Table 3.

EPHs/ephrins (bold) studied in solid tumors of the gastrointestinal tract, liver, biliary tract, and pancreas and correlations with clinicopathological parameters.

EPHs/Ephrins	Malignant Tissues	Benign Control Tissues	Methods	Results	Refs
ESOPHAGUS
EPHB3	141 Barrett’s carcinoma samples	20 healthy esophagi samples	IHC q PCR	EPHB3 and E-cadherin IHC expression was reduced in adenocarcinoma compared with dysplasia or healthy esophageal mucosa Simultaneous expression of E-cadherin and EPHB3 showed inverse correlation with the tumor stage E-cadherin mRNA expression reduced in adenocarcinoma compared with dysplasia	[37]
EPHB4	31 samples (GEJ and gastric carcinomas) from 30 patients (1 patient had 1 GEJ carcinoma and 1 gastric cancer in stomach corpus)	Paired normal samples	IHC q RT-PCR	EPHB4 overexpressed in: ○ preneoplastic gastroesophageal lesions ○ furtherly overexpressed in gastroesophageal cancers ○ advanced tumor stage ○ overexpression at the tumor invasion front and vascular endothelial cells	[38]
EPHB6	31 samples (GEJ and gastric carcinomas) from 30 patients	Paired normal samples	IHC q RT-PCR	EPHB6 was down-regulated, consistent with its tumor-suppressive properties in other cancers	[38]
STOMACH
EPHA2	176 gastric adenocarcinoma samples	Paired adjacent normal tissues	Real-time RT-PCR IHC Western blot	EPHA2 expression correlated with: ○ depth of tumor invasion ○ LN metastasis ○ TNM stage EPHA2 is an independent poor prognostic factor, and its overexpression is linked to poor OS	[39]
	46 adenocarcinomas, 2 adenosquamous carcinomas, 1 neuroendocrine carcinoma	Corresponding non-tumor samples	Semiquantitative RT-PCR IHC Western blot	EPHA2 expression linked to tumor invasion and tumors with undefined borders or that infiltrated diffusely No correlation between EPHA2 expression and tumor size/age/vessel infiltration/LN metastasis	[40]
	91 gastric adenocarcinomas	7 gastrointestinal stromal tumor samples	IHC Western blot	High EPHA2 expression correlated with: ○ advanced stage ○ tumor size ○ LN metastasis ○ lymphovascular invasion ○ TNM stage	[41]
	107 gastric adenocarcinoma samples (54 of them received adjuvant chemotherapy)	Normal paired gastric samples	Proteome analysis (LC-MS/MS) mRNA (real-time RT-PCR) IHC	EPHA2 expression was 2-fold higher in GCSC than GSC Cases that showed intermingling of EPHA2 (+) cells and GCSCs showed ○ more frequent relapse ○ shorter relapse-free survival	[44]
EPHA4	24 paired fresh gastric adenocarcinoma samples, 74 fresh frozen paraffin embedded gastric adenocarcinoma samples, 55 gastric adenocarcinoma samples in tissue microarrays	Adjacent non-tumor samples from 24 gastric adenocarcinoma specimens	Real-time RT-PCR RT-PCR IHC	Overexpression of EPHA4 in IHC was observed in cancer tissues EPHA4 protein levels associated with: ○ depth of invasion ○ recurrence EPHA4 (+) cancer tissues showed shorter OS than EPHA4 (-) cancers	[42]
EPHA5	107 gastric adenocarcinoma samples (54 of them received adjuvant chemotherapy)	Normal paired gastric samples	Proteome analysis (LC-MS/MS) mRNA (real-time RT-PCR) IHC	EPHA5 expression was 2-fold higher in GCSC than GSC	[44]
EPHA8	206 gastric cancer samples	32 normal gastric mucosa, 60 paracancerous samples	IHC Western blot	EPHA8 expression associated with: ○ differentiation level ○ TNM stage ○ depth of infiltration ○ distant metastasis ○ poor OS	[43]
