【作者】 陈敏丰；

【导师】 齐琳；

【作者基本信息】 中南大学 ， 外科学， 2010， 博士

【摘要】 荧光原位杂交技术(fluorescence in situ hybridization, FISH)是20世纪80年代末在已有放射性原位杂交技术基础上发展起来的一种非放射性分子细胞遗传学技术。采用荧光素直接标记DNA制备探针,根据探针与被检测样本中序列的碱基互补配对,通过荧光显微镜观察细胞内的荧光信号来判断有无染色体数目和结构的改变。由于其操作简单、安全有效、具有很好的特异性和灵敏性的特点,适于临床医学检测。目前,已广泛用于遗传疾病的诊断及血液肿瘤、实体肿瘤等方面的检测。近年来,随着分子遗传学的迅猛发展,研究发现了尿路上皮癌、前列腺癌中一些常发生、高特异性的染色体畸变,为FISH技术应用于这些泌尿生殖系肿瘤的检测奠定了理论基础。本研究分为三个部分,分别探讨FISH技术在上尿路尿路上皮癌、膀胱尿路上皮癌和前列腺癌的临床应用价值。目的探索在中国人群中3号、7号、17号染色体及p16位点异常与膀胱尿路上皮癌发生、发展的关系。评估FISH诊断膀胱尿路上皮癌的可行性和有效性及对术后随访的临床应用价值。方法分别对100例膀胱尿路上皮癌患者和30例非尿路上皮癌对照人群的新鲜尿液,进行尿脱落细胞学检查及FISH检测3号、7号及17号染色体、9号染色体p16位点异常,比较两种方法的敏感性和特异性。统计分析各染色体畸变与肿瘤数目、大小、既往复发率、临床分期及病理分级等临床参数的相关性。对66例非肌层浸润性膀胱癌术后三个月的膀胱冲洗液进行FISH检测,根据随访结果分析FISH在监测膀胱癌复发的作用。结果FISH和细胞学两种检测方法诊断膀胱尿路上皮癌的敏感性分别为82%和36%,有显著性差异(p<0.001)；特异性分别为97%和100%,差异无统计学意义(p>0.05)。各染色体多倍性畸变率分别为63%(3)、72%(7)、31%(p16)、60%(17)；单体畸变率为53%(3)、49%(7)、47%(p16)、46%(17)；基因纯合性缺失仅见于p16位点为34%。各染色体畸变与患者的肿瘤数目、大小、既往复发率均未见显著相关性(p>0.05)；17号染色体多倍性畸变率与肿瘤分期、分级呈正相关性(p<0.05)；其余类型的染色体畸变与肿瘤分期、分级无明显相关性P>0.05)。根据术后平均随访22个月的结果,23例非肌层浸润性膀胱癌患者术后复发,FISH阳性患者术后复发率为54%,阴性患者复发率为13%,差异有显著统计学意义(p<0.001),术后FISH阴性患者无复发生存率明显优于FISH阳性患者。结论FISH诊断膀胱尿路上皮癌具有较高的特异性和敏感性,可望成为早期诊断和术后监测膀胱癌复发有效、安全的检查方法。目的探讨通过FISH检测3、7、17号染色体及P16位点畸变诊断上尿路上皮癌的可行性和有效性。方法分别对29例上尿路上皮癌患者和20例非尿路上皮癌对照人群的新鲜尿液,进行尿脱落细胞学检查及FISH检测3号、7号及17号染色体、9号染色体p16位点异常,比较两种方法的敏感性和特异性。并对FISH诊断膀胱尿路上皮癌和上尿路上皮癌进行比较。结果FISH和细胞学两种检测方法诊断膀胱尿路上皮癌的敏感性分别为90%和48%,有显著性差异(p<0.05)；特异性均为100%。FISH诊断膀胱尿路上皮癌和上尿路上皮癌的敏感性分别为82%和90%,差异无统计学意义(p>0.05)。结论FISH用于检测上尿路移行细胞癌的敏感性高于细胞学检查,而特异性无差异。FISH技术有可能成为诊断和鉴别诊断上尿路尿路上皮癌可靠、无创的诊断方法。目的探索在中国人群中TMPRSS2基因和ETS基因家族中ERG、ETV1、ETV4基因融合的遗传学改变与前列腺癌发生、发展的关系。评估FISH诊断和鉴别诊断前列腺癌的临床应用价值。方法：分别对20例良性前列腺增生(BPH)、5例重度上皮内瘤样病变(HGPIN)和53例前列腺癌(Pca)石蜡切片进行FISH检测TMPRSS2基因和ERG、TV1、ETV4基因融合现象,比较不同前列腺疾病发生基因融合的情况。分析前列腺癌标本中ERG基因改变和Gleason评分、PSA、临床分期之间的关系。结果在53例前列腺癌标本中32例(60%)检测到ERG基因改变,提示TMPRSS2/ERG融合基因,5例(9%)检测到TMPRSS2/ETV1融合基因,1例(2%)检测到ERG基因改变。5例高级别上皮内瘤样病变标本中检测到1例(20%)发生了ERG基因改变。20例良性前列腺增生标本未检测到基因融合现象。FISH诊断前列腺癌的敏感性为72%,特异性为96%。ERG基因改变与前列腺癌Gleason评分、临床分期呈正相关(p<0.05),与PSA水平无明显相关性(p>0.05)。结论FISH诊断前列腺癌具有较高的特异性和敏感性,有助于前列腺癌的诊断和鉴别诊断。TMPRSS2基因和ETS家族基因的基因融合有助于前列腺癌的病因研究和探索,对后期治疗及预后有指导作用。更多还原

【Abstract】 Fluorescence in situ hybridization(FISH) is a kind of technology of non-radioactive in situ Hybridization, which was deVeloped on the basis of radioactiVe hybridization in the later 1980s. The fluorescence molecules were labeled to genetic material to obtain the probes. Probes have the ability to selectively block target gene according to base-pair complementary, and then the quantity and structure change of the hybridized chromosome could be observed by the fluorescence signal under fluorescence microscope. Because of the advantages of simple operation, safety, effective, high security and specificity, fluorescence in situ hybridization has widely been used in the detection of genetic disease, hematological malignancy, solid tumor and so on. With the rapid development of molecular genetics, some specific chromosome aberrat-ions have been often found in urothelial cell carcinoma and prostate cancer, laying theoretical foundation for the application of fluorescence in situ hybridizatio