Elsevier

Leukemia Research

Volume 38, Issue 8, August 2014, Pages 964-969
Leukemia Research

Impaired NK cells and increased T regulatory cell numbers during cytotoxic maintenance therapy in AML

https://doi.org/10.1016/j.leukres.2014.05.014Get rights and content

Highlights

  • We studied immunomodulatory effects of cyclic cytotoxic maintenance therapy in AML.

  • Patients in CR had low NK, TH and Treg counts and a reduced NK activation capacity.

  • After maintenance therapy, NK counts declined, while TH and Treg cells increased.

  • The proliferative potential of TH cells was reduced after maintenance therapy.

  • Immunotherapeutic approaches in post-remission have to consider these impairments.

Abstract

Cyclic cytotoxic maintenance therapy can be applied to patients with AML in post-remission. We studied the immune status of AML patients in complete remission and the effect of maintenance therapy on different immune cell populations. Patients in complete remission had reduced NK, TH and Treg counts and a reduced NK activation capacity. In the course of cytotoxic maintenance therapy, NK counts further declined, while TH and Treg cells increased, with lower proliferative potential of TH cells. We conclude that immunotherapeutic approaches in post-remission have to consider reduced NK cell function and further impairment of cellular immune responses during cytotoxic therapy.

Introduction

Curative treatment approaches for patients with acute myeloid leukemia (AML) are based on intensive induction chemotherapy consisting of cytarabine and anthracyclines. Despite high initial complete remission (CR) rates, clinical outcome is still poor, with an age-dependent 5-year overall survival rate of 15–40% [1], [2]. Relapse remains the biggest clinical problem; therefore, the choice of post-remission therapy is critical. One post-remission therapy approach developed by the AML Cooperative Group (AML-CG) has been prolonged cyclic maintenance therapy consisting of monthly courses of cytarabine in combination with various second agents (daunorubicin, tioguanine, or cyclophosphamide) in a rotating sequence for up to two years (Supplementary Fig. S1). It was shown that the addition of this treatment to consolidation therapy was able to improve the remission-free survival in AML patients compared to consolidation therapy alone [3], [4]; therefore, this post-remission treatment is regularly used within the clinical centers associated with the AML-CG. However, the mechanism for the clinical benefit of cyclic maintenance therapy has not been elucidated. There is no data available on potential conversion of minimal residual disease during this treatment. Importantly, hemato-oncologists are becoming increasingly aware that the effect of conventional chemotherapeutic agents has to be evaluated in the context of the patients’ immune system [5], [6].

So far, there are only few studies that have analyzed the immune status of AML patients in CR after intensive induction chemotherapy. In one of the first studies, a reduction of overall lymphocytes and specifically T helper (TH) cells was found [7]. Two reports showed that natural killer (NK) cell counts [8] as well as TC1 and TH1 levels [9] are reduced compared to healthy controls. In contrast, frequency and function of T regulatory (Treg) cells were shown to be elevated in CR, albeit to a lower degree compared to primary diagnosis [10]. Similarly, an early expansion of Treg cells after induction timed sequential chemotherapy was demonstrated [11].

Even less is known about the influence of cytotoxic maintenance therapy on the immune system. In patients with various solid tumors, cyclophosphamide has been repeatedly described to induce a temporary reduction in frequency and suppressor function of Treg cells when given iteratively in low doses (“metronomically”) [12], [13], [14]. A different study demonstrated differentiation of Th17 cells after cyclophosphamide therapy [15]. Effects of cyclophosphamide therapy in AML patients and of antimetabolite or anthracycline treatment on the different immune cell populations have not been described in detail. Thus, it is not known what effects the cyclic maintenance therapy has on the immune system of AML patients.

In the present study, we set out to first analyze the immune status of AML patients in CR after intensive induction and consolidation chemotherapy, focusing on frequency and function of different NK and T cell populations including Treg cells. We then for the first time provide data on immunomonitoring of AML patients that received cyclic cytotoxic maintenance therapy within the AML-CG 2008 study. This data will be of great interest for future considerations of immunotherapeutic strategies in post-remission and for possible combinatorial approaches with cytotoxic therapy for AML.

Section snippets

Peripheral blood samples

After written informed consent in accordance with the Declaration of Helsinki and approval by the Institutional Review Board of the Ludwig-Maximilians-Universität (Munich, Germany), peripheral blood (PB) was obtained from 15 healthy donors (HDs) and 17 AML patients treated in our department within the German AML-CG trial 2008 or analogous to the study protocol. All patients received intensive induction and consolidation chemotherapy and achieved CR as defined by cytomorphology of bone marrow

AML patients prior to maintenance therapy have reduced absolute NK, TH, and Treg cell counts

PB was obtained from 13 AML patients (#1–13; Supplementary Table S1) before the application of maintenance chemotherapy and from 6 HDs. PBMCs were isolated, stained with antibodies for T and NK cell markers, as specified in Section 2, and analyzed by flow cytometry. Absolute cell counts of different cell populations were calculated by multiplying their percentage within the lymphocyte gate with the absolute lymphocyte count according to the hemogram of the respective blood donor.

The total T

Discussion

Multiple trials have been conducted on the efficacy of cytotoxic regimens for the treatment of AML, but the immunomodulatory effect of these treatments has rarely been addressed. However, there is mounting evidence that the effect of conventional chemotherapeutic agents cannot be completely evaluated without considering their effect on the patient's immune system [5], [6]. Besides, there is a high interest in the development of novel immunotherapeutic strategies for post-remission therapy to

Funding

This work was supported by funds from BayImmuNet, the Bavarian Immunotherapy Network (http://www.bayimmunet.de). The funders had no role in study design, data collection, analysis and interpretation, decision to publish, or preparation of the manuscript.

Conflict of interest statement

Katharina Gellhaus is employed by Epiontis. All other authors declare no conflict of interest.

Acknowledgments

The authors thank Patrick Palluch for his excellent technical assistance.

Contributions: Research design: FSL, WH, BB, MS; performance of the experiments and collection of the data: FSL, RL, KG, BB; analysis and interpretation of the data: FSL, RL, BB, MS; writing of the manuscript: FSL, MS; supervision of the project: MS.

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