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

Delphine Rea, Guylaine Henry, Zena Khaznadar, Gabriel Etienne, François Guilhot, Franck Nicolini, Joelle Guilhot, Philippe Rousselot, Françoise Huguet, Laurence Legros, Martine Gardembas, Viviane Dubruille, Agnès Guerci-Bresler, Aude Charbonnier, Frédéric Maloisel, Jean-Christophe Ianotto, Bruno Villemagne, François-Xavier Mahon, Hélène Moins-Teisserenc, Nicolas Dulphy, Antoine Toubert, Delphine Rea, Guylaine Henry, Zena Khaznadar, Gabriel Etienne, François Guilhot, Franck Nicolini, Joelle Guilhot, Philippe Rousselot, Françoise Huguet, Laurence Legros, Martine Gardembas, Viviane Dubruille, Agnès Guerci-Bresler, Aude Charbonnier, Frédéric Maloisel, Jean-Christophe Ianotto, Bruno Villemagne, François-Xavier Mahon, Hélène Moins-Teisserenc, Nicolas Dulphy, Antoine Toubert

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 CD56dim subset than had relapsing patients, while CD56bright natural killer cells, T cells and their subsets did not differ significantly. Furthermore, the CD56dim 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).

Copyright© 2017 Ferrata Storti Foundation.

Figures

Figure 1.
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.
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−CD56dim NK-cell counts for each individual non-relapsing and relapsing patient; median values (horizontal bars) are also shown. (D) Scatter dot plots represent CD3−CD56bright 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.
Figure 3.
Molecular relapse-free survival after discontinuation of imatinib according to clinico-biological factors. (A) Sokal risk group, (B) imatinib treatment duration, (C) CD56dim 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.
Figure 4.
Expression of natural killer cell receptors in CD56dim and CD56bright 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.
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.

