PBRM1 Expression Research Articles

Dynamic and immunocytochemistry analysis of circulation tumor cells (CTCs) in blood samples from patients with advanced ccRCC starting first-line treatment in a Brazilian Cancer Center.

704 Background: Treatment of advanced clear cell renal carcinoma (ccRCC) improved dramatically in the last 20 years, but biomarkers development lagged behind. Circulating tumor cell (CTC) is used as a prognostic and predictive tool in many solid tumors but is poorly studied in ccRCC. Objective: Our aim was to evaluate CTC counts in serial blood samples from patients with advanced ccRCC that started first-line treatment and analyze the protein expression of PBRM1, BAP1, PD-L1 and CD133 in these cells. Methods: Blood samples (10mL) were collected in EDTA tubes at three different timepoints, 30 days apart, after treatment start. We used a filtration technique (ISET system, Rarecells/France) to isolate and collect CTCs. Protein expression was evaluated by immunocytochemistry. Results: Twelve patients were included. All had detectable CTCs at baseline (1st blood draw), with a median of 1.5 CTCs/mL. Patients with CTCs above the median had ≥2 metastatic sites (p=0.015) and worse progression free survival (PFS) (19.7 vs 31.1 months, p=0.35), although the difference was not statistically different. Favorable CTCs kinetics at the time of first follow-up (2nd blood draw) indicated better PFS (24.76 months) versus unfavorable (6.65 months; p=0.014). PBRM-1, BAP-1 and PD-L1 expression in CTCs was associated with better overall survival (OS), although without statistical significance. CD133 expression in CTCs at baseline was associated with worse OS (p=0.08). Conclusions: CTCs isolation was feasible in advanced ccRCC patients starting first-line treatment and were frequently detected by ISET method. CTC counts at baseline and at 30 days after treatment initiation had prognostic implication, as well as its dynamic evaluation after 30 days of treatment with a favorable kinetics associated with better outcome.

Evaluation of PBRM1, PD-L1, CD31, and CD4/CD8 ratio as a predictive signature of response to VEGFR-TKI–based therapy in patients with metastatic renal cell carcinoma (mRCC) with IMDC intermediate prognosis: Results from the APAChE-I Study.

714 Background: Intermediate IMDC group is the largest and most heterogeneous group of mRCC. Current first-line (1L) therapy options for these patients (pts) are based on either an anti-angiogenic agent (VEGFR-TKI) combined with immunotherapy (IO), or a combo of IO (ipilimumab+nivolumab [I/N]). No biomarkers (BM) for selecting the most effective regimen have been identified so far. Methods: Immunohistochemical expression of PBRM1, PD-L1, CD31, and CD4/CD8 ratio was evaluated on histological samples of intermediate-risk mRCC pts treated with VEGFR-TKI monotherapy, and then in pts receiving a VEGFR-TKI-based therapy or the immune doublet I/N. PBRM1 positivity score was based on the percentage of positive cells and on the intensity of nuclear expression; PD-L1 positivity was defined as CPS≥10; CD31 high-density had moderate to strong nuclear staining; and the CD4/CD8 ratio cut-off for positivity was >0.2. Cox model was used to assess the correlation between BM and outcomes; PFS and OS were estimated by Kaplan-Meier method. Results: After screening of tumor tissues from 150 pts, a total of 111 were included in the final analysis (Table). In pts treated with VEGFR-TKI monotherapy, a significant correlation with PFS was observed with loss of PBRM1 expression (HR 0.58, p=0.035), PD-L1 negativity (HR 0.44, p=0.048), and high CD4/CD8 ratio (HR 0.62, p=0.073). CD31 density did not significantly correlate with PFS. A profile potentially predictive of angiogenesis (AP+) was defined based on the PBRM1 loss, PD-L1 negative, and high CD4/CD8. In pts treated with VEGFR-TKI monotherapy, tumors with the AP+ (43% of all cases) had a significantly longer median PFS (mPFS 23.8 vs. 11.8 months, p=0.003) and mOS (41.5 vs. 26.9 months, p=0.024) compared to the others. The AP+ retained its significant correlation with PFS (mPFS 23.8 vs. 11.1 months, p<0.001) and OS (41.5 vs. 24.9, p=0.006) in pts receiving VEGFR-TKI-based therapies. The rate of AP+ tumors was 55.6% and 32.7% in pts with one or two IMDC risk factors, respectively (p=0.022). In the small cohort of pts treated with I/N, no differences were observed in PFS (p=0.64) and OS (p=0.75) between AP+ and AP-negative. Conclusions: The AP+ signature (loss of PBRM1, PD-L1 negative, and CD4/CD8 high ratio) was associated with improved clinical outcomes in mRCC pts at IMDC intermediate prognosis treated with VEGFR-TKI-based therapy; this correlation was significant regardless from the addition of IO to VEGFR-TKI monotherapy. Prospective validation of this signature is required for guiding the selection of the most appropriate 1L therapy. [Table: see text]

