Blood Reviews 27 (2013) 209–216

Contents lists available at ScienceDirect

Blood Reviews

j ourna l homepage: www.e lsev ie r .com/ locate /b l re

REVIEW

Immunotherapeutic strategies for relapse control in acutemyeloid leukemia

Anna Martner, Fredrik Bergh Thorén, Johan Aurelius, Kristoffer Hellstrand ⁎Sahlgrenska Cancer Center, University of Gothenburg, Box 405, 40530 Gothenburg, Sweden

⁎ Corresponding author at: Sahlgrenska Cancer CentBox 425, 40530 Gothenburg, Sweden. Tel.: +46 706 2

E-mail address: kristoffer.hellstrand@gu.se (K. Hells

0268-960X/$ – see front matter © 2013 Elsevier Ltd. Alhttp://dx.doi.org/10.1016/j.blre.2013.06.006

a b s t r a c t

a r t i c l e i n f o

Keywords:

Acute myeloid leukemiaMaintenance therapyLeukemia-related immunosuppression

Despite that the initial phases of chemotherapy induce disappearance of leukemic cells in many patients withacute myeloid leukemia (AML), the prevention of life-threatening relapses in the post-remission phaseremains a significant clinical challenge. Allogeneic bone marrow transplantation, which is available for a mi-nority of patients, efficiently prevents recurrences of leukemia by inducing immune-mediated elimination ofleukemic cells, and over the past decades, numerous immunotherapeutic protocols have been developedaiming to mimic the graft-versus-leukemia reaction for the prevention of relapse. Here we review past andpresent strategies for relapse control with focus on overcoming leukemia-related immunosuppressionin AML. We envisage future treatment protocols, in which systemic immune activators, such as vaccines,dendritic cell-based therapies, engineered variants of IL-2, or IL-15, are combined with agents that counterimmunosuppression mediated by, e.g., the PD/PDL interaction, CTLA-4, CD200, reactive oxygen species, IDOexpression, CXCR4, or the KIR/class I interaction, based on characteristics of the prevailing malignant clone.This combinatorial approach may pave the way for individualized immunotherapy in AML.

© 2013 Elsevier Ltd. All rights reserved.

1. Introduction

Acute myeloid leukemia (AML) is the most common form of acuteleukemia in adults with an incidence of approximately 4/100,000[1,2]. Upon diagnosis, patients receive induction chemotherapyaiming to achieve complete remission (CR), which is defined as ab-sence of microscopically detectable malignant cells along with the re-turn of normal hematopoiesis. The post-remission therapy comprisesconsolidation chemotherapy with the goal of eradicating remainingundetectable leukemic cells [3,4]. This phase of therapy is critical fora favorable long-term outcome as nearly all patients will experiencerecurrence of leukemia (relapse) if consolidation is not provided [5].

The risk of relapse varies with several well-defined prognosticfactors, in particular the patients' age and genetic aberrations of the leu-kemic clone, as reviewed in detail elsewhere [6–8]. Despite successfulinduction therapy and completed consolidation therapy, relapse occursin 60–70% of patients within five years [9]. In the first relapse, the like-lihood of achieving a renewed CR is lower than at onset of disease,and the duration the second CR is almost invariably shorter than thefirst [10]. The median survival of relapsing patients is in the range of6 months with few long-term survivors [11]. Relapse is a significantreason why the overall prognosis of adult patients with AML remainspoor with a 20–30% likelihood of 5-year survival after diagnosis [1,12].

er, University of Gothenburg,04358.trand).

l rights reserved.

Patients with unfavorable genetic aberrations in leukemic cells arecandidates for allogeneic stem cell transplantation, which is used in theconsolidation phase or as intensification therapy. Allo-transplantationwas earlier offered primarily to younger otherwise healthy patients, butin recent years the development of protocols using reduced intensity con-ditioning has enabled its use also among patients in their 70's and amongthosewith significant co-morbidities [13,14]. For themajority of AML pa-tients however, i.e. thosewho are not candidates for allo-transplantation,the consensual standard of care has been no further anti-leukemic treat-ment. An exception is that several centers in Germany practice repeatedcourses of chemotherapy also in the post-consolidation phase, which ap-pears beneficial in terms of relapse protection in elderly patients [15,16];however the toxicity of this treatment has limited its wide-spread use.

A relapse is assumed to result from expansion of malignant cells thathave escaped the cytotoxic action of chemotherapy, and the goal ofAML treatment beyond consolidation is to eradicate these remainingcells (“minimal residual disease”, MRD) to cure patients. In this overview,we present past and present strategies to maintain CR in the post-consolidation phase of AML with particular focus on the utility of immu-notherapy to achieve immune-mediated elimination of residual leukemiccells.

2. Immune reactivity in AML

Measurable adaptive immune responses evolve inmost patients withAML, as evidenced by the demonstration of antibodies [17–19] as well asby CD4+andCD8+T cell responses directed against leukemia-related an-tigens (reviewed in [20]). In addition, AML cells are frequently sensitive

210 A. Martner et al. / Blood Reviews 27 (2013) 209–216

to the cytotoxic action of natural killer (NK) cells [21–23]. While theimpact of humoral immune response for the outcome of AML remainsunknown, a large body of evidence suggests that T and NK cell functionsare pivotal for leukemia-free survival after the completion of chemother-apy. In the following section we review the purported role of these im-mune effector cellswith focus on their impact for remissionmaintenance.

2.1. T cells in immune surveillance

The surveilling role of T cells is founded on experiences fromallo-transplantation in AML and related diseases. For example, severalstudies demonstrate that transplanted patients with low-grade orabsent T cell-dependent graft-versus-host disease show a higher prob-ability of relapse [24,25]. In addition, elimination of T cells from thetransplant increases the relapse risk in AML [4,26], and the re-infusionof donor T cells can induce CR in a small but significant fraction ofpatients [23].

