EphA2 overexpression correlates with poor prognosis in esophageal squamous cell carcinoma

EphA2 is a member of the Eph family of receptor tyrosine kinases, which interact with cell-bound ligands known as ephrins. EphA2 expression was investigated by immunohistochemistry with an anti-EphA2 monoclonal antibody in 80 patients with esophageal squamous cell carcinoma (ESCC) who had undergone surgery. EphA2 overexpression was positive in 40 of the 80 patients (50%). A significant correlation was observed between EphA2 expression and regional lymph node metastasis (p=0.023), number of lymph node metastases (p=0.011) and poor degree of tumor differentiation (p=0.004). The survival rates of EphA2-positive patients were poorer than those of EphA2-negative patients (p=0.014). The 5-year survival rate of patients without EphA2 overexpression was 68%, whereas that of patients with EphA2 overexpression was 29%. EphA2 expression was also investigated in 7 ESCC cell lines (TE-1, -2, -8, -13, -15, TT and TTn) and 1 immortalized human esophageal keratinocyte cell line (CHEK-1). Western blotting revealed different levels of EphA2 expression in the 8 cell lines. EphA2 was expressed at a high level in the ESCC cell lines compared to CHEK-1. EphA2 phosphorylation was demonstrated in all cell lines. Northern blot analysis showed that EphA2 mRNA expression in TE-1 was greater than that in the other ESCC cell lines. The observation of small gaps on Western blot analysis of the ESCC cell lines suggests that there may be a mechanism for EphA2 regulation at the point of translation. In conclusion, EphA2 overexpression appears to be related to poor degree of tumor differentiation and lymph node metastasis in ESCC. Consequently, patients with EphA2 overexpression have a poorer prognosis than those without. EphA2 is a potential target to prevent ESCC cells spreading into the lymphatic drainage.     

High expression of TACC3 in esophageal squamous cell carcinoma correlates with poor prognosis

To analyze the expression of the transforming acidic coiled-coil protein 3 (TACC3) in esophageal squamous cell carcinoma (ESCC) samples, and to identify whether TACC3 can serve as a biomarker for the diagnosis and prognosis of ESCC, qPCR, western blotting and immunohistochemistry staining (IHC) were utilized to detect the expression of TACC3. Furthermore, cell growth, colony formation, migration ability and the epithelial-mesenchymal transition markers of ESCC cells in which TACC3 were knocked-down were measured. The mRNA and protein levels of TACC3 were higher in ESCC specimens compared to non-tumorous esophageal epithelial tissues. IHC results revealed TACC3 expression was significantly correlated to differentiation (p = 0.017) and lymphoid nodal status (p = 0.028). The patients with high-expression of TACC3 had a significantly poor prognosis compared to those of low-expression (p = 0.017), especially in the patients at stages I–II (p = 0.028). Multivariate analysis indicated that TACC3 expression was an independent prognostic factor for ESCC patients (p = 0.025). Knockdown of TACC3 inhibited the ability of cell proliferation, colony formation and migration. This study first identifies TACC3 not only as a useful biomarker for diagnose and prognosis of ESCC, but also as a potential therapeutic target for patients with ESCC.     

Vascular endothelial growth factor C expression correlates with lymphatic involvement and poor prognosis in patients with esophageal squamous cell carcinoma

Vascular endothelial growth factor C (VEGF-C), a member of VEGF gene family, is considered to induce both lymph node metastasis and lymphatic involvement in various cancers related to lymphangiogenesis. We examined the relationship between VEGF-C expression and clinicopathological features in 112 Japanese patients with esophageal squamous cell carcinoma (ESCC) who underwent esophagectomy and reconstruction without preoperative treatment. VEGF-C expression was examined using immunohistochemical staining and RT-PCR in surgically resected tissues. Regarding VEGF-C there were positive findings in 44 (39.3%) cases. VEGF-C expression in ESCC significantly correlated with the depth of tumor (p=0.0131), lymph node metastasis (p=0.0211), lymphatic involvement (p<0.0001) and pathological stage (p=0.0164). The prognosis for the VEGF-C positive group was poorer than that for the VEGF-C negative group (p=0.038). Multivariate analysis indicated that VEGF-C expression in ESCC is an independent factor only in cases of lymphatic involvement. VEGF-C expression in ESCC may play a great key role in lymphatic spread and this may be a consideration in terms of deciding the most appropriate treatment.     

