Serological landscape of cytokines in cutaneous melanoma

Abstract

BACKGROUND:

To date, serological markers to monitor melanoma progression and response to therapy are lacking. In this context cytokines appear to be promising biomarkers of the disease.

OBJECTIVE:

To compare cytokine and chemokine levels in melanoma patients and in healthy controls and to assess possible variations according to melanoma stage.

METHODS:

Serum chemokine and cytokine levels were determined by ELISA in 34 patients diagnosed histologically of malignant melanoma. Seven healthy volunteers were used as controls.

RESULTS:

We found a subset of cytokines (CCL3, CCL4, IFN- $\gamma$ and IL-10) to be significantly higher in melanoma patients than in control group, thus confirming the importance of the inflammation in cancer. While CCL3 increased with tumor progression, IFN- $\gamma$ and IL-10 showed higher levels in stage I patients. Moreover, we noticed a direct correlation between CCL3 level and the presence of ulceration in the primary tumor; on the contrary, CCL4, IL-10 and IFN- $\gamma$ were lowered down in patients with ulcerated melanoma.

CONCLUSIONS:

These results expand and confirm observations made in other studies focusing on a more limited number of molecules. This extended panel of cytokines examines the potential roles of type2 cytokines (such as IL-4) and many chemokines (mainly CCL3) as biomarkers in melanoma progression.

1. Introduction

Melanoma is one of the most aggressive forms of human cancer and its incidence has significantly increased worldwide over the last decades [1, 2, 3]. Every year, more than 100,000 melanomas occur worldwide. The lifetime risk of developing a melanoma is higher for whites than for other ethnic groups: nearly 2.4% for caucasians, 0.1% for blacks, and 0.5% for Hispanics [4, 5]. Intermittent UV exposure, especially during childhood, personal or family history of melanoma, presence of numerous melanocytic nevi and skin phototype I and II are important risks factors [6, 7, 8]. While surgery is the mainstay of therapy for localized disease, generalized forms of the disease have a poorer prognosis and were once considered incurable [9, 10, 11]. Melanoma staging has recently been revised by the American Joint Committee on Cancer [12, 13]. The most reliable outcome predictors, apart from the stage, are in fact several clinical and histological features of the primary tumor, which include not only the Breslow thickness and the presence of ulceration, but also the mitotic rate and the peri-tumoral inflammatory infiltrate [14, 15, 16]. Many authors characterized the peritumoral inflammatory infiltrate with a particular attention to tumor-infiltrating lymphocytes (TILs) [17, 18, 19, 20, 21]. Host-immune response evasion plays a key role in tumor growth and metastatization, as also confirmed by many studies both in animal models and in humans, with all the tested vaccines promoting an antigen-specific anti-tumor T-cell response [22, 23, 24, 25, 26, 27]. Patients with metastatic disease experience profound systemic immunosuppression due to tumor burden, documented to be IL-2 dependent [28, 29, 30]. Global cytokine suppression in the host, together with a selective increase in specific chemokines, constitute a predominant feature of certain melanoma metastatic localizations, such as uveal, lung or brain [31, 32, 33, 34, 35].

Nowadays new therapies boosting the anti-tumor immune response are available both as adjuvant therapies for stage III and as systemic treatments for stage IV disease [36, 37, 38]. These therapies demonstrated higher efficacy than traditional chemotherapy and are today considered first-choice treatments. Efficacy of those treatments (PD1- and CTLA4-inhibitors in particular) gives reason to the central role of the immune system in tumor growth and metastasis, meaning that serological tumor markers should be revisited [39, 40, 41]. Serological markers have not widely been introduced yet in routine clinical practice due to their insufficient diagnostic sensitivity and specificity [42, 43, 44]. Many studies identified possible cytokines subset to use as biomarkers, but they still are not routinely used [45, 46, 47]. In melanoma patients, cytokine profiles seem to be directly related to clinical course and a cytokine imbalance could be associated to tumor progression [48, 49]. However, no validated immune markers are today available so far for prognostic setting and monitoring of patients affected by melanoma [50, 51, 52]. We took advantage of serum-saving pre-arranged multiplexed arrays in order to explore the immunological landscape of cytokines with inflammatory and/or regulatory activities, with particular attention to chemokines, in melanoma patients at different stages.

