Standardization of the liquid biopsy for pediatric diffuse midline glioma using ddPCR

Daphne Li,Erin R Bonner,Carl Koschmann,Rishi Lulla,Kyle Wierzbicki,Amanda M Saratsis,Eshini Panditharatna,Javad Nazarian,Sabine Mueller,Tina Huang

doi:10.1038/s41598-021-84513-1

Abstract

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 poor 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 levels of ctDNA 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 c.83A > T (H3.3K27M) mutations in DMG CSF, plasma, and primary tumor specimens across three different institutions. 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 A > T H3F3A mutation detection. Mutation allelic frequency (MAF) was determined and validated through parallel analysis of matched H3.3K27M tissue specimens (n = 3). We determined technical nuances between ddPCR instruments, and optimized sample preparation and sequencing protocols for H3.3K27M mutation detection and quantification. We observed 100% sensitivity and specificity for mutation detection in matched DMG tissue and CSF across assays, platforms and institutions. ctDNA is reliably and reproducibly detected in the liquid biome using ddPCR, representing a clinically feasible, reproducible, and minimally invasive approach for DMG diagnosis, molecular subtyping and therapeutic monitoring.

Highlights

Diffuse midline glioma (DMG) is a highly morbid pediatric brain tumor
We optimized our digital droplet PCR (ddPCR)-based technique for H3.3K27M detection using matched DMG tissue and liquid biopsy specimens, and validated our approach across three academic institutions using two leading ddPCR instruments
For cerebrospinal fluid (CSF) circulating tumor DNA (ctDNA) analysis, we detected fewer false positive droplets with Assay A on the BioRad platform (Fig. 1A,B), which may be attributed to the shorter length of Assay A probes improving the specificity of probe-target DNA hybridization

Summary

Introduction

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 poor prognosis. 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 c.83A > T (H3.3K27M) mutations in DMG CSF, plasma, and primary tumor specimens across three different institutions. Mounting evidence in the literature, demonstrate that liquid biopsy is a viable tool for clinicians to diagnose and monitor pediatric CNS tumors[20] Clinical implementation of this approach requires exquisite test reliability, sensitivity and specificity. Poor access to specimens further exacerbates the challenge of validating and optimizing these analytic methods for rare pediatric CNS tumors To address these challenges, we optimized our ddPCR-based technique for H3.3K27M detection using matched DMG tissue and liquid biopsy specimens, and validated our approach across three academic institutions using two leading ddPCR instruments. We show high test sensitivity, specificity, and reproducibility for detecting and quantifying H3.3K27M mutant ctDNA across institutions and platforms, which is essential for clinical implementation of this powerful new approach

Methods

Results

Conclusion