尿路肿瘤细胞多重荧光PCR检测微卫星不稳定

目的：研究膀胱肿瘤尿路脱落细胞的微卫星状态及其临床病理关系。方法：35例膀胱肿瘤尿液脱落细胞应用多重荧光PCR检测尿路的微卫星状态。结果：35例尿液样本：膀胱肿瘤组15例,有6例MSI-H,8例MSI-L,1例MSS,诊断阳性敏感度达93.33%;膀胱癌术后复查组6例,血尿及尿路上皮轻度异型组7例,正常体检组7例中,在血尿及轻度异型组发现1例MSI-L,其余均为MSS,阴性特异率达95%。结论：尿路脱落细胞的微卫星检测可作为膀胱肿瘤诊断和监测复发的有效非侵袭性检测方法。     

RT-PCR法检测尿脱落细胞中XIAP的表达在膀胱癌诊断中的价值

目的:比较膀胱癌患者尿液脱落细胞中XIAP表达的RT-PCR检测法和常规尿脱落细胞病理学检测在膀胱癌诊断中的临床价值。方法:采用逆转录聚合酶链反应技术(RT-PCR)检测51例膀胱尿路上皮癌患者尿液脱落细胞中XIAP-mRNA的表达,同时行常规尿脱落细胞病理学检测,20例非肿瘤人员作为对照组。结果:实验组51例尿脱落细胞XIAP-mRNA RT-PCR检测阳性27例(53%),尿脱落细胞学病理学检测阳性12例(24%),对照组20例尿脱落细胞XIAP-mRNA检测阳性1例(5.0%),对照组尿脱落细胞病理学检测阳性0例(0%)。实验组RT-PCR检测膀胱尿路上皮癌患者尿脱落细胞中XIAP表达的敏感性高于尿脱落细胞病理学检测,差异有极显著统计学意义(P<0.01),实验组RT-PCR检测膀胱尿路上皮癌患者尿中XIAP表达的敏感性显著高于非肿瘤对照组,差异有极显著统计学意义(P<0.01)。结论:膀胱尿路上皮癌患者尿脱落细胞中XIAP表达的RT-PCR检测法较常规尿脱落细胞病理学检测更敏感,临床上作为膀胱癌的筛选方法,有一定的临床价值。     

RT-PCR法检测尿脱落细胞中XIAP的表达在膀胱癌诊断中的价值

目的：比较膀胱癌患者尿液脱落细胞中XIAP表达的RT-PCR检测法和常规尿脱落细胞病理学检测在膀胱癌诊断中的临床价值。方法：采用逆转录聚合酶链反应技术（RT-PCR）检测51例膀胱尿路上皮癌患者尿液脱落细胞中XIAP-mRNA的表达,同时行常规尿脱落细胞病理学检测,20例非肿瘤人员作为对照组。结果：实验组51例尿脱落细胞XIAP-mRNA RT-PCR检测阳性27例（53%）,尿脱落细胞学病理学检测阳性12例（24%）,对照组20例尿脱落细胞XIAP-mRNA检测阳性1例（5.0%）,对照组尿脱落细胞病理学检测阳性0例（0%）。实验组RT-PCR检测膀胱尿路上皮癌患者尿脱落细胞中XIAP表达的敏感性高于尿脱落细胞病理学检测,差异有极显著统计学意义（P〈0.01）,实验组RT-PCR检测膀胱尿路上皮癌患者尿中XIAP表达的敏感性显著高于非肿瘤对照组,差异有极显著统计学意义（P〈0.01）。结论：膀胱尿路上皮癌患者尿脱落细胞中XIAP表达的RT-PCR检测法较常规尿脱落细胞病理学检测更敏感,临床上作为膀胱癌的筛选方法,有一定的临床价值。     

