PURPOSE:Pediatric recurrent medulloblastoma and atypical teratoid/rhabdoid tumor (ATRT) are largely incurable and warrant novel therapies. PNOC005 is a phase I clinical trial investigating the safety and tolerability of intratumoral or intrathecal administration of oncolytic measles virus (MV-NIS) in children and young adults with recurrent medulloblastoma or ATRT. PATIENTS AND METHODS:We investigated (i) the safety of a measles virus variant, MV-NIS, in a pediatric phase I study and (ii) the mechanisms of MV-NIS and the potential benefit of combination with immune checkpoint inhibition (ICI). Pediatric patients with recurrent medulloblastoma or ATRT were treated with intratumoral injections for local recurrence or via lumbar puncture for disseminated recurrence. We evaluated local immune responses to MV-NIS with and without ICI via single-cell and bulk RNA sequencing in an intracranial, immunocompetent, syngeneic murine model. RESULTS:MV-NIS given intratumorally or via repeat intrathecal dosing was safe. MV-NIS prolonged survival in murine models but did not demonstrate an additive benefit with ICI. No changes in tumor-infiltrating immune cell composition or activation were observed in response to MV-NIS treatment; however, MV-NIS induced local expression of neutralizing antibodies, complement cascade, and phagocytosis-related genes. CONCLUSIONS:This is the first trial investigating intratumoral as well as repeated intrathecal delivery of MV-NIS in children with medulloblastoma and ATRT. We show that the therapy is safe and well tolerated, with minimal adverse effects. Immune markers and biological correlates preliminarily indicate antiviral effects in tumors.

Supplementary Figure S5. Phenotypic states and cell type composition of MV-NIS-infected cells.

Supplementary Table S1. PNOC005 clinical trial sites.

Abstract BACKGROUND PNOC005 is a phase 1 clinical trial investigating the safety and tolerability of intratumoral or intrathecal administration of oncolytic measles virus (MV-NIS) in children and young adults with recurrent medulloblastoma (MB) or atypical teratoid/rhabdoid tumor (ATRT). METHODS Patients with recurrence of MB or ATRT were stratified into Stratum A (local recurrence) or B (disseminated recurrence). Stratum A patients received MV-NIS directly into the tumor bed at time of surgical resection. Stratum B patients received a single dose of MV-NIS via lumbar puncture. After safety was demonstrated in Stratum A and B, Stratum C was added with repeat dosing on Day 0 and 7 for patients with recurrent disseminated MB. Specimens for viral shedding were collected. Blood was collected on days 0, 4, 7, 14, and 28. RNA-sequencing deconvolution and time-series analysis were performed to estimate the composition and phenotypes of peripheral blood mononuclear cells (PBMCs). RESULTS Thirty-four patients (median age 8.5, range 2-31) enrolled between February 2017 and April 2021, with 23 evaluable patients with MB and 4 with ATRT. One patient experienced a dose-limiting toxicity (grade 3 alanine aminotransferase increase). There were four MV-NIS-related adverse events grade 3 or greater across all strata. Viral shedding was detected in 5 patients, all of which cleared by end-of-treatment. We found a similar gene-expression program in PMBCs that correlated across patients (n=18), which was upregulated at days 4-7 post treatment and consistent with an antiviral response. Concomitant changes in B, T, and natural-killer cell composition and activation were consistent with this interpretation. CONCLUSION This is the first trial investigating intratumoral as well as repeated intrathecal delivery of MV-NIS in children with MB and ATRT. We show that therapy is safe and well-tolerated with minimal adverse effects. Immune markers and biologic correlates preliminarily indicate anti-viral effects in tumors.

