Survival In ESCC Patients Research Articles

Abstract 6705: Molecular detection of circulating cancer cells with cancer stem cell or mesenchymal characteristics in esophageal squamous cell carcinoma

Abstract Esophageal squamous cell carcinoma (ESCC) is a clinically challenging disease that requires a multidisciplinary approach. Unfortunately, the scarcity of ESCC genomic data hinders the understanding of ESCC biology, disease progression and rational therapy design. Circulating tumor cells (CTCs), which are shed from the primary and metastatic tumors and then circulate within the peripheral vasculature, reflect the existing tumor heterogeneity. Here, we utilized the non-biased size-based CTC enrichment strategy in combination with the real-time RT-PCR to molecularly characterize the CTCs with cancer stem cell (CSC) or mesenchymal properties in ESCC. Comprehensive data from ESCC cell lines, mouse ESCC xenograft models and clinical ESCC peripheral blood samples emphasize the importance of TWIST1 (Twist Family BHLH Transcription Factor 1) in ESCC progression. Gain-of-function and loss-of-function analyses demonstrate that TWIST1 promotes cell migration, invasion and colony formation in ESCC cells. Moreover, positive TWIST1 expression is responsible for the chemoresistance of ESCC cells to Cisplatin and is correlated with poor overall survival in ESCC patients. In addition, we show that TWIST1 promotes malignant potential, including tumor growth, invasion and chemoresistance via the TWIST1-TGFBI-ZEB1 signaling pathway in ESCC. Meanwhile, the TWIST1-TGFBI-ZEB1 signaling pathway confers immunosuppressive conditions to the tumor microenvironment, which in turn contributes to EMT to promote tumor progression. Collectively, these findings indicate that TWIST1 has a novel role in ESCC carcinogenesis by regulating tumorigenicity and cancer stem cell properties that may be useful as a prognostic monitoring biomarker and a promising therapeutic target in clinical ESCC treatment. Citation Format: Zhen Tan, Josephine Mun-Yee Ko, Valen Zhuoyou Yu, Hoi Yan Ng, Simon Law, Maria Li Lung. Molecular detection of circulating cancer cells with cancer stem cell or mesenchymal characteristics in esophageal squamous cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6705.

CircCYP24A1 facilitates esophageal squamous cell carcinoma progression through binding PKM2 to regulate NF-κB-induced CCL5 secretion

BackgroundEsophageal squamous cell carcinoma (ESCC) is a common gastrointestinal malignant tumor, while the molecular mechanisms have not been fully elucidated. Multiple circular RNAs have been reported to involve in the onset and progression of malignant tumors through various molecular mechanisms. However, the clinical significance and functional mechanism of most circRNAs involved in the progression of ESCC remains obscure.MethodsRNA-Seq was used to explore potential circRNAs in participated in 5 pairs of ESCC and their corresponding normal esophageal tissues. The up-regulated circCYP24A1 was selected. Fluorescence in situ hybridization was cunducted to verificated the expression and intracellular localization of circCYP24A1 by using the tissue microarray. The Kaplan–Meier method and Cox proportional hazards model was used to examine the potential prognostic value of circCYP24A1 on overall survival of ESCC patients. The biological function were confirmed by gain- and loss-of-function approaches in vivo. mRNA expression profile microarray was proformed to investigate the downstream signaling pathways involved in circCYP24A1. RNA pull-down assay and mass spectrometry were performed to identify the proteins associated with circCYP24A1. Rescue experiments were carried out to identified hypothetical regulatory role of circCYP24A1 on ESCC progression in vivo and in virto.ResultsIn this study, we identified circCYP24A1 in ESCC tissues by RNA sequencing, which is up-regulated in 114 cases of ESCC tissues and acts as a novel prognosis-related factor. Moreover, circCYP24A1 promoted the ability of proliferation, migration, invasion and clone formation in vitro, as well as tumor growth in vivo. Mechanistically, chemokine (C-Cmotif) ligand 5 (CCL5) is functional downstream mediator for circCYP24A1, which is screened by mRNA microarray. Moreover, circCYP24A1 physically interacts with M2 isoform of pyruvate kinase (PKM2). Rescue experiments showed that PKM2 knockdown partly reverses the promotional effects of circCYP24A1. It was revealed that circCYP24A1 increases secretion of CCL5 through the mechanism mainly by interacting with PKM2, an activator of NF-κB pathway, and thereby accelerate malignant progression of ESCC.ConclusionsUp-regulated circCYP24A1 could activate NF-κB pathway by binding PKM2, which promotes the secretion of CCL5 and accelerate malignant progression of ESCC. Our fndings recommended a novel function for circCYP24A1 as a potential effective biomarker for judging prognosis and a therapeutic target in ESCC.

