BAP1 Expression Research Articles

EZH2 Expression is increased in BAP1-mutant renal clear cell carcinoma and is related to poor prognosis.

Aim: BAP1 is frequently mutated in clear cell renal cell carcinoma (ccRCC) with a definitive role still unclear.Methods: In silico analysis of BAP1-mutant and wild-type gene enrichment and functional annotation in TCGA-KIRC dataset was performed. Target gene was studied based on functional clustering and was knowledge-based. Validation using in-house pathological sections were performed immunohistochemically. In vitro and in vivo studies on target gene were performed.Results: The TCGA ccRCC dataset included 534 ccRCC samples. BAP1 was frequently mutated and more frequently downregulated in ccRCC compared to normal kidney tissue or benign renal tumors. In the analysis between samples with BAP1 mutation (N = 33) and pan-negative (N = 33), we found that cancers with BAP1 mutation was significantly enriched for 14 pathways, of which 3 were DNA repair pathways, in which EZH2 played a role. CcRCC patients with lower BAP1 expression had poor prognosis and showed higher EZH2 expression, which also conferred worsened survival. Genetic and pharmaceutical inhibition of EZH2 not only inhibited BAP1-mutatn ccRCC cell viability and invasion but also abrogated genetic replenishing of BAP1 expression. Validation cohort encompassing 62 ccRCC samples confirmed the worsened phenotype for cases with higher EZH2 expression and significant positive correlation between expressions of EZH2 and BAP1. EZH2 inhibitor also inhibited tumor growth in xenograft mouse model with BAP1-mutated ccRCC cells with unremarkable toxicity.Conclusion: CcRCC with decreased BAP1 level has poor prognosis and is associated with higher EZH2 expression. Inhibition of EZH2 in BAP1-mutated entity holds promise for further investigation.

The epithelioid BAP1-negative and p16-positive phenotype predicts prolonged survival in pleural mesothelioma.

Mesothelioma is a relatively uncommon but highly malignant neoplasm. Most patients die of disease within 1 year of diagnosis, but some have prolonged survival. Prospective identification of these longer-term survivors may help to guide treatment. We therefore sought to investigate the role of p16 immunohistochemistry (IHC) both alone and in combination with other markers as a potential predictor of prolonged survival in mesothelioma. P16 IHC was performed on unselected pleural mesotheliomas biopsied from 1991 to 2014; 153 of 208 (74%) cases were p16-negative, which correlated significantly with poor overall survival in both univariate (median survival 7.6 versus 13.6 months; P = 0.001) and multivariate analysis [hazard ratio (HR): 1.632; 95% confidence interval (CI): 1.103-2.415; P = 0.014]. Other independent factors associated with prolonged survival included loss of expression of BAP1 and epithelioid morphology. We therefore stratified patients further based on these three independent prognostic variables and demonstrated an unusually prolonged survival in mesotheliomas which were epithelioid, BAP1 IHC negative and p16 IHC positive (12% of cases, median survival 31.7 months, P < 0.0001). In conclusion, p16 IHC is an independent prognostic biomarker in pleural mesothelioma. When used in combination with BAP1 IHC and morphological subtyping, patients with exceptionally prolonged survival can potentially be identified.

Modulating BAP1 expression affects ROS homeostasis, cell motility and mitochondrial function.

The tumor suppressor BAP1 associates with ASXL1/2 to form the core Polycomb complex PR-DUB, which catalyzes the removal of mono-ubiquitin from several substrates including histone H2A. This complex also mediates the poly-deubiquitination of HCFC1, OGT and PCG1-α, preventing them from proteasomal degradation. Surprisingly, considering its role in a Polycomb complex, no transcriptional signature was consistently found among BAP1-inactivated tumor types. It was hypothesized that BAP1 tumor suppressor activity could reside, at least in part, in stabilizing proteins through its poly-deubiquitinase activity. Quantitative mass spectrometry and gene expression arrays were used to investigate the consequences of BAP1 expression modulation in the NCI-H226 mesothelioma cell line. Analysis of differentially expressed proteins revealed enrichment in cytoskeleton organization, mitochondrial activity and ROS management, while gene expression analysis revealed enrichment in the epithelial-to-mesenchymal transition pathway. Functional assessments in BAP1 inactivated, BAP1 wild-type and BAP1 catalytically dead-expressing NCI-H226 and QR mesothelioma cell lines confirmed alteration of these pathways and demonstrated that BAP1 deubiquitinase activity was mandatory to maintain these phenotypes. Interestingly, monitoring intracellular ROS levels partly restored the morphology and the mitochondrial activity. Finally, the study suggests new tumorigenic and cellular functions of BAP1 and shows for the first time the interest of studying the proteome as readout of BAP1 inactivation.

