pRb2/p130 localizes to the cytoplasm in diffuse gastric cancer.

pRb2/p130 is a key tumor suppressor, whose oncosuppressive activity has mainly been attributed to its ability to negatively regulate cell cycle by interacting with the E2F4 and E2F5 transcription factors. Indeed, pRb2/p130 has been found altered in various cancer types in which it functions as a valuable prognostic marker. Here, we analyzed pRb2/p130 expression in gastric cancer tissue samples of diffuse histotype, in comparison with their normal counterparts. We found a cytoplasmic localization of pRb2/p130 in cancer tissue samples, whereas, in normal counterparts, we observed the expected nuclear localization. pRb2/p130 cytoplasmic delocalization can lead to cell cycle deregulation, but considering the emerging involvement of pRb2/p130 in other key cellular processes, it could contribute to gastric tumorigenesis also through other mechanisms. Our data support the necessity of further investigations to verify the possibility of using pRb2/p130 as a biomarker or potential therapeutic target for diffuse gastric cancer.

Tumor Cellularity Assessment of Breast Histopathological Slides via Instance Segmentation and Pathomic Features Explainability.

The segmentation and classification of cell nuclei are pivotal steps in the pipelines for the analysis of bioimages. Deep learning (DL) approaches are leading the digital pathology field in the context of nuclei detection and classification. Nevertheless, the features that are exploited by DL models to make their predictions are difficult to interpret, hindering the deployment of such methods in clinical practice. On the other hand, pathomic features can be linked to an easier description of the characteristics exploited by the classifiers for making the final predictions. Thus, in this work, we developed an explainable computer-aided diagnosis (CAD) system that can be used to support pathologists in the evaluation of tumor cellularity in breast histopathological slides. In particular, we compared an end-to-end DL approach that exploits the Mask R-CNN instance segmentation architecture with a two steps pipeline, where the features are extracted while considering the morphological and textural characteristics of the cell nuclei. Classifiers that are based on support vector machines and artificial neural networks are trained on top of these features in order to discriminate between tumor and non-tumor nuclei. Afterwards, the SHAP (Shapley additive explanations) explainable artificial intelligence technique was employed to perform a feature importance analysis, which led to an understanding of the features processed by the machine learning models for making their decisions. An expert pathologist validated the employed feature set, corroborating the clinical usability of the model. Even though the models resulting from the two-stage pipeline are slightly less accurate than those of the end-to-end approach, the interpretability of their features is clearer and may help build trust for pathologists to adopt artificial intelligence-based CAD systems in their clinical workflow. To further show the validity of the proposed approach, it has been tested on an external validation dataset, which was collected from IRCCS Istituto Tumori "Giovanni Paolo II" and made publicly available to ease research concerning the quantification of tumor cellularity.

The role of unpaired image-to-image translation for stain color normalization in colorectal cancer histology classification.

BACKGROUND: Histological assessment of colorectal cancer (CRC) tissue is a crucial and demanding task for pathologists. Unfortunately, manual annotation by trained specialists is a burdensome operation, which suffers from problems like intra- and inter-pathologist variability. Computational models are revolutionizing the Digital Pathology field, offering reliable and fast approaches for challenges like tissue segmentation and classification. With this respect, an important obstacle to overcome consists in stain color variations among different laboratories, which can decrease the performance of classifiers. In this work, we investigated the role of Unpaired Image-to-Image Translation (UI2IT) models for stain color normalization in CRC histology and compared to classical normalization techniques for Hematoxylin-Eosin (H&E) images. METHODS: Five Deep Learning normalization models based on Generative Adversarial Networks (GANs) belonging to the UI2IT paradigm have been thoroughly compared to realize a robust stain color normalization pipeline. To avoid the need for training a style transfer GAN between each pair of data domains, in this paper we introduce the concept of training by exploiting a meta-domain, which contains data coming from a wide variety of laboratories. The proposed framework enables a huge saving in terms of training time, by allowing to train a single image normalization model for a target laboratory. To prove the applicability of the proposed workflow in the clinical practice, we conceived a novel perceptive quality measure, which we defined as Pathologist Perceptive Quality (PPQ). The second stage involved the classification of tissue types in CRC histology, where deep features extracted from Convolutional Neural Networks have been exploited to realize a Computer-Aided Diagnosis system based on a Support Vector Machine (SVM). To prove the reliability of the system on new data, an external validation set composed of N = 15,857 tiles has been collected at IRCCS Istituto Tumori "Giovanni Paolo II". RESULTS: The exploitation of a meta-domain consented to train normalization models that allowed achieving better classification results than normalization models explicitly trained on the source domain. PPQ metric has been found correlated to quality of distributions (Fréchet Inception Distance - FID) and to similarity of the transformed image to the original one (Learned Perceptual Image Patch Similarity - LPIPS), thus showing that GAN quality measures introduced in natural image processing tasks can be linked to pathologist evaluation of H&E images. Furthermore, FID has been found correlated to accuracies of the downstream classifiers. The SVM trained on DenseNet201 features allowed to obtain the highest classification results in all configurations. The normalization method based on the fast variant of CUT (Contrastive Unpaired Translation), FastCUT, trained with the meta-domain paradigm, allowed to achieve the best classification result for the downstream task and, correspondingly, showed the highest FID on the classification dataset. CONCLUSIONS: Stain color normalization is a difficult but fundamental problem in the histopathological setting. Several measures should be considered for properly assessing normalization methods, so that they can be introduced in the clinical practice. UI2IT frameworks offer a powerful and effective way to perform the normalization process, providing realistic images with proper colorization, unlike traditional normalization methods that introduce color artifacts. By adopting the proposed meta-domain framework, the training time can be reduced, and the accuracy of downstream classifiers can be increased.

