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Background: Proton-based, definitive chemoradiotherapy (P-CRT) for esophageal squamous cell carcinoma (ESCC) previously showed comparable survival outcomes with the surgery-based therapy, i.e., neoadjuvant chemotherapy followed by esophagectomy (NAC-S), in a single-institutional study. This study aimed to validate this message in a Japanese multicenter study., Methods: Eleven Japanese esophageal cancer specialty hospitals have participated. A total of 518 cases with clinical Stage I-IVA ESCC between 2010 and 2019, including 168 P-CRT and 350 NAC-S patients, were enrolled and long-term outcomes were evaluated. Propensity-score weighting analyses with overlap weighting for confounding adjustment were used., Results: The 3-year overall survival (OS) of the P-CRT group was equivalent to the NAC-S group (74.8% vs. 72.7%, hazard ratio [HR]: 0.87, 95% confidence interval [CI]: 0.61-1.25). Although, the 3-year P-CRT group progression-free survival (PFS) was inferior to the NAC-S group (51.4% vs. 59.6%, HR 1.39, 95% CI 1.04-1.85), the progression P-CRT group cases showed better survival than the NAC-S group (HR 0.58, 95% CI 0.38-0.88), largely because of salvage surgery or endoscopic submucosal dissection for local progression. The survival advantage of P-CRT over NAC-S was more pronounced in the cT1-2 (HR 0.61, 95% CI 0.29-1.26) and cStage I-II (HR 0.50, 95% CI 0.24-1.07) subgroups, although this trend was not evident in other populations, such as cT3-4 and cStage III-IVA., Conclusions: Proton-based CRT for ESCC showed equivalent OS to surgery-based therapy. Especially for patients with cT1-2 and cStage I-II disease, proton-based CRT has the potential to serve as a first-line treatment., (© 2024. The Author(s), under exclusive licence to The Author(s) under exclusive licence to The Japan Esophageal Society.)

Background: Tumor cells (TC) participate in tumor progression by altering the immune responses in the tumor microenvironment. However, the clinical relevance and prognostic effect of PD-L1 expression and tumor-infiltrating lymphocytes (TILs) in esophageal squamous cell carcinoma (ESCC) are unknown. The purpose of this study was to investigate the interactions and clinical significance of PD-L1 expression and TILs in ESCC., Methods: Tissue specimens were collected from 126 patients with ESCC who underwent curative esophagectomy. Immunohistochemical analysis and multiplex immunofluorescence for CD4, CD8, CD25, FOXP3, and PD-L1 in the tumor were used to identify multiple tumor-infiltrating immune cells (TIIC), Tregs, and TC., Results: PD-L1 was expressed in tumor cells (PD-L1 TC). PD-L1 TIIC and PD-L1 TC affected the biological behavior of TC. The positive expression rate of PD-L1 TC and CD8 + TILs was 27.8% (35/126) and 31.7% (40/126), respectively. Kaplan-Meier analysis showed that overall survival (OS) was significantly associated with decreased CD8 + TILs and PD-L1 TC-positive expression, which promote ESCC progression and metastasis., Conclusion: Tumor depth, CD8, and PD-L1 TC were independent prognostic factors in ESCC, and a predictive nomogram with these three risk factors improved the accuracy of predicting OS in patients with ESCC after surgical resection. The conjoint analysis of multiple immune-related factors is beneficial for stratifying patient survival risk., (© 2024 The Author(s). Cancer Medicine published by John Wiley & Sons Ltd.)

Introduction: Identifying accurate biomarkers for predicting response to chemoradiotherapy (CRT) in patients with esophageal squamous cell carcinoma (ESCC) is a critical challenge. The protein SIRT1, recognized for its implications in longevity, has been associated with tumor promotion in ESCC. However, data regarding its correlation with CRT sensitivity remain unreported. Therefore, in this study, we aimed to investigate the relationship between SIRT1 expression and CRT sensitivity and concurrently assess the effect of SIRT1 knockdown on CRT sensitivity in ESCC., Methods: This study included 73 patients who underwent radical esophagectomy after CRT. SIRT1 expression in pre-treatment endoscopic biopsies was assessed through immunostaining, followed by a comparative analysis of CRT effects on surgical specimens. Small interfering RNA was used to attenuate SIRT1 expression in TE5 and TE10 cells, which were then subjected to cisplatin treatment at varying doses and concentrations and irradiation with X-rays, respectively., Results: High SIRT1 tissue expression was significantly associated with CRT resistance. Multivariate analysis identified high SIRT1 expression as an independent biomarker for poor CRT response. In TE-5 and TE-10 cells, SIRT1 knockdown significantly decreased cell viability and increased sensitivity to cisplatin and radiation treatment compared to that of the negative control., Conclusion: Our study results demonstrate the potential of SIRT1 as a predictive biomarker for CRT response in ESCC, highlighting the heightened sensitivity to CRT upon the transcriptional inactivation of SIRT1. Targeting SIRT1 emerges as a promising strategy for enhancing the efficacy of CRT for ESCC., (© 2024 S. Karger AG, Basel.)

