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miRNA panels as biomarkers for bladder cancer
Aim: Specific miRNA profiles have been identified for several samples from patients with bladder cancer. The results are not always congruent and partly contradictory. A comparison of published data was performed to select potential markers. Materials & methods: A literature search in PubMed identified 79 articles published prior to June 2013. Reports regarding the detection of miRNAs in urine and blood have rarely been published; to date, nine respectively three articles are available. Results: The comparison of published data proved the utility of miRNAs as diagnostic and prognostic indicators of bladder cancer. In urine samples from bladder cancer patients, seven miRNAs were concordantly expressed with tumor tissues. Conclusion: Standardization is strictly required in pre-analytics and methods of miRNA measurements.
Angelika Tölle*,1, Nadine Ratert1,2 & Klaus Jung1,2 Department of Urology, Charité– Universitätsmedizin Berlin, 10117 Berlin, Germany 2 Berlin Institute for Urologic Research, 10117 Berlin, Germany *Author for correspondence: angelika.toelle@ charite.de 1
Keywords: biomarker • bladder cancer • miRNAs • serum • tissue • urine
Background Characteristics of bladder cancer & the need for new biomarkers
Urinary bladder cancer (BC) is the fifth most common malignancy. In the USA, an incidence of 72,570 new cases and 15,210 cancer deaths has been projected for 2013 [1] . Males are more often affected than females, with a ratio of 3:1. At the moment of diagnosis, 85% of bladder tumors are limited to the organ, 10% are extended regionally and 5% are metastatic. The most prevalent histological type of bladder cancer in Western countries is transitional cell carcinoma (TCC), accounting for up to 95% of all cases. At initial diagnosis, approximately 70% of bladder cancer presents as nonmuscle invasive (pTa–pT1) bladder tumors (NMIBC), whereas the remaining cases have muscle invasive (pT2–pT4) tumors (MIBC) [2] . Most patients with NMIBC are treated with transurethral resection; however, such tumors recur in up to 70% of cases within 5 years [3] and develop into muscle-invasive tumor. This progression is combined with a marked decrease in 5-year disease-specific survival [4] .
10.2217/BMM.14.26 © 2014 Future Medicine Ltd
Bladder cancer is a malignancy with a complicated, multifactorial etiology, involving both genetic and environmental factors. Two molecular pathways characterize the two types of tumors mentioned above and underline their different biological behavior. For the NMIBC form, the most common genetic alteration is an inactivating mutation in the FGFR3 gene [5] . The FGFR3 gene belongs to a growth factor receptor family related to the tyrosine kinase signaling pathway, which has an important role in embryogenesis, development, angiogenesis, wound healing, tissue homeostasis, tumorigenesis, proliferation migration and apoptosis. For the MIBC form, mutations in P53, Rb1 and PTEN are typical [6] . P53, Rb1, and PTEN are traditional tumor suppressor genes in which mutations and loss of function are related to the loss of proliferation control, apoptosis and an increase of genetic instability, which leads to MIBC tumors [6] . To date, the diagnostic ‘gold standard’ in cases of urinary BC is cystoscopy, which is an invasive and relatively expensive procedure. It is combined with sampling of tissue pieces at suspicious locations and the subse-
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Review Tölle, Ratert & Jung quent histopathological examination of biopsy samples. Urine cytology still remains the standard noninvasive method for the detection and follow-up of bladder cancer. However, there are minor morphological abnormalities in low-grade tumors meaning that the detection rate is between 50 and 90% depending on the tumor grade [7] . Extensive laboratory research led to the development of numerous urine-based tests [8] for BC detection, such as bladder tumor antigen (BTA), nuclear matrix protein 22 (NMP22), UroVysion and ImmunCyt; these are all commercially available. However, their good sensitivity is often combined with an insufficient specificity, leading to a high rate of false-positive results. For that reason, none of these markers have generally been accepted for standard diagnostic and follow-up examination [2,9] . Furthermore, the currently used prognostic indices for recurrence or progression, such as tumor grade, tumor stage, number of lesions, tumor size, previous recurrence rate and presence of concomitant carcinoma in situ do not always accurately reflect the clinical outcome. Therefore, the identification of new biomarkers for bladder cancer is urgently needed to improve disease management regarding the diagnosis, prognosis and prediction of treatment for bladder cancer. The identification of new biomarkers for diagnosis, prognosis and prediction of bladder cancer is of urgent need. An ideal marker is produced by the tumor itself and is absent in healthy people or benign conditions, meaning it could then be used for a screening program. Usually marker values increase with disease progression and decrease with remission. The establishment of reference and predictive values are necessary before a wide application will be possible. In principle diagnostic biomarkers are indicators of a particular disease state, whereas prognostic biomarkers allow a statement about chances of recovery and/or disease progression. Predictive biomarkers indicate the probability to develop a certain disease or help to assess the most likely response to a particular therapy. Until now there are no common guidelines on how to evaluate biomarkers. Soletormos et al. propose a four-phase panel analogous to that for new drugs (verifying safety, detection potential, advantages to established markers, and what kind of additional information is needed) [10] . It is precisely in this regard that a new class of small molecules, the so called miRNAs, exhibit promising diagnostic, prognostic and/or predictive potential for cancer in general and also for bladder cancer in particular. In the following sections some important biochemical properties of miRNAs and analytical detection methods as well as articles of special interest to miRNAs used as biomarkers in bladder cancer are summarized.
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What are miRNAs?
For a long time, small noncoding RNAs were regarded as ‘trash’ of transcription. Beside miRNAs, the group of small RNAs with T2. A research group analyzed the expression of miR21, miR-30b, miR-141, miR-205, and miR-200c in NMIBC and MIBC FFPE bladder tumor samples [45,61] . The ratio of miR-21 to miR-205 reached a sensitivity and specificity of 100% and 78%, respec-
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Table 1. Overview of miRNA profiling studies in bladder cancer tissue. Number of patients: tumor vs nonmalignant
Validated miRNAs† by RT-qPCR
Regulation
Reference gene for normalization
Diagnostic and/or prognostic impact
Ref.
25/2
Not reported
[56]
106/11
.
