Response Of Esophageal Squamous Cell Carcinoma Research Articles

Identification of m5C-related lncRNAs signature to predict prognosis and therapeutic responses in esophageal squamous cell carcinoma patients

Esophageal squamous cell carcinoma (ESCC) has a dismal prognosis because of atypical early symptoms and heterogeneous therapeutic responses. 5-methylcytosine (m5C) modification plays an important role in the onset and development of many tumors and is widespread in long non-coding RNA (lncRNA) transcripts. However, the functions of m5C and lncRNAs in ESCC have not been completely elucidated. Herein, this study aimed to explore the role of m5C-related lncRNAs in ESCC. The RNA-seq transcriptome profiles and clinical information were downloaded from the TCGA-ESCC database. Pearson analysis was used to identify m5C-related lncRNAs. Then we established the m5C-related lncRNAs prognostic signature (m5C-LPS) using univariate Cox and least absolute shrinkage and selection operator (LASSO) regression analysis. Then, the prognostic value of m5C-LPS was evaluated internally and externally using the TCGA-ESCC and GSE53622 databases through multiple methods. We also detected the expression of these lncRNAs in ESCC cell lines and patient tissues. Fluorescence in situ hybridization (FISH) was used to detect the prognostic value of specific lncRNA. In addition, clinical parameters, immune status, genomic variants, oncogenic pathways, enrichment pathways, and therapeutic response features associated with m5C-LPS were explored using bioinformatics methods. We constructed and validated a prognostic signature based on 9 m5C-related lncRNAs (AC002091.2, AC009275.1, CAHM, LINC02057.1, AC0006329.1, AC037459.3, AC064807.1, ATP2B1-AS1, and UBAC2-AS1). The quantitative real-time polymerase chain reaction (qRT-PCR) revealed that most lncRNAs were upregulated in ESCC cell lines and patient tissues. And AC002091.2 was validated to have significant prognostic value in ESCC patients. A composite nomogram was generated to facilitate clinical practice by integrating this signature with the N stage. Besides, patients in the low-risk group were characterized by good clinical outcomes, favorable immune status, and low oncogenic alteration. Function enrichment analysis indicated that the risk score was associated with mRNA splicing, ncRNA processing, and DNA damage repair response. At the same time, we found significant differences in the responses to chemoradiotherapy between the two groups, proving the value of m5C-LPS in treatment decision-making in ESCC. This study established a novel prognostic signature based on 9 m5C-related lncRNAs, which is a promising biomarker for predicting clinical outcomes and therapeutic response in ESCC.

Delta-radiomics based on CT predicts pathologic complete response in ESCC treated with neoadjuvant immunochemotherapy and surgery.

Unnecessary surgery can be avoided, and more appropriate treatment plans can be developed for patients if the efficacy of neoadjuvant immunochemotherapy for esophageal cancer (EC) can be predicted before surgery. The purpose of this study was to evaluate the ability of machine learning models based on delta features of immunochemotherapy CT images to predict the efficacy of neoadjuvant immunochemotherapy in patients with esophageal squamous cell carcinoma (ESCC) compared with machine learning models based solely on postimmunochemotherapy CT images. A total of 95 patients were enrolled in our study and randomly divided into a training group (n = 66) and test group (n = 29). We extracted preimmunochemotherapy radiomics features from preimmunochemotherapy enhanced CT images in the preimmunochemotherapy group (pregroup) and postimmunochemotherapy radiomics features from postimmunochemotherapy enhanced CT images in the postimmunochemotherapy group (postgroup). We then subtracted the preimmunochemotherapy features from the postimmunochemotherapy features and obtained a series of new radiomics features that were included in the delta group. The reduction and screening of radiomics features were carried out by using the Mann-Whitney U test and LASSO regression. Five pairwise machine learning models were established, the performance of which was evaluated by receiver operating characteristic (ROC) curve and decision curve analyses. The radiomics signature of the postgroup was composed of 6 radiomics features; that of the delta-group was composed of 8 radiomics features. The area under the ROC curve (AUC) of the machine learning model with the best efficacy was 0.824 (0.706-0.917) in the postgroup and 0.848 (0.765-0.917) in the delta group. The decision curve showed that our machine learning models had good predictive performance. The delta group performed better than the postgroup for each corresponding machine learning model. We established machine learning models that have good predictive efficacy and can provide certain reference values for clinical treatment decision-making. Our machine learning models based on delta imaging features performed better than those based on single time-stage postimmunochemotherapy imaging features.

RAD18 confers radioresistance of esophagus squamous cell carcinoma through regulating p-DNA-PKcs.

BackgroundRadiotherapy has recently become more common for the treatment of esophageal squamous cell carcinoma (ESCC). Radioresistance, on the other hand, continues to be a major issue because it interferes with the effectiveness of ESCC radiation. It has been demonstrated that RAD18, an E3 ubiquitin‐protein ligase that regulates translesion DNA synthesis (TLS), is implicated in the regulation of genomic integrity and DNA damage response.MethodsIn the present study, immunohistochemical staining and western blotting were utilized to determine RAD18 expression in ESCC tissues and cells. ESCC cell proliferation was determined using a colony formation assay. Immunofluorescence staining, comet assay, and homologous recombination (HR)/non‐homologous end‐joining (NHEJ) assays were conducted to examine the effect of RAD18 on the DNA damage response in ESCC cells.ResultsWe found that high RAD18 expression was positively associated with a poorer prognosis in patients with ESCC who received radiotherapy. Downregulation of RAD18 expression significantly increased the sensitivity of ESCC cells to irradiation. Moreover, RAD18 knockdown prolonged the repair kinetics of γH2AX foci and resulted in longer comet tails. Furthermore, loss of RAD18 expression markedly decreased non‐homologous end‐joining (NHEJ) activity, but it did not affect homologous recombination (HR)‐mediated double‐strand break repair in ESCC cells. RAD18 upregulated p‐DNA‐dependent protein kinase complex (p‐DNA‐PKc) expression in vivo and in vitro.ConclusionsThese data indicated that RAD18 may regulate radioresistance by facilitating NHEJ via phosphorylation of DNA‐PKcs in ESCC cells, providing a novel radiotherapy target for ESCC.

