825 Systematic in vitro evaluation of survivin-directed antisense oligodeoxynucleotides in bladder cancer cells

825

824 CLINICAL, CHARACTERIZATION

HISTOPATHOLOGICAL OF EARLY

ONSET

AND BLADDER

MOLECULAR CANCER

Seitz M.‘, Zaak D.‘, Siebels M.‘, HartmannA.‘, StBhr R.‘, Filbeck Mohren W.‘, Urban H.“, Poremba C.‘, HeinmBller E.R, Helpap Mihatsch M.J.“‘, Giedl J.“, Kniichel R.“, HofstHdter F.’ ‘University Pathology, Regensburg, 5Hospital Pathology, Germany, Pathology, ’ ‘University

SYSTEMATIC ANTISENSE CELLS

T.‘, Wiinsch P.‘, B.‘, Sauter G.‘“,

of Munich, Urology, Munich, Germany, ‘University of Regensburg, Regensburg, Germany, 3University of Regensburg, Urology, Germany, YJniversity of Niimberg, Pathology, Niirnberg, Gemlany, Deggendorf, Pathology, Deggendorf, Germany, 6Hospital Weiden, Weiden, Gelmany, ‘University of Diisseldorf, Pathology, Diisseldorf, 8Hospital Kassel, Pathology, Kassel, Germany, ‘Hospital Singen, Singen, Germany, ‘OUniversity of Bale, Pathology, Bale, Switzerland, of Aachen, Pathology, Aachen, Germany

INTRODUCTION & OBJECTIVES: We conducted a clinico-pathological and molecular study to investigate the natural history and prognosis and the molecular alterations of bladder cancer in young patients. MATERIAL & METHODS: 160 early-onset bladder cancer patients (diagnosis 545 years) were collected through database search of cancer registries and eight pathologies in Germany. This cohort was compared with 129 bladder cancer patients (mean age 71.8 years) from a prospective study of molecular cancer diagnosis in urine. Deletions of chromosome 9p/q and 17 were determined by LOH analyses. P53 was directly sequenced and expression of p53, MIB-1 and CK20 was evaluated by immunohistochemistry (IHCH). RESULTS: The young patient cohort did not differ from the control group in grade, stage, recurrence rate, deletion frequency at chromosomes 9p/q and 17~ and expression of MIB-1 and XK20. There was a higher rate of tumours in relatives and a higher frequency of smokers in the young patients. The rate of p53-positive tumours by IHCH was significantly higher in the young patients (68%) compared to the control (34%, piO.0001). This was however not due to p53 mutation which were rare in these tumours (~21 and MDM2 over expression). CONCLUSIONS: In contrast to other studies, young bladder cancer patients not differ in the clinicopathological features from patients diagnosed later in However, the higher tumour rate in family history, the high frequency of p53 expression (wild-type) and the high percentage of smokers suggest polymorphisms could play the important role in early onset bladder cancer.

did life. over that

IN VITRO EVALUATION OLIGODEOXYNUCLEOTIDES

Fuessel S.‘, Kueppers B.‘, Meye A. ’ , Wirth M.P. ’ ‘Technical “Technical

University University

Ning

S. I, Kotzsch

OF

SURVIVIN-DIRECTED IN BLADDER CANCER

M.2, Kraemer

I(.‘,

Schmidt

Dresden, Department of Urology, Dresden, Germany, Dresden, Institute of Pathology, Dresden, Germany

INTRODUCTION & OBJECTIVES: The rather poor responses to conventional treatment of bladder cancer (BCa) require novel. specific therapy approaches. The down-regulation of BCa-associated genes may represent a new option to specifically inhibit BCa cell growth and to induce cell death. Survivin, an inhibitor of apoptosis upregulated in the vast majority of malignancies including BCa provides an attractive target for molecular therapies such as treatment with specific antisense oligodeoxynucleotides (AS-ODN). MATERIAL & METHODS: We used a mRNA secondary structure prediction to design survivin-directed AS-ODN. After lipid-mediated transfection with 30 selected anti-survivin AS-ODN inhibitory effects on cell growth properties (viability, clonogenic survival, induction of apoptosis, cell cycle alterations) were measured. Survivin expression was analyzed by a quantitative real-time PCR assay and ELISA techniques, respectively. RESULTS: Three out of 30 tested constructs remarkably impaired growth characteristics of four BCa cell lines. Detailed analysis of the cell line EJ28 treated with the constructs SVV261, SVV264 and SVV286 revealed an impressive reduction of viability (down to 35%) and long-term proliferation (down to 14%) which were caused by a cell cycle arrest and a dramatic increase of apoptosis (from 19.5% to 51.3% maximum). This strong inhibition of tumour cell growth was associated with the down-regulation of survivin expression of up to 60-80%. Interestingly, all three evolved AS-ODN are directed against the putative single stranded survivin mRNA motif between 274-285 nt identified by secondary structure prediction. The reported accessibility of this motif to other nucleic acid based inhibitors such as ribozymes and small interfering RNAs emphasizes the rationale of a systematic selection of mRNA target sites. CONCLUSIONS: The survivin-directed AS-ODN shown to inhibit remarkably the proliferation of BCa cells in the present study, may provide suitable adjuvant therapeutic agents for the specific local treatment of BCa.

826 METHYLATION OF THE E-CADHERIN AND POSITIVE PROGNOSTIC MARKERS FOR MUSCLE INVASIVE BLADDER CARCINOMA Friedrich M., Liang G., Siegmund Huland H., Jones P. University

Hospital

Hamburg,

Urology.

