Increased CCL17 serum levels are associated with ... - Springer Link

Cancer Immunol Immunother (2015) 64:1075–1082 DOI 10.1007/s00262-015-1714-4

ORIGINAL ARTICLE

Increased CCL17 serum levels are associated with improved survival in advanced melanoma Benjamin Weide1,2 · Nicolas Allgaier3 · Andreas Hector3 · Andrea Forschner2 · Ulrike Leiter2 · Thomas K. Eigentler2 · Claus Garbe2 · Dominik Hartl3

Received: 16 February 2015 / Accepted: 8 May 2015 / Published online: 20 May 2015 © Springer-Verlag Berlin Heidelberg 2015

Abstract Background Prognostic factors of melanoma patients with distant metastases remain poorly established. This study aimed to compare the prognostic impact of putative serum biomarkers, namely S100B, YKL-40 or CCL17, in stage IV melanoma patients. Patients and methods Serum concentrations were analyzed by ELISA. Disease-specific survival of 80 patients according to S100B, YKL-40 or CCL17 and clinical factors were calculated by univariate Kaplan–Meier survival and multivariate analysis. Results Low serum levels of S100B, high concentrations of CCL17 and female gender correlated with improved survival. A trend for favorable prognosis was observed for the M categories M1a/b versus M1c according to the AJCC classification. No correlation with survival was evident for YKL-40 serum levels and age. In multivariate analysis, S100B (HR 2.1; p = 0.005) and CCL17 (HR 1.8; p = 0.029) had independent prognostic impact. Patients with a combination of normal S100B and high CCL17 had a high chance for long-term survival, which was 43 % after 3 years. Conclusion Serum levels of CCL17 and S100B represent independent prognostic markers for melanoma patients with distant metastases. These biomarkers were more

* Dominik Hartl [email protected]‑tuebingen.de 1

Department of Immunology, University of Tübingen, Tübingen, Germany

2

Department of Dermatology, University of Tübingen, Tübingen, Germany

3

Department of Pediatrics I, University of Tübingen, Hoppe‑Seyler‑Strasse 1, 72076 Tübingen, Germany

powerful than the M category according to the AJCC classification to indicate overall survival. CCL17 represents a promising biomarker upon immune checkpoint blockade in melanoma. Keywords Advanced melanoma · Prognosis · Biomarker · S100B · YKL-40 · CCL17 Abbreviations AJCC American Joint Committee on Cancer CCL17 Chemokine (C–C motif) ligand 17 CCR4 C–C chemokine receptor type 4 CMMR Central Malignant Melanoma Registry IQR Inter-quartile range Tregs Regulatory T cells LDH Lactate dehydrogenase

Introduction Serum lactate dehydrogenase (LDH) is the most widely used prognostic biomarker in melanoma and was incorporated in the American Joint Committee on Cancer (AJCC) staging system for melanoma patients with distant metastases since 2001 [1]. LDH has been identified as an independent prognostic factor before for patients with unresectable disease by different research groups [2–4]. Moreover, serum concentrations of S100B are widely used in Europe to screen patients without evidence of disease to detect recurrences early [5]. A meta-analysis by Mocellin et al. [6] summarized the evidence on the suitability of serum S100B to predict patients’ survival. Twenty-two series enrolling 3393 patients comprising all stages were included in this analysis. Serum S100B positivity was associated with significantly poorer survival in patients of all stages as well

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as in the subgroup of stage I to stage III patients (hazard ratio 2.28; p ULN) were detected by the Elecsys S100 electrochemiluminescence immunoassay (Roche Diagnostics AG, Rotkreuz, Switzerland) according to the instructions of the manufacturers. YKL-40 and CCL17 serum levels were quantified in triplicates using commercial ELISA kits (R&D systems). Statistical analysis Serum levels of YKL-40 or CCL17 were dichotomized according to the median serum level of each protein and age according to the median age of patients. S100B was categorized as elevated for patients with measured levels above the upper limit of normal (0.10 µg/l) versus normal. Follow-up time was defined from the date of blood draw to the date of last follow-up or death. Estimates of cumulative survival probabilities according to Kaplan–Meier were described together with 95 % confidence intervals (CIs) and compared using two-sided log-rank test statistics. Median survival times (MSTs) are presented. For the analysis of overall survival, patients who were alive at the last follow-up were censored, while patients who had died were considered an “event”. Multivariable Cox proportional hazard models were used to determine independent prognostic factors. Patients with missing values were excluded from multivariate testing. Results of the Cox models were described by means of hazard ratios, and p values were based on the Wald test. All statistical analyses were carried out using the SPSS version 20 (IBM SPSS, Chicago, Illinois, USA).

