Assessment of circulating levels of microRNA-326, microRNA-424, and microRNA-511 as biomarkers for hepatocellular carcinoma in Egyptians

Table 1 Descriptive data of the studied hepatocellular carcinoma patients

			HCC
			N = 70
Demographic data	Age (yr)		62.0 ± 7.6
	Gender (%)	Female	22 (31.4)
		Male	48 (68.6)
	Smoker (%)	No	56 (80.0)
		Yes	14 (20.0)
	DM	No	52 (74.3)
	(%)	Yes	18 (25.7)
	BMI (kg/m2)		27.7 (23.6- 31.9)
Laboratory investigation	Hb (g/dL)		13.2 ± 1.9
	WBC (× 103/µL)		6.0 (4.6-8.0)
	Platelets (× 103/µL)		169.5 ± 77.9
	ALT (U/mL)		52.5 (32.0- 74.3)
	AST (U/mL)		60.5 (38.0- 83.3)
	Alb (g/dL)		3.7 ± 0.5
	Bil.T (mg/dL)		0.9 (0.6- 1.4)
	AFP (ng/dL)		38.8 (10.0- 370.0)
	Creatinine (mg/dL)		0.9 ± 0.2
BCLC (%)	Early		34 (48.6)
	Intermediate		23 (32.9)
	Late		13 (18.6)
Performance status (%)	0		43 (61.4)
	1		24 (34.3)
	2		2 (2.9)
	3		1 (1.4)
Child grade (%)	A		58 (82.9)
	B		11 (15.7)
	C		1 (1.4)
Child score			5.8 ± 1.0
Number of HCCs (%)	Single		49 (70.0)
	Two		12 (17.1)
	≥ 3		9 (12.9)
Site of HCC (%)	Right lobe		59 (84.3)
	Left lobe		7 (10.0)
	Both lobes		4 (5.7)
HCC size (cm)			4.0 (2.7- 5.0)
Portal vein	Patent		65 (92.9)
(%)
	Thrombosed		5 (7.1)
Abdominal lymphadenopathy	No		65 (92.9)
(%)	Yes		5 (7.1)
Decision of treatment	Best supportive care		7 (10.0)
	Combined therapy		1 (1.4)
(%)	Hepatectomy		3 (4.3)
	Microwave ablation		20 (28.5)
	Radioembolization		3 (4.3)
	Sorafinib		3 (4.3)
	TACE		29 (41.4)
Response to treatment according to mRECIST (%)	Stationary		4 (7.8)
	Partial response		7 (13.7)
	Complete response		34 (66.7)
	Progressive disease		6 (11.8)
Clinical decompensation (%)	Yes		9 (12.9)
	No		61 (54.5)
Alive or dead (%)	Dead		34 (48.6)
	Alive		36 (51.4)
Survival (days)			367.1 ± 173.9
miRNA-326			35.0 (12.5- 162.3)
miRNA-511			1.2 (0.3- 3.2)
miRNA-424			5.1 (2.9- 10.3)

DM: Diabetes mellitus; Hb: Hemoglobin; WBCs: White blood cells; ALT: Alanine aminotransferase; AST: Aspartate aminotransferase; INR: International nationalized ratio; AFP: α-fetoprotein; HCC: Hepatocellular carcinoma; BCLC: Barcelona clinic liver cancer; MWA: Microwave ablation; TACE: Trans-arterial chemoembolization; mRECIST: Modified response evaluation criteria in solid tumors; miRNA: MicroRNA.

Table 2 Studied miRNA associations with different parameters in patients with hepatocellular carcinoma

	miRNA-326	miRNA-511	miRNA-424
Gender
Male 48 (68.6%)	33.6 (11.3-157.4)	1.1 (0.4-3.4)	5.4 (3.2-10.4)
Females 22 (31.4%)	42.3 (11.9-168.2)	1.1 (0.4-3.4)	4.4 (1.9-8.3)
P value	0.7	0.8	0.3
Smoker
No 56 (80.0%)	27.4 (8.4-159.0)	1.1 (0.3-2.7)	5.4 (2.9-10.6)
Yes 14 (20.0%)	67.0 (28.0-177.0)	1.2 (0.7-5.9)	4.1 (2.7-7.0)
P value	0.3	0.3	0.3
Diabetes mellitus
No 52 (74.3%)	29.4 (9.1- 119.1)	0.9 (0.3-2.5)	4.6 (2.6-9.0)
Yes 18 (25.7%)	70.4 (16.6- 218.4)	1.5 (0.9-5.1)	6.5 (4.1-13.7)
P value	0.1	0.08	0.04a
Child grade
A 58 (82.9%)	64.4 (15.3-177.0)	1.2 (0.4-3.8)	5.1 (3.0-10.3)
B 11 (15.7%)	16.1 (4.1-110.7)	0.9 (0.0-1.4)	5.2 (2.6-11.9)
C 1 (1.4%)	5.7 (5.7-5.7)	0.03 (0.03-0.03)	0.9 (0.9-0.9)
P value
B vs A
C vs A	0.03a	0.1	0.7
C vs B	0.2	0.1	0.1
	0.7	0.3	0.3
HCC stages
Early	32.0 (7.5-121.0)	1.0 (0.3-2.6)	4.5 (2.3-8.0)
Intermediate	69.8 (16.1-185.0)	1.4 (0.6-4.6)	5.3 (3.9-13.2)
Late	26.2 (14.5-179.8)	0.9 (0.1-2.9)	5.9 (2.5-10.1)
P value
Intermediate vs Early	0.2	0.1	0.04a
Late vs Early	0.6	0.8	0.5
Late vs Intermediate	0.7	0.2	0.6
Performance status
0; 43 (61.4%)	64.4 (15.8-153.3)	1.2 (0.5-3.5)	5.7 (3.1-10.2)
1; 24 (34.3%)	18.9 (7.1-184.5)	0.6 (0.0-2.5)	4.0 (1.5-10.4)
2; 2 (2.9%)	7.2 (0.5-7.8)	3.1 (0.4-3.5)	5.3 (4.8-5.5)
3; 1 (1.4%)	160.9 (160.9- 160.9)	9.6 (9.6-9.6)	22.0 (22.0-22.0)
P value	0.2	0.2	0.2

Studied miRNAs are represented as Median with Interquartile range (25% -75%), the data were analyzed by Mann-Whitney U test except for the Studied miRNAs associations with Performance status which is analysed by the Kruskal Wallis Test. HCC: Hepatocellular carcinoma.

