Diagnosis Of Esophageal Squamous Cell Carcinoma Research Articles

Circulating tumour associated cells in esophageal cancers are resistance educated per previous chemo treatments

Abstract Background Innate and acquired chemoresistance to anticancer therapies are a well-known phenomenon in Esophageal Squamous Cell Carcinomas (ESCC). There are presently no viable approaches for real-time monitoring of resistance in ESCC. We used a novel method for chemo-interrogation (CI) by harvesting sufficient number of Circulating-Tumor Associated Cells (C-TACs) which are defined as apoptosis-resistant cells of tumorigenic origin and are positive for Epithelial Cell Adhesion Molecule (EpCAM) and pan-cytokeratins (pan-CK) irrespective of CD45 status. Methods Peripheral blood was collected from 80 patients with confirmed diagnosis of ESCC, among whom 52 were recently diagnosed therapy-naive, while 28 were pretreated patients. Peripheral blood mononuclear cells (PBMCs) were harvested by centrifugation and treated with commercially available stabilizing agents, by a proprietary protocol, to stabilize apoptosis resistant C-TACs. Surviving C-TACs were confirmed by immunostaining for EpCAM, pan-CK and CD45. Harvested C-TACs were treated in vitro with a panel of conventional cytotoxic anticancer agents and fraction of surviving cells were estimated to determine resistance profiles. Results Among the 52 cases of recently diagnosed therapy naive ESCC, innate chemoresistance was observed towards platinum agents in 40.8% samples (unique patient-drug combinations), taxanes in 34.6% samples, topoisomerase inhibitors in 42.9% samples and antimetabolites in 34.6% samples. Among the 28 cases of previously treated ESCC, resistance towards previously administered systemic agents was observed in 87% of all samples, which included resistance towards platinum agents in 87.5% samples, taxanes in 82.1% samples, topoisomerase inhibitors in 100% samples and antimetabolites in 85.7% samples, respectively. Conclusions We show for the first time that sufficient C-TACs can be harvested for meaningful CI in newly diagnosed treatment naive ESCC as well as refractory ESCCs. Post-treatment chemoresistance being an order of magnitude higher than the untreated cohort, indicates that C-TACs in ESCC are resistance-educated by previous treatments and can guide treatment strategy in ESCC. Legal entity responsible for the study The authors. Funding Datar Cancer Genetics Limited. Disclosure D. Patil: Full / Part-time employment: Datar Cancer Genetics Limited. D. Akolkar: Full / Part-time employment: Datar Cancer Genetics Limited. V. Datta: Full / Part-time employment: Datar Cancer Genetics Limited. S. Schuster: Full / Part-time employment: Datar Cancer Genetics Limited. C. Sims: Full / Part-time employment: Datar Cancer Genetics Limited. R. Patil: Full / Part-time employment: Datar Cancer Genetics Limited. A. Srinivasan: Full / Part-time employment: Datar Cancer Genetics Limited. S. Khan: Full / Part-time employment: Datar Cancer Genetics Limited. S. Patil: Full / Part-time employment: Datar Cancer Genetics Limited. V. Mhase: Full / Part-time employment: Datar Cancer Genetics Limited. S. Apurwa: Full / Part-time employment: Datar Cancer Genetics Limited. R. Datar: Shareholder / Stockholder / Stock options, Licensing / Royalties, Officer / Board of Directors: Datar Cancer Genetics Limited. All other authors have declared no conflicts of interest.

The Association between Functional Polymorphisms of COX-2 and Serum PGE2 Level in ESCC Patients in North of Iran

Background: Esophageal cancer is recognized as one of the most fatal diseases around the world. Many factors are involved in the development of esophageal cancer, including genetic factors and inflammation. Cyclooxygenase-2 (COX-2) and its downstream signaling are the most important proinflammatory factors contributing to cancer. The present study aimed to evaluate the relationship between the polymorphisms and expression of COX-2 and prostaglandin-E2 (PGE2) level in patients with esophageal squamous cell carcinoma (ESCC) in Golestan Province (Iran), situated on the “esophageal cancer belt”.
 
 Methods: In this case-control study, blood and biopsy samples were obtained from ESCC patients and healthy controls. The COX-2 polymorphisms for -1195, -1290, -765, and +8473 SNPs were assayed using PCR-RFLP assay, while the level of PGE2 was measured using an ELISA kit. In addition, real-time PCR assay and immunohistochemistry (IHC) were performed to assay mRNA and protein expression of COX-2, respectively.
 
 Results: An association was found between 8473TC genotype and risk of ESCC (OR= 5.417, P= 0.036). In addition, mRNA and protein expression of COX-2 in ESCC patients was higher than the controls (P=0.001 and P=0.048, respectively). Based on the findings, the level of PGE-2 was significantly higher in ESCC patients, compared to the controls (P= 0.045). However, ROC curve analysis revealed PGE2 is a weak biomarker for diagnosis of ESCC. There was a significant relationship between the level of PGE2 and 8473CC, 8473TC, -765CC, and -1290AA genotypes (P= 0.028, P= 0.022, P= 0.024, and P= 0.011, respectively).
 