Oncolytic virotherapy translational research in the current era is heavily focused on the interaction of the immune system and tumor microenvironment with oncolytic viruses. Preclinical xenograft studies using human cells in immunodeficient mouse models does not serve this purpose. As a consequence, developing syngeneic immunocompetent murine cancer models sensitive to infection and growth of specific oncolytic viruses is required. The group 3 subtype of medulloblastoma, among the four molecular subgroups-WNT, SHH, Group 3, and Group 4, has the worst prognosis and the poorest outcome. Sadly, current treatments cause long-term toxicity and morbidity to survivors adversely affecting their quality of life. Alternate effective therapy with less side effects is urgently needed. We have shown that oncolytic measles virus (MV) is effective against localized as well as CSF-disseminated medulloblastoma in immunodeficient mouse models. To study the interaction of immune system with oncolytic measles virotherapy, we have developed a murine group 3 medulloblastoma cell line (CSCG) that is infectible by MV, is killed by MV, allows replication of MV, and is tumorigenic in the brain of syngeneic transgenic immune-competent mice. Intratumoral injection of MV results in significant prolongation of survival in mice bearing CSCG tumors in the brain. This model provides the first suitable platform to examine therapeutic regimens of MV therapy for MB tumors in the presence of intact immune system. Here, we describe our lab protocols to develop this cell line and the mouse model.

Brain tumors are the most common solid tumors of childhood, and the genetic drivers and optimal therapeutic strategies for many of the different subtypes remain unknown. Here, we identify that bithalamic gliomas harbor frequent mutations in the EGFR oncogene, only rare histone H3 mutation (in contrast to their unilateral counterparts), and a distinct genome-wide DNA methylation profile compared to all other glioma subtypes studied to date. These EGFR mutations are either small in-frame insertions within exon 20 (intracellular tyrosine kinase domain) or missense mutations within exon 7 (extracellular ligand-binding domain) that occur in the absence of accompanying gene amplification. We find these EGFR mutations are oncogenic in primary astrocyte models and confer sensitivity to specific tyrosine kinase inhibitors dependent on location within the kinase domain or extracellular domain. We initiated treatment with targeted kinase inhibitors in four children whose tumors harbor EGFR mutations with encouraging results. This study identifies a promising genomically-tailored therapeutic strategy for bithalamic gliomas, a lethal and genetically distinct brain tumor of childhood.

The FGFR1 gene encoding fibroblast growth factor receptor 1 has emerged as a frequently altered oncogene in the pathogenesis of multiple low-grade neuroepithelial tumor (LGNET) subtypes including pilocytic astrocytoma, dysembryoplastic neuroepithelial tumor (DNT), rosette-forming glioneuronal tumor (RGNT), and extraventricular neurocytoma (EVN). These activating FGFR1 alterations in LGNET can include tandem duplication of the exons encoding the intracellular tyrosine kinase domain, in-frame gene fusions most often with TACC1 as the partner, or hotspot missense mutations within the tyrosine kinase domain (either at p.N546 or p.K656). However, the specificity of these different FGFR1 events for the various LGNET subtypes and accompanying genetic alterations are not well defined. Here we performed comprehensive genomic and epigenomic characterization on a diverse cohort of 30 LGNET with FGFR1 alterations. We identified that RGNT harbors a distinct epigenetic signature compared to other LGNET with FGFR1 alterations, and is uniquely characterized by FGFR1 kinase domain hotspot missense mutations in combination with either PIK3CA or PIK3R1 mutation, often with accompanying NF1 or PTPN11 mutation. In contrast, EVN harbors its own distinct epigenetic signature and is characterized by FGFR1-TACC1 fusion as the solitary pathogenic alteration. Additionally, DNT and pilocytic astrocytoma are characterized by either kinase domain tandem duplication or hotspot missense mutations, occasionally with accompanying NF1 or PTPN11 mutation, but lacking the accompanying PIK3CA or PIK3R1 mutation that characterizes RGNT. The glial component of LGNET with FGFR1 alterations typically has a predominantly oligodendroglial morphology, and many of the pilocytic astrocytomas with FGFR1 alterations lack the biphasic pattern, piloid processes, and Rosenthal fibers that characterize pilocytic astrocytomas with BRAF mutation or fusion. Together, this analysis improves the classification and histopathologic stratification of LGNET with FGFR1 alterations.

