Nuclear BRCA1-associated Protein-1 Research Articles

Rare germline heterozygous missense variants in BRCA1-associated protein 1, BAP1, cause a syndromic neurodevelopmental disorder

Nuclear deubiquitinase BAP1 (BRCA1-associated protein 1) is a core component of multiprotein complexes that promote transcription by reversing the ubiquitination of histone 2A (H2A). BAP1 is a tumor suppressor whose germline loss-of-function variants predispose to cancer. To our knowledge, there are very rare examples of different germline variants in the same gene causing either a neurodevelopmental disorder (NDD) or a tumor predisposition syndrome. Here, we report a series of 11 de novo germline heterozygous missense BAP1 variants associated with a rare syndromic NDD. Functional analysis showed that most of the variants cannot rescue the consequences of BAP1 inactivation, suggesting a loss-of-function mechanism. In Tcells isolated from two affected children, H2A deubiquitination was impaired. In matching peripheral blood mononuclear cells, histone H3 K27 acetylation ChIP-seq indicated that these BAP1 variants induced genome-wide chromatin state alterations, with enrichment for regulatory regions surrounding genes of the ubiquitin-proteasome system (UPS). Altogether, these results define a clinical syndrome caused by rare germline missense BAP1 variants that alter chromatin remodeling through abnormal histone ubiquitination and lead to transcriptional dysregulation of developmental genes.

Prognostic Factor Utility of BAP1 Immunohistochemistry in Uveal Melanoma: A Single Center Study in Spain.

Simple SummaryAs uveal melanoma metastasis rates are still very high, the mechanisms by which it spreads need to be evaluated. Our research sought to determine which pathological and clinical features were correlated with the prognosis of uveal melanoma in a Spanish community. BAP1 (BRCA1-Associated Protein 1) gene mutation is one of the strongest predictors for metastasis in uveal melanoma. The BAP1 protein has a tumor suppressor function and the presence of the BAP1 protein can be shown using immunohistochemical staining. Our study showed that nuclear BAP1 immunostaining had a significant correlation with survival rate in our sample, and patients with a lack of nuclear BAP1 immunostaining should be considered high-risk and receive a close follow-up. This stain can be used as routine technique in the pathological examination of uveal melanoma.Even today, the mortality rate for uveal melanoma (UM) remains very high. In our research, we sought to determine which pathological and clinical features were correlated with the prognosis of UM. BAP1 (BRCA1-Associated Protein 1) gene mutation has been analyzed as one of the strongest predictors for metastasis in UM. The BAP1 gene codifies the BAP1 protein which has a tumor suppressor function. The presence of this protein can be determined by BAP1 immunohistochemical staining. Eighty-four uveal melanoma patients and forty enucleated eyeballs were examined. Metastasis was present in 24 patients. Nuclear BAP1 staining was low in 23 patients. The presence of a higher large basal diameter tumor (p < 0.001), tumor infiltrating lymphocytes (p = 0.020), and a lack of nuclear BAP1 immunostaining (p = 0.001) ocurred significantly more often in the metastatic group. Metastasis-free survival was lower in patients with low nuclear BAP1 staining (p = 0.003). In conclusion, to the best of our knowledge, this is the first time that BAP1 staining has been studied in uveal melanoma in a Spanish community. We believe that this technique should become routine in the pathological examination of uveal melanoma in order to allow adequate classification of patients and to establish an individual follow-up plan.

Large-scale analysis of BAP1 expression reveals novel associations with clinical and molecular features of malignant pleural mesothelioma.

