AbstractSomatic mutations in cancer driver genes can help diagnosis, prognosis and treatment decisions. Formalin-fixed paraffin-embedded (FFPE) specimen is the main source of DNA for somatic mutation detection. To overcome constraints of DNA isolated from FFPE, we compared pyrosequencing and ddPCR analysis for absolute quantification of BRAF V600E mutation in the DNA extracted from FFPE specimens and compared the results to the qualitative detection information obtained by Sanger Sequencing. Sanger sequencing was able to detect BRAF V600E mutation only when it was present in more than 15% total alleles. Although the sensitivity of ddPCR is higher than that observed for Sanger, it was less consistent than pyrosequencing, likely due to droplet classification bias of FFPE-derived DNA. To address the droplet allocation bias in ddPCR analysis, we have compared different algorithms for automated droplet classification and next correlated these findings with those obtained from pyrosequencing. By examining the addition of non-classifiable droplets (rain) in ddPCR, it was possible to obtain better qualitative classification of droplets and better quantitative classification compared to no rain droplets, when considering pyrosequencing results. Notable, only the Machine learning k-NN algorithm was able to automatically classify the samples, surpassing manual classification based on no-template controls, which shows promise in clinical practice.
Analysis of BRAF V600E mutation status – concordance of results from circulating tumor DNA and tissue-based testing and impact on prediction of the clinical course in patients undergoing BRAFi therapy