EPHB2	107 gastric adenocarcinoma samples (54 of them received adjuvant chemotherapy)	Normal paired gastric samples	Proteome analysis (LC-MS/MS) mRNA (real-time RT-PCR) IHC	EPHB2 expression was 2-fold higher in GCSC than GSC	[44]
EPHB4	107 gastric adenocarcinoma samples (54 of them received adjuvant chemotherapy)	Normal paired gastric samples	Proteome analysis (LC-MS/MS) mRNA (real-time RT-PCR) IHC	EPHB4 expression was 2-fold higher in GCSC than GSC	[44]
	31 gastric and GEJ carcinomas	Paired normal samples	Quantitative real-time RT-PCR IHC	EPHB4 expression: ○ high in Barrett esophagus ○ high in gastroesophageal cancers ○ associated with advanced tumor stages ○ high at the tumor invasion front	[38]
EPHB6	31 gastric and GEJ carcinomas	Paired normal samples	Quantitative real-time RT-PCR IHC	EPHB6 downregulated in tumor tissues	[38]
	152 gastric carcinoma samples		IHC	EPHB6 expression: Positively associated with tumor differentiation Negatively associated with ○ LN metastasis ○ tumor stage ○ female sex Showed no association with age/tumor location/depth of invasion	[46]
ephrin-A1	176 gastric adenocarcinoma samples	Paired adjacent normal samples	Real-time RT-PCR IHC Western blot	ephrinA1 expression correlated with: ○ TNM stage ○ LN metastasis	[39]
	46 adenocarcinomas, 2 adenosquamous carcinomas, 1 neuroendocrine carcinoma	Paired non-tumor samples	Semiquantitative RT-PCR IHC Western blot	No correlation between ephrin-A1 expression and tumor size/age/vessel infiltration/LN metastasis	[40]
ephrin-B1	29 gastric carcinoma samples	Matched normal samples	Semiquantitative RT-PCR	ephrin-B1 expression more frequent in poorly differentiated adenocarcinomas than well-differentiated ones	[45]
COLON
EPHA1	53 CRC samples	adjacent normal samples	IHC q real-time PCR	EPHA1 expression increased in CRC tissues compared with normal ones EPHA1 downregulation was prevalent in more advanced TNM stages	[48]
	125 CRC specimens, 53 paired normal colon-CRC samples	18 normal colon samples, paired normal samples from the 53 CRC patients	q real-time PCR Flow cytometry Western blot IHC	EPHA1 upregulated in 50% of the cases EPHA1 overexpression more prevalent in stage 2 compared with stage 3 CRC Low EPHA1 expression correlated with poor OS	[49]
EPHA2	53 CRC samples	adjacent normal samples	IHC q real-time PCR	EPHA2 expression increased in CRC tissues compared with normal ones EPHA2 downregulation was prevalent in more advance TNM stages	[48]
	37 CRC samples	37 paired normal samples	IHC Semi-quantitative RT-PCR	EPHA2 overexpressed: ○ in tumor tissues compared to adjacent normal tissue ○ early stage cancers compared to late stage cancers ○ in smaller tumors (<5 cm)	[50]
EPHA3	53 CRC samples	adjacent normal tissues	IHC q real-time PCR	EPHA3 showed loss of expression in 96% of cases	[48]
	68 CRC samples	paired adjacent normal mucosa	IHC	EPHA3 highly expressed in CRC tissues compared with normal mucosa EPHA3 expression level associated with: ○ age ○ tumor differentiation ○ LN metastasis	[51]
EPHA7	53 CRC samples	adjacent normal samples	IHC q real-time PCR	EPHA7 showed loss of expression in 96% of cases	[48]
EPHB2	345 primary CRCs 98 LN metastasis 82 liver metastasis	100 adenomas 111 normal mucosa	IHC	EPHB2 expressed in: ○ 100% normal colon crypt base cells ○ 78% adenomas ○ 55.4% CRCs ○ 37.8% LN metastasis ○ 32.9% liver metastasis EPHB2 expression negatively correlated with: ○ tumor stage ○ poor differentiation ○ poor OS ○ poor DFS From tumor to metastasis, CRC carcinogenesis shows a progressive loss of EPHB2 expression	[54]
