european urology 50 (2006) 1347–1356 available at www.sciencedirect.com journal homepage: www.europeanurology.com From Lab to Clinic Partially Degraded RNA from Bladder Washing is a Suitable Sample for Studying Gene Expression Profiles in Bladder Cancer Lourdes Mengual a, Moise`s Burset a, Elisabet Ars a, Marı´a Jose´ Ribal b, Juan Jose´ Lozano c, Bele´n Minana c, Lauro Sumoy c, Antonio Alcaraz b,* a Molecular Biology Laboratory, Fundacio´ Puigvert, Universitat Auto`noma de Barcelona, Spain Department of Urology, Hospital Clı´nic, Institut d’Investigacions Biome`diques August Pi i Sunyer (IDIBAPS), Universitat de Barcelona, Spain c Microarray Laboratory, Program in Bioinformatics and Genomics, Centre de Regulacio´ Geno`mica, Barcelona, Spain b Article info Abstract Article history: Accepted May 29, 2006 Published online ahead of print on June 15, 2006 Objectives: To determine the impact of different levels of RNA degradation on gene expression measurements and to ascertain if the gene expression profile obtained from bladder washing (BW) correlates to that obtained from the related bladder tumour (BT). Methods: BT and BW RNAs from the same patient were heat shocked to obtain three RNA degradation states, which were compared with intact RNAs from healthy bladders by using complementary DNA (cDNA) microarrays. All samples were amplified by means of a T3N9-based transcription method. In addition, four of the differentially expressed genes in microarrays related to bladder cancer (KRT20, IGF2, GSN, and CCL2) were analyzed in 36 tumoural and 14 control BW samples by quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR). Results: A high percentage of overlapping differentially expressed genes were detected between BT arrays (85–91%) and between BW arrays (78–93%). Furthermore, the similarity between BW and BT arrays was relatively high and independent of the RNA degradation state (52–60%). Finally, expression differences for the four selected genes were confirmed in the vast majority of extended BW samples tested by qRT-PCR. Conclusions: Our results showed that partially degraded RNA samples analyzed by cDNA microarrays yielded gene expression profiles comparable to those obtained using intact RNA. Moreover, BW RNA exhibited gene expression patterns similar to those identified in the BT, indicating that BW is an appropriate sample for studying gene expression profiles of BT using cDNA microarrays. In addition, qRT-PCR results further support the suitability of BW for gene expression profiling and its potential use for routine diagnostics. # 2006 European Association of Urology. Published by Elsevier B.V. All rights reserved. Keywords: Bladder neoplasms Gene expression profiling Microarray analysis Molecular diagnostic techniques Reverse transcriptase polymerase chain reaction * Corresponding author. Hospital Clı´nic, Villarroel, 170, 08036 Barcelona, Spain. Tel. + 34 93 2275545; Fax: +34 93 2275545. E-mail address: [email protected] (A. Alcaraz). 0302-2838/$ – see back matter # 2006 European Association of Urology. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.eururo.2006.05.039 1348 1. european urology 50 (2006) 1347–1356 Introduction Optimal diagnosis of bladder cancer currently relies on cystoscopy, which is an invasive method. Urinary cytology supplements but cannot replace endoscopic evaluation [1]. As a noninvasive alternative for diagnosing bladder cancer, gene expression levels of several molecular tumour markers have been recently studied in voided urine or bladder washing (BW) [2–6]. Unfortunately, these studies have been performed with a small number of genes, and further confirmation is needed before their clinical application. The development of DNA microarray technologies is likely to facilitate the generation of novel diagnostic tools for bladder cancer detection. Thus, the study of bladder fluids by this technology could provide a noninvasive diagnostic approach as well as improve the reliability of current bladder cancer diagnostic methods. However, application of microarray technology is limited by the requirement of large amounts of high quality RNA. This requisite is especially difficult to achieve in some human samples because of the limited