Adjuvant immunotherapy with autologous dendritic cells for hepatocellular carcinoma, randomized phase II study

Figures & data

Figure 1. Study design and CONSORT diagram of patient flow. (A) Study design for vaccination and evaluation. CR, complete remission; Pts, patients; SR, surgical recession; RFA, radiofrequency ablation; PEI, percutaneous ethanol injection; TACE, transarterial chemoembolization; ICF, Informed consent form; LKP, leukaperisis; DC, dendritic cell; RFS, recurrence-free survival; CT, computed tomography; MRI, magnetic resonance imaging; TTR, time-to-recurrence; mOS, median overall survival. (B) Patient flow from random assignment *excluded according to ITT analysis-based criteria. Of the enrolled patients, 23 did not meet inclusion criteria and thus were excluded from random assignment. In addition, 12 more patients were excluded from the efficacy analysis because they were found to violate inclusion criteria after randomization.

Table 1. Baseline demographics and disease characteristics.

Variable	Immunotherapy(n = 69)	Control group(n = 75)	p value
Male sex, n (%)	48 (69.6)	61 (81.3)	0.10^d
Age years, mean (SD)	57.1 ± 9.5	58.1 ± 10.8	0.58^e
Cause of liver disease, n (%)			0.33
HBV infection only	51 (73.9)	53 (70.7)
HCV infection only	8 (11.6)	8 (10.7)
HBV and HCV co-infection	2 (2.9)	0 (0)
Others	8 (11.6)	14 (18.6)
Treatment modality, n (%)			1.00^f
Surgical resection	30 (43.5)	32 (42.7)
RFA	29 (42.0)	32 (42.7)
TACE	5 (7.3)	5 (6.7)
PEI	1 (1.5)	2 (2.7)
Combination ^a	4 (5.8)	4 (5.3)
HCC stage, n (%)^b			0.83^d
Stage I	20 (29.0)	24 (32.0)
Stage II or III	49 (71.0)	51 (68.0)
Histological confirmation, n (%)			0.62^d
No	15 (21.7)	19 (25.3)
Yes	54 (78.3)	56 (74.7)
ECOG performance status, n (%)^c			0.85^d
0	45 (65.2)	50 (66.7)
1	24 (34.8)	25 (33.3)
α-fetoprotein, ng/mL			0.45^e
Median	4.5	4.5
Range	0.9–242.4	0.4–731.5
PIVKA-II, mAU/mL			0.85^e
Median	19.0	20.0
Range	6.0–275.0	6.0–177.0
Aspartate aminotransferase, IU/L			0.15^e
Median	32.0	31.0
Range	15.0–362.0	13.0–127.0
Alanine aminotransferase, IU/L			0.11^e
Median	23.0	24.0
Range	5.0–512.0	6.0–104.0
Alkaline phosphatase, IU/L			0.92^e
Median	93.0	91.0
Range	47.0–629.0	48.0–362.0
Albumin, g/dL			0.85^e
Median	4.0	4.0
Range	2.8–5.1	2.9–4.8
Total bilirubin, mg/dL			0.77^e
Median	0.7	0.8
Range	0.2–2.4	0.3–4.5
Creatinine, mg/dL			0.19^e
Median	0.8	0.8
Range	0.5–1.2	0.4–1.4
Platelet, ×10^3/mm^3			0.09^e
Median	133.0	148.0
Range	54.0–314.0	45.0–500.0
Prothrombin time (INR)			0.14^e
Median	1.1	1.1
Range	1.0–1.4	0.9–1.7
Child-Pugh score			0.83^e
Mean(SD)	5.3(0.5)	5.3(0.6)
Median	5.0	5.0
Range	5.0–7.0	5.0–7.0

Abbreviations: ECOG, Eastern Cooperative Oncology Group; HCC, hepatocellular carcinoma; PEI, percutaneous ethanol injection; RFA, radiofrequency ablation; TACE, transarterial chemoembolization..

Note. Data are expressed as n (%), mean ± SD, or median with range.

^a The combination therapy indicates 3 cases with TACE/RFA and 1 with TACE/surgical resection in the immunotherapy group and 3 with TACE/RFA and 1 with RFA/surgical resection in the control group.

^b HCC staging was done according to the modified UICC (mUICC) staging system.

^c ECOG performance status assesses the daily living abilities of the patient, on a scale ranging from 0 (fully active) to 5 (dead).

^d By Chi-square test.

^e By Student's t-test.

^f By Fisher's exact test.

Figure 2. Kaplan–Meier estimates of recurrence-free survival (RFS) in (A) overall patients, (B) patients of non-RFA group, (C) patients of RFA group, (D) patients of surgical resection group and (E) patients of surgical resection group (PP analysis), (F) Kaplan–Meier estimates of RFS in patients of TACE group, (G) Kaplan-Meier estimates of RFS in non-RFA group patients with stage 2 and 3 diseases. RFS was computed on all patients included in the ITT population (except E). Patients who had not progressed or died were censored on data cut-off. p values calculated by log-rank test (and by Wilcoxon test). Note: HR, hazard ratio; CI, confidence interval.

