Burkhard Jandrig

PBRM1 and VHL expression correlate in human clear cell renal cell carcinoma with differential association with patient’s overall survival

OBJECTIVE To identify the clinicopathological association of PBRM1 (Polybromo-1 gene) and VHL (von Hippel-Lindau gene) expression at mRNA and protein levels in clear cell renal cell carcinoma (ccRCC) and its role in tumor progression. PATIENTS AND METHODS Immunohistochemical analysis, Western blotting and qPCR analysis of PBRM1 and VHL were performed on fresh-frozen ccRCC and adjacent normal tissue obtained from 70 patients who underwent radical nephrectomy. In addition, a tissue microarray (TMA) from specimens of 326 ccRCC patients was used to evaluate the effect of loss of PBRM1 and VHL immunohistological expression on clinicopathological features as well as patient survival. RESULTS In frozen tissue, PBRM1 and VHL mRNA were significantly down-regulated in most ccRCC tumors (77.6%/80.6%). Simultaneous weak PBRM1 and VHL protein expression was observed in 21.4% of frozen tumors. In the TMA samples, weak PBRM1 and VHL immunohistochemical staining was observed in 60.4% of the cases and was correlated (P<0.001). The association of PBRM1 and VHL immunohistochemical expression with clinicopathological parameters depicts a variable picture: predominantly weak PBRM1 and VHL expression were significantly associated with higher Fuhrman grade (P = 0.012 and 0.024, respectively) but only weak VHL expression was associated with a higher pT stage (P = 0.023). PBRM1 expression did not affect the overall survival, whereas weak VHL expression was associated with decreased patient overall survival (P = 0.013). CONCLUSIONS Our data suggest that reduced expression of PBRM1 and VHL is correlated with an increased tumor aggressiveness. Low VHL expression was identified as a risk factor for worse patient overall survival, independently from PBRM1 expression pattern.

Autoantibodies Directed Against the Endothelin A Receptor in Patients With Benign Prostatic Hyperplasia

Over‐stimulation of G‐protein coupled receptors (GPCRs) such as α1‐adrenergic, muscarinic, endothelin, and AT1 receptors is considered to drive benign prostatic hyperplasia (BHP) which is often associated with lower urinary tract syndrome (LUTS). However, in addition to physiologic GPCR ligands, there is a new class of autoantibodies called functional autoantibodies that target the same GPCRs (GPCR‐AABs) for over‐stimulation, thus, presenting pathogenic potency. We hypothesize that patients with BPH/LUTS could carry GPCR‐AABs representing potential targets for treatment.

Irreversible Electroporation of Prostate Cancer: Patient-Specific Pretreatment Simulation by Electric Field Measurement in a 3D Bioprinted Textured Prostate Cancer Model to Achieve Optimal Electroporation Parameters for Image-Guided Focal Ablation

Irreversible electroporation (IRE) of localized prostate cancer (PCA) for curatively intended treatment is still considered experimental, though first study results confirm its high developmental potential as an organand functionpreserving focal therapy. Current limitations thus far include exact calculation of the ablation field, congruence between tumor localization and extension of the ablation field, and organ confinement of the ablation field with sparing of structures/organs at risk. Van den Bos et al. [1], for example, described the ablation field as being two-tothree times larger than expected and extending beyond the prostatic capsule into the neurovascular bundle with the corresponding risks of stress incontinence and erectile dysfunction. Two important factors are discussed. For one thing, electric field configuration strongly depends on tissue heterogeneity and conductivity [2]. The aging prostate with PCA is a very inhomogeneous tissue or organ (PCA, nodular hyperplasia, inflammatory infiltrates, cysts, prostatoliths, urethra, anatomic zones, and capsule). IRE planning with the NanoKnife system, however, developmentally assumes the target tissue to be homogeneous and not organ specific. This limits individual tissue-texturerelated prostate-specific IRE ablation planning. For another thing, a spheroidal IRE field coaxially aligned with the needle electrodes in the longitudinal axis is generated in transperineal grid-directed IRE of the prostate. However, the prostate displays pyramidal-to-spheroid asymmetry. Moreover, PCA is often characterized by multifocal, peripheral, asymmetric, nonspheroidal, and capsule-infiltrating or transmural growth (apex, not capsule). This

Crosstalk between Akt signaling and cold shock proteins in mediating invasive cell phenotypes

