Original article: The expression of CFL1 and N‐WASP in esophageal squamous cell carcinoma and its correlation with clinicopathological features

Cofilin1 (CFL1) is an actin‐modulating protein, which belongs to the ADF/Cofilin family. Neural Wiskott–Aldrich syndrome protein (N‐WASP) is the key regulator of the actin cytoskeleton, a member of Wiskott‐Aldrich syndrome protein family. They have been suggested to be involved in cancer cell invasion and metastasis. In this study, the expression patterns of CFL1 and N‐WASP in normal esophageal mucosa and esophageal squamous cell carcinoma (ESCC) and their correlation with clinical characteristics were investigated. Immunohistochemical staining showed that CFL1 was expressed in nuclear and cytoplasm of cancer cells. However, N‐WASP was mainly found in the cytoplasm of the cancer cells. There were significant evidences that proved that CFL1 is correlated with clinicopathological factors in ESCC, such as infiltration depth, lymph node metastasis and pathological staging (P < 0.05). It is also proved that N‐WASP is related to lymph node metastasis and pathological staging in ESCC (P < 0.05). Kaplan–Meier analysis showed that there was no correlation between CFL1 and N‐WASP protein expression and survival (P > 0.05). Moreover, the mRNA expression of CFL1 and N‐WASP was detected by quantitative real time PCR in 70 tissue specimens. The results showed that CFL1 mRNA level was over‐expressed in ESCC tissue (P < 0.05), while N‐WASP mRNA expression level was not different between cancerous tissues and adjacent normal esophageal mucosa (P > 0.05). Also, CFL1 mRNA expression was significantly associated with regional lymph node metastasis and pathological staging (P < 0.05). Kaplan–Meier analysis showed that there was no correlation between CFL1 and N‐WASP mRNA expression and survival (P > 0.05). Our findings suggested that CFL1 and N‐WASP may play an important role in the tumorigenesis of ESCC, and to be the candidate novel biomarkers for the diagnosis and prognosis of ESCC. These findings may have implications for targeted therapies in patients with ESCC.     

Protein kinase D1 mRNA level may predict cancer‐specific survival in heavy smokers with esophageal squamous cell cancers

Protein kinase D1 (PRKD1) is a kinase that regulates various pathways, which involve in cell proliferation, apoptosis, cell adhesion and invasion. Although PRKD1 expression has been observed in many cancers, its role in esophageal squamous cell cancer (ESCC) has not been well reported. As its dysregulation in cancers is organ specific, we sought to investigate the potential role of PRKD1 in the progression of ESCC. Samples were collected from 178 patients with completely resected ESCCs at Sun Yat‐sen University Cancer Center, including 47 pairs of tumorous and non‐tumorous tissues. PRKD1 mRNA expression was investigated by quantitative real‐time polymerase chain reaction. Receiver operating characteristic (ROC) curve analysis was used to search for a feasible cut‐off point of PRKD1 mRNA levels for predicting cancer‐specific survival. Kaplan–Meier and multivariate Cox regression analysis were used to assess the prognostic value of PRKD1 mRNA level in ESCC patients. In result, upregulation of PRKD1 mRNA was detected in 55.3% (26/47) of ESCC tissues compared with paired non‐tumorous ones (P = 0.011). ROC analysis indicated 3.28 as a cut‐off point, and thus 72 and 106 tumors with low and high PRKD1 mRNA expression were categorized. High‐PRKD1 mRNA expression in tumors appeared with more frequency in heavy smokers (P = 0.002) and patients with advanced pathological T category (P = 0.034). Kaplan–Meier analysis indicated that patients with low‐PRKD1 mRNA had a longer cancer‐specific survival than the ones with high‐PRKD1 level (P = 0.044). Multivariate analysis showed that tumorous PRKD1 mRNA expression was an independent prognostic factor (hazard ratio: 1.538, 95% confidence interval: 1.018–2.323, P = 0.041) in resected ESCC. Subgroup analysis revealed that the discernibility of PRKD1 mRNA level on ESCC outcomes was only pronounced in heavy smokers (P = 0.002), but not in non‐heavy smokers (P = 0.870). PRKD1 might play a potential oncogenic role in ESCC. It might be an independent biomarker to predict prognosis in heavy smokers with ESCC.     

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, Kendall's 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.     

