Search

The prognostic importance of transcription factors promoting epithelial-mesenchymal transition (EMT) and angiogenesis has not been well explored in prostate cancer patients with long follow-up, nor the interplay between these factors. The objective of this study was to assess the individual protein expression and co-expression of Twist, Slug (Snai2), Snail (Snai1), and hypoxia-inducible factor-1 alpha (Hif-1α) in prostate cancer in relation to EMT, angiogenesis, hypoxia, tumour features, disease recurrence, and patient survival. Immunohistochemical staining was performed on tissue microarray sections from 338 radical prostatectomies with long follow-up. In addition, 41 cases of prostatic hyperplasia, 33 non-skeletal metastases, 13 skeletal metastases, and 33 castration-resistant prostate carcinomas were included. Our findings were validated in external gene expression data sets. Twist was overexpressed in primary prostate cancer and markedly reduced in distant metastases (p < 0.0005). Strong expression of Twist and Slug was associated with Hif-1α in localised prostate cancer (p ≤ 0.001), and strong Twist was associated with Hif-1α in castration-resistant carcinomas (p = 0.044). Twist, Slug, and increased Snail at the tumour stromal border were associated with vascular factors (p ≤ 0.045). Each of the three EMT-regulating transcription factors were associated with aggressive tumour features and shorter time to recurrence and cancer-specific death. Notably, the co-expression of factors demonstrated an enhanced influence on outcome. In the subgroup of E-cadherin low carcinomas, strong Slug was associated with shorter time to all end points and was an independent predictor of time to multiple end points, including cancer-specific death (hazard ratio 3.0, p = 0.041). To conclude, we demonstrate an important relation between EMT, hypoxia, and angiogenesis and a strong link between the investigated EMT regulators and aggressive tumour features and poor patient outcome in prostate cancer. Despite the retrospective nature of this long-term study, our findings could have a significant impact on the future treatment of prostate cancer, where tailored therapies might be directed simultaneously against epithelial-mesenchymal phenotypes, angiogenesis, and tumour hypoxia., (© 2021 The Authors. The Journal of Pathology: Clinical Research published by The Pathological Society of Great Britain and Ireland & John Wiley & Sons, Ltd.)

Background: TWIST1 and CD105, which contribute to tumor malignancy, are overexpressed in cancers. Accordingly, TWIST1 enhances epithelial-to-mesenchymal transition (EMT) and promotes the formation of cancer stem cells (CSCs). Also, CD105 is a neoangiogenesis marker in endothelial cells, which is introduced as a CSC marker in tumoral epithelial cells in several types of cancers. The present study was aimed to investigate expressions of TWIST1 and CD105 in colorectal cancer (CRC) patients., Methods: Expressions of TWIST1 and CD105 in 250 CRC tissue samples were evaluated using immunohistochemistry on tissue microarrays (TMAs). In this regard, TWIST1 expression was investigated in the subcellular locations (cytoplasm and nucleus), while CD105 was mapped in endothelial cells and cytoplasmic tumor cells of CRC tissues. The association between the expression of these markers and clinicopathological parameters, as well as survival outcomes were analyzed., Results: Results indicate a statistically significant association between higher nuclear expression levels of TWIST1 and distant metastases in CRC (P = 0.040) patients. In addition, it was shown that the increased nuclear expression of TWIST1 had a poor prognostic value for disease-specific survival (DSS) and progression-free survival (PFS) (P = 0.042, P = 0.043, respectively) in patients with CRC. Moreover, analysis of CD105 expression level has revealed that there is a statistically significant association between the increased expression of CD105 in tumoral epithelial cells and more advanced TNM stage (P = 0.050)., Conclusions: Our results demonstrate that nuclear TWIST1 and cytoplasmic CD105 expressions in tumor cells had associations with more aggressive tumor behavior and more advanced diseases in CRC cases.

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.

Undifferentiated carcinoma (UC) and undifferentiated carcinoma with osteoclast-like giant cells (UCOGC) are peculiar variants of pancreatic ductal adenocarcinoma (PDAC), characterized by hypercellularity and absence of glandular patterns. The inflammatory microenvironment is peculiar in UCOGC, since it is dominated by macrophages and osteoclast-like giant cells. However, from a molecular point of view, both UC and UCOGC are very similar to conventional PDAC, sharing alterations of the most common genetic drivers. Clinically, UC usually show a worse prognosis, whereas UCOGC may show a better prognosis if it is not associated with a PDAC component. To highlight potential biological differences between these entities, we investigated the role of the epithelial to mesenchymal transition (EMT) in UC and UCOGC. Specifically, we analyzed the immunohistochemical expression of three well-known EMT markers, namely Twist1, Snai2, and E-cadherin, in 16 cases of UCOGC and 10 cases of UC. We found that EMT is more frequently activated in UC (10/10 cases) than in UCOGC (8/16 cases; p = 0.05). Furthermore, in UCOGC, EMT was activated with a higher frequency in cases with an associated PDAC component. Snai2 was the most frequently and strongly expressed marker in both tumor types (10/10 UC, 8/16 UCOGC), and its expression was higher in UC than in UCOGC (mean immunohistochemical score: 4.8 in UC vs. 2.1 in UCOGC, p < 0.01). Our results shed new light on the biology of UC and UCOGC: EMT appeared as a more important process in UC, and Snai2 emerged as a central EMT effector in this setting.

Introduction: We have reported previously that fecal DNA testing of TWIST1 methylation in combination with the fecal immunochemical test for hemoglobin (FIT) (combination test) is useful for colorectal neoplasia screening. In this study, using larger sample sizes, we studied the clinical performance of the combination test for the detection of colorectal neoplasia and, especially, advanced colorectal adenoma., Methods: We performed a prospective study in which FIT, fecal DNA testing of TWIST1 methylation, and colonoscopy were performed on 372 patients with colorectal neoplasia and 71 subjects without colorectal neoplasia. We assessed the individual clinical performance of each of FIT and fecal DNA testing of TWIST1 methylation and of the combination test for the detection of colorectal neoplasia including advanced adenoma based on morphologic subtypes., Results: The FIT alone had a sensitivity of 7.5% (3/40) for nonadvanced adenoma, 32.3% (41/127) for advanced adenoma, and 93.7% (192/205) for colorectal cancer and a specificity of 87.3% (62/71). The combination test had a sensitivity of 35.0% (14/40) for nonadvanced adenoma, 68.5% (87/127) for advanced adenoma, and 95.6% (196/205) for colorectal cancer and a specificity of 80.3% (57/71). For morphological subtypes of advanced adenoma, the sensitivity of FIT was only 28.2% (20/71) for polypoid type and 16.1% (5/31) for nonpolypoid type, whereas the combination test increased the sensitivities to 64.8% (46/71) and 71.0% (22/31), respectively., Discussion: The combination of the fecal DNA test with FIT seemed to be useful to detect colorectal neoplasia and, especially, advanced adenoma of the nonpolypoid type.

Several subtypes of high-grade endometrial carcinomas (ECs) contain an undifferentiated component of non-epithelial morphology, including undifferentiated and dedifferentiated carcinomas and carcinosarcomas (CSs). The mechanism by which an EC undergoes dedifferentiation has been the subject of much debate. The epithelial-mesenchymal transition (EMT) is one of the mechanisms implicated in the transdifferentiation of high-grade carcinomas. To improve our understanding of the role of EMT in these tumors, we studied a series of 89 carcinomas including 14 undifferentiated/dedifferentiated endometrial carcinomas (UECs/DECs), 49 CSs (21 endometrial, 29 tubo-ovarian and peritoneal), 17 endometrioid carcinomas (grade 1-3), and 9 high-grade serous carcinomas of the uterus, using a panel of antibodies targeting known epithelial markers (Pan-Keratin AE1/AE3 and E-cadherin), mesenchymal markers (N-cadherin), EMT transcription factors (TFs) (ZEB1, ZEB2, TWIST1), PAX8, estrogen receptors (ER), progesterone receptors (PR), and the p53 protein. At least one of the three EMT markers (more frequently ZEB1) was positive in the sarcomatous component of 98% (n = 48/49) of CSs and 98% (n = 13/14) of the undifferentiated component of UEC/DEC. In addition, 86% of sarcomatous areas of CSs and 79% of the undifferentiated component of UEC/DEC expressed all three EMT-TFs. The expression of these markers was associated with the loss of or reduction in epithelial markers (Pan-keratin, E-cadherin), PAX8, and hormone receptors. In contrast, none of the endometrioid and serous endometrial carcinomas expressed ZEB1, while 6% and 36% of endometrioid and 11% and 25% of serous carcinomas focally expressed ZEB2 and TWIST1, respectively. Although morphologically different, EMT appears to be implicated in the dedifferentiation in both CSs and UEC/DEC. Indeed, we speculate that the occurrence of EMT in a well differentiated endometrioid carcinoma may consecutively lead to a dedifferentiated and undifferentiated carcinoma, while in a type II carcinoma, it may result in a CS.

