Peripheral Mononuclear Cells Surface Markers Evaluation in Different Stages of Hepatocellular Carcinoma; in a Trial for Early and Accurate Diagnosis in Patients with Post-Hepatitis Liver Cirrhosis and Unremarkable Raised AFP

Figures & data

Figure 1 Lymphocyte gating with CD3⁺ T-cells and subdivided T helper CD4⁺ and cytotoxic CD8⁺ cells.

Table 1 Clinical and Imaging Characteristics of the Included HCC and Liver Cirrhosis Patients

Clinical and Imaging Characteristics	HCC Patients (N=56)	Liver Cirrhosis Patients (N=56)
Age years, Median (IQR)	58.0 (50, 65)	59.0 (51, 66)
Male gender, N (%)	41 (73.2)	43 (76.8)
Hypertension, N (%)	14 (25.0)	16 (28.6)
Diabetes mellitus, N (%)	29 (51.8)	16 (28.6)
Smoking, N (%)	15 (26.8)	13 (23.2)
CHC Status (Patients), N (%)
Positive PCR HCV RNA	15 (26.8)	7 (12.5)
Sustained virologic response (SVR)	33 (58.9)	38 (67.86)
CHB Status (Patients)
Positive HBsAg and PCR HBV DNA	8 (14.3)	11 (19.64)
Child class, N (%)
Score A	12 (21.4)	11 (19.64)
Score B	22 (39.3)	21 (37.5)
Score C	22 (39.3)	24 (42.86)
AFP (ng/mL), Median (IQR)	21.9 (6.3, 61.25)	–
BCLC staging, N (%)
A	13 (23.2)
B	10 (17.9)
C	14 (25.0)	–
D	19 (33.9)
TNM staging, N (%)
I a / I b	2 (3.6) / 14 (25.0)	–
II	3 (5.4)
III a / III b	8 (14.3) / 29 (51.8)
Ascites, N (%)	49 (87.5)	41 (73.21)
Splenomegaly, N (%)	40 (71.4)	34 (60.71)
Site of focal lesion, N (%)
Right lobe	30 (53.6)	–
Left lobe	18 (32.1)
Right and left lobe	8 (14.3)
Number of focal lesions, N (%)
Single	38 (67.9)	–
Multiple lesions	18 (32.1)
Portal vein thrombosis, N (%)	30 (53.6)	–

Abbreviations: CHC, chronic hepatitis C; CHB, chronic hepatitis B; BCLC, Barcelona Clinic Liver Cancer; TNM, tumor/node/metastasis.

Table 2 Routine Laboratory Data of the Study Groups

Variables	HCC Group (N=56)	Cirrhosis Group (N=56)	P value
Hgb (g/dL), Median (IQR)	10.6 (9.8, 11.7)	11.0 (10.0, 12.5)	0.16
Platelets (10^3/mm^3), Median (IQR)	109.0 (88.0, 136.3)	129.5 (100.4, 164.8)	0.97
WBC (×10^3/mm^3), Median (IQR)	5.1 (4.1, 7.2)	5.8 (4.1, 9.9)	0.68
Neutrophils, Median (IQR)	3.6 (2.5, 5.0)	4.0 (2.4, 5.9)	0.61
Lymphocytes, Median (IQR)	0.8 (0.3, 1.4)	0.9 (0.3, 1.5)	1
Monocytes, Median (IQR)	0.69 (0.10, 1.22)	0.8 (0.2, 1.1)	0.65
Basophil, Median (IQR)	0.08 (0.02, 0.23)	0.1 (0.03, 0.3)	0.11
Eosinophil, Median (IQR)	0.06 (0.02, 0.23)	0.1 (0.02, 0.3)	1
RDW, Median (IQR)	14.8 (13.8, 16.9)	14.6 (13.6, 16.6)	1
MPV, Median (IQR)	8.4 (7.2, 9.1)	8.0 (7.2, 9.0)	1
INR, Median (IQR)	1.3 (1.2, 1.7)	1.3 (1.2, 1.5)	1
Creatinine (mg/dL), Median (IQR)	1.2 (0.8, 1.7)	1.1 (0.8, 1.6)	1
ALT (IU/L), Median (IQR)	55.5 (38.5, 76.0)	42.5 (29, 60.8)	0.002
AST (IU/L), Median (IQR)	68.0 (44.8, 99.0)	48.0 (35.8, 64.0)	<0.0001
Total bilirubin (mg/dL), Median (IQR)	2.6 (1.8, 3.4)	1.9 (1.5, 2.5)	0.07
Albumin (g/dL), Median (IQR)	2.7 (2.4, 3.2)	2.9 (2.5, 3.4)	0.08
ALP (U/L), Median (IQR)	72.5 (25.5, 226.0)	47.0 (25.0, 78.0)	<0.0001

