TP53 mutation is frequent in mantle cell lymphoma with EZH2 expression and have dismal outcome when both are present.

Enhancer of zeste homolog 2 (EZH2) expression is found in about 40% of mantle cell lymphoma (MCL) patients, which is associated with aggressive histology, high Ki-67 proliferation rate, p53 mutant pattern and inferior overall survival (OS). We conducted 11-gene (ATM, BIRC3, CCND1, KMT2C, KMT2D, NOTCH1, NOTCH2, RB1, TP53, TRAF2 and UBR5) next generation sequencing panel to shed more light on MCL with EZH2 expression (EZH2+ MCL). EZH2+ MCL more frequently harbor TP53 mutation compared to EZH2(-) MCL (41.2% vs. 19.1%, respectively, p = 0.045). TP53 mutation and EZH2 expression demonstrated overlapping features including aggressive histology, high Ki-67 proliferation rate and p53 mutant pattern by immunohistochemistry. Comparative analysis disclosed that EZH2 expression correlates with high Ki-67 proliferation rate irrespective of TP53 mutation. Aggressive histology is associated with EZH2 expression or TP53 mutation, possibly via independent mechanisms. p53 mutant pattern is due to TP53 mutation. MCL patients with EZH2 expression or TP53 mutation show inferior outcome and when both are present, patients have dismal outcome.

Elevated Expression of DNA Methyltransferases and Enhancer of Zeste Homolog 2 in Helicobacter pylori - Gastritis and Gastric Carcinoma.

AIM: The aim of this study was to study the role of key epigenetic regulators pertaining to DNA methylation and histone-modification systems in Helicobacter pylori (HP)-associated gastritis and gastric carcinogenesis. METHODS: The expression of DNA methyltransferase (DNMT-1, 3A, and 3B) and the catalytic subunit of polycomb repressive complex-2 (enhancer of zeste homolog 2 [EZH2]) in gastric carcinomas (n = 104), mucosa adjacent to carcinoma (n = 104), HP-associated gastritis (n = 95), and histologically normal mucosa (n = 31) was assessed by immunohistochemistry and qRT-PCR. RESULTS: The expression of all 3 DNMTs and EZH2 was significantly higher in HP-associated gastritis and carcinoma cases than in those with adjacent and normal mucosa. The expression of DNMT-1 and 3B was maximum in HP-associated gastritis. DNMT-3A showed higher expression in carcinoma-adjacent mucosa than in normal mucosa. Interestingly, the expression of EZH2 was higher in cases of HP-associated gastritis with metaplasia than in those without metaplasia and also in cases of intestinal type of adenocarcinoma. Significant positive correlation of EZH2 was identified with DNMT-1, DNMT-3A, and DNMT-3B. However, none of these markers was associated with survival outcome. CONCLUSION: This study establishes an important role of the key epigenetic regulators in the pathogenesis of both HP-associated gastritis and gastric carcinoma. Higher expression of all the epigenetic markers in the gastritis and their persistence in the carcinoma point toward their implications in HP-driven gastric carcinogenesis. Further, an inter-relation between the 2 arms of epigenetics, namely, DNA methylation and histone-modification in the pathogenesis of gastric carcinoma, is also documented. Given the reversibility of epigenetic phenomenon, these molecules may be of important therapeutic use.

EZH2 expression is associated with inferior overall survival in mantle cell lymphoma.

Enhancer of zeste homolog 2 (EZH2) is a catalytic component of the polycomb repressive complex 2 (PRC2) which reduces gene expression via trimethylation of a lysine residue of histone 3 (H3K27me3). Expression of EZH2 has not been assessed systematically in mantle cell lymphoma (MCL). Expression of EZH2 was assessed by immunohistochemistry in 166 patients with MCL. We also assessed other PRC2 components and H3K27me3. Fifty-seven (38%) of MCL patients were positive for EZH2 using 40% cutoff. EZH2 expression was associated with aggressive histologic variants (65% vs. 29%, p < 0.001), high Ki-67 proliferation rate (median, 72% vs. 19%, p < 0.001), and p53 overexpression (43% vs. 2%, p < 0.001). EZH2 expression did not correlate with expression of other PRC2 components (EED and SUZ12), H3K27me3, MHC-I, and MHC-II. Patients with EZH2 expression (EZH2+) had a poorer overall survival (OS) compared with patients without EZH2 expression (EZH2-) (median OS: 3.9 years versus 9.4 years, respectively, p < 0.001). EZH2 expression also predicted a poorer prognosis in MCL patients with classic histology (median OS, 4.6 years for EZH2+ and 9.6 years for EZH2-negative, respectively, p < 0.001) as well as aggressive histology (median OS, 3.7 years for EZH2+ and 7.9 years for EZH2-negative, respectively, p = 0.046). However, EZH2 expression did not independently correlate with overall survival in a multivariate analysis. Gene expression analysis and pathway enrichment analysis demonstrated a significant enrichment in cell cycle and mitotic transition pathways in MCL with EZH2 expression. EZH2 expression detected by immunohistochemistry is present in 38% of MCL cases and it is associated with high proliferation rate, p53 overexpression, aggressive histologic variants, and poorer OS. Based on gene expression profiling data, EZH2 expression could potentiate cell cycle machinery in MCL. These data suggest that assessment of EZH2 expression could be useful to stratify MCL patients into low- and high-risk groups.

