Expression of Notch pathway components (Numb, Itch, and Siah-1) in colorectal tumors: A clinicopathological study

Table 1 Distribution of immunohistochemical expression of Notch, Numb, Itch, and Siah-1 among the three groups, tissues adjacent to carcinoma, tissues adjacent to adenoma, and subtypes of adenomas

	Controls	Adenomas	Carcinomas	P value	Tissues adjacent to carcinomaa	Tissues adjacent to adenomaa	Subtypes of adenomasa
							Tubular	Tubulovillous	Villous
Notch expression
Negative (%)	15 (75)	12 (40)	18 (36)	0.02	25 (50)	16 (53)	4 (40)	5 (50)	3 (30)
Positive (%)	5 (25)	18 (60)	32 (64)		25 (50)	14 (47)	6 (60)	5 (50)	7 (70)
Numb expression
Negative (%)	6 (30)	21 (70)	30 (60)	0.008	26 (52)	14 (47)	6 (60)	7 (70)	8 (80)
Positive (%)	14 (70)	9 (30)	20 (40)		24 (48)	16 (54)	4 (40)	3 (30)	2 (20)
Itch expression
Negative (%)	8 (40)	24 (80)	30 (60)	0.01	27(54)	19 (63)	9 (90)	7 (70)	8 (80)
Positive (%)	12 (60)	6 (20)	20 (40)		23 (46)	11 (37)	1 (10)	3 (30)	2 (20)
Siah-1 expression
Negative (%)	5 (25)	19 (63.3)	29 (58)	0.024	31(62)	21 (70)	7 (70)	5 (50)	7 (70)
Positive (%)	15 (75)	11 (36.7)	21 (42)		19 (38)	9 (30)	3 (30)	5 (50)	3 (30)

^aP < 0.05.

Table 2 Correlations between immunohistochemical expression of Notch, Numb, Itch, and Siah-1 and clinicopathological parameters

