The Diffuse Large B-Cell Lymphoma Infiltrating Macrophage Transcriptome Signature Is Enriched for Both M1 and M2 Genes and Provides an Excellent Platform for Functional Validation of Macrophage Biology in DLBCL

Abstract

AbstractAbstract 790Several investigators have defined gene expression profiling (GEP) signatures in Diffuse Large B-cell Lymphoma (DLBCL) enriched for macrophage and stromal genes, suggesting an active innate immune response against lymphoma. We hypothesize that the malignant B-cells drive tumor-associated macrophage (TAM) dysfunction in a subset of patients with DLBCL which is relevant for their biology and prognosis. We performed GEP on TAM from diagnostic DLBCL in order to recognise key genes and pathways open to functional validation.Single cell suspensions from 8 DLBCL and 8 reactive lymph nodes were used in this study. TAMs were flow sorted using CD36 expression. cDNA synthesis and amplification was performed using the Nugen Ovation Pico WTA system and the Affymetrix GeneChip human gene 1.0 ST platform was used. We used bioinformatics analysis of GEP data from DLBCL whole tumors, DLBCL purified B-cells, in vitro manipulated macrophages and other immune cells in order to define macrophage-enriched genes as well as specific M1/M2 signatures.We identified a 221 gene signature that significantly distinguished DLBCL TAMs from control macrophages, with 165 genes upregulated and 56 genes downregulated. Moreover, a comparative transcriptome analysis of 22 diverse immune cell phenotypes/activation states (IRIS: GSE22886) revealed that 26% of these genes are highly macrophage-enriched.Gene Ontology analysis revealed an over-representation for transcripts involved in inflammatory response (p 6.8×10−23), wound healing (p 1.9×10−20), chemotaxis (p 3.2 ×10−9), and cell motility (p 1.7×10−7).Upregulated genes in TAMs included well known M1 (complement components, CXCL9 or CXCL10) as well as M2 genes (MSR, CD163 or MARCO) (Table 1). In our signature, there was enrichment for M1 compared to M2 genes as defined by bioinformatics analysis.TAMs showed overexpression of the CSF1R gene as well as the chemokines CCL2 and CCL5, suggesting an autocrine feed-back loop of macrophage chemotaxis and survival in DLBCL. Moreover, TAMs showed upregulation of the lymphocyte attractants CCL20, CXCL9 and CXCL10, together with T-cell immunosupressants indoleamine 2,3-dioxygenase 1 and PD-L1, which would support a role for macrophages in T-cell recruitment and dysfunction in DLBCL. We also saw strong upregulation of 7 metallothionein isoforms in TAMs. These are proteins known to be expressed in macrophages and linked to response to oxidative damage, modulation of inflammation and cell proliferation. However their role in cancer microenvironment is unclear.We describe for the first time the GEP from DLBCL TAMs. The TAM transcriptome has partial overlapping genes with both M1 and M2 gene signatures, but also has a characteristic GEP potentially driven by their presence in the DLBCL microenvironment. Although further molecular and functional validation is required, this data provides a platform of genes which serve as excellent candidates for future exploration to understand DLBCL pathogenesis and to define new therapeutic targets.Table 1:Genes of particular interest represented in our TAM signature. Probe collapse was done using the highest expression. The Benjamini-Hochberg multiple hypothesis test was used to determine significance (FDR 0.05).Gene IDsGene descriptionLog2 Fold ChangeAdjusted p valueMT1E-H, L, M, X, MT2Ametallothionein 1E-H, 1M, 1X, 2A2.3 – 4.40.00145 - 0.00017C1QA, B and Ccomplement component 1, q subcomponent, A, B and C chains3.1 – 3.60.00136 - 0.00255C2complement component 23.20.00136CCL2, 4, 5, 8, 20chemokine (C-C motif) ligand 2, 4, 5, 8 and 202.4 - 3.70.046 - 0.00130CD163CD163 molecule4.60.00025CD14CD14 molecule3.40.00091CD274CD274 molecule3.70.00447CSF1Rcolony stimulating factor 1 receptor2.10.00886CXCL9-11chemokine (C-X-C motif) ligand 9, 10 and 114.2 – 4.40.012 - 0.00158FCGR1BFc fragment of IgG, high affinity Ib, receptor (CD64)4.90.00091FCGR2AFc fragment of IgG, low affinity IIa, receptor (CD32)3.30.00139FCGR3AFc fragment of IgG, low affinity IIIa, receptor (CD16a)5.20.00011IDO1indoleamine 2,3-dioxygenase 14.80.00232MARCOmacrophage receptor with collagenous structure2.60.02029MSR1macrophage scavenger receptor 13.70.01467 Disclosures:Gribben:Celgene: Honoraria; Roche: Honoraria; Pharmacyclics: Honoraria; GSK: Honoraria; Mundipharma: Honoraria; Gilead: Honoraria.