Prognostic: miR-29c; miR-129; miR-133b; miR-518*
[38]
34/0
Diagnostic: miRNA-cluster for Ta, T1, and >T2 Prognostic: miR-452, and miR-452* (positive patients with lymph node metastasis)
[57]
52/10/10 healthy
RNU6B, RNU44, RNU48
Diagnostic and prognostic: miR-99a/miR-100 miR-21/ miR-373 (low- and high-grade discrimination and association with clinical outcome)
[37]
37/9
10; 182; 183; 196a; 203; 244
Up
RNU6B
Not reported
[39]
1; 29c; 101; 127; 143; 145
Down
51/51 (matched)
182; 183; 200a
Up
RNU6B
Not reported
143; 195
Down
104/31
30a-3p; 125b; 133a; 145; 195; 199a*
Down
RNU6B
Diagnostic
[42]
26/26
200a; 200c; 210
Up
RNU6B
Not reported
[43]
125a; 125b; 143; 145; 199b Down
25/25 (matched)
200b; 708
Up
RNU6B
Not reported
1; 99a; 100; 125b; 133a; 133b; 143; 145
Down
23/10
1; 133a; 139-5p; 204; 370; 574-3p
Down
RNU48
Not reported
[52]
7/7
129
Up
β-actin
Not reported
[53]
hsa-let-7a; 30c
Down
94/11
145
Down
RNU6B, RNU43
Not reported
[46]
9/3
143-145 cluster
Down
RNU43, RNU48
Not reported
[48]
28/10
218
Down
RNU48
Not reported
[51]
152/11
9; 138; 182; 200b
Up
RNU6B, RNU44
Prognostic: miR-9; miR-182; miR-200b
[58]
1; 133a; 133b; 143; 145; 199a; 199b; 204; 451; 921; 1281
Down
40/17
20a; 106b; 130b; 141; 200a; 200a*; 205
Up
miR-101, miR-141b, miR-125a-5p,
Diagnostic: miR-130b; miR-141; miR-199a-3p; miR-205
100; 125b; 130a; 139-5p; 145*; 199a-3p; 214; 222
Down
miR-151-5p
Prognostic: miR-141; miR-205
Fresh frozen
[40]
[47]
The name of miRNAs have been abridged and only the numerals are mentioned. FFPE: Formalin-fixed paraffin-embedded; RT-qPCR: Reverse-transcriptase, quantitative real-time PCR. †
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[59]
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Table 1. Overview of miRNA profiling studies in bladder cancer tissue (cont.). Number of patients: tumor vs nonmalignant
Validated miRNAs† by RT-qPCR
Regulation
Reference gene for normalization
Diagnostic and/or prognostic impact
Ref.
FFPE
137/19
Prognostic: miR-29*
[55]
10/10 (matched primary tumor and lymph node metastasis)
29b; 142-5p
Up
RNU48
Not reported
[35]
143; 145; 320
Down
15/15
27a; 193a-5p; 296-5p; 492; Down 624; 944
miR-193b
Predictive: response to cisplatin therapy
[60]
12/0
21; 205
Up
RNU6B
Diagnostic: miR-21/miR-205
[45]
57/0
21; 99a
Up
miR-222
Diagnostic and prognostic: miR-30b; miR-141; miR-200c
[61]
30b; 31; 200a; 200b; 200c; Down 205; 426
The name of miRNAs have been abridged and only the numerals are mentioned. FFPE: Formalin-fixed paraffin-embedded; RT-qPCR: Reverse-transcriptase, quantitative real-time PCR. †
tively, with an AUC of 0.89 to predict invasion events. Additionally, the three-miRNA signature of miR30b, miR-141, and miR-200c showed a sensitivity of 100% and a specificity of 96.2%. In the multivariate Cox regression analysis, miR-30b exhibited prognostic potential for cancer specific survival. In the study of Baffa et al., 326 miRNAs were screened and a global miRNA signature was reported that discriminated between primary tumors and the corresponding metastasis [35] . miR-143, miR-145, and miR-320 were significantly downregulated whereas miR-10b, miR-29b, miR-142-5p were found to be upregulated in primary bladder tumor samples compared with their metastases. An in situ hybridization against miR-200c on a tissue microarray (TMA) composed of 100 T1 bladder tumor samples showed a loss of miR-200c, which was associated with higher risk of disease progression to muscle-invasive lesions [63] . Another study reported on the ability of miR-886-3p, miR-923, and miR-944 expression to predict the cisplatin treatment response and survival [60] . For bladder cancer tissue, miRNAs are promising biomarkers, which may improve the diagnosis, prognosis, and prediction of therapy response. A concordant downregulation for nine miRNAs (miR-7; miR-99a; miR-100; miR-125b; miR-133a; miR33b; miR-143; miR-145; miR-145b) in bladder tumor tissues was reported by more than four authors and an accordant upregulation of five miRNAs (miR-21; miR-141; miR-173; miR-200a; miR-205) was observed more than three authors (Table 2) . But these results must be interpreted cautiously due to the lack of internal vali-
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dation, for example, leave-one-out, k-fold, or repeated random-split cross validation, as well as external validation, for example, using external data from completely different studies, of many of the mentioned studies. In order to develop a generalizable predictive model involving biomarkers to wider populations it is of urgent need to be aware of proper validation of the obtained results [64] . miRNA analysis in urine
miRNAs in easily accessible urine could reflect bladder tissue situation and are therefore of great diagnostic and prognostic interest in cases of BC. However, the composition of urine is very heterogeneous and the miRNAs measured could result from the kidney, from the tumor or from shed normal cells of the bladder, as well as from inflammatory cells, and as part of the exosomes and protein–miRNA complexes. In the case of urine, three specimens are available, namely the native urine without centrifugation, the urine sediment and the supernatant after centrifugation. Reports regarding the detection of miRNAs in urine have rarely been published; to date, nine articles are available. The essential results are represented in Table 3. Native urine samples were analyzed in three studies. In the first report, Hanke et al. investigated miRNAs in urine as markers for urinary bladder cancer [41] . A total of 157 miRNAs were analyzed. An RT-qPCRbased screening identified 40 differentially expressed miRNAs in urine of BC patients in comparison to healthy donors. These results were validated and miR126 and miR-182 were favored for the detection of BC from urine. RNU6B was not detectable in all samples,
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Table 2. Concordant reports on down- and upregulation of miRNAs in bladder tumor tissues. miRNA
Ref.
Downregulation miR-1
[39–40,47,52,58]
miR-99a
[37,40,42,47]
miR-100
[37,40,42,47,59]
miR-125b
[42–43,47,59]
miR-133a
[40,42,47,52,58]
miR-133b
[42,47,52,58]
miR-143
[35,38–40,43,47–48,53,58]
miR-145
[35,38–40,42–43,46–48,58]
miR-145b
[38,40,42,47]
Upregulation miR-21
[37,45,61–62]
miR-141
[40,47,53,59]
miR-173
[40,42,47]
miR-200a
[40,43,59]
miR-205
[38,45,47,56,59]
and was therefore not considered suitable for normalization; as a result, miR-152 was used as a normalizer. The ratio of miR-126 to miR-152 reached a specificity of 82% and a sensitivity of 72% with an AUC of 0.768. In comparison, the sensitivity of the ratio of miR-182 to miR-152 was lower (55%) with a specificity of 82% and an AUC of 0.799. In the second study Snowdon et al. [65] , four miRNAs were selected based on published data [43] of miRNA expression profiles in tissue samples from bladder tumors. Two miRNAs (miR-125b and miR-126) showed different expression levels between cancer patients and healthy controls. In the urine of cancer patients, miR-125b was decreased, while miR-126 was increased in comparison to the control group. The increased expression level of miR126 is in accordance with the findings of Hanke et al. [41] in native urine. The authors of this article recently described a miRNA profiling study on 754 miRNAs with a following RT-qPCR validation approach of differentially expressed miRNA in a separate patient group [66] . The validation experiment included six differentially expressed miRNAs and identified two new urine miRNAs (miR-618 and miR-1255b-5p) as markers for BC. The miR-1255b-5p reached 68% specificity and 85% sensitivity in the diagnosis of invasive tumors and discriminated between superficial and invasive tumors. The previously mentioned miRNAs in the other two studies were not confirmed as differentially expressed miRNAs. One reason may be the different normalization approaches used.