Parameters	n	Notch		Numb		Itch		Siah-1
		Negative (%)	Positive (%)	Negative (%)	Positive (%)	Negative (%)	Positive (%)	Negative (%)	Positive (%)
Age		18	32	30	20	30	20	29	21
64 ± 12.35 ≥	24	8 (44.3)	16 (66.7)	13 (54.1)	11 (45.9)	14 (58.3)	10 (41.7)	16 (66.7)	8 (33.3)
64 ± 12.35 <	26	10 (38.5)	16 (61.5)	17 (65.3)	9 (34.7)	16 (61.5)	10 (38.5)	13 (50)	13 (50)
Gender
Female	17	6 (35.3)	11 (66.7)	10 (58.8)	7 (41.2)	10 (58.8)	7 (42.2)	7 (41.2)	10 (58.8)
Male	33	12 (36.4)	21 (63.6)	20 (60.6)	13 (39.4)	20 (60.6)	13 (39.4)	22 (66.7)	11 (33.3)
Localization
Caecum	8	3 (37.59)	5 (62.5)	6 (75)	2 (25)	5 (62.5)	3 (37.5)	4 (50)	4 (50)
Colon	16	5 (31.3)	11 (68.7)	9 (56.3)	7 (43.7)	8 (50)	8 (50)	10 (62.5)	6 (37.5)
Sigmoid	10	4 (40)	6 (60)	7 (70)	3 (30)	7 (70)	3 (30)	5 (50)	5 (50)
Rectum	16	6 (37.5)	10 (62.5)	8 (50)	8 (50)	10 (62.5)	6 (37.5)	10(50)	6 (50)
Differentiationa
WD	18	16 (88.8)	2 (11.2)	3 (16.6)	15 (83.4)	5 (27.8)	13 (72.8)	8 (44.5)	10 (55.5)
MD	15	1 (6.6)	14 (93.4)	12 (80)	3 (20)	10 (66.7)	5 (33.3)	10 (66.6)	5 (33.4)
PD	17	1 (5.8)	16 (94.2)	15 (88.2)	2 (11.8)	15 (88.2)	2 (11.8)	11 (64.8)	6 (35.2)
Invasion
T1	7	6 (85.7)	1 (14.3)	4 (57.1)	3 (42.9)	2 (28.5)	5 (71.5)	3 (48.3)	4 (51.7)
T2	10	6 (60)	4 (40)	7 (70)	3 (30)	7 (70)	3 (30)	6 (60)	4 (40)
T3	23	4 (17.4)	19 (82.6)	12 (52.1)	11 (47.9)	15 (65.2)	8 (34.8)	14 (60.9)	9 (39.1)
T4	10	2 (20)	8 (80)	7 (70)	3 (30)	6 (60)	4 (40)	6 (60)	4 (40)
LNMa
Absent	17	1 (5.3)	17 (94.7)	5 (29.4)	12 (70.6)	7 (41.1)	10 (58.9)	4 (23.6)	13 (76.4)
Present	33	17 (61.39)	21(38.7)	25 (75.8)	8 (24.2)	23 (69.6)	10 (30.4)	25 (75.8)	8 (24.2)
Lymphatic invasion
Absent	37	17 (45.9)	20 (54.1)	22 (59.4)	15 (40.6)	23 (62.1)	14(37.9)	25 (67.6)	12 (32.4)
Present	13	1 (7.6)	12 (92.4)	8 (61.5)	5 (38.5)	7 (53.8)	6 (45.2)	4 (30.8)	9 (69.2)
Vascular invasion
Absent	42	16 (38.1)	26 (61.9)	25 (61.9)	17(38.1)	27 (64.3)	15 (35.7)	26 (61.9)	16 (38.1)
Present	8	2 (25)	6 (75)	5 (62.5)	3 (37.5)	3 (62.5)	5 (37.5)	3 (37.5)	5 (62.5)
Perineural invasion
Absent	31	11 (35,4)	20 (64.6)	21 (67.7)	10 (32.3)	20 (64.5)	11 (35.5)	18 (58.1)	13 (41.9)
Present	19	7 (36.8)	12 (63.2)	9 (47.4)	10 (52.6)	10 (52.6)	9 (47.4)	11 (57.9)	8 (42.1)
Stagea
1 + 2	24	13 (54.1)	11 (45.9)	8 (33.4)	16 (66.6)	10 (41.6)	14 (58.4)	10 (41.7)	14 (58.3)
3 + 4	26	5 (19.2)	21 (80.8)	22 (84.6)	4 (15.4)	20 (76.9)	6 (23.1)	19 (73.1)	7 (26.9)
Distant metastasis
Absent	26	8 (30.7)	18 (69.3)	15 (57.6)	11 (42.4)	13 (50)	13 (50)	15 (57.7)	11(42.3)
Present	24	10 (41.6)	14 (58.4)	15 (62.5)	9 (37.5)	17 (70.8)	7 (29.2)	14 (58.4)	10 (41.6)
Survival (mo)a
60.62 ± 26.68 ≥	26	2 (7.6)	24 (92.4)	20 (76.9)	6 (23.1)	22 (84.6)	4 (15.4)	19 (73.1)	7 (26.9)
60.62 ± 26.68 <	24	16 (66.6)	8 (33.4)	10 (41.7)	14 (58.3)	8 (33.4)	16 (66.6)	10 (41.7)	14 (58.3)

^aP < 0.05. WD: Well-differentiated; MD: Moderately differentiated; PD: Poorly differentiated; LNM: Lymph node metastasis.

Table 3 Distribution of Notch-1, Numb, Itch, and Siah-1 expression levels assessed by qPCR analysis in adenoma and carcinoma groups

Gene expression levels	Adenomas	Carcinomas	P value
Notch
mean ± SD	3.1016 ± 1.0452	10.2628 ± 5.8043	0.03
Median	2.6992	1.5305
Normal (%)	0	12 (24)	0.0001
(↑) (%)	15 (50)	22 (44)
(↓) (%)	15 (50)	16 (32)
Numb
mean ± SD1	8.1362 ± 1.6299	2.3153 ± 2.2267	0.03
Median	4.0287	-2.2616
Normal (%)	4 (13.3)	0	0.0001
(↑) (%)	18 (60)	24 (58)
(↓) (%)	8 (26.7)	26 (52)
Itch
mean ± SD	9.2899 ± 2.7369	2.1982 ± 1.0461	0.006
Median	2.0422	1.5933
Normal (%)	11 (36.7)	12 (32)	0.0001
(↑) (%)	15 (50)	18 (38)
(↓) (%)	4 (13,3)	20 (30)
Siah-1
mean ± SD	5.9024 ± 1.18671	0.8358 ± 1.3831	0.03
median	1.4031	-2.0322
Normal (%)	6 (20)	10 (20)	0.001
(↑) (%)	11 (36.7)	13 (26)
(↓) (%)	13 (43.3)	27 (54)

¹Fold increase 2^-∆∆Ct. (↑): Upregulation; (↓): Downregulation.

