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Background: Malignant mesothelioma is an aggressive cancer with poor prognosis and few effective therapies. Since mesothelioma is derived from the mesothelium of the lung, we hypothesize that immune cells in the tumor microenvironment (TME) may behave differently than other solid tumors. In our previous studies, utilizing multi-plexed immunofluorescence, we did not find immune phenotypes associated with improved patient survival. Here we describe a novel combination of two technologies to spatially characterize the interface between mesothelioma cells, stroma and immune cells in the TME in a high-plex capacity.

Methods: Ten FFPE mesothelioma tumors were characterized by Definiens’ Immune-Oncology Profiling (IOP) and NanoString Digital Spatial Profiling (DSP). Three alternating sequential sections were stained with Definiens’ IOP (CD8/PD-1/FOXP3, CD68/PD-L1/CD3, Granzyme B). Definiens analysis was combined to identify localization of each marker in the tumor center, invasive margin or stroma. Twelve regions-of-interest (ROIs) were then selected based on the Definiens analysis for high-plex analysis on DSP on the interleaving slide: 4 CD68-enriched, 6 CD8- enriched and 2 CD3-low. For DSP analysis, each slide was stained with a combination of fluorescent-labeled antibodies (pan-cytokeratin, CD3, CD68) and a panel of 38-antibodies each conjugated to a unique UV-photocleavable DNA barcode. ROIs from Definiens’ defined analysis were overlayed on DSP fluorescent scans, followed by UV excitation of the defined ROIs, which releases the DNA barcodes for downstream quantitation on the NanoString nCounter® platform.

Results: We found strong correlation between Definiens and NanoString analysis of T cell and macrophage markers in selected regions. Generally, patients with longer survival (>6 months) had increased density of immune infiltrates including higher density of T cells, T-cell activation markers (PD-1), higher cytokeratin levels and decreased Ki67 in the tumor center and increased tertiary lymphoid structure makers (B cells) in the invasive margin. Furthermore, STING and VISTA were highly expressed across all mesotheliomas. However, the patient with the longest survival (>31 months) expressed an immune-excluded phenotype. Co-localization analysis revealed that high CD68 density was tightly correlated to PD-L1 expression and in at least one case additional suppressive macrophage markers, including CD163 and B7-H3.

Conclusions: Already this small data set demonstrates that integration of two novel high-plex spatial analysis techniques separates distinct immune mechanisms in the TME. Our analysis suggests that macrophages are highly associated with expression of immune-inhibitory signals in mesothelioma. Therefore, we hypothesize that analysis of additional mesotheliomas may guide the development of combination immunotherapy trials that will be effective against this incurable disease.

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Clinical Institute





Earle A. Chiles Research Institute


Cancer, immunotherapy, multispectral imaging, tumor microenvironment, T cells, immuno-oncology, mesothelioma; genomics




Poster presented at Society for Immunotherapy of Cancer Annual Meeting, Washington, D.C., November 7 – 11, 2018.

Integrative spatially-resolved, high-plex digital profiling enables characterization of complex immune biology in the tumor microenvironment of mesothelioma

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