amounts available and their inherent low RNA quality. Protocols based on the linear T7dT-based in vitro transcription method described by Van Gelder and Eberwine [7,8] can amplify small amounts of mRNA without significantly distorting the information content of the sample [9], but they can be sensitive to RNA degradation. Xiang and colleagues [10] developed a new procedure that is able to amplify degraded RNA [10]. This alternative is based on the use of random nonamer primers modified by the addition of an upstream T3 promoter sequence (T3N9) to prime the initial round of reverse transcription. In this report, we have used the T3N9-based in vitro transcription method to analyze the suitability of partially degraded RNA from BW as a sample for bladder cancer diagnostics with the use of cDNA microarrays, and we have validated the differential expression detected by microarrays for four bladder cancer–related genes on an extended set of BW samples by quantitative RT-PCR (qRT-PCR). 2. Methods 2.1. Samples and RNA preparation All samples used in this study were collected between June 2003 and January 2005. Individual tumour tissue and bladder washing samples (T0 and BW0, respectively) were obtained from a patient with bladder cancer pathologically diagnosed as pT2 high-grade (HG) transitional cell carcinoma [11,12] during therapeutical surgery. Four urothelium specimens from individuals without a history of urothelial cancer were obtained intrasurgery as control tissue. Tissue samples (tumour and controls) were immediately frozen after collection in liquid nitrogen and stored at 80 8C until processed. Fifty BW, 36 from bladder cancer patients (8 pTa low grade [LG], 5 pTa HG, 3 pT1 LG, 5 pT1 HG, 4 pTis, 9 pT2 HG and 2 pT4 HG) [11,12] and 14 from patients without a history of bladder cancer were collected. None of the bladder cancer patients were treated with BCG therapy prior to sample collection. The hospital ethics committee approved this study and the patient and controls provided their informed consent before participating in the study. For washings, 100 ml of physiologic NaCl solution was flushed through the bladder about five times via a urethral catheter with a syringe. Ice-cooled BW samples were mixed with 1/25 volumes of 0.5 mol/l EDTA, pH 8.0, and were centrifuged at 1000 g for 10 min. The cell pellets were resuspended in 1 ml of TRIzol reagent (Invitrogen, Carlsbad, CA, USA) and frozen at 80 8C until RNA extraction. Tissue and BW RNAs were extracted with the use of TRIzol reagent according to the manufacturer’s instructions. Total RNA was quantified by spectrophotometric analysis at 260 nm. Equal amounts of intact RNA from T0 and BW0 were incubated for 15 (T1 and BW1), 30 (T2 and BW2) and 60 (T3 and BW3) minutes at 80 8C as described [10], with the exception that RNAse-free water was used instead of base buffer (Table 1). The four RNAs obtained from the healthy urothelium specimens were pooled in an equimolar proportion (C0). One microliter of each intact and partially degraded RNA sample was analyzed with the use of the Agilent 2100 bioanalyzer (Agilent Technologies, Waldbronn, Germany) [13] to assess the degree of degradation (Fig. 1). 2.2. T3N9-based in vitro amplification and direct labelling method Five micrograms of RNA templates were used for RNA amplification as previously described by Xiang et al. [10,14]. Probes were synthesized by a direct labelling method [15] and were purified with QIAquick PCR purification columns (Qiagen, Valencia, CA, USA). Labelling efficiency was calculated by quantification with NanoDrop (NanoDrop Technologies, Wilmington, DE, USA), obtaining between 40–50 pmol/ml of Cy3or Cy5-labelled probes. 