References

    1. Druker BJ, Guilhot F, O’Brien SG, et al. ; IRIS Investigators. Five-year follow-up of patients receiving imatinib for chronic myeloid leukemia. N Engl J Med. 2006;355(23):2408–2417.
    1. Kalmanti L, Saussele S, Lauseker M, et al. Safety and efficacy of imatinib in CML over a period of 10 years: data from the randomized CML-study IV. Leukemia. 2015;29(5): 1123–1132.
    1. Gambacorti-Passerini C, Antolini L, Mahon FX, et al. Multicenter independent assessment of outcomes in chronic myeloid leukemia patients treated with imatinib. J Natl Cancer Inst. 2011;103(7):553–561.
    1. Sasaki K, Strom SS, O’Brien S, et al. Relative survival in patients with chronic-phase chronic myeloid leukaemia in the tyrosine-kinase inhibitor era: analysis of patient data from six prospective clinical trials. Lancet Haematol. 2015;2(5):e186–e193.
    1. Corbin AS, Agarwal A, Loriaux M, Cortes J, Deininger MW, Druker BJ. Human chronic myeloid leukemia stem cells are insensitive to imatinib despite inhibition of BCR-ABL activity. J Clin Invest. 2011;121(1):396–409.
    1. Hamilton A, Helgason GV, Schemionek M, et al. Chronic myeloid leukemia stem cells are not dependent on Bcr-Abl kinase activity for their survival. Blood. 2012;119(6):1501–1510.
    1. Baccarani M, Deininger MW, Rosti G, et al. European LeukemiaNet recommendations for the management of chronic myeloid leukemia: 2013. Blood. 2013;122(6):872–884.
    1. Chomel JC, Bonnet ML, Sorel N, et al. Leukemic stem cell persistence in chronic myeloid leukemia patients in deep molecular response induced by tyrosine kinase inhibitors and the impact of therapy discontinuation. Oncotarget. 2016;7(23):35293–35301.
    1. Mahon FX, Réa D, Guilhot J, et al. ; Intergroupe Français des Leucémies Myéloïdes Chroniques. Discontinuation of imatinib in patients with chronic myeloid leukaemia who have maintained complete molecular remission for at least 2 years: the prospective, multicentre Stop Imatinib (STIM) trial. Lancet Oncol. 2010;11(11): 1029–1035.
    1. Ross DM, Branford S, Seymour JF, et al. Safety and efficacy of imatinib cessation for CML patients with stable undetectable minimal residual disease: results from the TWISTER study. Blood. 2013;122(4):515–522.
    1. Ross DM, Branford S, Seymour JF, et al. Patients with chronic myeloid leukemia who maintain a complete molecular response after stopping imatinib treatment have evidence of persistent leukemia by DNA PCR. Leukemia. 2010;24(10):1719–1724.
    1. Richter J, Söderlund S, Lübking A, et al. Musculoskeletal pain in patients with chronic myeloid leukemia after discontinuation of imatinib: a tyrosine kinase inhibitor withdrawal syndrome. J Clin Oncol. 2014;32(25):2821–2823.
    1. Mori S, Vagge E, le Coutre P, et al. Age and dPCR can predict relapse in CML patients who discontinued imatinib: the ISAV study. Am J Hematol. 2015;90(10):910–914.
    1. Lee SE, Choi SY, Song HY, et al. Imatinib withdrawal syndrome and longer duration of imatinib have a close association with a lower molecular relapse after treatment discontinuation: the KID study. Haematologica. 2016;101(6):717–723.
    1. Rohon P. Biological therapy and the immune system in patients with chronic myeloid leukemia. Int J Hematol. 2012;96(1):1–9.
    1. Barrett AJ, Ito S. The role of stem cell transplantation for chronic myelogenous leukemia in the 21st century. Blood. 2015;125(21):3230–3235.
    1. Khaznadar Z, Henry G, Setterblad N, et al. Acute myeloid leukemia impairs natural killer cells through the formation of a deficient cytotoxic immunological synapse. Eur J Immunol. 2014;44(10):3068–3080.
    1. Etienne G, Guilhot J, Rea D, et al. , on behalf of the Intergroupe Français des Leucémies Myéloïdes Chroniques (Fi-LMC). Long-term follow-up of the French Stop Imatinib Study (STIM1) in chronic myeloid leukemia patients. J Clin Oncol. 2017;35(3):298–305.
    1. Carrillo-Bustamante P, Ke mir C, de Boer RJ. The evolution of natural killer cell receptors. Immunogenetics. 2016;68(1):3–18.
    1. Björkström NK, Riese P, Heuts F, et al. Expression patterns of NKG2A, KIR, and CD57 define a process of CD56dim NK-cell differentiation uncoupled from NK-cell education. Blood. 2010;116(19):3853–3864.
    1. Nielsen CM, White MJ, Goodier MR, Riley EM. Functional significance of CD57 expression on human NK cells and relevance to disease. Front Immunol. 2013;4(422):1–8.
    1. Alter G, Malenfant JM, Altfeld M. CD107a as a functional marker for the identification of natural killer cell activity. J Immunol Methods. 2004;294(1–2):15–22.
    1. Cebo C, Da Rocha S, Wittnebel S, et al. The decreased susceptibility of Bcr/Abl targets to NK cell-mediated lysis in response to imatinib mesylate involves modulation of NKG2D ligands, GM1 expression, and synapse formation. J Immunol. 2006;176(2):864–872.
    1. Salih J, Hilpert J, Placke T, et al. The BCR/ABL-inhibitors imatinib, nilotinib and dasatinib differentially affect NK cell reactivity. Int J Cancer. 2010;127(9):2119–2128.