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Urine sediments were analyzed in four studies. At first, Yamada et al. reported the miRNA detection in urine sediments [49] . Based on a miRNA profile of BC tissues Ichimi et al., three preselected miRNAs were investigated [42] . The expression levels of miR-96 and miR-183 in the sediments were significantly higher in BC patients than in healthy controls. These miRNAs showed a good sensitivity and specificity for distinguishing BC patients from non-BC-patients (miR-96, 71% and 89.2%, AUC 0.831; miR-183, 74.0% and 77.3%, AUC 0.817). The sensitivity of miR-96 was improved in combination with urine cytology. Furthermore, the miRNA level was normalized after surgery. The overexpressed miR-96 and miR-183 correlated to tumor stage and grade. Wang et al. published a further study of miRNA expression in urine sediments and supernatants; however, they selected miRNAs for the quantification in urine under the aspect of epithelial–mesenchymal transition (EMT) [67] . The patients with BC had a lower expression of the miR-200 family, miR-192, and miR155 in the urinary sediment. Expression of the miR-200 family and miR-192 correlated with the urinary expression of EMT markers. The reduction of miR-200c and miR-141 in urinary sediment was reversible after surgery. Miah et al. studied the problem of a reference for the relative quantification of miRNAs [68] . The small nucleolar RNA RNU48 was detected in all urinary sediments, showing high expression and low variability and was therefore used for normalization. Expression differences between normal and tumor samples of 15 miRNAs were measured using RT-qPCR afterwards. Significant differences were observed for nine of the 15 miRNAs. MiRNA-1224-3p alone had the best result with specificity, positive and negative predictive value and concordance of 83, 83, 75 and 77%, respectively. A combination of three miRNAs (miR-15b, miR-135b, miR-1224-3p) resulted in high sensitivity (94.1%), low specificity (51%) and a correct classification rate of 86%. Mengual et al. performed extensive miRNA screening in urine sediments and the subsequent RT-qPCR validation of differentially expressed miRNAs in a large cohort of samples [69] . miR-103 and miR-30c were used for normalization and six miRNAs (miR-18a*, m iR-25, miR-140-5p, miR-142-3p, m iR-187, miR-204) were identified as capable of classifying BC cases. A sensitivity of 84.8%, specificity of 86.5% and AUC of 0.92 were obtained using these six miRNAs. Furthermore, the authors identified miR-92a and miR-125b as predictors. They discriminated between low-grade and highgrade tumors with a sensitivity of 84.9%, a specificity of 74.1% and an AUC of 0.83. Yun et al. reported on cell-free miRNAs in urine [70] . In a group of 207 patients and 144 controls, they found that miRNA-145 was able to discriminate BC
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miRNA panels as biomarkers for bladder cancer
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Table 3. Different miRNA expression in urinary and blood samples from patients with bladder cancer and controls. Number of patients: tumor vs nonmalignant
Validated miRNAs† by RT-qPCR
Regulation
Reference gene for normalization
Diagnostic or prognostic impact
Ref.
29/11
126; 182
Up
miR-152
Diagnostic
[41]
5/3
125b
Down
RNU6B
Diagnostic marker: miR-125b
[65]
126
Up
36/19
618; 1255-5p
Up
1 ng urine RNA
Diagnostic
[66]
100/49
96; 183
Up
RNUB6
Diagnostic; increase with tumor grade
[49]
51/24
141; 155; 192; 200a; 200b; 200c; 429
Down
RNU48
Correlation to EMT markers
[67]
68/53
15a; 15b; 24-1; 27b; 100; 203; 212; 328; 1224-3p
Down
RNU48
Diagnostic marker: miR-15b; miR-135b; miR-1224-3p
[68]
135b
Up
151/126
18a*; 25; 92a; 187
Up
miR-103,miR-30c
Diagnostic marker: miR-18a*; miR-25; miR-187; miR-140-5p; miR-142-3p; miR-204
125b; 140-5p; 142-3p; 204
Down
Prediction of aggressiveness: miR-92a; miR-125b
207/144
145; 200a
Down
snRNA U6
Diagnostic; decrease with tumor grade and correlation to recurrence
[70]
51/24
155
Up
RNU48
None
[67]
192
Down
None
145; 205
Down
RNU6B
Diagnostic
[71]
141; 639
Differences were not confirmed
cel-miR-39
none
[72]
20/18
148b; 200b; 487; 541; 566
Up
snRNA U6
Diagnostic
[73]
25; 33b; 92a; 92b; 302; 1290
Down
miR-1290; miR-92b correlate with BC state
snRNA U6
Diagnostic
Native urine
Urine sediments
[69]
Urine supernatant
Urine smears 10/15 Serum 126/105
Plasma
Whole blood 38/20
26b-5p; 144-5p; 374b-5p Up
[66]
The name of miRNAs was abridged and only the numerals were mentioned. RT-qPCR: Reverse-transcriptase, quantitative real-time PCR. †
patients from controls. The discrimination reached a sensitivity and specificity for NMIBC of 77.8% and 61.1%, and for MIBC of 84.1% and 61.1%. In addition, miR-200a was proven to be an independent pre-
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dictor of NMIBC recurrence. Previously, miRNA-145 decrease was described in bladder cancer tissues [43] and changes in the miR-200 family were reported in urine sediments [67] . Urinary supernatants were also
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Review Tölle, Ratert & Jung analyzed by Wang et al. [67] . A decreased expression of miR-192 and miR-155 in the supernatants was observed in 51 patients. However, the same article reported the altered expression of miR-192 and miR-155 in urinary supernatants. Simonato et al. used archival cytology urine smears as a new material for miRNA analysis for the first time [71] . Prestained urine smears with >5000 urothelial cells and 1000 nm observed in the plasma supernatant. Nanoparticles of this size contain exosomes and protein complexes with miRNAs. This observation emphasizes the importance of preanalytical factors. In summary, the investigation of miRNA profiles in whole blood as well as in the serum and plasma of BC patients stands at the beginning. However, the potential of this approach is obvious for the improvement of bladder cancer diagnostic and/or prognostic techniques, even in a noninvasive manner. The miRNA patterns which were identified in any blood material showed concordance with a miRNA (miR-200b) observed in bladder cancer tissue (Figure 2B) . Conclusion miRNAs are a new class of molecular biomarkers presented here. miRNAs provide new opportunities to understand bladder cancer at the molecular level due to their involvement in gene-expression regulation. In the case of urinary bladder cancer, both tissue samples and samples from body fluids are useful for analysis. Tissue samples are used as fresh-frozen tissue and/or FFPE material. Urine as native urine, supernatant and
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sediment after centrifugation as well as blood as whole blood, serum or plasma are the body fluid specimens used. Analytical methods, such as microarray, nextgeneration sequencing, bead-based flow cytometry and RT-qPCR, deliver specific, sensitive and reliable miRNA expression results. However, standardized normalization approaches are still lacking. Divergent results are caused by differences in the sample size and clinical characteristics of patients in the studies, in pre-analytics, methods of miRNA measurements and data evaluation. The current results can be summarized as follows: in bladder cancer tissue, miR-141 and miR-205 exhibited diagnostic and/or prognostic potential either as single markers or in different combinations with other miRNAs. Although miR-141 was also described in plasma and serum of patients with prostate cancer [26,79–80] and colon cancer [81] , in bladder cancer miR-141 was exclusively detectable in tissue samples and not body fluids. This fact underlines the great potential of miR-141 for use as diagnostic and/or prognostic biomarker in bladder cancer. Furthermore, seven differentially expressed miRNAs (miR-96, miR-100, miR-125b, miR-145, miR-182, miR-183, and miR-204) were concordantly identified in tumor tissues and urine samples as markers. Future perspective In future there will be more efforts to extend existing guidelines regarding the sample acquisition, handling, preparation, and quantification methods [33] . Additionally, development of synthetic materials as quality control, for example, for sample preparation, and/or as cali-
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Review Tölle, Ratert & Jung brators, for example, for relative quantification, may be led to more comparability as well as reproducibility of results of multicenter studies. Despite these challenges, it was already shown that changes of miRNA expression may be an early marker for diseases still before morphologic changes were detectable. Due to the fact that miRNAs are closely linked with the molecular development of diseases aberrant miRNA levels allow a deep insight in the reason of the disease. In the future it will be even more sensitive detection methods for miRNAs which may facilitate to detect the cancer in very early stage. Thereby the treatment options and the chance of recovery will be better. Already now it is possible to differentiate between response and nonresponse to therapeutic agents with
the help of miRNAs. This fact is a good condition for an individualized medicine. The knowledge about the molecular mechanism of diseases, including miRNAs uncloses new ways for the development of more effective therapeutic agents. Financial & competing interests disclosure The authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties. No writing assistance was utilized in the production of this manuscript.