BRCA1‐associated protein‐1 (BAP1) expression is commonly lost in several tumors including malignant pleural mesothelioma (MPM). Presence or absence of immunohistochemical BAP1 nuclear staining in tumor cells is currently used for differential diagnosis of MPM. In this study, a large cohort of 596 MPM tumors with available clinical data was analyzed to examine associations of BAP1 staining pattern with clinical and molecular features that may reflect the impact of BAP1 mutation on MPM biology. Cases were classified according to the BAP1 staining pattern of tumor cells. Exome and RNA‐sequencing data were available for subsets of cases. Levels of mRNA encoding claudin 15 (CLDN15) and vimentin (VIM) were determined using RT‐qPCR on 483 cases to estimate the relative proportions of epithelial‐like and mesenchymal‐like components in each tumor. Four BAP1 staining patterns were observed: single‐pattern nuclear staining (36%), single‐pattern cytoplasmic staining (25%), single‐pattern absent staining (12%), and combinations of these staining patterns (27%). This study confirmed prior reports that nuclear BAP1 is more frequently associated with wild‐type BAP1 and sarcomatoid histology. However, no associations between BAP1 staining pattern(s) and mutations in specific protein domains and/or mutation type were observed. BAP1 staining patterns were significantly associated (p < 0.001) with BAP1 gene expression, MPM histologic subtypes, molecular clusters, and markers of epithelial‐to‐mesenchymal transition. Frequent observation of combinations of BAP1 staining patterns in MPM tumors indicated intra‐tumoral heterogeneity of BAP1 status. Cytoplasmic BAP1 staining was identified as a putative indicator of favorable prognosis in non‐epithelioid MPM. In conclusion, novel significant associations among different BAP1 staining patterns and subgroups of MPM tumors were observed, suggesting that the role of BAP1 in tumor progression may be more complex than its presumed tumor suppressor function. Cytoplasmic staining was identified as a putative indicator of favorable prognosis in non‐epithelioid MPM, potentially addressing a critical need in clinical decision‐making in this disease. © 2020 The Authors. The Journal of Pathology published by John Wiley & Sons, Ltd. on behalf of The Pathological Society of Great Britain and Ireland.

Nuclear expression of BAP-1 in transvitreal incisional biopsies and subsequent enucleation of eyes with posterior choroidal melanoma

BackgroundAs a majority of patients with choroidal melanoma do not undergo enucleation, tumour tissue for prognostic testing has to be obtained with alternate methods. Transvitreal incisional biopsies enable histological examination...

Sensitivity of Mesothelioma Cells to PARP Inhibitors Is Not Dependent on BAP1 but Is Enhanced by Temozolomide in Cells With High-Schlafen 11 and Low-O6-methylguanine-DNA Methyltransferase Expression.

BRCA1-associated protein-1 (BAP1), a nuclear deubiquitinase thought to be involved in DNA double-strand break repair, is frequently mutated in mesothelioma. Because poly(adenosine diphosphate-ribose) polymerase inhibitors (PARPIs) induce synthetic lethality in BRCA1/2 mutant cancers, we evaluated whether BAP1 inactivating mutations confer sensitivity to PARPIs in mesothelioma and if combination therapy with temozolomide (TMZ) would be beneficial. A total of 10 patient-derived mesothelioma celllines were generated and characterized for BAP1 mutation status, protein expression, nuclear localization, and sensitivity to the PARPIs, olaparib, and talazoparib, alone orin combination with TMZ. BAP1 deubiquitinase (DUB) activity was evaluated by ubiquitin with 7-amido-4-methylcoumarin assay. BAP1 knockout mesothelioma cell lines were generated by CRISPR-Cas9. Because Schlafen 11 (SLFN11) and O6-methylguanine-DNA methyltransferase also drive response to TMZ and PARPIs, we tested their expression and relationship with drug response. BAP1 mutations or copy-number alterations, or both were present in all 10 cell lines. Nonetheless, four cell lines exhibited intact DUB activity and two had nuclear BAP1 localization. Half maximal-inhibitory concentrations of olaparib and talazoparib ranged from 4.8 μM to greater than 50 μM and 0.039 μM to greater than 5 μM, respectively, classifying them into sensitive (two) or resistant (seven) cells, independent of their BAP1 status. Cell lines with BAP1 knockout resulted in the loss of BAP1 DUB activity but did not increase sensitivity to talazoparib. Response to PARPI tended to be associated with high SLFN11 expression, and combination with temozolomide increased sensitivity of cells with low or no MGMT expression. BAP1 status does not determine sensitivity to PARPIs in patient-derived mesothelioma cell lines. Combination of PARPI with TMZ may be beneficial for patients whose tumors have high SLFN11 and low or no MGMT expression.

