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Objective: The involvement of epithelial-mesenchymal transition (EMT) in esophageal squamous cell carcinoma (ESCC) has not been fully elucidated. Here, we aimed to identify EMT-related genes associated with TGF-β in ESCC and to clarify the role of these genes in the progression of ESCC., Methods: EMT-related genes associated with TGF-β expression were identified in patients with ESCC using microarray analysis and public datasets. The effects of ubiquitin-like with PHD and ring finger domains 2 (UHRF2) expression were analyzed in ESCC cell lines. Cell proliferation and invasion were measured using MTT and invasion assays, respectively. UHRF2 mRNA expression was also analyzed in 75 ESCC specimens to determine the clinical significance of UHRF2 in ESCC., Results: Treatment of ESCC cell lines with TGF-β increased UHRF2 expression. UHRF2 overexpression increased CDH1 (E-cadherin) expression and decreased invasive capacity. The 75 ESCC specimens were divided into the UHRF2 high-expression group (n = 61) and the UHRF2 low-expression group (n = 14). Low UHRF2 expression was significantly correlated with vascular invasion (p = 0.034) and was an independent prognostic factor for poor prognosis (p = 0.005)., Conclusion: UHRF2 may be a negative regulator of EMT and a novel prognostic biomarker for ESCC., (© 2018 S. Karger AG, Basel.)

Programmed death-ligand 1 (PD-L1) plays a crucial role in the host immune system in cancer progression. The gene promoter region of PD-L1 also contains a binding site for ZEB1, a transcription factor related to epithelial-mesenchymal transition (EMT). The purpose of this study was to clarify the relationship between PD-L1 and EMT and its clinical importance in esophageal squamous cell carcinoma (ESCC). PD-L1 and ZEB1 expression at the tumor invasive front was examined by immunohistochemistry in resected specimens from 90 patients with ESCC who underwent surgery without preoperative therapy, and their expression and clinicopathological factors were compared. ZEB1 and PD-L1 expression was determined in TE8 cells, which demonstrate the EMT phenotype, following ZEB1 knockdown by siZEB1. TE5, TE6 and TE11 cells with non-EMT phenotype were also used for studies of TGF-β1-dependent EMT induction and ZEB1 and PD-L1 expression. In cases of high PD-L1 expression at the invasive front, significantly greater depth of tumor invasion, EMT, and less CD8 + lymphocyte infiltration were observed. High PD-L1 expression was also associated with worse overall and relapse-free survival. A correlation was observed between PD-L1 and ZEB1 expression. In TE8 cells, siZEB1 suppressed PD-L1 and promoted E-cadherin mRNA and protein expression. TGF-β1 induced EMT and surface expression of PD-L1 in TE5, TE6 and TE11 cell lines. PD-L1 expression at the ESCC invasive front was related to ZEB1 expression, EMT and poor prognosis. We suggest that a cooperative mechanism bridging between tumor immune avoidance and EMT contributes to tumor malignancy in ESCC., (© 2017 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association.)

Objectives: Esophageal squamous cell carcinoma (ESCC) is one of the most aggressive malignancies owing to the high frequency of tumor recurrence. The identification of markers for early ESCC diagnosis and prediction of recurrence is expected to improve the long-term prognosis. Therefore, we searched for associations between tumor recurrence and cell-free DNA (cfDNA) mutations in blood plasma, which contains genetic markers for various cancer types., Experimental Design: Genomic DNA from tumors and cfDNA from plasma were obtained from 13 patients undergoing treatment for newly diagnosed ESCC. Next-generation sequencing of cfDNA in plasma was performed to identify mutations in 53 cancer-related genes, in which recurrent mutations were previously detected in ESCC. cfDNA mutational profiles were compared before and after tumor resection in four patients. Furthermore, somatic mutations in serial plasma samples were monitored after treatment in four patients., Results: We identified multiple concordant somatic mutations in cfDNA and primary tumor samples from 10 patients (83.3%) and in cfDNA and metastatic tumor samples from one patient (100%). Furthermore, the allele frequency of the concordant mutations in cfDNA changed concomitantly with tumor burden and increased approximately 6 months earlier than the detection of tumor recurrences by imaging tests in two patients. Conventional biomarkers, such as SCC and p53-Ab, did not reflect tumor recurrences., Conclusions: The present multigene panel, which enabled the diagnosis of tumor recurrence with greater accuracy than did using standard tumor markers or imaging methods, is expected to greatly facilitate standard, postoperative follow-up monitoring in ESCC., Competing Interests: None of the authors have any potential conflicts of interest.

