Abstract
Aims: Squamous cell/adenosquamous carcinomas (SC/ASC) are rare subtypes of gallbladder cancers (GBCs). Clinical characteristics of SC/ASC have not been well documented, and no biological markers of GBC carcinogenesis, progression and prognosis are available. Methods: We detected EphA10 and EphB3 expression in 69 SC/ASCs and 146 adenocarcinomas (ACs) with EnVision immunohistochemistry. Results: The percentage of cases with a patient age of > 45 years, lymph node metastasis and invasion was significantly higher in the SCs/ASCs compared with the ACs (P < 0.05). The positive expression of EphA10 and negative expression of EphB3 were significantly higher in the cases of SC/ASC and AC than in chronic cholecystitis (P < 0.01). The positive expressions of EphA10 and negative expression of EphB3 were significantly higher in the cases of poorly differentiation, large tumor size, high TNM stage, lymph node metastasis, invasion and no resection (only biopsy) of SC/ASC and AC. The negative correlation was found between EphA10 and EphB3 expression in SC/ASC and AC (P < 0.01). The univariate Kaplan-Meier analysis showed that positive EphA10 and negative EphB3, differentiation, tumor size, TNM stage, lymph node metastasis, invasion and surgical curability, is closely associated with a decreased overall survival in SC/ASC and AC patients (P < 0.05 or P < 0.01). The multivariate Cox regression analysis identified that positive EphA10 and negative EphB3 expression are independent factors for a poor-prognosis in SC/ASC and AC patients. The AUC for EphA10 and EphB3 showed might have role for carcinogenesis and progression of SC/ASC and AC. Conclusions: The present study indicates that positive EphA10 and negative EphB3 expression are closely associated with the pathogenesis, clinical, pathological and biological behaviors, and poor prognosis in gallbladder cancer.
Keywords: Gallbladder neoplasm, squamous cell carcinoma, adenosquamous carcinomas, EphA10, EphB3, immunohistochemistry
Introduction
Gallbladder cancers (GBCs) are relatively uncommon human cancers, and they have a particular geographical distribution in central and South America, central and Eastern Europe, Japan and northern India [1,2]. GBC is increasing worldwide, and prognosis is extremely poor. Early diagnosis may be impossible because such cancers are asymptomatic [3]. More than 90% of GBC patients are diagnosed at an inoperable stage, when tumours are invasive and metastatic [4]. Most GBCs are adenocarcinomas (AC; > 90%) [5]. In contrast, squamous cell/adenosquamous carcinoma (SC/ASC) is rare, representing 1-12% of GBCs [2,6]. The clinicopathological characteristics of SC/ASC have not been well documented, and therapeutic interventions for these tumours are not established [7]. Although biomarkers for the prognosis of AC are currently under investigation, no proposed markers have been applied clinically [8]. No biomarkers relating to the progression and prognosis of SC/ASC have been identified.
The 14 members of the Eph family of receptors make up the largest family of receptor tyrosine kinases in humans [9,10]. These receptors are associated with 8 members of a class of ligands known as ephrins. These receptors and ligands are divided into A and B classes based on their sequence homology as well as their affinity for the corresponding receptor/ligand [10]. Many Eph receptors and ephrin ligands are known to be involved in the development or progression of certain cancers [11]. EphA10 which we identified as a novel breast cancer-related protein is hardly expressed in normal human tissues [12,13]. Furthermore, it also showed that an in-house developed anti-EphA10 mAb inhibited breast cancer cell proliferation at both in vitro and in vivo levels [13]. Nagano, et al. found that EphA10 expression might play a role in tumor progression and metastasis. It will help elucidate the role of EphA10 in clinical breast can cer progression [14]. These findings suggest that EphA10 is a promising target for breast cancer therapy. Fujimori, et al. found that EphA10 could promote tumor proliferation in athymic nude mice with xenograft cholangiocarcinoma [15]. Nagano, et al. found that the overexpression of EphA10 in prostate cancers might have a potential as a target for prostate cancer therapy [16].
In tumorigenesis, signaling by the protein tyrosine kinases Ephrin type-B receptor 2 (EphB2) and 3 (EphB3) represents a powerful barrier against tumor-cell spreading and the onset of metastasis, the main cause for cancer-related mortality [17,18]. Through repulsive interactions between cells expressing EphB receptors and cells presenting EphrinB ligands, EphB/EphrinB signaling compartmentalizes tumors n molecule E-cadherin, thereby adding to the stabilization of a noninvasive epithelial-cell phenotype [19,20]. EphB3 and locally confines their growth [19,20]. In addition, EphB/EphrinB signaling affects the function of the cell-cell adhesiois a direct target gene of Wnt/β-catenin and Notch signaling [21,22] and in agreement with the pivotal role of these pathways in tumor initiation [23]. EphB3 is strongly upregulated in colorectal adenomas [24]. However, this surge in EphB3 expression at early stages of tumorigenesis is followed by secondary downregulation in up to 30% of carcinomas. Ronscha et al. identify EphB3 as a novel target of Snail1 and suggest that disabling EphB3 signaling is an important aspect to eliminate a roadblock at the onset of EMT processes [25]. Recent studies have demonstrated a relatioship between EphB3 expression and colorectal cancer [17-19], gastric cancer [26], NSCLC [27], breast cancer [28], ovarian cancer [29] and prostate cancer [30], the experimental results suggested that the low expression of EphB3 had close relationship to pathogenesis, progresion and biological behaviors of above malignant lesions.
As far as we know, the role of EphA10 and EphB3 in gallbladder SC/ASC and AC remains to be clarified. Thus, we evaluated EphA10 and EphB3 expression in surgically resected specimens, including 146 ACs and 69 SC/ASCs, using immunohistochemistry. We then correlated their expression with the clinicopathological characteristics and prognosis of patients with AC and SC/ASC.
Materials and methods
Case selection
This study was pre-approved by the Ethics Committee for Human Research, Central South University. A total of 69 SC/ASC samples resulting from surgical resection or biopsy were collected from January 2001 to December 2013. According to the recommendations of the American Joint Committee on Cancer, tumours with a squamous component ≥ 10% were considered to represent adenosquamous carcinomas. The 69 SC/ASCs comprised 5.5% of 1248 GBCs. Of these 66, 16 (of 200 GBCs) were collected from Xiangya Hospital, 31 (of 628 GBCs) from Second Xiangya Hospital, 10 (of 200 GBCs) from Third Xiangya Hospital, 5 (of 70 GBCs) from Hunan Provincial People Hospital, 5 (of 100 GBCs) from Hunan Provincial Tumor Hospital, and one each from Changde Central Hospital and Loudi Central Hospital (two of 50 GBCs). A total of 146 AC samples derived from surgical resection or biopsy at Second Xiangya Hospital and Third Xiangya Hospital were collected between January 2008 and December 2013.
