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Pediatric diffuse midline glioma (DMG) with histone H3 mutations (80%), are highly morbid tumors with poor response to therapy. We previously detected H3 mutations in circulating tumor DNA (ctDNA) from CSF derived from children with DMG and high grade glioma. Here, we describe a high-throughput, sensitive and specific approach for H3 mutation detection and quantification in plasma and CSF specimens, validated across multiple centers. METHODS DNA extracted from tissue-validated H3.3K27M specimens (4 tumor, 5 CSF, 4 plasma) and pediatric glioma cell lines (1 wild-type, 1 H3.3K27M) were used to standardize ddPCR workflows. ctDNA extracted from 500uL of CSF or plasma was pre-amplified using sequence-specific primers, then analyzed on RainDance and BioRad ddPCR systems using two sets of custom primers and fluorescent LNA probes (Assays A and B). H3 mutation allelic frequency (MAF) was determined across specimens, and validated through parallel analysis of additional matched H3.3K27M tumor tissue, CSF and plasma specimens (n=3). RESULTS ctDNA processing and detection was standardized across three institutions and two platforms with sensitive, specific and reproducible H3 mutation detection. H3K27M mutations were detected in as little as 0.3-2pg input DNA on both platforms, with comparable MAFs across instruments and assays. RainDance yielded greater overall positive droplet number detection (increased sensitivity). Differences in optimal sample input volume between platforms were circumvented by vacuum concentration or dilution to maintain equal input DNA. Assay A yielded superior specificity so was used for subsequent matched specimen analysis. Mutation detection in plasma, as in previous studies, remained challenging due to low [ctDNA], while CSF analysis yielded reliable results across assays and platforms. CONCLUSIONS We demonstrate the utility of liquid biopsy for identifying H3K27M mutations in plasma and CSF via ddPCR with low starting [DNA], representing a rapid, minimally invasive method for diagnosis and therapeutic monitoring of DMG.

PDTM-42. CROSS-PLATFORM PROFILING OF ctDNA USING ddPCR: STANDARDIZATION OF THE LIQUID BIOPSY FOR PEDIATRIC DIFFUSE MIDLINE GLIOMA