ddPLEX EGFR/KRAS/BRAF: A highly multiplexed droplet digital PCR (ddPCR) panel for ultra-sensitive NSCLC biomarker detection

Biomarker testing for actionable oncogenic driver mutations is recommended in national and international guidelines based on the improved outcomes observed with use of targeted therapies in eligible patients with metastatic NSCLC. Here, we demonstrate the performance of the ddPLEX EGFR/KRAS/BRAF Mutation Detection Assay Kit, a research use only kit developed for the QX600 TM Droplet Digital TM PCR (ddPCR) System. This kit is designed for the detection, discrimination and quantification of 37 actionable variants in the EGFR , KRAS , and BRAF genes in a single ddPCR well. It also has a companion total quantification well that quantifies EGFR , KRAS and BRAF genes in a mutation agnostic manner and allows for variant allele frequency (VAF) calculation. It provides same day results with a streamlined workflow, includes positive and internal controls and is compatible with plasma and formalin-fixed paraffin-embedded (FFPE) samples. The analytical performance of the ddPLEX EGFR/KRAS/BRAF Mutation Detection Assay Kit was evaluated internally, and the findings are presented here. The results demonstrated an analytical sensitivity of 0.025%-0.1% in a background of 132 ng of human genomic DNA for all targeted variants. The assay had an analytical specificity of 100% when tested with 1000 copies input of each variant, one at a time, in a total of 132 ng of human genomic DNA. The mutant assay did not cross-react with any of the commonly reported mutations located in the same amplicon as KRAS G12C and BRAF V600E. The mutant assay was linear from 800 copies to 4 copies per reaction with a R 2 in the range of 0.94-0.99. The total quantification assay was also linear in the range of 1.3 ng to 53 ng of total DNA input with a R 2 >0.99 for all three targets. With previously extracted and archived cell-free DNA from plasma and FFPE DNA that were tested with alternative PCR based methods, the ddPLEX kit had a concordance of 100% and 96.5%, respectively. In conclusion, the ddPLEX EGFR/KRAS/BRAF Mutation Detection Assay Kit is sensitive, accurate, and efficient in time and cost for detecting clinically relevant NSCLC variants. Multiplexing maximizes information and reduces sampling bias from specimens with low nucleic acid quantities such as FFPE and plasma.