In-depth characterization of NK cell markers from CML patients who discontinued tyrosine kinase inhibitor therapy

Abstract

Introduction: Treatment-free remission (TFR) in patients with chronic myeloid leukemia in chronic phase is considered a safe option if suitable molecular monitoring is available. However, the question arises as to which factors can contribute to the maintenance of TFR, and immunologic surveillance of the remaining leukemic cells is believed to be one of them. Argentina Stop Trial is an open-label, single-arm, multicenter trial assessing TFR after tyrosine kinase inhibitors interruption, that after more than 4 years showed a successful TFR rate of 63%.

Methods: In this context, we set up an immunological study by flow cytometry in order to analyze specific NK cell subsets from peripheral blood patient samples both at the time of discontinuation as well as during the subsequent months.

Results: At the time of discontinuation, patients show a mature NK cell phenotype, probably associated to TKI treatment. However, 3 months after discontinuation, significant changes in several NK cell receptors occurred. Patients with a higher proportion of CD56dim NK and PD-1+ NK cells showed better chances of survival. More interestingly, non-relapsing patients also presented a subpopulation of NK cells with features associated with the expansion after cytomegalovirus infection (expression of CD57+NKG2C+), and higher proportion of NKp30 and NKp46 natural cytotoxicity receptors, which resulted in greater degranulation and associated with better survival (p<0.0001).

Discussion: This NK cell subset could have a protective role in patients who do not relapse, thus further characterization could be useful for patients in sustained deep molecular response.

Keywords: NK cells; chronic myeloid leukemia; degranulation; immunophenotype; treatment free remission.

Figure 1

Organization of the study and characteristics of the patients. (A) Flowchart summarizing the distribution of patients according to timepoints and clinical status. (B) Molecular recurrence-free survival after TKI discontinuation (N = 46). (C) Patients baseline characteristics.

Figure 2

Comparison between NK cell markers from the time of discontinuation and three months after. Wilcoxon tests were performed (**** p<0.0001, *** p<0.001, ** p<0.01, *p<0.05).

Figure 3

Phenotypic characterization of NK cell markers from relapsing and non-relapsing patients. (A) Percentage of CD56^dim NK cells in relapsed vs non-relapsed patients at the time of discontinuation. (B) Molecular recurrence-free survival according to percentage of CD3^-CD56^dim NK cells at the time of discontinuation. (C) Percentage of PD-1⁺ NK cells in relapsed vs non-relapsed patients at the time of discontinuation. (D) Molecular recurrence-free survival according to percentage of PD-1⁺NK cells at the time of discontinuation. (E) Percentage of CD56^dim NK cells in relapsed vs non-relapsed patients at month 3 after stopping TKI. (F) Molecular recurrence-free survival according to percentage of CD3^-CD56^dim NK cells after 3 months of TKI discontinuation. (G) Percentage of PD-1⁺ NK cells in relapsed vs non-relapsed patients at month 3 after stopping TKI. (H) Molecular recurrence-free survival according to percentage of PD-1⁺ NK cells after 3 months of TKI discontinuation. (I) Percentage of NKp44⁺ NK cells in relapsed vs non-relapsed patients at month 3 after stopping TKI. (J) Molecular recurrence-free survival according to percentage of NKp44⁺ NK cells after 3 months of TKI discontinuation. Mann-Whitney and Log-rank tests were performed (* p<0.05, ns: non-significant).

Figure 4

NK cell subsets analysis in relapsed vs non-relapsed patients at the time of discontinuation. (A) Percentage of CD56^dimNKG2C⁺CD57⁺ (HCMV-NK cells). (B) Representative gating strategy of a patient with CD56^dimNKG2C⁺CD57⁺ NK cell subset higher than 5%. (C) Percentage of CD56^dimCD57⁺NKG2C⁺NKp30⁺ NK cells (NKp30⁺HCMV-NK cells). (D) Percentage of CD56^dimCD57⁺NKG2C⁺NKp46⁺ NK cells (NKp46⁺HCMV-NK cells). (E) Percentage of CD56^dimCD57⁺NKG2C⁺NKp30⁺NKp46⁺ NK cells (NKp30⁺NKp46⁺HCMV-NK cells). (F) Molecular recurrence-free survival according to percentage of NKp30⁺NKp46⁺HCMV-NK cells. Mann-Whitney test and Log-rank test were performed when appropriate. P-values lower than 0.05 were considered statically significant. ns, non-significant.

Figure 5

Functional role of CD56^dimNKG2C⁺NKp46⁺ NK cells. (A) Representative gating strategy used for degranulation (CD107a) and IFNγ production assays. (B) Percentage of CD107a⁺ cells in the NKG2C⁺NKp46⁺ NK subpopulation at the time of discontinuation in relapsing and non-relapsing patients. (C) Molecular recurrence-free survival according to percentage of CD107a⁺ cells in the NKG2C⁺NKp46⁺ NK subpopulation. (D) Percentage of NKG2C⁺NKp46⁺CD107a⁺ cells as a proportion of CD56^dim NK cells at the time of discontinuation in relapsing and non-relapsing patients. (E) Molecular recurrence-free survival according to percentage of NKG2C⁺NKp46⁺CD107a⁺ cells as a proportion of CD56^dim NK cells. Mann-Whitney and Log-rank tests were performed (* p<0.05; ns, non-significant).