Int J Clin Exp Pathol 2018;11(2):512-525 www.ijcep.com /ISSN:1936-2625/IJCEP0068151

Original Article EphA10 and EphB3 are prognostic markers in gallbladder squamous /adenosquamous carcinomas and adenocarcinomas

Hai-Xiong Yan1*, Qing-Long Li1*, Wen-Wu Cai1, Da Tang2, Biao Wang1, Xiang Liu1, Zhi-De Liu1, Zhu-Lin Yang1

1Department of General Surgery, The Second Xiangya Hospital, Central South University, Changsha, Hunan, China; 2Department of General Surgery, The Third Xiangya Hospital, Central South University, Changsha, Hunan, China. *Equal contributors. Received October 29, 2017; Accepted November 13, 2017; Epub February 1, 2018; Published February 15, 2018

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 car- cinogenesis, 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. Con- clusions: 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, im- munohistochemistry

Introduction ASC have not been well documented, and therapeutic interventions for these tumours Gallbladder cancers (GBCs) are relatively are not established [7]. Although biomarkers uncommon human cancers, and they have a for the prognosis of AC are currently under particular geographical distribution in central investigation, no proposed markers have been and South America, central and Eastern applied clinically [8]. No biomarkers relating to Europe, Japan and northern India [1, 2]. GBC is the progression and prognosis of SC/ASC have increasing worldwide, and prognosis is extre- been identified. mely poor. Early diagnosis may be impossible because such cancers are asymptomatic [3]. The 14 members of the Eph family of receptors More than 90% of GBC patients are diagnosed make up the largest family of receptor tyrosine at an inoperable stage, when tumours are inva- kinases in humans [9, 10]. These receptors are sive and metastatic [4]. Most GBCs are adeno- associated with 8 members of a class of ligands carcinomas (AC; > 90%) [5]. In contrast, squa- known as . These receptors and ligands mous cell/adenosquamous carcinoma (SC/ are divided into A and B classes based on their ASC) is rare, representing 1-12% of GBCs [2, 6]. sequence homology as well as their affinity for The clinicopathological characteristics of SC/ the corresponding receptor/ligand [10]. Many EphA10 and EphB3 in gallbladder cancers

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)

Eph receptors and ligands are known to cer progression [14]. These findings suggest be involved in the development or progression that EphA10 is a promising target for breast of certain cancers [11]. EphA10 which we iden- cancer therapy. Fujimori, et al. found that tified as a novel breast cancer-related is EphA10 could promote tumor proliferation in hardly expressed in normal human tissues [12, athymic nude mice with xenograft cholangio- 13]. Furthermore, it also showed that an in- carcinoma [15]. Nagano, et al. found that the house developed anti-EphA10 mAb inhibited overexpression of EphA10 in prostate cancers breast cancer cell proliferation at both in vitro might have a potential as a target for prostate and in vivo levels [13]. Nagano, et al. found that cancer therapy [16]. EphA10 expression might play a role in tumor progression and metastasis. It will help eluci- In tumorigenesis, signaling by the protein tyro- date the role of EphA10 in clinical breast can sine kinases Ephrin type-B receptor 2 (EphB2)

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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], ovari- an cancer [29] and prostate cancer [30], the experimental results suggested that the low expression of EphB3 had close relationship to patho- genesis, progresion and bio- logical behaviors of above malignant lesions.

