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Purpose: This study characterizes intratumoral macrophage populations within baseline melanoma biopsies from patients with advanced melanoma who received either anti-PD-1 monotherapy or a combination with anti-CTLA-4. Particularly, FcγRIIIa (CD16)-expressing macrophage densities were investigated for associations with response and progression-free survival. Experimental Design: Patients with advanced melanoma who received either anti-PD-1 monotherapy or combination anti-PD-1 and anti-CTLA-4 were retrospectively identified. Macrophage populations were analyzed within baseline melanoma biopsies via multiplex IHC in relation to treatment outcomes. Results: Patients who responded to combination immune checkpoint inhibitor contained higher CD16+ macrophage densities than those who did not respond (196 vs. 7 cells/mm2; P = 0.0041). There was no diffidence in CD16+ macrophage densities in the PD-1 monotherapy-treated patients based on response (118 vs. 89 cells/mm2; P = 0.29). A significantly longer 3-year progression-free survival was observed in combination-treated patients with high intratumoral densities of CD16+ macrophages compared with those with low densities (87% vs. 42%, P = 0.0056, n = 40). No association was observed in anti-PD-1 monotherapy-treated patients (50% vs. 47%, P = 0.4636, n = 50). Melanoma biopsies with high densities of CD16+ macrophages contained upregulated gene expression of critical T-cell recruiting chemokines (CXCL9, CXCL10, and CXCL11). Conclusions: Our data demonstrate that tumor microenvironments enriched with CD16+ macrophages are favorable for response to combination anti-PD-1 and anti-CTLA-4 therapy but not anti-PD-1 monotherapy. These data provides a potential biomarker of response for combination immunotherapies in patients with metastatic melanoma.

C1Q and PD-1 expression on CD16+ macrophages in on treatment biopsies of melanoma patients treated with PD-1 or PD-1+CTLA-4 ICB. Dynamics between pretreatment and early-during treatment biopsies on (a) CD16+ macrophages as a proportion of all macrophages (b) CD16+ C1q+ macrophages as a proportion of all C1q+ macrophages (c) PD-1+ CD16+ macrophages as a proportion of PD-1+ macrophages (d) PD-1+ CD16+ C1q+ macrophages as a proportion of all macrophages (e) C1q+ CD16+ macrophages as a proportion of CD16+ macrophages (f) PD-1+ CD16+ macrophages as a proportion of CD16+ macrophages. (g) Log2 RNA expression of C1qa-c in pretreatment (PRE) and early during treatment (EDT) biopsies of responding and non-responding patients treated with PD-1 or PD-1+CTLA-4 ICB. (h) Flow cytometry analysis of ipilimumab (IgG1) and/or secondary anti-IgG1 binding to CD16+ and CD16- macrophages in a melanoma patient's tumor dissociate. FMO, a no-secondary control.

Differential expression analysis gene list of FACS sorted CD16+ compared to CD16- macrophages from human melanoma dissociates.

Purpose:This study characterizes intratumoral macrophage populations within baseline melanoma biopsies from patients with advanced melanoma who received either anti-PD-1 monotherapy or a combination with anti-CTLA-4. Particularly, FcγRIIIa (CD16)-expressing macrophage densities were investigated for associations with response and progression-free survival.Experimental Design:Patients with advanced melanoma who received either anti-PD-1 monotherapy or combination anti-PD-1 and anti-CTLA-4 were retrospectively identified. Macrophage populations were analyzed within baseline melanoma biopsies via multiplex IHC in relation to treatment outcomes.Results:Patients who responded to combination immune checkpoint inhibitor contained higher CD16+ macrophage densities than those who did not respond (196 vs. 7 cells/mm2; P = 0.0041). There was no diffidence in CD16+ macrophage densities in the PD-1 monotherapy-treated patients based on response (118 vs. 89 cells/mm2; P = 0.29). A significantly longer 3-year progression-free survival was observed in combination-treated patients with high intratumoral densities of CD16+ macrophages compared with those with low densities (87% vs. 42%, P = 0.0056, n = 40). No association was observed in anti-PD-1 monotherapy-treated patients (50% vs. 47%, P = 0.4636, n = 50). Melanoma biopsies with high densities of CD16+ macrophages contained upregulated gene expression of critical T-cell recruiting chemokines (CXCL9, CXCL10, and CXCL11).Conclusions:Our data demonstrate that tumor microenvironments enriched with CD16+ macrophages are favorable for response to combination anti-PD-1 and anti-CTLA-4 therapy but not anti-PD-1 monotherapy. These data provides a potential biomarker of response for combination immunotherapies in patients with metastatic melanoma.

