, Li Nan, Kyung-Don Kang, Stacie Totsch, Charles Schlappi, Allison M. Martin, Devang Pastakia, Sameer Farouk Sait, Yasmin Khakoo, Matthias A. Karajannis, Karina Woodling, Joshua D. Palmer, Diana S. Osorio, Jeffrey Leonard, Mohamed S. Abdelbaki, Avi Madan-Swain, T. Prescott Atkinson, Richard J. Whitley, John B. Fiveash, James M. Markert, G. Yancey Gillespie. Phase I immunovirotherapy trial of oncolytic HSV-1 G207 alone or combined with radiation in pediatric high-grade glioma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr CT018.

Cancer treatment strategies targeting the tumor microenvironment and corresponding immune cell infiltrate (i.e., immunotherapy) have recently proven effective against many solid tumors. In pediatric brain cancers, there is little known about the immune tumor microenvironment. We aim to characterize immune cell populations of pediatric brain tumors, which will better inform us about immune cell infiltration in different brain cancer subtypes and may indicate potential for patient response to immunotherapy. The Institute for Genomic Medicine at Nationwide Children’s Hospital has recently initiated a translational protocol to evaluate tumor specimens from pediatric patients with rare or refractory brain cancers in a focused N-of-1 manner. We performed RNA-seq and NanoString™ PanCancer Immune expression profiling on extracted tumor RNA from 22 patients comprising eight distinct brain cancer subtypes. We used CIBERSORT algorithm for deconvolution of RNA-seq global gene expression data and NanoString™ expression profiling to investigate methods of quantifying tumor-infiltrating leukocytes (TIL) and expression of immune genes. Our analyses identified a subset of pediatric brain tumors with evidence of immune infiltration. Using CIBERSORT, four gliomas were predicted to harbor significant TIL populations. Overall, we observed high correlation (Pearson R2 = 0.884) between expression values derived from RNA-seq and NanoString™ methods. Similarly, the two methods produced comparable predictions for TIL composition in the glioma tumors. Further genomic analysis identified one patient, with a diagnosis of glioblastoma and Neurofibromatosis type 1 (NF1), with marked infiltration of macrophages and neutrophils. Overexpression of tumor-associated macrophage/microglia genes (CD14, CD163, CD68, ITGAM, PTPRC) and other immune markers (PD-L1, CCL2/MCP-1, IL1B) was observed relative to all other samples. More specifically, overexpression of CD204, CD206, and CD163 suggested the infiltrating macrophages represent the M2/protumor phenotype. CIBERSORT allows more “granularity” in macrophage population prediction and indicated an equal representation of M2 and M0/uncommitted macrophages. Given the M2 phenotype, it is likely that the infiltrating macrophages contributed to tumor progression and radiation resistance observed in this patient. If confirmed, the presence of M0 macrophages and/or overexpression of PD-L1 could indicate potential for immunotherapy in this patient (e.g., oncolytic viral therapy, strategies focusing on M0 to M1/antitumor polarization, or combination targeted therapy with PD-L1 inhibitor). In summary, we identified a patient with progressive glioblastoma tumor growth and significant macrophage tumor infiltration as a likely candidate for immunotherapy. Citation Format: Katherine E. Miller, Kathleen Schieffer, James Fitch, Vincent Magrini, Amy Wetzel, Anthony R. Miller, Daniel R. Boue, Jeffrey Leonard, Jonathan L. Finlay, Diana S. Osorio, Mohamed S. AbdelBaki, Christopher R. Pierson, Annie Drapeau, Jonathan Pindrik, Kristen Leraas, Elizabeth Varga, Devon Dishman, Lauren Shoemaker, Nicole Ross, Jeremy Pitts, Julie Gastier-Foster, Peter White, Catherine E. Cottrell, Richard K. Wilson, Elaine R. Mardis. Expression profiling-based characterization of immune cell populations in pediatric brain cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4551.

Medulloblastoma is a pediatric embryonal tumor that can be classified into four molecular subgroups, each derived from a different progenitor cell. It is estimated that about 30-40% of patients will relapse, typically with recurrence at the primary site and of the same molecular subgroup. We present paired tumor/normal genomic analysis of an 18 year-old male who presented with non-Wnt/non-SHH medulloblastoma at age 12 and relapsed with metastatic disease of the falx cerebri 3 years later. Combination surgery, chemotherapy, and radiation were used in treatment of the primary and recurrent tumor. At a timepoint 6 years from original diagnosis, the patient presented with a cerebellar tumor histologically described as “consistent with recurrent medulloblastoma” with comment recommending genomics to confirm. The diagnosis was made based on near identifical morphology and retention of Neu-N and Synaptophysin in the tumor (confirmed by subsequent genetic analysis). The primary tumor and the tumor occurring 6 years after the primary diagnosis were analyzed by whole exome sequencing (blood and tumor tissues) to assess for germline variants, somatic mutation, and copy number variation. We observed no pathogenic germline variants in cancer predisposition genes. The tumor mutational profiles were distinct, with only 6 (1.8%) shared somatic variants between tumors. Specimen provenance was verified by germline variation and SRY coverage. Two targetable mutations within the RAS-MAPK pathway (PTPN11 p.Glu76Lys and PIK3CA p.Gly1007Arg) were present only in the new CNS tumor. Although the primary tumor harbored isochromosome 17q and a gain of chromosome 4, these somatic chromosomal aberrations were not detected in the new CNS tumor. RNA-seq was performed on both tumors and compared to pediatric CNS tumors from the University of California Santa Cruz Treehouse Initiative (n=434). The primary tumor clustered with the medulloblastoma patients by principal component analysis while the new CNS tumor clustered with a group of gliomas and non-medulloblastoma embryonal tumors. The primary tumor displayed evidence of overexpression of Group 4 medulloblastoma genes (e.g. EOMES, RBM24, SNCAIP, and UNC5D). These genes were not overexpressed in the new CNS tumor. Enrichment of genes commonly found in gliomas (e.g. BCAN, CHI3L2, PDGFRA, and SOX2) were noted in the new CNS tumor only. In summary, tumor genomic profiling of a primary medulloblastoma and the new CNS tumor arising 6 years later revealed two distinct sets of somatic mutations suggestive of second malignancy rather than recurrence in this patient. While second malignancy in the setting of medulloblastoma is a rare event, it has been documented, both in a time period consistent with that described in our patient and in the form of glioma. Thus, tumor profiling refined diagnosis in this patient allowing for a more accurate assessment of treatment and management options. Citation Format: Kathleen M. Schieffer, Katherine E. Miller, Daniel R. Boue, Daniel C. Koboldt, Patrick Brennan, Benjamin J. Kelly, Gregory Wheeler, Vincent Magrini, Amy Wetzel, Elizabeth Varga, Devon Dishman, Kristen Leraas, Vibhuti Agarwal, Mohamed S. AbdelBaki, Jonathan L. Finlay, Jeffrey R. Leonard, Peter White, Julie M. Gastier-Foster, Catherine E. Cottrell, Elaine R. Mardis, Richard K. Wilson. Molecular profiling identifies a second malignancy in a patient with medulloblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 484.