An immunohistochemical panel of insulin-like growth factor II mRNA-binding protein 3 (IMP3), enhancer of zeste homolog 2 (EZH2), and p53 is useful for a diagnosis in bile duct biopsy.

Bile duct biopsy is being increasingly performed in number for a definite diagnosis of cholangiocarcinoma. However, difficulties are associated with a histopathological diagnosis because of the limited small amount of specimen obtained and crash artifact. The aim of the present study was to identify useful diagnostic immunohistochemical markers in bile duct biopsy that support a histological diagnosis. Fifty-one bile duct biopsy samples, including 26 samples taken from patients with cholangiocarcinoma, 11 with intraductal papillary neoplasm of the bile duct (IPNB), and 14 with benign bile duct lesions, were examined. Histology and the immunohistochemical expression of insulin-like growth factor II mRNA-binding protein 3 (IMP3), enhancer of zeste homolog 2 (EZH2), and p53 were assessed. They were then evaluated for their usefulness as diagnostic markers of malignancy. The diagnostic sensitivity and accuracy of the institutional histological diagnosis were 53.8% and 70.0%, respectively. The diagnostic sensitivity and accuracy of IMP3, EZH2, and p53 were 69.2% and 80.0%, 76.9% and 85.0%, and 50.0% and 67.5%, respectively. Immunohistochemical staining for EZH2; the combination of either 2 of IMP3, EZH2, and p53; or the combination of IMP3, EZH2, and p53 significantly increased sensitivity and accuracy over those of the institutional histological diagnosis (p<0.05). In conclusion, an immunohistochemical panel consisting of IMP3, EZH2, and p53 increases the diagnostic sensitivity and accuracy of bile duct biopsy for the diagnosis of cholangiocarcinoma.

Enhancer of Zeste Homolog 2 (EZH2) in Malignant Progression of Gallbladder Carcinoma.

PURPOSE: Data related to the role of epigenetic modifications in gallbladder carcinoma (GBC) is limited. We intended to assess the role of crucial epigenetic modifiers pertaining to histone modification and DNA-methylation system in gallbladder carcinogenesis. METHODS: The expression of EZH2, H3K27me3, and DNA methyltransferases (DNMTs) was analyzed by immunohistochemistry in cases of GBC (n = 39), gallbladder dysplasia (GBD, n = 12), and benign mucosa (BM, n = 16). A semi-quantitative scoring system was used for assessing the immunohistochemical expression. RESULTS: The expression of EZH2 was significantly higher in cases of GBC than GBD (p value 0.001). The cases of BM were negative. Its expression was also higher in poorly differentiated tumors and positively correlated with the proliferative activity (MIB-1 labeling index) (p value 0.03 and 0.01, respectively). There was no significant difference in the expression levels of H3K27me3, DNMT-1, and DNMT-3B among GBC, GBD, and BM cases. Although GBC cases with strong EZH2 expression showed a shorter overall survival, the difference was not statistically significant. CONCLUSION: This study highlights the crucial role of the key epigenetic regulators EZH2 in the pathobiology and evolution of gallbladder carcinogenesis. Given the reversibility of epigenetic alterations, EZH2 may be a novel therapeutic target for gallbladder carcinogenesis.

Prognostic Value of TWIST1 and EZH2 Expression in Colon Cancer.

BACKGROUND: Colorectal carcinoma (CRC) is the third most common human cancer. Twist, a basic helix-loop-helix (bHLH) transcription factor, is an epithelial-mesenchymal transition ((EMT) inducer that has been involved in carcinogenesis and chemoresistance. Also, the enhancer of Zeste homolologue2 (EZH2), a member of the polycomb group of genes, had been associated with poor prognosis in several malignancies. OBJECTIVE: To evaluate the expression of Twist1 and EZH2 in colon carcinoma in Egyptian patients and its relation to clinicopathological parameters, prognosis, and survival. METHODS: Twist1 and EZH2 expressions were evaluated immunohistochemically in 50 cases of colorectal tumors (12 colon adenomas and 38 colon carcinomas) and 20 samples from normal colonic mucosa. RESULTS: The expression of Twist1 and EZH2 was significantly higher in colon adenoma and carcinoma than that in normal colonic mucosa (P < 0.05). Twist1 and EZH2 expressions were associated with decreased tumor differentiation, advanced stage, and lymph node metastasis. Twist1 and EZH2 expressions were significantly related to 3-year disease-free survival (P = 0.005 and 0.002 respectively) and 3-year overall survival (P = 0.045 and 0.039, respectively). CONCLUSIONS: Twist1 and EZH2 may serve as prognostic predictors for colon carcinoma and may have a potential role as therapeutic targets in patients with colon carcinoma in the future.

