BRCA1-associated Protein 1 Expression Research Articles

P3.03-012 Tumor-Infiltrating Lymphocytes, PDL-1, BAP-1, VEGFR-2 and IGF-1R Expression in Malignant Pleural Mesothelioma: Topic: Mesothelioma Transitional

to investigate whether there was any relationship between survival and the expression of tumor infiltrating lymphocytes (TILs), programmed cell-death-ligand-1 (PDL-1), BAP-1 (BRCA1-Associated Protein 1), VEGFR-2 (vascular endothelial growth factor receptor 2) and IGF-1R (Insulin-Like Growth Factor 1 Receptor) in malignant pleural mesothelioma (MPM). 63 cases of MPM were identified. All tissues were obtained at the time of diagnosis. There were 40 males; mean age was 70.4 years. 34 patients were smokers and 40 had a certain history of asbestos exposure. All histological slides were revised; there were 30 epithelioid subtypes, 20 biphasics and 13 sarcomatoids. The presence of TILs was scored as absent, weak, moderate and strong according to a quantitative assessment on hematoxylin and eosin slides. The expression of BAP-1, VEGFR-2, PDL-1 and IGF-1R was analyzed by immunohistochemistry. The impact of asbestos exposure, tobacco consumption and histological subtypes on survival were also assessed. The survival analysis was analyzed by Kaplan Meier curve. TILs were present in 89% of cases and were found to be a favorable prognostic factor (p=0.009) although related with histological subtypes (p=0.008). The absence of TILs was higher in biphasic and sarcomatoid subtypes (90.9%, 30/33) compared to epithelioid MPM (53.3%, 16/30 p<0.001). Median survival in TILs and non-TILs patients was 28 months and 11 months, respectively. The expression of PDL-1 in tumor cells (cut-off: 10%, p=0.028) and VEGFR-2 in TILs (p=0.003) were related with survival, but they were differently expressed in histological subtypes. Using a logistic regression model, TILs, PDL-1 and VEGFR-2 in TILs correctly classified 21/30 epithelioid subtypes (70%) and 29/33 biphasic and sarcomatoid subtypes (87.9%). IGF-1R was overexpressed in 82% of the tumors (21 epitheliods and 31 sarcomatoids) and in 25% of TILs (7 epitheliods and 3 sarcomatoids) and was a favorable prognostic factor (p=0.023) independently of the histological subtype. Median survival was 4 and 13 months in patients not overexpressing and overexpressing IGF-1R, respectively. In a Cox regression model including both IGF-1R and histological subtype, IGF-1R remained significant [p=0.006, HR=0.41 (0.20-0.84)]. Tobacco, asbestos exposure, age and BAP-1 expression were not significantly related with survival. the histological subtype is an important prognostic factor in MPM and it’s related to different histological markers: the presence of TILs, PDL-1 and VEGFR-2 in TILs. Moreover, the overexpression of IGF-1R is an independent favorable prognostic factor. Therefore, histological markers may improve the prognostic assessment of MPM and provide mechanistic clues for new therapeutic strategies.

P3.03-023 High Incidence of Somatic BAP1 Alterations in Sporadic Malignant Mesothelioma from Turkey: Topic: Mesothelioma Transitional

BRCA1-associated protein 1 (BAP1) gene is located at chromosome region 3p21.1, a genomic region that is deleted in several human malignancies, including approximately 30-60% of mesotheliomas(1). In this study, we retrospectively investigated BAP1 status in 41 unrelated patients with mesothelioma who had a history of environmental fibrous mineral exposure (erionite or asbestos). We have also reviewed histological type and clinical characteristics of the analyzed patients. A total of 41 malignant mesothelioma cases were reviewed histopathologically. Representative areas were selected and 4-mm-diameter tissue microarrays were composed from paraffin blocks. Immunohistochemistry (IHC) was performed on paraffin tissue sections prepared from microarrays with a monoclonal antibody against BAP1. Cases with loss of nuclear staining were considered as loss of BAP1 expression. Satisfactory results were obtained in 37 patients (25 females, 12 males; mean age 56 yrs). Thirty-one cases were pleural, 5 cases were peritoneal and 1 case was paratesticular mesotheliomas. Histologically, 31 cases were epithelioid, and 6 cases were biphasic type. Overall all loss of BAP1 expression was 31/37 [83, 8% (87, 1% pleural, 80% peritoneal, 0 paratesticular)]. Histologically, all biphasic types and 25/31 (80, 6%) epithelioid types showed BAP1 expression loss. Loss of BAP1 expression seems to be a frequent event in Turkish malignant mesotheliomas. However, in our small cohort, no significant correlation was found between tumor type and localization. We need to demonstrate both somatic and germ-like mutations in familial cases especially from erionite villages.