2.3. Array processing and data analysis Two-colour microarray hybridizations were performed, confronting the four progressively degraded RNAs from each of the two types of cancer-derived samples against the intact pool of control bladder RNA, used as common reference in all hybridizations (Table 1). Oncochip version 2 glass human cDNA microarrays produced at the Centro Nacional de Investigaciones Oncolo´gicas (http://grupos.cnio.es/ing/) were used. Fluorescent images were obtained with the use of the Agilent G2565BA Microarray Scanner System (Agilent Technologies) and TIFF images were quantified with the use of the Spot program (http://experimental.act.cmis.csiro.au/Spot/index.php) under N/A: not available. C0 refers to the intact pool of RNA obtained from four healthy bladder tissues (control sample). T0 and BW0 are the intact RNAs isolated from BT and BW samples, respectively, from the same bladder cancer patient. b 28S/18S rRNA ratios, % of total 28S and 18S area, and RNA integrity numbers (RINs) are used to quantify the state of RNA degradation. c Each specific RNA comparison was performed in duplicate, with dye swapping for a total of 16 hybridization experiments (as a matter of convenience, when we refer to the array name shown in thetable, we consider the mean values of the array and its dye swap duplicate). d Log 2 ratio limits of the 100 most differentially expressed genes of each array. BW0/C0 BW1/C0 BW2/C0 BW3/C0 36 34 31 23 8.6 7.2 5.8 3.6 24.92 12.17 4.51 0 16.04 12.99 9.13 4.43 1.55 0.94 0.49 0 0 15 30 60 BW0 BW1 BW2 BW3 Cancerous bladder washing a 1.68 1.86 1.64 2.27 1.64 1.79 1.64 2.25 0.98 1.06 0.99 1.03 0.97 1.06 1.00 1.00 N/A N/A N/A 4 T0 T1 T2 T3 Bladder tumour 0 15 30 60 1.9 1.26 0.75 0 11.47 10.17 7.86 4.43 21.74 12.78 5.9 0 32 31 26 24 T0/C0 T1/C0 T2/C0 T3/C0 – – 27 8.4 23.44 15.2 1.5 0 C0 Control bladder 28S/18S ratiob RNA degradation time (80 8C) (min) RNA/array namea RNA source Table 1 – Summary of RNA sample preparation and processing % of total 18S areab % of total 28S areab RINb RNA yield (mg) Samples cohybridizedc Log 2 ratio limitsd european urology 50 (2006) 1347–1356 1349 the R environment (http://www.r-project.org). The resulting raw values were filtered and an intensity cutoff was applied, selecting those points with a foreground median/background median >3 in at least one channel. The final intensity measure for each spot was calculated as previously suggested (http:// www.stat.berkeley.edu/users/terry/zarray/html/image.html); intensity values <150 were considered as background intensities and were converted to 150 for further calculations (publicly available in Gene Expression Omnibus database; series GSE3192). The mean of all considered good spots was obtained as a final clone intensity measure. Logarithmic ratio (log 2 ratio) was calculated between both channels (log 2 [R/G]). Of 4269 clones with at least one valid expression ratio from direct and dye swap duplicates, only those with an intensity value 400 for at least one channel were considered (1111 clones). The 100 most differentially expressed clones of each array were selected to compare between arrays, and the percentage of overlapping genes was calculated (Fig. 2A). Common (which appeared differentially expressed in at least 12 of 16 arrays) and unique nonoverlapping genes, exclusive of bladder tumour (BT) or BW, were grouped according to the biologic processes on the basis of gene ontology (GO) functional annotation (http://david.niaid. nih.gov/david/version2/index.htm) [16]. Unsupervised hierarchical cluster analysis was performed for all clones contained in the microarray using unweighted pair group method with arithmetic averages and Pearson correlation distance. 2.4. Quantitative RT-PCR To validate gene expression data from microarrays (technical validation of microarray data), 10 of the most differentially expressed genes (ANXA1, CD74, CDH11, EGR1, HIST1H2AC, IGF2, ITGA5, KRT20, PPAP2B and VAV3) were tested by qRT-PCR on intact and partially degraded RNA obtained from BT and BW. On the other hand, to demonstrate that the differences between tumoural and control microarrays were mainly due to the presence of a bladder carcinoma (biologic validation of the microarray results), we selected four differentially expressed genes related to bladder cancer according to the literature (KRT20, IGF2, GSN, and CCL2) [17–20] to be tested in 50 BW (36 cancerous and 14 controls). Two of the selected genes for the technical validation (KRT20 and IGF2) were also chosen for the biologic validation since they are involved in bladder carcinogenesis. cDNA was synthesized from 1 mg of total RNA with the use of the High-Capacity cDNA Archive Kit (Applied Biosystems, Foster City, CA, USA) according to the manufacturer’s