Expression patterns of immune checkpoints in acute myeloid leukemia

1To the Editor,

2Immune checkpoint (IC) blockade by inhibitors of the programmed cell death 1 (PD-1) and PD-1 ligand 1 (PD-L1) has significantly improved clinical outcome for a variety of solid tumors, while little is known about the role of ICs in leukemia. Previous reports have shown that higher numbers of PD-1 + T cells are related to poor outcome for patients with acute myeloid leukemia (AML). Clinical trials using PD-1 inhibitors are ongoing to treat patients with a high risk for AML relapse. However, the response rate varies widely, ranging from 22 to 72%, which may be due to heterogeneity in the IC expression level as well as distinct dominant IC expression patterns in different AML cases. Therefore, it is worth studying the expression patterns of ICs in AML. In this study, we first explored the prognostic value of ICs in AML patients through analyzing RNA-seq and mutation data from the Cancer Genome Atlas (TCGA) database and further validated the results by quantitative real-time PCR analysis of AML bone marrow (BM) samples from our clinical center.

3A total of 176 de novo AML patients from the TCGA database and 62 AML BM samples were used for overall survival (OS) analysis and validation. Higher expression of PD-1, PD-L1, and PD-L2 correlated with poor OS in the TCGA database analysis (3-year OS 23% vs 38%, 19% vs 46%, and 15% vs 40%, respectively, P < 0.05). This result was confirmed in the validation group (3-year OS 40% vs 68%, 22% vs 64%, and 42% vs 68%, respectively, P < 0.05, Fig. 1a, b). We further analyzed the expression patterns of PD-1, PD-L1, and PD-L2 with other important ICs. Subsequently, with Pearson’s correlation analysis, we found that the expression of PD-1, PD-L1, or PD-L2 was positively associated with the expression of cytotoxic T-lymphocyte associated protein 4 (CTLA-4) (r = 0.259, P < 0.001; r = 0.435, P < 0.001; r = 0.269, P < 0.001, respectively) and lymphocyte activation gene-3 (LAG-3) (r = 0.275, P < 0.001; r = 0.276, P < 0.001; r = 0.160, P = 0.033, respectively) in the TCGA group (Fig. 1c). This concomitant expression pattern was again confirmed in the validation group (Fig. 1e), showing the possibility of concomitant expression of PD-1, PD-L1, or PD-L2 with CTLA-4 (r = 0.373, P = 0.003; r = 0.998, P < 0.001; r = 0.998, P < 0.001, respectively) and LAG3 (r = 0.372, P = 0.003; r = 0.994, P < 0.001; r = 0.994, P < 0.001, respectively). AML patients with high expression of CTLA-4 and LAG-3 were found to have poor OS (3-year OS 9% vs 36% and 13% vs 40% respectively) (Fig. 1d). This result was again confirmed in the validation group (Fig. 1f) (3-year OS: CTLA-4 34% vs 66%, LAG-3 33% vs 70%).

4Combination of IC inhibitors (ICIs) has the potential to improve responses. We analyzed expression patterns of ICs and found that pairwise combinations of PD-1, PD-L1, and PD-L2 and CTLA-4 and LAG-3 correlated with poor OS in AML patients (P < 0.05, Figure S1). Furthermore, among AML patients with high expressions of PD-1 or PD-L2, concomitant high expression of CTLA-4 correlated with poor OS in both the TCGA database (3-year OS: PD-1highCTLA-4low vs PD-1highCTLA-4high 30% vs 0%, 20% vs 0%) and validation group (3-year OS: PD-L2highCTLA-4low vs PD-L2highCTLA-4high 57% vs 31%, 57% vs 33%) (P < 0.05, Fig. 2a, b). AML with PD-L1highCTLA-4high correlated with poor OS in the TCGA dataset (3-year OS 24% vs 0%, P < 0.001); however, OS was not significantly different in the validation group (3-year OS 33% vs 20%, P = 0.353, Fig. 2a, b). In addition, high expression of LAG-3 with PD-1high, PD-L1high, or PD-L2high failed to correlate with OS in the TCGA and validation groups (Figures S2A - B).

5To obtain the effects of PD-1, PD-L1, and PD-L2 on the prognosis of AML patients with genetic mutations, we analyzed OS of the top ten AML patients with a recurrent mutation (Figures S3), including FLT3mut, RUNX1mut, or TET2mut. Interestingly, in comparison with AML patients without such mutations, high co-expressions of PD-1/PD-L1 (P = 0.029), PD-1/PD-L1/PD-L2 (P = 0.003), and PD-1/LAG-3 (P < 0.001) were found to be associated with poor OS in AML patients with FLT3mut, RUNX1mut, or TET2mut (1-year OS 0% vs 58% vs 49%, 0% vs 56% vs 100%, and 0% vs100% vs 63%, respectively) (Fig. 2c).

6To the best of our knowledge, we for the first time described that high co-expressions of PD-1/CTLA-4 and PD-L2/CTLA-4 correlated with poor OS of AML patients. Moreover, high co-expressions of PD-1/PD-L1, PD-1/PD-L1/PD-L2, and PD-1/LAG-3 were associated with poor OS of AML patients with FLT3mut, RUNX1mut, or TET2mut, respectively (Fig. 2d). These co-expression patterns might be potential immune biomarkers for designing novel AML therapy.