Executive summary Methods for the analysis of miRNAs & their pitfalls • In the case of urinary bladder cancer, both tissue samples and samples from body fluids are useful for analysis. • Analytical methods such as microarray, reverse-transcription, quantitative real-time PCR and others deliver specific, sensitive and reliable miRNA expression results. However, standardized normalization approaches are still lacking.
miRNA analysis in urinary bladder tissues • Divergent results are caused by differences in the sample size and clinical characteristics of patients in the studies, in pre-analytics, methods of miRNA measurements, and data evaluation. • In bladder cancer tissue, miR-141 and miR-205 exhibited diagnostic and/or prognostic potential either as single markers or in different combinations with other miRNAs. Furthermore, seven differentially expressed miRNAs were concordantly identified in tumor tissues and urine samples as markers.
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Jung M, Schaefer A, Steiner I et al. Robust microRNA stability in degraded RNA preparations from human tissue and cell samples. Clin. Chem. 56, 998–1006 (2010).
26
Mitchell PS, Parkin RK, Kroh EM et al. Circulating microRNAs as stable blood-based markers for cancer detection. Proc. Natl Acad. Sci. USA 105, 10513–10518 (2008).
••
Demonstrates that miRNAs are detectable in urine.
42
Ichimi T, Enokida H, Okuno Y et al. Identification of novel microRNA targets based on microRNA signatures in bladder cancer. Int. J. Cancer 125, 345–352 (2009).
•
Presents the possibility of miRNAs in exosomes to be the reason for their stability.
43
27
Liu CG, Calin GA, Meloon B et al. An oligonucleotide microchip for genome-wide microRNA profiling in human and mouse tissues. Proc. Natl Acad. Sci. USA 101, 9740–9744 (2004).
Lin T, Dong W, Huang J et al. MicroRNA-143 as a tumor suppressor for bladder cancer. J. Urol. 181, 1372–1380 (2009).
44
Lin Y, Wu J, Chen H et al. Cyclin-dependent kinase 4 is a novel target in micoRNA-195-mediated cell cycle arrest in bladder cancer cells. FEBS Lett. 586, 442–447 (2012).
45
Neely LA, Rieger-Christ KM, Neto BS et al. A microRNA expression ratio defining the invasive phenotype in bladder tumors. Urol. Oncol. 28, 39–48 (2010).
46
Ostenfeld MS, Bramsen JB, Lamy P et al. miR-145 induces caspase-dependent and -independent cell death in urothelial cancer cell lines with targeting of an expression signature present in Ta bladder tumors. Oncogene 29, 1073–1084 (2010).
28
Lu J, Getz G, Miska EA et al. MicroRNA expression profiles classify human cancers. Nature 435, 834–838 (2005).
29
Schmittgen TD, Jiang J, Liu Q et al. A high-throughput method to monitor the expression of microRNA precursors. Nucleic Acids Res. 32, e43 (2004).
30
Chen C, Ridzon DA, Broomer AJ et al. Real-time quantification of microRNAs by stem-loop RT-PCR. Nucleic Acids Res. 33, e179 (2005).
31
Benes V, Castoldi M. Expression profiling of microRNA using real-time quantitative PCR, how to use it and what is available. Methods 50, 244–249 (2010).
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Song T, Xia W, Shao N et al. Differential miRNA expression profiles in bladder urothelial carcinomas. Asian Pac. J. Cancer Prev. 11, 905–911 (2010).
32
van Rooij E. The art of microRNA research. Circ. Res. 108, 219–234 (2011).
48
33
Bustin SA, Benes V, Garson JA et al. The MIQE guidelines: minimum information for publication of quantitative realtime PCR experiments. Clin. Chem. 55, 611–622 (2009).
Villadsen SB, Bramsen JB, Ostenfeld MS et al. The miR143/-145 cluster regulates plasminogen activator inhibitor-1 in bladder cancer. Br. J. Cancer 106, 366–374 (2012).
49
34
Ratert N, Meyer HA, Jung M et al. Reference miRNAs for miRNAome analysis of urothelial carcinomas. PLoS ONE 7, e39309 (2012).
Yamada Y, Enokida H, Kojima S et al. MiR-96 and miR183 detection in urine serve as potential tumor markers of urothelial carcinoma: correlation with stage and grade, and comparison with urinary cytology. Cancer Sci. 102, 522–529 (2011).
••
Identifies at first reference miRNAs in bladder cancer tissues.
50
Hirata H, Hinoda Y, Ueno K et al. MicroRNA-1826 targets VEGFC, beta-catenin (CTNNB1) and MEK1 (MAP2K1) in human bladder cancer. Carcinogenesis 33, 41–48 (2012).
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Review Tölle, Ratert & Jung 51
Tatarano S, Chiyomaru T, Kawakami K et al. miR-218 on the genomic loss region of chromosome 4p15.31 functions as a tumor suppressor in bladder cancer. Int. J. Oncol. 39, 13–21 (2011).
66
Tolle A, Jung M, Rabenhorst S et al. Identification of microRNAs in blood and urine as tumour markers for the detection of urinary bladder cancer. Oncol. Rep. 30(4), 1949–1956 (2013).
52
Yoshino H, Chiyomaru T, Enokida H et al. The tumoursuppressive function of miR-1 and miR-133a targeting TAGLN2 in bladder cancer. Br. J. Cancer 104, 808–818 (2011).
67
Wang G, Chan ES, Kwan BC et al. Expression of microRNAs in the urine of patients with bladder cancer. Clin. Genitourin. Cancer 10, 106–113 (2012).
53
Wang G, Zhang H, He H et al. Up-regulation of microRNA in bladder tumor tissue is not common. Int. Urol. Nephrol. 42, 95–102 (2010).