Purpose: The gut microbiome plays an important role in cancer pathogenesis and therapy. Some studies have reported that specific bacteria in tumor tissues may contribute to the prognosis and treatment of esophageal squamous cell carcinoma (ESCC). However, there is limited evidence that the gut microbiome is associated with ESCC. This study assessed the utility of the gut microbiome as a predictive marker of the therapeutic effect in patients with ESCC undergoing chemo-radiotherapy (CRT)., Patients and Methods: Fecal samples were collected from 51 patients with ESCC who had never undergone treatment between April 2021 and May 2022 in the Department of Frontier Surgery, Chiba University. The gut microbiome was analyzed using 16S metagenomics sequencing. The association between the gut microbiome composition and stage according to the TNM classification (American Joint Committee on Cancer 7.0) and CRT response according to the RECIST criteria was evaluated., Results: The relative abundance of Fusobacteriaceae was enriched in cStage III-IVb group. Among the 27 patients who received CRT, the relative abundance of Lactobacillaceae was enriched in those with a partial and complete response. Lactobacillaceae also did not correlate with any clinical data, but the high Lactobacillales group had a higher LMR (P = 0.032) and lower PLR (P = 0.045) than in the low Lactobacillales group., Conclusions: In conclusion, we found that the relative abundance of Lactobacillaceae was enriched in patients with a partial or complete response among CRT those with ESCC, thus suggesting that the relative abundance of Lactobacillaceae can predict the effect of CRT., (© 2023. The Author(s) under exclusive licence to The Japan Esophageal Society.)

Background: Esophageal squamous cell carcinoma (ESCC) is a malignant cancer with a poor prognosis. Chemoradiotherapy is one of the most important strategies for patients with locally advanced unresectable ESCC; however, its therapeutic effect is unsatisfactory. Tumor-initiating cells (TICs) have been reported to be resistant to conventional chemotherapy and radiotherapy so far. Therefore, we aimed to develop a treatment strategy targeting TICs in ESCC to improve radiosensitivity., Methods: First, we validated aldehyde dehydrogenase 1 (ALDH1) as a TIC marker and investigated its ability to mediate resistance in human ESCC cell lines using flow cytometry, Western blotting, and functional analyses. Then, we focused on disulfiram (DSF), an aldehyde dehydrogenase inhibitor, used to treat alcohol use disorder. We investigated the effect of DSF and copper (II) D-gluconate (Cu) on the radiosensitivity of ESCC in xenograft mouse models., Results: ALDH1-positive cells showed an upregulation of SOX2 and Nanog, exhibiting much stronger tumor-initiating properties than ALDH1-negative cells. Furthermore, inhibition of ALDH1 attenuated the tumor-initiating properties of ESCC cell lines. Our results also showed that ALDH1-positive cells were resistant to chemotherapy and radiotherapy, and the inhibition of ALDH1 led to the mitigation of therapeutic resistance. Our in vitro and in vivo studies revealed that the DSF/Cu complex could radiosensitize ALDH1-positive ESCC cells and downregulate the phosphoinositide 3-kinase/Akt pathway., Conclusion: ALDH1 inhibition by the DSF/Cu complex enhances the radiosensitivity of TICs in ESCC. The drug repositioning approach using disulfiram is a potential treatment option to overcome radioresistance in patients with locally advanced ESCC., (© 2022. The Author(s) under exclusive licence to The Japan Esophageal Society.)

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., (© 2022. The Author(s), under exclusive licence to Springer Nature Limited.)

Background: 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., Methods: 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., Results: 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., Conclusions: 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., (© 2021. The Author(s) under exclusive licence to The Japan Esophageal Society.)

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., (© 2022 S. Karger AG, Basel.)