Overexpression of nuclear NHERF1 in advanced colorectal cancer: association with hypoxic microenvironment and tumor invasive phenotype.

Over 57% of colorectal cancer (CRC) patients have regional or distant spread of their disease at the time of diagnosis. Despite recent advances, there is a compelling need to better characterize prognostic markers for advanced CRC. The present study investigates protein expression of NHERF1, HIF-1α and TWIST1 and their relationship in distant normal mucosa (DNM), tumor (T) and adjacent normal mucosa (ANM), lymph node metastasis (LNM) and liver metastasis (LM), determining their role as potential markers in advanced stages of human CRC. Overexpression of nuclear NHERF1 was shown in 47% of tumors, which exhibited a significant association with poor histological grade (P=0.0346). Nuclear NHERF1 showed a higher expression in T, LNM and LM than both DNM (P<0.0001) and ANM (P<0.05). Nuclear HIF-1α was significantly higher in T, LNM and LM than DNM and ANM (P<0.05, P<0.001, P<0.0001, respectively). A positive correlation between nuclear NHERF1 and nuclear HIF-1α was found in LNM (r=0.331, P=0.020), where an extended co-localization of the two proteins was demonstrated. TWIST1 was more expressed in T than DNM and ANM (P<0.0001) and was higher in T than LNM and LM (P<0.0001). Moreover, nuclear NHERF1 was directly correlated to TWIST1 (r=0.339, P=0.015) in T samples, where a high co-expression of the two proteins was demonstrated both in no longer polarized epithelial cells and in invasive mesenchymal elements adjacent to hypoxic and perinecrotic colonic areas. Overall, nuclear NHERF1 expression was associated with poorer differentiation grade and with higher expression both of HIF-1α in lymphatic metastasis and TWIST1 in invasive front of tumor. Our results support the oncogenic role of NHERF1 and promote nuclear NHERF1 as a potential new biomarker of advanced CRC.

Cytometry and DNA ploidy: clinical uses and molecular perspective in gastric and lung cancer.

Flow cytometry is one of the most powerful and specific methods used for the integrated study of the molecular and morphological events occurring during cell proliferation. Many methods have been described for investigating this process. Several cell cycle regulators controlling the correct entry and progression through the cell cycle are altered in tumors. In fact, in most, if not all, human cancers there is a deregulated control of G1 phase progression, the period when cells decide if they will start proliferation or stay quiescent. Cytometry (flow and image) is able to analyze DNA content thanks to the use of the same "molecule" conjugates with a fluorochrome that permits to identify DNA content of single cell in a sample. Most important results of studies on DNA ploidy have been reviewed during the last years and as a result the analyses of DNA ploidy in cancer may provide clinically useful information on diagnostic, therapeutic and prognostic aspects. In fact, aneuploid cancer has a high proliferative activity and a metastatic or invasive potential, markers of a poor prognosis. Multiparametric flow cytometry should allow the simultaneous determination of morphology, phenotype, intracellular protein expression, and status of chromatin and DNA. Evaluating if a particular protein is responsible for the aggressiveness of cancer, or the alteration of DNA content, or if the activation of its state is the cause of rapid growth of cancer cells, is very important and it can facilitate the clinical treatment of patients.