in urogenital cancers. This study is divided into three parts and the clinical application of fluorescence in situ hybridizatio in bladder urothelial carcinomas, upper tract urothelial carcinomas or prostate cancer was evaluated in this study respectively.Purpose To research the relations between aberrations of chromosome 3,7,17, p16 and bladder urothelial carcinoma, by the fluorescence in situ hybridization. To assess the clinical utility of fluorescence in situ hybridization as a non-invasive method in the diagnosis and postoperative follow-up of bladder urothelial carcinoma in Chinese group.Methods Urine samples of both 100 patients with bladder urothelial carcinoma and 30 non-cancer controls were analyzed by means of cytology and fluorescence in situ hybridization. Fluorescence in situ hybridization were used to detect the abnormalities of chromosome 3, 7,17 and p16. We compared the sensitivity and specificity between fluorescence in situ hybridization and cytology and analyzed whether the rates of abnormalities in chromosome 3,7,17, p16 have significant associations with tumor size, tumor number recurrence, clinical and pathologic tumor stages statistically. Bladder washings collected from 66 patients with non-muscle invasive bladder urothelial carcinomas three months after operation were analyzed by the fluorescence in situ hybridization to assess the role of fluorescence in situ hybridization in surveillance of recurring bladder cancer.Result The sensitivity of fluorescence in situ hybridization and cytology was 82% and 36%, and fluorescence in situ hybridization was significantly more sensitive than cytology (p< 0.001). The specificity of fluorescence in situ hybridization and cytology was 96%and 100%, and there was no significant difference between the two groups(p>0.05). The polysomic aberration rates were 63%(3),72%(7),31%(p16),60%(17); the monosomic aberration rates 53%(3),49%(7),47%(p16),46%(17); 34% presented a homozygous deletion of chromosome 9p21. There were no associations between abnormalities of chromosomes and tumor size, tumor number recurrence(p>0.05). The polysomic aberration rates of chromosome 17 were significantly associated with clinical and pathologic tumor stages(p< 0.05), while the other patterns of Chromosome aberrations were not(p>0.05). After a median follow-up of 22 months, 23 patients with non-muscle invasive bladder urothelial carcinomas developed tumor recurrence. The recurrence rate was significantly higher in patients with positive results of fluorescence in situ hybridization (54%) than in patients with negative results(13%) (p<0.001).Conclusions Fluorescence in situ hybridization assay of chromosomes 3,7,17 and p16 is an effective method to detect bladder urothelial carcinomas with with high sensitivity and specificity. FISH might contribute to not only an effective and invasive diagnostic approach to bladder urothelial carcinomas, but also an available tool for predicting tumor recurrence. Purpose To evaluate the possibility and validity of detecting the aberrations of chromosome 3,7,17and p16 by fluorescence in situ hybridization