Aim: To evaluate the clinical properties of three subpopulations of circulating tumor cells (CTCs) undergoing epithelial-mesenchymal transition (EMT) in pancreatic ductal adenocarcinoma (PDAC) patients., Methods: We identified CTCs for expression of the epithelial cell marker cytokeratin or epithelial cell adhesion molecule (EpCAM) (E-CTC), the mesenchymal cell markers vimentin and twist (M-CTC), or both (E/M-CTC) using the CanPatrol system. Between July 2014 and July 2016, 107 patients with PDAC were enrolled for CTC evaluation. CTC enumeration and classification were correlated with patient clinicopathological features and outcomes., Results: CTCs were detected in 78.5% of PDAC patients. The number of total CTCs ranged from 0 to 26 across all 107 patients, with a median value of six. CTC status correlated with lymph node metastasis, TNM stage, distant metastasis, blood lymphocyte counts, and neutrophil-to-lymphocyte ratio (NLR). Kaplan-Meier survival analysis showed that patients with ≥ 6 total CTCs had significantly decreased overall survival and progression-free survival compared with patients with < 6 total CTCs. The presence of M-CTCs was positively correlated with TNM stage ( P < 0.01) and distant metastasis ( P < 0.01). Additionally, lymphocyte counts and NLR in patients without CTCs were significantly different from those in patients testing positive for each CTC subpopulation ( P < 0.01)., Conclusion: Classifying CTCs by EMT markers helps to identify the more aggressive CTC subpopulations and provides useful evidence for determining a suitable clinical approach., Competing Interests: Conflict-of-interest statement: The authors do not have any conflict of interest to disclose.

Objectives: Lung cancer is the leading cause of cancer-related death worldwide. The 5-year survival rate for patients after curative surgery with pathological N0 non-small-cell lung cancer (NSCLC) is as low as 56%, which is due to recurrence and metastasis. Emerging evidence suggests that epithelial-mesenchymal transition is important for cancer metastasis. Twist and Snail are epithelial-mesenchymal transition regulators that induce metastasis by down-regulating E-cadherin. The aim of this study was to evaluate the prognostic value of Twist, Snail and E-cadherin expression in patients with resectable pathological N0 NSCLC., Methods: The expression levels of Twist, Snail and E-cadherin in 78 patients with resected pathological N0 NSCLC were assessed using immunohistochemistry. The association between the expression of Twist/Snail/E-cadherin and overall survival (OS) and recurrence-free survival (RFS) was investigated., Results: High expression of Twist, Snail and E-cadherin was detected in 18%, 21% and 53% of NSCLC samples, respectively. High expression of Twist and Snail and low expression of E-cadherin were associated with worse RFS [hazard ratio (HR) 2.29, 95% confidence interval (CI) 1.07-4.87, P = 0.026; HR 2.54, 95% CI 1.24-5.20, P = 0.008 and HR 2.41, 95% CI 1.23-4.73, P = 0.007, respectively] and worse OS (HR 2.26, 95% CI 1.01-5.04, P = 0.040; HR 2.56, 95% CI 1.20-5.43, P = 0.011 and HR 2.42, 95% CI 1.18-4.95, P = 0.012, respectively). Co-expression of at least 2 markers from the combination of high Twist/high Snail/low E-cadherin expression predicted poor RFS and OS (HR 4.12, 95% CI 2.08-8.16, P < 0.001 and HR 4.28, 95% CI 2.08-8.77, P < 0.001, respectively), and it was an independent predictor of RFS and OS (HR 3.99, 95% CI 1.89-8.44, P < 0.001 and HR 4.16, 95% CI 1.88-9.18, P < 0.001, respectively)., Conclusions: Co-expression of at least 2 markers from the combination of high Twist/high Snail/low E-cadherin expression was a significant prognostic predictor in patients with pathological N0 NSCLC.

Background: Metastasis-associated in colon cancer 1 (MACC1) has been reported to promote tumor cell invasion and metastasis. Cancer stem cells and epithelial-mesenchymal transition (EMT) have also been reported to promote tumor cell proliferation, invasion, and metastasis. KiSS-1, a known suppressor of metastasis, has been reported to be down-regulated in various tumors. However, the associations of MACC1, CD44, Twist1, and KiSS-1 in colonic adenocarcinoma (CAC) invasion and metastasis remain unclear. The purpose of this study is to investigate the roles of MACC1, CD44, Twist1, and KiSS-1 in CAC invasion and metastasis and their associations with each other and with the clinicopathological characteristics of CAC patients., Methods: Immunohistochemistry and multivariate analysis were carried out to explore the expression of MACC1, CD44, Twist1, and KiSS-1 in 212 whole-CAC-tissue specimens and the corresponding normal colon mucosa tissues. Demographic, clinicopathological, and follow-up data were also collected., Results: The results of this study showed MACC1, CD44, and Twist1 expression to be up-regulated, and KiSS-1 expression was down-regulated in CAC tissues. Positive expression of MACC1, CD44, and Twist1 was found to be positively correlated with invasion, tumor grades, and lymph- node-metastasis (LNM) stages and tumor-node-metastasis (TNM) stages for patients with CAC. Positive expression of KiSS-1 was inversely associated with invasion, tumor size, LNM stage, and TNM stage. The KiSS-1-positive expression group had significantly more favorable OS than did the KiSS-1-negative group. Univariate analysis indicated that overexpression of MACC1, CD44, and Twists1 was negatively associated with longer overall survival (OS) time, and there was a positive relationship between KiSS-1-positive expression and OS time for patients with CAC. Multivariate Cox analysis demonstrated that overexpression of MACC1, CD44, Twist1, and low expression of KiSS-1 and LNM and TNM stages were independent predictors of prognosis in patients with CAC., Conclusions: The results in this study indicated that levels of expression of MACC1, CD44, Twist1, and KiSS-1 are related to the duration of OS in patients with CAC. MACC1, CD44, Twist1, and KiSS-1 may be suitable for use as biomarkers and therapeutic targets in CAC.

We have earlier found abnormal expression of the mitochondrial ribosomal protein S18-2 (MRPS18-2, S18-2) in endometrial cancer, compared to the expression in hyperplasia and in normal endometrium. Here we report that expression of S18-2 was increased with disease progression in clinical specimens of prostate cancer (PCa). The level of induction of epithelial to mesenchymal cell transition (EMT) correlated with the expression level of S18-2 in PCa cell lines. Moreover, cells acquired increased ability of migration upon S18-2 overexpression, as was evaluated in zebrafish embryo model and in trans-well assay. We found that this is due to increased CXCR4 cell surface expression. Neutralizing CXCR4 protein or abrogating S18-2 expression in cells significantly reduced their migratory ability directed toward CXCL12. The mRNA expression of TWIST2, encoding one of transcription factors that induce EMT upon CXCR4 increase, positively correlated with the S18-2 protein level. Together, these data suggest that the S18-2 protein induces EMT through the TWIST2/E-cadherin signalling and, consequently, CXCR4-mediated migration of PCa cells.

In human placenta, the multinucleated syncytiotrophoblast (ST) allows all the exchanges between the maternal and fetal circulation and is also the site of placental hormonal functions. Absence or disturbances of ST formation are associated with a defect or pathologies of pregnancy such as preeclampsia (PE) and intrauterine growth retardation (IUGR). All along pregnancy, the ST is regenerated by fusion of underlying mononucleated villous cytotrophoblasts (VCT). The protocol described here provides details on how GATA3 or TWIST1 immunostaining and analysis can be used to easily assess the in vitro differentiation of human placental cytotrophoblast.

The BCR-ABL specific tyrosine kinase inhibitors (TKI) changed the outcome of chronic myeloid leukemia (CML), turning a life-threatening disease into a chronic illness. However, TKI are not yet curative, because most patients retain leukemic stem cells (LSC) and their progenitors in bone marrow and relapse following treatment cessation. At diagnosis, deregulation of the bone morphogenetic protein (BMP) pathway is involved in LSC and progenitor expansion. Here, we report that BMP pathway alterations persist in TKI-resistant patients. In comparison with patients in complete cytogenetic remission, TKI-resistant LSC and progenitors display high levels of BMPR1b expression and alterations of its cellular localization. In vitro treatment of immature chronic phase CML cells with TKI alone, or in combination with interferon-α, results in the preferential survival of BMPR1b + cells. We demonstrated persistent and increasing BMP4 production by patients' mesenchymal cells with resistance. Patient follow-up revealed an increase of BMPR1b expression and in BMP4 expression in LSC from TKI-resistant patients in comparison with diagnosis, while remaining unchanged in sensitive patients. Both leukemic and nonleukemic cells exhibit higher BMP4 levels in the bone marrow of TKI-resistant patients. Exposure to BMP2/BMP4 does not alter BCR-ABL transcript expression but is accompanied by the overexpression of TWIST-1, a transcription factor highly expressed in resistant LSC. By modulating BMP4 or BMPR1b expression, we show that these elements are involved in TKI resistance. In summary, we reveal that persistence of BMP alterations and existence of an autocrine loop promote CML-primitive cells' TKI resistance., (© 2017 by The American Society of Hematology.)

