The Study On The Effect Of ALK Inhibitor Crizotinib On Human Lymphocytes

Non Small Cell Lung Cancer (NSCLC) is one of the leading causes of tumor-related deaths in the world. New generation of Tyrosine Kinase Inhibitor (TKI) including Erlotinib and Gefitinib, which block driver oncogenic mutations have been showing unprecedented high response rates in clinical trials.Despite the initial high response rate to crizotinib therapy, patients develop resistance. Novel immunotherapy that build on the durable responses achieved with a variety of immune stimulating agents are also providing significant advances in the clinic. It is reasonable to postulate that agents that specifically impact on the target tumor cells and prepare them for the immune effector cell attack induced by immunotherapy strategies may increase the effectiveness of tumor immunotherapy for NSCLC. In2007, the EML4-ALK fusion gene which was combination of ALK gene and EML4(Echinoderm Microtubule-associated protein-Like4) was discovered in a subset of NSCLC. In a Phase I study, crizotinib, a dual ALK and MET inhibitor, showed an objective response rate (ORR) of60%and a median progression-free Survival (PFS) at6months of72%in patients carrying the ALK translocation. A Phase III trial comparing crizotinib with second line standard chemotherapy (docetaxel or pemetrexed) showed an improvement in terms of PFS and ORR for crizotinib compared with second line standard chemotherapy. Many intracellular pathways blocked by TKIs are critical for signaling of immune response, so potential adverse effects of blocking these pathways should be tested.Thus, it is necessary to evaluate the effect of crizotinib on Human PBMCs before combining it with immunotherapy.First, we determined whether PBMCs of NSCLC and healthy donors could have been the target of crizotinib and expression of ALK or c-Met was detected by RT-PCR and Western Blotting.Then we tested its effects on lymphocyte viability, apoptosis, cell cycle, proliferation and ability to response to antigen encounter or cytokine stimulation via Flow Cytometry and ELISA.Our studies showed that PBMCs expressed the ALK and p-ALK was also detected on CD3/CD28stimulated PBMCs. At average plasma concentrations of crozitinib, it did not negatively impact the viability or function of PBMCs, such as apoptosis, cell cycle, proliferation and immune response.The maximum plasma concentration of crizotinib had no effect on both resting and proliferating PBMCs, while it induced apoptosis of proliferating PBMCs significantly. The maximum plasma concentration of crizotinib inhibited the cell cycle of PBMCs significantly while it didn’t inhibit the proliferataion of PBMCs.These studies showed that the PBMCs had slightly negative impact on PBMCs and supplied the evidence of combination of crizotinib and immunotherapy.