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Background The liver is a known site of resistance to immunotherapy and the presence of liver metastases is associated with shorter progression-free and overall survival (OS) in melanoma, while lung metastases have been associated with a more favorable outcome. There are limited data available regarding the immune microenvironment at different anatomical sites of melanoma metastases. This study sought to characterize and compare the tumor immune microenvironment of liver, brain, lung, subcutaneous (subcut) as well as lymph node (LN) melanoma metastases.Methods We analyzed OS in 1924 systemic treatment-naïve patients with AJCC (American Joint Committee on Cancer) stage IV melanoma with a solitary site of organ metastasis. In an independent cohort we analyzed and compared immune cell densities, subpopulations and spatial distribution in tissue from liver, lung, brain, LN or subcut sites from 130 patients with stage IV melanoma.Results Patients with only liver, brain or bone metastases had shorter OS compared to those with lung, LN or subcutaneous and soft tissue metastases. Liver and brain metastases had significantly lower T-cell infiltration than lung (p=0.0116 and p=0.0252, respectively) and LN metastases (p=0.0116 and p=0.0252, respectively). T cells were further away from melanoma cells in liver than lung metastases (p=0.0335). Liver metastases displayed unique T-cell profiles, with a significantly lower proportion of programmed cell death protein-1+ T cells compared to all other anatomical sites (p

Background The profound disparity in response to immune checkpoint blockade (ICB) by cutaneous melanoma (CM) and uveal melanoma (UM) patients is not well understood. Therefore, we characterized metastases of CM and UM from the same metastatic site (liver), in order to dissect the potential underlying mechanism in differential response on ICB.Methods Tumor liver samples from CM (n=38) and UM (n=28) patients were analyzed at the genomic (whole exome sequencing), transcriptional (RNA sequencing) and protein (immunohistochemistry and GeoMx Digital Spatial Profiling) level.Results Comparison of CM and UM metastases from the same metastatic site revealed that, although originating from the same melanocyte lineage, CM and UM differed in somatic mutation profile, copy number profile, tumor mutational burden (TMB) and consequently predicted neoantigens. A higher melanin content and higher expression of the melanoma differentiation antigen MelanA was observed in liver metastases of UM patients. No difference in B2M and human leukocyte antigen-DR (HLA-DR) expression was observed. A higher expression of programmed cell death ligand 1 (PD-L1) was found in CM compared with UM liver metastases, although the majority of CM and UM liver metastases lacked PD-L1 expression. There was no difference in the extent of immune infiltration observed between CM and UM metastases, with the exception of a higher expression of CD163 (p

Immunotherapy, particularly immune checkpoint inhibitors, have become widely used in various settings across many different cancer types in recent years. Whilst patients are often treated on the basis of the primary cancer type and clinical stage, recent studies have highlighted disparity in response to immune checkpoint inhibitors at different sites of metastasis, and their impact on overall response and survival. Studies exploring the tumor immune microenvironment at different organ sites have provided insights into the immune-related mechanisms behind organ-specific patterns of response to immunotherapy. In this review, we aimed to highlight the key learnings from clinical studies across various cancers including melanoma, lung cancer, renal cell carcinoma, colorectal cancer, breast cancer and others, assessing the association of site of metastasis and response to immune checkpoint inhibitors. We also summarize the key clinical and pre-clinical findings from studies exploring the immune microenvironment of specific sites of metastasis. Ultimately, further characterization of the tumor immune microenvironment at different metastatic sites, and understanding the biological drivers of these differences, may identify organ-specific mechanisms of resistance, which will lead to more personalized treatment approaches for patients with innate or acquired resistance to immunotherapy.

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.