Normal Esophageal Tissues Research Articles

Microenvironmental changes co-occur with mosaic somatic clonal expansions in normal skin and esophagus tissues.

The presence of somatic mutations, previously identified in cancers, are being increasingly recognized in normal tissues. While the role of microenvironment (ME) in tumor progression is well understood, the changes that occur in the microenvironment of normal tissues that harbor somatic mutations has not been systematically studied. Here, using normal RNA-Seq data accrued from 6544 samples across 27 tissue types from Genotype-Tissue Expression (GTEx) project, we studied the association of microenvironmental changes in the presence of somatic clonal expansions of previously implicated cancer genes. We focused our analysis on skin and esophagus since they have the highest number of samples and mutation burden together. We observed changes in microenvironmental cell-types previously implicated in tumor progression including endothelial cells, epithelial cells, pericytes, fibroblasts, chondrocytes, among others. The Epithelial-Mesenchymal-Transition (EMT) pathway is dysregulated in both skin and esophagus, along with increased hypoxia scores in samples with clonal expansions. These results suggest that microenvironmental changes play an important role in clonal expansions and potentially the initiating stages of cancer progression. Studying these changes may provide new avenues for early intervention of cancer, for targeted therapies, or enhance activities of conventional therapies.

Comprehensive analysis of the relationship between cuproptosis-related genes and esophageal cancer prognosis.

Esophageal cancer is one of the most common malignant tumors of the digestive system, with a 5-year survival rate of 15% to 50%. Cuproptosis, a unique kind of cell death driven by protein lipoylation, is strongly connected to mitochondrial metabolism. The clinical implications of cuproptosis-related genes in esophageal cancer, however, are mainly unknown. To identify cuprotosis-related genes that are differentially expressed in esophageal cancer and investigate their prognostic significance. With |log fold change| > 1 and false discovery rate < 0.05 as criteria, the Wilcox test was used to evaluate the differentially expressed genes between 151 tumor tissues and 151 normal esophageal tissues. Cuproptosis-related genes were selected to be linked with prognosis using univariate Cox regression analysis. Genes were separated into high- and low- expression groups based on their cutoff value of gene expression, and the correlation between the two groups and overall survival or progression-free survival was investigated using the log-rank test. The C-index, calibration curve, and receiver operator characteristic (ROC) curve were used to assess a nomogram containing clinicopathological characteristics and cuproptosis-related genes. Pyruvate dehydrogenase A1 (PDHA1) was found to be highly correlated with prognosis in univariate Cox regression analysis (hazard ratio = 22.96, 95% confidence interval = 3.09-170.73; P = 0.002). According to Kaplan-Meier survival curves, low expression of PDHA1 was associated with a better prognosis (log-rank P = 0.0007). There was no significant correlation between PDHA1 expression and 22 different types of immune cells. Tumor necrosis factor superfamily member 15 (TNFSF15) (P = 3.2 × 10-6; r = 0.37), TNFRSF14 (P = 8.1 × 10-8; r = 0.42), H long terminal repeat-associating 2 (P = 6.0 × 10-8; r = 0.42) and galectin 9 (P = 3.1 × 10-6; r = 0.37) were all found to be considerably greater in the high PDHA1 expression group, according to an analysis of genes related to 47 immunological checkpoints. The low PDHA1 expression group had significantly lower levels of cluster of differentiation 44 (CD44) (P = 0.00028; R = -0.29), TNFRSF18 (P = 1.2 × 10-5; R = -0.35), programmed cell death 1 ligand 2 (P = 0.0032; R = -0.24), CD86 (P = 0.018; R = -0.19), and CD40 (P = 0.0047; R = -0.23), and the differences were statistically significant. We constructed a prognostic nomogram incorporating pathological type, tumor-node-metastasis stage, and PDHA1 expression, and the C-index, calibration curve, and ROC curve revealed that the nomogram's predictive performance was good. Cuproptosis-related genes can be used as a prognostic predictor for esophageal cancer patients, providing novel insights into cancer treatment.

MIR503HG promotes esophageal squamous cell carcinoma cell proliferation, invasion and migration via hsa-miR-503 pathway