Dysregulated calcium homeostasis and oxidative stress in chronic myeloid leukemia (CML) cells.

Chronic myeloid leukemia (CML) is a hematopoietic stem cell disorder caused by the oncogenic activity of the Bcr-Abl protein, a deregulated tyrosine kinase. Calcium may act directly on cellular enzymes and in conjunction with other cellular metabolites, such as cyclic nucleotides, to regulate cell functions. Alteration in the ionized calcium concentration in the cytosol has been implicated in the initiation of secretion, contraction, and cell proliferation as well as the production of reactive oxygen species (ROS) has been correlates with normal cell proliferation through activation of growth-related signaling pathways. In this study we evaluated in peripheral blood leukocytes from CML patients the role of the balance between intracellular calcium and oxidative stress in CML disease in order to identify possible therapeutic targets in patients affected by this pathology. Our results demonstrated that peripheral blood mononuclear cells derived from CML patients displayed decreased intracellular calcium [Ca(2+)](i) fluxes both after InsP(3) as well as ATP and ionomycin (IONO) administration. CML cells showed lower levels of superoxide dismutase (SOD) activity and significantly higher malondialdehyde levels (MDA) than peripheral blood mononuclear cells derived from control patients. Finally we showed that resveratrol is able to down-regulate InsP3 and ATP effects on intracellular calcium [Ca(2+)](i) fluxes as well as the effects of ATP and IONO on oxidative stress in CML cells.

P53-regulated miR-320a targets PDL1 and is downregulated in malignant mesothelioma.

Malignant pleural mesothelioma (MPM) is an aggressive cancer, related to asbestos exposure, which has a dismal prognosis. MPM diagnosis is late and often challenging, suggesting the need to identify more reliable molecular biomarkers. Here, we set out to identify differentially expressed miRNAs in epithelioid, biphasic, and sarcomatoid MPMs versus normal mesothelium and explored specific miRNA contribution to mesothelial tumorigenesis. We screened an LNA™-based miRNA-microrray with 14 formalin-fixed paraffin-embedded (FFPE) MPMs and 6 normal controls. Through real-time qRT-PCR we extended the analysis of a miRNA subset and further investigated miR-320a role through state-of-the-art techniques. We identified 16 upregulated and 32 downregulated miRNAs in MPMs versus normal tissue, including the previously identified potential biomarkers miR-21, miR-126, miR-143, miR-145. We showed in an extended series that miR-145, miR-10b, and miR-320a levels can discriminate tumor versus controls with high specificity and sensitivity. We focused on miR-320a because other family members were found downregulated in MPMs. However, stable miR-320a ectopic expression induced higher proliferation and migration ability, whereas miR-320a silencing reduced these processes, not supporting a classic tumor-suppressor role in MPM cell lines. Among putative targets, we found that miR-320a binds the 3'-UTR of the immune inhibitory receptor ligand PDL1 and, consistently, miR-320a modulation affects PDL1 levels in MPM cells. Finally, we showed that p53 over-expression induces the upregulation of miR-320a, along with miR-200a and miR-34a, both known to target PDL1, and reduces PDL1 levels in MPM cells. Our data suggest that PDL1 expression might be due to a defective p53-regulated miRNA response, which could contribute to MPM immune evasion or tumorigenesis through tumor-intrinsic roles.

Correction: P53-regulated miR-320a targets PDL1 and is downregulated in malignant mesothelioma.

Author Francesca Pentimalli was incorrectly associated with Histopathological Unit, IRCCS-Istituto Tumori "Giovanni Paolo II", Viale Orazio Flacco 65, 70124 Bari, Italy. The author's actual affiliation is Cell Biology and Biotherapy Unit, Istituto Nazionale Tumori-IRCCS-Fondazione G. Pascale, I-80131 Napoli, Italy.

The antiretroviral nucleoside analogue Abacavir reduces cell growth and promotes differentiation of human medulloblastoma cells.

