PBRM1 Expression Research Articles

Prognostic impact of concomitant loss of PBRM1 and BAP1 protein expression in early stages of clear cell renal cell carcinoma

PurposeTo evaluate the prognostic impact of immunohistochemical expression of BAP1 and PBRM1 in patients with early stage (pT1–pT2N0M0) clear cell renal cell carcinoma (ccRCC). Patients and methodsA total of 441 consecutive patients treated surgically for stages I and II (TNM-AJCC 2010) ccRCC between 1990 and 2016 were selected. All cases were reviewed for uniform reclassification and the most representative tumor areas were selected for the construction of a tissue microarray. Sixty-two patients had frozen tumoral tissue available in the tumor bank of our institution for quantitative real-time reverse transcriptase polymerase chain reaction analysis. ResultsOf the 441-immunostained ccRCC specimens, 91 (20.6%) and 107 (24.3%) showed negative-expression of PBRM1 and BAP1, respectively. Fifty-eight (13.2%) showed negative-expression of both markers (PBRM1-/BAP-). There was an association between both markers expression pattern and classical parameters, such as pT stage (P<0.001), tumor size (P<0.001), and tumor grade (P<0.001). Both independent PBRM1 and BAP1 negative-expression were associated with lower rates of disease-specific survival and recurrence-free survival. When patients were grouped into presence of positive expression of one or both markers vs. PBRM1-/BAP1- patients, disease-specific survival and rates were 95.3% vs. 77.6%, respectively (P<0.001). PBRM1-/BAP1-group presented a higher risk of cancer specific death (hazard ratio = 2.722, P = 0.007) and disease recurrence (hazard ratio = 2.467, P = 0.004) in multivariate analysis. ConclusionPatients with early stage tumors that present concomitant loss of both PBRM1 and BAP1 demonstrated worse survival rates and represent a relevant risk group for tumor recurrence and death.

Prognostic role of BAP1 and PBRM1 expression in intrahepatic cholangiocarcinoma

Prognostic role of BAP1 and PBRM1 expression in intrahepatic cholangiocarcinoma

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

ObjectiveTo 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 methodsImmunohistochemical 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. ResultsIn 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). ConclusionsOur 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.

BAP1 and PBRM1 in metastatic clear cell renal cell carcinoma: tumor heterogeneity and concordance with paired primary tumor

BackgroundBAP1 and PBRM1 are frequently mutated in primary clear cell renal cell carcinoma (ccRCC) tumors; however, the frequency and clinical relevance of these mutations in metastatic ccRCC tumors is unknown. Additionally, while intra-tumor heterogeneity has been shown to be common in primary ccRCC, little is known regarding heterogeneity in metastatic ccRCC tumors.Materials and methodsWe analyzed BAP1 and PBRM1 loss of protein expression in patient-matched primary and metastatic tumors from 97 patients. Expression was determined using a validated immunohistochemistry assay, which has been shown to be correlated with mutation status.ResultsOf the 97 patients evaluated, 20 and 57% showed loss of BAP1 and PBRM1 in their primary tumors, respectively. Comparing expression across patient-matched primary-metastatic tumor pairs, 98 and 90% had concordant BAP1 and PBRM1 expression, respectively. Both patients who demonstrated discordant BAP1 expression showed loss of BAP1 expression during progression to metastatic ccRCC. Similarly, seven of the ten patients that demonstrated discordant PBRM1 expression showed loss of PBRM1 expression during progression to metastatic ccRCC. We evaluated intra-metastatic tumor heterogeneity using 12 patients who had multiple blocks available from the same tumor with representative pathology; 100 and 92% showed concordant BAP1 and PBRM1 expression, respectively. Amongst 32 patients who had serial metastatic tumors available, both BAP1 and PBRM1 had 97% concordant expression.ConclusionsWe observed minimal intra- and inter- tumor heterogeneity in metastatic ccRCC tumors. Patients with discordant BAP1 or PBRM1 expression across their matched primary and metastatic tumors usually showed loss of expression during progression to metastatic ccRCC.

