Dual-parameter liquid biopsy using plasma miR-21-5p and T cell LAIR-1 mean fluorescence intensity for hepatocellular carcinoma diagnosis in a high-risk Egyptian cohort

Table 1 Baseline demographic and routine biochemical characteristics, n = 211

Parameter	HCC (n = 70)	Cirrhosis (n = 60)	Control (n = 81)	P value
Age in years	66 (6)	60 (6)	57.5 (7)	< 0.0011
Female sex	24 (34.3)	20 (33.3)	31 (38.3)	0.3502
AFP in ng/mL	305 (720)	18 (32)	4.5 (3)	< 0.0011
ALT in U/L	65 (38)	58 (35)	28 (10)	< 0.0011
AST in U/L	75 (42)	62 (38)	25 (9)	< 0.0011
Albumin in g/dL	3.1 (0.6)	3.4 (0.5)	4.2 (0.3)	< 0.0011

¹Kruskal-Wallis test.

²χ² test.

Data are median (interquartile range) or n (%). AFP: Α-fetoprotein; ALT: Alanine aminotransferase; AST: Aspartate aminotransferase; HCC: Hepatocellular carcinoma.

Table 2 Tumor characteristics in patients with hepatocellular carcinoma, n = 70

Tumor feature	n (%)
Solitary lesion (< 1.9 cm; no invasion)	33 (47.1)
Multiple lesions (no vascular invasion)	20 (28.6)
Multiple lesions (with vascular invasion)	17 (24.3)
Regional lymph-node metastasis	42 (60.0)
Distant metastasis	30 (42.9)

Table 3 Biomarker levels stratified by Barcelona Clinic Liver Cancer stage in the hepatocellular carcinoma cohort

Biomarker	BCLC stage 0/A (early) (n = 38)	BCLC stage B/C (advanced) (n = 32)	P value
hsa-miR-21-5p	28.4 (25.8-34.1)	39.2 (31.5-48.9)	0.021
LAIR-1 MFI	39.8 (36.2-44.3)	46.5 (41.8-50.1)	0.007

P values were calculated using the Mann-Whitney U test. Values are presented as median (interquartile range). BCLC: Barcelona Clinic Liver Cancer; LAIR-1: Leukocyte-associated immunoglobulin-like receptor-1; MFI: Mean fluorescence intensity.

Table 4 Circulating microRNA expression levels across the study groups

Biomarker	HCC (n = 70)	Cirrhosis (n = 60)	Control (n = 81)	P value
hsa-miR-21-5p	34.0 (32.5)	6.0 (5.25)	0.6 (0.6)	< 0.001
hsa-miR-155-5p	5.1 (2.2)	2.35 (1.23)	0.9 (0.5)	< 0.001

Values are presented as median (interquartile range). P values were calculated using the Kruskal-Wallis test. Relative expression was calculated using the 2^-ΔΔCt method with SNORD68 as the endogenous control. HCC: Hepatocellular carcinoma.

Table 5 Leukocyte-associated immunoglobulin-like receptor-1 expression on peripheral blood T cells across the study groups

Parameter	HCC (n = 70)	Cirrhosis (n = 60)	Control (n = 81)	P value
LAIR-1+ T cell %	56.7 (31.33)	33.0 (21.1)	11.75 (5.33)	< 0.001
LAIR-1 MFI	42.3 (7.95)	30.0 (16.0)	22.4 (8.13)	< 0.001

Values are presented as median (interquartile range). P values were calculated using the Kruskal-Wallis test. HCC: Hepatocellular carcinoma; LAIR-1: Leukocyte-associated immunoglobulin-like receptor-1; MFI: Mean fluorescence intensity.

Table 6 Receiver operating characteristic performance (hepatocellular carcinoma vs cirrhosis)

Biomarker	AUC (bootstrap 95%CI)	Optimal cutoff	Sensitivity (%)	Specificity (%)
hsa-miR-21-5p	0.990 (0.974-1.000)	> 14.0	98.6	96.7
hsa-miR-155-5p	0.874 (0.809-0.930)	> 3.8	75.7	93.3
LAIR-1+ T cell %	0.833 (0.767-0.895)	> 55.9	52.9	100.0
LAIR-1 MFI	0.997 (0.990-1.000)	> 29.3	100.0	98.3

Optimal cutoff values were determined by maximizing the Youden index (J). Analysis based on 70 hepatocellular carcinoma cases vs 60 cirrhosis cases. AUC: Area under the curve; CI: Confidence interval; LAIR-1: Leukocyte-associated immunoglobulin-like receptor-1; MFI: Mean fluorescence intensity.