As far as we know, the role of EphA10 and EphB3 in gall- bladder SC/ASC and AC re- mains to be clarified. Thus, we evaluated EphA10 and EphB3 expression in surgically resect- ed specimens, including 146 ACs and 69 SC/ASCs, using Figure 1. EphA10 and EphB3 expression. EnVision immunohistochemistry, immunohistochemistry. We th- original magnification ×200. EphA10 and EphB3 expression was localized in en correlated their expression the cytoplasm. A. Positive expression of EphA10 in poor differentiated SC. B. with the clinicopathological Negative expression of EphA10 in well differentiated SC. C. Positive expres- characteristics and prognosis sion of EphB3 in moderately differentiated ASC. D. Negative expression of of patients with AC and SC/ EphB3 in poorly differentiated SC. ASC. and 3 (EphB3) represents a powerful barrier Materials and methods against tumor-cell spreading and the onset of metastasis, the main cause for cancer-related Case selection mortality [17, 18]. Through repulsive interac- tions between cells expressing EphB receptors This study was pre-approved by the Ethics and cells presenting EphrinB ligands, EphB/ Committee for Human Research, Central South EphrinB signaling compartmentalizes tumors n University. A total of 69 SC/ASC samples result- molecule E-cadherin, thereby adding to the sta- ing from surgical resection or biopsy were col- bilization of a noninvasive epithelial-cell pheno- lected from January 2001 to December 2013. type [19, 20]. EphB3 and locally confines their According to the recommendations of the growth [19, 20]. In addition, EphB/EphrinB sig- American Joint Committee on Cancer, tumours naling affects the function of the cell-cell adhe- with a squamous component ≥ 10% were con- siois a direct target of Wnt/β-catenin and sidered to represent adenosquamous carcino- Notch signaling [21, 22] and in agreement with mas. The 69 SC/ASCs comprised 5.5% of 1248 the pivotal role of these pathways in tumor ini- GBCs. Of these 66, 16 (of 200 GBCs) were col- tiation [23]. EphB3 is strongly upregulated lected from Xiangya Hospital, 31 (of 628 GBCs) in colorectal adenomas [24]. However, this from Second Xiangya Hospital, 10 (of 200 surge in EphB3 expression at early stages of GBCs) from Third Xiangya Hospital, 5 (of 70 tumorigenesis is followed by secondary down- GBCs) from Hunan Provincial People Hospital, 5 regulation in up to 30% of carcinomas. Ronscha (of 100 GBCs) from Hunan Provincial Tumor et al. identify EphB3 as a novel target of Snail1 Hospital, and one each from Changde Central and suggest that disabling EphB3 signaling is Hospital and Loudi Central Hospital (two of 50 an important aspect to eliminate a roadblock at GBCs). A total of 146 AC samples derived from

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Among the 146 patients with ACs, invasion was found in 74 (50.7%); 66 (45.2%) had re- gional lymph node metasta- sis; and 68 (46.6%) had gall- stones. According to tumour- node-metastasis (TNM) stag- ing, 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, palli- ative 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 sur- Figure 2. EphA10 and EphB3 expression. EnVision immunohistochemistry, original magnification ×200. EphA10 and EphB3 expression was localized vived longer than 2 years were in the cytoplasm. A. Positive expression of EphA10 in moderately differenti- included in the analysis as ated adenocarcinoma. B. Negative expression of EphA10 in well differenti- censored cases. Of the AC ated adenocarcinoma. C. Positive expression of EphB3 in well differentiated patients, 58 survived more adenocarcinoma. D. Negative expression of EphB3 in poorly differentiated than 1 year (26 more than 2 adenocarcinoma. years), and 88 survived < 1 year. Of the SC/ASC patients, surgical resection or biopsy at Second Xiangya 17 survived more than 1 year (7 more than 2 Hospital and Third Xiangya Hospital were col- years) and 52 survived < 1 year. lected between January 2008 and December 2013. EnVision immunohistochemistry

Clinicopathological data is summarized in Table Four-micrometer-thick sections were cut from 1. Among the 69 SC/ASC samples, 47 were routinely paraffin-embedded tissues. The rab- from female patients (F/M = 2.14) and patient bit anti-human EphA10 and EphB3, and HRP- ages ranged from 35 to 80 (53.8 ± 10.2) years. conjugated anti-rabbit second antibody were Among the 146 patients with AC, 92 patients purchased from Santa Cruz Biotechnology were female (F/M = 1.77), with an age range of (Santa Cruz, CA, USA). EnVisionTM Detection 33 to 78 (52.4 ± 9.6) years. Of the 69 SC/ASCs, Kit was purchased from Dako Laboratories (CA, the squamous cell component was well differ- USA). The staining of EphA10 and/or EphB3 entiated in 19 (27.5%), moderately differentiat- was carried out according to the manufacture’s ed in 33 (47.8%), and poorly differentiated in protocol. Briefly, the sections were deparaf- 17 (24.6%). Of the 146 ACs, 51 were well dif- finized and then incubated withperoxidase ferentiated (34.9%), 54 were moderately differ- inhibitor (3% H2O2) in the dark for 15 minutes, entiated (37.0%) and 41 were poorly differenti- followed by EDTA-trypsin digestion for 15 min- ated (28.1%). Among the SC/ASC patients, utes. The sections were incubated with primary invasion of tissues and organs surrounding antibody for 120 minutes after being soaked the gallbladder was observed in 45 patients with PBS for 3 × 5 minutes. Solution A was (65.2%); 42 (60.7%) had regional lymph node added to the sections for 30 minutes followed metastasis; and 38 (55.1%) had gallstones. by DAB staining and hematoxylin counter-stain-