Abacavir is one of the most efficacious nucleoside analogues, with a well-characterized inhibitory activity on reverse transcriptase enzymes of retroviral origin, and has been clinically approved for the treatment of AIDS. Recently, Abacavir has been shown to inhibit also the human telomerase activity. Telomerase activity seems to be required in essentially all tumours for the immortalization of a subset of cells, including cancer stem cells. In fact, many cancer cells are dependent on telomerase for their continued replication and therefore telomerase is an attractive target for cancer therapy. Telomerase expression is upregulated in primary primitive neuroectodermal tumours and in the majority of medulloblastomas suggesting that its activation is associated with the development of these diseases. Therefore, we decided to test Abacavir activity on human medulloblastoma cell lines with high telomerase activity. We report that exposure to Abacavir induces a dose-dependent decrease in the proliferation rate of medulloblastoma cells. This is associated with a cell accumulation in the G(2)/M phase of the cell cycle in the Daoy cell line, and with increased cell death in the D283-MED cell line, and is likely to be dependent on the inhibition of telomerase activity. Interestingly, both cell lines showed features of senescence after Abacavir treatment. Moreover, after Abacavir exposure we detected, by immunofluorescence staining, increased protein expression of the glial marker glial fibrillary acidic protein and the neuronal marker synaptophysin in both medulloblastoma cell lines. In conclusion, our results suggest that Abacavir reduces proliferation and induces differentiation of human medulloblastoma cells through the downregulation of telomerase activity. Thus, using Abacavir, alone or in combination with current therapies, might be an effective therapeutic strategy for the treatment of medulloblastoma.

Endothelial area and microvascular density in a canine non-Hodgkin's lymphoma: an interspecies model of tumor angiogenesis.

Experimental and clinical data indicate that tumor progression is associated with angiogenesis and that an increase in microvascular density (MVD) is associated with a poor prognosis, in both solid and hematological malignancies. No data have been published concerning the relationship between angiogenesis and malignancy grade in canine non-Hodgkin's lymphoma (NHL), which is a neoplasm that shares several biological and clinical characteristics with human NHL. In the present study, we evaluated this relationship in a series of 43 cases of canine NHL. The results demonstrate that both MVD and endothelial area (EA) were significantly higher in high-grade compared to low-grade lymphoma and a good statistical correlation was found between MVD and EA. These data indicate that increased angiogenesis paralleled with increased malignancy grade in canine NHL, which represents an interesting tumor model for studying the role of angiogenesis as an interspecies pathway of tumoral malignancy and biological aggressiveness.

Pharmacological targeting of p53 through RITA is an effective antitumoral strategy for malignant pleural mesothelioma.

Malignant mesothelioma, a very aggressive tumor associated to asbestos exposure, is expected to increase in incidence, and unfortunately, no curative modality exists. Reactivation of p53 is a new attractive antitumoral strategy. p53 is rarely mutated in mesothelioma, but it is inactivated in most tumors by the lack of p14(ARF). Here, we evaluated the feasibility of this approach in pleural mesothelioma by testing RITA and nutlin-3, two molecules able to restore p53 function through a different mechanism, on a panel of mesothelioma cell lines representing the epithelioid (NCI-H28, NCI-H2452, IST-MES 2), biphasic (MSTO-211H), and sarcomatoid (NCI-H2052) histotypes compared with the normal mesothelial HMC-hTERT. RITA triggered robust caspase-dependent apoptosis specifically in epithelioid and biphasic mesothelioma cell lines, both through wild-type and mutant p53, concomitant to p21 downregulation. Conversely, nutlin-3 induced a p21-dependent growth arrest, rather than apoptosis, and was slightly toxic on HMC-hTERT.   Interestingly, we identified a previously undetected point mutation of p53 (p.Arg249Ser) in IST-MES 2, and showed that RITA is also able to reactivate this p53 mutant protein and its apoptotic function. RITA reduced tumor growth in a MSTO-211H-derived xenograft model of mesothelioma and synergized with cisplatin, which is the mainstay of treatment for this tumor. Our data indicate that reactivation of p53 and concomitant p21 downregulation effectively induce cell death in mesothelioma, a tumor characterized by a high intrinsic resistance to apoptosis. Altogether, our findings provide the preclinical framework supporting the use of p53-reactivating agents alone, or in combination regimens, to improve the outcome of patients with mesothelioma.

Centromere 17 copy number alteration: negative prognostic factor in invasive breast cancer?

