Electronic DNA Analysis of CSF Cell-free Tumor DNA to Quantify Multi-gene Molecular Response in Pediatric High-grade Glioma.
Electronic DNA Analysis of CSF Cell-free Tumor DNA to Quantify Multi-gene Molecular Response in Pediatric High-grade Glioma.
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CSF无细胞肿瘤DNA的电子DNA分析，以量化小儿高级神经胶质瘤中的多基因分子反应。
DOI：
10.1158/1078-0432.ccr-20-2066
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发表时间：
2020-12-01
期刊：
影响因子：
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通讯作者：
Koschmann C
中科院分区：
文献类型：
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作者：
Bruzek AK;Ravi K;Muruganand A;Wadden J;Babila CM;Cantor E;Tunkle L;Wierzbicki K;Stallard S;Dickson RP;Wolfe I;Mody R;Schwartz J;Franson A;Robertson PL;Muraszko KM;Maher CO;Garton HJL;Qin T;Koschmann C
儿童高级别胶质瘤（pHGG）的诊断预示着预后不良，治疗监测仍然很困难。肿瘤将无细胞肿瘤DNA（cf-tDNA）释放到脑脊液（CSF）中，允许通过CSF采样检测肿瘤相关突变。我们假设，使用手持式平台（Oxford Nanopore MinION）对cf-tDNA进行直接电子分析可以定量患者特异性CSF cf-tDNA变体等位基因分数（VAF），与已建立的方法相比，速度和检测限有所提高。我们对cf-tDNA进行了超短片段（100-200个碱基对）PCR扩增，用于来自pHGG患者（n = 12）以及非肿瘤CSF（n = 6）的CSF和肿瘤样品中的临床上可操作的改变。PCR产物通过Oxford Nanopore Technology（Nanopore）进行基于扩增子的快速测序，并定量VAF。进行了与下一代测序（NGS）和液滴数字PCR（ddPCR）的额外比较。Nanopore在CSF样品（n = 127次重复）中显示出85%的灵敏度和100%的特异性，检测限为0.1飞摩尔DNA，结果为12小时，所有结果均优于NGS。多重分析提供了对非活检患者中H3 F3 A和HIST 1H 3B突变的同时分析，并通过ddPCR证实了结果。通过Nanopore进行的连续CSF cf-tDNA测序证明了临床试验中放射学反应的相关性，其中一名患者显示出预测长期临床反应的显著多基因分子反应。超短pHGG CSF cf-tDNA片段的纳米孔测序对于低输入样品是可行的、有效的和灵敏的，从而克服了限制在该患者群体中更广泛地使用CSF cf-tDNA诊断和监测的许多障碍。
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 to established methods. We performed ultra-short fragment (100–200 base pair) PCR amplification of cf-tDNA for clinically actionable alterations in CSF and tumor samples from patients with pHGG (n = 12) alongside non-tumor 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. Nanopore demonstrated 85% sensitivity and 100% specificity in CSF samples (n = 127 replicates) with 0.1 femtomol DNA limit of detection and 12-hour results, all of which compared favorably to NGS. Multiplexed analysis provided concurrent analysis of H3F3A and HIST1H3B mutations in a non-biopsied 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.