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Introduction: Immune cells in the tumour microenvironment are associated with prognosis and response to therapy. We aimed to comprehensively characterise the spatial immune phenotypes in the mutational and clinicopathological background of non-small cell lung cancer (NSCLC)., Methods: We established a multiplexed fluorescence imaging pipeline to spatially quantify 13 immune cell subsets in 359 NSCLC cases: CD4 effector cells (CD4-Eff), CD4 regulatory cells (CD4-Treg), CD8 effector cells (CD8-Eff), CD8 regulatory cells (CD8-Treg), B-cells, natural killer cells, natural killer T-cells, M1 macrophages (M1), CD163+ myeloid cells (CD163), M2 macrophages (M2), immature dendritic cells (iDCs), mature dendritic cells (mDCs) and plasmacytoid dendritic cells (pDCs)., Results: CD4-Eff cells, CD8-Eff cells and M1 macrophages were the most abundant immune cells invading the tumour cell compartment and indicated a patient group with a favourable prognosis in the cluster analysis. Likewise, single densities of lymphocytic subsets (CD4-Eff, CD4-Treg, CD8-Treg, B-cells and pDCs) were independently associated with longer survival. However, when these immune cells were located close to CD8-Treg cells, the favourable impact was attenuated. In the multivariable Cox regression model, including cell densities and distances, the densities of M1 and CD163 cells and distances between cells (CD8-Treg-B-cells, CD8-Eff-cancer cells and B-cells-CD4-Treg) demonstrated positive prognostic impact, whereas short M2-M1 distances were prognostically unfavourable., Conclusion: We present a unique spatial profile of the in situ immune cell landscape in NSCLC as a publicly available data set. Cell densities and cell distances contribute independently to prognostic information on clinical outcomes, suggesting that spatial information is crucial for diagnostic use., Competing Interests: Conflict of interest statement The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: K.L. is a board member of Cantargia AB, a company developing IL1RAP inhibitors. This does not alter the author's adherence to all guidelines for publication and does not present a conflict of interest. All authors otherwise declare no conflicts of interest., (Copyright © 2023 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Background: Cancer immunity is based on the interaction of a multitude of cells in the spatial context of the tumour tissue. Clinically relevant immune signatures are therefore anticipated to fundamentally improve the accuracy in predicting disease progression., Methods: Through a multiplex in situ analysis we evaluated 15 immune cell classes in 1481 tumour samples. Single-cell and bulk RNAseq data sets were used for functional analysis and validation of prognostic and predictive associations., Findings: By combining the prognostic information of anti-tumoural CD8 + lymphocytes and tumour supportive CD68 + CD163 + macrophages in colorectal cancer we generated a signature of immune activation (SIA). The prognostic impact of SIA was independent of conventional parameters and comparable with the state-of-art immune score. The SIA was also associated with patient survival in oesophageal adenocarcinoma, bladder cancer, lung adenocarcinoma and melanoma, but not in endometrial, ovarian and squamous cell lung carcinoma. We identified CD68 + CD163 + macrophages as the major producers of complement C1q, which could serve as a surrogate marker of this macrophage subset. Consequently, the RNA-based version of SIA (ratio of CD8A to C1QA) was predictive for survival in independent RNAseq data sets from these six cancer types. Finally, the CD8A/C1QA mRNA ratio was also predictive for the response to checkpoint inhibitor therapy., Interpretation: Our findings extend current concepts to procure prognostic information from the tumour immune microenvironment and provide an immune activation signature with high clinical potential in common human cancer types., Funding: Swedish Cancer Society, Lions Cancer Foundation, Selanders Foundation, P.O. Zetterling Foundation, U-CAN supported by SRA CancerUU, Uppsala University and Region Uppsala., Competing Interests: Declaration of interests A.M. and T.S. are co-inventors on a provisional patent application P42105124SE00 “Novel biomarker” regarding a method for the prognosis of survival time of a subject diagnosed with a cancer described herein. K.L. is a board member of Cantargia AB, a company developing IL1RAP inhibitors. This does not alter the Author's adherence to all guidelines for publication. No other funding except listed in the section Methods/Funders was involved. No other conflicts of interest were disclosed by the other authors., (Copyright © 2023 The Author(s). Published by Elsevier B.V. All rights reserved.)

