Tissue inhibitor of metalloproteinase-3 expression affects clinicopathological features and prognosis of aflatoxin B1-related hepatocellular carcinoma

Table 1 Clinicopathological features of patients with hepatocellular carcinoma

Variable	n	Percentage (%)
Total	182	100.0
Age (years)1
< 49	98	53.8
≥ 49	84	46.2
Gender
Male	135	74.2
Female	47	25.8
Race
Han	89	48.9
Others	93	51.1
Smoking
No	138	75.8
Yes	44	24.2
Drinking
No	135	74.2
Yes	47	25.8
HBsAg
Negative	63	34.6
Positive	119	65.4
Anti-HCV
Negative	149	81.9
Positive	33	18.1
AFP
Negative	64	35.2
Positive	118	64.8
AFB1 exposure
Low	75	41.2
High	107	58.8
Tumor size
≤ 5 cm	56	30.8
> 5 cm	126	69.2
ES grade
Low	94	51.6
High	88	48.4
TNM stage
I	30	16.5
II	78	42.9
III	74	40.7
Liver cirrhosis
No	56	30.8
Yes	126	69.2
MVD
Low	90	49.5
High	92	50.5
PVTT
Negative	127	69.8
Positive	55	30.2
MVI
Negative	116	63.7
Positive	66	36.3

¹Age was grouped according to the average age of patients with hepatocellular carcinoma (49.52 10.79 years old).

HCV: Hepatitis C virus; AFP: Alpha fetoprotein; AFB1: Aflatoxin B1; ES: Edmondson and Steiner; TNM: Tumor node metastasis; MVD: Microvessel density; PVTT: Portal vein tumor thrombus; MVI: Microvessel infiltration.

Table 2 Clinical pathological features of hepatocellular carcinoma according to tissue inhibitor of metalloproteinase-3 expression

Variable	LTE		HTE		P value
	n	Percentage (%)	n	Percentage (%)
Total	89	100.0	93	100.0
Age (years)					0.78
< 49	47	52.8	51	54.8
≥ 49	42	47.2	42	45.2
Gender					0.18
Male	70	78.7	65	69.9
Female	19	21.3	28	30.1
Race					0.89
Han	44	49.4	45	48.4
Others	45	50.6	48	51.6
Smoking					0.38
No	70	78.7	68	73.1
Yes	19	21.3	25	26.9
Drinking					0.50
No	68	76.4	67	72.0
Yes	21	23.6	26	28.0
HBsAg					0.13
Negative	26	29.2	37	39.8
Positive	63	70.8	56	60.2
Anti-HCV					0.47
Negative	71	79.8	78	83.9
Positive	18	20.2	15	16.1
AFP					4.66 × 10-13
Negative	8	9.0	56	60.2
Positive	81	91.0	37	39.8
Tumor size					2.54 × 10-4
≤ 5 cm	16	18.0	40	43.0
> 5 cm	73	82.0	53	57.0
ES grade					3.84 × 10-4
Low	34	38.2	60	64.5
High	55	61.8	33	35.4
TNM stage					1.18 × 10-4
I	6	6.7	24	25.8
II	35	39.3	43	46.2
III	48	53.9	26	28.0
Liver cirrhosis					0.14
No	32	36.0	24	25.8
Yes	57	64.0	69	74.2
MVD					2.97 × 10-3
Low	34	38.2	56	60.2
High	55	61.8	37	39.8
PVTT					2.32 × 10-5
Negative	49	55.1	78	83.9
Positive	40	44.9	15	16.1
MVI					2.99 × 10-4
Negative	45	50.6	71	76.3
Positive	44	49.4	22	23.7

HCV: Hepatitis C virus; AFP: Alpha fetoprotein; ES: Edmondson and Steiner; TNM: Tumor node metastasis; MVD: Microvessel density; PVTT: Portal vein tumor thrombus; MVI: Microvessel infiltration; LTE: Low TIMP3 expression; HTE: High TIMP3 expression.

Table 3 The risk effects of tissue inhibitor of metalloproteinase-3 expression in the cancerous tissues on the clinicopathological features of hepatocellular carcinoma

Risk effects	ORLTE/HTG1	95%CI	P value
Higher AFP	15.32	6.63-35.38	1.65 × 10-10
Higher tumor size	3.47	1.75-6.87	3.69 × 10-4
Tumor dedifferentiation	3.04	1.65-5.65	4.31 × 10-4
Higher TNM stage	2.60	1.64-4.07	4.21 × 10-5
Higher MVD	2.45	1.35-4.45	3.20 × 10-3
Higher risk of PVTT	4.38	2.18-8.83	3.52 × 10-4
Higher risk of MVI	3.24	1.71-6.14	3.20 × 10-4

¹OR_LTE/HTG represents the risk value of low TIMP3 expression (compared to high TIMP3 expression) on the knowledge clinicopathological features of hepatocellular carcinoma.

