Natural killer-cell counts are associated with molecular relapse-free survival after imatinib discontinuation in chronic myeloid leukemia: the IMMUNOSTIM study

Abstract

Despite persistence of leukemic stem cells, patients with chronic myeloid leukemia who achieve and maintain deep molecular responses may successfully stop the tyrosine kinase inhibitor imatinib. However, questions remain unanswered regarding the biological basis of molecular relapse after imatinib cessation. In IMMUNOSTIM, we monitored 51 patients from the French Stop IMatinib trial for peripheral blood T cells and natural killer cells. Molecular relapse-free survival at 24 months was 45.1% (95% CI: 31.44%-58.75%). At the time of imatinib discontinuation, non-relapsing patients had significantly higher numbers of natural killer cells of the cytotoxic CD56^dim subset than had relapsing patients, while CD56^bright natural killer cells, T cells and their subsets did not differ significantly. Furthermore, the CD56^dim natural killer-cell count was an independent prognostic factor of molecular-relapse free survival in a multivariate analysis. However, expression of natural killer-cell activating receptors, BCR-ABL1⁺ leukemia cell line K562-specific degranulation and cytokine-induced interferon-gamma secretion were decreased in non-relapsing and relapsing patients as compared with healthy individuals. After imatinib cessation, the natural killer-cell count increased significantly and stayed higher in non-relapsing patients than in relapsing patients, while receptor expression and functional properties remained unchanged. Altogether, our results suggest that natural killer cells may play a role in controlling leukemia-initiating cells at the origin of relapse after imatinib cessation, provided that these cells are numerous enough to compensate for their functional defects. Further research will decipher mechanisms underlying functional differences between natural killer cells from patients and healthy individuals and evaluate the potential interest of immunostimulatory approaches in tyrosine kinase inhibitor discontinuation strategies. (ClinicalTrial.gov Identifier NCT00478985).

Figure 1.

Evolution of BCR-ABL1 transcripts in relapsing patients and molecular relapse-free survival. (A) Scatter dot plots represent BCR-ABL1 IS % for each individual relapsing patient at first detection of relapse, relapse confirmation and at imatinib reintroduction; median values (horizontal bars) are also shown. (B) Kaplan-Meier estimate of molecular relapse-free survival defined as the time interval between imatinib discontinuation and first occurrence of molecular relapse or death, whichever came first. Data were censored at last molecular assessment for patients who were alive and had not relapsed.

Figure 2.

Natural killer cells at imatinib discontinuation in non-relapsing and relapsing patients. (A) Flow cytometry dot plot from a representative patient showing CD3⁻CD56⁺ NK cells in the upper left quadrant after lymphocyte gating using the side and forward scatter display. (B) Scatter dot plots represent CD3⁻CD56⁺ NK-cell counts for each individual non-relapsing and relapsing patient; median values (horizontal bars) are also shown. (C) Scatter dot plots represent CD3⁻CD56^dim NK-cell counts for each individual non-relapsing and relapsing patient; median values (horizontal bars) are also shown. (D) Scatter dot plots represent CD3⁻CD56^bright NK-cell counts for each individual non-relapsing and relapsing patient; median values (horizontal bars) are also shown. P values (by the Mann-Whitney U-test) are indicated for each panel.

Figure 3.

Molecular relapse-free survival after discontinuation of imatinib according to clinico-biological factors. (A) Sokal risk group, (B) imatinib treatment duration, (C) CD56^dim NK-cell counts at baseline, (D) age, (E) sex, and (F) prior exposure to IFN-α. For each survival plot, a corresponding log-rank P value is shown.

Figure 4.

Expression of natural killer cell receptors in CD56^dim and CD56^bright natural killer cell subsets. Scatter dot plots for healthy donors (black dots, n=44), non-relapsing patients (filled gray dots, n=19) and relapsing patients (empty black dots, n=27) with median values (horizontal bars) are shown. Overall P value (by the Kruskall-Wallis test) is indicated for each panel.

Figure 5.

Natural killer-cell function. (A) NK-cell degranulation assay against K562 or medium control. (B) Target-specific degranulation estimated by the CD107a ratio. (C) NK-cell activation estimated by CD137 expression in the presence of K562 or medium control. (D) Production of IFN-γ upon stimulation with IL-12 and IL-18. Scatter dot plots for healthy donors (black dots, n=43), non-relapsing patients (filled gray dots, n=15) and relapsing patients (empty black dots, n=25) at baseline with median values (horizontal bars) are shown. Overall P value (by the Kruskall-Wallis test) is indicated for each panel.