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BACKGROUND: This study aimed to clarify the significance of the crosstalk between hypoxia-inducible factor-1α (HIF-1α) and the Wnt/β-catenin pathway in oesophageal squamous cell carcinoma (ESCC). METHODS: The oncogenic role of HIF-1α in ESCC was investigated using in vitro and in vivo assays. The clinicopathological significance of HIF-1α, β-catenin and TCF4/TCF7L2 in ESCC were evaluated using quantitative real-time PCR and immunohistochemistry. RESULTS: The expression level of HIF-1α, β-catenin, and TCF4/TCF7L2 in T.Tn and TE1 cell lines were elevated under hypoxia in vitro. HIF-1α knockdown suppressed proliferation, migration/invasion and epithelial–mesenchymal transition (EMT) progression, induced G0/G1 cell cycle arrest, promoted apoptosis and inhibited 5-fluorouracil chemoresistance in vitro. In vivo assays showed that HIF-1α is essential in maintaining tumour growth, angiogenesis, and 5-fluorouracil chemoresistance. Mechanically, we identified the complex between HIF-1α and β-catenin, HIF-1α can directly bind to the promoter region of TCF4/TCF7L2. The mRNA level of HIF-1α, β-catenin and TCF4/TCF7L2 were increased in ESCC tumour tissues compared to the corresponding non-tumour tissues. High levels of HIF-1α and TCF4/TCF7L2 expression were correlated with aggressive phenotypes and poor prognosis in ESCC patients. CONCLUSIONS: HIF-1α serves as an oncogenic transcriptional factor in ESCC, probably by directly targeting TCF4/TCF7L2 and activating the Wnt/β-catenin pathway.

Recent progress of large-scale international studies has provided comprehensive catalogs of somatic mutations in cancers. Additionally, it has become evident that allelic imbalance in the abundance of somatic mutations between DNA and RNA were pervasive in various types of cancer. However, the allelic imbalance of the abundance of somatic mutations in esophageal squamous cell carcinoma (ESCC) has not been fully analyzed.We performed exome sequencing for 25 Japanese patients with ESCC to detect a comprehensive catalog of somatic mutations in ESCC. Additionally, we performed mRNA sequencing to evaluate the allelic imbalance of the identified somatic mutations at the transcriptional level by comparing the mutant allele frequencies between RNA and DNA.The exome sequencing showed that TP53 and ZNF750 were significantly mutated genes. The expression levels of TP53 and ZNF750 were different depending on the mutation status. In almost all the tumors with missense mutations in TP53 and ZNF750, the mutant allele frequencies were higher in the RNA sequencing than those in the exome sequencing, indicating that the mutant alleles were preferentially expressed. By examining the allelic imbalances for all the identified missense mutations, we demonstrated that genes showing preferential expressions of the mutant alleles were involved in the pathways including cell cycle, cell death, and chromatin modification.The results of this study suggest that the allelic imbalance of the abundance of somatic mutations plays important roles in the initiation and progression of ESCC by modulating cancer-related biological pathways.

Introduction: Antidiabetic drug metformin exerts various antitumor effects on different cancers. Esophageal squamous cell carcinoma (ESCC) is an intractable digestive organ cancer and new treatment strategy is required. In this study, we performed a comprehensive gene expression analysis of ESCC cell lines treated with metformin, which provided helpful information on the antitumor effects of metformin in ESCC. Next, we selected a promising gene among them and examined its effects on ESCC properties. Methods: We examined metformin-induced mRNA expression changes in two human ESCC cell lines by performing next-generation sequencing (NGS) and pathway analysis. Heat shock protein family A (Hsp70) member 6 (HSPA6) expression in surgical specimens obtained from 83 ESCC patients who underwent curative operations was evaluated immunohistochemically and analyzed. Results: Metformin upregulated mRNA expression of the many genes, including HSPA6, a cancer immune-related gene, and inhibited mRNA expression of the other many genes. Pathway analysis indicated major canonical pathways and upstream regulators related to metformin. The result indicated HSPA6 as a promising biomarker. HSPA6 expression correlated with disease-free survival (DFS) of the patients with all stage ESCC (p = 0.021), especially with stage I/II ESCC (p < 0.001). With stage III, low HSPA6 expression was not associated with poor DFS (p = 0.918). Multivariate analysis indicated that independent low HSPA6 expression was an independent poor prognostic factor of stage I/II ESCC (p < 0.001). However, HSPA6 expression did not correlate with the clinicopathological characteristics, including age, sex, tumor depth, lymph node metastasis, tumor stage, and tumor markers of the patients with stage I/II ESCC. Conclusions: This NGS analysis detected prospective candidate genes, including HSPA6. Our results indicate that HSPA6 is a promising biomarker of the recurrence risk of stage I/II ESCC. Further studies on HSPA6 would lead to better treatment.