Immune cells of the tumor microenvironment are central but erratic targets for immunotherapy. The aim of this study was to characterize novel patterns of immune cell infiltration in non-small cell lung cancer (NSCLC) in relation to its molecular and clinicopathologic characteristics. Lymphocytes (CD3+, CD4+, CD8+, CD20+, FOXP3+, CD45RO+), macrophages (CD163+), plasma cells (CD138+), NK cells (NKp46+), PD1+, and PD-L1+ were annotated on a tissue microarray including 357 NSCLC cases. Somatic mutations were analyzed by targeted sequencing for 82 genes and a tumor mutational load score was estimated. Transcriptomic immune patterns were established in 197 patients based on RNA sequencing data. The immune cell infiltration was variable and showed only poor association with specific mutations. The previously defined immune phenotypic patterns, desert, inflamed, and immune excluded, comprised 30, 13, and 57% of cases, respectively. Notably, mRNA immune activation and high estimated tumor mutational load were unique only for the inflamed pattern. However, in the unsupervised cluster analysis, including all immune cell markers, these conceptual patterns were only weakly reproduced. Instead, four immune classes were identified: (1) high immune cell infiltration, (2) high immune cell infiltration with abundance of CD20+ B cells, (3) low immune cell infiltration, and (4) a phenotype with an imprint of plasma cells and NK cells. This latter class was linked to better survival despite exhibiting low expression of immune response-related genes (e.g. CXCL9, GZMB, INFG, CTLA4). This compartment-specific immune cell analysis in the context of the molecular and clinical background of NSCLC reveals two previously unrecognized immune classes. A refined immune classification, including traits of the humoral and innate immune response, is important to define the immunogenic potency of NSCLC in the era of immunotherapy. © 2021 The Authors. The Journal of Pathology published by John Wiley & Sons, Ltd. on behalf of The Pathological Society of Great Britain and Ireland., (© 2021 The Authors. The Journal of Pathology published by John Wiley & Sons, Ltd. on behalf of The Pathological Society of Great Britain and Ireland.)

Gene amplification is considered to be one responsible cause for upregulation of Programmed Death Ligand-1 (PD-L1) in non-small cell lung cancer (NSCLC) and to represent a specific molecular subgroup possibly associated with immunotherapy response. Our aim was to analyze the frequency of PD-L1 amplification, its relation to PD-L1 mRNA and protein expression, and to characterize the immune microenvironment of amplified cases. The study was based on two independent NSCLC cohorts, including 354 and 349 cases, respectively. Tissue microarrays were used to evaluate PD-L1 amplification by FISH and PD-L1 protein by immunohistochemistry. Immune infiltrates were characterized immunohistochemically by a panel of immune markers (CD3, CD4, CD8, PD-1, Foxp3, CD20, CD138, CD168, CD45RO, NKp46). Mutational status was determined by targeted sequencing. RNAseq data was available for 197 patients. PD-L1 amplification was detected in 4.5% of all evaluable cases. PD-L1 amplification correlated only weakly with mRNA and protein expression. About  37% of amplified cases were negative for PD-L1 protein. PD-L1 amplification did not show any association with the mutational status. In squamous cell cancer, PD-L1 amplified cases were enriched among patients with high tumoral immune cell infiltration and showed gene expression profiles related to immune exhaustion. In conclusion, PD-L1 amplification correlates with PD-L1 expression in squamous cell cancer and was associated with an immune cell rich tumor phenotype. The correlative findings help to understand the role of PD-L1 amplification as an important immune escape mechanism in NSCLC and suggest the need to further evaluate PD-L1 amplification as predictive biomarker for checkpoint inhibitor therapy., (© 2021. The Author(s).)