BAP1 Immunoreactivity Correlates with Gene Expression Class in Uveal Melanoma

Background: Uveal melanoma (UM) is the most common intraocular tumour in adults. Currently there are different tests available to determine the risk of UM for metastasis, among which include BRCA1-associated protein-1 (BAP1) immunohistochemistry (IHC) and gene expression profiling (GEP). BAP1 is a deubiquitylating enzyme (DUB) that has tumour suppressor activity, the loss of which therefore is associated with higher risk for tumour growth and metastasis. Objectives: To compare and correlate the prognostic significance of BAP1-IHC staining patterns and GEP in the prediction of UM’s risk for metastasis. Methods: This is a retrospective chart review with prospective follow-up of patients with primary UM who underwent enucleation from the year 2008 to 2018. Clinical history, histopathologic findings, GEP classification, BAP1-IHC of the formalin-fixed paraffin-embedded tissues, and follow-up data for metastasis were collected and statistically analysed. Results: A total of 30 enucleated eyes with UM were included in the study. All class 1a tumours had high nuclear BAP1 expression and all class 2 had low nuclear BAP1 expression. Fifty percent of the class 1b tumours had low nuclear BAP1 expression. Among the tumours with low nuclear BAP1 expression, 68% developed metastasis, while 9% developed metastasis among high nuclear expression. Fifty-five percent developed metastasis in tumours with high cytoplasmic expression and 42% for low cytoplasmic expression. Predictive values for metastasis (positive predictive value, negative predictive value) are as follows: (1) nuclear BAP1-IHC (68%, 91%), (2) cytoplasmic BAP1-IHC (55%, 58%), and (3) GEP (73%, 80%). Nuclear BAP1-IHC and GEP had the same accuracy rate of 77% and cytoplasmic BAP1-IHC had an accuracy of 57%. Conclusion: Low nuclear BAP1-IHC strongly correlates with GEP class 2 and was equally accurate in the prediction of metastasis.

The role of BRCA1-associated protein 1 in the diagnosis of malignant mesothelioma in effusion and fine-needle aspiration cytology.

Malignant mesothelioma (MM) is a diagnostically challenging entity in cytology specimens due to the lack of architectural context and a cytomorphologic overlap between malignant and reactive mesothelial cells (RMCs). A diagnostic marker with excellent specificity is not currently available in clinical practice. The newly appreciated BRCA1-associated protein 1 (BAP1) antibody may help distinguish MM from RMC based on its immunohistochemical (IHC) staining pattern but its role in cytopathology is controversial. Immunohistochemistry with BAP1 antibody was performed on cell blocks from 39 cytology specimens including 13 cases of RMC and 26 cases of effusion and fine-needle aspiration specimens (FNAC) with tissue-specimen-proven MM. Cases were dichotomised into positive and negative cohorts. Positivity was defined as >50% loss of nuclear BAP1 IHC staining. Of the 26 MM cases, a slight majority (14/26, 54%) showed loss of BAP1 nuclear IHC staining, while all 13 RMC controls showed strong nuclear BAP1 IHC staining. MM was more likely to show loss of BAP1 than RMC (P < .001); and peritoneal MM was more likely to demonstrate loss of BAP1 than pleural MM (P = .04). There was perfect specificity at 1.0 and positive predictive value of 1.0 for loss of nuclear BAP1 IHC staining. However, only modest sensitivity at 0.52 and negative predictive value of 0.50 was seen. These data confirm that absence of BAP1 nuclear staining identifies malignant mesothelial cells. On the other hand, positive BAP1 nuclear staining can occur in both benign and malignant pleural effusions.

