Cell Biochem Biophys DOI 10.1007/s12013-015-0632-6
REVIEW PAPER
Recent Advances in the Diagnosis and Management of Bladder Cancer Xiaoying Zhang • Conghui Han • Jantai He
Ó Springer Science+Business Media New York 2015
Abstract The most common malignancy of urinary tract is bladder cancer. It is one of the most widespread cancers of the world and ranks nine among frequent malignancies existing in world. The only solution to above burning problem is timely diagnosis at earlier stage, and the cancer research is being forwarded in this direction. There are various prominent gene modifications responsible for growth of bladder cancer. The present review is focused on recent advances in the field of cancer makers involving, genetic, urinary, pathological, etc., approaches to contain the deadly process of carcinogenesis. The present review provides an insight on the emerging biomarkers that could be developed to boost current bladder cancer detection strategies. This shall help timely diagnosis of this deadly disease at early stage, thereby, helping bladder cancer patients to fight against this iniquity of life. Keywords
Bladder cancer Genetic modulations Genes
Introduction Bladder cancer is the second most common malignancy of urinary tract and is forth most frequent cancer in men in
Xiaoying Zhang and Conghui Han contributed equally as co-first authors. X. Zhang J. He (&) Xiangya Hospital of Central South University, Changsha 410008, Hunan, People’s Republic of China e-mail: [email protected] C. Han Department of Urology Surgery, Xuzhou Central Hospital, Xuzhou 221009, Jiangsu, People’s Republic of China
developed countries [1]. The well-reported form of bladder cancer is transitional cell carcinoma (TCC), constituting approximately 95 % of all cases [2]. The majority of these cases of bladder cancer arise from the urothelium, the epithelium lining the inside of the bladder, and these cases are thus called urothelial carcinomas. Squamous cell carcinoma of the urinary bladder is a rarer malignant neoplasm, and it accounts for only 3–5 % of bladder cancer in Western populations [3]. With respect to phenotypes, there are broadly two main types of tumors reported as a result of bladder cancer. These are non-muscle invasive tumors and muscle invasive tumors. An approximate ratio of 5:1 exits between non-muscle invasive and muscle invasive phenotypes, respectively [4]. The leading cause of bladder cancer is cigarette smoking. Besides this, other risk factors include exposure to industrial carcinogens and chronic infection with Schistosomiasis haematobium [5]. The prominent symptom (80–85 % of patients) of bladder cancer is hematuria. Also, other urinary tract symptoms like increased frequency of urination, urgency, and dysuria contribute toward 15–20 % of patients. As discussed before, there are two main phenotypes viz. non-muscle invasive tumors and muscle invasive tumors and are classified on the basis of tumor invasion into the bladder wall [6]. The tumors, which are restricted to the inner epithelial lining of the bladder without any interference with muscle wall of bladder, are non-muscle invasive tumors. These are also called superficial bladder cancers and include Tis, Ta, and T1 stages. Of the ‘‘non-muscle invasive’’ tumors, Stage Ta tumors are confined to the mucosa, while Stage T1 tumors superficially invade the lamina propria. T1 tumors are regarded as being more aggressive than Ta tumors. On the other hand, the tumors that invade muscle wall of bladder are called muscle invasive tumors and they include Stages T2, T3,
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and T4. Stage T2 is confined to the muscle layer, Stage T3 is into the perivesical fat layer beyond the muscle, and stage T4 is of with metastatic nature involving local nodes as well as distal nodes. Bladder cancer has proclivity to recur, requiring lifelong surveillance after diagnosis. Urinary markers of disease have been of extreme interest in this field in an effort to simplify surveillance schedules and improve early detection of tumors. Many markers have been described, but most remain investigational. However, some markers have undergone clinical trials and are approved for clinical use. In this review, bladder cancer markers and their application for screening and surveillance of bladder cancer are discussed. The present review is also focused on the latest developments in the management of bladder cancer.