Loss of BAP1 protein expression in the first metastatic site predicts prognosis in patients with clear cell renal cell carcinoma

ObjectivesTo investigate the intratumoral heterogeneity of BAP1 and PBRM1 expression at the primary site and metastatic sites and to evaluate whether BAP1 and PBRM1 expression in metastatic sites of clear cell renal cell carcinoma (ccRCC) has prognostic value. Methods and materialsWe collected paired samples from the primary site and the first metastatic site in 41 patients with ccRCC. Immunohistochemistry analyses were performed for the expression of BAP1 and PBRM1 proteins. We retrospectively analyzed the associations between the expression of BAP1 and PBRM1 and overall survival (OS). ResultsThe most common first metastatic sites were lung (68.3%) and lymph node (12.2%). BAP1 protein expression was negative in 8 (19.5%) primary sites and in 11 (26.8%) metastatic sites. PBRM1 protein expression was negative in 9 (22.0%) primary sites and in 11 (26.8%) metastatic sites. The incidences of intratumoral heterogeneity for BAP1 and PBRM1 protein expression in primary/metastatic sites were 9.8%/2.4% and 24.4%/7.3%, respectively. The concordance rates between primary and metastatic sites for BAP1 and PBRM1 protein expression were 82.9% and 63.4%, respectively. Median OS from the first occurrence of metastasis in patients with BAP1-positive and BAP1-negative metastatic sites were 97 months (95% CI: 58–136) and 51 months (95% CI: 13–82), respectively (P = 0.0077). Median OS in patients with PBRM1-positive and PBRM1-negative metastatic sites were 82 (95% CI: 42–97) and 120 (95% CI: 52–120) months, respectively (P = 0.25). ConclusionIntratumoral heterogeneity of BAP1 protein expression is more frequent in primary tumor than in metastatic sites. The loss of BAP1 protein expression in metastatic sites predicts poor prognosis in patients with ccRCC.

Intrahepatic cholangiocarcinoma frequently shows loss of BAP1 and PBRM1 expression, and demonstrates specific clinicopathological and genetic characteristics with BAP1 loss

BAP1 and PBRM1 expression loss has been observed in multiple cancers, including intrahepatic cholangiocarcinoma (ICC). We investigated BAP1 and PBRM1 expression in ICC using immunohistochemistry, and analysed its association with clinicopathological and genetic features, including two histological subtypes. Whole-section slides of 108 consecutive primary ICC cases were immunostained against BAP1 and PBRM1. Complete loss of BAP1 and PBRM1 was observed in 21 (19.4%) and 25 (23.1%) cases, respectively, and partial loss was identified in four (3.7%) and nine (8.4%) cases. In all cases, normal bile ducts were strongly and diffusely positive for both BAP1 and PBRM1. ICC with BAP1 loss showed lower serum CA19-9 levels, less perineural invasion, rare mucin production, weaker immunoreactivity against S-100P and stronger immunoreactivity against N-cadherin and NCAM. IDH mutations were identified more frequently in ICCs with BAP1 loss. All ICC with BAP1 loss corresponded to small-duct type ICC. Multivariate Cox regression analysis showed that BAP1 loss was an independent prognostic factor for both overall and recurrence-free survival (P < 0.05). Conversely, PBRM1 loss was found in both small-duct type and large-duct type ICC, and was not associated significantly with any specific characteristics, including prognosis. BAP1 and PBRM1 loss is seen frequently in ICC. ICC with BAP1 loss shares features of small-duct type ICC.

