Jin-Hui Shen

Expression and prognostic significance of THBS1, Cyr61 and CTGF in esophageal squamous cell carcinoma

BackgroundThrombospondin1 (THBS1), cystene-rich protein 61 (Cyr61) and connective tissue growth factor (CTGF) are all involved in the transforming growth factor-beta (TGF-β) signal pathway, which plays an important role in the tumorigenesis. The purpose of this study is to explore the expression and prognostic significance of these proteins in esophageal squamous cell carcinoma (ESCC).MethodsWe used immunohistochemistry and western blotting to examine the expression status of THBS1, Cyr61 and CTGF in ESCC. Correlations of THBS1, Cyr61 and CTGF over-expressions with various clinicopathologic factors were also determined by using the Chi-square test or Fishers exact probability test. Survival analysis was assessed by the Kaplan-Meier analysis and the log-rank test. Relative risk was evaluated by the multivariate Cox proportional hazards model.ResultsTHBS1, Cyr61 and CTGF were all over-expressed in ESCC. THBS1 over-expression was significantly associated with TNM stage (P = 0.029) and regional lymph node involvement (P = 0.026). Kaplan-Meier survival analysis showed that over-expression of THBS1, Cyr61 or CTGF was related to poor survival of ESCC patients (P = 0.042, P = 0.020, P = 0.018, respectively). Multivariate Cox analysis demonstrated that Cyr61 and CTGF were independent factors in prognosis of ESCC.ConclusionCyr61, CTGF and THBS1 were all over-expressed in ESCC and might be new molecular markers to predict the prognosis of ESCC patients.

Autoantibodies as Potential Biomarkers for the Early Detection of Esophageal Squamous Cell Carcinoma

OBJECTIVES:Esophageal squamous cell carcinoma (ESCC) is one of the most frequent causes of cancer death worldwide and effective diagnosis is needed. We assessed the diagnostic potential of an autoantibody panel that may benefit early diagnosis.METHODS:We analyzed data for patients with ESCC and normal controls in a test cohort and a validation cohort. Autoantibody levels were measured against a panel of six tumor-associated antigens (p53, NY-ESO-1, matrix metalloproteinase-7 (MMP-7), heat shock protein 70 (Hsp70), peroxiredoxin VI (Prx VI), and BMI1 polycomb ring finger oncogene (Bmi-1)) by enzyme-linked immunosorbent assay.RESULTS:We assessed serum autoantibodies in 513 participants: 388 with ESCC and 125 normal controls. The validation cohort comprised 371 participants: 237 with ESCC, and 134 normal controls. Autoantibodies to at least 1 of 6 antigens demonstrated a sensitivity/specificity of 57% (95% confidence interval (CI): 52–62%)/95% (95% CI: 89–98%) and 51% (95% CI: 45–57%)/96% (95% CI: 91–99%) in the test and validation cohorts, respectively. Measurement of the autoantibody panel could differentiate early-stage ESCC patients from normal controls (sensitivity 45% (95% CI: 32–59%) and specificity 95% (95% CI: 89–98%) in the test cohort; 46% (95% CI: 35–58%) and 96% (95% CI: 91–99%) in the validation cohort). In either cohort, no significant differences were seen when patients were subdivided by age, gender, smoking status, size of tumor, site of tumor, depth of tumor invasion, histological grade, lymph node status, TNM stage, or early-stage and late-stage groups.CONCLUSIONS:Measurement of an autoantibody response to multiple tumor-associated antigens in an optimized panel assay, to help discriminate early-stage ESCC patients from normal controls, may aid in early detection of ESCC.

