302 CLINICAL APPLICATIONS OF BLADDER CANCER MICROARRAY DATA: TOWARDS THE DEVELOPMENT OF NON INVASIVE DIAGNOSTIC TESTS

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Molecular mRNA markers to detect and monitor circulating tumour cells in peripheral blood from urothelial carcinoma patients

Clinical applications of bladder cancer microarray data: Towards the development of non invasive diagnostic tests

Marin-Aguilera M.1, Mengual L.1, Burset M.1, Rios J.2, Gazquez C.3, Ars E.4, Ribal M.J.5, Villavicencio H.6, Alcaraz A.5

Mengual L.1, Burset M.1, Ars E.2, Lozano J.J.3, Villavicencio H.4, Ribal M.J.5, Alcaraz A.5

Hospital Clinic - Fundacio Puigvert, Laboratory and Dept. of Urology, Barcelona, Spain, 2 Hospital Clinic, Statistics and Methodology Support Unit, Barcelona, Spain, 3Hospital Clinic, Laboratory of Urology, Barcelona, Spain, 4Hospital Clinic - Fundacio Puigvert, Laboratory of Molecular Biology, Barcelona, Spain, 5Hospital Clinic, Dept. of Urology, Barcelona, Spain, 6Hospital Clinic - Fundacio Puigvert, Dept. of Urology, Barcelona, Spain

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Introduction & Objectives: Detecting haematogenous spread of bladder urothelial carcinoma (BUC) still represents a major challenge in clinical practice. To test the efficiency of six mRNA bladder markers in detecting and monitoring BUC haematogenous dissemination. Material & Methods: Between 2002 and 2004, 317 peripheral blood (PB) samples were collected from 101 patients with BUC that underwent radical cystectomy and from 29 healthy controls. Sequential PB sampling was performed in patients at time of surgery and at 6, 12, 18 and 24 months afterwards. Median patients’ follow-up was 35 months. The presence of disseminated urothelial cells in PB was evaluated by measuring gene expression from pre-amplified (PA) cDNA of KRT20, FXYD3, C10orf116, UPK2, AGR2 and KRT19 genes, using quantitative RT-PCR (qRT-PCR). Gene expression data were correlated with patients’ follow-up and outcome.

Hospital Clinic - Fundacio Puigvert, Dept. of Laboratory and Urology, Barcelona, Spain, Fundacio Puigvert, Dept. of Laboratory, Barcelona, Spain, 3Hospital Clinic, Dept. of Ciberhed, Barcelona, Spain, 4Fundacio Puigvert, Dept. of Urology, Barcelona, Spain, 5 Hospital Clinic, Dept. of Urology, Barcelona, Spain 1

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Introduction & Objectives: There is a need in urological practice to identify new molecular markers of bladder cancer (BC) in order to further develop non-invasive diagnostic tests. Here, we have analyzed gene expression profiles of BC with the aim to determine the relevant differentially expressed genes and subsequently ascertain whether this differential expression is maintained in urine samples. Material & Methods: Fifty-five bladder tissue specimens (15 Ta low grade [LG], 13 T1 high grade [HG], 15 T2-4HG and 12 controls) and 49 urines from bladder washings (BW) (6 TaLG, 7 TaHG, 3 T1LG, 5 T1HG, 4 Tis, 11 T2-4HG, and 13 controls) were collected between September 2003 and December 2004. Tissue samples were analyzed using a sub-pooling strategy. Three pools for each of the 4 experimental groups (TaLG, T1HG, T2+HG and controls) were performed. Pools were composed for 4 or 5 independent tumour or control samples. DNA microarrays were used to identify differently expressed genes in the three different BC stages. Then, selected differentially expressed genes in microarray data were validated in an independent set of individual BW by real time quantitative RT-PCR (qRTPCR).

Results: The PA qRT-PCR method with selected markers in PB was able to detect 2 cells in between 106-107leucocytes, depending on the gene. Differential gene expression was found between patients and controls for FXYD3 (p=0.002), C10orf116 (p=0.001), AGR2 (p=0.003) and KRT19 (p<0.001) genes. In contrast, no differences were found between gene expression from metastasized and recurrence-free patients (p=ns). Molecular monitoring of clinically metastasized patients did not show an increased gene expression for selected markers in blood throughout the follow-up after surgery.

Results: Unsupervised cluster analysis from DNA microarray data showed a clear distinction between control and tumour samples as well as in LG versus HG tumours (Fig.1). Statistically significant genes with at least two fold differential expression in controls versus tumours (3126 probe-sets), and in LG versus HG tumours (674 probe-sets) were identified and ranked. Gene expression validation of microarray data was performed by qRT-PCR in an independent set of 49 BW. The six most differentially expressed genes in control versus tumours were confirmed for the two over-expressed genes tested. All of the eight selected differentially expressed genes in LG versus HG tumours were confirmed in BW samples.

Conclusions: Gene expression analysis of PB samples using six specific bladder mRNA markers can distinguish between BUC patients and controls. However, despite its high sensitivity, PA qRT-PCR with these potential markers is not a suitable approach for detecting and monitoring BUC haematogenous dissemination. Thus, PCR-based technology should be abandoned as a candidate method for detecting such tumour spread.

