Lymph Node Metastasis In Esophageal Squamous Cell Carcinoma Research Articles

SOX2/SALL4 stemness axis modulates Notch signaling genes to maintain self-renewal capacity of esophageal squamous cell carcinoma.

Stemness phenotype is considered as the centerpiece of cancer biology due to its potential in conventional chemo-radiotherapy resistance and tumor recurrence after clinical intervention. This feature in tumor mass belongs to activation of core regulatory stemness factors and different cell signaling pathways in cancer stem cells. We aimed in this study to elucidate contribution of Notch signaling pathway in stemness state of esophageal squamous cell carcinoma (ESCC) through their relevance with stem cell markers SOX2 and SALL4. 50 ESCC tumor and related margin normal tissues were considered and categorized based on SOX2/SALL4 expression pattern, and mRNA levels of Notch signaling genes including ligands, receptors, target genes, and transcriptional coactivator were analyzed in the selected groups using qRT-PCR. Concomitant overexpression of stem cell markers SOX2 and SALL4 in ESCCs upregulated the involved genes in Notch signaling pathway. Upregulation of Notch pathway genes associated with depth of tumor invasion and lymph node metastasis of ESCC. Based on biological function of SOX2 and SALL4 axis in stemness state potential, our results may suggest contribution of Notch signaling pathway in self-renewal capacity of ESCCs, as well as invasion and metastasis of the disease. To the best of our knowledge, this is the first report elucidating the crosstalk between SOX2/SALL4 stemness factors and Notch signaling pathway in cancer research.

Fibroblast Activation Protein-α Expressing Fibroblasts Promote Lymph Node Metastasis in Esophageal Squamous Cell Carcinoma.

Background and ObjectivesEsophageal squamous cell carcinoma (ESCC) remains one of the most common malignancies in China and has a high metastasis rate and poor prognosis. Fibroblast activation protein-α (FAP-α) is a serine peptidase the expression of which in cancer-associated fibroblasts has been associated with a higher risk of metastases and poor survival. This study aimed to analyze the correlation of FAP-α expression with the lymph node metastasis and prognostic significance in ESCC.MethodsFAP-α expression was examined in 121 resected ESCC specimens and 10 adjacent normal tissue using immunohistochemistry. FAP-α expression was scored in the stromal fibroblasts adjacent to neoplastic nests. A chi-square test was used to analyze the correlation between FAP-α expression in tumors stromal and lymph node metastasis of ESCC. The association between FAP-α expression and prognosis was evaluated using univariable and multivariable statistical modeling.ResultsFAP-α expression was absent in the benign controls. FAP-α expression was evident in the stromal 37% (45/121) of ESCC. Expression of FAP-α level is significantly associated with lymph node metastasis (p=0.023), but it is not correlated to age, gender, and tumor location in ESCC patients. Stromal FAP-α expression was significantly associated with poor survival in univariable (HR 2.009; 95% CI 1.259–3.205; p=0.003) and multivariable analysis (HR 1.833; 95% CI 1.144–2.937; p=0.012).ConclusionFAP-α may be an important regulator in lymph node metastasis of ESCC and may provide a novel therapeutic target in ESCC.

Expression of ADAM29 and FAM135B in the pathological evolution from normal esophageal epithelium to esophageal cancer: Their differences and clinical significance.

