Shahsiah Reza, DeKoning Jenefer, Samie Saeed, Latifzadeh Seyed Ziaeddin, Kashi Zahra Mehdizadeh
Cancer Research Center, Tehran University of Medical Sciences, Iran.
Kashi Clinical Laboratories, USA.
Pathol Res Pract. 2017 Feb;213(2):98-105. doi: 10.1016/j.prp.2016.11.016. Epub 2016 Dec 16.
Recent advances in sequencing technologies have enabled us to scrutinize the versatile underlying mechanisms of cancer more precisely. However, adopting these new sophisticated technologies is challenging for clinical labs as it involves complex workflows, and requires validation for diagnostic purposes. The aim of this work is towards the analytical validation of a next generation sequencing (NGS) panel for cancer hotspot mutation analysis. Characterized formalin-fixed paraffin-embedded (FFPE) samples including biopsy specimens and cell-lines were examined by NGS methods utilizing the Ion Torrent™ Oncomine™ Focus DNA Assay and the PGM™ platform. Important parameters for somatic mutations including the threshold for differentiation of a positive and a negative result, coverage, sensitivity, specificity, and limit of detection (LoD) were analyzed. Variant calls with coverage of <100x were found to be inaccurate. The limit of detection for identifying hotspot mutations was determined to be 4.3%. The sensitivity and specificity of the method were 96.1% and 97.8% respectively. No statistically significant difference was found between different gene targets in terms of performance of hotspot frequency measurement for the subset tested. In every validation study, the number of samples, the manner of sample selection, and the number and type of variants play a role in the outcome. Therefore, these parameters should be assessed according to the clinical needs of each laboratory undertaking the validation.
测序技术的最新进展使我们能够更精确地审视癌症多样的潜在机制。然而，采用这些新的复杂技术对临床实验室来说具有挑战性，因为这涉及复杂的工作流程，且出于诊断目的需要进行验证。这项工作的目的是对用于癌症热点突变分析的下一代测序（NGS）检测板进行分析验证。利用Ion Torrent™ Oncomine™ Focus DNA检测法和PGM™平台，通过NGS方法对包括活检标本和细胞系在内的特征明确的福尔马林固定石蜡包埋（FFPE）样本进行了检测。分析了体细胞突变的重要参数，包括阳性和阴性结果的区分阈值、覆盖率、灵敏度、特异性和检测限（LoD）。发现覆盖率<100x的变异检出不准确。确定识别热点突变的检测限为4.3%。该方法的灵敏度和特异性分别为96.1%和97.8%。在所测试的子集中，不同基因靶点在热点频率测量性能方面未发现统计学上的显著差异。在每项验证研究中，样本数量、样本选择方式以及变异的数量和类型都会影响结果。因此，应根据进行验证的每个实验室的临床需求评估这些参数。
