Investigation On Target Therapy And Resistance Mechanism In Non-small Cell Lung Cancer

The clinical application of epidermal growth factor receptor tyrosine kinase inhibition (EGFR-TKI) and anaplastic lymphoma kinase (ALK) inhibitor had prolonged the survival of patients with non-small cell lung cancer (NSCLC). However, the cost of target therapy is expensitive and drug resistance could not be avoided. This study aimed to detect ALK rearrangement and analyze its clinical significance in advanced NSCLC, to provide data reference for clinical practice of target therapy in NSCLC. In addition, this study would establish EGFR-TKI resistant cell line and analyze the bilogical character, and research on the antitumor efficiency of histone deacetylase inhibitor (HDACi) chidamide or in combination with EGFR-TKI icotinib in NSCLC cell lines to provide clues in exploring new therapeutic strategies in the treatment of NSCLC. The contents of this study are divided into two sections as followed:Section 1. The detection and clinical significance analysis of ALK rearrangement in advanced non-small cell lung cancer.Chapter 1:The detection of ALK rearrangement in advanced non-small-cell lung cancer. We used immunohistochemistry (IHC), qRT-PCR (quantitative real-time polymerase chain reaction) and fluorescence in situ hybridization (FISH) to detect ALK status in 139 samples of advanced NSCLC with non-squamous histology, and compared the sensitivity and specificity of IHC or qRT-PCR with FISH. The concordance rate between FISH and IHC was 96.9%, between FISH and qRT-PCR was 95.7%. The sensitivity and specificity of IHC were 97.7% and 96.6%, respectively, while the sensitivity and specificity of qRT-PCR were 89.2% and 98.7%, respectively. IHC could be a useful method in screening ALK rearrangements in clinical practice with qRT-PCR as an alternative diagnostic tool to clarify specific ALK variants.Chapter 2:Clinical significance analysis of ALK arrangement in advanced non-small cell lung cancer. ALK status was assessed by FISH, IHC and qRT-PCR in 173 selected advanced NSCLC patients. Clinicopathologic data, genotype status and survival outcomes were analyzed. Moreover, the association of ALK expression with clinical outcomes was evaluated in ALK FISH-positive crizotinib-treated patients. The positivity detection rate of ALK rearrangement by FISH, IHC and qRT-PCR was 35.5%(59/166), 35.7%(61/171), and 27.9%(34/122), respectively. ALK rearrangement was observed predominantly in young patients, never or light smokers, and adenocarcinomas, especially with signet ring cell features and poor differentiation. Median progression free survival (PFS) of crizotinib-treated patients was 7.6 months. The overall survival (OS) of these patients was longer compared with that of crizotinib-naive or wild-type cohorts, but there was no significant difference in OS compared with patients with EGFR mutation. In addition, moderate and strong ALK expression had a decreased risk of death (P=0.026). Selective enrichment according to clinicopathologic features in NSCLC patients could highly improve the positivity detection rate of ALK rearrangement for ALK-targeted therapy. IHC could provide more clues for clinical trial design and therapeutic strategies for ALK-positive NSCLC patients.Section 2. Investigation on resistance of target therapy in non-small cell lung cancerChapter1:Generation and biological character analysis of EGFR-TKI resistant cell line and research on exploring new therapeutic strategies against resistance. Cell line were exposed to gradually increased concentrations of icotinib to establish EGFR-TKI resistant cell, then analyzed the biological character of resistant cells, revealed the mechanisms of resistance, and explored new therapeutic strategies against resistance. Icotinib resistant NSCLC cell line harbored some different biological characters compared with parent cells, which showed high cross-resistance to other EGFR-TKIs. MET gene amplification was the important reason of resistance. Both parent and resistant cells were sensitive to chidamide, but only resistant cells were sensitive to crizotinib. Crizotinib or combined with icotinib could inhibit the viability of resistant cells, reduce the activation levels of MET protein and the downstream moleculars in MET signal pathway, induce cells to arrest at G2/M phase of the cell cycle, cause apoptosis, and inhibit HCC827IR xenograft growth in athymic nude mice. The results could provide experimental reference for clinical application of EGFR-TKI and for the choice of treatment after drug resistance, and also provide cell line model for the research on resistant mechanism of EGFR-TKI and for the exploration of new therapy to reverse resistance.Chapter 2:Investigation on antitumor activity of histone deacetylase inhibitor chidamide or in combination with epidermal growth factor receptor tyrosine kinase inhibitor icotinib in NSCLC. This study assessed the antitumor efficiency and mechanisms of chidamide or in combination with icotinib in NSCLC with various mutation status. Chidamide significantly reduced the viability of A549, HCC827, HCC827IR cell line, increased the sensitivity of icotinib synergistically in H1975 cell line. Chidamide or in combination with icotinib could inhibit the activation of RAS/MAPK, PI3K/AKT and/or JAK/STAT pathways, induce cell cycle arrest, activate caspase 3 and PARP, and cause apoptosis. Chidamide or in combination with icotinib could inhibit β-catenin expression in HCC827, HCC827IR and H1975 cell line. In addition, chidamide increased the sensitivity of H1975 cell line to icotinib by restoring E-cadherin expression. Futhermore, chidamide and in combination with icotinib could inhibit HCC827IR and H1975 xenograft growth in athymic nude mice, respectively, and there was no appreciable weight loss or other side effects. The results of this study could provide experimental data for clinical application of chidamide in NSCLC.In summary, this study demonstrated the application of different methods in the detection of ALK status and the clinical significance of ALK rearrangement in Chinese patients with NSCLC. Meanwhile, this study established NSCLC cell line model with EGFR-TKI resistance, analyzed the biological characters and resistant mechanisms of the resistant cells, and explored new therapeutic strategies against resistance. In addition, this study also demonstrated the antitumor efficiency and mechanisms of chidamide or in combination with icotinib in NSCLC in vitro and vivo. The findings of this study could provide clues for the clinical practice of target therapy and the exploration of new strategies for the treatment of NSCLC.