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

研究目的：肺癌或稱之為支氣管癌，係指的是源自於呼吸道或肺部實質組織內細胞生長失去控制的惡性疾病。依據WHO針對原發性肺癌進行分類，肺癌主要分為小細胞肺癌small cell lung cancer (SCLC)和非小細胞肺癌non-small cell lung cancer (NSCLC)，大約95%的肺癌均屬於這兩種類型，各佔了約13%和82%，而非小細胞肺癌又可再細分成鱗狀上皮細胞癌squamous cell carcinoma (SCC) (20%)，肺腺adenocarcinoma (ADC) (38%)，大細胞癌large cell carcinoma(5%)，和無法分類的非小細胞肺癌(18%)，其他類型的肺癌則只佔了大約5%。肺癌是目前全世界最常見的一種癌症疾病，在2008，據估計，約有1,600,000新診斷個案，和1,380,000病患死亡。在美國，於2012年，估計會有228,000新診斷個案，並且造成159,500人死亡。大約自1953年起，肺癌就成為男性因癌症死亡的第一名，而到了1985年，肺癌也成為女性癌症死因的第一位。雖然，肺癌在男性所導致的死亡開始呈現下降的趨勢，這似乎與抽菸率的下降有著密切的關聯；但是，肺癌在女性卻是持續的攀升當中，大約有一半的肺癌死亡發生在女性。自民國70年代起，肺癌即列居我國國民十大死因之第一位，台灣的肺癌發生率，在過去的30年，不論男性或女性，均增加了將近8倍，是所有癌症中增加最快速的。依據衛生署國民健康局癌症登記資料呈現，每五個死於癌症的病患，其中之一就是死於肺癌。因肺癌而死亡的患者，在過去20年的時間增加了3倍。過去數年的時間，肺癌和肝癌同時為男性死亡原因之第一位。在女性，肺癌則一直是高居所有癌症死亡原因的榜首。在最新的統計再次顯示，於2012年，肺癌仍是我國癌症死亡原因的榜首。其實不只是在台灣，肺癌在歐美等先進國家之中，也是發生率和死亡率最高的癌症疾病。非小細胞肺癌(NSCLC)，可分成兩大組織學型態: 鱗狀上皮細胞癌 squamous cell carcinoma (SCC) 與肺腺癌 adenocarcinoma (ADC)。其中，鱗狀上皮細胞癌的發生與抽菸有著密切的關聯性。台灣歷年以來，非小細胞肺癌所占的平均比率是88-90%，小細胞癌的比率則只有10-12%。肺癌的預後總體來說不是很好，在所有肺癌病人中，15%的病患在診斷後只能約存活5年。病患在診斷時的肺癌階段分類，通常已經是晚期，在發現時，大約30-40%的非小細胞肺癌病例已是第四期，而60%小細胞肺癌病例在被診斷時，也已經是處於廣泛期。不論是非小細胞肺癌或是小細胞肺癌，與疾病預後的相關因素有很多，其中最重要的因子是，在肺癌診斷之時的癌症分期。其他的相關因子尚包括，臨床參數特徵(clinical parameters)，組織病理學(histopathology)，分子特徵(molecular characterization)，正子掃描Positron emission tomography(PET)或合併電腦斷層掃描computerized tomography (CT) (PET-CT)和完整手術切除後的復發(recurrence after tumor resection)。逆轉錄聚合酶鏈式反應‎Reverse Transcriptase Polymerase Chain Reaction (RT-PCR)也一直被用來做為探測非小細胞肺癌淋巴腺的腫瘤指標。免疫系統是一個極其複雜的網路架構，其設計是用來保護宿主免於受到外來(例如細菌或病毒)的侵犯以及來自內在的威脅(例如癌症轉變)。細胞激素(cytokine)則是免疫反應的重要中介物，Cytokine是屬於類似於荷爾蒙的蛋白質(hormone like proteins)，亦是具有相當影響能力的醣蛋白質類。用來做為免疫細胞之間的溝通。他們在各種免疫反應包括起始(initiation)，持續(perpetuation)及後續減弱(down regulation)調節等，均扮演了重要的角色。Interleukin-10 (IL-10)又被稱做人類細胞激素合成抑制因子human cytokine synthesis inhibitor factor (CSIF)，是一種重要的免疫抑制激素(immunoinhibitory cytokine)又稱為抗發炎細胞激素(anti-inflammatory cytokine)，是屬於複雜且平衡免疫網路的一部分。白介素 (IL-10)啓動基因的多型現象與轉錄活性有其相關性。抽煙所導置IL-10的誘發，在肺部腫瘤的進展扮演一個重要角色。在一些過去的研究發現，白介素(IL-10)啓動基因(promoter gene)的(polymorphisms)多型現象(-1082A>G, -819C>T, -592C>A)與轉錄活性(transcription activities)有其相關聯性。