Loss of BRCA1-associated protein 1 (BAP1) expression is rare in non–small cell lung cancer

BRCA1-associated protein 1 (BAP1) is a tumor suppressor gene involved in regulation of the cell cycle, cellular differentiation, repair of DNA damage, and apoptosis. In the distinction of malignant mesothelioma from benign mesothelial proliferations, immunohistochemical loss of BAP1, the protein expressed by the BAP1 gene, has proven highly specific for malignant mesothelioma. However, few studies have investigated the rate of BAP1 loss in tumors that commonly metastasize to the pleura. Our objective is to determine the rate of BAP1 loss in non-small cell lung cancer (NSCLC). Immunohistochemistry for BAP1 was performed using tissue microarrays containing 133 confirmed cases of NSCLC (80 of lung adenocarcinoma and 53 of squamous cell carcinoma). Cases were interpreted as showing BAP1 loss if nuclear staining was completely absent in all tumor cells and present in stromal and inflammatory cells that served as internal controls. Cases showing no BAP1 staining in the internal controls were excluded. After exclusion of 32 cases for technical reasons, only 1 case of pulmonary adenocarcinoma of 101 cases of NSCLC (69 adenocarcinoma and 32 squamous cell carcinoma; 1.0% of cases) showed BAP1 loss. We conclude that loss of BAP1 expression is a rare event in NSCLC. Therefore, BAP1 is a potentially useful addition to the immunohistochemical markers used to distinguish mesothelioma from pleural metastasis of NSCLC.

BAP1 immunohistochemistry has limited prognostic utility as a complement of CDKN2A (p16) fluorescence in situ hybridization in malignant pleural mesothelioma

BRCA-associated protein 1 (BAP1) immunohistochemistry (IHC) and CDKN2A (p16) fluorescence in situ hybridization (FISH) have shown clinical utility in confirming the diagnosis of malignant pleural mesothelioma (MPM), but the role for using these 2 markers to guide clinical management is not yet clear. Although p16 loss is predictive of poor prognosis, there is controversy as to whether BAP1 loss is predictive of a more favorable prognosis; how these results interact with one another has not been explored. We performed CDKN2A FISH on a previously published tissue microarray on which we had performed BAP1 IHC, revealing combined BAP1/p16 status for 93 MPM cases. As expected, BAP1 IHC in combination with CDKN2A FISH resulted in high sensitivity (84%) and specificity (100%) for MPM, and p16 loss was an independent predictor of poor survival (hazard ratio, 2.2553; P = .0135). There was no association between BAP1 loss and p16 loss, as 26%, 28%, 30%, and 16% of overall cases demonstrated loss of BAP1 alone, loss of p16 alone, loss of both BAP1 and p16, or neither abnormality, respectively. Although multivariate analysis demonstrated that BAP1 IHC is not an independent predictor of prognosis, when viewed in combination with homozygous CDKN2A deletion, risk stratification was evident. More specifically, patients with CDKN2A disomy and loss of BAP1 expression had improved outcomes compared with those with CDKN2A disomy and retained BAP1 expression (hazard ratio, 0.2286; P = .0017), and this finding was notably evident among epithelioid cases. We conclude that BAP1 IHC provides prognostic information within the context of CDKN2A FISH that may have clinical utility beyond diagnosis.

BRCA1-associated protein 1 deficiency in lung adenocarcinoma predicts poor outcome and increased tumor invasion.

BackgroundThe major pathological type of non-small cell lung cancer is lung adenocarcinoma (LAC), which has a poor prognosis. BRCA1-associated protein-1 (BAP1) is a newly identified tumor suppressor that regulates a number of cellular functions in somatic malignancies. However, the impact of BAP1 expression in LAC has not been investigated.MethodsA total of 112 cases of LAC and 101 cases of non-neoplastic lung diseases were included in this study. The study focused on BAP1 expression in lung tissues and its relationship to patients’ clinical and pathological features. BAP1 expression was detected by immunohistochemistry. A human LAC cell line NCI-H1299 was transfected with lipofectamine p3xFLAG-BAP1. BAP1 gene expression was silenced in another LAC cell line NCI-H1650, in order to test the inhibitory effect of BAP1 on cell migration and invasion, as well as cell cycle regulation.ResultsBAP1 expression showed a negative correlation with tumorigenesis of LAC (p <0.001) and lymph node metastasis (p = 0.010). High expression of BAP1 predicted longer disease free survival (p = 0.040) and overall survival (p = 0.021) of LAC patients. In functional assays, BAP1 was found to inhibit the migration and invasion of LAC cells, and promoted their apoptosis and necrosis.ConclusionsWe identify BAP1 as a LAC precursor as well as a robust prognostic indicator in LAC patients. This study provides in vitro rationale for the further investigation of BAP1 in preclinical studies.

Positive nuclear BAP1 immunostaining helps differentiate non-small cell lung carcinomas from malignant mesothelioma.

