PICOT binding to the polycomb group protein, EED, alters H3K27 methylation at the MYT1 PRC2 target gene.

PICOT is a ubiquitous protein that has no functional redundant ortholog and is critical for mouse embryonic development. It is involved in the regulation of signal transduction in T lymphocytes and cardiac muscle, and in cellular iron metabolism and biogenesis of Fe/S proteins. However, very little is known about the physiological role of PICOT and its mechanism of action, and on its upstream regulators or downstream target molecules. In attempt to identify new PICOT interaction partners, we adopted the yeast two-hybrid system and screened a Jurkat T cell cDNA library using the full-length human PICOT cDNA as a bait. We found that PICOT interacts with embryonic ectoderm development (EED), a Polycomb Group (PcG) protein that serves as a core component of the Polycomb repressive complex 2 (PRC2) and contributes to the regulation of chromatin remodeling and cell differentiation. Using bead immobilized GST-PICOT and GST-EED fusion proteins in a pull-down assay and reciprocal coimmunoprecipitation studies we demonstrated that the interaction between PICOT and EED also occurs in human Jurkat T cells. In addition, immunofluorescence staining of Jurkat T cells revealed partial colocalization of PICOT and EED, predominantly in the cell nuclei. A pull-down assay using the GST-EED fusion protein and lysates of cells expressing different Myc-tagged truncation products of PICOT revealed that binding of EED is mediated by each of the two C-terminal PICOT homology domains and suggests that simultaneous interaction via both domains increases the binding affinity. Furthermore, PICOT knock-down in Jurkat T cells resulted in a reduced histone H3 lysine 27 trimethylation (H3K27me3) at the PRC2 target gene, myelin transcription factor 1 (MYT1), suggesting that PICOT binding to EED alters PRC2-regulated transcriptional repression, and potentially contributes to the epigenetic regulation of chromatin silencing and remodeling.

Aberrant differential expression of EZH1 and EZH2 in Polycomb repressive complex 2 among B- and T/NK-cell neoplasms.

The Polycomb repressive complex-2 members (EZH2, EED, SUZ12 and EZH1) are important regulators of haematopoiesis, cell cycle and differentiation. Over-expression of EZH2 has been linked to cancer metastases and poor prognosis. Detailed information on the expression of other members in normal and neoplastic lymphoid tissue remains to be elucidated. Immunohistochemical and immunofluorescent analyses of 156 samples from haematopoietic neoplasms patients and 27 haematopoietic cell lines were used. B-cell neoplasms showed a significant over-expression of EZH2, EED and SUZ12 in the aggressive subtypes compared to the indolent subtypes and normal tissue (p = 0.000-0.046) while expression of EZH1 was decreased in mantle cell lymphoma compared to normal tissue (p = 0.011). T/NK-cell neoplasms also showed significant over-expression of EZH2, EED and SUZ12 (p = 0.000-0.002) and decreased expression of EZH1 (p = 0.001) compared to normal cells. EZH2 and EZH1 have opposite expression patterns both in normal and neoplastic lymphoid tissues as well as an opposite relation to Ki-67. These results were supported by western blotting analyses. Immunofluorescent staining revealed a difference in the intracellular localisation of EZH1 compared to other members. These evidences suggest that EZH2 and EZH1 are important in the counter-balancing mechanisms controlling proliferation/resting of lymphoid cells. The disruption of the balanced EZH2/EZH1 ratio may play important roles in the pathogenesis of lymphomas.

EZH2 overexpression in different immunophenotypes of breast carcinoma and association with clinicopathologic features.

