Digital spatial profiling of diffuse large b‐cell lymphomas reveals sting as an immune‐related determinant of survival after r‐chop therapy

Background: Alterations in the immune infiltrate of Diffuse Large B-Cell Lymphoma (DLBCL) are known to influence survival. However, the relative prognostic importance of different immune components is not clear. Digital spatial profiling (DSP) enables highly multiplexed protein-based measurement of immune markers in a single tissue section. Here, we applied DSP to evaluate the prognostic significance of immune markers in R-CHOP treated DLBCL. Methods: We evaluated a custom NanoString immune panel in a cohort of R-CHOP treated DLBCL from the National University Hospital Singapore (n = 86) using DSP. Quantitative marker expression values of 29 immune features were correlated with clinicopathological features and with survival outcomes. Fluorescence-based quantitative immunohistochemistry (fIHC) was applied to an independent DLBCL cohort (MDA, n = 36) to validate the prognostic relevance of STING. We also analysed mRNA data sets from the following patient cohorts: GSE117556 (R-CHOP, n = 469/RB-CHOP, n = 459), GSE10846 (R-CHOP, n = 233/CHOP, n = 181), GSE32918 (R-CHOP, n = 141), GSE98588 (R-CHOP, n = 101) and GSE23501 (R-CHOP, n = 67). Results: In a multivariate cox proportional hazards model analysing DSP data, high levels of the monocytic/macrophagic marker CD163 were associated with poor progression free survival (PFS) and overall survival (OS) (HR progression 3.8, p = 0.008; HR death 5.4, p = 0.010) while high expression of the inflammatory mediator STING was associated with better PFS and OS (HR progression 0.33, p = 0.020; HR death 0.17, p = 0.007). Given the role of STING in mediating immune activation after genotoxic chemotherapy, we further evaluated the clinical relevance of pre-treatment STING expression. Quantitative fIHC of STING associated with improved survival in an independent cohort (n = 36, MDA). Furthermore, high expression of STING (TMEM173) correlated with favourable prognosis across multiple gene expression datasets: GSE117556, R-CHOP, HR death 0.58, p = 0.016; GSE117556, RB-CHOP, HR death 0.34, p = 0.0001; GSE10846, CHOP, HR death 0.46, p < 0.0001; GSE98588, R-CHOP, HR death 0.40, p = 0.019. Consistently, DLBCL cell lines activated immune signalling pathways when treated with chemotherapy in vitro, which suggested that STING levels could positively affect prognosis through inflammatory priming. In this setting, low STING expression could be reminiscent of an immunologically cold dark zone microenvironment imprint in DLBCL, being listed among genes that we found transcriptionally induced in the dark-zone to light-zone transition in reactive lymphoid follicles in situ. Conclusions: High expression of STING confers good prognosis in DLBCL, suggesting that STING signalling is important for chemotherapy induced immune clearance. STING agonists may thus have a potential application alongside chemotherapeutic approaches in DLBCL. The research was funded by: SBN and ADJ are supported by the Singapore Ministry of Health's National Medical Research Council Transition Awards (CSAINV17nov016 and NMRC/TA/0020/2013 and NMRC/TA/0052/2016). Work in ADJ's laboratory is funded by the Cancer Science Institute of Singapore, National University of Singapore through the National Research Foundation Singapore and the Singapore Ministry of Education under its Research Centres of Excellence initiative. This research is also supported by Singapore Ministry of Health's National Medical Research Council under its Singapore IYMPHoma translatiONal studY (SYMPHONY) (OFLCG18May-0028). Keywords: Tumor Biology and Heterogeneity, Diagnostic and Prognostic Biomarkers, Aggressive B-cell non-Hodgkin lymphoma Conflicts of interests pertinent to the abstract A. VanSchoiack Employment or leadership position: NanoString Technologies Inc A. D. Jeyasekharan Consultant or advisory role: Turbine Ltd, AstraZeneca, Janssen and MSD Research funding: Janssen and AstraZeneca Educational grants: Perkin Elmer