Increased proportion of mature NK cells is associated with successful imatinib discontinuation in chronic myeloid leukemia

Abstract

Recent studies suggest that a proportion of chronic myeloid leukemia (CML) patients in deep molecular remission can discontinue the tyrosine kinase inhibitor (TKI) treatment without disease relapse. In this multi-center, prospective clinical trial (EURO-SKI, NCT01596114) we analyzed the function and phenotype of T and NK cells and their relation to successful TKI cessation. Lymphocyte subclasses were measured from 100 imatinib-treated patients at baseline and 1 month after the discontinuation, and functional characterization of NK and T cells was done from 45 patients. The proportion of NK cells was associated with the molecular relapse-free survival as patients with higher than median NK-cell percentage at the time of drug discontinuation had better probability to stay in remission. Similar association was not found with T or B cells or their subsets. In non-relapsing patients the NK-cell phenotype was mature, whereas patients with more naïve CD56^bright NK cells had decreased relapse-free survival. In addition, the TNF-α/IFN-γ cytokine secretion by NK cells correlated with the successful drug discontinuation. Our results highlight the role of NK cells in sustaining remission and strengthen the status of CML as an immunogenic tumor warranting novel clinical trials with immunomodulating agents.

Figure 1

The proportion and absolute count of NK cells at the time of imatinib discontinuation. (a) Molecular relapse-free survival of imatinib treated patients at baseline based on the median NK-cell proportion (in all patients median 16%, range 5–48%). Log-rank test was used to analyze the statistical significance between the two groups. Hazard ratio is reported in Table 1. (b) Patients were dichotomized to low- and high-NK-cell groups according to receiver-operating characteristics (ROC) curves and the Youden index to find more optimal cutoff for groups than the median value (AUROC 0.568, 95% CI: 0.4543–0.6818). On the basis of these analyses 11.3% was used as a cutoff. Hazard ratio is reported in the Supplementary Table 2. (c) Clinical characteristics of patients according to their TKI discontinuation success. Patients were divided in three groups: non-relapsing (able to maintain remission for at least 12 months, n=49), early relapsing (relapse before 6 months, n=34) and late relapsing patients (relapse after 6 months, n=17). ICF, immune cell function. (d) The relative number of NK cells at baseline in patient groups and healthy volunteers (n=48). Median early relapsing 12.8%, late relapsing 20%, non-relapsing 17.8% and healthy 11.5%. (e) Absolute NK-cell count at baseline in patient groups and healthy volunteers. Median early relapsing 0.19 × 10⁹ cells per l, late-relapsing 0.31 × 10⁹ cells per l, non-relapsing 0.25 × 10⁹ cells per l and healthy 0.21 × 10⁹ cells per l. Box-and-whisker plots present 5–95 percentiles. One-way ANOVA was used for comparison between multiple groups (exact P-value reported in the figure), and Bonferroni's post test was used to compare selected pairs (only early-relapse group was compared with other groups to avoid multiple comparisons). Statistically significant differences between the groups are noted with asterisk (*P<0.05).

Figure 2

NK-cell phenotype at the time of imatinib discontinuation. Ficoll isolated fresh PB MNCs were analyzed with multicolor flow cytometry. NK cells were defined as CD3^-CD56⁺ lymphocytes. (a) Molecular relapse-free survival of imatinib treated patients at baseline based on the median proportion of CD56^bright NK cells. Log-rank test was used to analyze the statistical significance between the two groups. Hazard ratio is reported in Table 1. (b) Patients were dichotomized to low and high CD56^bright NK-cell groups according to ROC and the Youden index analyses (AUROC 0.6011; 95% CI: 0.4275–0.7748). 2.99% was used as a cutoff. Hazard ratio is reported in the Supplementary Table 2. (c) The proportion of CD56^dim cells of CD56+ NK cells in early (n=13), late (n=8) and non-relapsing (n=19) patients (d) The proportion of CD57+ NK cells. (e) The proportion of CD16+ NK cells. In c–e Box-and-whisker plots present 5–95 percentiles. One-way ANOVA was used for comparison between multiple groups (exact P-value reported in the figure), and Bonferroni's post test was used to compare selected pairs (only early relapse group was compared to other groups to avoid multiple comparisons). Statistically significant differences between the groups are marked with asterisk (*P<0.05, **P<0.01).