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

Abstract 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 Högner, 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 10: Concordance of BAP1, PBRM1, Ki-67, and TOPOIIa protein expression in primary clear cell renal cell carcinoma tumors and patient-matched metastatic tumors

Abstract Introduction: Efforts to improve clear cell renal cell carcinoma (ccRCC) mortality have focused primarily on evaluating prognostic biomarkers in primary tumors in order to identify indicators of response to treatment and targets for novel therapies. To date little attention has been paid to the status of these biomarkers in the more therapeutically-relevant metastatic tumor. Thus, we evaluated concordance of expression of four key prognostic ccRCC biomarkers (BAP1, PBRM1, Ki-67 and TOPOIIa) in a large cohort of patient-matched primary ccRCC and metastatic tumors. Methods: We identified 114 patients treated surgically for ccRCC between 1990 and 2005 who had synchronous (M1) or metachronous (M0) metastases, underwent metastasectomy for at least one metastatic lesion and had archived tissue available from the primary and at least one metastatic tumor. We performed IHC on all primary and metastatic tumors for BAP1, PBRM1, Ki-67, and TOPOIIa using previously-published protocols. BAP1 and PBRM1 expression was categorized as positive or negative and Ki-67 and TOPOIIa was quantified as the number of positive cells per mm2. For BAP1 and PBRM1, we calculated the concordance between patient-matched primary and metastatic tumors as the percentage of pairs where the primary and metastatic tumor had the same designation (i.e., positive or negative). For TOPOIIa and Ki-67, we assessed concordance using Spearman correlation coefficient. Analysis of variance was used to evaluate if concordance between primary-metastatic tumor pairs for TOPOIIa and Ki-67 was associated with metastatic site, metastatic timing (M0/M1), metastatic tumor grade, metastatic tumor necrosis, and metastatic tumor sarcomatoid differentiation. Results: There were a total of 161 patient-matched metastatic tumors available for the 114 patients: 78 patients had only a single metastasis while 36 patients had two or more metastases. While sites of metastases varied, pulmonary metastases were the most common (44% of all metastases). Loss of BAP1 expression was observed in19% of the primary ccRCC and 98.6% of the tumor pairs had concordant BAP1 status between the paired primary and metastatic tumors. Loss of PBRM1 was observed in 56% of the primary ccRCC tumors and 89.3% of tumor pairs showed concordant PBRM1 status. By comparison, median expression of Ki-67 was 65.0mm2 in the primary ccRCC tumors and the Pearson correlation with the patient-matched metastatic tumors was 0.43. The difference in Ki-67 expression between primary-metastatic tumor pairs was associated with necrosis in the metastatic tumor (p=0.014). Median expression of TOPOIIa was 2.6mm2 in the primary ccRCC tumors and the Pearson correlation with the patient-matched metastatic tumors was 0.25. The difference in TOPOIIa expression between primary-metastatic tumor pairs was associated with metastatic site (p=0.0006) and metastases to the heart were significantly different than pulmonary metastases (p&amp;lt;0.0001). Additionally, synchronous metastases had larger differences from their patient-matched primary tumors in comparison to metachronous metastases (p=0.012), as did necrotic metastases (p=0.028). Conclusion: We observed high concordance for loss of BAP1 and PBRM1 expression between primary ccRCC and metastatic tumors in the same patient. Conversely, we noted high discordance in protein expression of Ki-67 and TOPOIIa between patient-matched primary-metastatic tumor pairs. Moreover, this discordance varied by key pathological features. Our results suggest that using the status of a biomarker in primary ccRCC as a guide for the status in metastatic tumors is not always appropriate and needs to be evaluated on a marker-by-marker basis. Citation Format: Jeanette E. Eckel-Passow, Daniel J. Serie, John C. Cheville, Thai H. Ho, Sue M. Harrington, Tracy Hilton, Christine Lohse, Amanda Shreders, Payal Kapur, James Brugarolas, R Houston Thompson, Bradley C. Leibovich, Eugene D. Kwon, Richard W. Joseph, Alexander S. Parker. Concordance of BAP1, PBRM1, Ki-67, and TOPOIIa protein expression in primary clear cell renal cell carcinoma tumors and patient-matched metastatic tumors. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Integrating Clinical Genomics and Cancer Therapy; Jun 13-16, 2015; Salt Lake City, UT. Philadelphia (PA): AACR; Clin Cancer Res 2016;22(1_Suppl):Abstract nr 10.