 Conclusion: Based on our results, functional polymorphisms of COX-2 (8473CC, 8473TC, - 765CC, and -1290AA) increase PGE2 level and carriers of these polymorphisms might be more susceptible to ESCC.

2869 Retrospective Study of Patients With Esophageal Squamous Cell Carcinoma in an Urban Safety Net County Hospital

INTRODUCTION: Esophageal squamous cell carcinoma (ESCC) accounts for 90% of esophageal cancers worldwide but is less common in the developed world. Our aim was to report patient and tumor characteristics of individuals diagnosed with ESCC within the underserved Los Angeles County population. METHODS: Review of the hospital pathology database identified 65 cases of patients ages 18-80 with a pathologic diagnosis of ESCC from 2003-2015. A retrospective chart review of electronic health records was conducted excluding patients of unknown ethnicity leaving 61 patients. Patients were characterized by gender, age, alcohol and tobacco use, and family history. Endoscopy and pathology reports were reviewed to confirm tumor location, length, and pathological staging. Frequency counts and percentages were reported for categorical characteristics. A chi-square test or Fisher’s Exact test was performed to assess if characteristic variables differed by ethnicity. RESULTS: Patients identified with ESCC in our cohort were predominantly Hispanic (59%, n = 36) and 41% were non-Hispanic (n = 10 Asian, n = 10 African Americans, and n = 5 Caucasian). ESCC was more commonly diagnosed in males vs. females in Hispanics and non-Hispanics (83.3% and 80% vs 16.7% and 20%, P = 0.75). Age of diagnosis was between the ages of 18-60 years in nearly 56% and greater than 60 years in 44% of Hispanics and non-Hispanics (P = 0.97). Most Hispanics and non-Hispanics had exposure to alcohol (74.1% and 85%, P = 0.48) or tobacco (53.9% and 68.2%, P = .31). Most Hispanic and non-Hispanics did not have a family history of cancer (72% and 80%, P = 0.73). Tumor location was most often proximal in Hispanic and non-Hispanics (66.7% and 83.3%, P = 0.16) and of similar length (P = 0.29). Most tumors in both Hispanic and non-Hispanic patients were moderately/poorly differentiated (82.9% and 91.3%, P = 0.46). Hispanics were less likely to present with metastatic ESCC as compared to non-Hispanic patients (42.3% vs. 75%, P = 0.03). CONCLUSION: ESCC in both groups within the Los Angeles County population were diagnosed most often in the proximal esophagus of males with history of exposure to alcohol and/or tobacco. Despite similar patient and tumor characteristics, ESCC in Hispanics was less often metastatic at the time of diagnosis compared to non-Hispanics. Despite the small numbers, our study is one of the few to include a large cohort of Hispanics and support further investigation of factors in different ethnicities that may be protective against advanced disease.

Identification of microRNAs as novel biomarkers for esophageal squamous cell carcinoma: a study based on The Cancer Genome Atlas (TCGA) and bioinformatics.

MicroRNAs (miRNAs) have played important roles in the regulation of gene expression in many cancers, but their roles in esophageal squamous cell carcinoma (ESCC) are still unclear. The aim of this study was to determine the potential ESCC-specific key miRNAs from a large sample dataset in The Cancer Genome Atlas (TCGA). Integrative bioinformatics analysis was used to identify key ESCC-specific miRNAs related to the ESCC patients' tumor histological grade and lymphatic metastasis from TCGA. Next, these key miRNA potential gene regulatory functions and relationships with ESCC patients' clinical characteristics and overall survival were analyzed. Finally, three key miRNAs were selected randomly and quantificational real-time polymerase chain reaction (qRT-PCR) was used to validate in 51 newly diagnosed ESCC patients' tissues samples (collected from Nov. 2017 to Feb. 2019, in Wuwei, China) whether the bioinformatics analyses results were reliable and valid. Two-tailed Student's t test, Pearson Chi-squared test and Kaplan-Meier survival analysis were used in this study. Thirty-five ESCC-specific miRNAs from TCGA database were investigated (fold-change > 2.0, P < 0.05), and 28 participated in the miRNAs-mRNAs co-expression network construction, while 17 were related with ESCC patients' tumor histological grade, TNM stage, and lymphatic metastasis (P < 0.05). Meanwhile, six miRNAs (including miR-200b-3p, miR-31-5p, miR-15b-5p, miR-141-3p, miR-135b-5p, and miR-195-5p) were correlated with overall survival of ESCC patients (log-rank, P < 0.05). MiR-135b-5p, miR-15b-5p, and miR-195-5p were selected for verification of the expression levels in 51 ESCC patients' tissue samples by using qRT-PCR. We found that the fold-changes between qRT-PCR and TCGA were completely consistent. The results also suggested that miR-135b-5p, miR-15b-5p, and miR-195-5p were significantly correlated with tumor differentiation degrees (P < 0.05), miR-195-5p was significantly correlated with tumor TNM stage (P < 0.05), and miR-135b-5p was significantly correlated with lymph-node metastasis (P < 0.05). MiR-135b-5p, miR-15b-5p, and miR-195-5p expression levels, ESCC patient clinical features association analysis results and the aforementioned TCGA bioinformatics analyses were similar. This study identified key ESCC-related miRNAs. The key miRNAs are worthy of further investigation as potential novel biomarkers for diagnosis, classification, and prognosis of ESCC.

