Peripheral immune factors aiding clinical parameter for better early recurrence prediction of hepatocellular carcinoma after thermal ablation

Figures & data

Figure 1. Study design. PBMCs: peripheral blood mononuclear cells; ER: early recurrence; PIFs: peripheral immune factors; RFS: recurrence-free survival.

Table 1. Baseline characteristics of each cohort.

No. (%)	Discovery cohort n = 183 (75%)	Validation cohort n = 61 (25%)	*p Value
Age(years)			0.933
< 65	134 (73.22)	45 (73.77)
≥65	49 (26.78)	16 (26.23)
Sex			0.482
Female	44 (24.04)	12 (19.67)
Male	139 (75.96)	49 (80.33)
Etiology of cirrhosis			0.704
HBV + HCV	165 (90.16)	56 (91.80)
Other	18 (9.84)	5 (8.20)
Differentiation			0.372
Well	32 (17.49)	13 (21.31)
Moderate	62 (33.88)	15 (24.59)
Poor	14 (7.65)	8 (13.11)
NA	75 (40.98)	25 (40.98)
Tumor number			1.000
Single	135 (73.77)	45 (73.77)
2–3	48 (26.23)	16 (26.23)
Tumor size			0.941
≤2.5cm	89 (48.63)	30 (49.18)
>2.5cm	94 (51.37)	31 (50.82)
BCLC stage			0.904
0	29 (15.85)	10 (16.39)
A	113 (61.75)	39 (63.93)
B	41 (22.40)	12 (19.67)
Child-Pugh			0.751
A	172 (93.99)	58 (95.08)
B	11 (6.01)	3 (4.92)
AFP (ng/mL)			0.452
≤25	115 (62.84)	35 (57.38)
>25	68 (37.16)	26 (42.62)
ALT (U/L)			0.920
≤50U/L	154 (84.15)	51 (83.61)
>50U/L	29 (15.85)	10 (16.39)
AST (U/L)
≤40U/L	148 (80.87)	50 (81.97)	1.000
>40U/L	35 (19.13)	11 (18.03)
WBC(×10^^9/L)			0.881
≤4.8	77 (42.08)	25 (40.98)
>4.8	106 (57.92)	36 (59.02)
PLT (×10^9/L)			0.172
< 100	66 (36.07)	28 (45.90)
≥100	117 (63.93)	33 (54.10)
Lymphcyte (%)			0.880
≤32	74 (40.44)	24 (39.34)
>32	109 (59.56)	37 (60.66)
Neutrophil (%)			0.059
≤60	129 (70.49)	35 (57.38)
>60	54 (26.51)	26 (42.62)
ALB (g/L)			0.522
≤35	27 (14.75)	7 (11.48)
>35	156 (85.25)	54 (88.52)
TBil (umol/L)			0.818
≤17.1	117 (63.93)	38 (62.30)
>17.1	66 (36.07)	23 (37.07)

HBV: hepatitis B virus; HCV: hepatitis C virus; BCLC: Barcelona Clinic Liver Cancer; AFP: alpha fetoprotein; ALT: alanine aminotransferase; AST: aspartate aminotransferase; WBC: white blood cell; PLT: platelet; ALB: albumin; TBil: total bilirubin.

Table 2. Univariate and multivariate analyses of baseline clinical and immune characteristics associated with early recurrence.