A Disintegrin And Metalloprotease Domain 29 (ADAM29) and Family with sequence similarity 135 member B (FAM135B) genes have been reported to be associated with a carcinogenic risk of esophageal squamous cell carcinoma (ESCC). However, to the best of our knowledge, the expression of ADAM29 and FAM135B in the pathological evolution from normal esophageal epithelial cells to ESCC has not yet been investigated. The present study aimed to investigate the expression of ADAM29 and FAM135B in normal esophageal mucosal epithelium, low-grade and high-grade esophageal intraepithelial neoplasia, and ESCC. Furthermore, the present study aimed to investigate the role of ADAM29 and FAM135B in the development of esophageal lesions. Immunohistochemistry was performed in order to detect the expression levels of ADAM29 and FAM135B proteins in normal esophageal mucosa samples (40 cases), low-grade intraepithelial neoplasia samples (20 cases), high-grade intraepithelial neoplasia samples (20 cases) and ESCC samples (40 cases). The results of the present study demonstrated that the positive rates of ADAM29 and FAM135B proteins increased gradually from normal esophageal mucosal epithelium and esophageal intraepithelial neoplasia, to ESCC (P<0.05). Furthermore, the expression levels of ADAM29 and FAM135B proteins in ESCC were not associated with age and the tumor size (P>0.05); however, the protein levels were associated with the pathological stage, clinical stage and lymph node metastasis of ESCC (P<0.05). In addition, there was a significant association between the expression levels of ADAM29 protein and FAM135B protein (χ2=60.071; P<0.001). The results of the present study demonstrated that the expression levels of ADAM29 and FAM135B were associated with the tumor behavior characteristics and the progression of esophageal cancer, the expression of which could be used for the diagnosis of early esophageal cancer, and provide the basis for guiding individualized treatment.

Inter- and intratumor DNA methylation heterogeneity associated with lymph node metastasis and prognosis of esophageal squamous cell carcinoma.

Background: Esophageal squamous cell carcinoma (ESCC), one of the leading causes of cancer mortality worldwide, is a heterogeneous cancer with diverse clinical manifestations. However, little is known about the epigenetic heterogeneity and its clinical relevance for this prevalent cancer.Methods: We generated 7.56 Tb single-base resolution whole-genome bisulfite sequencing data for 84 ESCC and paired paraneoplastic tissues. The analysis identified inter- and intratumor DNA methylation (DNAm) heterogeneity, epigenome-wide DNAm alterations together with the functional regulators involved in the hyper- or hypomethylated regions, and their association with clinical features. We then validated the correlation between the methylation level of specific regions and clinical outcomes of 96 ESCC patients in an independent cohort.Results: ESCC manifested substantial inter- and intratumor DNAm heterogeneity. The high intratumor DNAm heterogeneity was associated with lymph node metastasis and worse overall survival. Interestingly, hypermethylated regions in ESCC were enriched in promoters of numerous transcription factors, and demethylated noncoding regions related to RXR transcription factor binding appeared to contribute to the development of ESCC. Furthermore, we identified numerous DNAm alterations associated with carcinogenesis and lymph node metastasis of ESCC. We also validated three novel prognostic markers for ESCC, including one each in the promoter of CLK1, the 3' untranslated region of ZEB2, and the intergenic locus surrounded by several lncRNAs.Conclusions: This study presents the first population-level resource for dissecting base-resolution DNAm variation in ESCC and provides novel insights into the ESCC pathogenesis and progression, which might facilitate diagnosis and prognosis for this prevalent malignancy.

Overexpression of VEGF-C and MMP-9 predicts poor prognosis in Kazakh patients with esophageal squamous cell carcinoma.

Vascular endothelial growth factor (VEGF) and Matrix metalloproteinases (MMPs) are believed to participate in infiltration of tumors. High mortality of esophageal squamous cell carcinoma (ESCC) related to its primary infiltration; however, it is not clear whether the expression of VEGF and MMPs is involved in this process. Screening of The Cancer Genome Atlas (TCGA) database showed that among the VEGF family and MMP9, VEGF-A, VEGF-C, and MMP-9 mRNA were overexpression in ESCC. This result was verified using the Oncomine database and in Kazakh patients with ESCC. Overexpression of VEGF-C and MMP-9 and positive association with advanced esophageal cancer and invading ESCC cells (Gene Expression Omnibus (GEO): GSE21293). Immunohistochemical staining revealed that VEGF-C and MMP-9 were overexpressed in Kazakh ESCCs. VEGF-C expression was related to invasive depth, tumor-node-metastasis (TNM) staging, lymphatic, and lymph node metastasis of ESCC. The linear association between them was further confirmed in TCGA database and the specimens from Kazakh patients with ESCC. Patients with both proteins expression had tumors with greater aggressiveness, suffered from poor prognosis compared with patients who did not express either protein or expressed protein alone. Both proteins expression predicted high invasiveness of ESCC, which is related to worse prognosis of Kazakh ESCCs.