Absence of Iodine Staining Associates With Progression of Esophageal Lesions in a Prospective Endoscopic Surveillance Study in China

Chromoendoscopy with iodine staining is used to identify esophageal squamous dysplasia and esophageal squamous cell carcinomas (ESCCs)-absence of staining indicates suspicious regions of dysplasia. However, screening detects precancerous lesions (mild and moderate dysplasia) that do not require immediate treatment; it is a challenge to which lesions are at risk for progression. We investigated the association between absence of iodine staining at chromoendoscopy screening and lesion progression using 6 years of follow-up data from a population-based randomized controlled trial in China. We then constructed and validated a model to calculate risk of progression to severe dysplasia, carcinoma in situ, or ESCC. We collected data from 1468 participants (45-69 years old) who were either negative for iodine staining at a baseline chromoendoscopy or found to have mild or moderate dysplasia in histologic analysis of biopsies in the Endoscopic Screening for Esophageal Cancer study in China, from January 2012 through September 2016; 788 of these participants were re-examined by endoscopy after a median interval of 4.2 years (development cohort). We investigated the association between absence of iodine staining and progression of esophageal lesions using Cox prediction models, considering corresponding baseline pathology findings and patient answers to a comprehensive questionnaire. Patients who did not receive a follow-up examination (n= 680) was used as the validation cohort; outcome events in these patients were identified by annual door to door active interviews or linkage with local electronic registry data. The primary outcome was incident esophageal severe dysplasia, carcinoma in situ, or ESCC. In the development cohort, 11 lesions that did not stain with iodine but were classified as not dysplastic in the histology analysis were found to be severe dysplasia, carcinoma in situ, or ESCC at the follow-up evaluation. These lesions accounted for 39.3% of all progressed lesions (n= 28). In the validation cohort, 6 patients with lesions did not stain with iodine but were classified as not dysplastic by histology had a later diagnosis of ESCC, determined from medical records; these patients accounted for 50.0% of all patients with lesion progression (n= 12) until the closing date of this study. We developed a model based on patient age, body mass index, pathology findings, and baseline iodine staining to calculate risk for severe dysplasia, carcinoma in situ, or ESCC. It identified patients for severe dysplasia, carcinoma in situ, or ESCC in the development set with an area under the curve of 0.868 (95% CI, 0.817-0.920) and in the validation set with an area under the curve of 0.850 (95% CI, 0.748-0.952). Almost no cases would be missed if subjects determined to be high or intermediate-high risk subjects by the model were included in surveillance. Absence of iodine staining at baseline chromoendoscopy identifies esophageal lesions at risk of progression with a high level of sensitivity. A model that combines results of iodine chromoendoscopy with other patient features identifies patients at risk of lesion progression with greater accuracy than histologic analysis of baseline biopsies.

Discovery and Validation of a Serologic Autoantibody Panel for Early Diagnosis of Esophageal Squamous Cell Carcinoma.

Esophageal squamous cell carcinoma (ESCC) accounts for the highest incidence rate worldwide and is responsible for the fourth leading cause of cancer-related death. Currently, serologic biomarkers for early ESCC diagnosis are needed for timely treatment. The performance of a four-autoantibody panel (i.e., anti-TP53, HRAS, CTAG1A, and NSG1) was evaluated by ELISA for the early diagnosis of ESCC with 569 retrospective serum samples. A training set comprising 129 patients with early-stage ESCC, 130 patients with esophageal benign lesion (EBL), and 150 healthy controls (HC) was used to develop an early ESCC predictive model. Data obtained from an independent validation set were used to evaluate and validate the predictive model to distinguish the early ESCC from the controls (EBL+HC). Finally, a multiplexed assay based on the Luminex xMAP technology platform was developed to enable simultaneous detection of the four-autoantibody panel using the validation set. The four-autoantibody panel significantly discriminated early ESCC cases from the controls with 62.8% sensitivity at 88.9% specificity in the training set and with 58.0% sensitivity at 90.0% specificity in the independent validation set. The results of the multiplexed assay using xMAP technology for early ESCC showed a significant correlation with that of the ELISA assays with 66.0% sensitivity at 90.9% specificity. A four-autoantibody panel showed good performance for early ESCC diagnosis with ELISA and could be further developed into a multiplex assay using the Luminex xMAP technology. The four-autoantibody panel could be used for serologic screening for early ESCC.

