南台灣移形細胞癌p53腫瘤抑制基因的突變

p53腫瘤抑制基因的突變一直廣泛地被報導盛行於人類的許多腫瘤中，由其與泌尿道腫瘤之間的關係。早期偵測抗癌基因p53的突變可提供我們在臨床上處理泌尿道腫瘤一項有用的資料，為了確認p53基因在泌尿道移形細胞癌的角色，我們設計了這個實驗。我們利用聚合脢連鎖反應(polymerase chain reaction, PCR)，單股序列型態多形性(single-strand conformation polymorphism, SSCP)和序列分析(sequencing analysis)分析南台灣地區泌尿道腫瘤組織中抗癌基因p53的突變情形。我們利用DNeasyTM Tissue Kit萃取腫瘤組織樣本中的去氧核醣核酸(DNA)，並設計引子(primer)分別針對第4，5，6，7和8的位置之表現基因(exon)進行基因放大。利用電泳產生單位(Genephor Electrophoresis Unit)做單股序列型態多形性的分析，並經由銀染色而看出。在所有的病人中，序列分析是根據原始p53基因聚合脢連鎖反應的產物。統計分析採取商業電腦軟體SPSS 10.0. 卡方檢定(Chi-square test)和t檢定(t-test)方法來描述p53基因突變和腫瘤分期(stage)，腫瘤分級(grade)，性別和年齡之間的關係。 P≤ 0.05認為有統計上的意義。從1992年3月至2003年7月，在高雄醫學大學附設醫院，共取得94個手術檢體，最後有75位病人檢體加入本實驗。男性病人有54位，女性病人有21位。平均年齡是66.85歲。膀胱癌的病人有58位，輸尿管癌的病人有8位，腎盂癌的病人則有9位。在病理的診斷上，低分級(low grade)的腫瘤有37例，高分級(high grade)的腫瘤有38例。關於腫瘤分期(stage)，≤pT1分期有29位，≥pT2分期有46位。75位病人中，47位病人佔62.7%有p53基因的突變。更進一步來說，46個侵犯性的腫瘤(invasive tumor)中，有33位病人佔71.7%基因有突變的情形。統計分析指出，分級、年齡和性別並不是p53基因突變重要的危險因子(P≥0.05)。相反地，侵犯性的腫瘤則和p53基因突變在統計上有顯著的意義。表現基因4 (exon 4)是exon 4-8中最常見的突變基因，75位病人中有20位病人有基因突變的情形(26.7%)，且呈現出多個突變位置。另外，表現基因5有5位病人(6.7%)；表現基因6有12位病人(16%)；表現基因7有17位病人(22.7%)；和表現基因8有9位病人(12%)有基因突變的情形，這些表現基因亦皆呈現出多個變化位置。同時，75位病人中有14位病人有超過一個exon以上的突變。關於p53基因exon 4的突變，20例侵犯性的腫瘤有15位佔75 %，表淺性的腫瘤只有5位佔25%；高分級的腫瘤有9位佔45%，低分級的腫瘤有11位佔55%。這20位exon 4 的基因突變中，有6位病人合併有codon 72的異常，有14位則未與codon72的異常有關。密碼子(codon)72位置產生異常有20例，雖然這是正常的多型性現象(polymorphism)，但是其中有6位病人合併有exon 4 其他codon 的突變，有14位病人則是單獨存在。同時這20位codon 72異常的病人中，有16位是侵犯性的腫瘤佔80%，14位是高分級的腫瘤佔70%，且兩者皆達到統計學上的意義。總結，我們發現移形細胞癌的病人中，有62.7%有p53基因的突變。侵犯性的腫瘤較容易產生p53基因的突變，同時p53基因突變似乎可以作為移形細胞癌的標幟物(marker)。目前看來，利用聚合脢連鎖反應和單股序列型態多形性來做p53基因的簡單分析，對於偵測移形細胞癌p53基因的異常是適合的。另外，在p53基因上的表現基因exon 4-8皆有許多個突變位置，exon 4的突變率是最高且codon 72多型性現象比例也很高，與腫瘤之間的關係，需要進一步加以研究。