Original article: Upregulation of caspase‐3 expression in esophageal cancer correlates with favorable prognosis: an immunohistochemical study from a high incidence area in northern China

Caspase‐3 plays an important role as the key effector during apoptosis, but there are very few studies of caspase‐3 in esophageal squamous cell carcinoma (ESCC). The purpose of this study was to investigate the expression and prognostic significance of caspase‐3 in ESCC from Linzhou City, a high incidence area in northern China. All 64 patients underwent esophagectomy for ESCC between January 2002 and December were enrolled in this study. Caspase‐3 expression was assessed by immunohistochemistry (IHC) in primary ESCC and paired normal esophageal epithelium. The positive rate of caspase‐3 expression was higher in ESCC than in normal esophageal epithelium (79.7% vs. 50.0%, Chi‐square = 12.372, P= 0.001). Caspase‐3 expression was correlated with tumor cell differentiation (Phi = 0.717, P < 0.001), tumor infiltration depth (Phi =?0.334, P= 0.008), and pathologic TNM (pTNM) staging (rs =?0.268, P= 0.032). Patients in caspase‐3 positive group had a significantly better 5‐year overall survival than those in the negative group (77.4% vs. 35.9%, χ²= 7.344, P= 0.007). Our results showed that caspase‐3 expression was upregulated in ESCC compared with normal esophageal epithelium in population of Chinese high incidence area, and patients with caspase‐3 positive expression had better prognosis. Therefore, caspase‐3 immunostaining could be a simple and useful tool for predicting survival in ESCC patients.     

Prognostic relevance and therapeutic implications of centromere protein F expression in patients with esophageal squamous cell carcinoma

Centromere protein F (CENP‐F), a cell cycle‐regulated centromere protein, has been shown to affect numerous tumorigenic processes. This study aimed to clarify the prognostic significance of CENP‐F expression in patients with esophageal squamous cell carcinoma (ESCC). The levels of CENP‐F messenger RNA and protein were higher in ESCC cell lines than in the normal tissues. An immunohistochemical analysis of paired tissue specimens showed that the CENP‐F expression was higher in tumorous tissues than in the adjacent non‐tumorous tissues (P < 0.001). Moreover, there was a significant correlation between CENP‐F expression and gender (P = 0.012), clinical stage (P = 0.039), and T classification (P = 0.026). Patients with higher CENP‐F expression had shorter overall survival than those with lower CENP‐F expression (P = 0.009). Multivariate Cox analysis indicated that CENP‐F expression is an independent prognostic factor for overall survival (hazard ratio = 0.582, 95% confidence interval = 0.397–0.804, P = 0.041). Importantly, it was found that zoledronic acid (ZOL) could significantly enhance the chemotherapeutic sensitivity of ESCC cell lines with high CENP‐F expression to cisplatin, although ZOL alone only exhibited a minor inhibitory effect to ESCC cells. In summary, these findings demonstrate that CENP‐F may serve as a valuable molecular marker for predicting the prognosis of ESCC patients. In addition, the data indicate a potential benefit of combining ZOL with cisplatin in ESCC, suggesting that CENP‐F expression may have therapeutic implications.     

Expression of klotho and β‐catenin in esophageal squamous cell carcinoma,and their clinicopathological and prognostic significance

Esophageal carcinoma is one of the most common types of cancers in the world; the molecular mechanism underlying its tumorigenesis is still not well understood. This study was aimed at investigating the expression of klotho and β‐catenin in patients with esophageal squamous cell carcinoma (ESCC) and analyzing their association with clinicopathological variables and their effects on prognosis. The expression patterns of klotho and β‐catenin were determined by tissue microarray and immunohistochemical technique in ESCC and normal tissues, and their correlations with clinicopathological characteristics were investigated using univariate and multivariate analysis. The serum klotho levels in 40 ESCC patients and controls were measured by sandwich enzyme‐linked immunosorbent assay system (ELISA). The expression level of klotho was significantly lower in ESCC than in the adjacent noncancerous tissues (30 vs. 50%, P < 0.000), and the protein level was negative correlated with clinical staging, histological grade, lymph node metastasis, and invasion depth (P < 0.05). Whereas, the expression of β‐catenin was much higher in ESCC than their corresponding normal mucosa tissues (78.3 vs. 11.5%, P < 0.000), and the level of protein correlated only with histological grade and invasion depth (P < 0.05). Correlation analysis showed the expression level of klotho inversely correlated with that of β‐catenin (r = ?0.214, P < 0.01). Patients with klotho‐positive tumors had longer survival than those with klotho‐negative tumors (P < 0.01). Cox proportional hazards model analysis demonstrated that positive expression of klotho was an important factor indicating good prognosis (hazard ratio, 0.371; 95% confidence interval, 0.201–0.685; P < 0.01). ELISA showed that the level of serum klotho was markedly higher (461.50 ± 43.30 pg/mL) than control group (239.37 ± 20.65 pg/mL) (P < 0.001). Receiver operating characteristic analysis gave a cut‐off value of 327.031 of serum klotho with a sensitivity of 81.3% and specificity of 81.2% (P < 0.000). Our present study demonstrated for the first time that klotho might be a novel biomarker candidate for predicting progression and prognosis in patients with ESCC.     