Differential Expression of EZH2 and H3K27me3 in Oral Verrucous Carcinoma and Oral Verrucous Hyperplasia.

Enhancer of zeste homolog 2 (EZH2), a component of the polycomb repressive complex 2 that catalyzes trimethylation of H3K27 (H3K27me3), has been shown to promote tumor development and progression. Expression of EZH2 is associated with cell cycle regulation and cell proliferation in various neoplasms. Oral verrucous hyperplasia (OVH) and Oral verrucous carcinoma (OVC) are rare entities and share several clinical and histopathologic features. Problems distinguishing these lesions are added by a lack of adjacent normal tissue of the biopsy samples and poorly oriented tissue sections. The aim of this study was to investigate the expression of EZH2 and H3K27me3 in OVH and OVC and comparing the expression with normal oral mucosa and oral squamous cell carcinoma (OSCC). Seventy-eight samples, including 25 cases of OVC, 8 cases of OVH, 35 cases of OSCC and 10 cases of normal oral mucosa, were retrieved and submitted for immunohistochemical staining. The results demonstrated that the mean labeling indices (LIs) of EZH2 and H3K27me3 expression were highest in OSCC, followed by the OVC, OVH, and normal mucosa. Statistical differences in EZH2 LI were observed among these lesions whereas H3K27me3 LI was significantly different among OSCC, OVH and normal mucosa. EZH2 LI was found to have a sensitivity of 72.00% and specificity of 87.50% in distinguishing OVH from OVC, and a sensitivity of 57.14% and specificity of 84.00% in distinguishing OVC from OSCC. A positive correlation between EZH2 and H3K27me3 expression was significantly found in OVC but not in OVH and OSCC. These findings highlight the involvement of epigenetic regulation by EZH2-mediated H3K27me3 in the pathogenesis of OVH and OVC, and EZH2 expression indicates disease progression of these verrucous lesions. Diagnostic test analysis further suggests that EZH2 may be used as an additional test for differentiating OVH from OVC in questionable cases.

High level of EZH2 expression is linked to high density of CD8-positive T-lymphocytes and an aggressive phenotype in renal cell carcinoma.

PURPOSE: Enhancer of zeste homolog 2 (EZH2), the catalytic part of the Polycomb repressive complex 2 (PRC2), has a prognostic role in renal cell carcinoma (RCC) and was recently shown to modulate the immune response by reducing tumor cell immunogenicity. METHODS: To investigate whether the prognostic role of EZH2 might be driven by a modified immune environment, more than 1800 RCCs were analyzed in a tissue microarray for EZH2 expression and CD8 positive lymphocytes were quantitated by automated digital imaging. RESULTS: EZH2 positivity was found in 75.2% of 1603 interpretable tumors. In clear cell RCC, high EZH2 expression was significantly linked to high ISUP, Furmann, and Thoenes grade (p < 0.0001 each), advanced stage (p < 0.0001), nodal (p = 0.0190) and distant metastasis (p < 0.0001) as well as shortened overall (p < 0.0027) and recurrence free survival (p < 0.0001). The density of CD8+ cells varied from 0 to 5048 cells/mm2 (Median 120 cells/mm2). A high CD8+ count was significantly associated with high ISUP, Fuhrmann, and Thoenes grade (p < 0.0001 each), advanced tumor stage (p = 0.0041), distant metastasis (p = 0.0026) as well as reduced overall survival (p = 0.0373) and recurrence free survival (p = 0.0450). The density of CD8+ cells continuously increased with raising EZH2 levels (p < 0.0001). CONCLUSION: Our data support a striking prognostic role of both EZH2 expression and the density of CD8+ cells in RCC. The tight relationship of EZH2 expression and CD8+ cell counts in RCC is consistent with models suggesting that EZH2 overexpression can be caused by high lymphocyte content in certain tumor types. Such a mechanism could explain the unique finding of high lymphocyte counts driving poor prognosis in RCC patients.

High EZH2 expression in ductal carcinoma in situ diagnosed on breast core needle biopsy is an independent predictive factor for upgrade on surgical excision.