While the role of transplanted T cells for remission maintenance iswell established, relatively few investigators have addressed the clin-ical relevance of anti-leukemic T-cell responses in non-transplantedpatients. In a study of pediatric patients with AML, who had either re-ceived only chemotherapy or an autologous transplant, Montagna andco-workers showed that the emergence of anti-leukemic cytotoxicT cell precursors (CTLp's) in bone marrow and blood was associatedwith long-term remission maintenance: no relapses were observedamong 8 non-transplanted patients in CR in whom CTLp's weredetected, whereas 7/8 patients with absence of CTLp's experienced re-lapse. Interestingly, in patients with absence of anti-leukemic CTLp'ssizeable levels of EBV-specific CTLs were detected, thus suggesting aspecific deficiency to mount an anti-leukemic T cell response [27].Similarly, Greiner et al. demonstrated a significant correlation be-tween a strong mRNA expression of leukemia-related T cell antigensand survival of patients with AML [28].

Despite the relative scarcity of clinical correlates between T-cell func-tion and outcome in AML, several studies point to an impaired cytotoxicT-cell response in AML, partly due to a deficiency of antigen exposure onMHC class I by leukemic cells. AML blasts thus have been reported to dis-play a skewed balance of co-inhibitory vs. co-stimulatory receptors [29]and recent data also imply a deficiency of T cell/AML blast immunesynapse formation [30]. A striking, albeit indirect support for the roleof T cells for outcome was provided in a report by Chamuleau andco-workers in their study of the indoleamine 2,3-dioxygenase (IDO)immunosuppressive pathway. IDO degrades tryptophan, which isessential for T cell expansion, and high expression of INDO mRNA byleukemic blastswas shown to herald poor prognosis. High INDO expres-sion was associated with both a higher frequency of relapse amongpatients in CR and a significantly lower rate of CR, thus implying thatINDO/IDO determines the outcome of AML by multiple mechanisms[31].

2.2. NK cells in immune surveillance

The role of autologous NK cells as potential anti-leukemic effectorcells in AML has been studied extensively, as reviewed in detail else-where [32]. The number of circulating NK cells, an intact anti-tumorcytotoxicity of NK cells, a maintained cytokine-producing functionof NK cells, and a preserved expression of activating receptors byNK cells all herald a favorable outcome of AML [23,33–38]. The corre-lation between NK cell functional integrity and outcome largelyreflects a dysfunction of the NK cell population in AML. While themechanisms are only partly understood, convincing data point tothe possibility that NK cells may be inactivated by cells of the leuke-mic clone, which have been reported to secrete various solubleNK-cell inhibitory factors such as TGF-beta and reactive oxygen spe-cies [35,39–42] as well as to inhibit NK cells by cell-to-cell interaction

[43–45]. Table 1 summarizes aspects on lymphocyte dysfunction inAML.

The lytic activity of NK cells is governed by their expression of inhib-itory and activating receptors [46–48]. Inhibitory killer immunoglobulin-like receptors (KIRs) recognize HLA-A, HLA-B andHLA-C proteins leadingto inactivation of NK cells that encounter self-HLA molecules on putativetarget cells. Thismechanism of NK cell inactivation is proposed to explainhowNK cells avoid attacking autologous cells, but is assumed to have rel-evance also for the outcome of allo-transplantation in AML. Retrospectiveanalyses of the KIR status of donor NK cells vs. HLA class I molecules ofthe recipient thus revealed thatmismatches of KIR/class I –which predictallo-reactivity of grafted NK cells – were strongly associated with a re-duced risk of relapse after allo-transplantation [49]. Similar results werereported in a subsequent study [50] but disputed by others [51–53].Additional studies are warranted to define the value of KIR ligandmismatching in AML (for further review, see [32,48,54]).

Recent studies propose a protective role also for activating KIRs inAML in the context of allo-transplantation. KIR2DS1 recognizesHLA-C2 molecules, and NK cells expressing KIR2DS1 that are derivedfrom HLA-C1 subjects (HLA-C1/C1 or C1/C2) secrete interferon-γ andefficiently lyse target cells, in particular those expressing HLA-C2. Incontrast KIR2DS1+ NK cells from subjects who are homozygous forHLA-C2 (HLA-C2/C2) – thus expressing the ligand for KIR2DS1 as aself-molecule – are hyporesponsive in terms of cytotoxicity andcapacity to produce cytokines. With this background, Venstrom andco-workers [55] studied the impact of KIR2DS1 on the relapse rateof allo-transplanted patients. It was observed that the risk of relapsewas significantly lower when donors were KIR2DS1+, but onlywhen donors also were homo- or heterozygous for HLA-C1. Patientsreceiving KIR2DS1+ transplants from HLA-C2/C2 donors thus showeda frequency of relapse similar to that observed with KIR2DS1− trans-plants. These findings are consistent with the notion that KIR2DS1is an activating KIR in HLA-C1/C1 or HLA C1/C2 donors, and thatKIR2DS1-expressing NK cells are tolerized in HLA-C2/C2 donors[56–58]. Importantly, the results support that donor NK cells signifi-cantly contribute to the graft-versus-leukemia reaction with ensuingreduction of relapse risk.

3. Immunotherapy for remission maintenance

The notion that immune cells participate in surveillance of leukemiccells in AML, specifically that (1) endogenous T cells and NK cells areendowed with significant anti-leukemic function in AML and (2) bothof these lymphocyte subsets significantly contribute to the graft-versus-leukemia effect after allo-transplantation, has inspired the eval-uation of systemic T cell and NK cell activating agents for relapse con-trol. In addition, the favorable effects of monoclonal antibodies againstantigens expressed by malignant cells, in particular in the treatmentof B cell malignancies [59–62], have encouraged the development oftherapeutic antibodies against AML cells. A potential advantage ofimmunotherapy is that LSCs, which due to their non-proliferatingstate ostensibly escape chemotherapy, may be targeted by antibodiesand cytotoxic immune cells.

