Natural Killer Defective Maturation Is Associated with Adverse Clinical Outcome in Patients with Acute Myeloid Leukemia

Abstract

Accumulating evidence highlights natural killer (NK) cell parameters as potential prognostic factors in cancer patients, which provides a strong rationale for developing therapeutic strategies aiming at restoring NK cell. However, reaching this point warrants better characterization of tumor-induced NK cell alterations. Our group recently reported heterogeneous NK maturation in acute myeloid leukemia (AML) patients. However, the clinical significance of such observations remained to be assessed on a larger cohort of patients. NK maturation based on expression of CD56, CD57, and KIR was assessed by flow cytometry in newly diagnosed AML patients (N = 87 patients from GOELAMS-LAM-IR-2006 multicenter trial). Clinical outcome was evaluated with regard to NK maturation profiles. Unsupervised integrated analysis of NK maturation markers confirmed the existence of three distinct groups of patients [hypomaturation (24.1%), intermediate maturation (66.7%), and hypermaturation (9.2%)]. In univariate analysis, significant differences in overall survival (OS) (P = 0.0006) and relapse-free survival (RFS) (P < 0.0001) were observed among these different groups. Patients with hypomaturation profile had reduced OS, with 3-year OS rates of 12.5 vs 57.1 and 57.4% for patients with intermediate and hypermaturation, respectively. Consistently, patients with hypomaturation profile had reduced RFS, with 3-year RFS rates of 0 vs 52.6 and 73.3% for patients with intermediate and hypermaturation, respectively. In multivariate Cox regression models, NK hypomaturation remained significantly associated with reduced OS and RFS, independent of other factors [hazard ratio (HR) = 4.15, P = 0.004 and HR = 8.23, P = 0.003, respectively]. NK maturation defects were further explored by mass cytometry and revealed that NK hypomaturation profile is associated with a reduced frequency of memory-like NK cells. In conclusion, besides classical alterations of NK triggering and inhibitory receptors expression in AML, we confirm that the homeostasis of NK maturation can be modified in the context of AML, notably with a deep maturation blockade in almost 10% patients.

Keywords: acute myeloid leukemia; mass cytometry; natural killer; natural killer maturation; prognostic biomarkers.

Figure 1

Unsupervised classification of acute myeloid leukemia patients according to natural killer (NK) maturation profiles. Patients and HVs were classified according to the percentage of NK cells represented in the CD56^bright, KIR⁻/CD57⁻, KIR⁺/CD57⁻, KIR⁻/CD57⁺, and KIR⁺/CD57⁺ clusters using hierarchical clustering (Euclidian distance). HVs, healthy volunteers.

Figure 2

Maturation profiles in the peripheral blood are representative of natural killer (NK) maturation profiles in the bone marrow (BM). (A) Examples of maturation profiles on appariated peripheral blood and BM samples by flow cytometry. (B) Correlation between the frequencies of NK cells in the different NK maturation clusters (Pearson correlation). Hyper, hypermaturation; Hypo, hypomaturation; Interm, intermediate maturation.

Figure 3

Defective natural killer (NK) maturation impacts clinical outcome. Kaplan–Meier curves of overall survival (OS) (A) and event-free survival (B) by NK maturation profile at diagnosis. Kaplan–Meier curves (C,D) display OS and relapse-free survival (RFS) by NKp30 status refined by NK maturation status: patients were classified in two groups according to NKp30 expression. Among patients with high NKp30 expression, patients were divided into two groups according to NK maturation.

Figure 4

Natural killer (NK) hypomaturation profile is associated low frequency of memory-like NK cells. NK alterations associated with the different maturation phenotypes were explored by mass cytometry. Peripheral blood mononuclear cells from 17 additional patients with newly diagnosed acute myeloid leukemia and 7 healthy volunteers (HVs) were analyzed by mass cytometry. Memory-like NK cells were defined as CD56^dim/CD57⁺/NKG2C⁺ NK cells. (A) Analysis of the percentage of memory-like NK cells by NK maturation group. (B) Gating strategy of memory-like NK cells on a Spade tree. (C) Representative Spade trees of patients and HVs. The first column represents CD56 expression, which defines the cluster of CD56^bright NK cells. The second column displays CD57 expression, which divides CD56^dim NK cells into CD57⁺ and CD57⁻ NK cells. The third column displays NKG2C expression, which enables to identify the cluster of memory-like NK cells, defined as CD56^dim/CD57⁺/NKG2C⁺ NK cells.