Fc fragment of immunoglobulin G receptor IIa (FCGR2A) as a new potential prognostic biomarker of esophageal squamous cell carcinoma.

To the Editor: Esophageal squamous cell carcinoma (ESCC) is one of the most aggressive cancer types and places a heavy burden on human health. The early diagnosis and prognosis monitoring of ESCC is important for therapy. Despite recent progress in treatment regimens for ESCC, the prognosis of ESCC remains poor.[1] Therefore, it is important to find new molecular therapeutic targets and prognostic monitoring biomarkers for ESCC patients. In this study, we aimed to explore new prognostic biomarkers for ESCC. Based on the two public Illumina HiSeq platform RNA sequencing datasets downloaded from the Gene Expression Omnibus (GEO) database (https://www.ncbi.nlm.nih.gov/geo/), we collected the expression data of 33 ESCC tissues and 33 adjacent normal tissues from GSE130078 and GSE149609 to investigate the protein-coding differentially expressed genes (DEGs), using DESeq2 (http://www.bioconductor.org/packages/release/bioc/html/DESeq2.html) under the threshold of |log2 Fold-Change| ≥ 1 and the adjusted P value of ≤0.05. As a result, a total of 3885 and 3477 DEGs were identified in dataset GSE130078 and GSE149609, respectively. Then, a total of 1914 overlapping DEGs in these two datasets were detected. To explore the interactive relationships among these DEGs, protein-protein interaction (PPI) network analysis was performed for the 1914 DEGs using STRING database (http://string-db.org/cgi/input.pl). Then, Cytoscape (version 3.5.1, https://cytoscape.org/) was used to construct the PPI network, and the Molecular Complex Detection (MCODE) app in Cytoscape was used to analyze the modules in the PPI network (degree cutoff = 2, max. depth = 100, k-core = 2, and node score cutoff = 0.200). The module criteria were a MCODE score >20 and the number of nodes >10. Consequently, the first module (MCODE score = 29.156), which included 46 hub genes, was chosen. Specifically, the node degree of Fc fragment of immunoglobulin G receptor IIa (FCGR2A) was 19.863, which indicated that FCGR2A might play an important role in maintaining the structure and function in the ESCC PPI network. In addition, Kyoto Encyclopedia of Genes and Genomes pathway results for the 46 hub genes indicated that they were mainly enriched in cytokine–cytokine receptor interactions, the interleukin (IL)-17 signaling pathway, and the chemokine signaling pathway. These pathways have been reported to be correlated with the cancer survival or prognosis, which suggested that these 46 hub genes might be linked to ESCC prognosis. Afterward, the expression of the 46 hub genes was validated with 182 ESCC tissues and 286 adjacent tissues from The Cancer Genome Atlas (TCGA) and Genotype Tissue Expression (GTEx) databases. As a result, only 23 genes, including FCGR2A [Figure 1A], were found to be significantly and highly expressed in ESCC tissues compared with normal tissues [Supplementary Figure 1, https://links.lww.com/CM9/A768]. Then, we assessed the effect of the 23 hub genes on disease-free survival (DFS) in ESCC based on TCGA data using Gene Expression Profiling Interactive Analysis (GEPIA) (http://gepia.cancer-pku.cn/index.html). Cox proportional hazards regression models with estimations of hazard ratios (HR) and 95% confidence intervals (95% CI) were used to evaluate the impact of genes on DFS, and hub genes that were associated with significantly worse DFS (P < 0.05) were selected. The Kaplan–Meier curve with log-rank test results [Figure 1B] revealed that ESCC patients with higher mRNA expression levels of FCGR2A (HR: 1.700, 95% CI: 1.110–7.605, P = 0.032), fibronectin 1 (FN1) (HR: 2.000, 95% CI: 1.663–5.368, P = 0.007) and secreted phosphoprotein 1 (SPP1) (HR: 2.300, 95% CI: 2.150–3.796, P = 0.001) had significantly lower DFS [Supplementary Figure 2, https://links.lww.com/CM9/A768]. In addition, the expression of the three significant hub genes in ESCC at different stages was analyzed based on one-way analysis of variance test. Consequently, compared with stage I, FCGR2A (F = 2.980, P = 0.033) [Figure 1C], FN1 (F = 4.840, P = 0.003) and SPP1 (F = 5.710, P < 0.001) [Supplementary Figure 2, https://links.lww.com/CM9/A768] were found to be more significantly expressed in the advanced clinical stages of ESCC (stage II, III, and IV). These results demonstrated that overexpression of the three genes was associated with the poor prognosis in ESCC.Figure 1: FCGR2A as a potential prognostic biomarker of ESCC. (A) The expression level of FCGR2A in tumor tissues (n = 182) and normal tissues (n = 286) in ESCC from TCGA and GTEx databases. ESCC tissues and normal tissues are shown in red and black, respectively. ∗ P < 0.05. (B) An increased expression level of FCGR2A was associated with a poor DFS in ESCC. The samples with gene highly expressed or gene lowly expressed are shown in red and blue, respectively. The P value was verified by the log-rank test. (C) FCGR2A expression varied significantly in different clinical stages of ESCC (P < 0.05). The F value and P value were calculated by one-way ANOVA test. (D) Spearman correlations between FCGR2A expression and infiltration level of TIICs. (E–G) Correlations between the expression of FCGR2A and the gene markers in each subtype of macrophages, including (E) M2 macrophages, (F) M1 macrophages, and (G) TAMs. (H) Correlation between the expression of FCGR2A and TAMs associated marker CD204. ANOVA: Analysis of variance; CCL2: C-C motif chemokine ligand 2; CD: Cluster of differentiation; DFS: Disease-free survival; ESCC: Esophageal squamous cell carcinoma; FCGR2A: Fc fragment of immunoglobulin G receptor IIa; GTEx: Genotype Tissue Expression; HR: Hazard ratio; IL10: Interleukin 10; IRF5: Interferon regulatory factor 5; MS4A4A: Membrane spanning 4-domains A4A; NOS2: Nitric oxide synthase 2; PTGS2: Prostaglandin-endoperoxide synthase 2; TAMs: Tumor-associated macrophages; TCGA: The Cancer Genome Atlas; TIIC: Tumor-infiltrating immune cells; TPM: Transcripts per million; VSIG4: V-set and immunoglobulin domain-containing 4.More importantly, the Gene Ontology analysis of FCGR2A was analyzed using KEGG Orthology Based Annotation System (KOBAS) (http://kobas.cbi.pku.edu.cn/kobas3) with a corrected P value of <0.05. Results indicated that FCGR2A may be associated with the immune response in ESCC. To explore its potential correlation with the immune environment in ESCC, the relationships between FCGR2A and tumor-infiltrating immune cells (TIICs) were determined using the Tumor Immune Estimation Resource (https://cistrome.shinyapps.io/timer/) database because some TIICs have been reported to predict poor prognosis in ESCC patients.[2,3] Our results [Figure 1D] revealed that the expression of FCGR2A was most positively correlated with the infiltration level of macrophages in ESCC (R = 0.656, P < 0.001), while it had lower correlations with the level of tumor purity and infiltrating B cells, CD8+ T cells, CD4+ T cells, neutrophils, and dendritic cells (R < 0.500). Specifically, we focused on the correlation between FCGR2A and macrophages. Macrophages have two different phenotypes, the M1 phenotype, which is tumor suppressive, and the M2 phenotype, which is tumor supportive.[4] Furthermore, tumor-associated macrophages (TAMs) differentiate into the M2 phenotype and contribute to progression of the disease. An increase in the number of infiltrating CD204+ TAMs has been reported to be correlated with the poor prognosis in ESCC.[3] To detect the relationship between FCGR2A and diverse immune infiltrating cells, we also analyzed correlations between FCGR2A and the gene markers of various immune cells in ESCC. Regarding the correlation between FCGR2A and macrophages, the expression of FCGR2A was significantly and positively correlated with the expression of CD163 molecule (CD163) (R = 0.831, P < 0.001), V-set and immunoglobulin domain-containing 4 (R = 0.817, P < 0.001) and membrane spanning 4-domains A4A (R = 0.849, P < 0.001), which are markers of M2 macrophages [Figure 1E]. However, FCGR2A was negligibly associated with nitric oxide synthase 2, interferon regulatory factor 5, and prostaglandin-endoperoxide synthase 2 in M1 macrophages [Figure 1F]. In addition, the expression of FCGR2A was significantly and positively correlated with the expression of C-C motif chemokine ligand 2 (R = 0.609, P < 0.001), CD68 molecule (R = 0.433, P < 0.001), and IL-10 (R = 0.567, P < 0.001), which are markers of TAMs [Figure 1G]. The expression of FCGR2A was also significantly and positively correlated with the expression of CD204 molecule (R = 0.855, P < 0.001), which is a TAMs associated marker [Figure 1H]. These results showed that FCGR2A was correlated with immune infiltrates in ESCC and significantly positively correlated with M2 macrophages and TAMs. In the present study, we identified 1914 DEGs and 46 hub genes in ESCC using ESCC cohorts from GEO and we validated the 46 hub genes with ESCC cohorts from TCGA and GTEx databases. Among them, a higher expression level of FCGR2A had a significantly lower DFS. Moreover, the expression of FCGR2A was significantly and positively correlated with the expression of both CD163 and CD204. Strikingly, the elevated number of infiltrating CD204+ TAMs and CD163-positive M2 macrophages were reported to be correlated with the poor prognosis in ESCC,[2,3] which indicated that overexpression of FCGR2A might be associated with a poor prognosis and correlated with immune infiltrates in ESCC. To the best of our knowledge, the present study is the first to explore the association between FCGR2A and TIICs and the prognostic value of FCGR2A in ESCC via bioinformatics methods. However, our bioinformatics results should be confirmed by biological experiments in the future. Despite its limitations, our findings provide direct evidence for research on prognostic biomarkers of ESCC. In conclusion, FCGR2A may be a novel potential prognostic biomarker of ESCC. Funding This study was supported by the grants from National Key Development Plan for Precision Medicine Research, China (No. 2017YFC0910004), Sichuan Science and Technology Program, China (No. 2017HH0044), and Chengdu Science and Technology Program, China (No. 2017-CY02-00017-GX). Conflicts of interest None.