關鍵字

移形細胞癌；腫瘤抑制基因

並列摘要

Mutation of the tumor suppressor gene p53 has been reported occurring prevalently in a wide range of human tumors including urothelial tumor and colorectal cancer. Early detection of a mutated p53 gene is thought to provide useful information for the clinical treatment of urothelial tumor. To identify the role of the p53 gene in transitional cell carcinoma of urinary tract, we design this study. In this study, a rapid screening test for mutation of the p53 gene by Polymerase Chain Reaction / Single-Strand Conformation Polymorphism (PCR/SSCP) and sequencing analysis in transitional cell carcinoma of urinary tract was performed.The genomic DNA of the specimens were purified using DNeasyTM Tissue Kit. Primer sets were designed to amplify fragments within the exon 4, 5, 6, 7, and 8 of p53 gene. SSCP analysis was carried out on a Genephor Electrophoresis Unit and visualized by silver staining. Sequencing was done on original PCR products of p53 in all patients. The statistical analysis adopts commercial computer software SPSS 10.0. Chi-square test and t-test are adopted to describe the relationship between p53 gene mutation and tumor stage, tumor grade, sex and age. P≤0.05 is defined as statistical significance. From March 1992 to July of 2003, 94 surgical specimens collected from Kaohsiung Medical University hospital. A total of seventy-five patients were enrolled in this study finally. Fifty-four patients were male and 21 patients were female. The mean age was 66.85 years old. Fifty-eight patients had bladder cancer, 8 patients had ureteral cancer and 9 patients had renal pelvis cancer. At the time of pathological diagnosis, 37 tumors were classified as low grade and 38 tumors as high grade. As for tumor stage, ≤pT1 stages were noted in 29 cases and ≥pT2 were 46. Totally, 47 of 75 patients (62.7%) have p53 gene mutation. Moreover, 33 of 46 (71.7%) patients were found to have genetic alterations of p53 in invasive tumors. The analyzed results indicate that tumor grade, age and gender are not the important risk factors associated with p53 gene mutation (p>0.05). Conversely, tumors with invasive stage showed statistical significance with p53 gene mutation (p<0.05). Exon 4 presented with multiple mutation sites is the most common mutation gene among exon 4 -8 of p53 genes which was noted in 20/75(26.7%) patients. Besides, exon 5 mutation was noted in 5/75(6.7%) patients, exon 6 mutation was noted in 12/75(16%) patients, exon 7 mutation was noted in 17/75(22.7%) patients and exon 8 mutation was noted in 9/75(12%) patients. All of these exons also include multiple mutation sites. In addition, 14/75 patients with more than one exon mutation of p53 genes were also found in this study. When talking about the exon 4 mutation of p53 genes, 15 patients (75%) with invasive tumor , 5 patients (25%) with superficial tumor , 9 patients(45%) with high grade tumor and 11 patients(55%) with low grade tumor were noted. Among 20 patients with exon 4 gene mutation, 6 patients coexisted with codon 72 polymorphism, but the others did not. Twenty patients with codon 72 normal polymorphism were found in this study. However, only 6/20 codon 72 polymorphism presented with other codon change of exon 4 and the others (14/20) existed independently. Among 20 codon 72 polymorphisms, 16 patients (80%) with invasive tumor and 14 patients (70%) with high grade tumor were found. Both of these two findings reached statistical significance. In conclusion, we found that 62.7% of transitional cell carcinoma patients had p53 mutation. Invasive tumors are easy to be found to have p53 gene mutation. The p53 gene mutation may be used as a marker of transitional cell carcinoma. The simple analysis of p53 gene using PCR/SSCP is suitable for screening p53 abnormalities in transitional cell carcinoma at present. Besides, the relationship among multiple mutation sites existed in exon 4-8, highest exon 4 mutation rates, high codon 72 polymorphism and cancer risk need for further investigation.