In collaboration with Todd Golub at the Broad Institute, we have performed a luminex assay to quantitate tyrosine phosphorylation in tyrosine kinases. Active tyrosine kinases are also generally associated with increased overall tyrosine phosphorylation. Our assay captured more than 150 tyrosine kinases in a multiplex assay and detected the level of tyrosine phosphorylation in all these kinases using a pan-phosphotyrosine antibody (4G10). We used 150 human lung adenocarcinoma tumor tissue and corresponding adjacent normal lung tissue to profile tyrosine phosphorylation of these tyrosine kinases. We identified around 30 specific tyrosine kinases differentially phosphorylated in tumor tissue compared to adjacent normal lung. Examples of such potentially active tyrosine kinases are NTRK1, EPHA2, DDR. We then completed a nanostring assay in collaboration with Dr. Marc Ladanyi at MSKCC to measure transcript expression of majority of tyrosine kinases in a multiplex assay. Interestingly, as we had predicted, expression of several tyrosine kinases do not change at least at the level of trasnscription, however there is significant change in tyrosine phosphorylation, which is generally a surrogate of activation. In collaboration with Dr. Paul Meltzer in the NCI intramural program, we have sequenced around 1300 cancer-related genes, including the tyrosine kinases used in our luminex assay to identify specific mutations in a targeted manner. We have analyzed the sequencing data to identify somatic changes in the genes selected. The somatic mutation data has been correlated with the luminex data to identify possible somatic changes in kinases resulting in increased activity. We have identified, as expected EGFR mutations in patients with hyperphosphorylation of EGFR by luminex assay. However for most other kinases, no specific mutation has been identified that correlates with hyperphosphorylaiton. This underscores the importance of examining signaling pathways using tyrosine phosphorylation as a surrogate of kinase activity. We are currently identifying human lung adenocarcinoma cell lines in which tyrosine phosphorylation of kinases is high, but there is no identified mutation in those kinases. These cell lines will be treated with the specific kinase inhibitors to determine if the cell line is defendant on the specific kinase signaling pathway for survival and proliferation.