68
Miah S, Dudziec E, Drayton RM et al. An evaluation of urinary microRNA reveals a high sensitivity for bladder cancer. Br. J. Cancer 107, 123–128 (2012).
54
Sanders I, Holdenrieder S, Walgenbach-Brunagel G et al. Evaluation of reference genes for the analysis of serum miRNA in patients with prostate cancer, bladder cancer and renal cell carcinoma. Int. J. Urol. 19, 1017–1025 (2012).
69
Mengual L, Lozano JJ, Ingelmo-Torres M et al. Using microRNA profiling in urine samples to develop a noninvasive test for bladder cancer. Int. J. Cancer 133(11), 2631–2641 (2013).
55
Rosenberg E. Predicting progression of bladder urothelialcarcinoma using microRNA expression. BJU Int. 112(7), 1027–1034 (2013).
••
Uses a miRNA pattern in urine for diagnostic of bladder cancer.
70
56
Gottardo F, Liu CG, Ferracin M et al. Micro-RNA profiling in kidney and bladder cancers. Urol. Oncol. 25, 387–392 (2007).
Yun SJ, Jeong P, Kim WT et al. Cell-free microRNAs in urine as diagnostic and prognostic biomarkers of bladder cancer. Int. J. Oncol. 41, 1871–1878 (2012).
•
Discusses miRNAs found in bladder cancer tissues as biomarkers of this disease.
71
57
Veerla S, Lindgren D, Kvist A et al. MiRNA expression in urothelial carcinomas: important roles of miR-10a, miR222, miR-125b, miR-7 and miR-452 for tumor stage and metastasis, and frequent homozygous losses of miR-31. Int. J. Cancer 124, 2236–2242 (2009).
Simonato F, Ventura L, Sartori N et al. Detection of microRNAs in archival cytology urine smears. PLoS ONE 8, e57490 (2013).
72
Scheffer AR, Holdenrieder S, Kristiansen G et al. Circulating microRNAs in serum: novel biomarkers for patients with bladder cancer? World J. Urol. 32(2), 353–358 (2012).
73
Adam L, Wszolek MF, Liu CG et al. Plasma microRNA profiles for bladder cancer detection. Urol. Oncol. 31(8), 1701–1708 (2013).
74
Kenney PA, Wszolek MF, Rieger-Christ KM et al. Novel ZEB1 expression in bladder tumorigenesis. BJU Int. 107, 656–663 (2011).
75
Ratert N, Meyer HA, Jung M et al. miRNA profiling identifies candidate miRNAs for bladder cancer diagnosis and clinical outcome. J. Mol. Diagn. 15, 695–705 (2013).
Keller A, Leidinger P, Bauer A et al. Toward the blood-borne miRNome of human diseases. Nat. Methods 8, 841–843 (2011).
76
Nordentoft I, Birkenkamp-Demtroder K, Agerbaek M et al. miRNAs associated with chemo-sensitivity in cell lines and in advanced bladder cancer. BMC. Med. Genomics 5, 40 (2012).
Wang LG, Gu J. Serum microRNA-29a is a promising novel marker for early detection of colorectal liver metastasis. Cancer Epidemiol. 36, e61–e67 (2012).
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Mahn R, Heukamp LC, Rogenhofer S et al. Circulating microRNAs (miRNA) in serum of patients with prostate cancer. Urology 77, 1265–1216 (2011).
78
Zheng XH, Cui C, Zhou XX et al. Centrifugation: an important pre-analytic factor that influences plasma microRNA quantification during blood processing. Chin J. Cancer 32(12), 667–672 (2013) (2013).
••
Identifies by unsupervised hierarchical clustering miRNA clusters associated with pTa, PT1 and with pT2-pT4 cancer.
58
Pignot G, Cizeron-Clairac G, Vacher S et al. MicroRNA expression profile in a large series of bladder tumors: Identification of a 3-mirna signature associated with aggressiveness of muscle-invasive bladder cancer. Int. J. Cancer 132(11), 2479–2491 (2012).
59
60
••
Identifies miRNAs which predict the cisplatin treatment response.
61
Wszolek MF, Rieger-Christ KM, Kenney PA et al. A microRNA expression profile defining the invasive bladder tumor phenotype. Urol. Oncol. 29(6), 794–801.e1 (2009).
62
Dip N, Reis ST, Timoszczuk LS et al. Stage, grade and behavior of bladder urothelial carcinoma defined by the microRNA expression profile. J. Urol. 188, 1951–1956 (2012).
79
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63
Wiklund ED, Bramsen JB, Hulf T et al. Coordinated epigenetic repression of the miR-200 family and miR-205 in invasive bladder cancer. Int. J. Cancer 128, 1327–1334 (2011).
80
64
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81
Cheng H, Zhang L, Cogdell DE et al. Circulating plasma MiR-141 is a novel biomarker for metastatic colon cancer and predicts poor prognosis. PLoS ONE 6, e17745 (2011).
65
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Biomarkers Med. (2014) 8(5)
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miRNA panels as biomarkers for bladder cancer
Aim: Specific miRNA profiles have been identified for several samples from patients with bladder cancer. The results are not always congruent and partly contradictory. A comparison of published data was performed to select potential markers. Materials & methods: A literature search in PubMed identified 79 articles published prior to June 2013. Reports regarding the detection of miRNAs in urine and blood have rarely been published; to date, nine respectively three articles are available. Results: The comparison of published data proved the utility of miRNAs as diagnostic and prognostic indicators of bladder cancer. In urine samples from bladder cancer patients, seven miRNAs were concordantly expressed with tumor tissues. Conclusion: Standardization is strictly required in pre-analytics and methods of miRNA measurements.
Angelika Tölle*,1, Nadine Ratert1,2 & Klaus Jung1,2 Department of Urology, Charité– Universitätsmedizin Berlin, 10117 Berlin, Germany 2 Berlin Institute for Urologic Research, 10117 Berlin, Germany *Author for correspondence: angelika.toelle@ charite.de 1
Keywords: biomarker • bladder cancer • miRNAs • serum • tissue • urine
Background Characteristics of bladder cancer & the need for new biomarkers
Urinary bladder cancer (BC) is the fifth most common malignancy. In the USA, an incidence of 72,570 new cases and 15,210 cancer deaths has been projected for 2013 [1] . Males are more often affected than females, with a ratio of 3:1. At the moment of diagnosis, 85% of bladder tumors are limited to the organ, 10% are extended regionally and 5% are metastatic. The most prevalent histological type of bladder cancer in Western countries is transitional cell carcinoma (TCC), accounting for up to 95% of all cases. At initial diagnosis, approximately 70% of bladder cancer presents as nonmuscle invasive (pTa–pT1) bladder tumors (NMIBC), whereas the remaining cases have muscle invasive (pT2–pT4) tumors (MIBC) [2] . Most patients with NMIBC are treated with transurethral resection; however, such tumors recur in up to 70% of cases within 5 years [3] and develop into muscle-invasive tumor. This progression is combined with a marked decrease in 5-year disease-specific survival [4] .
