Identification of Common Differentially Expressed Genes in Urinary Bladder Cancer

BACKGROUND: Current diagnosis and treatment of urinary bladder cancer (BC) has shown great progress with the utilization of microarrays. PURPOSE: Our goal was to identify common differentially expressed (DE) genes among clinically relevant subclasses of BC using microarrays. METHODOLOGY/PRINCIPAL FINDINGS: BC samples and controls, both experimental and publicly available datasets, were analyzed by whole genome microarrays. We grouped the samples according to their histology and defined the DE genes in each sample individually, as well as in each tumor group. A dual analysis strategy was followed. First, experimental samples were analyzed and conclusions were formulated; and second, experimental sets were combined with publicly available microarray datasets and were further analyzed in search of common DE genes. The experimental dataset identified 831 genes that were DE in all tumor samples, simultaneously. Moreover, 33 genes were up-regulated and 85 genes were down-regulated in all 10 BC samples compared to the 5 normal tissues, simultaneously. Hierarchical clustering partitioned tumor groups in accordance to their histology. K-means clustering of all genes and all samples, as well as clustering of tumor groups, presented 49 clusters. K-means clustering of common DE genes in all samples revealed 24 clusters. Genes manifested various differential patterns of expression, based on PCA. YY1 and NFκB were among the most common transcription factors that regulated the expression of the identified DE genes. Chromosome 1 contained 32 DE genes, followed by chromosomes 2 and 11, which contained 25 and 23 DE genes, respectively. Chromosome 21 had the least number of DE genes. GO analysis revealed the prevalence of transport and binding genes in the common down-regulated DE genes; the prevalence of RNA metabolism and processing genes in the up-regulated DE genes; as well as the prevalence of genes responsible for cell communication and signal transduction in the DE genes that were down-regulated in T1-Grade III tumors and up-regulated in T2/T3-Grade III tumors. Combination of samples from all microarray platforms revealed 17 common DE genes, (BMP4, CRYGD, DBH, GJB1, KRT83, MPZ, NHLH1, TACR3, ACTC1, MFAP4, SPARCL1, TAGLN, TPM2, CDC20, LHCGR, TM9SF1 and HCCS) 4 of which participate in numerous pathways. CONCLUSIONS/SIGNIFICANCE: The identification of the common DE genes among BC samples of different histology can provide further insight into the discovery of new putative markers

Mutations and expression analysis of RAS family genes and BRAF gene in transitional cell carcinoma of the bladder