Low expression of IGFBP-3 predicts poor prognosis in patients with esophageal squamous cell carcinoma

Previous studies have suggested that insulin-like growth factor binding protein-3 (IGFBP-3) acts as a tumor suppressor in human esophageal squamous cell carcinoma (ESCC). The present study was designed to investigate the clinical and prognostic significance of IGFBP-3 in ESCC patients. In this study, IGFBP-3 was detected by immunohistochemistry (IHC) in paraffin-embedded tissues from 110 ESCC patients, of which 110 were from primary cancer sites and 56 from matched adjacent non-malignant sites. Differences in IGFBP-3 expression and clinical characteristics were compared by χ² test. Correlations between prognostic outcomes and with IGFBP-3 expression were investigated using Kaplan–Meier analysis and the Cox proportional hazards model. Among adjacent non-malignant tissues, 83.9% of individual tissue staining was scored as either high for IGFBP-3. However, among ESCC cases, only 51.8% of the cancer tissues were scored as high IGFBP-3 expression. In addition, IGFBP-3 expression inversely correlated with pathological classification (P < 0.05 for T, N, and M classifications) and clinical staging (P = 0.006). Furthermore, patients with higher levels of IGFBP-3 had prolonged overall survival (P < 0.001). In conclusion, reduced IGFBP-3 expression may be a risk factor for advanced clinicopathological classification and poor patient survival. These findings suggest that IGFBP-3 may serve as a useful marker for the prognostic evaluation of ESCC patients.     

Low p27 expression correlates with poor prognosis for patients with oral tongue squamous cell carcinoma

BACKGROUND: p27, a cyclin-dependent kinase inhibitor, regulates progression from G1 to S phase. There have been a few clinical reports of low p27 expression associated with poor survival among patients with cancer; however, there have been no reports of such an association in cases of head and neck cancer. The authors investigated whether p27 expression in patients with oral tongue squamous cell carcinoma was associated with their prognosis. METHODS: Ninety-four patients with oral tongue squamous cell carcinoma were analyzed. The authors performed p27 immunohistochemistry on all patients and Western blot analysis on 19 available patients. Cox proportional hazards regression analysis that included gender, history of smoking and alcohol usage, presence of multiple primary cancers, stage, histologic grade, and p27 status was used to identify the multivariate predictive value of prognostic factors. RESULTS: Twenty-six patients had high p27 expression (> or =50% tumor cell nuclei positive), and 68 patients had low p27 expression (<50%) by immunohistochemistry. In those with low p27 expression, N(+) and advanced T (T3 or T4) were significantly higher than in those with high p27 expression (P = 0.02 and 0.04). The 5-year survival rate in the low p27 group was 44%, whereas that in the high p27 group was 68%, indicating a significant difference (P = 0.04). p27 expression was inferred from Western blot analysis, and an arbitrary quantity (<1, 1-5, or > or =5) from the ratio of tumor to normal tissue density was used to characterize, resulting in 8 (42%), 3 (16%), and 8 (42%) patients in the low (<1-fold), intermediate (1-5-fold), and high (> or =5-fold) groups, respectively. Results of immunohistochemical analysis for p27 were significantly correlated with those of Western blot analysis (P = 0.02). Multivariate analysis revealed that low intensity of p27 expression and advanced stage (Stage III or IV) were predictors of reduced survival (P = 0.02 and 0.001). CONCLUSIONS: Low p27 expression was associated with increasing lymph node metastasis and stage of tumor and resulted in a poor prognosis for patients with oral tongue squamous cell carcinoma. p27 is apparently a significant predictor of survival.     