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Table 2. Correlations of EphA10 and EphB3 protein expression with the clinicopathological character- istics of gallbladder SC/ASC EphA10 EphB3 CPC Case No. 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) ing. The slides were dehydrated with different 13.0). The inter-relationship of EphA10 or concentrations (70%-100%) of alcohol,and EphB3 expression with histology or clinical fac- soaked in xylene for 3 × 5 minutes and finally tors was analyzed using X2 or Fisher’s exact mounted with neutral balsam. Ten random test. Kaplan-Meier and time series test (log- fields were examined per section. The percent ranktest) were used for Univariate survival to positively stained cells relative to the total analysis. Cox proportional hazards model was number of cells was determined. Next, the used for multivariate analysis and to determine strength of staining was rated on a scale of 1 to the 95% confidence interval. ROC of Diagonal 3. A score of 1 represents little to no positive segments is produced by ties. staining or uncertainly weak staining; a score of 2 represents weak to moderate staining; and a Results score of 3 represents moderate to strong stain- ing. A section is determined as positive for Comparison of EphA10 and EphB3 expression EphA10 or EphB3 when the percent of positive- and clinicopathological characteristics in SC/ ly stained cells was ≥ 10% and staining strength ASC and AC ≥ 2. The few sections where percent positive As shown in Table 1, the percentage of cases staining was 5% to 10% and staining strength with a patient age of > 45 years, lymph node was 3 were also regarded as positive. metastasis and invasion was significantly high- Statistical analysis er in the SCs/ASCs compared with the ACs (P < 0.05). Correlations between other clinicopatho- Data was analyzed using the statistical pack- logical characteristics and the percentage of age for the Social Sciences Version 13.0 (SPSS positive EphA10 or EphB3 expression were not

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Table 3. Correlations of EphA10 and EphB3 protein expression with the clinicopathological character- istics of gallbladder AC EphA10 EphB3 CPC Case No. 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) significant. The majority of EphA10- and EphB3- Association of clinicopathological characteris- positive reactions were localized in the cyto- tics and EphA10 and EphB3 expression in SC/ plasm of the SC/ASCs (Figure 1) and ACs (Fig- ASC and AC patients ure 2), as observed using EnVision immunohis- tochemistry (Dako Laboratories). A significantly lower association was apparent between the percentage of cases with EphB3 EphA10 and EphB3 expression in adenocarci- positive expression in the SC/ASC samples with noma of gallbladder, and gallbladder epitheli- poorly-differentiation, a large tumor mass size, um with chronic cholecystitis no gallstone, high TNM stage, lymph node metastasis, invasion and no resection (biopsy The percentage of cases with positive EphA10 only) compared with the cases of well- + was significantly higher in SC/ASC (50.9%) and Moderately-differentiation, small tumor size, AC (52.1%) than that in the gallbladder epithe- gallstone, low TNM stage, no lymph metastasis, lium with chronic cholecystitis (10.0%, ps = no invasion and radical resection (P < 0.05 or 0.000). The percentage of cases with positive P < 0.01; Table 2). A significantly higher asso- EphB3 expression was significantly lower in ciation was apparent between the percentage SC/ASC (47.8%) and AC (50.7%) than that in the of cases with EphA10 positive expression in the gallbladder epithelium with chronic cholecysti- SC/ASC samples with poorly-differentiation, a mpared with the cases of well differentiation, tis (90.0%, ps = 0.000). The epithelium of small tumor size, no gallstone, low TNM stage, chronic cholecystitis with high EphA10 or lower no lymph metastasis, no invasion and radical EphB3 expression showed moderate to severe resection (P < 0.05 or P < 0.01; Table 3). dysplasia. This suggests that both EphA10 and EphB3 could be markers to evaluate the pre- For AC tumors, the percentage of EphB3 potive malignant changes. expression was significantly lower in the cases

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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) with poor differentiation, large tumor mass 28 EphA10 negative cases, EphB3 was nega- size, high TNM stage, lymph node mettasis, tively expressed in 10 cases, suggesting a high invasion and no resection (biopsy only), com- inconsistency between these two markers in pared with the well-differentiated cases, small SC/ASC (X2 = 5.116, P < 0.05). Of the 76 tumor mass, low TNM stage, no lymph node EphA10 positive cases, EphB3 was positively metastasis, no invasion and radical resection expressed in 26 cases, while of the 70 EphA10 (P < 0.01; Table 3). The percentage of EphA10 negative cases, EphB3 was negatively ex- positive expression was significantly higher in pressed in 22 cases, suggesting a high in- the cases with poor differentiation, large tumor consistency between these two markers in AC mass size, high TNM stage, lymph node metas- (X2 = 17.212, P < 0.01). tasis, invasion and no resection (biopsy only), compared with the well-differentiated cases, Correlation between survival rates and EphA10 small tumor mass, low TNM stage, no lymph or EphB3 expression in patients with SC/ASC node metastasis, no invasion and radical and AC resection (P < 0.01; Table 3). Survival data for the 69 patients with SC/ASC Of the 41 EphA10 positive cases, EphB3 was and the 146 patients with AC was obtained positively expressed in 15 cases, while of the through letters and phone calls. The follow-up

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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 95% CI Groups Factors B SE Wald P RR 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.