Basic Routine Diagnostic Assays for Bladder Cancer In 50–70 % of bladder cancer patients, especially with nonmuscle invasive tumor types, tumors recur despite conservative measures. It is because of this natural history that bladder cancer requires lifelong surveillance [7]. Cystoscopy and urine cytology are the most gold standards for the diagnosis and follow-up of bladder cancer. Researchers are finding other alternatives, either to reduce the frequency of cystoscopy, or improve its sensitivity for detection of tumors. Cystoscopy is a minimally traumatic office procedure accompanied with significant patient discomfort, stress, and anxiety [8, 9]. However, the procedure is successful in identifying most bladder tumors but has a false negative results due to operator error, or from small areas of sessile tumor (carcinoma in situ), which may be difficult to detect [10, 11]. On the other hand, urine cytology has been reported to be successful in diagnosis of high grade muscle invasive as well as non-muscle invasive tumors [12]. However, one major limitation of urine cytology is its low sensitivity for the detection of low-grade tumors, at approximately 4–31 % [13]. Consequently, there is a need for an accurate marker of disease in order to decrease patient discomfort as well as the cost associated with surveillance. Moreover, an accurate marker would also have the added benefit of improving quality of life by potentially minimizing the number of invasive endoscopic evaluations.
Urinary Tumor Markers The stringent need of a ideal screening and surveillance method for efficient management of bladder cancer patients led to development of non-invasive, rapid, and easily accessible makers in the form of urinary tumor markers.
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Many markers have been investigated, but most of them are at pre-clinical stage and few have undergone clinical trials and are approved for clinical use (Table 1). Urine tumor markers have been proposed for use as diagnostic aids in patients who present with hematuria, as predictive indicators of disease recurrence and survival, and as early detectors of recurrent disease in the monitored patient. There are several potential applications of urine tumor marker tests in patient surveillance, including (a) serial testing to detect recurrent disease earlier, (b) as an adjunct to urine cytology in order to improve the detection of disease recurrence, (c) providing a less expensive and more objective alternative to urine cytology, and (d) directing the frequency of cystoscopy evaluation in the follow-up of patients with bladder cancer. The prime urinary marker tests approved by FDA for use as diagnostic markers for bladder cancer are BTA stat and BTA trak test, respectively. BTA stat provides semiquantitative detection of complement factor H and complement factor H-related proteins antigens using a double monoclonal antibody. It has 50–60 % sensitivity for both superficial (non-muscle invasive tumors viz. Tis, Ta, T1) and invasive (T2–T4) tumors and a specificity of 72 % BTA trek test. Further, BTA trek test provides complete quantitative analyses of both complement factor H and complement factor H-related proteins and is highly sensitive as confirmed by Mattioli et al. [14]. The quantitative estimation of nuclear mitotic apparatus (MUMA) protein in urine using double monoclonal antibody is called NMP-22 test (nuclear matrix protein). This component of the nuclear matrix is over expressed by bladder cancer and is released into the urine in increased quantity. NMP-22 has better specificity and efficiency in diagnosing bladder cancer as confirmed by various reports [15–17]. Bladder-chek test is the improvised version of the above test with better specificity and efficiency [18]. Further, the ImmunoCyt test detects bladder cancer markers namely carcinoembryonic antigen and mucin present on exfoliated cells using a cocktail of fluorescent antibodies [19]. Multi-target fluorescence in situ hybridization (FISH) detects cancer cells based on the aneuploidy of selected chromosomes, and the test is known as UroVysion test [20].
Marker Gene Mutations and Chromosome Aberrations In order to achieve better specificity as well as efficiency in bladder cancer detection, various molecular markers including genes and chromosomal aberrations have been considered. These molecular makers have direct association with development and progression of bladder cancer. Different methods to detect copy number changes, such as classical cytogenetics, interphase fluorescence in situ hybridization (FISH), Southern
Cell Biochem Biophys Table 1 Approved urinary tumor markers for bladder cancer Sr. No.