Bladder cancer is the fifth most common malignancy in men in Western society. We determined RAS codon 12 and 13 point mutations and evaluated mRNA expression levels in transitional cell carcinoma cases. Mutational activation of the MAP kinase pathway is frequently found in many types of cancer. Recently, activating mutations in the BRAF gene, an important activator of this pathway, have been described in several tumor types including melanoma, colorectal and papillary thyroid cancer. The most frequent mutation in exon 15 (V600E) as well as several other mutations within exons 11 and 15 result in constitutive activation of the oncoprotein. Samples from 30 human bladder cancers and 30 normal tissues were analyzed by polymeRASe chain reaction/restriction fragment length polymorphism and direct sequencing to determine the occurrence of mutations in codons 12 and 13 of RAS family genes. Moreover, we used real-time reverse transcriptase-polymeRASe chain reaction to evaluate the expression profile of RAS genes in bladder cancer specimens compared to that in adjacent normal tissues. Furthermore, we investigated BRAF mutations in 30 human bladder tumors and their adjacent normal tissues. The V600E mutation was screened by PCR/RFLP and exons 11, 14 and 15 of BRAF including intron-exon boundaries were sequenced. Overall H-RAS mutations in codon 12 were observed in 9 tumor samples (30%). Two of the 9 patients (22%) had invasive bladder cancer and 7 (77%) had noninvasive bladder cancer. One H-RAS mutation (11%) was homozygous and the remaining 89% were heterozygous. All samples were WT for K and N-RAS oncogenes. Moreover, 23 of 30 samples (77%) showed over expression in at least 1 RAS family gene compared to adjacent normal tissue. K and N-RAS had the highest levels of over expression in bladder cancer specimens (50%), whereas 27% of transitional cell carcinomas demonstrated H-RAS over expression relative to paired normal tissues. We detected two tumor specimens bearing two different mutations, both of which were found in exon 15. One sample showed the T1799A (V600E) and the other the G1798T (V600L) mutation. The first specimen was stage pT1a and grade II, whereas the second was stage pT2b and grade III. No mutations within the coding region of exons 11, 14, 15 and the intron-exon junctions for the remaining samples were found. Our results underline the importance of H-RAS activation in human bladder cancer by codon 12 mutations. Moreover, they provide evidence that increased expression of all 3 RAS genes is a common event in bladder cancer that is associated with disease development. Furthermore, our results suggest that involvement of BRAF mutations in the development of transitional cell carcinoma of the bladder is infrequent.Μεταλλάξεις στα μέλη της οικογένειας των γονιδίων RAS ανευρίσκονται σε μεγάλοαριθμό ανθρώπινων νεοπλασμάτων. Οι περισσότερες από αυτές αφορούν σημειακέςμεταλλάξεις στα κωδικόνια 12, 13 ή 61 που μετατρέπουν τα γονίδια RAS σε ογκογονίδια.Πρόσφατα ενεργείς μεταλλάξεις του γονιδίου BRAF, ενός σημαντικού διαμεσολαβητή της οδούRAS/RAF/MEK/ERK, περιγράφηκαν σε διάφορους όγκους συμπεριλαμβανομένων κακοήθουςμελανώματος, παχέος εντέρου και θηλώδους καρκίνου θυρεοειδούς. Η συχνότερη μετάλλαξηπου αφορά το εξόνιο 15 (V600E) καθώς και διάφορες άλλες στα εξόνια 11 και 15 οδηγούν σειδιοσυστατική ενεργοποίηση της ογκοπρωτεḯνης BRAF.Στην παρούσα μελέτη διερευνήθηκε η συχνότητα και η προγνωστική αξία τωνσημειακών μεταλλάξεων των γονιδίων της οικογένειας RAS σε άτομα με καρκίνο ουροδόχουκύστης. Δείγματα από ουροθηλιακό καρκίνο και φυσιολογικό ουροθήλιο 30 ασθενώνμελετήθηκαν με τις μεθόδους PCR/RFLP (αλυσιδωτή αντίδραση πολυμεράσης/ανάλυσηπολυμορφισμού μεγέθους περιοριστικών θραυσμάτων) και Direct Sequencing (άμεσηαλληλούχιση) για την παρουσία μεταλλαγών των γονιδίων RAS στα κωδικόνια 12 και 13.Επιπλέον, με την τεχνική real time RT-PCR (αντίστροφη μεταγραφή-αλυσιδωτή αντίδρασηπολυμεράσης σε πργματικό χρόνο) μελετήθηκε το επίπεδο έκφρασης των ογκογονιδίων RASστο επίπεδο του mRNA στον ουροθηλιακό καρκίνο και συγκρίθηκε με τα αντίστοιχα επίπεδαέκφρασης στο παρακείμενο φυσιολογικό ουροθήλιο. Στο δεύτερο σκέλος της παρούσαςδιατριβής διερευνήθηκε η συχνότητα των σημειακών μεταλλάξεων του γονιδίου BRAF σταδείγματα νεοπλασματικού και φυσιολογικού ουροθηλίου των παραπάνω ασθενών. Ημετάλλαξη V600E μελετήθηκε με τη μέθοδο PCR/RFLP και τα εξόνια 11, 14 και 15συμπεριλαμβανομένων των σημείων συνάντησης ιντρονίων/εξονίων υποβλήθηκαν σε άμεσηαλληλούχιση.Μεταλλάξεις του ογκογονιδίου H-RAS ανευρέθηκαν σε 9 ασθενείς (30%). Δύο από τους9 ασθενείς είχαν διηθητική νόσο (22%) και οι υπόλοιποι 7 (77%) επιφανειακή. Μία από τιςμεταλλάξεις ήταν ομόζυγη (11%) και οι υπόλοιπες (89%) ετερόζυγες. Σε κανένα δείγμα δενανιχνεύτηκαν μεταλλάξεις των ογκογονιδίων K και N-RAS (αγρίου τύπου). Yπερέκφραση σεένα τουλάχιστον μέλος της οικογένειας RAS παρατηρήθηκε στο 77% των δειγμάτων μεουροθηλιακό καρκίνο σε σύγκριση με τα παρακείμενα φυσιολογικά δείγματα. Τα υψηλότεραμεταγραφικά επίπεδα παρατηρήθηκαν στα Κ και N-RAS ογκογονίδια (50%) ενώ μόνο 27% τωνκαρκινικών ιστών έδειξαν υπερέκφραση του γονιδίου H-RAS συγκρινόμενοι με τουςαντίστοιχους φυσιολογικούς. Δεν διαπιστώθηκε στατιστικά σημαντική συσχέτιση μεταξύμεταλλάξεων και υπερέκφρασης των ογκογονιδίων RAS και κλινικοπαθολογικώνχαρακτηριστικών των όγκων. Δύο δείγματα έφεραν δύο διαφορετικές μεταλλάξεις στο εξόνιο15 του γονιδίου BRAF. Ενα δείγμα έφερε την μετάλλαξη V600E (νουκλεοτιδική αλλαγήT1799A) και το άλλο τη μετάλλαξη V600L (νουκλεοτιδική αλλαγή G1798T). Το πρώτο δείγμαήταν σταδίου pT1a και grade II ενώ το δεύτερο σταδίου pT2b και grade III. Καμμίαμεταλλαγή δεν ανευρέθηκε στα εξόνια 11, 14 και 15 καθώς και στα σημεία συνάντησηςιντρονίων/εξονίων των υπολοίπων δειγμάτων.Τα αποτελέσματά μας υπογραμμίζουν την σημασία της ενεργοποίησης, διαμέσουμεταλλάξεων στο κωδικόνιο 12, του ογκογονιδίου H-RAS στον καρκίνο της ουροδόχου κύστης.Επίσης, παρέχουν αποδείξεις ότι η υπερέκφραση και των τριών γονιδίων RAS είναι ένα κοινόγεγονός στο ουροθηλιακό καρκίνο που σχετίζεται με την ανάπτυξη της νόσου. Τέλος,αποδεικνύεται ότι η συμμετοχή των BRAF μεταλλαγών είναι ασυνήθης στον καρκίνο τηςουροδόχου κύστη