Background/aim: The transcription factors Twist, Snail, Slug, ZEB1 and ZEB2 regulate epithelial-mesenchymal transition (EMT) and their expression has been associated with a poor prognosis in several cancer entities. The aim of this analysis was to investigate in parallel the expression of all of these transcription factors in head and neck squamous cell carcinomas (HNSCCs) in order to gain insight into their possible co-expression., Materials and Methods: Tumor tissue samples were immunohistochemically stained using antibodies against these transcription factors. The staining intensity and cellular distribution of the immunoreactivity was recorded., Results: In general, transcription factor immunoreactivity was noted in the nucleus of both cancer and stromal cells. The highest immunoreactivity was observed for Twist. Snail, Slug, ZEB1 and ZEB2 showed a much lesser immunoreactivity in cancer cells and they were expressed independently from each other., Conclusion: Twist is the major transcription factor active in HNSCC; the other transcription factors of EMT seem to be of less importance in this tumor entity., (Copyright© 2017, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

Background/aim: As a result of activation of transcription factors engaged in epithelial-mesenchymal transition (EMT), such as Twist, inhibition of epithelial markers and an increased expression of mesenchymal markers are observed. One of the specific markers of cancer-associated fibroblasts is podoplanin (PDPN) - a mucin-type membrane glycoprotein. The aim of this work was to study the localisation and intensity of expression of Twist and PDPN on the mRNA and protein level in cases of invasive ductal breast carcinoma (IDC), and its association with patients' clinico-pathological data., Materials and Methods: The study included archival material in a form of 80 paraffin IDC blocks and 11 IDC fragments frozen in liquid nitrogen. Immunohistochemical expression of Twist and PDPN was evaluated using light microscope and semiquantitative scale for evaluation of nuclear expression or immunoreactive scale (IRS) for evaluation of cytoplasmic expression. Material was isolated from frozen IDC fragments using laser micro-dissection (from cancer and stromal cells, separately) and was used to perform real-time PCR., Results: Twist expression was higher in stromal cells in comparison to cancer cells. Analysis of patients' survival rate showed, that higher expression of Twist in cancer cells was associated with shorter overall survival time and shorter event-free survival time. The expression of PDPN was also higher in stromal cells in comparison with cancer cells. In addition, positive correlation was observed between expression of Twist and PDPN in stromal cells of IDC (r=0.267; p<0.05)., Conclusion: The relationship between the higher expression of Twist in both cancer and stromal cells and shorter patients' survival indicates Twist as a potential useful prognostic marker in IDC. Positive correlation of Twist and PDPN expression may indicate the role of PDPN in EMT in IDC., (Copyright© 2017, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

Aim: Malignant melanoma is an aggressive disease and its incidence is increasing worldwide. Genetic predisposition and exposure to environmental factors, especially sunlight, are risk factors. Histopathologic distinction between nevi and melanomas can be difficult. It is anticipated that the evaluation of the immunohistochemical expression of some genes could contribute to the differential diagnosis of questionable histologically lesions. The objective of this study was to investigate wether the evaluation of the immunohistochemical expression of genes CADM1, TWIST1 and CDH1 (E-cadherin), that take part in mechanisms of cell adhesion and epithelial-mesenchymal transition, contributes to the differential diagnosis of melanocytic lesions difficult to diagnose., Materials and Methods: Retrospective cross-sectional study based on immunohistochemistry analysis of samples of 30 dysplastic compound melanocytic nevi, 30 melanomas less than 1.0mm thick and 30 melanomas more than 1.0mm thick, diagnosed between 2013 and 2016. A score was used to evaluate color intensity and percentage of cells stained., Result: There were significant reductions in the expression of the genes CADM1 and CDH1 in melanomas (below and above 1.00mm of thickness) and in melanomas more than 1.0mm thick, respectively, compared to dysplastic compund melanocytic nevi. There was also lower expression of the genes CADM1 and CDH1 in melanomas greater than 1.0mm thick compared to melanomas less than 1.0mm. The gene TWIST1 showed no significant difference in expression between groups., Conclusion: These findings allow us to conclude that the immunohistochemical expression of CADM1 has the potential to contribute as an auxiliary tool to the differential diagnosis between nevi and melanomas., (Copyright © 2017 Elsevier GmbH. All rights reserved.)

Tumor budding is thought to reflect the epithelial-mesenchymal transition (EMT). However, the molecular mechanism linking tumor buds and the EMT remains unclear. Here, we examined the induction of tumor budding and EMT and their association with EMT-related proteins (ZEB1, TWIST, SNAIL, and SLUG) in colorectal cancer (CRC). Immunohistochemical expression of pan-cytokeratin was examined for identification of tumor budding in 101 CRCs. Grading of tumor budding was classified into low- and high-grade groups. Tissue microarray was conducted to identify tumor budding sites. The expression of E-cadherin, ZEB1, TWIST, SNAIL, and SLUG was examined in areas of tumor budding and the surrounding tumor stroma using a double-immunostaining method. Specifically, pan-cytokeratin and EMT-related proteins were assessed by double immunostaining. Low or no expression of E-cadherin was found in areas of tumor budding. Moreover, ZEB1, TWIST, SNAIL, and SLUG were not expressed in regions of tumor budding. However, the expression level of ZEB1 in the stromal cells surrounding tumor budding was significantly more frequent than that of TWIST, SNAI, and SLUG. In addition, the expression of EMT-related proteins in surrounding stromal cells was significantly greater in areas of high-grade tumor budding than in low-grade areas. Our present results suggest that EMT-related proteins play a minor role in forming tumor buds. In addition, our findings suggest the existence of subtypes of stromal cells in CRC with phenotypical and functional heterogeneity., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Objective: There is an urgent need to identify molecular biomarkers rather than clinical markers to distinguish aggressive prostate cancer from the indolent majority for proper treatment and accurate prediction of prognosis. We aimed to investigate the immunohistochemical expression of epithelial-mesenchymal transition (EMT)-related molecules (Twist-1 and E-cadherin) and the stem cell marker EZH2 in prostate cancer and to assess their ability to identify the high-risk patients, in a trial to explore their prognostic implications., Material and Method: Immunohistochemical expression of Twist-1, E-cadherin and EZH2 in 50 specimens of prostate cancer and 20 cases of benign prostatic hyperplasia were studied. The relationship between their expression and the clinicopathological variables, biochemical recurrence, and biochemical progression-free survival were investigated., Results: Our results revealed that high Twist-1, as well as high EZH2 expression, was strongly associated with higher pre-treatment PSA level, Gleason score ≥7, advanced tumor stage, lymph node involvement, distant metastasis and biochemical progression. Aberrant E-cadherin expression was significantly associated with higher pre-treatment PSA level, Gleason score ≥7, advanced tumor stage, lymph node involvement, and distant metastasis. A significant positive correlation between Twist-1 and EZH2 expression was found (p < 0.001), while E-cadherin expression showed a negative correlation with both markers (p < 0.001). A significant association was found between high Twist-1, high EZH2& aberrant E-cadherin expression, and shorter biochemical progression-free survival., Conclusion: The high Twist-1 expression, aberrant E-cadherin and high EZH2 expression in primary prostate cancer are considered as adverse prognostic markers of an aggressive tumor with high metastatic potential. Assessment of their expression level would contribute to the accurate prediction of biochemical progression.

Background: DNMT3A mutations are frequently discovered in acute myeloid leukemia (AML), associated with poor outcome. Recently, a relapse case report of AML extramedullary disease has showed that AML cells harboring DNMT3A variation were detected in the cerebral spinal fluid. However, whether a causal relationship exists between DNMT3A mutation (D3Amut) and extramedullary infiltration (EMI) is unclear., Methods: We took advantage of DNMT3A (R882C) mutation-carrying AML cell strain, that is, OCI-AML3, assessing its migration ability in vitro and in vivo. By RNA interfering technology and a xenograft mouse model, we evaluated the effect of DNMT3A mutation on cell mobility and explored the possible mechanism., Results: OCI-AML3 displayed extraordinary migration ability in vitro and infiltrated into meninges of NOD/SCID mice after intravenous transfusion. We found that this leukemic migration or infiltration capacity was significantly compromised by the knockdown of DNMT3A mutant. Notably, TWIST1, a critical inducer of epithelial-mesenchymal transition, which underlies the metastasis of carcinomas, was highly expressed in association with R882 mutations. Abrogation of TWIST1 in DNMT3A mutated cells considerably weakened their mobility or infiltration., Conclusions: Our results demonstrate that D3Amut in OCI-AML3 strain enhances leukemic aggressiveness by promoting EMI process, which is partially through upregulating TWIST1.

