Beatrice Stubendorff

Abstract 1358: Promoter hypermethylation of KISS1R, KiSS1, SEPT9 and CSAD as a prognostic biomarker panel to assess the metastatic potential of muscle invasive bladder tumors

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Introduction: Adjuvant chemotherapy improves the outcome of bladder cancer patients at risk for the development of metastases. However, there are no parameters available that allow an individual risk assessment. The aim of this project is to identify a specific DNA methylation pattern that provides a reliable tool for prognosis assessment of muscle invasive bladder tumors and to understand metastasis associated processes regulated by DNA methylation. Materials & methods: Based on our previous microarray analysis we validated differentially methylated genes in a larger cohort of primary bladder tumors (n=100) in correlation to metastasis. Candidate promoter methylation and gene expression were assessed by quantitative analysis of DNA methylation using real-time PCR and qRT-PCR. Protein expression was determined by immunohistochemical analysis for KiSS1 and KISS1R on tissue microarrays from 2 independent patient cohorts comprising 290 muscle-invasive bladder carcinomas. For analyzing the gene function in tumor cell behavior, gene expression was knocked down by siRNA transfection of bladder cancer cells. Changes in proliferation and migration were measured using real-time cell monitoring (xCELLigence, Roche). Invasion assay was performed using matrigel coated cell culture inserts. Results: Methylation differences were confirmed for KISS1R, KiSS1, SEPT9 (p<0.05) and CSAD (p<0.01). mRNA expression differences between patient groups were detected for KISS1R and SEPT9v3. Protein expression of KISS1R was significantly reduced in correlation to positive lymph nodes (p<0.001) and distant metastases (p<0.05). Knock down of SEPT9v3 resulted in increased cell proliferation (77%), migration (15%) and invasion (39%) compared to negative control. Conclusion: Muscle-invasive bladder tumors show significant differences in the DNA methylation pattern in correlation to the metastatic potential. KISS1R is characterized by strongest association with the metastatic risk of primary tumors on methylation as well as RNA and protein expression level. Reduced expression of SEPT9v3 seems to induce cell proliferation, migration and invasion. The combination of candidate genes KISS1R, KiSS1, CSAD and SEPT9 could serve as prognostic marker panel for determination of the metastatic risk of primary bladder tumors. Citation Format: Beatrice Stubendorff, Kerstin Wilhelm, Kathleen Posselt, James Catto, Arndt Harmann, Susanne Fuessel, Vladimir Novotny, Mieczyslaw Gajda, Heiko Wunderlich, Marc-Oliver Grimm, Kerstin Junker. Promoter hypermethylation of KISS1R, KiSS1, SEPT9 and CSAD as a prognostic biomarker panel to assess the metastatic potential of muscle invasive bladder tumors. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1358. doi:10.1158/1538-7445.AM2014-1358

2134 DIFFERENCES IN PROTEIN PROPERTIES OF POSTOPERATIVE URINE SAMPLES ENABLE THE PREDICTION OF EARLY ALLOGRAFT REJECTION

failure rate to convert computer-identified KPD matches into transplants due to a variety of reasons. Understanding these reasons has allowed us to modify our software to incorporate a centralized tissue typing laboratory and develop a process of offering preliminary combinations for transplant center evaluation prior to optimizing a final solution. Thus, only acceptable and crossmatch negative one-way combinations are used to construct more robust final offers. As a result, we have substantially improved our conversion rate and the number of transplants so that as of November 2011 we have performed a total of 110 transplants.

Abstract 5014: Differences in DNA methylation pattern of primary bladder tumours correlate with their metastatic potential

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Introduction: The prognosis for patients with metastatic bladder cancer is poor with a 5-year survival rate of only 6%. Early prediction of the metastatic risk in primary tumours can lead to improvements in prognosis and therapy. Currently, there are no parameters available that enable an individual risk assessment for patients with metastatic bladder cancer. Therefore, identification of new reliable prognostic markers for early prediction of tumour spread is required. The aim of this project is to determine whether changes in DNA methylation correlate with the metastasis risk and to identify a specific DNA methylation pattern that provides a reliable tool for prognosis assessment. Materials & methods: Genomic DNA was isolated from 23 invasive bladder tumour tissues with and without lymph node metastases. For enrichment of methylated fragments genomic DNA was incubated with 5-Methylcytosin antibody. Input and IP were labelled with Cy3 and Cy5. Labelled DNA was hybridizes onto CpG island microarrays. Cluster analysis enabled selection of differentially methylated candidate genes. The actual methylation state was confirmed by cleavage of genomic DNA with methylation dependent restriction enzyme (McrBc) followed by quantitative PCR. Results: In total, 201 promoter CpG islands show highly significant differences in DNA methylation between metastasised and non-metastasised primary bladder tumours (p<0,01). In metastasised tumours 74 CpG islands are hypermethylated and 127 CpG islands are hypomethylated. Data base research for identification of candidate genes resulted in 61 hypermethylated and 58 hypomethylated genes that can clearly assigned to the identified promoter associated CpG islands. Validation of methylation level of candidate gene RASSF1 resulted in higher methylation in metastatic tumours (mean methylation: 38%) than in non-metastatic tumours (mean methylation: 26%). RASSF1 methylation shows strong correlation to T stage (r=0,65). Methylation of candidate gene KISS1R shows significant differences between patient groups (p=0,001). Mean methylation in metastatic tumours is 48% and in non-metastatic tumours 22%. Conclusion: Primary bladder tumours show significant differences in their DNA methylation pattern in correlation to their metastatic potential. KISS1R methylation is strongly associated with the metastatic risk of primary tumours. Further analysis will demonstrate the correlation between changes in DNA methyaltion and expression of corresponding genes. The impact of DNA methylation on tumour behaviour shall be determined in functional analysis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5014. doi:1538-7445.AM2012-5014