Abstract 2798: High density of CD68+HLA-DR- macrophages in the stroma of primary melanoma correlates with an unfavorable immune microenvironment as assessed by Digital Spatial Profiling
Authors: Rizk Emanuelle M. , Chen Andrew , Silverman Andrew M. , Marks Douglas K. , Rabadan Raul , Fuhrman Kit , VanSchoiack Alison , Liang Yan , Beechem Joseph , Saenger Yvonne M. , Gartrell Robyn D. 
Published: August 2019

Abstract
Introduction: The role of macrophages (Mϕ) in melanoma progression is controversial, as they have been shown to both favor and inhibit anti-tumor immunity. Density of Mϕ at the leading tumor edge has been shown to be a marker of poor prognosis. In previous work, we used quantitative multiplexed immunofluorescence (qmIF) to discover the ratio of CD8+ T lymphocytes (CTLs) to CD68+ Mϕ in the peritumoral stroma predicts a favorable prognosis. Further, we found that increased proximity of HLA-DR- Mϕ to CTLs in the stroma predicts poor prognosis. These findings highlight the importance of the location of Mϕ within the tumor microenvironment (TME). Here, we further analyze and compare the TME of patients with high and low stromal densities of HLA-DR- Mϕ.
Methods: From a cohort of 104 patients with primary stage II-III melanoma and known survival information, we selected 8 patients for Digital Spatial Profiling (DSP) analyses. Of this subcohort, 4 patients had a high density of HLA-DR- Mϕ and close proximity of HLA-DR- Mϕ to CTLs in the stroma while the other 4 had a high CTL to Mϕ ratio with low HLA-DR- Mϕ density, as determined by qmIF (methods published). FFPE slides were stained with antibodies conjugated to UV-photocleavable DNA barcodes and specific to 34 proteins, including CD45, CD4, CD8, CD68, PD-1, and PD-L1. Twelve regions of interest (ROIs) per patient were selected based on high Mϕ density as determined by qmIF. ROIs were then analyzed using UV excitation, which releases DNA barcodes for downstream quantitation on the nanoString nCounter® platform. Protein expression was compared between patients and statistical analysis performed using Mann-Whitney test.
Results: We found that ROIs from patients with higher density of HLA-DR- Mϕ had a lower immune infiltration overall as assessed by quantitation of CD45 per ROI (p&lt;0.0001). As expected, the ratio of CD68 to CD45 was higher in these patients than in patients with lower density of HLA-DR- Mϕ (p&lt;0.0001). Interestingly, patients with higher density of HLA-DR- Mϕ also had a significantly higher ratio of CD4 to CD45 (p&lt;0.0001), but a similar CD8A to CD45 ratio. Patients with a higher HLA-DR- Mϕ density had a higher CD4 to CD8A ratio (p&lt;0.0001). Further, PD-L1 and PD1 levels per CD45 were significantly higher in patients with higher HLA-DR- Mϕ density (p&lt;0.0001 and p=0.0002, respectively).
Conclusion: Patients with higher densities of HLA-DR- Mϕ in the stroma and increased proximity of HLA-DR- Mϕ to CTLs in the tumor stroma have lower levels of CD45, a higher ratio of CD4 to CD8, and a higher ratio of PDL1 and PD1 to CD45 by assessment of Mϕ-rich areas using DSP. These findings highlight the close relation between Mϕ and the local immune microenvironment in primary stage II-III melanoma.
Citation Format: Emanuelle M. Rizk, Andrew Chen, Andrew M. Silverman, Douglas K. Marks, Raul Rabadan, Kit Fuhrman, Alison VanSchoiack, Yan Liang, Joseph Beechem, Yvonne M. Saenger, Robyn D. Gartrell. High density of CD68+HLA-DR- macrophages in the stroma of primary melanoma correlates with an unfavorable immune microenvironment as assessed by Digital Spatial Profiling [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2798.