A Subset of Malignant Mesotheliomas in Young Adults Are Associated With Recurrent EWSR1/FUS-ATF1 Fusions.

Malignant mesothelioma (MM) is a rare, aggressive tumor often associated with asbestos exposure and characterized by complex genetic abnormalities, including deletions of chromosome 22. A gene fusion involving EWSR1 and YY1 gene on 14q32 has been reported in 2 patients over the age of 60 with peritoneal MM. However, the incidence of EWSR1 rearrangements in MM and the spectrum of its fusion partners remain unknown. We recently encountered 2 MM cases with EWSR1-ATF1 fusions and sought to investigate the prevalence and clinicopathologic features associated with this abnormality. As both index cases occurred as intra-abdominal tumors in young adults, we searched our files for pleural and peritoneal MM occurring in adults younger than age of 40. All cases were tested by fluorescence in situ hybridization using custom bacterial artificial chromosomes probes for EWSR1, FUS, and ATF1 genes. When available, immunohistochemistry for BAP1 was performed. A total of 25 MM from patients aged 40 or less were screened, either from peritoneum (n=13) or pleura (n=12), with a median age of 31 (range: 7 to 40 y). Two additional ATF1-rearranged tumors were identified at pleural and peritoneal sites with EWSR1 and FUS as fusion partners, respectively, for a total of 4 cases (16%, 4/25). The fusion-positive cases displayed classic epithelioid morphology, immunoreactivity for cytokeratins and WT1, and negativity for S100. BAP1 expression was retained in the 3 fusion-positive cases with available material, and in 80% (12/15) of the fusion-negative cases. Our results expand the spectrum of tumor types harboring EWSR1/FUS-ATF1 gene fusions to include a subgroup of conventional epithelioid MM. Other features of this unique MM subset include young age at presentation, lack of asbestos exposure and retained BAP1 expression.

Abstract 5222: A landscape of deubiquinating enzymes (DUB) in lung cancer using activity based protein profiling (ABPP)

Abstract Dysregulation of ubiquitin signaling has been linked to many diseases, including cancer. Numerous DUBs are implicated in key pro-cancer pathways, including regulation of HIF1α, control of RAS signaling, signaling through both canonical and non-canonical NFκB pathways, and regulation of p53 activity. Overexpression of USP14, USP17 and USP28 is linked to promoting non-small cell lung cancer (NSCLC). OTUB1 has shown to trigger lung cancer progress by inhibiting RAS monoubiquitination in KRAS mutant NSCLC. Notably, BAP1 (tumor suppressor) expression decreases in lung cancer tissues although its mRNA levels did not reflect it. Based on these findings, we hypothesized that profiling DUB proteins and their activity in lung cancer cells and tissues could uncover new therapeutic targets. Since proteogenomics studies have revealed that mRNA expression is not a reliable predictor of the protein expression, we have used ABPP assays to profile DUB. ABPP, which uses chemical probes directed against the active sites of enzymes to interrogate the functional state of enzymes in biological samples, can serve as an important tool to nominate DUB targets. HA-tagged active site-directed ubiquitin probe, HA-Ub-VME was used to evaluate the DUBs profile of KRAS and EGFR mutant cell lines. Cell lysate was labeled in presence or absence of DUB inhibitors (DUBi) and the modified enzymes were visualized by anti-HA immunoblotting. Labeled proteome was enriched and analyzed using mass spectrometry and data was analyzed using APOSTL. In addition to DUB profiling, we screened lung cancer cell lines to DUBi, including small cell lung cancer, KRAS mutant, EGFR mutant, and models of EGFR TKI resistance. Immunoblotting in various KRAS and EGFR mutant cell lines displayed diverse profiles of DUBs. Enrichment of DUBs in A549 cells showed 23 DUBs and competition with bAP15, a well-known DUBi for USP14 resulted in reduced probe binding to USP14. We optimized the approach to capture maximum DUBs by examining parameters such as protein/probe ratio, probe incubation time and probing different lung cancer cells that may harvest a larger or complementary repertoire of DUBs. A preliminary screen of DUBi across 20 SCLC and NSCLC cell lines lead to a panel of DUBi (IC50=100nM-5µM), with broad or selective activity in particular cell lines. These results are being used for competitive DUB profiling using ABPP to comprehend target engagement and resulting changes in cell viability induced by DUBi. These results enable future studies to profile the DUB landscape in lung cancer cell lines, PDX tumor cohorts, and tumor tissues from patients. DUB ABPP can identify important DUBs in lung cancer cells to both identify new drug targets, as well as discern mechanism of action of drugs identified from compound screening campaigns. Citation Format: Shikha Mahajan, Bin Fang, Fumi Kinose, Eric B. Haura. A landscape of deubiquinating enzymes (DUB) in lung cancer using activity based protein profiling (ABPP) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5222. doi:10.1158/1538-7445.AM2017-5222

