Use of high-throughput DNA microarrays to identify biomarkers for bladder cancer

BACKGROUND: Numerous markers have been described to correlate to some extent with tumor stage and prognosis of patients with bladder cancer. The power of many of these biomarkers in detecting superficial disease or predicting the clinical outcome of individual tumors is limited, and alternative markers are still in demand. High-throughput microarrays represent novel means for cancer research and tumor marker discovery. APPROACH: The aim of this report was to discuss the application of DNA technologies to provide novel biomarkers for bladder cancer. CONTENT: Specific bladder tumor subtypes have distinct gene expression profiles. The use of high-throughput DNA microarrays allows identification of the most prevalent and relevant alterations within bladder tumors. Clusters of differentially expressed genes will become biomarkers to discriminate subgroups of patients with different histopathology or clinical outcome. Additionally, the identified individual molecular targets might be further validated and developed into novel serum or urinary biomarkers for the diagnosis and/or as prognostic factors to be applied in clinical practice. The diagnosis and prognosis of bladder cancer would be enhanced by the use of such markers, and the marker itself may constitute a therapeutic target when studied in appropriate patients and control groups. SUMMARY: Expression profiling with high-throughput DNA microarrays has the potential of providing critical clues for the management of bladder cancer patients. As the quality, standardization, and ease of use of the technology increase and the costs decrease, DNA microarrays will move from being a technology restricted to research to clinical laboratories in the near future.     

Analytical and clinical evaluation of a new urinary tumor marker: bladder tumor fibronectin in diagnosis and follow-up of bladder cancer.

Bladder cancer is the fourth most common malignant neoplasm in men and the tenth most common in women. Cystoscopy presents the gold standard for detection and monitoring of bladder cancer. However, it is an invasive and expensive procedure. Therefore, development of biomarkers for the purposes of screening, diagnosis and prediction of the prognosis in bladder cancer is required. Bladder tumor fibronectin is one of the new urinary tumor markers. The aim of this study is to evaluate the diagnostic performance of urinary bladder tumor fibronectin in detecting and monitoring bladder cancer. A total of 75 patients with the diagnosis of bladder cancer, 20 patients with the diagnosis of benign prostatic hyperplasia, 7 patients with the diagnosis of prostate cancer between the years 1996-2000, and 28 age-matched healthy individuals, were enrolled in the study. The patients were diagnosed by cystoscopy, with histopathological evaluation of the tumor, as having superficial or invasive bladder cancer. Patients were followed-up clinically with data pertinent to disease recurrence and progression. Bladder tumor fibronectin (BTF; ng/ml) was determined by solid phase, two-site chemiluminescent immunometric commercial diagnostic assay developed for the Immulite automated immunoassay system (Diagnostic Products Corporation, Los Angeles, CA, USA). All measured values were normalized by urinary creatinine, and the obtained data were evaluated by receiver-operating characteristics (ROC) curve analysis. Optimal cut-off was established at 43.4 ng/mg. This cut-off rendered overall sensitivities of 72% and specificity of 82.1%. The analytical evaluation of the BTF test displayed promising results in terms of a non-invasive in vitro test in the diagnosis of bladder cancer. Although it was not satisfactory in prediction of recurrence or progression of the disease, it correlated well with the stage, one of the most reliable prognostic factors. In conclusion, the urinary bladder tumor fibronectin test warrants further clinical evaluation.     

Urinary markers of bladder carcinoma

Purpose: Transitional cell carcinoma of bladder is one of the most common tumors of genitourinary tract. Cystoscopy along with cytology is the mainstay for diagnosing bladder cancer. Cytology is specific for diagnosis of bladder carcinomas but less sensitive particularly in low-grade disease. Cystoscopy on the other hand is invasive and relatively costly technique and may also be inconclusive at times particularly in case of cystitis. A simple noninvasive marker for detecting bladder cancer would be helpful. A clinically useful urinary marker should be easy to perform, should have minimum requirements for sample preparations and should have high sensitivity and specificity in diagnosis. In this review we discussed the various urinary markers and their role in detection of bladder cancer. Materials and methods: We reviewed the literature on urinary markers and tests that may have clinical usefulness. Results: Several urinary markers and tests such as BTA Stat, BTA TRAK, NMP22, telomerase, HA and HAse tests, Immunocyt, Quanticyt, FDP, BLCA-4, FISH, CYFRA-21-1 have enough potential for future clinical use. BTA stat, NMP22 (bladder check)and AccuDX (FDP) tests are presently point of care tests. The rest of the tests are laboratory-based and may need trained technicians. Majorities of the urinary markers have higher sensitivity and specificity than cytology. However, voided urinary cytology has the highest specificity. Conclusions: Till now there is no urinary marker or test that can replace the need of cystoscopy. However, some markers have the potential for future clinical use.     