DEPDC1B collaborates with GABRD to regulate ESCC progression

BackgroundEsophageal squamous cell carcinoma (ESCC) is the leading cause of cancer-related death worldwide with a poor prognosis. Given that DEPDC1B plays a key role in multiple cancers, the role of this molecule in ESCC was explored to identify potential targets for ESCC patients.MethodThe expression level of DEPDC1B in ESCC was revealed based on the TCGA database and immunohistochemical experiments on clinical tissues. The correlation between DEPDC1B and survival of ESCC patients was analyzed by Kaplan–Meier method. Small hairpin RNA (shRNA)-mediated silencing of DEPDC1B expression in ESCC cells and performed a series of in vitro and in vivo functional validations.ResultDEPDC1B was overexpressed in ESCC. High expression of DEPDC1B was significantly negatively correlated with overall survival in patients with ESCC. Moreover, knockdown of DEPDC1B inhibited ESCC cell proliferation, clone formation, migration, tumor formation and promoted apoptosis. Furthermore, knockdown of DEPDC1B leaded to significant downregulation of GABRD in ESCC cells. Meanwhile, GABRD expression was upregulated in ESCC, and its silencing can inhibit the proliferation and migration of the tumor cells. Interestingly, there was a protein interaction between DEPDC1B and GABRD. Functionally, GABRD knockdown partially reversed the contribution of DEPDC1B to ESCC progression. In addition, GABRD regulated ESCC progression may depend on PI3K/AKT/mTOR signaling pathway.ConclusionDEPDC1B collaborated with GABRD to regulate ESCC progression, and inhibition of this signaling axis may be a potential therapeutic target for ESCC.

Fusobacterium nucleatum induces MDSCs enrichment via activation the NLRP3 inflammosome in ESCC cells, leading to cisplatin resistance

Background To analyse the regulatory effect of Fusobacterium nucleatum (Fn) on NOD-like receptor protein 3 (NLRP3) and myeloid-derived suppressor cells (MDSCs) in oesophageal squamous cell carcinoma (ESCC) as well as its effect on cisplatin (CDDP) therapy and to explore its clinical significance. Methods Fn infection, NLRP3 expression and MDSCs infiltration in ESCC tissues were detected by RNAscope and immunohistochemistry (IHC). The correlation between these three factors and the clinicopathological features and survival of ESCC patients was analysed. A coculture system of human peripheral blood monocytes (PBMCs) and ESCC cells was established to simulate the tumour microenvironment. In vitro and in vivo models were used to analyse the effects of Fn on the percentage of MDSCs in the coculture system and the NLRP3 expression level and CDDP sensitivity of ESCC cells. Results Fn infection was consistent with high NLRP3 expression and MDSCs enrichment in ESCC tissues. Moreover, the survival time of ESCC patients was significantly shortened under Fn infection, high NLRP3 expression and MDSCs enrichment. In the in vitro and in vivo models, Fn induced abundant enrichment of MDSCs by inducing high expression of NLRP3 in ESCC cells and reducing the sensitivity of ESCC cells to CDDP. Conclusions Fn infection can induce high expression of NLRP3 in ESCC, lead to MDSCs enrichment, weaken the body's antitumour immunity, and lead to CDDP treatment resistance. The effective elimination of Fn and the inhibition of MDSCs enrichment may provide new strategies and treatments for ESCC. Highlights The survival of ESCC patients with Fn infection, high NLRP3 expression and MDSCs enrichment was significantly shortened. Fn infection could cause CDDP resistance in ESCC. Fn could induce the enrichment of MDSCs in the tumour microenvironment by activating NLRP3 in ESCC cells.

Low expression of LncRNA-CAF attributed to the high expression of HIF1A in esophageal squamous cell carcinoma and gastric cancer patients.

Cancer-associated fibroblasts (CAFs) are major components of tumor microenvironment that stimulate ESCC and GC progression. The LncRNA-CAF, FLJ22447, is located in the vicinity of HIF1A, while their association remains unclear. This study aims to assess the FLJ22447 expression in the ESCC and GC patients and evaluate its association with the HIF1A gene. Fresh ESCC and GC tumor samples and their adjacent non-tumor tissues were collected from patients who underwent surgery in Imam Khomeini Hospital, Tehran, Iran. The expression of FLJ22447, HIF1A, and VEGF was evaluated using qRT-PCR test. The association of their expression with tumor clinicopathological features in ESCC patients was assessed. System biology tools were then applied for the possible biological subsequences of the FLJ22447. A significant reduction in FLJ22447 expression was observed in ESCC and GC tissues than adjacent non-tumor tissues, while, the expression of HIF1A and VEGF were increased. Low expression of FLJ22447 was significantly correlated with HIF1A (P = 2.4e-73, R = 0.63) and VEGF (P = 0.00019, R = 0.15) expression. A significant relationship was detected between the high expression of HIF1A and tumor stages (I-II) and it was related to the reduced survival of ESCC patients. Conversely, increased VEGF expression was linked to the advanced stages (III-IV) and metastasis in ESCC. The analysis of FLJ22447-interacted proteins showed that MYC, JUN, SMRCA4, PPARG, AR, FOS, and CEBPA are the hub genes. These proteins were implicated in the cancer related pathways. Among them, SPI1, E2F1, TCF7L2, and STAT1 were significantly expressed in esophageal and gastric cancers that were functionally involved in the proliferation, apoptosis, and angiogenesis pathways in cancer. The results suggested that FLJ22447 may have a regulatory function on the HIF1A expression. We identified the FLJ22447-interacted proteins and their molecular function in cancer pathogenesis. Further research emphasis is to realize the association of FLJ22447 with its protein partners in progression of cancer. These may provide an insight into the FLJ22447 activity that could introduce it as a potential value in tumor gene therapy.

