Jimsgene Sanjmyatav

A Specific Gene Expression Signature Characterizes Metastatic Potential in Clear Cell Renal Cell Carcinoma

PURPOSE The discovery of metastasis markers in clear cell renal cell carcinoma is of critical importance to define individual metastatic risk and select patients for new targeted therapies. We identified potential biomarkers for metastatic clear cell renal cell carcinoma by gene expression analysis. MATERIALS AND METHODS We performed transcriptional profiling of 16 primary metastatic and 18 nonmetastatic clear cell renal cell carcinomas with PIQOR™ microarrays. Differentially expressed genes were validated by quantitative real-time polymerase chain reaction. RESULTS Genes discriminating between metastatic and nonmetastatic tumors were identified at q <0.001 by significance analysis of microarrays. The metastatic signature contained 127 transcripts. In metastatic samples a greater than 4-fold decrease in expression was detected for the genes CD151 and IKBA (t/F statistic p <0.0001) while the genes MMP16, B7-H1, BCL2L2 and FRA2 showed greater than 4-fold increase of expression in metastatic primary tumors (p <0.0001). Quantitative real-time polymerase chain reaction revealed significant differences in expression among all metastatic tumors, including synchronously and metachronously metastasized tumors, and nonmetastatic tumors for FRA2 (p = 0.032) and CD151 (p = 0.005). In addition, the genes B7-H1 (p = 0.040), FRA2 (p = 0.035), CD151 (p = 0.004) and BCL2L2 (p = 0.035) showed significantly higher expression in early metastasized than in nonmetastatic tumor samples. Different B7-H1 (p = 0.002) and BCL2L2 (p = 0.007) expression levels were found in samples with late metastasis compared to those in synchronously metastasized tumors. CONCLUSIONS We determined a metastatic signature of clear cell renal cell carcinoma by microarray analysis. Our data provide the possibility of defining the metastatic potential of primary clear cell renal cell carcinoma based on a select number of genes even in a localized situation.

Identification of Genomic Alterations Associated With Metastasis and Cancer Specific Survival in Clear Cell Renal Cell Carcinoma

PURPOSE We identified regions of DNA copy number changes that are significantly associated with metastasis and clinical outcome in patients with clear cell renal cell carcinoma. MATERIALS AND METHODS We analyzed 53 primary clear cell renal cell carcinomas, including 31 metastasized and 22 nonmetastasized tumors, by array comparative genomic hybridization with a median resolution of 1 to 1.5 Mbp. To validate copy number aberrations with potential prognostic value we performed fluorescence in situ hybridization analysis using commercially available fluorescent probes. RESULTS We identified 5 recurrent chromosomal aberrations that were significantly associated with metastasis, including gains of 1q21.3, 12q13.12, 12q13.3q14.1 and 20q11.21q13.2, and loss of 9p21.3p24.1. The most prominent of them with the highest OR for metastatic risk were loss of 9p21.3p24.1, and gains of 1q21.3 and 20q11.21q13.32. Eight alterations involving chromosomes 7, 9, 12, 16 and 20 significantly correlated with shortened cancer specific survival. The lowest p values on Kaplan-Meier analysis showed losses of 9p21.3p24.1 and 9q32q33.1, and gains of 7q36.3 and 20q11.21q13.32. Fluorescence in situ hybridization done in the same cohort for the 4 select regions 1q21.3, 7q36.3, 9p21.3p24.1 and 20q11.21q13.32 clearly confirmed the results of array comparative genomic hybridization. CONCLUSIONS Data suggest that specific chromosomal alterations in clear cell renal cell carcinoma can be used to predict metastasis and cancer specific survival in patients with clear cell renal cell carcinoma. It seems possible to design a combined fluorescence in situ hybridization assay based on these genetic targets for outcome prediction, which can be used for routine diagnostics.