Intracranial mesenchymal tumors with FET‐CREB fusions are a recently described group of neoplasms in children and young adults characterized by fusion of a FET family gene (usually EWSR1, but rarely FUS) to a CREB family transcription factor (ATF1, CREB1, or CREM), and have been variously termed intracranial angiomatoid fibrous histiocytoma or intracranial myxoid mesenchymal tumor. The clinical outcomes, histologic features, and genomic landscape are not well defined. Here, we studied 20 patients with intracranial mesenchymal tumors proven to harbor FET‐CREB fusion by next‐generation sequencing (NGS). The 16 female and four male patients had a median age of 14 years (range 4–70). Tumors were uniformly extra‐axial or intraventricular and located at the cerebral convexities (n = 7), falx (2), lateral ventricles (4), tentorium (2), cerebellopontine angle (4), and spinal cord (1). NGS demonstrated that eight tumors harbored EWSR1‐ATF1 fusion, seven had EWSR1‐CREB1, four had EWSR1‐CREM, and one had FUS‐CREM. Tumors were uniformly well circumscribed and typically contrast enhancing with solid and cystic growth. Tumors with EWSR1‐CREB1 fusions more often featured stellate/spindle cell morphology, mucin‐rich stroma, and hemangioma‐like vasculature compared to tumors with EWSR1‐ATF1 fusions that most often featured sheets of epithelioid cells with mucin‐poor collagenous stroma. These tumors demonstrated polyphenotypic immunoprofiles with frequent positivity for desmin, EMA, CD99, MUC4, and synaptophysin, but absence of SSTR2A, myogenin, and HMB45 expression. There was a propensity for local recurrence with a median progression‐free survival of 12 months and a median overall survival of greater than 60 months, with three patients succumbing to disease (all with EWSR1‐ATF1 fusions). In combination with prior case series, this study provides further insight into intracranial mesenchymal tumors with FET‐CREB fusion, which represent a distinct group of CNS tumors encompassing both intracranial myxoid mesenchymal tumor and angiomatoid fibrous histiocytoma‐like neoplasms.

Author(s): Sloan, Emily A; Hilz, Stephanie; Gupta, Rohit; Cadwell, Cathryn; Ramani, Biswarathan; Hofmann, Jeffrey; Kline, Cassie N; Banerjee, Anu; Reddy, Alyssa; Oberheim Bush, Nancy Ann; Chang, Susan; Braunstein, Steve; Chang, Edward F; Raffel, Corey; Gupta, Nalin; Sun, Peter P; Kim, John YH; Moes, Gregory; Alva, Elizabeth; Li, Rong; Bruggers, Carol S; Alashari, Mouied; Wetmore, Cynthia; Garg, Shipra; Dishop, Megan; Van Ziffle, Jessica; Onodera, Courtney; Devine, Patrick; Grenert, James P; Lee, Julieann C; Phillips, Joanna J; Pekmezci, Melike; Tihan, Tarik; Bollen, Andrew W; Berger, Mitchel S; Costello, Joseph F; Perry, Arie; Solomon, David A

Rosette-forming glioneuronal tumor (RGNT) is an uncommon CNS tumor originally described in the fourth ventricle characterized by a low-grade glial neoplasm admixed with a rosette-forming neurocytic component. We reviewed clinicopathologic features of 42 patients with RGNT. Targeted next-generation sequencing was performed, and genome-wide methylation profiling is underway. The 20 male and 22 female patients had a mean age of 25 years (range 3–47) at time of diagnosis. Tumors were located within or adjacent to the lateral ventricle (n=16), fourth ventricle (15), third ventricle (9), and spinal cord (2). All 31 tumors assessed to date contained FGFR1 activating alterations, either in-frame gene fusion, kinase domain tandem duplication, or hotspot missense mutation in the kinase domain (p.N546 or p.K656). While 7 of these 31 tumors harbored FGFR1 alterations as the solitary pathogenic event, 24 contained additional pathogenic alterations within PI3-kinase or MAP kinase pathway genes: 5 with additional PIK3CA and NF1 mutations, 4 with PIK3CA mutation, 3 with PIK3R1 mutation (one of which also contained focal RAF1 amplification), 5 with PTPN11 mutation (one with additional PIK3R1 mutation), and 2 with NF1 deletion. The other 5 cases demonstrated anaplastic features including hypercellularity and increased mitotic activity. Among these anaplastic cases, 3 harbored inactivating ATRX mutations and two harbored CDKN2A homozygous deletion, in addition to the FGFR1 alterations plus other PI3-kinase and MAP kinase gene mutations seen in those RGNT without anaplasia. Independent of ventricular location, RGNT is defined by FGFR1 activating mutations or rearrangements, which are frequently accompanied by mutations involving PIK3CA, PIK3R1, PTPN11, NF1, and KRAS. Whereas pilocytic astrocytoma and ganglioglioma are characterized by solitary activating MAP kinase pathway alterations (e.g. BRAF fusion or mutation), RGNT are genetically more complex with dual PI3K-Akt-mTOR and Ras-Raf-MAPK pathway activation. Rare anaplastic examples may show additional ATRX and/or CDKN2A inactivation.

