PD1 and TIM3 Expression is Associated with Very Early Hepatocellular Carcinoma Recurrence After Percutaneous Thermal Ablation

Figures & data

Figure 1 Flowchart of the study.

Table 1 Patients’ Characteristics

Variables	Prolonged Recurrence-Free Group (n=22)	Very Early Recurrence Group (n=9)	P value
Age (years), median [IQR]	70 [66–75]	81 [73–82]	0.09
Sex, n (%)
Male	17 (77.3)	9 (100)	0.31
Female	5 (22.7)	0 (0)
Etiology, n (%)
Alcohol	9 (40.9)	4 (44.5)	0.18
NASH	6 (27.3)	2 (22.2)
Viral	5 (22.7)	1 (11.1)
Hemochromatosis	0 (0)	2 (22.2)
None	2 (9.1)	0 (0)
Fibrosis stage Metavir, median [IQR]	3 [2–4]	3 [3–4]	0.71
BMI, median [IQR]	28.4 [25.3–32.5]	28.3 [26.6–30.5]	>0.99
Albumin (g/dL), median [IQR]	3.9 [3.7–4.3]	3.9 [3.7–4.1]	0.68
Bilirubin (mg/dL), median [IQR]	0.9 [0.5–1.4]	0.8 [0.7–0.8]	0.74
PT (%), median [IQR]	81 [73–91]	81 [68–91]	0.75
Creatinine (mg/dL), median [IQR]	0.8 [0.7–1.0]	0.9 [0.7–0.9]	0.97
AFP (ng/mL), median [IQR]	6.0 [4.4–17]	6.3 [4.2–29]	0.98
Child Pugh Score, n (%)
A5	19 (86.4)	9 (100)	0.63
A6	1 (4.5)	0 (0)
B7	1 (4.5)	0 (0)
Tumor size (mm), median [IQR]	22 [18.3–28.8]	20 [18–26]	0.83
Number of nodules, n (%)
One	21 (95.5)	8 (88.9)	0.50
Two	1 (4.5)	1 (11.1)
BCLC staging, n (%)
0	5 (22.7)	1 (11.1)	0.45
A	17 (77.3)	8 (88.9)
Ablation procedure, n (%)
RFA	10 (45.5)	2 (22.2)	0.42
MWA	12 (54.5)	7 (77.8)

Abbreviations: n, number; IQR, interquartile range; NASH, Non-Alcoholic SteatoHepatitis; BMI, body mass index; PT, prothrombin time; AFP, alphafetoprotein; BCLC, Barcelona clinic liver cancer; RFA, radiofrequency ablation; MWA, microwave ablation.

Figure 2 Immune signature associated with very early HCC recurrence after PTA. (A) Recurrence-free survival of thirty-one patients included in the study: 9 patients (29%) were later categorized into the very early recurrence group and 22 patients (71%) into the prolonged recurrence-free group. The median time between ablation procedure and intrahepatic distant recurrence was 4.5 months [IQR:3.1–9] in the very early recurrence group. (B) The frequencies of circulating PD1+ T cells compared between “prolonged free recurrence group” and “very early recurrence group”. The frequencies of PD1+ T cells, (C) in nontumoral and (D) in tumoral biopsies. (E) The frequencies of circulating TIM3+ T cells. The frequencies of TIM3+ T cells, (F) in nontumoral and (G) in tumoral biopsies. The violin plots show the distribution of the data, with median and quartiles.

Table 2 The Baseline Frequency of T Cells Expressing Immune Checkpoint Molecules in Peripheral Blood, Nontumoral and Intratumoral Liver Tissue