CD31, EDNRB and TSPAN7 are promising prognostic markers in clear-cell renal cell carcinoma revealed by genome-wide expression analyses of primary tumors and metastases†

Currently used clinicopathological parameters are insufficient for a reliable prediction of metastatic risk and disease‐free survival (DFS) of patients with clear‐cell renal cell carcinoma (ccRCC). To identify prognostic genes, the expression profiles of primary ccRCC obtained from patients with different DFS — eight synchronously, nine metachronously and seven not metastasized tumors — were determined by genome‐wide expression analyses. Synchronously and metachronously metastasized primary ccRCC differed in the expression of 167 genes. Thirty‐six of these genes were also differentially expressed in synchronously vs. metachronously developed pulmonary metastases analyzed in a previous study. Because of their DFS‐associated deregulation that is concordant in metastases and primary ccRCC, these genes are potentially functionally involved in metastatic tumor growth and are also prognostically useful. A prognostic impact was confirmed for the genes CD31, EDNRB and TSPAN7 at the mRNA level (n = 86), and for TSPAN7 at the protein level (n = 106). Patients with a higher gene expression of EDNRB or TSPAN7, or with TSPAN7‐positive vessels in both cores investigated on tissue microarrays had a significantly longer DFS and tumor‐specific survival (TSS). Patients with a higher CD31 gene expression showed a significantly longer TSS. EDNRB was an independent prognostic marker for the DFS. CD31, EDNRB and TSPAN7 had an independent impact on the TSS. In summary, comparative analysis of primary tumors and metastases is appropriate to identify independent prognostic markers in ccRCC. Gene expression of CD31 and EDNRB, and endothelial TSPAN7 protein level are potentially useful to improve outcome prediction because of their independent prognostic impact.

Establishment of a Multicolour Fluorescence In Situ Hybridisation-based Assay for Subtyping of Renal Cell Tumours

MATERIAL & METHODS: We composed 2 different multicolour FISH assays. Probe set 1 contained VHL gene region labelled in green, red-labelled chromosomal region 1p12, goldlabelled centromeric region of chromosome 7 and blue-labelled centromere of chromosome 17. Probe set 2 was composed of blue labelled chromosomal 2q11, gold-labelled centromere of chromosome 6 and green/orange labelled translocation probe for region 11q13. Overall 61 tumors were analyzed by FISH: 26 clear cell renal cell carcinomas (ccRCC), 10 papillary (pRCC), 13 chromophobe (chRCC) tumors and 12 renal oncocytomas (ROs). As reference to FISH analysis we carried out CGH for all tumors.

Reply to Jérôme Verine, Christophe Leboeuf and Philippe Ratajczak's Letter to the Editor re: Jimsgene Sanjmyatav, Sven Hauke, Mieczyslaw Gajda, et al. Establishment of a Multicolour Fluorescence In Situ Hybridisation-based Assay for Subtyping of Renal Cell Tumours. Eur Urol 2013;64:689–91

We appreciate the comments by Verine and colleagues regarding our paper [1]. The authors correctly state that a recent online survey by the International Society of Urological Pathology (ISUP) demonstrated that fluorescence in situ hybridization (FISH) is not commonly used in the diagnostic algorithms of practicing pathologists [2]. However, wewould conclude that this fact underscores the need to prove the impact of genetic analysis based on FISH tests, as we performed in our study. Does FISH help to overcome the limitations of pathologic classification? Because the paper was accepted as a short communication, it was impossible to present detailed data of our FISH results in each tumor sample. In our retrospective validation study, we used cases with unequivocal histopathologic diagnosis. As we discussed in the paper, it is especially useful to identify specific genetic alterations to distinguish poorly differentiated clear cell renal cell carcinoma (RCC) from variants of papillary type 2 RCC. Although there was a genetic overlap between these entities, a clear diagnosis could be made in the majority of cases on the basis of our FISH analysis. An overlap was also found between chromophobe RCC and oncocytomas. Histologically defined chromophobe RCCs were correctly identified based on FISH. In some oncocytomas, however, multiple chromosomal losses were detected in small numbers of cell nuclei in contrast to chromophobe RCCs, with >50% of altered nuclei per tumor. The oncocytomas with several chromosomal losses were independently evaluated by three pathologists, showing some interobserver variability in the classification of such tumors with equivocal FISH results. Further studies are necessary to characterize this specific subgroup of oncocytomas, which seems to be in a close relationshipwith chromophobe RCC. At the Vancouver ISUP conference, consensus was that such hybrid oncocytic chromophobe tumors should be placed, for the time being, in the chromophobe RCC category [3].