Abbreviations: Hgb, hemoglobin; WBCs, white blood cells; RDW, red cell distribution width; MPV, mean platelet volume; INR, international normalization ratio; ALT, alanine transaminase; AST, aspartate aminotransferase; ALP, alkaline phosphatase.

Table 3 Immunological Data of the Study Groups (All Data are Presented as Percentage)

Variables	HCC Group (N=56)	Liver Cirrhosis Group (N=56)	P value
CD3^+, Median (IQR)	50.4 (8.6, 61.1)	50.9 (32.5, 77.0)	<0.001
CD4^+, Median (IQR)	13.9 (4.3, 36.4)	24.2 (16.0, 49.2)	<0.001
CD8^+, Median (IQR)	14.9 (8.3, 27.8)	26.9 (16.3, 36.4)	<0.001
CD19^+, Median (IQR)	5.4 (2.4, 14.6)	34.8 (11.1, 59.6)	<0.001
CD56^+, Median (IQR)	14.3 (9.3, 21.1)	24.0 (16.6, 58.0)	<0.001
CD14^+ CD16^− Classical, Median (IQR)	11.7 (5.1, 20.5)	38.4 (17.9, 55.7)	<0.001
CD14^++ CD16^+ Intermediate, Median (IQR)	4.0 (2.1, 9.5)	18.3 (14.0, 33.5)	<0.001
CD14^−+ CD16^++ Non-Classical, Median (IQR)	3.5 (0.5, 9.0)	39.9 (19.8, 68.7)	<0.001
CD16^+ CD36^+, Median (IQR)	6.0 (3.8, 7.6)	46.9 (38.2, 71.2)	<0.001
CD36^+ HLA DR^+, Median (IQR)	10.0 (7.7, 23.7)	60.4 (30.0, 78.8)	<0.001
CD36^+ CD16^−, Median (IQR)	15.4 (6.4, 20.3)	13.7 (7.2, 18.8)	0.79
CD11C^+, Median (IQR)	78.6 (68.9, 87.8)	74.1 (66.4, 96.6)	0.67
CD33^+, Median (IQR)	80.7 (68.4, 88.7)	77.8 (27.5, 94.2)	0.33
HLA DR^+ lymphocytes, Median (IQR)	15.1 (7.3, 26.0)	32.9 (19.0, 36.9)	<0.001
CD16^+ HLA DR^+, Median (IQR)	3.6 (2.5, 12.3)	58.1 (37.4, 77.7)	<0.001

Figure 2 Monocyte subsets (classical, intermediate, and non-classical monocytes) on gated monocytes. The upper part of the figure shows marked increase in non-classical monocytes. The lower part of the figure shows decreased intermediate and absent non-classical monocytes.

