荧光原位杂交在尿路上皮癌及前列腺癌中的应用研究

【作者】 丁雪飞；

【导师】 李汉忠； 纪志刚； 毛全宗；

【作者基本信息】 中国协和医科大学 ， 泌尿外科学， 2010， 博士

【摘要】 目的评价FISH用于诊断泌尿系尿路上皮癌的临床应用价值。方法收集北京协和医院泌尿外科尿路上皮癌患者共241例,其中膀胱尿路上皮癌183例,上尿路上皮癌58例,行FISH检查及尿细胞学检查。同时以健康志愿者20例,建立本实验室FISH的正常阈值,以非尿路上皮癌患者40例为对照。FISH探针采用随机引物法标记3，7，17号染色体着丝粒及9p21(p16)区带。结果1、以健康志愿者建立FISH正常阈值。2、FISH对于膀胱尿路上皮癌敏感性共为85.4%(145/158),特异性为90%(36/40)。在低级别尿路上皮癌与高级别之间敏感性有统计学差异(P=0.002)；非肌层浸润性尿路上皮癌与肌层浸润性之间无统计学差异(P=0.487)。细胞学检查敏感性为41.1%(65/158),特异性为92.5%(37/40)。3、FISH;对于UUC-UT的敏感性高于尿细胞学检查(P=0.0001)；两者阳性预测值无明显差异(P=0.94),阴性预测值则有统计学差异(P=0.02)。两者对于非肌层浸润性UUC-UT患者两者检查分别是71.1%和24.4%(P<O.0001),肌层浸润性肿瘤分别为81.8%和48.9%(P=0.016)；在低级别肿瘤的敏感性分别是50%和12.5%(P=0.027),高级别肿瘤为87.0%和44.4%(P＜0.01)。尿细胞学和FISH检查的特异性无明显差别,P=0.548。探针中3，7,17染色体非整倍性扩增分别为53.8%、56.4%和39.7%，p16杂合性缺失丢失25.6%。结论1、FISH法诊断尿路上皮癌灵敏度比尿脱落细胞学高、特异性相似,是一种较为理想的无创性检查方法。2、FISH结果显示其敏感性与尿路上皮癌病理分级具有相关性,并且随着病理分级的增加敏感性增加；与临床分期无相关性。3、在膀胱尿路上皮癌术后随访中由于肿瘤临床分期较早,尿细胞学检查的敏感性更低,相比较而言FISH检查仍具有较高的敏感性和特异性,适合术后随访工作。4、p16杂合性缺失丢失出现频率明显低于其他三组探针,可能与其正常阈值较高有关。目的：1、评价在会阴模板定位下行经会阴前列腺穿刺活检术有效性及安全性；2、应用FISH技术检测前列腺活检组织中TMPRSS2基因与ETS家族基因ERG、ETV1、ETV4之间融合的表达情况,进而评价FISH诊断早期前列腺癌的可行性。方法：1、选择我院437例接受经直肠超声引导下模板定位经会阴前列腺穿刺活检术患者。年龄平均70.4±14.35岁(33-86岁)。PSA 0.2-5000ng/ml,平均80.3±12.96ng/ml。前列腺体积10-200ml,平均57.6±14.72ml。2、采用随机法合成FISH探针：TMPRSS2与ERG、ETV1、ETV4融合基因。