	159 samples of stage 3 CRC		IHC	Decrease of EPHB2 expression correlated independently with recurrence	[55]
	28 primary CRCs 39 metastasis 342 paired primary CRCs and normal samples in tissue microarrays	28 normal colon samples 148 colorectal adenomas	IHC	EPHB2 expressed in normal crypts, colorectal adenomas, primary cancers, and metastasis High EPHB2 expression associated with longer OS in CRC	[56]
	4000 samples from 138 different tumor types, 1476 samples of colon cancer	76 different normal samples	IHC	EPHB2 expression predominantly found in intestinal epithelium Loss of EPHB2 expression associated with: ○ high pT tumor stage ○ nodal positivity ○ infiltrative tumor margin ○ shorter OS	[57]
	36 CRCs	30 Dysplastic aberrant foci, (dACF) 31 small (<5 mm) adenomas, 12 large (>5 mm) adenomas	IHC	100% of dACFs and 61% of small adenomas retained EPHB2 expression All CRCs showed extensive loss of EPHB2 expression (>50%) and 25% of CRCs were entirely negative EPHB2 downregulation associated with higher histological grade	[58]
EPHB3	610 CRC samples formalin-fixed 32 fresh colorectal cancer samples		IHC q real-time PCR	EPHB3 higher in CRC tissues than in normal mucosa EPHB3 declined during transformation from adenoma to carcinoma and as the tumor invaded into deeper tissues Budding cancer cells at the invasive tumor fronts showed reduction of EPHB3 expression EPHB3 expressed in 24% of 610 CRCs EPHB3 expression negatively associated with: ○ tumor differentiation ○ lympho-vascular invasion ○ TNM stage EPHB3 expression positively associated with MSI EPHB3 expression correlated with better clinical outcomes, but was not and independent prognostic marker	[59]
EPHB4	20 colon carcinoma samples	adjacent uninvolved mucosa	IHC RT-PCR	EPHB4 frequently expressed on the luminal surface	[53]
	200 CRC samples	50 adjacent non-tumor samples	IHC	EPHB4 strongly expressed in CRC cells but minimal to no expression in normal colon mucosa EPHB4 associated with: ○ invasion depth ○ LN metastasis ○ distant metastasis ○ TNM stage	[60]
ephrin-A1	53 CRC samples	adjacent normal samples	IHC q real-time PCR	Ephrin-A1 expression increased in CRC compared with normal tissue in 51% of cases	[48]
	37 CRC samples	37 paired normal samples	IHC Semi-quantitative RT-PCR	Ephrin-A1 overexpressed: ○ in tumor tissues compared with adjacent normal tissue ○ early stage cancers compared with late stage cancers ○ in smaller tumors (<5 cm)	[50]
ephrin-A5	53 CRC samples	adjacent normal samples	IHC Quantitative real-time PCR	EphrinA5 expression higher in CRC tissues compared to its high affinity receptors	[48]
	72 colon malignancies (68 adenocarcinomas, 4 non-Hodgkin lymphomas)	10 normal colon samples 14 benign colon lesions (adenomas and polyps)	IHC Western blot q real-time PCR RT-PCR	EphrinA5 mRNA and protein levels significantly reduced in colon cancer compared to normal colon EphrinA5 negatively associated with: ○ tumor differentiation ○ clinical stage	[52]
ephrin-B2	20 colon carcinoma samples	adjacent uninvolved mucosa	IHC RT-PCR	Ephrin-B2 higher expression in CRC than normal mucosa Ephrin-B2 frequently expressed on the luminal surface	[53]
	200 CRC samples	50 adjacent non-tumor samples	IHC	EphrinB2 expression was the same in CRC and normal tissues	[60]
LIVER
EPHA1	20 HCC samples	Non-cancerous adjacent tissues	IHC Northern blot	EPHA1 and AFP strongly expressed in cancerous tissues	[61]