Prognostic value of BAP1 expression in clear cell renal cell carcinoma with inferior vena cava tumor thrombosis.

e16080 Background: Renal cell carcinoma (RCC) is characterized by a propensity for extension into the renal vein and inferior vena cava (IVC), and affected patients have a poor prognosis. BAP1 mutation, which occurs in about 15% of patients with clear cell RCC (ccRCC), also predicts a poor prognosis. The aim of this study was to elucidate the association between BAP1 expression and clinicopathologic outcomes in patients with ccRCC with an IVC tumor thrombus. Methods: Thirty-nine patients with ccRCC with an IVC tumor thrombus who underwent radical nephrectomy and tumor thrombectomy at our institution from 1999 to 2010 were retrospectively evaluated. Immunohistochemical analyses were performed for the expression of BAP1, and the associations between the expression of BAP1 and clinical outcomes were assessed. Survival analyses were performed using the Kaplan–Meier method and log-rank test. Multivariate analyses of the associations between overall survival (OS) and clinical variables were performed using a Cox proportional hazard model. For all analyses, P &lt; 0.05 was considered statistically significant. Results: The median follow-up time was 58.8 months (range, 2–130 months). The median age was 66 years (range, 37–80 years). Four patients (10.3%) had lung metastasis at the initial diagnosis. The primary tumor was right-sided in 27 (69.2%) patients and left-sided in 12 (30.8%). The IVC tumor thrombus extended above and below the diaphragm in 11 (28.2%) and 28 (71.8%) patients, respectively. The KPS score was &gt; 80 in 23 patients (59.0%). BAP1 expression was positive in 26 (67.0%) cases and negative in 13 (33.0%). BAP1-negative tumors were associated with a significantly shorter OS than BAP1-positive tumors (median OS, 42.0 vs. 81.5 months, respectively; P = 0.019). The median disease-free survival in BAP1-negative and -positive tumors was 10.0 and 19.0 months, respectively (P = 0.019). Multivariate analysis showed that only a BAP1-negative status was significantly associated with shorter OS (P = 0.026). Conclusions: A BAP1-negative tumor status was significantly associated with a poor prognosis in patients with ccRCC with an IVC tumor thrombus who underwent radical nephrectomy and tumor thrombectomy.

Association of loss of nuclear BAP1 expression with prognosis in oral mucosal melanoma.

e21021 Background: The prognostic factors of oral mucosal melanoma (OMM),a rare and aggressive neoplasm, remain to be determined. BAP1 is a tumor suppressor that has been associated with the outcome of melanomas and other malignancies. In this study, we investigated the genetic alterations in BAP1 and the prognostic potential of BAP1 protein expression in oral mucosal melanoma. Methods: DNA sequence analysis of BAP1 from 12 OMM patient samples revealed missense mutations in the tissues from four patients. Results: Based on immunohistochemical staining, loss of nuclear BAP1 expression was associated with poor overall survival (P &lt; 0.001, Log-rank = 21.308) and distant metastasis (P = 0.034, OR = 0.320). Multivariate analysis showed BAP1 to be an independent prognostic factor (P = 0.027, HR = 0.479). Conclusions: It thus appears that loss of nuclear BAP1 expression is an independent prognostic factor of poor overall survival and associated with distant metastasis in OMM.

Prognostic role of BAP1 in pT1 clear cell carcinoma in partial nephrectomy specimens.