Basal cell adenomas and basal cell adenocarcinomas show marked histomorphologic similarity and are separated microscopically primarily by the invasive characteristics of the adenocarcinomas. We wished to explore potential differences in the expression of epithelial-mesenchymal transition associated proteins in these two tumor types. A tissue microarray was constructed utilizing 29 basal cell adenomas and 16 basal cell adenocarcinomas. Immunohistochemical expression of E-cadherin, beta-catenin, Twist 1 and vimentin were investigated. Both tumors expressed all proteins in a relatively similar manner. Nuclear beta-catenin was essentially limited to the abluminal cell populations in both tumor types. E-cadherin was limited largely to luminal locations but was more prevalent in the adenocarcinomas as compared to the adenomas. Primarily abluminal expression for vimentin was seen, sometimes present in an apical dot-like pattern. Distinct populations of cellular expression of these four markers of epithelial mesenchymal transition were present but were similar in locations in both tumors with no patterns discerned to separate basal cell adenoma from basal cell adenocarcinoma. Given these findings, the mechanisms by which basal cell adenocarcinoma is able to invade while its counterpart, basal cell adenoma can not, may be more complex than in other tumor types.

Background: Aldehyde dehydrogenase 1 (ALDH1) is a marker of breast cancer stem cells, and the expression of ALDH1 may be a prognostic factor of poor clinical outcome. The epithelial-mesenchymal transition may produce cells with stem-cell-like properties promoted by transcription factors. We investigated the expression of ALDH1 and transcription factors in both primary and metastatic lesions, and prognostic value of them in breast cancer patients with axillary lymph node metastasis (ALNM)., Method: Forty-seven breast cancer patients with ALNM who underwent surgery at Okayama University Hospital from 2002 to 2008 were enrolled. We retrospectively evaluated the levels of ALDH1 and transcription factors, such as Snail, Slug and Twist, in both primary and metastatic lesions by immunohistochemistry., Results: In primary lesions, the positive rate of ALDH1, Snail, Slug and Twist was 19, 49, 40 and 26%, respectively. In lymph nodes, that of ALDH1, Snail, Slug and Twist was 21, 32, 13 and 23%, respectively. The expression of ALDH1 or transcription factors alone was not significantly associated with a poor prognosis. However, co-expression of ALDH1 and Slug in primary lesions was associated with a shorter DFS (P = 0.009)., Conclusions: The evaluation of the co-expression of ALDH1 and transcription factors in primary lesions may be useful in prognosis of node-positive breast cancers.

Unlabelled: Globally, most patients are at late-stage when they have been diagnosed with ovarian cancer. Investigating the potential mechanisms involved in tumor progression and prognosis is essential for improving treatment options, outcomes, and survival., Objective: This study elucidated the clinico-pathological significance of TWIST2 and the relationship of TWIST2, E-cadherin, and Vimentin expression in the progression and prognosis of epithelial ovarian cancer (EOC)., Materials and Methods: Immunohistochemical staining was used to quantify the expression and relevance of TWIST2, E-cadherin, and Vimentin in 103 ovarian specimens, including 30 cases of benign ovarian tumors, 30 cases of borderline ovarian tumors, and 43 cases of EOC., Results: The expression of TWIST2 in the cytoplasm may help to maintain characteristics of epithelial cancer cells with E-cadherin normal membranous expression, while nuclear TWIST2 induces tumor translation front with membranous expression of Vimentin, which eventually promotes cancer metastasis. Moreover, the upregulation of TWIST2 was also related to the aberrant expression of E-cadherin and the increased expression of Vimentin, which were reported as important indicators of epithelial-mesenchymal transition (EMT)., Discussion: The data suggested that co-expression of TWIST2/Vimentin was an independent prognostic indicator for both overall survival and disease-free survival by multivariate Cox proportional hazards model. TWIST2 regulates EMT by depriving the epithelial cell phenotype of E-cadherin and endowing the mesenchymal cell phenotype with Vimentin, which may be involved in the progression and prognosis of ovarian cancer, and TWIST2/Vimentin co-expression might be a novel indicator with prognostic potential in EOC patients.

Objective: Twist is an important transcription factor that induces epithelial-mesenchymal transition and therefore plays an important role in tumor progression. There are a few studies on Twist expression in salivary gland carcinomas. The aim of this study was to evaluate and compare the expression of Twist in the most common benign and malignant salivary gland tumors and to determine whether its expression was correlated with any tumor characteristics., Material and Method: In this retrospective cross-sectional study, 45 cases including 11 cases of normal salivary gland, 12 pleomorphic adenomas, 12 adenoid cystic carcinomas and 10 mucoepidermoid carcinomas were enrolled. The mean and intensity of Twist expression were evaluated immunohistochemically and were compared using statistical analysis., Results: The expression of Twist was higher in malignant salivary gland tumors in comparison with normal glands and benign tumors (p= 0.03). It was also higher in pleomorphic adenomas in comparison with normal tissue. Adenoid cystic carcinomas and mucoepidermoid carcinomas showed no significant difference in Twist expression (p= 0.50). There was no correlation with the size, stage or grade of tumor., Conclusion: The findings showed that Twist might play a role in the formation of salivary gland neoplasm and also may affect malignant transformation and tumoral invasion. The exact mechanism of this marker and the possibility of using it as a therapeutic target require further investigation.

Background: Epithelial-mesenchymal transition (EMT) is considered to play a critical role in cancer progression and metastasis. However, the impact of EMT on the prognosis of hepatocellular carcinoma (HCC) is still elusive. In this study, we examined the relationship between the expression of EMT markers and recurrence-free survival (RFS) and overall survival (OS) in HCC patients after hepatic resection., Summary: The mRNA expression of 15 genes related to EMT was assessed by quantitative real-time polymerase chain reaction in cancerous tissues from 72 patients who underwent hepatic resection of HCC between January 2005 and December 2010 at our hospital. The upregulation of TWIST and the downregulation of tight junction protein ZO-1 (TJP1) were significantly associated with shorter RFS as well as OS. Increased levels of TWIST and decreased levels of TJP1 should be predictive markers for poor prognosis in patients with HCC after hepatectomy; those could serve as potential biomarkers for the treatment of HCC. Key Messages: A low level of TJP1 and high level of TWIST expression were prognostic factors predicting HCC after hepatic resection., (© 2016 S. Karger AG, Basel.)

Aims and Methods: Epithelial-mesenchymal transition (EMT) has previously been linked to glioma invasion and progression. To determine whether EMT regulators, Twist and Zeb1, had clinical significance in astrocytic gliomas, the association of Twist and Zeb1 with clinicopathological and molecular factors was studied in 269 astrocytoma samples., Results: Twist and Zeb1 were widely expressed in astrocytic gliomas, but the expression of the former did not correlate with that of the latter. Stronger Twist expression levels were associated with higher WHO grades (p=0.001), whereas Zeb1 did not correlate with WHO grades. We found no association between Twist and proliferation activity (Ki67/MIB-1), p53 status, epidermal growth factor receptor (EGFR) amplification or neural cell adhesion molecule (NCAM) expression. There was no significant difference in Twist or Zeb1 expression when primary and secondary gliomas were analysed. Tumours with high Twist expression were IDH1 negative (p=0.009). High hypoxia-inducible factor-1α expression correlated significantly with positive Twist expression (p<0.001), whereas it was not associated with Zeb1 expression. Zeb1 expression did not correlate with proliferation, EGFR or IDH1. Nevertheless, we did find a correlation between high Zeb1 expression and low p53 expression levels (p=0.027). Positive NCAM expression was significantly associated with Zeb1 positivity (p=0.022). Zeb1 had no association with patient survival, whereas positive Twist expression predicted poor survival for patients in both univariate (p<0.001) and multivariable analyses (p=0.027)., Conclusions: EMT regulators, Twist and Zeb1, are common features of infiltrating astrocytomas, and Twist is upregulated in glioblastomas in particular. Twist may be a novel marker for poor prognosis in glioma patients., (Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://group.bmj.com/group/rights-licensing/permissions.)

