Expression, purification and characterization of the second DUSP domain of deubiquitinase USP20/VDU2.

Deubiquitinase USP20/VDU2 (VHL-interacting Deubiquitinating Enzyme 2) has been proved to play vital roles in multiple cellular processes by controlling the life-span of substrate proteins including hypoxia-inducible factor HIF1α, ß2-adrenergic receptor, and type 2 iodothyronine deiodinase etc. USP20 contains four distinct structural domains, which include the N-terminal zinc-finger ubiquitin binding domain (ZnF-UBP), the catalytic domain (USP domain), and two tandem DUSP domains (DUSP1 and DUSP2). Here in this study, we report the setting up of the production approach for USP20 DUSP2, and the NMR characterization of the produced target protein. With the assistance of GB1 tag and glycerol, both the solubility and stability of USP20 DUSP2 are significantly enhanced. And by using the optimized protein production procedure, monomeric and stable 15N, 13C-labeled USP20 DUSP2 sample for NMR data acquisition was obtained. The secondary structural elements of USP20 DUSP2 were then revealed by the analysis of recorded NMR spectra, and USP20 DUSP2 forms an AB3 fold in solution. The production protocol and NMR characterization results reported in this manuscript could be utilized in the extended structural and functional studies of USP20 DUSP2.

UCH-L3 promotes non-small cell lung cancer proliferation via accelerating cell cycle and inhibiting cell apoptosis.

Ubiquitin C-terminal hydrolase-L3 (UCH-L3) is a deubiquitinase that has a crucial role in oncogenesis. This study was aimed to explore the biological function of UCH-L3 in non-small cell lung cancer (NSCLC). Bioinformatics analysis was used to detect UCH-L3 expression in NSCLC tissues and normal lung tissues, and to analyze the relationship between UCH-L3 expression and survival of patients. qRT-PCR and western blotting assays were used to detect UCH-L3 expression in NSCLC tumor tissues and adjacent normal tissues. CCK-8 assay was performed to examine the effect of UCH-L3 on NSCLC cell proliferation. Flow cytometry assay was conducted to examine the effect of UCH-L3 on NSCLC cell cycle and apoptosis. The expression of UCH-L3 in NSCLC tissues was markedly higher than in normal lung tissues, and high expression of UCH-L3 was positively associated with the poor survival of patients. UCH-L3 knockdown significantly inhibited the proliferation of NSCLC cells, whereas UCH-L3 overexpression had the opposite effect. Moreover, UCH-L3 promoted NSCLC cells proliferation via accelerating cell cycle and inhibiting cell apoptosis. UCH-L3 is upregulated in NSCLC and positively associated with the poor survival, and its expression contributes to NSCLC cell proliferation by accelerating cell cycle and inhibiting cell apoptosis.

Gene Expression Analysis of Pediatric Acute Myeloid Leukemia Identified a Hyperactive ASXL1/BAP1 Axis Linked with Poor Prognosis and over Expressed Epigenetic Modifiers.

Genetic aberrations in the epigenome are rare in pediatric AML, hence expression data in epigenetic regulation and its downstream effect is lacking in childhood AML. Our pilot study screened epigenetic modifiers and its related oncogenic signal transduction pathways concerning clinical outcomes in a small cohort of pediatric AML in KSA. RNA from diagnostic BM biopsies (n = 35) was subjected to expression analysis employing the nCounter Pan-Cancer pathway panel. The patients were dichotomized into low ASXL1 (17/35; 49%) and high ASXL1 (18/35; 51%) groups based on ROC curve analysis. Age, gender, hematological data or molecular risk factors (FLT3 mutation/molecular fusion) exposed no significant differences across these two distinct ASXL1 expression groups (P > 0.05). High ASXL1 expression showed linkage with high expression of other epigenetic modifiers (TET2/EZH2/IDH1&2). Our data showed that high ASXL1 mRNA is interrelated with increased BRCA1 associated protein-1 (BAP1) and its target gene E2F Transcription Factor 1 (E2F1) expression. High ASXL1 expression was associated with high mortality {10/18 (56%) vs. 1/17; (6%) P < 0 .002}. Low ASXL1 expressers showed better OS {740 days vs. 579 days; log-rank P= < 0.023; HR 7.54 (0.98-54.1)}. The association between high ASXL1 expression and epigenetic modifiers is interesting but unexplained and require further investigation. High ASXL1 expression is associated with BAP1 and its target genes. Patients with high ASXL1 expression showed poor OS without any association with a conventional molecular prognostic marker.