BAP1 is a gene situated on chromosome 3p in a region that can be modified in renal cell carcinomas (RCCs). Mutations that cause loss of expression of BAP1 frequently occur in primary clear cell renal carcinoma (ccRCC). In a previous work, we observed that loss of nuclear BAP1 expression was crucial in ccRCC progression; in the current study, we investigated BAP1 expression in a large series of small conventional ccRCCs treated with partial nephrectomy, to assess a possible role as biomarker and the prognostic value in terms of patients' survival at long-term follow-up. One hundred sixty-two patients with single pT1 ccRCC were selected from those who underwent surgery at our Institute of Urology between 1987 and 2000. The features considered in this study were gender, age, tumor size, grade, incidence of metastasis, and patient-specific survival; they were correlated with immunohistochemical BAP1 nuclear expression in tumoral tissue. Median follow-up was 197.24months (range 19 to 274); median survival was 125.34months (range 5 to 274months). None of our pT1 ccRCCs showed total loss of nuclear BAP1 staining; we found a significant negative correlation between nuclear BAP1 expression and tumor size and between nuclear BAP1 expression and grade. In small ccRCCs, nuclear BAP1 staining was not correlated with disease-specific 5-year survival.Our data confirm the established role of BAP1 as a tumor suppressor protein. This is the first report where BAP1 has been studied in pT1 ccRCC tumors, but nuclear BAP1 expression cannot help identify patients having high-risk disease in these patients.

Genetic evolution of uveal melanoma guides the development of an inflammatory microenvironment

Uveal melanoma (UM) is characterized by a number of genetic aberrations that follow a certain chronology and are tightly linked to tumor recurrence and survival. Loss of chromosome 3, bi-allelic loss of BAP1 expression, and gain in chromosome 8q have been associated with metastasis formation and death, while loss of chromosome 3 has been associated with the influx of macrophages and T cells. We used a set of genetically-classified UM to study immune infiltration in the context of their genetic evolution. We show in two independent cohorts that lack of BAP1 expression is associated with an increased density of CD3+ T cells and CD8+ T cells. The presence of extra copies of chromosome 8q in disomy 3 tumors with a normal BAP1 expression is associated with an increased influx of macrophages (but not T cells). Therefore, we propose that the genetic evolution of UM is associated with changes in the inflammatory phenotype. Early changes resulting in gain of chromosome 8q may activate macrophage infiltration, while sequential loss of BAP1 expression seems to drive T cell infiltration in UM.

Dichotomy in intrahepatic cholangiocarcinomas based on histologic similarities to hilar cholangiocarcinomas.

Intrahepatic cholangiocarcinomas were classified into two types based on their microscopic appearance. Tumors with histologic similarities to hilar cholangiocarcinomas (predominantly ductal adenocarcinomas with minor tubular components, if present, restricted to the invasive front) were defined as the perihilar type, whereas the others were classified as peripheral cholangiocarcinomas. Among the 47 cases examined in the present study, 26 (55%) were classified as the perihilar type, whereas 21 (45%) were the peripheral type. The perihilar type had higher pT stages and more frequently showed a periductal-infiltrating gross appearance and microscopic perineural infiltration than peripheral cholangiocarcinomas. The presence of low-grade biliary intraepithelial neoplasia in the adjacent bile ducts was only found in perihilar cholangiocarcinomas (6/21, 29%). The immunophenotype also differed between the two types with MUC5AC and MUC6 being more commonly expressed in the perihilar type. One-third of perihilar cholangiocarcinomas lacked the expression of SMAD4, suggesting SMAD4 mutations, whereas the loss of BAP1 expression and IDH1 mutations were almost restricted to the peripheral type (35 and 15%, respectively). Patients with perihilar cholangiocarcinoma had worse overall survival than those with peripheral cancer (P=0.027). A multivariate analysis identified the histologic classification as an independent prognostic factor (P=0.005, HR=3.638). Comparisons between intrahepatic and hilar cholangiocarcinomas also revealed that the molecular features and prognosis of perihilar cholangiocarcinomas were very similar to those of hilar cholangiocarcinomas. In conclusion, this histology-based classification scheme of intrahepatic cholangiocarcinomas will be useful and clinically relevant because it represents different underlying molecular features and has an independent prognostic value.

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

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

Loss of nuclear BAP1 expression is associated with poor prognosis in oral mucosal melanoma.