Exploration of 68Ga-labelled prostate-specific membrane antigen-11 PET/CT parameters for identifying PBRM1 status in primary clear cell renal cell carcinoma

To investigate the predictive value of 68Ga-labelled prostate-specific membrane antigen-11 (68Ga-PSMA-11) integrated positron-emission tomography (PET)/computed tomography (CT) in PBRM1-deficient clear cell renal cell carcinoma (ccRCC). A total of 41 patients with ccRCC, were enrolled retrospectively and underwent 68Ga-PSMA-11 PET/CT preoperatively. Radiological parameters, including CT attenuation value and maximum standard uptake value (SUVmax), were derived. Immunohistochemical and multiple immunofluorescences staining were performed to evaluate the PBRM1 status and immune response. The predictive value of imaging factors was analysed using a receiver operator characteristic curve analysis. Univariate and multivariate logistic regression analyses were used to investigate the relationship between clinical and radiological variables and PBRM1 status. A total of 41 patients were included in this study, with 14 patients having PBRM1-deficient status. The tumour diameter on imaging and SUVmax differed significantly in patients with different PBRM1 expression statuses and no difference in CT attenuation was identified. Univariate and multivariate logistic regression analyses showed SUVmax was an obvious predictor for identification of PBRM1-deficient tumours. In addition, PBRM1-deficient tumours tended to be accompanied by greater cytotoxic T-cell infiltration, although most of them were in an exhausted state. 68Ga-PSMA-11 PET/CT could be used to discriminate invasive PBRM1-deficient ccRCC.

PBRM1 presents a potential prognostic marker and therapeutic target in duodenal papillary carcinoma.

BackgroundDue to its rarity, duodenal papillary carcinoma (DPC) is seldom studied as a unique disease and no specific molecular features or treatment guidelines are provided.MethodsWhole‐exome sequencing was performed to gain new insights into the DPC mutation landscape and to identify potential signalling pathways and therapeutic targets. Mechanistically, immunohistochemistry (IHC), immunofluorescence, RNA‐seq, ATAC‐seq and in vitro cell function experiments were performed to confirm the underlying mechanisms.ResultsWe described the mutational landscape of DPC for the first time as a group of rare tumours with a high frequency of dysregulation in the chromatin remodelling pathway, particularly PBRM1‐inactivating mutations that are significantly higher than duodenal adenocarcinomas and ampullary adenocarcinoma (27% vs. 0% vs. 7%, p < .01). In vitro cell experiments showed that downregulation of PBRM1 expression could significantly promote the cancer progression and epithelial‐to‐mesenchymal transition via the PBRM1‐c‐JUN‐VIM axis. The IHC data indicated that PBRM1 deficiency (p = .047) and c‐JUN expression (p < .001) were significantly associated with poor prognosis. Meanwhile, the downregulation of PBRM1 expression in HUTU‐80 cells was sensitive to radiation, which may be due to the suppression of c‐JUN by irradiation.ConclusionsOur findings define a novel molecular subgroup of PBRM1‐inactivating mutations in DPC. PBRM1 play an important role in DPC progression and may serve as a potential therapeutic target and prognostic indicator.

Establishing a Prognostic Model Based on Three Genomic Instability-related LncRNAs for Clear Cell Renal Cell Cancer.

Among all types of renal cell cancer (RCC), clear cell renal cell cancer (ccRCC) is the most common and aggressive one. Emerging evidence uncovers that long non-coding RNAs (lncRNAs) are involved in genomic instability, which correlates to the clinical outcomes of patients who suffer from various kinds of cancers. We gathered expression profiles of transcriptome RNA and clinical information about ccRCC from The Cancer Genome Atlas (TCGA) and The Gene Expression Omnibus (GEO) database. The lncRNA expression profiles and somatic mutation data were combined to identify genome instability-related lncRNAs (GILncRs) by significance analysis of T test. By means of univariate and multivariate cox regression analyses, 3 GILncRs strongly associated with patient prognosis were screened out to build a genomic instability-related risk score (GIRS) model. We use R-version 4.0.4 to draw Kaplan-Meier plots and ROC curves for survival prediction. The somatic mutation count was higher in genomic unstable group. PBRM1 showed lower expression in genomic unstable group. Three lncRNAs such as LINC00460, AC156455.1, LINC01606 were included in the GIRS model. Patients had poorer prognosis with higher risk score of GIRS model. The somatic mutation count was higher in patients with higher risk score while PBRM1 expression was lower. The GIRS model was independent from other clinical factors. The GIRS model was superior to other 2 published lncRNA signatures in survival prediction.

