Methodological Challenges of Digital PCR Detection of the Histone H3 K27M Somatic Variant in Cerebrospinal Fluid.
Methodological Challenges of Digital PCR Detection of the Histone H3 K27M Somatic Variant in Cerebrospinal Fluid.
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DOI：
10.3389/pore.2022.1610024
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发表时间：
2022
影响因子：
2.8
通讯作者：
Druy, Alexander
中科院分区：
文献类型：
--
作者：
Zaytseva, Margarita;Usman, Natalia;Salnikova, Ekaterina;Sanakoeva, Agunda;Valiakhmetova, Andge;Chervova, Almira;Papusha, Ludmila;Novichkova, Galina;Druy, Alexander
体液中的无细胞DNA（cfDNA）对于癌症诊断是无价的。尽管液体活检用于诊断中枢神经系统（CNS）肿瘤的潜力令人印象深刻，但许多挑战阻止将这种方法引入常规实验室实践。在这项研究中，我们采用了一种方案，敏感检测H3 K27 M体细胞变异的脑脊液（CSF），通过使用数字聚合酶链反应（dPCR）。逐步进行方案的优化，包括H3靶区域的预扩增和dPCR条件的调整。优化的方案允许从低至9皮克的DNA量开始检测突变等位基因。使用具有已知H3 K27 M状态的肿瘤组织样品的代表性组测试分析特异性，并且未检测到假阳性病例。将该方案应用于使用两种替代dPCR平台QX 200 Droplet Digital PCR系统（Bio-Rad）和QIAcuity Digital PCR系统（Qiagen）从CNS肿瘤患者（n = 18）收集的一系列CSF样品。从H3 K27 M阳性弥漫性中线胶质瘤患者采集的4份CSF标本中有3份，两种平台均允许检测突变等位基因。使用脑室入路采集CSF似乎是首选，因为腰椎CSF样本可能产生不明确的结果。从H3野生型肿瘤患者采集的所有CSF样本均被鉴定为H3 K27 M阴性。用这两个平台获得的定量数据的高度一致性证明了该方法的普遍性。
Cell-free DNA (cfDNA) in body fluids is invaluable for cancer diagnostics. Despite the impressive potential of liquid biopsies for the diagnostics of central nervous system (CNS) tumors, a number of challenges prevent introducing this approach into routine laboratory practice. In this study, we adopt a protocol for sensitive detection of the H3 K27M somatic variant in cerebrospinal fluid (CSF) by using digital polymerase chain reaction (dPCR). Optimization of the protocol was carried out stepwise, including preamplification of the H3 target region and adjustment of dPCR conditions. The optimized protocol allowed detection of the mutant allele starting from DNA quantities as low as 9 picograms. Analytical specificity was tested using a representative group of tumor tissue samples with known H3 K27M status, and no false-positive cases were detected. The protocol was applied to a series of CSF samples collected from patients with CNS tumors (n = 18) using two alternative dPCR platforms, QX200 Droplet Digital PCR system (Bio-Rad) and QIAcuity Digital PCR System (Qiagen). In three out of four CSF specimens collected from patients with H3 K27M-positive diffuse midline glioma, both platforms allowed detection of the mutant allele. The use of ventricular access for CSF collection appears preferential, as lumbar CSF samples may produce ambiguous results. All CSF samples collected from patients with H3 wild-type tumors were qualified as H3 K27M-negative. High agreement of the quantitative data obtained with the two platforms demonstrates universality of the approach.
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影响因子：
4.6
作者：
Dong L;Meng Y;Sui Z;Wang J;Wu L;Fu B
通讯作者：
Fu B
DOI：
10.3390/diagnostics11040681
发表时间：
2021-04-09
期刊：
Diagnostics (Basel, Switzerland)
影响因子：
--
作者：
On J;Natsumeda M;Watanabe J;Saito S;Kanemaru Y;Abe H;Tsukamoto Y;Okada M;Oishi M;Yoshimura J;Kakita A;Fujii Y
通讯作者：
Fujii Y
影响因子：
16.6
作者：
De Mattos-Arruda L;Mayor R;Ng CKY;Weigelt B;Martínez-Ricarte F;Torrejon D;Oliveira M;Arias A;Raventos C;Tang J;Guerini-Rocco E;Martínez-Sáez E;Lois S;Marín O;de la Cruz X;Piscuoglio S;Towers R;Vivancos A;Peg V;Ramon y Cajal S;Carles J;Rodon J;González-Cao M;Tabernero J;Felip E;Sahuquillo J;Berger MF;Cortes J;Reis-Filho JS;Seoane J
通讯作者：
Seoane J
影响因子：
3.9
作者：
Connolly ID;Li Y;Pan W;Johnson E;You L;Vogel H;Ratliff J;Hayden Gephart M
通讯作者：
Hayden Gephart M
影响因子：
4.1
作者：
Jackson, Jennifer B.;Choi, Daniel S.;Godfrey, Tony E.
通讯作者：
Godfrey, Tony E.