Oral mucosal melanoma (OMM) is an aggressive neoplasm with an extremely poor prognosis. BAP1 is a tumor suppressor that has been associated with the outcome of melanomas and other malignancies. In this study, we investigated the genetic alterations in BAP1 and the prognostic potential of BAP1 protein expression in oral mucosal melanoma. DNA sequence analysis of BAP1 from 12 OMM patient samples revealed missense mutations in the tissues from four patients. Based on immunohistochemical staining, loss of nuclear BAP1 expression was associated with poor overall survival (P < 0.001, Log-rank = 21.308) and distant metastasis (P = 0.034, OR = 0.320). Multivariate analysis showed BAP1 to be an independent prognostic factor (P = 0.027, HR = 0.479). It thus appears that loss of nuclear BAP1 expression is an independent prognostic factor of poor overall survival and associated with distant metastasis in OMM.

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

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

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

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

Diagnostic utility of BAP1 and EZH2 expression in malignant mesothelioma.

Malignant mesothelioma is a highly aggressive cancer that is usually diagnosed at advanced stages; thus, highly sensitive and specific markers are necessary for its early definitive diagnosis. The aim of this study was to evaluate the diagnostic utility and prognostic significance of BAP1 and EZH2 in malignant mesothelioma. The expression of BAP1 and EZH2 was investigated by immunohistochemistry in 32 malignant mesotheliomas and 44 benign mesothelial proliferative lesions, including well-differentiated papillary mesothelioma (n = 4), mesothelial inclusion cyst (n = 22), and reactive mesothelial hyperplasia (n = 18). BAP1 loss and high EZH2 expression were observed in 17 (53%) and 22 (66%) malignant mesothelioma cases, respectively, whereas none of the benign lesions showed BAP1 loss or high EZH2 expression. The combination of BAP1 loss and high EZH2 expression as markers to differentiate epithelioid/biphasic malignant mesothelioma from benign mesothelial lesions was highly sensitive (90%) and specific (100%). There were no statistically significant associations between parameters such as age and sex of patients, tumour location, asbestos exposure, treatment, histology, and BAP1 or EZH2 expression. Survival analysis revealed that BAP1 loss, but not high EZH2 expression, was associated with a better prognosis. BAP1 loss and high EZH2 expression were highly specific to malignant mesothelioma in differentiating it from benign mesothelial proliferations, and the combination of these two markers improved the diagnostic accuracy.

Malignant Mesothelioma, BAP1 Immunohistochemistry, and VEGFA: Does BAP1 Have Potential for Early Diagnosis and Assessment of Prognosis?

Malignant mesothelioma (MM) is an aggressive malignancy of the serosal membranes. Early diagnosis and accurate prognostication remain problematic. BAP1 is a tumour suppressor gene commonly mutated in MM. Germline BAP1 mutation has been associated with early onset and less aggressive disease compared with sporadic MM. Sporadic BAP1 mutations are common and are associated with improved survival in MM, contrary to other malignancies. This study investigated the prognostic role of BAP1 in matched cytology and surgical specimens and aimed to investigate the association between BAP1 and the established prognostic marker VEGFA from a cohort of 81 patients. BAP1 mutation was found in 58% of histology and 59% of cytology specimens. Loss of BAP1 expression in both surgical and cytology specimens was significantly associated with poorer survival in a multivariate analysis when controlling for known prognostic indicators. Increased levels of VEGFA in pleural effusions were associated with poor survival. We conclude that the prognostic significance of BAP1 mutations in MM cannot be determined in isolation of other prognostic factors, which may vary between patients. Pathologists should employ caution when commenting on prognostic implications of BAP1 status of MM patients in diagnostic pathology reports, but it may be useful for early diagnosis.

P3.03-013 BAP1 Immunostaining and FISH Analysis of p16 Help Making Distinction among Subtypes of Mesothelioma: Topic: Mesothelioma Transitional