Digital morphometry of tumor nuclei correlates to BAP-1 status, monosomy 3, gene expression class and survival in uveal melanoma.

Cytologic features such as the shape and size of tumor cells can predict metastatic death in uveal melanoma and other cancers but suffer from poor reproducibility. In this study, we investigate the interobserver concordance of digital morphometry, and correlate the results with BRCA associated protein-1 (BAP-1) expression and BAP-1 gene mutation status, monosomy 3, gene expression classifications and patient survival in uveal melanoma. The average number of cells analyzed in each of 107 tumors, was 1957 (SD 349). Mean time consumption was less than 2.5 min per tumor. Identical morphometric classification was obtained for ≥85% of tumors in all twelve evaluated morphometric variables (κ 0.70-0.93). The mean nucleus area, nucleus perimeter, nucleus max caliper and nucleus to cell area ratio were significantly greater in tumors with low BAP-1 expression and gene expression class 2. Patients had significantly shorter survival if their tumors had low BAP-1 (Log-Rank p = 0.002), gene expression class 2 (p = 0.004), long nucleus perimeters (p = 0.031), long nucleus max calipers (p = 0.029) and high mean nucleus to cell area ratios (p = 0.041) as defined in a training cohort and then tested in a validation cohort. Long nucleus perimeters and long nucleus max calipers correlated with monosomy 3 (Pearson Chi-Square p = 0.006 and p = 0.009, respectively). Long nucleus perimeters also correlated with BAP-1 mutation (p = 0.017). We conclude that digital morphometry can be fast and highly reproducible, that for the first time, morphometry parameters can be objectively quantitated in thousands of cells at a time in sub-µm resolutions, and that variables describing the shape and size tumor nuclei correlate to BAP-1 status, monosomy 3, gene expression class as well as patient survival.

Ubiquitin-Specific Protease 22/Silent Information Regulator 1 Axis Plays a Pivotal Role in the Prognosis and 5-Fluorouracil Resistance in Hepatocellular Carcinoma.

BACKGROUND: Ubiquitin-specific protease 22 (USP22) is described as a key subunit of the Spt-Ada-Gcn5 acetyl transferase complex, which plays an important role in the prognosis and resistance to chemotherapy drugs in hepatocellular carcinoma (HCC). Silent information regulator 1 (SIRT1) is a member of the sirtuin family that is deubiquitinated by USP22. However, it is still unknown whether USP22 and SIRT1 co-expression is associated with disease progression and 5-Fluorouracil (5-FU) resistance in HCC. METHODS: 141 patients who received hepatectomy at our hospital from January 2010 to December 2014 were enrolled in this study. The expression of USP22 and SIRT1 was detected by immunohistochemical staining. Clinicopathological features, including age, gender, tumor number, tumor size, tumor differentiation, tumor stage, alpha-fetoprotein and microscopic vascular invasion, were assessed. Further experiments confirmed the role of SIRT1 in 5-FU drug resistance in vivo. RESULTS: Immunohistochemical staining showed that the high expression of USP22 and SIRT1 was frequently observed in HCC tissues relative to normal liver tissues. Overexpression of USP22 is associated with microscopic vascular invasion (MVI). Further analysis showed that the co-expression of USP22 and SIRT1 was more effective in predicting the prognosis of HCC. The SIRT1 inhibitor EX-527 dramatically inhibited the expression of Cyclin B1 and resistance-associated protein 3 (MRP3) to reduce 5-FU drug resistance in vivo. CONCLUSION: These findings suggest that the co-expression of USP22 and SIRT1 is significantly associated with unfavorable HCC progression. The inhibition of SIRT1 in vivo could be valuable in improving 5-FU drug sensitivity and inhibiting tumor cell proliferation and inducing apoptosis.