Development of immune gene pair-based signature predictive of prognosis and immunotherapy in esophageal cancer.

BackgroundEsophageal cancer (EC) is one of the deadliest solid malignancies, mainly consisting of esophageal squamous cell carcinoma (ESCC) and adenocarcinoma (EAC). Robust biomarkers that can improve patient risk stratification are needed to optimize cancer management. We sought to establish potent prognostic signatures with immune-related gene (IRG) pairs for ESCC and EAC.MethodsWe obtained differentially expressed IRGs by intersecting the Immunology Database and Analysis Portal (ImmPort) with the transcriptome data set of The Cancer Genome Atlas (TCGA)-ESCC and EAC cohorts. A novel rank-based pairwise comparison algorithm was applied to select effective IRG pairs (IRGPs), followed by constructing a prognostic IRGP signature via the least absolute shrinkage and selection operator (LASSO) regression model. We assessed the predictive power of the IRGP signatures on prognosis, tumor-infiltrating immune cells, and immune checkpoint inhibitor (ICI) efficacy in EC. Kaplan-Meier survival analysis and receiver operating characteristic curves (ROC) were used to evaluate the clinical significance of IRGPs. Univariate and multivariate Cox regression analyses were performed to investigate the association of overall survival (OS) with IRGPs and clinical characteristics.ResultsWe built a 19-IRGP signature for ESCC (n=75) and a 17-IRGP signature for EAC (n=78), with an area under the ROC curve (AUC) of 0.931 and 0.803, respectively. IRGP signature-derived risk scores stratified patients into low- and high-risk groups with significantly different OS in ESCC and EAC (P<0.001). Nomogram and decision curve analysis were used to evaluate the clinical relevance of the prognostic signatures, achieving a C-index of 0.973 in ESCC and 0.880 in EAC. The risk scores were associated with immune and ESTIMATE (Estimation of STromal and Immune cells in MAlignant Tumor tissues using Expression data) scores and the composition of immune cells in the tumor microenvironment. The association between risk score and human leukocyte antigens (HLAs), mismatch repair (MMR) genes, and immune checkpoint molecules demonstrated its predictive value for ICI response. Differential immune characteristics and predictive value of the risk score were observed in EAC.ConclusionsThe established immune signatures showed great promise in predicting prognosis, tumor immunogenicity, and immunotherapy response in ESCC and EAC.