Clinicopathological Significance, Related Molecular Changes and Tumor Immune Response Analysis of the Abnormal SWI/SNF Complex Subunit PBRM1 in Gastric Adenocarcinoma.

Background: PBRM1 gene abnormalities were recently found to play a role in tumor development and tumor immune activity. This article will explore the clinicopathological and molecular changes and tumor immune activity of the abnormal SWI/SNF complex subunit PBRM1 in gastric adenocarcinoma (GAC) and its significance. Methods: The cBioPortal, LinkedOmics and TISIDB datasets were used to analyze the abnormality of the PBRM1 gene in GAC and its relationship with prognosis, related molecular changes and tumor-infiltrating lymphocytes (TILs). In addition, 198 GAC cases were collected to further study the relationship between the loss/attenuation of PBRM1 expression and clinicopathology, prognosis, microsatellite stability, PD-L1 expression and TIL in GAC. DNA whole-exome sequencing was performed on 7 cases of gastric cancer with loss of PBRM1 expression. Results: The cBioPortal data showed that PBRM1 deletion/mutation accounted for 7.32% of GAC and was significantly associated with several molecular changes, such as molecular subtypes of GAC. The LinkedOmics dataset showed that PBRM1 mutation and its promoter DNA methylation showed lower PBRM1 mRNA expression, and PBRM1 mutation cases showed significantly higher mRNA expression of PD-L1 (CD274). TISIDB data showed that PBRM1 abnormalities were significantly positively associated with multiple TILs. In our group of 198 cases, the loss/attenuation of PBRM1 expression was significantly positively correlated with intra-tumoral tumor infiltrating lymphocytes (iTILs) and deficient MMR and PD-L1 expression. Kaplan–Meier survival analysis showed that the overall survival of GAC patients with loss/attenuation of PBRM1 expression was significantly better (p = 0.023). iTIL was an independent prognostic factor of GAC. Loss of PBRM1 expression often co-occurs with mutations in other SWI/SNF complex subunit genes, and there are some repetitive KEGG signaling changes. Conclusion: Abnormality of the PBRM1 gene may be related to the occurrence of some GACs and can affect tumor immune activity, thereby affecting clinicopathology and prognosis. It may be a potentially effective predictive marker for immunotherapy and a novel therapeutic approach associated with synthetic lethality.

Abstract 5714: Characterization of SWI/SNF complex gene mutations, protein expression and tumor immune microenvironment in cholangiocarcinoma

Abstract Introduction: Cholangiocarcinoma (CCA) is a rare tumor accounting for less than 2% of all human malignancies. Mutations in the mammalian Switch/Sucrose Non-Fermentable (SWI/SNF) chromatin remodeling complex have been identified in approximately 20% of all human cancers and more than 40% of CCA. If the role of SWI/SNF on the tumor biology and microenvironment is being uncovered in other tumor types, it remains vastly unknown in CCA. Here, we wanted to investigate, using clinical samples, the association between SWI/SNF defects, the molecular landscape and tumor immune microenvironment in CCA. Material and Methods: Mutation profiling of 103 patients with CCA from Gustave Roussy (GR) was assessed with FoundationOne® CDx 324-gene NGS panel. Formalin-fixed paraffin-embedded tumor tissues from 11 patients with CCA were analyzed for SWI/SNF (PBRM1, ARID1A, SMARCB1 and SMARCA4) and Polycomb-DUB (BAP1) subunits, as well as lymphocytic markers (CD4 and CD8). Stainings were performed using a VENTANA BenchMark ULTRA. Absolute count of positive cells per mm2 was determined by digital image analysis with the Definiens Developer XD™ by selecting at least 5 independent tumoral zones. Pairwise comparisons were performed using the Mann-Whitney test. Results: Among the patients for whom NGS data was available, the most frequently altered genes were TP53 (27%), CDKN2A (17%), KRAS (16%), ARID1A (12%), FGFR2 (12%) and PBRM1 (10%). Subunits of the SWI/SNF complex and BAP1 were mutated in 25% and 10% of cases, respectively. Tumor samples with ARID1A mutations (n=5) showed strongly decreased ARID1A expression compared to WT tumor samples (n=2) (mean = 308.03 positive nuclei (p.n)/mm2 versus 6096.82 p.n/mm2 respectively; P&amp;lt;0.0001) but unchanged PBRM1, SMARCB1 and SMARCA4 expression. PBRM1 mutations (n=4) were associated with decreased expression of PBRM1 (mean = 309.6 p.n/mm2 versus 4980.2 p.n/mm2 in WT samples; P&amp;lt;0.0001) and ARID1A (mean=1196.15 p.n/mm2; P&amp;lt;0.001), but unchanged SMARCB1 and SMARCA4 expression. BAP1 mutations (n=3) were also associated with decreased BAP1 and ARID1A expression (ARID1A mean = 457.70 p.n/mm2; P&amp;lt;0.001). ARID1A and PBRM1-altered tumor samples showed a poorly infiltrated microenvironment, with decreased CD4+ T cell (159.48 positive cells (p.c)/mm2 and 188.66 p.c/mm2 respectively versus 1126.52 p.c/mm2 for WT; P&amp;lt;0.0001) and CD8+ T cell density (72.38 and 76.54 p.c/mm2 versus 548.58 p.c/mm2 for WT; P&amp;lt;0.0001). Conclusion: Mutations in the ARID1A and PBRM1 subunits of the SWI/SNF complex are associated with a loss of protein expression. In contrast to what reported in other tumor types, ARID1A and PBRM1 mutations correlated with poorly lymphocytic TIME in CCA. Decreased PBRM1 expression is further associated with decreased ARID1A expression. Independent revalidation and further study in larger tumor series are warranted. Citation Format: Clémence Astier, Jean-Yves Scoazec, Virginie Marty, Olivia Bawa, Nicolas Signolle, Carine Ngo, Francesco Facchinetti, Antoine Hollebecque, Sophie Postel-Vinay. Characterization of SWI/SNF complex gene mutations, protein expression and tumor immune microenvironment in cholangiocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5714.