BAP-1 Expression Status by Immunohistochemistry in Cellular Blue Nevus and Blue Nevus-like Melanoma.

The family of blue nevi includes the common blue nevus (BN), cellular blue nevus (CBN), and atypical BN, while melanomas with BN-like morphology can either arise in association with a blue nevus (MABN) or in the de novo setting mimicking cellular blue nevus (MMCBN). Recent molecular and immunohistochemical studies have demonstrated loss of BAP-1 in MABN/MMCBN but not in BN/CBN, suggesting that loss of BAP-1 correlates with a malignant phenotype in these lesions. In this study, we applied anti-BAP-1 antibodies to a series of CBN/BN (n = 11) and MABN/MMCBN (n = 4). Nuclear BAP-1 expression was detected in the majority of CBN/BN (n = 10/11) but was lost in 1 case. Most cases of MABN/MMCBN showed loss of nuclear BAP-1 expression (n = 3/4), with one case of MMCBN showing preserved BAP-1 expression. Demonstration of BAP-1 loss in a single case of CBN and preservation of BAP-1 expression in 1 case of MMCBN may indicate that detection of alterations in BAP-1 protein expression by immunohistochemistry may not be a completely reliable biomarker for the distinction of BN/CBN from MABN/MMCBN. Further investigation of the significance of BAP-1 loss/preservation in BN-like tumors is warranted.

Expression of BAP1 and ATM proteins: Association with AJCC tumor category in uveal melanoma.

BACKGROUND: Our aim is to detect the association of BAP1 with ATM protein with AJCC tumor category and its prognostic significance. METHODS: Based on AJCC tumor category, 69 patients samples were categorized into group A (LBD > 15 mm & tumor thickness ≥ 8 mm) and group B (LBD ≤ 15 mm & tumor thickness < 8 mm) subjected to immunohistochemistry to assess the nuclear expression of ATM and BAP1 proteins. Mutational analysis of BAP1 was performed on five samples from each group. RESULTS: Group A tumors showed insertion mutation of BAP1 gene while there was no mutation seen in group B tumor. At translational level loss of ATM and BAP1 was found in 65% and 66% of cases respectively. Loss of ATM with BAP1 was seen in 55% of cases which was more frequent in group A which was statically significant with metastasis (p = 0.006), advanced tumor staging (p = 0.021) and reduced metastasis-free survival (p = 0.048). On multivariate analysis loss of ATM along with BAP1 came out to be an independent prognostic marker (p = 0.035). CONCLUSION: Our data suggest that loss of BAP1 along with ATM might serve as a potential prognostic indicator in patients with an advanced AJCC tumor category, which leads to an increased risk of metastasis.

USP9X promotes LPS-induced pulmonary epithelial barrier breakdown and hyperpermeability by activating an NF-κBp65 feedback loop.

