Cross-platform Profiling of ctDNA Using ddPCR: Standardization of the Liquid Biopsy for Pediatric Diffuse Midline Glioma

Background Diffuse midline glioma (DMG) is a highly morbid pediatric brain tumor. Up to 80% of DMGs harbor mutations in histone H3-encoding genes, associated with poorer prognosis. We previously showed the feasibility of detecting H3 mutations in circulating tumor DNA (ctDNA) in the liquid biome of children diagnosed with DMG. However, detection of low ctDNA concentrations is highly dependent on platform sensitivity and sample type. To address this, we optimized ctDNA detection sensitivity and specificity across two commonly used digital droplet PCR (ddPCR) platforms (RainDance and BioRad), and validated methods for detecting H3F3A mutations in DMG CSF, plasma, and primary tumor specimens across three different institutions. Methods DNA was extracted from H3.3K27M mutant and H3 wildtype (H3WT) specimens, including H3.3K27M tumor tissue (n=4), CSF (n=6), plasma (n=4), and human primary pediatric glioma cells (H3.3K27M, n=2; H3WT, n=1). ctDNA detection was enhanced via PCR pre-amplification and use of distinct custom primers and fluorescent LNA probes for c.83 AàT H3F3A mutation detection. Mutation allelic frequency (MAF) was determined and validated through parallel analysis of matched H3.3K27M tissue specimens (n=3). Results We determined technical nuances between ddPCR instruments, and optimized sample preparation and sequencing protocols for H3F3A mutation detection and quantification. We observed 100% sensitivity and specificity for mutation detection in matched DMG tissue and CSF across assays, platforms and institutionsConclusion Our study demonstrates that ctDNA is reliably and reproducibly detected in ctDNA using ddPCR, representing a clinically feasible and reproducible minimally invasive approach for DMG diagnosis, molecular subtyping and therapeutic monitoring.