Introduction: The epithelial to mesenchymal transition, a well-known and re-emerging model in pathology, has not been completely investigated in the field of gestational pathology. This study aims at improving the comprehension of this process in molar disease, even looking for new possible immunohistochemical markers., Materials and Methods: We have analysed the immunohistochemical expression of Twist1 and Snai2, two of the most important transcription factors involved in epithelial to mesenchymal transition, in formalin-fixed paraffin-embedded samples of 23 spontaneous abortive pregnancies, 22 molar pregnancies (10 partial and 12 complete) and 7 term placentas., Results: Twist1 and Snai2 were highly expressed in stromal villi cells of molar disease. Particularly, Twist1 was highly expressed in complete moles compared to both abortive pregnancies (p < 0.001) and partial moles (p < 0.05). Also Snai2 was more expressed by complete moles, differentiating them from non-molar abortions (p < 0.05)., Discussion: On the basis of the known cadherins and claudins expression in these pathologies, our new findings reinforce the hypothesis of the involvement of epithelial to mesenchymal transition in early molar pregnancies and above all in complete moles. Furthermore, we highlighted that in molar disease not only the trophoblast, but even the villi stromal cells, are involved. Thanks to their specificity, furthermore, these Twist1 and Snai2 could be used as additional immunohistochemical tool in the diagnosis of complete molar disease, with Twist1 as the first choice., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Aggressive tumor cells can mimic embryonic vasculogenic networks and form vasculogenic mimicry (VM). Preliminary studies demonstrated that hypoxia can promote VM formation; however, the underlying mechanism remains unclear. The present study aimed to investigate the role of the Twist1‑Bmi1 connection in hypoxia‑induced VM formation and the underlying mechanism. In the in vitro experiments, western blot analysis demonstrated that hypoxia upregulated the expression of Twist1, Bmi1, epithelial‑mesenchymal transition (EMT) markers, stem cell markers and VM‑associated markers. The 3D culture assay showed that hypoxia promoted VM formation in hepatocellular carcinoma (HCC) cell lines. Using transfection and in vitro cell experiments, the Twist1‑Bmi1 connection was confirmed to have an important role in inducing EMT, cell stemness and VM formation. In the in vivo experiments, the murine hypoxia models were established via incomplete femoral artery ligation and the mechanism by which hypoxia promoted Twist1 and Bmi1 expression and led to VM formation was demonstrated by immunohistochemistry staining and endomucin/periodic acid Schiff double‑staining. In conclusion, hypoxia upregulate the expression of Twist1 and Bmi1, and these two proteins have an important role in inducing EMT and cancer cell stemness, which contributed to VM formation.

The incidence of esophageal adenocarcinoma has increased dramatically over recent years and Barrett's esophagus is considered the most established risk factor for its development. Endoscopic surveillance of Barrett's esophagus is therefore recommended but hinges on histological interpretation of randomly taken biopsies which is poorly reproducible. The use of biomarkers presents an opportunity to improve our ability to risk-stratify these patients.We examined three biomarkers namely p504s, CD133, and Twist in the setting of Barrett's esophagus, low-grade dysplasia, and esophageal adenocarcinoma to evaluate differential expression between benign, dysplastic, and malignant Barrett's tissue in an exploratory cross-sectional study. Twenty-five cases each of Barrett's esophagus, low-grade dysplasia, and esophageal adenocarcinoma were included along-with 25 cases of esophagectomy resections for Barrett's adenocarcinoma. The biomarkers were immunostained on automated Ventana(®) immunostainer. The biopsies were assessed for biomarker expression by two independent observers. Granular cytoplasmic staining of p504s was observed in dysplastic Barrett's biopsies and esophageal adenocarcinoma but not in Barrett's esophagus. Apical and membranous CD133 expression was also observed in dysplastic Barrett's and esophageal adenocarcinoma. Nuclear Twist expression was seen predominantly in stromal cells. There was increased p504s expression in dysplastic Barrett's esophagus and esophageal adenocarcinoma compared with controls. CD133 expression was detected for the first time in esophageal adenocarcinoma and dysplastic Barrett's esophagus. Twist expression was not convincing enough to be labeled as Barrett's biomarker. p504s and CD133 have the potential to differentiate benign from malignant Barrett's tissue in this exploratory study. Their validity should be established in prospective longitudinal studies., (© 2014 International Society for Diseases of the Esophagus.)

Epithelial-mesenchymal transition (EMT) is a process which is associated with a loss of intercellular adhesion, acquired mesenchymal shape, and increased motility by epithelial cells. Twist is one of the key regulators of EMT.In view of the distinct clinical behavior of odontogenic lesions, the objective of the present study was to investigate the immunohistochemical expression of Twist in these lesions. In this study, 70 formalin-fixed, paraffin-embedded tissue blocks of odontogenic lesion consisting of 16 unicystic ameloblastomas (UA), 17 solid ameloblastomas (SA), 18 odontogenic keratocysts (OKC), and 19 dentigerous cysts (DC) were reviewed using immunohistochemistry for Twist staining. In this study, Twist immunostaining was evident in all groups of the specimens except the dentigerous cyst group. Twist expression was seen in 58.8 % (10/17) of SA, 50 % (8/16) of UA, and 44.4 % (8/18) of OKCs. 23.5 % of SA, 18.8 % of UA, and 16.7 % of OKCs showed Twist expression in more than 50 % of cells. Statistical analysis showed that Twist expression levels were significantly higher in ameloblastomas (SA and UA) and OKCs than dentigerous cysts (P = 0.002). There were no significant differences between Twist expression in SAs, UAs, and OKCs (P > 0.05). The results of this study propose that the high expression rate of Twist plays a role in the pathogenesis of ameloblastomas and OKCs and might be one of the reasons for the aggressive behavior of ameloblastomas and high recurrence of OKCs and could reinforce the classification of OKC as an odontogenic tumor.

Upstream stimulating factor 1 (USF1) is a basic helix-loop-helix transcription factor that specifically binds to E-box DNA motifs, known cis-elements of key oocyte expressed genes essential for oocyte and early embryonic development. However, the functional and regulatory role of USF1 in bovine oocyte and embryo development is not understood. In this study, we demonstrated that USF1 mRNA is maternal in origin and expressed in a stage specific manner during the course of oocyte maturation and preimplantation embryonic development. Immunocytochemical analysis showed detectable USF1 protein during oocyte maturation and early embryonic development with increased abundance at 8-16-cell stage of embryo development, suggesting a potential role in embryonic genome activation. Knockdown of USF1 in germinal vesicle stage oocytes did not affect meiotic maturation or cumulus expansion, but caused significant changes in mRNA abundance for genes associated with oocyte developmental competence. Furthermore, siRNA-mediated depletion of USF1 in presumptive zygote stage embryos demonstrated that USF1 is required for early embryonic development to the blastocyst stage. A similar (USF2) yet unique (TWIST2) expression pattern during oocyte and early embryonic development for related E-box binding transcription factors known to cooperatively bind USF1 implies a potential link to USF1 action. This study demonstrates that USF1 is a maternally derived transcription factor required for bovine early embryonic development, which also functions in regulation of JY1, GDF9, and FST genes associated with oocyte competence., (© 2015 Society for Reproduction and Fertility.)

Epithelial-mesenchymal transition (EMT) has been implicated in the development of a number of cancers. An important EMT inducer, TWIST, has been detected to be over-expressed in a variety of tumors, but rarely been studied in nasopharyngeal carcinoma (NPC). This study aimed to examine TWIST expression and its association with clinicopathological factors and prognosis in NPC. A total of 65 NPC and 20 normal samples were involved in the present study. RT-PCR and immunohistochemistry were used to examine the mRNA and protein expressions of TWIST in NPC and normal tissues. The relationship of TWIST expression levels with clinical features and prognosis of NPC patients were analyzed. The positive rate of TWIST expression was markedly higher in NPC tissues than that in normal tissues. Over-expression of TWIST was correlated with N stage and the presence of distant metastasis. Patients with positive TWIST expression had a significantly shorter overall survival time relative to patients with negative TWIST expression. The data suggest that TWIST over-expression has a correlation with lymphatic and distant metastasis in NPC. Moreover, it might be a novel biomarker for prediction of advanced tumor progression and a potential unfavorable prognostic factor as well as a potential treatment target for NPC.

Aims: Twist has been reported to play crucial roles for malignant aggressiveness; however, detailed pathological significance of Twist in renal cell carcinoma (RCC) is not fully understood. The present study was to clarify clinical significance and molecular functions of Twist in patients with RCC., Methods: Twist expression was examined by immunohistochemical techniques in 156 formalin-fixed specimens. Cell proliferation, angiogenesis, and apoptosis were measured as the percentage of Ki-67-positive cells (proliferation index, PI), CD31-stained vessels (microvessel density, MVD), and TUNEL-positive cells (apoptotic index, AI). In addition, semi-quantification of matrix metalloproteinase (MMP)-2 was performed. Macrophages were identified with anti-CD68 antibody, and the tumor associated macrophage (TAM) density was calculated as CD68-positive cells per high-power field., Results: Twist expression was positively associated with grade, pT stage, and metastasis (p<0.001). We also noticed that its expression was considerably higher in cancer cells of sarcomatoid RCC and in those at the edge of the tumors. Twist expression was positively correlated with PI, MVD, MMP2 expression, and TAM density (P<0.001), but not with AI, and MMP-2 expression and TAM density were independently correlate by multi-variate analyses. Kaplan-Meir survival curves showed high Twist expression was a worse predictor for cause-specific survival (P<0.001)., Conclusions: Twist plays important roles in tumor growth, progression, and survival in patients with RCC patients. Such pathological mechanisms are significantly associated with increased cancer cell proliferation, angiogenesis, MMP2 expression, and macrophage recruitment. These findings are important information for discussion of treatment and observation strategies in these patients.

