Multiplexed quantitation of growth factor receptors and pathway activation in FFPE tumor tissue from the COIN trial.
Adams R., Hembrough TA., Thyparambil S., Krizman D., Darfler M., Jasani B., Maughan T., Kaplan RS., Burrows J.
e14117 Background: The epidermal growth factor receptor (EGFR) is the target of antibody therapeutics, with cetuximab approved for use in metastatic colorectal carcinoma (mCRC) among other indications. While KRAS mutation is a negative predictor of cetuximab response, patients with wtKRAS may show low response rates through a number of resistance mechanisms. Co-expression of receptor tyrosine kinases, such as IGF1R or cMet, can confer resistance by providing alternative ways to activate tumor growth pathways. Additionally, current diagnostic assays cannot quantitate receptor or pathway activation in tissue. Characterization of these various resistance mechanisms are critical to maximizing response rates to EGFR targeted therapies. METHODS: In order to address these issues, we used mass spectrometry to develop a multiplexed SRM (selective reaction monitoring) assay to measure levels of multiple protein targets on FFPE tumor tissue. These assays are based on the Liquid Tissue-SRM technology platform. This approach enables relative and absolute quantification of proteins and their phosphorylation status directly in formalin fixed paraffin embedded (FFPE) tissue. RESULTS: Using these multiplexed SRM assays we measured expression of EGFR, IGF1R and cMet, in 32 colorectal carcinoma samples from the COIN trial. EGFR expression in these tumors, ranged from less than 100amol to 1,200amol/ug of tumor protein. Of particular interest was the expression of potential resistance markers. Of 32 tumors characterized, 4 tumors showed no expression of EGFR, 19 expressed either cMet or IGF1R, and 4 tumors expressed both receptors. We are continuing analysis on a second cohort of 40 tumors, and will reconcile these results with clinical outcome data. Since active EGFR is necessary for the response to these therapies, we have initiated a phospho-SRM mass spectrometry strategy to quantitate activation of EGFR, other candidate resistance receptors and signaling pathway components in these same COIN trial tissues. This data will be presented. CONCLUSIONS: The ability to absolutely quantify and compare critical biomarkers in the same tumor cells represents a profound step forward for the diagnostic space.