Development and Validation of Liquid Biopsy ddPCR and Next Generation Sequencing Assays for Diffuse Midline Glioma, K27M Variant

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PROJECT SUMMARY/ABSTRACT Diffuse intrinsic pontine glioma (DIPG), affecting an estimated 200-300 children in the United States per year, is one of the deadliest cancers of any type. The median survival for children with DIPG is less than one year from diagnosis; which has not changed in three decades. The diagnosis of DIPG is typically made using clinical symptoms and magnetic resonance (MR) scans. Because of the location of the tumors in the brainstem, diagnostic biopsies are typically avoided, although MR-guided, stereotactic needle biopsies are available in a few specialized centers. Missense mutations in Histone 3 genes H3.3 (H3F3A K27M) or H3.1 (HIST1H3B K27M) are present in most DIPGs and represent an early tumorigenic event. The World Health Organization (WHO) now classifies DIPG and non-pontine midline gliomas together as a distinct pathologic group known as diffuse midline glioma, H3K27 mutant (DMG). Children with H3F3A mutant tumors are more resistant to radiotherapy and relapse earlier than HIST1H3B/C mutant tumors. Documentation of either H3K27 mutation determines eligibility for clinical trial use of H3-K27 demethylase and histone deacetylases. Further, a preliminary study published by collaborators in this proposal showed that 16/20 (80%) of children with H3K27 mutant tumors had detectable circulating tumor DNA (ctDNA) in plasma and mutant allele frequency in plasma correlated with tumor volume measured by MR scans, decreased with initial response to therapy, and increased upon tumor progression. ResourcePath is developing DMG-Dx, a digital droplet PCR (ddPCR) assay for diagnosis, genotyping and monitoring of midline malignant glial neoplasms of childhood in cerebrospinal fluid and/or plasma samples, i.e. liquid biopsy. The ddPCR assay uses advanced, locked-nucleic-acid probe chemistry to achieve exquisitely sensitive and specific histone H3.3 (H3F3A K27M) or histone H3.1 (HIST1H3B/C K27M) hotspot mutations which define DMG. Our long-term goal is to develop DMG-Dx as non-invasive and precise diagnostic tests that will improve clinical management and support drug development for pediatric brainstem gliomas. This liquid biopsy will serve three unmet needs: 1) obtain diagnostic information without having to perform invasive and risky biopsies available only at special centers; 2) determine eligibility for clinical trials and 3) serve as a therapeutic response biomarker providing objective information important to clinical trial of novel agents including immunotherapy. In this SBIR Phase I the aims are: 1) Perform analytic validation of DMG-Dx ddPCR assays for sensitive detection of H3K27 mutations for diagnosis and 2) Determine reliability of these assays for quantitative measurement in tumor monitoring indications. In Phase II we will clinically validate the DMG-Dx in the context of multiple clinical trials. DMG-Dx will be marketed by direct outreach to the international community of pediatric oncologists and neurologists who care for individuals with diffuse midline gliomas.