Distinction between mesothelioma and reactive mesothelial proliferation is difficult because of morphological overlap between mesothelioma cells and reactive mesothelial cells. It is often difficult to draw the line between epithelioid mesothelioma and biphasic mesothelioma with atypical stromal proliferation. However, separation of biphasic mesothelioma from epithelioid mesothelioma is important because therapeutic option and prognosis is different between two subtypes of mesothelioma. We collected 143 cases with malignant mesotheliomas (83 epithelioid, 22 biphasic and 38 sarcomatoid) and 33 cases with reactive mesothelial proliferation. Immunostaining was performed with anti-BAP1 antibody. Fluorescence in situ hybridization (FISH) analysis was performed with BAP1 probe and with p16 probe. BAP1 loss and deletion of p16 was separately analyzed in 19 biphasic mesotheliomas. We analyzed 76 cases with BAP1 immunostaining, 87 cases with p16 FISH, and 37 cases with BAP1 FISH. BAP1 loss with immunohistochemistry was observed in 55% of epithelioid and 37% of biphasic mesotheliomas, but not in sarcomatoid mesotheliomas. Homozygous deletion (HD) of BAP1 was observed in 50% of epithelioid and 11% of biphasic mesotheliomas, but not in sarcomatoid mesotheliomas. HD of p16 was observed in 64% of epithelioid, 91% of biphasic, and 100% of sarcomatoid mesotheliomas. Concordance of BAP1 loss and HD of p16 between epithelioid and sarcomatoid components of 19 biphasic mesotheliomas was 100%. Four of epithelioid mesotheliomas were difficult to differentiate from biphasic mesothelioma with histology alone because of florid proliferation of atypical stromal cells; however, BAP1 loss and HD of p16 were not observed in atypical stromal proliferation and diagnosis of epithelioid mesothelioma could be made. There was a significant difference in overall survival according to histologic subtype (epithelioid 24M, biphasic 15M, sarcomatoid 4.5M). Mesotheliomas with loss of BAP1 expression showed increased survival (20M vs 8M) and HD of p16 showed poor survival (9.5M vs 28M). However, if only epithelioid cases were analyzed, there was no trend toward increased survival with BAP1 loss (24M vs 22M) while HD of p16 still showed poor survival (17M vs 28M). Most of biphasic mesotheliomas and all sarcomatoid mesotheliomas harbor HD of p16. BAP1 loss and HD of BAP1 are observed in epithelioid and biphasic mesotheliomas, but not in sarcomatoid mesotheliomas. BAP1 immunostaining and FISH analysis of p16 help making distinction between epithelioid and biphasic mesothelioma as well as between benign and malignant mesothelial proliferation. p16 is a prognostic factor for patients with epithelioid mesothelioma, but BAP1 is not.

Cytogenetic or molecular analysis for prognosis?

SummaryPrognostication in uveal melanoma can have several functions: to advice the patient, to council patients with regard to monitoring, and to stratify patients in trials. Scientifically, determination of the parameters that define the development of metastases is of interest to understand tumour behaviour and develop treatments.The behaviour of tumor cells is related to changes at the DNA level, which lead to differences in mRNA, lncRNA, miRNA, and protein expression. A variety of tests using different technical platforms identify these differences, and can, after validation, be used for prognostication. Commonly‐used tests use the chromosome status (chrom 3, 8q), or the RNA expression pattern (Class 1 and 2). Expression of BAP1 can also be used. Some patients with low risk uveal melanoma also develop metastases, and these have been associated with the presence of extra copies of chromosome 8q, and with a specific marker, PRAME.The biological pathways that lead to metastases are still under investigation, although it is clear that a combination of loss of one chromosome 3 and loss of expression of BAP1 on the other chromosome 3 plays an important role in this pathway.

BAP1 immunohistochemistry and p16 FISH results in combination provide higher confidence in malignant pleural mesothelioma diagnosis: ROC analysis of the two tests.

Differentiation of malignant pleural mesothelioma (MPM) from benign mesothelial proliferation remains problematic. Loss of nuclear staining of BRCA1-associated protein 1 (BAP1; detected using immunohistochemistry (IHC)) and homozygous deletion (HD) of p16 (detected using fluorescence in situ hybridization (FISH)) are useful for differentiation of MPM from reactive mesothelial hyperplasia (RMH), but the correlation between BAP1 expression loss and p16 HD has not been fully described. We performed BAP1 IHC and p16-specific FISH for 40 MPM and 20 RMH cases, and measured proportions of cells showing BAP1 expression and p16 HD for each case. The diagnostic accuracy for MPM and the cut-off values of the two methods were assessed using receiver operating characteristic (ROC) analysis. BAP1 expression loss, p16 HD and coexistence of both were present in 27 (67.5 %), 27 (67.5 %) and 17 (42.5 %) MPM cases, respectively. Three MPM cases (7.5 %) and all 20 RMH cases had neither BAP1 loss nor p16 HD. There was no correlation between the results of the two methods. Their combination showed higher sensitivity (92.5 %, 37/40) and estimated probability than BAP1 IHC and p16-specific FISH used alone. BAP1 IHC and p16-specific FISH have independent diagnostic value, and have increased reliability when used in combination, for MPM diagnosis.

BAP1 Expression Is Preserved in BRCA-Associated Serous Carcinoma of the Ovary

BAP1 Expression Is Preserved in BRCA-Associated Serous Carcinoma of the Ovary