PURPOSE: Approximately 25 % of DCIS diagnosed on breast core needle biopsy (CNB) is upgraded to invasive carcinoma on surgical excision. Risk factors to predict the upgrade on excision are not well established, leading many patients to be over or under-treated. EZH2 was shown to be associated with aggressive behavior of cancer from many sites, including breast cancer. We aimed to analyze EZH2 expression and tumor infiltrating lymphocytes (TILs) in DCIS as predictive factors for an upgrade on excision. METHODS: We assessed EZH2 expression in 34 DCIS cases diagnosed on CNB and upgraded to invasive carcinoma on excision. Then, we compared these cases with 60 control cases that were not upgraded on excision. A staining score for DCIS (0-12) was obtained by multiplying the staining intensity (0-3) and the percentage of positive cells (1-4). The nuclear staining score ≥6 was considered as 'high' expression. RESULTS: 46 of 94 (49 %) DCIS on CNB showed high EZH2 expression. EZH2 expression was directly correlated with TILs density, nuclear grade, HER2 expression, Ki-67 index and negative ER status. On univariate analysis, upgrade on excision was associated with high EZH2 expression, high TILs density, negative ER status and high Ki-67 index. Multivariate analysis revealed the high EZH2 expression as the only independent predictive factor for upgrade on excision. CONCLUSIONS: Our study revealed the high EZH2 expression as the only independent predictive factor for an upgrade on excision. Future studies should focus on the evaluation of EZH2 expression in tumor-microenvironment interaction in terms of diagnostic, treatment and prognostic purposes.

Diagnostic Value of the EZH2 Immunomarker in Malignant Effusion Cytology.

BACKGROUND: Differentiating reactive mesothelial cells from metastatic carcinoma in effusion cytology is a challenging task. The application of at least 4 monoclonal antibodies including 2 epithelial markers (Ber-EP4, MOC-31, CEA, or B72.3) and 2 mesothelial markers (calretinin, WT-1, CK5/6, or HBME-1) are often useful in this distinction; however, it is not readily available in many resource-limited developing countries. Aberrant immunoexpression of enhancer of zeste homolog 2 (EZH2), a transcriptional repressor involved in cancer progression, is observed widely in various malignancy. In this study, we evaluate the diagnostic value of EZH2 as a single reliable immunomarker for malignancy in effusion samples. METHODS: A total of 108 pleural, peritoneal, and pericardial effusions/washings diagnosed as unequivocally reactive (n = 41) and metastatic carcinoma (n = 67) by cytomorphology over 18 months were reviewed. Among the metastatic carcinoma cases, 54 were adenocarcinoma and others were squamous cell carcinoma (n = 1), carcinosarcoma (n = 1), and carcinoma of undefined histological subtypes (n = 11). Cell block sections were immunostained by EZH2 (Cell Marque, USA). The percentages of EZH2-immunolabeled cells over the total cells of interest were calculated. Receiver operating characteristic (ROC) curve analysis was performed to determine the optimal cut-off score to define EZH2 immunopositivity. RESULTS: A threshold of 8% EZH2-immunolabeled cells allows distinction between malignant and reactive mesothelial cells, with 95.5% sensitivity, 100% specificity, 100% positive predictive value, and 93.2% negative predictive value (p < 0.0001). The area under the curve was 0.988. CONCLUSION: EZH2 is a promising diagnostic biomarker for malignancy in effusion cytology which is inexpensive yet trustworthy and could potentially be used routinely in countries under considerable economic constraints.

Inhibition of MELK Protooncogene as an Innovative Treatment for Intrahepatic Cholangiocarcinoma.

Background and Objectives: Intrahepatic cholangiocarcinoma (iCCA) is a pernicious tumor characterized by a dismal outcome and scarce therapeutic options. To substantially improve the prognosis of iCCA patients, a better understanding of the molecular mechanisms responsible for development and progression of this disease is imperative. In the present study, we aimed at elucidating the role of the maternal embryonic leucine zipper kinase (MELK) protooncogene in iCCA. Materials and Methods: We analyzed the expression of MELK and two putative targets, Forkhead Box M1 (FOXM1) and Enhancer of Zeste Homolog 2 (EZH2), in a collection of human iCCA by real-time RT-PCR and immunohistochemistry (IHC). The effects on iCCA growth of both the multi-kinase inhibitor OTSSP167 and specific small-interfering RNA (siRNA) against MELK were investigated in iCCA cell lines. Results: Expression of MELK was significantly higher in tumors than in corresponding non-neoplastic liver counterparts, with highest levels of MELK being associated with patients' shorter survival length. In vitro, OTSSP167 suppressed the growth of iCCA cell lines in a dose-dependent manner by reducing proliferation and inducing apoptosis. These effects were amplified when OTSSP167 administration was coupled to the DNA-damaging agent doxorubicin. Similar results, but less remarkable, were obtained when MELK was silenced by specific siRNA in the same cells. At the molecular level, siRNA against MELK triggered downregulation of MELK and its targets. Finally, we found that MELK is a downstream target of the E2F1 transcription factor. Conclusion: Our results indicate that MELK is ubiquitously overexpressed in iCCA, where it may represent a prognostic indicator and a therapeutic target. In particular, the combination of OTSSP167 (or other, more specific MELK inhibitors) with DNA-damaging agents might be a potentially effective therapy for human iCCA.