微卫星状态与术后大肠癌患者的临床及病理特征相关性研究

  目的  分析微卫星状态与术后大肠癌患者的临床及病理特征的相关性。  方法  选取南京大学医学院附属鼓楼医院2014年6月至2017年6月病理诊断确诊的572例大肠癌患者的临床及病理资料,并对其手术切除标本进行微卫星状态与KRAS、NRAS突变状态的检测,同时采用免疫组织化学法检测组织中Ki-67、EGFR、MGMT及Lgr5的表达情况。按照微卫星不稳定性（microsatellite instability,MSI）状态,分为高度微卫星不稳定（high-frequency MSI,MSI-H）大肠癌组与微卫星稳定状态（microsatellite stability,MSS）和低度微卫星不稳定（low-frequency MSI,MSI-L）大肠癌组,比较组间的临床、病理等资料的差异。  结果  572例大肠癌患者中40例（7.0%）为MSI-H,532例（93.0%）为MSS/MSI-L。与MSS/MSI-L组大肠癌病例相比MSI-H组具有以下特征:1）病灶常位于右半结肠;2）不同年龄段的发病比例相当;3）肿瘤分期较早,即Ⅰ/Ⅱ期分期比例较大（P=0.003）;4）淋巴结转移比例较低（P= 0.023）;5）形成癌结节比例较低（P=0.005）;6）KRAS第2外显子突变率较低（P=0.004）,NRAS未突变。两组在MGMT、EGFR、Lgr5、Ki-67免疫组织化学阳性率检测未见显著性差异。  结论  微卫星状态与大肠癌患者的年龄相关,与MSS/MSI-L的大肠癌患者相比,MSI-H的患者具有其特殊的临床病理特征,且NRAS及KRAS第2外显子的突变率低,MGMT甲基化率高,可为大肠癌的个体化诊断和治疗提供依据。      

微卫星不稳定性在膀胱癌复发诊断中的价值

背景与目的:评估尿脱落细胞中微卫星不稳定性(Microsatelliteinstability,MSI)在膀胱癌复发诊断中的价值及可能的临床意义。材料与方法:对60份膀胱癌术后随访尿液标本及20份正常对照标本进行微卫星不稳定性的检测。分析参数包括有无血尿、肿瘤数目、大小、WHO分级及5个微卫星位点标志物。结果:20例复发者中17例MSI阳性(85.00%),且15例为血尿标本,检测术后随访者尿脱落细胞中MSI诊断膀胱癌复发的敏感性、特异性及阳性预报值分别为85.00%、82.50%及83.33%,MSI与肿瘤分级间无明显相关性。结论:MSI可以作为膀胱癌术后随访病人尤其伴有血尿者监测肿瘤复发的良好标志物。     

尿液P16基因甲基化对浅表性膀胱移行细胞癌早期诊断的意义

目的探讨P16基因甲基化在浅表性膀胱移行细胞癌发生早期的临床意义。方法取42例膀胱移行细胞癌组织及配对尿液标本,以其中9例的对侧正常膀胱黏膜组织作对照,采用甲基化特异性PCR方法检测P16基因CpG岛甲基化状态。结果35例肿瘤组织标本,18例P16基因CpG岛存在甲基化状态,占51.4%（18/35）;尿液标本中,16例P16基因CpG岛存在甲基化状态,占45.7%（16/35）。Log istic回归分析显示年龄、性别、是否吸烟、合并其他疾病、肿瘤的临床分期和病理分型等因素对P16基因甲基化无影响（P〉0.05）。20例浅表性膀胱移行细胞癌患者的尿液标本中发现P16基因甲基化10例,其配对的浅表性膀胱移行细胞癌组织中均检出P16基因甲基化,正常人和非尿路肿瘤患者尿液中未检出到P16基因甲基化,尿液P16基因甲基化在浅表性膀胱移行细胞癌中的阳性预测值和特异度均为100%,假阳性为0%,灵敏度为90.91%。正常人组和非尿路肿瘤组与浅表性膀胱移行细胞癌组尿液中P16基因甲基化比较差异有统计学意义（P〈0.001）,9例正常组织对照均未发现P16基因CpG岛甲基化。结论P16基因甲基化与年龄、性别、肿瘤的分级分期无相关性;尿液P16基因异常甲基化可成为浅表性膀胱移行细胞癌早期诊断的分子生物学标志。     