Development and Validation of a Prognostic Gene Signature Correlated With M2 Macrophage Infiltration in Esophageal Squamous Cell Carcinoma.

BackgroundEsophageal squamous cell carcinoma (ESCC) is the most common type of esophageal cancer and the seventh most prevalent cause of cancer-related death worldwide. Tumor microenvironment (TME) has been confirmed to play an crucial role in ESCC progression, prognosis, and the response to immunotherapy. There is a need for predictive biomarkers of TME-related processes to better prognosticate ESCC outcomes.AimTo identify a novel gene signature linked with the TME to predict the prognosis of ESCC.MethodsWe calculated the immune/stromal scores of 95 ESCC samples from The Cancer Genome Atlas (TCGA) using the ESTIMATE algorithm, and identified differentially expressed genes (DEGs) between high and low immune/stromal score patients. The key prognostic genes were further analyzed by the intersection of protein–protein interaction (PPI) networks and univariate Cox regression analysis. Finally, a risk score model was constructed using multivariate Cox regression analysis. We evaluated the associations between the risk score model and immune infiltration via the CIBERSORT algorithm. Moreover, we validated the signature using the Gene Expression Omnibus (GEO) database. Within the ten gene signature, five rarely reported genes were further validated with quantitative real time polymerase chain reaction (qRT-PCR) using an ESCC tissue cDNA microarray.ResultsA total of 133 up-regulated genes were identified as DEGs. Ten prognostic genes were selected based on intersection analysis of univariate COX regression analysis and PPI, and consisted of C1QA, C1QB, C1QC, CD86, C3AR1, CSF1R, ITGB2, LCP2, SPI1, and TYROBP (HR>1, p<0.05). The expression of 9 of these genes in the tumor samples were significantly higher compared to matched adjacent normal tissue based on the GEO database (p<0.05). Next, we assessed the ability of the ten-gene signature to predict the overall survival of ESCC patients, and found that the high-risk group had significantly poorer outcomes compared to the low-risk group using univariate and multivariate analyses in the TCGA and GEO cohorts (HR=2.104, 95% confidence interval:1.343-3.295, p=0.001; HR=1.6915, 95% confidence interval:1.053-2.717, p=0.0297). Additionally, receiver operating characteristic (ROC) curve analysis demonstrated a relatively sensitive and specific profile for the signature (1-, 2-, 3-year AUC=0.672, 0.854, 0.81). To identify the basis for these differences in the TME, we performed correlation analyses and found a significant positive correlation with M1 and M2 macrophages and CD8+ T cells, as well as a strong correlation to M2 macrophage surface markers. A nomogram based on the risk score and select clinicopathologic characteristics was constructed to predict overall survival of ESCC patients. For validation, qRT-PCR of an ESCC patient cDNA microarray was performed, and demonstrated that C1QA, C3AR1, LCP2, SPI1, and TYROBP were up-regulated in tumor samples and predict poor prognosis.ConclusionThis study established and validated a novel 10-gene signature linked with M2 macrophages and poor prognosis in ESCC patients. Importantly, we identified C1QA, C3AR1, LCP2, SPI1, and TYROBP as novel M2 macrophage-correlated survival biomarkers. These findings may identify potential targets for therapy in ESCC patients.

PRDM5 suppresses oesophageal squamous carcinoma cells and modulates 14-3-3zeta/Akt signalling pathway.

Dysregulation of PR (PRDI-BF1 and RIZ) domain protein 5 (PRDM5) expression has been shown to be associated with the progression of many malignancies. Nevertheless, the role and underlying mechanism of PRDM5 in oesophageal squamous cell carcinoma (ESCC) remain elusive. qRT-PCR was performed to analyze PRDM5 mRNA expression, and western blot was used to determine protein expression of PRDM5, MMP-2, MMP-9, 14-3-3zeta, pan-Akt and phosphorylated Akt expression. CCK-8 staining was employed to evaluate cell proliferation, while wound scratch assay and Transwell assay were carried out to detect cell migration. A tumour xenograft model of ESCC was also established to validate the effect of PRDM5. PRDM5 expression was downregulated in ESCC tissues and positively correlated with the overall survival of ESCC patients. Silencing PRDM5 expression promoted cell proliferation in ESCC cells, while overexpressing PRDM5 inhibited cell proliferation. Moreover, the migratory abilities of ESCC cells were promoted by PRDM5 knockdown but were attenuated by PRDM5 overexpression. Importantly, 14-3-3zeta expression, along with the phosphorylation of Akt, was suppressed by PRDM5 in ESCC cells. In the established tumour xenograft model, PRDM5 regulated ESCC tumour growth as well as the expression of 14-3-3zeta and phosphorylation of Akt protein. In conclusion, PRDM5suppresses ESCC cell proliferation and migration and negatively regulates 14-3-3zeta/Akt signalling pathway in vitro and in vivo.