藉由抑制T-cell的免疫能力進而阻擋腫瘤免疫偵測(tumor immune-surveillance)，可以用來解釋IL-10於肺癌發展中的角色。此外也有一些研究顯示，IL-10啓動基因的多型現象亦與多種癌症具有相關性，包括肺癌而進一步的研究也發現，IL-10啓動基因的多型現象也和其他癌症，例如瀰漫型B細胞淋巴癌diffuse B-cell lymphoma，non-Hodgkin’s lymphoma，乳癌，胃癌，大腸癌，骨髓癌，腎臟移植後皮膚上皮細胞癌，和黑色素細胞瘤。非小細胞肺癌特別是麟狀上皮細胞癌與抽菸具有密切的關聯，有些研究發現，一種自香菸萃取的(procarcinogen)主要致癌物質(NNK)Nicotine-derived nitrosamine ketone,這是一種存在於香菸中的亞硝胺(nitrosamine)，可誘發IL-10的製造。抽煙所導致IL-10的誘發，在肺部腫瘤的進展扮演一個重要角色。肺癌的預後總體來說不是很好，我們希望利用對於已知的免疫機轉和分子生物醫學技術，期望針對肺癌的治療和預後的結果，能更精確的診斷和評估。白介素interleukin (IL-10)啓動基因的多型現象與轉錄活性有其相關性。抽煙所導置IL-10的誘發，在肺部腫瘤的進展扮演一個重要角色。我們預期可以發現，IL-10 haplotypes對於鱗狀上皮細胞肺癌squamous cell carcinoma(SCC)和肺腺癌adenocarcinoma(ADC)病患整體的存活overall survival(OS)與無復發的存活relapse free survival(RFS)時間，會有其不同之影響。研究方法及資料：在肺癌病患切除的腫瘤相鄰的正常肺部組織收集後，利用聚合鏈反應-限制酵素切割長度多樣性Polymerase Chain Reaction-Restriction Fragment Length Polymorphism(PCR-RFLP)進行直接排序，已定續IL-10 haplotypes。 Kaplan-Meier和多變相回歸分析(Cox regression)，用以評估IL-10 haplotype對於對於肺癌病患整體的存活overall survival(OS)與無復發的存活relapse free survival(RFS)時間，所造成的不同影響。研究結果：從聚合鏈反應-限制酵素切割長度多樣性(PCR-RFLP)進行直接排序發現，Non-ATA haplotype與淋巴結轉移(N1, N2)，相較於沒有淋巴轉移(N0)的肺癌病患，其關聯性較為顯著。這樣的現象似乎只存在於鱗狀上皮細胞肺癌(SCC)，而在肺腺癌(ADC)則無此發現。帶有non-ATA haplotype的鱗狀上皮細胞肺癌病患，其整體的存活(OS)與無復發的存活(RFS)時間，較帶有ATA haplotype的病患要來的顯著的短。結論與建議：經由本篇研究可推測，IL-10 haplotype可能可以用來做為預測鱗狀上皮細胞肺癌(SCC)病患的整體存活(OS)與無復發的存活(RFS)時間的生物指標。

關鍵字

細胞介素-10 (IL-10)啟動子基因多型性；預後；鱗狀上皮細胞肺癌；肺腺癌；肺癌

並列摘要

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 <0.05 were considered to be statistically significant. All statistical analyses were performed using the SPSS statistical software program (version 13.0) Results：Two IL-10 haplotypes (ATA and non-ATA) were categorized by three IL-10 promoter polymorphisms (-1082A>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.

並列關鍵字

IL-10 haplotype ； prognosis ； squamous cell carcinoma ； adenocarcinoma ； lung cancer

參考文獻

Muthukuru M, Schanbacher CF, Edwards V, Miller DM, Kim JE,

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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

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