P27kipl、CyclinD1在膀胱癌中的表达及其意义

目的:通过检测P27kipl与CyclinD1在膀胱癌中的表达,以探讨两者在膀胱癌发生发展中的作用及其与临床病理特征的关系.方法:对80例标本进行SP免疫组织化学方法检测P27kipl、CyclinD1的表达水平,其中正常组织28例,膀胱癌组织52例.结果:P27kipl、CyclinD1在正常膀胱组织中的表达率和在膀胱癌组织中的表达差异均具有显著性(P＜0.01).P27kipl蛋白在低分化组中的表达水平显著低于高分化组(P＜0.01)和中分化组(P＜0.01),CyclinD1表达水平在低分化组中显著高于高分化组(P＜0.01),在中、低分化组间差异无显著性(P＞0.05).P27kipl的表达与临床分期无关,而CyclinD1的表达与临床分期相关.P27kipl与CyclinD1的相互关系呈负相关.结论:P27kipl蛋白的低表达或缺失在膀胱癌的发生中起重要作用,是膀胱癌发生中的早期分子事件.CyclinD1的过表达参与了膀胱癌的发生,并与其发展密切相关,可作为评价预后的指标之一.膀胱癌的发生发展与P27kipl、CyclinD1蛋白的异常表达密切相关,联合测定P27kipl、CyclinD1蛋白的表达对膀胱癌的诊断、治疗有一定的指导意义,可作为膀胱癌诊断和预后评价的重要参数.     

尿液膀胱肿瘤抗原水平检测在膀胱癌诊断中的意义

目的 　评估尿液膀胱肿瘤抗原（BTA）水平在膀胱癌诊断中的价值。方法　收集98 例膀胱癌患者、57 例非膀 胱癌患者以及68 例健康体检者晨尿标本,利用化学发光分析仪进行各样本中BTA 水平的检测。通过ROC 曲线下面积 计算,分析BTA 对膀胱癌的诊断灵敏度、特异度及最佳临界值。结果　膀胱癌患者尿液中BTA 水平[486.56（66.27, 2 013.81）] U/ml 明显高于健康对照组[25.71（10.40,53.39）] U/ml 及非膀胱癌组[55.47（26.55,296.24）] U/ml,差异 具有统计学意义（χ2=64.172,P <0.01）;BTA 诊断膀胱癌的ROC 曲线下面积为0.816,与参考线下面积相比,差异具 有统计学意义（P <0.01）,BTA 诊断膀胱癌的最佳临界值为84.38U/ml,诊断敏感度为70.8%,特异度为93.5%。结论　 尿液BTA 的检测在膀胱癌的诊断筛查中具有较高的临床价值,可用于膀胱癌的早期筛查。     

Measurements of complement factor H-related protein (BTA-TRAK assay) and nuclear matrix protein (NMP22 assay)--useful diagnostic tools in the diagnosis of urinary bladder cancer?