Objective: To explore the function and mechanism of long non-coding RNA MIR503HG in esophageal squamous cell carcinoma (ESCC). Methods: The MIR503HG expression data in 60, 119 and 23 cases of ESCC and their paired adjacent tissues were chosen from three ESCC datasets GSE53622, GSE53624 and GSE130078, respectively. The expression data of MIR503HG in 81 ESCC tissues and 271 unpaired normal esophageal tissues were screened from the combined dataset of Cancer Genome Atlas and Genotype-Tissue Expression Database (TCGA+ GTEx). The MIR503HG knockdown plasmid was constructed, packaged into lentivirus. The lentivirus was used to infect with esophageal squamous cell carcinoma cell lines KYSE30 and KYSE510 to screen out the stable MIR503HG knockdown cell lines. ESCC cell line KYSE30 was transiently transfected with miRNA mimics to overexpress hsa-miR-503-3p and hsa-miR-503-5p.The expression levels of MIR503HG, hsa-miR-503-3p and hsa-miR-503-5p were detected by quantitative real-time polymerase chain reaction. The proliferation ability of the cells was detected by cell counting kit 8 and clone formation assay. The invasion and migration ability of the cells were detected by Transwell assay. Cell cycle was detected by flow cytometry. The effect of MIR503HG on the proliferation of ESCC was detected by xenograft experiment in BALB/c-nu/nu mice. Results: Both GEO and TCGA+ GTEx databases showed that the expression of MIR503HG in ESCC tissues was higher than that in adjacent tissues and normal esophageal tissues (P<0.01). Compared with shNC group, the proliferation rates of KYSE30 and KYSE510 cells after knockdown of MIR503HGwere significantly inhibited (P<0.001). The colony formation numbers of KYSE30 cells in shMIR503HG1 group and shMIR503HG2 group were (2.00±1.41) and (1.33±0.47), respectively, significantly lower than that of the shNC group (P=0.002). The clone formation numbers of KYSE510 cells in shMIR503HG1 group and shMIR503HG2 group were (174.67±15.97) and (80.33±6.34), respectively, significantly lower than that of the shNC group (P<0.001). The invasive numbers of KYSE30 cells in shMIR503HG1 group and shMIR503HG2 group were 75.33±6.02 and 45.67±7.59, significantly lower than that of the shNC group(P<0.001). The migrating number of KYSE30 cells in shMIR503HG1 group and shMIR503HG2 group were 244.00±10.23 and 210.67±13.52, significantly lower than that of the shNC group(P<0.001), and the cell cycle was arrested in G(0)/G(1) phase. The xenograft experiment showed that the subcutaneous tumor in shMIR503HG group was significantly smaller than that in shNC group, and the tumor weight in shMIR503HG group was (0.097±0.026) g, which was lower than (0.166±0.021) g in shNC group (P<0.001). After knockdown of MIR503HG, the relative expression levels of hsa-miR-503-3p in KYSE30 cells of shMIR503HG1 group and shMIR503HG2 group were 0.66±0.02 and 0.58±0.00, respectively, the relative expression levels of hsa-miR-503-5p were 0.64±0.00 and 0.68±0.03, respectively, which were all lower than those in shNC group (P<0.01). After knockdown of MIR503HG, overexpression of hsa-miR-503-3p and hsa-miR-503-5p attenuated the inhibitory effects of knockdown of MIR503HG on proliferation (P<0.001), invasion (P<0.01) and migration (P<0.001) of KYSE30 cells. Conclusions: MIR503HG promotes the proliferation, invasion and migration of ESCC cells by regulating hsa-miR-503 pathway and can be used as a new potential target for targeted therapy of ESCC.

Long-Term Excessive Selenium Supplementation Affects Gene Expression in Esophageal Tissue of Rats.

Esophageal cancer is one of the leading causes of cancer death and the seventh most prevalent cancer worldwide. Considering the positive association of high selenium with the prevalence of esophageal cancer, we have investigated the effect of high doses of selenium on gene expression in the normal esophageal tissue of rats. Twenty male rats were randomly divided into four groups: control group, group 2mg Se/L, 10mg Se/L, and 20mg Se/L rats fed with a basal basic diet and 2, 10, and 20mg Se/L as sodium selenite in drinking water, respectively, for 20weeks. Serum malondialdehyde and glutathione peroxidase activity were measured. Moreover, the expression and concentration of the cyclin D1, cyclin E, KRAS, p53, NF-kB, TGF-β, and MGMT in the esophageal tissue were analyzed and compared between the four groups. In normal esophageal tissue, selenium supplementations (2, 10, and 20mg Se/L) increased the mRNA levels of cyclin D1, P53, KRAS, NF-κB p65, and MGMT and decreased the mRNA level of TGFß1. The concentrations of cyclin D1 and MGMT were also significantly increased by selenium supplementations. Selenium supplementations had no significant effect on serum MDA but significantly increased GPX activity. The present study suggests that selenium supplementation (2, 10, and 20mg Se/L) affects gene expression related to inflammation, Cell proliferation, and apoptosis in the normal esophageal tissue. However, there were no observed abnormalities other than reduced growth with supplementation of 20mg/L as Na2SeO3 in rats.

S419 Discovery of Methylated DNA Biomarkers for the Potential Non-Endoscopic Detection of Barrett's Esophagus (BE)

Introduction: Barrett’s esophagus (BE) is a known precursor of esophageal adenocarcinoma (EAC), the type of esophageal cancer that accounts for over 80% of all esophageal cancer cases in the United States. The current gold standard for diagnosis of BE is endoscopic biopsy with histologic examination. Unfortunately, most individuals in the United States do not undergo endoscopy and thus BE is not diagnosed, resulting in progression toward fatal EAC in most undiagnosed BE cases. For this reason, minimally invasive sponge or balloon devices have been studied in conjunction with various types of biomarkers. However, most markers used in current BE diagnostic biomarker panels are outdated, lacking in sufficient specificity and sensitivity, and warrant substantial improvement. Methods: Using a novel approach, we accessed and integrated 6 Infinium HumanMethylation450 BeadChip datasets from various research groups within the Gene Expression Omnibus (GEO) database, from which we selected probes that were highly methylated in Barrett’s (beta ≥ 0.30) and mostly unmethylated in normal esophageal and gastric tissues (beta ≤ 0.05) yielding 30 candidate BE-specific markers. All 30 were identified from research groups who used microdissection and/or careful histopathologic review of each tissue biopsy. We then analyzed 248 BE, 184 normal esophageal, and 101 normal gastric tissue samples from our archives. After designing qMSP primers and probes, and further testing, we assayed 14 candidate markers in 21 matched normal-BE tissue pairs, 8 matched normal-BE-tumor tissue triplets, and 17 matched normal-tumor tissue pairs. Results: All 14 biomarkers tested exhibited significantly higher methylation levels in BE DNAs vs. matched normal DNAs (p < 0.01 by Wilcoxon rank-sum test). 4 of the 14 markers showed significantly higher methylation levels in tumor DNAs vs. matched normal DNAs (P < 0.01). 3 markers were statistically significantly different across matched normal, BE, and tumor tissue triplets (Kruskal-Wallis test, p < 0.05), and these 3 markers were used to develop a diagnostic panel for future testing on minimally invasively obtained cytologic DNAs from sponge-capsule samples. Conclusion: This discriminatory biomarker panel shows potential for BE diagnosis using an inexpensive, minimally invasive sampling technique and thus merits further study in case-control sponge studies. Due to our systematic and rigid method of selecting these markers, these genes are expected to be extremely important for the diagnosis of BE.

