Serum Level of Soluble CD163 May Be a Predictive Marker of the Effectiveness of Nivolumab in Patients With Advanced Cutaneous Melanoma

Antibodies against programmed cell death protein 1, such as nivolumab and pembrolizumab, are widely used for treating various cancers, including advanced melanoma. Nivolumab significantly prolongs survival in patients with metastatic melanoma, and sequential administration with lipilimumab may improve outcomes when switched at the appropriate time. Biomarkers are therefore needed to evaluate nivolumab efficacy soon after first administration. This study analyzed serum levels of soluble cluster of differentiation 163 (sCD163) in 59 cases of advanced cutaneous melanoma and 16 cases of advanced mucosal melanoma treated using nivolumab. Serum levels of sCD163 were significantly increased after 6 weeks in responders compared to non-responders after initial administration of nivolumab for cutaneous melanoma. In contrast, no significant difference between responders and non-responders was seen among patients with non-cutaneous melanoma. These results suggest that sCD163 may be useful as a biomarker for selecting patients with advanced cutaneous melanoma most likely to benefit from anti-melanoma immunotherapy.


INTRODUCTION
The programmed cell death protein 1/programmed death-ligand 1 (PD-1/PD-L1) pathway plays a critical role in tumor immune response. Anti-PD-1 antibodies such as nivolumab and pembrolizumab are thus in wide use for the treatment of various cancers, including advanced melanoma (1,2). Nivolumab significantly prolongs survival in patients with metastatic melanoma (1), and co-administration with ipilimumab also leads to improved outcomes (1)(2)(3). Ipilimumab is a fully humanized immunoglobulin (Ig)G1 monoclonal antibody that blocks cytotoxic T-lymphocyte antigen (CTLA-4) to activate and increase T cells, and suppress the function of regulatory T cells (Tregs) (4). Previous reports have suggested ipilimumab is useful for treating advanced melanoma, particularly in combination with other anti-melanoma reagents (1)(2)(3)5). Moreover, combination therapy using nivolumab and ipilimumab reportedly increases the response rate for untreated metastasis of melanoma to the brain (6). However, the efficacy of ipilimumab in patients with nivolumab-resistant melanoma is extremely low after objective tumor progression (7). Because both coadministration of nivolumab and ipilimumab and sequential administration of nivolumab and ipilimumab with a planned switch leads to a high frequency of immune-related adverse events (irAEs) in patients with advanced melanoma (1,3), determining the efficacy of nivolumab monotherapy before the planned switch from nivolumab to ipilimumab is important.
Tumor-associated macrophages (TAMs) are characterized by their heterogeneity and plasticity, and may be functionally reprogrammed to polarized phenotypes by exposure to cancerrelated factors, stromal factors, or infection, leading to the production of various chemokines that play significant roles in maintaining the tumor microenvironment (8)(9)(10)(11)(12)(13). Because PD-1 expression in TAMs is one of the key factors in M2 macrophage polarization (14), administration of an anti-PD1 antibody might repolarize TAMs, leading to TAM activation in melanoma patients. Because the main population of TAMs in skin cancer is CD163 + M2 macrophages, and soluble cluster of differentiation 163 (sCD163) is a TAM marker that appears in the serum as a result of proteolytic shedding (15), we hypothesized that serum sCD163 may offer a predictive marker for the efficacy of nivolumab in the early stage of disease. This study analyzed serum levels of sCD163 in 59 cases of advanced cutaneous melanoma and 16 cases of advanced mucosal melanoma treated with nivolumab.

Ethics Statement For Human Experiments
The protocol for this human study was approved by the ethics committee of Tohoku University Graduate School of Medicine, Sendai, Japan (Permit No: 2017-1-064). All methods were performed in accordance with the relevant guidelines and regulations. All patients provided written informed consent.

Patients
Data from patients treated with nivolumab were collected from eight clinical sites in Japan. Patients were eligible if they had unresectable stage III melanoma, the tumor was resectable but the patient had declined resection, or if the patient had stage IV melanoma with accessible cutaneous, subcutaneous, and/or nodal lesions (staging was performed according to the AJCC Staging Manual, 7th edition, 2011). All patients received 2 mg/kg of nivolumab followed by a 3-week rest period or 3 mg/kg of nivolumab followed by 2 weeks of rest, both of which are approved dosing schedules in Japan. Serum from patients was obtained on days 0 and 42.

Serum Levels of sCD163
On days 0 and 42 after nivolumab administration, we stored the serum and analyzed serum levels of soluble sCD163 by enzymelinked immunoassay (ELISA) according to the protocol provided by the manufacturer (catalog number DY1607; R&D Systems, Minneapolis, MN). Data from each donor were obtained as the mean of duplicate assays. Serum levels on day 42 were compared to baseline (day 0) and statistically analyzed.

Statistical Methods
Receiver operating characteristic (ROC) curves were applied to calculate cut-off values for serum levels of sCD163 and areas under the curve (AUCs). Cut-offs were determined using Youden's index (16) (sensitivity + specificity −1) to determine the point of maximum index value. ROC curves were established to evaluate serum levels of sCD163 in patients administered nivolumab. For a single comparison between two groups, the Mann-Whitney U-test was used. Levels of significance were set at p < 0.01. All statistical analyses were performed using JMP version 14.1 software (SAS Institute, Tokyo, Japan).