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

526 ESTABLISHMENT OF AN INTERPHASE FISH TEST FOR PREDICTION OF PROGNOSIS IN CLEAR CELL RENAL CELL CARCINOMA

INTRODUCTION AND OBJECTIVES: By precise mapping of genomic imbalances of 56 clear cell renal cell carcinomas using arrayCGH in our previous studies we identified specific chromosomal aberrations which are significantly associated both with metastasis and cancer specific survival. In multivariate analysis gains of 1q21.3 and of 20q11.21–q13.2 as well as loss of 9p21.3–p24.1 were independent predictors for metastasis. Multivariate Cox-regression analysis revealed gains of 7q36.3 and of 20q11.21–q13.32 and a loss of 9p21.3– p24.1 as independent prognostic factors for outcome of patients. The aim of this study was to prove the prognostic value of these aberrations using FISH probes on these critical regions in order to develop a routine diagnostic tool. METHODS: FISH experiments were performed on isolated cell nuclei from tumor tissues. The same cohort of previous array-CGH analysis was used for these experiments (32 metastasized and 24 non-metastasized). For each critical chromosomal region (1q21.3, 7q36.3, 9p21.3–p24.1 und 20q11.21–q13.32) we hybridized a mixture of three different commercially available FISH probes: region specific probe in red dye, centromeric probe of corresponding chromosome or probe close to centromere in green dye and centromeric probe of chromosome 2 in blue colour as control. 100 nuclei were scored in each case. RESULTS: Loss of 9p21.3–p24.1 (Fisher’s exact test; p 0.017) and gains of 1q21.3 (p 0.030), 7q36.3(p 0.019) and 20q11.21–q13.32 (p 0.008) displayed significant correlations with metastasis occurrence. Our analysis revealed significant correlation of gains 7q36.3 (p 0.001) and 20q11.21–q13.32 (p 0.05) as well as loss of 9p21.3-p24.1 (p 0.00002) with cancer specific death. Patients with tumours harbouring these aberrations in more than 30% of cells have a poor outcom. CONCLUSIONS: FISH results confirmed our previous findings of array-CGH and showed that these regions play an important role in cancer progression. Based on these genetic targets it seems possible to design a combined FISH assay which can be used in routine diagnostics for outcome prediction of patients with ccRCC.

Abstract 1142: A specific miRNA signature characterizes metastasis in renal cell carcinoma

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL Background: MicroRNAs (miRNAs) as small non-coding RNAs are regulators of gene expression in tumor development and progression. However, its specific role in metastasis of clear cell renal cell carcinoma (ccRCC) is almost unknown. Therefore, we aimed in defining specific miRNA expression pattern of metastatic ccRCC and identifying cellular functions of specific miRNAs deregulated in metastatic process. Methods: We isolated total RNA of 29 ccRCC samples (including 13 non-metastatic and 16 metastatic tumors) and from 16 metastases using snap frozen tissue. Single channel microarray analysis was performed for a global miRNA expression profiling in 22 ccRCC samples. The validation of the differentially expressed miRNAs in metastatic ccRCC compared to non-metastatic tumor samples and furthermore in metastases of ccRCC was performed by using qRT-PCR. Results: We identified a miRNA expression pattern that distinguishes between metastatic and non-metastatic ccRCC. Furthermore, we found a group of miRNAs with altered expression in early-metastatic compared to late metastatic and non-metastatic tumors. These microarray results were verified by qRT-PCR. By analyzing the expression of four of these differentially expressed miRNAs (26a, 30c, 126, 451) in metastases tissue by qRT-PCR we detected the same expression behavior as in the primary metastatic tumor counterparts. Conclusion: Our findings indicate that miRNAs are involved in the metastatic process. Thus, we were able to identify a specific miRNA expression pattern in metastatic ccRCC. Metastases of ccRCC are characterized by the same expression value of specific miRNAs as the primary metastatic tumor tissue. These investigations showed that specific miRNAs are possible prognostic markers for metastatic disease in RCC patients. Further studies have to define the functional role of the miRNAs in the metastatic process. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1142. doi:10.1158/1538-7445.AM2011-1142

Abstract 2246: Identification of genomic alterations associated with metastasis and poor prognosis in clear cell renal cell carcinoma