The differential diagnosis between pleural malignant mesothelioma (MM) and lung cancer is often challenging. Immunohistochemical (IHC) stains used to distinguish these malignancies include markers that are most often positive in MM and less frequently positive in carcinomas, and vice versa. However, in about 10–20% of the cases, the IHC results can be confusing and inconclusive, and novel markers are sought to increase the diagnostic accuracy.We stained 45 non-small cell lung cancer samples (32 adenocarcinomas and 13 squamous cell carcinomas) with a monoclonal antibody for BRCA1-associated protein 1 (BAP1) and also with an IHC panel we routinely use to help differentiate MM from carcinomas, which include, calretinin, Wilms Tumor 1, cytokeratin 5, podoplanin D2-40, pankeratin CAM5.2, thyroid transcription factor 1, Napsin-A, and p63. Nuclear BAP1 expression was also analyzed in 35 MM biopsies. All 45 non-small cell lung cancer biopsies stained positive for nuclear BAP1, whereas 22/35 (63%) MM biopsies lacked nuclear BAP1 staining, consistent with previous data. Lack of BAP1 nuclear staining was associated with MM (two-tailed Fisher's Exact Test, P = 5.4 × 10−11). Focal BAP1 staining was observed in a subset of samples, suggesting polyclonality. Diagnostic accuracy of other classical IHC markers was in agreement with previous studies. Our study indicated that absence of nuclear BAP1 stain helps differentiate MM from lung carcinomas. We suggest that BAP1 staining should be added to the IHC panel that is currently used to distinguish these malignancies.

BRCA1-Associated Protein 1 (BAP1) Immunohistochemical Expression as a Diagnostic Tool in Malignant Pleural Mesothelioma Classification: A Large Retrospective Study

IntroductionMalignant pleural mesothelioma (MPM) is a highly aggressive disease with limited therapeutic options. Histological subtype remains among the most reliable prognostic factors, because the epithelioid subtype associated with the best prognosis and the sarcomatoid subtype with the worst. The biphasic subtype has an intermediate prognosis, but its definitive histological diagnosis may be challenging owing to the difficulty of assessing the neoplastic nature of the stromal component. Recent data identified BRCA1-associated protein 1 gene (BAP1) as one of the most frequently mutated genes in MPM. Immunohistochemical testing for BRCA1-associated protein 1 (BAP1) has been proposed to be predictive for the detection of BAP1 mutation in neoplastic cells. The aim of the present study was to define the diagnostic usefulness of immunohistochemical determination of BAP1 in MPM, with clinicopathological correlation. MethodsA series of 143 MPMs were investigated for BAP1 protein expression in correlation with clinical and pathological data, including with a newly proposed nuclear grade. A pilot series of 20 selected cases were also investigated for BAP1 mutational status. ResultsNegative nuclear staining for BAP1 occurred in 62% of MPMs (including 27% with a cytoplasmic pattern) and was significantly associated with the presence of BAP1 mutation, epithelioid subtype, and a better prognosis. In a subgroup of cases, the pattern of expression of BAP1 in stromal cells supported their distinction as reactive versus neoplastic, thus helping achieve the correct classification of biphasic histological subtype. ConclusionsWe showed that BAP1 protein determination is a diagnostic tool to correctly distinguish biphasic MPM from epithelial subtypes with an atypical/activated reactive stroma and an independent prognostic parameter in MPM.

Apport de l’expression protéique de BRCA1 associated protein 1 (BAP1) dans le diagnostic des mésothéliomes malins diffus pleuraux : une analyse cytologique et histologique comparative sur une série de 50 patients

Diffuse malignant mesothelioma (MMD) is a rare disease. The diagnosis is difficult and needs an antibody panel. The tumor suppressor gene BRCA1 associated protein 1 (BAP1) is involved in several cancers, including MMD. Loss of BAP1 expression is correlated with BAP1 somatic or constitutional genetic defects. Our work assesses the value of integrating BAP1 in the panel of antibodies used for the diagnosis of MMD. Immunohistochemical techniques were performed on cytological and histological specimens of MMD and adenocarcinoma pleural metastasis. Of the 26 patients with MMD and the 24 patients with adenocarcinoma pleural metastasis, loss of BAP1 expression was observed in 11 (48%) and one adenocarcinoma (6%) on cytological specimens and in 12 MMD (48%) and in one adenocarcinoma (5%) on biopsy specimens. The concordance between immunocytochemistry and immunohistochemistry was 100%. The specificity of BAP1 was 100% on cytological and biopsy specimen for the diagnosis of malignancy in case of mesothelial proliferation. Loss of BAP1 expression is an indicator of MMD in a context of mesothelial proliferation. This immunohistochemistry could be integrated in the panel of immunostaining used for MMD diagnosis, either on histological or cytological samples. Furthermore, loss of BAP1 expression guides the patient to an oncology genetic counseling in order to eliminate a MMD developed as part of a constitutional genetic defect.