Human leukocyte antigen-G expression is associated with a poor prognosis in patients with esophageal squamous cell carcinoma

Human leukocyte antigen (HLA)-G inhibits functions of immune component cells and promotes malignant cells evading from antitumor immunity. We investigated the clinical relevance of HLA-G expression in esophageal squamous cell carcinoma (ESCC). In our study, HLA-G expression in 79 primary ESCC lesions and corresponding adjacent normal tissues were analyzed with immunohistochemistry. Soluble HLA-G (sHLA-G) in plasma was detected with enzyme-linked immunosorbent assay (ELISA) in 41 ESCC patients (including 19 case-matched lesions and plasma samples) and in 153 normal healthy controls. HLA-G expression was observed in 65.8% (52/79) of the ESCC lesions but not in adjacent normal esophageal tissues. HLA-G expression was more frequently observed in patients with advanced disease stage (III/IV vs. I/II, p = 0.01). Patients with HLA-G expression had a significantly worse survival, and HLA-G could be an independent prognostic factor. sHLA-G levels in plasma were significantly increased in patients compared to normal controls (median: 152.4 U/ml vs. 8.9 U/ml, p < 0.001). The area under receiver-operating characteristic (ROC) curve for sHLA-G in plasma was 0.992. However, no significant correlation was found between sHLA-G in plasma and clinical parameters studied. In conclusion, our findings indicated that HLA-G expression in ESCC is associated with poor survival and could be a prognostic indicator. Furthermore, increased levels of sHLA-G in plasma might be a useful preoperative biomarker for diagnosis.     

Overexpression of Tiam1 predicts poor prognosis in patients with esophageal squamous cell carcinoma

Accumulating evidence has demonstrated that T-cell lymphoma invasion and metastasis 1 (Tiam1) plays an important role in the occurrence and development of several different tumors; however, to date, little research has been done to verify the potential role of Tiam1 as a prognostic marker for esophageal squamous cell carcinoma (ESCC). In the present study, we examined the expression of Tiam1 in ESCC tissues by immunohistochemistry, in situ hybridization, semi-quantitative RT-PCR and Western blotting methods and investigated the correlation between Tiam1 levels and prognosis of patients with ESCC. Tiam1 exhibited high expression in ESCC tissues, whereas the normal esophageal tissues showed negative or weak Tiam1 expression. Additionally, Tiam1 mRNA and protein expression levels were both significantly correlated with histology grade, clinical staging and lymph node metastasis (all P<0.05), but not related to age and gender (both P>0.05). Further, ESCC patients with strong Tiam1 mRNA (P=0.000) and protein (P=0.000) expression had a poorer prognosis than those with weak expression. These findings demonstrate that Tiam1 may be used as molecular marker for predicting the prognosis of patients with ESCC.     

Cyclooxygenase-2 expression is related to prognosis in patients with esophageal squamous cell carcinoma

Aim

Esophageal carcinoma is one of the most aggressive malignancies. Many studies have examined various biological factors associated with the malignant potential of esophageal carcinoma. Cyclooxygenase (COX)-2 is overexpressed in various types of human malignancies, including esophageal carcinomas. Although some groups have described COX-2 expression in esophageal adenocarcinoma, few studies have reported COX-2 expression in esophageal squamous cell carcinoma (ESCC).

Methods

We immunohistochemically investigated relationships between COX-2 overexpression in surgical specimens of primary tumors in 228 patients with ESCC. Relationships between COX-2 expression and clinicopathological factors, including prognosis, were analyzed. COX-2 expressions were classified into 4 criteria: Score 0, no staining; Score 1, <10% staining; Score 2, 10–90% staining; and Score 3, >90% staining.

Results

Scores of COX-2 immunoreactivity in 228 patients were as follows: Score 0, 21 of 228; Score 1, 71of 228; Score 2, 117 of 228; and Score 3, 19 of 228, respectively. COX-2 expression was significantly correlated with depth of invasion and tumor stage (p = 0.03 and p = 0.04, respectively). The 5-year survival rate of patients decreased significantly with increased expression of COX-2 (p = 0.005). Multivariate regression analysis indicated COX-2 expression as an independent prognostic factor for ESCC.

