Developing New Diagnostic and Therapeutic Strategies for Lung Cancer with ROS1, RET and NTRK1 Mutations in Patients without EGFR, KRAS and ALK Mutations

EGFR mutations, KRAS mutations and ALK fusions, the three most common genetic abnormalities in non-small cell lung cancer (NSCLC) in Taiwan are critical in the treatment selection. NSCLC without the three mutations are called triple-negative lung cancer in this proposal. In the triple-negative lung cancer, about 10% of them contain either ROS1, RET or recently identified NTRK1 fusion proteins. These fusion proteins have been shown to be driver mutations for tumorigenesis. NSCLC with these mutations are sensitive to specific targeted agents. Because of the rarity and complexity of these mutations, it has been difficult to detect these mutations. In addition, our understanding in the cancer with these mutations has been limited by the availability of model system. To further elucidate the mechanisms of how these mutations cause carcinogenesis and develop resistance to targeted agents, we plan to develop a multiplex PCR method to isolate the mutations from patient-derived cancer tissues (specific aim 1), clone out the full-length fusion proteins to establish in vitro and in vivo model systems (specific aim 2) and elucidate the resistance mechanism to specific targeted agents (specific aim 3). To achieve specific aim 1, we have conducted preliminary studies to demonstrate the feasibility of multiplex PCR, and attempted to clone out the full-length cDNA. The multiplex PCR method intends to screen the ROS1, RET and NTRK1 mutations in triple-negative NSCLC. We will further explore multiplex PCR-DHPLC method to expedite the screening process for future clinical use. To achieve specific aim 2, we will clone out the full-length fusion proteins and establish 32D surrogate system for future drug screening platform. In addition, we hope to establish passable cell lines with these mutations from patient-derived cancer tissues and implanted into NOD/SCID mice for in vivo studies. The in vivo system will be used for subsequent cancer biology studies and as drug development platform. To achieve specific aim 3, we will use the model systems established in specific aim 2 to elucidate resistance mechanisms to clinically available targeted agents by focusing on the emergence of new mutations in the fusion proteins. Furthermore, we hope to identify the most active agents for cancer with different variants. This proposal will allow us to build a repertoire of rare mutations in NSCLC, which can be used as a basis for further development of rapid diagnositic kits. It can also build up in vitro and in vivo model systems for subsequent drug discovery program. Furthermore, the elucidation of resistance mechanisms can further improve our understanding in cancer biology and serve for development of 2nd generation pharmaceuticals by local industry.