AFP: Alpha fetoprotein; TNM: Tumor node metastasis; MVD: Microvessel density; PVTT: Portal vein tumor thrombus; MVI: Microvessel infiltration; CI: Confidential interval; OR: Odds ratio.

Table 4 Univariable Kaplan-Meier survival analysis

	OS			RFS
	De, n (%)	MST (95%CI)	P value	Re, n (%)	MRT (95%CI)	P value
Age (years)			0.71			0.97
< 49	79 (80.6)	27.00 (22.12-31.88)		68 (69.4)	27.00 (20.88-33.12)
≥ 49	71 (84.5)	23.00 (19.04-26.96)		56 (66.7)	24.00 (18.84-29.16)
Gender			0.68			0.55
Male	113 (83.7)	26.00 (2299-29.01)		91 (67.4)	24.00 (17.96-30.05)
Female	37 (78.7)	26.00 (21.71-30.29)		33 (70.2)	25.00 (17.96-30.04)
Race			0.76			0.26
Han	74 (83.1)	27.00 (22.55-31.76)		65 (73.0)	25.00 (19.01-30.99)
Others	76 (81.7)	26.00 (21.88-30.81)		59 (63.4)	25.00 (15.63-34.37)
Smoking			0.06			0.01
No	114 (82.6)	24.00 (20.33-27.67)		101 (73.2)	24.00 (18.62-29.38)
Yes	36 (81.8)	27.00 (19.67-34.43)		23 (52.3)	33.00 (15.27-50.74)
Drinking			0.10			0.03
No	110 (81.5)	24.00 (20.17-27.83)		97 (71.9)	24.00 (18.44-29.56)
Yes	40 (85.1)	27.00 (22.53-31.47)		27 (57.4)	30.00 (15.73-44.27)
HBsAg			0.26			0.05
Negative	44 (69.8)	28.00 (24.38-31.62)		34 (54.0)	29.00 (18.01-40.00)
Positive	106 (89.1)	24.00 (19.47-28.53)		90 (75.6)	22.00 (16.52-27.48)
Anti-HCV			0.45			0.88
Negative	123 (82.6)	24.00 (19.69-28.31)		99 (66.4)	24.00 (17.81-30.19)
Positive	27 (81.8)	28.00 (23.80-32.20)		25 (75.8)	28.00 (20.61-35.39)
AFP			1.85 × 10-9			1.03 × 10-6
Negative	44 (68.7)	41.00 (29.46-52.54)		35 (54.7)	40.00 (30.18-49.82)
Positive	106 (89.8)	20.00 (17.24-22.77)		89 (75.4)	17.00 (13.10-20.90)
AFB1 exposure			1.31 × 10-9			9.39 × 10-10
Low	58 (77.3)	36.00 (26.75-45.25)		38 (50.7)	43.00 (33.01-52.99)
High	92 (86.0)	20.00 (17.22-22.78)		86 (80.4)	18.00 (14.25-21.75)
Tumor size			5.52 × 10-8			5.3 × 10-6
≤ 5 cm	37 (66.1)	39.00 (27.27-50.73)		31 (55.4)	40.00 (31.01-48.99)
> 5 cm	113 (89.7)	19.00 (15.84-22.16)		93 (73.8)	16.00 (12.48-19.52)
ES grade			3.82 × 10-4			9.07 × 10-3
Low	75 (79.8)	33.00 (28.08-37.92)		61 (64.9)	30.00 (25.23-34.77)
High	75 (85.2)	20.00 (17.07-22.93)		63 (71.6)	19.00 (13.83-24.17)
TNM stage			7.40 × 10-17			4.47 × 10-11
I	24 (80.0)	45.00 (35.63-54.37)		19 (63.3)	37.00 (28.77-45.23)
II	59 (75.6)	27.00 (23.79-30.21)		47 (60.3)	29.00 (21.89-36.11)
III	67 (90.5)	12.00 (8.63-15.37)		58 (78.4)	10.00 (6.26-13.75)
Liver cirrhosis			0.93			0.43
No	45 (80.4)	27.00 (21.49-32.52)		34 (60.7)	27.00 (21.56-32.44)
Yes	105 (83.3)	25.00 (21.51-28.49)		90 (71.4)	24.00 (19.69-28.31)
MVD			7.60 × 10-11			1.07 × 10-11
Low	68 (75.6)	34.00 (31.65-36.35)		49 (54.4)	36.00 (27.49-44.51)
High	82 (89.1)	15.00 (11.87-18.13)		75 (81.5)	13.00 (9.00-16.01)
PVTT			1.50 × 10-6			9.99 × 10-7
Negative	103 (81.1)	29.00 (24.61-33.39)		80 (63.0)	30.00 (25.71-34.29)
Positive	47 (85.5)	12.00 (7.96-16.04)		44 (80.0)	8.00 (5.09-10.91)
MVI			6.65 × 10-8			1.55 × 10-7
Negative	95 (81.9)	29.00 (24.69-33.31)		73 (62.9)	30.00 (25.90-34.10)
Positive	55 (83.3)	11.00 (6.66-15.34)		51 (77.3)	8.00 (4.38-11.62)
TIMP3			3.91 × 10-11			1.89 × 10-6
LTE	86 (96.6)	18.00 (16.64-21.36)		70 (78.7)	16.00 (12.31-19.69)
HTE	64 (68.8)	36.00 (28.20-43.81)		54 (58.1)	32.00 (22.79-41.21)

