The Immune Checkpoint Receptors PD-1, PD-L1, TIM-3 and LAG-3 in Lymphoma: Tumor Cell and Tumor Infiltrating Lymphocyte (TIL) Expression, Patient Prognostication, and Identification of Rational Therapeutic Targets

Introduction: Programmed cell death-1 (PD-1) based immunotherapy represents a significant advance in the treatment of hematologic malignancies, including lymphoma. Mechanisms of resistance and potential methods to overcome this treatment paradigm are a critical focus of research. Signaling through additional immune checkpoint receptors, such lymphocyte-activation gene-3 (LAG-3) and T cell immunoglobulin and mucin domain-3 (TIM-3), may lead to T-cell exhaustion and be a mechanism of immune escape for tumors. However, the expression patterns of TIM-3 and LAG-3 in lymphoma, including DLBCL and HL, are largely unknown. Furthermore, potential override of resistance to PD-1 blockade via rational co-targeting of checkpoint receptors represents a novel therapeutic strategy.

Methods: Tissue sections (whole sections and tissue microarrays) of newly diagnosed patient cases of diffuse large B-cell lymphoma (DLBCL, n=123) and Hodgkin lymphoma (HL, n=30) were stained with antibodies to PD-L1, PD-1, TIM-3, and LAG-3. Immunohistochemical (IHC) expression of each marker on tumor cells and on tumor infiltrating lymphocytes (TILs) were scored. Staining patterns were correlated with additional pathologic and clinical parameters as well as patient outcome (i.e., progression free survival (PFS) and overall survival (OS)) among n=59 newly diagnosed advanced-stage DLBCL patients treated from 2005 to 2013 with rituximab-CHOP therapy. 4-year survival rates were estimated by Kaplan-Meier with differences assessed by log rank test. Univariate analysis and multivariate regression according to the Cox proportional hazards regression model were used (for International Prognostic Index (IPI) and TIM-3). In addition, the effects of in vitro checkpoint blockade were evaluated using investigational anti-PD-1, anti-TIM-3 or anti-LAG-3 compounds with tumor-primed T cells and Raji or SUDHL10 as target DLBCL cells. Cell viability was assessed using AcellaTox assay and IL-2 release quantified by ELISA.

Results: TIM-3 showed strong, membranous staining (H-score >80) on tumor cells in over 1/3 of DLBCL (39%, 48/123) and the majority of HL cases (70%, 21/30). Furthermore, PD-L1 was expressed (>30% cells tumor cells positive) in essentially all HL cases (100%, 30/30) and in a subset of DLBCL cases (15%, 19/126). This is similar to our and others' previously reported data (Xing W et al. O ncotarget 2016). Both PD-1 and LAG-3 in the current study were positive on tumor cells in only a minority of DLBCL cases (8% and 7%, respectively), and rarely in HL tumor cells. Conversely, PD-1 and LAG-3 were widely expressed on the TILs found in DLBCL in 75% and 94% of cases, respectively. With a median follow-up of 44 months (range 5-85), 4-year PFS and OS rates were significantly inferior among DLBCL patients with high vs low/negative TIM-3 expression (i.e., PFS: 23% (95% CI 7% to 46%) vs 60% (95% CI 43% to 74%), respectively, P=0.008; and OS: 30% (95% CI 10% to 53%) vs 74% (95% CI 58% to 85%), respectively, P=0.006) (seeFigure 1A/1B). Furthermore, when controlling for the IPI, both survival endpoints remained significant with high TIM-3 predicting increased risk of progression (PFS HR 2.60 (95% CI 1.22-5.53, P=0.013) and OS predicting increased risk of death (OS HR 3.36, 95% CI 1.52-7.42, P=0.003) on multivariate Cox regression analyses. In addition, we examined these checkpoint receptors as potential therapeutic targets. Co-culture of PD-L1 expressing Raji cells with tumor-dendritic cell primed T cells in the presence of anti-LAG-3 induced a potent dose-dependent increase in in vitro cell death with 15nM IC₅₀ and IL-2 secretion (120pg/ml) as determined by AcellaTox & ELISA based assays (see Figure 1C).

Conclusions: Altogether, IHC analyses showed strong expression of the TIM-3 immune checkpoint receptor in over 1/3 of DLBCL and in the majority of HL tumors. Furthermore, analysis of patient outcomes identified that DLBCL patients with high TIM-3 expression (i.e., H-score >80) had significantly inferior survival rates. PD-1 and LAG-3 were consistently expressed in the TILs of DLBCL and HL, but were infrequently expressed by tumor cells. Therapeutically, we identified a dose-dependent increase in Raji-targeted cell death and increased T cell activity with a novel anti-LAG-3 compound. Continued examination of mechanistic studies of these immune checkpoint receptors and associated rational treatment studies are warranted.

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