NF-κB is a central regulator of inflammatory and immune responses and has been shown to regulate transcription of several inflammatory factors as well as promote acute lung injury. However, the regulation of NF-κB signaling in acute lung injury has yet to be investigated. Human pulmonary alveolar epithelial cells (HPAEpiC) were treated with LPS to establish an acute lung injury model in vitro in which LPS stimulation resulted in pulmonary epithelial barrier breakdown and hyperpermeability. Cell viability was measured by CCK-8, and the transepithelial permeability was examined by measurement of transepithelial electrical resistance (TEER) and the transepithelial flux. Expression of ubiquitin-specific peptidase 9 X-linked (USP9X), zonula occludens (ZO-1), occludin and NF-κBp65, and the secretion of TNF-α and IL-1ß were measured by Western blotting and ELISA, respectively. For in vivo studies, mice were intraperitoneally injected with LPS and/or NF-κB inhibitor pyrrolidine dithiocarbamate (PDTC). Lung tissues were harvested for hematoxylin-eosin staining and Western blotting, and bronchoalveolar lavage fluid (BALF) was harvested for ELISA. We found that treatment with LPS in HPAEpiC inhibited cell viability and induced the expression of USP9X. Interestingly, knockdown of USP9X and treatment with PDTC suppressed LPS-induced HPAEpiC injury. USP9X overexpression promoted NF-κB activation, while NF-κB inactivation inhibited USP9X transcription and HPAEpiC injury induced by USP9X overexpression. Furthermore, LPS also induced the expression of USP9X in lungs, which was inhibited by PDTC. Taken together, these results demonstrate a critical role of USP9X-NF-κBp65 loop in mediating LPS-induced acute lung injury and may serve as a potential therapeutic target in acute lung injury.

Prognostic significance of BAP1 expression in high-grade upper tract urothelial carcinoma: a multi-institutional study.

PURPOSE: To evaluate the prognostic value of BRCA1-associated protein-1 (BAP1) expression in upper tract urothelial carcinoma (UTUC), as BAP1 mutations have been associated with prognostic implications in urologic and non-urologic malignancies. METHODS: We reviewed a multi-institutional cohort of patients who underwent radical nephroureterectomy (RNU) for high-grade UTUC from 1990-2008. Immunohistochemistry (IHC) for BAP1 was performed on tissue microarrays. Staining intensity was graded from 0-3, with BAP1 loss defined as an average intensity of < 1. Clinicopathologic characteristics and oncologic outcomes [recurrencefree (RFS), cancer-specific (CSS), and overall survival (OS)] were stratified by BAP1 status. The prognostic role of BAP1 was assessed using Kaplan-Meier (KM) and Cox regression analysis. Significance was defined as p < 0.05. RESULTS: 348 patients were included for analysis and 173 (49.7%) showed BAP1 loss. Median follow-up was 36.0 months. BAP1 loss was associated with papillary architecture and absence of tumor necrosis or CIS. On univariable analysis, BAP1 loss was associated with improved RFS (HR 0.60, p = 0.013) and CSS (HR 0.55, p = 0.007), although significance was lost on multivariable analysis (HR 0.71, p = 0.115 and HR 0.65, p = 0.071; respectively) after adjusting for other significant parameters. BAP1 expression was not significantly associated with OS. CONCLUSIONS: BAP1 loss was associated with favorable pathologic features and better oncologic outcomes in univariate but not multivariate analysis in patients with high-grade UTUC. In contrast to renal cell carcinoma, loss of BAP1 expression appears to confer a better prognosis in high-grade UTUC. The role of the BAP1 pathway in UTUC pathogenesis remains to be further elucidated.

Overexpression of the Ubiquitin-Specific Peptidase 9 X-Linked (USP9X) Gene is Associated with Upregulation of Cyclin D1 (CCND1) and Downregulation of Cyclin-Dependent Inhibitor Kinase 1A (CDKN1A) in Breast Cancer Tissue and Cell Lines.