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Table 6. Relationship between EphA10 and EphB3 expression clinicopathological characteristics and average survival of AC patients Averagesurvival Clinicopathological characteristics Sample (n) X2 P value (month) 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) time the present study was 2 years, and positive patients was significantly lower than patients who survived longer than 2 years were that of the patients with a negative result for included in the analysis as censored cases. Of EphA10 (P < 0.01; Table 4 and Figure 3). The the 146 AC patients, 58 survived more than 1 average survival time of the EphB3 positive year (26 more than 2 years), and 57 survived < patients was significantly higher than that of 1 year. Of the 69 SC/ASC patients, 17 survived the patients with a negative result for EphB3 (P more than 1 year (7 more than 2 years) and 52 < 0.01; Table 4 and Figure 3). Cox’s multiva- survived < 1 year. riate analysis demonstrated that the differen- tiation, tumor size (≥ 3 cm), TNM stage, inva- Evaluation of the SC/ASC patients using a sion, surgical procedure, EphA10-positive and Kaplan-Meier survival analysis demonstrated EphB3-negative expression were negatively that differentiation, tumor size, TNM stage, correlated with overall survival, indicating that lymph node metastasis, invasion, surgical pro- the positive expression of EphA10 and negative cedure and gallstone were significantly associ- expression of EphB3 is a risk factor of SCs/ ated with average survival time (P < 0.01, Table ASCs (Table 5). Finally, we calculated the AUC 4). The average survival time of the EphA10 for EphA10 (AUC = 0.747, 95% CI: 0.648-0.864),

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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 95% CI Groups Factors B SE Wald P RR 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 or EphB3 (AUC = 0.711, 95% CI: 0.607-0.805) tor of AC (Table 7). Finally, we calculated the in SCs/ASCs, respectively (Figure 4). AUC for EphA10 (AUC = 0.710, 95% CI: 0.621- 0.799), or EphB3 (AUC = 0.697, 95% CI: 0.606- The Kaplan-Meier survival analysis of the AC 0.787) in AC, respectively (Figure 6). patients revealed similar results as for the SC/ ASC patients (Table 6). The average survival Discussion time of the EphA10 positive AC patients was significantly lower than patients exhibiting neg- The current knowledge on the clinicopathologi- ative EphA10 expression (P < 0.01; Table 6 and cal characteristics of SC/ASC has mainly been Figure 5). The average survival time of the obtained from individual case studies or analy- EphB3 positive AC patients was significantly ses of small case series. Therefore, accurate higher than patients exhibiting negative EphB3 understanding of the differences between rare expression (P < 0.01; Table 6 and Figure 5). SC/ASC tumors and ordinary AC is not po ssible Cox’s multivariate analysis demonstrated that without further studies. The reported incidence the differentiation, tumor size (≥ 3 cm), TNM of squamous differentiation is 1-12% in gall- stage, invasion, surgical procedure, EphA10- bladder malignancies [4, 7], and in the present positive and EphB3-negative expression were study 4.34% SCs/ASCs were observed. A previ- negatively correlated with overall survival, indi- ous study identified that the occurrence of SC/ cating that the positive expression of EphA10 ASC is predominant in females (F/M, 3.8) [31]; and negative expression of EphB3 is a risk fac- however in the present study there was no sig-