Clinicopathological data is summarized in Table 1. Among the 69 SC/ASC samples, 47 were from female patients (F/M = 2.14) and patient ages ranged from 35 to 80 (53.8 ± 10.2) years. Among the 146 patients with AC, 92 patients were female (F/M = 1.77), with an age range of 33 to 78 (52.4 ± 9.6) years. Of the 69 SC/ASCs, the squamous cell component was well differentiated in 19 (27.5%), moderately differentiated in 33 (47.8%), and poorly differentiated in 17 (24.6%). Of the 146 ACs, 51 were well differentiated (34.9%), 54 were moderately differentiated (37.0%) and 41 were poorly differentiated (28.1%). Among the SC/ASC patients, invasion of tissues and organs surrounding the gallbladder was observed in 45 patients (65.2%); 42 (60.7%) had regional lymph node metastasis; and 38 (55.1%) had gallstones. Among the 146 patients with ACs, invasion was found in 74 (50.7%); 66 (45.2%) had regional lymph node metastasis; and 68 (46.6%) had gallstones. According to tumour-node-metastasis (TNM) staging, 29 of the 69 SC/ASCs were stage I+II and 40 were stage III+IV. Among the 146 ACs, 77 were stage I+II and 69 were stage III+IV. Surgery included radical resection for 27 SC/ASCs and 75 ACs, palliative surgery for 28 SC/ASCs and 50 ACs, and no operation for 14 SC/ASCs and 21 ACs which were only biopsied. Survival data for the 69 patients with SC/ASC and the 146 patients with AC was obtained through letters and telephone calls. The follow-up time for both groups was 2 years, and patients who survived longer than 2 years were included in the analysis as censored cases. Of the AC patients, 58 survived more than 1 year (26 more than 2 years), and 88 survived < 1 year. Of the SC/ASC patients, 17 survived more than 1 year (7 more than 2 years) and 52 survived < 1 year.
Table 1.
Comparison of gallbladder SC/ASC and AC clinicopathological features and EphA10 and EphB3 expression status
| Clinicopathological characteristics | SC/ASC (n = 69) | AC (n = 146) | X2 | P value |
|---|---|---|---|---|
| Gender, n (%) | ||||
| Male | 25 (36.2) | 61 (41.8) | 0.601 | 0.438 |
| Female | 44 (63.8) | 85 (58.2) | ||
| Age, n (%) | ||||
| ≤ 45 years | 3 (4.3) | 20 (13.7) | 4.289 | 0.038 |
| > 45 years | 66 (95.7) | 126 (86.3) | ||
| Differentiation, n (%) | ||||
| Well | 19 (27.5) | 51 (34.9) | ||
| Moderate | 33 (47.8) | 54 (37.0) | 2.235 | 0.308 |
| Poor | 17 (24.6) | 41 (28.1) | ||
| Maximum tumor diameter, n (%) | ||||
| ≤ 3 cm | 39 (56.5) | 90 (61.6) | 0.512 | 0.474 |
| > 3 cm | 30 (43.5) | 56 (38.4) | ||
| Cholecystolithiasis, n (%) | ||||
| (-) | 31 (44.9) | 78 (53.4) | 1.353 | 0.245 |
| (+) | 38 (55.1) | 68 (46.6) | ||
| TNM stages, n (%) | ||||
| I+II | 29 (42.0) | 77 (52.7) | 2.151 | 0.143 |
| III+IV | 40 (58.0) | 69 (47.3) | ||
| Lymph node metastasis, n (%) | ||||
| (-) | 27 (39.1) | 80 (54.8) | 4.599 | 0.032 |
| (+) | 42 (60.9) | 66 (45.2) | ||
| Locoregional invasion, n (%) | ||||
| (-) | 24 (34.8) | 72 (49.3) | 4.004 | 0.045 |
| (+) | 45 (65.2) | 74 (50.7) | ||
| Surgical methods, n (%) | ||||
| Radical | 27 (39.1) | 75 (51.4) | ||
| Palliative | 28 (40.6) | 50 (34.2) | 3.002 | 0.223 |
| Without resection | 14 (20.3) | 21 (14.4) | ||
| EphA10 | ||||
| - | 28 (40.6) | 70 (47.9) | 1.025 | 0.311 |
| + | 41 (59.4 ) | 76 (52.1) | ||
| EphB3 | ||||
| - | 27 (39.1) | 69 (47.3) | 1.253 | 0.263 |
| + | 42 (60.9) | 77 (52.7) | ||
EnVision immunohistochemistry
Four-micrometer-thick sections were cut from routinely paraffin-embedded tissues. The rabbit anti-human EphA10 and EphB3, and HRP-conjugated anti-rabbit second antibody were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). EnVisionTM Detection Kit was purchased from Dako Laboratories (CA, USA). The staining of EphA10 and/or EphB3 was carried out according to the manufacture’s protocol. Briefly, the sections were deparaffinized and then incubated with peroxidase inhibitor (3% H2O2) in the dark for 15 minutes, followed by EDTA-trypsin digestion for 15 minutes. The sections were incubated with primary antibody for 120 minutes after being soaked with PBS for 3 × 5 minutes. Solution A was added to the sections for 30 minutes followed by DAB staining and hematoxylin counter-staining. The slides were dehydrated with different concentrations (70%-100%) of alcohol,and soaked in xylene for 3 × 5 minutes and finally mounted with neutral balsam. Ten random fields were examined per section. The percent to positively stained cells relative to the total number of cells was determined. Next, the strength of staining was rated on a scale of 1 to 3. A score of 1 represents little to no positive staining or uncertainly weak staining; a score of 2 represents weak to moderate staining; and a score of 3 represents moderate to strong staining. A section is determined as positive for EphA10 or EphB3 when the percent of positively stained cells was ≥ 10% and staining strength ≥ 2. The few sections where percent positive staining was 5% to 10% and staining strength was 3 were also regarded as positive.
Statistical analysis
Data was analyzed using the statistical package for the Social Sciences Version 13.0 (SPSS 13.0). The inter-relationship of EphA10 or EphB3 expression with histology or clinical factors was analyzed using X2 or Fisher’s exact test. Kaplan-Meier and time series test (log-ranktest) were used for Univariate survival analysis. Cox proportional hazards model was used for multivariate analysis and to determine the 95% confidence interval. ROC of Diagonal segments is produced by ties.
Results
Comparison of EphA10 and EphB3 expression and clinicopathological characteristics in SC/ASC and AC
As shown in Table 1, the percentage of cases with a patient age of > 45 years, lymph node metastasis and invasion was significantly higher in the SCs/ASCs compared with the ACs (P < 0.05). Correlations between other clinicopathological characteristics and the percentage of positive EphA10 or EphB3 expression were not significant. The majority of EphA10- and EphB3-positive reactions were localized in the cytoplasm of the SC/ASCs (Figure 1) and ACs (Figure 2), as observed using EnVision immunohistochemistry (Dako Laboratories).