instructions, except that the final volume of the reaction was 50 ml. The gene GUSB was used as endogenous control. PCRs were performed with the use of Assays-on-Demand gene expression products in an ABI Prism 7000 SDS (Applied Biosystems) according to the manufacturer’s recommendations, except that the final volume of the reaction was 20 ml. All samples were amplified in duplicate and the CT mean was obtained for further calculations. Duplicates with SD 0.38 were excluded, and these samples were reamplified. The DDCt method (ABI Prism 7700 Sequence Detection System User Bulletin #2: relative quantification of gene expression P/N 1350 european urology 50 (2006) 1347–1356 Fig. 1 – Agilent 2100 bioanalyzer electropherograms from intact and partially degraded RNA samples from bladder tumour (BT), bladder washing (BW), and control samples. Intact RNAs from tumour and bladder washing samples (T0 and BW0) were heat shocked at 80 8C for different time periods to obtain three degradation states. The numbers 0, 1, 2, and 3 were assigned to samples in order of increasing degradation; the same number was chosen for samples with comparable RNA quality. european urology 50 (2006) 1347–1356 1351 Fig. 2 – Percentage of differentially expressed genes in common between pairs of arrays. (A) Semimatrix comparison between pairs of arrays of the 100 most differentially expressed genes of each array. The orange-shaded upper and lower parts of the semimatrix show the percentage of differentially expressed genes identified in common between pairs of BT or BW arrays, respectively, hybridized with RNA at different degradation states. The white part of the table corresponds to the percentage of differentially expressed genes identified in common between pairs of tumour and bladder washing arrays. (B) Unsupervised hierarchical cluster analysis for all clones contained in the microarray, including the respective dye swap duplicates. DS: dye swapping. 4303859) was used to quantify the relative amount of mRNA in each tumoural sample in comparison with the C0 sample (technical validation) or to the mean of the 14 control samples (biologic validation). Next, 2 DDCt of each gene was transformed into logarithmic scale (log 2 ratio), and comparisons to the microarray results were performed by means of linear regression. The analysis of variance test was used to determine if the expression levels measured by qRT-PCR of intact and partially degraded RNA were significantly different. The SPSS software version 12.0 (SPSS Inc, Chicago, IL, USA) was used for all statistical calculations. 3. Results 3.1. Effect of degraded RNA on microarray profiling Intact RNA from tumour (T0) and bladder washing (BW0) samples were heat shocked to obtain three controlled degradation states (Fig. 1 and Table 1). We cohybridized the four progressively degraded RNA aliquots from BT and BW with the intact control bladder RNA. A high percentage of overlapping differentially expressed genes were detected between BT arrays (85–91%) and between BW arrays (78–93%) (Fig. 2A). Interestingly, the percentage of genes in common identified between the two arrays hybridized with different RNA degradation states (e.g., BW0 and BW1) was, sometimes, higher than the percentage between dye swap duplicates of the same array (e.g., BW0 vs BW0-DS; Fig. 2B). When we analyzed the suitability of BW for diagnosing bladder cancer using cDNA microarrays, we found that the similarity between BW and BT was relatively high and independent of the RNA degradation state (52–60%; Fig. 2A). Among the differentially expressed genes, we detected 48 genes in common between BW and BT samples (see Materials and Methods for details). These genes showed enrichment in ‘‘growth’’ and ‘‘differentiation’’ go categories. On the other hand, we detected 33 and 35 differentially expressed genes exclusive to BW and BT, respectively. The BW-specific genes were enriched in ‘‘immune response’’ and ‘‘catabolism,’’ whereas tumour-related genes were, once again, enriched in ‘‘growth.’’ 