Conclusions

COX-2 overexpression was significantly correlated with depth of invasion, tumor stage and survival in ESCC. Evaluation of COX-2 expression should be useful for determining tumor properties, including prognosis, in patients with ESCC.     

Association of trypsin expression with recurrence and poor prognosis in human esophageal squamous cell carcinoma

BACKGROUND: Overexpression of the matrix serine proteinase (MSP) trypsin has been implicated in tumor growth, invasion, and metastasis. The objective of this study was to clarify the clinicopathologic and prognostic significance of trypsin expression in esophageal squamous cell carcinomas (SCC). METHODS: Production of trypsin in tissue extracts was analyzed by immunoblotting and gelatin zymography. The authors analyzed the association between immunohistochemically detected trypsin expression in esophageal SCC and clinicopathologic characteristics, and they investigated whether trypsin is a predictor of recurrence and/or survival. RESULTS: Overproduction and activation of trypsin was observed in 6 of 10 tumor extracts. The trypsin immunoreactivities at the invasive front were more intense than those at the superficial layer. Sections with immunostaining signals in greater than 30% of carcinoma cells at the invasive front, which were observed in 52 (52%) cases, were judged to be positive for trypsin. Trypsin positivity was significantly correlated with the depth of invasion (P < 0.0001), lymph node metastasis (P = 0.0048), advanced pTNM classification (P = 0.0006), recurrence (P = 0.0003), and recurrence within the first postoperative year (P = 0.0005). Patients with trypsin positive carcinoma had significantly shorter disease free and overall survival times than did those with trypsin negative carcinoma (P < 0.0001 and P < 0.0001, respectively). Trypsin retained its significant predictive value for disease free and overall survival in multivariate analysis that included conventional clinicopathologic factors (P = 0.0029 and P = 0.0006, respectively). Patients with concomitant overexpression of trypsin and matrilysin at the invasive front, in which they often were colocalized, had the worst prognosis. CONCLUSIONS: The authors' results suggest that trypsin plays a key role in the progression of esophageal carcinoma. Detection of trypsin expression as well as matrilysin is useful for the prediction of recurrence and poor prognosis.     

Cyclin D1 overexpression correlates with poor prognosis in patients with tongue squamous cell carcinoma

Tongue squamous cell carcinoma makes up a large percentage of head and neck cancers, and the incidence among young patients is increasing. The aim of this study was to reveal the correlation between cyclin D1 (CCND1) expression and clinical and histologic features. We performed an immunohistochemical study on the level of CCND1 expression in tumor specimens obtained from 94 patients with tongue squamous cell carcinoma. The relationship between the expression and the following features such as age, sex, smoking and alcohol intake history, T, N, histologic grade, and multiple primary cancer was analyzed. Eighteen patients (19%) showed CCND1 overexpression (tumor cell nuclei positivity >/=50%). The 5-year survival rate of high CCND1 expressors was 39%, which was significantly poor (p=0.04). N classification correlated with CCND1 expression. CCND1 overexpression is associated with poor survival associated with progression of lymph node spread in patients with tongue squamous cell carcinomas. CCND1 expression may be a useful biologic marker for prognosis.     