Figure 1.
EphA10 and EphB3 expression. EnVision immunohistochemistry, original magnification ×200. EphA10 and EphB3 expression was localized in the cytoplasm. A. Positive expression of EphA10 in poor differentiated SC. B. Negative expression of EphA10 in well differentiated SC. C. Positive expression of EphB3 in moderately differentiated ASC. D. Negative expression of EphB3 in poorly differentiated SC.
Figure 2.
EphA10 and EphB3 expression. EnVision immunohistochemistry, original magnification ×200. EphA10 and EphB3 expression was localized in the cytoplasm. A. Positive expression of EphA10 in moderately differentiated adenocarcinoma. B. Negative expression of EphA10 in well differentiated adenocarcinoma. C. Positive expression of EphB3 in well differentiated adenocarcinoma. D. Negative expression of EphB3 in poorly differentiated adenocarcinoma.
EphA10 and EphB3 expression in adenocarcinoma of gallbladder, and gallbladder epithelium with chronic cholecystitis
The percentage of cases with positive EphA10 was significantly higher in SC/ASC (50.9%) and AC (52.1%) than that in the gallbladder epithelium with chronic cholecystitis (10.0%, ps = 0.000). The percentage of cases with positive EphB3 expression was significantly lower in SC/ASC (47.8%) and AC (50.7%) than that in the gallbladder epithelium with chronic cholecystitis (90.0%, ps = 0.000). The epithelium of chronic cholecystitis with high EphA10 or lower EphB3 expression showed moderate to severe dysplasia. This suggests that both EphA10 and EphB3 could be markers to evaluate the pre-malignant changes.
Association of clinicopathological characteristics and EphA10 and EphB3 expression in SC/ASC and AC patients
A significantly lower association was apparent between the percentage of cases with EphB3 positive expression in the SC/ASC samples with poorly-differentiation, a large tumor mass size, no gallstone, high TNM stage, lymph node metastasis, invasion and no resection (biopsy only) compared with the cases of well- + Moderately-differentiation, small tumor size, gallstone, low TNM stage, no lymph metastasis, no invasion and radical resection (P < 0.05 or P < 0.01; Table 2). A significantly higher association was apparent between the percentage of cases with EphA10 positive expression in the SC/ASC samples with poorly-differentiation, a mpared with the cases of well differentiation, small tumor size, no gallstone, low TNM stage, no lymph metastasis, no invasion and radical resection (P < 0.05 or P < 0.01; Table 3).
Table 2.
Correlations of EphA10 and EphB3 protein expression with the clinicopathological characteristics of gallbladder SC/ASC
| CPC | Case No. | EphA10 | EphB3 | ||||
|---|---|---|---|---|---|---|---|
|
|
|
||||||
| Pos No. (%) | X2 | P value | Pos No. (%) | X2 | P value | ||
| Differentiation | |||||||
| Well | 19 | 9 (47.4) | 7.854 | 0.020 | 11 (57.9) | 5.391 | 0.068 |
| Moderately | 33 | 17 (51.5) | 18 (54.5) | ||||
| Poorly | 17 | 15 (88.2) | 4 (23.5) | ||||
| Tumor size | |||||||
| ≤ 3 cm | 30 | 10 (33.3) | 14.980 | 0.004 | 26 (86.7) | 32.089 | 0.000 |
| > 3 cm | 39 | 31 (79.5) | 7 (17.9) | ||||
| Gallstone | |||||||
| No | 31 | 24 (77.4) | 7.563 | 0.006 | 10 (32.3) | 5.467 | 0.019 |
| Yes | 38 | 17 (44.7) | 23 (60.5) | ||||
| Lymph node metastasis | |||||||
| No | 27 | 8 (29.6) | 16.326 | 0.000 | 21 (77.8) | 15.947 | 0.000 |
| Yes | 42 | 33 (78.6) | 12 (28.6) | ||||
| Invasion | |||||||
| No | 24 | 9 (37.5) | 10.386 | 0.001 | 20 (83.3) | 18.594 | 0.000 |
| Yes | 45 | 33 (73.3) | 13 (28.9) | ||||
| TNM stage | |||||||
| I+II | 29 | 10 (34.5) | 12.902 | 0.000 | 23 (79.3) | 19.873 | 0.000 |
| III+IV | 40 | 31 (77.5) | 10 (25.0) | ||||
| Surgery | |||||||
| Radical | 27 | 10 (37.0) | 7.070 | 0.027 | 22 (81.5) | 21.328 | 0.000 |
| Palliative | 28 | 20 (71.4) | 9 (32.1) | ||||
| Biopsy | 14 | 11 (78.6) | 2 (14.3) | ||||
Table 3.
Correlations of EphA10 and EphB3 protein expression with the clinicopathological characteristics of gallbladder AC
| CPC | Case No. | EphA10 | EphB3 | ||||
|---|---|---|---|---|---|---|---|
|
|
|
||||||
| Pos No. (%) | X2 | P value | Pos No. (%) | X2 | P value | ||
| Differentiation | |||||||
| Well | 51 | 18 (35.3) | 14.947 | 0.001 | 38 (74.5) | 19.947 | 0.000 |
| Moderately | 54 | 27 (50.0) | 24 (44.4) | ||||
| Poorly | 41 | 31 (75.6) | 12 (29.3) | ||||
| Tumor size | |||||||
| ≤ 3 cm | 90 | 38 (42.2) | 9.089 | 0.003 | 57 (63.3) | 15.018 | 0.000 |
| > 3 cm | 56 | 38 (67.9) | 17 (30.4) | ||||
| Gallstone | |||||||
| No | 78 | 42 (53.8) | 0.215 | 0.358 | 38 (48.7) | 0.259 | 0.611 |
| Yes | 68 | 34 (50.0) | 36 (52.9) | ||||
| Lymph node metastasis | |||||||
| No | 80 | 23 (28.8) | 38.511 | 0.000 | 56 (70.0) | 26.414 | 0.000 |
| Yes | 66 | 53 (80.3) | 18 (27.3) | ||||
| Invasion | |||||||
| No | 72 | 21 (29.2) | 29.817 | 0.000 | 53 (73.6) | 29.872 | 0.000 |
| Yes | 74 | 55 (74.3) | 21 (28.4) | ||||
| TNM stage | |||||||
| I+II | 77 | 20 (26.0) | 44.405 | 0.000 | 57 (74.0) | 35.512 | 0.000 |
| III+IV | 69 | 56 (81.2) | 17 (24.6) | ||||
| Surgery | |||||||
| Radical | 75 | 23 (30.7) | 33.334 | 0.000 | 56 (74.7) | 38.475 | 0.000 |
| Palliative | 50 | 33 (66.0) | 16 (32.0) | ||||
| Biopsy | 21 | 20 (95.2) | 2 (9.5) | ||||
For AC tumors, the percentage of EphB3 potive expression was significantly lower in the cases with poor differentiation, large tumor mass size, high TNM stage, lymph node mettasis, invasion and no resection (biopsy only), compared with the well-differentiated cases, small tumor mass, low TNM stage, no lymph node metastasis, no invasion and radical resection (P < 0.01; Table 3). The percentage of EphA10 positive expression was significantly higher in the cases with poor differentiation, large tumor mass size, high TNM stage, lymph node metastasis, invasion and no resection (biopsy only), compared with the well-differentiated cases, small tumor mass, low TNM stage, no lymph node metastasis, no invasion and radical resection (P < 0.01; Table 3).
