In PNOC003, children and young people with a recent diagnosis of diffuse intrinsic pontine glioma (DIPG) will participate in a multi-center precision medicine trial. Patients were enrolled between the ages of 3 and 25 if they had imaging findings suggestive of DIPG. The biopsy material was taken for mRNA and whole-exome sequencing. Based on the recommendations of the molecular tumor board, patients were given up to 4 FDA-approved medications to use after radiotherapy (RT). The levels of H3K27M-mutant circulating tumor DNA (ctDNA) from tumors were tracked over time. Using whole-genome sequencing and DNA methylation profiling, researchers evaluated tumor tissue and corresponding primary cell lines. Results were double-checked in a separate cohort from the Children’s Brain Tumor Network (CBTN) when possible. The molecular tumor board considered 28 out of 38 enrolled patients (median age 6, 10 females). Around 19 of them really took the prescribed medication. Patients who followed the advice and those who didn’t fare similarly in terms of overall survival (OS), with a median of 13.1 months (95% CI, 11.2-18.4). About 60% of cases with baseline testing positive for H3K27M-mutant ctDNA were linked to RT response and survival. Around 11 different cell lines were derived from the initial tumor, each of which was completely faithful to the original mutations in the essential somatic driver genes. OS was shorter in H3K27-altered DIPGs with TP53 mutations (TP53mut 11.1 mo; 95% CI, 8.7-14; TP53wt 13.3 mo; 95% CI, 11.8-NA; P=3.4e2), as was genomic instability (P=3.1e3) and resistance to RT (P=6.4e4). A link between TP53 mutant status, genomic instability, and clinical outcome was verified in the CBTN cohort. Molecular abnormalities and prognostic indicators for H3K27-altered DIPGs can be better understood with an upfront treatment-naive biopsy.