Characteristics	Discovery cohort (n = 183)
	Univariate analysis			Multivariate analysis
	HR	95%CI	p	HR	95%CI	p
Age, years (>65 vs. ≤65)	1.135	(0.587, 2.194)	0.707
Sex (male vs. female)	1.409	(0.700, 2.836)	0.337
Tumor number (multiple vs. single)	1.338	(0.690, 2.595)	0.389
Tumor size, cm (>2.5 vs.≤2.5)	2.718	(1.467, 5.037)	0.001**	4.023	(1.944, 8.327)	0.000***
BCLC stage (B–A vs. 0)	1.804	(0.772, 4.214)	0.173
Child-Pugh (B vs. A)	0.757	(0.214, 2.681)	0.666
AFP, ng/mL (>25 vs.≤25)	2.387	(1.296, 4.396)	0.005**	2.279	(1.102, 4.716)	0.026*
ALT, U/L (>50 vs.≤50)	0.794	(0.351, 1.793)	0.578
AST, U/L (>40 vs.≤40)	1.168	(0.557, 2.450)	0.681
ALB, g/L (≤35 vs.>35)	0.917	(0.403, 2.087)	0.836
TBil, umol/L (>17.1 vs.≤17.1)	0.896	(0.486, 1.652)	0.725
WBC,×10^^9/L (>4.8 vs.≤4.8)	1.078	(0.595, 1.953)	0.804
Lymphocyte,% (≤0.32 vs.>0.32)	2.198	(1.202, 4.019)	0.011*
Neutrophil,% (>0.60 vs.≤0.60)	2.933	(1.522, 5.653)	0.001**	3.374	(1.509, 7.546)	0.003**
PLT,×10^^9/L (≤100 vs.>100)	2.365	(1.276, 4.383)	0.006**	2.561	(1.225, 5.354)	0.012*
CD3^+ % (≤69 vs.>69)	1.019	(0.567, 1.831)	0.949
CD4^+ % (≤44 vs.>44)	1.063	(0.588, 1.922)	0.839
CD8^+ % (≤21 vs.>21)	0.980	(0.544, 1.763)	0.945
CD4^+/CD8^+ (>2.2 vs.≤2.2)	0.807	(0.441, 1.478)	0.488
NK % (>7 vs.≤7)	1.814	(0.936, 3.515)	0.078
NKT % (>10 vs.≤10)	5.314	(2.009, 14.056)	0.001**	5.137	(1.657, 15.929)	0.005**
CD19^+ % (≤15 vs.>15)	1.033	(0.575, 1.856)	0.914
Treg % (>7.5 vs.≤7.5)	1.301	(0.721, 2.349)	0.382
CD4^+CD45RA % (>12.5 vs.≤12.5)	0.991	(0.549, 1.789)	0.976
CD4^+CD45RO % (≤30 vs.>30)	0.933	(0.519, 1.679)	0.817
CD4^+CD45RA/CD4^+CD45RO (>0.45 vs.≤0.45)	0.937	(0.340, 2.581)	0.899
CD8^+CD28^+ % (≤1.1 vs.>1.1)	1.339	(0.733, 2.447)	0.342
CD8^+CD28^- % (>12 vs.≤12)	3.067	(1.648, 5.707)	0.000***	3.246	(1.574, 6.695)	0.001**
CD8^+CD28^+/CD8^+CD28^- (≤0.5 vs.>0.5)	1.458	(0.712, 2.986)	0.303

*p < 0.05; **p < 0.01; ***p < 0.001. BCLC: Barcelona Clinic Liver Cancer; AFP: alpha fetoprotein; ALT: alanine aminotransferase; AST: aspartate aminotransferase; ALB: albumin; TBil: total bilirubin; WBC: white blood cell; PLT: platelet.

Figure 2. Performances of Model^Clin and Model^PIFs-Clin. (a–b) The AUC and 95%CI of Model^Clin and Model^PIFs-Clin for ER prediction in two cohorts. AUC: overall area under the receiver operating characteristic curve; CI: confidence interval; PIFs: peripheral immune factors; ER: early recurrence.

Figure 3. Model^PIFs-Clin risk categories in discovery and validation cohorts. The cumulative incidence of ER in Model^PIFs-Clin-high subgroup compared with Model^PIFs-Clin-low, with 78.0 and 32.2% (p < 0.001) in discovery cohort (a), and and with 54.29 versus 42.31% (p = 0.355) in validation cohort (b). The recurrence free survival (RFS) in Model^PIFs-Clin different subgroups of discovery cohort (c) and validation cohort (d). ns: not significant; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.

Figure 4. Model^PIFs-Clin reclassified the ER patients who were underestimated by Model^Clin as low-risk. (a) Among those ER patients who were underestimated as low-risk by Model^Clin, Model^PIFs-Clin could screened out 48.39% (15/31) as high-risk ER patients in discovery cohort. (b) In validation cohort, Model^PIFs-Clin screened out 43.75(7/16) the ER patients who were classified as low-risk by Model^Clin.

Table 3. Prognostic performance of the models.

Model	Discovery cohort			Validation cohort
	Model^Clin	Model^PIFs	Model^PIFs-Clin	Model^Clin	Model^PIFs	Model^PIFs-Clin
AUC	0.664 (0.588–0.740)	0.737 (0.666–0.809)	0.801 (0.734–0.867)	0.645 (0.509–0.781)	0.700 (0.569–0.831)	0.737 (0.608–0.865)
p Value	–	0.192^#	<0.001*	–	0.524^#	0.100*
Sensitivity	0.603	0.603	0.731	0.533	0.633	0.833
Specificity	0.638	0.762	0.752	0.742	0.645	0.516
PPV	0.553	0.653	0.687	0.667	0.633	0.625
NPV	0.684	0.721	0.790	0.622	0.645	0.762

^#Compared the AUC between Model^Clin and Model^PIFs.

*Compared the AUC between Model^Clin and Model^PIFs-Clin.

Figure 5. Differences of peripheral immune factors in Model^PIFs-Clin subgroups. According to the optimal cutoff by Youden’s Index, we divided all patients into two subgroups, for whose predictive value of Model^PIFs-Clin were below the cutoff were classified in Model^PIFs-Clin-low subgroup, and those above it were classified in Model^PIFs-Clin-high subgroup. The immune contexture was distinct between the two subgroups. ns: not significant; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.