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, 11 (5), 1220-1224

Measuring KRAS Mutations in Circulating Tumor DNA by Droplet Digital PCR and Next-Generation Sequencing

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Measuring KRAS Mutations in Circulating Tumor DNA by Droplet Digital PCR and Next-Generation Sequencing

Christina Demuth et al. Transl Oncol.

Abstract

Measuring total cell-free DNA (cfDNA) or cancer-specific mutations herein has presented as new tools in aiding the treatment of cancer patients. Studies show that total cfDNA bears prognostic value in metastatic colorectal cancer (mCRC) and that measuring cancer-specific mutations could supplement biopsies. However, limited information is available on the performance of different methods. Blood samples from 28 patients with mCRC and known KRAS mutation status were included. cfDNA was extracted and quantified with droplet digital polymerase chain reaction (ddPCR) measuring Beta-2 Microglobulin. KRAS mutation detection was performed using ddPCR (Bio-Rad) and next-generation sequencing (NGS, Ion Torrent PGM). Comparing KRAS mutation status in plasma and tissue revealed concordance rates of 79% and 89% for NGS and ddPCR. Strong correlation between the methods was observed. Most KRAS mutations were also detectable in 10-fold diluted samples using the ddPCR. We find that for detection of KRAS mutations in ctDNA ddPCR was superior to NGS both in analysis success rate and concordance to tissue. We further present results indicating that lower amount of plasma may be used for detection of KRAS mutations in mCRC.

Figures

Figure 1
Figure 1
Comparison of the NGS and ddPCR ctDNA analyses. (A) Linear regression from the comparison of NGS and ddPCR AFs (R2 = 0.91). Results related to the regression are presented below the graph. (B) Bland-Altman plot of the differences (NGS (AF) − ddPCR (AF)).
Figure 2
Figure 2
Comparison of log-transformed B2M measurements from cohort 2. Constant and slope for the regression and mean difference and limits of agreements are found in the figure. (A) Linear regression on measurements from cohort 2. The regression resulted in an R2 value of 0.994. (B) Bland-Altman plot of the differences (log(B2M 2 ml) − log(B2M 200 μl)).

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