Evaluation of the Prognostic Significance and Effect of NT5E-Adenosine Axis, a Novel Immune Checkpoint, in Diffuse Large B-Cell Lymphoma

Background: The NT5E-adenosine axis constitutes one of the most promising immunosuppressive pathways in immune-oncology. NT5E is also named as CD73, which catalyzes the conversion of AMP to adenosine, and then the extracellular adenosine within tumor microenvironment bindings to A2a adenosine receptor (A2aR) to further dampen T cell-mediated immune responses and promote tumor immune escape. Several anti-CD73 or anti-A2aR antibodies are being evaluated in different types of malignancies in clinical trials. However, the prognostic significance and effect of NT5E-adenosine axis in diffuse large B-cell lymphoma (DLBCL) remain unclear.

Methods: Multiplexed immunofluorescence staining, and a professionally and automatically assessed computer-assisted platform were applied to localize and quantify the markers from the DLBCL tumor and microenvironment cells. Gene expression status was analyzed according to the microarray data. The associations among marker expression patterns or their correlations with clinicopathological characteristics were estimated with the χ² test and two-tailed Spearman analyses. Co-culture system of CD73 positive DLBCL cells and primary CD8 positive T cells was applied to evaluate the effect of NT5E-adenosine axis in DLBCL.

Results: We found that CD73 was widely expressed on tumor and immune cells in DLBCL tissue. The CD73 positive rate on tumor cells was 52.31%, and CD73 expression on tumor cells was significantly associated with several clinicopathologic parameters, including IPI score (p = 0.020), LDH levels (p = 0.016) and Ki67 expression (p = 0.014). The A2aR positive rate on tumor-infiltrating lymphocytes (TILs) was 43.08%, and A2aR expression on TILs was significantly correlated with several clinicopathologic parameters, including IPI score (p = 0.036), clinical stage (p = 0.011), B symptom (p = 0.024) and Ki67 expression (p = 0.036). No significant difference in OS was observed in 233 DLBCL patients stratified by their CD73 gene expression status according to the microarray data (p = 0.267). There was also no significant effect of total CD73 protein expression on OS for the patients. However, a significant difference in OS was found when the patients were stratified by the CD73 expression on tumor cells, and the median survival times of the patients with CD73⁺/Pax-5⁺ and those with CD73^-/Pax-5⁺ were 57.8 months (95% CI: 46.4-69.3) and 73.5 months (95% CI: 65.9-81.2), respectively. Patients with CD73⁺/Pax-5⁺ experienced significantly poorer outcomes than those with CD73^-/Pax-5⁺ (p = 0.027). Furthermore, the median survival times of the patients with A2aR⁺ TILs and those with A2aR^- TILs were respectively 53.3 months (95% CI: 40.6-66.0) and 74.5 months (95% CI: 67.5-81.5), and patients with A2aR⁺ TILs also had a significantly shorter survival time than those with A2aR^- TILs (p = 0.003). Spearman's analysis revealed that CD73⁺/Pax-5⁺ tumor cells were positively correlated with A2aR⁺ TILs (R=0.395, p = 0.001). In a cohort, we found that 24 exhibited CD73^-/Pax-5⁺ as well as A2aR^- TILs, and 21 exhibited CD73⁺/Pax-5⁺ as well as A2aR⁺ TILs. Patients with CD73⁺/Pax-5⁺ and A2aR⁺TILs had poorer survival than those with CD73^-/Pax-5⁺ and A2aR^- TILs (p = 0.001). Residual patients were further classified into 2 groups, in which 7 exhibited CD73^-/Pax-5⁺ as well as A2aR⁺ TILs, and 13 displayed CD73⁺/Pax-5⁺ as well as A2aR^- TILs. There was a significant difference in survival among these four groups, and patients with CD73⁺/Pax-5⁺ and A2aR⁺ TILs had the worst outcome, with a 5-year OS rate of 57.1% (p = 0.017). We also found that CD73 positivity on tumor cells weakened the favorable prognosis for patients, which was correlated with the presence of CD8⁺ TILs. Patients with CD73⁺/Pax-5⁺ and CD8⁺ TILs^low had the worst clinical outcome, with a 5-year OS rate of 50%. In contrast, patients with CD73^-/Pax-5⁺ and CD8⁺ TILs^high had the best clinical outcome, with a 5-year OS rate of 100% (p = 0.002). Co-culture system displayed that CD73 being expressed on the DLBCL cells suppressed the growth of CD8 positive T cells through A2aR, but not A2aR negative T cells.

Conclusions: Our findings uncovered that DLBCL patients with CD73⁺ on tumor cells as well as A2aR⁺ on TILs exhibited inferior survival and NT5E-adenosine axis inhibited the growth of CD8 positive T cells, supporting the potential combination strategies using CD73/A2aR immunosuppressive blockades as treatment options for DLBCL patients.

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