Shuhei Ito

Alcohol drinking, cigarette smoking, and the development of squamous cell carcinoma of the esophagus: molecular mechanisms of carcinogenesis

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.

p53 Mutation Profiling of Multiple Esophageal Carcinoma Using Laser Capture Microdissection to Demonstrate Field Carcinogenesis

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.

Lymph node metastasis from cancer of the esophagogastric junction, and determination of the appropriate nodal dissection

PurposeBoth squamous cell carcinomas and adenocarcinomas can develop in the esophagogastric junction. To clarify the appropriate lymph node dissection range, lymph node metastases from cancers in the esophagogastric junction were investigated.MethodsThe nodal metastases were analyzed in 64 patients with squamous cell carcinoma and 129 with adenocarcinoma according to Siewert’s classification, which is based on topographic anatomical criteria for adenocarcinoma.ResultsThe squamous cell carcinomas located above the esophagocardial junction had more frequent metastasis to the lower and middle mediastinal lymph nodes in proportion to the depth of the tumor. Nodal metastasis was also often detected in the abdominal lymph nodes. In contrast, adenocarcinomas metastasized less frequently to the mediastinal lymph nodes, and the metastatic rates in the abdominal nodes were higher than those from squamous cell carcinoma.ConclusionEsophagectomy with mediastinal and abdominal lymph node dissection is considered to be an appropriate approach for surgical resection of squamous cell carcinomas, whereas transhiatally extended gastrectomy with lower mediastinal and abdominal lymph node dissection is recommended for the treatment of adenocarcinomas.

Clinical and biological significance of circulating tumor cells in cancer.

During the process of metastasis, which is the leading cause of cancer‐related death, cancer cells dissociate from primary tumors, migrate to distal sites, and finally colonize, eventually leading to the formation of metastatic tumors. The migrating tumor cells in circulation, e.g., those found in peripheral blood (PB) or bone marrow (BM), are called circulating tumor cells (CTCs). CTCs in the BM are generally called disseminated tumor cells (DTCs). Many studies have reported the detection and characterization of CTCs to facilitate early diagnosis of relapse or metastasis and improve early detection and appropriate treatment decisions. Initially, epithelial markers, such as EpCAM and cytokeratins (CKs), identified using immunocytochemistry or reverse transcription polymerase chain reaction (RT‐PCR) were used to identify CTCs in PB or BM. Recently, however, other markers such as human epidermal growth factor receptor 2 (HER2), estrogen receptor (ER), and immuno‐checkpoint genes also have been examined to facilitate detection of CTCs with metastatic potential. Moreover, the epithelial‐to‐mesenchymal transition (EMT) and cancer stem cells (CSCs) have also received increasing attention as important CTC markers owing to their roles in the biological progression of metastasis. In addition to these markers, researchers have attempted to develop detection or capture techniques for CTCs. Notably, however, the establishment of metastasis requires cancer‐host interactions. Markers from host cells, such as macrophages, mesenchymal stem cells, and bone marrow‐derived cells, which constitute the premetastatic niche, may become novel biomarkers for predicting relapse or metastasis or monitoring the effects of treatment. Biological studies of CTCs are still emerging. However, recent technical innovations, such as next‐generation sequencing, are being used more commonly and could help to clarify the mechanism of metastasis. Additionally, biological findings are gradually being accumulated, adding to our body of knowledge on CTCs. In this review, we will summarize recent approaches to detect or capture CTCs. Moreover, we will introduce recent studies of the clinical and biological importance of CTCs and host cells.

Decreased Expression of Fructose-1,6-bisphosphatase Associates with Glucose Metabolism and Tumor Progression in Hepatocellular Carcinoma

Fructose-1,6-bisphosphatase (FBP1), the rate-limiting enzyme in gluconeogenesis, is reduced in expression in certain cancers where it has been hypothesized to act as a tumor suppressor, including in hepatocellular carcinoma (HCC). Here, we report functional evidence supporting this hypothesis, providing a preclinical rationale to develop FBP1 as a therapeutic target for HCC treatment. Three independent cohorts totaling 594 cases of HCC were analyzed to address clinical significance. Lower FBP1 expression associated with advanced tumor stage, poor overall survival, and higher tumor recurrence rates. In HCC cell lines, where endogenous FBP1 expression is low, engineering its ectopic overexpression inhibited tumor growth and intracellular glucose uptake by reducing aerobic glycolysis. In patient specimens, promoter methylation and copy-number loss of FBP1 were independently associated with decreased FBP1 expression. Similarly, FBP1 downregulation in HCC cell lines was also associated with copy-number loss. HCC specimens exhibiting low expression of FBP1 had a highly malignant phenotype, including large tumor size, poor differentiation, impaired gluconeogenesis, and enhanced aerobic glycolysis. The effects of FBP1 expression on prognosis and glucose metabolism were confirmed by gene set enrichment analysis. Overall, our findings established that FBP1 downregulation in HCC contributed to tumor progression and poor prognosis by altering glucose metabolism, and they rationalize further study of FBP1 as a prognostic biomarker and therapeutic target in HCC patients. Cancer Res; 76(11); 3265-76. ©2016 AACR.

Chromosomal Instability Associated with Global DNA Hypomethylation is Associated with the Initiation and Progression of Esophageal Squamous Cell Carcinoma

BackgroundGlobal 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.MethodsUsing 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.ResultsThe 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.ConclusionsOur 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.

Programmed death-ligand 1 expression at tumor invasive front is associated with epithelial-mesenchymal transition and poor prognosis in esophageal squamous cell carcinoma

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.

MicroRNAs as Biomarkers in Colorectal Cancer

MicroRNAs (miRs) are small RNAs that repress mRNA translation, resulting in the degradation of mRNAs and regulation of the expression levels of various genes. Recent studies have shown that aberrant miR expression has a functional role in the initiation and progression of various malignancies, including colorectal cancer (CRC), which is one of the leading causes of cancer-related death worldwide. miRs have also been shown to have applications as diagnostic, prognostic, and predictive biomarkers because of their high tissue specificity, stability, and altered expression in tumor development. In this report, we examined the role of miRs as biomarkers in CRC through a review of meta-analyses and large-scale analyses having strong statistical confidence in the study outcomes. We also discuss current issues in the clinical application of these miRs.

Somatic mutations in plasma cell-free DNA are diagnostic markers for esophageal squamous cell carcinoma recurrence

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.

Technical Improvement of Total Pharyngo-Laryngo-Esophagectomy for Esophageal Cancer and Head and Neck Cancer

AbstractPurposeTotal pharyngo-laryngo-esophagectomy (PLE) is highly invasive, and the subsequent reconstruction is difficult. The purpose of this study was to clarify the techniques that can decrease the surgical stress and allow for safe reconstruction after this operation. MethodsThe surgical method and clinical outcomes of total PLE were reviewed in 12 patients with either cervicothoracic esophageal cancer or double cancer of the esophagus and pharynx. Microscopic venous anastomosis was principally performed, and arterial anastomosis was added, if needed.ResultsA narrow gastric tube was used in ten patients, including two patients who underwent free jejunal interposition, while the colon was used as the main reconstructed organ in two other patients. Staged operations were performed in three high-risk patients. All six patients treated after 2010 were able to undergo thoracoscopic and/or laparoscopic surgery. No critical postoperative complications developed, although minor anastomotic leakage developed in two patients who were successfully treated conservatively.ConclusionWhen performing PLE, it is important to decrease the surgical stress and ensure a reliable reconstruction by adopting techniques that are appropriate for each case, such as thoracoscopic and laparoscopic surgery, staged operations, microvascular anastomosis, and muscular flaps.