689 MMS22L EXPRESSION AS A PREDICTIVE BIOMARKER FOR THE EFFICACY OF NEOADJUVANT CHEMORADIOTHERAPY IN ESOPHAGEAL SQUAMOUS CELL CARCINOMA

Abstract   Long-term survival in esophageal squamous cell carcinoma (ESCC) is related with pathological response after neoadjuvant chemoradiation therapy (NCRT) followed by surgery. However, effective biomarkers for prediction of pathologic response is still lacking. Therefore, a systematic analysis focusing on genes associated with efficacy of chemoradiotherapy in ESCC will provide valuable insights to understand the molecular features. Methods We collected genes associated with efficacy of chemoradiotherapy by screening publications deposited in PubMed. A specific subnetwork was constructed using Steiner minimum tree algorithm. Then the correlations between gene expression and prognosis of ESCC patients was analyzed using Kaplan–Meier Plotter online resources. The key gene expression was evaluated by quantitative real-time polymerase chain reaction, western blotting analysis and immunohistochemical staining. The predictive performance of biomarkers was estimated using receiver operating characteristic (ROC) curve analysis. Results We identified 101 genes associated with efficacy of chemoradiotherapy. Moreover, specific molecular network included some potential related genes, such as CUL3, MUC13, MMS22L, MME, UBC, VAPA, CYP1B1 and UGDH. MMS22L mRNA expression level had most significant association with the ESCC patient outcome (P &amp;lt; 0.01). Furthermore, MMS22L was downregulated at both the mRNA (P &amp;lt; 0.01) and protein levels in tumor compared with normal tissues. Lymph node metastasis was significantly associated with low MMS22L expression (P &amp;lt; 0.001). MMS22L levels were inversely correlated with NCRT response in ESCC (P &amp;lt; 0.01). MMS22L yielded an area under the ROC curve (AUC) of 0.847 (P &amp;lt; 0.01). Conclusion Low MMS22L expression could be an effective predictive biomarker for resistance to NCRT in ESCC.

Detention and Identification of Cancer Stem Cells in Esophageal Squamous Cell Carcinoma.

Cancer stem cells (CSCs) are a small subpopulation of cells associated with cancer initiation, progression, metastasis, therapy resistant, and recurrence. In esophageal squamous cell carcinoma (ESCC), several cell surface and intracellular markers, for example, CD44, ALDH, Pygo2, MAML1, Twist1, Musashi1, side population (SP), CD271, and CD90, have been proposed to identify CSCs. In addition, stem cell markers such as ALDH1, HIWI, Oct3/4, ABCG2, SOX2, SALL4, BMI-1, NANOG, CD133, and podoplanin were associated with pathological stages of cancer, cancer recurrence, prognosis, and therapy resistance of patients with ESCC. Identification and isolation of CSCs could play an important part of improved cancer management regime in ESCC. Furthermore, CSCs may be used as the predictive tool for chemoradiotherapy response in ESCC. Different methods such as in vitro functional assays, cell sorting using various intracellular, and cell surface markers and xenotransplantation techniques are frequently used for the identification and isolation of CSCs in different cancers, including ESCC. However, none of these methods solely can guarantee complete isolation of CSC population. Therefore, a combination of methods is used for reliable detection and isolation of CSCs. Herein, we describe the identification and isolation of CSCs from ESCC cells by cell sorting after Hoechst 33342 staining followed by in vitro functional assays and in vivo mouse xenotransplantation techniques.

Comprehensive Exploration to Identify Predictive DNA Markers of ΔNp63/SOX2 in Drug Resistance in Human Esophageal Squamous Cell Carcinoma.

OBP-801 is a novel histone deacetylase inhibitor being developed as an anticancer drug. In this study, we explored genes to predict drug resistance in human cancer. OBP-801 resistance was assessed in 37 strains of human cancer cell lines. Expression microarrays harboring 54,675 genes were used to focus on candidate genes, which were validated for both functional and clinical relevance in esophageal squamous cell carcinoma (ESCC). OBP-801 is sensitive to esophageal, gastric, and thyroid cancer, and resistant to some esophageal and colorectal cancers. We therefore used ESCC to explore genes. Comprehensive exploration focused on ΔNp63/SOX2, which were both genetically and epigenetically overexpressed in ESCC. Genomic amplifications of ΔNp63/SOX2 were tightly correlated each other (r = 0.81). Importantly, genomic amplification of ΔNp63/SOX2 in the resected tumors after neoadjuvant chemotherapy was significantly associated with histological grade of response (G1). Forced expression of either of these two genes did not induce each other, suggesting that their functional relevances were independent and showed robust drug resistance in OBP-801, as well as 5-fluorouracil. Furthermore, ΔNp63 could exert a potent oncogenic potential. RNA interference of ΔNp63 supported its oncological properties, as well as drug resistance. Comprehensive exploration of genes involved in anticancer drug residence could identify critical oncogenes of ΔNp63/SOX2 that would predict chemotherapy response in ESCC.