Name of the test (commercial)
Commercial analyte
Use
1
BTA Stat
Bladder tumor associated antigen
Diagnosis/monitoring
2
BTA TRAK
Bladder tumor associated antigen
Diagnosis/monitoring
3
NMP 22
NUMA1 (nuclear mitotic apparatus protein 1)
Diagnosis/monitoring
4
Immunocyte
High-MW form of glycosylated Chronic Embryonic Antigen (CEA)
Diagnosis/monitoring
5
UroVysion
Detection of aneuploidy for chromosomes 3, 7, and 17, and loss of the 9p21 locus
Diagnosis/monitoring
blot analysis, quantitative polymerase chain reaction (PCR)based assays, and comparative genomic hybridization (CGH), have been utilized [21]. Reports have confirmed the role of activating mutations of oncogenes such as HRAS [22] and FGFR3 [23] during bladder cancer. Gain-of-function mutations affecting RAS and FGFR3 and loss-of-function mutation affecting RB, PTEN, and TP53 have also been associated with the pathological stage of bladder cancer [24, 25]. Further, in chromosomal alterations, loss of genetic material on chromosome 9 is one of the most frequent alteration in bladder cancer, with 9p and 9q, often both, lost entirely or in part [26, 27]. A recent report characterized a common amplification at chromosomal region 1p21-22 in bladder cancer cell lines [28].
DNA Markers The requirement of an approach that could query multiple gene targets and identify specific patterns associated with bladder cancer led to investigation of DNA methylation patterns of different genes specific for bladder cancer. The DNA methylation patterns were studied in DAPK, RARb, E-cadherin, and p16 genes of bladder cancer patients and produced a sensitivity of 91 % and a specificity of 76 % [29, 30]. Dumiel et al. [31] achieved 87 % sensitivity and 100 % specificity by monitoring hypermethylation of the APC, RASSF1A, and p14 (ARF) genes. A quantitative PCR approach showed that the TWIST1 and NID2 genes are frequently methylated in urine samples collected from BCa patients. Numerous other genes have been proposed as valuable methylation biomarkers in various studies [32– 34]. Scher et al. [34] developed a nested methylationspecific PCR assay to detect bladder cancer by assay of BCL2 gene in small volumes of patient urine.
RNA Markers The key transcriptional regulators of gene expression comprise transcribed, non-protein-coding microRNA (miRNA) molecules [35]. There is rough estimate of about
1500 human miRNAs known, to date and are characterized to some extent. Each miRNA controls the expression of multiple genes, so this molecular family may represent an opportunity to identify biomarkers of a higher order for diagnosis of bladder cancer. Studies on miRNAs have confirmed and identified them as potential makers associated with bladder cancer and some of them have been confirmed in urine samples too. [36–39]. In a group sample of 47 bladder cancer patients, Hanke et al. [40] examined the expression of 157 miRNAs in exfoliated urothelial cells using quantitative RT-PCR and reported that the ratio of miR-126 to miR-182 achieved 72 % sensitivity and 82 % specificity. Further, a combination of three miRNAs (135b/ 15b/1224-3p) has been reported to effective diagnostic in 15 bladder cancer patients with a high sensitivity (94.1 %), using quantitative PCR [41]. Also, another combination of miR-222 and miR-452 has been noticed to be cooperative in tumor stratification in bladder cancer [38]. Moreover, combination of miRNAs, viz. miR-96 and miR-183, has been utilized successfully to correlate advancing tumor grade and stage in bladder cancer patients [39].
Recent Advances in Bladder Cancer Markers Scientific fraternity is extensively involved in research to find new ideas to effectively as well as timely diagnose this deadly disease called bladder cancer. As timely and early detection is the ultimate solution for effective management and treatment of this disease. High-throughput experimental platform technologies ranging from genomic sequencing to proteomic and metabolomic profiling are now being used for molecular characterization of clinical phenotypes of bladder cancer [42–46]. For the advancement of scientific knowledge, a variety of datasets have become available, e.g., in Array Express/Gene Expression Omnibus (GEO) for transcriptomics, Human Proteinpedia for proteomics, or in large data consolidation platforms such as GeneCards [47]. Moreover, omics-based profiling has significantly advanced bladder cancer research, providing the basis for an integrative analysis approach in
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Cell Biochem Biophys
delineating a more comprehensive overview of molecular processes and pathways of bladder cancer. It is clear from the above discussion that scientific community is working on various avenues for the better management of bladder cancer patients. The hopes are high so as to study and develop new robust tests that can be informative across the broad range of clinical presentation. Moreover, these advanced diagnostic assays may enter the clinical setting to augment, or eventually even replace, cystoscopy and/or cytology for diagnosis, and recurrence monitoring of bladder cancer patients. Moreover, this will ultimately improvise the quality of life of bladder cancer patients indeed.