荧光原位杂交在尿路上皮癌诊断中的应用

目的:检测尿路上皮肿瘤患者尿液脱落细胞染色体的缺失和非整倍异常,探讨FISH技术作为尿路上皮肿瘤患者无创诊断方法的价值.方法:收集可疑尿路上皮肿瘤患者和健康对照人群的新鲜尿液,同步进行细胞形态学分析及荧光原位杂交(Fluorescencein situ hybridization,FISH)检测3号、7号及17号染色体、9号染色体p16位点异常.共入选可疑尿路上皮肿瘤患者100例,正常健康对照组20例,采用正常对照组患者各染色体异常数据设定阈值用于肿瘤患者的实验室诊断.根据检验结果与病理结果对照分别计算FISH和脱落细胞的敏感度和特异度并进行统计学分析.结果:与正常对照组相比,尿路上皮肿瘤患者尿液脱落细胞染色体异常明显增多.尿脱落细胞学的敏感度和特异度分别为71%和80%,FISH的敏感度和特异度分别为88%和80%(P<0.01).根据两种检测方法的敏感度和特异度绘制的接受者工作特征(ROC)曲线显示尿脱落细胞学和FISH的曲线下面积分别为0.758和0.842.结论:对可疑尿路上皮肿瘤的患者进行FISH检测是一种有价值的无创检测方法.FISH的总体敏感度高于尿脱落细胞学,特异度与尿脱落细胞学相当.     

Survivin分子信标诊断膀胱肿瘤的临床应用研究

目的:探讨分子信标检测尿脱落细胞Survivn mRNA的可行性,寻找一种能够早期诊断膀胱肿瘤的方法。方法:分子信标检测膀胱肿瘤5637、J82细胞Survivin mRNA的表达,并通过Western bolt方法验证,并对35例膀胱移行细胞癌患者和35名正常健康成人行分子信标检测尿脱落细胞,Western bolt检测组织中的Survivin含量,同时行尿脱落细胞学检查。结果:Survivin分子信标检测肿瘤细胞内的Survivin表达且具有高特异性。以随机100个细胞中60个以上的细胞为阳性做为阳性标准,确定MB-cy3的阳性率为80%(28/35),特异性为77.1%(27/35);Western bolt检测的阳性率为71.4%(25/35)。两种实验方法对细胞和蛋白质中Survivin的检测结果具有一致性。尿脱落细胞学的敏感性为28.6%(10/35),特异性为100.00%(35/35)。结论:使用分子信标检测尿液脱落细胞中Survivin mRNA具有可行性,分子信标有可能成为在膀胱肿瘤的早期临床诊断和术后随访的有效工具。     

微卫星不稳定结直肠癌临床病理特征及生存预后

目的 国内外已有学者提出微卫星不稳定(microsatellite instability,MSI)状态可能是影响结直肠癌(colorectal cancer,CRC)患者预后的因素,同时提出微卫星不稳定结直肠癌患者存在较为特殊的临床病理特征,本研究旨在探讨微卫星不稳定CRC的临床病理特征及生存预后.方法 应用免疫组织化学方法检测2010-03-24-2015-12-24济南市第四人民医院60例CRC组织中人MutL蛋白同系物1(human mutl homologue 1,hMLH1)、人MutS蛋白同系物2(human muts homologue 2,hMSH2)及人MutS蛋白同系物6(human muts homologue 6,hMSH6)3种DNA错配修复蛋白表达缺失情况,判断肿瘤微卫星不稳定状态,并分析高度微卫星不稳定(microsatellite instability-high,MSI-H)和低度微卫星不稳定(microsatellite instability-low,MSI-L)/微卫星稳定(microsatellite stable,MSS)不同组别间的临床病理特征及生存预后情况;应用Cox风险比例模型对可能影响CRC患者预后的因素进行多因素分析.结果 60例CRC患者的肿瘤组织中MSI-H为40.0%(24/60),MSI-L为31.7%(19/60),MSS为28.3%(17/60).MSI-H的CRC患者,与MSS和MSI-L患者相比,好发于右半结肠(χ2=6.279,P=0.043),黏液腺癌多见(χ2=6.025,P=0.049);3组在性别、年龄、分期、肿瘤浸润深度、淋巴结转移和分化程度差异无统计学意义.MSI-H患者的中位无病生存期(disease-free survival,DFS)为21个月,明显长于MSS的11个月及MSI-L的13个月,χ2=7.994,P=0.018.多因素Cox分析结果显示,淋巴结转移(P=0.013)和MSI(P=0.018)为CRC患者DFS的独立预后因素.结论 MSI-H的CRC患者与MSI-L及MSS相比,具有独特的临床病理特征且预后相对较好.检测MSI状态对提高CRC治疗水平,及改善预后有重要的临床意义.     