TGF-\u03b2-induced PLEK2 promotes metastasis and chemoresistance in oesophageal squamous cell carcinoma by regulating LCN2

Oesophageal squamous cell carcinoma (ESCC) has a relatively unfavourable prognosis due to metastasis and chemoresistance. Our previous research established a comprehensive ESCC database (GSE53625). After analysing data from TCGA database and GSE53625, we found that PLEK2 predicted poor prognosis in ESCC. Moreover, PLEK2 expression was also related to the overall survival of ESCC patients undergoing chemotherapy. Repression of PLEK2 decreased the proliferation, migration, invasion and chemoresistance of ESCC cells in vitro and decreased tumorigenicity and distant metastasis in vivo. Mechanistically, luciferase reporter assay and chromatin immunoprecipitation assay suggested that TGF-β stimulated the process that Smad2/3 binds to the promoter sequences of PLEK2 and induced its expression. RNA-seq suggested LCN2 might a key molecular regulated by PLEK2. LCN2 overexpression in PLEK2 knockdown ESCC cells reversed the effects of decreased migration and invasion. In addition, TGF-β induced the expression of LCN2, but the effect disappeared when PLEK2 was knockdown. Moreover, AKT was phosphorylated in all regulatory processes. This study detected the major role of PLEK2 in driving metastasis and chemoresistance in ESCC by regulating LCN2, which indicates the potential use of PLEK2 as a biomarker to predict prognosis and as a therapeutic target for ESCC.

72 PHASE II ADJUVANT CANCER-SPECIFIC VACCINE THERAPY FOR ESOPHAGEAL CANCER PATIENTS CURATIVELY RESECTED AFTER PREOPERATIVE THERAPY WITH PATHOLOGICALLY POSITIVE NODES

Abstract Esophageal squamous cell carcinoma (ESCC) patients with pathologically positive nodes have a high risk for postoperative recurrence, despite curative resection after preoperative therapy. Subclinical micrometastases are an appropriate target for cancer vaccine. To elucidate the efficacy of adjuvant vaccine monotherapy using three HLA-A*24-restricted tumor-specific peptide antigens for ESCC, up-regulated lung cancer 10, cell division cycle associated 1 and KH domain-containing protein overexpressed in cancer 1, we conducted an exploratory prospective phase II clinical trial (UMIN000003557). Methods Patients with ESCC who underwent curative resection after preoperative therapy and were found pathologically positive lymph node metastasis were enrolled from December 2009 to September 2014 and allocated into the control and vaccine groups (CG and VG) based on the HLA-A*2402 positive or negative. One mg each of three epitope peptides mixed with 1 mL of Montanide ISA 51 VG was administered the first 10weekly injections followed by 10 additional biweekly injections to VG. No other adjuvant therapy was given until recurrence occurred. The primary endpoint was relapse-free survival (RFS) and the secondary endpoint was esophageal cancer-specific survival (ECSS). Results Thirty were in the CG and 33 in the VG. No significant difference was observed in RFS between the CG and VG (5-year RFS: 32.5% vs. 45.3%, p = 0.205 (one-sided)), but the recurrence rate significantly decreased with the number of peptides which induced antigen-specific cytotoxic T lymphocytes. The VG showed a significantly higher 5-year ECSS than the CG (60.0% vs. 32.4%, p = 0.045 (one-sided)) probably due to the better clinical responses in patients of the VG to the post-recurrence therapy and this difference was more prominent in patients with CD8+ and programmed death-ligand 1 double negative tumor (68.0% vs. 17.7%, p = 0.010 (two-sided)). Conclusion Our results suggested that the cancer peptide vaccine suppressed the postoperative recurrence, enhanced the post-recurrence therapy and improved the survival of ESCC patients, particularly in the cases without CD8 infiltration and PD-L1 expression. A phase III randomized controlled study has been conducted in response to these results, and the results are waiting to be published.

TGFBR3 is an independent unfavourable prognostic marker in oesophageal squamous cell cancer and is positively correlated with Ki-67.

The transforming growth factor beta(TGF-β) superfamily plays an important role in cancer development. One aspect of this is that the transforming growth factor beta receptor III (TGFBR3) is frequently overexpressed in some tumours. However, the role of TGFBR3 in oesophagealsquamous cell carcinoma (ESCC) has not been explored as yet. In this study, we aimed to determine the role of TGFBR3 in the development and prognosis of ESCC and the correlation between TGFBR3 expression and Ki-67 and p53. Immunohistochemistry was performed to investigate the expression of TGFBR3 in the tumour tissue microarray consisting of ESCC tissues and matched adjacent normal tissues (n=80). Only ESCC tissues (n=20) were also used in our analysis. The association between TGFBR3 expression and clinicopathological characteristics, such as Ki-67 and p53, was analysed by Spearman's rank correlation coefficient analysis. The association between TGFBR3 expression and prognosis of ESCC was analysed using Kaplan-Meier analysis and log-rank tests. The expression levels of TGFBR3 in oesophageal cancer tissues were markedly higher than in matched adjacent normal tissues. Furthermore, TGFBR3 overexpression was significantly associated with tumour-node-metastasis (TNM) stage, lymph node metastasis (N stage) and Ki-67 expression. However, TGFBR3 overexpression was not significantly related to age, sex or p53. In univariate analysis, overall survival of ESCC patients was significantly associated with high TGFBR3 expression, sex, T stage, N stage and TNM stage. Moreover, ESCC patients with high TGFBR3 expression had poorer overall survival than those with low TGFB R3 expression. Our findings showed that TGFBR3 was upregulated in the development of human ESCC and high TGFBR3 expression was associated with high expression of Ki-67 and poor prognosis of ESCC. Therefore, TGFBR3 may be a valuable prognostic marker and a novel therapeutic target for ESCC.

