Abstract 2087: Detection of KRAS mutations in circulating tumor cells (CTCs) and in formalin-fixed, paraffin-embedded (FFPE) tissue using castPCR method
Authors: Thobe Megan N. , Um Suzane L. , Peek Victoria L. , Ballard Darryl W. , Konicek Bruce W. , Credille Kelly M. , Ebert Philip J. , Donoho Gregory P. , Yan S. Betty 
Published: August 2012

Abstract
CTCs are tumor cells in circulation in the blood and are believed to be shed from both the primary and metastatic tumors. CTC counts are prognostic of survival in metastatic breast, colorectal, and prostate cancers. It is postulated that these cells are genetically similar to cells comprising the patient's tumor(s). The presence of these cells in blood provides a minimally invasive source of nucleic acid for serial monitoring of tumors for disease progression and emergence of drug treatment resistance without the risks of traditional solid tumor biopsy. Activating mutations in the KRAS gene are frequently found in human cancers (up to 30% of cancer cases), and are present in a large portion of non-small cell lung cancer (NSCLC), pancreatic, and colorectal cancers. Reported mutations are mostly single point mutations in codons 12, 13 and 61, with the majority of mutations present in codon 12. Importantly, KRAS mutation status can be predictive of response to treatment, and may be used to determine the best therapeutic strategy. Therefore, it is critical to develop a robust method for detecting mutations from a limited number of enriched tumor cells (e.g. CTCs or fine needle aspirate) in a high background of normal cells. Our studies utilize competitive allele-specific PCR (castPCR) for detecting mutant KRAS from human cancer cells spiked into and retrieved from blood, and from fixed cells embedded in paraffin. This technology is reportedly capable of detecting one mutant allele molecule in 10 million wild-type molecules. KRAS is mutated in human NSCLC H441 cells (G12V) and in KP-4 pancreatic cells (G12D). After spiking H441 and KP-4 cells into human blood, cells were captured/enriched and enumerated by the Veridex CellTracks™ system. Both CXC and Profile kits (Veridex) were used. Recovered cells were then lysed overnight with Arcturus® PicoPure® DNA extraction lysis buffer, and the resulting DNA was analyzed for mutant KRAS with the castPCR TaqMan® Mutation Detection Assay (Applied Biosystems) specific for the mutation found in H441 cells (G12V). Under these conditions, we detected KRAS mutation in H441 cells with robust sensitivity (20 spiked cells into 7.5ml of blood; KP-4 cells were negative for the G12V mutation). To further examine if this method could be used for detecting mutations in human tissue, FFPE tumor cells were analyzed. The WaxFreeTM DNA kit (TrimGen) was used to extract DNA from FFPE cells, and castPCR was performed. H441 mutant KRAS was detected from as little as ∼200 pg DNA (100-fold dilution of DNA). Ongoing studies involve correlating mutation status between CTCs and tumor FFPE tissue. These data will aid in clinical biomarker development used in clinical trials and improve methods to determine the most beneficial treatment options for patients. Importantly, this approach may enable patients to be serially monitored for tumor mutation status with blood draws.
Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2087. doi:1538-7445.AM2012-2087