尿脱落细胞Lewis X检测诊断膀胱尿路上皮癌的价值

目的探讨Lewis X抗原在膀胱尿路上皮癌非侵袭性诊断中的应用价值.方法采用EnVision免疫细胞化学方法,检测52例膀胱尿路上皮癌和16例非肿瘤患者尿脱落细胞标本中Lewis X抗原的表达情况,并与细胞病理学检测结果相比较.结果尿路上皮癌诊断的敏感性和特异性分别为84.6%和87.5%,其敏感性显著高于细胞病理学.结论尿脱落细胞Lewis X抗原免疫染色,是检测膀胱尿路上皮癌可行的较敏感的非侵袭性方法.     

Microsatellite instability in transitional cell carcinoma of the urinary tract and its relationship to clinicopathological variables and smoking

To determine whether microsatellite instability is involved in the development of transitional cell carcinoma (TCC) of the urinary tract, a microsatellite instability assay was carried out using PCR with 9 microsatellite loci. Thirty-eight TCC samples (30 patients with bladder cancer, 5 with renal pelvic tumors and 3 with ureteral tumors) and 1 lymph node with metastasis were examined. Microsatellite instability was found in 8 of 38 tumors examined, and 3 showed alterations in more than 2 microsatellite loci. All 8 tumors were beyond grade 2 and stage pT2 advanced tumors. Stages pT1-2 and pT3-4 patients differed significantly. Microsatellite instability was greater in smokers than non-smokers, but the differences were not significant. Microsatellite instability in TCC of the urinary tract is rare in superficial tumors but more common in invasive tumors. Microsatellite alterations would thus appear to occur, and possibly be importantly involved, in the tumorigenesis of urinary tract TCC. © 1996 Wiley-Liss, Inc.     

Distinct patterns of microsatellite instability are seen in tumours of the urinary tract

To date, two forms of microsatellite instability (MSI) have been described in human cancer. MSI typical of hereditary nonpolyposis colon cancer (HNPCC), is due to deficient DNA mismatch repair (MMR) and is defined with mono- and dinucleotide repeat microsatellites. A second variety of instability is best seen at selective tetranucleotide repeats (EMAST; elevated microsatellite alterations at select tetranucleotides). While MSI occurs infrequently in bladder cancers, EMAST is common. Sporadic tumours with the largest proportion showing MSI are those found most frequently in HNPCC kindreds. While bladder cancer is not frequently seen in HNPCC, upper urinary tract tumours (UTTs) are. Having previously found a low frequency of MSI in bladder cancer, we sought to determine the relative levels of MSI and EMAST in transitional cell carcinoma (TCC) of the upper and lower urinary tracts. Microsatellite analysis was performed at 10 mono- and dinucleotide and eight tetranucleotide loci, in 89 bladder and 71 UTT TCC. Contrasting patterns of instability were seen in urinary tumours. In bladder cancer, MSI was rare and EMAST was common. The presence of EMAST was not related to tumour grade, stage, subsequent outcome or immunohistochemical expression of the MMR proteins. In UTT, while MSI occurred frequently, EMAST was seen less frequently than in bladder cancer. When TCC of the upper and lower urinary tracts are compared, MSI-H is more frequent in UTT and EMAST more frequent in bladder cancer. Our findings show that, as for colorectal cancer, the pattern of MSI varies with location in the urinary tract. In addition, we have confirmed that MSI and EMAST are discrete forms of MSI, and that the presence of EMAST does not affect tumour phenotype.     