Of the 41 EphA10 positive cases, EphB3 was positively expressed in 15 cases, while of the 28 EphA10 negative cases, EphB3 was negatively expressed in 10 cases, suggesting a high inconsistency between these two markers in SC/ASC (X2 = 5.116, P < 0.05). Of the 76 EphA10 positive cases, EphB3 was positively expressed in 26 cases, while of the 70 EphA10 negative cases, EphB3 was negatively expressed in 22 cases, suggesting a high inconsistency between these two markers in AC (X2 = 17.212, P < 0.01).
Correlation between survival rates and EphA10 or EphB3 expression in patients with SC/ASC and AC
Survival data for the 69 patients with SC/ASC and the 146 patients with AC was obtained through letters and phone calls. The follow-up time the present study was 2 years, and patients who survived longer than 2 years were included in the analysis as censored cases. Of the 146 AC patients, 58 survived more than 1 year (26 more than 2 years), and 57 survived < 1 year. Of the 69 SC/ASC patients, 17 survived more than 1 year (7 more than 2 years) and 52 survived < 1 year.
Evaluation of the SC/ASC patients using a Kaplan-Meier survival analysis demonstrated that differentiation, tumor size, TNM stage, lymph node metastasis, invasion, surgical procedure and gallstone were significantly associated with average survival time (P < 0.01, Table 4). The average survival time of the EphA10 positive patients was significantly lower than that of the patients with a negative result for EphA10 (P < 0.01; Table 4 and Figure 3). The average survival time of the EphB3 positive patients was significantly higher than that of the patients with a negative result for EphB3 (P < 0.01; Table 4 and Figure 3). Cox’s multivariate analysis demonstrated that the differentiation, tumor size (≥ 3 cm), TNM stage, invasion, surgical procedure, EphA10-positive and EphB3-negative expression were negatively correlated with overall survival, indicating that the positive expression of EphA10 and negative expression of EphB3 is a risk factor of SCs/ASCs (Table 5). Finally, we calculated the AUC for EphA10 (AUC = 0.747, 95% CI: 0.648-0.864), or EphB3 (AUC = 0.711, 95% CI: 0.607-0.805) in SCs/ASCs, respectively (Figure 4).
Table 4.
Relationship between EphA10 and EphB3 expression, clinicopathological characteristics and average survival of SC/ASC patients
| Clinicopathological characteristics | Sample (n) | Average survival (month) | X2 | P value |
|---|---|---|---|---|
| Differentiation | ||||
| Well | 19 | 13.68 (5-24) | ||
| Moderately | 33 | 11.58 (4-24) | 20.815 | 0.000 |
| Poorly | 17 | 6.12 (2-14) | ||
| Tumor siz | ||||
| ≤ 3 cm | 30 | 14.57 (6-24) | 21.493 | 0.000 |
| > 3 cm | 39 | 7.44 (2-24) | ||
| Gallstones | ||||
| No | 31 | 8.26 (3-18) | 7.125 | 0.008 |
| Yes | 38 | 12.90 (2-24) | ||
| TNM stage | ||||
| I+II | 29 | 16.31 (3-24) | ||
| III+IV | 40 | 6.83 (2-14) | 46.137 | 0.000 |
| Lymph node metastasis | ||||
| No | 27 | 16.04 (3-24) | 29.663 | 0.000 |
| Yes | 42 | 7.45 (2-15) | ||
| Invasion | ||||
| No | 24 | 17.25 (3-24) | 36.974 | 0.000 |
| Yes | 45 | 7.38 (2-20) | ||
| Surgery | ||||
| Radical | 27 | 16.93 (5-24) | ||
| Palliative | 28 | 7.32 (2-12) | 54.660 | 0.000 |
| Biopsy | 14 | 6.00 (4-8) | ||
| EphA10 | ||||
| - | 28 | 15.57 (7-24) | 23.269 | 0.000 |
| + | 41 | 7.56 (-128) | ||
| EphB3 | ||||
| - | 36 | 6.67 (2-11) | 42.016 | 0.000 |
| + | 33 | 15.33 (6-24) | ||
Figure 3.
EphA10 and EphB3 expression and survival in patients with SC/ASC of gallbladder. A. Kaplan-Meier plots of overall survival in patients with EphA10-positive and -negative tumours. B. Kaplan-Meier plots of overall survival in patients with EphB3-positive and -negative tumours.
Table 5.
Multivariate Cox regression analysis of survival rate in SC/ASC patients
| Groups | Factors | B | SE | Wald | P | RR | 95% CI | |
|---|---|---|---|---|---|---|---|---|
|
| ||||||||
| Lower | Upper | |||||||
| Differentiated degree | Well/moderately/poorly | 0.537 | 0.215 | 6.254 | 0.012 | 1.711 | 1.123 | 2.605 |
| Tumor size | ≤3 cm/>3 cm | 0.886 | 0.384 | 5.312 | 0.021 | 2.425 | 1.142 | 5.151 |
| Gallstone | No/yes | 0.739 | 0.313 | 5.573 | 0.018 | 2.095 | 1.134 | 3.870 |
| TNM stage | I+II/III+IV | 1.270 | 0.597 | 4.529 | 0.033 | 3.560 | 1.105 | 11.461 |
| Lymph node metastasis | No/yes | 1.159 | 0.464 | 6.229 | 0.013 | 3.187 | 1.283 | 7.920 |
| Invasion | No/yes | 1.337 | 0.573 | 5.433 | 0.020 | 3.806 | 1.237 | 11.710 |
| Surgery | Radical/Palliative/Biopsy | 0.765 | 0.292 | 6.883 | 0.009 | 2.149 | 1.213 | 3.805 |
| EphA10 | -/+ | 1.192 | 0.493 | 5.853 | 0.016 | 3.294 | 1.254 | 8.652 |
| EphB3 | -/+ | -1.339 | 520 | 6.634 | 010 | 0.262 | 0.095 | 0.726 |
Figure 4.