Author(s): Lee, Julieann C; Mazor, Tali; Lao, Richard; Wan, Eunice; Diallo, Alpha B; Hill, Nicholas S; Thangaraj, Naina; Wendelsdorf, Katherine; Samuel, David; Kline, Cassie N; Banerjee, Anuradha; Auguste, Kurtis; Raffel, Corey; Gupta, Nalin; Berger, Mitchel; Raleigh, David R; Shai, Anny; Phillips, Joanna J; Bollen, Andrew W; Tihan, Tarik; Perry, Arie; Costello, Joseph; Solomon, David A

Though diffuse gliomas arising in the thalamus of children frequently harbor K27M mutation in the histone H3 genes H3F3A or HIST1H3B, children with diffuse gliomas involving the bilateral thalami at presentation often lack histone H3 mutations (Broniscer et al, Brain Pathology 2018). In order to investigate the molecular pathogenesis of pediatric bithalamic diffuse gliomas, we performed comprehensive genetic profiling of seven cases (ages 3–11 years). Six tumors (86%) harbored mutations in the EGFR oncogene in the absence of accompanying EGFR amplification. Four of these EGFR mutations were small in-frame insertions/duplications within exon 20 that encodes a portion of the intracellular tyrosine kinase domain, where mutations are common in lung adenocarcinoma and fibrous hamartoma of infancy but are not typically present in gliomas. The other two cases with EGFR mutations were located in the extracellular ligand-binding domain, where mutations and rearrangements are common in IDH-wildtype glioblastomas in the cerebral hemispheres of adults. Additional pathogenic alterations included TP53 mutation (n=4 cases), CDKN2A homozygous deletion (1), CDK6 amplification (1), CDKN2C mutation (1), TERT promoter mutation (1), HIST1H3B K27M mutation (1), and truncating mutations involving the BCOR (1), BCORL1 (2), LZTR1 (1), and CREBBP (1) transcriptional regulatory genes. No tumors harbored pathogenic alterations involving the H3F3A, HIST1H3C, SETD2, PDGFRA, MET, PPM1D, ACVR1, FGFR1, BRAF, PTPN11, IDH1, IDH2, MYB, and MYBL1 genes. In comparison, EGFR mutations are rare (<1% of 982 cases) in pediatric high-grade gliomas in general (Mackay et al, Cancer Cell 2017). Together, these findings identify pediatric bithalamic diffuse gliomas as a unique subtype of IDH-wildtype and mostly histone H3-wildtype high-grade glioma characterized by frequent EGFR kinase domain mutations, with implications for precision medicine treatment using small molecule kinase inhibitors such as poziotinib, a CNS-penetrant agent with selective activity against exon 20 mutant EGFR.

