LAUSR

Abstract Background Malignant mesothelioma is an aggressive cancer with poor prognosis and few effective therapies. Due to its derivation from the mesothelium of the lung, immune cells in the tumor microenvironment (TME) may behave differently than in other solid tumors. We describe the combination of two techniques to spatially characterize the tumor-immune profile in mesothelioma. Methods 47 FFPE mesothelioma tumors were characterized by Definiens’ Immune-Oncology Panel (IOP) and NanoString’s GeoMx™ Digital Spatial Profiling (DSP). Three alternating sequential sections were stained with IOP (CD8/PD-1/FOXP3, CD68/PD-L1/CD3, Granzyme B (GRZMB)). DSP was performed on 12 IOP-derived regions-of-interest (ROIs) on the interleaving slide by a combination of a fluorescent antibodies stain (pan-CK, CD3, CD68) and a panel of 38 UV-photocleavable DNA barcoded antibodies quantified on the nCounter® platform. Gene expression was assessed using NanoString’s PanCancer IO 360™ (IO360) assay. Results Cluster analysis based on the IOP revealed 6 distinct immune groups. Two of these had high IOP CD68 density, distinguished by an inhibitory phenotype (PD-L1+) and activated macrophage profile. Subsequent DSP analysis showed that e.g. VISTA, B7-H4 amongst others were higher in the active vs. inhibitory macrophage group. Overall survival (OS) was not associated with density of T cell marker expression measured by IOP/DSP. The IO360 analysis confirmed that and showed tumor-related gene signatures (e.g. proliferation, hypoxia), but not lymphoid-related ones were upregulated in patients with 12 months OS. Conclusions Based on a novel combination of two high-plex spatial analysis techniques, we propose mesothelioma subtypes where macrophages act as immune inhibitory or activated with the latter displaying a possible T-cell excluded phenotype. We also show that deep-profiling of biology within the TME identifies prognostic markers of OS. Thus, we hypothesize that this approach may guide a better understanding and help to develop effective immunotherapies. Legal entity responsible for the study Bernard A. Fox. Funding Nanostring and Definiens. Disclosure T. Herz: Full / Part-time employment: Definiens. S.E. Church: Shareholder / Stockholder / Stock options, Full / Part-time employment: Nanostring. M. Widmaier: Full / Part-time employment: Definiens. A. Budco: Full / Part-time employment: Definiens. D. Medrikova: Full / Part-time employment: Definiens. I. Kanchev: Full / Part-time employment: Definiens. A. Spitzmueller: Full / Part-time employment: Definiens. A. Shaepe: Full / Part-time employment: Definiens. A. White: Shareholder / Stockholder / Stock options, Full / Part-time employment: Nanostring. J. Reeves: Shareholder / Stockholder / Stock options, Full / Part-time employment: Nanostring. A.H. Sullivan: Shareholder / Stockholder / Stock options, Full / Part-time employment: Nanostring. M. Bailey: Shareholder / Stockholder / Stock options, Full / Part-time employment: Nanostring. R. Sanborn: Advisory / Consultancy: CellDex. C. Bifulco: Advisory / Consultancy: Ventana/Roche; Advisory / Consultancy: BMS; Advisory / Consultancy: HalioDx; Advisory / Consultancy, Shareholder / Stockholder / Stock options: PrimeVax. S. Warren: Shareholder / Stockholder / Stock options, Full / Part-time employment: Nanostring. J.M. Beechem: Shareholder / Stockholder / Stock options, Full / Part-time employment: Nanostring. B. Fox: Advisory / Consultancy: Argos; Advisory / Consultancy: AstraZeneca/MedImmune; Advisory / Consultancy, Research support: Bristol-Myers Squibb; Advisory / Consultancy: Bayer; Advisory / Consultancy: CellDex; Advisory / Consultancy: Clearlight; Advisory / Consultancy, Research support: Definiens; Advisory / Consultancy, Research support: Janssen; Advisory / Consultancy, Research support: Macrogenics; Advisory / Consultancy, Research support: Nanostring; Advisory / Consultancy, Research support: OncoSec; Advisory / Consultancy, Research support: PerkinElmer/Akoya; Advisory / Consultancy, Shareholder / Stockholder / Stock options: PrimeVax; Advisory / Consultancy, Research support: Quanterix; Advisory / Consultancy, Research support: Shimadzu; Advisory / Consultancy: Ultivue; Advisory / Consultancy, Research support: Ventana/Roche; Advisory / Consultancy, Research support: Viralytics/Merck; Leadership role, Shareholder / Stockholder / Stock options: UbiVac. All other authors have declared no conflicts of interest.