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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.

Cohort and dataset overview

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.

Multiplex immunofluorescence staining enables the simultaneous detection of multiple immune markers in a single tissue section, and is a useful tool for the identification of specific cell populations within the tumour microenvironment. However, this technology has rarely been validated against standard clinical immunohistology, which is a barrier for its integration into clinical practice. This study sought to validate and investigate the accuracy, precision and reproducibility of a multiplex immunofluorescence compared with immunohistochemistry (IHC), including tissue staining, imaging and analysis, in characterising the expression of immune and melanoma markers in both the tumour and its microenvironment. Traditional chromogenic IHC, single-plex immunofluorescence and multiplex immunofluorescence were each performed on serial tissue sections of a formalin-fixed paraffin-embedded (FFPE) tissue microarray containing metastatic melanoma specimens from 67 patients. The panel included the immune cell markers CD8, CD68, CD16, the immune checkpoint PD-L1, and melanoma tumour marker SOX10. Slides were stained with the Opal™ 7 colour Kit (Akoya Biosciences) on the intelliPATH autostainer (Biocare Medical) and imaged using the Vectra 3.0.5 microscope. Marker expression was quantified using Halo v.3.2.181 (Indica Labs). Comparison of the IHC and single-plex immunofluorescence revealed highly significant positive correlations between the cell densities of CD8, CD68, CD16, PD-L1 and SOX10 marker positive cells (Spearman’s rho = 0.927 to 0.750, p < 0.0001). Highly significant correlations were also observed for all markers between single-plex immunofluorescence and multiplex immunofluorescence staining (Spearman’s rho >0.9, p < 0.0001). Finally, correlation analysis of the three multiplex replicates revealed a high degree of reproducibility between slides (Spearman’s rho >0.940, p < 0.0001). Together, these data highlight the reliability and validity of multiplex immunofluorescence in accurately profiling the tumour and its associated microenvironment using FFPE metastatic melanoma specimens. This validated multiplex panel can be utilised for research evaluating melanoma and its microenvironment, such as studies performed to predict patient response or resistance to immunotherapies.

Purpose Patients with melanoma liver metastases have significantly reduced overall response and survival when treated with immune checkpoint inhibitors (ICI) compared to those without liver metastases. Melanoma liver metastases are less likely to respond to ICI compared to other sites of metastases, and the presence of liver metastases has also been associated with reduced responses to ICI at other metastatic sites. We aimed to profile circulating and tumor immune profiles of melanoma patients with versus without liver metastases to elucidate the factors behind this observation. Methods Pre-treatment PBMCs from 37 advanced melanoma patients (Cohort A) were profiled using mass cytometry (CyTOF) spanning 46 markers. Expression of specific immune cells were compared between those with (n = 8) vs without (n = 29) liver metastases. In a separate independent cohort of 93 untreated metastatic melanoma patients (Cohort B), FFPE tumour samples comprised of subcutaneous, lymph node (LN) and brain metastases were stained for myeloid and T cell markers using multiplex IHC. Immune cell populations in cohort B tissues were compared between patients with (n=40) vs without (n=53) liver metastases. Results PBMCs from patients in Cohort A with liver metastases had an increased proportion of a myeloid population characterised as HLA-DR+CD14+CD16- compared to patients without liver metastases (p = 0.035). However, a difference in CD68+CD14+CD16- myeloid cells was not seen in cohort B melanoma tissues (subcut, LN, brain metastases) between patients with vs without liver metastases. In subcutaneous tissues, there was a significantly reduced density of T cells in patients with liver metastases (median = 59 cells/mm2) compared to those without (median = 217 cells/mm2; p = 0.037). This trend was also observed in LN and brain metastases but did not reach significance. In brain metastases, a higher proportion of FoxP3+ T cells was observed in patients with liver metastases (p = 0.003). An increase in this population was also observed in the LN and subcutaneous metastases in the presence (vs absence) of liver metastases, however this did not reach significance. LN metastases also showed a reduced proportion of both TIM3+ (p = 0.049) and CD103+ (p = 0.048) T cells in patients with liver metastases (vs absence), and a similar trend was observed in subcutaneous metastases. In contrast, brain metastases showed the opposite trend as well as higher proportion of PD1+ T cells in patients with liver metastases vs those without (p = 0.033). Conclusion These data provide insights into the differences in the anti-tumor immune profiles of patients with versus without liver metastases; the presence of liver metastases may have a specific impact at different metastatic sites. This highlights the need for further validation and investigation into the mechanism by which the presence of liver metastases may exert this effect. Citation Format: Jordan W. Conway, Felix Marsh-Wakefield, Kazi J. Nahar, Serigne N. Lo, Ismael A. Vergara, Tuba N. Gide, Grace H. Attrill, Jorja Braden, Matteo S. Carlino, Robyn P. Saw, John F. Thompson, Andrew J. Spillane, Kerwin F. Shannon, Brindha Shivalingam, Alexander M. Menzies, Umaimainthan Palendira, James S. Wilmott, Georgina V. Long, Richard A. Scolyer, Ines Pires Da Silva. The association between melanoma liver metastases (mets) and the systemic anti-tumor immune profile [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 81.