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Figure 6. ROC of Diagonal segments are produced by ties of EphA10 and EphB3 in AC. nificant difference (F/M, 2.14). It was also significant increase in EphA10 and significant apparent in the present study that the preva- decrease in EphB3 expression in AC and SC/ lence of SC/ASC was more significant in older ASC tumors was observed. Positive EphA10 patients compared with AC. In previous studies, and negative EphB3 expressions are associat- it has been demonstrated that the proliferation ed with TNM stages, invasion, metastasis, and of SC occurs at a higher rate than AC, whereas poor prognosis of AC and SC/ASC. the prevalence of squamous tumors is less fre- quent with lymph node metastasis [32, 33]. The roles of Eph receptors and ephrin ligands Observations from the present study revealed have been described in normal development the percentage of cases with lymph node [34, 35], and aberrant patterns of their expres- metastasis and invasion was significantly high- sion have been linked to a variety of human er in the SCs/ASCs compared with the ACs (P < cancer types [9, 36]. Eph receptor family with 0.05). No differences in the occurrence of dif- 14 distinct RTKs constitutes an important class ferentiated degrees, tumor size, TNM stages, of cell surface . Among these, EphA10 surgical methods, EphA10 and EphB3 expres- is a kinase-deficient protein [37]. Some pre- sive rates between AC and SC/ASC. In total, clinical and laboratory studies have established 60.9% of SC/ASC and 48.6% of AC patients the correlation of Eph RTKs in tumor growth, were diagnosed at a later stage; however, for metastasis, and the formation of functional the remaining patients it was apparent that microvascular networks in cancer [38-40]. The radical resection was a good prognostic factor investigations of breast cancer cell lines with for AC and SC/ASC. There was no significant dif- differing phenotypes have illustrated the ference in the post-operative survival time involvement of a variety of cell surface proteins between cases of AC and SC/ASC. These obser- in the progression of breast carcinoma [41, 42]. vations indicated that the clinicopathological While EphA10 was recently correlated with presentations of SC/ASC might have strong poor prognosis and metastasis, higher expres- invasive and metastaticpotential compared to sion levels of EphA10 were found in invasive ordinary AC. breast carcinoma cells [14, 15, 41-43]. Given its lack of kinase activity, the biochemical The expression of EphA10 and EphB3 in AC and mechanisms underlying EphA10 activation SC/ASC has not been previously reported, have, thus, become relevant to explaining its although their expressions have been associ- involvement in breast tumorigenesis. ated with the progression and prognosis of a variety of tumors. This study investigated The 14 members of the Eph family of receptors EphA10 and EphB3 protein expression in AC make up the largest family of receptor tyrosine and SC/ASC using immunohistochemistry. A kinases in humans [33]. These receptors are

