PATH-13. MOLECULAR AND BIOLOGY CORRELATIVE RESULTS FROM THE PHASE 1B TRIAL OF UNESBULIN (PTC596) WITH RADIOTHERAPY IN CHILDREN NEWLY-DIAGNOSED WITH DIFFUSE INTRINSIC PONTINE GLIOMA (DIPG) AND HIGH-GRADE GLIOMA (HGG): A REPORT FROM THE COLLABORATIVE N

Abstract BACKGROUND Within the CONNECT1702 trial of unesbulin (tubulin-binding agent causing BMI-1 modulation) with radiotherapy in patients with DIPG/HGG, tumor tissue was analyzed at diagnosis and/or autopsy for biology correlatives, enabling comprehensive, longitudinal molecular characterization. METHODS Whole genome sequencing, RNA-sequencing, and Western blot analyses were performed on tumor, normal brain, and peripheral blood mononuclear cells (PBMCs). RESULTS Tumor tissue from diagnosis (n=11) and/or autopsy (n=7) was analyzed for 16 patients (DIPG=9; HGG=7). Western blot analyses demonstrated 1) BMI-1 overexpression in baseline tumor compared to normal brain and 2) primary on-target effect of BMI1-modulation causing M-phase mitotic arrest when comparing pre- and post-treated PBMCs (increased H3S10-phosphorylation). Among 9 DIPG patients, alterations in the following genes were identified: H3-3A (H3.3) (n=5), H3C2 (H3.1) (n=2, both with co-occurring ACVR1 and PIK3CA mutations), TP53 (n=5), ATRX (n=2), and PPM1D (n=2). Tumors from two longer DIPG survivors were H3.1-, ACVR1-mutant (overall survival [OS]=26 months) and MET-amplified (OS=18 months); one patient with H3.3K27M-DIPG had germline MSH6 mutation (Lynch syndrome) (OS=12.6 months). Among 7 HGG patients, alterations in the following genes were identified: H3-3A (n=5), TP53 (n=3), NF1 (n=3), PDGFRA (n=1), EGFR (n=1), and PIK3CA (n=1). A BRAFV600E mutation was observed in the thalamic HGG patient (H3.3K27M) with greatest progression-free survival (>23 months). Early leptomeningeal metastases occurred in one DIPG patient with BCOR alteration and one HGG patient, with MYCN amplification identified in the spinal metastasis, but not intracranial disease. From gene set enrichment analyses, pathways enriched in patients with longer survival included telomerase regulation and ATP synthesis, whereas pathways enriched in patients with shorter survival included interneuron development, neuron semaphorin signaling, and gamma aminobutyric signaling. CONCLUSIONS Comprehensive molecular profiling of diagnostic and post-mortem tumor tissue provides valuable early biological insight, elucidating potential genomic biomarkers predictive of outcome and response to M-phase alteration and BMI-1 modulation in DIPG/HGG.