Clear Cell Renal Cell Carcinoma Subtypes Identified by BAP1 and PBRM1 Expression

In clear cell renal cell carcinoma BAP1 and PBRM1 are 2 of the most commonly mutated genes (10% to 15% and 40% to 50%, respectively). We sought to determine the prognostic significance of PBRM1 and BAP1 expression in clear cell renal cell carcinoma. We used immunohistochemistry to assess PBRM1 protein expression in 1,479 primary clear cell renal cell carcinoma tumors that were previously stained for BAP1. A centralized pathologist reviewed all cases and categorized tumors as positive or deficient for PBRM1 and BAP1. Kaplan-Meier and Cox regression models were used to evaluate association of PBRM1 and BAP1 expression with the risk of death from renal cell carcinoma and the risk of metastasis after adjustment for age and the Mayo Clinic SSIGN (stage, size, grade and necrosis) score. PBRM1 and BAP1 expression was PBRM1+ BAP1+ in 40.1% of tumors, PBRM1- BAP1+ in 48.6%, PBRM1+ BAP1- in 8.7% and PBRM1- BAP1- in 1.8%. The incidence of PBRM1 and BAP1 loss in the same tumor was significantly lower than expected (actual 1.8% vs expected 5.3%, p <0.0001). Compared to patients with PBRM1+ BAP1+ tumors those with PBRM1- BAP1+ lesions were more likely to die of renal cell carcinoma (HR 1.39, p = 0.035), followed by those with PBRM1+ BAP1- and PBRM1- BAP1- tumors (HR 3.25 and 5.2, respectively, each p <0.001). PBRM1 and BAP1 expression did not add independent prognostic information to the SSIGN score. PBRM1 and BAP1 expression identified 4 clinical subgroups of patients with clear cell renal cell carcinoma who had divergent clinical outcomes. The clinical value of these biomarkers will be fully realized when therapies targeting pathways downstream of PBRM1 and BAP1 are developed.

Abstract 3416: IHC assessment of PBRM1 loss in colon and lung carcinomas

Abstract PBRM1 (also known as BAF 180) is a chromatin remodeling polybromodomain protein recently identified as the second most frequently mutated gene in renal cell carcinoma (RCC). Loss of PBRM1 protein expression is most frequently observed in clear cell RCC and correlated with tumor progression. Approximately 40-50% of patients with RCC have mutations in PBRM1, and loss of PBRM1 was also reported in 28% of melanocytic neoplasms. However, loss of PBRM1 has not been studied in other tumors using patient tissues. The purpose of this study was to assess the protein expression of PBRM1 in a wide-ranging collection of normal and tumor tissues. Here we report our findings of PBRM1 loss in colon and lung carcinomas using a newly developed rabbit monoclonal antibody (RabMAb), Clone EP342. The antibody was generated by immunization of rabbits with PBRM1 recombinant protein. Sera collected from immunized rabbits were screened by ELISA, WB and IHC. After fusion, antibody from final hybridoma cell line was characterized using ELISA and Western blot (WB). A 180 KDa PBRM1 protein was detected by WB in A431 and MCF7 cell lysates. The antibody was further characterized by extensive IHC testing using formalin-fixed and paraffin-embedded (FFPE) human normal and tumor tissue microarray (TMA). IHC analysis on human tissue microarray (TMA) comprising seventeen types of normal tissues showed that PBRM1 antibody stained positively in the nuclei of all normal cells except cardiac muscle cells. In tumor TMAs comprising of fifteen types of tumors (2-4 cases for each type), negative staining was observed in carcinomas of the lung and colon, as well as clear cell RCC. Further analysis of specific types of tumor TMAs showed that PBRM1 was lost in 5 out of 30 colon carcinomas (17%), 8 out of 35 (14%) lung carcinomas, and 8 out of 1844%) clear cell RCC. In summary, the RabMAb PBRM1 antibody, clone EP342 specifically labels normal cells and most tumor cells. It is a useful tool in identifying cancers with loss of PBRM1 expression on FFPE tissues. The rate of PBRM1 loss in clear cell RCC (44%) detected by this clone is consistent with literature reported. Our study showed that PBRM1 protein was lost in colon and lung carcinomas, but less frequent than that of clear cell RCC. Further studies on colon and lung carcinomas are needed to clarify the role of PBRM1 in tumor progression in these tumors. Citation Format: Aihua Li, Yongfu Gao, Yuekai Zhang, Hongyang Pan, Jackie K. Chan, Ximing J. Yang, Taiying Chen. IHC assessment of PBRM1 loss in colon and lung carcinomas. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3416. doi:10.1158/1538-7445.AM2015-3416

