Dataset Information

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.

SUBMITTER: Bruzek AK

PROVIDER: S-EPMC7710567 | biostudies-literature | 2020 Dec

REPOSITORIES: biostudies-literature

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Publications

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

Bruzek Amy K AK Ravi Karthik K Muruganand Ashwath A Wadden Jack J Babila Clarissa May CM Cantor Evan E Tunkle Leo L Wierzbicki Kyle K Stallard Stefanie S Dickson Robert P RP Wolfe Ian I Mody Rajen R Schwartz Jonathan J Franson Andrea A Robertson Patricia L PL Muraszko Karin M KM Maher Cormac O CO Garton Hugh J L HJL Qin Tingtin T Koschmann Carl C

Clinical cancer research : an official journal of the American Association for Cancer Research 20201021 23

<h4>Purpose</h4>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 limi ...[more]

PMID: 33087334

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Project description:Abstract Diffuse midline glioma (DMG) with the H3K27M mutation is a lethal childhood brain cancer, with patients rarely surviving 2 years from diagnosis. We conducted a multi-site Phase 1 trial of the imipridone ONC201 for children with H3K27M-mutant glioma (NCT03416530). Patients enrolled on Arm D of the trial (n=24) underwent serial lumbar puncture (baseline, 2, 6 months) for cell-free tumor DNA (cf-tDNA) analysis at time of MRI. Additionally, patients on all arms of the trial at the University of Michigan underwent serial plasma collection. CSF collection was feasible in this cohort, with no procedural complications. We collected 96 plasma samples and 53 CSF samples from 29 patients, including those with H3F3A (H3.3) (n=13), HIST13HB (H3.1) (n= 4), and unknown H3 status/not biopsied (n=12) [range of 0–8 CSF samples and 0–10 plasma samples]. We performed digital droplet polymerase chain reaction (ddPCR) analysis and/or amplicon-based electronic sequencing (Oxford Nanopore) of cf-tDNA samples and compared variant allele fraction (VAF) to radiographic change (maximal 2D tumor area on MRI). Preliminary analysis of samples demonstrates a correlation between changes in tumor size and H3K27M cf-tDNA VAF, when removing samples with concurrent bevacizumab. In multiple cases, early reduction in CSF cf-tDNA predicts long-term clinical response (>1 year) to ONC201, and does not increase in cases of later-defined pseudo-progression (radiation necrosis). For example, a now 9-year old patient with thalamic H3K27M-mutant DMG underwent treatment with ONC201 after initial radiation and developed increase in tumor size at 4 months post-radiation (124% baseline) of unclear etiology at the time. Meanwhile, her ddPCR declined from baseline 6.76% VAF to <1%, which has persisted, with now near complete response (15% tumor reduction) at 30 months on treatment from diagnosis. In summary, we present the feasibility and utility of serial CSF/plasma monitoring of a promising experimental therapy for DMG.

Dataset Information

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

Purpose

Experimental design

Results

Conclusions

Publications

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

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