Identification of potentially active tyrosine kinases in lung cancer

与Broad研究所的Todd Golub合作，我们进行了luminex测定，以定量酪氨酸激酶中的酪氨酸磷酸化。活性酪氨酸激酶通常也与酪氨酸整体磷酸化增加有关。我们的实验在多重分析中捕获了150多种酪氨酸激酶，并使用泛磷酸酪氨酸抗体（4G10）检测所有这些激酶中酪氨酸磷酸化的水平。我们使用150人肺腺癌肿瘤组织和相应的邻近正常肺组织来分析这些酪氨酸激酶的酪氨酸磷酸化。我们发现肿瘤组织中约有30种酪氨酸激酶与邻近正常肺组织的磷酸化差异。这些具有潜在活性的酪氨酸激酶有NTRK1、EPHA2、DDR。然后，我们与MSKCC的Marc Ladanyi博士合作完成了一项纳米链检测，以在多重检测中测量大多数酪氨酸激酶的转录物表达。有趣的是，正如我们所预测的那样，几种酪氨酸激酶的表达至少在转录水平上没有变化，然而酪氨酸磷酸化有显著变化，酪氨酸磷酸化通常是激活的替代品。与NCI内部项目的Paul Meltzer博士合作，我们已经对大约1300个癌症相关基因进行了测序，包括在luminex检测中使用的酪氨酸激酶，以靶向方式识别特定突变。我们已经开始分析测序数据，以确定所选基因的体细胞变化。随后，我们将把突变状态与酪氨酸磷酸化的luminex检测结果联系起来。

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 started analyzing the seqeuncing data to identify somatic changes in the genes selected. Subsequently, we will correlate the mutation status with the luminex assay results for tyrosine phosphorylation.