Purpose: Therapeutic blockade of immune checkpoints has revolutionized cancer treatment. Durable responses, however, occur in less than half of those treated, and efforts to improve treatment efficacy are confounded by a lack of understanding of the characteristics of the cells that initiate antitumor immune response.Patients and Methods: We performed multiparameter flow cytometry and quantitative multiplex immunofluorescence staining on tumor specimens from immunotherapy-naïve melanoma patients and longitudinal biopsy specimen obtained from patients undergoing anti–PD-1 therapy.Results: Increased numbers of CD69+CD103+ tumor-resident CD8+ T cells were associated with improved melanoma-specific survival in immunotherapy-naïve melanoma patients. Local IL15 expression levels strongly correlated with these tumor-resident T-cell numbers. The expression of several immune checkpoints including PD-1 and LAG3 was highly enriched in this subset, and these cells significantly expanded early during anti–PD-1 immunotherapy.Conclusions: Tumor-resident CD8+ T-cell numbers are more prognostic than total CD8+ T cells in metastatic melanoma. In addition, they are likely to initiate response to anti–PD-1 and anti–LAG-3 treatments. We propose that the immune profile of these cells prior to treatment could inform strategies for immune checkpoint blockade. Clin Cancer Res; 24(13); 3036–45. ©2018 AACR.

Supplementary data on flow cytometry analysis

Supplementary table with clinical data

TPS9599 Background: Anti-PD1-based immunotherapies have been approved for many cancer types and are now a standard therapy for advanced melanoma. Despite this, ̃50% of advanced melanoma patients (pts) fail to respond or eventually progress after response. It is therefore critical to identify pts with a low likelihood of response to anti-PD1-based therapy and efficiently assess activity of rationally-selected alternative novel immunotherapies. Methods: We designed this investigator-initiated phase II PIP-Trial to evaluate two consecutive biomarker testing platforms, followed by the activity of rationally selected 5 novel agents in pts with advanced melanoma. Two separate pt populations are included: Part-A) treatment-naïve pts predicted to be resistant to either anti-PD-1 alone or combined with ipilimumab using Biomarker Test-1); and Part-B) pts who had progressed on 1 prior line of PD1-based therapy. Part-A) is a Bayesian adaptive multi-arm multi-stage design using response adaptive randomisation after a burn-in period where pts are randomised to the existing arms with equal probability. From then on, regular interim analyses will be carried out with the objective to either drop poorly performing arms or continue. Part-B) is an open platform without control that combined a selection and an expansion phase to identify best novel agent(s) as second-line therapy. Expansion phase decisions will be based on enrichment for biomarker Test-2. Dropping an arm occurs when the posterior probability of observing a clinically significant effect on the primary outcome (i.e. 6-month RECIST objective response rate (ORR)) is low. The operational characteristics of the design were investigated through simulations considering 4 plausible scenarios with 40% ORR in the control arm (anti-PD1 + Anti-CTLA4). Simulations were based on the upcoming R package BATS. Part-A has at least 85% power to detect a 30% absolute improvement in ORR with respect to the control arm (with a max N = 216 – Table below). Part-B will be able to select two promising treatments in the expansion phase and formally test their efficacy against a minimum ORR of 25% at 80% power (max N = 150). [Table: see text]