| Summary: | 博士 === 國立陽明大學 === 臨床醫學研究所 === 95 === Abstract
Lung cancer has become the leading cause of cancer death in many industrialized countries; in Taiwan, lung cancer clams more than 7,000 lives annually. In recent years much attention has been focused on the rapidly increasing incidence of primary lung cancer in non-smoking patients. Some gender differences exist in the distribution of lung cancer, including histological type and exposure to tobacco. Although 80% of female lung cancer patients worldwide have smoked, less than 10% of Taiwanese women with lung cancer have ever smoked. The low smoking status and high incidence rate of adenocarcinoma constitute distinctive characteristics of Taiwanese female lung cancers. Some authors suggested that environmental and/or genetic factors might be involved in the development of lung cancer in women. Molecular biological studies have demonstrated that overt cancers carry multiple genetic and epigenetic alterations, indicating inactivation of tumor suppressor genes and activation of dominant oncogenes during the process of carcinogenesis and subsequent progression of cancers. Alteration analysis in the genes controlling acquired somatic mutations, such as genes involved in the DNA repair system, may explain some of the observed significance in susceptibility to various conditions caused by environmental factors in non-smoking female lung cancer.
hMLH1 and hMSH2 are two of the genes known to be implicated in the DNA mismatch repair (MMR) system. Inactivation of these two genes by promoter hypermethylation has been reported to be associated with some human cancers. Some studies showed that DNA methylation associated with transcriptional silencing of hMLH1 is the underlying cause of MMR defects in most sporadic colorectal cancers. In the previous report from our laboratory, we examined the protein expression and the status of promoter hypermethylation of hMLH1 and hMSH2 genes in 77 non-small cell lung cancer (NSCLC) tumors and found that protein expression of hMLH1 and hMSH2 in the tumor specimens from female patients with lung cancer was higher than in specimens from male patients, although only a few female tumor samples were examined. Following this finding, we aimed to collect more tumor specimens, focusing on non-smoking female NSCLC patients, to investigate the protein expression of hMLH1 and hMSH2 and promoter hypermethylation of these two genes in these tumor tissues. We also analyzed the clinical association and prognostic significance of both genes in the non-smoking female patients with lung cancer. In this study, we found that protein expression alteration and promoter hypermethylation were observed in 66-67% and 30-34% of tumor specimens for hMLH1 and hMSH2 genes, respectively. Loss of hMLH1and hMSH2 protein expression was significantly associated with their promoter hypermethylation (P<0.0001 and P=0.049). The overall survival and cancer-specific survival rates were significantly lower in patients with promoter hypermethylation of hMSH2 gene than in those without hypermethylation (P=0.038 and P=0.004). The poor prognosis was still especially significant in adenocarcinoma (P=0.035 and P=0.061) and early-staged NSCLC patients (P=0.067 and P=0.041). The data suggest that hMLH1 is the major altered mismatch repair gene involved in non-smoking NSCLC tumorigenesis, and that promoter methylation is the predominant mechanism in hMLH1 and hMSH2 deregulation. In addition, promoter methylation of the hMSH2 gene may be a potential prognostic factor in non-smoking female lung cancer.
In another study, we focused on genetic polymorphisms of these two mismatch repair genes. Although cigarette smoking is the major etiological factor of lung cancer, only a small fraction of smokers develop the disease, suggesting that genetic factors may be of importance in determining individual susceptibility to tobacco carcinogens. This genetic susceptibility may result from inherited polymorphisms in genes involved in carcinogen metabolism and repair of DNA damage. DNA repair systems are fundamental to the maintenance of genomic integrity in the face of replication errors, environmental carcinogens and the cumulative effects of age, and their inactivation can dramatically increase the susceptibility to cancer. Some studies showed that genetic polymorphisms of hMLH1 and hMSH2 genes are associated with increased risk of colorectal cancer and lymphoma. The clinical significance of genetic polymorphism in lung cancer patients has never been reported in the literature. To elucidate the relation between genetic polymorphism of hMLH1 and hMSH2 genes and lung cancer, we investigated the frequencies of genetic polymorphisms of hMLH1 and hMSH2 genes in the lung cancer patients and normal population in Taiwan. The correlation between the results and the clinical association and prognostic significance of the patients with lung cancer was also analyzed. In the study of hMSH2 Gene (gISV12–6T>C), we found that the variant (C) allele was represented at a significantly higher frequency in the general Taiwanese population than in non-Asian populations (P<0.0001, χ2 test). No significant difference in hMSH2 genotype distribution was found between NSCLC patients and cancer-free controls (P=0.255, multivariate logistic regression). However, the homozygous wild-type T/T genotype was significantly associated with a poor prognosis (P=0.007, log-rank test). Our study showed that the frequency of the variant C allele was significantly higher in the general Taiwanese population than in non-Asian populations and the T/T genotype of hMSH2 gIVS12–6T>C polymorphism was a poor prognostic factor in NSCLC patients.
In the future, we will investigate the significance of hMLH1 and hMSH2 protein expression in the female patients with lung cancer who had received chemoirradiation for adjuvant therapy after surgical resection and we will continue to study the distribution of hMLH1 (G-93A) and hMSH2 (C-181T) polymorphism in normal population and lung cancer patients in Taiwan and their significance in patients with lung cancer. In addition, to evaluate if plasma methylation markers can be used for risk assessment or early detection of lung cancer, we have checked the promoter methylation status of BLU, CDH13, FHIT, p16, RARβ, and RASSF1A genes in corresponding samples of plasma, tumor, and normal lung tissue from lung cancer patients and in plasma samples from cancer-free individuals matched for age, sex, and smoking habit. In the preliminary result, we found that the concordance of methylation in tumor and plasma from the same patient was 86% for BLU, 87% for CDH13, 80% for FHIT, 75% for p16, 76% for RARβ, and 84% for RASSF1A. Multiple logistic regression analysis showed that odds ratio for having lung cancer was 10.204 (P=0.013) and 9.952 (P=0.019) for individuals with p16 methylation and RASSF1A methylation, respectively. After several training tests, we established methylation of more than two of the six markers as the criterion of elevated risk. Our comparison yielded a sensitivity of 73%, specificity of 82%, and concordance of 75% between the methylation pattern in tumor and the corresponding plasma. The detection rate was relatively high in advanced smokers with squamous cell lung cancer. Five cancer-free individuals met this criterion and were therefore considered to have an elevated risk. The data suggest that using multiple epigenetic markers in plasma may be useful for detecting lung cancer. In combination with conventional diagnostic tools, this methylation marker panel should improve detection or risk assessment of lung cancer.
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