Objective: The goal of this study was to utilize long-term patient follow-up to determine whether epithelial-to-mesenchymal transition (EMT)-related markers can predict bladder cancer patient survival and progression of disease., Materials and Methods: This study included 121 patients with bladder cancer. Sixty-four of these patients presented with non-muscle invasive (NMI, stage T1) bladder cancer and 57 with muscle invasive (MI, stage T2, T3). The patients were diagnosed and treated between May 1998 and July 2012. The EMT markers E-cadherin, Twist, and Vimentin were detected via immunohistochemistry. Univariate and multivariate/Cox analyses were then utilized to determine whether these EMT markers could be useful prognostic markers for predicting bladder cancer patient outcomes., Results: Analysis of the 121 bladder cancer patients in this study revealed that the frequency of E-cadherin expression was 59.5% (72/121), Twist was 54.5% (66/121), and Vimentin was 24.8% (30/121). Twist and Vimentin were found to have statistically significant correlations with grade, recurrence, and progression but not with stage, whereas E-cadherin was associated with stage but not with the other parameters. In the univariate analysis, grade (p = 0.02) was the only significant predictor for progression-free survival (PFS). Stage, grade, and expression of E-cadherin, Vimentin and Twist were included in the multivariate analysis of predicting PFS. In this analysis, grade (p = 0.01) and Vimentin expression (p = 0.001) were found to be significant prognostic factors in predicting PFS., Conclusions: Grade and Vimentin are potential independent indicators in predicting bladder cancer progression and survival.

Background: Epithelial-to-mesenchymal transition (EMT) via the mechanism of transcription repression is a crucial process for the induction of invasiveness in many human tumors. Ameloblastoma is a benign odontogenic epithelial neoplasm with a locally infiltrative behavior. Twist, an EMT promoter, has been implicated in its invasiveness. The roles of the other transcription factors remain unclarified., Materials and Methods: Four transcription factors, namely Snail, Slug, SIP1, and Twist, were examined immunohistochemically in 64 ameloblastoma [18 unicystic (UA), 20 solid/multicystic (SA), 4 desmoplastic (DA), and 22 recurrent (RA)]., Results: All four transcription factors were differentially expressed in ameloblastoma [Snail: n = 60/64 (94%); Slug: n = 21/64 (33%); SIP: n = 18/64 (28%); Twist: n = 26/64 (41%)] (P < 0.05). Their distribution patterns were heterogeneous and were not significantly different between the tumor invasive front and central area (P > 0.05). Intracellular protein localization was predominantly nuclear for Snail, cytoplasmic>nuclear for Slug and SIP1, and cytoplasmic/nuclear for Twist. Overexpression of Snail in most subsets (UA = 18/18; SMA = 19/20; DA = 4/4; RA = 19/22) compared with the other transcription factors (P < 0.05) and selective expression for Slug, SIP1, and Twist in squamous/keratinous foci and at sites of epithelial cystic degeneration were among the main observations made. Stromal cells surrounding immunoreactive tumor cells tended to stain positive., Conclusions: Present findings suggest that these transcription factors probably play differential roles in mediating local invasiveness in ameloblastoma. Overexpression of Snail in most subsets suggests that this molecule is most likely the prototype transcription factor involved in inducing EMT in the ameloblastoma., (© 2013 John Wiley & Sons A/S. Published by Blackwell Publishing Ltd.)

Objective: To study the expression of E-cadherin, N-cadherin, TGF-β1 and Twist protein and investigate its significance in the occurrence and development of prostate cancer., Methods: The expression of E-cadherin, N-cadherin, TGF-β1 and Twist protein in 59 prostate cancer tissues and 21 adjacent tissues were detected by immunohistochemical SABC staining, and the correlation with clinicopathological features was analyzed., Results: Positive rates of E-cadherin, N-cadherin, TGF-β1 and Twist were 32.2%, 54.2%, 71.2% and 74.6%, respectively, in prostate cancer tissues and 85.7%, 9.52%, 19.0% and 9.52%, respectively, in cancer-adjacent tissues, with significant differences between the two groups (P<0.05). The reduced expression of E-cadherin was related to the differentiation of prostate cancer tissues and PSA level, but was not associated with clinical stage, lymph node metastasis, bony metastasis and age. The increased expression of N-cadherin, TGF-β1 and Twist was related to the differentiation of prostate cancer tissues, clinical stage, lymph node metastasis, bony metastasis, but not to age. The difference in positive expression of N-cadherin and TGF-β1 was significant between PSA≤20 μg/L group and PSA>20μg/L group, but the positive expression of Twist was not significant between groups. The expression of E-cadherin was highly negatively correlated with that of N-cadherin and also highly negatively correlated with that of Twist. The expression of TGF-β1 was correlated with those of E-cadherin, N-cadherin and Twist., Conclusions: The reduced expression of E-cadherin, abnormal expression of N-cadherin, transformation form E-cadherin to N-cadherin and the increased expression of TGF-β1 and Twist play an important role in the occurrence and development of prostate cancer., (Copyright © 2014 Hainan Medical College. Published by Elsevier B.V. All rights reserved.)

Objective: Intratumoral hypoxia promotes angiogenesis, invasion and epithelial-mesenchymal transition, a pivotal event in tumor metastasis. TWIST is a master regulator of multiple developmental processes and has recently been shown to be the key factor responsible for cancer metastasis via the inhibition of E-cadherin expression, a hallmark of epithelial-mesenchymal transition. This study aimed to determine the expression of hypoxia-inducible factor 1α, TWIST and E-cadherin in patients with endometrioid endometrial carcinoma and to examine their clinical significance in endometrioid endometrial carcinoma progression., Methods: Using immunohistochemical and tissue microarray approaches, we evaluated the expression of hypoxia-inducible factor 1α, TWIST and E-cadherin in normal endometrial (n = 35), atypical hyperplasia (n = 28) and endometrioid endometrial carcinoma samples (n = 124). Furthermore, we statistically analyzed the association between these markers, as well as their correlation with clinicopathologic variables., Results: The expression of hypoxia-inducible factor 1α and TWIST were markedly increased, whereas E-cadherin was decreased, as lesions progressed from normal endometrium to atypical hyperplasia to carcinoma (P < 0.01). Among various clinical parameters, the expression of hypoxia-inducible factor 1α and TWIST was strikingly elevated with aggressive tumor characteristics, including higher pathologic grade, deep myometrial invasion and lymph node involvement (P < 0.05). More importantly, overexpression of hypoxia-inducible factor 1α positively correlated with enhanced TWIST expression in endometrioid endometrial carcinoma samples (r = 0.249, P < 0.01); however, statistical analysis showed a negative relationship between TWIST upregulation and E-cadherin downregulation (r = -0.183, P = 0.042)., Conclusions: These results demonstrated for the first time that the hypoxia-inducible factor 1α/TWIST/E-cadherin pathway may play a critical role in invasion and metastasis of endometrioid endometrial carcinoma. The combined evaluation of these markers may be useful in predicting aggressive phenotypes and thus prognosis in patients with endometrioid endometrial carcinoma.

Purpose: Transcription factor Twist1 is known to play a vital role in cancer development, progression and metastasis. However, regulation mechanisms beneath Twist1 expression, as well as the correlation between its expression and bladder urothelial carcinoma (BUC), are still under investigation. Herein, we tried to investigate the expression of Twist1 in BUC specimens and non-cancerous mucosas and illustrate their relationships with clinicopathological features., Methods: The expression of Twist1 mRNA in 42 fresh BUC specimens and 13 paired non-cancerous mucosas was detected by real-time fluorescence quantitative reverse transcription polymerase chain reaction (RFQ-RTPCR). Immunohistochemistry (IHC) was used to detect the expression of Twist1 protein in 40 paraffin embedded BUC specimens and 14 paired non-cancerous mucosas, and their relationships with clinicopathological features., Results: The expression levels of Twist1 mRNA in 13 paired BUC specimens were significantly lower than the non-cancerous mucosas. The positive expression rate of Twist1 protein in BUC specimens (90.0%; 36/40) was significantly higher than the non-cancerous mucosas (7.14%; 1/14). Twist1 protein was mainly distributed in the nucleus, and expressed obviously in the mesenchymal cells of several specimens (13.9%;5/36). However, expressions of Twist1 protein were not associated with TNM stage and grade. It was also shown that the expression tendency of Twist1 protein was distinct from Twist1 mRNA, and both were not correlated with age, gender, and smoking history., Conclusion: As a probable potential biomarker for BUC, Twist1 gene may play a role as an oncogene during the tumorigenesis and development of BUC. Its abnormal protein expression may be associated with disordered regulations after transcription.