Cold shock proteins are up-regulated by cellular stress and orchestrate inflammatory responses, cell proliferation, and differentiation. Enhanced cold shock protein expression promotes malignant cell transformation; up-regulation is detected in most cancers and associated with poor prognosis. Akt1, a serine/threonine kinase, is a potent oncogene, which activates pro-proliferative and anti-apoptotic signaling pathways, and phosphorylates the cold shock domain. Unexpectedly, chicken-YB-1 abrogates PI3K-Akt-dependent oncogenic cell transformation in embryonic fibroblasts. Here, we addressed the question whether chicken and human Y-box binding protein-1 (YB-1) act differently on cell transformation, and how a related protein, DNA-binding protein-A (DbpA) behaves in comparison. NIH3T3 cells were transduced with lentiviral vectors encoding for myristoylated (constitutive active) Akt1, YB-1, DbpA, or shRNA targeting YB-1 expression. Colony formation assays showed that human YB-1 acts similar to chicken on Akt-dependent cell transformation. This activity was not titratable. Given the correlation of nuclear YB-1 and upregulated DbpA expression in a series of clear cell renal cell carcinomas (n = 40) the colony formation assay was extended to include ectopic DbpA expression. DbpA alone prominently induced cell transformation, which was enhanced when constitutive active Akt1 or concomitant YB-1 expression was present. Notably, co-expression of DbpA together with YB-1 abrogated the repressive effect on Akt1 signaling observed with YB-1 alone. Macroscopically, some colonies yielded a remarkable “invasive” phenotype. Thus, cold shock proteins may convey profound anti- and pro-oncogenic effects on Akt-dependent cell transformation. DbpA is able to overcome the anti-oncogenic effects seen with combined YB-1 and Akt signaling in an in vitro model of colonial growth.

Abstract 1843: Intratumoral heterogeneity of renal cancer is related to differences in drug response and development of therapy resistance

Patients with advanced renal cell cancer (RCC) have a poor prognosis not least because of resistance towards standard drugs. Recently, pronounced intratumoral heterogeneity (ITH) in RCC was shown. We were interested whether this ITH is a potential cause for treatment failure. We developed a large panel of patient-derived xenograft (PDX) models from RCC, including subsets of models from different regions of one individual patient tumor. The PDX models were evaluated for response to targeted standard therapeutics. To better understand correlations between interand intratumoral heterogeneity and treatment response, an explorative analysis of gene expression and panel sequencing data was performed.

Abstract 3181: Expression changes of the chromatin modifier PBRM1 in human renal cell carcinomas in relation to histopathological features

Recent next-generation sequencing studies of clear cell renal cell carcinoma (ccRCC) have identified point mutations in chromatin-modifying genes, among them PBRM1 which is now the second most frequently mutated gene in RCCs (in up to 41% ccRCCs cases) and belongs to the m1 TCGA (The Cancer Genome Atlas) subset. The occasional presence of PBRM1 mutations in the absence of VHL mutations indicates, that PBRM1 might be another driver tumor suppressor gene in ccRCCs. We were interested in examining PBRM1 expression at mRNA and protein levels and correlated these findings to histopathological features of RCC patients. Relative gene expression (RGE) data were obtained by Q-PCR from matched tumor and adjacent normal fresh frozen ccRCC tissues from 57 patients who underwent radical nephrectomy at our clinic. Papillary (pRCC) as well as chromophobic cases (chRCC) were added to analyze the expression of splice variants of PBRM1. Comparative Western blot analyses as well as semi-quantitative immuno-histochemical staining (IHC) for PBRM1 and VHL were performed on all ccRCC cases. In 78.9% of cases (45/57) PBRM1 mRNA was downregulated at least 1.5-fold, 7% (4/57) showed PBRM1 upregulation, and 14% (8/57) displayed no obvious expression changes between tumor and corresponding tumor tissue. Interestingly, 21 of 45 ccRCCs tumor tissues expressed splice variant 4 more abundantly when compared to normal tissue, whereas the normal tissue preferentially expressed splice variant 1 that includes an additional exon. Remarkably, this differential expression picture is completely reversed in pRCC and chRCC cases. The majority of 57 ccRCCs displayed weak nuclear PBRM1 staining (52.6%), whereas 31.6% showed moderate and 15.8% strong staining. However, we were not able to demonstrate a significant correlation of IHC expression levels, neither to tumor staging nor to Fuhrman grading. The observed high frequency of decreased expression of the chromatin-remodeling gene PBRM1 on mRNA (78.9%) and protein levels (52.6%), respectively, as wells as a high mutation rate (about 15% in our data set) indicate a substantial role of PBRM1 in the tumorigenesis of ccRCCs. Preferences in the expression of different PBRM1 splice variants warrant further investigation with regard to renal cell carcinoma development. Citation Format: Hans Krause, Anica Hogner, Burkhard Jandrig, Ergin Kilic, Carsten Kempkensteffen. Expression changes of the chromatin modifier PBRM1 in human renal cell carcinomas in relation to histopathological features. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3181.