369. GENOMIC ALTERATIONS AND SURVIVAL AFTER TREATMENT OF EAC BY SURGERY WITHOUT CHEMO- OR RADIOTHERAPY

Abstract Prognostic biomarkers for esophageal adenocarcinoma (EAC) remain elusive but may be helpful in treatment planning, as TNM staging often fails to accurately predict survival. Advanced stage patients are treated by peri-operative chemo/radiotherapy; biomarkers associated with survival are thus likely to represent both true prognostic markers as well as markers of response to chemo/radiotherapy. Here we present first-pass genomic findings for a cohort of optimally staged EAC patients treated by surgery alone. DNA was extracted from fresh frozen EAC and matching normal squamous esophageal tissues obtained at esophagectomy from 65 consenting patients who did not receive any neo- or adjuvant chemo- or radiotherapy due to institutional preference at the time of tissue collection. Library preparation and sequencing for a targeted 600 cancer-associated gene panel designed using the Roche/NimbleGen EZ Choice Library NimbleDesign platform were conducted on Illumina-based platforms followed by analysis with an accredited bioinformatics pipeline. The panel design incorporated the entire coding region plus 10 bp of flanking intron of each gene. Mean age 70 yrs; 88% male. All patients underwent an en bloc esophagectomy (mean 35 lymph nodes). TNM (7th ed.) stages were 61% Stage 3, 18% stage 2, 14% stage 1, 6% stage 4. At median 9.5 years follow-up, 20 (31%) patients remained alive. Copy number changes were common, reflecting chromosomal instability. Less than 8% of patients had high tumour mutational burden (&amp;gt;10mut/MB), associated with mutations in DNA repair genes. Commonest somatic mutations included TP53 (HR: 0.43) and LRP1B (HR: 1.7). Pathways implicated included RTK signalling (PIK3CA, EGFR, KRAS), DNA damage (BRCA1, PALB2, MGMT) and histone remodelling (ARID1A, ARID2, KMT2D). Peri-operative treatment means linking genomic changes independent of stage to clinical outcomes is difficult. Neoadjuvant and/or adjuvant treatment is now standard for T2–4 or node positive EAC. This cohort of 65 patients, most of whom had stage 3 disease and all of whom underwent optimal staging with en bloc esophagectomy, represents a unique opportunity to analyze characteristics of EAC without confounding by peri-operative treatment. An EAC-specific DNA sequencing panel approach offers potential for personalized therapy.

Potentially functional genetic variants of the notch signaling pathway genes predict survival of Chinese patients with esophageal squamous cell carcinoma.

The Notch signaling pathway is involved in the progression of esophageal squamous cell carcinoma (ESCC), although the roles of single nucleotide polymorphisms (SNPs) of the Notch signaling pathway genes in the process remain unknown. The present study included 1009 patients with histopathologically diagnosed ESCC at Fudan University Shanghai Cancer Center. Two-stage multivariate Cox proportional hazards regression analysis was used to estimate associations between 13,248 SNPs in 103 Notch signaling pathway genes and overall survival of the patients. We found that overall survival of the patients was significantly associated with genotypes of HDAC9 rs1729318 (AT+TT vs. AA: hazard ratio =1.44, 95% confidence interval =1.16-1.80, pcombined =0.001) and HDAC9 rs1339555498 (GT + TT vs. GG: hazard ratio =1.38, 95% confidence interval =1.10-1.74, pcombined =0.005). Further receiver operator characteristic (ROC) curve analysis indicated that the model with both available clinical factors and these two SNPs improved the area under the ROC curve compared to the model with clinical factors only (1-year: 0.66 vs. 0.64, p= 0.034). Additional expression quantitative trait loci analysis showed that the rs1729318 T variant genotypes were associated with increased mRNA expression levels of HDAC9 in normal esophageal muscular tissue (p= 0.003). The results suggest that these two potential functional SNPs on HDAC9 may serve as biomarkers for predicting survival of ESCC patients. However, further studies are needed to confirm these findings.

IPO5 Mediates EMT and Promotes Esophageal Cancer Development through the RAS-ERK Pathway.