Epithelial-mesenchymal transition is an important mechanism of epithelial tumor progression, local invasion and metastasis. The E-cadherin (CDH1) repressor SLUG (SNAI2) and the basic helix-loop-helix transcription factor TWIST1 inhibit CDH1 expression in poorly differentiated malignancies as inducers of epithelial-mesenchymal transition. Epithelial-mesenchymal transition has been implicated in progression from well to poorly differentiated/anaplastic thyroid carcinoma but the expression of SNAI2 and TWIST1 proteins and their phenotypic association in human thyroid cancers has not been extensively studied. We examined the expression of SNAI2, TWIST1 and CDH1 by immunohistochemistry in a panel of well-differentiated and anaplastic thyroid cancers and by qRT-PCR in thyroid cell lines. Ten normal thyroids, 33 follicular adenomas, 56 papillary thyroid carcinomas including 28 follicular variants, 27 follicular carcinomas and 10 anaplastic thyroid carcinomas were assembled on a tissue microarray and immunostained for SNAI2, TWIST1 and CDH1. Most (8/10) anaplastic thyroid carcinomas demonstrated strong nuclear immunoreactivity for SNAI2 with associated absence of CDH1 in 6/8 cases (75%). TWIST1 was expressed in 5/10 anaplastic thyroid carcinomas with absence of CDH1 in 3/5 (60%) cases. These findings were confirmed in whole sections of all anaplastic thyroid carcinomas and in a separate validation set of 10 additional anaplastic thyroid carcinomas. All normal thyroids, follicular adenomas, papillary and follicular thyroid carcinomas were negative for SNAI2 and TWIST1 (P<0.0001) and all showed strong diffuse immunoreactivity for CDH1 (P=0.026). Expression of SNAI2, TWIST1 and CDH1 mRNA varied in a normal thyroid, papillary carcinoma and two anaplastic thyroid carcinoma cell lines tested, but the highest levels of CDH1 mRNA were detected in the normal thyroid cell line while the anaplastic thyroid carcinoma cell line demonstrated the highest levels of SNAI2 and TWIST1 mRNA. Our findings support the role of epithelial-mesenchymal transition in the development of anaplastic thyroid carcinoma.

The anti-apoptosis factor Bcl-2 is known to contribute to tumorigenesis and metastasis. Epithelial-mesenchymal transition (EMT) may also participate in tumor invasion and metastasis. This study investigated the relationship between coexpression profiles of Bcl-2 and EMT regulators in hepatocellular carcinoma (HCC) tumor samples and clinical outcome. The nuclear (Nu) and cytoplasmic (Cyt) expression of Bcl-2 and the EMT regulators Twist-1, Twist-2, and Snail were determined by immunohistochemical staining in tumor tissue isolated from 97 HCC patients. The clinical prognostic values of both individual protein expression and various expression combinations were investigated using univariate and multivariate survival analysis. Results showed that patients with nuclear expression of Bcl-2 had worse clinical outcomes than patients exhibiting cytoplasmic expression. Overall survival was significantly shorter in HCC patients individually expressing Bcl-2-Nu, Twist-1-Nu, Twist-1-Cyt, and Snail (all P<0.05). Patients coexpressing Bcl-2-Nu with Twist-1-Nu, Twist-1-Cyt, Twist-2, or Snail had even worse prognoses than those expressing no biomarker or any one biomarker alone (all P<0.05). Multivariate analysis showed that HCC patients coexpressing Bcl-2-Nu with Twist-1-Cyt had the worst prognosis. This study provides clinical evidence that nuclear expression of Bcl-2 combined with cytoplasmic expression of Twist-1 is a predictor of very poor prognosis in HCC. Coexpression profiles of Bcl-2 and EMT regulators might aid in the selection of the most efficacious therapy for patients with HCC.

Twist, a basic helix-loop-helix transcription factor, and E-cadherin are both correlated with the metastatic progression of several types of cancer. However, it is currently unknown whether their activations have relevance to the progression of osteosarcoma. The purpose of the present study was to investigate the clinicopathological and prognostic value of Twist and E-cadherin in osteosarcoma. Twist and E-cadherin expressions were determined by immunohistochemistry. Patient survival rates were determined by Kaplan-Meier method and log-rank test. Cox regression was adopted for multivariate analysis of prognostic factors. The positive rates of Twist and E-cadherin in 107 osteosarcoma specimens were 31.8 % (34/107) and 20.6 % (22/107), respectively. Twist expression was significantly correlated with that of E-cadherin (r = -0.209, P = 0.031). The positive expression of Twist and E-cadherin was significantly associated with metastasis in 107 osteosarcoma specimens (both P < 0.05). Patients with positive Twist expression had significantly poorer overall survival (OS; P < 0.05) and disease-free survival (DFS, P < 0.05) when compared with patients with the negative expression of Twist. Patients with positive expression of E-cadherin had significantly poorer OS (P < 0.05) when compared with patients with negative E-cadherin expression, but not a significantly poorer DFS (P = 0.081). On multivariate analysis, Twist expression and age were found to be independent prognostic factors for OS (both P < 0.05) and DFS (both P < 0.05). Our results suggest that Twist was expressed significantly more and E-cadherin significantly less in osteosarcoma with metastasis, and expression of both might be related to the prediction of metastasis potency and poor prognosis for patients with osteosarcoma.

The molecules Indian hedgehog (IHH), SP7 (also known as osterix), sex-determining region Y-box 9 (SOX9), runt-related transcription factor 2 (RUNX2) and TWIST1 regulate the normal differentiation of osteo- and chondrogenic cells from precursors during skeletal development and remodeling. The aberrant function of the same molecules has been implicated in the pathogenesis of bone tumors. Preliminary studies suggest that antibodies against these molecules have practical, diagnostic or prognostic utility in tumors. However, a comprehensive analysis of the expression of these molecules in a large, diverse set of bone tumors has yet to be reported. The goals of this study were to compare the immunohistochemical profiles of IHH, SP7, SOX9, RUNX2 and TWIST1 among bone tumors and to determine the optimum panel for diagnostic utility. Tissue microarrays prepared from 206 undecalcified tumors (71 osteosarcomas, 26 osteoblastomas/osteoid osteomas, 50 giant cell tumors, 5 chondromyxoid fibromas and 54 chondroblastomas) were stained with antibodies to IHH, SP7, SOX9, RUNX2 and TWIST1. The stains were scored for intensity (0-3+) and distribution. The results were analyzed by cluster analysis. Optimum antibody panels for diagnostic sensitivity and specificity were calculated. Analysis revealed six main clusters that corresponded well to tumor types and suggested a close relationship between the stromal cells of giant cell tumor and the osteoblasts of osteosarcoma. The expression profile of chondromyxoid fibroma and chondroblastoma also suggested related differentiation. The distribution of osteoblastomas and osteoid osteomas was more heterogeneous. RUNX2, SOX9 and TWIST1 represented the most sensitive and specific immunohistochemical panel to distinguish among these diagnoses with the limitation that no result could discriminate between chondroblastoma and chondromyxoid fibroma. IHH and SP7 did not yield additional utility.

Background: To examine the expression of signal transducer and activator of transcription 3 (STAT3) and its activated form (p-STAT3), Twist, and E-cadherin in hepatocellular carcinoma (HCC), and explore their correlations with HCC progression and prognosis., Materials and Methods: The expression profiles of STAT3, p-STAT3, Twist, and E-cadherin were assessed on 100 clinical HCC samples and 10 normal liver tissues by using an immunohistochemical staining method, and their correlations with clinicopathologic parameters and survival of HCC patients were statistically analyzed., Results: The results demonstrated that the positive rate of STAT3, p-STAT3, and Twist in HCC was significantly higher than that in normal liver tissues; furthermore, 52% of HCC lesions showed reduced E-cadherin expression. Correlation analysis indicated that p-STAT3 was positively correlated with Twist expression, whereas Twist was negatively correlated with E-cadherin expression; p-STAT3, Twist, or E-cadherin expression was significantly associated with HCC invasion and metastasis. Survival analysis showed that HCC patients with p-STAT3, Twist positive expression, or reduced E-cadherin expression had a significantly shorter survival duration than those with p-STAT3, Twist negative expression, or those with normal E-cadherin expression. Multivariate analysis identified p-STAT3, Twist, or E-cadherin expression as an independent prognostic factor for overall survival of HCC patients after surgery., Conclusions: By this study, we suggest that activated STAT3 signal may associate with Twist and E-cadherin expression and mediate HCC invasiveness and metastasis; abnormal p-STAT3/Twist/E-cadherin signal axis may predict poor prognosis of HCC patients., (Copyright © 2012 Elsevier Inc. All rights reserved.)