Retrospective response analysis of BAP1 expression to predict the clinical activity of systemic cytotoxic chemotherapy in mesothelioma

Introduction BRCA1 associated protein-1 (BAP1) is a key tumor driver in mesothelioma and a potential biomarker predicting response to several targeted therapies in clinical testing. Whether it also modulates response to cytotoxic chemotherapy is undetermined. This study used retrospective response analysis of BAP1 expression in archival tumor biopsies taken from patients in the MS01 trial (NCT00075699). We aimed to determine if BAP1 expression correlated with overall survival within the three treatment arms in this trial, namely active symptom control (ASC); ASC plus mitomycin, vinblastine and cisplatin (MVP); and ASC plus vinorelbine.Materials and methods We used immunohistochemical analysis of tumor samples from the MS01 trial to identify subgroups with and without nuclear BAP1 expression. We performed correlative analysis of clinical characteristics (age at diagnosis, sex and histological subtype) and overall survival within treatment arms with nuclear BAP1 expression.Results 89 tumor samples from the 409 patients originally in the trial were available for analysis. Of these, 60 samples harbored a positive internal control, in the form of positive staining of inflammatory cells for BAP1, and were carried forward for analysis. Correlative analysis suggested no significant association between loss of nuclear BAP1 expression and age at diagnosis, sex and histological subtype. Kaplan Meier survival analysis revealed a small, though non-significant, overall survival disadvantage associated with BAP1 expression in tumors from patients treated with vinorelbine.Discussion This exploratory analysis suggests BAP1 expression may modify response to vinorelbine in MPM, possibly due to prevention of mitotic microtubule formation. We suggest ongoing and planned clinical studies of vinorelbine in MPM assess BAP1 expression as a predictive biomarker of response.

Loss of Nuclear BAP1 Expression Is Associated with High WHO/ISUP Grade in Clear Cell Renal Cell Carcinoma.

BackgroundBRCA1-associated protein 1 (BAP1) mutations are frequently reported in clear cell renal cell carcinoma (ccRCC); however, very few studies have evaluated the role of these mutations in other renal cell carcinoma (RCC) subtypes. Therefore, we analyzed BAP1 protein expression using immunohistochemistry in several RCC subtypes and assessed its relationship with clinicopathological characteristics of patients.MethodsBAP1 expression was immunohistochemically evaluated in tissue microarray blocks constructed from 371 samples of RCC collected from two medical institutions. BAP1 expression was evaluated based on the extent of nuclear staining in tumor cells, and no expression or expression in < 10% of tumor cells was defined as negative.ResultsLoss of BAP1 expression was observed in ccRCC (56/300, 18.7%), chromophobe RCC (6/26, 23.1%), and clear cell papillary RCC (1/4, 25%), while we failed to detect BAP1 expression loss in papillary RCC, acquired cystic disease-associated RCC, or collecting duct carcinoma. In ccRCC, loss of BAP1 expression was significantly associated with high World Health Organization (WHO)/International Society of Urological Pathology (ISUP) grade (p = .002); however, no significant correlation was observed between loss of BAP1 expression and survival in ccRCC. Loss of BAP1 expression showed no association with prognostic factors in chromophobe RCC.ConclusionsLoss of BAP1 nuclear expression was observed in both ccRCC and chromophobe RCC. In addition, BAP1 expression loss was associated with poor prognostic factors such as high WHO/ISUP grade in ccRCC.