Objective In the development of many tumors, IPO5, as a member of the nuclear transporter family, exerts a significant function. Also, IPO5 is used as a therapeutic target for tumors based on some reports. By studying IPO5 expression in esophageal cancer tissues, the mechanism associated with IPO5 improving esophageal cancer development was explored in this study. Methods To gain differentially expressed genes, this study utilized mRNA microarray and TCGA database for comprehensive analysis of esophageal cancer tissues and normal esophageal cancer tissues, and then the differentially expressed gene IPO5 was screened by us. To assess esophageal cancer patients' prognosis, this study also applied the Kaplan-Meier analysis, and we also conducted the GSEA enrichment analysis to investigate IPO5-related signaling pathways. This study performed TISIDB and TIMER online analysis tools to study the correlation between IPO5 and immune regulation and infiltration. We took specimens of esophageal cancer from patients and detected the expression of IPO5 in tumor and normal tissues by immunohistochemistry. The IPO5 gene-silenced esophageal cancer cell model was constructed by lentivirus transfection. Through the Transwell invasion assay, CCK-8 assay, and cell scratch assay, this study investigated the effects of IPO5 on cell propagation, invasion, and transfer. What is more, we identified the influences of IPO5 on the cell cycle through flow cytometry and established a subcutaneous tumor-forming model in nude mice. Immunohistochemistry was used to verify the expression of KI-67, and this study detected the modifications of cell pathway-related proteins using Western blot and applied EMT-related proteins to explain the mechanism of esophageal cancer induced by IPO5. Results According to database survival analysis, IPO5 high-expression patients had shorter disease-free survival than IPO5 low-expression patients. Compared to normal tissues, the IPO5 expression in cancer tissues was significantly higher in clinical trials (P < 0.05). Through TISIDB and TIMER database studies, we found that IPO5 could affect immune regulation, and the age of IPO5 expression grows with the increase of immune infiltration level. The IPO5 expression in esophageal cancer cells was higher than normal, especially in ECA109 and OE33 cells (P < 0.01). After knocking out IPO5 gene expression, cell proliferation capacity and invasion capacity were reduced (P < 0.05) and decreased (P < 0.01) in the IPO5-interfered group rather than the negative control group. The growth cycle of esophageal carcinoma cells was arrested in the G2/M phase after IPO5 gene silencing (P < 0.01). Tumor-forming experiments in nude mice confirmed that after IPO5 deletion, the tumor shrank, the expression of KI67 decreased, the downstream protein expression level of the RAS pathway decreased after sh-IPO5 interference (P < 0.01), and the level of EMT marker delined (P < 0.05). Conclusion In esophageal cancer, IPO5 is highly expressed and correlates with survival rate. Esophageal cancer cell growth and migration were significantly affected by the inhibition of IPO5 in vitro and in vivo. IPO5 mediates EMT using the RAS-ERK signaling pathway activation and promotes esophageal cancer cell development in vivo and in vitro.

FBXO39 predicts poor prognosis and correlates with tumor progression in cervical squamous cell carcinoma

BackgroundCancer/testis antigen (CTA) is a class of antigen molecules mainly expressed in the germinal epithelium of testis and some tumor tissues. FBXO39, also known as F-box protein 39, is a crucial CTA molecule. F-box protein 39 (FBXO39) is overexpressed in cervical squamous cell carcinomas (CESCs), however its function in cancer development and clinical significance are still unknown. MethodsWe used paraffin-embedded tumor tissues from 124 patients and fresh-harvested and paired adjacent normal esophageal tissues from 15 CESC patients who underwent primary surgical resection in Xijing Hospital between 2015 and 2020. The expression level of FBXO39 was evaluated through immunohistochemistry, Western Blot and q-PCR. Prognostic and survival analyses were conducted using univariate/multivariate analysis and log-rank analysis with SPSS 23.0. CCK-8, wound-healing and Transwell assays were applied to demonstrate that FBXO39 promoted the proliferation, migration and invasion. Finally, we constructed a xenografts model of the C-33A cell lines to observe the effect of FBXO39 on tumorigenesis in vivo. ResultsImmunohistochemical results showed that FBXO39 was highly expressed in cancer tissues than in corresponding non-cancer tissues. Similarly, we proved this result at protein and mRNA level by Western-Blotting and q-PCR. Prognostic and OS analyses showed that the FBXO39 expression level was an individual prognostic factor in CESC patients. CCK-8, wound-healing and Transwell assays proved that the overexpression of FBXO39 in Si-Ha cells promoted the proliferation, migration and invasion of the cells. Knocking down FBXO39 in C-33A cells inhibited the proliferation, migration and invasion of cells. The experimental results of xenografts model in nude mice showed that the knockdown of FBXO39 in C-33A cells slowed down the growth of tumor. ConclusionFBXO39 is a poor prognostic factor of cervical squamous cell carcinoma, which may provide a novel therapeutic target for CESC.

KIFC3 promotes proliferation, migration and invasion of esophageal squamous cell carcinoma cells by activating EMT and β-catenin signaling.

BACKGROUNDEsophageal squamous cell carcinoma (ESCC) is one of the most common malignancies. A total of 45 kinesin superfamily proteins (KIFs) have been identified in humans, among which several family members have demonstrated varied functions in tumor pathobiology via different mechanisms, including regulation of cell cycle progression and metastasis. KIFC3 has microtubule motor activity and is involved in cancer cell invasion and migration, as well as survival. However, the role of KIFC3 in ESCC is still unknown. AIMTo evaluate the role of KIFC3 in ESCC and the underlying mechanisms.METHODSExpression of KIFC3 was evaluated in ESCC tissues and adjacent normal esophageal tissues. The prognostic value of KIFC3 was analyzed using Kaplan–Meier Plotter. Colony formation, EdU assays, cell cycle analysis, Transwell assay, immunofluorescence, and western blotting were performed in ESCC cell lines after transfection with pLVX-Puro-KIFC3-shRNA- and pLVX-Puro-KIFC3-expressing lentiviruses. A xenograft tumor model in nude mice was used to evaluate the role of KIFC3 in tumorigenesis. Inhibitor of β-catenin, XAV-939, was used to clarify the mechanism of KIFC3 in ESCC. To analyze the differences between groups, t test and nonparametric tests were used. P < 0.05 was considered statistically significant.RESULTSImmunohistochemical staining indicated that KIFC3 was upregulated in ESCC tissues compared with adjacent normal tissues. Kaplan–Meier Plotter revealed that overexpressed KIFC3 was associated with poor prognosis in ESCC patients. Colony formation and EdU assay showed that KIFC3 overexpression promoted cell proliferation, while KIFC3 knockdown inhibited cell proliferation in ESCC cell lines. In addition, cell cycle analysis showed that KIFC3 overexpression promoted cell cycle progression. KIFC3 knockdown suppressed ESCC tumorigenesis in vivo. Transwell assay and western blotting revealed that KIFC3 overexpression promoted cell migration and invasion, as well as epithelial–mesenchymal transition (EMT), while KIFC3 knockdown showed the opposite results. Mechanistically, KIFC3 overexpression promoted β-catenin signaling in KYSE450 cells; however, the role of KIFC3 was abolished by XAV-939, the inhibitor of β-catenin signaling.CONCLUSIONKIFC3 was overexpressed in ESCC and was associated with poor prognosis. Furthermore, KIFC3 promoted proliferation, migration and invasion of ESCC via β-catenin signaling and EMT.