1352 european urology 50 (2006) 1347–1356 Fig. 3 – Comparison of microarray and quantitative RT-PCR (qRT-PCR) expression data of 10 selected differentially expressed genes. Positive values indicate overexpression in BT or BW, compared with a pool of healthy bladder tissue (C0). The down arrows on BW indicate that these samples do not contain enough CDH11 cDNA for an accurate comparison with the control bladder tissue. Therefore, the stated expression level should be interpreted as the minimum value of differential expression for BW samples. The numbers 0, 1, 2, and 3 correspond to the RNA degradation states in dicated in Table 1. Fig. 4 – Differential expression of four bladder cancer-related genes between tumoral and control BW by qRT-PCR. Positive values indicate overexpression in tumoral BW compared with the control BW samples. For the control reference the mean of the 14 BW controls tested was obtained. Samples are arranged by tumoral stage and grade; low grade (LG) superficial tumours (8 pTa LG and 3 pT1 LG), high grade (HG) superficial tumours (5 pTa HG, 5 pT1 HG, and 4 pTis), and muscle invasive tumours (9 pT2 and 2 pT4). european urology 50 (2006) 1347–1356 3.2. Validation of microarray data using qRT-PCR We were able to verify the 10 tested genes in BW samples (100%). In the tumour samples, we could validate 90% (9 of 10) of tested genes (except HIST1H2AC) (Fig. 3). Perhaps cross-hybridization processes that are much more common in microarrays than in qRT-PCR could account for these differences. The comparison of microarray and qRTPCR results for the 10 selected genes showed a high correlation between the two methods (r = 0.84, p < 0.0005), although differences in the magnitude of change between microarrays and qRT-PCR were found; 78% of samples had greater gene expression in qRT-PCR than in microarrays. Otherwise, qRTPCR analysis showed no significant differences in relative differential expression levels between intact and partially degraded RNA samples ( p = 0.99). Gene expression validation of KRT20, GSN, IGF2 and CCL2 by means of qRT-PCR in 36 individual BWs from bladder cancer patients is shown in Fig. 4. The percentage of BW samples that confirmed microarray results was 81% for KRT20, 89% for GSN, 64% for IGF2, and 89% for CCL2. 4. Discussion In this study, cDNA microarrays were used to determine if gene expression profiles derived from low-quality RNAs obtained from a cancerous BW sample correlated with those obtained from the related BT. Using a controlled RNA degradation procedure, we obtained different RNA degradation states that could represent the quality of the vast majority of the RNA that we generally isolate from BW samples. Subsequently, we selected the linear T3N9-based in vitro transcription method to amplify the limiting RNA quantities usually isolated from bladder fluids. We found that the quality of the starting RNA was directly related to the quantity and quality of the RNA produced by the T3N9-based transcription method (Table 1). Nevertheless, microarray expression data did not seem to be practically affected by the initial RNA quality. Thus, we were able to identify a high percentage of differentially expressed genes in common between the different degraded RNA states of both BT (85–91%) and BW (78–93%) samples (Fig. 2A). However, even though the RNA degradation patterns of our RNAs are very similar to those obtained by other authors who used more physiologic RNA degradation methods, such as endogenous tissue ribonucleases [21] or RNAse A [22], it remains to be shown whether physiologic RNAse degradation 1353 would have an effect on the reproducibility of measurements on equivalent samples. Therefore, our data suggest that small amounts of partially degraded RNA amplified with the T3N9based method may be used in cDNA microarray studies without a great reduction in the ability to detect differentially expressed genes. Moreover, it is also of note that relative gene expression levels measured by qRT-PCR do not seem to be affected by RNA degradation either, as previously described [22]. This observation opens up the possibility of performing qRT-PCR studies on inherently degraded RNA samples such as those extracted from urine, archival paraffin-embedded specimens, faeces, and so forth. In the present study, we also sought to determine whether gene expression profiles found in BW reflected those obtained in the related tumour. The comparison