Subtotal gastrectomy (SG) has become a general option for distal gastric cancer. However, the availability of an organ-preserving approach for the treatment of remnant gastric cancer (RGC) is still controversial. Thus, the objective of the present study was to assess the safety and efficacy of SG for RGC by integrating data from published articles. We searched the PubMed, Cochrane Library, and Web of Science databases for studies that compared SG versus total gastrectomy (TG) for RGC published from the inception of the databases until May 2020. A meta-analysis was performed using the Review Manager Version 5.0 software program from the Cochrane Collaboration. Three retrospective cohort studies with 144 patients were included. The meta-analysis revealed that the operative time of the SG group was significantly shorter than that in the TG group (MD: -34.84. 95% CI: -59.97- -9.71, P = 0.007). There was no significant difference in intraoperative blood loss (MD: -109.19. 95% CI: -240.37-21.99, P =0.10), length of postoperative hospital stay (MD: 0.40. 95% CI: -3.03-3.83, P = 0.82), postoperative complications (RR: 1.41. 95% CI: 0.76-2.63, P = 0.28), or recurrence (RR: 2.33, 95% CI: 0.48-11.44, P = 0.30). SG for RGC tended to be correlated with favorable 5-year overall survival; however, the association was not statistically significant (HR: 0.89, 95% CI: 0.63-1.26, P = 0.51). Organ-preserving approaches such as SG may be a safe and feasible treatment option for early-stage RGC.

In actual surgical research, case-matched studies are frequently conducted as an alternative to randomized controlled trials (RCTs). However, it is still unclear what differences there are between RCTs and case-matched studies in upper gastrointestinal surgery, and clarifying them is a very important clinical issue. Thus, the purpose of this study was to investigate estimated treatment effects between RCTs, case-matched studies, and cohort studies regarding laparoscopic distal gastrectomy (LDG) for advanced gastric cancer (AGC).We searched the PubMed, Cochrane Central Register of Controlled Trials, and Web of Science databases for studies that compared LDG versus open distal gastrectomy for AGC published from the inception of the databases until July 2021. A meta-analysis was performed using the Review Manager version 5.3 software program from the Cochrane Collaboration, and six short-term outcomes and three long-term outcomes were assessed.Twenty-three studies with 13698 patients were included. There was no difference in estimated treatment effects between RCTs and case-matched studies for all outcomes except for the number of retrieved lymph nodes and postoperative complications. In terms of intraoperative blood loss, postoperative hospital stay, number of retrieved lymph nodes, and recurrence, observational studies tended to overestimate the treatment effects.The estimated treatment effects of LDG for AGC in the case-matched study were almost the same as in the RCTs. However, to assess the true magnitude of the treatment effect, the design and actual implementation of the analysis must be critically evaluated.

We determined the serum concentrations of Programmed cell death-1 (PD-1) and its ligands (PD-L1 and PD-L2) in patients with esophageal squamous cell carcinoma (ESCC). Blood samples were collected from 85 patients with histologically proved ESCC. Serum levels of PD-1, PD-L1, and PD-L2 were measured using enzyme linked immunosorbent assays. Correlations between serum PD-1, PD-L1, and PD-L2 concentration and tumor depth, number of lymph node metastases, organ metastasis status, or disease stage were assessed and five-year survival rates according to clinicopathological characteristics were calculated. The concentration of PD-1 was not differed according to tumor progression. On the other hand, the average concentration of PD-L1 in patients with T3/T4 disease was 15.6 (12.2–18.3) pg/mL (25–75%), and this was significantly higher than that in patients with Tis/T1/T2 disease (p = 0.020). Similarly, PD-L1 levels were significantly higher in patients with positive lymph nodes than in cases with negative lymph node involvement (p = 0.006) and were higher in patients with organ metastasis (p = 0.123) and in more advanced stage (p = 0.006). Similar tendency was observed regarding PD-L2 concentrations. PD-L2 concentration was higher in T3, T4 cases (p = 0.008), in LN positive cases (p = 0.032), and in more advanced stage (p = 0.024). Our data showed that a concentration of PD-L1 in peripheral blood was high in advanced cancer and high concentration of PD-L1 predicted disease progression and also poor survival in patients with ESCC.