Overexpression of cyclooxygenase‐2 is associated with chemoradiotherapy resistance and prognosis in esophageal squamous cell carcinoma patients

Our objective was to investigate whether cyclooxygenase‐2 (COX‐2) expression can predict the patient's response to chemoradiotherapy (CRT) and ensuing prognosis in esophageal squamous cell carcinoma (ESCC). The clinicopathological and follow‐up data of 112 patients with ESCC who underwent CRT from January 2001 to June 2006 were analyzed retrospectively. The immunohistochemical expression level of COX‐2 was examined for all biopsy specimens of primary tumors, and the correlation of COX‐2 expression with the patient's response to CRT and prognosis was examined. COX‐2 positive immunostaining was detected in 111 (99.1%) of the patients, including overexpression in 54 (48.2%) patients and low expression in 58 (51.8%) of the patients. The response of tumors with a low level expression of COX‐2 (70.7%, 41/58) was significantly higher than that of tumors with COX‐2 overexpression (42.6%, 23/54; P = 0.003). Patients with a low level of COX‐2 expression had a higher downstaged rate than those with a high level of COX‐2 expression (9/13 vs 2/8), but the difference was not statistically significant (P = 0.08). In the definitive CRT group (91 cases), COX‐2 overexpression was significantly associated with poor 3‐year overall survival (P = 0.028). Multivariate analysis showed that only metastatic stage (nonregional node metastasis) was an independent prognosis factor. The assessment of COX‐2 status may provide additional information to identify ESCC patients with poor chances of response to CRT and potential candidates for more individualized treatment.     

Pilot study special AT‐rich sequence‐binding protein 1 investigating as a potential biomarker for esophageal squamous cell carcinoma

In the present study, we aimed to evaluate the expression of special AT‐rich sequence‐binding protein 1 (SATB1) in esophageal squamous cell carcinoma (ESCC) and assess the correlation between its expression and the clinicopathological features and prognosis of the disease. SATB1 expression in ESCC tissue was determined by using immunohistochemical analysis, quantitative real‐time polymerase chain reaction, and western blot analysis. The relationship between SATB1 expression and clinicopathological features was examined by using the chi‐squared test, and the survival rate was calculated by using the Kaplan–Meier survival curve. The correlation between the indicators and patient survival was estimated by using a Cox regression analysis. High SATB1 expression in was detected in 48.3% and 7.8% of ESCC and normal esophagus tissues (P < 0.05), respectively. SATB1 expression did not significantly correlate with clinicopathological features. The Kaplan–Meier curve indicated that patients with high SATB1 expression had significantly shorter survival than those with low SATB1 expression. In a multivariate Cox regression model, high SATB1 expression was identified as an independent prognostic factor for patients with ESCC. In conclusion, these results suggest that high SATB1 expression is predictive of poor prognosis in ESCC and may be a promising new candidate for targeted therapies for ESCC.     

Serum soluble E‐cadherin is a potential prognostic marker in esophageal squamous cell carcinoma

E‐cadherin is a well‐documented tumor suppressor with downregulated expression in many cancer types. Upon proteolytic cleavage, a soluble form of 80‐kDa degradation fragment, known as soluble E‐cadherin (s‐Ecad), is present in circulation; its level in sera of cancer patients is significantly associated with metastasis, recurrence, and prognosis in some malignancies. The present study investigated the association of s‐Ecad with clinicopathological characteristics of patients with esophageal squamous cell carcinoma (ESCC) and its prognostic significance. A cohort of 97 patients who underwent surgery alone (n= 56) or neoadjuvant chemoradiation therapy and surgery (CRT) (n= 41) was recruited for this study. Serum samples were collected at operation (surgery group) and pre‐ and post‐CRT treatment (CRT group) for measurement of s‐Ecad protein by enzyme linked immunosorbent assay. Serum s‐Ecad levels were correlated with clinicopathological parameters as well as survival. Univariate analysis showed no significant relationship between serum s‐Ecad level and clinicopathological parameters for all sets of samples. Survival analysis showed that in patients who had surgical resection only, those with s‐Ecad levels equal to or below the median value survived significantly longer than those with levels above the median (median survival 25.6 vs. 14.1 months, P= 0.012). Multivariate analysis showed that pathological N stage, M stage, R category, and serum s‐Ecad level were significant independent prognostic factors for ESCC patients who underwent surgery only. The hazard ratio for s‐Ecad was 1.104 (95% CI: 1.026–1.187) and P= 0.008. Serum s‐Ecad was detected in ESCC patients and its potential as an independent prognostic marker requires further investigation.     