BACKGROUND: Enhancer of zest homolog 2 (EZH2), a histone 3 methyltransferase, is associated with aggressive behavior of many tumors and is a promising target of molecular therapy. METHODS: To better elucidate the relevance of EZH2 in breast cancer subtypes, we evaluated EZH2 expression in 226 invasive breast carcinomas with four distinct immunophenotypes and in association with clinicopathological features. RESULTS: Of these cases, 138 (61.1 %) were defined as EZH2-overexpressing with a multiplicative score > 3. EZH2 expression was inversely related to the status of ER and PR (Chi-square, p < 0.001), and it was significantly associated with HER2 positivity, high proliferative index, and high histologic grade (Chi-square, p < 0.05). ER-positive breast carcinoma with low proliferative index (Ki67 < 14 %) showed the lowest expression and triple-negative breast carcinoma showed the highest overexpression of EZH2, 18.5 % (10/54) versus 90.9 % (50/55) (Chi-square, p < 0.001). Intriguingly, 88 % (44/50) cases of grade 3 triple-negative breast carcinoma showed uniformly strong EZH2 expression with a multiplicative score of 9. The percentage of EZH2 overexpression in ER-positive breast carcinoma with a high proliferative index or HER2-positive cases were 61.2 and 74 %, respectively. Furthermore, EZH2 expression was significantly elevated in high-grade DCIS compared to benign lesions (90 % versus 0, p < 0.001). However, there is no association between EZH2 expression and the status of histone 3 lysine 27 trimethylation or other clinicopathologic features. CONCLUSION: In summary, triple-negative breast carcinoma showed the highest overexpression of EZH2. EZH2 overexpression is associated aggressive pathologic features including high nuclear grade, high proliferative index, and positivity of HER2 of breast carcinoma.

Megakaryocytic hyperplasia in myeloproliferative neoplasms is driven by disordered proliferative, apoptotic and epigenetic mechanisms.

AIMS: Myeloproliferative neoplasms (MPN) are a heterogeneous group of clonal proliferative bone marrow diseases characterised by extensive megakaryocytic hyperplasia and morphological atypia. Despite knowledge of genomic defects, the pathobiological processes driving these megakaryocytic abnormalities in MPN remain poorly explained. We have explored the proliferative, apoptotic and epigenetic profiles of megakaryocytes in human MPN. METHODS: Immunohistochemical staining was performed on bone marrow trephine biopsies of 81 MPN (with and without JAK2(V617F) and CALR mutations) and 15 normal controls to assess the megakaryocytic expression of biomarkers associated with proliferation (Ki67), apoptosis (Bcl-XL, BNIP-3) and epigenetic regulation (EZH2, SUZ12). RESULTS: Myeloproliferative megakaryocytes showed significantly greater expression of proliferative Ki67 and anti-apoptotic Bcl-XL, reduced pro-apoptotic BNIP-3 and increased SUZ12 compared with controls. In essential thrombocythaemia, large-giant megakaryocytes with hyperlobated nuclei showed a trend towards a proliferative signature. In contrast, myelofibrotic megakaryocytes with condensed nuclear chromatin, and cases with CALR mutations, had significant reductions in pro-apoptotic BNIP-3. CONCLUSIONS: Uncontrolled megakaryocytic expansion in MPN results from a combination of increased proliferation, attenuated apoptosis and defective epigenetic regulation with CALR mutations favouring apoptotic failure. The higher platelet counts reported to be seen in MPN with CALR mutations may be due to greater dysregulation of megakaryocyte apoptosis.

R loops regulate promoter-proximal chromatin architecture and cellular differentiation.

Numerous chromatin-remodeling factors are regulated by interactions with RNA, although the contexts and functions of RNA binding are poorly understood. Here we show that R loops, RNA-DNA hybrids consisting of nascent transcripts hybridized to template DNA, modulate the binding of two key chromatin-regulatory complexes, Tip60-p400 and polycomb repressive complex 2 (PRC2) in mouse embryonic stem cells (ESCs). Like PRC2, the Tip60-p400 histone acetyltransferase complex binds to nascent transcripts; however, transcription promotes chromatin binding of Tip60-p400 but not PRC2. Interestingly, we observed higher Tip60-p400 and lower PRC2 levels at genes marked by promoter-proximal R loops. Furthermore, disruption of R loops broadly decreased Tip60-p400 occupancy and increased PRC2 occupancy genome wide. In agreement with these alterations, ESCs partially depleted of R loops exhibited impaired differentiation. These results show that R loops act both positively and negatively in modulating the recruitment of key pluripotency regulators.

Cancer stem cell markers in prostate cancer: an immunohistochemical study of ALDH1, SOX2 and EZH2.

The aims of this study were to investigate the immunohistochemical expression and potential prognostic significance of putative cancer stems cell markers ALDH1, EZH2 and SOX2 in prostate cancer.A total of 142 consecutive radical prostatectomies submitted to one laboratory with a diagnosis of prostatic adenocarcinoma between 2008 and 2012 were retrieved and retrospectively studied. Immunohistochemistry for the three markers was performed in each case and both univariate and multivariate analyses were undertaken to evaluate the correlation between the staining patterns and known histopathological prognostic features.ALDH1 showed a statistically significant association with tumour stage p < 0.001), extraprostatic extension (p < 0.001) and lymphovascular invasion (p = 0.001). EZH2 correlated with Gleason score (p = 0.044) and lymph node metastases (p = 0.023). SOX2 showed a statistically significant correlation with lymphovascular invasion only (p = 0.018) in both univariate and multivariate analyses.Cancer stem cell markers are variably expressed in prostate adenocarcinoma and immunohistochemical staining for ALDH1 and EZH2 may have a role in predicting tumour aggressiveness before treatment of prostate cancer.

