https://scholars.lib.ntu.edu.tw/handle/123456789/188146
標題: | 以比較基因雜交法和微衛星體分析研究與胰臟癌有關的基因變化特徵(3/3) | 其他標題: | Characterization of genomic alterations associated with pancreatic cancer by comparative genomic hybridization and microsatellite analysis(3/3) | 作者: | 林肇堂 | 關鍵字: | 胰臟癌;基因變化;免疫組織化學染色法;雷射捕捉顯微切割系統;比較基因雜交法;微衛星體分析;Pancreatic cancer;Genetic alterations;Laser capture microdiseection;Comparative genomic hybridization;Microsatellite analysis | 公開日期: | 2002 | 出版社: | 臺北市:國立臺灣大學醫學院內科 | 摘要: | 胰臟癌約90%是從胰管長出的腺癌,它們和從膽管或十二指腸附近所發生 的腺癌,及黏液性囊腺癌在臨床表現及治療結果上有明顯差異。目前有愈來愈 多証據指出胰臟癌的多步驟致癌過程中累積許多基因變化,而且認為胰臟癌在 腫瘤生物學上的差異可能與其基因變化之種類不同有關。因此,釐清參與胰臟 癌發生及進展的基因,將可能反映其致癌原因及臨床病理的差異性。雖然如此, 過去有關胰臟癌的基因變化研究,由於受限於傳統顯微分離技術及細胞遺傳方 法上的限制,欠缺系統性的了解。本研究的目的在於進一步(1)探討胰臟癌基因 體的整體變化(2)尋找胰臟致癌相關之基因表現(3)釐清胰臟癌發生與進展時的 基因變化(4)闡明基因變化與胰臟癌的臨床病理特徵關係。 在第一年的計劃中,吾人首先以回溯性及前瞻性方式有系統地收集包括胰 臟及胰臟附近的癌症病例,為了取得較純檢體做進一步的DNA ,有15 例胰臟 癌以雷射捕捉顯微切割系統(LCM)取得癌細胞,並順利抽取DNA 分析k-ras 基 因變化,結果發現有9 例在codon 12 上發生GGTàGAT 的點突變(表一),証實 此套系統可成功運用於胰臟癌的研究(詳見已發表論文1)。而k-ras 的基因變化 在不同的研究報告中(參考文獻13),由於所使用的方法與檢體不同,所得之結 果亦異。尤其在胰臟癌的腫瘤當中,有許多的基質細胞(stromal cell)可能干擾到 對胰臟癌基因研究的結果,所以目前之趨勢,均建議應以“純粹”的癌細胞來 研究。而吾人之報告,即為文獻中首次應用雷射捕捉顯微系統(LCM)來分析k-ras 在胰臟癌之變化情形。此外,在西方的文獻中,也注意到可能有一類以野生型 (wildtype) ras 基因之突變為主的胰臟癌,名之為medullary carcinoma of pancreas ,暗示可能由於不同的致癌過程,k-ras 之變化與否也有所差異(參考文獻14)。在第二年的計劃中,吾人運用免疫組織化學染色法分析與細胞週期蛋白 (cell cycle protein)常見的基因變化,包括p16, Rb, cylin D 與p53 。結果發現在 30 例確定為胰臟癌的檢體當中,70%(21/30)有Rb 的變化;40%(12/30)有p53的突變;26.7%(8/30)有p16 的變化;26.7%(8/30)有cyclin D 的變化。而在25 例 確定為十二指腸乳頭癌之病例當中,68%(17/25)有Rb 的變化,24%(6/25)有p53 的變化,4%(2/25)有p16 的變化56%(14/25)有cyclin D1 的變化(表二)。依初步 之統計分析,這些細胞週期蛋白的變化,在分辨胰臟癌或十二指腸乳頭癌上, 不具統計學上的差異。至於更一步的了解,將配合後續對於這些基因之變化分 析予以釐清。 在第三年的計劃中,吾人以比較基因雜交法分析胰臟癌的基因變化,發現 在染色體有gain 的包括1p (36.4%), 2p(36.4%), 2q(36.4%), 3q(45.5%), 5p(63.6%), 7p(36.4%), 8q(45.5%), 9p(36.4%), 13q(54.5%), 18q(36.4%), 21q(36.4%), Xp(31.4%)。而在染色體1p(36.4%), 1q(27.3%), 2q(27.3%), 3p(27.3%), 7p(27.3%), 11q(36.4%), 16q(27.3%), 17p(63.6%), 17q(27.3%), 18q(45.5%), 19q(36.4%)則有不 同程度的loss(表三)。 在此計劃當中,吾人利用LCM 取得胰臟癌細胞的DNA ,以過去吾人在胃 癌已建立的微衛星體分析和比較基因雜交法(參考文獻15-20)等技術探討更詳 細的基因變化的區段及其在胰臟癌的臨床病理的意義:由於所分析的是 genomic DNA ,所以在一些石蠟包埋組織或是保存包埋不妥切的組織,無法得 到較好的DNA 來分析。故吾人僅將檢體收集得宜並得到較佳品質的組織與 DNA 予以分析,亦為後續之研究建立基礎,之後亦將善用此次之經驗繼續收集 並研究胰臟癌之基因變化。 About 90% of pancreatic tumors are adenocarcinomas with a ductal phenotype. They differ from adenocarcinoma of the distal bile duct, ampulla of Vater and mucinous cystic carcinoma in terms of clinical manifestations and prognosis. Increasing evidence indicates that pancreatic cancer (PC) development is a multistep event proceeding from normal, preneoplastic lesions, to highly malignant tumor accompanied by accumulations of multiple genetic alterations. Collectively, it was believed that variability in the biologic characteristics of PC may be related to the profile of genetic alterations. Delineating genes involved in development and progression of PC can reflect the heterogeneity of their causes and subtypes. However, genetic changes underlying the initiation and progression of PC lack systemic data. This is partly attributable to the limitation of current research techniques such as manual microdissection and conventional cytogenetics. This project has been designed to further investigate the chromosomal aberrations of PC, identify target genes for DNA amplifications and losses in PC, clarify different genetic alterations in the development of PC, and elucidate the relationship between