Electronic DNA Analysis of CSF Cell-free Tumor DNA to Quantify Multi-gene Molecular Response in Pediatric High-grade Glioma

Abstract

Purpose: Pediatric high-grade glioma (pHGG) diagnosis portends poor prognosis and therapeutic monitoring remains difficult. Tumors release cell-free tumor DNA (cf-tDNA) into cerebrospinal fluid (CSF), allowing for potential detection of tumor-associated mutations by CSF sampling. We hypothesized that direct, electronic analysis of cf-tDNA with a handheld platform (Oxford Nanopore MinION) could quantify patient-specific CSF cf-tDNA variant allele fraction (VAF) with improved speed and limit of detection compared with established methods. Experimental Design: We performed ultra-short fragment (100–200 bp) PCR amplification of cf-tDNA for clinically actionable alterations in CSF and tumor samples from patients with pHGG (n = 12) alongside nontumor CSF (n = 6). PCR products underwent rapid amplicon-based sequencing by Oxford Nanopore Technology (Nanopore) with quantification of VAF. Additional comparison to next-generation sequencing (NGS) and droplet digital PCR (ddPCR) was performed. Results: Nanopore demonstrated 85% sensitivity and 100% specificity in CSF samples (n = 127 replicates) with 0.1 femtomole DNA limit of detection and 12-hour results, all of which compared favorably with NGS. Multiplexed analysis provided concurrent analysis of H3.3A (H3F3A) and H3C2 (HIST1H3B) mutations in a nonbiopsied patient and results were confirmed by ddPCR. Serial CSF cf-tDNA sequencing by Nanopore demonstrated correlation of radiological response on a clinical trial, with one patient showing dramatic multi-gene molecular response that predicted long-term clinical response. Conclusions: Nanopore sequencing of ultra-short pHGG CSF cf-tDNA fragments is feasible, efficient, and sensitive with low-input samples thus overcoming many of the barriers restricting wider use of CSF cf-tDNA diagnosis and monitoring in this patient population.

Cell-Free Tumor DNA (cf-tDNA) Liquid Biopsy: Current Methods and Use in Brain Tumor Immunotherapy

“…Nanopore sequencing is a relatively new technology ( 57 ) that has been used for liquid biopsies ( 58 , 59 ). Nanopore sequencers work by feeding DNA strands through small pores embedded in a membrane.…”

“…Nanopore devices are low-cost, can sequence any length DNA strand, have a small form factor, and offer a rapid time to result making them ideal devices for liquid biopsy. However, the device’s error rate has traditionally prevented it from being applied to liquid samples where allele frequencies are less than ~2% ( 58 ). Our group previously analyzed CSF samples from 12 pediatric high grade glioma patients and found that nanopore had a 85% sensitivity and 100% specificity in CSF samples ( 58 ), which compared favorably to Illumina-based targeted sequencing.…”

“…However, the device’s error rate has traditionally prevented it from being applied to liquid samples where allele frequencies are less than ~2% ( 58 ). Our group previously analyzed CSF samples from 12 pediatric high grade glioma patients and found that nanopore had a 85% sensitivity and 100% specificity in CSF samples ( 58 ), which compared favorably to Illumina-based targeted sequencing. More recent improvements to basecaller accuracy, and also the use of circular consensus sequencing ( 59 – 61 ) have improved accuracy to <0.05%, comparable with ddPCR-based approaches ( 59 ).…”

“…In addition to tumor evolution, several studies have shown the utility of serial cf-tDNA sampling for treatment response monitoring and resolution of pseudoprogression in gliomas ( 19 , 37 , 58 , 62 , 68 , 69 , 74 , 86 ). One of the largest studies thus far from Panditharatna et al.…”

Ultra-Rapid Somatic Variant Detection via Real-Time Threshold Sequencing

“…PCR primer sets were drawn from multiple sources and selected or designed to flank the HIST1H3B K27M hotspot mutation. The 187 bp primer set was taken from prior work 39 . The 260 bp primer set was designed using Primer3 v4.1.0 and tailed at the 5' ends with ONT handshake sequences to enable potential interoperability with ONT's four-primer library preparation protocol.…”