EZH2 is a sensitive marker of malignancy in salivary gland tumors.

BACKGROUND: The immunohistochemical detection of Enhancer of zeste homologue 2 (EZH2) proved to be a useful tool to recognize the malignant nature of tumors in a wide variety of neoplasms. The histological diagnostics of salivary gland tumors is a challenging task, and a reliable marker of malignancy would be extremely helpful. METHODS: EZH2 expression was investigated in 54 malignant and 40 benign salivary gland tumors of various histological types by standard immunohistochemistry. RESULTS: The majority (n = 52) of the malignant tumors stained positively, while all the investigated benign tumors were negative for EZH2. CONCLUSIONS: EZH2 expression in salivary gland tumors, similarly to the tumors of other organs is not characteristic for any tumor type, but is a solid marker of the malignant nature of the tumors.

Clinical and prognostic relevance of EZH2 in breast cancer: A meta-analysis.

The polycomb group protein enhancer of zeste homolog 2 (EZH2) is regarded as a tightly linking oncogene in many types of cancer. However, the prognostic role of EZH2 in breast cancer (BC) still remains controversial. Our study aimed to evaluate the clinical and prognostic relevance of EZH2 in BC patients based on published studies. 11 studies totally containing 2330 patients (1052 EZH2-positive and 1278 EZH2-negative) were included in our meta-analysis. Our data showed that EZH2 over-expression was significantly associated with estrogen receptor (ER) negativity [OR=0.227, 95% CI=0.174-0.297, P=0.000], progesterone receptor (PR) negativity [OR=0.454, 95% CI=0.300-0.687, P=0.000], human epidermal growth factor receptor type 2 (HER-2) positivity [OR=1.846, 95% CI=1.366-2.496, P=0.000], invasive ductal cancer (IDC) [OR=2.237, 95% CI=1.489-3.361, P=0.000], race (Caucasian) [OR=0.707, 95% CI=0.522-0.957, P=0.025], high histological grade [OR=3.177, 95% CI=2.012-5.014, P=0.000] and triple-negative status (TNBCs) [OR=5.380, 95% CI=1.065-27.187, P=0.042], which led to a poor OS rate in BC [RR=2.193, 95% CI=1.495-3.217, P=0.000]. In conclusion, EZH2 participated in the progression of BC as a putative factor, and over-expression of EZH2 was distinctly correlated with a poor patient survival. EZH2 may serve as a prognostic biomarker and target in BC patients.

Epigenetic modifications in cutaneous malignant melanoma: EZH2, H3K4me2, and H3K27me3 immunohistochemical expression is enhanced at the invasion front of the tumor.

Cancer stem cells and the misregulation of epigenetic modifications have been identified to possess a determinative role in carcinogenesis. The purpose of this study was to investigate the expression profile of EZH2 and H3K4me2 and H3K27me3, which constitute stem cell-like "bivalent" domains, in cutaneous malignant melanoma. A comparative analysis of their immunohistochemical expression between the invasion front (IF) and the inner tumor mass was also evaluated. Immunohistochemical methodology was performed on sections of 89 melanoma lesions from 79 patients. The 3 markers studied were identified in the cell nuclei of melanoma cells, nevus cells, and normal epidermal keratinocytes. A specific distribution pattern of H3K4me2 and H3K27me3 was found, as stronger levels were localized at the IF of the tumor (P = 0.034 and P < 0.01, respectively). In general, H3K4me2 and H3K27me3 levels were lower in metastatic with respect to primary melanoma cases (P = 0.0065 and P = 0.027, respectively). Advanced melanoma demonstrated significantly lower H3K4 immunohistochemical expression than did cases of lowest Clark level (I) (P = 0.038) or low Breslow depth (≤1 mm; P < 0.001). Furthermore, EZH2 expression in melanoma cells was higher compared with that in nevus cells (P = 0.02). A positive correlation between EZH2-H3K27me3 (P = 0.03) and H3K4me2-H3K27me3 (P < 0.01) in melanoma cells was also found. Our results suggest the possibility that combined immunohistochemical expression of EZH2, H3K4me2, and H3K27me3 might identify cancer cells with potential stem cell properties, particularly at the IF of this malignancy. This hypothesis should be further investigated, as many of the epigenetic changes are reversible via pharmacologic manipulations and new therapies, overpassing the resistance of advanced melanoma, may be developed.