ROC of Diagonal segments are produced by ties of EphA10 and EphB3 in SC/ASC.
The Kaplan-Meier survival analysis of the AC patients revealed similar results as for the SC/ASC patients (Table 6). The average survival time of the EphA10 positive AC patients was significantly lower than patients exhibiting negative EphA10 expression (P < 0.01; Table 6 and Figure 5). The average survival time of the EphB3 positive AC patients was significantly higher than patients exhibiting negative EphB3 expression (P < 0.01; Table 6 and Figure 5). Cox’s multivariate analysis demonstrated that the differentiation, tumor size (≥ 3 cm), TNM stage, invasion, surgical procedure, EphA10-positive and EphB3-negative expression were negatively correlated with overall survival, indicating that the positive expression of EphA10 and negative expression of EphB3 is a risk factor of AC (Table 7). Finally, we calculated the AUC for EphA10 (AUC = 0.710, 95% CI: 0.621-0.799), or EphB3 (AUC = 0.697, 95% CI: 0.606-0.787) in AC, respectively (Figure 6).
Table 6.
Relationship between EphA10 and EphB3 expression clinicopathological characteristics and average survival of AC patients
| Clinicopathological characteristics | Sample (n) | Averagesurvival (month) | X2 | P value |
|---|---|---|---|---|
| Differentiation | ||||
| Well | 51 | 16.69 (5-24) | ||
| Moderately | 54 | 12.33 (2-24) | 55.112 | 0.000 |
| Poorly | 41 | 6.49 (1-24) | ||
| Tumor size | ||||
| ≤ 3 cm | 90 | 14.60 (1-24) | 23.174 | 0.000 |
| > 3 cm | 56 | 8.38 (1-24) | ||
| Gallstones | ||||
| No | 78 | 12.19(2-24) | 0.001 | 0.980 |
| Yes | 68 | 12.24 (1-24) | ||
| TNM stage | ||||
| I+II | 77 | 16.99 (3-24) | ||
| III+IV | 69 | 6.88 (1-24) | 87.485 | 0.000 |
| Lymph node metastasis | ||||
| No | 80 | 16.35 (2-24) | 71.402 | 0.000 |
| Yes | 66 | 7.20 (1-24) | ||
| Invasion | ||||
| No | 72 | 18.08 (4-24) | 124.522 | 0.000 |
| Yes | 74 | 6.50 (1-14) | ||
| Surgery | ||||
| Radical | 75 | 17.84 (6-24) | ||
| Palliative | 50 | 6.86 (1-14) | 150.255 | 0.000 |
| Biopsy | 21 | 4.86 (1-9) | ||
| EphA10 | ||||
| - | 70 | 16.50 (3-24) | 49.650 | 0.000 |
| + | 76 | 8.26 (1-24) | ||
| EphB3 | ||||
| - | 72 | 7.71 (1-24) | 62.779 | 0.000 |
| + | 74 | 16.60 (2-24) | ||
Figure 5.
EphA10 and EphB3 expression and survival in patients with AC of gallbladder. A. Kaplan-Meier plots of overall survival in patients with EphA10-positive and -negative tumours. B. Kaplan-Meier plots of overall survival in patients with EphB3-positive and -negative tumours.
Table 7.
Multivariate Cox regression analysis of survival rate in AC patients
| Groups | Factors | B | SE | Wald | P | RR | 95% CI | |
|---|---|---|---|---|---|---|---|---|
|
| ||||||||
| Lower | Upper | |||||||
| Differentiated degree | Well/moderately/poorly | 0.434 | 0.164 | 7.017 | 0.008 | 1.543 | 1.119 | 2.127 |
| Tumor size | ≤ 3 cm/> 3 cm | 0.786 | 0.324 | 5.870 | 0.015 | 2.194 | 1.162 | 4.144 |
| Gallstone | No/yes | 0.410 | 0.210 | 3.790 | 0.052 | 1.506 | 0.997 | 2.275 |
| Lymph node metastasis | No/yes | 0.921 | 0.361 | 6.519 | 0.011 | 2.513 | 1.239 | 5.096 |
| Invasion | No/yes | 1.748 | 0.426 | 16.835 | 0.000 | 5.741 | 2.49 | 13.229 |
| TNM stage | I+II/III+IV | 0.952 | 0.414 | 5.286 | 0.022 | 2.590 | 1.151 | 5.829 |
| Surgery | Radical/Palliative/Biopsy | 0.813 | 0.268 | 9.182 | 0.002 | 2.255 | 1.333 | 3.815 |
| EphA10 | -/+ | 0.833 | 0.273 | 3.9328 | 0.002 | 2.301 | 1.346 | 9.286 |
| EphB3 | -/+ | 0.771 | 0.272 | 8.009 | 0.005 | 0.463 | 0.271 | 0.789 |
Figure 6.
ROC of Diagonal segments are produced by ties of EphA10 and EphB3 in AC.
Discussion
The current knowledge on the clinicopathological characteristics of SC/ASC has mainly been obtained from individual case studies or analyses of small case series. Therefore, accurate understanding of the differences between rare SC/ASC tumors and ordinary AC is not po ssible without further studies. The reported incidence of squamous differentiation is 1-12% in gallbladder malignancies [4,7], and in the present study 4.34% SCs/ASCs were observed. A previous study identified that the occurrence of SC/ASC is predominant in females (F/M, 3.8) [31]; however in the present study there was no significant difference (F/M, 2.14). It was also apparent in the present study that the prevalence of SC/ASC was more significant in older patients compared with AC. In previous studies, it has been demonstrated that the proliferation of SC occurs at a higher rate than AC, whereas the prevalence of squamous tumors is less frequent with lymph node metastasis [32,33]. Observations from the present study revealed the percentage of cases with lymph node metastasis and invasion was significantly higher in the SCs/ASCs compared with the ACs (P < 0.05). No differences in the occurrence of differentiated degrees, tumor size, TNM stages, surgical methods, EphA10 and EphB3 expressive rates between AC and SC/ASC. In total, 60.9% of SC/ASC and 48.6% of AC patients were diagnosed at a later stage; however, for the remaining patients it was apparent that radical resection was a good prognostic factor for AC and SC/ASC. There was no significant difference in the post-operative survival time between cases of AC and SC/ASC. These observations indicated that the clinicopathological presentations of SC/ASC might have strong invasive and metastaticpotential compared to ordinary AC.
The expression of EphA10 and EphB3 in AC and SC/ASC has not been previously reported, although their expressions have been associated with the progression and prognosis of a variety of tumors. This study investigated EphA10 and EphB3 protein expression in AC and SC/ASC using immunohistochemistry. A significant increase in EphA10 and significant decrease in EphB3 expression in AC and SC/ASC tumors was observed. Positive EphA10 and negative EphB3 expressions are associated with TNM stages, invasion, metastasis, and poor prognosis of AC and SC/ASC.