Semirigid ureteroscopy prior retrograde intrarenal surgery (RIRS) helps to select the right ureteral access sheath

Objective: To evaluate ureteral compliance through semirigid ureteroscopy (sURS) in order to select the proper ureteral access sheath (UAS) size for retrograde intrarenal surgery (RIRS). Patients and methods: In a prospective study, 100 consecutive patients selected for elective sURS or RIRS were recruited. Each patient, initially underwent 9.5 Fr sURS with a safety guidewire 3Fr, in order to estimate ureteral compliance. If the ureter was compliant, a gently passage of a 12/14Fr UAS was attempted. If the ureter was not deemed compliant, passage of either a smaller UAS or a smaller semirigid 7Fr or a flexible 7.5Fr or a digital 8.5Fr scope with and without safety guidewire, was attempted. Age, gender, disease location, prestenting, previous RIRS and/or stone elimination, hydronephrosis, ureteral strictures, unsuccessful procedures, and complications, were analyzed as possible correlated factors of ureteral compliance. Results: In 77 patients the ureter was deemed compliant ≥ 14Fr. Of the preoperative factors that were examined, stent placement before RIRS (P < 0.002), previous RIRS (P = 0.000) and previous stone elimination (P = 0.004), correlated with ureter ≥ 14Fr. Ureteral lithiasis (P < 0.001), ureteral strictures (P < 0.05), unsuccessful procedures (P < 0.005) and complications (P = 0.01) correlated with ureter < 14Fr. The complication rate was 10% (10 patients) with ureteral injuries grade I in 9 patients and grade III in 1 patient according to the endoscopic grading system. Age, gender, hydronephrosis and urothelial carcinoma (UC) had no influence. Conclusions: sURS performed before RIRS allows selection of the right ureteral access sheath (UAS) and avoidance of major complications. Pre-stenting, previous RIRS and stone elimination history are all factors correlating with a compliant ureter

The tumour suppressor RhoB is an independent prognostic factor for metastasis in urinary bladder cancer.