Bivalent histone modifications during tooth development.

Histone methylation is one of the most widely studied post-transcriptional modifications. It is thought to be an important epigenetic event that is closely associated with cell fate determination and differentiation. To explore the spatiotemporal expression of histone H3 lysine 4 trimethylation (H3K4me3) and histone H3 lysine 27 trimethylation (H3K27me3) epigenetic marks and methylation or demethylation transferases in tooth organ development, we measured the expression of SET7, EZH2, KDM5B and JMJD3 via immunohistochemistry and quantitative polymerase chain reaction (qPCR) analysis in the first molar of BALB/c mice embryos at E13.5, E15.5, E17.5, P0 and P3, respectively. We also measured the expression of H3K4me3 and H3K27me3 with immunofluorescence staining. During murine tooth germ development, methylation or demethylation transferases were expressed in a spatial-temporal manner. The bivalent modification characterized by H3K4me3 and H3K27me3 can be found during the tooth germ development, as shown by immunofluorescence. The expression of SET7, EZH2 as methylation transferases and KDM5B and JMJD3 as demethylation transferases indicated accordingly with the expression of H3K4me3 and H3K27me3 respectively to some extent. The bivalent histone may play a critical role in tooth organ development via the regulation of cell differentiation.

The prognostic role of EZH2 expression in rectal cancer patients treated with neoadjuvant chemoradiotherapy.

BACKGROUND: Neoadjuvant chemoradiotherapy (nCRT) combined with surgery has been implemented as a standard treatment strategy in locally advanced rectal cancer (LARC). However, there is a wide spectrum of response to nCRT. The aim of this study was to determine whether enhancer of zeste homologue 2 (EZH2 ) expression could predict response to nCRT and outcomes for patients in LARC. METHOD: The study examined the EZH2 expression in 112 biopsies by immohistochemistry. The associations between EZH2 and clinical characters were analyzed. RESULTS: EZH2 expression in biopsy tissue was significantly related to increased tumor cell proliferation, as assessed by Ki-67 expression with a cutoff value of 37% (p <0.001). High EZH2 expression was correlated closely with low differentiation (p = 0.029), high CEA level (p = 0.041), T4 status (p = 0.011) and node metastasis (p =0.045). By univariate and multivariate analysis, we observed low EZH2 expression could reliably and independently predict the good response to nCRT ( p = 0.026 and p = 0.023) and down-staging ( p = 0.021 and p = 0.027). In univariate analysis, high EZH2 expression was significantly associated with poor 5-year disease-free survival (p = 0.025) and 5-year overall survival (p = 0.032). In multivariate analysis, EZH2 was a prognostic factor for 5-year DFS (HR = 2.287; 95% CI 1.137-4.602, p = 0.020) but not for 5-year OS (HR = 2.182; 95% CI 0.940-5.364, p = 0.069). CONCLUSION: Our study revealed that low EZH2 expression in biopsy tissue might be a useful predictive factor of good tumor response to nCRT and longer 5-year DFS in patients with LARC. However this is a relatively small retrospective study, to further validate the role of EZH2 in rectal cancer, large consistent cohort studies are needed.

EZH2 expression correlates with locoregional recurrence after radiation in inflammatory breast cancer.