SNHG8 is upregulated in esophageal squamous cell carcinoma and directly sponges microRNA-411 to increase oncogenicity by upregulating KPNA2.

BackgroundThe long noncoding RNA, small nucleolar RNA host gene 8 (SNHG8), is upregulated in multiple human cancer types. However, whether SNHG8 is aberrantly expressed in esophageal squamous cell carcinoma (ESCC) and its biological functions have yet to be elucidated. Thus, we aimed to determine the expression status of SNHG8 in ESCC, explore the effects of SNHG8 on the oncogenicity of ESCC, and investigate the potential underlying mechanisms.MethodsSNHG8 expression in ESCC tissues and cell lines was determined via reverse-transcription quantitative polymerase chain reaction. The actions of SNHG8 on the malignant characteristics of ESCC were explored using CCK-8 assay, flow-cytometric analysis, Transwell migration and invasion assays, and tumor xenografts in nude mice.ResultsSNHG8 expression was significantly higher in ESCC tissues and cell lines. High SNHG8 expression was revealed to closely correlate with primary tumor invasion depth, lymph node metastases, TNM stage, and worse overall survival among patients with ESCC. Functional investigation showed that ablation of SNHG8 notably restricted ESCC cell proliferation, migration, and invasion while inducing apoptosis in vitro and hindered tumor growth in vivo. In the meantime, SNHG8 acted as a molecular sponge of microRNA-411 (miR-411) in ESCC. Furthermore, miR-411 exerted a tumor-suppressive effect on ESCC cells, and karyopherin alpha 2 (KPNA2) turned out to be a direct target gene of miR-411. Restoring KPNA2 expression neutralized the inhibitory effects of miR-411 overexpression on the malignant behaviors of ESCC cells. Moreover, silencing of miR-411 abrogated the influence of SNHG8 downregulation in ESCC cells.ConclusionSNHG8 may play oncogenic roles in the malignancy of ESCC by sponging miR-411 to increase KPNA2 expression. The SNHG8–miR-411–KPNA2 pathway may be a novel target for the treatment of patients with ESCC and offer potential biomarkers for the diagnosis and prognosis of ESCC.

The prediction of molecular functions for three novel miRNAs in esophageal squamous cell carcinoma

In order to find novel biomarkers for the diagnosis and treatment of esophageal squamous cell carcinoma (ESCC), qRT-PCR was used to detect the expression of three unknown miRNAs (miR-3914, miR-8082 and miR-4770) in ESCC. GO (gene ontology) and Kyoto encyclopedia of genes and genomes (KEGG) enrichment analyses were performed to evaluate and predict biological functions of targeted genes in differentially expressed miRNAs. Westernblot was used to analyzecancer-related protein expression after miRNA mimic transfection. Results showed that miR-3914 and miR-8082 were significantly decreased in ESCC tissues/cells compared with normal esophageal tissues/cells (<italic>P</italic>&lt;0.05). Data of GO enrichment analysis revealed that miR-3914 and miR-8082 could participate in RNA polymerase II promoter transcription, protein binding, cell apoptosis and other biological processes. KEGG pathway analysis displayed that both miR-3914 and miR-8082 were associated with pathway in cancer, simultaneously regulated to <italic>LAMC</italic>2, <italic>CCDC</italic>6 and <italic>PDGFB</italic> genes. After transfection of miR-3914 and miR-8082 mimic, the expression of p53 protein was increased and accompanied with the decreases of c-Myc and Bcl-2proteins in ESCC cells compared to untreated cells. Taken together, both miR-3914 and miR-8082 may become novel potential biomarkers for helping early diagnosis and precision treatment of ESCC.

CCL18-induced HOTAIR upregulation promotes malignant progression in esophageal squamous cell carcinoma through the miR-130a-5p-ZEB1 axis.

Accumulating evidence indicates that CCL18 and the long non-coding RNA, HOTAIR, have critical roles in cancer progression and metastasis, but the correlation between CCL18 and HOTAIR in esophageal squamous cell carcinoma (ESCC) and their downstream molecular mechanisms remain unclear. Overexpression of CCL18 in ESCC tissues was associated with a worse survival in patients with ESCC. CCL18 enhanced the invasiveness of ESCC cells in a dose-dependent manner, whereas CCL18 knockdown inhibited their invasiveness. In particular, CCL18 expression was positively associated with HOTAIR expression in ESCC tissues. Furthermore, CCL18 upregulated the expression of HOTAIR, and knockdown of HOTAIR alleviated the CCL18-induced invasiveness of ESCC cells. HOTAIR may act as a competing endogenous RNA and could effectively becoming a sponge for miR-130a-5p, thereby modulating the derepression of ZEB1 and promoting epithelial-mesenchymal transition in ESCC. Our study suggests that CCL18 contributes to the malignant progression of esophageal cancer by upregulating HOTAIR expression. HOTAIR overexpression may promote tumor invasiveness and progression in ESCC, given that HOTAIR functions as a miR-130a-5p sponge, positively regulating ZEB1. This provides new therapeutic targets for early diagnosis and treatment of ESCC.