Predictive value of intravoxel incoherent motion imaging for pathologic response to neoadjuvant chemotherapy in locally advanced esophageal squamous cell carcinoma

Objective To explore the predictive value of intravoxel incoherent motion (IVIM) imaging for the pathologic response to neoadjuvant chemotherapy in locally advanced esophageal squamous cell carcinoma (ESCC). Methods The prospective study was conducted. The clinicopathological data of 33 patients with locally advanced ESCC who were admitted to Affiliated Hospital of Zhengzhou University from September 2015 to October 2017 were collected. Patients received magnetic resonance imaging (MRI) and IVIM imaging examination before and after neoadjuvant chemotherapy. Two radiologists read the imaging together, manually delineated the region of interest in the diffusion-weighted imaging, and the apparent diffusion coefficient (ADC), diffusion coefficient (D), perfusion coefficient (D*), and perfusion score of the tumor (f) were automatically measured. Patients underwent neoadjuvant chemotherapy with paclitaxel plus cisplatin, and underwent radical surgery for esophageal cancer after 2 cycles of chemotherapy. Observation indicators: (1) comparison of IVIM imaging parameters before and after neoadjuvant chemotherapy in patients with ESCC; (2) comparison of change value and change rate of IVIM imaging parameters before and after neoadjuvant chemotherapy in patients with different tumor regression grade (TRG); (3) predictive efficacy of change value and change rate of IVIM imaging parameters before and after neoadjuvant chemotherapy for TRG. Measurement data with normal distribution were presented as Mean±SD, and comparison before and after neoadjuvant chemotherapy was done using the paired t test, and comparison between different TRG patients was done using the t test. Measurement data with skewed distribution were presented as M(P25, P75), and comparison before and after neoadjuvant chemotherapy and between different TRG patients were done using the Wilcoxon rank sum test. The receiver operating characteristic (ROC) curve was used to evaluate predictive value of IVIM imaging parameters. Results Thirty-three patients were screened for eligibility, including 26 males and 7 females, aged from 44 to 74 years, with an average age of 60 years. All the 33 patients were diagnosed as ESCC by pathological examination. (1) Comparison of IVIM parameters before and after neoadjuvant chemotherapy in patients with ESCC: 33 patients with ESCC showed a significant difference in the ADC, D, and f value after neoadjuvant chemotherapy [ADC: (1.95±0.56)×10-3 mm2/s vs. (2.54±0.50)×10-3 mm2/s, t=-6.98; D: (1.26×10-3 mm2/s (0.81×10-3 mm2/s, 2.44×10-3 mm2/s) vs. 1.68×10-3 mm2/s (0.83×10-3 mm2/s, 2.27×10-3 mm2/s), Z=-3.96; f: 0.33%±0.14% vs. 0.42%±0.15%, t=-3.13, P<0.05]. (2) Comparison of change value and change rate of IVIM imaging parameters before and after neoadjuvant chemotherapy in different TRG patients: of 33 patients, 15 were in TRG 2 and 18 were in TRG 3. The ADC change value, ADC change rate, D change value, D change rate were (0.85±0.52)×10-3 mm2/s, 52.91%±32.51%, 0.64×10-3 mm2/s (0.05×10-3 mm2/s, 1.41×10-3 mm2/s) , 48.20%(3.03%, 16.95%) of TRG 2 patients, and (0.38±0.35)×10-3 mm2/s, 21.94%±19.08%, 0.26×10-3 mm2/s (-1.43×10-3 mm2/s, 0.81×10-3 mm2/s), 20.18%(-58.61%, 77.14%) of TRG 3 patients, respectively, with significant differences between two groups (t=3.09, 3.41, Z=-3.04, -2.93, P<0.05). (3) Predictive efficacy of change value and change rate of IVIM imaging parameters before and after neoadjuvant chemotherapy for TRG: ROC curve analysis showed that ADC change value exhibited an area under curve (AUC) of 0.798, a sensitivity of 66.7% and a specificity of 94.4% in predicting TRG, when 0.86×10-3 mm2/s was used as the cut-off value. With 43.3% as the cut-off value, ADC change rate had an AUC of 0.793, a sensitivity of 66.7% and a specificity of 88.9% in predicting TRG. With 0.35×10-3 mm2/s as the cut-off value, D change value had an AUC of 0.809, a sensitivity of 73.3% and a specificity of 77.8% in predicting TRG. With 25.9% as the cut-off value, D change rate had an AUC of 0.800, a sensitivity of 80.0% and a specificity of 72.2% in predicting TRG. Conclusions The change value and change rate of ADC and D values before and after neoadjuvant chemotherapy are potential predictors of pathologic response in ESCC. The significantly increased ADC and D values after neoadjuvant chemotherapy are prone to good pathologic response. The change value and change rate of D values show a better predictive value for pathologic response to neoadjuvant chemotherapy in ESCC compared with those of ADC values. Key words: Esophageal neoplasms; Esophageal cancer; Magnetic resonance imaging; Intravoxel incoherent motion; Diffusion weighted imaging; Neoadjuvant chemotherapy

Lymphopenia in Esophageal Squamous Cell Carcinoma: Relationship to Malnutrition, Various Disease Parameters, and Response to Concurrent Chemoradiotherapy.

