Prospective study of genetic mutations in matched tumor and plasma specimens in colorectal cancer patients.

Abstract

356 Background: Circulating free DNA (cfDNA) represents a minimally-invasive resource for detecting mutations in advanced cancer patients. The primary objective of this study was to determine if cfDNA is representative of tumor tissue for multiplex mutation detection utilizing Sequenom MassARRAY. Methods: Samples were spiked with dilutions (10ng/μl to 0.01ng/μl) of HCT116 DNA containing KRAS G13D mutations to determine assay sensitivity and specificity. Metastatic colorectal cancer patients referred to the Drug Development Unit at the Royal Marsden Hospital between 9/09-8/10 gave their consent to provide DNA from matched archival formalin fixed paraffin-embedded (FFPE) tumor and plasma. Samples had ∼200 described gene mutations genotyped using Sequenom MassARRAY (OncoCarta Panel). Results: Serial dilution spiking experiments revealed that the KRAS G13D mutation was reproducibly detectable to 40ng/mL of HCT116 DNA; 26 patient samples were then analyzed. KRAS, BRAF and PIK3CA mutations were detected in 8 (31%), 3 (12%) and 3 (12%) tumor specimens respectively; 100% concordance for KRAS status was observed between multiple FFPE biopsies from the same patient and analysis by Amplification Refractory Mutation System (ARMS)-Scorpion PCR. The median quantity of cfDNA was 353ng/ml (range 106-4603). Concordance between matched FFPE and cfDNA was 88% for KRAS and 100% for BRAF mutations. No patients with wildtype KRAS or BRAF tumor genotypes had mutations in their respective cfDNA confirming the high specificity of cfDNA analysis. Three patients had detectable PIK3CA mutations; 1 patient had a E346K mutation detected in both FFPE tissue and plasma; 1 patient had E545K detected only in FFPE and the other had E542K detected in a liver metastasis but not in the colorectal primary or plasma. The recently reported oncogenic AKT1 E17K mutation was detected in 1 patient in tissue and plasma. No mutations in any of the other tested oncogenes studied were detected. Conclusions: A high concordance in detected mutations was observed between FFPE tumor and matched cfDNA. cfDNA is representative of tumor DNA and may be used for the prospective selection of cancer patients for treatment with targeted therapeutics. No significant financial relationships to disclose.