Abstract 1715: Phenotypic characterization of spatial immune infiltration niches in non-small cell lung cancer
Authors: Gerdtsson Anna Sandström , Mezheyeuski Artur , Micke Patrick , Ek Sara 
Published: June 2022

Abstract
Introduction: Prolonged response to checkpoint inhibitors is still rare in patients with non-small cell lung cancer (NSCLC) and the performance of current predictive biomarkers is limited. Thus, more accurate biomarkers and alternative treatment targets are needed to optimize immunotherapy. The objective of this study was to advance the understanding of cancer immunity by characterization of spatially resolved compartments of immune infiltration.
Material and methods: Using the GeoMx Digital Spatial Profiler (NanoString), 49 proteins were measured in 183 regions of interest (ROIs) from FFPE tissue sections of 33 NSCLC patients. The pan-leukocyte marker CD45 was used to define region of interests (ROIs) of three spatial phenotypes: (1) tumor infiltrating leukocytes (n=39), (2) tertiary lymphoid structures (TLS, n=19) and (3) tumor stroma without specific leukocyte aggregation (n=125). Differential protein expression (FDR&lt;0.05) was determined using generalized linear mixed models with patient as random effect, to account for multiple samples per patient.
Results: Normalized levels of lymphocyte phenotypic markers (CD3, CD8, CD4, and CD20) did not differ between stroma and tumor infiltrates, however the infiltrates had higher expression of markers of immune suppression (PD-L1, B7-H3, CTLA-4, IDO-1, PD-L2, 4-1BB, LAG3), regulatory T-cells (FOXP3), macrophages (CD68), and antigen presentation (CD40, OX40L, CD11c). The stromal ROIs were characterized by markers of fibroblast activation (Fibronectin, SMA, FAP alpha), and higher levels of VISTA and CD27 compared to infiltrating regions, and VISTA, ARG1 and CD66b compared to TLS. In comparison, TLS had higher levels of lymphocyte phenotypic markers (CD20, CD3, CD4, CD8), markers of activation (CD45RO, CD40, ICOS), proliferation (Ki67), and antigen presentation (HLA-DR, B2M, CD11c). In general, markers for macrophages (CD68), neutrophils (CD66b), regulatory T-cells (FOXP3) and immune suppression (B7-H3, IDO1, PD-L1, CTLA4) were more abundant in the vicinity of tumor cells, while CD27, ICOS, SMA and CD3 increased with longer distance to tumor cells. PD-L1 and IDO1 protein expression was higher in tumors that were immunohistochemical positive for PD-L1.
Conclusions: Spatially resolved protein profiling showed that NSCLC TLS are characterized by activated, proliferating lymphocytes and antigen presentation. We identified a general immunosuppressive microenvironment and indication of T cell exhaustion in the tumor cell compartment and its close vicinity. The clear discrimination of different immune infiltration niches independent of the overall level of leukocytes in the sampled regions illustrates the value of spatial profiling for deconvoluting how immune cells can dictate tumors, and to identify predictive immune signatures in the context of immunomodulatory treatment of NSCLC patients.
Citation Format: Anna Sandström Gerdtsson, Artur Mezheyeuski, Patrick Micke, Sara Ek. Phenotypic characterization of spatial immune infiltration niches in non-small cell lung cancer [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 1715.