of microarrays hybridized with both intact RNA samples (T0 vs BW0) yielded an important percentage of overlapping differentially expressed genes (59%), indicating the suitability of the BW sample to reflect tumour gene expression profiles. It is remarkable that the percentage of differentially expressed genes in common among all the microarrays hybridized with different RNA degradation states is practically constant (52–60%). On the other hand, although our aim was not to identify differentially expressed genes in bladder cancers, we determined that the common differentially expressed genes between BT and BW were mainly related to cancer (the GO functional annotations were growth and differentiation). It is important to emphasize that some of the genes identified (e.g., UPK1A, KRT20, IGF2, GSN, ANXA1, CCL2, NRAS, etc) had been previously described as bladder cancer-related genes [17–20,23–26] and, interestingly, some of them had already been suggested as bladder cancer markers in urine or BW [18,27,28]. To confirm some differentially expressed genes detected in our microarrays in a larger set of BW, we evaluated KRT20, IGF2, GSN, and CCL2 gene expressions in 36 individual BW samples by qRTPCR. The concordance of microarray data of a single patient and qRT-PCR results in an extended series of BW strongly suggest that our findings were not due to the particular gene expression profile of the analyzed patient. Finally, we focused on gene expression differences between BT and BW. These gene expression variations may reflect differences in tissue composition, since BW represent primarily urotheliumderived cells, whereas the tumour sample may contain other cell types such as smooth muscle, mesenchymal fibroblasts, and endothelial cells [29]. 1354 european urology 50 (2006) 1347–1356 We found the growth biologic process category overrepresented within the list of tumour-specific genes. Specifically, we identified many genes involved in nucleic acid metabolism such as EGR1, ERF, HIST1H1C, or ENTPD5. This observation could be due to the enrichment of cancer cells in the BT, compared with the BW sample, which must be composed by a mix of normal and cancerous cells producing a dilution effect. When we analyzed genes expressed exclusively in BW, we found that functional annotations for immune response and catabolism were overrepresented. For example, we have identified genes such as HLA-G, IGLL1, or IFITM1, which are strongly related with immunity and defence processes, and genes such as MALT1, TIMP3, MMP2, or TIMP2, which are involved mainly in proteolysis and protein metabolism. A possible explanation for these findings could be the need for a constant defence of the urothelial mucosa against external agents, which would be reflected in the BW. In addition, catabolism processes could be occurring in the damaged cells present in the bladder. 5. Conclusions In this study, we demonstrated that a small amount of partially degraded RNA isolated from BW, processed with the T3N9-based in vitro amplification method, is suitable for inferring gene expression profiles in the related BT by microarray expression analysis. Our results provide information that could lead to encouraging the development of a noninvasive method to diagnose and provide a prognosis in BT, which is based on gene expression patterns derived from bladder fluid cellular fractions. This alternative diagnostic method could become available in the future once appropriate molecular bladder cancer biomarkers are selected. The next step should be to test if similar results can be obtained with the use of RNA isolated from urine samples. Acknowledgements This work was supported by Laboratorios Indas S.A. and by a grant from Fondo de Investigaciones Sanitarias (FIS04/2630). The authors would like to thank Manel Ferna´ndez, Lidia Sedano, and David Otero for their excellent technical assistance and Helena Kruyer for correction of the manuscript. We are also grateful to Dr Artur Oliver for his help with the statistical analysis and to Dr Ferran Algaba (Section of Pathology) and Dr Humberto Villavicencio (Department of Urology) for their clinical support. References [1] Little B. Non-invasive methods of bladder cancer