Esophageal squamous cell carcinoma (ESCC) is an intractable digestive organ cancer that has proven difficult to treat despite multidisciplinary therapy, and a new treatment strategy is demanded. Metformin is used for type 2 diabetes mellitus and its antitumor effects have been reported recently. Metformin exerts antitumor effects in various respects, such as inhibiting inflammation, tumor growth and epithelial-mesenchymal transition (EMT). However, few reports have described the efficacy of metformin on ESCC, and their findings have been controversial. We analyzed the antitumor effects of metformin and clarified its effects on anti-inflammation, growth suppression and EMT inhibition. Activation of nuclear factor kappa B (NF-κB), the major transcription factor induced by inflammation, was investigated by immunostaining. We found that localization of NF-κB in the nucleus was reduced after metformin treatment. This suggests that metformin inhibited the activation of NF-κB. Metformin inhibited tumor growth and induced apoptosis in ESCC cell lines. Associated with EMT, we examined cell motility by a wound healing assay and the epithelial marker E-cadherin expression of various ESCC cell lines by western blotting. Metformin inhibited cell motility and induced E-cadherin expression. In conclusion, metformin showed multiple antitumor effects such as growth suppression, invasion inhibition, and control of EMT by inhibiting NF-κB localization on ESCC. Further exploration of the marker of treatment efficacy and combination therapy could result in the possibility for novel treatment to use metformin on ESCC.

Objective: ZNF750, an epidermal differentiation regulator, has been suggested to act as a tumor suppressor of esophageal squamous cell carcinoma (ESCC). Although a correlation between the epidermal differentiation gene and resistance to chemoradiotherapy (CRT) has been posited, no data regarding the ZNF750 status in ESCC have been reported. The aim of the present study was to evaluate the relationship between ZNF750 expression and response to CRT in ESCC. Methods: Eighty-seven patients who had been pathologically diagnosed with ESCC were evaluated in the present study. All patients underwent neoadjuvant CRT, followed by curative esophagectomy. The expression of ZNF750 in pretreatment biopsy samples was immunohistochemically investigated and compared to the histopathological effectiveness of CRT in surgical specimens. Results: High expression of ZNF750 was closely correlated with good sensitivity to CRT (p = 0.016). A univariate analysis showed that high/intermediate expression of ZNF750 was a significant predictive factor for good sensitivity to CRT (p = 0.006). High/intermediate expression of ZNF750 (30% or more) remained an independent predictive factor for sensitivity to CRT in a multivariate analysis (p = 0.033). Conclusions: ZNF750 expression predicts sensitivity to CRT and can be a biomarker that reliably predicts the response of ESCC to CRT.

Genome medicine has been attractingmuch of attention in Japan. The combination of molecular targetingdrug s and somatic mutations has been developed for cancer treatment, which was introduced clinically with evidence by cancer type. Several cancer somatic mutations can be identified in a single test inexpensively using next-generation sequencing(NGS). Drug approval not based on organs but on cancer genome analysis has been practiced mainly in the United States, and is also being implemented in Japan. However, cancer treatment strategies using molecular targeting drugs and the associated diagnosis are limited in each type of cancer. Furthermore, the benefit of NGS, which is an improved and inexpensive technique, is still insignificant in Japan. However, the clinical biobank system was initiated in 2011 to prepare the era of cancer genome medicine in our department. The quality of biological samples was strictly controlled by the standardized sampling procedures, which can be used by the researchers accordingto their convenience. Furthermore, the cooperative research involvingcommercial corporations has been started.