Loss of BAP1 Expression Is Very Rare in Pancreatic Ductal Adenocarcinoma.

BackgroundPancreatic cancer is both common and highly lethal and therefore new biomarkers or potential targets for treatment are needed. Loss of BRCA associated protein-1 (BAP1) expression has been found in up to a quarter of intrahepatic cholangiocarcinomas. Given the close anatomical relationship between intrahepatic cholangiocarcinoma and pancreatic ductal adenocarcinoma, we therefore sought to investigate the frequency of loss of BAP1 expression in pancreatic ductal adenocarcinoma.MethodsThe records of the department of Anatomical Pathology Royal North Shore Hospital, Sydney, Australia, were searched for cases of pancreatic ductal adenocarcinoma diagnosed between 1992 and 2014 with material available in archived formalin fixed paraffin embedded tissue blocks. Immunohistochemistry for BAP1 was performed on tissue microarray sections and if staining was equivocal or negative it was confirmed on whole sections. Negative staining for BAP1 was defined as loss of expression in all neoplastic nuclei, with preserved expression in non-neoplastic cells which acted as an internal positive control.ResultsLoss of BAP1 expression was found in only 1 of 306 (0.33%) pancreatic ductal adenocarcinomas. This case was confirmed to demonstrate diffuse loss of expression throughout all neoplastic cells in multiple blocks, consistent with BAP1 loss being an early clonal event. All other cases demonstrated positive expression of BAP1.ConclusionWe conclude that, in contrast to intrahepatic cholangiocarcinoma, loss of expression of BAP1 occurs very rarely in pancreatic ductal adenocarcinoma. Therefore BAP1 inactivation is unlikely to be a frequent driver abnormality in pancreatic adenocarcinoma.

The impact of PBRM1 and BAP1 expression on outcomes of patients with metastatic renal cell carcinoma (mRCC) treated with VEGF-targeted therapy (TT).

616 Background: Polybromo-1 (PBRM1) and BRCA1 associated protein-1 (BAP1) are genes commonly mutated in clear cell RCC (ccRCC) and have been associated with clinical outcome. This work aims to evaluate the impact of PBRM1 and BAP1 expression by IHC in mRCC patients treated with VEGF-TT. Methods: PBRM1 and BAP1 expression was evaluated by IHC in a TMA including 146 mRCC patients. PBRM1 and BAP1 IHC scores were dichotomized as a binary variable: negative (-) or positive (+) (weak positivity was excluded). The associations of PBRM1 or BAP1 expression with baseline clinico-pathological characteristics were evaluated, as well as with overall survival (OS) and time to treatment failure (TTF) usingCox proportional hazards models. Results: Out of 146 patients, 116 and 109 samples had available results for PBRM1 and BAP1 staining, respectively. Overall, 90% (n = 131) patients had ccRCC. 70/116 samples (60%) were PBRM1- and 26/109 patients (24%) were BAP1-. Only 12% (n = 13) of patients had simultaneous negative PBRM1 and BAP1. While there was no association between PBRM1 expression status and clinical factors, BAP1- samples were associated with poor IMDC prognostic risk score (p = 0.004) and higher Fuhrman grade (p = 0.012). PBRM1+ patients showed a trend towards an increased risk of death and a shorter TTF compared to PBRM1- patients (OS: HR = 1.38, 95%CI: 0.92-2.07, p = 0.1; TTF: HR = 1.39, 95%CI: 0.94-2.06, p = 0.1). BAP1 expression was not independently associated with OS or TTF. Conclusions: Loss of PBRM1, but not BAP1 expression showed a trend towards longer TTF and OS in mRCC patients treated with VEGF-TT.

Loss of BAP1 Expression Occurs Frequently in Intrahepatic Cholangiocarcinoma.

BRCA1-associated protein 1 (BAP1) is a deubiquitinating enzyme that functions as a tumor suppressor gene. Double hit BAP1 inactivation has been reported in a range of tumor types, including intrahepatic cholangiocarcinoma (ICC), sometimes in association with germline mutation.We performed immunohistochemistry for BAP1 on a well-characterized cohort of 211 ICC patients undergoing surgical resection with curative intent at 3 institutions based in 3 different countries. The median age at diagnosis was 65 years (range, 36.5–86) and 108 (51%) were men. Negative staining for BAP1 (defined as completely absent nuclear staining in the presence of positive internal controls in nonneoplastic cells) occurred in 55 ICCs (26%). BAP1 loss predicted a strong trend toward improved median survival of 40.80 months (95% CI, 28.14–53.46) versus 24.87 months (95% CI, 18.73–31.01), P = 0.059). In a multivariate model including age, sex, BAP1 status, tumor stage, tumor grade, lymphovascular invasion, and tumor size, female sex was associated with improved survival (hazard ratio [HR] 0.54; 95% CI, 0.34–0.85), while advanced tumor stage and lymphovascular invasion (HR 1.89; 95% CI, 1.09–3.28) correlated with decreased survival. In a multivariate analysis, high grade tumors were associated with BAP1 loss (odds ratio [OR] 3.32; 95% CI, 1.29–8.55), while lymphatic invasion was inversely associated with BAP1 loss (OR 0.36; 95% CI, 0.13–0.99).In conclusion, we observed a trend toward improved prognosis in ICC associated with absent expression of BAP1 and an association of BAP1 loss with higher histological grade and absent lymphatic invasion. Female sex was associated with improved survival while advanced tumor stage and lymphatic invasion were associated with decreased survival.