Table 4 Mean and median values of relative gene expression levels of Notch-1, Numb, Itch and Siah-1 in tumors with different clinicopathological parameters (qPCR analysis)

Parameters	n	Notch-1		Numb		Itch		Siah-1
		mean ± SD1	Median	mean ± SD	Median	mean ± SD	Median	mean ± SD	Median
Age
64 ± 12.35 ≥	24	11.23 ± 5.23	-1.18	1.08 ± 1.03	-1.03	1.73 ± 2.34	-1.22	0.81 ± 0.32	-1.22
64 ± 12.35 <	26	9.20 ± 4.34	1.67	3.28 ± 4.32	-2.26	3.35 ± 1.64	1.99	0.85 ± 0.42	-2.99
Gender
Female	17	10.21 ± 6.02	1.38	1.74 ± 1.34	-2.43	1.93 ± 1.34	1.35	0.23 ± 0.03	-3.19
Male	33	10.58 ± 5.03	1.67	3.42 ± 3.42	4.67	2.70 ± 2.10	-1.21	2.00 ± 0.93	-1.22
Localization
Caecum	8	13.10 ± 5.23	12.39	1.17 ± 1.45	1.31	1.85 ± 1.62	2.43	2.40 ± 1.11	0.41
Colon	16	7.99 ± 7.32	-1.50	2.60 ± 2.34	1.20	3.33 ± 2.03	2.30	0.26 ± 0.23	-2.56
Sigmoid	10	15.03 ± 6.23	20.79	3.69 ± 2.45	3.03	-1.03 ± 0.3	-1.75	-0.77 ± 0.22	-1.49
Rectum	16	8.37 ± 3.41	1.16	1.72 ± 1.65	-2.96	2.63 ± 1.22	1.67	1.62 ± 0.51	-3.48
Differentiationa
WD	18	3.92 ± 2.45	-3.08	6.64 ± 3.46	9.92	5.26 ± 2.42	7.05	3.67 ± 1.53	-1.22
MD	15	9.85 ± 4.46	21.67	1.26 ± 1.04	-2.26	-1.20 ± 0.84	-2.41	-1.66 ± 0.66	-3.24
PD	17	17.28 ± 9.03	22.54	-3.62 ± 1.81	-4.45	-0.03 ± 0.01	-1.86	-1.91 ± 0.65	-4.21
Invasion
T1	7	5.39 ± 5.93	-7.70	9.40 ± 4.72	14.62	10.04 ± 5.80	12.06	2.75 ± 1.36	-3.89
T2	10	10.01 ± 6.82	4.45	3.63 ± 2.45	-3.06	1.41 ± 0.56	-2.26	1.43 ± 0.92	-3.86
T3	23	11.38 ± 7.02	1.67	1.63 ± 1.85	-2.26	1.39 ± 0.8	-1.91	1.87 ± 0.23	-1.16
T4	10	11.75 ± 6.88	9.59	-2.40 ± 2.03	-3.39	-1.65 ± 0.3	-1.51	-3.51 ± 1.48	-2.94
LNMa
Absent	17	2.45 ± 2.67	-4.54	8.66 ± 4.82	13.40	5.91 ± 2.20	7.84	5.77 ± 2.81	-1.16
Present	33	15.17 ± 6.89	19.90	-1.57 ± 1.24	-2.35	-1.07 ± 0.51	-1.65	-2.19 ± 1.08	-3.54
Lymphatic invasion
Absent	37	10.84 ± 6.36	1.67	1.54 ± 3.42	-2.26	1.68 ± 0.83	1.35	0.45 ± 0.23	-3.19
Present	13	8.92 ± 5.32	1.38	4.51 ± 3.83	3.79	3.67 ± 1.43	1.38	1.90 ± 0.92	-1.35
Vascular invasion
Absent	42	11.77 ± 8.43	1.67	1.92 ± 1.65	-2.26	1.44 ± 0.96	1.07	-0.48 ± 0.28	-3.22
Present	8	12.96 ± 7.32	-2.98	9.63 ± 5.32	13.13	6.17 ± 3.02	5.66	7.77 ± 3.82	5.50
Perineural invasion
Absent	31	8.92 ± 5.32	-1.01	2.58 ± 2.48	-2.26	2.86 ± 1.32	2.00	1.22 ± 0.32	-3.22
Present	19	12.65 ± 6.02	17.79	1.87 ± 1.82	2.27	1.17 ± 0.61	-1.21	0.20 ± 0.80	5.50
Stagea
1 + 2	24	4.45 ± 4.02	-9.81	12.85 ± 5.63	7.53	5.24 ± 2.31	6.89	1.24 ± 0.51	-1.64
3 + 4	26	15.78 ± 7.94	-8.25	9.72 ± 4.63	-2.29	-1.61 ± 0.22	-1.88	0.21 ± 0.03	-2.42
Distant metastasis
Absent	26	9.83 ± 7.34	-1.18	3.42 ± 1.86	1.99	3.17 ± 1.24	1.77	0.99 ± 0.23	-2.45
Present	24	10.89 ± 6.53	1.67	1.11 ± 1.02	-2.86	1.14 ± 0.82	-1.15	0.66 ± 0.23	-2.02
Survival (mo)a
60.62 ± 26.68 ≥	26	17.16 ± 6.04	21.22	-5.47 ± 2.24	-4.90	-2.03 ± 1.06	-2.47	-4.63 ± 2.07	-4.05
60.62 ± 26.68 <	24	2.95 ± 2.02	-5.33	10.75 ± 5.21	13.13	6.78 ± 2.83	8.51	6.76 ±	3.15