Neutrophil gelatinase-associated lipocalin and its receptor: independent prognostic factors of oesophageal squamous cell carcinoma

Aim Previous studies have shown that neutrophil gelatinase-associated lipocalin (NGAL) is overexpressed in oesophageal squamous cell carcinoma (ESCC) and closely associated with the invasiveness of ESCC cells. Recently, NGAL receptor (NGALR) was identified from ESCC cells, and was also found to be increased in ESCC. The purpose of this study was to reveal the clinical significance of NGAL and/or NGALR in ESCC. Methods Tissue microarray was performed to detect expression of NGAL and NGALR in 222 ESCC specimens. Pearson χ2 test was used to analyse correlations between NGAL and/or NGALR expression and clinicopathological features. Kaplan–Meier survival curves and the Cox proportional hazards regression model were used to evaluate the effect of NGAL and/or NGALR expression on prognosis of patients with ESCC. Results NGAL and NGALR were highly expressed in ESCC. χ2 test results showed no significant correlations between NGAL or NGALR expression and clinicopathological features. However, NGAL/NGALR coexpression correlated with histological differentiation grade (p=0.033). Survival analysis showed that positive expression of NGAL or NGALR was significantly associated with a poor prognosis for patients with ESCC (p=0.000 or p=0.002). Patients with positive expression of both NGAL and NGALR had a shorter survival time than those with negative expression of both (p=0.048). Multivariate analysis showed that both NGAL and NGALR were independent prognostic factors. Conclusion These results indicate that both NGAL and NGALR may be involved in the progression of ESCC and can be considered as independent prognostic factors of ESCC.

Phosphorylation of Fascin Decreases the Risk of Poor Survival in Patients With Esophageal Squamous Cell Carcinoma

Phosphorylation of fascin at serine 39 (phospho-S39-fascin) could inhibit its actin-binding and actin-bundling activities and decrease filopodia formation. However, the relationship between phospho-S39-fascin expression and clinicopathological parameters in tumors is still unknown. Here, Western blot analysis and IHC applied to tissue microarray technology were performed to examine the expression status of non-phosphorylated fascin (fascin) and phospho-S39-fascin and their impacts on the prognosis of patients with esophageal squamous cell carcinoma (ESCC). Fascin and phospho-S39-fascin expressions were tested by cytoplasmic staining. Among the 254 patients, 90 cases showed high expression of fascin and 87 cases showed high expression of phospho-S39-fascin. Survival analysis showed that high expression of fascin was significantly associated with a poor prognosis of the patients with ESCC (p = 0.004). In contrast, high expression of phospho-S39-fascin correlated significantly with an improved outcome of patients (p = 0.020). Multivariate analysis showed that both fascin and phospho-S39-fascin were independent prognostic factors. In a combined analysis, the patients with high expression of fascin and low expression of phospho-S39-fascin tumors had a shorter overall survival than those with high expression of both fascin and phospho-S39-fascin tumors (5-year overall survival rate: 28.7% vs 48.3%, p = 0.068). Our results suggest that high expression of fascin correlates with poor outcome and that high expression of phospho-S39-fascin decreases the risk of poor prognosis in ESCC. This manuscript contains online supplemental material at http://www.jhc.org. Please visit this article online to view these materials.

A three‐gene signature and clinical outcome in esophageal squamous cell carcinoma

It is increasingly apparent that cancer development depends not only on genetic alterations, but also on epigenetic changes involving histone modifications. GASC1, member of the histone demethylases affecting heterochromatin formation and transcriptional repression, has been found to be dysregulation in many types of cancers including breast cancer, prostate cancer, metastatic lung sarcomatoid carcinoma, and leukemia. In this study, we examined the expression of GASC1 and certain GASC1‐targeted genes (KLF4, MYC, SOX2, PPARG, MDM2, and NANOG) and identified a three‐gene prognostic signature (PPARG, MDM2, and NANOG), using risk scores based on immunohistochemical analyses of 149 tumor specimens from patients with esophageal squamous cell carcinoma (ESCC). The presence of a high‐risk three‐gene signature in the ESCC tumors was significantly associated with decreased overall survival (OS) of the patients. We validated the predictive value of the three‐gene signature in a second independent cohort of 101 patients with ESCC in order to determine whether it had predictive value. The results were similar to those in 149 patients. According to multivariate Cox proportional hazards analyses, the predictive model of a three‐gene signature was an independent predictor for OS (p = 0.005 in cohort 1, p = 0.025 in cohort 2). In addition, ROC analysis indicated that the predictive ability of the three‐gene model was more robust than that of a single biomarker. Therefore, our three‐gene signature is closely associated with OS among patients with ESCC and may serve as a predictor for the poor prognosis of ESCC patients.