Decreased PBRM1 expression predicts unfavorable prognosis in patients with clear cell renal cell carcinoma

AimsPBRM1 is one of the histone and chromatin regulators. A mutation in PBRM1 was recently identified in clear cell renal cell carcinoma (ccRCC). The aim of this study was to determine the clinicopathologic and prognostic significance of PBRM1 expression in ccRCC. Methods and resultsImmunohistochemistry was performed for PBRM1 in 657 ccRCC cases. The number of positive cells was determined using image analyzer after virtual microscope scanning. There was a strong correlation between decreased PBRM1 expression and old age, increased tumor size, higher Fuhrman grade, higher pT stage, and higher stage (all P <0.001). Patients with decreased PBRM1 expression showed significantly worse cancer-specific survival (CSS) and progression-free survival (PFS) (both P<0.001). In multivariate analysis, PBRM1 expression was an independent predictor of shorter PFS (P = 0.007). In lower-stage group (stages I and II), decreased expression of PBRM1 exhibited significantly worse CSS and PFS (both P<0.001) but not in higher-stage group (stages III and IV). In multivariate analysis of lower-stage group, decreased expression of PBRM1 was significantly associated with both poor CSS and PFS (P = 0.038 and 0.003, respectively). ConclusionsDecreased expression of PBRM1 predicts unfavorable clinical outcome in patients with ccRCC.

The Impact of PBRM1 Expression as a Prognostic and Predictive Marker in Metastatic Renal Cell Carcinoma

PBRM1, a SWI/SNF chromatin remodeling complex gene, is the second most frequently mutated gene in clear cell renal cell carcinoma. Although previous studies showed that loss of PBRM1 expression was associated with poor prognosis of renal cell carcinoma, these studies were performed on earlier stage, surgically resected renal cell carcinoma. Accordingly we investigated the PBRM1 expression profile in patients with stage IV renal cell carcinoma to determine theprognostic and predictive value of PBRM1. A total of 53 patients with stage IV or recurrent renal cell carcinoma were included in analysis. Immunohistochemistry was performed for PBRM1 using formalin fixed, paraffin embedded tissue microarrays. Overall 25 of 53 patients (47%) had high PBRM1 expression in the tumor. On analysis comparing survival rates and treatment responses of patients with renal cell carcinoma high PBRM1 expression in the tumor was associated with reduced overall survival (mean ± SD 45.0 ± 4.8 vs 23.0 ± 8.3months, p = 0.022, and for clear cell renal cell carcinoma 46.0 ± 4.1 vs 27.0 ± 6.7 months, p = 0.053). Regarding treatment outcome higher PBRM1 expression tended to indicate a poorer response to mTOR inhibitor (median progression-free survival 3.0 ± 0.2 vs 1.9 ± 2.3 months, p = 0.101). On subgroup analysis according to the Heng score this trend was more significant in the higher risk group than in the low risk group (progression-free survival for low risk mean 11.6 ± 1.2 vs 7.4 ± 7.4 months, p = 0.157, and for intermediate risk 7.1 ± 24.7 vs 2.9 ± 0.9 months, p = 0.060). Our study shows that the PBRM1 expression level is a potential prognostic marker for advanced renal cell carcinoma. We suggest that determining the tumor PBRM1 expression level may be used to inform the prognosis and treatment of metastatic renal cell carcinoma.