BAP1 Loss Results in EZH2-Dependent Transformation in Myelodysplastic Syndromes

Recurrent somatic loss-of-function mutations in ASXL1 (Addition of sex combs-like 1) are common genetic events in a spectrum of myeloid malignancies and these alterations demarcate patients with poor outcome. ASXL1 forms a chromatin regulatory complex with the ubiquitin hydrolase BAP1 (BRCA1 associated protein-1), a protein that has been found to be transcriptionally repressed in MDS patients. These data are consistent with BAP1 having tumor suppressive activity in MDS; however, the mechanism by which disruption of the ASXL1-BAP1 axis leads to transformation is not well understood. We conditionally deleted Bap1 in the murine hematopoietic system utilizing Mx1-Cre (hereafter referred to as Bap1 KO). One hundred percent of mice with confirmed Bap1 deletion developed a fully penetrant MDS-like disease characterized by leukocytosis, anemia, and splenomegaly. Bap1 KO mice have an expansion of the granulocyte macrophage progenitor compartment (GMP; Lin- c-Kit+ Sca1- CD34+ Fcϒ+). Given the role of BAP1 in epigenetic regulation, we investigated the effect of Bap1 loss on chromatin state and transcriptional output.We first assessed epigenetic changes in Bap1 KO mice by performing histone mass spectometry in control and Bap1 KO hematopoietic stem and progenitor cells (HSPCs, c-Kit+ enriched). Bap1 loss increased H3K27me2/3 at the expense of H3K27me0/1. We confirmed that H3K27me3 was increased in Bap1 KO bone marrow cells by completing H3K27me3 ChIP-Sequencing in HSPCs. Enumeration of H3K27me3 peaks in Bap1 KO versus control cells indicated an increase in H3K27me3 domains (Figure A). We next overlaid RNA-Sequencing from GMP sorted Bap1 KO bone marrow cells with genes marked by H3K27me3, as indicated by ChIP-Sequencing. We found that Bap1 loss resulted in a global decrease in gene expression (68% downregulated, 657/968 genes, p-adj <0.01) and that increased H3K27me3 identified genes with reduced expression after Bap1 loss (NES=-1.39, FDR<0.001) (Figure A). Gene set enrichment analyses (GSEA) revealed that genes that were altered following depletion of Bap1 corresponded to differentiation, hematopoietic lineage specification, and proliferation pathways. Combined, these data suggest that Bap1 depletion results in increased H3K27me3 and represses gene targets implicated in normal and malignant hematopoiesis.Given the alterations in H3K27me3 in Bap1 KO mice, we investigated whether Bap1- deficient transformation could be rescued by abrogation of PRC2-mediated gene repression. We developed a genetic model with compound deletion of Bap1 and Ezh2, the catalytic component of the PRC2 complex. Co-deletion of Bap1 and Ezh2 resulted in a phenotypic rescue of Bap1 KO associated splenomegaly (spleen weights, Bap1 KO avg. 541.6 mg, Bap1/Ezh2 KO avg. 157.0 mg, p<0.005) (Figure B), leukocytosis (white blood cells counts, Bap1 KO avg. 51 K/uL, Bap1/Ezh2 KO avg. 8 K/uL, p <0.005), anemia (hematocrit, Bap1 KO avg. 28.2%, Bap1/Ezh2 KO avg. 46.0%, p<0.005). Importantly, the increased H3K27me3 levels in Bap1 KO mice were reduced in Bap1/Ezh2 KO mice (Figure B), suggesting that loss of Bap1 leads to Ezh2-dependent malignant transformation.EZH2 small molecule inhibitors have proven effective in EZH2-dependent models of B cell lymphoma. To determine if Ezh2 inhibition was efficacious in the setting of Bap1 loss, we treated a cohort of Bap1 KO mice with either vehicle (NaCMC) or 500 mg/kg EPZ011989, an EZH2 inhibitor with in vivo activity. Treatment of Bap1 KO mice for 16 days resulted in significant reduction of splenomegaly (spleen weights, vehicle avg. 522.0, EPZ011989 treated avg. 216.2, p<0.005) (Figure C) and anemia (white blood cell counts, vehicle avg. 61.7 K/uL, EPZ011989 treated avg. 14.5 K/uL, p <0.005), consistent with the phenotype of our genetic Bap1/Ezh2 compound deletion model. These data suggest that decreased BAP1 expression could serve as a biomarker for sensitivity to EZH2 inhibition. [Display omitted] DisclosuresKnutson:Epizyme, Inc: Employment. Campbell:Epizyme, Inc: Employment. Keilhack:Epizyme: Employment, Equity Ownership. Melnick:Janssen: Other: Research; ROCHE: Other: Research; Genentech: Speakers Bureau; Celgene: Consultancy; Eli Lilly: Consultancy; Epizyme: Consultancy. Armstrong:Epizyme, Inc: Consultancy. Levine:Foundation Medicine: Consultancy; CTI BioPharma: Membership on an entity's Board of Directors or advisory committees; Loxo Oncology: Membership on an entity's Board of Directors or advisory committees.