Development and Validation of an Esophageal Squamous Cell Carcinoma Detection Model by Large-Scale MicroRNA Profiling

Patients with late-stage esophageal squamous cell carcinoma (ESCC) have a poor prognosis. Noninvasive screening tests using serum microRNAs (miRNAs) to accurately detect early-stage ESCC are needed to improve mortality. To establish a model using serum miRNAs to distinguish patients with ESCC from noncancer controls. In this case-control study, serum miRNA expression profiles of patients with ESCC (n = 566) and control patients without cancer (n = 4965) were retrospectively analyzed to establish a diagnostic model, which was tested in a training set and confirmed in a validation set. Patients histologically diagnosed as having ESCC who did not receive prior therapy or have a past or concurrent cancer other than ESCC were enrolled from the National Cancer Center Hospital in Tokyo, Japan. Between October 2010 and November 2015, control samples were collected from the National Cancer Center Biobank, the Biobank of the National Center for Geriatrics and Gerontology, and the general population undergoing routine health examination. Data analysis was performed between August 2015 and October 2018. Serum samples were randomly divided into discovery and validation sets. The expression of 2565 miRNAs was assessed in each sample. The discriminant model (named the EC index) was evaluated in the training set using Fisher linear discriminant analysis with a greedy algorithm. Receiver operating characteristic curve analysis evaluated the diagnostic ability of the model in the validation set. In the training set, 283 patients with esophageal cancer (median age, 67 years [range, 37-90 years]; 83.4% male) were compared with 283 control patients (median age, 54 years [range, 22-100 years]; 43.1% male), and the EC index was constructed using 6 miRNAs (miR-8073, miR-6820-5p, miR-6794-5p, miR-3196, miR-744-5p, and miR-6799-5p). The area under the receiver operating characteristic curve was 1.00, with sensitivity of 1.00 and specificity of 0.98. The validation set included 283 patients (median age, 66 years [range, 42-87 years]; 83.0% male) and 4682 control patients (median age, 68 years [range, 20-98 years]; 44.7% male), and the area under the receiver operating characteristic curve for the EC index was 1.00, with sensitivity of 0.96 and specificity of 0.98. What appears to be novel serum miRNA discriminant model was developed for the diagnosis of ESCC. A multicenter prospective study is ongoing to confirm the present observations.

Impact of Brush Cytology Analysis for the Diagnosis of Esophageal Squamous Cell Carcinoma: The Quality of Liquid-Based Preparation of Cytological Slides

Objectives: The diagnostic performance of cytology in esophageal squamous cell carcinoma (ESCC) is meticulously described. Methods: Cytological and biopsy specimens were prospectively taken during esophagogastroduodenoscopy of 123 individuals in 2013 and 2014. Cytology samples were maintained in preservative fluid until processing and biopsies were formalin-fixed and paraffin-embedded. Results: Based on endoscopic biopsy results, 70 cases were positive for ESCC whilst 53 were negative for cancer. In addition, brush cytology showed high sensitivity and specificity (98.57 and 96.23%, respectively) in detecting the disease, and high accuracy (97.5%) comparable to that provided by histopathology which is the accepted gold standard. Conclusion: Brush cytology specimens preserved in liquid medium may be a good alternative for ESCC diagnosis.

Research on relationship between the circulating tumor cells and esophageal squamous cell carcinoma clinicopathological characteristics