EIF6 is potential diagnostic and prognostic biomarker that associated with 18F-FDG PET/CT features and immune signatures in esophageal carcinoma

BackgroundAlthough eukaryotic initiation factor 6 (eIF6) is a novel therapeutic target, data on its importance in the development of esophageal carcinoma (ESCA) remains limited. This study evaluated the correlation between eIF6 expression and metabolic analysis using fluorine-18 fluorodeoxyglucose (18F-FDG) -Positron emission tomography (PET) and immune gene signatures in ESCA.MethodsThis study employed The Cancer Genome Atlas (TCGA) to analyze the expression and prognostic value of eIF6, as well as its relationship with the immune gene signatures in ESCA patients. The qRT-PCR and Western blot analyses were used to profile the expression of eIF6 in ESCA tissues and different ESCA cell lines. The expression of tumor eIF6 and glucose transporter 1 (GLUT1) was examined using immunohistochemical tools in fifty-two ESCA patients undergoing routine 18F-FDG PET/CT before surgery. In addition, the cellular responses to eIF6 knockdown in human ESCA cells were assessed via the MTS, EdU, flow cytometry and wound healing assays.ResultsOur data demonstrated that compared with the normal esophageal tissues, eIF6 expression was upregulated in ESCA tumor tissues and showed a high diagnostic value with an area under curve of 0.825 for predicting ESCA. High eIF6 expression was significantly correlated with shorter overall survival of patients with esophagus adenocarcinoma (p = 0.038), but not in squamous cell carcinoma of the esophagus (p = 0.078). In addition, tumor eIF6 was significantly associated with 18F-FDG PET/CT parameters: maximal and mean standardized uptake values (SUVmax and SUVmean) and total lesion glycolysis (TLG) (rho = 0.458, 0.460, and 0.300, respectively, p < 0.01) as well as GLUT1 expression (rho = 0.453, p < 0.001). A SUVmax cutoff of 18.2 led to prediction of tumor eIF6 expression with an accuracy of 0.755. Functional analysis studies demonstrated that knockdown of eIF6 inhibited ESCA cell growth and migration, and fueled cell apoptosis. Moreover, the Bulk RNA gene analysis revealed a significant inverse association between eIF6 and the tumor-infiltrating immune cells (macrophages, T cells, or Th1 cells) and immunomodulators in the ESCA microenvironment.ConclusionOur study suggested that eIF6 might serve as a potential prognostic biomarker associated with metabolic variability and immune gene signatures in ESCA tumor microenvironment.

Overexpression of GNA13 correlates with poor prognosis in esophageal squamous cell carcinoma after esophagectomy.

This study aimed to explore the expression and clinical implication of guanine nucleotide-binding protein alpha 13 (GNA13) in esophageal squamous cell carcinoma (ESCC). We first employed western blot analysis to test the GNA13 protein expression level in ESCC tissues. Subsequently, we used immunohistochemistry assays to detect the GNA13 in ESCC specimens from 173 patients who underwent esophagectomy. Survival analysis was performed to define the impact of GNA13 expressions on the prognosis of the ESCC patients based on the clinical and follow-up data. The GNA13 protein was shown to be considerably higher in ESCC tissues than in normal esophageal tissues. The level of expression was closely related to the tumor, node, TNM stage, and tumor size. More importantly, ESCC patients with high GNA13 expression carried an increased risk of tumor recurrence compared to those with low GNA13 expression. In addition, a high GNA13 expression level could independently predict worse overall survival and disease-free survival in ESCC. GNA13 could be a novel prognostic biomarker for ESCC patients after esophagectomy.