PBRM1 suppresses bladder cancer by cyclin B1 induced cell cycle arrest

Growing evidence indicates that dys-regulation of PBRM1 contributes to tumorigenesis. However, little is known about the biological function of PBRM1 in the development or progression of bladder cancer. In this study, we aimed to elucidate the pathophysiological role of PBRM1 in bladder cancer. We assessed the expression of PBRM1 in 64 bladder cancer tissue samples with matching normal tissues. We explored the biological functions of PBRM1 both in vitro and in vivo. Mutational status of PBRM1 was analyzed. Effect of PBRM1 on cell cycle was evaluated. qRT-PCR and Western blot were carried out to evaluate the expression of cyclins affected by PBRM1. Our results showed that PBRM1 expression was significantly reduced in bladder cancer cells and tissues compared to their normal counterparts. The reduced expression of PBRM1 was associated with advanced tumor stage, low differentiation grade and worse patient outcome. Further functional analysis demonstrated that PBRM1 suppressed bladder cancer cell proliferation, migration, colony formation in vitro and tumorigenicity in vivo. Genetic alteration analysis showed no amino-acid sequence altering mutations. We found that PBRM1 could block the G2/M transition by repressing cyclin B1. Our data indicated that PBRM1 functions as a tumor suppressor in bladder cancer by repressing cyclin B1 expression.

High concordance of BAP1 and PBRM1 expression in patient-matched primary and metastatic ccRCC tumors.

507 Background: Clear cell renal cell carcinoma (ccRCC) is a well-described molecularly heterogeneous tumor. Herein, we assessed the concordance of two of the most commonly mutated genes in ccRCC, PBRM1 (~50%), and BAP1 (~15%), in patient-matched primary and metastatic tumors. Methods: One pathologist (PK) assessed PBRM1 and BAP1 protein expression using immunohistochemistry (IHC) in 99 patients with a primary and at least one metastatic ccRCC tumor available for analysis. All available metastatic tumors were analyzed. Results: A total of 99 patients (48 M0 and 51 M1) had both a primary tumor and at least one metastatic tumor available for analysis. There were a total of 158 metastases with one patient having up to 7 metastases available for analysis. The concordance between primary and patient-matched metastasis was 87% for PBRM1 and 99% for BAP1. We observed a similar concordance between patients with M0 versus M1 disease. Conclusions: While ccRCC is molecularly heterogeneous, PRBM1, and BAP1 are largely concordant between primary and metastatic lesions suggesting that PBRM1 and BAP1 are genetically truncal events in the molecular pathogenesis of ccRCC.

Multiplatform molecular analysis of biomarkers in renal cell carcinoma.

501 Background: Predictive biomarkers of response to targeted therapy are lacking in renal cell carcinoma (RCC). We evaluated a cohort of RCC patients referred for molecular profiling to identify potentially actionable recurrent molecular aberrations. Methods: 166 consecutive renal cases referred to Caris Life Sciences over 2 yrs were evaluated with central pathology review. Cases were subtyped into clear cell (ccRCC), n=100, papillary (PRCC), n=20, sarcomatoid, n=10, translocation, n=6, medullary, n=4 or unclassified, n=26. 63% of cases were metastatic. Testing included a combination of sequencing (Sanger or next generation sequencing [NGS]), protein expression (immunohistochemistry [IHC]), and/or gene amplification (CISH or FISH). Results: ccRCC had a 52% loss of PTEN, while PRCC had a 21% loss (p value=0.02). 100% of ccRCC with sarcomatoid features (4) showed aberrant expression of PD-L1 and were infiltrated with PD-1+ tumor infiltrating lymphocytes; 100% of 10 non-ccRCC sarcomatoids also had aberrant expression of PD-L1. Loss of PBRM1 expression was observed in 60% of ccRCC. Loss of histone 3 lysine 36 trimethylation (H3K36me3), which is associated with SETD2 mutations, was observed in 30% of ccRCC. TOP2A was overexpressed in ccRCC at 30% and in non-ccRCC at 50%. 100% of ccRCC and PRCC overexpressed EGFR; a single unclassified renal cell carcinoma did not overexpress EGFR. 50% of cases had cMET overexpression, amplification, or mutation. HER2 was not amplified or overexpressed in any RCC; a single rare HER2 mutation (R816H) was found in a collecting duct carcinoma. VHL mutations were identified in 50% of ccRCC tumors. We observed lower rates of TP53 (14%), ATM (6%), and PIK3CA(4% ccRCC, 7% PRCC) mutations compared to other cancers. Conclusions: Multiplatform molecular profiling of renal cell carcinoma identifies potential predictive biomarkers in ccRCC. The documented activity of everolimus in ccRCC (RECORD-1 trial) may be attributable to PIK3CA pathway alterations. RCC with sarcomatoid features may respond to PD1/PD-L1 targeted immunotherapies. The impact of molecular profiling in ccRCC to predict responses to currently available targeted therapy has important implications for trial design and patient selection.