Objective To evaluate the diagnostic value of circulating tumor cells (CTCs) with expressing epithelial-mesenchymal transitions (EMT) biomarkers in esophageal squamous cell carcinoma (ESCC) patients and investigated the association with their clinicopathological parameters. Methods We included 174 patients with ESCC as the observation group and 40 healthy volunteers as negative control group. According to the different treatment stages, blood samples from ESCC patients were analyzed qualitatively and quantitatively for CTCs in peripheral blood. Results (1) The total CTCs in 174 ESCC patients was higher than that in 40 healthy volunteers (21 vs. 0), and the positive rate was also higher than that of healthy volunteers (99.4% vs. 35.0%), the area under receiver operating characteristic curve (ROC) of epithelial CTCs, mesenchymal CTCs and total CTCs were 0.813, 0.782 and 0.991, respectively. The clinical diagnostic critical point of each phenotypes of CTCs were 2, 0 and 3, respectively; (2) For ESCC patients, the more the mesenchymal CTCs, the later the clinical stage; (3) Follow-up blood specimens were obtained from neoadjuvant chemotherapy in ESCC patients. Complete response (CR), partial response (PR), stable disease (SD) and progressive disease (PD) after treatment were seen in 2, 20, 13 and 4 patients, respectively. Patients with a negative mesenchymal CTCs status after two cycles of chemotherapy had a good chemosensitivity (P<0.05), and the positive rate of mesenchymal CTCs in SD/PD (70.6%) was significantly higher than that in CR/PR (36.4%) group; (4) We compare the changes in CTCs between preoperative and postoperative of 47 ESCC patients, and the analysis revealed that the total CTCs of postoperative patients were decreasing (P<0.01), however, mesenchymal CTCs were increasing dramatically (P<0.01). Conclusion Mesenchymal CTCs as a biochemical marker of ESCC has a certain value in diagnosis of ESCC, which is significantly correlated with the clinical stage and chemosensitivity of the tumor. Key words: Esophageal cancer; Circulating tumor cells; Epithelial-mesenchymal transition; Vimentin

Identification of the Germline Mutation Profile in Esophageal Squamous Cell Carcinoma by Whole Exome Sequencing.

Background: Esophageal squamous cell carcinoma (ESCC) is associated with poor prognosis and occurs with high frequency in China. The germline mutation profile in ESCC remains unclear, and therefore, the discovery of oncogenic alterations in ESCC is urgently needed. This study investigates the germline mutation profile and reveals associations among genotype-environment interactions in ESCC.Methods: Whole exome sequencing and follow-up analysis were performed in 77 matched tumor-normal ESCC specimens to examine the germline profiles. Additionally, associations among genotype-environment interactions were investigated.Results: We identified 84 pathogenic/likely pathogenic mutations and 51 rare variants of uncertain significance (VUS). Twenty VUS with InterVar evidence of a score of moderate pathogenicity (PM) 2/PM2+ supporting pathogenicity (PP) 1 were found to have pathogenic significance. CYP21A2 was the most frequently mutated gene, and the p.Gln319* variant was identified in 6.5% (5/77) of patients. The TP53 p.V197E mutation, located within the DNA binding domain, was found in 1.3% (1/77) of patients. In total, the 11.7% (9/77) of individuals with homologous recombination (HR) VUS were more likely to have well-differentiated tumors than those without (P = 0.003). The degree of lymph node metastasis was correlated with homologous recombination deficiency (HRD) and VUS group (P < 0.05). Moreover, the 10.4% (8/77) of individuals with mismatch repair (MMR) VUS had a higher tumor mutational burden (TMB), although the correlation was not significant.Conclusions: Our study identified the germline mutation profiles in ESCC, providing novel insights into the molecular pathogenesis of this disease. Our results may also serve as a useful resource for the exploration of the underlying mechanism of ESCC and may provide information for the prevention, diagnosis and risk management of ESCC.

Identification of Serum MicroRNAs as Novel Biomarkers in Esophageal Squamous Cell Carcinoma Using Feature Selection Algorithms.

Introduction: Circulating microRNAs (miRNAs) are promising molecular biomarkers for the early detection of esophageal squamous cell carcinoma (ESCC). We investigated the serum miRNA expression profiles from microarray-based technologies and evaluated the diagnostic value of serum miRNAs as potential biomarkers for ESCC by using feature selection algorithms.Methods: Serum miRNA expression profiles were obtained from 52 ESCC patients and 52 age- and sex-matched controls via performing a high-throughput microarray assay. Five representative feature selection algorithms including the false discovery rate procedure, family-wise error rate procedure, Lasso logistic regression, hybrid huberized support vector machine (SVM), and SVM using the squared-error loss with the elastic-net penalty were jointly carried out to select the significantly differentially expressed miRNAs based on the miRNA profiles.Results: Three miRNAs including miR-16-5p, miR-451a, and miR-574-5p were identified as the powerful biomarkers for the diagnosis of ESCC. The diagnostic accuracy of the combination of these three miRNAs was evaluated by using logistic regression and the SVM. The averages of the area under the receiver operating curve and classification accuracies based on different classifiers were more than 0.80 and 0.79, respectively. The cross-validation results suggested that the three-miRNA-based classifiers could clearly distinguish ESCC patients from healthy controls. Moreover, the classifying performance of the miRNA panel persisted in discriminating the healthy group from patients with ESCC stage I-II (AUC > 0.76) and patients with ESCC stage III-IV (AUC > 0.80).Conclusions: These results in this study have moved forward the identification of novel biomarkers for the diagnosis of ESCC.