Abstract 2805: Mass spectrometry detects lower frequency of routine markers and consistent overexpression of novel disease drivers in Barrett's-related esophageal cancer

Abstract Introduction: The incidence of esophageal adenocarcinoma (EAC) has increased more than any other cancer in our lifetime. Barrett’s esophagus (BE) is the only known precursor to EAC. At present, HER-2 and anti-PD-L1 therapies are FDA-approved for EAC, and off-label use of EGFR-directed therapy has been reported. Our study aims to use liquid chromatography mass spectrometry (LC/MS) to measure the expression of oncoproteins that are currently approved-drug targets in EAC and explore new promising targets for drug development and diagnostic applications for EAC and BE. Methods: EAC patients who underwent esophagectomy without chemo-radiation were identified. BE patients with subsequent progression to EAC and those without progression for &amp;gt;10 years were identified. Normal esophageal mucosa tissues with no history of progression were analyzed for baseline expression. Tissue sections were microdissected to isolate pure EAC, BE and normal mucosal cells from stroma. These biopsies were solubilized for LC/MS-based quantification of 80+ clinically relevant tumor markers including novel markers which are believed to play a role in the disease progression from BE to EAC. The expression trends of these markers were scrutinized by immunofluorescence (IF) in a subset of specimens/biomarkers to confirm mass spec results. Results: A total of 159 tissues were analyzed via LC-MS. In 55 EAC tissues - HER2, PD-L1 and EGFR were overexpressed in 16.3%, 0%, and 0% of patients, respectively. New markers DAD1 and S100P were noted to have high (7,634.0 and 27,222.6 attomoles/ug) and consistent (98.1% and 94.5%) overexpression in EAC samples. Expression of HER2, S100P and DAD1 were significantly higher in EAC compared to 23 normal esophageal mucosa specimens. There was no statistical difference in the expression of PD-L1 and EGFR between EAC and healthy mucosal samples. 45 BE biopsies progressed to EAC within a mean of 380 days, while the remaining 36 BE specimens did not progress over a 10-year period. 8 novel biomarkers were expressed at a higher level in BE tissue that progressed to cancer when compared to esophageal tissue that remained stable over 10 years. The expression trends observed via LC/MS were confirmed via IF in DAD1, S100P and two other novel markers. Discussion: LC-MS revealed low prevalence of target proteins in currently approved EAC therapies and high levels of newly discovered disease drivers that should be explored as possible therapy targets or diagnostic markers. The differential overexpression of the novel markers in progressive and non-progressive BE tissues, as well as the consistent overexpression of these markers in EAC tumors, demonstrates the potential for early detection of esophageal carcinogenesis and/or reveals potential therapeutic programs for an indication that lacks highly efficacious targeted therapy options. Citation Format: Christopher Hartley, Ajay Bansal, Catherine Hagen, Olivia Driscoll, Seyun Oh, Joe Abdo, Sumeet K. Mittal. Mass spectrometry detects lower frequency of routine markers and consistent overexpression of novel disease drivers in Barrett's-related esophageal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2805.

Identification of Tumor Antigens and Immune Subtypes of Esophageal Squamous Cell Carcinoma for mRNA Vaccine Development.

Purpose: The applicability of mRNA vaccines against esophageal squamous cell carcinoma (ESCC) remains unclear. Here, we identified potential antigens for developing mRNA vaccines against ESCC and characterized immune subtypes to select appropriate patients for vaccination. Methods: RNA-seq, genetic alteration data, and corresponding clinical information of ESCC patients were obtained from the Cancer Genome Atlas (TCGA) database. The RNA-seq data of normal esophageal tissue were obtained from the Genotype-Tissue Expression (GTEx) database. Potential tumor antigens were screened by analyzing differentially expressed and mutated genes and potential antigens with significant differences in prognosis were screened using the Kaplan-Meier method. The proportion of immune cell infiltration in the tumor microenvironment was estimated using CIBERSORT and MCPcounter, and the correlation of potential antigens with antigen-presenting cells and major histocompatibility complex class II was analyzed. Subsequently, immune subtypes were constructed using consensus clustering analysis and characterized by single-sample gene set enrichment analysis and weighted gene co-expression network analysis (WGCNA). The Genomics of Drug Sensitivity in Cancer (GDSC) database was used to analyze the drug sensitivity of different immune subtypes. Results: Four overexpressed and mutated tumor antigens associated with antigen presentation and poor prognosis were identified in ESCC, including NLRC5, FCRL4, TMEM229B, and LCP2. By consensus clustering, we identified two immune-associated ESCC subtypes, immune subtype 1 (IS1) and immune subtype 2 (IS2); the prognosis of the two subtypes was statistically different. In addition, the two immune subtypes had distinctly different cellular, molecular, and clinical characteristics. IS1 patients have a distinct immune “hot” phenotype with strong immune tolerance, whereas patients with IS2 have an immune “cold” phenotype. Differential expression of immune checkpoints and immunogenic cell death modulators was observed between the different immune subtypes. Finally, we found that IS1 and IS2 patients showed different drug sensitivities to common anti-tumor drugs, possibly facilitating the development of individualized treatment regimens for patients. Conclusion: NLRC5, LCP2, TMEM229B, and FCRL4 are potential antigens for ESCC mRNA vaccines, and such vaccines may be more suitable for IS2 patients. This study provides a theoretical basis for mRNA vaccines against ESCC, by identifying the critical characteristics to predict ESCC prognosis and select suitable patients for vaccination.