Table 2. Subgroup analysis of recurrence rates between control and immunotherapy groups over 2 y.

	Treat. Mod	Sur. Resec.		RFA		TACE		Combination		PEIT		Overall^a		Non-RFA^b
Pt. Stage	DC therapy	Pt #	Rec. #(%)	Pt #	Rec. #(%)	Pt #	Rec. #(%)	Pt #	Rec. #(%)	Pt #	Rec. #(%)	Pt #	Rec. #(%)	Pt #	Rec. #(%)
I	Control	2	0	18	4	2	1	1	1	1	1	24	7	6	3
			(0.0)		(22.2)		(50.0)		(100)		(100)		(29.2)		(50.0)
	DC therapy	3	1	14	7	2	0	0	0	1	1	20	9	6	2
			(33.3)		(46.7)		(0.0)		(NA)		(100)		(45.0)		(33.3)
	Benefit (%)^c		(−33.3)		(−24.5)		(+50.0)		(N/A)		(0.0)		(-15.8)		(+16.7)
II + III	Control	30	17	14	5	3	3	3	2	1	1	51	28	37	23
			(55.7)		(35.7)		(100)		(66.7)		(100)		(54.9)		(62.2)
	DC therapy	27	10	15	12	3	0	4	2	0	0	49	24	34	12
			(37.0)		(80.0)		(0.0)		(50.0)		(0.0)		(49.0)		(29.4)
	Benefit (%)^c		(+18.7)		(-44.3)		(+100)		(+16.7)		(N/A)		(+5.9)		(+32.8)
Total	Control	32	17	32	9	5	4	4	3	2	2	75	35	43	26
			(53.1)		(28.1)		(80.0)		(75.0)		(100)		(46.7)		(60.5)
	DC therapy	30	11	29	19	5	0	4	2	1	1	69	33	40	14
			(36.7)		(65.5)		(0.0)		(50.0)		(100)		(47.8)		(35.0)
	Benefit (%)^c		(+16.4)		(−37.4)		(+80.0)		(+25.0)		(0.0)		(-1.1)		(+25.5)

Abbreviation: N/A, not applicable; Pt #, patient number; Rec.# recurrent patient number; Treat Mod, treatment modality.

^a All subgroup analyses were assembled at the time of the data cut-off date.

^b Subgroups excluding patients who underwent RFA treatment.

^c % improvement of recurrence inhibition (control group recurrence rate – DC therapy group recurrence rate); (+) or (-) indicates improved or unimproved, respectively, by DC immunotherapy.

Figure 3. Overall survival Kaplan–Meier estimates of OS in overall patients. OS was computed on all patients included in the ITT population.

Figure 4. Immune responses after DC vaccination Changes in immune responses before and after DC vaccination against a tumor-associated antigen mixture (AFP, GPC-3, and MAGE-1). Data are represented after subtraction with the count of no Ag. (A) IFNγ ELISPOT assays were performed with PBMCs obtained from each patient at the indicated time points after the start of DC vaccination (0 w). Analysis was performed by paired t-test (GraphPad PRISM, ver 6.07) with the indicated number of patients. (B) Antigen-specific lymphocyte proliferation assays were performed after DC vaccinations using autologous PBMCs at each time point. Proliferation was determined by ³H-thymidine incorporation (DPM, disintegrations per minute) using a liquid scintillation counter and the results were analyzed using the same method as for the ELISPOT assay. **p < 0.01, ***p < 0.001, ****p < 0.0001.

Table 3. Treatment-emergent adverse events (safety population).