detection. Int Urol Nephrol 2003;35:331–43. [2] Siracusano S, Niccolini B, Knez R, et al. The simultaneous use of telomerase, cytokeratin 20 and CD4 for bladder cancer detection in urine. Eur Urol 2005;47:327–33. [3] de Kok JB, van Balken MR, Roelofs RW, van Aarssen YA, Swinkels DW, Klein Gunnewiek JM. Quantification of hTERT mRNA and telomerase activity in bladder washings of patients with recurrent urothelial cell carcinomas. Clin Chem 2000;46:2003–7. [4] Schultz IJ, Kiemeney LA, Karthaus HF, et al. Surviving mRNA copy number in bladder washings predicts tumor recurrence in patients with superficial urothelial cell carcinomas. Clin Chem 2004;50:1425–8. [5] Chiu AW, Huang YL, Huan SK, et al. 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In: Eble JN, Sauter G, Epstein JI, Sesterhenn IA, editors. Pathology and genetics of tumours of the urinary system and male genital organs. World Health Organization classification of tumours. Lyon: IARC Press; 2004. p. 89–157. [12] Sobin LH, Wittekind CH. TNM classification of malignant tumours. International Union Against Cancer. 6th ed. New York: John Wiley & Sons; 2002. [13] Imbeaud S, Graudens E, Boulanger V, et al. Towards standardization of RNA quality assessment using userindependent classifiers of microcapillary electrophoresis traces. Nucleic Acids Res 2005;33:E56. [14] Xiang CC, Chen M, Kozhich OA, et al. Probe generation directly from small numbers of cells for DNA microarray studies. Biotechniques 2003;34:386–93. [15] Richter A, Schwager C, Hentze S, Ansorge W, Hentze MW, Muckenthaler M. Comparison of fluorescent tag DNA european urology 50 (2006) 1347–1356 [16] [17] [18] [19] [20] [21] [22] labeling methods used for expression analysis by DNA microarrays. Biotechniques 2002;33, 620–8, 630. Dennis Jr G, Sherman BT, Hosack DA, et al. DAVID: Database for annotation, visualization, and integrated discovery. Genome Biol 2003;4:3. Fichera E, Liang S, Xu Z, Guo N, Mineo R, Fujita-Yamaguchi Y. A quantitative reverse transcription and polymerase chain reaction assay for human IGF-II allows direct comparison of IGF-II mRNA levels in cancerous breast, bladder, and prostate tissues. Growth Horm IGF Res 2000;10:61–70. Parekattil SJ, Fisher HA, Kogan BA. Neural network using combined urine nuclear matrix protein-22, monocyte chemoattractant protein-1 and urinary intercellular adhesion molecule-1 to detect bladder cancer. J Urol 2003;169:917–20. Simoneau M, Aboulkassim TO, Larue H, Rousseau F, Fradet Y. Four tumor suppressor loci on chromosome 9q in bladder cancer: evidence for two novel candidate regions at 9q22.3 and 9q31. Oncogene 1999;18:157–63. Ribal MJ, Mengual L, Marin M, et al. Molecular staging of bladder cancer with RT-PCR assay for CK20 in peripheral blood, bone marrow and lymph nodes: comparison with standard histological staging. Anticancer Res 2006;26: 411–20. Schoor O, Weinschenk T, Hennenlotter J, et al. Moderate degradation does not preclude microarray analysis of small amounts of RNA. Biotechniques 2003;35:1192–201. Tong D, Schneeberger C, Leodolter S, Zeillinger R. Quantitative determination of gene expression by competitive Editorial Comment Morgan Roupreˆt, Urology Department, Hospital Pitie´-Salpe´trie`re, University Paris VI, France [email protected] Bladder cancer is the fourth commonest malignancy amongst men in the western world. The majority of these tumors are of the superficial phenotype and most patients (>50%) develop more than one carcinoma in their lifetime [1]. Approximately 90% of bladder transitional cell carcinomas are superficial (pTa, pT1, pTis) at initial diagnosis. The rate of tumor recurrence ranges 50–80% in the couple of years following tumor resection and progression to invasive disease accounts for 15–23% of recurred cases [1]. Because tumor recurrence, if not detected, may progress and invade the musculature, early detection of tumor recurrence is of primary importance and requires close surveillance for successful treatment. Follow-up is essentially based on cystoscopy: the gold-standard method, despite being invasive and uncomfortable [1]. Non-invasive ancillary methods, less expensive, with high sensitivity and sensibility would be of great help in the diagnosis and follow-up [23] [24] [25] [26] [27] [28] [29] 1355 reverse transcription-polymerase chain reaction in degraded RNA samples. Anal Biochem 1997;251:173–7. Olsburgh J, Harnden P, Weeks R, et al. Uroplakin gene expression in normal human tissues and locally advanced bladder cancer. J Pathol 2003;199:41–9. Sanchez-Carbayo M, Socci ND, Lozano JJ, et al. Gene discovery in bladder cancer progression using cDNA microarrays. Am J Pathol 2003;163:505–16. Przybojewska B, Jagiello A, Jalmuzna P. H-RAS, K-RAS, and N-RAS gene activation in human bladder cancers. Cancer Genet Cytogenet 2000;121:73–7. Christoph F, Muller M, Schostak M, Soong R, Tabiti K, Miller K. Quantitative detection of cytokeratin 20 mRNA expression in bladder carcinoma by real-time reverse transcriptase-polymerase chain reaction. Urology 2004; 64:157–61. Eissa S, Kenawy G, Swellam M, El Fadle AA, Abd El-Aal AA, El Ahmady O. Comparison of cytokeratin 20 RNA and angiogenin in voided urine samples as diagnostic tools for bladder carcinoma. Clin Biochem 2004;37: 803–10. Larsson PC, Beheshti B, Sampson HA, Jewett MA, Shipman R. Allelic deletion fingerprinting of urine cell sediments in bladder cancer. Mol Diagn 2001;6:181–8. Sanchez-Carbayo M, Saint F, Lozano JJ, Viale A, CordonCardo C. Comparison of gene expression profiles in lasermicrodissected, nonembedded, and OCT-embedded tumor samples by oligonucleotide microarray analysis. Clin Chem 2003;49:2096–100. procedures in the daily urological practice. It is widely accepted that human neoplasms including bladder cancer arise from the accumulation of multiple genetic events leading to activation of proto-oncogenes or inactivation of tumor suppressor genes. New noninvasive methods for the diagnosis and surveillance of urothelial carcinomas are required to replace cystoscopy. Analysis of the DNA extracted from exfoliated cells can be used for noninvasive tumour detection. A noninvasive diagnostic molecular method based on the detection of loss of heterozygosity (LOH) or microsatellite instability (MSI) in cells exfoliated in urine has been reported previously [2]. However, most molecular biomarkers analysed to date lack sufficient specificity or sensitivity for their widespread use and require extensive genetic analysis preventing their routine clinical use [1,2]. The present article from Mengual et al. is a study about technical feasibility and ability of bladder washing to detect or identify early recurrence in bladder cancer. Although the aim of the study is not novel, it is quite important to determine a suitable way to detect urothelial tumors regarding these new biological tools. The current results are very interesting because this 1356 european urology 50 (2006) 1347–1356 team has decided to focus on exclusive RNA material. The effect of RNA degradation is apparently less important than the type of sample that is being analyzed. Moreover, gene expression analysis by microarrays using small amounts of RNA is becoming more and more popular against the background of advances and increasing importance of small-sample acquisition methods like laser microdissection techniques [3]. The quality of RNA preparations from such samples constitutes a frequent issue in this context [3]. If the results from the next step of the study (i.e., RNA isolated from urine samples) are conclusive, then this should be one of the methods of choice for these analyses (instead of DNA). One important issue is to make basic research more understandable for clinicians and urologists and to involve deeply our community in the ability to use new, accurate and reliable methods of diagnosis. These results are a good example and will probably be of clinical relevance in the daily urological practice for the nearly future. References [1] Chopin DK, Gattegno B. Superficial bladder tumors. Eur Urol 2002;42:533–41. [2] Amira N, Mourah S, Rozet F, Teillac P, Fiet J, Aubin P, et al. Non-invasive molecular detection of bladder cancer recurrence. Int J Cancer 2002;101:293–7. [3] Schoor O, Weinschenk T, Hennenlotter J, Corvin S, Stenzl A, Rammensee HG, et al. Moderate degradation does not preclude microarray analysis of small amounts of RNA. Biotechniques 2003;35:1192–201.
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