Authors: Amanda Muhs Saratsis, MD; Erin Bonner, BS; Eshini Panditharatna, PhD; Daphne Li, MD; Julia Pantalone, BA; Tina Huang, MS; Rishi Lulla; Carl Koschmann, MD; Javad Nazarian, PhD; Amanda Saratsis, MD (Chicago, IL)

Introduction Pediatric diffuse midline glioma (DMG) is a highly morbid tumor not amenable to surgical resection. Mutations in histone H3 encoding genes occur in 80% of cases, and portend a worse clinical prognosis. We previously detected H3 mutations in circulating tumor DNA (ctDNA) in CSF derived from children with DMG and high grade glioma. Here, we describe a more sensitive and specific approach for H3 mutation detection and quantification in plasma and CSF with low starting [ctDNA], with validation performed across multiple centers.

Methods Plasma (n= 7) and CSF (n=4) specimens were collected at diagnosis and submitted for ddPCR. H3 mutation status was assessed by calculating mutation allelic frequency (MAF, mutant/mutant + wild type droplet counts). To validate our approach, CSF from patients who received tumor biopsies (n=8) and one healthy control was also analyzed, with H3 mutation status determined via tissue sequencing (H3K27M n=5, H3WT n=4). Isolated ctDNA was pre-amplified using sequence-specific primers, then analyzed using RainDance and BioRad ddPCR workflows with custom primers and fluorescent LNA probes. Primers and probes were validated using DNA from H3K27M mutant and wild-type pediatric glioma cell lines.

Results Thermal gradient analysis identified optimal annealing temperatures of 50.7°C-52.1°C for H3 K27M mutant and H3 WT probes, and 58°C for primers. 83.5 copies of H3.3K27M DNA/uL was detected with 1.0 ng loaded DNA, and 7.66 copies of H3.3K27M DNA/uL for 0.01 ng loaded DNA derived from mutant glioma cell lines. H3.3K27M mutations were robustly detected using ctDNA isolated from as little as 1 mL plasma or 500µL CSF.

Conclusions We demonstrate the utility of liquid biopsy for identifying H3K27M mutations in plasma and CSF with low starting [DNA], representing a rapid, minimally invasive method for diagnosis and therapeutic monitoring of pediatric brain tumors. ddPCR of additional pediatric glioma liquid biopsy specimens is currently underway.

Columbia Softball Pediatric Award