Clinicopathological significance of altered Notch signaling in extrahepatic cholangiocarcinoma and gallbladder carcinoma

Table 1 Antibodies used for immunohistochemistry

Antibody	Dilution	Company	Non-neoplastic tissue	Aberrant expression in cancer
Notch 1	0.111	Santa Cruz	Variable	Cytoplasm/nucleus
Notch 2	0.111	Santa Cruz	Variable	Cytoplasm/nucleus
Notch 3	0.111	Santa Cruz	Variable	Cytoplasm/nucleus
Notch 4	1:50	Santa Cruz	Variable	Cytoplasm/nucleus
DLL4	1:50	Sigma	Variable	Cytoplasm/nucleus

DLL4: Delta-like ligand-4.

Table 2 Expression rates of Notch receptors 1-4 and delta-like ligand-4 in extrahepatic cholangiocarcinoma and gallbladder carcinoma n (%)

	Negativity	Positivity		Total
		Low grade	High grade
Notch 1	14 (12.7)	55 (50.0)	41 (37.3)	110 (100)
Notch 2	13 (11.8)	60 (54.6)	37 (33.6)	110 (100)
Notch 3	13 (11.8)	56 (50.9)	41 (37.3)	110 (100)
Notch 4	7 (6.4)	61 (55.4)	42 (38.2)	110 (100)
DLL4	26 (23.6)	63 (57.3)	21 (19.1)	110 (100)

DLL4: Delta-like ligand-4.

Table 3 Correlation between cytoplasmonuclear coexistent localization of Notch receptors 1-4 and delta-like ligand-4 expression, and clinicopathological parameters

	Notch1			Notch 2			Notch 3			Notch 4			DLL4
Nuclear localization	-	+	P value	-	+	P value	-	+	P value	-	+	P value	-	+	P value
Total	78	18		57	40		65	32		4	99		36	48
Differentiation			0.545			0.418			0.556			0.036			0.175
Well	37	11		29	18		32	17		2	47		17	20
Moderate	34	6		22	20		27	14		0	43		14	26
Poorly	7	1		6	2		6	1		2	9		5	2
T stage			0.846			0.594			0.269			0.365			0.053
T1	12	4		11	5		8	9		0	16		5	9
T2	43	8		25	23		33	13		1	51		15	26
T3	18	5		18	10		20	8		3	26		11	13
T4	5	1		3	2		4	2		0	6		5	0
N stage			0.100			0.408			0.027			0.34			0.558
N0	52	16		44	27		41	28		2	70		27	32
N1	26	2		13	13		24	4		2	29		9	16

P by χ² test. DLL4: Delta-like ligand-4.

Table 4 Correlation between expression of Notch receptors 1-4 and Delta-like ligand-4, and clinicopathological parameters

		Notch 1				Notch 2				Notch 3				Notch 4				DLL4
Total (n = 110)		-	+	++	P value	-	+	++	P value	-	+	++	P value	-	+	++	P value	-	+	++	P value
Gender					0.207				0.005				0.2				0.144				0.517
Male	47	9	21	17		11	22	14		8	20	19		5	22	20		13	24	10
Female	63	5	34	24		2	38	23		5	36	22		2	39	22		13	39	11
Age					0.251				0.34				0.107				0.761				0.873
< 60	34	3	21	10		5	15	14		1	21	12		1	20	13		7	20	7
≥ 60	76	11	34	31		8	45	23		12	35	29		6	41	29		19	43	14
Differentiation					0.391				0.266				0.101				0.414				0.095
Well	53	5	30	18		6	29	18		4	28	21		4	29	20		16	31	6
Moderate	46	6	20	20		4	24	18		5	23	18		3	23	20		6	27	13
Poorly	11	3	5	3		3	7	1		4	5	2		0	9	2		4	5	2
T stage					0.103				0.285				0.017				0.294				0.614
T1	17	1	8	8		1	11	5		0	13	4		1	12	4		3	12	2
T2	55	4	32	19		7	31	17		9	26	20		3	25	27		14	31	10
T3	32	9	11	12		4	13	15		4	11	17		3	19	10		8	15	9
T4	6	0	4	2		1	5	0		0	6	0		0	5	1		1	5	0
N stage					0.999				0.223				0.065				0.795				0.301
N0	78	10	39	29		7	46	25		9	45	24		6	42	30		19	47	12
N1	32	4	16	12		6	14	12		4	11	17		1	19	12		7	16	9
M stage					0.467				0.443				0.999				0.999				0.221
M0	103	13	53	37		13	57	33		13	52	38		7	57	39		25	60	18
M1	7	1	2	4		0	3	4		0	4	3		0	4	3		1	3	3
TNM stage					0.043				0.144				0.052				0.898				0.36
I	51	2	29	20		4	31	16		6	30	15		3	27	21		11	33	7
II	45	11	20	14		8	21	16		7	16	22		4	24	17		12	22	11
III	6	0	4	2		1	5	0		0	6	0		0	5	1		1	5	0
IV	8	1	2	5		0	3	5		0	4	4		0	5	3		2	3	3

P by χ² test. TNM: Tumor, node, metastasis.