Mutational analysis of circulating tumor cells using the IsoFlux™ System and QX100™ Droplet Digital™ PCR Platform (MMTC 2012)

Abstract

Here we developed a start to finish assay for quantitative mutational analysis of CTCs. We ran analytical validation of both the CTC isolation process and the digital mutation detection using a model CTC system (MDA-MB-231, heterozygous for KRAS G13D mutation, spiked into healthy donor blood). The IsoFlux System was shown to be effective in recovering CTCs from the model system. The digital mutation detection assay was characterized for sensitivity and linearity using titration studies with purified G13D mutant gDNA in a wild-type gDNA background.

The assay was shown to be linear across the range tested and sensitive down to 0.06% mutant with wild-type background. Model CTC samples were tested in the range of 10-100 CTCs per 7mL blood. Mutations were detected with high probability across this entire range.

The results of these experiments indicate that the IsoFlux System can be combined with the QX100 ddPCR System to provide quantitative mutational analysis of CTCs captured from a simple blood draw. This assay workflow is also amenable to other mutations and biomarkers relevant to oncology.

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