De: Death patients; Re: Patients with tumor recurrence; OS: Overall survival; RFS: Recurrence-free survival; HCV: Hepatitis C virus; AFP: Alpha fetoprotein; ES: Edmondson and Steiner; TNM: Tumor node metastasis; MVD: Microvessel density; PVTT: Portal vein tumor thrombus; MVI: Microvessel infiltration; LTE: Low TIMP3 expression; HTE: High TIMP3 expression; AFB1: Aflatoxin B1; MST: Median overall survival time; MRT: Median tumor reoccurrence-free survival time.

Table 5 Univariable Cox proportional hazard regression survival analysis

	OS		RFS
Variable	HR (95%CI)	P value	HR (95%CI)	P value
Age (years), ≥ 49 vs < 49	1.06 (0.77-1.47)	0.72	1.01 (0.71-1.44)	0.97
Gender, male vs female	0.93 (0.64-1.35)	0.68	0.89 (0.59-1.32)	0.55
Race, Han vs others	0.95 (0.69-1.31)	0.76	0.80 (0.58-1.32)	0.27
Smoking, yes vs no	0.70 (0.48-1.03)	0.07	0.57 (0.36-0.89)	0.01
Drinking, yes vs no	0.74 (0.51-1.07)	0.11	0.63 (0.41-0.96)	0.03
HBV, positive vs negative	1.22 (0.86-1.74)	0.27	1.48 (0.99-2.19)	0.05
HCV, positive vs negative	0.85 (0.56-1.30)	0.46	0.97 (0.62-1.50)	0.89
AFP, positive vs negative	2.93 (2.03-4.24)	1.18 × 10-8	2.63 (1.75-3.95)	3.04 × 10-6
AFB1 exposure, high vs low	3.00 (2.06-4.36)	8.43 × 10-9	3.41 (2.25-5.18)	8.67 × 10-9
Tumor size (cm), > 5 vs ≤ 5	2.70 (1.85-3.93)	2.63 × 10-7	2.50 (1.65-3.77)	1.37 × 10-5
ES grade, III-IV vs I-II	1.79 (1.29-2.49)	5.55 × 10-4	1.60 (0.79-2.30)	0.01
TNM stage, II vs I	1.57 (0.97-2.53)	0.06	1.35 (0.79-2.30)	0.27
TNM stage, III vs I	5.95 (3.56-9.94)	1.01 × 10-11	4.29 (2.48-7.41)	1.92 × 10-7
Liver cirrhosis, yes vs no	1.02 (0.72-1.44)	0.93	1.17 (0.79-1.74)	0.43
MVD, high vs low	2.87 (2.06-4.02)	6.84 × 10-10	3.40 (2.34-4.96)	1.82 × 10-10
PVTT, positive vs negative	2.30 (1.62-3.28)	4.06 × 10-6	2.43 (1.68-3.53)	2.95 × 10-6
MVI, positive vs negative	2.48 (1.75-3.50)	2.56 × 10-7	2.53 (1.76-3.64)	5.63 × 10-7
TIMP3, LTE vs HTE	2.92 (2.19-4.09)	4.10 × 10-10	2.34 (1.63-3.38)	4.80 × 10-6

OS: Overall survival; HR: Hazard ratio; RFS: Recurrence-free survival; HCV: Hepatitis C virus; HBV: Hepatitis B virus; AFP: Alpha fetoprotein; ES: Edmondson and Steiner; TNM: Tumor node metastasis; MVD: Microvessel density; PVTT: Portal vein tumor thrombus; MVI: Microvessel infiltration; LTE: Low TIMP3 expression; HTE: High TIMP3 expression; AFB1: Aflatoxin B1.