in order to diagnose upper tract urothelial carcinoma.Methods Urine samples of both 23 patients with upper tract urothelial carcinomas and 20 patients without urothelial carcinomas served as control group were analyzed by means of cytology and fluorescence in situ hybridization. The mixtures of fluorescent labeled probes to the centromeres of chromosomes 3,7 and 17, and p16 were used to detect chromosomal abnormalities. Sensitivity and specificity of both methods were determined and cpmpared.The utility of FISH in upper tract urothelial carcinomas were compared to the utility in bladder urothelial carcinomas through statistical analysis.Result The sensitivity of fluorescence in situ hybridization and cytology was 90% and 48%, fluorescence in situ hybridization was significantly more sensitive than cytology (p<0.001). The specificity of both methods wae 100%. The sensitivities of fluorescence in situ hybridization were no significant difference between upper tract urothelial carcinoma and bladder urothelial carcinoma.Conclusions The sensitivity of fluorescence in situ hybridization for the detection of upper tract urothelial carcinomas is superior than that of cytology whilst maintaining a similar specificity, fluorescence in situ hybridization might become a very useful tool in the diagnosis and differential diagnosis of upper tract urothelial carcinoma. Purpose To research the relations between fushion of TMPRSS2 and ETS transcription factor genes (ERG, ETV1, ETV4) and prostater caner, by the fluorescence in situ hybridization. To assess the clinical utility of fluorescence in situ hybridization in the diagnosis and differential diagnosis of prostater caner in Chinese group.Methods The prostate specimens including 53 prostate cancers,20 benign prostatic hyperplasias (BPH),5 high grade prostatic intrae-pithelial neoplasias (HGPIN) were separetely analyzed by means of fluorescence in situ hybridization to assess the gene fushions indiative of malignancy. The relationship between aberration of ERG gene and Gleason’s scores, PSA and clinical stage was analysed respectively.Result Of 53 prostate cancer specimens,TMPRSS2/ERG fusion was detected in 32cases(60%); TMPRSS2/ETV1 fusion was detected in 5 cases(9%) and TMPRSS2/ETV1 fusion in 1 case(2%). 1case(20%) of HGPIN was detected with aberration of ERG gene. None of the three types of gene fushion was detected in all 20 cases of BPH. The sensitivity and specificity of FISH for the detection of prostate cancer was 72% and 96%. Aberration of ERG gene in prostate cancer was significantly associated with Gleason’s scores and pathological stage(p<0.05), while not associated with PSA(p>0.05).Conclusions Fluorescence in situ hybridization assay is an effective method to detect prostate cancer with high sensitivity and specificity, contributing to improving the current diagnosis and differential diagnosis of prostate cancer. The gene fushions of TMPRSS2 and ETS transcription factor genes could be a new approach for the study on etiology of prostate cancer, and an important direction in treatment and prediction of the prognosis.更多还原