Table 4 Immunological Data of the Different Barcelona Clinic Liver Cancer (BCLC) Scoring Systems of HCC (All Data are Presented as Percentage)

Variables	BCLC (A and B) (N=23)	BCLC (C and D) (N=33)	P value
CD3^+, Median (IQR)	16.5 (1.3, 61.7)	50.9 (10.3, 61.1)	0.55
CD4^+, Median (IQR)	16.3 (1.3, 39.3)	11.4 (4.8, 21.3)	0.68
CD8^+, Median (IQR)	9.4 (0.6, 24.0)	15.5 (9.4, 29.1)	0.35
CD19^+, Median (IQR)	2.8 (0.6, 13.2)	8.3 (3.2, 15.5)	0.07
CD56^+, Median (IQR)	10.9 (4.7, 21.1)	14.7 (10.6, 27.8)	0.06
CD14^+ CD16^− Classical, Median (IQR)	9.2 (4.7, 21.2)	14.2 (5.5, 18.4)	0.79
CD14^++ CD16^+ Intermediate, Median (IQR)	3.9 (2.6, 9.5)	4.1 (2.0, 10.1)	0.89
CD14^−+ CD16^++ Non-Classical, Median (IQR)	3.5 (0.4, 9.0)	2.6 (0.5, 22.0)	0.91
CD16^+ CD36^+, Median (IQR)	6.0 (4.0, 7.7)	6.0 (3.8, 7.3)	0.38
CD36^+ HLA DR^+, Median (IQR)	9.9 (7.1, 23.7)	10.0 (7.7, 23.7)	0.33
CD36^+ CD16^−, Median (IQR)	7.1 (6.3, 20.3)	15.4 (7.1, 20.3)	0.28
CD11C^+, Median (IQR)	78.6 (68.9, 94.2)	69.1 (57.4, 87.8)	0.47
CD33^+, Median (IQR)	74.9 (60.0, 85.6)	83.9 (74.9, 88.7)	0.05
HLA DR^+ lymphocytes, Median (IQR)	13.1 (1.6, 25.0)	21.8 (14.1, 31.0)	0.04
CD16^+ HLA DR^+, Median (IQR)	49.0 (37.4, 77.7)	69.4 (35.2, 77.7)	0.93

Table 5 Immunological Data of the Different TNM Scoring Systems of HCC (All Data are Presented as Percentage)

Variables	TNM (I and II) (N=19)	TNM (III) (N=37)	P value
CD3^+, Median (IQR)	56.1 (11.9, 61.7)	16.5 (7.2, 58.7)	0.07
CD4^+, Median (IQR)	17.8 (4.3, 37.2)	11.4 (3.9, 27.9)	0.42
CD8^+, Median (IQR)	23.9 (9.4, 30.3)	9.4 (6.4, 24.0)	0.06
CD19^+, Median (IQR)	4.9 (2.4, 14.6)	5.8 (2.4, 13.9)	0.77
CD56^+, Median (IQR)	14.3 (10.3, 34.5)	14.3 (9.0, 21.1)	0.50
CD14^+ CD16^− Classical, Median (IQR)	9.1 (1.6, 15.0)	14.9 (7.2, 40.3)	0.04
CD14^++ CD16^+ Intermediate, Median (IQR)	4.4 (1.6, 20.8)	3.9 (2.5, 9.2)	0.59
CD14^−+ CD16^++ Non-Classical, Median (IQR)	3.5 (0.4, 58.1)	3.4 (0.5, 9.0)	0.68
CD16^+ CD36^+, Median (IQR)	4.0 (2.2, 28.6)	6.0 (4.0, 7.3)	0.42
CD36^+ HLA DR^+, Median (IQR)	7.7 (6.4, 12.1)	10.0 (7.7, 23.7)	0.04
CD36^+ CD16^−, Median (IQR)	7.1 (1.7, 15.4)	15.4 (6.8, 20.3)	0.03
CD11C^+, Median (IQR)	78.6 (68.9, 96.6)	78.6 (63.1, 87.8)	0.31
CD33^+, Median (IQR)	74.9 (68.4, 83.9)	81.0 (71.3, 88.7)	0.27
HLA DR^+ lymphocytes, Median (IQR)	18.5 (6.2, 25.0)	15.1 (10.9, 26.4)	0.99
CD16^+ HLA DR^+, Median (IQR)	69.4 (49.0, 89.8)	49.0 (33.0, 77.7)	0.18