MLL2 promotes cancer cell lymph node metastasis by interacting with RelA and facilitating STC1 transcription

Esophageal squamous cell carcinoma (ESCC) presents with lymph node metastasis in the early stages, limiting the opportunities for curative local resection, including endoscopic submucosal dissection (ESD). ESD is regarded as the standard treatment for early-stage ESCCs. However, radical surgery is recommended when lymph node metastasis risk exists. More efforts are needed to find the markers for early prediction and clarify the molecular mechanism underlying the pathogenesis of lymph node metastasis. Recently, aberrant regulation of gene expression by histone methylation modifiers has emerged as an important mechanism for cancer metastasis. Herein, we demonstrated that mixed-lineage leukemia 2 (MLL2) positively regulates gene expression programs associated with ESCC cell migration. MLL2 interacts with RelA in the nucleus to enhance transcription of stanniocalcin-1 (STC1) and to facilitate cancer metastasis. Meanwhile, MLL2 knockdown resulted in a significant decrease in the migration of ESCC cells. Clinically, high level of MLL2 was significantly associated with early-stage ESCC lymph node metastasis. In summary, these findings discovered a previously unidentified molecular pathway underlying the coordinated regulation of metastasis-related STC-1 expression by MLL2 and RelA and highlighted the critical role of MLL2 in ESCC.

LncRNA MNX1-AS1 promotes progression of esophageal squamous cell carcinoma by regulating miR-34a/SIRT1 axis

BackgroundLong non-coding RNAs (lncRNAs) are powerful factors influencing the tumorigenesis and metastasis of multiple carcinomas. LncRNA MNX1-AS1 plays critical roles in the progression of tumor formation according to recent research, while its roles in esophageal squamous cell carcinoma (ESCC) remains unknown. MethodsThe expression levels of lncRNA MNX1-AS1 were examined in ESCC tissues by quantitative reverse transcriptase polymerase chain reaction (qRT-PCR). The role of lncRNA MNX1-AS1 was performed by WST-1 proliferation assays, migration and invasion assays. Besides, the molecular mechanism of lncRNA MNX1-AS1 was verified by online bioinformatics, qRT-PCR and rescue assays. ResultsMNX1-AS1 was signifcantly upregulated in ESCC tissues. It was conformed that high MNX1-AS1 expression was associated with ESCC lymph node metastasis. Moreover, we found that knockdown of MNX1-AS1 apparently suppressed the cell proliferation, migration, and invasion capacity. Flow cytometry analysis showed MNX1-AS1 regulated ESCC cell cycle and apoptosis progression. Mechanism analysis revealed that miR-34a inhibitor could rescue the influence of inhibiting MNX1-AS1 on ESCC cells migration by serving as competing endogenous RNA (ceRNAs). Furthermore, we found that miR-34a specifically targeted SIRTI. ConclusionsTaken together, we demonstrated that lncRNA MNX1-AS1/miR-34a/SIRT1 regulatory axis could play an important role in ESCC progression, and MNX1-AS1 may act as a novel potential biomarker for esophageal squamous cell carcinoma.

Expression and Significance of TRIM 28 in Squamous Carcinoma of Esophagus

Tripartite motif-containing protein 28 (TRIM28) has been proved to accelerate cell proliferation and metastasis in a variety of human cancers. However, the role of TRIM28 in esophageal squamous cell carcinoma (ESCC) remains unclear. In this study, to compare the biological effect and significance of TRIM28 expression in ESCC, immunohistochemistry (streptavidin-perosidase, S-P) method was used firstly to examine the expression of TRIM28 in 136 cases of ESCC, 35 cases of high grade intraepithelial neoplasia (HGIN), 29 cases of low grade intraepithelial neoplasia (LGIN) and 37 cases of normal esophageal epithelium (NEE). Then the associations of TRIM28 expression with clinicopathological data and overall survival (OS) were also analyzed. Western blot was performed to evaluate TRIM28 protein in a total of 20 matched human ESCC and NEE tissues. Moreover, the localization of TRIM28 protein in ESCC and NEE tissues was also detected by immunofluorescence. TRIM28 protein was mainly distributed in the nucleus of ESCC. The expression of TRIM28 increased progressively from NEE to LGIN, to HGIN, and to ESCC, and it was also related to invasive depth, pTNM stage and lymph node metastasis in ESCC (P < 0.05). The results of western blot and immunofluorescence all showed that the relative expression of TRIM28 protein was markedly upregulated in ESCC compared with the NEE tissues (P < 0.01). However, prognostic analysis showed that TRIM28 may not be a prognostic factor of patients with ESCC. In conclusion, the overexpression of TRIM28 may play an important role for development and metastasis in ESCC.