3.1. Monoclonal antibodies

Strategies to target leukemic cells using monoclonal antibodieshave significantly improved treatment of chronic lymphatic leukemia(CLL) and lymphoma, where CD20 antibodies such as rituximab areroutinely used [59–62]. Similar strategies are currently evaluatedalso in AML. Antibodies binding to leukemic cells are assumed to facil-itate clearance of the malignant cells via antibody-dependent cellularcytotoxicity (ADCC), presumably exerted by CD16+ NK cells, or com-plement activation. Antibodies may also be conjugated to cytotoxicdrugs and radioisotopes for directed actions on leukemic cells.

Table 1Leukemia-related immunosuppression in AML.

Immunosuppressivemechanism

Mode of action and rationale Therapy Clinical development Reference

Reactive oxygenspecies (ROS)

ROS produced by malignant andnon-malignant myeloid cells inactivecytotoxic lymphocytes in a PARP-1/AIF-dependent fashion with ensuinglymphocyte apoptosis.

Histamine dihydrochloride(HDC), a ROS formationinhibitor

Post-consolidation immunotherapy withHDC in conjunction with low-dose IL-2significantly improved leukemia-freesurvival in a phase III trial. Post hoc analysessuggested pronounced clinical benefit inmonocytic leukemias, where malignant cellsexpress histamine H2-receptors andROS-producing enzymes. HDC/IL-2 isapproved for relapse prevention withinEU and in Israel.

[39,112,113,115,117]

CTLA-4 CTLA-4 (CD152) on Tregs impairs T-cellfunction, partly by down-regulating CD80and CD86 on APCs. CTLA-4 also negativelyregulates CD4+ and CD8+ T cells.

Ipilimumab (anti-CTLA-4) Early-stage trials are ongoing. [79,83,84]

Programmed celldeath-1 (PD-1)

PD-1 down-modulates T cell function afterinteraction with PD-L1, which is expressedby a fraction of AML cells.

Anti-PD-L1 Early-stage trials are ongoing. [43,76]

Indoleamine2,3-dioxygenase(IDO)

IDO depletes tryptophan, which is essentialfor T cell expansion. IDO also induces Tregsvia the tryptophan metabolite 3-HAA. Highexpression of INDO (encoding IDO) predictspoor prognosis in AML.

Preclinical stage. [133,134,31]

Fas-ligand (FasL) FasL is expressed by subgroups of AML cellsand triggers apoptosis of Fas+ T cells in vitro.FasL is also present in immunosuppressivetumor-derived exosomes (TEX) obtainedfrom AML patients.

Preclinical stage. [135,136]

CD200 CD200R on T cells/NK cells transducesimmunosuppressive signals by targetingCD200 expressed by AML cells. CD200expression by AML cells entails poorprognosis. NK cells from patients withCD200-positive AML are reduced in numberand function.

Anti-CD200 Preclinical stage. [45,91,137,138]

Killer-cellimmunoglobulin-likereceptors (KIR)

Inhibitory KIRs suppress NK cells by trans-mitting a “self”-signal after binding to HLAclass I. Mismatches of KIR/class I may be apositive prognostic marker after allo-BMT.Blocking of KIR using IPH2101 has shownpromise in a phase I trial.

1. SCTwithmismatched KIR/class I2. Anti-KIR (IPH2101)

Early-phase clinical trials. [48,49,55,85]

211A. Martner et al. / Blood Reviews 27 (2013) 209–216

3.1.1. Anti-CD33Most studies ofmonoclonal antibodies in AML therapy have focused

on CD33 as the target antigen. CD33 is expressed on85–90%of leukemicblasts as well as by multipotent myeloid precursors, monocytes andneutrophils. The antigen is absent from pluripotent hematopoieticstem cells. Unconjugated anti-CD33 antibodies have shown little clini-cal benefit in CD33+ non-APL AML [63], but conjugation of anti-CD33antibodies to cytotoxic drugs or radioisotopes has proven a more effi-cacious strategy (reviewed in [64]). Gemtuzumab ozogamicin (GO),where anti-CD33 is conjugated to the cytotoxic agent calicheamicin,was approved in 2000 by the FDA as a single-agent induction therapyin elderly patients with relapsed AML [65]. In 2010, GO was withdrawnfrom the market due to post-approval clinical trials that failed to verifysufficient clinical benefit along with liver toxicity [66,67].

GO is currently undergoing further clinical evaluation, and the use offractionated lower doses of GO was recently reported to allow the safedelivery of higher cumulative doses along with significantly improvedoutcome [68]. In APL, cells of the leukemic clone homogenously expressCD33, and GO appears to efficiently target these leukemic cells [69,70].

3.1.2. Antibodies targeting LSCA disadvantage of anti-CD33 antibodies is that less differentiated

leukemic cells, not expressing CD33, are not targeted. Althoughcontroversies still exist, accumulating evidence indicates that AMLcells are organized as a cellular hierarchy, initiated and maintained byself-renewing leukemic stem cells (LSC) [71]. Thus, the LSC most prob-ably need to be eliminated for a permanent cure of AML.

The phenotype, maturation stage and expression of cell-surface mol-ecules on the LSC are likely to differ between subtypes of AML. Studiesaiming at identifying cell-surface antigens preferentially expressed byLSC, but not by normal hematopoietic stem cells (HSC), have revealedseveral promising targets including the adhesion molecule CD44, theanti-phagocytic molecule CD47 and the high affinity IL-3 receptorIL3RAP or CD123 (reviewed in [72]). Monoclonal antibodies targetingCD44 [73], CD47 [74] and CD123 [75] have demonstrated efficacyagainst LSC expressing these surface antigens inmurine xenotransplan-tation models, and anti-CD123 antibodies are currently undergoinginvestigation in a phase I trial (NCT01632852).