Conclusions: We demonstrate that the analysis by DNA microarray technology of BC tissues samples provides candidate mRNA markers suitable for detection in urine. Therefore, many of the genes contained in our lists of differentially expressed genes may be useful for the development of a non-invasive method of bladder cancer diagnosis and/or prognosis. Further studies are warranted to narrow the list of genes of interest.





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Endovesical therapy in high risk non muscle invasive bladder cancer: A new chemosensitivity test De Berardinis E.1, Busetto G.M.1, Antonini G.1, Sciarra A.1, Di Silverio F.1, Gazzaniga P.2 Policlinico Umberto I, Dept. of Urology, Rome, Italy, 2Policlinico Umberto I, Dept. of Molecular Medicine, Rome, Italy 1

Introduction & Objectives: The treatment choice for high risk NMIBC is still controversial, and no markers are still available to guide the urologist in the individualization of therapy. Although intravesical BCG treatment represents the gold standard in adjuvant setting after TURB, the percentage of recurrence and progression under treatment is still high. We here describe the design of a new chemosensitivity assay based on the expression of genes involved in the resistance to standard intravesical regimens. Material & Methods: 35 patients with high risk NMIBC have been enrolled, all candidates for TUR-B followed by intravesical treatment. All patients have been evaluated by cystoscopy 3 and 6 months after TUR-B. One mg of tumoral tissue from each patient was kept for molecular assay subjected to RNA extraction and RT-PCR amplifications with primers specific for MRP1, MRP2, hENT1, dCK, α5β1 integrin, used to trace a specific chemosensitivity profile to drugs commonly used in intravesical regimen: anthracyclines, mitomycin-c, gemcitabine and BCG. On the basis of densitometric analysis of the amplification bands obtained by normalisation with the GAPDH internal controls, we obtained for each patient a chemosensitivity molecular profile. We considered high, intermediate and low sensitivity to mitomycin c, epirubicin, and doxorubicin a ratio MRP/GAPDH <1, =1, >1 respectively. For gemcitabine resistance, we considered sensitivity, intermediate sensitivity and resistance a ratio hENT-dCK/GAPDH >1, =1 and <1 respectively. Sensitivity to BCG was evaluated as follows: high, intermediate, low sensitivity in the presence of α5β1/GAPDH >1; =1 ; <1 respectively. We then compared both the molecular profiles of chemosensitivity to the clinical response to the intravesical regimen adopted in the first 6 months of follow up. Results: This chemosensitivity test was able to predict response to treatment in 96% of patients. The assay is easy to perform with low costs and rapid time of execution. Conclusions: Our results are encouraging in the view of an individualised therapeutic approach, to provide a higher treatment success rate while sparing patients unnecessary toxicity from drugs that are not suited for their tumors.

Eur Urol Suppl 2009;8(4):196

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Novel intravesical therapy for non muscle invasive bladder cancer: Combination of a fusogenic glycoprotein, pro-drug activation and oncolytic herpes simplex virus Horvath A.1, Simpson G.R.1, Coffin R.S.2, Mostafid H.3, Pandha H.1 University of Surrey, Dept. of Oncology, Guildford, United Kingdom, 2Biovex Inc, Woburn, United States of America, 3North Hampshire Hospital, Dept. of Urology, Basingstoke, United Kingdom 1

Introduction & Objectives: In the UK approximately 12,000 new cases of bladder cancer are diagnosed every year. Approximately 80% of these cases are non muscle invasive bladder cancer (NMIBC) defined as stage Ta-T1, grade 1-3 and carcinoma in situ. Despite current operative and postoperative treatment options there is recurrence rate of 15-61% and significant risk of progression (1-17%). OncoVexGALV/CD is an oncolytic herpes simplex virus 1 (HSV-1), which has shown enhanced local tumour control by combining oncolysis with the expression of a highly potent pro-drug activating gene (yeast cytosine deaminase / uracil phospho-ribosyltransferase [Fcy::Fur]) and a fusogenic glycoprotein from gibbon ape leukemia virus (GALV). Effective use (enhanced cell killing and tumour shrinkage) of OncoVexGALV/CD vector has been previously described in vitro and in vivo other tumour sites but not the bladder. A version of this virus expressing GM-CSF has shown promising results in Phase I and II clinical trials. The present study aims to test in vitro and in vivo the OncoVexGALV/CD as an intravesical therapy for NMIBC. Material & Methods: In vitro tumour cell killing by OncoVexGALV/CD was assessed by Fusion/Prodrug MTS assays. For the in vivo tests we have developed a rat orthotopic bladder tumour model using acid and alkali to damage the bladder mucosa and using AY27 HVEM cells for implantation. In vivo efficacy of the OncoVexGALV/CD treatment is studied on this model through histology and qPCR. Results: Treatment of three human bladder carcinoma cell lines with the virus, resulted in higher tumour cell killing through oncolysis, pro-drug activation and glycoprotein fusion. Preliminary results on the orthotopic bladder tumour model showed larger tumour sizes in the control group and higher tumour free ratio in the OncoVexGALV/CD treated group. We are optimizing this model and evaluating the relative contribution of vector (oncolysis) ± prodrug activation. This approach would be suitable for clinical evaluation of refractory transitional cell cancer. Conclusions: Preliminary results on human bladder carcinoma cell lines and rat orthotopic bladder tumour model indicate that OncoVexGALV/CD may improve local tumour control within the bladder.