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15. Miyanaga, N., Akaza, H., & Tsukamoto, S. (2003). Usefulness of urinary NMP22 to detect tumor recurrence of superficial bladder cancer after transurethral resection. International Journal of Clinical Oncology, 8, 369–373. 16. Lahme, S., Bichler, K. H., Feil, G., Zumbragel, A., & Gotz, T. (2003). Comparison of cytology and nuclear matrix protein 22 (NMP22) for detection and follow-up of bladder-cancer. Advanced Experimental Medicine Biology, 539, 111–119. 17. Ponsky, L. E., Sharma, S., Pandrangi, L., Kedia, S., Nelson, D., Agarwal, A., et al. (2001). Screening and monitoring for bladder cancer, refining the use of NMP22. Journal of Urology, 166(1), 75–78. 18. Grossman, H., Messing, E., Soloway, M., Tomera, K., Katz, G., Berger, Y., et al. (2005). Detection of bladder cancer using a point-of-care proteomic assay. JAMA, 293, 810–816. 19. Mian, C., Pycha, A., Wiener, H., et al. (1999). Immunocyt: A new tool for detecting transitional cell cancer of the urinary tract. Journal of Urology, 161, 1486–1489. 20. Halling, K. C., King, W., Sokolova, I. A., Meyer, R. G., Burkhardt, H. M., Halling, A. C., et al. (2000). A comparison of cytology an fluorescence in situ hybridization for the detection of urothelial carcinoma. Journal of Urology, 164, 1768–1775. 21. Ho¨glund, M. (2012). The bladder cancer genome, chromosomal changes as prognostic makers, opportunities, and obstacles. Urology Oncology, 30, 533–540. 22. Jebar, A. H., Hurst, C. D., Tomlinson, D. C., Johnston, C., Taylor, C. F., & Knowles, M. A. (2005). FGFR3 and Ras gene mutations are mutually exclusive genetic events in urothelial cell carcinoma. Oncogene, 24(33), 5218–5225. 23. Tomlinson, D. C., Baldo, O., Harnden, P., & Knowles, M. A. (2007). FGFR3 protein expression and its relationship to mutation status and prognostic variables in bladder cancer. Journal of Pathology, 213(1), 91–98. 24. Cordon-Cardo, C. (2008). Molecular alterations associated with bladder cancer initiation and progression. Scand Journal of Urology and Nephrology, 42, 154–165. 25. Puzio-Kuter, A. M., Castillo-Martin, M., Kinkade, C. W., Wang, X., Shen, T. H., Matos, T., et al. (2009). Inactivation of p53 and Pten promotes invasive bladder cancer. Genes & Development, 23(6), 675–680. 26. Habuchi, T., Devlin, J., Elder, P. A., & Knowles, M. A. (1995). Detailed deletion mapping of chromosome 9q in bladder cancer, evidence for two tumour suppressor loci. Oncogene, 11, 1671–1674. 27. Kimura, F., Florl, A. R., Seifert, H. H., Louhelainen, J., Maas, S., Knowles, M. A., & Schulz, W. A. (2001). Destabilization of chromosome 9 in transitional cell carcinoma of the urinary bladder. British Journal of Cancer, 85(12), 887–893. 28. Scaravilli, M., Asero, P., Visakorpi, T., & Sarama¨ki, O. R. (2014). Mapping of the chromosomal amplification 1p21-22 in bladder cancer. BMC Research Notes, 7, 547. 29. Chan, M. W., Chan, L. W., Tang, N. L., Tong, J. H., Lo, K. W., Lee, T. L., et al. (2002). Hypermethylation of multiple genes in tumor tissues and voided urine in urinary bladder cancer patients. Clinical Cancer Research, 8(2), 464–470. 30. Friedrich, M. G., Weisenberger, D. J., Cheng, J. C., Chandrasoma, S., Siegmund, K. D., Gonzalgo, M. L., et al. (2004). Detection of methylated apoptosis-associated genes in urine sediments of bladder cancer patients. Clinical Cancer Research, 10(22), 7457–7465. 31. Dulaimi, E., Uzzo, R. G., Greenberg, R. E., Al-Saleem, T., & Cairns, p. (2004). Detection of bladder cancer in urine by a tumor suppressor gene hypermethylation panel. Clinical Cancer Research, 10(6), 1887–1893. 32. Reinert, T. (2012). Methylation markers for urine-based detection of bladder cancer, the next generation of urinary markers for
