細胞介素-10 (IL-10)啟動子基因多型性對麟狀上皮細胞肺癌及肺腺癌預後的不同影響之研究

Abstract

Objective：The term lung cancer or known as bronchogenic carcinoma,whichrefers to malignancies that originate in the airways or pulmonary parenchyma. Lung cancers are classified as small cell lung cancer (SCLC) (13%) or non-small cell lung cancer (NSCLC) (82%). These cell types of lung cancer comprise up to 95% of all lung cancer. Only 5% of lung cancers are comprised of other cell types. Non-small cell lung cancer can be further divided into three major cell types, including squamous cell lung cancer (SCC) (20%), adenocarcinoma (ADC) (38%) and large cell carcinoma (5%). Lung cancer is the most common cancer worldwide. In the year 2009, it was estimated to have 1,600,000 new cases and 1,380,000 deaths directly related with lung cancer occurred in the US. The absolute and relative frequency of lung cancer has increased dramatically. Lung cancer has became the most common cause of cancer deaths in men since the year of 1953 and has also became the most common cause of cancer deaths in women after the year 1985. Despite the cause of lung cancer deaths in male starts to decrease, but the trend of lung cancer deaths continues to increase in female. It was estimated that approximately half of cancer deaths occur in women. In Taiwan, lung cancer has also become the leading cause of deaths since the year 1980. In the past 30 years, lung cancer remains to be the leading cause of deaths. In both men and women, the incidence of lung cancer has increased 8 times and is also the most rapidly increased cancer among all cancers. The incidence of small cell lung cancer in Taiwan is approximately 88-90% and the incidence of small cell lung cancer is about 10-12%. The primary risk factor for the development is cigarette smoking, especially in squamous cell lung cancer. The overall survival and prognosis is poor. In all lung cancer patients, the overall 5 year survival rate is about 15%. Usually, patients are in late clinical staging at the time of cancer diagnosis. In non-small cell lung cancer, nearly 30-40% of cases were in stage four. Likewise, about 60% of small cell lung cancer patients were in disseminated stage. Many factors are related with prognosis in both small cell and non-small cell lung cancer, among these, cancer stage is the most important prognostic factor. Other related prognostic factors include, clinical parameters, histopathology, molecular characterization, positron- computerizedtomography (PET-CT) findings, and whether there is any recurrence after tumor resection. Reverse Transcriptase Polymerase Chain Reaction (RT-PCR) is also being utilized to be a diagnostic assay to detect whether there is any lymphatic metastasis in non-small cell lung cancer. The immune system is a complex and sophisticatenetwork, that is designed to protect the host from both external (such as bacteria and virus) and internal threats (such as malignant transformation). Cytokines are hormone-like proteins that enable immune cells to communicate and play an integral role in the initiating, perpetuation and subsequent downregulation of the immune response. Cytokines are as important to the termination of the immune response as they are to its initiation. Interleukine-10 (IL-10) is also known as a human cytokine synthesis inhibitor factor (CSIF). It is an important immunoinhibitory cytokine or function as an anti-inflammatory cytokine. It also plays an integral role in the complex but balanced immune network system. Previous studies have shown that interleukine-10 promoter polymorphisms at (-1082A>G, -819C>T, -592C>A) is closely related to its transcription activities. Blocking the tumor immune-surveillance by suppressing T-cell immunity could explain the important role of IL-10 in the progression of tumor. Several other studies have also observed that Il-10 promoter polymorphisms are also related to many other cancers, including diffuse B-cell lymphoma, non-Hodgkin’s lymphoma, breast cancer, gastric cancer, colon cancer, myeloma, advancedmelanoma and skin squmaous cell carcinoma occurred after renal transplantation. Squamous cell lung cancer is closely related to cigarette smoking, a procarcinogen extracted from cigarette (NNK)Nicotine-derived nitrosamine ketone, or 4-(methylnitrosamino)- 1-(3-pyridyl)-1-butanone, is a nitrosamine, which could induce the production of IL-10. It probably plays an important role in the development of lung cancer. In our previous study, we hypothesized that IL-10 haplotypes categorized by IL-10 promoter polymorphisms at (-1082A>G, -819C>T, -592C>A), might influence IL-10 expression and may be related with poor clinical outcomes and relapse in patients with non-small cell lung cancer. We have observed that lung tumors with non-ATA haplotypes had significantly higher mRNA level when compare with tumor