Soluble NKG2D ligands are biomarkers associated with the clinical outcome to immune checkpoint blockade therapy of metastatic melanoma patients

Figures & data

Table 1. Clinical-pathological features of melanoma patients treated with immune checkpoint blockade agents or with standard/targeted therapies.

	Control cohort(N = 65)		Immune checkpoint cohort(N = 162)		Immune checkpoint plus chemotherapy cohort ^a(N = 37)
Parameter	N	%	N	%	N	%
Men	42	65	92	57	24	65
Women	23	35	70	43	13	35
Median age (years)	59		62		55
PS
0	24	36.9	42	25.9	33	89.2
1	38	58.5	118	72.8	4	10.8
2	3	4.6	2	1.2	0	0
Median LDH	268		290		332
Pathological type
Cutaneous melanoma	59	91	137	85	37	100
Ocular melanoma	0	0	9	6	0	0
Mucosal melanoma	2	3	12	7	0	0
Acral melanoma	4	6	4	2	0	0
Clinical stage
III	12	18	7	4	1	3
IV	53	82	155	96	36	97
Therapeutic treatment
ipilimumab	0	0	132	81	0	0
ipilimumab + pembrolizumab	0	0	15	9	0	0
Nivolumab, Ipilimumab monotherapy or combination	0	0	15	9	0	0
Ipilimumab plus chemotherapy	0	0	0	0	37	100
Standard therapies alone	31	48	0	0	0	0
BRAF inhibitors	34	52	0	0	0	0
Clinical responses
DCR	29	59	64	40	24	65
Median OS (95% C.I.)	13 (7.7–18.3)		13.8 (10.0–17.6)		24.3 (18.0–29.7)

^a : Patients were treated with the combination of ipilimumab plus fotemustine; see Ref. [46];

PS: Performance status;

LDH: Lactate dehydrogenase

DCR: disease control rate which includes complete responses (CR), partial responses (PR), and stable disease (SD) according the immune-related response criteria assessment; see Refs. [45],[46].

Figure 1. sNKG2DLs in the serum of melanoma patients treated with immune checkpoint blockade agents. The presence of sMICA (Panel A), sMICB (Panel B), and sULBP-2, 3 (Panels D and E) in the serum at baseline (W1; black circle) and post-treatment (W12; black square) of melanoma patients (N = 162) treated with immune checkpoint blockade agents was measured by ELISA assay (see Material and methods). ULBP-1 determinations at pre- and post-treatment were performed in N = 131 and 128 patients, respectively (Panel C). Mean and error bars are shown in the graphs. As negative control the serum of N = 10 HD was used in ELISA assays (data not shown; see Ref. [25]).

Figure 2. Detection of sNKG2DLs in the serum of control group melanoma patients. The presence of soluble NKG2DLs (MICA, Panel A; MICB, Panel B; ULBP-1, Panel C; ULBP-2, Panel D; ULBP-3, Panel E) was measured by ELISA assay (see Material and methods) in the baseline serum (W1; black circle) of melanoma patients (N = 65) treated with either standard or BRAFi therapies. Mean and error bars are shown in the graphs. As negative control the serum of N = 10 HD was used in ELISA assays (data not shown; see Ref. [25]).

Table 2. Association between the levels at baseline serum of sNKG2DLs and the disease control of melanoma patients.

sNKG2DLs ^a		Immune checkpoint treatment ^b	DCR ^c	p ^d	Control ^e	DCR	p
MICA	+	22	45.7	0.99	16	32.5	0.24
	−	171	45.6		42	45.2
MICB	+	42	42.9	0.72	19	36.8	0.16
	−	151	46		39	56.4
ULBP-1	+	85	32.9	0.002	35	54.3	0.42
	−	77	57.1		23	43.5
ULBP-2	+	45	44.4	0.86	9	44.4	0.72
	−	148	45.9		49	51
ULBP-3	+	77	49.4	0.39	23	43.5	0.42
	−	116	43.1		35	54.3

^a : detection of sNKG2DLs (MICA, MICB, ULBP-1,2,3) in the pre-treatment serum of melanoma patients; positive (+) or negative (−) measurement (ng/mL) of sNKG2DLs in the patients' serum;

^b : Number of subjects from the cohort of patients treated with immune checkpoint blockade agents;

^c : disease control rate (DCR) expressed as the percentage of patients with complete responses (CR), partial responses (PR), and stable disease (SD) according the immune-related response criteria assessment; see Refs. [45],[46];

^d : p value obtained from chi-squared test analysis;

^e : number of patients from the control cohort.

Table 3. Association between the levels at baseline serum of sNKG2DLs and the OS of melanoma patients.

	sNKG2DLs ^a		Immune checkpoint treatment ^b	OS ^c	p ^d	Control ^e	OS	p
Baseline
	MICA	+	22	12.5	0.45	19	15.6	0.94
		−	172	16.4		46	12.0
	MICB	+	43	8.8	0.02	25	13	0.4
		−	151	21.6		40	12
	ULBP-1	+	85	12.1	0.01	36	15.6	0.85
		−	78	25.3		29	8.5
	ULBP-2	+	45	9.8	0.11	9	15.6	0.77
		−	149	20.2		56	12
	ULBP-3	+	77	13.8	0.10	25	16	0.65
		−	117	16.7		40	11.2
W12
	MICA	+	22	12.4	0.02	11f	11.1	0.18
		−	159	20.2		13	n.r.
	MICB	+	54	10.4	0.01	11	n.r.	0.05
		−	127	22.8		12	11.0
	ULBP-1	+	80	14.7	0.17	13	11.1	0.14
		−	70	26.3		10	n.r.
	ULBP-2	+	49	14.7	0.27	5	15.6	0.82
		−	132	21.6		12	24.3
	ULBP-3	+	70	16.8	0.57	5	n.r.	0.49
		−	106	16.7		18	13.1

^a : detection of sNKG2DLs (MICA, MICB, ULBP-1, 2, 3) in the pre- (baseline) or post- (W12) treatment serum of melanoma patients; positive (+) or negative (−) measurement (ng/mL) of sNKG2DLs are indicated;

^b : number of subjects from the cohort of patients treated with immune checkpoint blockade agents;

^c : OS: median overall survival expressed as months;

^d : p value obtained from log-rank test analysis;

^e : Number of patients from the control cohort.