Microsatellite alterations in urinary sediments from patients with cystitis and bladder cancer

Microsatellite instability (MIN) and loss of heterozygosity (LOH) in bladder cancer have been suggested for diagnosis and follow-up of bladder cancer based on urinary sediments, reflecting tumor alterations. We have examined 6 microsatellites in urine sediments from 11 patients with transitional-cell carcinomas (TCC) and in 31 patients with benign prostatic hyperplasia (BPH), 22 of whom had cystitis. In the TCC patients, tumor tissue was available for comparison with urine. Microsatellites were amplified by PCR and compared with leukocyte DNA from the same individual in silver-stained gels. Altered mobility of bands, new bands and loss of bands were scored. We found MIN and LOH at relatively high frequency in markers from chromosomes 8 and 14 in urine from patients with TCC, but also in BPH patients who had cystitis. Even control patients with BPH without cystitis showed some instability and some losses. Novel bands in urine occurred significantly more often among TCC patients than among BPH patients with or without cystitis (p < 0.001). Band shifts in urine appeared to be more associated with BPH plus cystitis than with TCC. The alterations we found in urine from patients with bladder cancer did not always reflect those found in their tumors, the occurrence of novel bands being significantly higher (p < 0.008) in tumor tissue than in corresponding urine. In conclusion, microsatellite alterations in urine are indicators not only of malignancy but also of inflammatory conditions.     

检测尿脱落细胞中微卫星不稳定性的临床意义

目的:探讨膀胱癌患者尿脱落细胞微卫星不稳定性及临床意义.方法:将42例患者尿液标本经适当处理后,采用PCR-PAGE-银染方法检测尿脱落细胞中微卫星不稳定性的情况,分析其与临床病理参数的关系.结果:膀胱癌患者尿中MSI阳性率为59.52%(25/42),浸润性肿瘤患者尿中阳性率较高,但无统计学意义(P＞0.05);复发病例中阳性率(80.00%)比初发者(40.91%)明显要高(P＜0.05).结论:检测尿脱落细胞微卫星不稳定性可以作为膀胱癌的一种无创诊断及筛查手段.     

尿脱落细胞微卫星DNA改变在膀胱癌早期诊断中的应用

目的 利用检测尿脱落细胞微卫星ＤＮＡ序列（ｍｉｃｒｏｓａｔｅｌｌｉｔｅ,ＭＳ）改变,建立早期诊断膀胱癌的方法。方法 选择１０对微卫星ＭＳ引物,利用聚合酶链反应（ＰＣＲ）方法,以自身外周血和膀胱癌组织为对照,检测２８例膀胱癌患者尿脱落细胞中ＭＳ的失杂合（ｌｏｓｓ ｏｆ ｈｅｔｅｒｏｚｙｇｏｓｉｔｙ,ＬＯＨ）和不稳定性（ｍｉｃｒｏｓａｔｅｌｌｉｔｅ ｉｎｓｔａｂｉｌｉｔｙ,ＭＩＮ）。结果 ２８例膀胱癌患者中,２４例（８５．７％）尿脱落细胞至少在１个ＭＳ位点存在ＬＯＨ或ＭＩＮ改变,３例（１０．７％）脱落细胞学检查阳性患者尿脱落细胞均检出ＬＯＨ或ＭＩＮ。同一患者尿脱落细胞与癌组织ＬＯＨ改变一致率为９４．１％。１５例正常人悄脱落细胞中未见ＭＳ的改变。结论 利用检测尿脱落细胞ＭＳ改变诊断膀胱癌,比常规的细胞学检查更敏感、更     

Alterations of microsatellite repeats in bladder-cancer

Investigation of the genomic instability of microsatellite repeats indicates a new mechanism for human carcinogenesis. This study was conducted to determine whether such alterations in microsatellite repeats are associated with the onset of bladder cancer. Thirty-two primary bladder cancer DNA samples were examined for genomic instability at (CA)n repeats on human chromosomes 5q (D5S107), 17p (D17S261) and 18q (DCC) by polymerase chain reaction (PCR) assay. Differences in unrelated microsatellites for tumor and normal DNA were detected in 6 of the 32 (18.8%) tumors examined. These six tumors were beyond grade 2 and stage pT2 invasive bladder tumors. However, only one of 32 (3.1%) showed alterations with more than 2 microsatellite probes. It follows that alterations of (CA)n microsatellite instability may be infrequent in the tumorigenesis of bladder cancer.     