Figure 3

Adaptive-like NK cells in treatment discontinuation. Frozen PB MNCs from 1-month time-point were analyzed with multicolor flow cytometry. (a) Adaptive-like NK cells based on the downregulation of EAT2, SYK, FCɛR1γ and PLZF expression at 1 month. EAT-2^– in non-relapsing group 2.1% (n=19), early relapsing 0.5% (n=13), late relapsing 1.1% (n=7) of lymphocytes, **P<0.01. SYK^– non-relapsing 3.3%, early relapsing 1.3%, late relapsing 1.3% of lymphocytes, P=NS. FcɛRγ^– non-relapsing 5.9%, early relapsing 1.5%, late relapsing 3.5% of lymphocytes, P=0.18. PLZF^– non-relapsing 3.7%, early relapsing 1.4%, late relapsing 5.0% of lymphocytes, P=0.11. Box-and-whisker plots present 5–95 percentiles. (b) The proportion of EAT2- adaptive-like NK cells at 1 month plotted according to the treatment-free remission time (n=39). For non-relapsing patients, the latest follow-up time is reported. Statistical significance was analyzed with non-parametric Mann–Whitney test (a) and with Spearman correlation (b).

Figure 4

NK-cell activation and cytokine secretion at the time of imatinib discontinuation. MNCs collected at the baseline before imatinib discontinuation were stimulated with K562 cells for 6 hours at +37C. After, the cells were collected and stained for surface and intracellular markers and analyzed with flow cytometry. (a) Molecular relapse-free survival of imatinib treated patients at baseline based on the median proportion of CD16- NK cells secreting TNF-α/IFN-γ. Log-rank test was used to analyze the statistical significance between the two groups. Hazard ratio is reported in Table 1. (b) Patients were dichotomized to low and high TNF-α/IFN-γ secretion groups according to ROC and the Youden index analyses (AUROC 0.7175; 95% CI: 0.5580–0.8770). 14.05% was used as a cutoff. Hazard ratio is reported in the Supplementary Table 2. (c) CD16 downregulation in CD56^dim NK cells after K562 stimulation. (d) The proportion of CD16- NK cells secreting TNF-α/IFN-γ in patient groups at baseline after K562 stimulation. Early relapse n=14, late relapse n= 6, non-relapse n=19, and healthy n=8. (c, d) Box-and-whisker plots present 5–95 percentiles. One-way ANOVA was used for comparison between multiple groups (exact P-value reported in the figure), and Bonferroni's post test was used to compare selected pairs (only early relapse group was compared to other groups to avoid multiple comparisons). Statistically significant differences between the groups are marked with asterisk (*P<0.05, **P<0.01).

Figure 5

T-cell immunophenotype and cytokine secretion analyzed with flow cytometry. (a) The proportion of naïve CD8⁺ CD45RA⁺CD62L⁺ T cells in early-relapsing patients (20.6%, n=13), late-relapsing patients (18.9%, n=8) and non-relapsing patients (12.5%, n=17). (b) PD-1 expressing central memory CD4+ T cells (CCR7+CD45RA-) in early relapsing (16.5%, n=9), late relapsing (16.0%, n=8) and non-relapsing patients at baseline (21.7% (n=12). (a, b) Box-and-whisker plots present 5–95 percentiles. One-way ANOVA was used for comparison between multiple groups (exact P-value reported in the figure), and Bonferroni's post test was used to compare selected pairs (only early relapse group was compared with other groups to avoid multiple comparisons). Statistically significant differences between the groups are marked with asterisk (*P<0.05). (c) Correlation between TNF-α/IFN-γ secretion of CD4+ T cells with NK-cell proportion in non-relapsing patients at baseline. (d) Correlation between TNF-α/IFN-γ secretion of CD4+ T cells and NK-cell proportion in relapsing patients (early and late) at baseline. Correlations were analyzed with the non-parametric Spearman test.