^f : W12 post-treatment data from melanoma patients treated with BRAFi; n.r. = not reached. Post-treatment from patients treated with standard therapies was not available.

Figure 3. Overall survival of melanoma patients treated with immunotherapy in relation with the presence or not of sNKG2DLs in serum. Kaplan–Meier plots of overall survival of melanoma patients treated with immune checkpoint blockade agents (Panels A and B) or with standard or BRAFi therapies (Panels C and D) in relation with the detection (black line) or not (dotted line) at baseline of sMICB (Panels A and C) and sULBP-1 (Panels B and D). The baseline serum levels of sMICB (Panel A) and ULBP-1 (Panel B) could discriminate melanoma patients with long-term survival (median OS = 21.6 and 25.3 mo, p = 0.02 and 0.01, respectively) from poor survivors (median OS = 8.8 and 12.1 mo, respectively) for the cohort of patients treated with immune checkpoint blockade agents. Panels C and D show the absence of association between the serum levels of these ligands and OS in the control group of patients (median OS = 12.0 vs. 13.1 and 8.5 vs.15.6 and p = 0.4 and 0.85, respectively).

Figure 4. Overall survival of melanoma patients treated with immunotherapy in association with the levels of sNKG2DLs in post-treatment serum. The absence (dotted line) in the post-treatment serum of sMICA (Panel A) and sMICB (Panel B) correlated with improved OS (median OS = 20.2 and 22.8 vs. 10.4 mo, p = 0.02 and 0.01, respectively) while detectable levels of these molecules were found in the serum of poor survivor patients (median OS = 12.4 and 10.4 mo, respectively) (black line).

Table 4. COX regression analysis to identify biomarkers influencing the clinical outcome of melanoma patients.

	Patient
	Immune checkpoint cohort				Control cohort
	Univariate analysis of factor		Multivariate analysis of factor		Univariate analysis of factor		Multivariate analysis of factor
Marker	HR (CI 95%)	p	HR (CI 95%)	p	HR (CI 95%)	p	HR (CI 95%)	p
Gender	0.96 (0.69–1.33)	0.80			1.26 (0.67–2.38)	0.47
Age	1.00 (0.99–1.01)	0.39			1.02 (0.99–1.04)	0.16	1.03 (1.01–1.05)	0.02
LDH	1.01 (1.01–1.02)	<0.0001	1.02 (1.01–1.03)	<0.0001	1.01 (1.01–1.02)	<0.0001	1.02 (1.01–1.03)	<0.0001
PS	1.25 (0.90–1.72)	0.18			2.02 (1.07–3.82)	0.03
Stage	2.63 (0.87–8.26)	0.10			6.20 (1.50–25.62)	0.01
MICA	1.25 (0.70–2.24)	0.45			0.97 (0.50–1.91)	0.94
MICB	1.67 (1.08–2.59)	0.02			0.75 (0.38–1.47)	0.40
ULBP-1	1.78 (1.14–2.77)	0.01	1.72 (1.09–2.71)	0.02	0.94 (0.50–1.76)	0.84
ULBP-2	1.42 (0.92–2.19)	0.11	1.91 (1.10–3.33)	0.02	0.88 (0.37–2.10)	0.77
ULBP-3	1.38 (0.94–2.04)	0.10	1.38 (0.94–2.04)	0.10	0.87 (0.47–1.61)	0.65

The markers were determined as the following: gender: male vs. female; LDH was categorized based on increment of 10 IU/L; PS: 1, 2 vs. 0; Stage: IV vs. III; MICA, MICB, ULBP-1, -2, and -3: positive detection vs. absence in the serum.

Di Giacomo AM, Ascierto PA, Pilla L, Santinami M, Ferrucci PF, Giannarelli D, Marasco A, Rivoltini L, Simeone E, Nicoletti SV et al. Ipilimumab and fotemustine in patients with advanced melanoma (NIBIT-M1): An open-label, single-arm phase 2 trial. Lancet Oncol 2012; 13(9):879-86; PMID: 22894884; https://doi.org/10.1016/S1470-2045(12)70324-8

 PubMed Web of Science ®Google Scholar

Wolchok JD, Hoos A, O'Day S, Weber JS, Hamid O, Lebbé C, Maio M, Binder M, Bohnsack O, Nichol G et al. Guidelines for the evaluation of immune therapy activity in solid tumors: Immune-related response criteria. Clin Cancer Res 2009; 15(23):7412-20; PMID: 19934295; https://doi.org/10.1158/1078-0432.CCR-09-1624

 PubMed Web of Science ®Google Scholar

Maccalli C, Giannarelli D, Capocefalo F, Pilla L, Fonsatti E, Di Giacomo AM, Parmiani G, Maio M. Immunological markers and clinical outcome of advanced melanoma patients receiving ipilimumab plus fotemustine in the NIBIT-M1 study. Oncoimmunology 2016; 5(2):e1071007; PMID: 27057436; https://doi.org/10.1080/2162402X.2015.1071007

 PubMed Web of Science ®Google Scholar