Research On Target Therapy And Clinical Significance Of Molecular Markers In Non-small Cell Lung Cancer

This study mainly researched on pognositic and predictive biomarkers and target therapy in the non-small cell lung cancer (NSCLC) which is a common type of malignant tumor in China. The contents are divided into two parts as followed:Part1. Research on detection and clinical significance of molecular biomarkers in non-small cell lung cancerChapter1. Detection of EGFR gene mutation in Chinese NSCLC patients.In this study, we investigated the mutation of epidermal growth factor receptor (EGFR) in tumor tissue and pleural effusion from advanced NSCLC patients with amplification refractory mutation system(ARMS) and direct sequencing, analyzed the relationship between the EGFR mutation and clinicopathologic characteristics, and assessd the concordance rate of the2detection methods. Formalin-fixed and paraffin-embedded tumor tissues and paired pleural effusion from advanced NSCLC patients were collected. EGFR somatic mutations were detected in215of417advanced NSCLC patients, and the mutation rate was51.6%; the test results of14paired specimens were entirely same; and the concordance rate of2different detection methods was94.6%(Kappa=0.89,P<0.001). We found that EGFR mutation rate was higher in female (55.9%) than that in male(42.3%), EGFR somatic mutations appear to occur frequently in Chinese NSCLC patients, ARMS is a sensitive method to detect EGFR mutation and is suitable for sreening patients who would likely respond to EGFR inhibitors therapy.Chapter2. Screening predictive and prognostic molecular markers for NSCLC in peripheral blood specimens. The aim of this study was to explore whether biomarkers detected in plasma are predictive for response to EGFR-TKIs and survival time of NSCLC patients. Tumor tissues and paired blood were collected from134advanced NSCLC patients treated with EGFR-TKIs. EGFR mutations in both types of specimens and expression of transforming growth factor-alpha and beta one (TGF-α and TGF-β1) were assessed in NSCLC patients. Concentrations of circulating free DNA were detected in plasma from both NSCLC patients and healthy subjects. The clinical significance of these markers was assessed in advanced NSCLC patients. EGFR mutations were detected in68tumor samples and17plasma samples of134NSCLC patients, the concordance rate of2specimens was59.0%(79/134, Kappa=0.19, P<0.001). The concentrations of circulating free DNA were higher in NSCLC patients than in healthy subjects (5.82ng/μL vs.4.37ng/μL, P<0.05). Patients with high TGF-β1level showed shorter overall survival and worse response to EGFR-TKI than patients with low TGF-β1level.Part2. Research on target therapy in non-small cell lung cancerChapter3. Analysis of genes potentially associated with gefitinib-resistance in non-small cell lung cancer. EGFR-tyrosine kinase inhibitors (TKIs), such as gefitinib and erlotinib, have been shown to remarkably suppress tumor development and have improved the survival of NSCLC patients. Unfortunately, the majority of patients who initially respond to EGFR-TKI treatments eventually develop resistance. Although several potential mechanisms of acquired resistance to EGFR-TKIs have been reported, there is still no explanation for about30%of cases. In this study, we applied next-generation sequencing and microarray-based whole-genome single nucleotide polymorphism (SNP) genotyping to investigate the transcriptome profile and mutation patterns of gefitinib resistance in NSCLC H1650cells. By comparing the gene expression profiles of H1650gefitinib-resistant cell line H1650GR and gefitinib-sensitive cell lines H1650, we identified28and41differently expressed genes in the MAPK and PI3K/AKT signaling pathways, respectively; and amplification of mitogen-activated protein kinase4(MAPK4) and myosin light chain kinase (MYLK) play important roles in the development of EGFR-TKI resistance. Further mechanistic studies are warranted to explore the involvement of these genes in gefitinib resistance in NSCLC.Chapter4. Anti-tumor effect of novel CRM1inhibitors in NSCLC. Chromosome Region Maintenance1(CRM1) is a nuclear exporter and its inhibitor has anti-tumor activity in various cancers. This study assessed the anti-tumor efficiency of the novel CRM1inhibitors on NSCLC. NSCLC cell lines were treated with KPT-185to assess changes in cell viability, cell cycle, apoptosis, and protein expressions. NOD-SCID mice carrying NSCLC cell xenografts were orally treated with KPT-276, a clinical analog of KPT-185, to examine the efficacy and side effects of KPT-276in vivo. We found that KPT-185significantly reduced the viability of six NSCLC cell lines in a time-and dose-dependent manner, including EGFR-TKI-resistant H1975and H1650GR cell lines. In addition, KPT-185induced these NSCLC cells to arrest at G1phase of the cell cycle and caused apoptosis in a dose-dependent manner. KPT-185treatment also reduced CRM1protein levels in six NSCLC cell lines, and the reduction could be completely abolished by the proteasome inhibitor bortezomib. KPT-185activated caspase3,8, and9, but inhibited survivin expression in NSCLC cells. In a mouse H1975cell xenograft model, tumor growth was significantly inhibited by oral KPT-276administration, and there was no significant mouse body weight loss or other side effects. The current study demonstrated the anti-tumor effects of KPT-185in NSCLC cells, including EGFR-TKI-resistant NSCLC cell lines. Further studies will assess KPT-276anti-tumor activity in a clinical trial for NSCLC patients.