Frequent co-inactivation of the SWI/SNF subunits SMARCB1, SMARCA2 and PBRM1 in malignant rhabdoid tumours.

Malignant rhabdoid tumours (MRTs) are highly aggressive malignancies of early infancy characterized by inactivation of SMARCB1, a core member of the SWI/SNF chromatin-remodelling complex. The aim of this study was to explore the status of multiple key subunits of the SWI/SNF complex in MRTs. We screened the key subunits of the SWI/SNF complex, including SMARCB1, SMARCA2, PBRM1, SMARCA4, and ARID1A, in four MRTs by immunohistochemistry, sequencing, and fluorescence in-situ hybridization (FISH). Complete loss of SMARCB1, SMARCA2 and PBRM1 expression and corresponding mutations in the same genes were observed in all cases. The mutations included seven missense, three same-sense, four frameshift and two truncating mutations. FISH revealed heterozygous deletion of SMARCB1 in one case, and monoploidy of chromosome 22, which harbours SMARCB1, in another case. Furthermore, trisomy of chromosome 9, which harbours SMARCA2, was observed in two cases. Abnormality of PBRM1 was not found in any case. We report, for the first time, co-inactivation and frequent mutations of SMARCB1, SMARCA2 and PBRM1 in MRTs. Multiple subunit abnormalities of the SWI/SNF complex potentially act together to contribute to the tumorigenesis of MRTs, which provides unique insights into this disease.

Loss of PBRM1 and BAP1 expression is less common in non–clear cell renal cell carcinoma than in clear cell renal cell carcinoma

BackgroundRecurrent mutations in polybromo-1 (PBRM1, ~40%) and BRCA1-associated protein-1 (BAP1, ~10%) occur in clear cell renal cell carcinoma (ccRCC), but their prevalence in non-ccRCC or renal oncocytoma (RO) is unknown. We evaluated loss of PBRM1 and BAP1 staining in ccRCC, papillary RCC (pRCC), chromophobe RCC (chRCC), and RO tumors using an immunohistochemistry assay in which negative staining was associated with loss-of-function mutations. MethodsWe identified 458 patients treated surgically for ccRCC, pRCC, chRCC, and RO between 2004 and 2012. We performed immunohistochemistry assays to evaluate PBRM1 and BAP1 protein expression to classify tumors as PBRM1 or BAP1 negative. We compared loss of staining of these 2 proteins in ccRCC and non-ccRCC using the Fisher exact test. ResultsFor the total cohort of 458 patients, we successfully stained both PBRM1 and BAP1 in 408 tumor samples. Consistent with the mutation rate, loss of PBRM1 and BAP1 staining occurred in 43% (80/187) and 10% (18/187) of ccRCC cases, respectively. However, loss of PBRM1 staining occurred in only 3% (2/59), 6% (1/17), and 0% (0/34) of pRCC, chRCC, and RO tumors, respectively (P<0.0001). BAP1 loss was not observed in any of the pRCC (n = 61), chRCC (n = 17), or RO (n = 34) tumors, (P = 0.00021). ConclusionOur data suggest that biallelic inactivation of PBRM1 or BAP1 is less common in non-ccRCC when compared with ccRCC tumors. These findings suggest that loss of PBRM1 or BAP1 are key events in ccRCC, whereas other pathways may support tumorigenesis in non-ccRCC subtypes.