Mantle Cell Lymphoma Involving Skin: A Clinicopathologic Study of 37 Cases.

Mantle cell lymphoma (MCL) rarely involves the skin and the histologic and immunohistochemical features of this neoplasm at this site are under described. In this study, we report 37 skin specimens involved by MCL, representing 1.4% of total MCL biopsy specimens in our institution. The median age at time of skin involvement was 66 years (range, 36 to 85 y) and there was a male predilection of 2.7 to 1. The most frequently involved site was the skin of extremities, in 59.3% of patients, and 30 (81.1%) patients had advanced stage (III/IV) disease. Eleven (29.7%) patients presented with skin lesions as the first manifestation of MCL and 26 (70.3%) patients presented as relapse or progression of previously documented MCL and despite therapy for systemic MCL. Multiple skin lesions were more common (81.8%) in the former group whereas a solitary skin lesion was more frequent (65.4%) in the relapse/progression group (P=0.01). Thirty (81.1%) patients had skin nodules. Microscopically, the epidermis was spared with a grenz zone in all cases. A diffuse pattern of involvement was the most common architectural pattern (66.7%). In 27 (72.9%) patients, the MCL was either blastoid or pleomorphic variant, in 9 (24.3%) patients classic variant, and the disease was not further classified in 1 (2.7%) patient. The Ki-67 proliferation rate was higher in aggressive variants as compared with classic variant MCL (median 90% vs. 20%, P <0.01). In patients who presented skin lesions as a manifestation of disease relapse or progression, 16 patients initially had classic variant MCL and in 10 of the patients the MCL evolved over time (median interval: 4.1 y) to an aggressive variant at progression or relapse. The overall survival of patients with aggressive variant MCH was inferior to that of patients with classic variant MCL (median: 59 vs. 155.8 mo, P<0.05). In summary, MCL rarely involves the skin and correlates with relapse or progression of disease, aggressive morphologic features, and a poorer prognosis.

Robust expression of EZH2 in endocervical neoplastic lesions.

The aim of this study was to evaluate the nuclear expression of histone methyltransferase enhancer of zeste homolog 2 (EZH2) in endocervical neoplastic lesions such as invasive endocervical adenocarcinoma (ECA) and cervical in situ adenocarcinoma (AIS) in comparison with normal endocervix and non-neoplastic counterparts. A total of 54 consecutive neoplastic cases (37 ECA, 17 AIS) and 32 non-neoplastic endocervical lesions (15 reactive atypia, 9 microglandular hyperplasia, 3 tuboendometrioid metaplasia, 3 tunnel cluster, 2 endometriosis) were included in the study with adjacent normal endocervix if present. EZH2 immunoreactivity was evaluated semiquantitatively by three independent experts in lesions and adjacent normal glandular epithelium as well. EZH2 expression was defined robust if at least two of the three experts rated partial or diffuse positivity. Robust EZH2 expression was statistically compared among the neoplastic, non-neoplastic, and normal glandular epithelium samples. Diagnostic test capability of robust EZH2 expression was calculated. Fifty-three out of the 54 neoplastic cases (98%) showed robust EZH2 expression. Robust EZH2 expression was significantly less often (4 out of 32 cases, 12.5%) found in the non-neoplastic endocervical lesions (p < 0.0001) and never (0 out of 66 samples) in the adjacent normal glandular epithelium. Robust EZH2 overexpression had a sensitivity and specificity of over 95% in detecting neoplastic lesions versus non-neoplastic lesions or normal glandular epithelium. EZH2 may play a role in the pathogenesis of endocervical neoplasia, and the detection of robust expression of EZH2 might be a useful differential diagnostic tool in problematic endocervical lesions in histology and cytology as well.

Clinical significance and EZH2, ERG and SPINK1 protein expression in pure and mixed ductal adenocarcinoma of the prostate.

BACKGROUND: Although ERG and SPINK1 molecular alterations have been studied in acinar and ductal adenocarcinoma of the prostate, EZH2 expression has not been previously evaluated in ductal adenocarcinoma. METHODS: We collected cases of pure and mixed ductal adenocarcinoma of the prostate and evaluated clinical significance and EZH2, ERG, and SPINK1 protein expression. RESULTS: We investigated 61 ductal adenocarcinomas, 22 pure and 39 mixed ductal/acinar. Except for tumor growth pattern, none of the clinical parameters studied significantly differed between pure and mixed tumors. Thirty-five percent of ductal adenocarcinomas were organ confined, 15% displayed seminal vesicle invasion. Lymph node and distal metastasis occurred in 13% and 24% of cases, respectively; 34% of patients experienced biochemical failure, 7% died of disease. Ninety-eight percent of tumors expressed EZH2; in 80% of cases >50% of tumor cells were positive. ERG and SPINK1 were expressed in 20% and 36% of cases, respectively. There was no difference in protein expression between pure and mixed ductal adenocarcinomas. ERG expression tended to be lower, and SPINK1 higher than reported for acinar tumors. Biochemical failure, metastasis and death did not differ between EZH2, ERG, and SPINK1 positive and negative patients, nor between <50% versus >50% expression of SPINK1 and EZH2, respectively. CONCLUSIONS: Pure and mixed ductal adenocarcinomas have similar clinical behavior and molecular alterations. Higher EZH2 and SPINK1 protein expression, compared to acinar prostatic adenocarcinoma, might account for the more aggressive clinical course of ductal adenocarcinoma.