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diagnosis and surveillance of bladder cancer. Advance Urology, 2012, 503271. Phe, V., Cussenot, O., & Roupret, m. (2009). Interest of methylated genes as biomarkers in urothelial cell carcinomas of the urinary tract. British Journal of Urology International, 104(7), 896–901. Scher, M. B., Elbaum, M. B., Mogilevkin, Y., Hilbert, D. W., Mydlo, J. H., Sidi, A. A., et al. (2012). Detecting DNA methylation of the BCL2, CDKN2A and NID2 genes in urine using a nested methylation specific polymerase chain reaction assay to predict bladder cancer. Journal of Urology, 188(6), 2101–2107. Catto, J. W., Alcaraz, A., Bjartell, A. S., De Vere, White R., Evans, C. P., Fussel, S., et al. (2011). MicroRNA in prostate, bladder, and kidney cancer, a systematic review. European Urology, 59(5), 671–681. Dyrskjot, L., Ostenfeld, M. S., Bramsen, J. B., Silahtaroglu, A. N., Lamy, P., Ramanathan, R., et al. (2009). Genomic profiling of microRNAs in bladder cancer, miR-129 is associated with poor outcome and promotes cell death in vitro. Cancer Research, 69(11), 4851–4860. Han, Y., Chen, J., Zhao, X., Liang, C., Wang, Y., Sun, L., et al. (2011). MicroRNA expression signatures of bladder cancer revealed by deep sequencing. PLoS One, 6(3), e18286. Puerta-Gil, P., Garcia-Baquero, R., Jia, A. Y., Ocana, S., AlvarezMugica, M., Alvarez-Ossorio, J. L., et al. (2012). miR-143, miR222, and miR-452 are useful as tumor stratification and noninvasive diagnostic biomarkers for bladder cancer. American Journal of Pathology, 180(5), 1808–1815. Yamada, Y., Enokida, H., Kojima, S., Kawakami, K., Chiyomaru, T., Tatarano, S., et al. (2011). MiR-96 and miR-183 detection in urine serve as potential tumor markers of urothelial carcinoma,
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correlation with stage and grade, and comparison with urinary cytology. Cancer Science, 102(3), 522–529. Hanke, M., Hoefig, K., Merz, H., Feller, A. C., Kausch, I., Jocham, D., et al. (2010). A robust methodology to study urine microRNA as tumor marker, microRNA-126 and microRNA-182 are related to urinary bladder cancer. Urology Oncology, 28(6), 655–661. Miah, S., Dudziec, E., Drayton, R. M., Zlotta, A. R., Morgan, S. L., Rosario, D. J., et al. (2012). An evaluation of urinary microRNA reveals a high sensitivity for bladder cancer. British Journal of Cancer, 107(1), 123–128. Mayer, P., Mayer, B., & Mayer, G. (2012). Systems biology, building a useful model from multiple markers and profiles. Nephrology Dial Transplant, 27, 3995–4002. Besaratinia, A., Cockburn, M., & Tommasi, S. (2013). Alterations of DNA methylome in human bladder cancer. Epigenetics, 8(10), 1013–1022. Chihara, Y., Kanai, Y., Fujimoto, H., Sugano, K., Kawashima, K., et al. (2013). Diagnostic markers of urothelial cancer based on DNA methylation analysis. BMC Cancer, 13, 275. Weiss, G. J., Liang, W. S., Demeure, M. J., Kiefer, J. A., Hostetter, G., et al. (2013). A pilot study using next-generation sequencing in advanced cancers, feasibility and challenges. PLoS One, 8, e76438. Alberice, J. V., Amaral, A. F. S., Armitage, E. G., Lorente, J. A., Algaba, F., et al. (2013). Searching for urine biomarkers of bladder cancer recurrence using a liquid chromatography-mass spectrometry and capillary electrophoresis-mass spectrometry metabolomics approach. Journal of Chromatography A, 1318, 163–170. GeneCards (2013) The Human Gene Compendium. Crown Hum Genome Center, Dep Mol Genet Weizmann Institute of Science.
