Biological Features of Response to Immune Checkpoint Inhibitor and Rituximab Priming in Diffuse Large B-Cell Lymphoma

Introduction: Immune checkpoint inhibitors (ICIs) show efficacy in a subset of patients with diffuse large B-cell lymphoma (DLBCL). Biomarkers of response and resistance to ICIs are needed to not only optimize their use but uncover novel biological insights into DLBCL. Here we perform whole transcriptomic analysis of pre-treatment tumour biopsies in DLBCL patients treated with ICI in the AvR-CHOP clinical trial (NCT03244176).

Methods: AvR-CHOP was a phase II window study in 28 newly diagnosed DLBCL patients (Manos et al. Leukaemia 2023). Immune priming with avelumab (PD-L1 inhibitor) and rituximab was followed by six cycles of R-CHOP chemotherapy and avelumab maintenance. The window study design allowed for investigation of response to immune priming without the confounding effects of chemotherapy. PET response parameters including total metabolic tumour volume (TMTV) were evaluated after immune priming and at key timepoints during therapy. To discover biomarkers, we performed bulk RNA sequencing of pre-treatment biopsies. Differences in gene expression were examined by stratifying patients according to a reduction in TMTV by 75% (TMTV75) after immune priming. Two patients with progressive disease with immune priming and R-CHOP chemotherapy were also assessed. RNA sequencing was analysed using differential gene expression (DGE) with DESeq2 (Love et al. Genome Biology 2014) and gene set enrichment analysis using gene ontology biological processes (GO GSEA) and Kyoto Encyclopedia of Genes and Genomes (KEGG). Transcriptomic analysis extended to classification of each sample into a lymphoma microenvironment (LME) and comparison of functional gene expression signatures (FGES) using procedures previously published (Kotlov et al. Cancer Discovery 2021).

Results: Whole transcriptomic analysis was performed in 14 patients with available RNA. Immunotherapy priming responders (n=7, 50%) with TMTV75 included 3 patients with primary mediastinal B-cell lymphoma and one patient with Epstein-Barr Virus positive DLBCL. DGE showed patients responding to immunotherapy priming with TMTV75 had significantly increased gene expression of PD-L1 (p<0.001) and cancer testis antigens (CTAs) such as PRAME and CT45A5 (p <0.001). GO GSEA showed activation of pathways pertaining to adaptive immune response, response to cytokines and innate immune response (p<0.05) whilst KEGG pathway analysis showed increased cytokine-cytokine receptor pathway interactions (p<0.05). Immunotherapy priming responders with TMTV75 belonged to the mesenchymal stromal (n=3, 42%), inflammatory (n=2, 29%) or immune depleted LMEs (n=2, 29%). Non-responders belonged to either germinal centre (n=3, 43%), mesenchymal stromal (n=1, 14%), inflammatory (n=1, 14%) or immune depleted (n=2, 29%) subtypes. Two patients with progressive disease with immune priming and R-CHOP chemotherapy had significantly decreased innate immune responses in GO GSEA analysis whilst KEGG analysis showed reduced antigen processing and presentation pathways (p<0.05). Consistent with this analysis, FGES in the two non-responders showed reduced MHC Class II gene signature as well as reduced T-cell traffic, and NK cell signatures (p<0.05, unadjusted).

Conclusions: Responders to immune priming with avelumab and rituximab stratified by TMTV75 had increased gene expression of CTAs and PD-L1 as well as enrichment in pathways related to adaptive immune response, response to cytokines and innate immune response. Two patients with progressive disease with immune priming and R-CHOP chemotherapy had significantly decreased antigen processing and presentation pathways, reduced innate immune responses and reduced gene expression signatures of MHC Class II pathways. These findings raise the hypothesis regarding whether patients with DLBCL treated with ICI are stimulating elements of adaptive and innate immunity and, in particular, neo-epitope specific T-cells to CTAs or other lymphoma neoantigens. These preliminary findings warrant further exploration in other cohorts of DLBCL patients treated with ICIs and other immune therapies.

Chong:Amgen, AstraZeneca, Bayer, Dizal Pharma, HUTCHMED, Incyte, Innate Pharma, Merck, Pfizer, Pharmacyclics, Roche: Research Funding; Bristol Myers Squibb, Regeneron Pharmaceuticals, Inc.: Consultancy, Research Funding; Takeda: Consultancy. Barraclough:Gilead: Honoraria; Roche: Honoraria; Novartis: Honoraria; Beigene: Honoraria. Wight:Sobi: Membership on an entity's Board of Directors or advisory committees. Swain:Limbic: Honoraria; Gilead: Honoraria. Hawkes:Novartis: Membership on an entity's Board of Directors or advisory committees; Antengene: Membership on an entity's Board of Directors or advisory committees; Regeneron Pharmaceuticals, Inc.: Membership on an entity's Board of Directors or advisory committees, Other: Trial Steering Committee, Speakers Bureau; AstraZeneca: Membership on an entity's Board of Directors or advisory committees, Other: Trial Steering Committee, Research Funding, Speakers Bureau; Sobi: Membership on an entity's Board of Directors or advisory committees; Merck Sharpe and Dohme: Membership on an entity's Board of Directors or advisory committees; Gilead: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Merck KGaA: Research Funding; Bristol Myer Squibb: Membership on an entity's Board of Directors or advisory committees, Other: Trial Steering Committee, Research Funding; Janssen: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Roche: Membership on an entity's Board of Directors or advisory committees, Research Funding. Keane:Gilead: Consultancy; Merck: Consultancy, Speakers Bureau; Roche: Consultancy; Takeda: Speakers Bureau; Astra Zeneca: Speakers Bureau.