Surgical excision with histologically clear margins results in cure of most melanoma patients. Involvement of the margins is associated with tumour recurrence and adverse outcomes. Determining whether or not the histological margins are involved by melanoma, particularly in situ melanoma, can be very difficult particularly in acral locations where insitu melanoma can be extremely subtle. This study sought to determine the proximity of acral melanoma in situ to the surgical excision margin using multiple methods including routine histopathological assessment, FISH (CCND1 amplification), and multiplex immunofluorescence (mIF: SOX10, p16, Cyclin D1 and PRAME) in primary acral melanomas with initially histopathologically reported clear excision margins of >0.3mm (n=85). The clear excision margins determined by each modality were compared between those melanomas which recurred (n=47) and those which remained recurrence-free at last follow-up (median = 46 months, range = 1-260 months) (n=35). Histopathologically confirmed excision margin width was smaller in the tumours of patients who recurred (mean = 7.083mm vs 9.964mm, p = 0.0811). Intratumoural CCND1 amplification was increased in recurrence-free patients in comparison to recurrence patients (16.2% vs 7.7%). FISH and mIF confirmed the correlation of intratumoural CCND1 amplification with Cyclin D1 expression SOX10+ melanoma cells (p=0.0347). No correlations between recurrence status and Cyclin D1, PRAME or p16 expression levels in the tumour or epidermis were identified. In histopathologically normal epidermal tissue as assessed on H&E sections,Cyclin D1+, p16- and PRAME+ cells were classified as malignant cells when co-expressed with SOX10. Upon measuring the distance from the tumour margin to the nearest malignant cell, we found that malignant cells were closer to the tumour in recurrence patients in comparison to recurrence-free patients (mean = 7.324mm vs 9.464mm, p = 0.2735). These distances closely correlated with the excision margin width as determined via histopathological assessment (R2 = 0.7424, p Citation Format: Grace Heloise Attrill, Teresa A. Bailey, Trina Lum, Jordan W. Conway, Georgina V. Long, James S. Wilmott, Richard A. Scolyer. Molecular analysis finds excision margin width predictive of recurrence in acral melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2221.

3024 Background: Liver metastasis is a poor prognostic factor in several cancers and is associated with poor response to immunotherapy (IO) in melanoma and lung cancer. VEGFi have activity in hepatocarcinoma (HCC) and is hypothesized to be due the hypoxic microenvironment. Whether this is also true for liver mets is unknown. We sought to assess the efficacy of VEGFi in liver mets utilizing randomized-controlled clinical trials (RCTs) testing the efficacy of VEGFi, regardless of primary cancer site. Methods: Systematic searches of PubMed, Cochrane CENTRAL, and Embase were conducted from January 1, 2000, to January 1, 2022. All RCTs that compared a backbone of systemic therapy (chemotherapy [chemo] and/or IO and/or targeted therapy [TT]) or best supportive care (BSC) with vs without VEGFi in patients (pts) with liver mets from any cancer were selected. HCC trials were excluded. Study design, cancer type, number of pts, lines of treatment, study drugs and hazard ratios (HRs) with 95% CIs for overall survival (OS) and progression-free survival (PFS) were extracted. Pooled effects of VEGFi in pts with liver mets across different cancer types were estimated using random effect model with inverse variance. Heterogeneity between studies was assessed by I2 statistics. Sensitivity analyses were performed considering prespecified subgroups of trials. Results: 3170 pts with liver mets from 19 RCTs were included in this meta-analysis: 8 colorectal cancer, 4 non-small cell lung cancer, 4 renal cell cancer & urothelial cancer, 1 pancreatic cancer, 1 GIST and 1 gastric cancer. Backbone systemic therapy in these trials included: chemo (11), TT (2), IO (2), chemo+IO (1), chemo+TT (1); and 2 BSC trials. Moderate heterogeneity between studies for both PFS (I2= 55%) and OS (I2= 46%) was seen. The addition of VEGFi to standard systemic therapy or BSC was associated with superior PFS (HR = 0.61; 95% CI, 0.50-0.74; p < 0.0001) and OS (HR = 0.86; 95%CI, 0.76-0.99; p = 0.0334) in pts with liver mets, regardless of whether pts had only liver mets or concurrent other sites of mets. In the subset of RCTs with data on pts without liver mets, the benefit with VEGFi was more pronounced in patients with liver mets (HR = 0.56) vs those without liver mets (HR = 0.64) for PFS, but not for OS. Conclusions: The addition of VEGFi to standard management improved survival outcomes in pts with liver mets across different cancer types and warrants further investigation. VEGFi added to IO may be effective in pts with resistant liver mets.

Purpose. To evaluate the feasibility of utilizing a system of SMP acrylates for a thrombectomy device by determining an optimal crosslink density that provides both adequate recovery stress for blood clot removal and sufficient strain capacity to enable catheter delivery. Methods. Four thermoset acrylic copolymers containing benzyl methacrylate (BzMA) and bisphenol A ethoxylate diacrylate (Mn∼512, BPA) were designed with differing thermomechanical properties. Finite element analysis (FEA) was performed to ensure that the materials were able to undergo the strains imposed by crimping, and fabricated devices were subjected to force-monitored crimping, constrained recovery, and bench-top thrombectomy. Results. Devices with 25 and 35 mole% BPA exhibited the highest recovery stress and the highest brittle response as they broke upon constrained recovery. On the contrary, the 15 mole% BPA devices endured all testing and their recovery stress (5 kPa) enabled successful bench-top thrombectomy in 2/3 times, compared to 0/3 for the devices with the lowest BPA content. Conclusion. While the 15 mole% BPA devices provided the best trade-off between device integrity and performance, other SMP systems that offer recovery stresses above 5 kPa without increasing brittleness to the point of causing device failure would be more suitable for this application.