CONTEXT: Chromosome 17 polysomy has been identified in 5% to 50% of invasive breast cancers; even though a relationship with human epidermal growth factor receptor 2 (HER2/ neu ) status has been reported, other studies have shown that coincident centromere 17 (Cep17) amplification may be the cause of an overestimation of chromosome 17 polysomy in fluorescence in situ hybridization (FISH) testing. OBJECTIVE: To evaluate polysomy/amplification of Cep17 in invasive breast cancer with relation to proliferative activity (Ki-67), estrogen receptor, progesterone receptor, and HER2/ neu status, in an attempt to identify a subgroup of patients with a worse prognosis. DESIGN: A total of 647 cases of invasive ductal breast cancer were collected and subjected to FISH analysis for HER2/neu gene and centromere 17 alteration, HercepTest for HER2/ neu protein expression, and routine immunohistochemistry for Ki-67 and hormone receptor status. RESULTS: Copy number gain of Cep17 was observed in 27.3% of cases. Within this group, HER2/neu gene amplification was detected in 14.1% of cases, whereas HER2/ neu expression was scored 3+ in 20.1% of cases; about half of the HER2/ neu overexpressing cases (9.8%) did not show amplification by FISH. Moreover, 69% of polysomic cases showed high Ki-67 index. CONCLUSIONS: (1) Centromere 17-altered cases are frequently HER2/ neu overexpressing but not amplified, resulting in HercepTest/FISH disagreement; (2) HER2/neu amplification is seen at a higher incidence in cases without Cep17 copy number alterations, which are therefore not necessarily due to chromosome 17 disorder; (3) proliferation index is significantly higher in aneusomic tumors. These data suggest that the presence of Cep17 alterations could identify a subset of breast cancers with more aggressive biological and clinical behavior, which may show nonresponsiveness to conventional therapy independently of HER2/neu amplification status.

Rb family proteins in gastric cancer (review).

Gastric cancer is one of the most diffuse neoplastic pathologies in the world whose environmental and molecular causes, although deeply investigated, have not been completely clarified. Besides some well-established etiological factors, such as Helicobacter pylori and E-cadherin mutations, investigations on other possible causes gave contrasting results. Rb family proteins (including pRb/p105, pRb2/p130 and p107) are involved in cell cycle regulation and their function and/or expression is often lost in various kinds of tumours such as lung, bladder, breast and brain cancer. The consequences of RB inactivation in tumours can be very different depending on the context and the type of cancer. Recent evidence indicates that Rb status correlates with a different therapeutic response according to the tumour type and the therapeutic agent. Studies performed on Rb family proteins in gastrointestinal tract tumours suggest that these proteins have an important role in these cancer types. However, owing to contrasting results, further investigation is required to assess whether the expression of Rb family proteins can potentially be used as a prognostic or predictive factor in gastric cancer.

Genes involved in regulation of stem cell properties: studies on their expression in a small cohort of neuroblastoma patients.

Cancer stem cells have been isolated from many tumors. Several evidences prove that neuroblastoma contains its own stem cell-like cancer cells. We chose to analyze 20 neuroblastoma tumor samples in the expression of 13 genes involved in the regulation of stem cell properties to evaluate if their misregulation could have a clinical relevance. In several specimens we detected the expression of genes belonging to the OCT3/SOX2/NANOG/KLF4 core circuitry that acts at the highest level in regulating stem cell biology. This result is in agreement with studies showing the existence of malignant stem cells in neuroblastoma. We also observed differences in the expression of some stemness-related genes that may be useful for developing new prognostic analyses. In fact, preliminary data suggests that the presence/absence of UTF1 along with differences in BMI1 mRNA levels could distinguish low grade neuroblastomas from IV stage tumors.

Immunohistochemical analysis of pRb2/p130, VEGF, EZH2, p53, p16(INK4A), p27(KIP1), p21(WAF1), Ki-67 expression patterns in gastric cancer.