Natural history of Von Hippel-Lindau disease-associated and sporadic clear cell renal cell carcinoma: a comparative study.

To compare the tumor growth kinetics between sporadic clear cell renal cell carcinoma (ccRCC) and Von Hippel-Lindau disease-associated renal cell carcinoma (VHL-associated RCC). To analyze predictive markers for the growth rate of these two types of RCC. The clinical data of patients with renal tumors who received active surveillance were collected retrospectively. Immunohistochemical staining was utilized to analyze the expression levels of VHL, PBRM1, H3K36me3, and BAP1 in the postoperative specimens. The age of the VHL group was significantly younger than that of the sporadic group (P < 0.0001). The mean linear growth rate (LGR) was significantly faster in the sporadic group (P = 0.0004). The tumors of those in the sporadic group tended to have a higher histologic grade (P = 0.0011). In the sporadic group, tumor histologic grade was an independent predictor for rapid mean LGR (P = 0.0022). In the VHL group, initial maximal tumor diameter (MTD) was the only independent predictor for rapid mean LGR (P < 0.0001). Tumors with low VHL expression and negative PBRM1 expression showed a faster growth rate in the sporadic group (P = 0.001 and P = 0.008, respectively). The expression levels of the four biomarkers showed no impact on the tumor growth rate in the VHL group. Sporadic ccRCC grew faster than VHL-associated RCC. High histologic grade, low VHL expression and negative PBRM1 expression were predictors of faster growth in sporadic ccRCC. A large initial MTD was a predictor of faster growth for VHL-associated RCC.

Mutational Analysis of PBRM1 and Significance of PBRM1 Mutation in Anti-PD-1 Immunotherapy of Clear Cell Renal Cell Carcinoma.

Renal cell carcinoma is a common solid tumor. PBRM1 is one of the most mutation-prone genes in clear cell renal cell carcinoma (ccRCC) with the occurrence of mutation in 40% of ccRCC patients. Mutations in PBRM1 have been correlated with the efficacy of immunotherapy. However, the mutation types of PBRM1 are not well characterized. The effects of PBRM1 expression levels in the tumor microenvironment are not well studied. In addition, the mechanism and effect of anti-PD-1 immunotherapy in ccRCC tumor microenvironments are not well clarified. In this study, using bioinformatics methods we analyzed the alternation frequency and expression levels of PBRM1 in various tumors. Next, we experimentally validated their expression levels in ccRCC tissues from human and mouse models. We attempted to clarify the mechanisms of anti-PD-1 immunotherapy in ccRCC with various PBRM1 expression levels. Our results showed that deficiency of PBRM1 protein is correlated with CD4 T cell reduction in human and mouse ccRCC tissues. We also showed that anti-PD-1 Immunotherapy can increase the infiltration of T cells in both PBRM1 high and PBRM1 low tumors but to different degrees. Our study indicates that the reduction of CD4 cells in tumor tissues with low expression of PBRM1 may explain the compromised efficacy of anti-PD-1 immunotherapy in patients with PBRM1 mutated ccRCC. Our study sheds light on the potential of PBRM1 as a therapeutic target in ccRCC.