Abstract LB-88: The role of PBRM1 as tumor suppressor gene in renal cell carcinomas

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Objective: The polybromodomain containing gene PBRM1 is thought to be the second major tumor suppressor gene in clear cell renal cell carinomas (ccRCCs) next to the VHL gene. The PBRM1 protein BAF180 belongs to the SWI/SNF complex and provides functional specificity to this class of chromatin remodelers by binding to acetylated histones via its bromodomains and to nucleosomal DNA by its HMG domain. Our aim was to study the expression of PBRM1 in RCC in comparison to the mutational status of PBRM1 and VHL as well as the expression of VHL. Methods: To assess the contribution of somatic mutations for PBRM1 downregulation we resequenced 31 exons of 96 tumor tissues from consenting patients who underwent radical tumor nephrectomy. In addition, the 3 exons of VHL were resequenced as well. Expression of PBRM1 and VHL was measured in 96 cases of paired normal and tumor tissue samples of RCC patients by Q-PCR using TaqMan specific probes. By immunohistochemistry BAF180 could be detected using a rabbit anti-BAF180 antibody. Results: Specific truncating mutations in the PBRM1 gene were detected in 13 (13.5%) of 96 tumor samples (15.5% of 84 ccRCC cases). No mutations could be observed in 12 cases of papillary tumors or tumors of the chromophobe type. Mutations in the VHL gene could be detected in 28/84 (33.3%) of the ccRCC samples. A marked downregulation of PBRM1 expression (> 2fold) was measured in 41/81 ccRCC cases (50.6%) whereas in 3/81 ccRCC cases (3.7%) PBRM1 was upregulated (>2fold). In 37/81 cases (45.7%) no expression differences between normal and tumor tissue could be seen. However, all cases with mutations in the PBRM1 gene showed a clear decrease of PBRM1 mRNA expression. Interestingly, in papillary carcinomas PBRM1 was strongly upregulated in comparison to the normal tissue showing a completely different expression behaviour. In addition, there was a significant difference in PBRM1 expression depending on the site of the tumor within the kidney (p=0.001). PBRM1 codes for different major transcripts. In our experiments five different transcripts could be observed although two transcripts gave only faint bands in PCR. Concurrent expression of three major transcripts (var.1, 2, and 4) is seen in all matched normal and tumor samples analyzed so far. In 22/46 samples (47.8%) we detected a dominance of var.1 (NM\_018165) in normal tissue when compared to the corresponding tumor sample, that preferentially expresses var.4 (NM\_181042) that might alter the properties of the HMG domain. Conclusions: Understanding the contribution of PBRM1 changes, existing at the genomic (mutation) and/or mRNA (differential splicing) and/or protein (cellular localization) level, to clinical disease progression and outcome in kidney cancer is an absolute prerequisite for the better understanding of renal carcinogenesis and the putative development of new therapeutic approaches. Citation Format: Burkhard Jandrig, Odiljon Ikromov, Anica Hoegner, Johann J. Wendler, Martin Schostak, Hans Krause. The role of PBRM1 as tumor suppressor gene in renal cell carcinomas. [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 LB-88. doi:10.1158/1538-7445.AM2014-LB-88

Abstract 1192: Establishment and characterization of a new patient-derived renal cell carcinoma xenograft panel