TAB3 promotes human esophageal squamous cell carcinoma proliferation and invasion via the NF‑κB pathway.

Esophageal squamous cell carcinoma (ESCC) has become one of the most common causes of cancer‑associated mortality worldwide. Transforming growth factor‑activated kinase (TAK1)‑binding protein3 (TAB3) is essential for activation of the NF (NF)‑κB pathway in response to TAK1 activation. The NF‑κB pathway serves important roles in tumor cell proliferation and migration; however, the clinical relevance of TAB3 and its biological function in ESCC progression remain elusive. The present study investigated the expression and function of TAB3 in ESCC tissues, and its association with the clinical prognosis of patients. The results demonstrated that TAB3 expression was significantly increased in human ESCC cell lines and tissue samples, and the expression of TAB3 was associated with ESCC lymph node metastasis, Tstage, pathological grade and Ki‑67 expression in 80ESCC samples, as determined by immunohistochemistry. Patients with ESCC and high TAB3 expression exhibited worse overall survival. Furthermore, knockdown of TAB3 by small interfering RNA inhibited the proliferation of ESCC cells, and reduced the migration and invasion of ESCC cells. In addition, knockdown of TAB3 decreased the expression of the NF‑κB pathway in TE‑1 cells. Taken together, these results demonstrated that TAB3 may be a promising therapeutic target for the treatment of ESCC.

Excision repair cross-complementing group 1 (ERCC1) overexpression inhibits cell apoptosis and is associated with unfavorable prognosis of esophageal squamous cell carcinoma.

Excision repair cross-complementing group 1 (ERCC1) functions as a nucleotide excision repair (NER) enzyme. Altered ERCC1 expression or function is closely associated with cancer development and progression. This study determined the association of ERCC1 expression with survivin expression, clinicopathological characteristics, and survival of esophageal squamous cell carcinoma (ESCC) patients after postoperative concurrent chemoradiotherapy.Tissue specimens from 102 resected ESCC patients were acquired for immunohistochemical analysis of ERCC1 and survivin protein expression.ERCC1 expression was detected in 62.7% of ESCC tissues and in 9.8% of normal squamous epithelium tissues (P < .01), while survivin expression was detected in 60.8% of ESCC tissues and in 19.6% of normal squamous epithelia (P < .01). ERCC1 overexpression associated with advanced tumor clinical stage and lymph node metastasis (P < .05), but not with tumor size, depth of invasion, or differentiation (P > .05). ERCC1 overexpression was also associated with survivin levels (r = 0.42, P < .01) and worse progression-free survival of ESCC patients after concurrent chemoradiotherapy. Multivariate analysis data revealed that ERCC1 and survivin protein expression were independent predictors of overall survival of ESCC patients after chemotherapy and/or radiotherapy (P < .05).ERCC1 overexpression is an important phenotype that is associated with ESCC lymph node metastasis and advanced tumor clinical stages. ERCC1 expression may also inhibit ESCC cell apoptosis via regulating survivin expression, and ERCC1 and survivin overexpression are independent predictors of prognosis for ESCC patients who receive chemotherapy and/or radiotherapy.

Radiomics nomogram outperforms size criteria in discriminating lymph node metastasis in resectable esophageal squamous cell carcinoma.

