Development and Interlaboratory Evaluation of a NIST Reference Material RM 8366 for EGFR and MET Gene Copy Number Measurements
Hua-Jun He, Biswajit Das, Megan H. Cleveland, Chen Li, Corinne Camalier, Liang-Chu Liu, Kara L. Norman, Andrew Fellowes, Christopher McEvoy, Steven P. Lund, Jamie L. Almeida, Carolyn R. Steffen, Chris Karlovich, P. M. Williams, Kenneth D. Cole
The National Institute Standard and Technology (NIST) Reference Material RM 8366 was developed to improve the quality of gene copy measurements of EGFR (epidermal growth factor receptor) and MET (proto-oncogene, receptor tyrosine kinase), important targets for cancer diagnostics and treatment. The reference material is composed of genomic DNA prepared from six human cancer cell lines with different levels of amplification of the target genes. The reference values for the ratios of the EGFR and MET gene copy numbers to the copy numbers of reference genes were measured using digital PCR. The digital PCR measurements were confirmed by two additional laboratories. The samples were also characterized using Next Generation Sequencing (NGS) methods including whole genome sequencing (WGS) at two levels of coverage (approximately 5X and greater than 30X), whole exome sequencing (WES), and two different pan-cancer gene panels. The WES data were analyzed using three different bioinformatic algorithms. At low and moderate amplification levels the NGS results were highly correlated with the digital PCR results. This study shows that NIST RM 8366 is a valuable reference material to validate assays that assess EGFR and MET gene copy number measurements.