Decreased plasma riboflavin is associated with poor prognosis, invasion, and metastasis in esophageal squamous cell carcinoma.

Riboflavin deficiency confers a predisposition for esophageal cancer. The role of plasma riboflavin levels in development and prognosis of individuals with digestive tract inflammation and ulcer (DTIU), digestive tract polyps (DTPs), and ESCC is not well understood. We performed a cross-sectional study, including 177 DTIU, 80 DTP, and 324 ESCC cases, to measure the plasma riboflavin levels among the three populations. Correlation between plasma riboflavin levels (categorized as ≥31.8, 6.5-31.8 and ≤6.5 nmol/L groups) and clinical characteristics, as well as survival of ESCC patients (556 cases) was analyzed. There was no difference in plasma riboflavin levels between DTIU, DTP, and ESCC cases (P > 0.05). Plasma riboflavin levels were inversely correlated with invasive depth (correlation coefficient = -0.09, P = 0.026) and lymph node metastasis (correlation coefficient = -0.11, P = 0.010) of ESCC, and ESCC patients with low riboflavin levels had poor recurrence-free survival (P = 0.035) and overall survival (P = 0.003). Decreased riboflavin was a prognostic factor for poor overall survival (HR = 1.91, 95% CI = 1.19-3.07, P = 0.007). Plasma riboflavin levels in DTIU, DTP, and ESCC patients are similar. Plasma riboflavin levels are associated with the development and prognosis of ESCC.

Up-Regulation of MicroRNA-21 Indicates Poor Prognosis and Promotes Cell Proliferation in Esophageal Squamous Cell Carcinoma via Upregulation of lncRNA SNHG1.

IntroductionMicroRNA-21 (miRNA-21) and lncRNA SNHG1 (small nucleolar RNA host gene 1) are known to be aberrantly upregulated and promote tumor progression in various cancers. Nevertheless, very few studies have determined the roles of tissue and circulating miRNA-21 and SNHG1 in ESCC patients. Particularly, knowledge about the characteristics of miRNA-21 and SNHG1 expression and their correlations with survival rates, as well as their interaction with each other remains inadequate in ESCC.MethodsThse expression level of miRNA-21 and SNHG1 of tissues, serum and cell lines were detected by qRT-PCR, and the characteristics of their expression and clinicopathology were analyzed. Then, the diagnostic and prognosis value of serum and tissue miRNA-21 and SNHG1 were evaluated, respectively. In addition, the interaction with each other between miRNA-21 and SNHG1, as well as the effect on ESCC cell proliferation were further clarified.ResultsThe expression level of miRNA-21 and SNHG1 are significantly upregulated in tissues, serum and cell lines of ESCC, and tissue miRNA-21 and SNHG1 significantly correlates with lymph node metastasis, TNM stage, tumor size, and poor overall survival in ESCC patients. The receiver operating characteristic (ROC) curves show that areas under the ROC curve (AUC) for serum miRNA-21 and SNHG1 are 0.928 and 0.850, respectively. Pearson correlation coefficient indicated that the expression levels of miRNA-21 and SNHG1 in frozen cancerous tissues are significantly associated with their respective serum levels. Further, Cox univariate and multivariate analyses reveal that miRNA-21 and SNHG1 are independent prognostic factors for overall survival (OS) and disease-free survival (DFS) in ESCC patients. In addition, our in vitro data revealed a novel regulatory pathway, in which miRNA-21 is probably a unidirectional upstream positive regulator of SNHG1 in ESCC cells, and the interaction between miRNA-21 and SNHG1 plays an important role in the proliferation of ESCC cells.DiscussionIn summary, our data show that SNHG1 may be a novel downstream target of miRNA-21 and not vice versa in ESCC cells and contributes significantly toward the proliferation of ESCC cells. These findings suggest that miRNA-21 and SNHG1 may serve as potential diagnostic, prognostic biomarkers and therapeutic targets for ESCC patients.