微卫星不稳定散发性大肠癌的临床病理特征和DNA倍体研究

目的:探讨微卫星不稳定的散发性大肠癌的临床病理特征及微卫星不稳定表型和DNA倍体类型的关系。方法:对71例散发性大肠癌行BAT25和BAT26两个位点的微卫星不稳定检测和流式细胞术倍体分析,探讨微卫星不稳定状态和临床病理特征及DNA倍体类型的关系。结果:微卫星不稳定的阳性率为9.86%(7/71),微卫星不稳定表型和发病部位、组织学类型及分化程度相关(P<0.05),而与性别、年龄、淋巴结转移和分期无关。微卫星不稳定的散发性大肠癌中右半结肠癌和低分化腺癌的比例高于微卫星稳定者。68例患者检出二倍体和异倍体分别为18和50例,微卫星不稳定表型者5例为二倍体,因此和DNA倍体类型显著相关(P=0.012)。结论:微卫星不稳定的散发性大肠癌好发于右半结肠,具有低分化腺癌的倾向,多为二倍体。     

Sensitive detection of transitional cell carcinoma of the bladder by microsatellite analysis of cells exfoliated in urine.

Transitional cell carcinoma (TCC) is the most common bladder tumor. Urine cytology can identify most high-grade tumors but sensitivity is lower if one includes lesions of all grades. Microsatellite marker alterations have been found in many tumor types including bladder cancer and have been used to detect cancer cells in body fluids including urine. The aim of our study is to further evaluate feasibility and sensitivity of microsatellite analysis to detect bladder cancer cells in urine. We studied 55 individuals: 21 with symptoms suggestive of bladder cancer, 23 patients with previous history of TCC and 11 healthy subjects. Genomic DNA was extracted from blood lymphocytes, urine sediment, bladder washings and tumor or normal bladder mucosa. Twenty highly informative microsatellite markers were analyzed for loss of heterozigosity (LOH) and microsatellite instability (MIN) by polymerase chain reaction. Microsatellite analysis of urine identified 33 of 34 (97%) patients with either primary or tumor recurrence, whereas urine cytology identified 27 of 34 (79%) patients (p = 0.0001). Detection of microsatellite abnormalities improved the sensitivity of detecting low-grade and/or stage bladder tumor: from 75-95% for grades G1-G2 and from 75-100% for pTis-pTa tumors. Bladder washings from 25 patients were also analyzed, and in all cases results were identical to those obtained from voided urine. None of the 16 patients without evidence of TCC showed LOH and/or MIN in urine samples or bladder washings. Interestingly, in a patient with persistent bladder mucosa abnormalities, microsatellite alterations were demonstrated 8 months before the histopathologic diagnosis of tumor recurrence. These results further indicate that microsatellite marker analysis is more sensitive than conventional urine cytology in detecting bladder cancer cells in urine and represents a potential clinical tool for monitoring patients with low-grade/stage TCC.     

Microsatellite alterations in superficial and locally advanced transitional cell carcinoma of the bladder.

Recent studies described the existence of genetic instability associated with bladder carcinogenesis. Alterations at microsatellite loci constitute a recognized tumor marker of genome instability. A series of 21 transitional cell carcinomas of the bladder (10 superficial and 11 invasive carcinomas) was analyzed for the presence of alteration in 12 microsatellite loci, in order to detect the role of microsatellite instability in genesis and progression of human bladder cancer. Our preliminary results indicate a trend to presence of microsatellite instability (MI) in invasive and undifferentiated tumors compared to superficial and differentiated forms. Eight out of 11 T2-T4 tumors presented a number of altered microsatellite >/=2 compared to one out of 10 Ta-T1 bladder carcinomas (p=0.008). Moreover, 9 out of 15 (60%) G2-G3 tumors had significantly more unstable microsatellites than those differentiated (0 out of 6) (p=0.019). Our results provide an insight into the potential usefulness of microsatellite analysis of bladder carcinoma to better understand which neoplastic forms will evolve to invasive progression and indicate that pronounced MI may be associated with more aggressive bladder carcinomas.