Objective: Patients with advanced kidney cancer have a poor prognosis. The majority of patients develops treatment resistance towards Standard of Care (SoC) drugs within months. The successful development of novel personalized therapies depends on the availability of preclinical models with high clinical relevance. Our aim was to establish a panel of patient-derived renal cell carcinoma xenograft models for translational research. Methods: Specimens from primary and metastatic renal cell carcinoma tumors from consenting patients were collected after surgery and transplanted subcutaneously to immunodeficient mice within 24 hours. Tumor engraftment was followed for an initial period of up to 4 months. Successful engrafted patient-derived tumors were subsequently passaged to further mice. HE stained tumor sections were histopathologically examined to assess morphological concordance between patient tumor and PDX model. Renal cell carcinoma marker expression was determined by immunohistochemistry. Stably growing xenografts were tested for sensitivity towards SoC drugs, such as Avastin, Sunitinib, Sorafenib and Everolimus. Response to treatment was evaluated by comparing growth inhibition of treated in relation to untreated tumors. Primary tumors and the derived xenografts are currently under characterization for common oncogenic mutations using the Illumina TruSeq Amplicon Cancer Panel. Results: Nearly 200 tumor samples were transplanted since 2011. To date, 30% were successfully engrafted in immunodeficient mice and further patient-derived tumors are presently under establishment. Engraftment rates of samples from pT3 and pT4 tumors were higher (34%) than those from pT1 and pT2 tumors (12%). Two tumor samples which were pre-treated before surgery engrafted so far (11%). We show that specific tumor histology and renal cell carcinoma marker expression remained preserved over the passages. The drug sensitivity testings revealed a heterogeneous response to SoC treatments with Sunitinib (79% significant growth inhibition) and Everolimus (64%) as most effective treatments in the PDX models. Avastin showed a significant growth inhibition in 57% whereas Sorafenib was effective in 36% of the RCC models. We are furthermore presenting the individual gene mutation status of each RCC model. Conclusions: Our newly established patient-derived renal cell carcinoma panel provides a novel tool for the investigation of mechanisms of drug resistance and sensitivity in kidney cancer, biomarker research and the development of new therapeutic approaches. Citation Format: Susanne Flechsig, Annika Wulf-Goldenberg, Christian Schmees, Burkhard Jandrig, Jorg Hennenlotter, Jens Bedke, Martin Schostak, Andrew Crockford, Marco Gerlinger, James Larkin, Charles Swanton, Zoltan Szallasi, Iduna Fichtner, Jens Hoffmann. Establishment and characterization of a new patient-derived renal cell carcinoma xenograft panel. [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 1192. doi:10.1158/1538-7445.AM2014-1192

Abstract 2170: The SWI/SNF nucleosome-remodeling gene PBRM1 - Another tumor suppressor gene in renal cell carcinomas

The polybromodomain containing gene PBRM1 was recently discovered to harbor 41% truncating somatic mutations in clear cell renal cell carinomas (ccRCC). Next to the VHL tumor suppressor gene this gene is thought to be the second major cancer gene in ccRCCs. PBRM1 (also called BAF180) belongs to the SWI/SNF-B or PBAF complex and provides functional specificity to this class of chromatin remodelers by binding to acetylated histones via its six bromodomains, two BAH domains, and one HMG domain (the latter binding to nucleosomal DNA). PBRM1 is located on chromosome 3p21 in close vicinity to VHL and SETD2 genes arguing for a putative gatekeeper function in cases where VHL loss alone is insufficient for ccRCCs tumorigenesis. PBRM1 codes for at least three major transcripts of about 7500nt in length whose expression was measured by Q-PCR in 49 cases of paired normal and tumor tissue samples of RCC patients who underwent radical tumor nephrectomy at our clinic between 06/2009 and 10/2010. 20/49 cases showed a marked upregulation of PBRM1 expression whereas in 29/49 cases PBRM1 was clearly downregulated. To assess the contribution of somatic mutations for PBRM1 downregulation we resequenced 29 PBRM1 exons of 48 tumor tissues. Specific truncating mutations were detected in 6 (15.4%) of 39 ccRCCs. These mutations include 5 deletions in exons 1, 10, 16, 23, and 24, as well as one insertion in exon 16. All these heterozygous mutations lead to a decrease of PBRM1 mRNA expression, that was verified by Q-PCR measurements. In addition, 6 coding SNPs (cSNPs) were revealed as well as 5 SNPs in intronic regions, one of the latter is in close vicinity to the splice acceptor site of exon 9. Concurrent expression of three major transcripts (var. 1,2, and 4) is seen in all 46 matched normal and tumor samples analyzed so far. In 22 (47.8%) samples we detected a dominance of var.1 (NM_018165) in normal tissue when compared to the corresponding tumor sample, that preferentially expresses var.4 (NM_181042). So far our data set of 15.4% truncating mutations in 39 cases of ccRCC could not match the exceptional high number of 41% reported in the initial report by the Sanger Institute. Truncating mutations of PBRM1 have been described in breast cancers but were absent in lung carcinomas, despite the fact that LOH at 3p21 is frequently seen in these cancers. Understanding the contribution of PBRM1 expression changes, be it on the genomic level (mutation) and/or mRNA (differential splicing), to clinical disease progression and outcome in kidney cancer needs further investigation. 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 2170. doi:1538-7445.AM2012-2170