Lymphopenia occurs commonly in esophageal squamous cell carcinoma (ESCC) and may influence treatment outcomes. We aimed to examine its association with treatment response and tumor progression in patients with locally advanced ESCC treated with concurrent chemoradiotherapy (CCRT). A total of 286 patients with stage II-IVa ESCC treated with CCRT between 2015 and 2017 were analyzed. Total lymphocyte counts were assessed at baseline, weekly, and 4 weeks after CCRT. Pretreatment lymphopenia was defined as total lymphocyte count <1,000 cells per mm3 at diagnosis, and treatment-related lymphopenia was defined as total lymphocyte count <200 cells per mm3 with 6 weeks after starting CCRT. Univariate and multivariate logistic regression methods were used to analyze factors associated treatment-related lymphopenia and treatment response. Lymphopenia was observed in 44 patients (15.4%) at initial diagnosis. Pretreatment lymphopenia was significantly associated with greater tumor length, worse T status, body mass index ≤18.5 kg/m2, and weight loss ≥3 kg in the previous 3 months. Six weeks after starting CCRT, 89 patients (31%) developed treatment-related lymphopenia. Tumor progression and cancer-related death were more frequently observed in treatment-related lymphopenia group than those without (76.4% vs. 52.8% and 58.4% vs. 39.6%). A complete response (CR) was achieved in 62 patients (21.7%). In multivariate analysis, treatment-related lymphopenia was significantly associated with lack of clinical CR, and older age, lower tumor location, greater tumor length, and larger planning target volume were independent predictors of treatment-related lymphopenia. Treatment-related lymphopenia during CCRT is an independent predictor for poor treatment response in ESCC. A total of 286 patients with locally advanced esophageal squamous cell carcinoma were treated with concurrent chemoradiotherapy (CCRT), and treatment-related lymphopenia occurred in 31% of patients within 6 weeks from the start of CCRT. Treatment-related lymphopenia was significantly associated with lack of treatment response, and older age, lower tumor location, greater tumor length, and larger planning target volume were independent predictors of treatment-related lymphopenia. Lymphocyte count is an inexpensive biomarker that may be easily used by clinicians to identify patients who are most likely to benefit from CCRT.

Transcriptional factor regulation network and competitive endogenous RNA (ceRNA) network determining response of esophageal squamous cell carcinomas to neoadjuvant chemoradiotherapy.

BackgroundNeoadjuvant chemoradiotherapy (nCRT) followed by surgery benefits survival for patients with esophageal squamous cell carcinomas (ESCC) compared with surgery alone, but the clinical outcomes of nCRT are heterogeneous. This study aimed to elucidate transcriptional factor (TF) regulation network and competitive endogenous RNA (ceRNA) network determining response of ESCC to nCRT.Materials and MethodsRNA microarray data of GSE59974 and GSE45670 were analyzed to investigate the significant changes of lincRNAs, miRNAs, mRNAs in responders and non-responders of nCRT in ESCC. Functional and enrichment analyses were conducted by clusterProfiler. The target lincRNAs and mRNAs of miRNAs were predicted by miRWalk. The ceRNA and TF regulatory networks were constructed using Cytoscape.ResultsDifferentially expressed genes between responders and non-responders mainly enriched in biological process including Wnt signaling pathway and regulation of cell development and morphogenesis involved in differentiation. Besides, these genes showed enrichment in molecular function of glycosaminoglycan binding, metalloendopeptidase inhibitor and growth factor activity. KEGG analysis enriched these genes in pathways of neurotrophin signaling pathway, cell adhesion molecules and Wnt signaling pathway. We also constructed ceRNA network and TF network regulating response of ESCC to nCRT. Core regulatory miRNAs were miR-520a, miR-548am, miR-3184, miR-548d, miR-4725, miR-148a, miR-4659a and key regulatory TFs included MBNL1, SLC26A3, BMP4, ZIC1 and ANKRD7.ConclusionWe identified significantly altered lincRNAs, miRNAs and mRNAs involved in the nCRT response of ESCC. In addition, the ceRNA regulatory network of lincRNA-miRNA-mRNA and TF regulatory network were constructed, which would elucidate novel molecular mechanisms determining nCRT response of ESCC, thus providing promising clues for clinical therapy.

High mobility group box 1 promotes radioresistance in esophageal squamous cell carcinoma cell lines by modulating autophagy

Resistance to radiotherapy results in relapse and treatment failure in locally advanced esophageal squamous cell carcinoma (ESCC). High mobility group box 1 (HMGB1) is reported to be associated with the radioresistance in bladder and breast cancer. However, the role of HMGB1 in the radiotherapy response in ESCC has not been fully elucidated. Here, we investigated the role of HMGB1 to radioresistance in ESCC clinical samples and cell lines. We found that HMGB1 expression was associated with tumor recurrence after postoperative radiotherapy in locally advanced ESCC patients. HMGB1 knockdown in ESCC cells resulted in increased radiosensitivity both in vitro and in vivo. Autophagy level was found depressed in HMGB1 inhibition cells and activation of autophagy brought back cell’s radioresistance. Our results demonstrate that HMGB1 activate autophagy and consequently promote radioresistance. HMGB1 may be used as a predictor of poor response to radiotherapy in ESCC patients. Our finding also highlights the importance of the utility of HMGB1 in ESCC radiosensitization.

DW-MRI for esophageal squamous cell carcinoma, correlations between ADC values with histologic differentiation and VEGF expression: A retrospective study.

The aim of the present study was to assess the correlations between diffusion-weighted magnetic resonance imaging (DW-MRI) features with the histologic differentiation and the expression of vascular endothelial growth factor (VEGF) in esophageal squamous cell carcinoma (ESCC). A total of 52 patients with ESCC included in the present study received radiotherapy, and all patients underwent contrast enhanced MRI and DW-MRI prior to and following radiotherapy. The diffusion sensitivity coefficient (b value) was set as 800 s/mm2. Apparent diffusion coefficient (ADC) values were automatically computed. VEGF expression was evaluated by immunohistochemical staining. The results demonstrated that the pathological grading of ESCC was positively correlated with ADC values (r=0.635, P=0.0007), and the VEGF expression was inversely correlated with ADC values (r=−0.321, P=0.008). However, no correlation was identified between the pathological grading and the VEGF expression (r=0.178, P=0.284). All patients were categorized as complete response (CR) or partial response (PR) and the ADC values were increased significantly following radiotherapy. The mean ADC values in the CR group were higher than the PR group prior to radiotherapy (t=5.156, P=0.0004). Therefore, we concluded that the DWI with ADC value measurement may represent the grade of tumor histologic differentiation and the degree of VEGF expression, and may also serve as a useful marker to predict radiotherapy and anti-VEGF response in ESCC. ADC value may be a substitution for assessing tumor angiogenesis and novel prognostic factor and contribute to the treatment of ESCC.

