Lab Med Qual Assur.  2021 Mar;43(1):31-36. 10.15263/jlmqa.2021.43.1.31.

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

• Hong J
• Kim JH
• Park S
• Lee SG
• Lee W
• Chun S
• Min WK

• KMID: 2514599
• DOI: http://doi.org/10.15263/jlmqa.2021.43.1.31

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.

Keyword

Genetic testing; Laboratory proficiency testing; Somatic variant