with ATA-haplotypes. The overall survival (OS) and relapse free survival (RFS) in tumors with non-ATA haplotypes was significantly shorter then tumors with ATA-haplotypes. Furthermore, T-cells collected from peripheral blood in healthy donors when co-cultured with cancer cells, were more susceptible to apoptosis and less cytoxic to tumor cells in patients with non-ATA haplotypes than in patients with ATA-hapoltypes. T-cells apoptosis could be increased and tumor cell apoptosis could be decreased by adding IL-10 recombinant protein. On the contrary, T-cell apoptosis could be decreased and tumor cell apoptosis could be increased by adding neutralizing antibody. This was also consistent with our hypothesis that IL-10 haplotypes categorized by IL-10 promoter polymorphisms at (-1082A>G, -819C>T, -592C>A), might influence IL-10 expression and may be related with poor clinical outcomes and relapse in patients with non-small cell lung cancer. The influence of IL-10 was further demonstrated in our animal experiment. In the histological examination of TC-1 tumors in lung following intravenous injection of TC-1 cells into experiment mice. Lung metastasis was found in mice injected with IgG antibody but not in mice injected with IL-10 neutralizing antibody. We have concluded that, IL-10 can promote tumor malignancy via promoting T-cell apoptosis and tumor cell survival, and IL-10 haplotypes may be used to predict survival and relapse in resected non-small cell lung cancer. The overall survival and prognosis of lung cancer is poor, therefore we are expecting to apply and utilize the state of the art molecular technology, in predicting the response of lung cancer treatment and overall survival. Interleukin-10 (IL-10) may play an importance role in the progression of lung cancer. The objective of this study is to evaluate the different impact of IL-10 haplotypes on prognosis, including overall survival (OS) and relapse-free survival (RFS) in lung squamous cell cancer (SCC) and adenocarcinoma (ADC). Methods and Materials：In this study, we included 439 non-small cell lung cancer (NSCLC) patients recruited from Taichung Veteran General Hospital (TCVGH). Study samples were collected from immediately frozen section during surgery. Surgically resected normal lung tissues adjacent to the lung tumor were examined. Genomic DNA was extracted by conventional methods, which was prepared by prokinase K digestion and phenol-chloroform extraction; followed by ethanol precipitation. The genotypes of IL-10 were determined by Polymerase Chain Reaction-Restriction Fragment Length Polymorphism (PCR-RFLP). Polymorphisms of IL-10 were determined by direct sequencing of Polymerase Chain Reaction(PCR) products amplified from the DNA of normal tissues adjacent to the tumors. For continuous or discrete data analysis, Student’s t-test and Chi-square test were applied. Kaplan-Meier method and log-rank test were used to determine the association between IL-10 promoter polymorphisms and patients’ survival. Cox regression models were used to adjust potential confounders. Statistical testing was conducted by using two-sided tests and p-values G, -819C>T, and =592G>A). The non-ATA haplotype in NSCLC is more prevalent both in nodal metastatic tumors than in non-nodal metastatic tumors (N0) (59.7% vs. 48.4%, p = 0.017). In our previous study, we have also shown that patients with non-ATA haplotypes had higher IL-10 mRNA expression levels than patients with ATA haplotypes. In tumor histology, IL-10 haplotypes also correlated with nodal metastasis (63.0% vs 40.5%, p < 0.01) and tumor stage in SCC (59.2% vs 46.3%, p = 0.047). This was not observed in ADC patients. The non-ATA haplotypes may be used as a biomarker for poor prognosis in surgically resected NSCLC. Kaplan-Meier and Cox regression analysis showed that patients with ATA haplotypes had poorer OS and RFS. (HR 1.522, 95% CI = 1.191-1.945, p=0.001 for OS; HR 1.611, 95% CI = 1.247-2.082, p < 0.01 for RFS). The median survival duration and five years survival rate of patients with non-ATA haplotypes were significantly shorter when compare with patients ATA (median OS = 25.8 vs. 42.9 months; median RFS =16.8 vs. 30.9 months; 5-year OS = 28.6% vs. 44.7% and RFS = 22.2% vs. 36.2%). SCC patient with non-ATA haplotypes had poorer OS and RFS than patients with ATA haplotypes. No prognostic value was observed in ADC patients. These results suggest that IL-10 haplotypes has different impacts on OS and RFS in SCC patients from those with ADC. Conclusion and Suggestion：The IL-10 haplotypes has been associated with lung cancer risk. A prognostic value of non-ATA haplotypes was observed for SCC, but not for ADC. Non-ATA haplotypes were associated with nodal metastasis and tumor stage. In summary, IL-10 may have a stronger influence on tumor progression in SCC than ADC.