E-cadherin (E-CD) inactivation with loss of E-CD-mediated cell adhesion is the hallmark of lesions of the lobular phenotype. E-CD is typically absent by immunohistochemistry in both lobular carcinoma in situ (LCIS) and invasive lobular lesions, suggesting it occurs early in the neoplastic process. In laboratory models, downstream post-transcriptional modifiers such as TWIST and SNAIL contribute to the dissociation of the intracellular component of the cadherin-catenin complex (CCC), resulting in tumor progression and invasion. We hypothesized that complete CCC dissociation may play a role in lobular neoplasia progression. Here we explore the relationship between loss of E-CD and dissociation of the CCC in pure LCIS and LCIS associated with invasive cancer. Fresh-frozen tissues were obtained from 36 patients undergoing mastectomy for pure LCIS (n = 11), LCIS with ILC (n = 18) or LCIS with IDC (n = 7). Individual lesions were subject to laser-capture microdissection and gene-expression analysis (Affymetrix HG-U133A 2.0). Immunohistochemistry for ER,PR,HER2, E-CD,N-CD,α-,β-, and phosphoβ-catenin, TWIST, and SNAIL were evaluated in normal, in situ, and invasive components from matched formalin-fixed paraffin-embedded samples (n = 36). CCC-dissociation was defined as negative membranous E-CD, α- and β-catenin expression. E-CD was negative in all LCIS and ILC lesions, and positive in all normal and IDC lesions. Membranous α and β-catenin expressions decreased with the transition from LCIS to ILC (pure LCIS 82%; LCIS w/ILC 28%; ILC 0%), while TWIST expression increased (pure LCIS low; LCIS w/ILC moderate; ILC high). Gene expression paralleled IHC-staining patterns with a stepwise downregulation of E-CD, α and β-catenins from normal to LCIS to invasive lesions, and increasing expression of TWIST from normal to LCIS to ILC. Loss of E-CD expression is an early event in lobular neoplasia. Decreasing membranous catenin expression in tandem with increasing levels of TWIST across the spectrum of lobular lesions suggests that CCC dissociation is a progressive process.

Purpose: To investigate the prognostic relevance of disseminated tumor cells (DTCs) in bone marrow (BM) assessed by a multimarker mRNA panel consisting of TWIST1, cytokeratin 19 (CK19) and human mammaglobin A (hMAM) mRNA, in patients with early breast cancer., Patients and Methods: TWIST1 (gene name: TWIST1), CK19 (gene name: KRT19), and hMAM (gene name: SCGB2A2) mRNA was quantitated in BM samples from 191 operable breast cancer patients by real-time reverse transcriptase polymerase chain reaction (RT-PCR)., Results: Using the highest relative mRNA concentration of TWIST1 in the control population as a cut-off, 5 of the 191 breast cancer patients showed elevated TWIST1 mRNA levels in their BM by real-time RT-PCR. Two of these patients experienced a systemic relapse during a median follow-up of 98 months. Combining these results with previous hMAM and CK19 mRNA quantifications in the same BM samples, 12 (40%) of the 30 patients with BM positive for at least 1 marker (multimarker positive) experienced a systemic relapse as compared with 18 (11%) of the 161 patients with multimarker-negative BMs. The patients with multimarker-positive BM had significantly shorter systemic recurrence-free survival (P < .001, log-rank test), breast cancer-specific survival (P < .001), and overall survival (P = .03). The prognostic relevance of BM multimarker detection appeared to be independent of adjuvant treatment, although the difference was not statistically significant in the subgroup of patients who received adjuvant chemotherapy. Multivariate analysis demonstrated the BM multimarker panel status to be a strong independent predictor of clinical outcome., Conclusion: Our results demonstrated the prognostic relevance of BM DTCs assessed by a multimarker mRNA panel consisting of TWIST1, CK19, and hMAM in operable breast cancer patients.

Objective: The objective of this study was to investigate the pattern of Twist expression in a series of ameloblastomas, and to study the possible role of Twist in the bone destruction and local invasiveness of ameloblastoma variants., Study Design: Immunohistochemical study was performed for Twist protein in 53 ameloblastomas (32 solid/multicystic [SA] and 21 unicystic [UA])., Results: The salient finding was that expression of Twist was related to the histological subtype of tumors, as there was a higher expression in SA (14/32, 43.75%) as compared to UA (4/21, 19.05%) (P < .05). Both nuclei and cytoplasm positivities were detected in positive cases, whereas cytoplasmic staining was diffused and predominant. Cases rich in stromal cells showed a higher percentage of positive cells than those with less stroma., Conclusions: Our results suggest that Twist expression might be associated with invasion in ameloblastoma variants, and stromal cells might play a regulatory role during tumor development.

Changes in the expression of E-cadherin have been reported to be important in the tumorigenesis and progression of epithelial ovarian carcinoma. To further examine the mechanisms regulating E-cadherin expression in ovarian tumorigenesis, we investigated the immunohistochemical expression of transcriptional repressors for E-cadherin, such as Snail, Slug, SIP1, and Twist, in the ovarian surface epithelium (OSE) and 95 cases of epithelial ovarian tumors. OSE cells were negative for SIP1 and Slug, whereas weak expression of Snail and Twist was observed in 8 (73%) and 3 (27%) cases, respectively. Of 95 ovarian tumors, the expression of Snail, Slug, SIP1, and Twist increased stepwise in benign, borderline, and malignant tumors. Among them, the expression of Snail showed significantly inverse correlation with that of E-cadherin. Regarding the FIGO stage classification, the expressions of Snail and Twist were significantly increased in advanced cases. The prognosis of ovarian carcinoma patients positive for Snail expression was poorer than that of negative patients. Our results indicate that the expression of E-cadherin transcriptional repressors increased with malignancy in ovarian epithelial neoplasms and that the expression of E-cadherin and its negative regulators is altered during ovarian cancer development and peritoneal dissemination.

Objectives: Expression of transcription factors that mediate epithelial-mesenchymal transition (EMT), such as Twist and Slug, is correlated with poor prognosis in many tumor types. Selected EMT markers were studied in a series of pancreatic ductal adenocarcinomas (PDAs) and benign pancreatic tissues to determine whether expression levels correlated with diagnosis, histologic grade, or patient outcome., Methods: Immunohistochemical stains for Twist, Slug, and N-cadherin were performed using a tissue microarray containing 68 PDAs and 38 samples of normal pancreas or chronic pancreatitis tissues., Results: Twist and Slug were identified in both the nucleus and cytoplasm of benign pancreatic ductal epithelium, chronic pancreatitis, and PDA. Compared with normal ductal epithelium, nuclear levels of Twist are decreased in PDA. None of the other EMT markers showed significant differences in staining indices among the diagnostic groups. There were no correlations between EMT marker expression and histologic grade. Epithelial-mesenchymal transition marker expression was not associated with N-cadherin expression, patient outcome, or duration of survival., Conclusions: Decreased expression of nuclear Twist is observed in malignant pancreatic epithelium. However, use of Twist as a diagnostic marker is precluded because decreased expression is also seen in chronic pancreatitis. None of the markers studied were predictive of patient outcome.

Background: Twist, a basic helix--loop-helix transcription factor, has been reported to be associated with the development and progression of human cancer. We examined the distribution and expression of Twist in cervical cancer to examine its clinical significance., Patients and Methods: We examined the distribution and expression of Twist in 101 cervical cancer specimens and determined the association between their expression and the clinico-pathological features observed, including patient outcome., Results: Of the 101 specimens, 55 cases were negative for Twist immuno-expression, whereas 46 were positive. When categorized into negative versus positive expression, Twist was not associated with any of the clinico-pathological parameters examined. Positive Twist expression significantly predicted poorer overall survival (OS) and progression-free survival (PFS) when compared with negative expression (P < 0.01). In the multivariate analyses, positive Twist expression was an independent prognostic factor for OS (P < 0.05)., Conclusions: Our data imply that positive Twist expression seems to be a useful marker in patients with cervical cancer likely to have an unfavorable clinical outcome.

Purpose: Epithelial to mesenchymal transitions are vital for tumor growth and metastasis. Several inducers of epithelial to mesenchymal transition are transcription factors that repress E-cadherin expression, such as Snail, Slug, and Twist. In this study, we aimed to examine the expression of these transcription factors in pancreatic cancer., Experimental Design: The expression of Snail, Slug, and Twist was detected by immunohistochemistry in tissue samples from patients with pancreatic ductal adenocarcinoma. Five human pancreatic cancer cell lines (AsPC-1, Capan-1, HPAF-2, MiaPaCa-2, and Panc-1) were analyzed by reverse transcription-PCR, real-time PCR, and Western blotting. An orthotopic nude mouse model of pancreatic cancer was applied for in vivo experiments., Results: Seventy-eight percent of human pancreatic cancer tissues showed an expression of Snail, and 50% of the patients displayed positive expression of Slug. Twist showed no or only weak expression. Snail expression was higher in undifferentiated cancer cell lines (MiaPaCa-2 and Panc-1) than in more differentiated cell lines (Capan-1, HPAF-2, AsPC-1). Expression of Slug was detected in all cell lines with different intensities. Twist was not expressed. After exposure to hypoxia, the Twist gene was activated in all five pancreatic cancer cell lines., Conclusions: The transcription factors Snail and Slug are expressed in pancreatic cancer but not in normal tissue, suggesting a role in the progression of human pancreatic tumors. Twist, activated by hypoxia, may play an important role in the invasive behavior of pancreatic tumors.