	Prolonged Recurrence-Free Group (n=22)		Very Early Recurrence Group (n=9)		P value
Blood
% of CD4^+ T cells, median [IQR]
PD1 CD4^+ T cells	14.00	[11.54–16.36]	24.30	[22.3–36.52]	<0.0001
TIM3 CD4^+ T cells	0.17	[0.11–0.33]	0.10	[0.07–0.79]	ns
LAG3 CD4^+ T cells	0.08	[0.05–0.77]	0.03	[0.02–0.1]	ns
CTLA4 CD4^+ T cells	0.24	[0.06–1.55]	0.18	[0.09–0.3]	ns
ICOS CD4^+ T cells	18.75	[14.33–21.93]	20.13	[12.66–39.42]	ns
OX40 CD4^+ T cells	1.77	[0.38–2.46]	0.65	[0.16–2]	ns
41BB CD4^+ T cells	0.53	[0.3–1.21]	0.62	[0.52–1.1]	ns
% of CD8^+ T cells, median [IQR]
PD1 CD8^+ T cells	16.85	[14.54–18.9]	25.55	[16.09–30.4]	ns
TIM3 CD8^+ T cells	0.83	[0.17–1.16]	16.48	[4.12–19.56]	0.0021
LAG3 CD8^+ T cells	0.63	[0.13–1.01]	0.20	[0.11–0.34]	ns
CTLA4 CD8^+ T cells	0.17	[0.07–2.01]	0.14	[0.05–0.36]	ns
ICOS CD8^+ T cells	7.47	[4.36–11.22]	22.88	[15.37–52.75]	0.0390
OX40 CD8^+ T cells	0.91	[0.33–1.48]	0.64	[0.12–1.23]	ns
41BB CD8^+ T cells	0.87	[0.34–1.48]	0.88	[0.24–2.21]	ns
Non-tumoral
% of CD4^+ T cells, median [IQR]
PD1 CD4^+ T cells	22.48	[18.03–29.87]	37.9	[35.98–50.00]	0.0004
TIM3 CD4^+ T cells	0.84	[0.6–1.13]	0.44	[0.05–1.72]	ns
LAG3 CD4^+ T cells	1.00	[0.67–1.89]	1.33	[0.58–1.43]	ns
CTLA4 CD4^+ T cells	1.99	[0.53–4.42]	0.44	[0.31–0.91]	ns
ICOS CD4^+ T cells	20.67	[12.68–24.19]	29.23	[22.64–31.90]	ns
OX40 CD4^+ T cells	3.82	[1.98–5.47]	2.48	[2.21–3.12]	ns
41BB CD4^+ T cells	2.07	[1.02–3.86]	1.89	[1.15–2.87]	ns
% of CD8^+ T cells, median [IQR]
PD1 CD8^+ T cells	31.62	[18.81–36.18]	33.33	[27.92–45.64]	ns
TIM3 CD8^+ T cells	1.61	[0.57–2.9]	6.78	[3.27–11.18]	0.0012
LAG3 CD8^+ T cells	1.14	[0.58–2.73]	0.73	[0.4–1.46]	ns
CTLA4 CD8^+ T cells	1.47	[0.25–2.89]	0.4	[0.37–0.82]	ns
ICOS CD8^+ T cells	8.56	[5.54–17.01]	23.66	[12.23–33]	0.0330
OX40 CD8^+ T cells	1.77	[0.65–2.87]	1.25	[0.58–1.97]	ns
41BB CD8^+ T cells	2.52	[1.25–5.42]	2.22	[1.35–4.44]	ns
Intra-tumoral
% of CD4^+ T cells, median [IQR]
PD1 CD4^+ T cells	24.04	[20.02–30.28]	37.57	[32.31–39.27]	0.0137
TIM3 CD4^+ T cells	0.96	[0.31–2.02]	1.48	[0.89–2.76]	ns
LAG3 CD4^+ T cells	1.31	[0.78–2.34]	0.59	[0.33–1.38]	ns
CTLA4 CD4^+ T cells	2.05	[1.14–2.89]	0.79	[0.37–1.71]	ns
ICOS CD4^+ T cells	24.13	[17.44–39.92]	25.98	[21.03–40.33]	ns
OX40 CD4^+ T cells	4.37	[2.63–6.57]	5.50	[3.24–8.29]	ns
41BB CD4^+ T cells	2.34	[1.1–6.26]	3.50	[2.27–4.61]	ns
% of CD8^+ T cells, median [IQR]
PD1 CD8^+ T cells	32.68	[18.26–40.26]	32.92	[26.47–50.66]	ns
TIM3 CD8^+ T cells	2.24	[1.2–5.37]	9.70	[8.83–13.59]	0.0239
LAG3 CD8^+ T cells	1.71	[1.25–3.32]	1.53	[0.32–1.58]	ns
CTLA4 CD8^+ T cells	1.47	[0.34–2.51]	0.41	[0.31–0.5]	0.024
ICOS CD8^+ T cells	14.3	[4.12–24.37]	24.48	[17.53–34.33]	ns
OX40 CD8^+ T cells	1.45	[0.85–2.69]	1.51	[1.28–2.07]	ns
41BB CD8^+ T cells	2.66	[1.71–6.71]	2.26	[1.77–3.45]	ns

Abbreviations: n, number; IQR, interquartile range; ns, not significant; PD1, Programmed cell death 1; TIM3, T cell immunoglobulin and mucin domain-containing protein 3; LAG3, Lymphocyte-activation gene 3; CTLA4, cytotoxic T-lymphocyte antigen 4; ICOS, Inducible T cell costimulatory.

Figure 3 Imaging and immunological profiles of representative patients. (A) Arterial phase axial abdominal CT scan in a 73-year-old man (patient No 13) shows a single 28-mm right liver HCC (white arrow) with peripheral perfusional disturbances at baseline. (B) Arterial phase axial abdominal CT scan of the same patient 45 months after percutaneous thermal ablation shows the ablated area (white arrowhead) without any recurrence. (C) and (E) Arterial phase axial abdominal MRI in a 66-year-old man (patient No 5) shows a single 18-mm left liver HCC (white arrow) at baseline. (D and F) Arterial phase axial abdominal CT scan in the same patient 9 months after percutaneous thermal ablation shows no recurrence at the ablated area (white arrowhead) but a very early intrahepatic distant recurrence in segment VIII (white circle). (G) Immunological profiles of patients No 13 and 5 at baseline. Results are expressed as a heat map-based frequency of positive cells compared with the mean frequency of a corresponding subpopulation of cells of the entire cohort; higher than the mean in dark blue.