3.1.3. Antibodies targeting PD-L1, PD-L2 and PD-1Expression of PD-L1 and PD-L2 by cancer cells has been proposed

to represent an immune escape mechanism as interactions betweenthese receptors and PD-1, expressed by T cells and NK cells, conveysinhibitory signals to these lymphocytes. In a murine model of AML,blocking of either PD-1 or PD-L1 led to improved T cell responses,rejection of PD-L1 expressing AML cells and increased survival [76].In a study of 79 AML patients, 18% had B7-H1+ (PD-L1+) blasts[43]; this study also demonstrated that the expression of PD-L1 onleukemic cells was inducible by IFN-γ, and that expression of PD-L1protected AML cells from CTL lysis [43]. No clinical trials evaluatingthe usefulness of antibodies inhibiting interactions between PD-L1/PD-L2 and PD-1 have been completed for AML patients, but trials inother forms of cancer (NCT00729664, NCT00730639) imply thattargeting the PD/PD-L interaction is a potentially efficacious strategy

212 A. Martner et al. / Blood Reviews 27 (2013) 209–216

[77,78]. Since expression of PD-L1 and PD-L2 is induced duringimmune responses, in part due to the secretion of by IFN-*by lymphocytes, antibodies targeting the PD-1/PD-L1/2 axis may beuseful in particular as adjuncts to immunostimulatory therapies. Anongoing trial in AML evaluates the efficacy of blocking PD-1 in con-junction with a DC vaccine (NCT01096602). However, treatmentsblocking interactions between PD-L1/PD-L2 and PD-1 entail consider-able toxicity that may limit its putative use for remission mainte-nance in AML [77,78].

3.1.4. Antibodies targeting CTLA-4In analogy with antibodies targeting PD-1, antibodies against the

negative T cell regulator CTLA-4 have been considered in the treatmentof AML. CTLA-4 inhibits T cell activation via two principal mechanisms;firstly, when T cells are activated these cells up-regulate expression ofCTLA-4, which conveys T cell inhibition upon interaction with its recep-tors CD80 and CD86. Secondly, CTLA-4 is constitutively expressed byregulatory T cells (Tregs) and plays a critical role for the immunosuppres-sion exerted by these cells [79]. Thus, Tregs down-modulate CD80 andCD86 by DCs in a CTLA-4 dependent manner, rendering the DCs less ca-pable of initiating immune responses [79,80]. The CTLA-4 antibodyipilimumab has been approved for treatment of metastatic malignantmelanoma on the basis of several trials showing improved overall surviv-al [81,82]. There are reports showing a correlation between high frequen-cies of Tregs and unfavorable outcome of AML [83,84] and early-stagetrials targeting CTLA-4 in AML are underway (NCT01757639,NCT00060372).

3.1.5. Anti-KIRAs discussed in Section 2.2, allo-transplantation with KIR-HLA-

mismatched donors has highlighted the importance of NK cell func-tion for outcome. A fraction of grafted NK cells cannot be inhibiteddue to the KIR/KIR ligand mismatch, which enhances NK cell cytotox-icity against leukemic cells. With this background, attempts are beingmade in non-transplanted patients to treat patients with antibodiesdirected against inhibitory KIRs on NK cells. In a phase I trial, the ad-ministration of the antibody IPH2101, which blocks the major inhib-itory KIRs, was shown to be feasible and potentially efficacious inelderly AML patients in first CR [85]. An ongoing randomized phaseII trial evaluates IPH2101 for remission maintenance (NCT01687387).

3.1.6. CXCR4 inhibitors and antibodiesThe interaction of myeloid leukemic blasts with the bone marrow

environment is postulated to keep leukemic cells in a dormant stage.The chemokine receptor CXCR4, which is expressed by approximately50% of AML blasts, binds to CXCL12 produced by bone marrow stromalcells [86]. Targeting the CXCR4/CXCL12 interaction therefore may favorstem cell mobilization and cell cycle entry, thus resulting in enhancedsensitivity to chemotherapy [87]. Early-stage trials have been performedusing the CXCR4 inhibitor plerixafor in combination with chemotherapy[88]. Preclinical studies have shown that also other smallmolecule antag-onists and humanized antibodies targeting CXCR4 exhibit potent CXCR4-dependent cytotoxicity [89,90], and a phase I study has been initiatedusing anti-CXCR4 in AML (NCT01120457).

3.1.7. Targeting CD200CD200 is an immunosuppressive glycoprotein that transduces

inhibitory signals upon interaction with CD200R. CD200 is expressedby several subsets of leukocytes including T cells, B cells and dendriticcells as well as by subsets of AML cells [44]. Over-expression of CD200by AML cells has been associated with increased relapse risk, in partic-ular in non-core binding factor leukemias [91]. NK cells from patientswith CD200hi AML cells are reduced in number and function [45] andin vitro analyses imply that CD200+ AML cells suppress NK cell func-tion, which is prevented by CD200 blockade [45]. Notably, anti-CD200of the IgG1, but not IgG2 or IgG4, isotypes yielded depletion of activated

human T cells in vitro by mediating antibody-dependent cellular cyto-toxicity by NK cells [92].

3.2. Immunotherapy with systemic cytokines

3.2.1. Cell cycle inductionA limitation of chemotherapy is that only dividing cells are targeted,

thus dormant leukemic stemcells (LSC)may escape the cytotoxic actionof such therapy. A means to enable targeting also of LSC may be to in-duce cell-cycle progression of resting AML cells. Induction therapy reg-imens in China and Japan frequently combine cytarabine and aclarubinwith G-CSF, where the latter drug aims to trigger proliferation of restingleukemic cells. A meta-analysis of thirty-five trials with a total of 1029AML and MDS patients showed that the CR rate of regimens whereG-CSF was included was significantly higher than regimens withoutG-CSF [93].

3.2.2. NK and T cell activatorsAn early form of immunotherapy in AML comprised stimulation

with bacillus Calmette-Guérin (BCG), which triggers the formation ofimmune-enhancing cytokines such as interleukin-2 (IL-2), IL-12 andIL-18 in addition to polyclonally activating subsets of T cells. No random-ized evaluation of the putative efficacy of BCG administration in thepost-chemotherapy phase of AML treatment has been performed, butmeta-analyses suggested that this treatment did not impact on relapserisk or survival [94]. More recent studies have used interferon-α,which efficiently activates NK cell-mediated cytotoxicity and improvesantigen presentation [95], for remission maintenance; a randomizedAML trial by Goldstone and co-workers did not, however, show a benefitin terms of relapse protection by this treatment [96]. In the late 1990'slinomide, a quinoline derivative with pleiotropic immunostimulatoryproperties, including NK cell activation, was evaluated for relapse pre-vention in AML in a randomized phase III trial, again with disappointingresults [97].