BACKGROUND: Enhancer of zeste homolog 2 (EZH2), a member of the polycomb group proteins, has been shown to promote cancer progression and breast cancer stem cell (CSC) expansion. Breast CSCs are associated with resistance to radiation in inflammatory breast cancer (IBC), a rare but aggressive variant of breast cancer. In this retrospective study, we examined the clinical role of EZH2 in locoregional recurrence (LRR) of IBC patients treated with radiation. PATIENTS AND METHODS: 62 IBC patients who received radiation (7 pre-operative, 55 post-operative) and had adequate follow up to assess LRR were the subject of this study. Positive EZH2 status was defined as nuclear immunohistochemical staining in at least 10% of invasive cancer cells. Association of EZH2 expression with clinicopathologic features were evaluated using the Chi-square statistic and actuarial LRR free survival (LRFS) was determined using the Kaplan-Meier method. RESULTS: The median follow-up for this cohort was 33.7 months, and the 5-year overall LRFS rate was 69%. Of the 62 patients, 16 (25.8%) had LRR, and 15 out of 16 LRR occurred in EZH2 expressing cases. Univariate analysis indicated that patients who had EZH2-positive IBC had a significantly lower 5-year locoregional free survival (LRFS) rate than patients who had EZH2-negative IBC (93.3% vs. 59.1%; P = 0.01). Positive EZH2 expression was associated significantly with negative ER status (97.1% in ER- vs 48.1% in ER+; P < 0.0001) and triple-negative receptor status (P = 0.0001) and all triple-negative tumors were EZH2-positive. In multivariate analysis, only triple negative status remained an independent predictor of worse LRFS (hazard ratio 5.64, 95% CI 2.19 - 14.49, P < 0.0001). CONCLUSIONS: EZH2 correlates with locoregional recurrence in IBC patients who received radiation treatment. EZH2 expression status may be used in addition to receptor status to identify a subset of patients with IBC who recur locally in spite of radiation and may benefit from enrollment in clinical trials testing radiosensitizers.

Histone-modifying genes as biomarkers in hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is the world's fifth most common cancer and second leading cause of cancer-related death in Taiwan. Over 600,000 HCC patients die each year worldwide despite recent advances in surgical techniques and medical treatments. Epigenetic regulations including DNA methylation and histone modification control gene expressions and play important roles during tumorigenesis. This study evaluates association between histone-modifying genes and prognosis of HCC to ferret out new diagnostic markers. We collected 50 paired HCC and adjacent non-cancerous tissues from Taiwanese patients for survey by RT-qPCR and tissue microarray-based immunohistochemistry (TMA-based IHC) staining. RT-qPCR data showed four of twenty-four genes over eightfold up-regulated in tumor tissues: e.g., histone phosphorylation gene-ARK2, methylation genes-G9a, SUV39H2, and EZH2 (n=50, all p<0.0001). Results of TMA-based IHC staining showed proteins of ARK2, EZH2, G9a, and SUV39H2 also overexpressed in tumor tissues. Staining intensity of SUV39H2 correlated with HCV infection (p=0.025). We further restricted the analysis only in tumor tissues, we found EZH2 staining intensity associated with tumor stage (p=0.016) and survival (p=0.007); SUV39H2 intensity associated with tumor stage (p=0.044). Our findings indicate overexpression of histone-modifying genes EZH2 and SUV39H2 associated with prognosis of HCC cases. EZH2 expression can serve as a novel prognostic biomarker during HCC progression among Taiwanese.

Immunostaining for polycomb group protein EZH2 and senescent marker p16INK4a may be useful to differentiate cholangiolocellular carcinoma from ductular reaction and bile duct adenoma.

Intrahepatic cholangiocarcinoma arising in chronic advanced liver disease sometimes contains a component of cholangiolocellular carcinoma. Bile duct adenoma, a benign tumor/tumorous lesion and ductular reaction, is also composed of bile ductular cells, and the differential diagnosis is sometimes difficult. We have previously reported that cholangiolocellular carcinoma showed overexpression of a polycomb group protein EZH2, which participates in bypass/escape from cellular senescence during carcinogenesis. In contrast, the ductular reaction showed high expression of senescence-associated p16(INK4a). In this study, we examined whether immunostaining for EZH2 and p16(INK4a) is useful for differential diagnosis among cholangiolocellular carcinoma, bile duct adenoma, and ductular reactions. Subjects included 33 patients with intrahepatic cholangiocarcinoma and combined hepatocellular-cholangiocarcinoma with components of cholangiolocellular carcinoma and 16 patients with bile duct adenoma. The expressions of EZH2 and p16(INK4a) were examined immunohistochemically. The expression of EZH2 was seen in all cases of cholangiolocellular carcinomas, but it was not observed in bile duct adenomas and ductular reactions, which were seen around carcinomas in 80% of cases. The extensive expression of p16(INK4a) was seen only in 4 cases of cholangiolocellular carcinomas (12%). In contrast, the expression of p16(INK4a) was seen in 13 cases (81%) of bile duct adenomas and in all cases of ductular reactions. The borderline between the component of cholangiolocellular carcinoma and the surrounding ductular reaction was clearly highlighted by the reverse expression pattern of EZH2 and p16(INK4a) in 69% of cases. In conclusion, immunostaining for EZH2 and p16(INK4a) may be useful for differential diagnosis among cholangiolocellular carcinomas, bile duct adenomas, and ductular reactions.