FBXO31 determines poor prognosis in esophageal squamous cell carcinoma

Cyclin D1 plays important roles in esophageal squamous cell carcinoma (ESCC) cases by amplification of the 11q13.3 locus. FBXO31 is a subunit of the SCF ubiquitin ligase, which targets cyclin D1 for degradation. In this study, we clarified the clinical significance of FBXO31 and characterized the association between cyclin D1 and FBXO31 in ESCC cases. Total RNA was extracted from tumor tissues obtained from 68 ESCC patients who underwent surgical resection. FBXO31 expression levels were determined by quantitative RT-PCR, and both FBXO31 and cyclin D1 protein expression and localization were evaluated by immunohistochemistry (IHC). Furthermore, using CGH and gene expression array data of another subset, we validated the association between cyclin D1 genomic amplification and FBXO31 expression levels. Higher FBXO31 expression levels significantly correlated with depth of tumor invasion and clinical stage (P<0.05). In addition, the FBXO31 high expression group showed a significantly poorer prognosis than the low expression group (P<0.001). Multivariate analysis indicated that FBXO31 expression was an independent prognostic factor [relative risk (RR): 1.79, confidence interval (CI): 1.14-3.01, P=0.01]. Using IHC, concordant expression was observed between cyclin D1 and FBXO31 in the nucleus and cytoplasm, respectively. CGH array indicated that cases having cyclin D1 with increased copy number were significantly associated with elevated FBXO31 expression levels (P<0.05). FBXO31 could be a novel and robust prognostic marker for ESCC.     

Prolyl isomerase Pin1 expression predicts prognosis in patients with esophageal squamous cell carcinoma and correlates with cyclinD1 expression

Esophageal carcinoma is one of the most lethal tumors, and identification of prognostic factors for patients with this disease is important. Propyl isomerase Pin1 is overexpressed in some human cancers and thought to be an important regulator of cyclinD1. However, the relationships between Pin1 expression and clinicopathologic features in patients with esophageal squamous cell carcinoma (SCC) have not been explored. Here, we investigated the role of Pin1 in association with cyclinD1 in esophageal SCC progression and its clinicopathological significance. The expressions of Pin1 and cyclinD1 were examined immunohistochemically in surgical specimens from 119 esophageal SCC patients. The expression levels of Pin1 and cyclinD1 in 6 esophageal SCC-derived cell lines were compared with those in an immortalized human esophageal cell line by western blotting. Pin1 overexpression was correlated with lymph node metastasis (P=0.0384), and its expression was related to cyclinD1 expression. Pin1 expression was correlated with poor prognosis in esophageal SCC patients (P=0.0044), and found to be an independent prognostic factor (P=0.0277). Pin1 was overexpressed in 5 of 6 esophageal SCC-derived cell lines compared with immortalized esophageal keratinocytes. Moreover, the Pin1 level was correlated with the cyclinD1 level in 4 of the 6 cell lines. In conclusion, Pin1 expression is correlated with cyclinD1 expression and may be a useful prognostic factor for esophageal SCC.     

Elevated expression of the c-myc oncoprotein correlates with poor prognosis in head and neck squamous cell carcinoma

We quantitated c-myc oncoprotein in 44 squamous cell carcinomas of the head and neck using an enzyme-linked immunosorbence assay. The clinicopathological parameters of these patients were followed up for between 3 and 60 months and analysed for any correlations with observed levels of c-myc protein using the Kruskal-Wallis one-way analysis of variance method. Although no statistical correlation was found between different clinicopathological parameters (patient age, sex, TNM staging, number of lymph nodes invaded, extracapsular rupture of the tumour, its histopathological differentiation, or its site), the survival periods of patients with tumours possessing elevated levels of c-myc protein were found to be statistically shorter than those with lower levels of c-myc expression, (P less than 0.02). This indicates that c-myc expression may be an effective prognostic indicator in head and neck cancer.     

High expression of survivin predicts poor prognosis in esophageal squamous cell carcinoma following radiotherapy

Radiotherapy is one of the main treatments for esophageal squamous cell carcinoma, but there are still no biomarkers to differentiate patients who will benefit from radiation. Although treatment with a combination of radiotherapy with chemotherapy, and/or surgery improves the prognosis of patients, no biomarkers can distinguish between the responses obtained with the combined therapies. Therefore, in this study, we selected patients treated with radiotherapy alone to evaluate survivin as a predictor for radiotherapy. One hundred two biopsy samples collected by endoscopy were immunostained by survivin antibody. Positive staining for survivin was obtained in 60.8% tumor samples. Survivin expression, metastasis, and clinical stage correlated significantly with overall survival. In multivariate analysis, survivin was an independent prognostic factor for predicting overall survival of patients with esophageal cancer. Moreover, in esophageal cancer cell lines, overexpression of survivin reduced the percentage of cell death induced by radiation. Our data indicate that survivin could be a potential predictor to define those patients with esophageal squamous carcinoma who would benefit from radiotherapy.     