10.2217/BMM.14.26 © 2014 Future Medicine Ltd
Bladder cancer is a malignancy with a complicated, multifactorial etiology, involving both genetic and environmental factors. Two molecular pathways characterize the two types of tumors mentioned above and underline their different biological behavior. For the NMIBC form, the most common genetic alteration is an inactivating mutation in the FGFR3 gene [5] . The FGFR3 gene belongs to a growth factor receptor family related to the tyrosine kinase signaling pathway, which has an important role in embryogenesis, development, angiogenesis, wound healing, tissue homeostasis, tumorigenesis, proliferation migration and apoptosis. For the MIBC form, mutations in P53, Rb1 and PTEN are typical [6] . P53, Rb1, and PTEN are traditional tumor suppressor genes in which mutations and loss of function are related to the loss of proliferation control, apoptosis and an increase of genetic instability, which leads to MIBC tumors [6] . To date, the diagnostic ‘gold standard’ in cases of urinary BC is cystoscopy, which is an invasive and relatively expensive procedure. It is combined with sampling of tissue pieces at suspicious locations and the subse-
Biomarkers Med. (2014) 8(5), 733–746
part of
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Review Tölle, Ratert & Jung quent histopathological examination of biopsy samples. Urine cytology still remains the standard noninvasive method for the detection and follow-up of bladder cancer. However, there are minor morphological abnormalities in low-grade tumors meaning that the detection rate is between 50 and 90% depending on the tumor grade [7] . Extensive laboratory research led to the development of numerous urine-based tests [8] for BC detection, such as bladder tumor antigen (BTA), nuclear matrix protein 22 (NMP22), UroVysion and ImmunCyt; these are all commercially available. However, their good sensitivity is often combined with an insufficient specificity, leading to a high rate of false-positive results. For that reason, none of these markers have generally been accepted for standard diagnostic and follow-up examination [2,9] . Furthermore, the currently used prognostic indices for recurrence or progression, such as tumor grade, tumor stage, number of lesions, tumor size, previous recurrence rate and presence of concomitant carcinoma in situ do not always accurately reflect the clinical outcome. Therefore, the identification of new biomarkers for bladder cancer is urgently needed to improve disease management regarding the diagnosis, prognosis and prediction of treatment for bladder cancer. The identification of new biomarkers for diagnosis, prognosis and prediction of bladder cancer is of urgent need. An ideal marker is produced by the tumor itself and is absent in healthy people or benign conditions, meaning it could then be used for a screening program. Usually marker values increase with disease progression and decrease with remission. The establishment of reference and predictive values are necessary before a wide application will be possible. In principle diagnostic biomarkers are indicators of a particular disease state, whereas prognostic biomarkers allow a statement about chances of recovery and/or disease progression. Predictive biomarkers indicate the probability to develop a certain disease or help to assess the most likely response to a particular therapy. Until now there are no common guidelines on how to evaluate biomarkers. Soletormos et al. propose a four-phase panel analogous to that for new drugs (verifying safety, detection potential, advantages to established markers, and what kind of additional information is needed) [10] . It is precisely in this regard that a new class of small molecules, the so called miRNAs, exhibit promising diagnostic, prognostic and/or predictive potential for cancer in general and also for bladder cancer in particular. In the following sections some important biochemical properties of miRNAs and analytical detection methods as well as articles of special interest to miRNAs used as biomarkers in bladder cancer are summarized.
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What are miRNAs?
For a long time, small noncoding RNAs were regarded as ‘trash’ of transcription. Beside miRNAs, the group of small RNAs with T2. A research group analyzed the expression of miR21, miR-30b, miR-141, miR-205, and miR-200c in NMIBC and MIBC FFPE bladder tumor samples [45,61] . The ratio of miR-21 to miR-205 reached a sensitivity and specificity of 100% and 78%, respec-
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Table 1. Overview of miRNA profiling studies in bladder cancer tissue. Number of patients: tumor vs nonmalignant
Validated miRNAs† by RT-qPCR
Regulation
Reference gene for normalization
Diagnostic and/or prognostic impact
Ref.
25/2
Not reported
[56]
106/11
.
Prognostic: miR-29c; miR-129; miR-133b; miR-518*
[38]
34/0
Diagnostic: miRNA-cluster for Ta, T1, and >T2 Prognostic: miR-452, and miR-452* (positive patients with lymph node metastasis)
[57]
52/10/10 healthy
RNU6B, RNU44, RNU48
Diagnostic and prognostic: miR-99a/miR-100 miR-21/ miR-373 (low- and high-grade discrimination and association with clinical outcome)
[37]
37/9
10; 182; 183; 196a; 203; 244
Up
RNU6B
Not reported
[39]
1; 29c; 101; 127; 143; 145
Down
51/51 (matched)
182; 183; 200a
Up
RNU6B
Not reported
143; 195
Down
104/31
30a-3p; 125b; 133a; 145; 195; 199a*
Down
RNU6B
Diagnostic
[42]
26/26
200a; 200c; 210
Up
RNU6B
Not reported
[43]
125a; 125b; 143; 145; 199b Down
25/25 (matched)
200b; 708
Up
RNU6B
Not reported
1; 99a; 100; 125b; 133a; 133b; 143; 145
Down
23/10
1; 133a; 139-5p; 204; 370; 574-3p
Down
RNU48
Not reported
[52]
7/7
129
Up
β-actin
Not reported
[53]
hsa-let-7a; 30c
Down
94/11
145
Down
RNU6B, RNU43
Not reported
[46]
9/3
143-145 cluster
Down
RNU43, RNU48
Not reported
[48]
28/10
218
Down
RNU48
Not reported
[51]
152/11
9; 138; 182; 200b
Up
RNU6B, RNU44
Prognostic: miR-9; miR-182; miR-200b
[58]
1; 133a; 133b; 143; 145; 199a; 199b; 204; 451; 921; 1281
Down
40/17
20a; 106b; 130b; 141; 200a; 200a*; 205
Up
miR-101, miR-141b, miR-125a-5p,
Diagnostic: miR-130b; miR-141; miR-199a-3p; miR-205
100; 125b; 130a; 139-5p; 145*; 199a-3p; 214; 222
Down
miR-151-5p
Prognostic: miR-141; miR-205
Fresh frozen
[40]
[47]
The name of miRNAs have been abridged and only the numerals are mentioned. FFPE: Formalin-fixed paraffin-embedded; RT-qPCR: Reverse-transcriptase, quantitative real-time PCR. †
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[59]
miRNA panels as biomarkers for bladder cancer
Review
Table 1. Overview of miRNA profiling studies in bladder cancer tissue (cont.). Number of patients: tumor vs nonmalignant
Validated miRNAs† by RT-qPCR
Regulation
Reference gene for normalization
Diagnostic and/or prognostic impact
Ref.