Identification of a serum-based miRNA signature for response of esophageal squamous cell carcinoma to neoadjuvant chemotherapy

BackgroundNeoadjuvant chemotherapy (NAC) has become the standard of care for resectable esophageal squamous cell carcinoma (ESCC) which is one of the most lethal cancers, to improve resectability and prognosis. On this basis, to provide individually optimized therapy for ESCC, a minimally-invasive biomarker for response to NAC is strongly desired. This study aimed to identify the miRNA signature in serum specimens taken from ESCC patients undergoing NAC through genome-wide microarray technology.MethodsComprehensive miRNA-expression profiles of serum specimens from ESCC patients before initial treatment were analyzed using microarray. A qPCR assay was performed to test the robustness of identified serum-based miRNA signature for discriminating response to NAC with serum specimens taken from 100 ESCC cases undergoing NAC.ResultsWe prioritized 62 miRNAs differentially expressed between responders and non-responders (absolute log2 fold change > 1.0, corresponding P < 0.05) and from the 62 miRNAs, we selected the miR-23a-5p, miR-193b-5p, and miR-873-3p, which were highly expressed in non-responders. Following qPCR analysis indicated the expression of miR-193b-5p and miR-873-3p in serum specimens were significantly higher in non-responders among three selected miRNAs (P = 0.004 and 0.001, respectively). Subsequently, we developed 2-miR-model (miR-193b-5p and miR-873-3p), 3-miR-model, and 2-miR + lymphatic invasion (ly) model based on logistic regression analysis, which achieved the better area under the receiver operating characteristic curves than those of single miRNAs as 2-miR-model, 0.70 (95% CI 0.57 to 0.82); 3-miR-model, 0.70 (95% CI 0.57 to 0.83); and 2-miR + ly, 0.73 (95% CI 0.60–0.86), respectively. Furthermore, we compared the detective power of the combined model: 2-miR + ly for discriminating non-responders to NAC, to other pretreatment clinical features. Consequently, 2-miR + ly model was superior to serum SCC antigen with great significance (P = 0.01) and to ly, and clinical T stage with marginal significance (P = 0.18, 0.07, respectively).ConclusionsCollectively, we demonstrated that the potential of a multi-miRNA biomarker for identifying NAC response in ESCC is realistic, and can be used in the clinic with the further validation.

LINC00657 played oncogenic roles in esophageal squamous cell carcinoma by targeting miR-615-3p and JunB.

The prognosis of esophageal squamous cell carcinoma (ESCC) is relatively poor due to the absence of efficient treatment. In this manuscript, we have investigated the specific roles and molecular mechanisms of LINC00657 to order to identify novel therapeutic targets for ESCC. The LINC00657 expression in ESCC tissues and cell lines were evaluated by quantitative real-time PCR. The expression of LINC00657 in ESCC cells was regulated by lentivirus transfection. Online bioinformatics analysis tools were used to predict the potential targets of LINC00657 and miR-615-3p. TCGA database was used to analyze the prognosis of ESCC patients. Transwell, wound healing assay and MTT were performed to investigate the ESCC cells' biological functions. JunB expression was evaluated by Western blot. LINC00657 was moderately increased in ESCC both in vivo and in vitro and up regulated by irradiation. LINC00657 knockdown could inhibit the migration and proliferation of ESCC cells. And downregulation of LINC00657 significantly enhanced the radio-sensitivity. Moreover, LINC00657 could act as a ceRNA to increase the expression of JunB by binding to miR-615-3p. Meanwhile, overexpression of miR-615-3p resulted in anti-tumor effects and led to the down-regulation of JunB. Survival analysis from TCGA indicated that ESCC patients with higher JunB expression had significant poorer prognosis. LINC00657 might be involved in regulating ESCC's response to radiation; and it functioned as an oncogene in ESCC by targeting miR-615-3p and JunB, providing novel potential therapeutic targets.

A subset of esophageal squamous cell carcinoma patient-derived xenografts respond to cetuximab, which is predicted by high EGFR expression and amplification.

Epidermal growth factor receptor (EGFR) is reportedly overexpressed in most esophageal tumors, but most targeted therapies showed no efficacy in non-selected patients. This study aims at investigating the adaptive cetuximab subset in a cohort of esophageal squamous cell carcinoma (ESCC) patient-derived xenografts (PDXs). A large panel of ESCC PDXs has been established. The copy number, mRNA expression and immunohistochemistry (IHC) of key EGFR pathways have been examined along with cetuximab response. A preclinical trial on a randomly selected cohort of 16 ESCC PDXs was conducted, and the genomic annotations of these models were compared against the efficacy readout of the mouse trial. The trial identified that 7 of 16 (43.8%) responded to cetuximab (ΔT/ΔC <0 as responders). The gene amplification and expression analysis indicated that EGFR copy number ≥5 (P=0.035), high EGFR mRNA expression (P=0.001) and IHC score of 2-3 (P=0.034) are associated with tumor growth inhibition by cetuximab, suggesting EGFR may function as a single predictive biomarker for cetuximab response in ESCC. Overall, our results suggest that an ESCC subtype with EGFR amplification and overexpression benefits from cetuximab treatment, which warrants further clinical confirmation.