EPHA2	139 HCC samples		Real-time q RT-PCR	EPHA2 expression correlated with microscopic portal invasion	[63]
	40 HCC samples	Cirrhotic non-tumorous samples	IHC Northern blot RT-PCR	EPHA2 expression correlated with: ○ decreased differentiation ○ poor OS	[62]
EPHA5	250 HCC samples	Paired normal samples	IHC	None of the kinases expression examined (ALK, FGFR, and EPHA5) individually showed any correlation with OS, but co-activation of all three kinases showed a worse prognosis	[64]
ephrin-A1	20 HCC samples	Non-cancerous adjacent samples	IHC Northern blot	Ephrin-A1 expression ○ lowest in normal liver tissues ○ elevated in cirrhotic tissues ○ further elevated in HCC Ephrin-A1 expression strongly correlates with AFP expression, which is associated with poor OS Ephrin-A1, and AFP strongly expressed in cancerous tissues	[61]
	139 HCC samples		Real-time q RT-PCR	Ephrin-A1 expression correlated with decreased DFS	[63]
	40 HCC samples	Cirrhotic non-tumorous samples	IHC Northern blot RT-PCR	Ephrin-A1 was overexpressed in HCC tissues compared with corresponding non tumor tissues	[62]
ephrin-A5	142 paired HCC and peritumoral liver samples	Paired peritumoral samples	Real-time q RT-PCR	Ephrin-A5s and ephrin-A5l down-regulated in HCC compared with peritumoral tissues Ephrin-A5s positively correlated with: ○ old age ○ histological grade Ephrin-A1s expression in peritumoral tissue had better DFS and OS	[65]
BILIARY TRACT
EPHA2	30 ICC samples, 5 LN metastasis from 5 of the above patients	Normal adjacent samples	PCR-based Sanger sequencing	EPHA2 frequently mutated in primary ICC tumors EPHA2 mutations more likely to occur in ICC with LN metastasis	[66]
EPHB2	50 cholangiocarcinoma samples		IHC	High EPHB2 expression EPHB2/ephrin-B1 co-expression EPHB2/ephrin-B2 co-expression correlated with metastasis status	[67]
EPHB4	50 cholangiocarcinoma patients		IHC	High EPHB4 and high MVD correlated with metastasis status	[67]
ephrin-B1	50 cholangiocarcinoma samples		IHC	High EPHB2/ephrin-B1 co-expression Ephrin-B1 expression and high MVD correlated with metastasis status	[67]
ephrin-B2	50 cholangiocarcinoma samples		IHC	High EPHB2/ephrin-B2 co-expression correlated with metastasis status	[67]
PANCREAS
ephrin-B2	179 PDAC samples (mRNA data)	171 normal pancreatic samples (mRNA data)	Statistical analysis	Data analysis of mRNA expression of the 179 PDAC and 171 normal pancreatic tissues showed that: Ephrin-B2 expression was higher in PDAC tissues than normal ones High ephrin-B2 expression correlated with: ○ shorter OS ○ shorter DFS	[68]
	54 PDAC samples	54 adjacent normal samples	IHC Quantitative real-time PCR Western blot	IHC, qRT-PCR and western blot analysis of the 54 PDAC tissues showed that: Ephrin-B2 expression was higher in PDAC tissues than normal ones High ephrin-B2 expression correlated with: ○ TNM staging ○ higher ki67 expression

q PCR: quantitative polymerase chain reaction, IHC: immunohistochemistry, q RT-PCR: quantitative reverse transcription-polymerase chain reaction, q real time PCR: quantitative real time transcription-polymerase chain reaction, GEJ: gastroesophageal junction, LN: lymph nodes, OS: overall survival, DFS: disease-free survival, MVD: microvessel density, GCSC: gastric cancer tissues stromal cells, GSC: normal gastric tissue stromal cells, ICC: intrahepatic cholangiocarcinoma, PDAC: pancreatic ductal adenocarcinoma.