CLINICAL SIGNIFICANCE OF T-CELL EXHAUSTION IN PATIENTS WITH DIFFUSE LARGE B-CELL LYMPHOMA

Introduction: Tumor microenvironment (TME) and limited immune surveillance play important role in lymphoma pathogenesis and survival. Immune checkpoint receptors, such as programmed cell death-1 (PD1), lymphocyte-activation gene-3 (LAG3) and T-cell immunoglobulin and mucin domain-3 (TIM3), mediate signals leading to T-cell exhaustion and immune escape. Here, we have characterized the immunological profiles of diffuse large B-cell lymphoma (DLBCL) and associated the findings with outcome. Methods: Gene expression analysis was conducted for 81 DLBCL samples utilizing the NanoString platform with a 770-gene PanCancer Immune panel. Multiplex immunohistochemistry (mIHC) with digital image analysis was used to characterize the T-cell phenotypes, including cytotoxic T-cells (CD8, GrB, OX40, Ki67), exhausted T-cells (CD3, CD4, CD8, PD1, TIM3, LAG3), Tregs and Th1 effector cells (CD3, CD4, FOXP3, TBET) of a total of 225 DLCBL samples. The findings were associated with patient demographics and survival and clinical significance validated in an independent patient cohort. Results: We observed a high degree of heterogeneity in transcriptome level among the DLBCL samples. Correlation matrix analysis identified gene expression signatures with highly correlating genes. The main signatures contained genes for cytolytic factors and immune checkpoint molecules (GZMB, PRF1, IFNG, TIM3, LAG3) and T-cells (CD3, CD2, CD28), together entitled as a T-cell signature, macrophages (CD68, CD163), B-cells (MS4A1, CD19, CD79A/B), and extracellular matrix (FN1, ITGA1/5/6, VEGFA). None of the gene expression signatures as such were associated with survival. However, immunophenotyping of the distinct T-cell subsets with mIHC revealed that a high proportion of exhausted T-cells (TIM3+ and/or LAG3+) was associated with unfavorable survival in a cohort of 51 young high risk DLBCL patients treated in a Nordic phase II (NLG-LBC-05) trial with dose-dense immunochemotherapy and systemic CNS prophylaxis (5-year OS 73% vs. 96%, p = 0.022 and 5-year PFS 74% vs. 93%, p = 0.064). In contrast, the number of cytotoxic T cells, Tregs or Th1 effector cells did not correlate with outcome. The adverse prognostic impact of exhausted T-cells on survival was validated in an independent cohort of 137 DLBCL patients treated with immunochemotherapy (5-year OS 66% vs. 79%, p = 0.029 and 5-year PFS 60% vs. 76%, p = 0.035). Conclusions: Our results demonstrate that the molecular immunological profile of DLBCL is heterogenic and that putative markers of T-cell exhaustion associate with unfavorable survival in patients with DLBCL. Keywords: diffuse large B-cell lymphoma (DLBCL); immunophenotype; T-cells.