To determine the value of radiomics in predicting lymph node (LN) metastasis in resectable esophageal squamous cell carcinoma (ESCC) patients. Data of 230 consecutive patients were retrospectively analyzed (154 in the training set and 76 in the test set). A total of 1576 radiomics features were extracted from arterial-phase CT images of the whole primary tumor. LASSO logistic regression was performed to choose the key features and construct a radiomics signature. A radiomics nomogram incorporating this signature was developed on the basis of multivariable analysis in the training set. Nomogram performance was determined and validated with respect to its discrimination, calibration and reclassification. Clinical usefulness was estimated by decision curve analysis. The radiomics signature including five features was significantly associated with LN metastasis. The radiomics nomogram, which incorporated the signature and CT-reported LN status (i.e. size criteria), distinguished LN metastasis with an area under curve (AUC) of 0.758 in the training set, and performance was similar in the test set (AUC 0.773). Discrimination of the radiomics nomogram exceeded that of size criteria alone in both the training set (p <0.001) and the test set (p=0.005). Integrated discrimination improvement (IDI) and categorical net reclassification improvement (NRI) showed significant improvement in prognostic value when the radiomics signature was added to size criteria in the test set (IDI 17.3%; p<0.001; categorical NRI 52.3%; p<0.001). Decision curve analysis supported that the radiomics nomogram is superior to size criteria. The radiomics nomogram provides individualized risk estimation of LN metastasis in ESCC patients and outperforms size criteria. • A radiomics nomogram was built and validated to predict LN metastasis in resectable ESCC. • The radiomics nomogram outperformed size criteria. • Radiomics helps to unravel intratumor heterogeneity and can serve as a novel biomarker for determination of LN status in resectable ESCC.

Autocrine CCL5 promotes tumor progression in esophageal squamous cell carcinoma in vitro

The pro-tumoral effects of CCL5 have been identified in numerous cancer types. We successfully cultivated 4 esophageal squamous cell carcinoma (ESCC) cell lines, including TWES-1, TWES-3 and a pair of cell lines derived from primary lesion (TWES-4PT) and metastatic lymph node (TWES-4LN) of the same patient. Whole genome screening showed that TWES-4LN expressed higher levels of CCL5 compared to that of TWES-4PT; quantification of protein secretion displayed comparable results, suggesting that CCL5 could be associated with lymph node metastasis in ESCC. CCL5 knockdown by siRNA significantly reduced basal growth rate, tumor migration and invasiveness in the paired cell lines; whereas this treatment induced cell apoptosis in TWES-1 and TWES-3. CCR5 antagonist maraviroc significantly inhibited tumor migration and invasion in the paired cell lines without affecting tumor growth. Collectively, these results suggest that CCL5 autocrine loop may promote ESCC progression; targeting the CCL5/CCR5 axis could be a potential therapeutic strategy for this deadly disease.

14-3-3ζ promotes esophageal squamous cell carcinoma invasion by repressing S1PR2 protein expression through NF-κB signaling

14-3-3ζ is overexpressed in several cancers, including esophageal squamous cell carcinoma (ESCC), and plays an important role in tumorigenesis. However, the mechanisms underlying its tumorigenesis remain unclear. Here we report that 14-3-3ζ was upregulated in ESCC tumors, compared with adjacent normal tissues; 14-3-3ζ levels were positively correlated with ESCC lymph node metastasis and recurrence. Overexpression of 14-3-3ζ promoted the tumor growth and invasion of ESCC in vitro and in vivo, whereas depletion of 14-3-3ζ suppressed these effects. Moreover, 14-3-3ζ reduces expression of genes mediating S1P/S1PR2 signaling, and this effect is mediated through activation of NF- κ B. Taken together, 14-3-3ζ contributes to ESCC tumorigenesis and progression through repressing S1PR2 signaling and may act as a new therapeutic target for ESCC.