EZH2, a unique marker of malignancy in effusion cytology.

Distinguishing reactive mesothelial cells from metastatic disease, typically adenocarcinoma, in effusion cytology can be challenging at times. We currently use a panel of immunocytochemical markers for select cases including MOC-31 and BerEp4, but difficulties still exist. Enhancer of zeste homologue 2 (EZH2) plays important roles in epigenetic silencing and cell cycle regulation and is upregulated in a wide variety of malignancies. Thus, we hypothesized that EZH2 immunocytochemistry, which to our knowledge has not yet been reported on cytology material, might serve as a unique marker of malignancy in morphologically equivocal effusion specimens by highlighting aberrant protein expression in malignant cells. A total of 96 (48 benign and 48 malignant) effusion cases were selected retrospectively from our department archives. All malignant cases were metastatic adenocarcinomas except for three high grade neuroendocrine carcinomas (two lungs and one ovary), one cervical squamous cell carcinoma, and one epithelioid endometrial stromal sarcoma. The 48 benign cases were all negative for EZH2, and 43 of 48 malignant effusions were positive. As a solitary marker, EZH2 exhibited a sensitivity of 90% and a specificity of 100% (P < 0.0001). EZH2 functioned as a unique and accurate marker of malignancy in this series of effusions. Relative to published data, EZH2 demonstrated a sensitivity comparable to MOC-31 and superior to BerEp4, and a specificity superior to both of these commonly used immunostains. Thus, EZH2 is likely to be of great value as an adjunct to morphology in diagnosing malignancy in effusion specimens.

Prognostic value of EZH2, paxillin expression and DNA ploidy of breast adenocarcinoma: correlation to pathologic predictors.

PURPOSE: The objective of this study was to examine the association of EZH2 and paxillin expression and DNA ploidy status with pathological parameters of breast cancer, aiming to correlate tumor phenotype with its malignant behavior. METHODS: EZH2 and paxillin expression and DNA ploidy were evaluated in imprint smear samples obtained from 105 breast tumors after surgical removal. RESULTS: Increased expression of paxillin was associated with p53 expression (p=0.005), Ki-67 expression (p=0.018) and EZH2 expression (p<0.0001). EZH2 expression correlated with estrogen receptor (ER) and progesterone receptor (PR) status (p=0.01 and p=0.035, respectively), and expression of p53 and Ki-67 (p=0.007 and p<0.0001, respectively). Aneuploid tumors were significantly correlated with poor differentiation (p=0.000), stage of disease (p=0.000), size of the primary tumor (p=0.015), presence of nodal metastasis (p=0.001), ER status (p=0.008), cerbB2 status (p=0.012), and expression of Ki-67 (p=0.001) and EGFR (p=0.018). Multivariate analysis of ploidy results using paxillin and EZH2 expression as dependent variables revealed that aneuploid tumors were associated with disease stage and grade of differentiation, cerbB2 expression and EZH2 expression. CONCLUSION: Our results show that aneuploid tumors, EZH2 expression and paxillin expression correlate with more aggressive phenotype of breast cancer.

Immunohistochemical analysis of E-cadherin and zeste homolog 2 expression in endoscopic ultrasound-guided fine-needle aspiration of pancreatic adenocarcinoma.

BACKGROUND: Recent studies have demonstrated that partial or complete loss of E-cadherin (EC) and nuclear accumulation of zeste homolog 2 (EZH2) are hallmarks of poorly differentiated pancreatic adenocarcinoma (PAC). Depletion of EZH2 sensitizes cancer cells to chemotherapy in vitro. The objective of this study was to determine EC and EZH2 expression by immunohistochemistry (IHC) in samples obtained by endoscopic ultrasound-guided fine-needle aspiration (EUS-FNA) as potential biomarkers for treatment and disease prognosis. METHODS: Thirty-eight EUS-FNA samples from patients with a PAC diagnosis were analyzed by IHC for EC and EZH2 expression. Seven corresponding surgical resection specimens were included in the study. The intensity of EZH2 and EC expression in PAC and in normal gastrointestinal pick-ups (internal positive control) was scored by using a 4-tier grading system. RESULTS: EC demonstrated a focal, weak-to-moderate decrease in 24 PAC samples. Complete loss of EC expression was observed in the poorly differentiated areas represented by single tumor cells. The average staining intensity of EC in samples of poorly differentiated PAC was significantly lower than that of moderately differentiated PAC samples (P = .0005). EZH2 was variably positive in 31 of 38 PAC samples. The average staining intensity of EZH2 in moderately and poorly differentiated PACs did not differ significantly (P = .81). CONCLUSIONS: EC and EZH2 expression was determined reliably by IHC on paraffin sections of EUS-FNA cell block specimens. The current results were consistent with prior reports indicating a decrease or loss of EC expression in poorly differentiated PAC. However, EZH2 expression did not always correlate inversely with EC expression and was more heterogeneous.