<p>Introduction & Objectives: Rho members may affect the process of tumourigenesis either by over-expression of some members of the family with oncogenic activity or by down-modulation of other members with suggested tumour suppressor activity.</p> <p>Materials & Methods: Rho gene mRNA expression was studied in 77 bladder cancer (BC) specimens. The Kaplan-Meier method was used to estimate survival as a function of time, and survival differences were assessed by the Log-rank test. Logistic regression (univariate and multivariate) analysis was performed to determine the potential predictors of survival, recurrence and metastasis.</p> <p>Results: The cases whose tumours exhibited increased levels of RhoB mRNA expression exhibited worse overall and cancer-specific survival rates, than those expressing decreased RhoB mRNA levels. Moreover, those cases whose tumours exhibited high Cdc42 mRNA expression showed a worse overall survival rate than those expressing low Cdc42 levels. High RhoC levels tended to correlate with better survival. Although univariate analysis, using the Cox proportional hazards model, showed that RhoB has a tendency for being an independent prognostic factor for overall survival (p=0.086), both univariate and multivariate analysis for RhoA, RhoC, Rac1 and Cdc42 did not exhibit the same tendency. Moreover, no gene was identified as independent prognostic factor for tumour recurrence. Finally, both univariate and multivariate analysis identified RhoB independent prognostic factors for metastasis (p=0.012 and p=0.050, respectively).</p> <p>Conclusions: Our results confirm a tumour suppressor role for RhoB in bladder cancer, opposing the positive functions of RhoA and RhoC. Moreover, our analysis identified RhoB as an independent prognostic factor for metastasis.</p

Transcription Factor Binding Motifs, Chromosome mapping and Gene Ontology analysis in Cross-platform microarray data from bladder cancer.

<p>We have previously analyzed the gene expression profile in urinary bladder cancer and determined the differentially expressed (DE) genes between cancer and healthy tissue. We aimed: 1) To identify the over-represented Transcription Factor Binding Motifs (TFBMs) in the promoters of the DE genes. 2) To map the DE genes on the chromosomal regions. 3) To gain more insight into the DE gene functions, using Gene Ontology (GO) analysis. We investigated the TFBMs in the Transcription Element Listening System Database (TELiS). The TRANSFAC TF database was used for the identification of TF binding sites. The Gene Ontology Tree Machine, WebGestalt web-tool and the Matlab ® (The Mathworks Inc.) computing environments were used for chromosome mapping. GO analysis was performed using the eGOn online tool. The WebGestalt web-tool was used for gene function classifications. Relations of the DE genes and the transcription factor binding motifs were further investigated using the Pubgene Ontology Database. The glucocorticoid receptor (GR) was predicted as one of the TFs in the common gene set. In order to find which gene was most commonly represented among the TFs, we plotted the incidence of each gene as a function of the times of appearance within the predicted TFs. The gene BMP4 (bone morphogenetic protein 4; ID: 652) exhibited the higher number of binding sites for the predicted TFs. The majority of the chromosomes in BC had inactivated (down-regulated) genes, compared to the normal tissue. However, two genes were significantly over-expressed: CDC20 (in chromosome 1) and HCCS (in chromosome X). Three main functions were outlined by GO for the DE genes: a) circulatory system regulation, b) reproductive organ and sex development, and c) catecholamine metabolism. This enrichment showed that the predicted gene set has more than a dual role. Through this study, we were able to identify several important factors that warrant further investigation both as prognostic markers and as therapeutic targets for bladder cancer. Such approaches may provide a better insight into tumorigenesis and tumor progression.</p

Cross-platform comparisons of microarray data. Elucidation of common differentially expressed genes in bladder cancer.