2. Methods

We enrolled 34 patients affected by melanoma, referred to the “SS. Annunziata” dermatology clinic (Chieti, Italy) between 2001 and 2017, and 7 healthy controls (HC) in our study (for patient and control characteristics, see Table 1).

Table 1

Stage, sex and age distribution of the subjects enrolled in the study

Stage	Patients			Age: mean $\pm$ SD (range)
	( $n$ ${}^{\circ }$ )
	TOT	F	M	F	M
I	10	5	5	39.8 $\pm$ 11.25 (29–52)	43 $\pm$ 13.5 (31–64)
II	12	9	3	48.7 $\pm$ 13.3 (27–63)	60 $\pm$ 12.1 (47–71)
III	6	2	4	49.5 $\pm$ 9.1 (43–56)	54 $\pm$ 19 (33–73)
IV	6	3	3	50.6 $\pm$ 22.5 (29–74)	65 $\pm$ 6.8 (60–73)
0 (HC)	7	4	3	51.5 $\pm$ 7.5 (43–60)	44 $\pm$ 15 (30–60)

HC: healthy controls; SD: standard deviation.

HC included 4 women and 3 men (M:F ratio 1:1.3), with a mean age of 48. Patients were stratified according to the 8th AJCC classification (American Joint Committee on Cancer Classification of cutaneous melanoma 2018) and included all the possible disease stages (stage I, II, III and IV). The study and consent procedure were approved by the involved Hospital’s Ethics Committee. We obtained appropriate institutional review board approval and have followed the principles outlined in the Declaration of Helsinki for all human experimental investigations. Patients and healthy subjects have provided their written informed consent. For each subject, both serum cytokines and the tumor inflammatory infiltrate were examined. We measured serum levels of a total of 12 cytokines (8 of them belonging to the chemokine family) in 34 patients diagnosed histologically with malignant melanoma just after excision and staging, before the beginning of any systemic therapy, and in 7 HC. Measurements included interleukin (IL)-2, interferon (IFN)- $\gamma$ , IL-4 and IL-10, Chemokine C-C Motif Ligand 3, CCL3 (or Macrophage Inflammatory Protein (MIP)-1 $\alpha$ ), CCL4 (MIP-1 $\beta$ ), CCL2 (or Monocyte Chemotactic protein (MCP)-1), CCL17 (or Thymus and Activation Regulated Chemokine, TARC), CCL1 (or I-309), CCL11 (or eotaxin), CCL22 (or Macrophage Derived chemo-attractant, MDC) and Chemokine (C-X-C motif) Ligand 8, CXCL8 (or IL-8).

Table 2

Patient characteristics: age, sex, features of the primary tumor (Breslow thickness, ulceration, regression, inflammatory infiltrate), staging (TNM and AJCC)