The significance of neoadjuvant chemoradiotherapy (NACRT) for esophageal squamous cell carcinoma (ESCC) remains controversial with regard to the pathological response and long-term survival. We herein review the current status of and future perspectives regarding NACRT followed by esophagectomy for locally advanced ESCC. Some studies have suggested that a pathological complete response with NACRT is more common in patients with ESCC than in those with adenocarcinoma and that NACRT provided a survival benefit limited to patients with ESCC. However, NACRT may increase the risk of postoperative complications after esophagectomy. It is obvious that a favorable pathological response is the most important factor for obtaining a survival benefit, although no established parameters have been implemented clinically to predict the response to NACRT. Prospective clinical studies and basic research studies to identify predictive biomarkers for the response to NACRT are needed to aid in the development of NACRT treatment strategies for patients with ESCC.

Background: Cardiac tamponade, due to bleeding in the pericardial space after esophagectomy for esophageal cancer, is an extremely rare complication and may be associated with sudden hemodynamic instability that can lead to death unless there is prompt diagnosis and appropriate treatment., Case Report: A 76-year-old man underwent sub-total esophagectomy via a cervico-right thoracoabdominal approach and reconstruction with a gastric tube through the retrosternal route. On postoperative day 4, the patient developed hypotension due to cardiac tamponade caused by bleeding into the pericardial space and he had a decreased level of consciousness. Pericardial resection and open drainage via a minimal left anterior thoracotomy was performed that resulted in hemodynamic improvement followed by an uneventful recovery., Conclusion: Cardiac tamponade due to postoperative bleeding, which is a rare but life-threatening complication, should be considered as a cause of hemodynamic instability in the early postoperative period after esophagectomy., (Copyright© 2015 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.)

Purpose: This study clarifies age differences in clinicopathologic characteristics and risk factor exposure of patients who have undergone esophagectomy for esophageal cancer (EC)., Methods: Clinical results of esophagectomy were compared between 22 patients younger than 50 years of age (Group I) and 327 patients older than 50 years of age (Group II) with esophageal squamous cell carcinoma., Results: The two groups did not significantly differ in clinicopathological characteristics, including prognosis. Postoperative pulmonary complication incidence rates were 4.2 % (Group I) and 14.4 % (Group II). In Group I, the incidence of multiple ECs was 36.4 %, and association with head and neck cancer was 31.8 %, which were significantly higher than in Group II (13.4 %, p = 0.021; and 9.2 %, p = 0.015, respectively). Furthermore, the patients in Group I with multiple cancers were almost all heavy smokers and/or users of alcohol., Conclusions: These results suggest that multiple upper aerodigestive tract cancers are associated with heavy exposure to risk factors in patients younger than 50 years of age.

Background: Global DNA hypomethylation is associated with increased chromosomal instability and plays an important role in tumorigenesis. The methylation status of the long interspersed nuclear element-1 (LINE-1) element is a useful surrogate marker for global DNA methylation. Although LINE-1 hypomethylation is recognized as a poor prognostic marker, the correlation of LINE-1 methylation level with tumor suppressor gene mutation, chromosomal instability, and clinical significance in esophageal squamous cell carcinoma (ESCC) remains unclear., Methods: Using resected tumor tissues and the corresponding normal esophageal mucosa from 105 patients with ESCC, bisulfite pyrosequencing analysis was performed to quantify the LINE-1 methylation levels. p53 mutations in exons two to ten were detected by polymerase chain reaction direct sequencing. Chromosomal instability was assessed by single nucleotide polymorphism array comparative genomic hybridization analysis., Results: The LINE-1 methylation level of ESCC was significantly lower than matched normal mucosa. LINE-1 methylation levels of normal mucosa from the esophagus had a significant inverse correlation with both cigarette smoking and alcohol consumption of the study subjects. LINE-1 hypomethylation of ESCC was significantly associated with lymph node metastasis, lymphovascular invasion, the frequency of p53 mutation and poor survivability. The LINE-1 methylation levels in ESCC had a significant inverse association with the percentage of copy number alterations in the whole genome, mirroring chromosomal instability., Conclusions: Our results suggested that whole genome hypomethylation caused by chronic inflammation could initiate carcinogenesis of esophageal squamous cells through chromosomal instability. In addition, chromosomal instability associated with the global hypomethylation might correlate highly with the progression of ESCC.