Between 1997 and 2000 we investigated in a prospective study the voided urine samples of all consecutive patients undergoing cystoscopy independent from their clinical background (n = 705) with the BTA-TRAK assay (Bard Diagnostics, Redmont, USA) detecting a complement factor H-related protein (CFHrP) and the NMP22 assay (Matritech, Newton, USA) measuring a nuclear matrix protein, which is supposed to be specific for bladder cancer. The individuals were divided into three groups concerning the clinical background: 233 patients had urological diseases, 268 patients had urinary bladder cancer and 150 patients had other urological malignancies. Based on the clinical findings we compared our results with well established diagnostic methods for urinary bladder cancer such as cytology and the detection of hematuria. In addition, we investigated urine samples from 30 healthy individuals and 24 patients with urinary tract infection without performing cystoscopy. Following the recommendations of the European Group on Tumor Markers we used 95% specificity for benign urological diseases and urinary tract infections, which resulted in a sensitivity of 17% for active bladder cancer for the BTA-TRAK assay and 31% for NMP22. We compared these results with the detection of hematuria (specificity: 72%) and cytology, which had a sensitivity of 64% and 89%, respectively. Subsequently, we calculated sensitivity and specificity for the detection of relapse of the disease. Again using 95% specificity, in this case for patients with no evidence of disease (NED), in patients with recurrent disease the BTA-TRAK assay showed 8% sensitivity as compared to 12% for the NMP22 assay. Due to an insufficient specificity and sensitivity, both tests can neither be clinically useful in screening of high risk patients, nor in primary diagnosis of bladder cancer. They cannot replace neither cystoscopy nor cytology. In the follow-up care more investigations may be necessary to prove the benefit of existing diagnostic strategies for the discrimination between active and inactive bladder cancer.     

尿核基质蛋白22是膀胱移行细胞癌诊断及检测复发的指标

目的：探讨检测尿中核基质蛋白22（nuclear matrix proteins22,NMP22）含量对膀胱移行细胞癌（BTCC）诊断及检测复发的意义。方法：用NMP22试剂盒（ELISA）检测24例BTCC患者手术前、后48份尿标本及15份泌尿系良性疾病患者的尿标本。结果：24例BTCC术前尿NMP22含量中位数为71．5U/ml；15例泌尿系良性疾病对照组为11．0U／ml，两者差异非常显著（P＜0．01）。以尿NMP22 18U/ml为cut off值，诊断BTCC的灵敏度为95．8％（23／24），特异性为86．7％（13／15）。24例BTCC患者术后尿NMP22中位数为45．0U/ml，与术前比较明显下降（P＜0．01）。随访发现术后肿瘤复发组和无复发组尿NMP22的中位数分别为60．0U／ml和12．0U/ml，两组差异显著（P＜0．01）。结论：尿NMP22测定是一种无创伤性、灵敏度高、特异性好的BTCC辅助诊断指标，并可望用于判断术后有无复发。     

64排螺旋CT尿路成像的临床应用价值探讨

目的：探讨64排螺旋CT尿路成像的临床应用价值。方法：对临床怀疑泌尿系统病变的52例患者行64排螺旋CT容积扫描及尿路三维重建。结果：52例中输尿管瘘1例，输尿管结石12例，输尿管癌5例，双肾盂2例，肾盂癌5例，膀胱癌2例，其他3例，正常22例。CT图像不仅能清晰地显示输尿管走行、腔内改变、梗阻部位及原因，还可以同时显示管壁及腔外情况。结论：64排螺旋CT可多方位动态观察肾盂、输尿管及膀胱的正常解剖形态及各种病理改变，并从三维立体图像上获得更丰富的诊断信息，是全面立体动态诊断泌尿系统病变较好的影像检查方法。     

Overactive bladder: achieving a differential diagnosis from other lower urinary tract conditions

Overactive bladder (OAB) is a debilitating condition characterised by an urgent need to urinate (urgency), often with urinary frequency and, in some cases, urgency incontinence and nocturnal frequency. Patients often adopt complex adaptive behaviours to cope with their symptoms as OAB can compromise all dimensions of a patient's quality of life. Most OAB patients present initially to their primary care physician. Diagnosis is based on presenting symptomatology and does not require any invasive tests. Direct questioning about symptoms is important in achieving a differential diagnosis. The most common condition to be considered when working towards a differential diagnosis is a urinary tract infection (UTI). However, some physicians have expressed concerns about identifying the small number of cases where bladder cancer is a potential underlying aetiology for the symptoms of OAB. In this review, we examine the prevalence and patient profiles for these bladder conditions and their presenting symptomatology. We also review tests that may be recommended to exclude a diagnosis of UTI or bladder cancer and present a diagnostic algorithm suitable for office-based primary care practice.     