145: THE USE OF MACHINE LEARNING FOR REAL-TIME DETECTION OF OESOPHAGEAL AND GASTRIC CANCER BASED ON DIFFUSE REFLECTANCE SPECTROSCOPY: A VALIDATION STUDY

Abstract Background and aim The lack of a fast and accurate intraoperative tumour margin assessment tool contributes to high positive circumferential resection margin rates for oesophageal and gastric cancers, which is associated with local recurrence and poor long-term survival. Diffuse reflectance spectroscopy (DRS) can provide non-invasive, accurate, and real-time tissue classification based on quantification of light reflectance in tissue, providing a unique optical fingerprint of the tissue. This study aimed to validate the use of DRS for discrimination between normal and cancerous tissues based on ex-vivo gastric and oesophageal cancer resection specimens. Methods Consecutive patients undergoing resections for gastric and oesophageal carcinomas at a tertiary referral centre in London between July 2020 and June 2021 were included. Reflectance data in the 468-720 nm spectral range were recorded on ex-vivo specimens within 15 minutes of surgical specimen excision. Based on video recordings of the data acquisition procedure and probe tip tracking, sampling sites were correlated with histological reports manually labeled as either normal or cancer (Figure 1). Following spectral data normalisation and feature selection, four supervised machine learning approaches were tested for classification using 5-fold cross-validation. Overall classification accuracy and Area Under the Curve (AUC) across machine learning approaches were compared. Results A total of 32 patients (median age 68, 75% male) were included. 11,862 mean spectra (2990 spectra for normal oesophagus, 4628 for normal stomach, 2305 for gastric cancer, and 1939 for oesophageal cancer) were collected. For oesophageal cancer, classification accuracy was 96.22 ± 0.50 and AUC was 99.24 ± 0.19, while for gastric cancer, the accuracy was 93.86 ± 0.66, and AUC 98.50 ± 0.28 for gastric cancer. For both malignancy types, Extreme Gradient Boosting was the best performing classifier. The differences in spectral data between patients receiving neoadjuvant treatment and treatment naïve were non-significant. Conclusion Machine learning can be used to accurately differentiate between normal and cancerous oesophageal and gastric tissues in an ex-vivo setting, based on DRS data. Features used for class discrimination should be investigated to find possible physiological correlates of spectral data differences. Future intraoperative validation is required to assess the utility of this technology for real-time tumour margin mapping and its effect on long-term outcomes.

Identification and Validation of a Prognostic Immune-Related Gene Signature in Esophageal Squamous Cell Carcinoma.

Esophageal carcinoma (EC) is a common malignant cancer worldwide. Esophageal squamous cell carcinoma (ESCC), the main type of EC, is difficult to treat because of the widespread morbidity, high fatality rates, and low quality of life caused by postoperative complications and no specific molecular target. In this study, we screened genes to establish a prognostic model for ESCC. The transcriptome expression profiles of 81 ESCC tissues and 340 normal esophageal mucosal epithelium tissues were obtained from The Cancer Genome Atlas (TCGA) and Genotype-Tissue Expression (GTEx) cohorts. The transcriptome expression datasets of 19 esophageal squamous carcinoma cell lines were downloaded from Cancer Cell Line Encyclopedia (CCLE). The R software Limma package was used to identify 6,231 differentially expressed genes and 647 differentially expressed immune-related genes between normal and ESCC tissues. Gene functional analysis was performed using Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG). Weighted gene co-expression network analysis (WGCNA) was used to screen out 18 immune-related prognostic genes. We then established the prognostic and risk signature using these genes, and the patients were divided into low-risk and high-risk groups. Compared with high-risk group patients, the low-risk group patients had longer overall survival. M1 macrophages and resting dendritic cells were differentially distributed between the low-risk and high-risk groups and were related to patient survival. We also examined the functional immune cell and immune molecule levels in low-risk and high-risk group patients, with significant differences in the tumor microenvironment between the two groups. To further verify the accuracy of the prognostic risk model, we performed area under the ROC curve (AUC) analysis. The AUC value was 0.931 for the prognostic risk, which was better than the microsatellite instability (MSI) and Tumor Immune Dysfunction and Exclusion (TIDE) scores. In conclusion, we found 18 immune-related prognostic genes related to the occurrence of ESCC and established a prognostic model for predicting disease severity.

PABPC1-induced stabilization of IFI27 mRNA promotes angiogenesis and malignant progression in esophageal squamous cell carcinoma through exosomal miRNA-21-5p

BackgroundEmerging evidence has demonstrated that RNA-binding protein dysregulation is involved in esophageal squamous cell carcinoma (ESCC) progression. However, the role of poly (A) binding protein cytoplasmic 1 (PABPC1) in ESCC is unclear. We therefore aimed to explore the functions and potential mechanisms of PABPC1 in ESCC progression.MethodsPABPC1 expression was characterized using immunohistochemistry and qRT-PCR in ESCC tissues and cell lines. Chromatin immunoprecipitation (ChIP) and luciferase reporter assays were used to detect histone acetylation in the promoter region of PABPC1. A series of in vitro and in vivo assays were further applied to elucidate the functions and underlying molecular mechanisms of PABPC1 in ESCC angiogenesis and malignant procession.ResultsPABPC1 expression was upregulated in ESCC tissues compared with in normal esophageal epithelial tissues. Elevated PABPC1 expression was correlated with tumor cell differentiation and poor prognosis in patients. Sp1 and p300 cooperated to increase the level of H2K37ac in the PABPC1 promoter. Functionally, PABPC1 overexpression enhanced esophageal squamous cell proliferation and invasion by activating the IFN/IFI27 signaling pathway. PABPC1 interacted with eIF4G to increase the stability of IFI27 mRNA by competing with RNA exosomes in ESCC. Furthermore, PABPC1/IFI27 could increase miR-21-5p expression to enable exosomal delivery of miR-21-5p to human umbilical vein endothelial cells to increase angiogenesis via inhibiting CXCL10.ConclusionPABPC1 plays a critical role in ESCC malignant progression by interacting with eIF4G to regulate IFI27 mRNA stability and promote angiogenesis via exosomal miR-21-5p/CXCL10. Taken together, our results suggest that PABPC1 is a promising therapeutic target for ESCC.