<p>INTRODUCTION: Parallel gene-expression monitoring is a powerful tool for analyzing relationships among tumors, discovering new tumor subgroups, assigning tumors to pre-defined classes, identifying co-regulated or tumor stage-specific genes and predicting disease outcome. Previous gene expression studies have focused on identifying differences between tumor samples of the same type.</p> <p>AIM OF STUDY: Using a reverse engineering approach, we searched for common expression profiles among tumor samples. We analyzed the gene expression profile of bladder cancer (BC) and determined the differentially expressed (DE) genes between cancer and healthy tissue, using cross-platform comparisons.</p> <p>MATERIALS AND METHODS: We performed cDNA microarray analysis, comprising both in-house experimental and publicly available GEO microarray data. In total, our pooled microarray analysis was composed of 17 control samples (n=5, for the CodeLink platform; and n=12, for the remaining microarray platforms) and 129 BC samples (n=10, for the CodeLink platform; and n=119, for the remaining microarray platforms). Tumor samples were separated into the following groups: Ta/T1 without CIS; Ta/T1 with CIS; Ta-grade 1; Ta-grade 3; T1-grade2; T1-grade 3; T2-grade 2-4. Each group was compared against all control samples and the DE genes were identified. Data were clustered with different algorithms.</p> <p>RESULTS:</p> <p>A two-sample T-test analysis for all tumor samples vs. all normal samples, revealed 434 DE genes between the two tissue groups. Hierarchical clustering (HCL) showed a clear distinction among tumor samples. In total, 17 genes appeared to be commonly expressed among all BC samples: BMP4, CRYGD, DBH, GJB1, KRT83, MPZ, NHLH1, TACR3, ACTC1, MFAP4, SPARCL1, TAGLN, TPM2, CDC20, LHCGR, TM9SF1 and HCCS. Three groups of genes were down-regulated in all samples: BMP4, CRYGD, DBH, GJB1, KRT83, MPZ, NHLH1, TACR3 in cluster 79; ACTC1, MFAP4, SPARCL1, TAGLN in cluster 81; and TPM2 in cluster 82. CDC20, TM9SF1 and HCCS appeared to be simultaneously over-expressed in all tumor groups. LHCGR was differentially expressed in 108/129 (83.7%) of the BC samples.</p> <p>DISCUSSION: Through this investigation we were able to identify several important factors that warrant further investigation both as prognostic markers and as therapeutic targets. Such approaches may provide a better insight into tumorigenesis and tumor progression.</p

Linear correlations in chromosomal-based gene expression in urinary bladder cancer

<p>Introduction & Objectives: Gene expression is a very tidy and well coordinated procedure. Consecutive genes are often similarly expressed. We hypothesized that correlations might exist between genes of the same chromosome, yet belonging to different urinary bladder cancer (BC) samples, in order to indicate a common regulation for genes following this pattern.</p> <p>Materials & Methods: We analyzed BC gene expression profiles, with emphasis in linear correlations of gene expression based on their chromosomal locations. Samples from 10 human BCs and 5 normal tissues were analyzed by whole genome microarrays, along with a computational approach, for their expression profiles. After raw data normalization and classification, differentially expressed genes (DE) were sorted according to their chromosome distributions and were further investigated for linear correlations among them. Chromosomal activity in terms of gene expression was measured by calculating the average expression of all DE genes for each chromosome, both for tumour and control samples.</p> <p>Results: Chromosome-based expression analysis predicted that among the most active chromosomes were chromosomes 9 and X. Similarly, control samples also manifested high expression activity on the X chromosome. The genes that exhibited significant linear correlations (p<0.05) among tumor samples on chromosomes 4, 8, 13, 21 and 22, were as follows: TACR3, RNF150, ANXA10, CENTD1, EXOC1, GRSF1 for chromosome 4; ANXA13, DENND3, FGF20, EFHA2, DNAJC5B, MRPS28, FABP5 for chromosome 8; ITGBL1, RXFP2, KL, MYCBP2, FARP1 for chromosome 13; KRTAP19-1, IFNAR1, SON for chromosome 21; MORC2, PLA2G6, ACO2, ARHGAP8 for chromosome 22; SERPINA7, TMEM164, ARHGAP6, APLN, FHL1, PNMA6A, UBL4A, PRDX4, POLA1, MXRA5 for chromosome X.</p> <p>Conclusions: Despite the fact that linear correlations occurred among distinct patients, the expression of the genes appeared to be correlated among them, in a similar manner. We have previously reported that there are hints of common mechanisms between BCs of different stage/grade, employing microarray analysis. Chromosomal correlation analysis comes to support our previous findings, since it revealed genes bearing common regulation among samples of different histology. Gene expression correlations can further assist us to understand more in-depth the mechanisms underlying tumour progression and biology.</p

MiR-21 can be used as independent prognostic factor for survival and metastasis in urinary bladder cancer.