BACKGROUND The role of the ubiquitin-specific peptidase 9 X-linked (USP9X) gene in breast cancer remains poorly understood. This study aimed to investigate the role of USP9X in breast cancer tissue and cell lines. MATERIAL AND METHODS Immunohistochemistry was used to examine the expression levels of USP9X in 102 breast cancer tissue samples and 41 normal breast tissue samples. Overexpression of USP9X in MCF-7 and MDA-MB-231 breast cancer cell lines were studied by USP9X lentivirus vector transfection. Clustered regularly interspaced short palindromic repeats (CRISPR)/caspase-9 USP9X gene knockout was performed. Cell proliferation, growth, and survival were examined using the cell counting kit-8 (CCK-8) assay, the colony formation assay, flow cytometry assays, and a tumor xenograft study. RESULTS Immunohistochemistry showed that USP9X was significantly overexpressed in 93 of 102 (91.1%) breast cancer tissue samples compared with 41 normal breast tissue samples and was associated with tumor size ≥5.0 cm (P<0.05). USP9X overexpression in MCF-7 and MDA-MB-231 breast cancer increased cell proliferation and survival, significantly reduced the number of cells in the G1-phase cells and increased the number of cells in the S-phase cells, which were reversed by CRISPR/caspase-9 USP9X gene knockout. Overexpression of USP9X upregulated the CCND1 gene encoding cyclin D1 and downregulated cyclin-dependent inhibitor kinase 1A (CDKN1A) gene in breast cancer cells, which were reversed by USP9X knockout. CONCLUSIONS Overexpression of USP9X was associated with upregulation of the CCND1 gene and downregulation of the CDKN1A gene in breast cancer tissue and cell lines.

Prognostic value of chromosomal imbalances, gene mutations, and BAP1 expression in uveal melanoma.

Uveal melanoma (UM) exhibits recurring chromosomal abnormalities and gene driver mutations, which are related to tumor evolution/progression. Almost half of the patients with UM develop distant metastases, predominantly to the liver, and so far there are no effective adjuvant therapies. An accurate UM genetic profile could assess the individual patient's metastatic risk, and provide the basis to determine an individualized targeted therapeutic strategy for each UM patient. To investigate the presence of specific chromosomal and gene alterations, BAP1 protein expression, and their relationship with distant progression free survival (DPFS), we analyzed tumor samples from 63 UM patients (40 men and 23 women, with a median age of 64 years), who underwent eye enucleation by a single cancer ophthalmologist from December 2005 to June 2016. UM samples were screened for the presence of losses/gains in chromosomes 1p, 3, 6p, and 8q, and for mutations in GNAQ, GNA11, BAP1, SF3B1, and EIF1AX. BAP1 protein expression was detected by immunohistochemistry (IHC). Multivariate analysis showed that the presence of monosomy 3, 8q gain, and loss of BAP1 protein were significantly associated to DPFS, while BAP1 gene mutation was not, mainly due to the presence of metastatic UM cases with negative BAP1 IHC and no BAP1 mutation detected by Sanger sequencing. Loss of BAP1 protein expression and monosomy 3 represent the strongest predictors of metastases, and may have important implications for implementation of patient surveillance, properly designed clinical trials enrollment, and adjuvant therapy.

Impairment of protein degradation and proteasome function in hereditary neuropathies.

In several neurodegenerative diseases in which misfolded proteins accumulate there is impairment of the ubiquitin proteasome system (UPS). We tested if a similar disruption of proteostasis occurs in hereditary peripheral neuropathies. In sciatic nerves from mouse models of two human neuropathies, Myelin Protein Zero mutation (S63del) and increased copy number (P0 overexpression), polyubiquitinated proteins accumulated, and the overall rates of protein degradation were decreased. 26S proteasomes affinity-purified from sciatic nerves of S63del mice were defective in degradation of peptides and a ubiquitinated protein, unlike proteasomes from P0 overexpression, which appeared normal. Nevertheless, cellular levels of 26S proteasomes were increased in both, through the proteolytic-activation of the transcription factor Nrf1, as occurs in response to proteasome inhibitors. In S63del, increased amounts of the deubiquitinating enzymes USP14, UCH37, and USP5 were associated with proteasomes, the first time this has been reported in a human disease model. Inhibitors of USP14 increased the rate of protein degradation in S63del sciatic nerves and unexpectedly increased the phosphorylation of eIF2α by Perk. Thus, proteasome content, composition and activity are altered in these diseases and USP14 inhibitors have therapeutic potential in S63del neuropathy.

Validation of the diagnosis of mesothelioma and BAP1 protein expression in a cohort of asbestos textile workers from Northern Italy.