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REVIEW PAPER
Recent Advances in the Diagnosis and Management of Bladder Cancer Xiaoying Zhang • Conghui Han • Jantai He
Ó Springer Science+Business Media New York 2015
Abstract The most common malignancy of urinary tract is bladder cancer. It is one of the most widespread cancers of the world and ranks nine among frequent malignancies existing in world. The only solution to above burning problem is timely diagnosis at earlier stage, and the cancer research is being forwarded in this direction. There are various prominent gene modifications responsible for growth of bladder cancer. The present review is focused on recent advances in the field of cancer makers involving, genetic, urinary, pathological, etc., approaches to contain the deadly process of carcinogenesis. The present review provides an insight on the emerging biomarkers that could be developed to boost current bladder cancer detection strategies. This shall help timely diagnosis of this deadly disease at early stage, thereby, helping bladder cancer patients to fight against this iniquity of life. Keywords
Bladder cancer Genetic modulations Genes
Introduction Bladder cancer is the second most common malignancy of urinary tract and is forth most frequent cancer in men in
Xiaoying Zhang and Conghui Han contributed equally as co-first authors. X. Zhang J. He (&) Xiangya Hospital of Central South University, Changsha 410008, Hunan, People’s Republic of China e-mail: [email protected] C. Han Department of Urology Surgery, Xuzhou Central Hospital, Xuzhou 221009, Jiangsu, People’s Republic of China
developed countries [1]. The well-reported form of bladder cancer is transitional cell carcinoma (TCC), constituting approximately 95 % of all cases [2]. The majority of these cases of bladder cancer arise from the urothelium, the epithelium lining the inside of the bladder, and these cases are thus called urothelial carcinomas. Squamous cell carcinoma of the urinary bladder is a rarer malignant neoplasm, and it accounts for only 3–5 % of bladder cancer in Western populations [3]. With respect to phenotypes, there are broadly two main types of tumors reported as a result of bladder cancer. These are non-muscle invasive tumors and muscle invasive tumors. An approximate ratio of 5:1 exits between non-muscle invasive and muscle invasive phenotypes, respectively [4]. The leading cause of bladder cancer is cigarette smoking. Besides this, other risk factors include exposure to industrial carcinogens and chronic infection with Schistosomiasis haematobium [5]. The prominent symptom (80–85 % of patients) of bladder cancer is hematuria. Also, other urinary tract symptoms like increased frequency of urination, urgency, and dysuria contribute toward 15–20 % of patients. As discussed before, there are two main phenotypes viz. non-muscle invasive tumors and muscle invasive tumors and are classified on the basis of tumor invasion into the bladder wall [6]. The tumors, which are restricted to the inner epithelial lining of the bladder without any interference with muscle wall of bladder, are non-muscle invasive tumors. These are also called superficial bladder cancers and include Tis, Ta, and T1 stages. Of the ‘‘non-muscle invasive’’ tumors, Stage Ta tumors are confined to the mucosa, while Stage T1 tumors superficially invade the lamina propria. T1 tumors are regarded as being more aggressive than Ta tumors. On the other hand, the tumors that invade muscle wall of bladder are called muscle invasive tumors and they include Stages T2, T3,
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Cell Biochem Biophys
and T4. Stage T2 is confined to the muscle layer, Stage T3 is into the perivesical fat layer beyond the muscle, and stage T4 is of with metastatic nature involving local nodes as well as distal nodes. Bladder cancer has proclivity to recur, requiring lifelong surveillance after diagnosis. Urinary markers of disease have been of extreme interest in this field in an effort to simplify surveillance schedules and improve early detection of tumors. Many markers have been described, but most remain investigational. However, some markers have undergone clinical trials and are approved for clinical use. In this review, bladder cancer markers and their application for screening and surveillance of bladder cancer are discussed. The present review is also focused on the latest developments in the management of bladder cancer.