Utility of Combined EZH2, p-ERK1/2, p-STAT, and MYC Expression in the Differential Diagnosis of EZH2-positive Hodgkin Lymphomas and Related Large B-Cell Lymphomas.

EZH2 is a methyltransferase that plays an important tumorigenic role in various neoplasms. We previously found that EZH2 is expressed in a range of aggressive B-cell lymphomas (ABCLs), T-cell lymphomas, and histiocytic neoplasms, with differential expression of intracellular signaling molecules p-ERK, MYC, and p-STAT3, potential regulators of EZH2 expression. We studied EZH2 expression in nodular lymphocyte predominant Hodgkin lymphoma (NLPHL), classic Hodgkin lymphoma (cHL), T-cell/histiocyte-rich large B-cell lymphoma (THRLBCL), and B-cell Lymphoma, unclassifiable, with features intermediate between diffuse large B-cell lymphomas and classic Hodgkin lymphoma (BCLu-DLBCL/cHL), as well as the coexpression of p-ERK, MYC, and p-STAT3 in these neoplasms. The neoplastic LP cells of NLPHL and Hodgkin/Reed-Sternberg cells of cHL were strongly positive for EZH2, as were the neoplastic cells in THRLBCL and BCLu-DLBCL/cHL. EZH2 expression correlated with proliferation rate, as assessed by Ki-67 staining. LP cells in NLPHL and Hodgkin/Reed-Sternberg cells in cHL were strongly positive for p-ERK, p-STAT3, and MYC, as were the neoplastic cells in THRLBCL and BCLu-DLBCL/cHL, in contrast to the differential expression of these molecules seen in ABCLs. These findings suggest that combined expression of p-ERK, MYC, and p-STAT3 is a useful immunohistochemical pattern for the diagnosis of EZH2-positive Hodgkin lymphomas and related lymphomas, in contrast to ABCLs. Furthermore, the overexpression of EZH2, in association with coexpression of tumorigenic signaling molecules, suggests an oncogenic role for this molecule in the development of Hodgkin lymphomas and related lymphomas. THRLBCL and BCLu-DLBCL/cHL appear to have a mechanism for the regulation of EZH2 expression that is similar to NLPHL and cHL and different from that of ABCLs. In addition, EZH2 and associated signaling cascades may serve as therapeutic targets for the treatment of Hodgkin lymphomas and related lymphomas.

Effect of lincRNA PVT1 associated with Enhancer of Zeste homolog 2 (EZH2) and with androgen receptor (AR) on the large-scale gene expression in LNCaP prostate cancer cells

O lincRNA PVT1 (Plasmacytoma Variant Translocation 1) é um RNA longo não codificador de proteínas (ncRNA) descrito como um oncogene sendo superexpresso em vários tipos de cânceres. LincRNA PVT1 está localizado na região genômica 8q24, também conhecida como 'gene desert'. O nível de expressão do lincRNA PVT1 está associado ao aumento do risco de câncer de próstata (PCa) e está correlacionado com os níveis de expressão do receptor de andrógeno (AR). No entanto, o mecanismo do envolvimento do lincRNA PVT1 com o AR no desenvolvimento de câncer de próstata ainda não está bem esclarecido. Aqui, nós testamos a hipótese que a formação do complexo AR-EZH2-PVT1 participa na regulação da expressão gênica em câncer de próstata, nas células LNCaP. A imunoprecipitação de ribonucleoproteínas seguida de PCR quantitativo (RIP-qPCR) revelou que o lincRNA PVT1 está associado fisicamente ao AR (12% do input) e à metiltransferase EZH2, proteína componente do complexo repressor Polycomb 2 (36% do input) sob condições suplementadas com andrógeno (+R1881). O lincRNA PVT1 também está associado fisicamente ao AR (10% de input) e à EZH2 (42% de input) em condições de privação de andrógeno (-R1881). Assim, a associação física entre lincRNA PVT1, AR e EZH2 é independente do hormônio andrógeno. Usando uma abordagem de estudo em larga-escala de perda e ganho de função, nossos resultados mostraram que o silenciamento do lincRNA PVT1 em células LNCaP na presença de andrógeno restaura a expressão parcialmente, totalmente ou causa superexpressão de 160 genes que tiveram a expressão inibida por andrógeno. Entre esses genes, destacamos genes envolvidos na regulação da diferenciação celular, em componentes da junção célula-célula, na inibição da migração e invasão celular e no desencadeamento da via apoptótica. Imunoprecipitação da cromatina seguida de PCR quantitativo (ChIP-qPCR), em cultura de células LNCaP suplementada com andrógeno sob silenciamento do lincRNA PVT1, mostrou aumento significativo na ocupação pela marca de histona ativadora H3K27Ac do promotor do gene NOV, um dos genes que tiveram sua expressão aumentada com o silenciamento de PVT1. O ChIP-qPCR também mostrou, após o silenciamento do lincRNA PVT1, um aumento significativo da marca H3K27me3 na região enhancer do gene NOV, uma característica de enhancers poised (prontos para ativação). Em conclusão, nós fornecemos a primeira evidência experimental para um mecanismo de ação do oncogene lincRNA PVT1 em células de câncer de próstata e demonstramos que sua ação inibidora da expressão afeta genes alvo que facilitam a proliferação e migração de células do câncer de próstata, sugerindo que o lincRNA PVT1 é um novo agente no complexo mecanismo de repressão transcricional envolvendo um RNA silenciador, o receptor de andrógeno (AR) e o potenciador de Zeste homólogo 2 (EZH2) no remodelamento da cromatina em células LNCaP

