Validation of a serum microRNA panel as biomarkers for early diagnosis of hepatocellular carcinoma post-hepatitis C infection in Egyptian patients

Table 1 Clinical data of hepatocellular carcinoma and hepatitis C virus patients

	Non-malignant chronic HCV (n = 250)		HCC (n = 224)	P value
	Moderate fibrosis (n = 150)	Late fibrosis (n = 100)
Age (years)	40 ± 8.6a	45 ± 6.5c	55 ± 9.3e	< 0.0001
ALP (IU/L)	102.3 ± 36.9a	119 ± 72.1a	182.5 ± 58c	< 0.0001
SGOT (IU/L)	58 ± 18a	62.5 ± 28.2a,b	85.4 ± 45.2c	0.001
SGPT (IU/L	52.1 ± 18a	68.46 ± 24.8a	120.4 ± 53.4c	< 0.0001
Bilirubin, total (mg/dL)	0.40 ± 0.17a	0.43 ± 0.16a	0.78 ± 0.69c	< 0.0009
Bilirubin, direct (mg/dL)	0.68 ± 0.28a	0.85 ± 0.37a	1.9 ± 1.2c	< 0.0001
Log AFP (ng/mL)	0.49 ± 0.9a	0.98 ± 0.4a	2.3 ± 0.97c	< 0.0001
Hemoglobin (g/dL)	13.6 ± 1.8a	14.1 ± 1.5a	10.7 ± 1.2c	< 0.0001
Platelet count × 103 (/mm3)	236.5 ± 21.6a	158.2 ± 52.6c	131.2 ± 75.5c	< 0.0001
leukocyte count × 103 (/mm3)	6.3 ± 2.22a	6.4 ± 2.41a	5.7 ± 2.4a	0.460
Prothrombin concentration (%)	103 ± 4.7a	84.4 ± 8.0c	69.9 ± 19.3e	< 0.0001
Albumin (g/dL)	4.7 ± 0.8a	3.9 ± 0.9c	3.28 ± 0.6e	< 0.0001

Data are expressed as mean ± SD. ANOVA was used to analyze the clinical data. Groups with different letters show a significant difference (P < 0.05), while those with same letters show no significant difference (P > 0.05). AFP: Alpha-fetoprotein; ALP: Alkaline phosphatase; HCC: Hepatocellular carcinoma; HCV: Hepatitis C virus; SGOT: Serum glutamic oxaloacetic transaminase; SGPT: Serum glutamic pyruvic transaminase.

Table 2 Clinical and pathological aspect for hepatocellular carcinoma patients

Factor	n
AFP level
Less than 20 ng/mL	56
Between 20 and 400 ng/mL	64
More than 400 ng/mL	104
Child-Pugh grade
A	128
B	80
C	16
Barcelona Clinic Liver Cancer staging
0	0
A	8
B	160
C	40
D	16
Focal lesions
Single	112
Multiple	112
Focal size by CT
< 3 cm	48
> 5 cm	64
Thrombosis in portal vein
Present	48
Absent	176

AFP: Alpha-fetoprotein; CT: Computed tomography.

Table 3 Diagnostic ability for studied miRNA to categorize hepatocellular carcinoma among hepatitis C virus-infected fibrotic patients and controls

Serum miRNA	AUC value	Sensitivity	Specificity	PPV	NPV	Accuracy
miR-214-5P
HCC against ctrl	0.721	61.0%	89.0%	81.0%	75.0%	77.0%
HCC against HCV	0.842	92.9%	75.5%	51.0%	97.5%	80.0%
miR-494
HCC against ctrl	0.813	77.0%	76%	69.0%	82.0%	76.0%
HCC against HCV	0.631	77.0%	56%	32.0%	90.0%	60.0%
miR-138p
HCC against ctrl	0.842	96.4%	78.4%	77.1%	96.0%	86.0%
HCC against HCV	0.642	68.2%	58.2%	34.0%	90.0%	62.0%
miR-125b
HCC against ctrl	0.702	66.7%	75.7%	66.7%	75.7%	72.0%
HCC against HCV	0.769	92.6%	55.4%	35.7%	96.6%	63.3%
miR-1269
HCC against ctrl	0.862	96.4%	78.4%	77.1%	96.7%	86.2%
HCC against HCV	0.691	78.6%	59.8%	34.9%	91.0%	63.8%
miR-145
HCC against ctrl	0.892	100.0%	89.2%	87.1%	100.0%	93.8%
HCC against HCV	0.624	81.5%	51.5%	30.6%	91.4%	57.7%
miR-375
HCC against ctrl	0.741	96.4%	59.5%	63.4%	95.7%	75.4%
HCC against HCV	0.811	96.4%	69.3%	46.6%	98.6%	75.2%

AUC: Area under curve, PPV: Positive predictive value, NPV: Negative predictive value.

Table 4 Regression analysis of the miRNAs studied

	Coefficient	Standard error	P value
Univariate analysis
miR-214-5p	-0.570	0.105	< 0.0001
miR-494	-0.005	0.025	0.825
miR-125b	-0.064	0.013	< 0.0001
miR-1269	0.184	0.044	< 0.0001
miR-145	0.0021	0.011	0.844
miR-375	0.066	0.011	< 0.0001
miR-138b	0.0019	0.013	0.852
Multivariate analysis
miR-214-5p	-1.116	0.229	< 0.0001
miR-125b	-0.076	0.028	0.004
miR-1269	0.569	0.121	< 0.0001
miR-375	0.052	0.013	0.002
Constant	-0.240

Table 5 Significant relations among enrolled circulating miRNAs in hepatocellular carcinoma patients

	miR-138b	miR-494	miR-145	miR-375	miR-214-5p	miR-1269	miR-125b
miR-138b		r = 0.471	r = 0.521	NS	r = 0.649	r = 0.862	r = 0.685
		P = 0.008	P = 0.006		P < 0.0002	P < 0.0001	P < 0.0001
miR-494	r = 0.471		r = 0.538	NS	NS	r = 0.418	NS
	P = 0.008		P = 0.003			P = 0.021
miR-145	r = 0.521	r = 0.538		r = 0.460	r = 0.449	r = 0.453	NS
	P = 0.006	P = 0.003		P = 0.038	P < 0.009	P = 0.019
miR-375	NS	NS	r = 0.460		r = 0.884	NS	NS
			P = 0.038		P < 0.0001
miR-214-5p	r = 0.649	NS	r = 0.449	r = 0.884		r = 0.621	r = 0.518
	P < 0.0002		P < 0.009	P < 0.0001		P = 0.0005	P = 0.005
miR-1269	r = 0.862	r = 0.418	r = 0.453	NS	r = 0.621		r = 0.602
	P < 0.0001	P = 0.021	P = 0.019		P = 0.0005		P = 0.001
miR-125b	r = 0.685	NS	NS	NS	r = 0.518	r = 0.602
	P < 0.0001				P = 0.005	P = 0.001

r is the Spearman rho coefficient. NS: Non-significant.