Results Patients’ characteristics are shown in Table 1. A total of 80 stage IV melanoma patients (67.5 % male) were analyzed. The median age was 59 years [inter-quartile range (IQR) 51–70 years]. The median follow-up for patients who died was 8 months and 46 months for patients who were censored. The median survival time according to

1

1.8 0.020

29.7 % (15.0 %; 44.4 %) Significant p values are in bold

15.4 % (4.0 %; 26.8 %) 38.5 % (23.2 %; 53.8 %)

56.8 % (40.9 %; 72.7 %) 15

8 87.2

73.0 48.7

51.3 39

>278 pg/ml

≤278 pg/ml CCL17

Survival analysis according to Kaplan–Meier

37

0.260

0.029

0.449

0.005

1 2.2 1 1.2 0.1 µg/l ≤19.7 pg/ml >19.7 pg/ml YKL40

M category

Gender

S100B

0.068 0.086

0.022

1 1.0 1.5 1 1 1.8 0.704

17.5 % (5.7 %; 29.3 %) 27.0 % (13.1 %; 40.9 %) 14.2 % (4.8 %; 23.6 %) 38.5 % (19.8 %; 57.2 %) 36.4 % (16.2 %; 56.6 %) 17.5 % (7.7 %; 27.3 %) 50.0 % (34.5 %; 65.5 %) 45.0 % (29.6 %; 60.4 %) 42.6 % (29.4 %; 55.8 %) 57.7 % (38.7 %; 76.7 %) 50.0 % (29.0 %; 71.0 %) 47.4 % (34.5 %; 60.3 %) 10 9 9 15 10 9 85.0 75.0 87.0 65.4 68.2 84.2 50.0 50.0 67.5 32.5 27.8 72.2 ≤59 Years >59 Years Male Female M1a/M1b M1c Age

40 40 54 26 22 57

HR Median survival (months)

1-Year survival rate (95 % confidence interval)

2-Year survival rate (95 % confidence interval)

Log-rank p value

Multivariate analysis Univariate analysis % Death % n Definition Factor

Table 1 Patients’ characteristics and survival analysis

0.159

Kaplan–Meier (MST) was 9 months. Cumulative survival rates were 47.5 % (1 year), 22.5 % (2 years), and 18.8 % (3 years). Based on the site of distant metastases and the serum LDH, the assignment to the M category was M1c in 72.2 %, M1b in 22.8 % and M1a in 5.1 % of patients. The M category was unknown for one patient with lung metastases and unavailable LDH analysis. An elevated S100B serum level was observed in 56.0 % of patient. YKL40 and CCL17 serum levels were below the detection limit in one and four patients, respectively. The median serum concentration was 19.7 pg/ml for YKL40 and 278 pg/ml for CCL17, respectively. LDH serum levels were available for 68 patients and were within normal limits in 45 and elevated in 23 patients. Ulceration of the primary melanoma was observed in 37 patients and was absent or not known for 28 and 15 patients, respectively. Analyzed serum samples derived from routine blood draw during patient appointments at the Department of Dermatology for surveillance and/or treatments at the time of diagnosis of distant metastases or later during the further course of disease.

0.974

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p

Cancer Immunol Immunother (2015) 64:1075–1082

An overview over 1- and 2-year survival probabilities according to all analyzed factors is given in Table 1. S100B was the strongest serum biomarker to indicate prognosis (p 600 pg/ ml) were compared to those with lower levels (Fig. 1c). Similar levels of CCL17 were observed in patients with ulcerated (mean 405 pg/ml; median 269 pg/ml) or nonulcerated primary melanoma (mean 386 pg/ml; median 278 pg/ml). In contrast, the YKL-40 serum concentration was not associated with prognosis (p = 0.260; Fig. 1d). Among clinical factors, age did not correlate with survival, but female patients had a better prognosis compared to male patients