超声检查在腺性膀胱炎诊断中的应用

目的探讨超声检查在腺性膀胱炎诊断中的临床价值。方法使用彩色超声诊断仪观察腺性膀胱炎患者膀胱壁及其周围情况,尤其要清晰显示膀胱三角区及膀胱颈部,总结其声像图特点。结果本组28例腺性膀胱炎经超声确诊24例,误诊为膀胱肿瘤4例,符合率85．7l％。结论超声检查方便、经济、对人体无伤害,可重复检查,并能直观显示膀胱内病灶的大小、部位、形态及病变处膀胱各层受侵犯程度,可作为腺性膀胱炎诊断的首选方法。     

膀胱肉瘤样癌误诊1例报告并文献复习

目的：探讨膀胱肉瘤样癌的组织学特性，提高对膀胱肉瘤样癌的诊断治疗水平。方法：报告1例膀胱肉瘤样癌患者的临床、病理资料，复习相关文献进行讨论。结果：术后病理检查见上皮和肉瘤样间质2种恶性成分，癌与肉瘤样区有移行，癌组织CK阳性，Vimentin阴性，肉瘤样组织CK及Vimentin阳性，诊断为膀胱肉瘤样癌。1个月后死于全身衰竭、多处转移。结论：膀胱肉瘤样癌具有浸润性生长的生物学特性，恶性程度高，预后不良，确诊依赖病理学及免疫组织化学检查；化疗、放疗都不太敏感，手术仍是首选治疗方式。     

Multiplexed methylation profiles of tumor suppressor genes in bladder cancer

Changes in DNA methylation of tumor suppressors can occur early in carcinogenesis and are potentially important early indicators of cancer. The objective of this study was to assess the methylation of 25 tumor suppressor genes in bladder cancer using a methylation-specific (MS) multiplex ligation-dependent probe amplification assay (MLPA). Initial analyses in bladder cancer cell lines (n = 14) and fresh-frozen primary bladder tumor specimens (n = 31) supported the panel of genes selected being altered in bladder cancer. The process of MS-MLPA was optimized for its application in body fluids using two independent training and validation sets of urinary specimens (n = 146), including patients with bladder cancer (n = 96) and controls (n = 50). BRCA1 (71.0%), WT1 (38.7%), and RARB (38.7%) were the most frequently methylated genes in bladder tumors, with WT1 methylation being significantly associated with tumor stage (P = 0.011). WT1 and PAX5A were identified as methylated tumor suppressors. In addition, BRCA1, WT1, and RARB were the most frequently methylated genes in urinary specimens. Receiver operating characteristic curve analyses revealed significant diagnostic accuracies in both urinary sets for BRCA1, RARB, and WT1. The novelty of this report relates to applying MS-MLPA, a multiplexed methylation technique, for tumor suppressors in bladder cancer and body fluids. Methylation profiles of tumor suppressor genes were clinically relevant for histopathological stratification of bladder tumors and offered a noninvasive diagnostic strategy for the clinical management of patients affected with uroepithelial neoplasias.     

彩色多普勒超声对腺性膀胱炎的鉴别诊断价值

目的 探讨彩色多普勒超声检查对腺性膀胱炎的鉴别诊断价值.方法 采用彩色多普勒超声检查39例腺性膀胱炎和192例膀胱癌患者,观察膀胱病变的位置、形态、边界、内部回声及其与膀胱壁的关系,并检查病变内部的血流分布,将其声像图表现进行对比分析.超声检查结果与膀胱镜活检或手术病理结果对照.结果 59.0%(23/39)腺性膀胱炎和52.9%(74/140)的单发膀胱癌病灶位于膀胱三角区及输尿管口周围;53.8%(21/39)的腺性膀胱炎表现为膀胱壁局限性增厚,72.9%(140/192)的膀胱癌为单发结节或团块,两者的病灶形态分布差异有统计学意义(P＜0.01);69.2%(27/39)的腺性膀胱炎病灶内部见囊状无回声区,而膀胱癌病变内均未见此声像改变;膀胱癌病灶边缘见点状强回声者占48.4%(93/192),而腺性膀胱炎仅为7.7%(3/39),二者差异有统计学意义(P＜0.01);彩色多普勒显示84.9%(163/192)膀胱癌病变内检出血流信号,其血流检出率明显高于腺性膀胱炎.彩色多普勒超声对腺性膀胱炎与膀胱癌的诊断准确率分别为84.6%(33/39)、95.3%(183/192).结论 彩色多普勒超声检查能清晰显示腺性膀胱炎的位置、边界、形态、内部回声及其与膀胱壁的关系,对腺性膀胱炎的鉴别诊断和临床随访有重要作用.     