Despite the failure of these earlier efforts to avoid relapses byboosting immunity in the post-chemotherapy phase, there were signif-icant expectations of successful AML immunotherapy in the form ofIL-2, a T cell-derived cytokine that activates several subsets of T cellsand, additionally, strongly boosts the cytotoxic activity of NK cells[98]. However, IL-2 monotherapy for relapse control has yielded disap-pointment: six randomized trials using IL-2 have failed to demonstratesignificant relapse prevention or prolongation of time in CR [99–104].The inefficiency of IL-2 to prevent relapse has been confirmed in twometa-analyses comprising a total of N1400 patients [105,106].

The lack of in vivo efficacy of NK and T stimulatory therapies may bedue to leukemia-related immunosuppressive mechanisms that counterthe desired induction of cell-mediated, anti-leukemic immunity. Severalimmunosuppressive pathways have been described in AML [33,34,40]and thus immunostimulatory agents may need to be combined withcounter-suppressive strategies to unravel their anti-leukemic potential.

A mechanism of immunosuppression of potential relevance tothe efficiency of IL-2 treatment in AML relates to the capacity ofmyeloid cells (i.e. normal and malignant monocyte/macrophages andgranulocytes) to produce reactive oxygen species (ROS) via the NADPHoxidase (NOX-2) [107–109]. Extracellular, myeloid cell-derived ROSdown-regulate functions of NK cells and T cells and trigger significantcell death in these lymphocyte subsets. A NOX-2 inhibitor, histaminedihydrochloride (HDC), acting via H2 type histamine receptors expressedbymyeloid cells, rescues NK cells and T cells fromROS-induced inhibitionand apoptosis, and promotes anti-leukemic functions of IL-2 [110–114].HDC has been used together with IL-2 for relapse prevention inAML in several clinical trials, and a phase III study of 320 patientsin the post-consolidation phase showed a significantly improvedleukemia-free survival in patients receiving HDC/IL-2 [115]. In 2009,the combination of HDC and IL-2 was approved within EU as mainte-nance remission therapy in AML [113,114,116].

213A. Martner et al. / Blood Reviews 27 (2013) 209–216

Recently, it was reported that the clinical benefit of HDC/IL-2may bepreferential to or restricted to subtypes of AML. Aurelius et al. performedpost hoc analyses of phase III trial results pointing to a pronouncedrelapse-protective among patients with monocytic AML (FAB classesM4 and M5), but no apparent benefit in FAB-M2 (myeloblastic) AML[117]. These authors also reported that FAB-M4/M5 AML cells, but notFAB-M2 cells, triggered extensive apoptosis in adjacent NK cells by pro-ducing ROS via NOX-2. FAB-M4/M5 cells expressed functional H2Rs thatmediated inhibition of ROS production by HDC, which rescued NKcells from AML cell-induced apoptosis. These findings thus imply thatmonocytic AML cells utilize ROS as a strategy to avoid destructionby NK cells, and that the value of HDC/IL-2 therapy may be explainedby H2R-dependent inhibition of ROS-production by leukemic cells, asdepicted in Fig. 1 [39,117].

A potential disadvantage with the use of IL-2 as an immuno-stimulatory agent is that this cytokine induces Tregs [79,118–121].These cells express the high-affinity IL-2 receptor CD25 (IL-2Rα),and the IL-2-induced expansion of Tregs in AML immunotherapymay thus compromise anti-leukemic efficacy. Recently, modified vari-ants of IL-2were developed that activate T cells irrespective of interactionwith CD25. One such IL-2 variant was superior to IL-2 in expanding thepopulation of cytotoxic T cells; it also showed improved anti-tumor re-sponses in vivo and elicited less expansion of Treg cells [122]. A similar ap-proach was adopted by Klein and co-workers, who developed an IL-2variant with abolished CD25 binding, fused to the C-terminus of atumor-specific IgG1 antibody [123].

IL-15 shares several immunostimulatory properties of IL-2, but doesnot induce Treg responses. Thus, IL-15 preferentially drives expansion ofCD8+ central and effector memory T cells and NK cells, with no apparenteffect on Tregs [124]. Two phase I trials are currently determining the safe-ty of IL-15monotherapy inmetastaticmalignantmelanoma and renal cellcarcinoma, and in combination with adoptive transfer of haploidenticalNK cells in AML (NCT01369888, NCT01021059, NCT01385423).

3.3. Strategies for vaccination in AML

As recently reviewed by Anguille and co-authors [20], a largenumber of T-cell antigens have been identified in AML. These anti-gens are leukemia-specific (LSA), i.e. uniquely expressed by AMLcells, or leukemia-associated (LAA), i.e. expressed at low levels alsoby non-malignant cells.

Fig. 1. Schematic representation of ROS-induced suppression of NK cells and its regulation bwhich triggers apoptotic cell death in adjacent NK cells. (B) HDC inhibits ROS production afunction, including responsiveness to IL-2.

Several of the LSAs are proteins that arise from chromosomaltranslocations and include fusion proteins such as AML1-ETO(t(8;21)), DEK-CAN (t(6;9)) and promyelocytic leukemia-retinoic re-ceptor alpha (PML-RARα; t (15;17)). Mutant proteins with propertiesof LSA have also been identified in Flt3-ITD+ and NPM1+ AML. The de-velopment of LSAs for AML immunotherapy has hitherto been ham-pered by the fact that these antigens typically are relevant to smallgroups of patients, and the current information on the clinical utilityof LSA-based immunotherapy is scarce. Recently, immunogenic proper-ties of the protein encoded by the mutated nucleophosmin gene(NPMmut) were described; this mutation is found in approximately30% of all patients with AML and in 50–60% of those with normal karyo-type. Greiner et al. [125] found that 30–40% of AML patients showed Tcell responses to NPMmut-derived epitopes, implying that this LSA mayconstitute a target for T-cell based therapy in a relatively large patientgroup.