Abstract 1599: SMARCA4 regulates tumor resistance to anti-tumor immune response

Abstract Chromatin remodelers regulate chromatin accessibility and translocation of the nucleosome to expose the underlying DNA sequence. SWI/SNF is a conserved chromatin remodeling complex required for transcriptional activation or repression. In fact, SWI/SNF is the most frequently mutated chromatin regulator in cancer. Though the general understanding is that subunits of the SWI/SNF complex mainly act as tumor suppressors, studies have shown that they can have a context-dependent oncogenic role. Moreover, the PBAF subunits of the complex, PBRM1 and ARID2 impart immunoresistance in B16 melanoma tumors. We perform a TCGA analysis of the subunits based on expression and see that high PBRM1 or SMARCA4 expression in SKCM melanoma correlates with lower survival. Interestingly, SMARCA4 is the conserved catalytic subunit of the complex, and is essential to hydrolyze the energy of the ATP to execute the remodeling function of the complex. On performing a knockdown of SMARCA4 in an in vitro model of B16 cells, a co-culture with antigen specific T cells shows an increase in T cell cytotoxicity. Furthermore, RNA sequencing reveals that knockdown of SMARCA4 upregulates the genes associated with interferon response and downregulates MYC targets. We show that SMARCA4 may mediate immunoresistance such that a knockdown of SMARCA4 in the tumor will sensitize it to anti-tumor immunity. We will next take an in vivo approach to examine if SMARCA4 knockdown will enhance the tumor immune response via an interferon related mechanism. Citation Format: Apoorvi Chaudhri, Yunfei Wang, Leilei Shi, Kunal Rai, Patrick Hwu. SMARCA4 regulates tumor resistance to anti-tumor immune response [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1599.

MUC1-C activates the PBAF chromatin remodeling complex in integrating redox balance with progression of human prostate cancer stem cells

The polybromo-associated PBAF (SWI/SNF) chromatin remodeling complex, which includes PBRM1, ARID2, and BRD7, regulates cell differentiation and genomic integrity. MUC1-C is an oncogenic protein that drives lineage plasticity in prostate cancer (PC) progression. The present work demonstrates that MUC1-C induces PBRM1, ARID2, and BRD7 expression by the previously unrecognized E2F1-mediated activation of their respective promoters. The functional significance of the MUC1-C→PBAF pathway is supported by demonstrating involvement of MUC1-C in associating with nuclear PBAF and driving the NRF2 antioxidant gene transcriptome in PC cells. Mechanistically, MUC1-C forms a complex with NRF2 and PBRM1 on the NRF2 target SLC7A11 gene that encodes the xCT cystine-glutamate antiporter, increases chromatin accessibility and induces SLC7A11/xCT expression. We also show that MUC1-C and PBRM1 are necessary for induction of other NRF2 target genes, including G6PD and PGD that regulate the pentose phosphate pathway. Our results further demonstrate that MUC1-C integrates activation of PBRM1 with the regulation of antioxidant genes, ROS levels, pluripotency factor expression and the cancer stem cell (CSC) state. These findings reveal a role for MUC1-C in regulating PBAF, redox balance and lineage plasticity of PC CSC progression. Our findings also uncover involvement of MUC1-C in integrating the PBAF and BAF pathways in cancer.

The SSPN Score, a Novel Scoring System Incorporating PBRM1 Expression, Predicts Postoperative Recurrence for Patients with Non-metastatic Clear Cell Renal Cell Carcinoma.

The loss of PBRM1 expression (as identified by immunohistochemistry) is associated with a high risk of postoperative recurrence for patients with clear cell renal cell carcinoma (ccRCC). The authors developed a scoring system to predict recurrence based on clinicopathologic factors incorporating PBRM1 expression. This study retrospectively reviewed 479 ccRCC patients who underwent radical surgery between 2006 and 2017. The study extracted a subset of 389 non-metastatic ccRCC patients for whom relevant clinicopathologic factors were available. The primary end point was recurrence-free survival (RFS). The Kaplan-Meier method and the Cox proportional hazards model were used for statistical analysis. Leibovich score, SSIGN score, and University of California, Los Angeles (UCLA) Integrated Staging System were included as conventional prediction models. Of the 389 patients, 53 (13.6%) experienced recurrence during a median period of 61months. Multivariable analyses showed that that the independent factors for RFS were ≥ pT3 (hazard ratio [HR] 3.64; P < 0.001), sarcomatoid or rhabdoid component (HR 3.29; P = 0.005), PBRM1 negativity (HR 3.39; P = 0.001), and necrosis (HR 3.60; P < 0.001). A scoring system calculated with these factors, named the SSPN (stage, sarcomatoid, PBRM1 expression, and necrosis) score, showed significant differences in RFS among the following four groups; low-risk group (0 factors), intermediate-risk group (1 factor), high-risk group (2 to 3 factors), and very high-risk group (4 factors) (P < 0.001). The authors' model also showed a greater predictive accuracy for 5-year RFS than the conventional models (0.841 vs 0.747-0.792). The SSPN score, which integrates clinicopathologic findings and PBRM1 expression, can accurately predict postoperative recurrence for patients with non-metastatic ccRCC after radical surgery.