Decreased expression of BRCA1-associated protein 1 predicts unfavorable survival in gastric adenocarcinoma.

BRCA1-associated protein 1 (BAP1) has been reported as a novel tumor suppressor, while in gastric adenocarcinoma, the function of this protein was still await to be uncovered. Based on a large group of patients with gastric adenocarcinoma, our study aimed to have a further understanding about the correlation of BAP1 expression and patients' clinical outcomes. We performed quantitative PCR and Western blot to examine BAP1 expression in 38 cases of gastric adenocarcinoma samples and adjacent non-cancerous tissues. Immunochemistry was used to evaluate BAP1 expression in a large cohort of 474 paraffin-embedded specimens. The clinical and prognostic significance of BAP1 expression was statistically analyzed. Postoperative survival between groups was using Kaplan-Meier analysis. BAP1 was overexpressed in paracancerous normal mucosa compared with gastric cancer. Decreased BAP1 expression was associated with higher histologic grade (P = 0.044), tumor infiltration (P < 0.001), metastasis status (P = 0.023), and TNM stage (P < 0.001). Patients with low expression of BAP1 had shorter overall survival compared with those with high expression (P < 0.001). Patients' survival in stage N0 could be stratified by the expression of BAP1. Multivariate analysis showed that in gastric adenocarcinoma, BAP1 expressing level was an independent prognostic factor (RR = 0.575, P < 0.001). Decreased expression of BAP1 suggests pessimistic prognosis for gastric adenocarcinoma patients. Further studies are warranted.

Stabilization of MCRS1 by BAP1 prevents chromosome instability in renal cell carcinoma

Characterization of the exome and genome of carcinoma (ccRCC) by next-generation sequencing identified numerous genetic alternations. BRCA1-associated protein-1 (BAP1) was identified as one of the most frequently mutated genes in ccRCC, suggesting that BAP1 is a potential key driver for ccRCC cancer initiation and progression. However, how BAP1 mutations contribute to ccRCC remains to be elucidated. BAP1 is a nuclear de-ubiquitinating enzyme and cleaves the ubiquitin chain from the substrates. Here, we identified MCRS1 as a bona fide substrate for BAP1. MCRS1 is a component of the centrosome proteins, and plays an essential role in spindle assembly. BAP1 binds to MCRS1 and stabilizes MCRS1 by de-ubiquitination. BAP1 contributes to chromosome stability partially via MCRS1. A positive correlation was identified between BAP1 and MCRS1 expression in ccRCC tissues. Both BAP1 loss and MCRS1 down-regulation in ccRCC were associated with adverse clinicopathological features. This study revealed a novel mechanism for BAP1 involved in MCRS1 stability regulation, and provided insight in understanding the relationship between BAP1 mutations and chromosome instability in ccRCC.

Somatic alteration and depleted nuclear expression of BAP1 in human esophageal squamous cell carcinoma.

BRCA1-associated protein 1 (BAP1) is a deubiquitinating enzyme that is involved in the regulation of cell growth. Recently, many somatic and germline mutations of BAP1 have been reported in a broad spectrum of tumors. In this study, we identified a novel somatic non-synonymous BAP1 mutation, a phenylalanine-to-isoleucine substitution at codon 170 (F170I), in 1 of 49 patients with esophageal squamous cell carcinoma (ESCC). Multiplex ligation-dependent probe amplification (MLPA) of BAP1 gene in this ESCC tumor disclosed monoallelic deletion (LOH), suggesting BAP1 alterations on both alleles in this tumor. The deubiquitinase activity and the auto-deubiquitinase activity of F170I-mutant BAP1 were markedly suppressed compared with wild-type BAP1. In addition, wild-type BAP1 mostly localizes to the nucleus, whereas the F170I mutant preferentially localized in the cytoplasm. Microarray analysis revealed that expression of the F170I mutant drastically altered gene expression profiles compared with expressed wild-type BAP1. Gene-ontology analyses indicated that the F170I mutation altered the expression of genes involved in oncogenic pathways. We found that one candidate, TCEAL7, previously reported as a putative tumor suppressor gene, was significantly induced by wild-type BAP1 as compared to F170I mutant BAP1. Furthermore, we found that the level of BAP1 expression in the nucleus was reduced in 44% of ESCC examined by immunohistochemistry (IHC). Because the nuclear localization of BAP1 is important for its tumor suppressor function, BAP1 may be functionally inactivated in a substantial portion of ESCC. Taken together, BAP1 is likely to function as a tumor suppressor in at least a part of ESCC.