MicroRNA-155 acts as a diagnostic and prognostic biomarker for oesophageal squamous cell carcinoma

MicroRNA-155 is over-expressed in many human cancers, but researches on its association with malignant oesophageal squamous cell carcinoma (ESCC) are limited. The aim of the present study was to evaluate the potential value of miR-155 as a biomarker for ESCC diagnosis and prognosis. In this study, we found that miR-155 was significantly increased in ESCC tissues compared with the paired adjacent tissues and healthy normal controls (p < .001), according to qRT-PCR, which suggested that miR-155 might act as an oncogene in ESCC. In addition, clinical features such as the depth of tumour invasion, tumour size, and TNM stage were all proved to impact the expression of miR-155 (p < .01). Then, ROC curve analysis, reaching an AUC of 0.870, and a sensitivity and specificity of 83.5% and 77.5%, respectively, revealed that miR-155 was a predictive factor for ESCC. As well, high expression of miR-155 was associated with poor overall survival of the patients (log-rank test, p = .004), according to Kaplan-Meier analysis. MiR-155 might be an independent predictor for overall survival in ESCC patients, manifested by Cox regression analysis (HR = 16.94, 95%CI = 3.33–86.12, p = .001). Taken together, miR-155 could be an independent diagnostic and prognostic biomarker for ESCC.

472P - Integration of expression rate and absolute cell counts of PD-1+ stromal tumour-infiltrating lymphocytes: Prognostic significance in esophageal squamous cell carcinoma

BackgroundEsophageal squamous cell carcinoma (ESCC) has a poor prognosis in Asian regions and the immunotherapy targeting PD-1 is on the stage of clinical trials. This study aims to investigate the prognostic effect of PD-1 expression in intratumoral and stromal tumor-infiltrating lymphocytes (TILs) on relapse and overall survival of ESCC patients. MethodsA retrospective cohort study was conducted with recruiting 142 ESCC patients who received surgical treatment. Intratumoral and stromal PD-1 expression was tested by immunohistochemistry (IHC). ResultsThe median follow-up time was 22 months and 21.1% patients were lost of follow-up. Cell counts and expression rate of intratumoral PD1+ TILs did not show any association with disease-free survival (DFS) and overall survival (OS). Expression rate of stromal PD1+ TILs did not have a significant relationship with DFS. The patients with expression rate of stromal PD1+ TILs >20% had the median OS being 19 months and the patients with expression rate ≤20% did not achieved median OS (p = 0.034). The adjusted HR of higher expression rate was 1.49 (95%CI 0.82, 2.60, p = 0.189) for OS. ESCC patients with ≤18 stromal PD1+ TILs/HPF had the median DFS being 10 months however the patients with >18 cells/HPF had the median DFS being 48 months (p = 0.037). The adjusted HR of > 18 stromal PD1+ TILs/HPF on DFS was 0.59 (95%CI 0.35, 1.01, p = 0.055). With reference to the patients of lower expression rate/higher cell counts of stromal PD1+ TILs, patients with lower expression rate/lower cell counts, higher expression rate/higher cell counts, and higher expression rate/lower cell counts, had the adjusted HR for DFS increased to 3.73, 3.36 and 3.99 (p for trend being 0.030) and the adjusted HRs for OS increased to 2.95, 3.64 and 3.82 (p for trend being 0.015), respectively. ConclusionsIntegration of expression rate and cell counts of stromal PD1+ TILs had a significant prognostic effect in terms of relapse and overall survival. Further studies are warranted to provide reference for immunotherapy. Legal entity responsible for the studyThe authors. FundingBeijing Hospitals Authority. DisclosureAll authors have declared no conflicts of interest.

Identification of hub genes and therapeutic drugs in esophageal squamous cell carcinoma based on integrated bioinformatics strategy

BackgroundEsophageal squamous cell carcinoma (ESCC) is one of leading malignant cancers of gastrointestinal tract worldwide. Until now, the involved mechanisms during the development of ESCC are largely unknown. This study aims to explore the driven-genes and biological pathways in ESCC.MethodsmRNA expression datasets of GSE29001, GSE20347, GSE100942, and GSE38129, containing 63 pairs of ESCC and non-tumor tissues data, were integrated and deeply analyzed. The bioinformatics approaches include identification of differentially expressed genes (DEGs) and hub genes, gene ontology (GO) terms analysis and biological pathway enrichment analysis, construction and analysis of protein–protein interaction (PPI) network, and miRNA–gene network construction. Subsequently, GEPIA2 database and qPCR assay were utilized to validate the expression of hub genes. DGIdb database was performed to search the candidate drugs for ESCC.ResultsFinally, 120 upregulated and 26 downregulated DEGs were identified. The functional enrichment of DEGs in ESCC were mainly correlated with cell cycle, DNA replication, deleted in colorectal cancer (DCC) mediated attractive signaling pathway, and Netrin-1 signaling pathway. The PPI network was constructed using STRING software with 146 nodes and 2392 edges. The most significant three modules in PPI were filtered and analyzed. Totally ten genes were selected and considered as the hub genes and nuclear division cycle 80 (NDC80) was closely related to the survival of ESCC patients. DGIdb database predicted 33 small molecules as the possible drugs for treating ESCC.ConclusionsIn summary, the data may provide new insights into ESCC pathogenesis and treatments. The candidate drugs may improve the efficiency of personalized therapy in future.

