MAGE-A3 及 PRAME 基因在台灣非小細胞肺癌病人中的表現量及單一核苷酸多型性盛行率與表皮生長因子受體突變相關性探討

The prevalence of SNPs and gene expression in MAGE-A3 and PRAME of Taiwan NSCLC patients with or without EGFR mutation

黃婷沅(Ting-Yung Huang)

指導教授 ： 楊泮池

共同指導教授 ： 張逸良(Yih-Leong Chang)

國立臺灣大學/醫學院/病理學研究所/碩士(2012年)

https://doi.org/10.6342/NTU.2012.00650

摘要

肺癌是全球癌症中致死率最高的一項疾病，依照其病理型態主要可區分為非小細胞肺癌及小細胞肺癌兩大類。目前非小細胞肺癌的治療主要是以手術切除配合化療或是放射治療為主，但是這些療法都會有明顯的副作用。因此開發新的治療策略，對於肺癌研究而言，是現階段許多科學家積極努力的一個重要方向；免疫療法便是最近幾年廣被研究人員所採取的一項具有潛力的治療策略，此種治療是利用病人的免疫系統，使其能夠辨識在癌細胞上特有的抗原進而殺死癌細胞。MAGE-A3 與PRAME是兩個與腫瘤細胞形成相關的抗原，一般在正常細胞中並不會表現。根據研究，大約有35~50%的肺癌病患會在其癌組織檢體中偵測到MAGE-A3的表現，同時這些病患也伴隨著較差的預後情形。根據荷商葛蘭素藥廠所進行的一個大型的三期臨床試驗 (MAGRIT) 顯示，對摘除腫瘤後的病人施以MAGE-A3 antigen specific immunotherapy (ASCI) 有助於避免病患癌症的再次復發。在這個臨床試驗中也同時發現MAGE-A3與PRAME基因的表現量在鱗狀上皮細胞肺癌和肺腺癌細胞中有明顯的不同；此外，在不同人種中這類基因的表現也有極大的差異，歐洲人的表現量是遠高於亞洲人種;是以這兩個基因成為現階對研究癌症免疫療法相當中要的兩個指標。考慮到MAGE-A3 及PRAME 在亞洲人的低表現和亞洲人的表皮生長因子接受器的高比例突變，在本篇研究中我們很有興趣探討這兩個基因的表現量在表皮生長因子接受器活化突變之間是否有所關連。近年來，有許多證據顯示許多單一核苷酸多型態現象(SNP)存在MAGE-A3 和PRAME中。在本篇研究裡，我們利用即時定量PCR的偵測方式分析兩個基因的表現量，同時也運用direct sequence 以及質譜分析的方式檢測檢體中產生SNP的機率；希望藉由我們的實驗釐清是否有特殊的SNP存在於MAGE-A3 及PRAME 這兩個基因，進而影響台灣地區非小細胞肺癌病患組織中此兩基因的表現量。本篇研究中，我們也同時分析SNP的存在是否與病人的EGFR status有關。實驗結果顯示，MAGE-A3與PRAME基因的表現量在台灣地區非小細胞肺癌病患中明顯偏低；有關SNP的分析也發現，有3個特殊的SNP 存在於PRAME基因中，但此情形在MAGE-A3的基因中並未發現；不僅如此，我們更找到一個PRAME的SNP與其基因本身的表現量及病人產生EGFR activating mutation相關有關。藉由我們的研究，希望未來可以找出相關的機制作為病人面臨治療過程選擇上，一個有效的篩選指標。

關鍵字

MAGE-A3 ； PRAME ； 表皮生長因子接受器突變 ； 單核酸多型態現象 ； 非小細胞肺癌

並列摘要

Lung cancer is the leading cause of cancer related death worldwide, and can be divided into non-small-cell lung cancer (NSCLC) and small-cell lung cancer (SCLC). Typically doctors will consider removing the cancer cell mass and combine this operation with chemo- or radio- therapy, but there may have many side effects of cancer patients. For this reason, developing a new strategy for lung cancer treatment has become an important topic for cancer research. One of them is to develop the method of immunotherapy. Scientists attempt to allow a patients’ immune system to have the ability to recognize the specific antigen of cancer cells and further to kill them. MAGE-A3 and PRAME are two tumor-associated antigens, both of them specifically expressed in cancer cells but not normal cells. According to the interim preliminary data of MAGRIT phase III trial, scientists at GlaxoSmithKline found that patients can have a better clinical outcome when they receive MAGE-A3 antigen specific immunotherapy (ASCI) after their operations; in addition, they also found that the expression levels of these two genes are significantly different between squamous cell carcinoma and adenocarcinoma, and also slightly different with regards to ethnicities. The expression level in Europeans is higher than in Asians. For these reasons, these two genes have become good candidates for studies related to cancer immunotherapy. Considering the low expression rate of MAGE-A3 and PRAME genes and highly mutation frequency of EGFR status in NSCLC patients in Asia especially Southeast Asia, it would be quite interesting to investigate whether the expression levels of MAGE-A3 are related to the status of EGFR. Recently, evidence has also indicated that several single nucleotide polymorphisms (SNPs) exist in both MAGE-A3 and PRAME genes; furthermore, differences with regards to MAGE-A3 or PRAME polymorphism between racial populations may potentially contribute to its regulation as well as the relevant differential mRNA expression and the relationship between the SNPs and the EGFR activation mutation status have become a very important study topics related to cancer immunotherapy in Asia. In this study, we will analyze expression levels by Real-time Quantitative RT-PCR and potential polymorphisms of MAGE-A3 by direct sequencing and PRAME by DNA Mass in NSCLC patients in Taiwan; In addition, we will also investigate whether their expressions are correlated with the EGFR status of these patients.

並列關鍵字

MAGE-A3 ； PRAME ； SNPs ； EGFR mutation ； NSCLC patients in Taiwan

參考文獻

1. Jemal A, Bray F, Center MM, Ferlay J, Ward E et al. (2011) Global cancer statistics. CA Cancer J Clin. 61 (2): 69-90.

2. Mirsadraee S, Oswal D, Alizadeh Y, Caulo A, van Beek EJ. (2012) The 7th lung cancer TNM classification and staging system: Review of the changes and implications. World J Radiol. 4 (4): 128-134.

3. Tyagi P, Mirakhur B (2009) MAGRIT: The largest-ever phase III lung cancer trial aims to establish a novel tumor-specific approach to therapy. Clin Lung Cancer. 10 (5): 371-4.

4. Janku F, Stewart DJ, Kurzrock R. (2010) Targeted therapy in non-small-cell lung cancer--is it becoming a reality? Nat Rev Clin Oncol. 7 (7): 401-14.

5. Tsai TH, Su KY, Wu SG, Chang YL, Luo SC et al. (2012) RNA is favourable for analysing EGFR mutations in malignant pleural effusion of lung cancer. Eur Respir J. 39 (3): 677-84.