尿膀胱癌抗原、透明质酸和存活素联合检测对膀胱癌的诊断运用探讨

目的探讨在对膀胱癌的诊断过程中,尿膀胱癌抗原、透明质酸和存活素的应用价值。方法膀胱癌患者74例,泌尿系统良性疾病患者20例,健康志愿者标本10例分别行膀胱癌抗原(UBC)、透明质酸(HA)、存活素(Sur-vivin)和尿脱落细胞学(UC)检查。结果 UBC、HA、Survivin和尿脱落细胞学的检测有利于膀胱癌的诊断,且UBC、HA、Survivin三者的有效性优于尿脱落细胞学的检测。UBC、HA、Survivin在检测膀胱癌的过程中,三者的敏感性和准确性高于尿脱落细胞学。UBC、HA、Survivin在肿瘤的各分级(G1、G2、G3)和各分期(pTa、pT1、≥pT2)中的敏感性明显高于尿脱落细胞学。结论尿膀胱癌抗原、透明质酸和存活素在诊断膀胱癌过程中应用价值优于尿脱落细胞学。     

磁共振扩散加权成像在膀胱病变鉴别诊断中的应用价值

目的：探讨磁共振扩散加权成像（DWI）和表观扩散系数（ADC）在膀胱病变鉴别诊断中的应用价值。方法：以53例膀胱癌及12例良性膀胱病变作为病例组,11例正常膀胱壁作为对照组。分析病例组及对照组的DWI并测量病变及正常膀胱壁的ADC值。结果：所有53例膀胱癌表现为扩散受限。12例良性病变中,2例轻度扩散受限,余10例无扩散受限。DWI上检出膀胱癌的敏感度为100%,特异度为83.3%,准确度为96.9%。膀胱癌的平均ADC值为（0.76±0.17）×10-3 mm2/s,显著低于良性病变的（1.21±0.20）×10-3 mm2/s,并显著低于对照组的（1.55±0.15）×10-3 mm2/s（F=89.04,P<0.001）。结论：DWI和ADC在膀胱良恶性病变的鉴别诊断中具有较好的应用价值。     

The fluorescent hybridization in situ as applied in diagnostics of urinary bladder cancer

The adaptation of techniques of molecular genetic analysis into clinical practice increased the possibilities of diagnostics of urinary bladder cancer at early stage of disease development. The article discusses the results of cytological and cytogenetic examination of urine cells in patients with diagnostics of urinary bladder cancer. The sampling consisted of 44 cytological and 25 cytogenetic tests. The results of both methods of diagnostics of urinary bladder cancer matched in 12 cases. Besides, in 2 tests the carcinoma of urinary bladder was confirmed only by using molecular genetic technique.     

Identification by proteomic analysis of calreticulin as a marker for bladder cancer and evaluation of the diagnostic accuracy of its detection in urine

BACKGROUND: New methods for detection of bladder cancer are needed because cystoscopy is both invasive and expensive and urine cytology has low sensitivity. We screened proteins as tumor markers for bladder cancer by proteomic analysis of cancerous and healthy tissues and investigated the diagnostic accuracy of one such marker in urine. METHODS: Three specimens of bladder cancer and healthy urothelium, respectively, were used for proteome differential display using narrow-pH-range two-dimensional electrophoresis. To evaluate the presence of calreticulin (CRT) as detected by Western blotting, we obtained 22 cancerous and 10 noncancerous surgical specimens from transurethral resection or radical cystectomy. To evaluate urinary CRT, we collected 70 and 181 urine samples from patients with and without bladder cancer, respectively. Anti-CRT COOH-terminus antibody was used to detect CRT in tissue and urine. RESULTS: Proteomic analysis revealed increased CRT (55 kDa; pI 4.3) in cancer tissue. Quantitative Western blot analysis showed that CRT was increased in cancer tissue (P = 0.0003). Urinary CRT had a sensitivity of 73% (95% confidence interval, 62-83%) at a specificity of 86% (80-91%) for bladder cancer in the samples tested. CONCLUSIONS: Proteomic analysis is useful in searching for candidate proteins as biomarkers and led to the identification of urinary CRT. The diagnostic accuracy of urinary CRT for bladder cancer appears comparable to that of Food and Drug Administration-cleared urinary markers, but further studies are needed to determine its diagnostic role.     