Role of co-expression of c-Myc, EZH2 and p27 in prognosis of prostate cancer patients after surgery.

BACKGROUND: c-Myc, EZH2 and p27 were defined to modulate the behavior of prostate cancer with pro-tumoral or anti-tumoral effects and had ability in predicting prostate cancer progression, but the research of their co-expression value of prognosis is rarely. This study aimed to investigate the prognostic value of combining tri-marker together in patients with intermediate-risk prostate cancer after surgery. METHODS: Expression levels of c-Myc, EZH2 and p27 in 129 patients with intermediate-risk prostate cancer were assessed using immunohistochemistry in a semi-quantitative manner. The expression profiles of these three markers were analyzed and investigated for association with biochemical recurrence. RESULTS: In all, fifty of 129 cases experienced biochemical recurrence during a median follow-up time of 31 months (range, 6 - 60 months). Of these relapse patients, one case without and 10 cases with any single positive marker were observed; 39 cases were detected with any two or all three positive markers (22 cases with any two and 17 cases with all three positive markers). Survival analysis showed that patients with over-expression of c-Myc or EZH2, and lower expression of p27 manifested significantly higher biochemical recurrence rates. Subsequent multivariate analysis revealed that c-Myc, EZH2 and p27 expression statuses showed potential in predicting relapse, respectively. Notably, combining three markers together as a "composite index" (0 or 1, vs. 2 or 3 positive markers) provided powerful prognostic value (HR 6.57, 95% CI 3.02 - 14.31, P < 0.001). There was a significant difference between the patient subgroups with 0 or 1 and those with 2 or 3 positive markers expression statuses, and tri-marker composite index was an independent risk factor for predicting relapse in patients with intermediate-risk prostate cancer after surgery. CONCLUSION: Composite index of c-Myc, EZH2, and p27 can be valued as powerful prognosis parameter for intermediate-risk prostate cancer patients after the surgery, and postoperative adjuvant therapy can be adopted accordingly.

Significance of cyclooxygenase 2, EZH-2 polycomb group and p53 expression in actinic keratosis and squamous cell carcinomas of the skin.

The development and progression of squamous cell carcinoma (SCC) of the skin is characterized by an accumulation of molecular changes. The aim of this study was to investigate the association of the immunohistochemical expression of cyclooxygenase-2 (COX-2), enhancer of zeste homolog 2 (EZH-2), and p53 in actinic keratosis (AK) and SCC and detect any differences between invasive and preinvasive squamous epidermal lesions. Forty-three cases with AK, 38 with SCC, and 9 with SCC arising on AK (SCC/AK) were studied. For COX-2 immunostaining, weak or no reaction was associated with AK (58.10% of cases), whereas moderate or strong reaction with SCCs (34.2% and 39.5%, respectively). Furthermore, 88.9% of the "mixed" SCC/AK specimens demonstrated moderate reaction (χ2 = 29.924, P < 0.0001). For EZH-2 immunostaining, a weak or no reaction was observed in 62.8% of AK cases, whereas a moderate reaction was observed in 42.1% of SCCs and 77.8% of "mixed" SCC/AK cases (χ2 = 18.91, P = 0.001). Weak immunoreactivity of p53 was associated with AK (58.1%), moderate with SCC (44.7%), and strong with SCC/AK lesions (66.7%) (χ2 = 15.999, P = 0.003). COX-2, p53, but mainly EZH-2 immune expression seems to be strongly associated with the biological potential of squamous epidermal cells and seems to be differentiating SCC by comparison to AK of the skin. The value of the combined expression of these markers is worth being further investigated as an additional tool for diagnostic, prognostic, and possibly, therapeutic use.