Table 7 Pairwise comparison of biomarker area under the curve values for discriminating hepatocellular carcinoma from cirrhosis

Comparison	AUC1	AUC2	z statistic	P value
hsa-miR-21-5p vs hsa-miR-155-5p	0.990	0.874	4.025	< 0.001
hsa-miR-21-5p vs LAIR-1 MFI	0.990	0.997	-1.218	0.223
hsa-miR-155-5p vs LAIR-1 MFI	0.874	0.997	-4.275	< 0.001

Pairwise comparisons of correlated receiver operating characteristic curves were performed using a bootstrap method (1000 replicates) to approximate the DeLong test. AUC: Area under the curve; LAIR-1: Leukocyte-associated immunoglobulin-like receptor-1; MFI: Mean fluorescence intensity.

Table 8 Multivariable logistic regression model for the diagnosis of hepatocellular carcinoma

Predictor	Odds ratio	95%CI	P value	Variance inflation factor
hsa-miR-21-5p (per unit)	1.013	0.831-1.235	0.899	4.8
hsa-miR-155-5p (per unit)	0.911	0.463-1.795	0.789	1.5
LAIR-1 MFI (per unit)	1.484	1.043-2.111	0.028	3.1
AFP (per ng/mL)	1.053	0.946-1.173	0.345	2.9

All predictors were included as continuous variables. The model was fitted to predict hepatocellular carcinoma (n = 70) vs cirrhosis (n = 60). A variance inflation factor value > 5 was considered indicative of significant multicollinearity. AFP: Α-fetoprotein; CI: Confidence interval; LAIR-1: Leukocyte-associated immunoglobulin-like receptor-1; MFI: Mean fluorescence intensity.

Table 9 Correlations between biomarkers in patients with hepatocellular carcinoma, n = 70

Correlation pair	Spearman’s ρ	P value
hsa-miR-21-5p vs AFP	0.812	< 0.001
hsa-miR-21-5p vs LAIR-1+ T cell %	0.654	< 0.001
hsa-miR-21-5p vs LAIR-1 MFI	0.627	< 0.001
LAIR-1+ T cell % vs LAIR-1 MFI	0.714	< 0.001

Analysis performed within the hepatocellular carcinoma group only. AFP: Α-fetoprotein; LAIR-1: Leukocyte-associated immunoglobulin-like receptor-1; MFI: Mean fluorescence intensity.

Table 10 Diagnostic performance of leading biomarkers in clinically challenging subgroups

Biomarker	Subgroup	AUC (95%CI)	Sensitivity (%)	Specificity (%)
hsa-miR-21-5p	Early-stage HCC (n = 33) vs cirrhosis	0.981 (0.949-1.000)	96.9	96.7
LAIR-1 MFI	Early-stage HCC (n = 33) vs cirrhosis	0.992 (0.975-1.000)	100.0	98.3
hsa-miR-21-5p	AFP-negative HCC (n = 22) vs cirrhosis	0.986 (0.961-1.000)	95.5	96.7
LAIR-1 MFI	AFP-negative HCC (n = 22) vs cirrhosis	0.998 (0.990-1.000)	100.0	98.3

Analysis based on the optimal cut-offs derived from the main cohort (Table 5). AFP: Α-fetoprotein; AUC: Area under the curve; CI: Confidence interval; HCC: Hepatocellular carcinoma; LAIR-1: Leukocyte-associated immunoglobulin-like receptor-1; MFI: Mean fluorescence intensity.

Table 11 Synthesis of biomarker performance and clinical relevance for hepatocellular carcinoma diagnosis

Biomarker	Diagnostic performance (AUC)	Key strength	Role in multivariable model	Correlation with disease severity
hsa-miR-21-5p	0.990 (exceptional)	Very high sensitivity (98.6%) and specificity (96.7%)	Not an independent predictor (P = 0.899)	Strong correlation with AFP (ρ = 0.812); associated with metastasis
hsa-miR-155-5p	0.874 (good)	Good specificity (93.3%)	Not an independent predictor (P = 0.789)	Not assessed in this context
LAIR-1+ T cell %	0.833 (good)	Perfect specificity (100%) as a rule-in test	Not tested in the primary model	Strong correlation with LAIR-1 MFI (ρ = 0.714)
LAIR-1 MFI	0.997 (exceptional)	Perfect sensitivity (100%) and high specificity (98.3%)	Sole independent predictor (OR = 1.48, P = 0.028)	Associated with vascular invasion and metastasis
Combined model	0.998 (near-perfect)	Superior net benefit for clinical decision-making	N/A	N/A

Diagnostic performance for discriminating hepatocellular carcinoma from liver cirrhosis. The combined model included hsa-miR-21-5p, hsa-miR-155-5p, leukocyte-associated immunoglobulin-like receptor-1, mean fluorescence intensity, and alpha-fetoprotein. AUC: Area under the curve; LAIR-1: Leukocyte-associated immunoglobulin-like receptor-1; MFI: Mean fluorescence intensity; AFP: α-fetoprotein; N/A: Not applicable; OR: Odds ratio.