genetic abnormalities and different subtypes of PC. In the first year grant period, we have collected the following cases retrospectively and prospectively. To obtain pure cancer cells for further DNA analysis, laser capture microdissection (LCM) was performed in 15 cases with PC. K-ras mutations were investigated in the subsequently extracted DNA and disclosed GGTàGAT point mutation at codon 12 in 9 cases (Table 1). These results indicated LCM could be successfully applied to investigate PC. Different data were reported about the k-ras mutation rate by using different methods or material in the literature (reference 13). However, abdance of stromal cell DNA will easily contaminate the analysis of genetic alterations of pancreatic cancers. Microdisseciton is strongly suggested in investigation of pancreatic cancers nowadays. Our report is the first one which analyzed the “true” k-ras mutation rate in pancreatic cancer using laser capture microdissection. Besides, the relative lower rate of k-ras mutation may echo the newly classified category of PC, medullary caninoma of pancreas, which imply a different caninogenesis (reference 14). In the second year grant period, we performed immunohistochemical analysis of cell cycle proteins, including p16, Rb, cyclin D and p53 in our specimens. The data showed 70%(21/30) of pc with Rb changes; 40%(12/30) with mutant p53; 26.7%(8/30) with p16 changes and 26.7%(8/30) with cyclin D changes. Besides, in the cancers of papilla of vater, 68%(17/25) was with Rb changes; 24%(6/25) with mutant p53; 4%(2/25) with p16 changes and 56%(14/25) with cyclin D changes (Table 2). There is no obvious statistical difference between above 2 groups noticed. However, further analysis of these genes is mandatory to elucidate the role of these genes in cancinogenesis of pancreatic cancer. In addition to continuously enrolling more specimens LCM-procured pure DNA of pancreatic cancer and noncancerous tissue were be further analyzed in the last year period. The comparative genomic hybridization which we have established in gastric cancer (references 15-20) was applied to study the genetic profiles of these samples and elucidate the clinicopathologic significance of genetic abnormalities in the third year grant period. We found that chromosomes gains occurred more frequently in 1p(36.4%), 2p(36.4%), 2q(36.4%), 3q(45.5%), 5p(63.6%), 7p(36.4%), 8q(45.5%), 9p(36.4%), 13q(54.5%), 18q(36.4%), 21q(36.4%), Xp(31.4%). In the other hands, chromosome loss occurred more frequently in chromosome 1p(36.4%), 1q(27.3%), 2q(27.3%), 3p(27.3%), 7p(27.3%), 11q(36.4%), 16q(27.3%), 17p(63.6%), 17q(27.3%), 18q(45.5%), 19q(36.4%) (Table 3). In this project, we also noticed that some genomic DNA extracted from paraffin embedded formalin fired tissues might be well- applicated by DOP-PCR followed by CGH. However, some cases even though dealt with OCT might not be feasible in such applications. Therefore, some valuable experiences in dealing with tissue observation and management also were obtained in this grant period. We will keep on collecting suitable specimens and completing more data based on the present results in the future. |
URI: | http://ntur.lib.ntu.edu.tw//handle/246246/23532 | 其他識別: | 902314B002142 | Rights: | 國立臺灣大學醫學院內科 |
顯示於: | 醫學系 |
檔案 | 描述 | 大小 | 格式 | |
---|---|---|---|---|
902314B002142.pdf | 127.21 kB | Adobe PDF | 檢視/開啟 |
在 IR 系統中的文件,除了特別指名其著作權條款之外,均受到著作權保護,並且保留所有的權利。