Patient	Age	Sex	Breslow	Ulceration	Regression	Inflammatory	TNM	Stage
			thickness			infiltrate
1	47	M	1.75 mm	Present	Absent	Present: brisk type	T2bN0M0	IIA
2	52	F	1.8 mm	Present	Absent	Present: brisk type	T2bN0M0	IIA
3	62	M	2.5 mm	Absent	Present	Absent	T3aN0M0	IIA
4	31	F	1.3 mm	Present	Absent	Absent	T2bN0M0	IIA
5	61	F	2.5 mm	Absent	Absent	Present: brisk type	T3aN0M0	IIA
6	63	F	1.8 mm	Present	Absent	Absent	T2bN0M0	IIA
7	56	F	3.5 mm	Present	Absent	Absent	T3bN2bM0	IIIB
8	63	F	2 mm	Present	Absent	Present: brisk type	T2bN0M0	IIA
9	33	M	3.8 mm	Absent	Absent	Absent	T3aN2bM0	IIIB
10	44	F	1.1 mm	Present	Present	Present: brisk type	T2bN0M0	IIA
11	43	F	3.4 mm	Present	Absent	Present: brisk type	T3bN2aM0	IIIC
12	73	M	3 mm	Present	Present	Absent	T3bN1aM0	IIIA
13	71	M	1.9 mm	Present	Absent	Present: band like type	T2bN0MO	IIA
14	54	F	1.5 mm	Present	Absent	Present: brisk type	T2bN0M0	IIA
15	33	F	0.8 mm	Absent	Absent	Absent	T1bN0M0	IB
16	64	M	1.1 mm	Absent	Absent	Absent	T2aN0M0	IB
17	43	M	4.8 mm	Present	Absent	Absent	T4bN2bM0	IIIC
18	52	F	0.36 mm	Absent	Absent	Absent	T1aN0M0	IA
19	34	M	0.31 mm	Absent	Absent	Present band like type	T1aN0M0	IA
20	60	M	8 mm	Present	Present	Present: brisk type	T4bN0M1b	IV
21	74	F	2 mm	Absent	Absent	Absent	T2aN2aM1c	IV
22	27	F	1.75	Present	Absent	Absent	T2bN0M0	IIA
23	31	M	0.60 mm	Absent	Present	Absent	T1aN0M0	IA
24	73	M	1.7 mm	Present	Absent	Present: brisk type	T2bN0M1c	IV
25	33	F	1.6 mm	Absent	Absent	Present: brisk type	T2aN0M0	IB
26	63	M	6 mm	Absent	Absent	Absent	T4aN2bM1b	IV
27	67	M	2 mm	Absent	Absent	Present: brisk type	T2aN1bM0	IIIB
28	49	F	5 mm	Absent	Absent	Present: band like type	T4aN0M1c	IV
29	36	M	0.30 mm	Absent	Present	Absent	T1aN0M0	IA
30	50	M	0.68 mm	Absent	Absent	Absent	T1aN0M0	IA
31	52	F	0.50 mm	Absent	Absent	Present: brisk type	T1aN0M0	IA
32	44	F	2.2 mm	Absent	Absent	Present: brisk type	T3aN0M0	IIA
33	29	F	1.6 mm	Absent	Absent	Absent	T2aN2bM1c	IV
34	29	F	0.6 mm	Absent	Present	Present: band like type	T1aN0M0	IA

Table 3

Cytokines serum levels expressed in pg/ml in patients at different stages of melanoma as median and IQR

	Stage
	I ( $n=$ 10)		II ( $n=$ 12)		III ( $n=$ 6)		IV ( $n=$ 6)		$p$ -value ${}^{\text{a}}$
CCL3	173.4	(159.0–246.6) pg/ml	297.5	(253.3–368.0) pg/ml	330.0	(272.0–438.0) pg/ml	222.0	(175.6–256.3) pg/ml	0.001
CCL4	130.7	(85.0–168.3) pg/ml	89.5	(70–110.4) pg/ml	143.0	(61.3–210.2) pg/ml	199.4	(171.3–297.5) pg/ml	0.011
CCL2	344.1	(208.9–875.5) pg/ml	447.5	(317.1–632.3) pg/ml	544.0	(363.7–728.3) pg/ml	595.6	(455.0–1322.2) pg/ml	0.378
CCL1	350.2	(293.7–618.9) pg/ml	284.0	(146.3–377.0) pg/ml	267.0	(165.0–526.5) pg/ml	466.2	(356.7–631.3) pg/ml	0.084
CCL17	95.25	(63.0–252.3) pg/ml	88.0	(56.5–293.8) pg/ml	207.4	(68.5–634.7) pg/ml	211.4	(101.8–338.3) pg/ml	0.740
CCL11	296.8	(242.0–659.8) pg/ml	313.5	(253.3–453.8) pg/ml	267	(204.5–443.5) pg/ml	359.8	(278.2–514.7) pg/ml	0.698
CCL22	461.9	(313.9–927.2) pg/ml	753.2	(547.5–925.8) pg/ml	729	(575.5–887.4) pg/ml	739.9	(569.5–895.6) pg/ml	0.347
IL-8	10.6	(6.8–28.2) pg/ml	10.0	(7.0–12.1) pg/ml	13.5	(5.5–24.9) pg/ml	29.8	(9.9–99.2) pg/ml	0.162
IL-2	51.5	(28.1–89.5) pg/ml	37.6	(23.0–58.7) pg/ml	61.5	(26.4–72.9) pg/ml	38.7	(24.8–64.2) pg/ml	0.339
IFN $\gamma$	59.5	(42.5–127.5) pg/ml	8.0	(4.0–11.1) pg/ml	11.5	(3.9–46.9) pg/ml	28.5	(3.8–71.0) pg/ml	0.003
IL-4	28.15	(2.4–61.4) pg/ml	6.0	(5.0–8.0) pg/ml	10.0	(3.1–17.1) pg/ml	1.3	(0.0–14.6) pg/ml	0.098
IL-10	29.4	(5.6–73.7)	3.0	(2.3–7.0)	6.0	(1.5–22.1)	2.8	(1.1–6.6)	0.004