FFPE
137/19
Prognostic: miR-29*
[55]
10/10 (matched primary tumor and lymph node metastasis)
29b; 142-5p
Up
RNU48
Not reported
[35]
143; 145; 320
Down
15/15
27a; 193a-5p; 296-5p; 492; Down 624; 944
miR-193b
Predictive: response to cisplatin therapy
[60]
12/0
21; 205
Up
RNU6B
Diagnostic: miR-21/miR-205
[45]
57/0
21; 99a
Up
miR-222
Diagnostic and prognostic: miR-30b; miR-141; miR-200c
[61]
30b; 31; 200a; 200b; 200c; Down 205; 426
The name of miRNAs have been abridged and only the numerals are mentioned. FFPE: Formalin-fixed paraffin-embedded; RT-qPCR: Reverse-transcriptase, quantitative real-time PCR. †
tively, with an AUC of 0.89 to predict invasion events. Additionally, the three-miRNA signature of miR30b, miR-141, and miR-200c showed a sensitivity of 100% and a specificity of 96.2%. In the multivariate Cox regression analysis, miR-30b exhibited prognostic potential for cancer specific survival. In the study of Baffa et al., 326 miRNAs were screened and a global miRNA signature was reported that discriminated between primary tumors and the corresponding metastasis [35] . miR-143, miR-145, and miR-320 were significantly downregulated whereas miR-10b, miR-29b, miR-142-5p were found to be upregulated in primary bladder tumor samples compared with their metastases. An in situ hybridization against miR-200c on a tissue microarray (TMA) composed of 100 T1 bladder tumor samples showed a loss of miR-200c, which was associated with higher risk of disease progression to muscle-invasive lesions [63] . Another study reported on the ability of miR-886-3p, miR-923, and miR-944 expression to predict the cisplatin treatment response and survival [60] . For bladder cancer tissue, miRNAs are promising biomarkers, which may improve the diagnosis, prognosis, and prediction of therapy response. A concordant downregulation for nine miRNAs (miR-7; miR-99a; miR-100; miR-125b; miR-133a; miR33b; miR-143; miR-145; miR-145b) in bladder tumor tissues was reported by more than four authors and an accordant upregulation of five miRNAs (miR-21; miR-141; miR-173; miR-200a; miR-205) was observed more than three authors (Table 2) . But these results must be interpreted cautiously due to the lack of internal vali-
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dation, for example, leave-one-out, k-fold, or repeated random-split cross validation, as well as external validation, for example, using external data from completely different studies, of many of the mentioned studies. In order to develop a generalizable predictive model involving biomarkers to wider populations it is of urgent need to be aware of proper validation of the obtained results [64] . miRNA analysis in urine
miRNAs in easily accessible urine could reflect bladder tissue situation and are therefore of great diagnostic and prognostic interest in cases of BC. However, the composition of urine is very heterogeneous and the miRNAs measured could result from the kidney, from the tumor or from shed normal cells of the bladder, as well as from inflammatory cells, and as part of the exosomes and protein–miRNA complexes. In the case of urine, three specimens are available, namely the native urine without centrifugation, the urine sediment and the supernatant after centrifugation. Reports regarding the detection of miRNAs in urine have rarely been published; to date, nine articles are available. The essential results are represented in Table 3. Native urine samples were analyzed in three studies. In the first report, Hanke et al. investigated miRNAs in urine as markers for urinary bladder cancer [41] . A total of 157 miRNAs were analyzed. An RT-qPCRbased screening identified 40 differentially expressed miRNAs in urine of BC patients in comparison to healthy donors. These results were validated and miR126 and miR-182 were favored for the detection of BC from urine. RNU6B was not detectable in all samples,
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Table 2. Concordant reports on down- and upregulation of miRNAs in bladder tumor tissues. miRNA
Ref.
Downregulation miR-1
[39–40,47,52,58]
miR-99a
[37,40,42,47]
miR-100
[37,40,42,47,59]
miR-125b
[42–43,47,59]
miR-133a
[40,42,47,52,58]
miR-133b
[42,47,52,58]
miR-143
[35,38–40,43,47–48,53,58]
miR-145
[35,38–40,42–43,46–48,58]
miR-145b
[38,40,42,47]
Upregulation miR-21
[37,45,61–62]
miR-141
[40,47,53,59]
miR-173
[40,42,47]
miR-200a
[40,43,59]
miR-205
[38,45,47,56,59]
and was therefore not considered suitable for normalization; as a result, miR-152 was used as a normalizer. The ratio of miR-126 to miR-152 reached a specificity of 82% and a sensitivity of 72% with an AUC of 0.768. In comparison, the sensitivity of the ratio of miR-182 to miR-152 was lower (55%) with a specificity of 82% and an AUC of 0.799. In the second study Snowdon et al. [65] , four miRNAs were selected based on published data [43] of miRNA expression profiles in tissue samples from bladder tumors. Two miRNAs (miR-125b and miR-126) showed different expression levels between cancer patients and healthy controls. In the urine of cancer patients, miR-125b was decreased, while miR-126 was increased in comparison to the control group. The increased expression level of miR126 is in accordance with the findings of Hanke et al. [41] in native urine. The authors of this article recently described a miRNA profiling study on 754 miRNAs with a following RT-qPCR validation approach of differentially expressed miRNA in a separate patient group [66] . The validation experiment included six differentially expressed miRNAs and identified two new urine miRNAs (miR-618 and miR-1255b-5p) as markers for BC. The miR-1255b-5p reached 68% specificity and 85% sensitivity in the diagnosis of invasive tumors and discriminated between superficial and invasive tumors. The previously mentioned miRNAs in the other two studies were not confirmed as differentially expressed miRNAs. One reason may be the different normalization approaches used.
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Urine sediments were analyzed in four studies. At first, Yamada et al. reported the miRNA detection in urine sediments [49] . Based on a miRNA profile of BC tissues Ichimi et al., three preselected miRNAs were investigated [42] . The expression levels of miR-96 and miR-183 in the sediments were significantly higher in BC patients than in healthy controls. These miRNAs showed a good sensitivity and specificity for distinguishing BC patients from non-BC-patients (miR-96, 71% and 89.2%, AUC 0.831; miR-183, 74.0% and 77.3%, AUC 0.817). The sensitivity of miR-96 was improved in combination with urine cytology. Furthermore, the miRNA level was normalized after surgery. The overexpressed miR-96 and miR-183 correlated to tumor stage and grade. Wang et al. published a further study of miRNA expression in urine sediments and supernatants; however, they selected miRNAs for the quantification in urine under the aspect of epithelial–mesenchymal transition (EMT) [67] . The patients with BC had a lower expression of the miR-200 family, miR-192, and miR155 in the urinary sediment. Expression of the miR-200 family and miR-192 correlated with the urinary expression of EMT markers. The reduction of miR-200c and miR-141 in urinary sediment was reversible after surgery. Miah et al. studied the problem of a reference for the relative quantification of miRNAs [68] . The small nucleolar RNA RNU48 was detected in all urinary sediments, showing high expression and low variability and was therefore used for normalization. Expression differences between normal and tumor samples of 15 miRNAs were measured using RT-qPCR afterwards. Significant differences were observed for nine of the 15 miRNAs. MiRNA-1224-3p alone had the best result with specificity, positive and negative predictive value and concordance of 83, 83, 75 and 77%, respectively. A combination of three miRNAs (miR-15b, miR-135b, miR-1224-3p) resulted in high sensitivity (94.1%), low specificity (51%) and a correct classification rate of 86%. Mengual et al. performed extensive miRNA screening in urine sediments and the subsequent RT-qPCR validation of differentially expressed miRNAs in a large cohort of samples [69] . miR-103 and miR-30c were used for normalization and six miRNAs (miR-18a*, m iR-25, miR-140-5p, miR-142-3p, m iR-187, miR-204) were identified as capable of classifying BC cases. A sensitivity of 84.8%, specificity of 86.5% and AUC of 0.92 were obtained using these six miRNAs. Furthermore, the authors identified miR-92a and miR-125b as predictors. They discriminated between low-grade and highgrade tumors with a sensitivity of 84.9%, a specificity of 74.1% and an AUC of 0.83. Yun et al. reported on cell-free miRNAs in urine [70] . In a group of 207 patients and 144 controls, they found that miRNA-145 was able to discriminate BC
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miRNA panels as biomarkers for bladder cancer
Review
Table 3. Different miRNA expression in urinary and blood samples from patients with bladder cancer and controls. Number of patients: tumor vs nonmalignant
Validated miRNAs† by RT-qPCR
Regulation
Reference gene for normalization
Diagnostic or prognostic impact
Ref.