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Fig. 1 Univariate analysis of overall survival. Kaplan–Meier survival curves according to elevated versus normal serum concentrations of S100B (a) or CCL17 serum levels (b, c). Patients are assigned to groups with CCL17 concentrations above median versus those with lower concentrations (b) or above 600 pg/ml versus patients with

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lower CCL17 concentrations (c). Overall survival is presented after dichotomization according to YKL-40 serum levels (d) for different AJCC M categories (e) and for different combinations of CCL17 and S100B serum levels (f). Censored events are indicated by vertical lines


(p = 0.022). There was only a trend for a correlation between the M category according to the AJCC-staging system for patients with distant metastasis (p = 0.086) even if patients with normal serum LDH and distant metastases limited to soft tissue and/or lung (M1a/M1b) had a much higher probability to survive the first 2 years after blood draw (36.4 % for M1a/M1b versus 17.5 % for M1c; Fig. 1e). Next, multivariate analysis was performed in 75 patients with complete data to compare the relative impact and to account for confounding among the analyzed prognostic factors. S100B and CCL17 serum concentrations were independently impacting prognosis, while gender failed to demonstrate additional prognostic impact if analyzed in combination with the serum markers (p = 0,159). Elevated S100B increased the relative risk to die from disease by 2.2-fold (p = 0.005). In addition, patients with low concentrations of CCL17 had a 1.8-fold increased risk of melanoma-related death (p = 0.029). Still, only a trend for an independent impact of the M category was observed after multivariate analysis in addition to S100B and CCL17 (p = 0.068). Finally, we analyzed in detail the relative impact of the serum markers LDH, YKL-40, CCL17 and S100B by multivariate analysis. In 64 patients with available results for all four factors, LDH was the strongest independent marker for prognosis with a hazard ratio (HR) of 2.2 (p = 0.013). S100B and CCL17 had both similar additional independent impact on survival (HR 1.9; p = 0.041 and HR 1.9; p = 0.031, respectively). As expected, YKL-40 did not add prognostic information if analyzed in combination with the other three markers.

Discussion We observed a strong impact of CCL17 on prognosis of melanoma patients with distant metastases. Serum levels above 278 pg/ml were significantly associated with longer overall survival in univariate analysis (p = 0.020), and an extraordinary rate of long-term survival was shown in 16 patients with CCL17 > 600 pg/ml. Their survival probability was as high as 43.8 % 3 years after blood draw. CCL17 still independently indicated prognosis in addition to LDH and S100B serum levels if analyzed in combination. The role of the CCL17 serum concentration as prognostic marker was rarely analyzed in solid tumor so far. Low baseline serum levels of CCL17 were associated with improved outcome after peptide vaccination in patients with renal cell cancer [11]. Our observation is in line with a study of Cornforth et al. [12] who reported that high serum CCL17 levels were associated with favorable progressionfree survival in advanced melanoma patients in response to a dendritic cell-based therapy.

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YKL-40 is a heparin-binding chitinase-like anti-apoptotic glycoprotein, which is expressed by macrophages and neutrophilic granulocytes [17] as well as by tumor cells [18]. In cancer, a role was reported for neovascularization, matrix degradation and re-modeling [18–20]. Elevated serum levels of YKL-40 were previously associated with poor clinical outcome in various solid cancers [21–23]. In stage I/II melanoma, YKL-40 was reported to serve as a prognostic factor for relapse-free and overall survival [24]. However, in advanced melanoma, controversial results about the prognostic impact of YKL-40 serum levels were reported [13, 14]. In our study, YKL-40 did not show any impact on prognosis. This is in line with the study of Egberts et al. [14] in which no prognostic impact could be established in 71 stage IV melanoma patients. In contrast, Schmidt et al. [13] observed a strong correlation between elevated YKL-40 serum concentrations and poor performance status or overall survival in 110 patients with metastatic melanoma. The divergent results might be based on differences between the cohorts, but also could be caused by methodological differences, for example different concentration cutoffs for stratification. S100B serum levels showed the highest degree of association with overall survival of advanced melanoma patients as a single marker as we and others reported before [7, 8, 14, 25, 26]. Moreover, normal S100B values were associated with long-term survival with more 40 % of patients alive after 3 years. S100B and CCL17 serum levels represented independent prognostic biomarkers in our study as shown by multivariate analysis with hazard ratios of 2.2 (p = 0.005) and 1.8 (p = 0.029), respectively. The consideration of both serum markers in combination allowed us to identify patients with extraordinary favorable outcome. In case of normal S100B and CCL17 above median, the chance to be alive 3 years after blood draw was as high as 44 % (Fig. 1f) and 67 % in nine patients with normal S100B and CCL17 levels above 600 pg/ml. The M category according to AJCC was still confounding the results in multivariate analysis, but no independent prognostic value in addition to both serum biomarkers was observed (p = 0.068). Moreover, in multivariate analysis available for a subcohort of patients (n = 64), LDH was the strongest independent marker for prognosis, suggesting that LDH, CCL17 and S100B may have distinct roles as potential biomarkers in melanoma depending on the sample size and cohort studied, warranting further comparative investigations of these markers in larger patient cohorts. The independent prognostic impact of S100B and CCL17 may be explained by differences in the origin of these serum proteins. S100B is physiologically expressed by melanocytes, but its function is still not yet clarified [27, 28]. In melanoma cells, which strongly express S100B, a functional role in the disease evolution was suggested due