Esophageal cancer is frequently associated with squamous cell carcinoma in the head and neck. Both cigarette smoking and alcohol consumption are risk factors for multiple cancers of the head and neck, as well as the esophagus. Routine screening and close follow-up for second cancers are important in patients with esophageal cancer or head and neck cancer. For this purpose, endoscopy with Lugol's staining, as well as narrow-band imaging combined with magnifying endoscopy, is a powerful tool for the early detection of esophageal cancer. Multimodal therapy is essential for patients with double cancers. When considering surgical treatment, the curability of both cancers must be carefully evaluated. If both tumors are potentially curable, each lesion should be treated individually. In patients with metachronous double cancers, the prior treatment of the first primary carcinoma often affects the treatment of the second cancer. Close cooperation among medical staff members is essential for complicated surgeries for double cancers. Techniques that are appropriate for each case must be adopted, such as careful dissection, staged operations, muscular flaps and microvascular anastomosis.

Esophageal cancer is the eighth most common incident cancer in the world and ranks sixth among all cancers in mortality. Esophageal cancers are classified into two histological types; esophageal squamous cell carcinoma (ESCC), and adenocarcinoma, and the incidences of these types show a striking variety of geographic distribution, possibly reflecting differences in exposure to specific environmental factors. Both alcohol consumption and cigarette smoking are major risk factors for the development of ESCC. Acetaldehyde is the most toxic ethanol metabolite in alcohol-associated carcinogenesis, while ethanol itself stimulates carcinogenesis by inhibiting DNA methylation and by interacting with retinoid metabolism. Cigarette smoke contains more than 60 carcinogens and there are strong links between some of these carcinogens and various smoking-induced cancers; these mechanisms are well established. Synergistic effects of cigarette smoking and alcohol consumption are also observed in carcinogenesis of the upper aerodigestive tract. Of note, intensive molecular biological studies have revealed the molecular mechanisms involved in the development of ESCC, including genetic and epigenetic alterations. However, a wide range of molecular changes is associated with ESCC, possibly because the esophagus is exposed to many kinds of carcinogens including alcohol and cigarette smoke, and it remains unclear which alterations are the most critical for esophageal carcinogenesis. This brief review summarizes the general mechanisms of alcohol- and smoking-induced carcinogenesis and then discusses the mechanisms of the development of ESCC, with special attention to alcohol consumption and cigarette smoking.

Background: Abnormal hypermethylation of the CDKN2A (p14ARF and p16INK4a) gene can lead to repression of gene expression and contribute to carcinogenesis and tumor progression., Materials and Methods: In esophageal squamous cell carcinoma (ESCC), the promoter methylation of the p14ARF and p16INK4a gene was investigated in 38 cases by methylation-specific PCR and the expression of each gene in 18 cases was quantified by real-time quantitative reverse transcription-PCR., Results: Aberrant methylation of p14ARF and of p16INK4a was detected in 23 (61%) and 29 (76%) cases, respectively. No relationship was found between clinicopathological variables and p14ARF or p161NK4a promoter methylation. A statistically significant association between p14ARF methylation status and mRNA expression was found (p=0.0441). Regarding p14ARF, a low expression group showed a significantly higher proportion of cases with deep invasion of tumor, lymph node metastasis, and a high TNM stage of disease (p=0.0474, 0.0474, and 0.0441, respectively), and a significantly poor prognosis (p=0.0402). Regarding p161NK4a, no relationship was found among the methylation status, mRNA expression and clinicopathological variables, including survival., Conclusion: Our results suggest that methylation is the predominant mechanism of inactivation of the p14ARF gene in ESCC. The decrease in p14ARF gene expression associated with invasive and metastatic phenotypes may be significant as an indicator of the malignant potential of human ESCC.