Gastric cancer (GC) with peritoneal metastasis remains difficult to treat. The anti-diabetic drug metformin exerts various antitumor effects via the 5'-adenosine monophosphate-activated protein kinase (AMPK) pathway and nuclear factor-kappa B (NF-ĸB). Therefore, we evaluated the antitumor effects of metformin for GC in vitro and on peritoneal metastasis.The human GC cell lines MKN1, MKN45, KATO-III and SNU-1 were used. The antiproliferative effect was evaluated in vitro with 0.5 mM or 25 mM glucose and in vivo using tumor xenograft peritoneal models of metastasis. The protein expression of AMPK, liver kinase B1 (LKB1) and NF-ĸB in tumors was examined by western blotting.Metformin inhibited cell proliferation in all GC lines and sensitivity was increased under low-glucose conditions in vitro. Metformin also suppressed peritoneal metastasis. In tumors, metformin reduced the numbers of proliferating cells and NF-ĸB expression, but a similar trend was not noted for AMPK.Metformin may be a useful drug for the treatment of GC with peritoneal metastasis.

Objective: ZNF750, a transcriptional regulator of epidermal differentiation, has been identified as a tumor suppressor in esophageal squamous cell carcinoma (ESCC). The aim of the present study was to investigate the clinical and prognostic significance of ZNF750 expression and to evaluate the effect of ZNF750 knockdown on cell proliferation, migration, and invasion in ESCC. Methods: A total of 124 patients with ESCC who underwent curative esophagectomy were evaluated in this study. The expression of ZNF750 in surgical specimens was immunohistochemically assessed and used in the analysis of clinicopathological features and overall survival (OS). The molecular role of ZNF750 was investigated by ZNF750 knockdown using small interfering RNA (siRNA) in ESCC cell lines. Results: Low ZNF750 expression had a significant correlation with positive lymph node metastasis (p = 0.028). Furthermore, there was a significant relationship between low expression of ZNF750 in ESCC and a poor OS, and a multivariate analysis showed that low ZNF750 expression was an independent prognostic factor (p = 0.020). The cell growth, migration, and invasion were significantly increased by downregulation of ZNF750. Conclusions: The low expression of ZNF750 was significantly associated with a poor prognosis, and ZNF750 expression may, therefore, be a reliable prognostic biomarker in ESCC.

Exosomes play important roles in cancer progression. Although its contents (e.g., proteins and microRNAs) have been focused on in cancer research, particularly as potential diagnostic markers, the exosome behavior and methods for exosome quantification remain unclear. In the present study, we analyzed the tumor-derived exosome behavior and assessed the quantification of exosomes in patient plasma as a biomarker for esophageal squamous cell carcinoma (ESCC). A CD63-GFP expressing human ESCC cell line (TE2-CD63-GFP) was made by transfection, and mouse subcutaneous tumor models were established. Fluorescence imaging was performed on tumors and plasma exosomes harvested from mice. GFP-positive small vesicles were confirmed in the plasma obtained from TE2-CD63-GFP tumor-bearing mice. Patient plasma was collected in Chiba University Hospital (n=86). Exosomes were extracted from 100 µl of the plasma and quantified by acetylcholinesterase (AChE) activity. The relationship between exosome quantification and the patient clinical characteristics was assessed. The quantification of exosomes isolated from the patient plasma revealed that esophageal cancer patients (n=66) expressed higher exosome levels than non-malignant patients (n=20) (P=0.0002). Although there was no correlation between the tumor progression and the exosome levels, exosome number was the independent prognostic marker and low levels of exosome predicted a poor prognosis (P=0.03). In conclusion, exosome levels may be useful as an independent prognostic factor for ESCC patients.

The current report presents the case of a 46-year-old woman with phyllodes tumor metastasis to the anterior chest wall treated by radiation therapy. Although the lesion was not controlled with surgery and chemotherapy, the tumor size markedly reduced after radiation therapy, and bleeding and foul odor from the tumor stopped. Radiation therapy for phyllodes tumor appears to be an effective treatment and should be recognized as one choice of palliative medicine.

A 63-year-old man who had been admitted to another institute with sepsis and renal failure was referred to our hospital after computed tomography (CT) findings showed thickening of the walls in the sigmoid colon and a defect in contrast enhancement in the portal and inferior mesenteric veins. Emergency sigmoid colon resection with D2 lymphadenectomy was performed after detection of perforation due to sigmoid colon cancer. The histopathological diagnosis was adenosquamous carcinoma, pSS, int, INF b, ly1, v0, pN2, pStage IIIband inferior mesenteric vein thrombosis. He was discharged on day 12, and we administered anticoagulant warfarin therapy.