Immunogenic LAAs, in particular those expressed by a wider range ofAML subtypes, have been more extensively studied. Induction of T cellimmunity has been achieved either by vaccination with LAA-associatedpeptides or observed in the context of allo-transplantation withCD8+ responses occurring against, e.g., BMI1, HOXA9, hTERT, MUC1,myeloperoxidase, proteinase 3, and RHAMM, but the informationabout the clinical relevance of induction of respective T cell subsetsis mostly anecdotal. The Wilm's tumor 1 antigen (WT1), which isoverexpressed by AML cells, is arguably the best characterized LAA. Theantigen is expressed across FAB classes of AML and expressed by LSCs,and several WT1-derived peptides have been assessed for in vivo immu-nogenicity in patients with AML [126] (reviewed in Angullie et al. [20]).Recently, Van Tendeloo et al. [127] introduced full-length WT1 mRNAinto dendritic cells (DCs) and observed reductions of WT1 transcriptlevels (as a marker of residual leukemia) in five WT1/DC-vaccinatedAML patients in parallel with the development of WT1-specific CD8+

T cells. Several clinical trials are in process to evaluate the potentialbenefit of WT1/DC immunotherapy in AML [128].

In addition to immunity evokedbyvaccinationor allo-transplantation,non-transplanted patients may display natural T and B cell reactivityagainst AML-derived antigens. A study by Greiner et al. [28] showedthat approximately one third of AML patients develop antibodies againstseveral AML antigens, and Scheibenbogen et al. [129] reported theinduction of CD8+ T cell responses to WT1 and proteinase 3 amongnewly diagnosed patients and those in CR. Additional studies from the

y HDC and IL-2. (A) Mature monocytic AML cells produce ROS via the NADPH-oxidase,cting via H2-receptors on monocytic AML cells and thus preserves NK cell viability and

214 A. Martner et al. / Blood Reviews 27 (2013) 209–216

Greiner group demonstrated that 7/10 patients in CR displayed T cellresponses against the oncofetal antigen preferentially expressed antigenof melanoma (PRAME), and that mRNA expression in AML cells of atleast 1 of the 3 LAAs RHAMM/HMMR, PRAME, or G250/CA9 was associ-ated with a significant survival advantage [28].

4. Future immunotherapeutic strategies

Most immunotherapeutic protocols in AML have been developed forrelapse prevention in the post-consolidation phase, but immune-basedstrategies appear to play a significant role also in earlier stages of dis-ease. The example of GO is likely to inspire further development of con-jugated antibodies, and the relicensing of GO for use in CD33+ AMLseems probable. An additional strategy, i.e. the adoptive transfer ofT cells retrovirally transfected to express an antibody against leukemiccells along with co-stimulatory molecules (chimeric antigen receptors,CAR) has shown promise in B cell malignancies, including refractoryALL [130], and most likely will be evaluated also in AML. Also, a morewidespread use of cell cycle inducers, such as G-CSF and/or the CXCR4inhibitor plerixafor, during induction therapy may result in higherrates of patients achieving complete remission and lower incidenceof relapses. In addition, the use of adoptively transferred NK cells totreat hematopoietic malignancies is currently expanding, as reviewedin [131].

There is a high demand for improved strategies to prevent relapses inthe post-remission stage, in particular for patients who are not eligiblefor allo-transplantation. HDC/IL-2 is currently the only approvedpost-remission treatment (in EU and Israel), but the current pace ofdevelopment is likely to bring about additional immunotherapeutics.Lessons from the past decades teach that single-agent NK cell- orT cell-activators are likely to be inefficacious, as exemplified by theunexpected failure of post-remission IL-2 therapy for relapse control.Future protocols may comprise a counter-suppressive componentaiming to reduce or neutralize leukemia-related immunosuppression(as listed in Table 1)with, for example, a T- or NK-cell activating compo-nent such as a vaccine, engineered variants of IL-2, IL-15, or an antibodymediatingADCC against leukemic cells. In addition, biomarkers that pre-dict efficacy need to be developed in parallel with the immunotherapies,not least because several counter-suppressive strategies, includinganti-PD/PDL or anti-CTLA-4, entail significant immune-related toxicity[78,132]. Ideally, this development results in personalized immunother-apy that controls relapse of AML as efficiently as allo-transplantation,but with reduced treatment-related morbidity.

Conflict of interest

Author KHholds issued and pending patents on the use of histaminedihydrochloride and related compounds in cancer.

References

[1] Tallman MS, Gilliland DG, Rowe JM. Drug therapy for acute myeloid leukemia.Blood 2005;106:1154–63.

[2] Astrom M, Bodin L, Tidefelt U. Adjustment of incidence rates after an estimateof completeness and accuracy in registration of acute leukemias in a Swedishpopulation. Leuk Lymphoma 2001;41:559–70.

[3] Schiller G, Gajewski J, TerritoM,Nimer S, LeeM, Belin T, et al. Long-termoutcomeofhigh-dose cytarabine-based consolidation chemotherapy for adults with acutemyelogenous leukemia. Blood 1992;80:2977–82.

[4] Kolb HJ. Graft-versus-leukemia effects of transplantation and donor lympho-cytes. Blood 2008;112:4371–83.

[5] Breems DA, Van PuttenWL, Huijgens PC, Ossenkoppele GJ, Verhoef GE, VerdonckLF, et al. Prognostic index for adult patients with acute myeloid leukemia in firstrelapse. J Clin Oncol 2005;23:1969–78.

[6] Estey EH. Acute myeloid leukemia: 2012 update on diagnosis, risk stratification,and management. Am J Hematol 2012;87:89–99.

[7] Martelli MP, Sportoletti P, Tiacci E, Martelli MF, Falini B. Mutational landscape ofAML with normal cytogenetics: biological and clinical implications. Blood Rev2013;27:13–22.