尿液中核基质蛋白22联合端粒酶活性检测在膀胱癌诊断中的作用

目的探讨尿液中核基质蛋白22（NMP22）联合端粒酶活性检测在膀胱癌诊断中的价值。方法收集38例膀胱癌和28例非膀胱癌患者新鲜尿液,用化学发光分析法检测尿NMP22水平,TRAP—PCR-ELISA法检测尿脱落细胞端粒酶活性,并同时行尿脱落细胞学检查。结果尿液中NMP22对膀胱癌诊断的敏感性为79％,特异性为64％;端粒酶敏感性为82％,特异性为71％;细胞学检查敏感性为32％,特异性为97％。NMP22联合端粒酶活性检测,两者均为阳性时诊断为阳性,否则为阴性,则联合检测的敏感性为76％,特异性为89％。结论联合检测可使在维持较高敏感性的前提下,大大提高诊断的特异性,其特异性与细胞学检测相差较小,而敏感性却远高于细胞学检测,因而联合检测较单一指标的检测对膀胱癌具有更高的诊断价值。     

多探针荧光原位杂交技术在膀胱尿路上皮癌诊断中的意义

目的探讨多探针荧光原位杂交技术（fluorescence in situ hybridization,FISH）在膀胱尿路上皮癌诊断的可行性,并探讨其与肿瘤临床分期和病理分级的关系。方法收集经膀胱镜检查诊断为膀胱移行细胞癌35例患者,及10例非肿瘤病人,10例正常人群的新鲜尿液标本,应用FISH检测膀胱尿路上皮癌患者尿液脱落细胞中的3、7、17、9号染色体异常,并进行尿细胞学检测（Urinary cytological examination,UCE）。统计学分析FISH和UCE诊断膀胱癌的灵敏性和特异性,并分析FISH检测染色体突变与膀胱肿瘤分期及分级的相关性。结果多探针联合FISH技术与UCE诊断膀胱癌的灵敏度分别为82.86%和37.14%（P〈0.05）,特异度分别为95.0%和100%（P〉0.05）。3、7及17号染色体着丝粒特异性探针及9号染色体长臂2区1带（9p21）的p16位点特异性探针联合应用诊断膀胱癌的灵敏度为82.86%,而单一探针诊断膀胱癌的灵敏度最高仅为65.71%。膀胱癌患者中发生3、7、17号染色体畸变及9号染色体p16基因畸变均与不同病理分级（G1-G3）有关（P〈0.05）;3、7号染色体畸变与临床分期（T0-T4）有相关性（P〈0.05）,而17号染色体及9号染色体p16基因畸变与临床分期无相关性（P〉0.05）。结论多荧光探针FISH诊断膀胱尿路上皮癌敏感性高、特异性强、无创,且与肿瘤分期和分级有一定相关性,临床应用价值高。     

Update on urine as a biomarker in cancer: a necessary review of an old story

ABSTRACT

Introduction: Cancer causes thousands of deaths worldwide each year. Therefore, monitoring of health status and the early diagnosis of cancer using noninvasive assays, such as the analysis of molecular biomarkers in urine, is essential. However, effective biomarkers for early diagnosis of cancer have not been established in many types of cancer.

Areas covered: In this review, we discuss recent findings with regard to the use of urine composition as a biomarker in eleven types of cancer. We also highlight the use of urine biomarkers for improving early diagnosis.

Expert opinion: Urinary biomarkers have been applied for clinical application of early diagnosis. The main limitation is a lack of integrated approaches for identification of new biomarkers in most cancer. The utilization of urinary biomarker detection will be promoted by improved detection methods and new data from different types of cancers. With the development of precision medicine, urinary biomarkers will play an increasingly important clinical role. Future early diagnosis would benefit from changes in the utilization of urinary biomarkers.