Altered Nuclear Expression of the Deubiquitylase BAP1 Cannot be Used as a Prognostic Marker for Canine Melanoma

SummaryBRCA1-associated protein-1 (BAP1) is a nuclear localized deubiquitylating enzyme that belongs to the ubiquitin c-terminal hydrolase subfamily. The encoded protein is highly homologous between man and dogs, suggesting a functional significance preserved by evolution. BAP1 has multiple properties, including tumour suppressor activity. Loss of BAP1 function is implicated in the oncogenesis of several types of cancers including uveal, mucosal and some cutaneous melanomas in humans, as well as in mesothelioma. In this study we investigate the significance of BAP1 in canine melanoma. Nuclear BAP1 protein was detected in five canine oral melanoma cell lines using an antibody commonly used for analysis of human tissues. BAP1 loss of function mutations often lead to loss of nuclear BAP1 (nBAP1) expression in humans; this is associated with a poorer prognosis in uveal and mucosal melanoma. Therefore, as a prelude to a study evaluating the prognostic significance of nBAP1 expression in dogs, immunohistochemistry (IHC) was used to assess cases of canine melanoma for nBAP1 expression. In 89 cases where tumour cells were identified by melan-A labelling, 100% of tumour cells were positive for nBAP1 expression, including eight uveal tract and 29 oral mucosal melanomas. This finding indicates that BAP1 IHC cannot be used as a prognostic marker in canine uveal and mucosal melanoma. Moreover, this observation suggests that either BAP1 has a different functional significance in canine melanoma or that loss of BAP1 function is achieved by a different route. This is a novel finding that warrants further investigation to determine the comparative biological relevance.

Uveal Melanoma Nuclear BRCA1-Associated Protein-1 Immunoreactivity Is an Indicator of Metastasis

To examine the BRCA1-associated protein-1 (BAP1) expression of primary uveal melanomas without and with metastasis, and to analyze the correlation between the BAP1 immunoreactivity of primary uveal melanoma and other clinicopathologic features. Retrospective case series. Forty patients with uveal melanoma (mean age, 57.98±14.75 years) were included in this analysis, of whom 20 had no metastatic disease and 20 had metastasis. Medical records and histology slides of patients with primary uveal melanoma treated by enucleation were reviewed. BAP1 expression was evaluated by immunohistochemical staining of formalin-fixed, paraffin-embedded sections. Immunoreactivity in the nucleus and cytoplasm were graded by estimating the percentage of primary tumor cells showing a positive staining of their nucleus or cytoplasm per 1 high-power field 200× (grades 0-3). Tumor size, histologic features, nuclear and cytoplasmic BAP1 immunoreactivity grade, and patient outcome, including development of metastasis. Significantly lower (P= 0.025) nuclear BAP1 immunoreactivity was observed in the metastatic melanoma group. Greater tumor thickness, basal diameter, and more advanced TNM stage were associated with an increased odds ratio of developing metastasis (P < 0.05). In addition, tumors with a higher proportion of cells expressing nuclear BAP1 had decreased odds of developing metastatic disease in a multivariate model (P=0.042). Metastasis-free survival was significantly longer in patients with uveal melanoma with high nuclear BAP1 stain (P= 0.004). Time to metastasis differs in patients with primary uveal melanoma with different grades of nuclear BAP1 immunoreactivity. Nuclear BAP1 stain is the only significant independent predictor of metastatic disease in this study. Our data support the role of BAP1 immunohistochemical staining of primary uveal melanoma to evaluate metastatic risk.

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.

Genomic profiling of the genes on chromosome 3p in sporadic clear cell renal cell carcinoma

