大腸直腸癌之分子遺傳學研究

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行政院國家科學委員會專題研究計畫成果報告

Molecular genetic study of color ectal cancer 計畫編號 : NSC 88-2314-B-002-274 執行期限 : 87 年 8 月 1 日至 88 年 7 月 31 日 主持人 : 楊 雅 倩 執行機構 : 國立臺灣大學醫學院醫技系 一、中文摘要 大腸直腸癌近年來皆為為臺灣地區十 大癌症死因之一。近來的研究已知有兩種大 腸直腸癌與家族的遺傳有關：一是家族性多 發性息肉症(FAP)，其乃導因於APC 基因的 突變。另一則是遺傳性非息肉性大腸直腸癌 (HNPCC)，其則主要是 DNA 錯誤配對修復因 基：hMSH2、hMLH1、hPMS1、hPMS2 和 hMSH6 之一發生突變；約有 95% HNPCC 腫瘤可發現 微 小 衛 星 體 不 穩 定 (microsatellite instability) 亦 稱 為 複 製 錯 誤 (replication error, RER)的現象。腫瘤的 發展過程中，某些基因發生雜接合子漏失 (loss of heterozygosity, LOH)以及複製 錯誤是很重要的現象，其與臨床之診斷和預 後亦或有相關。因此，本研究於大腸直腸癌 腫瘤中，針對其相關的基因：APC、 hMSH2、 hMLH1、DCC，以及抑癌基因：P53、NM23 和

致癌基因MET 等進行 LOH 和 RER 分析，並探

討此些基因變化與臨床病理特徵、腫瘤轉移 及預後之相關性，進而了解其在臨床上的應 用性。 關鍵詞 : 大腸直腸癌、複製錯誤、雜接合 子漏失 Abstract

Colorectal cancer is one of the most common malignancies in Taiwan. Recent advances in genetics have more clearly defined the impact of inheritance in the multistep process of the colorectal cancer. Researchers have identified single genes that confer a susceptibility to hereditary colorectal

cancer: familial adenomatous polyposis (FAP) and hereditary nonpolyposis colorectal cancer (HNPCC). FAP is caused by mutation of the adenomatous polyposis coli (APC)

gene. HNPCC is caused by mutation of one of several DNA mismatch repair genes including hMSH2, hMLH1, hPMS1, hPMS2

and hMSH6. Additionally, microsatellite

instability, also referred to as replication error (RER), is observed in most cancers from HNPCC patients. Loss of heterozygosity (LOH) and RER are important phenomena in tumor development, with diagnostic and prognostic relevance. Therefore, screening for LOH and RER is a desirable first step in the molecular analysis of tumors. This study was conducted to determine LOH and RER with a set of 10 microsatellite loci linked to

APC, hMSH2, hMLH1, DCC, P53, NM23, HPC1 and MET genes as well as tumor

suppressor gene(s) on 8p22 in colorectal tumors and to ascertain the significance of the genetic alteration at specific loci in the clinical pathology, chronological progression and prognosis of colorectal cancer.

Keywords: colorectal cancer, replication error, loss of heterozygosity 一、緣由與目的

Colorectal carcinogenesis is a multistep process in which the majority of carcinomas arise from adenomatous polyps through many consecutive steps. This involves the accumulation of genetic alterations: the mutational activation of oncogenes (e.g. K-ras) and the mutational inactivation of tumor

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suppressor genes (e.g. p53) [1,2]. About 80%

of patients with colorectal cancer have sporadic disease, with no evidence of having inherited the disorder. In the other 20% of patients, there appears to be a genetic link. There are two well known inherited colorectal cancer: familial adenomatous polyposis (FAP) and hereditary nonpolyposis colorectal cancer (HNPCC) [3]. The recent identification of specific genes associated with a predisposition to colorectal cancer has provided new insight, not only regarding inherited colorectal cancer, but also about the underlying mechanisms of sporadic tumor formation.

Microsatellite analysis is emerging as an important tool in the study of cancer. The addition of novel microsatellite alleles is referred to as replication error (RER) or microsatellite instability, indicating possible mutations in the cell's DNA repair mechanisms. Another phenotype, exhibited by chromosome deletions, often referred to as allelic loss or loss of heterozygosity (LOH), is commonly believed to represent the second genetic inactivation step consistent with the "two-hit" theory of carcinogenesis. The detection of either of these genetic changes demonstrates the presence of a clonal population of cells that share altered genetic information, which is characteristic of cancer cells.

The presence of RER has been shown in colorectal cancers, notably HNPCC and sporadic tumors in other tissues such as bladder and stomach [4,5]. Studying RER and LOH phenomena in tumor vs. normal tissues will help to elucidate which types of cancer exhibit these phenotypes and their possible relevance for future use as a diagnostic tool. Tumors of the proximal colon, which display RER, have been reported to have a better clinical prognosis [6]. Studies to date have also indicated that LOH of microsatellite markers near specific loci in colorectal cancer can have some prognostic significance [7]. Research in these areas is ongoing. The main aim of this study was to determine the incidence of LOH and RER at specific loci, its associations, and significance in the chronological course of

tumor progression in colorectal cancer. 三、結果與討論

Thirty-nine patients with sporadic colorectal cancer were accumulated. The stored frozen tissues were retrieved for simultaneous analyses of RER and LOH at 10 distinct chromosomal loci via an automated fluorescent microsatellite assay. RER was observed in 8/39 (20.5%) of the cases at one or more chromosomal loci. Five of RER+ tumors showed LOH at one to three microsatellite loci linked to APC, hMSH2, hMLH1, P53, NM23 and HPC1 genes as

well as tumor suppressor gene(s) on 8p22. RER+

phenotype was associated with LOH at the hMLH1 gene (P = 0.035). On the

other hand, LOH was detected at all microsatellite loci studied, with the exception of that linked to MET oncogene.

More than one third of informative tumors showed LOH at P53, DCC and APC genes

with the frequency 57.9%, 35.3% and 33.3%, respectively. LOH at hMSH2 and hMLH1 genes was present in 12.1% and

9.1% of informative tumors. Of 22 informative tumors, six showed LOH at marker D8S254 that was suspected near one or more tumor suppressor genes. The frequencies of LOH at NM23 and HPC1

genes were 18.5% and 7.4%, respectively. 四、計畫成果自評

We have established the automated procedures of LOH and RER assay system via ABI 377 automated sequencer as well as GeneScan and Genotyper software. The majority of sporadic colorectal cancer studied showed RER and/or LOH at the microsatellite loci linked to APC, hMSH2, hMLH1, DCC, P53, NM23 and HPC1 genes

as well as tumor suppressor gene(s) on 8p22. The significance of the genetic alterations at specific loci in the clinical pathology, chronological progression, tumor recurrence and prognosis of colorectal cancer is being ascertained.

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