522 Int J Clin Exp Pathol 2018;11(2):512-525 EphA10 and EphB3 in gallbladder cancers associated with 8 members of a class of ligands Disclosure of conflict of interest known as ephrins. These receptors and ligands are divided into A and B classes based on their None. sequence homology as well as their affinity for Address correspondence to: Zhu-Lin Yang, Depart- the corresponding receptor/ligand [33]. Many ment of General Surgery, The Second Xiangya Eph receptors and ephrin ligands are known to Hospital, Central South University, Changha be involved in the development or progression 410011, Hunan, China. Tel: +86-731-88187376; of certain cancers [44]. In CRC, EphB2 and Fax: +86-731-84898168; E-mail: yangzhulin8@csu. EphB3 are important tumor suppressors whose edu.cn activity effectively restricts tumor-cell spread- ing [17, 18]. However, the transition from nonin- References vasive adenoma to invasive carcinoma states is frequently paralleled by their transcriptional [1] Jemal A, Siegel R, Ward E, Hao Y, Xu J, Murray down-regulation [17, 18, 24]. EphB3 is sparsely T, Thun MJ. Cancer statistics, 2008. CA Cancer mentioned as a therapeutic target, but Eph J Clin 2008; 58: 71-96. family kinase receptors are associated with [2] Jayaraman S, Jarnagin WR. Management of gallbladder cancer. Gastroenterol Clin North gastrointestinal tract cancers [45]. Overex- Am 2010; 39: 331-342. pression in lung cancer has been reported, and [3] Reid KM, Ramos-Dela MA, Donohue JH. Diag- a correlation with metastasis has been sug- nosis and surgical management of gallbladder gested [46, 47]. cancer: a review. J Gastrointest Surg 2007; 11: 671-681. The role of EphA10 and EphB3 expressions in [4] Hawkins WG, DeMatteo RP, Jarnagin WR, Ben- gallbladder SC/ASC and AC remains to be clari- Porat L, Blumgart LH, Fong Y. Jaundice pre- fied. Our study first showed that the positive dicts advanced disease and early mortality in expressions of EphA10 and negative expres- patients with gallbladder cancer. Ann Surg On- sion of EphB3 were significantly higher in the col 2004; 11: 310-315. cases of SC/ASC and AC than in chronic chole- [5] Ootani T, Shirai Y, Tsukada K, Muto T. Relation- cystitis. The positive expressions of EphA10 ship between gallbladder carcinoma and the segmental type of adenomyomatosis of the were significantly higher in the cases of poorly gallbladder. Cancer 1992; 69: 2647-2652. differentiation, large tumor size, high TNM [6] Roa JC, Tapia O, Cakir A,Basturk O, Dursun N, stage, lymph node metastasis, invasion and no Akdemir D, Saka B, Losada H, Bagci P, Adsay resection (only biopsy) of SC/ASC and AC. The NV. Squamous cell and adenosquamous carci- positive expressions of EphB3 were significant- nomas of the gallbladder: clinicopathological ly lower in the cases of poorly differentiation, analysis of 34 cases identified in 606 carcino- large tumor size, high TNM stage, lymph node mas. Mod Pathol 2011; 24: 1069-1078. metastasis, invasion and no resection (only [7] Park SB, Kim YH, Rho HL, Chae GB, Hong SK. biopsy) of SC/ASC and AC. The univariate Primary carcinosarcoma of the gallbladder. J Korean Surg Soc 2012; 82; 54-58. Kaplan-Meier analysis showed that positive [8] Liu DC, Yang ZL. MTDH and EphA7 are markers EphA10 and negative EphB3 expression is for metastasis and poor prognosis of gallblad- closely associated with a decreased overall der adenocarcinoma. Diagn Cytopathol 2013; survival in SC/ASC and AC patients. The multi- 41: 199-205. variate Cox regression analysis identified that [9] Manning G, Whyte DB, Martinez R, Hunter T, positive EphA10 and negative EphB3 expres- Sundaisanam S. The protein kinase comple- sion are independent factors for a poor- ment of the . Science 2002; prognosis in SC/ASC and AC patients. The AUC 298: 1912-1934. for EphA10 and EphB3 showed might have [10] Fox BP, Tabone CJ, Kandpal RP. Potential clini- cal relevance of Eph receptors and ephrin li- role for carcinogenesis and progression of SC/ gands expressed in prostate carcinoma cell ASC and AC. lines. Biochem Biophy Res Commun 2006; 342: 1263-1272. In conclusion, EphA10 and EphB3 are involved [11] Surawska H, Ma PC, Salgia R. The role of eph- in the tumorigenesis and progression of SC/ rins and Eph receptors in cancer. Cytokine ASC and AC, and negative EphB3 and positive Growth Factor Rev 2004; 15: 419-433. EphA10 expressions were associated with poor [12] Imai S, Nagano K, Yoshida Y, Okamura T, Ya- prognosis in patients with SC/ASC and AC. mashita T, Abe Y, Yoshikawa T, Yoshioka Y, Ka-