a: Kruskall-Wallis test. MIP-1 $\alpha$ , MIP-1 $\beta$ , IFN- $\gamma$ , and IL-10 are significantly higher in melanoma patients compared to HC. A statistically significant increase in MIP-1 $\alpha$ , and a concomitant decrease in IFN- $\gamma$ , and IL-10 levels is evident between stage I and II; MIP-1 $\beta$ is particularly elevated in stage IV patients. IL-4 tends to decrease with disease progression, while IL-8 increases. IL-2 is typically very low in the stage IV group. However, data regarding IL-2, IL-4 and IL-8 are not statistically significant.

Figure 1.

CCL3, CCL4, IFN- $\gamma$ , and IL-10 serum levels (expressed in pg/ml) in melanoma patients (MM) compared to HC. All those cytokines showed significantly higher levels in MM when compared to HC. Kruskall-Wallis test was used to determine differences among different groups.

Figure 2.

Correlations between CCL3, CCL4, IFN- $\gamma$ , IL-10 levels (expressed in pg/ml) and melanoma stages. Panel a: CCL3 increases from stage I to stages II–III. Panel b: CCL4 is higher in stage IV patients when compared to stage I–III. Panel c: IFN- $\gamma$ is significantly higher in stage I patients rather than stages II–IV. Panel d: also IL-10 levels are higher in stage I and decrease in more advanced stages. Kruskall-Wallis test was used to determine differences among different groups.

Serum cytokine and chemokine levels were determined by enhanced ELISA-type assays, namely multiplex and protein microarray methods (Pierce Searchlight Proteome Arrays, Search Light Human Th1/Th2 Array 1 and 2, Boston, MA; Human Chemokine array 1, Pierce-Endogen, Rockford, IL, USA). Samples were processed according to the manufacturer’s instructions. Chemiluminescence was detected with an Image and Analysis System, equipped with Array Vision software and a cooled CCD camera (Perbio Science GmbH, Bonn, Germany).

We correlated the serum levels with known parameters affecting clinical outcome, such as Breslow thickness, ulceration status, and lymphocytic infiltrate, as well as stage of disease.

Statistical analysis was performed by Kruskall-Wallis test, Mann-Withney U test and Spearman’s rank test. In particular, correlation between cytokines serum levels and melanoma stage was measured with Kruskall-Wallis test, while Mann-Withney U test was performed to asses correlation of cytokine levels with ulceration; Spearman’s rank test was used to analyze possible correlations among cytokine serum levels. Significance was defined as $P<$ 0.05 when comparing various groups or parameters.