Expression and significance of ASPP2 in squamous carcinoma of esophagus

To study the significance of apoptosis stimulating protein of P53 2 (ASPP2) expression in esophageal squamous cell carcinoma (ESCC), immunohistochemistry S-P method was used to examine the expression of ASPP2 in 136 cases of ESCC, 35 cases of high grade intraepithelial neoplasia (HGIN), 29 cases of low grade intraepithelial neoplasia (LGIN) and 37 cases of normal esophageal epithelium (NEE). The associations of ASPP2 expression with clinicopathological data and overall survival (OS) were also analyzed. Quantitative real-time polymerase chain reaction (qRT-PCR) was performed to evaluate ASPP2 expression in a total of 20 matched human ESCC tumor tissues and normal adjacent tissues (NAT). In addition, EC109 cells were treated with cisplatin (CDDP) in vitro for 24 h (the intervention group) and the control group was set up at the same time. Western blot was used to examine the expression of ASPP2 protein between the two groups. The expression of ASPP2 decreased progressively from NEE to LGIN, to HGIN, and to ESCC, and it was related to TNM stage, histological differentiation and lymph node metastasis in ESCC (P < 0.05). ASPP2 was a protective factor of patients with ESCC (P = 0.008). The relative expression of ASPP2 mRNA was markedly downregulated in ESCC compared with the paired NAT (P < 0.01). Western blot results showed that cells in the intervention group could express ASPP2 while there was no expression of ASPP2 in the control group. Taken together, these results indicate that the abnormal expression of ASPP2 may play an important role for development and metastasis in ESCC.

Associations between the expression of MTA1 and VEGF-C in esophageal squamous cell carcinoma with lymph angiogenesis and lymph node metastasis.

The aim of the present study was to investigate the association between the expression levels of metastasis-related gene 1 (MTA1) and vascular endothelial growth factor C (VEGF-C) in esophageal squamous cell carcinoma (ESCC) with lymph angiogenesis and lymph node metastasis. The paraffin-embedded tissue samples of 107 cases of ESCC and 56 cases of normal esophageal tissues were collected from the Department of Cardiothoracic Surgery, Suining Central Hospital from March 2013 to January 2014. Immunohistochemical assays were performed to detect the expression levels of MTA1, VEGF-C and D2-40 in ESCC, and the micro-lymphatic vessel density (LVD) was evaluated. Their associations with various clinicopathological parameters were also analyzed. The protein expression levels of MTA1 and VEGF-C in ESCC were significantly higher compared with those in normal esophageal tissues (P<0.05); the high protein expression levels of MTA1 and VEGF-C in ESCC tissues at various tumor-node-metastasis stages exhibited statistically significant differences, as revealed by the Kruskal-Wallis test (P<0.05). The protein expression levels of MTA1 and VEGF-C in ESCC exhibited positive correlations (Spearman's ρ, r=0.512; P=0.000); the LVD level in the group with high expression of MTA1 and VEGF-C was significantly higher compared with in the low expression group (P<0.05). The comparison between MTA1 and VEGF-C protein expression levels in the group with a high rate of lymph node metastasis demonstrated statistically significant differences when compared with in the low lymph node metastasis group (P<0.05). The expression levels of MTA1 and VEGF-C in ESCC exhibited a positive correlation in ESCC, which may co-promote lymph angiogenesis and lymph node metastasis in ESCC; therefore, they may be used as biomarkers for determining the prognosis of ESCC.

Abstract 3293: Impact of the invasive depth on lymph node metastasis and survival in Chinese patients with esophageal squamous cell carcinoma