Association of matrilysin expression with recurrence and poor prognosis in human esophageal squamous cell carcinoma.

Matrix metalloproteinase-7 (matrilysin) has been implicated in tumor invasion and metastasis as well as tumor initiation and growth. In this study, we analyzed an association between immunohistochemically detected matrilysin expression at the invasive front in esophageal squamous cell carcinomas and clinicopathological characteristics and determined whether matrilysin predicts recurrence and/or survival Matrilysin expression at the invasive front was detected in 49% of 100 carcinoma tissues and was associated with the depth of invasion (P < 0.0001), advanced tumor stage (P = 0.0159), recurrences (P = 0.0002), and recurrences within the first postoperative year (P = 0.002). Patients with matrilysin-positive carcinoma had a significantly shorter disease-free and overall survival time than did those with a matrilysin-negative one (P < 0.0001). Matrilysin remained a significant predictive value for disease-free and overall survival in multivariate analysis, including conventional clinicopathological factors (P = 0.0007 and 0.0004, respectively). Our results suggest that matrilysin may play a key role in the progression of esophageal carcinoma and that its detection may be useful for the prediction of recurrence and poor prognosis and, possibly, for selecting patients for anti-matrix metalloproteinase therapy.     

Expression of cysteine-rich 61 is correlated with poor prognosis in patients with esophageal squamous cell carcinoma

Introduction

Cysteine-rich 61 (Cyr61), a secreted protein belonged to the CCN family, was involved in the progression of many cancers. The purpose of this study was to explore the clinical significance of Cyr61 expression in esophageal squamous cell carcinoma (ESCC).

Materials and methods

Cyr61 expression was detected on tissue microarrays of ESCC samples in 372 cases by using immunohistochemical staining. Survival analysis was assessed by the Kaplan-Meier analysis. Relative risk was evaluated by the multivariate Cox proportional hazards model.

Results

The staining pattern of Cyr61 was heterogeneous and varied from negative to intense expression in a cytoplasmic distribution. Kaplan-Meier analysis revealed that expression of Cyr61 was related to poor survival of ESCC patients (P = 0.001). Further analysis revealed that Cyr61 high-expression was related to poorer overall survival of patients in stage I/II (P = 0.001); but did not effect the overall survival of patients in stage III/IV. Univariate and multivariate analysis suggested that Cyr61 expression status was an independent prognostic factor for ESCC (P = 0.001).

Discussion

Cyr61 might play important roles in the progression of ESCC. Cyr61 is a new biomarker to predict the prognosis of ESCC patients.     

Loss of p27(KIP1) expression predicts poor prognosis in patients with esophageal squamous cell carcinoma

Immunohistochemical studies of cell cycle-regulating proteins were conducted on tissue samples from 106 patients with esophageal squamous cell carcinoma. Reduction of p27(KIP1) was observed in 41 (39%) cases and was significantly associated with a poor prognosis by univariate and multivariate analyses (p = 0.015). In contrast, p16(INK4) expression was reduced in 55 (52%) of the cases and was not correlated with prognosis. p27(KIP1) was not correlated with the PCNA index, while p16(INK4) expression was inversely correlated with the PCNA index (p = 0.01). The expression of these two proteins was not correlated with the clinicopathological parameters of the patients. Nodal status was shown by univariate analysis (p = 0.01) to be a prognostic factor, and poorly differentiated tumors were significantly associated with a poor prognosis by multivariate analysis (p = 0.027). Thus, reduction of p27(KIP1) plays an important role in the progression of esophageal squamous cell carcinomas and is considered to be an independent prognostic indicator of this disease.