CD51 is an independent unfavorable prognostic factor in esophageal squamous cell carcinoma

BackgroundEsophageal squamous cell carcinoma (ESCC) is a common cancer in East Asia and some other parts of the world with a dismal prognosis. CD51 (integrin αv),a transmembrane glycoprotein responsible for cell-to-matrix binding has been found to enhance tumor progression. However, its expression and clinicopathological significance in ESCC tumors are not fully understood. The purpose of this study was to investigate the expression level of CD51 and to explore its clinicopathological significance in ESCC. MethodsThe expression of CD51 in 122 ESCC samples was examined by immunohistochemistry and its clinicopathological significance was evaluated. ResultsThe expression of CD51 was observed in tumor cell membrane and/or cytoplasm, with a positive rate of 48.36% (59/122). High expression of CD51 was significantly associated with lymph node metastasis (P = 0.031), tumor size (P = 0.028) and invasive depth (P = 0.027). Kaplan-Meier analysis revealed that positive expression of CD51 was correlated with poor overall survival of ESCC patients (P = 0.015). Multivariate analysis suggested that CD51 was an independent prognositic factor for ESCC (hazard ration = 1.604; 95% CI, 1.086–2.368; P = 0.017). ConclusionThese data suggested CD51 was a predictor for the prognosis of ESCC patients.

BMI-1 suppression increases the radiosensitivity of oesophageal carcinoma via the PI3K/Akt signaling pathway.

B-cell‑specific Moloney murine leukaemia virus integration site-1 (BMI-1) contributes to the growth of tumour cells post-irradiation (IR). The aim of the present study was to characterize the effects of BMI-1 on cell viability, radiosensitivity and its mechanisms of action in oesophageal squamous cell cancer (ESCC). Western blotting and immunohistochemistry were employed to evaluate the protein expression of BMI-1 in ESCC cells and specimens, respectively. Additionally, the protein expression levels of BMI-1, H2AK119ub and γH2AX in ESCC cells were detected following different doses of IR and at different times after IR. The protein expression levels of MDC1 and 53BP1 were also measured. Flow cytometry and MTT assays were used to determine cell cycle progression, apoptosis and cell viability. The phosphatidylinositol 3-kinase inhibitor LY294002 and the agonist IGF-1 were employed to suppress or induce the phosphorylation of Akt to determine whether BMI-1 induces radioresistance in ESCC cells via activation of the PI3K/Akt pathway. The expression of BMI-1 was higher in ESCC tissues and cells compared with that in normal oesophageal tissues and cells. In addition, BMI-1 was positively related to tumour size and lymph node metastases and negatively to the overall survival of ESCC patients. IR induced the expression of BMI-1, H2AK119ub and γH2AX in a dose-and time-dependent manner. BMI-1 knockdown lowered the expression of γH2AX, MDC1 and 53BP1, suppressed cell viability and increased radiosensitivity. G2/Mphase arrest was eliminated; this was followed by an increased proportion of cells entering the G0/G1phase after IR and BMI-1 knockdown via the upregulation of P16 and downregulation of cyclinD2 and cyclin-dependent kinase-4. Moreover, BMI-1 knockdown increased cell apoptosis, downregulated MCL-1 and p-Akt and upregulated Bax. Additionally, the inhibitory effect of the downregulation of p-Akt by LY294002 on tumour cell viability was identical to that of BMI-1 knockdown, while the kinase agonist IGF-1 reversed the effects of BMI-1 knockdown on cell viability and radiosensitivity. Taken together, BMI-1 knockdown induces radiosensitivity in ESCC and significantly inhibits cell viability, which may contribute to an increased proportion of cells in the G0/G1phase and cell apoptosis via suppression of the PI3K/Akt signalling pathway.

Abstract 942: Role of IL-8/CXCR2 network in the tumor cell proliferation of esophageal squamous cell carcinoma

Abstract Background: IL-8, a pro-inflammatory cytokine, and its receptor, CXCR2, are expressed invarious cancer cells. The IL-8/CXCR2 network is believed to be involved in angiogenesis, tumor cell proliferation and invasion. However, its role inesophageal squamous cell carcinoma (ESCC) remains unclear. In this study, we investigated the role of the IL-8/CXCR2 network in the tumor microenvironment of ESCC. Methods: IL-8 and CXCR2 expression was investigated immunohistochemically in 63 primary ESCCs with R0 resection but without any preoperative treatment. We evaluated the intensity of the staining in four grades; grades 3 (strong) and 2 (medium) were deemed “positive”, and 1 (weak) and 0 (negative) as “negative”. The concentration of IL-8 and CXCR2 were investigated using the human ESCC cell lines TE1, 4, 5, 6, 8, 9, 10, 11, 14 and 15. IL-8/CXCR2 signaling was stimulated by IL-8 addition, and suppressed by SB225002 (Cayman Chemical) treatment, a selective antagonist of CXCR2, and IL-8 specific siRNA. Stable IL-8 over-expressing TE4 cells were established, and cell proliferation assays were performed. To evaluate tumor growth in vivo, we subcutaneously inoculated 1.0×106 TE4 cells or transfectant into nude mice, and half of the transfectant-inoculated mice received 1mg/kg of SB225002 by intraperitoneal injection thrice weekly. The size and incidence of subcutaneous tumors were recorded. Result: Eight (12.7%) patients were positive for IL-8/CXCR2 network immunostaining. IL-8/CXCR2 network expression was significantly related to poor survival in ESCC patients (P&amp;lt;0.05). IL-8 concentration was high in TE4, 5, 10 and 14 supernatant, and low in that of TE1, 6, 8, 9, 11 and 15. CXCR2 expression was high in TE1, 4, 5, 6, 8, and 9 cells, and low in TE10, 11, 14, and 15. We chose TE1 and TE4 cells for further investigation. Stimulation or inhibition of the IL-8/CXCR2 network resulted in significant enhancement or suppression of cell proliferation in both cell lines (P&amp;lt;0.05) in a dose-dependent manner. IL-8 over-expressing TE4 cells showed significantly higher proliferative activity than the parental line (P&amp;lt;0.05), which was markedly suppressed by inhibition of the IL-8/CXCR2 network using SB225002 (P&amp;lt;0.05). In vivo, tumor sizes were smaller in the SB225002-treated group than in the untreated group. Conclusion: Our results demonstrated that stimulation of the IL-8/CXCR2 network clearly enhanced ESCC cell proliferation, while its inhibition obviously suppressed ESCC cell proliferation in vitro. These results indicate that control of IL-8/CXCR2 network signaling may be a new therapeutic strategy for ESCC. Citation Format: Masazumi Inoue, Hiroya Takeuchi, Sachiko Matsuda, Tomohiko Nishi, Kazumasa Fukuda, Rieko Nakamura, Tsunehiro Takahashi, Norihito Wada, Hirofumi Kawakubo, Yuko Kitagawa. Role of IL-8/CXCR2 network in the tumor cell proliferation of esophageal squamous cell carcinoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 942.