Abstract The lymph node metastasis (LNM) has been well recognized as one of the major risk factors affecting the survival of the patients with esophageal squamous cell carcinoma (ESCC). However, it is a dilemma to determine the LNM status before surgery. The invasive depth (T stage) of ESCC could be determined either with computed tomography or ultrasound endoscopy and may reflect the severity of LNM. Thus, the present study was designed to determine the impact of tumor invasive depth on LNM status and survival in ESCC patients with T1-4N0M0. The enrolled 30,514 ESCC patients in this study were from the ESCC database in Henan Key Laboratory for Esophageal Cancer Research of the First Affiliated Hospital, Zhengzhou University. Of the patients, there were 18346 males with an mean age of 59.2±8.9, and 12168 females with an mean age of 59.7±8.7. All the patients had been performed radical esophagectomy without radiotherapy and chemotherapy before surgery, and the invasive depth records were retrieved from the medical records in hospitals after surgical resection. Based on the 6th version of UICC criteria, the invasive depth (T) was classified as Tis, and T1-4. Of the 30,514 ESCC patients, 20,288 cases were successfully followed until the end of 2015. The Logistic regression method were used to determine the correlation between T stage and LNM status. The Kaplan-Meier method was applied to analyze the survival in different groups. The results showed that, of the 30,514 ESCC patients, there were 3324 (10.9%) patients with Tis+T1, 9616 (31.5%) with T2, 17249 (56.5%) with T3 and 325 (1.1%) with T4. Accordingly, from Tis&amp;T1 to T4, the number of the patients with positive LNM were 554 (16.7%), 3392 (35.3%), 7737 (44.9%) and 205 (63.1%), respectively. Logistic regression analysis indicated the risk of LNM was dramatically increased from 2- to 8-folds with the T stages from Tis&amp;T1 to T4, with OR values of 2.725 (T2: 95%CI: 2.465-3.013), 4.067 (T3: 95%CI: 3.695-4.477) and 8.542 (T4: 95%CI: 6.699-10.892), respectively. Interestingly, seventeen percent of the patients with Tis&amp;T1 stages had occurred positive LNM, indicating that LNM may occur in very early stage of ESCC. Kaplan-Meier analysis showed that T staging was obviously associated with the survival in ESCC patients with negative LNM (P value &amp;lt; 0.05). Overall, the present results indicated a strong correlation between invasive depth and LNM status, suggesting that the invasion depth may be one of crucial markers to reflect the LNM in ESCC. The invasive depth could be used as an promising indicator for LNM status judgment in clinical TNM staging and prognosis for ESCC. [Supported by the Joint Funds of the National Natural Science Foundation of China (U1301227), the General Program of National Natural Science Foundation of China (81472323) and Correspondence to: Li Dong Wang, Email: ldwang2007@126.com] Note: This abstract was not presented at the meeting. Citation Format: Xin Song, Fu You Zhou, Hai Jun Yang, Xue Na Han, Tang Juan Zhang, Lian Qun Zhang, Ning Liu, Jing Li Ren, Zhi Wei Chang, Xin Tian, Hai Ling Wang, Hui Fang Tan, Li Dong Wang. Impact of the invasive depth on lymph node metastasis and survival in Chinese patients with esophageal squamous cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3293. doi:10.1158/1538-7445.AM2017-3293

Genome-wide screening of DNA methylation associated with lymph node metastasis in esophageal squamous cell carcinoma.

Lymph node metastasis (LNM) of esophageal squamous cell carcinoma (ESCC) is well-known to be an early event associated with poor prognosis in patients with ESCC. Recently, tumor-specific aberrant DNA methylation of CpG islands around the promoter regions of tumor-related genes has been investigated as a possible biomarker for use in early diagnosis and prediction of prognosis. However, there are few DNA methylation markers able to predict the presence of LNM in ESCC. To identify DNA methylation markers associated with LNM of ESCC, we performed a genome-wide screening of DNA methylation status in a discovery cohort of 67 primary ESCC tissues and their paired normal esophageal tissues using the Illumina Infinium HumanMethylation450 BeadChip. In this screening, we focused on differentially methylated regions (DMRs) that were associated with LNM of ESCC, as prime candidates for DNA methylation markers. We extracted three genes, HOXB2, SLC15A3, and SEPT9, as candidates predicting LNM of ESCC, using pyrosequencing and several statistical analyses in the discovery cohort. We confirmed that HOXB2 and SEPT9 were highly methylated in LNM-positive tumors in 59 ESCC validation samples. These results suggested that HOXB2 and SEPT9 may be useful epigenetic biomarkers for the prediction of the presence of LNM in ESCC.

Promoter hypermethylation of the RECK gene is associated with its low expression and poor survival of esophageal squamous cell carcinoma.