^aP < 0.05.

¹Fold increase (2^-∆∆Ct). WD: Well-differentiated; MD: Moderately differentiated; PD: Poorly differentiated; LNM: Lymph node metastasis.

Table 5 Correlations between expression levels of Notch, Numb, Itch, and Siah-1 and clinicopathological parameters in colorectal carcinoma

Levels	n	Notch			Numb		Itch			Siah-1
Parameters		(-)	(↓)	(↑)	(↓)	(↑)	(-)	(↓)	(↑)	(-)	(↓)	(↑)
		12	16	22	26	24	16	19	15	10	27	13
Age
64 ± 12.353 ≥	22	7 (31.8)	4 (18.2)	11 (50)	12 (54.5)	10 (45.5)	7 (31.8)	6 (27.3)	9 (40.9)	4 (18.2)	13 (59.1)	5 (22.7)
64 ± 12.353 <	28	5 (17.9)	12 ( 42 .9)	11 (39.3)	14 (50)	14 (50)	5 (17.9)	12 (42.9)	11(39.3)	6 (21.4)	14 (50)	8 (28.6)
Gender
Female	17	3 (17.6)	5 ( 29.5)	9 (52.9)	17 (51.5)	16 (48.5)	6 (35.3)	7 (41.2)	4 (23.4)	3 (17.6)	10 (58.8)	4 (23.5)
Male	33	9 (27.3)	11 (33.3)	13 (39.4)	9 (52.9)	8 (47.1)	6 (18.2)	11 (33.3)	16 (48.5)	7 (21.2)	17 (51.5	9 (27.3)
Localisation
Caecum	8	1 (12.5)	3 (37.5)	4 (50)	4 (50)	4(50)	0	3 (37.5)	5 (62.5)	2 (25)	4 (50)	2 (25)
Colon	16	3 (18.8)	3 (18.8)	10 (62.5)	9 (56.2)	7 (43.8)	5 (31.2)	5 (31.2)	6 (37.5)	3 (18.8)	9 (56.2)	4 (25)
Sigmoid	16	7 (43.8)	7 (43.8)	2 (12.5)	6 (60)	4 (40)	3 (30)	3 (30)	4 (40)	2 (12.5)	10 (62.5)	4 (25)
Rectum	10	1 (10)	3 (30)	6 (60)	7 (43.8)	9 (56.2)	4 (25)	7 (43.8)	5 (31.2)	3 (30)	4 (40)	3 (30)
Differentiationa
WD MD	24 13	6 (25) 4 (30.7)	13 (54.1) 2 (15.3)	5 (20.9) 7 (54)	8 (33.3) 8 (61.5)	16 (66.7) 5 (38.5)	5 (20.8) 3 (23.1)	5 (20.8) 5 (38.5)	14 (58.3) 5 (38.5)	7 (29.2) 2 (15.49	7 (29.2) 10 (76.9)	10 (41.7) 1 (7.7)
PD	13	2 (15.4)	1 (7.7)	10 (76.9)	10 (76.9)	3 (23.1)	4 (30.8)	8 (61.5)	1 (7.7)	1 ( 7.7)	10 (76.9)	2 (15.4)
Invasion
T1	7	1 (14.3)	1 (14.3)	5 (71.4)	1 (14.3)	6 (85.7)	2 (20)	0	5 (71.4)	2 (28.6)	3 (42.9)	2 (28.6)
T2	10	3 (30)	3 (30)	4 (40)	6 (60)	4 (40)	2 (20)	(30)	5 (50)	1 (10)	5 (50)	4 (40)
T3	23	6 (26.1)	7 (30.4)	10 (43.5)	15 (65.2)	8 (34.8)	5 (21.7)	11 (47.8)	7 (30.4)	5 (21.7)	5 (50)	4 (17.4)
T4	10	2 (20)	5 (50)	3 (30)	4 (40)	6 (60)	24 (30)	18 (36)	20 (40)	2 (20)	5 (50)	3 (30)
LNMa
Absent	19	3 (15.8)	13 (68.4)	3 (15.8)	6 (31.6)	13 (68.4)	5 (26.3)	2 (10.5)	12 (63.2)	6 (31.6)	5 (26.3)	8 (42.1)
