Abstract 5586: Interpatient differences in colon tumors reflected in kinase activity and inhibition profiles related to KRAS and BRAF mutation status.
Authors: van den Oord Rosanne , Pruijt Hans , van den Brule Adriaan , Nooijen Peet , de Wijn Rik , Mommersteeg Monique , Hilhorst Riet , Ruijtenbeek Rob 
Published: August 2014

Abstract
Background. Due to the biological heterogeneity, response to systemic therapy is variable in colon cancer. Chemotherapy improves median survival from 6-12 months to 18-24 months. Epidermal growth factor receptor (EGFR) antibodies have shown to improve survival, although modest, even further. KRAS mutations were identified as negative predictive markers for EGFR directed therapy. The response rate to EGFR targeted therapy remains low. Therefore there is an urgent need to find additional therapeutic options and accompanying biomarkers. The analysis of kinase activity in colon tumors may yield tumor-specific information on aberrant cell signaling pathways and may be usefull in a better patient stratification. Aim of this study is to generate overall kinase activity profiles from colon cancer tissues to explore if subtypes of this cancer show distinct kinase activity profiles. This can help to find new therapeutic options or predictive markers for response to therapy. Methods. Normal and tumorous colon tissue from patients who had given informed consent was fresh frozen after surgical resection. All tumor specimens were analyzed for BRAF and KRAS mutation status. Tissue cryosections were lysed in buffer with phosphatase and protease inhibitors. Tyrosine kinase (PTK) and serine/threonine kinase (STK) activity profiles of normal and tumor tissue were generated on PamChip® peptide microarrays comprising peptide sequences derived from known human phosphorylation sites. The ex vivo effect of kinase inhibitors on these kinase activity profiles was also determined. Peptide phosphorylation was monitored using fluorescently labeled antibodies. Data were analysed with Bionavigator software. Results. Tumor tissue has a higher PTK activity than normal tissue, whereas STK activity was equal to or lower than in normal tissue. STK activity profiles of 23 colon tumors could not be grouped according to mutation status, percentage of tumor tissue, differentiation grade or TNM classification. The STK activity profiles showed that within the group of wild type KRAS tumors, clearly different clusters were found. In the group with KRAS mutations (G12V, G12D, G12C, G12R) the STK activity profiles differed between the tumors. A subset of tumors, (two KRAS mutants, a BRAF mutant and a WT tumor) were tested ex vivo with a selection of 11 kinase inhibitors and showed different sensitivity to these inhibitors. Conclusions. Kinase activity and the response to inhibitors can be measured ex vivo in colon tumor tissue to capture the diversity in kinase activity and response to inhibitors between patients. The differences in kinase activity profiles of colon cancer samples could not be explained by subtypes according to mutation (KRAS, BRAF) status. Interesting subclusters could be identified using the kinase activity profiling providing a higher granularity than KRAS mutation scoring allows.
Citation Format: Rosanne van den Oord, Hans Pruijt, Adriaan van den Brule, Peet Nooijen, Rik de Wijn, Monique Mommersteeg, Riet Hilhorst, Rob Ruijtenbeek. Interpatient differences in colon tumors reflected in kinase activity and inhibition profiles related to KRAS and BRAF mutation status. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5586. doi:10.1158/1538-7445.AM2013-5586