Abstract 3419: Genomic characterization of mutation hotspots related to chemotherapy responses in esophageal squamous-cell carcinoma with archived formalin-fixed paraffin-embedded tissues

Abstract Introduction: Currently neoadjuvant chemoradiation therapy (CRT) prior to surgery is the most common treatment for Esophageal Squamous-cell Carcinoma (ESCC) patients. Given the fact that traditional TNM staging of the patients before treatment could not reflect their overall survival, a recent study conducted in Hong Kong demonstrated that the percentage of viable tumor cells remaining in the primary tumor after the CRT therapy was a prognostic marker for overall survival. Clinicians defined patients with no viable tumor cells (0%) as good responders, while the others having greater than 50% tumor cells remaining (50-100%) were considered poor responders. They observed that good responders show significantly better five-year survival rate. However, little is known about the mutation profiles between good and poor responders stratified in this way. Methods: Taking advantage of archived formalin-fixed paraffin-embedded (FFPE) tissues with comprehensive clinical outcomes, I apply a novel approach to systematically screen for possible somatic mutations that are associated with patients' responses to CRT in ESCC. I obtained 100 archived FFPE blocks from Queen Mary Hospital, Hong Kong, 50 each representing either good responders or poor responders. DNA libraries were constructed to target 131 cancer-related genes with TOMA Os-seq, which adopted single-stranded ligation to minimize the loss of input FFPE DNA. Then, libraries were sequenced on Illumina HiSeq X Ten. The final list of somatic mutations was filtered with in-house pipeline, excluding both germline variants from public databases and in-house Hong Kong database. Copy number variations (CNVs) were also identified with a pre-designed control sample in the same sequencing batch. Results: One test run on samples with good quality DNA showed high sensitivity and specificity up to 100% for pre-designed mutations. For CNV, the sensitivity was 87.5% and the specificity was 97.5%. Trials of sequencing with several ESCC FFPE samples revealed deleterious mutations from genes including TP53, BRCA2, and those mutations were successfully validated by Sanger sequencing. Groups of critical protein-altered mutations and CNVs that distinguish good and poor responders of ESCC will be presented in detail. Conclusion: In all, FFPE blocks are valuable sources to uncover the biomarkers for CRT response in ESCC. I expect those biomarkers can aid future evaluation of newly diagnostic patients to avoid unnecessary efforts in treating patients that are unsuitable for the traditional therapy and thus facilitate effective treatments. Acknowledgements: Theme-based Research Scheme grant provided by the Hong Kong Research Grants Council (T12-701/17-R to MLL). Citation Format: Chen Guo, Josephine Ko, Wei Dai, Lihua Tao, Simon Law, Anthony Wing Ip Lo, Maria Li Lung. Genomic characterization of mutation hotspots related to chemotherapy responses in esophageal squamous-cell carcinoma with archived formalin-fixed paraffin-embedded tissues [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3419.

The mutational landscape of circulating cell free DNA in patients with esophageal squamous cell carcinoma response and non-response to neoadjuvant chemotherapy.

e16082Background: This study was designed to explore the plasma cell free DNA (cfDNA) mutation profile in the patients with esophageal squamous cell carcinoma (ESCC) response and non-response to ne...

MicroRNA expression profiling for the prediction of resistance to neoadjuvant radiochemotherapy in squamous cell carcinoma of the esophagus

BackgroundMicroRNAs (miRNAs) play an important role in cancer biology. Neoadjuvant radiochemotherapy followed by surgery is a standard treatment for locally advanced esophageal squamous cell carcinoma (ESCC). However, a subset of patients do not respond. We evaluated whether miRNA profiles can predict resistance to radiochemotherapy.MethodsFormalin-fixed, paraffin-embedded pretherapeutic biopsies of patients treated by radiochemotherapy followed by esophagectomy were analyzed. The response was determined by histopathological tumor regression grading. miRNA profiling was performed by microarray analysis (Agilent platform) in 16 non-responders and 15 responders. Differentially expressed miRNAs were confirmed by real-time quantitative PCR (qRT-PCR) in an expanded cohort of 53 cases.ResultsThe miRNA profiles within and between non-responders and responders were highly similar (r = 0.96, 0.94 and 0.95). However, 12 miRNAs were differentially expressed (> twofold; p ≤ 0.025): non-responders showed upregulation of hsa-miR-1323, hsa-miR-3678-3p, hsv2-miR-H7-3p, hsa-miR-194*, hsa-miR-3152, kshv-miR-K12-4-3p, hsa-miR-665 and hsa-miR-3659 and downregulation of hsa-miR-126*, hsa-miR-484, hsa-miR-330-3p and hsa-miR-3653. qRT-PCR analysis confirmed the microarray findings for hsa-miR-194* and hsa-miR-665 (p < 0.001 each) with AUC values of 0.811 (95% CI 0.694–0.927) and 0.817 (95% CI 0.704–0.930), respectively, in ROC analysis.ConclusionsOur results indicate that miRNAs are involved in the therapeutic response in ESCC and suggest that miRNA profiles could facilitate pretherapeutic patient selection.

Effects of neutropenia and histological responses in esophageal squamous cell carcinoma with neo-adjuvant chemotherapy.

Neo-adjuvant chemotherapy (NAC) followed by radical esophagectomy has been shown to prolong survival in patients with locally advanced esophageal squamous cell carcinoma (ESCC). However, neutropenia, one of the major adverse events due to NAC, influences the therapeutic course. The aim of this study is to clarify the relationship between neutropenia and therapeutic response in ESCC with NAC. A total of 117 patients with clinical stage II/III ESCC who had undergone NAC followed by radical esophagectomy were retrospectively analyzed in terms of the relationship between neutropenia and clinicopathological features or outcomes. Neutropenia was the major adverse event observed in 56 % (66/117) and grade 3/4 neutropenia occurred in 29 % of patients. Grade 3/4 neutropenia correlated with a high histological response (Grade 1b-3) (p < 0.01). Correlative analysis identified grade 3/4 neutropenia and poor differentiation as independent predictors of a high histological response (odds ratio 5.13 and 3.25, p < 0.01 and p = 0.01, respectively). Survival analysis showed that patients with a high histological response had significantly longer survival than those with a low histological response (Grade 0-1a) (p = 0.03), whereas no significant differences were found for survival according to the grade of neutropenia (p = 0.45). In a subgroup analysis according to histological response, grade 3/4 neutropenia correlated with worse survival in patients with a low histological response (p = 0.05). Severe neutropenia due to NAC correlates with a high histological response in ESCC. However, severe neutropenia may also result in a worse prognosis for patients with a low histological response.