Table 4

Serum levels of CCL4, CCL3, IFN- $\gamma$ and IL-10 expressed in pg/ml

	CCL4		CCL3		IFN- $\gamma$		IL-10
Stage I	111	.5	173	.2	122	.5	75	.1
	81	.2	251	.8	142	.6	87	.4
	101	.8	160	.3	60	.6	17	.7
	149	.9	202	.8	112	.6	70	.3
	154	.3	259	.8	43		5	.7
	166	.3	155	.1	53	.9	11	.3
	174	.1	173	.6	40	.9	5
	155	.2	244	.8	208	.6	41	.1
	86	.3	162		20	.1	5	.3
	56	.2	132	.5	58	.4	73	.2
Stage II	74		303		5		3
	49		376		12		8
	75		376		4		2
	203		344		11		4
	73		341		10		3
	104		386		4		2
	69		284		4		4
	53		292		4		3
	105	.3	206	.3	11	.1	2
	105		260		7		3
	112	.1	181	.1	77	.9	20	.5
	123		251		9		9
Stage III	147		603		10		5
	47		383		4		2
	66		317		17		7
	252	.3	248	.8	3	.5	0
	139		343		13		9
	196	.2	279	.8	136	.4	61	.2
Stage IV	272	.2	293	.2	85	.3	3	.5
	165		244		20	.6	2
	192	.4	220	.7	36	.4	1	.5
	173	.4	223	.2	3		0
	373	.2	186	.1	4	.1	3	.5
	206	.3	143	.9	66	.2	15	.7
Subtot overall M	165	.06	264	.76	41	.81	16	.6
HC	216		165		4		2
	18		123		4		2
	13		59		4		2
	86		161		4		2
	19		119		4		2
	10		121		4		2
	118		180		4		2
Subtot HC	125	.71	132	.57	4		2

M: melanoma patients; HC: healthy controls.

3. Results

Patients had a mean age of 49.9 $\pm$ 14.7 and a male:female ratio of 1:1.2. Mean Breslow thickness was 2.2 mm. Ulceration was present in 15 cases, none of them classified as stage I. Regression was present in 7 primary tumors and half of the cases showed peritumoral inflammatory infiltrate, 4 being band-like type and 13 brisk-type. Patient characteristics are summarized in Table 2.

Cytokine and chemokine levels both in patients affected by melanoma and in healthy controls were measured. Results were then divided according to disease stage. As shown in Table 3, only results regarding CCL3, CCL4, IFN- $\gamma$ and IL-10 were considered significant and mainly correlated with disease stage. However, a significant principal difference ( $p<$ 0.05) between all melanoma patients and healthy controls (HC) was found: all the four above-mentioned cytokines showed lower levels in the control group, thus confirming the prominent role of inflammation in cancer [53, 54, 55] (see Fig. 1 and Table 4).

All the significant correlations between these four cytokines and melanoma stages are shown in Fig. 2. A statistically significant increase in CCL3, and a concomitant decrease in IFN- $\gamma$ , and IL-10 levels has been noticed between stage I and II (see Fig. 2, panels a, c, d). Moreover, IFN- $\gamma$ and IL-10 levels continue to be significantly lower in stage III and IV, while CCL3 is persistently increased.

Patients with stage-I disease show IL-10 and IFN- $\gamma$ serum levels significantly higher when compared to healthy controls, while IFN- $\gamma$ and IL-10 decrease together with disease progression. CCL4 was particularly elevated in stage IV patients, both when compared to healthy controls and stage I-III (see Fig. 2, panel b).

As shown in Table 3, IL-4 tends to decrease with disease progression, while IL-8 (a chemokine well known to be correlated to a metastatic phenotype) increases [56, 57]. IL-2 is typically very low in the stage IV group. However, data regarding IL-2, IL-4 and IL-8 are not statistically significant.