    1. Chen CI, Koschmieder S, Kerstiens L, et al. NK cells are dysfunctional in human chronic myelogenous leukemia before and on imatinib treatment and in BCR-ABL-positive mice. Leukemia. 2012;26(3):465–474.
    1. Zitvogel L, Rusakiewicz S, Routy B, Ayyoub M, Kroemer G. Immunological off-target effects of imatinib. Nat Rev Clin Oncol. 2016;13(7):431–446.
    1. Rea D, Rousselot P, Guilhot J, Guilhot F, Mahon FX. Curing chronic myeloid leukemia. Curr Hematol Malig Rep. 2012;7(2):103–108.
    1. Hughes TP, Ross DM. Moving treatment-free remission into mainstream clinical practice in CML. Blood. 2016;128(1):17–23.
    1. Saußele S, Richter J, Hochhaus A, Mahon FX. The concept of treatment-free remission in chronic myeloid leukemia. Leukemia. 2016;30(8): 1638–1647.
    1. Deininger MW. Molecular monitoring in CML and the prospects for treatment-free remissions. Hematology Am Soc Hematol Educ Program. 2015;(2015):257–263.
    1. Imagawa J, Tanaka H, Okada M, et al. ; DADI Trial Group. Discontinuation of dasatinib in patients with chronic myeloid leukaemia who have maintained deep molecular response for longer than 1 year (DADI trial): a multicentre phase 2 trial. Lancet Haematol. 2015;2(12):e528–e535.
    1. Ilander M, Olsson-Strömberg U, Schlums H, et al. Increased proportion of mature NK cells is associated with successful imatinib discontinuation in chronic myeloid leukemia. Leukemia. 2017;31(5):1108–1116.
    1. Salih HR, Antropius H, Gieseke F, et al. Functional expression and release of ligands for the activating immunoreceptor NKG2D in leukemia. Blood. 2003;102(4):1389–1396.
    1. Sconocchia G, Lau M, Provenzano M, et al. The antileukemia effect of HLA-matched NK and NK-T cells in chronic myelogenous leukemia involves NKG2D-target-cell interactions. Blood. 2005;106(10):3666–3672.
    1. Boissel N, Rea D, Tieng V, et al. BCR/ABL oncogene directly controls MHC class I chain-related molecule A expression in chronic myelogenous leukemia. J Immunol. 2006;176(8):5108–5116.
    1. Verheyden S, Demanet C. NK cell receptors and their ligands in leukemia. Leukemia. 2008;22(2):249–257.
    1. Hilpert J, Grosse-Hovest L, Grünebach F, et al. Comprehensive analysis of NKG2D ligand expression and release in leukemia: implications for NKG2D-mediated NK cell responses. J Immunol. 2012;189(3):1360–1371.
    1. Yong AS, Keyvanfar K, Hensel N, et al. Primitive quiescent CD34+ cells in chronic myeloid leukemia are targeted by in vitro expanded natural killer cells, which are functionally enhanced by bortezomib. Blood. 2009;113(4):875–882.
    1. Cervantes F, Pierson BA, McGlave PB, Verfaillie CM, Miller JS. Autologous activated natural killer cells suppress primitive chronic myelogenous leukemia progenitors in long-term culture. Blood. 1996;87(6): 2476–2485.
    1. Savani BN, Rezvani K, Mielke S, et al. Factors associated with early molecular remission after T cell-depleted allogeneic stem cell transplantation for chronic myelogenous leukemia. Blood. 2006;107(4):1688–1695.
    1. Pawelec G, Da Silva P, Max H, et al. Relative roles of natural killer- and T cell-mediated anti-leukemia effects in chronic myelogenous leukemia patients treated with interferon-alpha. Leuk Lymphoma. 1995;18(5–6):471–478.
    1. Meseri A, Delwail V, Brizard A, et al. Endogenous lymphokine activated killer cell activity and cytogenetic response in chronic myelogenous leukaemia treated with alpha-interferon. Br J Haematol. 1993;83(2):218–222.
    1. Degli-Esposti MA, Smyth MJ. Close encounters of different kinds: dendritic cells and NK cells take centre stage. Nat Rev Immunol. 2005;5(2):112–124.
    1. Walzer T, Dalod M, Robbins SH, Zitvogel L, Vivier E. Natural-killer cells and dendritic cells: “l’union fait la force”. Blood. 2005;106(7):2252–2258.
    1. Lichtfuss GF, Cheng WJ, Farsakoglu Y, et al. Virologically suppressed HIV patients show activation of NK cells and persistent innate immune activation. J Immunol. 2012;189(3):1491–1499.
    1. Takahashi N, Miura I, Saitoh K, Miura AB. Lineage involvement of stem cells bearing the philadelphia chromosome in chronic myeloid leukemia in the chronic phase as shown by a combination of fluorescence-activated cell sorting and fluorescence in situ hybridization. Blood. 1998;92(12):4758–4763.
    1. Mellqvist UH, Hansson M, Brune M, Dahlgren C, Hermodsson S, Hellstrand K. Natural killer cell dysfunction and apoptosis induced by chronic myelogenous leukemia cells: role of reactive oxygen species and regulation by histamine. Blood. 2000;96(5): 1961–1968.
    1. Park JS, Lee SE, Jeong SH, et al. Change of health-related profiles after Imatinib cessation in chronic phase chronic myeloid leukemia patients. Leuk Lymphoma. 2016;57(2):341–347.
    1. Ohyashiki K, Katagiri S, Tauchi T, et al. Increased natural killer cells and decreased CD3(+)CD8(+)CD62L(+) T cells in CML patients who sustained complete molecular remission after discontinuation of imatinib. Br J Haematol. 2012;157(2):254–256.
    1. Fehniger TA, Caligiuri MA. Interleukin 15: biology and relevance to human disease. Blood. 2001;97(1):14–32.

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