[8] Smith ML, Hills RK, Grimwade D. Independent prognostic variables in acutemyeloid leukaemia. Blood Rev 2011;25:39–51.

[9] Byrd JC, Mrozek K, Dodge RK, Carroll AJ, Edwards CG, Arthur DC, et al. Pretreat-ment cytogenetic abnormalities are predictive of induction success, cumulativeincidence of relapse, and overall survival in adult patients with de novo acutemyeloid leukemia: results from Cancer and Leukemia Group B (CALGB 8461).Blood 2002;100:4325–36.

[10] Forman SJ, Rowe JM. The myth of the second remission of acute leukemia in theadult. Blood 2013;121:1077–82.

[11] Rowe J, LI X, Cassileth PA. Very poor survival of patients with AML who relapseafter achieving a first complete remission: the Eastern Cooperative OncologyGroup Experience. ASH Annual Meeting Abstracts, 106; 2005. p. 546.

[12] Pulte D, Gondos A, Brenner H. Improvements in survival of adults diagnosedwith acute myeloblastic leukemia in the early 21st century. Haematologica2008;93:594–600.

[13] Gyurkocza B, Storb R, Storer BE, Chauncey TR, Lange T, Shizuru JA, et al.Nonmyeloablative allogeneic hematopoietic cell transplantation in patientswith acute myeloid leukemia. J Clin Oncol 2010;28:2859–67.

[14] Mohty M, de Lavallade H, Ladaique P, Faucher C, Vey N, Coso D, et al. The role ofreduced intensity conditioning allogeneic stem cell transplantation in patientswith acute myeloid leukemia: a donor vs no donor comparison. Leukemia2005;19:916–20.

[15] Buchner T, Hiddemann W, Wormann B, Loffler H, Gassmann W, Haferlach T,et al. Double induction strategy for acute myeloid leukemia: the effect ofhigh-dose cytarabine with mitoxantrone instead of standard-dose cytarabinewith daunorubicin and 6-thioguanine: a randomized trial by the German AMLCooperative Group. Blood 1999;93:4116–24.

[16] Buchner T, Urbanitz D, Hiddemann W, Ruhl H, Ludwig WD, Fischer J, et al. Inten-sified induction and consolidation with or without maintenance chemotherapyfor acute myeloid leukemia (AML): two multicenter studies of the GermanAML Cooperative Group. J Clin Oncol 1985;3:1583–9.

[17] Greiner J, Ringhoffer M, Taniguchi M, Schmitt A, Kirchner D, Krahn G, et al.Receptor for hyaluronan acid-mediated motility (RHAMM) is a new immuno-genic leukemia-associated antigen in acute and chronic myeloid leukemia. ExpHematol 2002;30:1029–35.

[18] Greiner J, Ringhoffer M, Taniguchi M, Hauser T, Schmitt A, Dohner H, et al.Characterization of several leukemia-associated antigens inducing humoral immuneresponses in acute and chronic myeloid leukemia. Int J Cancer 2003;106:224–31.

[19] Elisseeva OA, Oka Y, Tsuboi A, Ogata K, Wu F, Kim EH, et al. Humoral immuneresponses against Wilms tumor geneWT1 product in patients with hematopoieticmalignancies. Blood 2002;99:3272–9.

[20] Anguille S, Van Tendeloo VF, Berneman ZN. Leukemia-associated antigens andtheir relevance to the immunotherapy of acute myeloid leukemia. Leukemia2012;26:2186–96.

[21] Brune M, Hellstrand K. Remission maintenance therapy with histamine andinterleukin-2 in acute myelogenous leukaemia. Br J Haematol 1996;92:620–6.

[22] Smits EL, Berneman ZN, Van Tendeloo VF. Immunotherapy of acute myeloidleukemia: current approaches. Oncologist 2009;14:240–52.

[23] Lowdell MW, Koh MB. Immunotherapy of AML: future directions. J Clin Pathol2000;53:49–54.

[24] Appelbaum FR. Haematopoietic cell transplantation as immunotherapy. Nature2001;411:385–9.

[25] Damiani D, Tiribelli M, Geromin A, Cerno M, Sperotto A, Toffoletti E, et al. Donorcompatibility and performance status affect outcome of allogeneic haematopoieticstem cell transplant in patients with relapsed or refractory acute myeloid leukae-mia. Ann Hematol 2012;91:1937–43.

[26] Horowitz MM, Gale RP, Sondel PM, Goldman JM, Kersey J, Kolb HJ, et al.Graft-versus-leukemia reactions after bone marrow transplantation. Blood 1990;75:555–62.

[27] Montagna D,Maccario R, Locatelli F, Montini E, Pagani S, Bonetti F, et al. Emergenceof antitumor cytolytic T cells is associatedwithmaintenance of hematologic remis-sion in children with acute myeloid leukemia. Blood 2006;108:3843–50.

[28] Greiner J, Schmitt M, Li L, Giannopoulos K, Bosch K, Schmitt A, et al. Expression oftumor-associated antigens in acute myeloid leukemia: implications for specificimmunotherapeutic approaches. Blood 2006;108:4109–17.

[29] van Luijn MM, van den Ancker W, Chamuleau ME, Ossenkoppele GJ, van HamSM, van de Loosdrecht AA. Impaired antigen presentation in neoplasia: basicmechanisms and implications for acute myeloid leukemia. Immunotherapy2010;2:85–97.

[30] Le Dieu R, Taussig DC, Ramsay AG, Mitter R, Miraki-Moud F, Fatah R, et al.Peripheral blood T cells in acute myeloid leukemia (AML) patients at diagnosishave abnormal phenotype and genotype and form defective immune synapseswith AML blasts. Blood 2009;114:3909–16.

[31] ChamuleauME, van de Loosdrecht AA, Hess CJ, Janssen JJ, Zevenbergen A, Delwel R,et al. High INDO (indoleamine 2,3-dioxygenase) mRNA level in blasts of acutemyeloid leukemic patients predicts poor clinical outcome. Haematologica 2008;93:1894–8.