Somatic mutations of the BRCA1 associated protein-1 (BAP1) gene, which maps to 3p21, have been found in several tumors including malignant mesothelioma, uveal melanoma, and renal cell carcinoma (RCC). The role of BAP1 inactivation in tumor development remains unclear. It has been reported that Vhl knock-out mice did not develop RCC, but Vhl knock-out mice with single allele loss of Bap1 in nephron progenitor cells developed RCC, indicating that Bap1 inactivation may be essential in murine renal tumorigenesis. To clarify the role of BAP1 in human RCC development, we performed mutation analyses, including copy number detection of BAP1 and assessment of allelic imbalance using microsatellite polymorphisms on 3p, in 45 RCC samples derived from 45 patients without VHL or BAP1 germline mutation. Additionally, we analyzed the sequences of the VHL, PBRM1, and SETD2 genes, and examined promoter methylation of VHL. Using immunostaining, we also checked for expression of BAP1 protein, which is normally located in the nuclei. None of the RCCs had biallelic deletion of BAP1, but five (11.1%) showed a biallelic mutation (four with a sequence-level mutation with monoallelic loss and one with a biallelic sequence-level mutation); these cells were negative for nuclear BAP1 staining. These patients had worse recurrence-free survival than the patients without a biallelic mutation (p=0.046). However, there were no significant differences in worse outcome by multivariate analysis combined with age, T stage, histological subtype, infiltration and vascular invasion. In 35 RCCs (77.8%), monoallelic loss of BAP1 was accompanied by VHL biallelic mutation or VHL promoter hypermethylation. In five RCCs (11.1%), we detected 3p loss-of-heterozygosity, but the copy number of BAP1 was normal. Surprisingly, nuclear staining of BAP1 was negative in 10 out of 31 tumors (32.3%) with hemizygous normal BAP1, suggesting that haploinsufficiency may relate to RCC development.

Lack of BAP1 protein expression in uveal melanoma is associated with increased metastatic risk and has utility in routine prognostic testing.

Background:The absence of BRCA1-associated protein 1 (BAP1) expression in uveal melanoma (UM) is associated with metastatic progression and reduced survival. In this study, we examine nuclear BAP1 (nBAP1) protein expression in primary UMs (PUMs) that show both ‘typical' and ‘atypical' clinical courses according to their chromosome 3 status, and secondary hepatic metastatic UM (MUM), correlating the results with histological, clinical and survival data.Methods:Nuclear BAP1 expression was immunohistochemically assessed in tissue microarrays (TMAs) of: (a) 68 PUM patients, who had been treated surgically; and (b) 13 MUM patients, with 5 cases being paired with primary tumour tissue. All cases were fully annotated. The percentage of tumour cell nuclei staining positively for BAP1 was scored by independent observers.Results:Nuclear BAP1 protein expression was absent in 35 out of 68 (51%) PUM patients, correlating strongly with poor prognostic clinicopathological and genetic parameters and reduced survival (Log rank, P<0.001). Lack of nBAP1 expression importantly identified a subset of ‘atypical' PUM patients with disomy of chromosome 3 but with unexpected metastatic relapse. Nuclear BAP1 expression was absent in 10 out of 13 (77%) MUM and expression was concordant in all paired PUM and MUM patients.Conclusions:Absent nBAP1 protein expression is an independent survival predictor for UM patients, easily examined using immunohistochemistry.

Clumped perinuclear BAP1 expression is a frequent finding in sporadic epithelioid Spitz tumors

BRCA1-associated protein 1 (BAP1) represents a recently identified tumor suppressor protein. Loss of BAP1 has been observed in cutaneous epithelioid Spitz tumors. These cutaneous melanocytic tumors show a distinct histopathologic phenotype characterized by an intradermal sheet-like proliferation of epithelioid melanocytes. We retrospectively reviewed the clinical outcomes, histopathologic findings and immunophenotype in spitzoid melanocytic neoplasms with the morphologic features seen in BAP1 mutated Spitz tumors. Cases were obtained from our files. BAP1 immunohistochemistry was evaluated dichotomously for the presence of nuclear staining. Fifteen of 19 cases showed loss of nuclear BAP1 expression. Of the 15 cases displaying nuclear loss of BAP1, clumped perinuclear staining was observed in 8 cases while 7 cases showed complete loss. Follow up ranging from 0-45 months (mean 17 months) was uneventful. Our data are consistent with an indolent overall clinical course for epithelioid Spitz tumors with loss of BAP1. Furthermore, a large subset of epithelioid Spitz tumors display loss of nuclear expression but show a reproducible clumped perinuclear staining pattern.