偶發性大腸直腸癌在第三號染色體之刪除圖譜繪製

Abstract

針對第3號染色體的癌症研究方面，在前列腺癌發現3p12-22和3p24-26兩個區域經常有基因缺失的現象，在小細胞肺癌則發現3p14-21和3q28兩個區域有高頻率的基因缺失。進一步的研究中發現3p14.2的區域有一個FHIT ( fragile histidine triad )是肺癌相關的抑癌基因。而在大腸直腸癌方面，染色體3p21.3區域的hMLH1基因屬於DNA錯誤配對修復系統的一員，此基因的異常與遺傳性非息肉性大腸直腸癌( hereditary nonpolyposis colon cancer )的發生有相關，另外在肺癌中所發現的FHIT基因，對於大腸直腸癌的細胞分化、病程發展、淋巴轉移和5年存活率也有相關。本論文的研究目的：定義偶發性大腸直腸癌在第3號染色體之高頻率刪除區域，並分析DNA刪除與病人臨床結果之相關性，以了解是否有大腸直腸癌相關的抑癌基因。研究中，利用分佈在第3號染色體的23個微衛星標記，針對112對不同分期的大腸直腸癌檢體，以螢光-聚合酶鏈鎖反應( fluorescent-polymerase chain reaction )及自動DNA片段分析儀，檢測各微衛星標記在腫瘤DNA的變化。研究發現：112例腫瘤中有58例至少有一標記發生失異合性( Loss of heterozygosity，LOH )變化，其中3例可能為一整條第3號染色體缺失，因此在統計分析時剔除。利用各微衛星標記之LOH結果，定義出3p14.2-21.1、3p25-26和3q13三個區域有較高頻率的基因缺失現象，分別稱為最小刪除區域( minimal deletion region，MDR ) I、II、III。分別將23個微衛星標記之變異與病人的年齡、性別、腫瘤發生位置、腫瘤分化程度、癌症分期做單變項統計分析，結果為：(1)於 D3S1297、D3S1263、D3S2338、D3S1266和D3S1300發生缺失的病人，其發現癌症的年齡較年輕( p 值依序為0.01、0.037、0.033、0.047和0.029)；(2) D3S1614和D3S1311發生LOH的頻率，男性病人高於女性病人(p 值分別為0.029和0.028)；(3)D3S1300和D3S1311發生LOH變化之大腸直腸癌的發生位置多位於右側大腸( p 值分別為0.033和0.051 )；(4) D3S1297的LOH變化，與存活有相關( p 值為0.018)。而針對MDR I、II及III與臨床資料的統計分析結果顯示：腫瘤發生MDR I及II刪除的病人，年齡較年輕，其它對於性別、腫瘤分化程度、腫瘤發生位置、癌症分期及存活分析都無顯著影響，而腫瘤發生MDR III刪除之病人，在癌症分期早期( Dukes’ A及B )佔較高的比例( p值為0.022)，其它對於病人的年齡、性別、腫瘤發生位置、腫瘤分化程度及存活分析都無顯著影響。以multivariable Cox’s proportional hazards model分析disease-free survival rate及overall survival rate，發現D3S3681發生LOH變化對病人的disease-free survival rate較差( HR：3.22, 95% CI：0.89 – 11.66, p值為0.023 )，但年齡越大的病人，其disease-free survival rate較好( HR：0.966, 95% CI：0.935 – 0.999, p值小於0.0001 )，但再進一步分析時，年齡越大的病人在剛完成手術後的存活率較差，但是隨著時間的增加，其影響漸減 ( HR：1.35, 95% CI：1.172 – 1.569, p值小於0.0001 )。對overall survival rate的分析中，顯示Dukes’ D及疾病復發之病人，其存活率較差( HR分別為2.03 x 108及1.49 x 108 , 95% CI：1.8 * 105 – 2.32 * 108及1.3 * 105 – 1.64 * 108, p值都小於0.0001 )。

The research of cancer on chromosome 3 : the regions on 3p12-22 and 3p24-26 usually had allele loss in prostatic adenocarcinoma. And the regions 3p14-21 and 3q28 had high frequency allele loss in small cell lung cancer. To go deeper into the region 3p14.2, there was a lung tumor suppressor gene called FHIT ( fragile histidine triad ). In colorectal cncer ( CRC ), the region on 3p21.3 found hMLH1, which was a part of DNA mismatch repair system. When the hMLH1 is abnormal, it was related to hereditary non-polyposis colon cancer happened. FHIT also influenced differentiation grades, pathological stages, lymph node metastases and 5-year survival rate in CRC. The purpose of this study is to identify the high frequency deletion region on chromosome 3 in CRC. Then to analyze the relationship between DNA deletion and the patients’ clinical data, to discusses whether there are tumor suppressor gene on chromosome 3 in CRC. In this study, using 23 microsatellite markers spread on chromosome 3 analyzes different stage of CRC cases. Then to use fluorescent-polymerase chain reaction amplifies 23 microsatellite markers. After that, using capillary array electrophoresis analyzies the change of the microsatellite markers in CRC. The result : there are 58 cases at least one microsatellite marker had LOH change, and there are three cases which may identify to be monosomy in site. The three monosomy cases will reject before comparing with clinical data. Using the LOH change of microsatellite markers, there are three regions of high frequency allele loss 3p14.2-21.1, 3p25-26 and 3q13 and name minimal deletion region I, II, III. Then to use 23 microsatellite markers combine with 109 CRC cases’ age, gender, tumor location, tumor differentiation and pathological stage to analyze. (1) LOH at D3S1297, D3S1263, D3S2338, D3S1266, D3S1300, the CRC patients are related to younger onset-age of CRC ( p -value = 0.01, 0.037, 0.033, 0.047, 0.029 ). (2) The frequency of LOH change at D3S1614 and D3S1311 in male patients are higher than female ones ( p -value = 0.029, 0.028 ). (3) When D3S1300 or D3S1311 has LOH change, the tumor is more easy to grow on right colon. ( p -value = 0.033, 0.051 ) (4) LOH at D3S1297, the patients’ overall survival rate is lower ( p – value = 0.018 ). After that, to analyze the minimal deletion region ( MDR ) I, II, III combine with patients’ age, gender, tumor location, tumor differentiation and pathological stage. The MDR I & II are related to younger onset-age of CRC. Then gender, tumor location, tumor differentiation and pathological stage are no significance in statistics, and the patients of the deletion on the MDR III are more high frequency in Dukes’ A & B ( p – value = 0.022 ). But the deletion on the MDR III is no significance with the age, gender, tumor location and tumor differentiation. In multivariable Cox’s proportional hazard model, to analyze disease-free survival rate find that the LOH at D3S3681 is lower survival rate in CRC patients ( HR：3.22, 95% CI：0.89 – 11.66, p -value = 0.075 ), and the patients of older age is higher survival rate (HR：0.966, 95% CI：0.935 –0.999, p -value < 0.0001 ). Then to interact with the time, the age is higher risk factor after operation, but, however years as followed the risk of the age is lower ( HR：1.35, 95% CI：1.172 – 1.569, p – value < 0.0001 ). Then to analyze overall survival rate, the Duke’s D and relapse are higher risk factors in CRC patients ( HR: 2.03 x 108 & 1.49 x 108 , 95% CI：1.8 * 105 – 2.32 * 108 & 1.3 * 105 – 1.64 * 108, p -value < 0.0001 ).