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。      

端粒酶活性检测在膀胱肿瘤早期诊断中的应用

目的：探讨检测端粒酶活性在膀胱癌早期诊断中的临床意义。方法：采用TRAP-PCR-ELISA法检测32例膀胱癌患者尿液和膀胱冲洗液中脱落细胞、膀胱癌组织、20例正常膀胱组织及14例非膀胱肿瘤患者尿液脱落细胞中端粒酶活性，并行尿脱落细胞学检查。结果：32例膀胱癌患者尿液脱落细胞、膀胱冲洗液脱落细胞、膀胱癌组织中端粒酶活性阳性率分别为65．6％（21／32）、71．9％（23／32）和84．0％（27／32），20例正常膀胱组织端粒酶活性均为阴性，14例非膀胱肿瘤患者尿液中1例端粒酶活性阳性。端粒酶活性阳性表达与肿瘤的分级、分期之间差异无明显相关性（P〉0．05），敏感性明显高于脱落细胞病理学检查。结论：尿液、膀胱冲洗液脱落细胞端粒酶活性测定敏感性较高．可用于膀胱癌的早期诊断。     

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。     

尿脱落细胞LewisX检测诊断膀胱尿路上皮癌的价值

目的：探讨LewisX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LewisX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LewisX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。     

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。     

散发性结直肠癌微卫星不稳定状态与错配修复蛋白表达缺失及临床病理特征的相关性

目的： 探讨散发性结直肠癌微卫星不稳定(microsatellite instability,MSI)情况及其与错配修复 (mismatch repair,MMR) 蛋白MLH1、MSH2、MSH6、PMS2表达缺失的相关性,并总结MSI散发性结直肠癌的临床病理学特征。方法：多重荧光PCR法检测散发性结直肠癌肿瘤组织DNA的微卫星不稳定性,免疫组化(Immunohistochemistry,IHC)S-P法检测散发性结直肠癌肿瘤组织MLH1、MSH2、MSH6、PMS2蛋白的表达缺失,分析MSI发生与MMR蛋白表达缺失及临床病理特征的相关性。结果：75 例散发性结直肠癌检出MSI 21例(28%),包括 MSI-H 19例、MSI-L 2例,其他54例(72%)为MSS。检出MMR蛋白表达缺失16例(21.33%),其中15例(93.75%)为MSI-H、1例(6.25%)为MSS;MMR蛋白表达59例(78.67%),其中4例(6.78%)为MSI-H、2例(3.39%)MSI-L,其他53例为MSS。MSI组MMR蛋白缺失率(15/21,71.43%)显著高于MSS组(1/54,1.9%)（P<0.01）。MSI与患者年龄,是否黏液腺癌,肿瘤有无远处转移有关（P<0.01）,其中MSI-H好发于年龄>50岁、肿瘤无远处转移、MMR蛋白缺失人群,且类型以黏液腺癌为主。结论： 散发性结直肠癌肿瘤组织中MSI发生率高于MMR蛋白缺失率,并且MSI-H的散发性结直肠癌转移风险较低、预后较好。检测MSI 状态对提高结直肠癌的预防、诊断和治疗水平,降低结直肠癌的发病率和病死率有着重要意义。     

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.     

尿细胞角蛋白检测与尿脱落细胞学检查在膀胱癌诊断中的价值

目的:探讨尿细胞角蛋白检测与尿脱落细胞学检查在膀胱移行细胞癌诊断中的价值。方法:136例怀疑膀胱癌者,进行尿细胞角蛋白8和18的含量(UBC值)。检测与尿细胞学检查,其中87例经组织学证实为膀胱移行细胞癌。比较两者诊断膀胱癌的敏感性和特异性。结果:尿细胞角蛋白的敏感性为70.1%,特异性为73.3%;尿细胞学的敏感性为42.5%,特异性为83.7%。尿细胞角蛋白在膀胱癌不同分级和分期中的敏感性优于尿细胞学(P<0.05)。结论:尿细胞角蛋白的检测在早期诊断膀胱癌方面优于尿细胞学检查,可作为膀胱癌的早期检测指标。     

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.     

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     

Early diagnosis and monitoring of superficial transitional cell carcinoma by microsatellite analysis on urine sediment

It has recently been shown that allelic abnormalities, detected by microsatellite analysis of the DNA extracted from urine sediment, can be successfully used for the detection of transitional cell carcinoma (TCC) of the bladder. The diagnostic accuracy of urinary cytology, urinary bladder cancer (UBC) marker, bladder tumor antigen (BTA) and microsatellite sequence alterations was compared in 42 patients who were recruited for the study. Of them, 30 had been diagnosed with TCC at cystoscopy plus biopsy (group A). Seven patients without any apparent lesions after trans-urethral resection (TUR) and 6 subsequent weeks of endovesical administration of bacillus Calmette-Guerin (BCG), had irritative symptoms. None of them had positive cytology or TCC bladder mucosa biopsies (group B). In the control group were 5 other subjects who were affected by benign prostatic hypertrophy and candidates for prostatectomy (group C). Urine and blood samples were obtained from all of the patients before surgery. Tumor tissue and normal mucosa samples were taken from groups A and C during surgery. Different urinary sediment analyses were performed by using both nuclear medicine and molecular tests. UBC and BTA-t analyses were carried out using monoclonal antibody tests while microsatellite analyses were performed using extracted DNA and electrophoresis of polymerase chain reaction (PCR) products on 13 different primers. Urinary cytological examinations were carried out using the Autocyte Preparation System(R). Urinary cytology confirmed the presence of TCC in 13.3% of patients. The BTA-t marker allowed the identification of 73.3% of cancers with 50% specificity; the UBC marker identified 63.3% of the cases with 41.6% specificity. Microsatellite analysis permitted the identification of 83.3% of the tumors with 100% specificity. DNA analysis demonstrated high sensitivity in patients affected by superficial (81.4%) or G1 (80%) tumors, even when cytological studies demonstrated little or no sensitivity. Microsatellite analysis is a highly-sensitive and specific marker for TCC diagnosis and its monitoring, especially in patients with low-stage and low-grade tumors. Other testing procedures failed to increase urinary cytological diagnostic significance.     

