Department of Neurosurgery, Michigan Medicine, University of Michigan, Ann Arbor, Michigan.
Department of Pediatrics, Michigan Medicine, University of Michigan, Ann Arbor, Michigan.
Clin Cancer Res. 2020 Dec 1;26(23):6266-6276. doi: 10.1158/1078-0432.CCR-20-2066. Epub 2020 Oct 21.
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.
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 ( = 12) alongside nontumor CSF ( = 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.
Nanopore demonstrated 85% sensitivity and 100% specificity in CSF samples ( = 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.
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.
小儿高级别神经胶质瘤（pHGG）的诊断预后不良，治疗监测仍然困难。肿瘤会将无细胞肿瘤 DNA（cf-tDNA）释放到脑脊液（CSF）中，从而可以通过 CSF 取样检测到与肿瘤相关的突变。我们假设，与传统方法相比，通过手持式平台（Oxford Nanopore MinION）直接对 cf-tDNA 进行电子分析，可以快速且更灵敏地定量患者特异性 CSF cf-tDNA 变异等位基因分数（VAF）。
我们对 pHGG 患者（n=12）的 CSF 和肿瘤样本以及非肿瘤 CSF（n=6）中的 cf-tDNA 进行了超短片段（100-200bp）PCR 扩增，以检测有临床意义的改变。PCR 产物通过 Oxford Nanopore Technology（Nanopore）进行快速扩增子测序，对 VAF 进行定量。还与下一代测序（NGS）和液滴数字 PCR（ddPCR）进行了额外的比较。
在 CSF 样本（n=127 个重复）中，Nanopore 的灵敏度为 85%，特异性为 100%，DNA 检测下限为 0.1 飞摩尔，结果在 12 小时内得出，所有这些均优于 NGS。多重分析可同时分析非活检患者的 H3.3A（H3F3A）和 H3C2（HIST1H3B）突变，结果通过 ddPCR 得到了验证。Nanopore 对 CSF cf-tDNA 的连续测序显示与临床试验的影像学反应相关，一位患者表现出显著的多基因分子反应，这预示着长期的临床反应。
对 pHGG CSF cf-tDNA 片段进行 Nanopore 测序是可行、高效和敏感的，即使是低输入样本也能如此，从而克服了限制在该患者群体中更广泛应用 CSF cf-tDNA 诊断和监测的许多障碍。