Basic Routine Diagnostic Assays for Bladder Cancer In 50–70 % of bladder cancer patients, especially with nonmuscle invasive tumor types, tumors recur despite conservative measures. It is because of this natural history that bladder cancer requires lifelong surveillance [7]. Cystoscopy and urine cytology are the most gold standards for the diagnosis and follow-up of bladder cancer. Researchers are finding other alternatives, either to reduce the frequency of cystoscopy, or improve its sensitivity for detection of tumors. Cystoscopy is a minimally traumatic office procedure accompanied with significant patient discomfort, stress, and anxiety [8, 9]. However, the procedure is successful in identifying most bladder tumors but has a false negative results due to operator error, or from small areas of sessile tumor (carcinoma in situ), which may be difficult to detect [10, 11]. On the other hand, urine cytology has been reported to be successful in diagnosis of high grade muscle invasive as well as non-muscle invasive tumors [12]. However, one major limitation of urine cytology is its low sensitivity for the detection of low-grade tumors, at approximately 4–31 % [13]. Consequently, there is a need for an accurate marker of disease in order to decrease patient discomfort as well as the cost associated with surveillance. Moreover, an accurate marker would also have the added benefit of improving quality of life by potentially minimizing the number of invasive endoscopic evaluations.
Urinary Tumor Markers The stringent need of a ideal screening and surveillance method for efficient management of bladder cancer patients led to development of non-invasive, rapid, and easily accessible makers in the form of urinary tumor markers.
123
Many markers have been investigated, but most of them are at pre-clinical stage and few have undergone clinical trials and are approved for clinical use (Table 1). Urine tumor markers have been proposed for use as diagnostic aids in patients who present with hematuria, as predictive indicators of disease recurrence and survival, and as early detectors of recurrent disease in the monitored patient. There are several potential applications of urine tumor marker tests in patient surveillance, including (a) serial testing to detect recurrent disease earlier, (b) as an adjunct to urine cytology in order to improve the detection of disease recurrence, (c) providing a less expensive and more objective alternative to urine cytology, and (d) directing the frequency of cystoscopy evaluation in the follow-up of patients with bladder cancer. The prime urinary marker tests approved by FDA for use as diagnostic markers for bladder cancer are BTA stat and BTA trak test, respectively. BTA stat provides semiquantitative detection of complement factor H and complement factor H-related proteins antigens using a double monoclonal antibody. It has 50–60 % sensitivity for both superficial (non-muscle invasive tumors viz. Tis, Ta, T1) and invasive (T2–T4) tumors and a specificity of 72 % BTA trek test. Further, BTA trek test provides complete quantitative analyses of both complement factor H and complement factor H-related proteins and is highly sensitive as confirmed by Mattioli et al. [14]. The quantitative estimation of nuclear mitotic apparatus (MUMA) protein in urine using double monoclonal antibody is called NMP-22 test (nuclear matrix protein). This component of the nuclear matrix is over expressed by bladder cancer and is released into the urine in increased quantity. NMP-22 has better specificity and efficiency in diagnosing bladder cancer as confirmed by various reports [15–17]. Bladder-chek test is the improvised version of the above test with better specificity and efficiency [18]. Further, the ImmunoCyt test detects bladder cancer markers namely carcinoembryonic antigen and mucin present on exfoliated cells using a cocktail of fluorescent antibodies [19]. Multi-target fluorescence in situ hybridization (FISH) detects cancer cells based on the aneuploidy of selected chromosomes, and the test is known as UroVysion test [20].
Marker Gene Mutations and Chromosome Aberrations In order to achieve better specificity as well as efficiency in bladder cancer detection, various molecular markers including genes and chromosomal aberrations have been considered. These molecular makers have direct association with development and progression of bladder cancer. Different methods to detect copy number changes, such as classical cytogenetics, interphase fluorescence in situ hybridization (FISH), Southern
Cell Biochem Biophys Table 1 Approved urinary tumor markers for bladder cancer Sr. No.