PT174 - A novel scoring system integrating PBRM1 expression to predict recurrence in patients with non-metastatic clear cell renal cell carcinoma undergoing radical surgery

PT174 - A novel scoring system integrating PBRM1 expression to predict recurrence in patients with non-metastatic clear cell renal cell carcinoma undergoing radical surgery

Prognostic role of PBRM1 marker expression in clear-cell renal-cell carcinoma

Background . Clear-cell renal-cell carcinoma (CCRCC) is the most common histological type of cancer of this localization. Changes in 16 genes were identified as significant in carcinogenesis of CCRCC. After VHL suppressor gene, PBRM1 gene is the second by frequency of genetic abnormalities in CCRCC and it is mutated in 40—50 % cases of CCRCC. The study objective is to analyze the effect of abnormalities in PBRM1 protein expression on survival of patients with CCRCC. Materials and methods . The study included 137patients with newly diagnosed and histologically confirmed CCRCC. For all study participant, detailed medical history and questionnaire data were acquired. Prior to treatment, blood samples and tumor tissue removed during surgery were obtainedfrom all patients. All patients are annually followed up for current information on their life status, disease dynamics, treatment. Minimalfollow-up time is 22 months, maximal is 128 months, mean is 61.8 months, median is 48 months. Immunohistochemical (IHC) testing of PBRM1 expression was performed using standard technique with polyclonal rabbit antibodies PB1[N1N2] N-term (GeneTex 100781) with 1:50 dilution, DAB staining. Normally, protein product of the wild type PBRM1 gene is functioning and can be detected in the nucleus. Absence of nuclear expression of PBRM1 points to genetic or epigenetic abnormalities. Results. Renal cancer-specific survival is significantly lower in patients without expression of the PRBM1 protein in tumor cells. The longest 5- (84 %) and 10-year (84 %) survival was observed in patients with diffuse nuclear expression of the PBRM1 protein. Difference in survival of these patients compared to patients without PRBM1 protein expression is statistically significant (p = 0.004). We have performed an analysis of the association between survival of patients with CCRCC andfocal nuclear PBRM1 expression. In these patients, survival is lower than in patients with diffuse expression but higher than in patients without nuclear expression of PBRM1 (p = 0.02). Cytoplasmic expression of PBRM1 doesn’t affect survival. Conclusion. The obtained results point to prognostic value of PBRM1 gene activity which is abnormal in almost half of all CCRCC cases. IHC testing is an appropriate, reliable and affordable method for determination of PBRM1 protein expression and therefore can be used in practice. Favorable course and prognosis in patients with stage I—II CCRCC and preserved nuclear expression of the PBRM1 protein should be noted: 5-year survival for these patients is 100 %. This observation is crucial for making decisions on treatment of these patients.

Expression and Mutation Patterns of PBRM1, BAP1 and SETD2 Mirror Specific Evolutionary Subtypes in Clear Cell Renal Cell Carcinoma

Bi-allelic inactivation of the VHL gene on chromosome 3p is the characteristic feature in most clear cell renal cell carcinomas (ccRCC). Frequent gene alterations were also identified in SETD2, BAP1 and PBRM1, all of which are situated on chromosome 3p and encode histone/chromatin regulators. The relationship between gene mutation, loss of protein expression and the correlations with clinicopathological parameters is important for the understanding of renal cancer progression. We analyzed PBRM1 and BAP1 protein expression as well as the tri-methylation state of H3K36 as a surrogate marker for SETD2 activity in more than 700 RCC samples. In ccRCC loss of nuclear PBRM1 (68%), BAP1 (40%) and H3K36me3 (47%) expression was significantly correlated with each other, advanced tumor stage, poor tumor differentiation (P < .0001 each), and necrosis (P < .005) Targeted next generation sequencing of 83 ccRCC samples demonstrated a significant association of genetic mutations in PBRM1, BAP1, and SETD2 with absence of PBRM1, BAP1, and HEK36me3 protein expression (P < .05, each). By assigning the protein expression patterns to evolutionary subtypes, we revealed similar clinical phenotypes as suggested by TRACERx Renal. Given their important contribution to tumor suppression, we conclude that combined functional inactivation of PBRM1, BAP1, SETD2 and pVHL is critical for ccRCC progression.