Preoperative level of serum amyloid A is superior to C‐reactive protein in the prognosis of esophageal squamous cell carcinoma

Preoperative elevations in the levels of serum amyloid A (SAA) or C‐reactive protein (CRP) have been reported to be prognostic indicators in several malignancies. The aim of this study is to evaluate the serum levels of SAA and CRP in the prognosis of esophageal squamous cell carcinoma (ESCC). In total, 252 patients with ESCC who had undergone surgery with curative‐intent were retrospectively recruited. The specificity, sensitivity, and prognostic value of SAA or CRP levels were measured as the area under the receiver operating characteristic (ROC) curve (AUC). The clinical value of SAA and CRP levels as prognostic indicators was evaluated using Cox's proportional hazards model. The 1‐, 3‐, and 5‐year overall survival (OS) rates for the entire cohort of patients with ESCC were 71.0%, 61.0%, and 43.0%, respectively. The correlation between the levels of SAA and CRP was significant (r² = 0. 685, P < 0.001). The ROC analysis showed that the levels of CRP were associated with a significantly lower overall accuracy than were the SAA levels (AUC, 0.615 vs. 0.880; P < 0.001). For the complete cohort, the median OS was 52.0 months longer in patients with low preoperative serum levels of SAA (72.0 months) compared with patients who had high SAA levels (20.0 months, P < 0.001). The median OS among patients with low CRP levels was also longer compared with the patients who had high CRP levels (72.0 vs. 51.0 months, respectively; P < 0.001). Subgroup analyses showed that the preoperative elevated levels of SAA could find significant differences in OS for stage I, stage II, and stage III (P < 0.001, P = 0.001, and P < 0.001, respectively), whereas the increased levels of CRP could only find a difference in OS for stage II cancers. After a multivariate analysis, preoperative elevated level of SAA was found to be an independently and significant prognostic factor (P < 0.001). Our study indicates that the preoperative levels of SAA and CRP can act as prognostic factors, and that elevated levels of these proteins are associated with negative effects on the survival of patients with ESCC. SAA showed a higher prognostic value than CRP in both cohort and subgroup analysis.     

Expression of breast cancer anti‐estrogen resistance 1 in relation to vascular endothelial growth factor,p53, and prognosis in esophageal squamous cell cancer

The purpose of this study was to clarify the role of breast cancer anti‐estrogen resistance 1 (BCAR1) expression in relation to vascular endothelial growth factor (VEGF), p53, and proliferation in esophageal squamous cell cancer (ESCC). Expression of BCAR1, VEGF, p53, and the ki‐67 proliferative index were examined by tissue microarray and immunohistochemistry in 106 specimens with ESCC and matched adjacent normal tissues. Among them, 40 cases were simultaneously examined by Western blot. Both Western blot and immunohistochemistry showed that BCAR1 expression was substantially higher in ESCC than in adjacent normal tissues (P < 0.001). BCAR1 expression was significantly connected with degree of tumor differentiation, with poorly differentiated tumors showing higher BCAR1 expression (P < 0.001). BCAR1 expression was significantly and positively correlated with VEGF and p53 expression levels (r= 0.541, P < 0.001; r= 0.374; P < 0.001) but not proliferative index (r= 0.44; P= 0.066). Additionally, a significant relationship was also observed between VEGF and p53 (r= 0.321; P= 0.001). Kaplan–Meier survival analysis revealed that patients with high BCAR1 expression had significantly shorter survival times than those with low BCAR1 expression levels (median survival 40 months vs. 27 months, P= 0.09). Multivariate analysis also revealed that levels of BCAR1 expression (hazard ratio 2.250, P= 0.015) was a significant and independent prognostic indicator. High expression of BCAR1 is associated with elevated VEGF and p53 expression levels, as well as poor prognosis in ESCC. Therefore, BCAR1 may be a potential candidate for predicting prognosis and a new therapy target for ESCC.     