Upregulation of the long non-coding RNA BANCR correlates with tumor progression and poor prognosis in esophageal squamous cell carcinoma.

Esophageal squamous cell cancer was one of the most lethal tumors. The lack of effective early diagnosis and treatment ways brings its mortality quite high. Esophageal squamous dysplasia (ESD), is the only histopathology that predicts the development of ESCC. Recent evidence indicates that long noncoding RNAs (lncRNAs) play a critical role in the tumor progression and poor prognosis. BRAF activated non-coding RNA (BANCR) is a 693-bp transcript with a potential functional role in melanoma cell migration. The clinical significance of BANCR, and its' relationship with precancerous lesion of ESCC are unclear. Expression of BANCR was analyzed in 142 ESCC tissues and eight ESCC cell lines using quantitative polymerase chain reaction (qPCR) assays. We further analyzed the BANCR expression level in plasma and precancerous lesion of ESCC. In our results, BANCR levels in plasma were significantly higher in ESCC patients than in normal controls and correlated well with the presence of tumor status. Besides, BANCR expression increased significantly from low to high-grade intraepithelial neoplasia. Additionally, increased BANCR expression was associated with worse histologic grade, advanced TNM stage, more lymph node metastasis, and shorter survival of ESCC patients. The expression level of BANCR reverts to normal after the tumor has been resected, and the expression level of BANCR increased with the disease progression. Increased BANCR expression was found to be an independent prognostic factor for ESCC. In summary, BANCR may be a novel tumor biomarker for early detection, a prospective prognostic indicator and a potential therapeutic target for ESCC and ESD patients.

Prognostic value of volumetric PET parameters in unresectable and metastatic esophageal cancer

PurposeTo assess the prognostic value of volumetric parameters measured with PET/CT in patients with advanced or metastatic esophageal cancer (EC). Materials and methodsWe identified 71 patients (33 adenocarcinoma [AC] and 38 squamous cell carcinoma [ESCC]) with unresectable or metastatic EC who had PET/CT prior to palliative treatment. Volumetric parameters (metabolic tumor volume [MTV], total lesion glycolysis [TLG], tumor length [TL]) as well as maximum and mean standardized uptake (SUVmax, SUVmean) were obtained from 18F-FDG PET/CT studies. The correlation between overall survival (OS) and established clinical parameters was assessed using a Cox proportional hazards model. ResultsESCC patients had higher SUVmax and SUVmean compared to AC (p=0.002 and p<0.001, respectively). There was an association of lower SUVmax and SUVmean with metastatic compared to locally advanced tumors (e.g., median SUVmax stage IV: 14.9, 95% confidence interval [95% CI 4.4–35.5] vs. stage IIIA-C: 23.3 [9.2–40.6], p=0.017). TL, MTV and TLG showed an association to OS for all patients and for the subgroup of AC patients (AC; TL: Hazard ratio [HR] 3.23, [95% CI 1.03–10.11], p=0.044; MTV: HR 3.16, [95% CI 1.08-9.23], p=0.035). There was no correlation between PET parameters and survival in ESCC patients. Clinical nodal status was the only clinical variable associated to OS (HR 2.45 [95% CI 1.26–4.75], p=0.008) in AC patients. In a multivariate analysis, nodal status and MTV remained as independent factors associated to OS (N: HR 9.98, [95% CI 1.28–78.11], p=0.028; MTV: HR 1.02, [95% CI 1.01–1.03], p=0.003). ConclusionsMTV predicted poor OS in patients with advanced AC. No PET parameters were associated to OS in ESCC patients.