Mutational analysis of circulating tumor cells using the IsoFlux™ System and a high sensitivity TaqMan® assay (castPCR™)

Abstract

The IsoFlux System (Fluxion Biosciences) provides high recovery of CTCs in a format optimized for downstream analysis. Competitive Allele-Specic TaqMan® PCR (castPCR™) (Life Technologies) is a sensitive mutation detection assay designed to detecting rare mutations in a background of wild-type gDNA. When combined, these two technologies provide a sensitive detection platform for CTC mutations from a simple blood draw.
 

Here we present analytical and clinical validation of a KRAS mutation detection assay on CTC samples. The castPCR assay was rst characterized with titered amounts of gDNA to assess sensitivity and limits of detection. Model CTC samples were prepared using spiked cell lines to further qualify the assay. For clinical validation, whole blood samples from colorectal cancer patients (N=22) were collected and analyzed using the KRAS mutation assay as well as immunouorescence to conrm the presence of CTCs. Over 80% of the patient samples had CTC levels that exceeded the assay’s limit of detection, and 36% of the patient samples had a KRAS mutation (inline with reported prevalence from tissue analysis). The results of this study indicate that the IsoFlux System can be combined with castPCR to provide a sensitive ‘sample to answer’ detection platform for DNA mutations in CTCs drawn from a routine blood sample. The assay workow is also amenable to additional mutations relevant to clinical oncology.

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