The roles of Eph receptors and ephrin ligands have been described in normal development [34,35], and aberrant patterns of their expression have been linked to a variety of human cancer types [9,36]. Eph receptor family with 14 distinct RTKs constitutes an important class of cell surface proteins. Among these, EphA10 is a kinase-deficient protein [37]. Some pre-clinical and laboratory studies have established the correlation of Eph RTKs in tumor growth, metastasis, and the formation of functional microvascular networks in cancer [38-40]. The investigations of breast cancer cell lines with differing phenotypes have illustrated the involvement of a variety of cell surface proteins in the progression of breast carcinoma [41,42]. While EphA10 was recently correlated with poor prognosis and metastasis, higher expression levels of EphA10 were found in invasive breast carcinoma cells [14,15,41-43]. Given its lack of kinase activity, the biochemical mechanisms underlying EphA10 activation have, thus, become relevant to explaining its involvement in breast tumorigenesis.
The 14 members of the Eph family of receptors make up the largest family of receptor tyrosine kinases in humans [33]. These receptors are associated with 8 members of a class of ligands known as ephrins. These receptors and ligands are divided into A and B classes based on their sequence homology as well as their affinity for the corresponding receptor/ligand [33]. Many Eph receptors and ephrin ligands are known to be involved in the development or progression of certain cancers [44]. In CRC, EphB2 and EphB3 are important tumor suppressors whose activity effectively restricts tumor-cell spreading [17,18]. However, the transition from noninvasive adenoma to invasive carcinoma states is frequently paralleled by their transcriptional down-regulation [17,18,24]. EphB3 is sparsely mentioned as a therapeutic target, but Eph family kinase receptors are associated with gastrointestinal tract cancers [45]. Overexpression in lung cancer has been reported, and a correlation with metastasis has been suggested [46,47].
The role of EphA10 and EphB3 expressions in gallbladder SC/ASC and AC remains to be clarified. Our study first showed that the positive expressions of EphA10 and negative expression of EphB3 were significantly higher in the cases of SC/ASC and AC than in chronic cholecystitis. The positive expressions of EphA10 were significantly higher in the cases of poorly differentiation, large tumor size, high TNM stage, lymph node metastasis, invasion and no resection (only biopsy) of SC/ASC and AC. The positive expressions of EphB3 were significantly lower in the cases of poorly differentiation, large tumor size, high TNM stage, lymph node metastasis, invasion and no resection (only biopsy) of SC/ASC and AC. The univariate Kaplan-Meier analysis showed that positive EphA10 and negative EphB3 expression is closely associated with a decreased overall survival in SC/ASC and AC patients. The multivariate Cox regression analysis identified that positive EphA10 and negative EphB3 expression are independent factors for a poor-prognosis in SC/ASC and AC patients. The AUC for EphA10 and EphB3 showed might have role for carcinogenesis and progression of SC/ASC and AC.
In conclusion, EphA10 and EphB3 are involved in the tumorigenesis and progression of SC/ASC and AC, and negative EphB3 and positive EphA10 expressions were associated with poor prognosis in patients with SC/ASC and AC.
Disclosure of conflict of interest
None.
References
- 1.Jemal A, Siegel R, Ward E, Hao Y, Xu J, Murray T, Thun MJ. Cancer statistics, 2008. CA Cancer J Clin. 2008;58:71–96. doi: 10.3322/CA.2007.0010. [DOI] [PubMed] [Google Scholar]
- 2.Jayaraman S, Jarnagin WR. Management of gallbladder cancer. Gastroenterol Clin North Am. 2010;39:331–342. doi: 10.1016/j.gtc.2010.02.006. [DOI] [PubMed] [Google Scholar]
- 3.Reid KM, Ramos-Dela MA, Donohue JH. Diagnosis and surgical management of gallbladder cancer: a review. J Gastrointest Surg. 2007;11:671–681. doi: 10.1007/s11605-006-0075-x. [DOI] [PubMed] [Google Scholar]
- 4.Hawkins WG, DeMatteo RP, Jarnagin WR, Ben-Porat L, Blumgart LH, Fong Y. Jaundice predicts advanced disease and early mortality in patients with gallbladder cancer. Ann Surg Oncol. 2004;11:310–315. doi: 10.1245/aso.2004.03.011. [DOI] [PubMed] [Google Scholar]
- 5.Ootani T, Shirai Y, Tsukada K, Muto T. Relationship between gallbladder carcinoma and the segmental type of adenomyomatosis of the gallbladder. Cancer. 1992;69:2647–2652. doi: 10.1002/1097-0142(19920601)69:11<2647::aid-cncr2820691105>3.0.co;2-0. [DOI] [PubMed] [Google Scholar]
- 6.Roa JC, Tapia O, Cakir A, Basturk O, Dursun N, Akdemir D, Saka B, Losada H, Bagci P, Adsay NV. Squamous cell and adenosquamous carcinomas of the gallbladder: clinicopathological analysis of 34 cases identified in 606 carcinomas. Mod Pathol. 2011;24:1069–1078. doi: 10.1038/modpathol.2011.68. [DOI] [PubMed] [Google Scholar]
- 7.Park SB, Kim YH, Rho HL, Chae GB, Hong SK. Primary carcinosarcoma of the gallbladder. J Korean Surg Soc. 2012;82:54–58. doi: 10.4174/jkss.2012.82.1.54. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 8.Liu DC, Yang ZL. MTDH and EphA7 are markers for metastasis and poor prognosis of gallbladder adenocarcinoma. Diagn Cytopathol. 2013;41:199–205. doi: 10.1002/dc.21821. [DOI] [PubMed] [Google Scholar]
- 9.Manning G, Whyte DB, Martinez R, Hunter T, Sundaisanam S. The protein kinase complement of the human genome. Science. 2002;298:1912–1934. doi: 10.1126/science.1075762. [DOI] [PubMed] [Google Scholar]