Reduced nuclear and ectopic cytoplasmic expression of lysyl oxidase-like 2 is associated with lymph node metastasis and poor prognosis in esophageal squamous cell carcinoma ☆

Lysyl oxidase family members have various roles in cancer progression. The aim of this study was to investigate their expression and clinical significance in esophageal squamous cell carcinoma. We examined messenger RNA expression of lysyl oxidase family members including lysyl oxidase and lysyl oxidase-like proteins (lysyl oxidase L) in 10 esophageal squamous cell carcinoma cell lines and 83 pairs of tumor samples by quantitative real-time polymerase chain reaction. All except lysyl oxidase L3 were expressed at high levels in esophageal squamous cell carcinoma, but only lysyl oxidase L2 was associated with lymph node metastasis (P = .034). We examined lysyl oxidase L2 protein further by immunohistochemistry staining in 178 surgically resected esophageal squamous cell carcinoma tissue samples. The protein manifested decreased nuclear expression and increased cytoplasmic expression. Moreover, these 2 events both had significant correlation with the presence of lymph node metastasis (P = .001 and P < .001). Overall survival rates of the patients with esophageal squamous cell carcinoma with decreased nuclear expression or increased cytoplasmic expression of lysyl oxidase L2 were significantly lower than those of the patients with esophageal squamous cell carcinoma with the reverse expression pattern (P = .040 or P = .022). Multivariate analyses revealed that nuclear expression of lysyl oxidase L2 was an independent prognostic factor for esophageal squamous cell carcinoma. These results suggest that lysyl oxidase L2 exerts a critical effect on esophageal squamous cell carcinoma progression and can be a predictive marker of lymph node metastasis and outcome.

A molecular prognostic model predicts esophageal squamous cell carcinoma prognosis.

Background Esophageal squamous cell carcinoma (ESCC) has the highest mortality rates in China. The 5-year survival rate of ESCC remains dismal despite improvements in treatments such as surgical resection and adjuvant chemoradiation, and current clinical staging approaches are limited in their ability to effectively stratify patients for treatment options. The aim of the present study, therefore, was to develop an immunohistochemistry-based prognostic model to improve clinical risk assessment for patients with ESCC. Methods We developed a molecular prognostic model based on the combined expression of axis of epidermal growth factor receptor (EGFR), phosphorylated Specificity protein 1 (p-Sp1), and Fascin proteins. The presence of this prognostic model and associated clinical outcomes were analyzed for 130 formalin-fixed, paraffin-embedded esophageal curative resection specimens (generation dataset) and validated using an independent cohort of 185 specimens (validation dataset). Results The expression of these three genes at the protein level was used to build a molecular prognostic model that was highly predictive of ESCC survival in both generation and validation datasets (P = 0.001). Regression analysis showed that this molecular prognostic model was strongly and independently predictive of overall survival (hazard ratio = 2.358 [95% CI, 1.391–3.996], P = 0.001 in generation dataset; hazard ratio = 1.990 [95% CI, 1.256–3.154], P = 0.003 in validation dataset). Furthermore, the predictive ability of these 3 biomarkers in combination was more robust than that of each individual biomarker. Conclusions This technically simple immunohistochemistry-based molecular model accurately predicts ESCC patient survival and thus could serve as a complement to current clinical risk stratification approaches.

Reduced membranous and ectopic cytoplasmic expression of DSC2 in esophageal squamous cell carcinoma: an independent prognostic factor.