(1)在模板定位下经会阴前列腺分区穿刺活检术行穿刺活检,病理确定前列腺癌患者40例,为实验组,应用FISH技术检查前列腺癌患者组织中TMPRSS2-ERG、TMPRSS2-ETV1和TMPRSS2-ETV4融合基因。良性前列腺增生患者20例,为对照组。(2)分析融合基因与前列腺癌组织生物学特性中Gleason评分、前列腺体积、临床分期和PSA水平的关系。结果：1、将直肠超声下前列腺分为11各区,每区穿刺1-4针,每例平均18.6(11-44)针。活检阳性率38.5%(168/437)。2、活检率与前列腺穿刺相关参数之间关系：(1)与分区关系：不考虑PSA水平时,1-11区阳性检出率无统计学差异,P=0.16。当PSA<20 ng/ml时,前列腺11区的检出率为47.9%,高于其他区,P<0.05；(2)与前列腺体积关系：阳性检出率与前列腺体积成负相关；(3)与PSA水平关系：当PSA＞4ng/ml时,阳性检出率与PSA水平呈正相关,PSA<4ng/ml时,阳性检出率与PSA水平无相关性；(4)与前列腺前后区关系：前列腺前区(1、2、9、10)与后区(4、5、6、7)阳性检出率比较无统计学差异,P=0.45。3、穿刺1周内肉眼血尿发生率为29.7%(130/437),尿潴留发生率为1.1%(5/437),无严重感染及直肠感染等并发症。4、FISH用于前列腺癌诊断中的40例前列腺癌患者阳性率为80%(32/40),其中TMPRSS2-ERG探针为52.5%(21/40), TMPRSS2-ETV1为22.5%(9/40),TMPRSS2-ETV4为5%(2/40)。三组探针中有一组阳性者,其余两组即为阴性,无重复阳性患者。5.FISH结果与前列腺癌生物学特性关系：FISH阴性组与阳性组Gleason评分无统计学差异,P=0.874。FISH阴性组与阳性组PSA无统计学差异,P=0.141。FISH阴性组与阳性组临床分期无统计学差异,P＞0.05。实验组淋巴结pN0为35例,pN1-2为5例,FISH阴性组与阳性组与淋巴结转移无统计学差异，P＞0.05。根治性前列腺癌11例,切除后切缘情况：切缘阴性为8例,阳性为3例,FISH阴性组与阳性组切缘阳性与否无统计学差异，P＞0.05。结论：1、模板定位下经会阴前列腺分区穿刺活检阳性率较高,并与前列腺体积、血清PSA水平高具有相关性。2、当PSA<20 ng/ml时,11区的肿瘤检出率较高。3、模板定位下经会阴前列腺分区穿刺安全性高,手术并发症低。4、FISH三组探针可以检测出前列腺癌组织中的TMPRSS2-ERG和TMPRSS2-ETV1以及TMPRSS2-ETV4融合基因,出现频率以TMPRSS2-ERG最高。FISHT阳性组与阴性组与前列腺癌生物学特性,如Gleason分级、前列腺体积、临床分期和PSA水平之间无统计学差异。5、FISH检测前列腺癌TMPRSS2-ERG和TMPRSS2-ETV1以及TMPRSS2-ETV4融合基因有望成为早期诊断前列腺癌的一个新的有力手段。更多还原