29/11
126; 182
Up
miR-152
Diagnostic
[41]
5/3
125b
Down
RNU6B
Diagnostic marker: miR-125b
[65]
126
Up
36/19
618; 1255-5p
Up
1 ng urine RNA
Diagnostic
[66]
100/49
96; 183
Up
RNUB6
Diagnostic; increase with tumor grade
[49]
51/24
141; 155; 192; 200a; 200b; 200c; 429
Down
RNU48
Correlation to EMT markers
[67]
68/53
15a; 15b; 24-1; 27b; 100; 203; 212; 328; 1224-3p
Down
RNU48
Diagnostic marker: miR-15b; miR-135b; miR-1224-3p
[68]
135b
Up
151/126
18a*; 25; 92a; 187
Up
miR-103,miR-30c
Diagnostic marker: miR-18a*; miR-25; miR-187; miR-140-5p; miR-142-3p; miR-204
125b; 140-5p; 142-3p; 204
Down
Prediction of aggressiveness: miR-92a; miR-125b
207/144
145; 200a
Down
snRNA U6
Diagnostic; decrease with tumor grade and correlation to recurrence
[70]
51/24
155
Up
RNU48
None
[67]
192
Down
None
145; 205
Down
RNU6B
Diagnostic
[71]
141; 639
Differences were not confirmed
cel-miR-39
none
[72]
20/18
148b; 200b; 487; 541; 566
Up
snRNA U6
Diagnostic
[73]
25; 33b; 92a; 92b; 302; 1290
Down
miR-1290; miR-92b correlate with BC state
snRNA U6
Diagnostic
Native urine
Urine sediments
[69]
Urine supernatant
Urine smears 10/15 Serum 126/105
Plasma
Whole blood 38/20
26b-5p; 144-5p; 374b-5p Up
[66]
The name of miRNAs was abridged and only the numerals were mentioned. RT-qPCR: Reverse-transcriptase, quantitative real-time PCR. †
patients from controls. The discrimination reached a sensitivity and specificity for NMIBC of 77.8% and 61.1%, and for MIBC of 84.1% and 61.1%. In addition, miR-200a was proven to be an independent pre-
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dictor of NMIBC recurrence. Previously, miRNA-145 decrease was described in bladder cancer tissues [43] and changes in the miR-200 family were reported in urine sediments [67] . Urinary supernatants were also
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Review Tölle, Ratert & Jung analyzed by Wang et al. [67] . A decreased expression of miR-192 and miR-155 in the supernatants was observed in 51 patients. However, the same article reported the altered expression of miR-192 and miR-155 in urinary supernatants. Simonato et al. used archival cytology urine smears as a new material for miRNA analysis for the first time [71] . Prestained urine smears with >5000 urothelial cells and 1000 nm observed in the plasma supernatant. Nanoparticles of this size contain exosomes and protein complexes with miRNAs. This observation emphasizes the importance of preanalytical factors. In summary, the investigation of miRNA profiles in whole blood as well as in the serum and plasma of BC patients stands at the beginning. However, the potential of this approach is obvious for the improvement of bladder cancer diagnostic and/or prognostic techniques, even in a noninvasive manner. The miRNA patterns which were identified in any blood material showed concordance with a miRNA (miR-200b) observed in bladder cancer tissue (Figure 2B) . Conclusion miRNAs are a new class of molecular biomarkers presented here. miRNAs provide new opportunities to understand bladder cancer at the molecular level due to their involvement in gene-expression regulation. In the case of urinary bladder cancer, both tissue samples and samples from body fluids are useful for analysis. Tissue samples are used as fresh-frozen tissue and/or FFPE material. Urine as native urine, supernatant and
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sediment after centrifugation as well as blood as whole blood, serum or plasma are the body fluid specimens used. Analytical methods, such as microarray, nextgeneration sequencing, bead-based flow cytometry and RT-qPCR, deliver specific, sensitive and reliable miRNA expression results. However, standardized normalization approaches are still lacking. Divergent results are caused by differences in the sample size and clinical characteristics of patients in the studies, in pre-analytics, methods of miRNA measurements and data evaluation. The current results can be summarized as follows: in bladder cancer tissue, miR-141 and miR-205 exhibited diagnostic and/or prognostic potential either as single markers or in different combinations with other miRNAs. Although miR-141 was also described in plasma and serum of patients with prostate cancer [26,79–80] and colon cancer [81] , in bladder cancer miR-141 was exclusively detectable in tissue samples and not body fluids. This fact underlines the great potential of miR-141 for use as diagnostic and/or prognostic biomarker in bladder cancer. Furthermore, seven differentially expressed miRNAs (miR-96, miR-100, miR-125b, miR-145, miR-182, miR-183, and miR-204) were concordantly identified in tumor tissues and urine samples as markers. Future perspective In future there will be more efforts to extend existing guidelines regarding the sample acquisition, handling, preparation, and quantification methods [33] . Additionally, development of synthetic materials as quality control, for example, for sample preparation, and/or as cali-
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Review Tölle, Ratert & Jung brators, for example, for relative quantification, may be led to more comparability as well as reproducibility of results of multicenter studies. Despite these challenges, it was already shown that changes of miRNA expression may be an early marker for diseases still before morphologic changes were detectable. Due to the fact that miRNAs are closely linked with the molecular development of diseases aberrant miRNA levels allow a deep insight in the reason of the disease. In the future it will be even more sensitive detection methods for miRNAs which may facilitate to detect the cancer in very early stage. Thereby the treatment options and the chance of recovery will be better. Already now it is possible to differentiate between response and nonresponse to therapeutic agents with
the help of miRNAs. This fact is a good condition for an individualized medicine. The knowledge about the molecular mechanism of diseases, including miRNAs uncloses new ways for the development of more effective therapeutic agents. Financial & competing interests disclosure The authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties. No writing assistance was utilized in the production of this manuscript.
Executive summary Methods for the analysis of miRNAs & their pitfalls • In the case of urinary bladder cancer, both tissue samples and samples from body fluids are useful for analysis. • Analytical methods such as microarray, reverse-transcription, quantitative real-time PCR and others deliver specific, sensitive and reliable miRNA expression results. However, standardized normalization approaches are still lacking.
miRNA analysis in urinary bladder tissues • Divergent results are caused by differences in the sample size and clinical characteristics of patients in the studies, in pre-analytics, methods of miRNA measurements, and data evaluation. • In bladder cancer tissue, miR-141 and miR-205 exhibited diagnostic and/or prognostic potential either as single markers or in different combinations with other miRNAs. Furthermore, seven differentially expressed miRNAs were concordantly identified in tumor tissues and urine samples as markers.
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