BAP1 (BRCA1-associated protein 1) is a highly specific marker for differentiating mesothelioma from reactive mesothelial proliferations

The distinction between malignant mesothelioma and reactive mesothelial proliferation can be challenging both on histology and cytology. Recently, variants of the BRCA1-associated protein 1 (BAP1) gene resulting in nuclear protein loss were reported in hereditary and sporadic mesothelioma. Using immunohistochemistry, we evaluated the utility of BAP1 expression in the differential diagnosis between mesothelioma and other mesothelial proliferations on a large series of biopsies that included 212 mesotheliomas, 12 benign mesothelial tumors, and 42 reactive mesothelial proliferations. BAP1 stain was also performed in 70 cytological samples (45 mesotheliomas and 25 reactive mesothelial proliferations). BAP1 was expressed in all benign mesothelial tumors, whereas 139/212 (66%) mesotheliomas were BAP1 negative, especially in epithelioid/biphasic compared with sarcomatoid/desmoplastic subtypes (69% vs 15%). BAP1 loss was homogeneous in neoplastic cells except for two epithelioid mesotheliomas showing tumor heterogeneity. By fluorescence in situ hybridization, BAP1 protein loss was paralleled by homozygous deletion of the BAP1 locus in the vast majority of BAP1-negative tumors (31/41, 76%), whereas 9/10 BAP1-positive mesotheliomas were normal. In biopsies interpreted as reactive mesothelial proliferation BAP1 loss was 100% predictive of malignancy, as all 6 cases subsequently developed BAP1-negative mesothelioma, whereas only 3/36 (8%) BAP1-positive cases progressed to mesothelioma. On cytology/cell blocks, benign mesothelial cells were invariably positive for BAP1, whereas 64% of mesotheliomas showed loss of protein; all 6 cases showing BAP1 negativity were associated with histological diagnosis of BAP1-negative mesothelioma. BAP1 stain also showed utility in the differential of mesothelioma from most common pleural and peritoneal mimickers, such as lung and ovary carcinomas, with specificity and sensitivity of 99/70% and 100/70%, respectively. Our results show that BAP1 protein is frequently lost in mesothelioma, especially of epithelioid/biphasic subtype and is commonly associated with homozygous BAP1 deletion. BAP1 immunostain represents an excellent biomarker with an unprecedented specificity (100%) in the distinction between benign and malignant mesothelial proliferations. Finding BAP1 loss in mesothelial cells should prompt to immediately reevaluate the patient; moreover, it might be useful in mapping tumor extent and planning surgical resection.

BAP1 facilitates diagnostic objectivity, classification, and prognostication in malignant pleural mesothelioma

BRCA-associated protein 1 (BAP1) has emerged as a promising biomarker for malignant pleural mesothelioma (MPM). Loss of BAP1 expression can occur by a variety of mechanisms, but reports on incidence are variable and the clinical significance is unclear. In order to investigate the diagnostic and prognostic significance of BAP1, we constructed a tissue microarray consisting of 111 MPM cases and performed BAP1 immunohistochemistry. BAP1 was lost in 77% of epithelioid cases (n=58) but was retained in all sarcomatoid cases (n=10); 49% of biphasic cases showed loss (n=43), and BAP1-negative cases demonstrated loss of staining in both the epithelioid and sarcomatoid components. All non-neoplastic mesothelial tissues (n=20) retained BAP1, resulting in a sensitivity, specificity, positive predictive value, and negative predictive value of 61%, 100%, 100%, and 32%, respectively. Moreover, BAP1 expression in spindled mesothelium enabled discrimination of reactive and malignant cells, thus providing a more objective means of distinguishing epithelioid from biphasic morphology compared to histology alone. Nonetheless, BAP1 staining was patchy in some benign mesothelial neoplasms, which raises concern for using BAP1 in small biopsies. Kaplan-Meier analysis demonstrated a significant improvement in overall survival with BAP1 loss, but this did not reach significance in multivariate analysis accounting for histologic subtype. When only epithelioid cases were analyzed there was a trend toward increased survival, but it did not reach significance. We conclude that BAP1 loss is frequent in epithelioid MPM, which is in turn associated with improved survival, and that it can have additional clinical significance by facilitating histologic classification.