【Abstract】 Objectives:To evaluate the clinical utility of a Multiprobe FISH (fluorescence in situ hybridization, FISH) Assay in Voided Urine Specimens for the detection of Urothelial carcinomas (UC) comparing the results with those afforded by urinary cytology. Methods:Voided urine specimens from 241 patients who had Urothelial carcinomas,183 patients with bladder cancer and 58 consecutive patients with UUT(upper urinary trac) UC and 40 healthy controls were analyzed by means of cytology and FISH. FISH was performed using a mixture of fluorescent labeler DNA probes for the centromeric regions of chromosomes 3,7, and 17 and 9p21 region.Results 1.The normal range of FISH was blind by 20 health volunteers.2. Overall sensitivity of FISH for bladder cancer was 85.4%(145/158) and specificities of FISH was 90%(36/40). There was significantly difference between low grade and high grade tumors (P=0.002). However, there was no significantly difference between non and muscle-invasive bladder cancer (P=0.487). Urinary cytology affords an overall sensitivities of 41.1% (65/158) and specificities of 92.5%(37/40). Overall sensitivity of FISH was significantly higher than that of urine cytology (P= 0.0001). The positive values was no significantly of difference between the urine cytology and FISH(P=0.94), and the negative predictive values was significantly (P=0.02). The sensitivities of FISH and cytology were 71.1% and 24.4%(P<0.0001) in non muscle-invasive UUT, and were 81.8% and 48.9%(P=0.016) in muscle-invasive UUT. The sensitivities were 50% and 12.5%(P=0.027) in low grade cancers and 87.0% and 44.4%(P<0.01) in high grade tumors.Specificities for both FISH and cytology were similar results P=0.548. Of 58 patients with UUT UC, polysomies of chromosome 3,7 and 17 were observed in 52.3%,56.9% and 35.7%, respectively, and loss of the p16 gene in 27.3%.Conclusion:1. FISH assay of chromosomes 3,7,9, and 17 performed on exfoliated cells from voided urine specimens has greater sensitivity than cytology for detecting UUT-UC whilst maintaining a similar specificity.2. The sensitivities of FISH is significantly correlated to pathologic grade of UC, and With the increase in sensitivity to increased pathological grade. There is no correlated between the sensitivities of FISH and tumors stage.3. FISH has higher sensitivity for recurrent UC, and it could be useful for monitoring in the follow-up after surgery.4. The aberrations of chromosomes p16 is lower than of 3,7,17 chromosomes, and one of causes clould is relatively strict of normal range. Objective 1. To assess the feasibility and advantage of Systematic transperineal ultrasound guided template prostate biopsy.2. We analyzed genetic rearrangements between TMPRSS2 and ETS (ERG, ETV1 and ETV4) in prostate cancer, and evaluated the clinical utility of in patients treated with prostate biopsy detecting PCa by FISH (Fluorescence in situ hybridization), which of the probes of TMPRSS2-ERG, ETV1 and ETV4.Methods 1. In a prospective study, a total of 437 patients (33-86year old,mean age 70.4±14.35) who met the inclusion criteria underwent 11 regions systematic transperineal ultrasound guided template prostate biopsy. The median PSA level was 80.3±12.9612.96 ng/ml (range 0.2-5000ng/ml) and the mean prostate volume was 57.6±14.72ml (range 10-200ml).2. FISH was performed using a mixture of fluorescent labeler DNA probes for the TMPRSS2-ERG, ETV1 and ETV4 fusion. (1) 40 patients were identified by prostate pathology,who met the inclusion criteria underwent 11 regions systematic transperineal ultrasound guided template prostate biopsy. They was Experimental group and 20 patients of benign prostatic hyperplasia controls were analyzed by means of FISH. (2) We analyzed the fusion gene and the biological features of prostate cancer Gleason score, prostate volume, pTNM stage and PSA Levels.Results 1. Prostate cancer was detected in 168 of 486 (38.5%).2. Prostate needle biopsy rate and the parameters relationship:(1) Relationship with the areas:the positive rate of 1-11 areas was no significant difference, P=0.16. The mean positives for the cancer of regions 1-10 and region 11 (theapical region) were 35.6%vs.47.9% in patients whose PSA<20 ng/ml (P<0.05); (2)and prostate volume relationship:The positive rate was negatively correlated with prostate volume; (3) and PSA level of relationship:When PSA> 4ng/ml, the positive detection rate was positively correlated with PSA level, PSA<4ng/ml, the positive detection rate of PSA levels were not related; (4) The positives for cancer contained within the anterior (1,2,9,10) and posterior parts (4,5,6,7) were no insignificantly (P>0.05) in all patients. Incidence of gross in hematuria 29.7%(130/437), urinary retention rate 1.1%(5/437) after puncture 1 week, and no serious complication occurred during the procedure.3. In 40 patients, the prostate cancer positive rate of 80%(32/40) by FISH, in which TMPRSS2-ERG probe was 52.5%(21/40), TMPRSS2-ETV1 was 22.5%(9/40), TMPRSS2-ETV45%(2/40). Three probes in a positive, and the remaining two shall be negative. No repeat positive patients.4. The relationship between biological characteristics of prostate cancer and FISH:No significant association between the presence of the fusion gene and any clinical feature, such as Gleason score, prostate volume, pTNM stage and PSA Levels.Conclusions 1. Systematic transperineal ultrasound guided template prostate biopsy is accurate, and there is relevance between the rate of positive and Prostate volume, serum PSA level.2. Prostate carcinoma foci are more frequently localized in the apical region in patients with PSA<20 ng/ml.3. Systematic transperineal ultrasound guided template prostate biopsy has no serious complication occurred during the procedure.4. FISH probes can detect the fusion gene of prostate cancer in TMPRSS2-ERG, TMPRSS2-ETV1 and TMPRSS2-ETV4. There is are no significant difference between positive and negative group of FISH and the biological characteristics of prostate cancer, such as Gleason grade, prostate volume, clinical stage and the PSA level.5. FISH is expected to become a new powerful tool in early diagnosis of prostate cancer.更多还原