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

Abstract 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 (&amp;gt; 2fold) was measured in 41/81 ccRCC cases (50.6%) whereas in 3/81 ccRCC cases (3.7%) PBRM1 was upregulated (&amp;gt;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

Concurrent loss of INI1, PBRM1, and BRM expression in epithelioid sarcoma: implications for the cocontributions of multiple SWI/SNF complex members to pathogenesis.

The loss of INI1 (SMARCB1) expression, caused by SMARCB1 (INI1, SNF5L4, BAF47) inactivation, frequently occurs in epithelioid sarcoma (ES) and could aid in confirming the diagnosis. Except for INI1, the expression of switch in mating type/sucrose nonfermentation complex members in ES has never been examined. In this study, the expression of key subunits of this complex-INI1, BRG1 (SMARCA4), BRM (SNF2L2, SMARCA2), PBRM1 (hPB1, BAF180), and BAF155 (SMARCC1)-was analyzed in 23 ES cases: 15 conventional and 8 proximal type. All of the cases were reviewed and reclassified by hematoxylin-eosin staining and immunostaining for cytokeratin AE1/3, epithelial membrane antigen, CD34, vimentin, and INI1 expression. Of the 23 ES cases, 19 (82.6%) showed a loss of PBRM1, and 18 (78.3%), a loss of INI1. In most cases (17, 73.9%), loss of INI1 and PBRM1 expression was observed. The pattern of PBRM1 expression was similar to that of INI1, that is, not correlated with changes in cellular morphology. The concurrent loss of BRM, PBRM1, and INI1expression was detected in 2 cases with pure rhabdoid tumor features. The frequent observation of concurrent loss of INI1 and PBRM1 suggests that certain switch in mating type/sucrose nonfermentation complex components might act synergistically in the pathogenesis of ES by unknown mechanisms and that these components could provide new targets for therapy. The usefulness of PBRM1 as a biomarker of ES and its mechanism in ES require further investigation. Loss of BRM in ES with pure rhabdoid features suggests that BRM might be involved in the underlying mechanisms of this type of ES.

Biallelic germline and somatic mutations in malignant mesothelioma: Multiple mutations in transcription regulators including mSWI/SNF genes

We detected low levels of acetylation for histone H3 tail lysines in malignant mesothelioma (MM) cell lines resistant to histone deacetylase inhibitors. To identify the possible genetic causes related to the low histone acetylation levels, whole-exome sequencing was conducted with MM cell lines established from eight patients. A mono-allelic variant of BRD1 was common to two MM cell lines with very low acetylation levels. We identified 318 homozygous protein-damaging variants/mutations (18-78 variants/mutations per patient); annotation analysis showed enrichment of the molecules associated with mammalian SWI/SNF (mSWI/SNF) chromatin remodeling complexes and co-activators that facilitate initiation of transcription. In seven of the patients, we detected a combination of variants in histone modifiers or transcription factors/co-factors, in addition to variants in mSWI/SNF. Direct sequencing showed that homozygous mutations in SMARCA4, PBRM1 and ARID2 were somatic. In one patient, homozygous germline variants were observed for SMARCC1 and SETD2 in chr3p22.1-3p14.2. These exhibited extended germline homozygosity and were in regions containing somatic mutations, leading to a loss of BAP1 and PBRM1 expression in MM cell line. Most protein-damaging variants were heterozygous in normal tissues. Heterozygous germline variants were often converted into hemizygous variants by mono-allelic deletion, and were rarely homozygous because of acquired uniparental disomy. Our findings imply that MM might develop through the somatic inactivation of mSWI/SNF complex subunits and/or histone modifiers, including BAP1, in subjects that have rare germline variants of these transcription regulators and/or transcription factors/co-factors, and in regions prone to mono-allelic deletion during oncogenesis.