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Pathol Res Pract. 2018 May;214(5):713-719. doi: 10.1016/j.prp.2018.03.009. Epub 2018 Mar 29.

Use of the Ion AmpliSeq Cancer Hotspot Panel in clinical molecular pathology laboratories for analysis of solid tumours: With emphasis on validation with relevant single molecular pathology tests and the Oncomine Focus Assay.

Author information

1
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.
2
Department of Hospital Pathology, Seoul St. Mary's Hospital, College of Medicine, The Catholic University of Korea, Seoul, Republic of Korea.
3
Department of Hospital Pathology, St. Vincent's Hospital, College of Medicine, The Catholic University of Korea, Seoul, Republic of Korea.
4
Department of Hospital Pathology, St. Paul's Hospital, College of Medicine, The Catholic University of Korea, Seoul, Republic of Korea.
5
Department of Hospital Pathology, Yeouido St. Mary's Hospital, College of Medicine, The Catholic University of Korea, Seoul, Republic of Korea.
6
Department of Hospital Pathology, Seoul St. Mary's Hospital, College of Medicine, The Catholic University of Korea, Seoul, Republic of Korea. Electronic address: lys9908@catholic.ac.kr.

Abstract

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.

KEYWORDS:

High-throughput nucleotide sequencing; Malignant neoplasm; Molecular diagnostic techniques

PMID:
29615338
DOI:
10.1016/j.prp.2018.03.009
[Indexed for MEDLINE]

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