Although the considerable progress against gastric cancer, it remains a complex lethal disease defined by peculiar histological and molecular features. The purpose of the present study was to investigate pRb2/p130, VEGF, EZH2, p53, p16(INK4A), p27(KIP1), p21(WAF1), Ki-67 expressions, and analyze their possible correlations with clinicopathological factors. The expression patterns were examined by immunohistochemistry in 47 patients, 27 evaluated of intestinal-type, and 20 of diffuse-type, with a mean follow up of 56 months and by Western blot in AGS, N87, KATO-III, and YCC-2, -3, -16 gastric cell lines. Overall, stomach cancer showed EZH2 correlated with high levels of p53, Ki-67, and cytoplasmic pRb2/p130 (P < 0.05, and P < 0.01, respectively). Increased expression of EZH2 was found in the intestinal-type and correlated with the risk of distant metastasis (P < 0.05 and P < 0.01, respectively), demonstrating that this protein may have a prognostic value in this type of cancer. Interestingly, a strong inverse correlation was observed between p27(KIP1) expression levels and the risk of advanced disease and metastasis (P < 0.05), and a positive correlation between the expression levels of p21(WAF1) and low-grade (G1) gastric tumors (P < 0.05), confirming the traditionally accepted role for these tumor-suppressor genes in gastric cancer. Finally, a direct correlation was found between the expression levels of nuclear pRb2/p130 and low-grade (G1) gastric tumors that was statistically significant (P < 0.05). Altogether, these data may help shed some additional light on the pathogenetic mechanisms related to the two main gastric cancer histotypes and their invasive potentials.

Drug targets to pro-angiogenetic factors with special reference to primary peritoneal mesothelioma.

Angiogenesis is necessary for growth and the spread of human tumors. Animal studies also suggest that angiogenesis is an important interspecies biological mechanism of tumor development. Angiogenesis is a complex multistep cascade modulated by both positive soluble factors, such as vascular endothelial growth factor, thymidine phosphorylase, basic-fibroblast growth factor and negative soluble factors such as angiostatin and endostatin. From the imbalance of the above angiogenesis regulators, tumor endothelial cells may divide up to 50 times more frequently than endothelial cells of normal tissue. Published studies have suggested that the assessment of microvessel density (MVD) or endothelial area (EA) can be considered as surrogate markers of angiogenesis with biological and prognostic relevance. Literature data on angiogenesis of mesothelioma are inconclusive, with only a few studies performed in primary peritoneal mesothelioma (PPM) due to the rarity of the disease. We assessed immunohistochemically MVD and EA and their biological and clinical significance in a consecutive series of 23 PPM cases. MVD and EA were detected in "hot spots" by a computerized image analyzer. The mean value of MVD and EA was 27 +/- 14 and 26.04 +/- 8.35 x10(-2) micro(2) per field (400x), respectively. Patients with a high MVD or EA tumors showed a more clinical aggressiveness due to the presence of ascites and a shorter overall survival. Our results suggest that PPM is an angiogenesis-dependent neoplasia. Therefore, antiangiogenic compounds should be tested particularly in those patients with highly vascularized PPM.

Immunohistochemical evaluation of pRb2/p130, VEGF, EZH2, p53, p16, p21waf-1, p27, and PCNA in Barrett's esophagus.

Control of the G1/S-phase transition as well as angiogenic switch are two of the most studied mechanisms in cancer. The current study examined the correlation between the immunohistochemical expression of pRb2/p130, VEGF, EZH2, p53, p16, p21waf-1, p27, and PCNA in Barrett's esophagus (BE). Overall, p53 showed a much higher expression in BE patients (up to 50%) than in controls (1-10%) (P < 0.005). Also p21 showed a downregulation in BE when compared to normal esophagus (70% of cells vs. 65%), but the difference did not show any statistical significance (P = 0.45). pRb2/p130 was detected in 80% of cells in normal controls, but showed positive in only 20% of cells in BE biopsies. Additionally, Rb2/p130 expression was inversely correlated to that of VEGF, EZH2, and PCNA (P < 0.0001, P = 0.0032, P < 0.001, respectively). p27 stained more intensely and in a widespread manner (70%) cells in normal esophageal tissues but about only 30% in BE samples (P < 0.001). Lastly, in accordance with other reports, we also found p16 expressed by immunohistochemistry at high levels in normal controls and at low levels in BE (P < 0.001). In conclusion, p16, p21, p27, and p53 staining confirmed previously published data. Interestingly, pRb2/p130 expression was found significantly decreased in metaplastic epithelium compared to normal controls and showed significant inverse correlation with the expression of other markers, such as VEGF, EZH2, and PCNA. These data, taken together, indicate that these molecular events occurring in Barrett's metaplasia (BM) may represent one of the many steps taking place during esophageal malignant progression such as impairment of cell-cycle control, altered differentiation, and unbalanced angiogenesis.