Desmocollin 2, a desmosomal component, is a key membrane glycoprotein critically involved in cell-cell adhesion and the maintenance of normal tissue architectures in epithelia. Reports exploring the link of desmocollin expression to cancers are limited. The aim of this study was to investigate the expression of desmocollin 2 in esophageal squamous cell carcinoma and, in particular, to determine the extent to which the patterns of desmocollin 2 expression correlated with the clinical parameters. Desmocollin 2 expression was evaluated in 308 cases of esophageal squamous cell carcinoma using immunohistochemistry. Western blotting and reverse transcriptase polymerase chain reaction were performed to characterize the relative expression levels of desmocollin 2 isoforms. The results indicated that desmocollin 2 expression was reduced significantly in esophageal cancer in both protein and messenger RNA levels and that this reduction was associated with poor survival (P = .011). The expression of desmocollin 2 was prominent in normal esophageal epithelia and highly differentiated esophageal tumors, but was reduced or absent in poorly differentiated tumor specimens. Furthermore, in 74.7% of tumor tissues, desmocollin 2 immunoreactivity displayed an abnormal cytoplasmic localization that was correlated with poor tumor differentiation (P < .001), regional lymph node metastasis (P < .001), pathologic tumor-node-metastasis stages (P < .001), and poor prognosis (P = .048). Multivariate analysis showed that desmocollin 2 expression level was an independent prognostic factor for esophageal squamous cell carcinoma. These data suggest that desmocollin 2 is involved in the transformation and development of esophageal tumors and that desmocollin 2 expression level and intracellular localization may serve as a predictor for patient outcomes.

Association of mu‐opioid receptor expression with lymph node metastasis in esophageal squamous cell carcinoma

The mu-opioid receptor (MOR), a membrane-bound G protein-coupled receptor, is the main target for opioids in the nervous system. MOR1 has been found in several types of cancer cells and reported to be involved in tumor progression and metastasis. However, the expression and clinical significance of MOR1 in esophageal squamous cell carcinoma (ESCC) remain unclear. In our study, the expression of MOR1 was confirmed in ESCC cell lines (KYSE180, KYSE150, and EC109) by Western blot. MOR1 was also detected on tissue microarrays of ESCC samples in 239 cases using immunohistochemical staining. We found that MOR1 was mainly located in the cytoplasm and occasionally occurred in the membrane or nucleus of ESCC cells. Moreover, results indicated that MOR1 expression in the cytoplasm was associated with lymph node metastasis (R = 0.164, P = 0.008, Kendalls tau-b-test). No more associations were found between MOR1 expression status and other clinical parameters. However, no statistical significant differences were found between MOR1 expression in the cytoplasm, nucleus/membrane, and the overall survival of ESCC patients (P = 0.848; P = 0.167; P = 0.428, respectively, log-rank test). Our results suggest that the cytoplasmic MOR1 may be a high-risk factor for lymph node metastasis of ESCC patients. We also hypothesize that MOR1 agonists used in ESCC patients should be prudent, and opioid receptor antagonists may be novel therapeutic drugs for ESCC patients.

κ-Opioid receptor in the nucleus is a novel prognostic factor of esophageal squamous cell carcinoma

Opioid receptors, members of the G-protein-coupled receptor superfamily, appear to be involved in cancer progression. However, the expression and significance of opioid receptors in esophageal squamous cell carcinoma (ESCC) remain unclear. In this study, we demonstrated by flow cytometry that μ, δ, and κ-opioid receptors (MOR, DOR, and KOR) are expressed to various degrees in ESCC cell lines. The KOR protein was further examined by several methods in ESCC cell lines and tissues. Immunocytochemical staining localized KOR to the cell membrane in KYSE180 cells and the nucleus in EC109 cells, whereas no signal or weak staining of the cytoplasm was observed in KYSE150 cells. The expression of KOR was confirmed in ESCC cells by Western blotting. Furthermore, immunohistochemistry staining showed that KOR was up-regulated in ESCC tissues compared with nontumorous esophageal epithelium (P = .004, χ(2) test). Moreover, high nuclear KOR expression was significantly correlated with lymph node metastasis in 256 ESCC cases (R = 0.144; P = .030, Kendall τB test). Patients with high nuclear KOR expression in ESCC had a significantly poorer prognosis (P = .001, log-rank test). Multivariate Cox analysis revealed that KOR in the nucleus was an independent prognostic factor (hazard ratio, 1.789; 95% confidence interval, 1.177-2.720; P = .006). Our results suggest that KOR is involved in the carcinogenesis or progression of ESCC and that nuclear KOR may be indicative of prognosis.