Table 5

Cytokines serum levels in pg/ml in patients with and without ulceration as median and IQR (interquartile range)

	Ulceration
	Absent ( $n=$ 19)		Present ( $n=$ 15)		$p$ -value ${}^{\text{a}}$
CCL3	202.8	(162.0–251.8)	317.0	(284.0–376.0)	0.001
CCL4	149.9	(105.0–192.4)	75.0	(66.0–165.0)	0.043
CCL2	471.0	(313.1–573.6)	618.0	(329.0–852.1)	0.242
CCL1	380.3	(294.3–470.2)	251.0	(147.0–504.2)	0.066
CCL17	116.9	(71.6–246.3)	236.0	(58.0–294.0)	0.560
CCL11	304.9	(251.6–540.1)	278.0	(238.0–463.4)	0.372
CCL22	612.7	(412.6–829.6)	780.0	(616.0–971.0)	0.147
IL-8	11.0	(7.0–25.8)	11.0	(7.0–20.0)	0.632
IL-2	53.1	(27.6–84.0)	33.2	(23.0–64.0)	0.157
IFN- $\gamma$	53.9	(13.0–112.6)	10.0	(4.0–12.0)	0.001
IL-4	14.5	(2.0–35.3)	6.0	(4.0–9.0)	0.128
IL-10	11.3	(5.0–61.2)	3.0	(2.0–4.0)	0.001

a: Mann-Withney U test. MIP-1 $\alpha$ is significantly higher in patients with ulcerated melanomas, while MIP-1 $\beta$ , IL-10 and IFN- $\gamma$ are lowered down in this subgroup of patients.

Tumor thickness is certainly a major determinant in melanoma staging, especially for stage I and II, but ulceration plays also a key role in melanoma staging. Cytokine and chemokine levels were stratified according to the presence or absence of ulceration (see Table 2). None of the 8 patients with T1 (Breslow $⩽$ 1 mm) had histologically proven ulceration, while in T2 group (Breslow thickness between 1.1 and 2 mm), 9 patients out of 14 had ulcerated melanomas. Tumor thickness and ulceration are in fact two highly-associated variables: none of the patients with a melanoma $⩽$ 1 mm had ulceration, while the majority of tumors $>$ 1 mm was ulcerated. It is therefore quite impossible to evaluate the statistical significance of the two isolated variables. However, as shown in Table 5, CCL3 is significantly higher in patients with ulcerated melanomas, while CCL4, IL-10 and IFN- $\gamma$ are lowered down in this subgroup of patients.

Table 6

Correlation among cytokine serum levels

	Rho		$p$ -value ${}^{\text{a}}$
CCL3
CCL1	$-$	0.399		0.019
IFN $\gamma$	$-$	0.478		0.004
IL-10	$-$	0.378		0.028
CCL4
CCL2		0.422		0.013
CCL1		0.607	$<$	0.001
TARC		0.502		0.003
CCL11		0.536		0.001
CCL22		0.415		0.015
IL-8		0.657	$<$	0.001
CCL2
CCL4		0.422		0.013
TARC		0.398		0.020
CCL1
CCL3	$-$	0.399		0.019
CCL4		0.607	$<$	0.001
CCL17		0.432		0.011
CCL11		0.588	$<$	0.001
IL-8		0.780	$<$	0.001
CCL17
CCL4		0.502		0.003
CCL2		0.398		0.020
CCL1		0.432		0.011
CCL11		0.608	$<$	0.001
CCL22		0.598	$<$	0.001
IL-8		0.573	$<$	0.001
CCL11
CCL4		0.536		0.001
CCL1		0.588	$<$	0.001
CCL17		0.608	$<$	0.001
CCL22		0.387		0.024
IL-8		0.627	$<$	0.001
CCL22
CCL4		0.415		0.015
CCL17		0.598	$<$	0.001
CCL11		0.387		0.024
IL-8		0.384		0.025

Table 6, continued
	Rho		$p$ -value ${}^{\text{a}}$
IL-8
CCL3		0.657	$<$	0.001
CCL1		0.780	$<$	0.001
CCL17		0.573	$<$	0.001
CCL11		0.627	$<$	0.001
CCL22		0.384		0.025
IL-2
IFN $\gamma$		0.658	$<$	0.001
IL-4		0.652	$<$	0.001
IL-10		0.686	$<$	0.001
IFN $\gamma$
CCL3	$-$	0.478		0.004
IL-2		0.658	$<$	0.001
IL-4		0.705	$<$	0.001
IL-10		0.784	$<$	0.001
IL-4
IL-2		0.652	$<$	0.001
IFN $\gamma$		0.705	$<$	0.001
IL-10		0.818	$<$	0.001
IL-10
CCL3	$-$	0.378		0.028
IL-2		0.686	$<$	0.001
IFN $\gamma$		0.784	$<$	0.001
IL-4		0.818	$<$	0.001

a: Spearman’s rank test. An inverse correlation between MIP-1 $\alpha$ and IL-10 and/or IFN- $\gamma$ is present: IL-10 and IFN- $\gamma$ higher in stage I and slowly decrease in parallel to MIP-1 $\alpha$ increase.