Functional analysis of the mismatch repair system in bladder cancer

In bladder cancer the observed microsatellite instability indicates that mismatch repair deficiency could be a frequently involved factor in bladder cancer progression. To investigate this hypothesis we analysed extracts of seven bladder cancer cell lines and, as a novel approach, five clinical cancer samples for mismatch repair activity. We found that one cell line (T24) and three of the clinical samples had a reduced repair capacity, measured to approximately 20% or less. The T24 cell extract was unable to repair a G-G mismatch and showed reduced repair of a 2-base loop, consistent with diminished function of the MSH2-MSH6 heterodimer. The functional assay was combined with measurement for mutation frequency, microsatellite analysis, sequencing, MTT assay, immunohistochemical analysis and RT-PCR analysis of the mismatch repair genes MSH2, MSH3, MSH6, PMS1, PMS2 and MLH1. A >7-fold relative increase in mutation frequency was observed for T24 compared to a bladder cancer cell line with a fully functional mismatch repair system. Neither microsatellite instability, loss of repair nor mismatch repair gene mutations were detected. However, RT-PCR analysis of mRNA levels did detect changes in the ratio of expression of the Mut S and Mut L homologues. The T24 cell line had the lowest MSH6 expression level of the cell lines tested. Identical RT-PCR analysis of seventeen clinical samples (normal urothelium, 7; pTa low stage, 5; and pT1-4 high stage, 5) indicated a significant change in the expression ratio between MSH3/MSH6 (P< 0.004), MSH2/MSH3 (P< 0.012) and PMS2/MLH1 P< 0.005, in high stage bladder tumours compared to normal urothelium and low stage tumours. Collectively, the data suggest that imbalanced expression of mismatch repair genes could lead to partial loss of mismatch repair activity that is associated with invasive bladder cancer.     

联合检测UBC、HA和CK20诊断膀胱癌的临床价值

背景与目的:膀胱癌是最常见的泌尿系统肿瘤,尿脱落细胞学检查是无创性诊断的金标准,但敏感性较低.本研究探讨联合运用尿膀胱癌抗原(urinary bladder cancer,UBC)、透明质酸(hyaluronic acid,HA)和细胞角蛋白20(cytokeratin 20,CK20)诊断膀胱癌的临床价值.方法:对64例膀胱癌患者、20例泌尿系良性疾病患者,在膀胱镜检查之前留尿,分别采用酶链免疫吸附实验、放射免疫分析和逆转录聚合酶链反应检测UBC、HA和CK20在尿液中的表达,同时行脱落细胞学检查,分析比较4种方法诊断膀胱癌的临床价值.结果:UBC、HA和CK20诊断膀胱癌的敏感性分别为85.9%(55/64)、89.1%(57/64)、78.1%(50/64),与脱落细胞学(40.6%)检查比较,差异有统计学意义(P<0.01);4种方法诊断膀胱癌的特异性分别为85,0%(17/20)、80.0%(16/20)、80%(16/20)和95%(19/20).各分级和分期肿瘤UBC、HA和CK20的敏感性分别高于尿脱落细胞学检查,UBC值各分级和分期比较差异无统计学意义(P>0.05).HA检测G2、G3组较G1组明显增高(P<0.01),但G2、G3组间比较差异无统计学意义(P>0.05);各分期之间比较差异无统计学意义(P>0.05).CK20检测随肿瘤的分级与分期的增高,敏感性增高(P<0.01).联合运用UBC、HA和CK20敏感性可达96.9%,特异性达100%.结论:联合UBC、HA和CK20能提高诊断膀胱癌的敏感性和特异性,初步诊断能够代替膀胱镜检查.     

尿液中尿膀胱癌抗原和钙网蛋白联合检测对原发性膀胱癌的诊断价值

目的评价尿液中尿膀胱癌抗原（UBC）和钙网蛋白（CRT）联合检测对原发性膀胱癌的诊断价值。方法 76例膀胱癌患者、50例泌尿系统良性疾病患者均在膀胱镜检查前留取尿液,用ELISA法进行UBC、CRT定量检测,同时进行尿液中脱落细胞学检测。结果 UBC和CRT诊断膀胱癌的敏感性分别为89.47%和82.89%,高于脱落细胞学的51.32%（P〈0.05）;3种诊断方法对膀胱癌的诊断特异性分别为92.00%、78.00%和94.00%。联合检测UBC和CRT诊断膀胱癌的敏感性和特异性高达94.74%、94.00%。结论尿液中UBC和CRT是早期诊断膀胱癌较好的肿瘤标志物,而两者联合检测能进一步提高诊断效率。