Introduction: We determined the soluble programmed cell death-1 ligand-1 (sPD-L1) concentration in patients with esophageal squamous cell carcinoma (ESCC), and confirmed the PD-L1 expression in resected specimens., Methods: Blood samples were collected from 73 patients with histologically proven ESCC. The serum levels of sPD-L1 were measured using an enzyme-linked immunosorbent assay. The correlations between the sPD-L1 concentration and the expression of PD-L1 in tumor specimens and tumor depth, lymph node metastasis, disease stage, and various laboratory data were assessed., Results: sPD-L1 levels in patients with high PD-L1 expression levels in tumor tissue were significantly higher than in patients with low PD-L1 expression levels (p = 0.042). The OS of the sPD-L1-high group was significantly worse than that of the low group (p = 0.028). Similarly, patients in whom a tissue specimen was PD-L1-positive group showed significantly poorer OS., Conclusion: The sPD-L1 concentration was correlated with the PD-L1 expression in tissues. Patients with PD-L1-positive tissue specimens showed significantly higher sPD-L1 levels in comparison to PD-L1-negative cases. Furthermore, patients with high sPD-L1 expression levels had a significantly worse prognosis than those with low sPD-L1 expression levels, and patients with a PD-L1-positive tissue specimen had a significantly worse prognosis than patients in whom the tissue specimen showed a low PD-L1 expression level., (© 2021 S. Karger AG, Basel.)

Background: Intravoxel incoherent motion MRI (IVIM-MRI) can quantify micro-perfusion at the capillary level in the tissue. The purpose of this study is to measure tumor perfusion using IVIM-MRI, and evaluate its value as a biomarker to predict prognosis in esophageal squamous cell carcinoma (ESCC) patients., Methods: 109 ESCC patients (93 men and 16 women; median age: 72) who underwent IVIM-MRI prior to treatment between February 2018 and August 2020 were retrospectively investigated. Both mean apparent diffusion coefficient (ADC) value and mean perfusion-related parameter (PP) value of the primary tumor were measured using three b values of 0, 400, and 1000 s/mm 2 based on the IVIM model. We analyzed associations of these parameters with clinical stage and disease-specific survival (DSS)., Results: Lower ADC and PP values of the tumor were significantly associated with the higher clinical T stage (p < 0.0001, p < 0.0001, respectively). In Kaplan-Meier analyses, patients with lower PP value tumors (< 18.94, median) had significantly worse DSS (p < 0.0001), while tumor ADC value did not show a significant correlation with DSS. In a multivariate analysis, PP value of the tumor was an independent prognostic factor for DSS (p = 0.0027)., Conclusions: Quantification of tumor perfusion using IVIM-MRI can be a non-invasive prognostic biomarker of ESCC, reflecting clinical stage and survival., (© 2021. The Japan Esophageal Society.)

Our laboratory previously reported the usefulness as biomarkers of exosomes in the plasma of esophageal squamous cell carcinoma (ESCC) patients. However, the influence of tumor-derived exosomes on the tumor itself and underlying mechanisms remain unclear. We here report changes in the phenotype and gene expression when cancer cells exist in an environment with tumor-derived exosomes. The exosomes were isolated from the culture medium of human ESCC cells (TE2, T.Tn) by ultracentrifugation; cell proliferation assay, wound-healing assay, and fluorescence imaging of the cell cycle were performed to clarify the phenotypic changes in the high concentration of tumor-derived exosomes. Gene expression changes were also assessed by mRNA microarray, and the data were analyzed by gene set enrichment analysis (GSEA). The data revealed that the proliferation of both TE2 and T.Tn was inhibited, and cell migration ability was upregulated in the exosome exposure group (P < .05). Fluorescence imaging using a fluorescent ubiquitination-based cell cycle indicator expressing ESCC cells revealed that the ratio of G1-phase cells was significantly increased in the exosome exposure group (P < .05). Findings of the GSEA clarified that high-density exposure of cancer-derived exosomes to their parent cancer cells downregulated the expression of genes related to cell proliferation and cell cycle, and upregulated the expression of genes related to actin filament length and extracellular structure organization. In conclusion, an environment of high-density tumor-derived exosomes induces changes in the gene expression and phenotype of tumor cells and may lead to tumor progression or malignant transformation., (© 2020 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association.)

Recently, the interest in cancer genomic medicine has increased, owing to the powerful and cost-effective technology of next-generation sequencing(NGS), which allows rapid identification of a large number of gene mutations. TP53 mutations are frequently found in solid cancers, especially in esophageal squamous cell carcinoma(ESCC), wherein the frequency of TP53 mutation is considered to be 90% or more. However, there is no clinical targeted therapy as yet utilizing TP53. Here, we aimed to characterize TP53 mutations associated with ESCC, in order to assess its feasibility as a therapeutic target. We extracted DNA and RNA from specimens of ESCC patients and analyzed them using NGS, which revealed different TP53 mutations. Based on previous reports, it is considered that different TP53 mutations lead to different functions of the protein, and subsequently account for varied prognosis in squamous cell carcinoma of the head and neck. We also performed cell viability assay using ESCC cell lines with different TP53 mutations and 2 kinds of p53-targeted drug and found differences in the growth inhibition of the cell lines. Although individual treatment can be determined depending on the type of TP53 mutation, it would be necessary to further examine the interaction of TP53 with other genes to determine its therapeutic efficacy as a target.