Background: Diagnosis of mesothelioma based on death certificate is subject to misclassification, which may bias the results of epidemiology studies. A high proportion of mesothelioma harbor mutations in the BRCA1-associated protein 1 (BAP1) gene. Methods: We searched medical and pathology records and specimens for 127 workers from a textile-asbestos factory in Italy who died during 1963-2013 with a diagnosis of pleural or peritoneal neoplasm or mesothelioma on death certificate, to confirm the diagnosis with immunohistochemistry markers. We calculated the odds ratio of confirmation by selected characteristics and asbestos exposure variables. When sufficient pathology material was available, we analyzed BAP1 protein expression. Results: The diagnosis of mesothelioma was histologically confirmed for 35 cases (27.6%); 5 cases were classified as non-mesothelioma (3.9%), for 33 cases a mention of mesothelioma was found on record but no sufficient material was available for revision (26.0%); no records were available for 54 cases (death-certificate-only 42.5%). Diagnostic confirmation was not associated with sex, location of the neoplasm, age, or duration of employment; however, there was a significant association with time since first employment (P for linear trend 0.04). An association between duration of employment and time since first employment was observed for confirmed cases but not for death-certificate-only cases. BAP1 protein was lost in 18/35 cases (51.4%), without an association with sex, location, age, indices of asbestos exposure, or survival. Conclusions: We were able to confirm by immunohistochemistry a small proportion of mesothelioma diagnoses on certificates of deceased asbestos workers, and confirmation correlated with latency of asbestos exposure but not other characteristics. BAP1 protein loss is a frequent event in mesothelioma of asbestos-exposed workers, but does not correlate with exposure.

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

PURPOSE: 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. DESIGN: Retrospective case series. PARTICIPANTS: 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. METHODS: 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). MAIN OUTCOME MEASURES: Tumor size, histologic features, nuclear and cytoplasmic BAP1 immunoreactivity grade, and patient outcome, including development of metastasis. RESULTS: 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). CONCLUSIONS: 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.

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

AIMS: 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. METHODS AND RESULTS: 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). CONCLUSIONS: 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.

19q12 amplified and non-amplified subsets of high grade serous ovarian cancer with overexpression of cyclin E1 differ in their molecular drivers and clinical outcomes.

OBJECTIVES: Readily apparent cyclin E1 expression occurs in 50% of HGSOC, but only half are linked to 19q12 locus amplification. The amplified/cyclin E1hi subset has intact BRCA1/2, unfavorable outcome, and is potentially therapeutically targetable. We studied whether non-amplified/cyclin E1hi HGSOC has similar characteristics. We also assessed the expression of cyclin E1 degradation-associated proteins, FBXW7 and USP28, as potential drivers of high cyclin E1 expression in both subsets. METHODS: 262 HGSOC cases were analyzed by in situ hybridization for 19q12 locus amplification and immunohistochemistry for cyclin E1, URI1 (another protein encoded by the 19q12 locus), FBXW7 and USP28 expression. Tumors were classified by 19q12 amplification status and correlated to cyclin E1 and URI1 expression, BRCA1/2 germline mutation, FBXW7 and USP28 expression, and clinical outcomes. Additionally, we assessed the relative genomic instability of amplified/cyclin E1hi and non-amplified/cyclin E1hi groups of HGSOC datasets from The Cancer Genome Atlas. RESULTS: Of the 82 cyclin E1hi cases, 43 (52%) were amplified and 39 (48%) were non-amplified. Unlike amplified tumors, non-amplified/cyclin E1hi tumor status was not mutually exclusive with gBRCA1/2 mutation. The non-amplified/cyclin E1hi group had significantly increased USP28, while the amplified/cyclin E1hi cancers had significantly lower FBXW7 expression consistent with a role for both in stabilizing cyclin E1. Notably, only the amplified/cyclin E1hi subset was associated with genomic instability and had a worse outcome than non-amplified/cyclin E1hi group. CONCLUSIONS: Amplified/cyclin E1hi and non-amplified/cyclin E1hi tumors have different pathological and biological characteristics and clinical outcomes indicating that they are separate subsets of cyclin E1hi HGSOC.