Inactivation of the p53 tumor suppressor gene is one of the most frequent genetic alterations observed in human esophageal carcinomas. In patients with esophageal carcinoma, one of the significant pathological features of the tumor is the presence of multiple lesions within the esophagus. However, the molecular mechanisms involved in the occurrence of multiple lesions have remained elusive. To characterize p53 alterations in multiple esophageal carcinomas and to study their roles in carcinogenesis, we performed p53 immunohistochemical and p53 mutation analyses using laser capture microdissection on surgically resected human esophageal carcinomas from 11 patients: 9 patients with multiple esophageal carcinomas, 1 with an intramural metastasis lesion within the esophagus and 1 with an intraepithelial carcinoma lesion contiguous to the main lesion. In each of the patients with multiple esophageal carcinomas, we examined samples from 1 main lesion and 1 representative concomitant lesion. Molecular analyses of samples from fresh-frozen normal tissues and tumor tissues of the main lesion (whole tumor) were also performed by the same method. p53 protein accumulation was observed in 16 (72.7%) of 22 lesions from the 11 cases. No p53 mutation was found in normal esophageal tissues. In the 9 cases of multiple esophageal carcinomas, point mutations were detected in the whole tumor in 1 (11.1%) case, in the microdissected tumor samples of main lesions in 3 (33.3%) cases and in the microdissected tumor samples of concomitant lesions in 3 (33.3%) cases. For the microdissected tumor samples, there was a 54.5% (12/22) concordance rate between the results of immunostaining and molecular analysis. In the 8 cases of whole tumors in which a p53 mutation was not observed, 2 cases revealed p53 mutation in the microdissected tumor samples of the main lesion. All 6 cases of multiple esophageal carcinomas that showed a p53 mutation in the microdissected tumor sample had a discordant p53 mutational status between the main and concomitant lesions. In contrast, both the intramural metastasis lesion and the intraepithelial carcinoma contiguous to the main lesion showed p53 mutational patterns identical to those of the main lesions. In conclusion, the analysis of microdissected DNA by laser capture microdissection is useful for characterizing the heterogeneity of the p53 gene mutation in multiple carcinoma lesions that cannot be accurately analyzed in whole esophageal tumor DNA. The finding of different p53 gene mutations among multiple esophageal carcinoma lesions suggest further evidence of multicentric or field carcinogenesis of the human esophagus.

Dihydropyrimidine dehydrogenase (DPD) is the initial and rate-limiting enzyme involved in the degradation of 5-FU. DPD activity in peripheral blood mononuclear cells of 30 esophageal cancer patients treated with 5-FU and low-dose CDDP with irradiation was determined at the beginning of each cytostatic cycle, the objective being to determine if DPD activity is related to the occurrence of side-effects and responses to therapy. The DPD activity showed interpatient variability (mean: 325.5 pmol/min/mg protein). 5-FU-related side-effects tended to be registered more frequently in patients with low DPD activity. In particular, nausea occurred in 30.8% of patients in the high DPD activity group but, in 70.6% in those with low DPD activity (p < 0.05). The relationship between the histological response to therapy and DPD activity was nil. We propose that determination of DPD activity prior to initiation of 5-FU-based chemotherapy for patients with esophageal cancer could aid in identifying those at risk for toxicity.

Squamous epithelial dysplasia is frequently encountered in the cancerous esophagus. However, its biological and clinical significance have not yet been fully elucidated. Investigations in squamous cell dysplasia of the esophagus have been performed to date in our department. We consider dysplasia to be the earliest malignancy of the esophagus based on such biologic features as the histopathologic findings, the proliferative activity, and the altered expression of cancer-associated genes. It is essential to detect and treat these early lesions endoscopically. Hopefully the findings of further studies of dysplasia can help to elucidate the mechanism of carcinogenesis in esophageal squamous cell carcinoma.