Long non-coding RNA (lncRNA) plasmacytoma variant translocation 1 (PVT1) is an oncogene known to be overexpressed in various types of cancer. PVT1 lincRNA is located in the wellknown cancer-related genomic region 8q24, also known as 'gene desert. PVT1 lincRNA level of expression is associated with increased prostate cancer (PCa) risk and is correlated with androgen receptor (AR) expression levels. However, the mechanism of PVT1 and AR involvement in the development of prostate cancer is still unclear. Here, we tested the hypothesis that formation of the complex AR-EZH2-PVT1 participates in the regulation of gene expression in prostate cancer, in LNCaP cells. Ribonucleoprotein immunoprecipitation followed by quantitative PCR (RIP-qPCR) revealed that PVT1 lincRNA binds both the AR (12 % of PVT1 input) and the methyltransferase EZH2 from the Polycomb repressive complex 2 (36 % of input) under androgen-supplemented conditions (+R1881). PVT1 also binds both AR (10 % of input) and EZH2 (42 % of input) under androgen-deprived conditions (-R1881). Thus, PVT1 binding to AR and EZH2 is independent of the androgen hormone. Using a large-scale loss and gain of function approach, our results show that PVT1 knockdown (KD) in LNCaP in the presence of androgen restores the expression partially, fully or causes overexpression of 160 genes that are inhibited by androgen. Among these genes, we highlight genes involved in regulation of cell differentiation, in components of cell-cell junction, in inhibition of cell migration and invasion and in triggering of the apoptotic pathway. Chromatin immunoprecipitation followed by quantitative PCR (ChIP-qPCR) with LNCaP cells in androgen-supplemented cultures under PVT1 lincRNA knockdown showed a significant increase in occupancy by the histone activation mark H3K27Ac of the promoter region of the NOV gene, one of the genes that had an increased expression upon PVT1 silencing. ChIPqPCR also showed a significant increase upon PVT1 lincRNA silencing of the H3K27me3 histone mark in the enhancer region of the NOV gene, a distinct feature of poised enhancers. In conclusion, we provide first experimental evidence for a mechanism of action of PVT1 lincRNA oncogene in prostate cancer cells, and show that its inhibitory action affects targetgenes that facilitate proliferation and migration of prostate cancer cells, thus suggesting PVT1 lincRNA as a novel lncRNA player in the complex mechanism of transcriptional repression involving a silencer RNA, the androgen receptor (AR) and the Enhancer of zeste homolog 2 (EZH2) in chromatin remodeling in LNCaP cells

Morphoproteomics and Biomedical Analytics Identify the c-Myc, EZH2, Sirt1 and CXCR4 Pathways as Potential Blocks to Differentiation Leading to Proliferation and Metastatic Potential in Sinonasal Undifferentiated Carcinoma (SNUC) and Provide Therapeutic Options: A Case Study.

Sinonasal undifferentiated carcinoma (SNUC) is a highly malignant tumor in the nasal cavity or paranasal sinuses. Morphoproteomics has defined its biology to some degree, allowing the identification of targeted therapeutic options with clinical efficacy [1]. This study's objective was to identify putative SNUC pathways that are known to pose a block in differentiation both in early embryogenesis and in tumorigenesis or that might promote metastasis and recurrent disease. DESIGN: Morphoproteomic analysis of SNUC from a case study of this patient included immunohistochemical probes to detect c-Myc, EZH2, Sirt1 and CXCR4 protein analytes. Biomedical analytics schematically showed the interactions of these analytes with the morphoproteomic findings and illustrated targeted therapeutic options. RESULTS: Representative sections of this patient's tumor displayed plasmalemmal expression for CXCR4 and nuclear immunopositivity for c-Myc, EZH2, and Sirt1. This coincided with their block in differentiation and their proliferative state with progression into the mitotic phase. Biomedical analytics integrated the morphoproteomic findings with the undifferentiated and proliferative state of SNUC and depicted pharmacogenomic and other related factors that target the c-Myc, EZH2, Sirt1 and CXCR4 pathways. CONCLUSION: Morphoproteomics and biomedical analytics have identified c-Myc, EZH2, Sirt1 and CXCR4 pathways that collectively could contribute to the block in differentiation and increase the propensity for recurrence and metastasis in SNUC. This suggests that combinatorial therapies modulating these pathways could be used in a maintenance mode to minimize the risk of recurrent disease.