The present study aimed to investigate the association between the methylation status of the reversion-inducing cysteine-rich protein with kazal motifs (RECK) gene and its mRNA expression levels in patients with esophageal squamous cell carcinoma (ESCC). The methylation status of RECK was analyzed by methylation-specific polymerase chain reaction (PCR), and RECK mRNA expression levels were analyzed by quantitative PCR, in 310 paired ESCC tissues. The mean RECK methylation index (MI) was 0.65 in ESCCs and 0.49 in non-tumor samples. There was a significant association between RECK methylation and the American Joint Committee on Cancer stage and lymph node metastasis in ESCC (P<0.0001; P=0.001). The mRNA expression level of RECK was lower in ESCC tissues (mean-∆Cq=-4.66) compared with non-tumor tissues (mean-∆Cq=-2.79), and decreased RECK mRNA expression levels were associated with lymph node metastasis in ESCC. In addition, RECK mRNA levels were decreased in ESCC patients with hypermethylation of the RECK gene (∆MI >0.16; mean-∆∆Cq=-2.85) compared with those with hypomethylation of the RECK gene (∆MI ≤0.16; mean-∆∆Ct=-0.83), and there was a significant difference in the mRNA expression levels of RECK between those with N0-1 and N2-3 lymph node metastasis (P<0.0001). A significant correlation was observed between RECK mRNA expression levels, the MI of RECK and poor postoperative survival (P=0.0003; P<0.0001). The results of the present study suggested that promoter hypermethylation may be an important factor for loss of RECK mRNA expression and may be an indicator of poor survival in ESCC.

The increased number of tumor-associated macrophage is associated with overexpression of VEGF-C, plays an important role in Kazakh ESCC invasion and metastasis

Tumor associated macrophages (TAMs) play an important role in the growth, progression, and metastasis of tumors. The distribution of TAMs in Kazakh esophageal squamous cell carcinoma (ESCC) is not determined. We aimed to investigate the role of TAMs in the occurrence and progression of Kazakh ESCC. CD163 was used as the TAM marker, and immunohistochemistry (IHC) counts were used to quantify the density of TAMs in tumor nest and surrounding stroma. IHC staining was used to evaluate the expression of vascular endothelial growth factor C (VEGF-C) in Kazakh ESCC and cancer adjacent normal (CAN) tissues. The density of TAMs in Kazakh ESCCs tumor nest and stromal was significantly higher than that in CAN tissues. The increased number of CD163-positive TAMs in tumor nest and tumor stromal was positively associated with Kazakh ESCC lymph node metastasis and clinical stage progression. Meanwhile, the expression of VEGF-C in Kazakh ESCCs was significantly higher than that in CAN tissues. Overexpression of VEGF-C in Kazakh ESCCs was significantly associated with gender, depth of tumor invasion, lymph node metastasis and tumor clinical stage. The increased number of TAMs, either in the tumor nests or tumor stroma was positively correlated with the overexpression of VEGF-C, which may promote lymphangiogenesis and play an important role in the invasion and metastasis of Kazakh ESCC.

KIAA1377 is associated with lymph node metastasis in esophageal squamous cell carcinoma.

KIAA1377, of which there are few studies regarding cell biology and neurological diseases, has been found to be significantly amplified in esophageal squamous cell carcinoma (ESCC) with lymph node metastasis compared with ESCC without lymph node metastasis. This suggests that KIAA1377 may play a role in the lymph node metastasis of ESCC. There has, to the best of our knowledge, been no study performed to investigate the role of KIAA1377 in ESCC. In the present study, the expression of KIAA1377 was detected by immunohistochemistry, and its expression was statistically analyzed with clinicopathological parameters, using commercially obtained tissue arrays consisting of 86 cases of ESCC and 79 paired controls. KIAA1377 was knocked down ex vivo using transient transfection with specific small hairpin RNA (shRNA) vectors into ESCC TE-1 and EC9706 cell lines whose endogenous KIAA1377 level was highest. The variation of proliferation, migration and invasion were evaluated using methyl thiazolyl tetrazolium, wound healing and Transwell assay, respectively. It was found in vivo that KIAA1377 expression was significantly associated with lymph node metastasis and differentiation, and ex vivo that knockdown of KIAA1377 cannot significantly affect proliferation and mobility in the ESCC cell line TE-1. Overall, this is the first study suggesting that KIAA1377 may play a role in the lymph node micrometastasis of ESCC.