	Immunotherapy (n = 70)				Control (n = 76)
	All TEAE		Related TEAE		All AE		p value
Adverse event	Any grade	Grade 3 or 4	Any grade	Grade 3 or 4	Any grade	Grade 3 or 4	Any grade	Grade 3 or 4
Number of Pts	69 (98.6%)	10(14.2%)	65(92.9%)	1(1.4%)	39 (51.3%)	13 (17.1%)	<0.001^a	0.283^a
General disorders and administration site conditions (# of Pts)
Injection site pain	28 (40.0%)	0	28 (40.0%)	0	0	0	<.001^a	NA
Injection site erythema	24 (34.3%)	0	24 (34.3%)	0	0	0	<.001^a	NA
Injection site induration	23 (32.9%)	0	23 (32.9%)	0	0	0	<.001^a	NA
Influenza like illness	9 (12.9%)	0	9 (12.9%)	0	0	0	0.001^b	NA
Injection site mass	9 (12.9%)	0	9 (12.9%)	0	0	0	0.001^b	NA
Injection site swelling	9 (12.9%)	0	9 (12.9%)	0	0	0	0.001^b	NA
Injection site ulcer	1 (1,4%)	1 (1.4%)	1 (1.4%)	1 (1.4%)	0	0		NA
Pyrexia	8 (11.4%)	0	8 (11.4%)	0	0	0	0.002^b	NA
Fatigue	5 (7.1%)	0	1 (1.4%)	0	2 (2.6%)	0	0.260^b	NA
Induration	3 (4.3%)	0	3 (4.3%)	0	0	0	0.108^b	NA
Chills	3 (4.3%)	0	3 (4.3%)	0	0	0	0.108^b	NA
Gastrointestinal disorders (#of Pts)
Dyspepsia	4 (5.7%)	0	0	0	2 (2.6%)	0	0.427^b	NA
Epigastric discomfort	3 (4.3%)	0	0	0	3 (3.9%)	0	1.000^b	NA
Abdominal pain	4 (5.7%)	0	0	0	1 (1.3%)	0	0.194^b	NA
Abdominal pain upper	3 (4.3%)	0	0	0	1 (1.3%)	0	0.350^b	NA
Diarrhea	4 (5.7%)	0	0	0	0	0	0.050^b	NA
Musculoskeletal and connective tissue disorders (# of Pts)
Myalgia	20 (28.6%)	0	20 (28.6%)	0	0	0	<.001^a	NA
Pain in extremity	5 (7.1%)	0	2 (2.9%)	0	0	0	0.023^b	NA
Muscular weakness	3 (4.3%)	0	0	0	1 (1.3%)	0	0.350 ^b	NA
Investigations
AST increased	4 (5.7%)	3 (4.3%)	0	0	3 (3.9%)	1 (1.3%)	0.710^b	0.350^b
ALT increased	3 (4.3%)	1 (1.4%)	0	0	2 (2.6%)	1 (1.3%)	0.671^b	1.000^b
Metabolism and nutrition disorders
Hyperglycemia	9 (12.9%)	4 (5.7%)	0	0	5 (6.6%)	3 (3.9%)	0.198^a	0.710^b
Nervous system disorders
Headache	9 (12.9%)	1 (1.4%)	7 (10.0%)	0	1 (1.3%)	0	0.007^b	0.479^b
Infections and infestations
Upper resp. tract infect.	6 (8.6%)	0	1 (1.4%)	0	2 (2.6%)	0	0.153^b	NA
Influenza	3 (4.3%)	0	0	0	2 (2.6%)	1 (1.3%)	0.671^b	1.000^b
Respiratory, thoracic and mediastinal disorders
Cough	3 (4.3%)	0	0	0	3 (3.9%)	0	1.000^b	NA
Oropharyngeal pain	3 (4.3%)	0	1 (1.4%)	0	0	0	0.108^b	NA
Skin and subcutaneous tissue disorders
Pruritus	3 (4.3%)	0	0	0	2 (2.6%)	0	0.671^b	NA
Erythema	5 (7.1%)	0	4 (5.7%)	0	0	0	0.023^b	NA
Psychiatric disorders
Insomnia	3 (4.3%)	0	0	0	0	0	0.108^b	NA
Reproductive system and breast disorders
BPH	0	0	0	0	3 (3.9%)	0	0.246^b	NA

Abbreviations: AE, adverse events in control group; ALT, alanine aminotransferase; AST, aspartate aminotransferase; BPH, benign prostate hyperplasia; TEAE, treatment-emergent adverse events..

Note. Listed are adverse events, as defined by the National Cancer Institute Common Terminology Criteria (version 3.0), that were considered drug-related or that occurred in at least three patients in either study group regardless of relationship to drug. Data are expressed as number of patients and (%).

TEAE incidences and detailed number of TEAEs are summarized in Table S5.

^a By chi-square test.

^b By Fisher's exact test.

Figure 5. Baseline IL-15 level in the sera of healthy donors (HD) and patients with (Recur) or without (R-free) tumor-recurrence at the indicated time points after DC vaccination in mixed group patients (A) and resected patients (B). IL-15 levels were assessed by Luminex Bead-based Milliplex#x08E8; Multiplex Assay system (Millipore, St Charles, MO, USA). Assay sensitivity of the system; minimum detectable concentration was 0.6 pg/mL at a short protocol (n = 4 assays). Precision; intra-assay % coefficient of variance (CV) was 6.7 and extra-assay %CV was 9.5. Accuracy; 100.5 at spike recovery in serum matrix (6 Point Spikes). Statistical analysis was performed by unpaired t-test with the number (n) of patients: *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. (C) Baseline IL-15 level and RFS after DC immunotherapy. Twenty-eight patients from the DC vaccination group were assessed. IL-15(hi) and IL-15(lo) represent patients with baseline IL-15 >0.6 pg/mL (higher than minimal detectable concentration) and baseline IL-15<0.6 pg/mL (lower than minimal detectable concentration), respectively. HRs and p values were represented.