High Expression of Ubiquitin-Specific Protease 8 (USP8) Is Associated with Poor Prognosis in Patients with Cervical Squamous Cell Carcinoma.

BACKGROUND Cervical cancer is one of the most common female malignancies in the world. The ubiquitin-specific protease 8 (USP8) functions by removing ubiquitin from protein substrates, and its potential role in cancer development was recently uncovered in lung cancer. The aim of this study was to investigate the expression and function of USP8 in cervical squamous cell carcinoma (CSCC). MATERIAL AND METHODS Immunohistochemical staining and quantitative PCR were performed to explore the expression of USP8 in both CSCC tissues and adjacent normal cervical tissues. Univariate and multivariate analyses were conducted to evaluate the clinical significance of USP8 in CSCC. Proliferation, migration, and invasion abilities of 2 CSCC cell lines were assessed after overexpression or silencing USP8, respectively. RESULTS Both the RNA and protein levels of USP8 were upregulated in CSCC tissues compared to normal cervical tissues. High expression of USP8 was correlated with advanced tumor stage and high recurrence risk. Moreover, USP8 was identified as a novel independent prognostic factor for CSCC patients. Cellular studies showed that USP8 can enhance the proliferation, migration, and invasion abilities of CSCC cells, thereby promoting tumor progression. CONCLUSIONS High expression of USP8 is frequent in CSCC tissues, which promotes tumor proliferation and invasion, and is correlated with a poor overall survival. Targeting USP8 may be a novel direction for drug development for CSCC therapy.

USP6 activation in nodular fasciitis by promoter-swapping gene fusions.

Nodular fasciitis is a self-limited myofibroblastic lesion that can be misdiagnosed as a sarcoma as a result of its rapid growth, cellularity, and sometimes prominent mitotic activity. A recurrent translocation t(17;22) has been identified in nodular fasciitis, fusing the coding region of USP6 to the promoter region of MYH9, and resulting in increased USP6 expression. A subset of cases show USP6 rearrangement without the typical fusion variants by RT-PCR, or any MYH9 rearrangement by FISH. We sought to further characterize such tumors using molecular diagnostic assays. A novel RT-PCR assay was designed to detect the two known MYH9-USP6 fusion types in formalin-fixed paraffin-embedded and frozen tissue, and a break-apart FISH assay was designed to detect USP6 rearrangement. Twenty-six cases of nodular fasciitis diagnosed between 2002 and 2013 were retrieved from the pathology files of our institutions and were confirmed to be positive by FISH and/or RT-PCR. Seven samples showed USP6 rearrangement by FISH but were negative for MYH9-USP6 fusion by RT-PCR; these cases were subjected to a next-generation sequencing assay utilizing anchored multiplex PCR technology. This assay targets a single partner gene associated with fusions in bone and soft tissue tumors for agnostic detection of gene fusion partners. Novel fusion partners were identified in all seven cases and confirmed by RT-PCR. Structurally, all fusions consisted of the juxtaposition of the entire coding region of USP6 with the promoter of the partner gene, driving increased USP6 expression. This study confirms the neoplastic nature of nodular fasciitis, defines additional pathogenic fusion partners, and adds to the growing body of literature on USP6-associated neoplasia. Given the diagnostic challenges of these tumors, molecular assays can be useful ancillary tools; however, the prevalence of promoter swapping must be recognized when interpreting results.

Diagnostic utility of BAP1 and EZH2 expression in malignant mesothelioma.

AIMS: 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. METHODS AND RESULTS: 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. CONCLUSIONS: 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.

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

AIMS: 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. METHODS AND RESULTS: 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. CONCLUSION: BAP1 and PBRM1 loss is seen frequently in ICC. ICC with BAP1 loss shares features of small-duct type ICC.