Abstract 4513: KRAS mutation analysis using blood derived cell free tumor DNA for treatment monitoring of metastatic colorectal cancer patients
Authors: Maritschnegg Elisabeth , Dandachi Nadia , Stadelmeyer Elke , Speicher Michael R. , Heitzer Ellen , Samonigg Hellmut , Höfler Gerald , Pichler Martin , Otte Marcus , Bauernhofer Thomas 
Published: August 2012

Abstract
Currently used non-invasive diagnostic methods for monitoring patients undergoing anti-cancer treatment are limited. The applicability of diagnostic imaging is limited and lack of sensitivity and specificity of routinely used tumor markers hampers clinical application. Circulating cell free DNA (ccfDNA) has been shown to contain minute fractions of tumor-derived DNA. We tested in a proof of principal study the applicability of circulating mutant DNA as a tool to (1) evaluate the clinical sensitivity of cancer detection by analyzing KRAS2 mutations and (2) monitor therapy response in metastatic colorectal cancer (mCRC) patients. In our retrospective study we analyzed 50 serum samples of 18 patients with mCRC and confirmed KRAS2 mutation in the resected primary tumor (G12D, G12V, or G13D). Samples were collected at therapy start (t0), after four cycles of therapy (t1), which was on average after 8 weeks and if available, additional samples at later time points (t2, t3) to assess the longitudinal outcome (median follow up 23 weeks, range 8-248weeks). Therapy response and course of disease were assessed using RECIST criteria at all analyzed time points. The three most commonly occurring KRAS2 (G12D, G12V, or G13D) mutations were examined by Allele-specific PCR. The assay was established using defined tumor cell line DNA and its sensitivity proofed to be up to 0.1%. In 15/18 patients (83.3%), we could detect a KRAS2 mutation at start of therapy (t0) and could confirm diagnosis of mCRC by analysis of ccfDNA. In contrast only 8/18 (44.4%) patients and 7/18 (38.9%) patients showed increased levels of the tumor markers CEA and CA19-9, respectively. Therefore, the analysis of the KRAS2 status identified 5 additional patients as positive for colorectal cancer. To assess therapy response we quantified mutant KRAS2 levels in ccfDNA of 15 patients with detectable KRAS2 mutations. The changes of KRAS2 level were compared to the information from imaging, evaluated by RECIST criteria. A concordance in the assessment of therapy response after 4 cycles (t1) could be observed in 10/15 (66.7%) analyzed cases. In 12/15 patients the longitudinal outcome (t2, t3) was assessed and compared to t1. A concordance in the assessment of therapy response could be observed in 9/12 (75%) patients. Validation of data from patient sample analysis with Deep Sequencing is in progress.Our findings confirm the potential of ccfDNA as a biomarker for improved serum based relapse diagnosis and enables closed meshed monitoring of therapy response. A multiplex mutational analysis will be needed to translate this principle and include also other patient subpopulations (ie. KRAS2 negative). Therefore, we adapted the assay using whole genome amplification to amplify the limited amounts of ccfDNA that now allows the parallel testing of multiple mutations (work in progress).
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 4513. doi:1538-7445.AM2012-4513