Trends of different cytokines in patients affected by melanoma are strictly correlated one to each other. Table 6 summarizes all the statistically significant correlations among cytokines. It’s important to underline that our results confirm the inverse correlation between CCL3 and IL-10 and/or IFN- $\gamma$ that could be hypothesized from our data regarding melanoma stages. In fact, while CCL3 increases with disease progression from stage I to stage IV, IL-10 and IFN- $\gamma$ appear to be higher in stage I, and slowly decrease in parallel to CCL3 build up.

4. Discussion

Several studies in the past 15 years have concluded that multiplexed analysis of serum biomarkers may be useful for the evaluation of clinical outcome as well as potentially predictive of response to therapy [58, 59, 60]. However, the panels investigated varied markedly and the cytokine markers indicated differed among the studies, most of them evaluating longitudinal changes after treatment in just one or few molecules [61, 62, 63, 64]. An exception is represented by Neagu and coworkers who analyzed up to 6 cytokines in over 100 cases followed for up to 3 years [65].

Of the 12 analyzed in our study, 4 cytokines (IL-10, IFN- $\gamma$ , CCL3 and CCL4) were demonstrated to be significantly higher in melanoma patients when compared to HC. In particular, IL-10 levels, higher in melanoma patients than in HC, confirmed its role in the evasion of immune-surveillance [66, 67, 68, 69]. IFN- $\gamma$ decrease from stage I to stages II–IV reflects the importance of interferons in boosting the anti-tumor immune response [70, 71, 72]. Further studies are needed to assess whether both type I and type II endogenous interferons decrease together with disease progression and to justify the use of IFN-a as an adjuvant therapy for high-risk patients [73]. CCL-3 and CCL-4, also known as MIP-1 $\alpha$ and MIP-1 $\beta$ , are monokines with inflammatory and chemotactic properties [74]. They are generally produced by innate immunity cells, but CD8 $+$ T-cells can as well produce them as they were first discovered as HIV-suppressive factors [75]. Their role in cancer and in the anti-tumor response is still not well-known [76, 77].

IL2 and IL4 levels tend to decrease with disease progression. Despite not statistically significant, those results reflect the reduction in the anti-tumor T-cell response with disease progression, both Th1 and Th2 [57, 78]. IL8 is a chemokine well-known to be related to a metastatic phenotype: despite not statistically significant, it is increased in stage IV-patient sera [79, 80, 81].

Our results expand observations made in several previous studies which focused on a more limited number of molecules, and for those already reported confirms some observations (as for IFN- $\gamma$ and IL-8) regarding changes in different stages [82, 83]. This extended panel of cytokines examines for the first time the potential roles of type2 cytokines (such as IL-4) and many chemokines (mainly CCL3) in melanoma progression and for the assessment of the patients’ immune status. Data regarding MIP are of particular interest: if the $\beta$ version attracts mainly NK and monocytes, the $\alpha$ form attracts mainly neutrophils, our results emphasizing thus on the intense inflammatory status of the patients. Since the main limit of the study is probably the small number of patients screened, we strongly encourage the use of this particular panel of cytokines in a larger population.

More data are needed to confirm the trends of mean cytokine and chemokine levels observed among different disease stages and to better define cytokine role in cancer development and spreading [84].

Footnotes

Conflict of interest

none.

Supplementary materials

Supplementary Fig. 1.

Dot plot showing CCL4, CCL3, IFN- $\gamma$ and IL-10 serum levels of the single subjects enrolled in the study (measured in pg/ml).