Present	31	9 (29)	3 (9.7)	19 (61.3)	20 (64.5)	11 (35.5)	7 (22.6)	16 (88.9)	8 (25.8)	4 (12.9)	22 (71)	5 (16.1)
Lymphatic invasion
Absent	37	11(29.7)	13 (35.1)	13 (35.1)	20 (54.1)	17 (45.9)	7(18.9)	13 (35.1)	17 (45.9)	7 (18.9)	24 (64.9)	6 (16.2)
Present	13	1 (7.7)	3 (23.1)	9 (69.2)	6 (46.2)	7 (53.8)	5 (38.5)	5 (38.5)	3 (23.1)	3 (23.1)	3 (23.1)	7 (53.8)
Vascular invasion
Absent	42	12(28.6)	14(33.3)	16 (38.1)	23 (54.8)	19 ( 45.2)	8 (19)	16 (38.1)	18 (42.9)	8 (19)	25 (59.5)	9 (21.4)
Present	8	0	2 (25)	6 (75)	3 (37.5)	5 (62.5)	4 (50)	2 (25)	2 (25)	2 (25)	2 (25)	4 (50)
Perineural invasion
Absent present	31	6 (19.4)	12 (38.7)	13 (41.9)	15 (48.4)	16 (51.6)	8 (25.8)	10 (32.3)	13 (41.9)	5 (16.1)	17 (54.8)	9 (29)
	19	6 (31.6)	4 (21.1)	9 (47.4)	11 (57.9)	8 (42.1)	4 (21.1)	8 (42.1)	7 (36.8)	5 (26.3)	10 (52.6)	4 (21.1)
Stagea
1 + 2	24	8 (33.3)	15 (62.5)	9(4.2)	7 (29.2)	17 (70.8)	5 (20.8)	4 (16.7)	15 (62.5)	7 (29.2)	9 (37.5)	8 (33.3)
3 + 4	26	4 (15.4)	1(11.6)	19(73)	19 (73.1)	7 (26.9)	7 (26.9)	14 (53.8)	5 (19.1)	3 (11.5)	18 (69.2)	5 (19.2)
Distant metastasis
Absent	26	7 (26.9)	8 (30.8)	11 (42.3)	11 (42.3)	15 (57.7)	7 (26.9)	7 (26.9)	12 (46.2)	4 (15.4)	16 (61.5)	6 (23.1)
Present	24	12(20.8)	8 (33.3)	11 (45.8)	15 (62.5)	9 ( 37.5)	5 (20.8)	11 (45.8)	8 (33.3)	6 (25)	11 (45.8)	7 (39.2)
Survival (mo)a
60.62 ± 26.68 ≥	26	11 (42.3)	0	15 (57.7)	22 (84.6)	4 (15.4)	7 (26.9)	15 (57.6)	4 (42.4)	4 (15.5)	21 (80.7)	1 (3.8)
60.62 ± 26.68 <	24	1 (4.2 )	16 (66.6 )	7 (29.2)	4 (16.7)	20 (83.3)	5 (20.8)	3 (12.5)	16 (87.5)	6 (25)	6 (25)	12 (50)

^aP < 0.05. Downregulation of Notch and upregulation of Numb, Itch, and Siah-1 are associated with better prognosis. (↑): Upregulation, (↓): Downregulation; WD: Well-differentiated; MD: Moderately differentiated; PD: Poorly differentiated; LNM: Lymph node metastasis.

Table 6 Independent prognostic factors according to Cox regression analysis

Parameter	Regression coefficient	P value	Relative risk
LN metastasis	2.406	0.001	11.088
Notch expression levels	1.579	0.03	3.677
Numb expression levels	-1.166	0.01	0.561