<p>Introduction & Objectives: Our goal was to correlate the expression of 12 micro-RNAs with the corresponding expression of FGF2, OPN and VEGFA. Gene expression was correlated with the overall and cancer-specific survival of patients suffering from urinary bladder cancer (BC), as well as with recurrence and metastasis.</p> <p>Materials & Methods: Gene expression were acquired by qPCR, from 77 BC specimens. Correlation of the gene expression with survival, recurrence and metastasis was employed by SPSS.</p> <p>Results: High expression of miR-21 correlated with worse overall survival (p=0.0099). Univariate analysis showed that miR-21 and miR-210 can be used as independent prognostic factors for overall survival (p=0.015 and p=0.049, respectively). Moreover, univariate analysis revealed that miR-21 can be used as independent prognostic factor for metastasis (p=0.049). Multivariate analysis revealed that miR-21, miR-210 and miR-378_1 can be used as independent prognostic factors for overall survival (p=0.005, p=0.033 and p=0.012, respectively); miR-21 and miR-378_1 can be used as independent prognostic factors for recurrence (p=0.030 and p=0.031, respectively); and miR-21 can be used as independent prognostic factors for metastasis (p=0.049). FGF2 was positively correlated with the majority of the miRs both in BC and normal tissue (p<0.001). OPN was positively correlated with miR-145_1 (p=0.015) in BC, and with miR-296-5p (p=0.017) in normal tissue. VEGFA was positively correlated with miR-21 in BC (p=0.043), and with miR-205_1 (p=0.045), FGF2 (p=0.004) and OPN (p<0.001) in normal tissue.</p> <p>Conclusions: miR-21 can be used as independent prognostic factor both for overall patient survival and metastasis of BC. miR-210 is an independent prognostic factor for overall survival.</p

Expression profile of oncomiRs and tumor-suppressor miRs in urothelial carcinoma of the bladder.

<p>Introduction & Objectives: Micro-RNAs are small, regulatory molecules approximately 21-24 nucleotides in length. They function at the post-transcriptional level by controlling the expression of more than 50% of human protein-coding genes and play an essential role in cell signaling pathways. Our goal was explore the expression profile of oncomiRs and tumor-suppressor miRs, and to define their possible correlations in urothelial carcinoma of the bladder (BC).</p> <p>Materials & Methods: Seventy-seven primary BCs, along with 77 matched tumor-associated normal samples were investigated for the expression of 12 micro-RNAs using qPCR. Relationships between the expression of miR-10b, miR19a, miR19b, miR-21, miR-122a, miR-145_1, miR-205_1, miR-210, miR-221, miR-222, miR-378-1 and miR-296-5p and the pathologic features of the tumors were also examined.</p> <p>Results: The majority of the micro-RNAs exhibited down-regulation in BC vs. normal tissue [miR-10b (p=0.0007), miR-19a (p=0.012), miR-19b (p=0.0361), miR-126_1 (p=0.0021), miR-145_1 (p<0.0001), miR-221 (p<0.0001), miR-296-5p (p<0.0001), miR-378-1 (p<0.0001)]. miR-21, miR-205_1 and miR-210 expression levels did not present difference between BC and normal tissue. However, we noticed a great range in the x-fold expression values of all micro-RNAs. The median x-fold expression (range) was as follows: miR-10b, 0.45 (0-12.58); miR-19a, 0.56 (0-25.63); miR-19b, 0.50 (0-18.90); miR-21, 0.96 (0-52.95); miR-126_1, 0.36 (0-42.62); miR-145_1, 0.04 (0-56.36); miR-205_1, 1.07 (0.01-36.42); miR-210, 1.09 (0-44.43); miR-221, 0.32 (0-33.51); miR-296-5p, 0.08 (0-75.24); miR-378-1, 0.17 (0-3.66). Significant correlations among all of the studied microRNAs were scored both in BC and control tissue.</p> <p>Conclusions: Different micro-RNAs are deregulated in BC through down-regulation. A synergistic involvement of these genes in the development of BC is implied.</p