- 10.Fox BP, Tabone CJ, Kandpal RP. Potential clinical relevance of Eph receptors and ephrin ligands expressed in prostate carcinoma cell lines. Biochem Biophy Res Commun. 2006;342:1263–1272. doi: 10.1016/j.bbrc.2006.02.099. [DOI] [PubMed] [Google Scholar]
- 11.Surawska H, Ma PC, Salgia R. The role of ephrins and Eph receptors in cancer. Cytokine Growth Factor Rev. 2004;15:419–433. doi: 10.1016/j.cytogfr.2004.09.002. [DOI] [PubMed] [Google Scholar]
- 12.Imai S, Nagano K, Yoshida Y, Okamura T, Yamashita T, Abe Y, Yoshikawa T, Yoshioka Y, Kamada H, Mukai Y, Nakagawa S, Tsutsumi Y, Tsunoda S. Development of an antibody proteomics system using a phage antibody library for efficient screening of biomarker proteins. Biomaterials. 2011;32:162–169. doi: 10.1016/j.biomaterials.2010.09.030. [DOI] [PubMed] [Google Scholar]
- 13.Nagano K, Maeda Y, Kanasaki S, Watanabe T, Yamashita T, Inoue M, Higashisaka K, Yoshioka Y, Abe Y, Mukai Y, Kamada H, Tsutsumi Y, Tsunoda S. Ephrin receptor A10 is a promising drug target potentially useful for breast cancers including triple negative breast cancers. J Control Release. 2014;189:72–79. doi: 10.1016/j.jconrel.2014.06.010. [DOI] [PubMed] [Google Scholar]
- 14.Nagano K, Kanasaki S, Yamashita T, Maeda Y, Inoue M, Higashisaka K, Yoshioka Y, Abe Y, Mukai Y, Kamada H, Tsutsumi Y, Tsunoda S. Expression of Eph receptor A10 is correlated with lymph node metastasis and stage progression in breast cancer patients. Cancer Med. 2013;2:972–7. doi: 10.1002/cam4.156. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 15.Fujimori T, Kato K, Fujihara S, Iwama H, Yamashita T, Kobayashi K, Kamada H, Morishita A, Kobara H, Mori H, Okano K, Suzuki Y, Masaki T. Antitumor effect of metformin on cholangiocarcinoma: in vitro and in vivo studies. Oncol Rep. 2015;34:2987–96. doi: 10.3892/or.2015.4284. [DOI] [PubMed] [Google Scholar]
- 16.Nagano K, Yamashita T, Inoue M, Higashisaka K, Yoshioka Y, Abe Y, Kamada H, Tsutsumi Y, Tsunoda S. Eph receptor A10 has a potential as a target for a prostate cancer therapy. Biochem Biophys Res Commun. 2014;450:545–9. doi: 10.1016/j.bbrc.2014.06.007. [DOI] [PubMed] [Google Scholar]
- 17.Batlle E, Bacani J, Begthel H, Jonkheer S, Gregorieff A, van de Born M, Malats N, Sancho E, Boon E, Pawson T, Gallinger S, Pals S, Clevers H. EphB receptor activity suppresses colorectal cancer progression. Nature. 2005;435:1126–1130. doi: 10.1038/nature03626. [DOI] [PubMed] [Google Scholar]
- 18.Chiu ST, Chang KJ, Ting CH, Shen HC, Li H, Hsieh FJ. Over-expression of EphB3 enhances cell-cell contacts and suppresses tumor growth in HT-29 human colon cancer cells. Carcinogenesis. 2009;30:1475–1486. doi: 10.1093/carcin/bgp133. [DOI] [PubMed] [Google Scholar]
- 19.Cortina C, Palomo-Ponce S, Iglesias M, Fernández-Masip JL, Vivancos A, Whissell G, Humà M, Peiró N, Gallego L, Jonkheer S, Davy A, Lloreta J, Sancho E, Batlle E. EphB-ephrin-B interactions suppress colorectal cancer progression by compartmentalizing tumor cells. Nat Genet. 2007;39:1376–1383. doi: 10.1038/ng.2007.11. [DOI] [PubMed] [Google Scholar]
- 20.Jäglea S, Rönscha K, Timme K, Andrlová H, Bertrand M, Jäger M, Proske A, Schrempp M, Yousaf A, Michoel T, Zeiser R, Werner M, Lassmann S, Hecht A. Silencing of the EPHB3 tumor-suppressor gene in human colorectal cancer through decommissioning of a transcriptional enhancer. Proc Natl Acad Sci U S A. 2014;111:4886–4891. doi: 10.1073/pnas.1314523111. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 21.Batlle E, Bacani J, Begthel H, Jonkheer S, Gregorieff A, van de Born M, Malats N, Sancho E, Boon E, Pawson T, Gallinger S, Pals S, Clevers H. EphB receptor activity suppresses colorectal cancer progression. Nature. 2005;435:1126–1130. doi: 10.1038/nature03626. [DOI] [PubMed] [Google Scholar]
- 22.Jagle S, Ronsch K, Timme S, Andrlova H, Bertrand M, Jager M, Proske A, Schrempp M, Yousaf A, Michoel T, Zeiser R, Werner M, Lassmann S, Hecht A. Silencing of the EPHB3 tumor-suppressor gene in human colorectal cancer through decommissioning of a transcriptional enhancer. Proc Natl Acad Sci U S A. 2014;111:4886–4891. doi: 10.1073/pnas.1314523111. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 23.Fre S, Pallavi SK, Huyghe M, Lae M, Janssen KP, Robine S, Artavanis-Tsakonas S, Louvard D. Notch and Wnt signals cooperatively control cell proliferation and tumorigenesis in the intestine. Proc Natl Acad Sci U S A. 2009;106:6309–6314. doi: 10.1073/pnas.0900427106. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 24.Ronsch K, Jager M, Schopflin A, Danciu M, Lassmann S, Hecht A. Class I and III HDACs and loss of active chromatin features contribute to epigenetic silencing of CDX1 and EPHB tumor suppressor genes in colorectal cancer. Epigenetics. 2011;6:610–622. doi: 10.4161/epi.6.5.15300. [DOI] [PubMed] [Google Scholar]
- 25.Ronscha K, Jaglea S, Rosea K, Seidl M, Baumgartnere F, Freihena V, Yousaf A, Metzger E, Lassmann S, Schüle R, Zeiser R, Michoel T, Hecht A. SNAIL1 combines competitive displacement of ASCL2 and epigenetic mechanisms to rapidly silence the EPHB3 tumor suppressor in colorectal cancer. Mol Oncol. 2015;9:335–354. doi: 10.1016/j.molonc.2014.08.016. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 26.Shinmura K, Kiyose S, Nagura K, Igarashi H, Inoue Y, Nakamura S, Maeda M, Baba M, Konno H, Sugimura H. TNK2 gene amplification is a novel predictor of a poor prognosis in patients with gastric cancer. J Surg Oncol. 2014;109:189–97. doi: 10.1002/jso.23482. [DOI] [PubMed] [Google Scholar]