Up-regulation of circ-SMAD7 inhibits tumor proliferation and migration in esophageal squamous cell carcinoma

BackgroundIncreasing evidences demonstrate that circular RNAs (circRNAs) play an important role in the development and progression of human cancers. Nevertheless, the functions and molecular mechanism of circRNAs in esophageal squamous cell carcinoma (ESCC) tumorigenesis are largely unknown. The purpose of this research is to investigate the expression and potential role of a new circular RNA named circ-SMAD7 on ESCC carcinogenesis. Materials and methodsQuantitative real-time polymerase chain reaction (qRT-PCR) was used to measure circ-SMAD7 expression amount in both ESCC plasmas and tissues. Then the correlation between the expression of circ-SMAD7 and clinicopathological features was analyzed. Furthermore, by loss-of-function and gain-of-function experiments in ESCC cells, a functional analysis of circ-SMAD7 on ESCC cell proliferation and migration was performed. ResultsThe expression of circ-SMAD7 was validated to be significantly down-regulated in ESCC plasmas in comparison with normal controls, showing a high negative correlation with TNM stage (P = 0.000) and lymphatic metastasis (P = 0.000). Moreover, circ-SMAD7 was significantly down-regulated in ESCC tissues compared to adjacent normal tissues. Furthermore, Loss-of-function and gain-of-function experiments revealed that the expression level of circ-SMAD7 affected the proliferation and migration of ESCC cell lines. ConclusionOur study firstly exposed that over-expressioncirc-SMAD7, an influential regulator in cancer progression, can inhibit tumor proliferation and migration in ESCC and have the potential of becoming a biomarker for the diagnosis and therapy of ESCC.

Clinical and translational advances in esophageal squamous cell carcinoma.

Esophageal squamous cell carcinoma (ESCC) is among the most deadly forms of human malignancy characterized by late stage diagnosis, metastasis, therapy resistance and frequent recurrence. Clinical management of ESCC remains challenging and the disease presently lacks approved targeted therapeutics. However, emerging data from recent clinical and translational investigations hold great promise for future progress toward improving patient outcomes in this deadly disease. Here, we review current clinical perspectives in ESCC epidemiology, pathophysiology, and clinical care, highlighting recent advances with potential to impact ESCC prevention, diagnosis and management. We further provide an overview of recent translational investigations contributing to our understanding of the molecular mechanisms underlying ESCC development, progression and therapy response, including insights gained from genetic studies and various murine model systems. Finally, we discuss future perspectives in the clinical and translational realms, along with remaining hurdles that must be overcome to eradicate ESCC.

Epigenetic deregulations of Wnt/β-catenin and transforming growth factor beta-Smad pathways in esophageal cancer: Outcome of DNA methylation.

Promoter methylation of tumor suppressor genes (TSGs) is a well-reported portent in carcinogenesis; hence, it is worthy to investigate this in high-risk Northeast population of India. The study was designed to investigate methylation status of 94 TSGs in esophageal squamous cell carcinoma (ESCC). Further, the effect of OPCML promoter methylation on gene expression was analyzed by immunohistochemistry. Moreover, in silico protein-protein interactions were examined among 8 TSGs identified in the present study and 23 epigenetically regulated genes reported previously by our group in ESCC. Methylation profiling was carried out by polymerase chain reaction array and OPCML protein expression was examined by tissue microarray-based immunohistochemistry. OPCML, NEUROG1, TERT, and WT1 genes were found hypermethylated and SCGB3A1, CDH1, THBS1, and VEGFA were hypomethylated in Grade 2 tumor. No significant change in OPCML expression was observed among control, Grade 1, and Grade 2 tumor. Conclusively, hypermethylation of the studied OPCML promoter in Grade 2 tumor produced no effect on expression. Unexpectedly, OPCML expression was downregulated in Grade 3 tumor in comparison to other groups signifying that downregulation of OPCML expression may lead to higher grade of tumor formation at the time of diagnosis of ESCC in patients. Significant interactions at protein level were found as VEGFA:PTK2, CTNNB1:CDH1, CTNNB1:VEGFA, CTNNB1:NEUROG1, CTNND2:CDH1, and CTNNB1:TERT. These interactions are pertinent to Wnt/β-catenin and TGF-β-Smad pathways. Deranged OPCML expression may lead to high-grade ESCC as well as epigenetically regulated genes, that is, CDH1, CTNNB1, CTNND2, THBS1, PTK2, WT1, OPCML, TGFB1, and SMAD4 may alter the Wnt/β-catenin and TGF-β-Smad pathways in ESCC. Further study of these genes could be useful to understand the molecular pathology of ESCC with respect to epithelial-mesenchymal transition (EMT) mediated by Wnt/β-catenin and TGF-β signaling pathways.

