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Development of a Method for Manufacturing External Quality Assessment Material for Genetic Testing of Solid Tumors Using Mutant and Wild-Type Cell Lines

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Abstract
Background: DNA extracted from mutant cell lines is frequently used as an external quality assessment (EQA) material for genetic testing of solid tumors because it is easy to obtain. However, the proportion of mutations in cell lines is different from that in actual tumor samples. In this study, mixtures of mutant DNA and wild-type DNA mimicking patient samples were analyzed to optimize the amount of mutant DNA in EQA specimens.
Methods: Four cell lines harboring the selected mutation were cultured, and genomic DNA was extracted from cultured cells. Wild-type cell line DNA was prepared in the same manner. Diluted samples were prepared by mixing each mutant cell line DNA and wild-type cell line DNA at different ratios. Sanger sequencing of target variants was performed. For reliability, sequencing was repeated three times and read by two readers. The cutoff was based on the lowest proportion of mutant DNA that was determined to be positive in all three experiments.
Results: The cutoffs of mutant cell line DNA ratios were 10%, 5%, 25%, and 25% for KRAS G12C, EGFR exon 19 deletion, EGFR T790M, and BRAF V600E, respectively. For the cell lines harboring EGFR T790M and BRAF V600E, the mutant fraction was not 100%.
Conclusions: When manufacturing EQA material for solid tumor genetic testing, consistent results can be obtained if the mutant proportion is 10% or more. In addition, the mutant allele frequency of the cell line should be checked in advance to guarantee that EQA materials contain enough mutant DNA.
Author(s)
김지현민원기박승만이상곤이우창전사일홍진영
Issued Date
2021
Type
Article
Keyword
Genetic testingLaboratory proficiency testingSomatic variant
DOI
10.15263/jlmqa.2021.43.1.31
URI
https://oak.ulsan.ac.kr/handle/2021.oak/8585
https://ulsan-primo.hosted.exlibrisgroup.com/primo-explore/fulldisplay?docid=TN_cdi_nrf_kci_oai_kci_go_kr_ARTI_9765936&context=PC&vid=ULSAN&lang=ko_KR&search_scope=default_scope&adaptor=primo_central_multiple_fe&tab=default_tab&query=any,contains,Development%20of%20a%20Method%20for%20Manufacturing%20External%20Quality%20Assessment%20Material%20for%20Genetic%20Testing%20of%20Solid%20Tumors%20Using%20Mutant%20and%20Wild-Type%20Cell%20Lines&offset=0&pcAvailability=true
Publisher
Laboratory Medicine and Quality Assurance
Location
대한민국
Language
한국어
ISSN
2384-2458
Citation Volume
43
Citation Number
1
Citation Start Page
31
Citation End Page
36
Appears in Collections:
Medicine > Medicine
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