Loss of BAP1 Expression in Basal Cell Carcinomas in Patients With Germline BAP1 Mutations.

Patients with heterozygous germline mutations in BRCA1-associated protein 1 (BAP1), a tumor suppressor gene, develop a tumor predisposition syndrome (OMIM 614327) with increased risk of uveal and cutaneous melanomas, cutaneous atypical and epithelioid melanocytic lesions, lung adenocarcinoma, clear cell renal cell carcinoma, and other tumors. Early recognition of this syndrome is of clinical importance. In addition, screening for BAP1 mutation, loss, and inactivation by performing BAP1 immunohistochemistry on cutaneous lesions would be a simple method for screening patients suspected of having germline BAP1 mutations. We investigated BAP1 expression in seven basal cell carcinomas (BCCs) in two patients with germline BAP1 mutation and a family history of uveal melanoma. Six lesions were from the head and neck region and one from the shoulder. Thirty-one sporadic BCCs were included as controls. All seven BCCs in the patients with germline BAP1 mutations exhibited loss of BAP1 nuclear staining, while 30 (97%) of 31 sporadic BCCs exhibited positive BAP1 nuclear staining. Loss of BAP1 expression could be associated with the development of BCC in patients with germline BAP1 mutations. These results suggest that BCC may be a component of the expanding category of tumors associated with this syndrome.

Loss of expression of BAP1 predicts longer survival in mesothelioma

BRCA1-associated protein 1 (BAP1) is a tumour suppressor gene frequently inactivated in mesothelioma, rarely also in association with germline mutation. BAP1 mutations have been associated with improved prognosis and distinct clinicopathological features. We sought to determine the clinicopathological significance of BAP1 immunohistochemistry (IHC) in mesothelioma.We performed IHC on a tissue microarray (TMA) cohort comprising all available thoracic mesotheliomas encountered during the period 1991-2014 at our institution (n = 229). All cases were independently reviewed to confirm the diagnosis and subclassify as epithelioid, sarcomatoid or biphasic. The median age at diagnosis was 72 years; 188 (82.1%) were male; 120 (52.4%) were epithelioid (median survival 13.0 months), 67 (29.3%) sarcomatoid (median survival 5.6 months) and 42 (18.3%) biphasic (median survival 10.6 months). Negative staining for BAP1 (defined as completely absent nuclear staining in the presence of positive internal controls in non-neoplastic cells) occurred in 106 (46.3%) mesotheliomas. There was complete interobserver concordance for BAP1 IHC status. BAP1 loss was strongly associated with younger age at onset (p < 0.01) and epithelioid differentiation (p < 0.01). BAP1 loss predicted an improved median survival of 16.11 months (95% CI 12.16-20.06) versus 6.34 months (95% CI 5.34-7.34), p < 0.01. In a multivariate model including age, gender and histological type, BAP1 loss, younger age and epithelioid differentiation remained protective (all p < 0.01).If our results are confirmed by others, BAP1 IHC may have a role to predict prolonged survival or triage formal genetic testing for germline BAP1 mutation in patients presenting with mesothelioma.

Loss of the deubiquitylase BAP1 alters class I histone deacetylase expression and sensitivity of mesothelioma cells to HDAC inhibitors.

Histone deacetylases are important targets for cancer therapeutics, but their regulation is poorly understood. Our data show coordinated transcription of HDAC1 and HDAC2 in lung cancer cell lines, but suggest HDAC2 protein expression is cell-context specific. Through an unbiased siRNA screen we found that BRCA1-associated protein 1 (BAP1) regulates their expression, with HDAC2 reduced and HDAC1 increased in BAP1 depleted cells. BAP1 loss-of-function is increasingly reported in cancers including thoracic malignancies, with frequent mutation in malignant pleural mesothelioma. Endogenous HDAC2 directly correlates with BAP1 across a panel of lung cancer cell lines, and is downregulated in mesothelioma cell lines with genetic BAP1 inactivation. We find that BAP1 regulates HDAC2 by increasing transcript abundance, rather than opposing its ubiquitylation. Importantly, although total cellular HDAC activity is unaffected by transient depletion of HDAC2 or of BAP1 due to HDAC1 compensation, this isoenzyme imbalance sensitizes MSTO-211H cells to HDAC inhibitors. However, other established mesothelioma cell lines with low endogenous HDAC2 have adapted to become more resistant to HDAC inhibition. Our work establishes a mechanism by which BAP1 loss alters sensitivity of cancer cells to HDAC inhibitors. Assessment of BAP1 and HDAC expression may ultimately help identify patients likely to respond to HDAC inhibitors.