Concomitant 1p36 deletion and TNFRSF14 mutations in primary cutaneous follicle center lymphoma frequently expressing high levels of EZH2 protein.

Primary cutaneous follicle center lymphoma (PCFCL) is an indolent variant of follicular lymphoma (FL) with limited information available on the genetic background of the disease. The genetic hallmark of nodal FL, the t(14;18) translocation, affecting the BCL2 gene, is rare in PCFCL. Loss of 1p36, the most common secondary chromosomal abnormality in nodal FL, has been recently reported in 16.7% of PCFCL cases. In order to further characterize PCFCL, 21 cases were analyzed using interphase fluorescence in situ hybridization with BCL2 break apart and 1p36/1q25 dual color probes. Sanger sequencing was used to investigate TNFRSF14 and EZH2 mutations and immunohistochemistry to assess BCL2, EZH2 protein expressions.1p36 deletion occurred in 22% (5/21), BCL2 gene break in 10% (2/20) of the PCFCL cases. Mutations of the candidate tumor suppressor gene of the 1p36 region, TNFRSF14 mutations were detected in 4/17 (23.5%) cases with 2 cases presenting with concurrent 1p36 deletion. EZH2 hotspot mutations at Y641, A682, and A692 were not found. High EZH2 protein expression associated with a BCL2 negative phenotype was observed in 43% (9/21) of the cases. BCL2 gene break or 1p36 deletion did not impact the prognosis; however, they showed association with advanced stages at diagnosis (p = 0.016) and a tendency with shorter event free survival (p = 0.052).In conclusion, 1p36 deletion co-occurs with acquired TNFRSF14 mutations, suggesting a role of this tumor suppressor gene in the development of a subgroup of PCFCL. High EZH2 protein expression associated with BCL2 negative phenotype is common and might represent an ideal therapeutic target.

Estrogen-related receptors alpha, beta and gamma expression and function is associated with transcriptional repressor EZH2 in breast carcinoma.

BACKGROUND: Orphan nuclear receptors ERRα, ERRß and ERRγ that belong to NR3B or type IV nuclear receptor family are well studied for their role in breast cancer pathophysiology. Their homology with the canonical estrogen receptor dictates their possible contributing role in mammary gland development and disease. Although function and regulation of ERRα, ERRγ and less about ERRß is reported, role of histone methylation in their altered expression in cancer cells is not studied. Transcriptional activity of nuclear receptors depends on co-regulatory proteins. The present study for the first time gives an insight into regulation of estrogen-related receptors by histone methylation specifically through methyltransferase EZH2 in breast cancer. METHODS: Expression of ERRα, ERRß, ERRγ and EZH2 was assessed by immunohistochemistry in four identical tissue array slides that were prepared as per the protocol. The array slides were stained with ERRα, ERRß, ERRγ and EZH2 simultaneously. Array data was correlated with expression in MERAV expression dataset. Pearson correlation coeficient r was calculated from the partial matrix expression values available at MERAV database to study the strength of association between EZH2 and three orphan nuclear receptors under study. By western blot and real time PCR, their correlated expression was studied in breast cancer cell lines MCF-7, MDA-MB-231, T47D and MDA-MB-453 including normal breast epithelial MCF-10A cells at both protein and RNA level. Regulation of ERRα, ERRß, ERRγ by EZH2 was further investigated upon overexpression and silencing of EZH2. The interaction between ERRs and EZH2 was validated in vivo by CHIP-qPCR. RESULTS: We found a negative correlation between estrogen-related receptors and Enhancer of Zeste Homolog 2, a global repressor gene. Immunohistochemistry in primary breast tumors of different grades showed a correlated expression of estrogen-related receptors and EZH2. Their correlated expression was further validated using online MERAV expression dataset where a negative correlation of variable strengths was observed in breast cancer. Ectopic expression of EZH2 in low EZH2-expressing normal breast epithelial cells abrogated their expression and at the same time, its silencing enhanced the expression of estrogen-related receptors in cancerous cells. Global occupancy of EZH2 on ERRα and ERRß was observed in-vivo. CONCLUSION: Our findings identify EZH2 as a relevant coregulator for estrogen-related receptors in breast carcinoma.