Serum Levels of sCD163
To determine whether serum levels of sCD163 may predict early response in melanoma patients treated with nivolumab, we evaluated levels in 75 patients with advanced melanoma treated using nivolumab (Supplemental Figure 1). Compared to baseline (day 0), serum levels of sCD163 at day 42 were significantly increased in the group showing objective response (p < 0.0001; Figure 1A) among patients with advanced cutaneous melanoma, whereas no significant difference in serum sCD163 levels was seen among patients with advanced mucosal melanoma (Figure 2A). Increases in serum sCD163 and efficacy at 3 months after the first administration of nivolumab in each patient are described in Tables 1, 2. The thresholds for sCD163 change between day 42 and day 0 to distinguish responders from non-responders were respectively 3.07 ± 0.07 ng/mL and 0.47 ± 0.05 ng/mL in cutaneous and non-cutaneous melanoma. In the cutaneous melanoma cohort, the mean change ratio in sCD163 serum level was 144.6% for those patients showing objective response, compared to 101.0% for non-responders ( Table 1). In the mucosal melanoma cohort, the mean change ratio in sCD163 serum level was 84.1% for those patients showing objective response, and 122.3% for nonresponders ( Table 2). The threshold for increased serum sCD163 in cutaneous melanoma was 3.07 ± 0.07 ng/mL, whereas that in non-cutaneous melanoma was 0.47 ± 0.05 ng/mL. The sensitivity and specificity of serum sCD163 in cutaneous melanoma were 84.6 and 87.0%, respectively (p = 0.0030; Figure 1B), whereas the sensitivity and specificity of serum sCD163 in non-cutaneous melanoma were 100 and 66.7%, respectively (p = 0.3154; Figure 2B).

DISCUSSION
Because nivolumab shows higher efficacy than other antimelanoma drugs (e.g., ipilimumab and dacarbazine) (1,17), and induces a longer duration of anti-tumor response than BRAF/MEK inhibitors (e.g., vemurafenib, dabrafenib, trametinib) (18,19), oncologists have been particularly interested in combining nivolumab with agents that enhance anti-tumor immune responses in patients with metastatic melanoma (1)(2)(3)20). The efficacy of nivolumab appears significantly increased when combined with ipilimumab (57.7%), but the rate of severe treatment-related AEs (Grade 3 or 4) is unfortunately also significantly increased with this particular combination (59.0%) (1,2). To avoid severe AEs caused by ipilimumab, predictive biomarkers are needed to evaluate the efficacy of nivolumab monotherapy at 2-3 months after first administration, to prepare for any planned switch from nivolumab to ipilimumab.
CD163 is a member of the scavenger receptor cysteinerich family, and is exclusively expressed on cells of the monocyte/macrophage lineage (15). As previously reported, in metastatic melanoma, a substantial number of CD163 + TAMs are present in metastatic melanoma and are activated by stromal factors, leading to an increase in serum levels of sCD163 as a result of proteolytic shedding (21-23). In addition, Jensen et al. (21) previously reported sCD163 and CD163 + TAMs as a prognostic marker for early-stage cutaneous melanoma. Notably, as Gordon et al. (14) reported, PD-1 expression is a key factor in maintaining TAMs as M2-polarized, and blockade of PD-1/PD-L1 leads to conversion of TAMs into M1-polarized activated macrophages. Because TAMs in melanoma comprise a heterogeneous, mainly immunosuppressive population (9,23), and because repolarization of TAMs into the activated subtype by immunomodulatory drugs has been reported to significantly suppress melanoma growth in a spontaneous mouse melanoma model (8), we hypothesized that one of the targets for nivolumab in melanoma is activated CD163 + TAMs.
To prove our hypothesis, we analyzed serum levels of sCD163 in 59 cases of advanced cutaneous melanoma and 16 cases of advanced mucosal melanoma treated with nivolumab. Serum levels of sCD163 were significantly increased 6 weeks after initial administration of nivolumab in the response group compared to the non-response group in cutaneous melanoma. In contrast, no significant difference between nivolumab responders and non-responders was seen for non-cutaneous melanoma. Interestingly, in non-cutaneous melanoma, serum levels of sCD163 in non-responders tended to be even higher than those in responders. This discrepancy might be explained by differences in cancer stroma that could stimulate TAMs in the tumor microenvironment of each organ. Such studies should be performed in the future.
According to the present results, sCD163 may represent a predictive biomarker for evaluating the efficacy of nivolumab at 3 months after first administration for advanced cutaneous melanoma. Because sequential administration of ipilimumab followed by nivolumab is only effective for advanced melanoma before the melanoma develops tolerance for nivolumab (7), and administration of ipilimumab leads to the development of irAEs more frequently than nivolumab (2, 3), predicting the efficacy of nivolumab before first tumor estimation is crucial to determining whether the patient will successfully adapt to the planned switch from nivolumab to ipilimumab therapy. The present study suggested that sCD163 may be a useful biomarker for the selection of those cutaneous melanoma patients most likely to benefit from anti-melanoma immunotherapy using nivolumab and ipilimumab. Because this was a pilot study, future independent studies with larger patient cohorts are needed to confirm our findings. TFuj designed the research study. TFuj, YS, KT, and YK  performed and analyzed the ELISA data. TFuj, YK, AO, YF, KY,  SM, TFun, HH, YY, HU, YN, RT, MA, KI, HO, NW, HI, TH, and AH treated the patients and acquired the clinical data and samples. TFuj wrote the manuscript. TFuj and SA supervised the study.

FUNDING
This study was partially funded by Ono Pharmaceutical Co., Ltd. and Bristol-Myers Squibb.