- 27.Li G, Ji XD, Gao H, Zhao JS, Xu JF, Sun ZJ, Deng YZ, Shi S, Feng YX, Zhu YQ, Wang T, Li JJ, Xie D. EphB3 suppresses non-small-cell lung cancer metastasis via a PP2A/RACK1/Akt signalling complex. Nat Commun. 2012;3:667. doi: 10.1038/ncomms1675. [DOI] [PubMed] [Google Scholar]
- 28.Casimiro MC, Wang C, Li Z, Di Sante G, Willmart NE, Addya S, Chen L, Liu Y, Lisanti MP, Pestell RG. Cyclin D1 determines estrogen signaling in the mammary gland in vivo. Mol Endocrinol. 2013;27:1415–28. doi: 10.1210/me.2013-1065. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 29.Wu Q, Lind GE, Aasheim HC, Micci F, Silins I, Tropé CG, Nesland JM, Lothe RA, Suo Z. The EPH receptor Bs (EPHBs) promoters are unmethylated in colon and ovarian cancers. Epigenetics. 2007;2:237–43. doi: 10.4161/epi.2.4.5406. [DOI] [PubMed] [Google Scholar]
- 30.Fox BP, Tabone CJ, Kandpal RP. Potential clinical relevance of Eph receptors and ephrin ligands expressed in prostate carcinoma cell lines. Biochem Biophys Res Commun. 2006;342:1263–72. doi: 10.1016/j.bbrc.2006.02.099. [DOI] [PubMed] [Google Scholar]
- 31.Li L, Liao J, Ruland J, Mak TW, Cohen SN. A TSG101/MDM2 regulatory loop modulates MDM2 degradation and MDM2/p53 feedback control. Proc Natl Acad Sci U S A. 2001;98:1619–1624. doi: 10.1073/pnas.98.4.1619. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 32.Kondo M, Dono K, Sakon M, Shimizu J, Nagano H, Nakamori S, Umeshita K, Wakasa K, Monden M. Adenosquamous carcinoma of the gallbladder. Hepatogastroenterology. 2002;49:1230–1234. [PubMed] [Google Scholar]
- 33.Muzio G, Maggiora M, Paiuzzi E, Oraldi M, Canuto RA. Aldehyde dehydrogenases and cell proliferation. Free Radic Biol Med. 2012;52:735–746. doi: 10.1016/j.freeradbiomed.2011.11.033. [DOI] [PubMed] [Google Scholar]
- 34.Holder N, Klein R. Eph receptors and ephrins: effectors of morphogenesis. Development. 1999;126:2033–2044. doi: 10.1242/dev.126.10.2033. [DOI] [PubMed] [Google Scholar]
- 35.Barrios A, Poole RJ, Durbin L, Brennan C, Holder N, Wilson SW. Eph/Ephrin signaling regulates the mesenchymal-to-epithelial transition of the paraxial mesoderm during somitemorphogenesis. Curr Biol. 2003;13:1571–1582. doi: 10.1016/j.cub.2003.08.030. [DOI] [PubMed] [Google Scholar]
- 36.Pasquale EB. Eph-ephrin bidirectional signaling in physiology and disease. Cell. 2008;133:38–52. doi: 10.1016/j.cell.2008.03.011. [DOI] [PubMed] [Google Scholar]
- 37.Murai KK, Pasquale EB. ‘Eph’ective signaling: forward, reverse and crosstalk. J Cell Sci. 2003;116:2823–2832. doi: 10.1242/jcs.00625. [DOI] [PubMed] [Google Scholar]
- 38.Pasquale EB. Eph receptors and ephrins in cancer: bidirectional signalling and beyond. Nat Rev Cancer. 2010;10:165–180. doi: 10.1038/nrc2806. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 39.Brantley-Sieders D, Schmidt S, Parker M, Chen J. Eph receptor tyrosine kinases in tumor and tumor microenvironment. Curr Pharm Des. 2004;10:3431–3442. doi: 10.2174/1381612043383160. [DOI] [PubMed] [Google Scholar]
- 40.Brantley-Sieders DM, Chen J. Eph receptor tyrosine kinases in angiogenesis: from development to disease. Angiogenesis. 2004;7:17–28. doi: 10.1023/B:AGEN.0000037340.33788.87. [DOI] [PubMed] [Google Scholar]
- 41.Fox BP, Kandpal RP. Invasiveness of breast carcinoma cells and transcript profile: Eph receptors and ephrin ligands as molecular markers of potential diagnostic and prognostic application. Biochem Biophys Res Commun. 2004;318:882–892. doi: 10.1016/j.bbrc.2004.04.102. [DOI] [PubMed] [Google Scholar]
- 42.Nagaraja GM, Othman M, Fox BP, Alsaber R, Pellegrino CM, Zeng Y, Khanna R, Tamburini P, Swaroop A, Kandpal RP. Gene expression signatures and biomarkers of noninvasive and invasive breast cancer cells: comprehensive profiles by representational difference analysis, microarrays and proteomics. Oncogene. 2006;25:2328–2338. doi: 10.1038/sj.onc.1209265. [DOI] [PubMed] [Google Scholar]
- 43.Bai YQ, Zhang JY, Bai CY, Xu XE, Wu JY, Chen B, Wu ZY, Wang SH, Shen J, Shen JH, Yao XD, Gao LZ, Wu B, Gu HL, Liu XH, Li X, Li EM, Xu LY. Low EphA7 expression correlated with lymph node metastasis and poor prognosis of patients with esophageal squamous cell carcinoma. Acta Histochem Cytochem. 2015;48:75–81. doi: 10.1267/ahc.14054. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 44.Fox BP, Tabone CJ, Kandpal RP. Potential clinical relevance of Eph receptors and ephrin ligands expressed in prostate carcinoma cell lines. Biochem Biophys Res Commun. 2006;342:1263–1272. doi: 10.1016/j.bbrc.2006.02.099. [DOI] [PubMed] [Google Scholar]
- 45.Sugimura H, Wang JD, Mori H, Tsuboi M, Nagura K, Igarashi H, Tao H, Nakamura R, Natsume H, Kahyo T, Shinmura K, Konno H, Hamaya Y, Kanaoka S, Kataoka H, Zhou XJ. EPH-EPHRIN in human gastrointestinal cancers. World J Gastrointest Oncol. 2011;2:421–8. doi: 10.4251/wjgo.v2.i12.421. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 46.Ji XD, Li G, Feng YX, Zhao JS, Li JJ, Sun ZJ, Shi S, Deng YZ, Xu JF, Zhu YQ, Koeffler HP, Tong XJ, Xie D. EphB3 is overexpressed in non-small-cell lung cancer and promotes tumor metastasis by enhancing cell survival and migration. Cancer Res. 2011;71:1156–66. doi: 10.1158/0008-5472.CAN-10-0717. [DOI] [PubMed] [Google Scholar]
- 47.Kiyose S, Nagura K, Tao H, Igarashi H, Yamada H, Goto M, Maeda M, Kurabe N, Suzuki M, Tsuboi M, Kahyo T, Shinmura K, Hattori N, Sugimura H. Detection of kinase amplifications in gastric cancer archives using fluorescence in situ hybridizationpion. Pathol Intern. 2012;62:477–484. doi: 10.1111/j.1440-1827.2012.02832.x. [DOI] [PubMed] [Google Scholar]