Table 6 Effects of aflatoxin B1 exposure and tissue inhibitor of metalloproteinase-3 expression on survival of patients with aflatoxin B1-related hepatocellular carcinoma

	OS		RFS
Variable	MST (95%CI)	HR (95%CI/P value)	MRT (95%CI)	HR (95%CI/P value)
AETE1	47.00 (43.20-50.80)	1.00	49.98 (42.03-57.93)1	1.00
AETE2	28.00 (21.25-34.75)	2.51 (1.48-4.24/6.44 × 10-4)	37.00 (21.45-52.55)	1.84 (0.95-3.57/0.07)
AETE3	26.00 (21.29-30.71)	2.59 (1.53-4.40/4.04 × 10-4)	28.00 (21.60-34.40)	2.90 (1.63-5.15/2.86 × 10-4)
AETE4	16.00 (11.72-20.28)	7.41 (4.49-12.23/4.77 × 10-15)	12.00 (7.65-16.35)	6.38 (3.68-11.05/3.85 × 10-11)

¹Because median tumor reoccurrence-free survival time for AETE1 cannot be calculated using the Kaplan-Meier survival method, it was replaced by the average survival time for AETE1.

AETE1: Low aflatoxin B1 exposure plus high TIMP3 expression; AETE2: Low aflatoxin B1 exposure plus low TIMP3 expression; AETE3: High aflatoxin B1 exposure plus high TIMP3 expression; AETE4: High aflatoxin B1 exposure plus low TIMP3 expression; OS: Overall survival; HR: Hazard ratio; RFS: Recurrence-free survival; CI: Confidential interval.

Table 7 Mulvariable Cox proportional hazard regression analysis for survival

	OS		RFS
Variable	HR (95%CI)	P value	HR (95%CI)	P value
AFP, positive vs negative	2.85 (1.96-4.15)	5.06 × 10-8	2.48 (1.64-3.73)	1.44 × 10-5
AFB1 exposure, high vs low	2.92 (1.99-4.27)	3.71 × 10-8	3.23 (2.12-4.94)	5.78 × 10-8
Tumor size (cm), > 5 vs ≤ 5	2.93 (1.99-4.31)	4.53 × 10-8	2.79 (1.83-4.25)	2.00 × 10-6
ES grade, III-IV vs I-II	1.81 (1.29-2.55)	6.19 × 10-4	1.63 (1.12-2.37)	0.01
TNM stage, II vs I	1.61 (1.02-2.42)	0.03	1.75 (1.04-2.45)	0.02
TNM stage, III vs I	6.22 (3.70-10.48)	6.21 × 10-12	4.43 (2.54-7.71)	1.47 × 10-7
MVD, positive vs negative	2.88 (2.05-4.04)	1.07 × 10-9	3.34 (2.29-4.89)	4.71 × 10-10
PVTT, positive vs negative	2.30 (1.59-3.32)	8.73 × 10-6	2.34 (1.60-3.42)	1.16 × 10-5
MVI, positive vs negative	2.48 (1.74-3.54)	5.63 × 10-7	2.44 (1.68-3.53)	2.42 × 10-6
TIMP3, LTE vs HTE	2.85 (2.04-4.00)	1.17 × 10-9	2.26 (1.57-3.26)	1.26 × 10-5

OS: Overall survival; HR: Hazard ratio; RFS: Recurrence-free survival; CI: Confidential interval; AFP: Alpha fetoprotein; AFB1: Aflatoxin B1; ES: Edmondson and Steiner; TNM: Tumor node metastasis; MVD: Microvessel density; PVTT: Portal vein tumor thrombus; MVI: Microvessel infiltration; LTE: Low TIMP3 expression; HTE: High TIMP3 expression.

Table 8 Correlation between aflatoxin B1 exposure and tissue inhibitor of metalloproteinase-3 expression in hepatocellular carcinoma

	Low AFB1 exposure		High AFB1 exposure		r	P value
	n	Percentage (%)	n	Percentage (%)
LTE	27	36.0	62	57.9	-0.216	0.003
HTE	48	64.0	45	42.1

AFB1: Aflatoxin B1; LTE: Low TIMP3 expression; HTE: High TIMP3 expression.