Introduction: Clear cell renal cell carcinoma (ccRCC) is the most frequently encountered renal malignancy with a high metastatic potential and the prognosis of patients in this subgroup is very poor. Prognostic parameters which can be used for an individual prognostic evaluation are still not available, So the aim of our study was high resolution screening of genomic imbalances of metastasized and non-metastasized primary ccRCC by array CGH in order to identify regions of DNA copy number changes significantly associated with metastasis and clinical outcome of patients. Methods: We conducted genome-wide copy number profiles in 56 primary ccRCC including 32 metastasized and 24 non-metastasized tumours by array CGH with a median resolution of 2Mbp. The median follow up of the patients was 47 months. Results: Array CGH identified 6 recurrent chromosomal aberrations: gains of 1q21.3, 12q13.11, 12q13.2 and 20q11.21q13.2 and losses of 8p11.23p12 and 9p21.3p24.1, which were significantly associated with metastasis occurrence. In multivariate analysis including these aberrations gains of 1q21.3 and of 20q11.21q13.2 and loss of 9p21.3p24.1 attained as independent predictors for metastasis in our cohort with 81.5% sensitivity and 95.8% specificity of 81.5% and 95.8%, respectively. Kaplan-Meier survival analysis showed that gains on chromosomes 7, 12, 16 and 20q and losses on chromosome 9 have a significant correlation with disease specific survival. Multivariate Cox-regression analysis revealed gains of 7q36.3 and of 20q11.21q13.2 and a loss of 9p21.3p24.1 as independent prognostic factors for patients9 outcome of patients. Conclusions: Our data suggest that specific common copy number alterations in ccRCC could serve as independent predictors for metastasis occurrence and cancer specific survival in patients with ccRCC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2246.

Molekulares Tumorprofiling von Nierenzelltumoren: Relevanz für Diagnostik und Therapie

ZusammenfassungMolekularbiologische Tumormarker und Prognoseparameter sind Voraussetzung für eine differenzierte Diagnostik und daraus resultierend für eine individuelle Prognosebewertung und Therapiewahl auch für Patienten mit Nierenzelltumoren (NZK). Es ist heute möglich, Tumoren sehr komplex unter Einsatz von Hochdurchsatzverfahren zu charakterisieren. Durch genetische Analysen konnten die einzelnen histopathologisch definierten Subtypen der Nierenzelltumoren als eigenständige Tumorentitäten definiert werden. Die genetischen Merkmale der einzelnen Subtypen können heute zur Diagnosesicherung am Tumormaterial, aber auch an der Biopsie eingesetzt werden. Darüber hinaus ist es möglich, metastasierungsspezifische molekulare Muster zu definieren, die zukünftig die Bewertung des Metastasierungspotenzials bereits am Primärtumor erlauben. Durch Proteomanalysen im Serum wurden spezifische Proteinmuster im Serum definiert und daraus ableitend erste Kandidatenproteine, die als Biomarker dienen könnten, identifiziert. Hierzu zählt das SAA-1, welches bei Patienten mit klarzelligen Nierenzellkarzinomen erhöht vorliegt. Da neue Therapeutika für Patienten mit metastasierten NZK zur Verfügung stehen, ist es notwendig, Patienten für die effektivste Therapie zu selektieren und frühzeitig das Therapieversagen zu erkennen. Es konnten Biomarker im Tumorgewebe und im Serum identifiziert werden, die mit dem Therapieansprechen korrelieren.AbstractMolecular biological tumor markers and prognostic parameters are necessary for differential diagnosis, individual prognosis, and therapy in patients with renal cell tumors. By using high throughput technologies, it is possible to characterize tumor samples comprehensively. Based on specific genetic alterations, histopathological subtypes were defined as independent tumor entities. Genetic characteristics can be used for diagnosis of primary tumor samples and also of biopsies. Furthermore, specific molecular patterns of metastatic tumors are known, allowing the determination of the primary tumor’s metastatic potential. The specific protein patterns of serum samples of tumor patients were analyzed, and several candidate proteins have been identified. One of these is SAA-1, which is elevated in patients with clear cell renal cell carcinomas (RCC). New therapeutic options are now available for patients with metastatic RCC. Therefore, it is necessary to select the best therapy for each patient and to detect therapy resistance very early. Biomarkers in tumor tissue and serum were found to correlate with therapy response.