Circ-TTC17 Promotes Proliferation and Migration of Esophageal Squamous Cell Carcinoma

BackgroundCircular RNAs (circRNAs), a special class of noncoding RNAs with the characteristic of covalent closed-loop structure, have been widely found in various organisms. Growing evidence has shown that circRNAs play a crucial role in regulating biological functions of cancers. However, the specific role of circRNAs in esophageal squamous cell carcinoma (ESCC) remains largely unknown.AimThe present study aims to investigate the effects of circ-TTC17 in ESCC clinical samples as well as cells.MethodsSanger sequencing and agarose gel electrophoresis were used to verify the specificity of circ-TTC17. Expression levels of circ-TTC17 in ESCC cells, plasma, and tissues were measured by quantitative real-time polymerase chain reaction. A colony formation experiment, CCK-8 assay, and wound-healing assay were applied to detect the functions of circ-TTC17 in KYSE30 and KYSE450 cells. A nucleus–cytoplasm fractionation experiment was used to probe the location of circ-TTC17 in ESCC cells. Finally, a network of circ-TTC17 with its targeted miRNAs interactions and corresponding mRNAs was analyzed and framed by bioinformatics.ResultsThe expression level of circ-TTC17 was found to be significantly higher in ESCC cells, plasma, and tissues compared with normal cases. In vitro experiments indicated that circ-TTC17 promoted proliferation and migration of ESCC cells. Bioinformatics predictions showed that circ-TTC17 might regulate progress of ESCC by acting as a sponge for microRNAs (miRNAs).ConclusionsThe results of this study demonstrate that upregulated circ-TTC17 plays a key role in promoting proliferation and migration of ESCC cells and has potential to become a novel biomarker for diagnosis, treatment, and prognosis of ESCC in the future.

Circulating tumor cells detected using fluid-assisted separation technique in esophageal squamous cell carcinoma.

Esophageal squamous cell carcinoma (ESCC) is one of the aggressive gastrointestinal tract cancers. Detection of circulating tumor cells (CTCs) in peripheral blood from patients with various malignancies has been reported to have diagnostic, prognostic, and therapeutic implications. We aimed to evaluate CTCs in patients with ESCC and assess the clinical significance of CTCs in the early diagnosis of ESCC. Peripheral blood samples for CTCs analyses were prospectively obtained from 73 patients with ESCC prior to treatment between March 2015 and June 2018. CTCs were detected using a centrifugal microfluidic system with a new fluid-assisted separation technique. Blood samples from 31 healthy volunteers were used as controls. After creating a receiver operating characteristic curve to determine the optimal CTC threshold to differentiate patients with ESCC from healthy controls, sensitivity and specificity were most optimized at a CTC threshold of two per 7.5mL of blood. Among 66 subjects with ≥2 CTCs per 7.5mL of blood, 63 (95.5%) had ESCC. Among 38 subjects with <2 CTCs per 7.5mL of blood, 28 (73.7%) were healthy controls. When using this threshold, the sensitivity and specificity for differentiating patients with ESCC from healthy controls were 86.3% and 90.3%, respectively. CTC count was associated with tumor-node-metastasis stage, especially lymph node metastasis, but there was no correlation with any other relevant clinicopathologic variable. Our results suggest that CTCs detected using fluid-assisted separation technique could be helpful for early diagnosis of ESCC. Further large-scale prospective studies are warranted to validate our findings.

Expression of human leukocyte antigen-G in esophageal squamous cell carcinoma and its significance

Objective To investigate the expression of human leukocyte antigen-G (HLA-G) in patients with primary esophageal squamous cell carcinoma (ESCC) and its significance in the diagnosis and prognosis of ESCC. Methods Immunohistochemical SP method was used to detect the expression of HLA-G in 121 patients with primary ESCC admitted to Sichuan Provincial People's Hospital from January 2013 to January 2018. The relationship between expression of HLA-G and clinicopathological parameters and host immune response was compared by χ2 test and analysis of variance. Kaplan-Meier method was used for survival analysis, and Cox regression model was used for multivariate analysis. Results The expression rate of HLA-G in patients with primary ESCC was 90.9% (110/121). The expression of HLA-G had relationship with tissue differentiation (χ2 = 41.695, P = 0.001), the depth of tumor invasion (χ2 = 120.432, P = 0.001), lymph node metastasis (χ2 = 6.297, P = 0.043), clinical TNM stage (χ2 = 110.394, P = 0.001), and there was a statistically significant difference in host immune response among patients with different HLA-G expression levels (F = 3.190, P = 0.048). The survival rate of HLA-G-negative patients was significantly higher than that of HLA-G-positive patients [72.7% (8/11) vs. 33.9% (20/59)], and the difference was statistically significant (χ2 = 10.036, P = 0.001). The expression of HLA-G and TNM stage were independent factors affecting the prognosis of patients with primary ESCC (HR=2.99, 95% CI 1.52-5.88, P = 0.002; HR=1.95, 95% CI 1.47-3.13, P = 0.049). Conclusion HLA-G is highly expressed in ESCC tissues, and HLA-G expression is associated with the prognosis of patients with ESCC. Key words: Esophageal neoplasms; Carcinoma, squamous cell; HLA antigens; Immunohistochemistry