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Electronic DNA analysis of CSF cell-free tumor DNA to quantify multi-gene molecular response in pediatric high-grade glioma

Publication

Date: 22nd October 2020 | Source: Clincal Cancer Research

Authors: Amy K. Bruzek, Karthik Ravi, Ashwath Muruganand, Jack Wadden, Clarissa May Babila, Evan Cantor, Leo Tunkle, Kyle Wierzbicki, Stefanie Stallard, Robert P. Dickson, Ian Wolfe, Rajen Mody, Jonathan Schwartz, Andrea Franson, Patricia L. Robertson, Karin M. Mu.

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.

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