Expression of Bmi-1 and PAI-1 in esophageal squamous cell carcinoma

AIM:To determine the correlation between invasiveness,migration and prognosis in esophageal squamous cell carcinoma(ESCC)and expression of the B-cellspecific Moloney leukemia virus insert site 1(Bmi-1)and plasminogen activator inhibitor-1(PAI-1).METHODS:Eighty previously untreated patients who underwent surgical excision of ESCC were included.The expression of Bmi-1 and PAI-1 was examined immunohistochemically in formalin-fixed paraffinembedded primary tissue specimens.The relationships between the expression of Bmi-1 and PAI-1,the clinicopathologic features of ESCC,and the survival rate of ESCC patients were also discussed.The correlation between Bmi-1 and PAI-1 protein expression in ESCC was analyzed.The relationship between Bmi-1 and PAI-1expression and ESCC prognosis was evaluated using a Cox regression model and Kaplan-Meier survival curve analysis.RESULTS:The rates of positive Bmi-1 and PAI-1 expression in ESCC were higher than those in normal esophageal tissue(P<0.05).The expression of Bmi-1and PAI-1 was correlated with depth of invasion and lymph node metastasis(P<0.05),but not with patient age,tumor size or nationality(P>0.05).The expression of Bmi-1 was positively correlated with that of PAI-1(P<0.05).The 10-year overall survival rate for all patients was 20%(16∕80).Univariate KaplanMeier survival analysis showed that patients with high expression of esophageal PAI-1 and Bmi-1 had lower survival,however,the difference was not statistically significant.Cox multivariate analysis showed that PAI-1and Bmi-1 were not independent factors for survival rate,while the depth of tumor invasion and metastasis were independent factors affecting patient survival.CONCLUSION:The expression of Bmi-1 and PAI-1plays a role in ESCC progression,and may be used as a prognostic marker in ESCC.     

Prognostic significance of gamma‐glutamyltransferase in patients with resectable esophageal squamous cell carcinoma

Gamma‐glutamyltransferase (GGT) is a membrane‐bound enzyme involved in the glutathione metabolism. Studies suggested that GGT was a marker of apoptotic balance and modulated tumor progression, invasion and drug resistance. Recently, GGT was shown to be associated with the progression of high‐grade esophageal epithelial dysplasia to invasive carcinoma. This study was conducted to investigate the value of pre‐therapeutic serum GGT levels as prognostic parameter in esophageal squamous cell carcinoma. Six hundred thirty‐nine resectable esophageal squamous cell carcinoma patients were recruited in this study and were stratified into two GGT risk groups. The association of pre‐therapeutic serum GGT levels and clinical–pathological parameters was examined. Univariate and multivariate survival analyses were performed. GGT serum levels were associated with gender, smoking status, TNM stage and lymph node involvement. Higher pre‐therapeutic serum GGT was found in males, smoker, advanced TNM stage and lymph node positive patients. Patients assigned to the low‐risk group had higher 5‐year overall survival rate (53.1% vs. 33.0%, P < 0.01) and disease‐free survival rate (45.2% vs. 23.4%, P < 0.01) than the high‐risk group. Patients with high‐risk group of GGT had 1.568 (95% confidence interval [CI], 1.259 ∼ 1.952) times the risk of death and 1.582 (95% CI, 1.286 ∼ 1.946) times the risk of disease recurrence contrast with those with low‐risk group of GGT. The pre‐therapeutic serum GGT is a novel independent prognostic parameter for disease‐free survival and overall survival in resectable esophageal squamous cell carcinoma.     

Amplification of the telomerase RNA component gene as a new genetic marker for disease progression and prognosis in esophageal squamous cell carcinoma

Amplification of the human telomerase RNA component (TERC) gene was found in esophageal squamous cell carcinoma (ESCC). However, its roles in the progression and prognosis of ESCC have not been well understood. The amplification of TERC in normal mucosa, low‐grade and high‐grade intraepithelial neoplasia, and invasive ESCC samples were evaluated using a fluorescence in situ hybridization assay. The amplification of TERC invariably occurred in high‐grade intraepithelial neoplasia and invasive ESCC, partially occurred in low‐grade intraepithelial neoplasia specimens, and seldom occurred in normal mucosa. The average signal ratio of TERC to chromosome 3 centromere‐specific probe (TERC/CSP3) was 1.00 ± 0.01 (average ± standard deviation) in normal mucosas, 1.01 ± 0.08 in low‐grade intraepithelial neoplasias, 1.39 ± 0.26 in high‐grade intraepithelial neoplasias, and 1.56 ± 0.41 in invasive ESCC. High TERC/CSP3 ratio was positively associated with lymph node metastasis (P = 0.005) and advanced tumor stage (P = 0.045). Patients with high amplification of TERC had poor survival (P = 0.01). The amplification of TERC could be used as a new genomic marker for disease progression and prognosis of ESCC. The amplified TERC gene may be a potential therapeutic target for ESCC.