High-grade neuroepithelial tumor with BCOR exon 15 internal tandem duplication (HGNET BCOR ex15 ITD) is a recently proposed tumor entity of the central nervous system (CNS) with a distinct methylation profile and characteristic genetic alteration. The complete spectrum of histologic features, accompanying genetic alterations, clinical outcomes, and optimal treatment for this new tumor entity are largely unknown. Here, we performed a comprehensive assessment of 10 new cases of HGNET BCOR ex15 ITD. The tumors mostly occurred in young children and were located in the cerebral or cerebellar hemispheres. On imaging all tumors were large, well-circumscribed, heterogeneous masses with variable enhancement and reduced diffusion. They were histologically characterized by predominantly solid growth, glioma-like fibrillarity, perivascular pseudorosettes, and palisading necrosis, but absence of microvascular proliferation. They demonstrated sparse to absent GFAP expression, no synaptophysin expression, variable OLIG2 and NeuN positivity, and diffuse strong BCOR nuclear positivity. While BCOR exon 15 internal tandem duplication was the solitary pathogenic alteration identified in six cases, four cases contained additional alterations including CDKN2A/B homozygous deletion, TERT amplification or promoter hotspot mutation, and damaging mutations in TP53, BCORL1, EP300, SMARCA2 and STAG2. While the limited clinical follow-up in prior reports had indicated a uniformly dismal prognosis for this tumor entity, this cohort includes multiple long-term survivors. Our study further supports inclusion of HGNET BCOR ex15 ITD as a distinct CNS tumor entity and expands the known clinicopathologic, radiographic, and genetic features.

INTRODUCTION: Oncolytic viruses selectively replicate in tumor cells, and may attract immune cells into the microenvironment to stimulate a systemic antitumor response. We have shown efficacy of oncolytic measles virus (MV) against medulloblastoma (MB) in preclinical models. To study the effect of immune system on MV therapy, we have developed the first immunocompetent MV-sensitive murine model of Group 3 MB (CSCG model) and showed that intratumoral MV treatment of tumor-bearing mice significantly prolonged survival. Herein, we study the interaction of MV therapy and tumor immune microenvironment in this model. METHODS: Naive and MV-preimmunized mice were injected with 200,000 CSCG tumor cells in caudate putamen and tumor-bearing mice were treated identically with intratumoral MV injections (n=5 per group). Tumor growth was monitored by bioluminescence imaging and RT-PCR was performed to assess infiltrating CD4+ and CD8+ T cells in MV-treated and untreated control tumors. RESULTS: RT-PCR of untreated tumors shows an average of 43.4 ±1.45-fold and 13.24 ±1.34-fold increase in infiltrating CD8+T cells and CD4+T cells, respectively, compared to normal brain (n=3). Tumors that responded to MV therapy, but recurred had a dramatic decrease in infiltrating CD8+T cells (2.66 ±0.8-fold higher than normal brain). No significant difference in survival was observed between MV-naive and MV-immunized mice (p= 0.773), indicating that anti-measles immunity does not affect MV efficacy, an important point given that almost all patients have been vaccinated with MV. Long-term survivors, both naïve and MV-immune, remained tumor-free for at least 150 days post treatment. These mice (n=3) were re-challenged with a 3-fold higher number of tumor cells than the original injection, but tumor failed to grow for at least 50 days post-injection. CONCLUSION: This finding strongly suggests MV treatment induces anti-tumor immunity. Ongoing studies are being performed to dissect this immune response and to explore the possibility of enhancing it.

Author(s): Sloan, Emily A; Cooney, Tabitha; Oberheim Bush, Nancy Ann; Buerki, Robin; Taylor, Jennie; Clarke, Jennifer L; Torkildson, Joseph; Kline, Cassie; Reddy, Alyssa; Mueller, Sabine; Banerjee, Anu; Butowski, Nicholas; Chang, Susan; Mummaneni, Praveen V; Chou, Dean; Tan, Lee; Theodosopoulos, Philip; McDermott, Michael; Berger, Mitchel; Raffel, Corey; Gupta, Nalin; Sun, Peter P; Li, Yi; Shah, Vinil; Cha, Soonmee; Braunstein, Steve; Raleigh, David R; Samuel, David; Scharnhorst, David; Fata, Cynthia; Guo, Hua; Moes, Gregory; Kim, John YH; Koschmann, Carl; Van Ziffle, Jessica; Onodera, Courtney; Devine, Patrick; Grenert, James P; Lee, Julieann C; Pekmezci, Melike; Phillips, Joanna J; Tihan, Tarik; Bollen, Andrew W; Perry, Arie; Solomon, David A