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mada H, Mukai Y, Nakagawa S, Tsutsumi Y, H. EphB receptor activity suppresses colorec- Tsunoda S. Development of an antibody pro- tal cancer progression. Nature 2005; 435: teomics system using a phage antibody library 1126-1130. for efficient screening of biomarker proteins. [22] Jagle S, Ronsch K, Timme S, Andrlova H, Ber- Biomaterials 2011; 32: 162-169. trand M, Jager M, Proske A, Schrempp M, [13] Nagano K, Maeda Y, Kanasaki S, Watanabe T, Yousaf A, Michoel T, Zeiser R, Werner M, Lass- Yamashita T, Inoue M, Higashisaka K, Yoshio- mann S, Hecht A. Silencing of the EPHB3 tu- ka Y, Abe Y, Mukai Y, Kamada H, Tsutsumi Y, mor-suppressor gene in human colorectal Tsunoda S. A10 is a promising cancer through decommissioning of a tran- drug target potentially useful for breast can- scriptional enhancer. Proc Natl Acad Sci U S A cers including triple negative breast cancers. J 2014; 111: 4886-4891. Control Release 2014; 189: 72-79. [23] Fre S, Pallavi SK, Huyghe M, Lae M, Janssen [14] Nagano K, Kanasaki S, Yamashita T, Maeda Y, KP, Robine S, Artavanis-Tsakonas S, Louvard Inoue M, Higashisaka K, Yoshioka Y, Abe Y, Mu- D. Notch and Wnt signals cooperatively control kai Y, Kamada H, Tsutsumi Y, Tsunoda S. Ex- cell proliferation and tumorigenesis in the in- pression of Eph receptor A10 is correlated with testine. Proc Natl Acad Sci U S A 2009; 106: lymph node metastasis and stage progression 6309-6314. in breast cancer patients. Cancer Med 2013; [24] Ronsch K, Jager M, Schopflin , Danciu M, Lass- 2: 972-7. mann S, Hecht A. Class I and III HDACs and [15] Fujimori T, Kato K, Fujihara S, Iwama H, Ya- loss of active chromatin features contribute to mashita T, Kobayashi K, Kamada H, Morishita epigenetic silencing of CDX1 and EPHB tumor A, Kobara H, Mori H, Okano K, Suzuki Y, Masa- suppressor in colorectal cancer. Epi- ki T. Antitumor effect of metformin on cholan- genetics 2011; 6: 610-622. giocarcinoma: in vitro and in vivo studies. On- [25] Ronscha K, Jaglea S, Rosea K, Seidl M, col Rep 2015; 34: 2987-96. Baumgartnere F, Freihena V, Yousaf A, Metzger [16] Nagano K, Yamashita T, Inoue M, Higashisaka E, Lassmann S, Schüle R, Zeiser R, Michoel T, K, Yoshioka Y, Abe Y, Kamada H, Tsutsumi Y, Hecht A. SNAIL1 combines competitive dis- Tsunoda S. Eph receptor A10 has a potential placement of ASCL2 and epigenetic mecha- as a target for a prostate cancer therapy. Bio- nisms to rapidly silence the EPHB3 tumor chem Biophys Res Commun 2014; 450: 545- suppressor in colorectal cancer. Mol Oncol 9. 2015; 9: 335-354. [17] Batlle E, Bacani J, Begthel H, Jonkheer S, Gre- [26] Shinmura K, Kiyose S, Nagura K, Igarashi H, gorieff A, van de Born M, Malats N, Sancho E, Inoue Y, Nakamura S, Maeda M, Baba M, Boon E, Pawson T, Gallinger S, Pals S, Clevers Konno H, Sugimura H. TNK2 gene amplifica- H. EphB receptor activity suppresses colorec- tion is a novel predictor of a poor prognosis in tal cancer progression. Nature 2005; 435: patients with gastric cancer. J Surg Oncol 1126-1130. 2014; 109: 189-97. [18] Chiu ST, Chang KJ, Ting CH, Shen HC, Li H, [27] Li G, Ji XD, Gao H, Zhao JS, Xu JF, Sun ZJ, Deng Hsieh FJ. Over-expression of EphB3 enhances YZ, Shi S, Feng YX, Zhu YQ, Wang T, Li JJ, Xie D. cell-cell contacts and suppresses tumor growth EphB3 suppresses non-small-cell lung cancer in HT-29 human colon cancer cells. Carcino- metastasis via a PP2A/RACK1/Akt signalling genesis 2009; 30: 1475-1486. complex. Nat Commun 2012; 3: 667. [19] Cortina C, Palomo-Ponce S, Iglesias M, Fernán- [28] Casimiro MC, Wang C, Li Z, Di Sante G, Willmart dez-Masip JL, Vivancos A, Whissell G, Humà M, NE, Addya S, Chen L, Liu Y, Lisanti MP, Pestell Peiró N, Gallego L, Jonkheer S, Davy A, Lloreta RG. Cyclin D1 determines estrogen signaling in J, Sancho E, Batlle E. EphB-ephrin-B interac- the mammary gland in vivo. Mol Endocrinol tions suppress colorectal cancer progression 2013; 27: 1415-28. by compartmentalizing tumor cells. Nat Genet [29] Wu Q, Lind GE, Aasheim HC, Micci F, Silins I, 2007; 39: 1376-1383. Tropé CG, Nesland JM, Lothe RA, Suo Z. The [20] Jäglea S, Rönscha K, Timme K, Andrlová H, EPH receptor Bs (EPHBs) promoters are un- Bertrand M, Jäger M, Proske A, Schrempp M, methylated in colon and ovarian cancers. Epi- Yousaf A, Michoel T, Zeiser R, Werner M, Lass- genetics 2007; 2: 237-43. mann S, Hecht A. Silencing of the EPHB3 tu- [30] Fox BP, Tabone CJ, Kandpal RP. Potential clini- mor-suppressor gene in human colorectal can- cal relevance of Eph receptors and ephrin li- cer through decommissioning of a transcrip- gands expressed in prostate carcinoma cell tional enhancer. Proc Natl Acad Sci U S A 2014; lines. Biochem Biophys Res Commun 2006; 111: 4886-4891. 342: 1263-72. [21] Batlle E, Bacani J, Begthel H, Jonkheer S, Gre- [31] Li L, Liao J, Ruland J, Mak TW, Cohen SN. A gorieff A, van de Born M, Malats N, Sancho E, TSG101/MDM2 regulatory loop modulates Boon E, Pawson T, Gallinger S, Pals S, Clevers MDM2 degradation and MDM2/p53 feedback