^aP value < 0.05 is significant.

Table 3 Studied miRNAs in healthy controls vs hepatocellular carcinoma

	Controls	HCC patients	P value
	N = 25	N = 70
miRNA-326	1.2 (0.3- 30.8)	35.0 (12.5- 162.3)	0.001b
miRNA-511	2.1 (0.9- 7.7)	1.2 (0.3- 3.2)	0.02a
miRNA-424	6.1 (0.7- 12.4)	5.1 (2.9- 10.3)	0.4

Studied miRNAs are represented as median with interquartile range (25% -75%), the data were analyzed by the Mann-Whitney U test. HCC: Hepatocellular carcinoma.

^aP value < 0.05 is significant.

^bP value < 0.01 is highly significant.

Table 4 Diagnostic performance of the studied miRNAs

	Cut off	Sensitivity	Specificity	PPV	NPV	Accuracy	AUC	95%CI	P value
miRNA-326	> 1.165	0.971	0.52	0.85	0.867	0.853	0.784	67.7-89.1	< 0.001b
miRNA-511	< 2.063	0.714	0.6	0.833	0.429	0.684	0.654	53.1-77.7	0.01a
miRNA-424	> 2.462	0.829	0.48	0.817	0.5	0.737	0.559	40.4-71.5	0.4

PPV: Positive predictive value; NPV: Negative predictive value; AUC: Area under curve; 95%CI: 95%Confidence interval.

^aP value < 0.05 is significant.

^bP value < 0.01 is highly significant.

Table 5 Survival analysis

		Dead, N = 34	Median (95%CI)of the EstimatedSurvival Time	Log rank (Mantel-Cox)	P value
Gender	Female (%)	9 (26.5)	315.0 (134.4- 495.6)	0.094	0.8
	Male (%)	25 (73.5)	441.0 (329.7- 552.3)
Smoker	No (%)	29 (85.3)	395.0 (299.9- 490.1)	1.595	0.2
	Yes (%)	5 (14.7)	581.0 (336.4- 825.6)
DM	No (%)	27 (79.4)	413.0 (341.7- 484.3)	0.259	0.6
	Yes (%)	7 (20.6)	429.0 (257.4- 600.6)
BCLC stages	Early (%)	12 (35.3)	532.0 (401.2- 662.8)	5.594	0.05a
	Intermediate (%)	14 (41.2)	393.0 (225.7- 560.3)
	Late (%)	8 (23.5)	284.0 (196.6- 371.4)
Performance status	0 (%)	21 (61.8)	395.0 (314.6 - 475.4)	1.188	0.6
	1 (%)	12 (35.3)	516.0 (404.0- 628.0)
	2 (%)	1 (2.9)	278.0 (278.0- 278.0)
	3	0 (0.0)	-
Child score	A (%)	26 (76.5)	429.0 (316.6- 541.4)	0.574	0.8
	B (%)	7 (20.6)	375.0 (241.9- 508.1)
	C (%)	1 (2.9)	516.0 (516.0- 516.0)
Response to treatment mRECIST	Stationary	4 (11.8%)	284.0 (215.4- 352.6)	2.9	0.4
	Partial response	1 (2.9%)	412.0 (412.0- 412.0)
	Complete response	19 (55.9%)	456.0 (346.7- 565.3)
	Progressive disease	5 (14.7%)	128.0 (0.0- 285.2)
miRNA-326 >1.165			441.0 (336.7- 545.3)	17.1	0.001b
miRNA-511<2.063			441.0 (329.5- 552.5)	0.626	0.4
miRNA-424>2.462			395.0 (300.7- 489.3)	2.707	0.1

mRECIST: Modified Response Evaluation Criteria in Solid Tumors; BCLC: Barcelona Clinic Liver Cancer; DM: Diabetes mellitus; 95%CI: 95%Confidence interval.

^aP value < 0.05 is significant.

^bP value < 0.01 is highly significant.

Table 6 Studied miRNA associations with response to treatment according to modified response evaluation criteria in solid tumors

Response to treatment mRECIST	Stationary, n = 4 (5.7%)	Partial response, n = 7 (10.0%)	Complete response, n = 34 (48.6%)	Progressive disease, n = 6 (8.6%)	P value
miRNA-326	11.5 (2.1- 142.6)	31.1 (6.9- 209.4)	27.4 (10.0- 155.2)	69.6 (17.7- 197.9)	0.5
miRNA-511	3.8 (0.8- 10.5)	1.2 (0.3- 4.2)	1.0 (0.2- 1.5)	0.6 (0.1- 1.3)	0.05a
miRNA-424	4.9 (3.0- 8.6)	4.8 (2.1- 5.1)	5.4 (2.6- 13.1)	4.6 (3.1- 15.2)	0.8

mRECIST: Modified Response Evaluation Criteria in Solid Tumors.

^aP value < 0.05 is significant.