Risk Prediction Tool for Aggressive Tumors in Clinical T1 Stage Clear Cell Renal Cell Carcinoma Using Molecular Biomarkers

Some early-stage clear cell renal cell carcinomas (ccRCCs) of ≤7 cm are associated with a poor clinical outcome. In this study, we investigated molecular biomarkers associated with aggressive clinical T1 stage ccRCCs of ≤7 cm, which were used to develop a risk prediction tool toward guiding the decision of treatment. Among 1069 nephrectomies performed for ccRCC of ≤7 cm conducted between January 2008 and December 2014, 177 cases with available formalin-fixed paraffin-embedded tissue were evaluated. An aggressive tumor was defined as a tumor exhibiting synchronous metastasis, recurrence, or leading to cancer-specific death. Expression levels of six genes (FOXC2, CLIP4, PBRM1, BAP1, SETD2, and KDM5C) were measured by reverse-transcription polymerase chain reaction (qRT-PCR) and their relation to clinical outcomes was investigated. Immunohistochemistry was performed to validate the expression profiles of selected genes significantly associated with clinical outcomes in multivariate analysis. Using these genes, we developed a prediction model of aggressive ccRCC based on logistic regression and deep-learning methods. FOXC2, PBRM1, and BAP1 expression levels were significantly lower in aggressive ccRCC than non-aggressive ccRCC both in univariate and multivariate analysis. The immunohistochemistry result demonstrated the significant downregulation of FOXC2, PBRM1, and BAP1 expression in aggressive ccRCC. Adding immunohistochemical staining results to qRT-PCR, the aggressive ccRCC prediction models had the area under the curve (AUC) of 0.760 and 0.796 and accuracy of 0.759 and 0.852 using the logistic regression method and deep-learning method, respectively. Use of these biomarkers and the developed prediction model can help stratify patients with clinical T1 stage ccRCC.

Loss of BAP1 expression in metastatic tumor tissue is an event of poor prognosis in patients with metastatic clear cell renal cell carcinoma

PurposeTo evaluate the prognostic impact of the protein expression of both PBRM1 and BAP1 in metastatic tissue of patients with metastatic clear cell renal cell carcinoma (ccRCC). Patients and methodsIn all 124 consecutive cases of metastatic ccRCC, who underwent metastasectomy or biopsy of metastatic tumor tissue between 2007 and 2016 were selected from the medical records of our institution. Additionally, 38 paired cases with tissue from the primary tumor involving radical or partial nephrectomy for ccRCC were also selected. All cases were reviewed for uniform reclassification and the most representative tumor areas were selected for the construction of a tissue microarray. ResultsPBRM1 nuclear staining of the 124-immunostained metastases of ccRCC specimens showed that 98 (79.0%) had negative expression and 26 (21.0%) positive expression of PBRM1. Regarding BAP1 expression, we observed that 77 (62.1%) specimens were negative and 47 (37.9%) showed positive nuclear staining. When we compared the expression of both markers on primary tumor and tumor metastasis, we found disagreement in half of the cases. Five-year overall survival rates in patients with positive expression and negative expression of BAP1 were 53.2% and 35.1%, respectively (P = 0.004). Five-year progression-free survival rates in patients with positive expression and negative expression of BAP1 were 14.9% and 3.9%, respectively (P = 0.003). Conversely, PBRM1 expression did not significantly influence either overall survival or progression-free survival rates. In multivariate analysis, negative expression of BAP1 tumors also presented higher risks of death (hazard ratio (HR) = 1.913, P = 0.041) and disease progression (HR = 1.656, P = 0.021). ConclusionThe use of prognostic biomarkers identified in the primary tumor tissue might be not reliable in the metastatic disease scenario. Patients with metastatic ccRCC that present loss of BAP1 expression in metastatic tissue demonstrated poor survival rates and represent a relevant risk group for tumor recurrence and death.

Prognostic role of BAP-1 and PBRM-1 expression in intrahepatic cholangiocarcinoma.