Name of the test (commercial)
Commercial analyte
Use
1
BTA Stat
Bladder tumor associated antigen
Diagnosis/monitoring
2
BTA TRAK
Bladder tumor associated antigen
Diagnosis/monitoring
3
NMP 22
NUMA1 (nuclear mitotic apparatus protein 1)
Diagnosis/monitoring
4
Immunocyte
High-MW form of glycosylated Chronic Embryonic Antigen (CEA)
Diagnosis/monitoring
5
UroVysion
Detection of aneuploidy for chromosomes 3, 7, and 17, and loss of the 9p21 locus
Diagnosis/monitoring
blot analysis, quantitative polymerase chain reaction (PCR)based assays, and comparative genomic hybridization (CGH), have been utilized [21]. Reports have confirmed the role of activating mutations of oncogenes such as HRAS [22] and FGFR3 [23] during bladder cancer. Gain-of-function mutations affecting RAS and FGFR3 and loss-of-function mutation affecting RB, PTEN, and TP53 have also been associated with the pathological stage of bladder cancer [24, 25]. Further, in chromosomal alterations, loss of genetic material on chromosome 9 is one of the most frequent alteration in bladder cancer, with 9p and 9q, often both, lost entirely or in part [26, 27]. A recent report characterized a common amplification at chromosomal region 1p21-22 in bladder cancer cell lines [28].
DNA Markers The requirement of an approach that could query multiple gene targets and identify specific patterns associated with bladder cancer led to investigation of DNA methylation patterns of different genes specific for bladder cancer. The DNA methylation patterns were studied in DAPK, RARb, E-cadherin, and p16 genes of bladder cancer patients and produced a sensitivity of 91 % and a specificity of 76 % [29, 30]. Dumiel et al. [31] achieved 87 % sensitivity and 100 % specificity by monitoring hypermethylation of the APC, RASSF1A, and p14 (ARF) genes. A quantitative PCR approach showed that the TWIST1 and NID2 genes are frequently methylated in urine samples collected from BCa patients. Numerous other genes have been proposed as valuable methylation biomarkers in various studies [32– 34]. Scher et al. [34] developed a nested methylationspecific PCR assay to detect bladder cancer by assay of BCL2 gene in small volumes of patient urine.
RNA Markers The key transcriptional regulators of gene expression comprise transcribed, non-protein-coding microRNA (miRNA) molecules [35]. There is rough estimate of about
1500 human miRNAs known, to date and are characterized to some extent. Each miRNA controls the expression of multiple genes, so this molecular family may represent an opportunity to identify biomarkers of a higher order for diagnosis of bladder cancer. Studies on miRNAs have confirmed and identified them as potential makers associated with bladder cancer and some of them have been confirmed in urine samples too. [36–39]. In a group sample of 47 bladder cancer patients, Hanke et al. [40] examined the expression of 157 miRNAs in exfoliated urothelial cells using quantitative RT-PCR and reported that the ratio of miR-126 to miR-182 achieved 72 % sensitivity and 82 % specificity. Further, a combination of three miRNAs (135b/ 15b/1224-3p) has been reported to effective diagnostic in 15 bladder cancer patients with a high sensitivity (94.1 %), using quantitative PCR [41]. Also, another combination of miR-222 and miR-452 has been noticed to be cooperative in tumor stratification in bladder cancer [38]. Moreover, combination of miRNAs, viz. miR-96 and miR-183, has been utilized successfully to correlate advancing tumor grade and stage in bladder cancer patients [39].
Recent Advances in Bladder Cancer Markers Scientific fraternity is extensively involved in research to find new ideas to effectively as well as timely diagnose this deadly disease called bladder cancer. As timely and early detection is the ultimate solution for effective management and treatment of this disease. High-throughput experimental platform technologies ranging from genomic sequencing to proteomic and metabolomic profiling are now being used for molecular characterization of clinical phenotypes of bladder cancer [42–46]. For the advancement of scientific knowledge, a variety of datasets have become available, e.g., in Array Express/Gene Expression Omnibus (GEO) for transcriptomics, Human Proteinpedia for proteomics, or in large data consolidation platforms such as GeneCards [47]. Moreover, omics-based profiling has significantly advanced bladder cancer research, providing the basis for an integrative analysis approach in
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delineating a more comprehensive overview of molecular processes and pathways of bladder cancer. It is clear from the above discussion that scientific community is working on various avenues for the better management of bladder cancer patients. The hopes are high so as to study and develop new robust tests that can be informative across the broad range of clinical presentation. Moreover, these advanced diagnostic assays may enter the clinical setting to augment, or eventually even replace, cystoscopy and/or cytology for diagnosis, and recurrence monitoring of bladder cancer patients. Moreover, this will ultimately improvise the quality of life of bladder cancer patients indeed.
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