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to its capacity to interact with p53 and to activate STK38/ NDR1, a protein kinase involved in cell survival and proliferation [28, 29]. The appearance of S100B in the serum of melanoma patients is most likely a consequence of tumor cell death or lysis, releasing the intracellular protein into the circulation. Therefore, the serum level may mainly reflect tumor cell turnover and tumor burden. In contrast, CCL17 is a chemokine, which is actively secreted by immune cells, binds to the G-protein coupled C–C chemokine receptors type 4 (CCR4) and 8 (CCR8) and induces the activation and migration of T lymphocytes [30]. CCR4 is predominantly expressed by Th2 and regulatory T cells (Tregs) [31, 32], which are important for tumor immune evasion. Recently, CCL17 has further been involved in CCR4-mediated recruitment of cytotoxic T lymphocytes [10] and has been reported to serve as a mediator of CD8+ T cell activation by dendritic cells [33]. In vivo, CCL17 was shown to enhance anti-tumor immunity [34]. Therapeutically, interfering with the CCL17/CCR4 axis could have a beneficial effect on melanoma progression by inhibiting CCR4-expressing Tregs, as shown previously for lung metastasis [35]. Recently, CCR4+ Tregs were found to be predominant among tumor-infiltrating FOXP3+ T cells in melanoma tissues, in a substantially higher frequency compared to peripheral blood [36]. Depletion of CCR4+ T cells and subsequent stimulation with the cancer antigen NY-ESO-1 induced NY-ESO1-specific CD4+ T cells and enhanced the induction of NY-ESO-1-specific CD8+ T cells in melanoma patients. Therefore, targeting CCL17 and/or CCR4 could represent a novel promising immunotherapy approach for patients with melanoma. Immune system-related factors, such as CCL17, represent promising biomarker candidates for various cancers. The breakthroughs in the last years in the field of melanoma immunotherapies, as documented by clinical responses in up to 70 % after adoptive T cell transfer [37] or improved survival upon immune checkpoint blockade using CTLA-4, PD-1 or PD-L1 antibodies [38–41], impressively demonstrate the capacity of the immune system to efficiently combat cancer. These immunotherapies act indirectly through immune cells, cytokines and chemokines [42]. Therefore, immune-related factors might not only being prognostic, but may also serve as predictive markers for clinical response and outcome upon immunotherapies [43]. However, CCL17 needs to be validated prospectively, before embarking into a predictive biomarker evaluation in the context of checkpoint blockers, as, so far, there is not sufficient experimental evidence to support a role for this chemokine in immunotherapies. A limitation of our study is that serum samples derived from different time points after diagnosis of distant metastasis. Influences of therapeutic interventions on serum

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levels of the exploratory biomarkers can therefore not be excluded and need to be clarified in future studies. In conclusion, serum levels of CCL17 and S100B represented independent prognostic markers for melanoma patients with distant metastases and were superior to the M category of the AJCC classification to indicate overall survival. Both markers in combination allowed us to identify and stratify patients with a high chance for long-term survival. CCL17 as an immune-based biomarker represents a promising candidate to be tested as predictive marker for response upon immune checkpoint blockade in melanoma. Acknowledgments This work was supported by the collaborative research center (CRC/SFB) 685 of the German Research Foundation (DFG) to Dominik Hartl. Conflict of interest All authors declare that no conflict of interest exists.

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40.

41.

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