DIPG-06. RAPID, ULTRA-DEEP SEQUENCING OF PEDIATRIC DIPG FROM CEREBROSPINAL FLUID USING A NOVEL HAND-HELD ELECTRONIC DNA ANALYSIS PLATFORM

INTRODUCTION: Brain tumors release tumor DNA (tDNA) into cerebrospinal fluid (CSF), allowing for detection and serial monitoring of tumor-associated genetic mutations by CSF sampling. For midline and brainstem tumors such as diffuse intrinsic pontine glioma (DIPG), surgical biopsy poses possible severe neurological deficits. As such, liquid biopsy is needed. Current platforms for CSF tDNA analysis are limited by their requirement for assay development for each mutation (digital droplet PCR), or cost and timeliness (Illumina sequencing). We hypothesized that direct, electronic analysis of DNA with a novel hand-held platform (Oxford Nanopore) could provide real-time, ultra-deep (>1,000x reads) sequencing of DIPG CSF tDNA. METHODS: We performed amplicon-based PCR on DNA from normal brain (n=6), normal CSF (n=13), tumor brain (n=3), and tumor CSF (n=10) to amplify wildtype and mutant H3F3A K27M, HIST1H3B K27M, and ACVR1 G328E genes from normal controls and pediatric patients with DIPG. We performed parallel barcoded sequencing of multiple samples run in duplicate or triplicate using NanoPore MinION technology and determined variant allele fraction (VAF) of each amplicon via an expeditious pipeline using MinKNOW, Albacore, MiniMap2, and Integrated Genome Browser. RESULTS: NanoPore sequenced 30 amplicons with average depth of 17,244 reads per amplicon in under 80 minutes. H3F3A K27M VAF was 49.14% (S.D. 0.49) in tumor tissue and 13.63% (S.D. 0.35) in CSF. VAF was 10.42% (S. D. 1.11) for HIST1H3B K27M and 7% (S.D. 0.46) for ACVR1 G328E in CSF. VAF were comparable to biopsy results by Illumina. Sensitivity and specificity of NanoPore were 100% and 98.8%, respectively, with p-values of < 0.05 for normal versus tumor samples. CONCLUSIONS: NanoPore MinION technology rapidly, reliably, and efficiently sequences tumor mutations in pooled tissue and CSF from patients with DIPG with outstanding sensitivity and specificity. NanoPore is more efficient, and cost and time effective than digital droplet PCR and Illumina.