Lee Ahwon, Lee Sung-Hak, Jung Chan Kwon, Park Gyungsin, Lee Kyo Young, Choi Hyun Joo, Min Ki Ouk, Kim Tae Jung, Lee Eun Jung, Lee Youn Soo
Department of Hospital Pathology, Seoul St. Mary's Hospital, College of Medicine, The Catholic University of Korea, Seoul, Republic of Korea; Cancer Research Institute, College of Medicine, The Catholic University of Korea, Seoul, Republic of Korea.
Department of Hospital Pathology, Seoul St. Mary's Hospital, College of Medicine, The Catholic University of Korea, Seoul, Republic of Korea.
Pathol Res Pract. 2018 May;214(5):713-719. doi: 10.1016/j.prp.2018.03.009. Epub 2018 Mar 29.
Targeted application of next-generation sequencing (NGS) technology allows detection of specific mutations that can provide treatment opportunities for cancer patients. We evaluated the applicability of the Ion AmpliSeq Cancer Hotspot Panel V2 (CHV2) using formalin-fixed, paraffin-embedded (FFPE) tissue of clinical specimens. Thirty-five FFPE tumour samples with known mutational status were collected from four different hospitals and sequenced with CHV2 using an Ion Chef System and Ion S5 XL system. Out of 35 cases, seven were sequenced with Oncomine focus Assay Panel for comparison. For the limit of detection test, we used an FFPE reference standard, a cell line that included an engineered 50% EGFR T790 M in an RKO cell line background. Coverage analysis results including number of mapped reads, on target percent, mean depth, and uniformity were not different according to hospitals. Sensitivity for mutation detection down to 3% was demonstrated. NGS results showed 100% concordance with the results from single molecular pathology tests Assay in 30 cases with 24 known positive mutations and 14 known negative mutations, and another NGS panel of the Oncomine focus in seven cases. The CHV2 NGS test for solid tumours using Ion chef system and S5 XL system in clinical molecular pathology laboratories for analysis of solid tumours could be routinely used and could replace some single molecular pathology tests after a stringent and thorough validation process.
靶向应用下一代测序（NGS）技术能够检测特定突变，为癌症患者提供治疗机会。我们使用临床标本的福尔马林固定石蜡包埋（FFPE）组织评估了Ion AmpliSeq癌症热点区域Panel V2（CHV2）的适用性。从四家不同医院收集了35份已知突变状态的FFPE肿瘤样本，并使用Ion Chef系统和Ion S5 XL系统用CHV2进行测序。在35例样本中，7例使用Oncomine聚焦检测Panel进行测序以作比较。对于检测限测试，我们使用了一种FFPE参考标准品，即一种在RKO细胞系背景中包含工程化50%表皮生长因子受体（EGFR）T790M的细胞系。根据医院不同，包括映射读数数量、靶向百分比、平均深度和均匀性在内的覆盖分析结果并无差异。检测到低至3%的突变敏感性。在30例有24个已知阳性突变和14个已知阴性突变的病例中，NGS结果与单分子病理学检测结果显示100%一致，在7例病例中与Oncomine聚焦的另一个NGS Panel结果一致。在临床分子病理学实验室中，使用Ion chef系统和S5 XL系统对实体瘤进行CHV2 NGS检测以分析实体瘤，经过严格全面的验证过程后可常规使用，并可替代一些单分子病理学检测。