524 Int J Clin Exp Pathol 2018;11(2):512-525 EphA10 and EphB3 in gallbladder cancers

control. Proc Natl Acad Sci U S A 2001; 98: [42] Nagaraja GM, Othman M, Fox BP, Alsaber R, 1619-1624. Pellegrino CM, Zeng Y, Khanna R, Tamburini P, [32] Kondo M, Dono K, Sakon M, Shimizu J, Nagano Swaroop A, Kandpal RP. Gene expression sig- H, Nakamori S, Umeshita K, Wakasa K, Mon- natures and biomarkers of noninvasive and den M. Adenosquamous carcinoma of the gall- invasive breast cancer cells: comprehensive bladder. Hepatogastroenterology 2002; 49: profiles by representational difference analy- 1230-1234. sis, microarrays and proteomics. Oncogene [33] Muzio G, Maggiora M, Paiuzzi E, Oraldi M, Ca- 2006; 25: 2328-2338. nuto RA. Aldehyde dehydrogenases and cell [43] Bai YQ, Zhang JY, Bai CY, Xu XE, Wu JY, Chen B, proliferation. Free Radic Biol Med 2012; 52: Wu ZY, Wang SH, Shen J, Shen JH, Yao XD, Gao 735-746. LZ, Wu B, Gu HL, Liu XH, Li X, Li EM, Xu LY. Low [34] Holder N and Klein R. Eph receptors and eph- EphA7 expression correlated with lymph node rins: effectors of morphogenesis. Develop- metastasis and poor prognosis of patients with ment 1999; 126: 2033-2044. esophageal squamous cell carcinoma. Acta [35] Barrios A, Poole RJ, Durbin L, Brennan C, Hold- Histochem Cytochem 2015; 48: 75-81. er N, Wilson SW. Eph/Ephrin signaling regu- [44] Fox BP, Tabone CJ, Kandpal RP. Potential clini- lates the mesenchymal-to-epithelial transition cal relevance of Eph receptors and ephrin li- of the paraxial mesoderm during somitemor- gands expressed in prostate carcinoma cell phogenesis. Curr Biol 2003; 13: 1571-1582. lines. Biochem Biophys Res Commun 2006; [36] Pasquale EB. Eph-ephrin bidirectional signal- 342: 1263-1272. ing in physiology and disease. Cell 2008; 133: [45] Sugimura H, Wang JD, Mori H, Tsuboi M, Na- 38-52. gura K, Igarashi H, Tao H, Nakamura R, Nat- [37] Murai KK and Pasquale EB. ‘Eph’ective signal- sume H, Kahyo T, Shinmura K, Konno H, Hama- ing: forward, reverse and crosstalk. J Cell Sci ya Y, Kanaoka S, Kataoka H, Zhou XJ. 2003; 116: 2823-2832. EPH-EPHRIN in human gastrointestinal can- [38] Pasquale EB. Eph receptors and ephrins in cers. World J Gastrointest Oncol 2011; 2: 421- cancer: bidirectional signalling and beyond. 8. Nat Rev Cancer 2010; 10: 165-180. [46] Ji XD, Li G, Feng YX, Zhao JS, Li JJ, Sun ZJ, Shi [39] Brantley-Sieders D, Schmidt S, Parker M and S, Deng YZ, Xu JF, Zhu YQ, Koeffler HP, Tong XJ, Chen J. Eph receptor tyrosine kinases in tumor Xie D. EphB3 is overexpressed in non-small- and tumor microenvironment. Curr Pharm Des cell lung cancer and promotes tumor metasta- 2004; 10: 3431-3442. sis by enhancing cell survival and migration. [40] Brantley-Sieders DM and Chen J. Eph receptor Cancer Res 2011; 71: 1156-66. tyrosine kinases in angiogenesis: from devel- [47] Kiyose S, Nagura K, Tao H, Igarashi H, Yamada opment to disease. Angiogenesis 2004; 7: 17- H, Goto M, Maeda M, Kurabe N, Suzuki M, 28. Tsuboi M, Kahyo T, Shinmura K, Hattori N, Sug- [41] Fox BP, Kandpal RP. Invasiveness of breast car- imura H. Detection of kinase amplifications in cinoma cells and transcript profile: Eph recep- gastric cancer archives using fluorescence in tors and ephrin ligands as molecular markers situ hybridizationpion.Pathol Intern2012; 62: of potential diagnostic and prognostic applica- 477-484. tion. Biochem Biophys Res Commun 2004; 318: 882-892.

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