K., Cheng

THE TIMP-3 PATIENTS

J., Weisenberger

Hamburg,

GENES ARE WITH NOND., Toma

M.,

Germany

INTRODUCTION & OBJECTIVES: There is increasing evidence for the role of epigenetic gene silencing in superficial bladder cancer. The aim of the current studv was to investigate the uromlostic value of eoigenetic alterations in Inatients with non-muscle invisive bladder carcinoma. We checked the methylation status of 20 cancer associated genes @14ARE ~16 CDKNZA, STAT-I, SOCS-I, DR-3, DR-6, PIG-7, BCL-2, H-TERZ BAX EDNRB, DAPK, RASSF-IA, FADD, TMS-I, E-Cadherin, ICAM-I. TIMP-3. MLH-I, COX2) for DNA methvlation. We analyzed micro dissected tumour samples from f22 patients with primary non muscle invasive bladder carcinoma. I

-

I

827 ENVIRONMENTAL PRELIMINARY

FACTORS IN SUPERFICIAL REPORT OF A PROSPECTIVE

Serretta V.‘, Altierl

V.“; Morgia G.3. Allegro

La110 A.‘, Gal10 A.6, Saita A.‘, Urologici (GSTU), Italy

Motta

M.‘,

BLADDER STUDY

R.‘, Pavane Macaluso Salrano L.8, Zito A.“:

CANCER.

M.‘, Melloni Gruppo

D.4, DI Studi Tumori

‘Umversity, Urology, Palermo, Italy, ‘University, Urology, Napoli, Italy, YJniversity. Urology. Sass& Italy, 4Unwersity, Urology, Messina, Italy, ‘Civic Hospital, Urology. Campobasso. Italy, ‘lnt Pascale, Urology, Napoh, Italy, 7University, Urology, Catama. Italy, lOsped& Rummo, Urology. Benevento, Italy, 90spedale Civile, Urology, Terre de1 Greco, Italy

”

MATERIAL & METHODS: Quantitative methylation analysis of CpG sites in the promoter region of the genes was performed with methylation specific real time PCR (Methylight). Genomic DNA (Promega, USA) that was treated with SssI was used as a fully methylated reference. The percentage of fully methylated DNA was calculated as the ratio between a sample and the standards. Each set of primers and probe were designed to amplify a bisulfite-converted sequence of the promoter region overlapping at least five CpG sites. RESULTS: DR-3, ICAM-1, and COX-2 were methylated in >70% of the samples. SOCS-I, EDNRB, RASSF-IA, BCL-2 displayed methylation in 40% - 69% of the tumour samples. STAT-I, p16CDKN2A, H-TERI: DAPK, TIMP-3, and MLH-I showed methylation in 10% - 39% of the turnours, whereas pl4ARE DR.6, TMS1, PIG-7(LITAF), Ba FADD, and E-Cadherin were methylated in none of the turnours, or only in single cases. We could identify 7 genes (SOCS-1, STAT-l, BCL-2, DAPK, TIMP-3, E-Cadherin, COX-2), that showed an association with tumour recurrence. In a Kaplan-Meier analysis TIMP-3 showed a significant and E-Cadherin showed a borderline significant association with recurrence free survival. Methylation of both genes predicted a prolonged disease free interval. CONCLUSIONS: of gene methylation genes (TIMP-3 and favourable outcome aggressive adjuvant

U.‘.

We present a comprehensive analysis on prognostic relevance in non-muscle invasive bladder cancer. We could identify two E-Cadhwin) in which methylation was associated with a more possibly identifying a subpopulation that requires a less therapy and follow-up.

INTRODUCTIOK & OBJECTIVES: Although the pathogenetic role in bladder cancer of cigarette smoking and employment is well defined, other environmental factors have not been extensively studied. Case-control studies require high numbers and are expensive. As a preliminary approach to define the object of larger case-control studies, the distribution of potential risk factors coming from envmxunental pollution among patients affected by superficial bladder cancer (TCCB) has been prospectively analysed. MATERIAL & METHODS: The analysis was limited to patients affected by medium risk superficial TCCB. Patients with primary single Ta Cl-2, Tis or TlG3 turnours were excluded. Forty Italian urolog$al centres joined the study. Detalled information about age, sex, urban or extra-urban residency, employment, active and passive cigarette smoking, water resource, hair-dye use were centralised. All patients underwent TUR and early intravesical chemotherapy. The distribution of the above mentioned environmental factors was analysed in relation to tumour characteristics such as multiplicity and prevmus natural history. RESULTS: Until now today 474 patients have been recruited, 182 (38.4%) with primary tumours and 293 (61.8%) with multiple lesions. Over 80% of the patients lived in urban areas: 20% were employed in industry and 8% used hair dye. Forty percent of the patients were smokers, with a median smoking permd of 30 years. One third has smoked cigarettes in the past. Bottled water was the only water resource for 42% of the patients. At multivariate statistical analysis a significant correlation between tumour multiplicity and employment in industry (~~0.01) and between past natural history and period of cigarette smoking (p
CONCLUSIONS: Industry employment and period of cigarette smoking were statistically related to multiplicity and previous histoly of the tumour. In non-smokers water resource can be implied as an environmental risk factor. Further and larger case-control studies are required. Acknowledgements: The study was supported in part by IMinistero Italian0 dell’lstruzione. Universitit e Ricerca (MIUR). European

Urology

Supplements

3 (2004)

No. 2, pp.

209