Overexpression of OTX1 promotes tumorigenesis in patients with esophageal squamous cell carcinoma

BackgroundEsophageal squamous cell carcinoma (ESCC) is a multifactorial disease and the sixth leading cause of death from cancer worldwide. Patients with ESCC usually have a short survival period due to the late stage at diagnosis. Incidence rates of ESCC remain high among the elderly. With recent advances, it has been demonstrated that ESCC tumors display a unique genetic profile. This study aimed to examine the possible function of OTX1 in ESCC. MethodsWe collected paraffin-embedded tumor tissues from 107 patients originally diagnosed with ESCC at Xijing Hospital and fresh-harvested and paired adjacent normal esophageal tissues from 15 ESCC patients undergoing curative resection. The expression level of OTX1 was evaluated through immunohistochemistry and western blot. Prognostic and survival analyses were conducted using univariate/multivariate analysis and log-rank analysis with SPSS 23.0. Cell models and xenograft models were used to examine the overexpression of OTX1 in vivo and in vitro. ResultsOTX1 was overexpressed in ESCC tissues compared with normal esophageal tissues. Both the mRNA expression level and protein level of OTX1 were higher than they were in paired normal tissue. Prognostic and OS analyses showed that the OTX1 expression level was an individual prognostic factor in ESCC patients. Cell viability was significantly promoted when OTX1 was overexpressed in ESCC cell, Furthermore, downregulating OTX1 in EC109 cell significantly attenuated the cell proliferation migration and invasion. Flow cytometric detection showed that cells overexpressing OTX1 were predominantly in the S and G2&M phases. In the xenograft model, both tumor size and weight in the OTX1 overexpression group were significantly larger than those in the control group. ConclusionOTX1 is an independent prognostic factor of ESCC and contributes to tumorigenesis both in vivo and in vitro.

CD98hc has a pivotal role in maintaining the immuno-barrier integrity of basal layer cells in esophageal epithelium

ObjectivesThe current study aims to find the linker between esophageal epithelial carcinogenesis and chronic inflammation and the origin of hyperproliferative cells in precancerous lesions of esophageal squamous cell carcinoma (ESCC).Materials and methodsTwenty one normal esophageal tissues from cadavers and 180 paired tissues from 60 surgical resected ESCC specimens were utilized for immunohistochemistry staining against CK14, CK6, CD98hc and Ki67. NE6 cell line was treated with H2O2 to mimic chronic inflammation microenvironment and TPA for malignant orientated transformation. Cell proliferation and CD98hc mRNA were assessed by CCK8 assay and RT-qPCR.ResultsCD98hc expression was correlated with chronic inflammation severity, precancerous lesion stage, and epithelial cell proliferative activity. CD98hc expression and proliferation rate of NE6 were up regulated by low dose H2O2 treatment and long term TPA treatment. The proliferating cells in hyperplastic and dysplastic tissues could be divided into two patterns by the expression of CK14, CD98hc, CK6 and Ki67: CK14+CD98hc+CK6−Ki67− in basal cells with CK14−CD98hc−CK6+Ki67+ in proliferating cells and CK14+CD98hc+CK6+Ki67+ in both basal cells and proliferating cells.ConclusionsOur study revealed that CD98hc was a marker of cells originated from basal cell in esophagus, ectopic expression of CD98hc in hyperplastic/dysplastic cells by chronic inflammation stimulation crippled the linkage between basal cell and basement membrane, sabotaged the integrity of the barrier in between lamina propria and epithelium, subsequentially initiate carcinogenesis.

Estimating the prognosis of esophageal squamous cell carcinoma based on The Cancer Genome Atlas (TCGA) of m6A methylation-associated genes.

N6-methyladenosine (m6A) mRNA modification is the most prevalent in certain tumors. However, its expression profile and prognostic value in human esophageal squamous cell carcinoma (ESCC) remains unknown. Herein, we performed an extensive investigation of the m6A-associated gene expression profile and determined its significance in the prognosis of ESCC. We received the RNA expression profiles of 81 ESCC tissues and one normal esophageal tissue from The Cancer Genome Atlas (TCGA) database. Kaplan-Meier (KM) survival analysis was used to assess the predictability of m6A methylation-associated gene expression in ESCC prognosis. In addition, univariate and multivariate Cox regression, as well as least absolute shrinkage and selection operator (LASSO) regression models were employed for the establishment of prognostic signatures. Lastly, KM survival analysis, proportional hazard models, and receiver operating characteristic (ROC) curves were used to verify the prognostic value. Moreover, we also investigated the associations among the m6A prognostic signature, immune cell infiltration, and programmed cell death-ligand 1 (PD-L1) expression. We demonstrated that YTHDF3 [hazard ratio (HR): 0.910; 95% confidence interval (CI): 0.832-0.995; P=0.038], RBM15 (HR: 0.721; 95% CI: 0.549-0.948; P=0.019), KIAA1429 (HR: 0.801; 95% CI: 0.664-0.967; P=0.021), and ALKBH5 (HR: 0.948; 95% CI: 0.895-1.003; P=0.0.064) overexpression predicted better overall survival (OS) of ESCC patients. Furthermore, based on prognostic factors, the high-risk (H-R) cohort was found to have worse survival than the low-risk (L-R) cohort (P<0.001). We revealed three m6A methylation-associated genes that were closely correlated with enhanced survival in ESCC patients. In addition, we generated an independent prognostic signature based on the expression of YTHDF3, RBM15, KIAA1429, and ALKBH5 genes. The results revealed significantly higher proportions of CD8+ T cells and higher expression of PD-L1 in the H-R group.