A 70-year-old woman was admitted for difficulty in swallowing. Esophageal cancer (MtLt, type 3, T4N3M0, cStage IVa) was diagnosed in May 2010. The cancer was unresectable, and chemoradiotherapy (CRT) with TS-1 was initiated in June 2010, and a partial response (PR) was observed. After CRT, TS-1 was continued, but a brain metastasis was detected owing to the development of right hemiplegia in April 2012. Craniotomy and tumorectomy were performed, and the right hemiplegia improved. Pathological examination of the brain tumor indicated squamous cell carcinoma. Because of a recurrence of brain metastasis, a gamma knife procedure was performed in May 2012. Subsequently, several recurrences of brain metastases were diagnosed, and a total of 7 gamma knife procedures were performed up to January 2014. Although systemic chemotherapy (5-fluorouracil and cisplatin [FP], 5 courses)was administered, the patient showed progressive lung metastases in February 2013. The chemotherapy regimen was changed from FP to docetaxel (TXT), but the lung metastases continued to progress up to June 2013. The patient died in March 2014. Patients with esophageal cancer and metastases to the brain have poor prognosis, but the present patient survived approximately 2 years after first diagnosis of metastases to the brain after multidisciplinary therapy.

A 63-year-old man was admitted for an abdominal mass. Computed tomography revealed an abscess (21 × 20 cm) in the abdominal wall and a tumor in the sigmoid colon. Thus, cancer of the sigmoid colon complicated by an abscess of the abdominal wall was diagnosed. The abscess was drained and transverse colostomy was performed with curative intent. After the intervention, chemotherapy (XELOX×3) was administered. Three months later, sigmoidectomy was performed and the stoma was closed. Macroscopic and microscopic examination of the resected specimen detected no remnants of cancer. In patients with advanced colon cancer and abdominal wall involvement, a two-stage operation and preoperative chemotherapy may be considered essential when curative resection is performed.

Roux-en-Y reconstruction after distal gastrectomy can result in delayed gastric emptying, called Roux stasis syndrome (RSS). The cause of RSS has not been completely identified. This study retrospectively investigated the development of RSS.Between April 2008 and March 2014, we performed 138 procedures with distal gastrectomy using Roux-en-Y reconstruction. The development of RSS was analyzed and examined for correlations with the length of the operation, amount of blood loss, and surgical procedure.RSS was observed in 16 of the 138 patients. There were no relationships between the length of the operation or amount of blood loss and the development of RSS according to the Mann-Whitney U test. There were also no significant differences in the development of RSS between the patients treated with laparotomy and laparoscopic surgery, end-to-side, side-to-side, or end-to-end anastomosis or isoperistaltic or antiperistaltic anastomosis, as determined using the chi-square test. However, the development of RSS tended to lower in the patients who underwent laparoscopic surgery, side-to-side anastomosis, and isoperistaltic anastomosis. In addition, there was a significant difference between the patients who received antecolic and retrocolic reconstruction (p=0.005).Our findings suggest that antecolic reconstruction correlates with a lower likelihood of developing RSS.

We report a case of patient who is alive 10 years after total gastrectomy and partial liver resection for gastric cancer and liver metastasis, respectively. The patient is 69-year-old man who was examined in another hospital for nausea, tarry stool, and weight loss. Diagnosis by stomach fluoroscopy revealed gastric cancer and he was admitted to our hospital in September 2002. Computed tomography (CT) scan and ultrasonography (US) revealed liver metastasis in S7. Distant metastases were not detected. The patient underwent distal gastrectomy, transverse colon partial resection, and a hepatic partial resection in October 2002. Pathological findings showed SI, N1, and M1. Though adjuvant chemotherapy was given, a local recurrence was detected in the liver in February 2003. Hepatic arterial infusion (HAI) was started in April 2003. After 8 courses, HAI was discontinued because of hepatic artery obstruction. No additional distant metastases were found. Therefore, transdiaphragmatic radiofrequency ablation (RFA) was performed by thoracotomy. Currently, the patient is treatment free and remains alive after 10 years with no recurrence.