Intrahepatic cholangiocarcinoma (ICC) has universally poor outcome, mainly due to its late clinical presentation. Identification of specific biomarkers and development of effective treatment are still urgently required. Mutations in PBRM-1 and BAP-1 genes, and the expression of S100P have been related to survival in ICC. miR-31 seems also to play important regulatory functions in ICC and it directly regulates BAP-1 expression in lung cancer. In this study, tissue expression of BAP-1, PBRM-1, S100P, and miR-31 was investigated in ICC and correlated with clinical-pathological features. Sixty-one consecutive patients who underwent curative hepatic resection for ICC were enrolled. None received any therapy prior to surgery. Immunostaining for BAP-1, PBRM-1, and S100P, and in situ hybridization for miR-31 were performed, using tissue microarray slides. A strong retained expression of BAP-1 and PBRM-1 was associated with a reduced overall (p = 0.04 and p = 0.002, respectively) and disease-free survival (p = 0.05 and p = 0.02, respectively). An overexpression of S100P was related to a reduced overall survival (p = 0.005). The multivariate analyses identified the presence of perineural invasion and the retained PBRM-1 expression as independent predictors of worse overall [p = 0.02, hazard ratio (HR) = 2.25 (1.16-4.39) and p = 0.001, HR = 3.13 (1.56-6.28), respectively] and disease-free survivals [p = 0.03, HR = 2.43 (1.09-5.4) and p = 0.03, HR = 2.51 (1.11-5.67), respectively]. An overexpression of S100P was predictive of a worse overall survival [p = 0.02, HR = 1.66 (1.08-2.55)]. High levels of miR-31 were significantly associated to a low expression of BAP-1 protein (p = 0.03). In ICC, a retained expression of BAP-1 and PBRM-1, and an overexpression of S100P are related to a poor prognosis.

Prognostic and clinicopathological value of PBRM1 expression in renal cell carcinoma

BackgroundThe prognostic value of PBRM1 expression in renal cell carcinoma (RCC) had been investigated in previous studies; however, the results remain inconclusive. We investigated the prognostic value and clinicopathological significance of PBRM1 protein expression in RCC. MethodsPubMed, Embase, Web of Science, and Cochrane database were searched for studies investigating the relationships between PBRM1 expression and outcomes in RCC. Hazard ratios (HRs) for survival outcomes and odds ratios (ORs) for clinical parameters were extracted from eligible studies. Heterogeneity was assessed using the I2 value. The fixed-effects model was used if there was no evidence of heterogeneity; otherwise, the random-effects model was used. Publication bias was evaluated using Begg's funnel plots and Egger's regression test. ResultsA total of 2942 patients from 7 studies were included in the meta-analysis. The results showed that decreased expression of PBRM1 is associated with poor overall survival (OS) (HR = 2.11, 95% CI: 1.52–2.96), cancer-specific survival (CSS) (HR = 1.32, 95% CI: 1.10–1.58), and progression-free survival/ recurrence-free survival (PFS/RFS) (HR = 1.57, 95%CI: 1.34–1.85) in RCC. In addition, PBRM1 positive expression was significantly associated with earlier TNM stage (III/IV vs. I/II, OR = 0.53, 95% CI: 0.30–0.94), primary tumor stage (pT3/4 vs. pT1/2, OR = 0.32, 95% CI: 0.20–0.52), and Fuhrman grade (3/4 vs. 1/2, OR = 0.69, 95% CI: 0.46–1.02), but not related to Necrosis or Sex. ConclusionsDecreased expression of PBRM1 is correlated with poor prognosis and advanced clinicopathological features in patients with RCC.

SWI/SNF subunit expression heterogeneity in human aplastic anemia stem/progenitors

Acquired aplastic anemia (AA) is a bone marrow (BM) failure associated with autoimmune destruction of hematopoietic stem cells (HSCs). Although somatic mutations have been identified in AA patients, mutations alone do not explain AA pathophysiology. SWI/SNF is an evolutionarily conserved, multi-subunit, ATP-dependent chromatin-remodeling protein complex that plays an important role in mammalian hematopoiesis. Herein, gene expression analysis identified a significant loss of the SWI/SNF core component SMARCC1, along with ARID1B, ACTL6A, and SMARCD1, in human AA BM CD34+ HSCs and hematopoietic stem and progenitor cells (HSPCs) compared with normal HSPCs. However, expression of SMARCA4, SMARCB1, SMARCD3, and DPF2 remained intact in our AA cohort. PBRM1, BRD7, and SMARCA2 expression were significantly upregulated in both untreated and follow-up AA patients. Clonal hematopoiesis in AA is associated with evolution to late clonal disorders, including myelodysplastic syndromes (MDS). Apart from SMARCD1 loss, we did not observe significant alteration of SWI/SNF expression in MDS HSPCs, indicating SWI/SNF differential expression in AA and MDS. In addition, except for ACTL6A, SWI/SNF expression was unaltered in aged HSPCs. Importantly, our results provide evidence for loss of SWI/SNF in AA, and may implicate AA HSPC-autonomous defective SWI/SNF regulation as an integral component of BM failure, in addition to autoimmune destruction of AA HSCs. These findings illustrate for the first time SWI/SNF subunit expression heterogeneity in human AA HSPCs and require prognostic validation in a larger cohort.