[32] Lion E, Willemen Y, Berneman ZN, Van Tendeloo VF, Smits EL. Natural killer cellimmune escape in acute myeloid leukemia. Leukemia 2012;26:2019–26.

[33] Costello RT, Sivori S, Marcenaro E, Lafage-Pochitaloff M, Mozziconacci MJ, Reviron D,et al. Defective expression and function of natural killer cell-triggering receptors inpatients with acute myeloid leukemia. Blood 2002;99:3661–7.

[34] Fauriat C, Just-Landi S, Mallet F, Arnoulet C, Sainty D, Olive D, et al. Deficientexpression of NCR in NK cells from acute myeloid leukemia: evolution duringleukemia treatment and impact of leukemia cells in NCRdull phenotype induc-tion. Blood 2007;109:323–30.

215A. Martner et al. / Blood Reviews 27 (2013) 209–216

[35] Orleans-Lindsay JK, Barber LD, Prentice HG, Lowdell MW. Acute myeloid leukaemiacells secrete a soluble factor that inhibits T andNK cell proliferation but not cytolyticfunction—implications for the adoptive immunotherapy of leukaemia. Clin ExpImmunol 2001;126:403–11.

[36] Sanchez-Correa B, Morgado S, Gayoso I, Bergua JM, Casado JG, Arcos MJ, et al.Human NK cells in acute myeloid leukaemia patients: analysis of NK cell-activatingreceptors and their ligands. Cancer Immunol Immunother 2011;60:1195–205.

[37] Tajima F, Kawatani T, EndoA, KawasakiH. Natural killer cell activity and cytokine pro-duction as prognostic factors in adult acute leukemia. Leukemia 1996;10:478–82.

[38] Costello RT, Fauriat C, Sivori S, Marcenaro E, Olive D. NK cells: innate immunityagainst hematological malignancies? Trends Immunol 2004;25:328–33.

[39] Aurelius J, Thoren FB, Akhiani A, BruneM, Palmqvist L, HanssonM, et al. MonocyticAML cells inactivate anti-leukemic lymphocytes: role of NADPH oxidase/gp91phoxexpression and the PARP-1/PAR pathway of apoptosis. Blood 2012;119:5832–7.

[40] Buggins AG, Milojkovic D, Arno MJ, Lea NC, Mufti GJ, Thomas NS, et al. Microenvi-ronment produced by acute myeloid leukemia cells prevents T cell activation andproliferation by inhibition of NF-kappaB, c-Myc, and pRb pathways. J Immunol2001;167:6021–30.

[41] GrohV,Wu J, Yee C, Spies T. Tumour-derived solubleMIC ligands impair expressionof NKG2D and T-cell activation. Nature 2002;419:734–8.

[42] Vesely MD, Kershaw MH, Schreiber RD, Smyth MJ. Natural innate and adaptiveimmunity to cancer. Annu Rev Immunol 2011;29:235–71.

[43] Berthon C, Driss V, Liu J, Kuranda K, Leleu X, Jouy N, et al. In acute myeloidleukemia, B7-H1 (PD-L1) protection of blasts from cytotoxic T cells is inducedby TLR ligands and interferon-gamma and can be reversed using MEK inhibitors.Cancer Immunol Immunother 2010;59:1839–49.

[44] Barclay AN, Wright GJ, Brooke G, Brown MH. CD200 and membrane proteininteractions in the control of myeloid cells. Trends Immunol 2002;23:285–90.

[45] Coles SJ, Wang EC, Man S, Hills RK, Burnett AK, Tonks A, et al. CD200 expressionsuppresses natural killer cell function and directly inhibits patient anti-tumorresponse in acute myeloid leukemia. Leukemia 2011;25:792–9.

[46] Lanier LL. NK cell recognition. Annu Rev Immunol 2005;23:225–74.[47] Long EO, Sik Kim H, Liu D, Peterson ME, Rajagopalan S. Controlling natural killer

cell responses: integration of signals for activation and inhibition. Annu RevImmunol 2013;31:227–58.

[48] Babor F, Fischer JC, Uhrberg M. The role of KIR genes and ligands in leukemiasurveillance. Front Immunol 2013;4:27.

[49] Ruggeri L, Capanni M, Urbani E, Perruccio K, Shlomchik WD, Tosti A, et al. Effec-tiveness of donor natural killer cell alloreactivity in mismatched hematopoietictransplants. Science 2002;295:2097–100.

[50] Giebel S, Locatelli F, Lamparelli T, Velardi A, Davies S, Frumento G, et al. Survivaladvantage with KIR ligand incompatibility in hematopoietic stem cell transplan-tation from unrelated donors. Blood 2003;102:814–9.

[51] Miller JS, Cooley S, Parham P, Farag SS, Verneris MR, McQueen KL, et al. MissingKIR ligands are associatedwith less relapse and increased graft-versus-host disease(GVHD) following unrelated donor allogeneic HCT. Blood 2007;109:5058–61.

[52] Hsu KC, Gooley T, Malkki M, Pinto-Agnello C, Dupont B, Bignon JD, et al. KIRligands and prediction of relapse after unrelated donor hematopoietic cell trans-plantation for hematologic malignancy. Biol Blood Marrow Transplant 2006;12:828–36.

[53] Davies SM, Ruggieri L, DeFor T, Wagner JE, Weisdorf DJ, Miller JS, et al. Evalua-tion of KIR ligand incompatibility in mismatched unrelated donor hematopoietictransplants. Killer immunoglobulin-like receptor. Blood 2002;100:3825–7.

[54] Verheyden S, Demanet C. NK cell receptors and their ligands in leukemia. Leukemia2008;22:249–57.

[55] Venstrom JM, Pittari G, Gooley TA, Chewning JH, Spellman S, Haagenson M, et al.HLA-C-dependent prevention of leukemia relapse by donor activating KIR2DS1.N Engl J Med 2012;367:805–16.

[56] Sivori S, Carlomagno S, Falco M, Romeo E, Moretta L, Moretta A. Natural killercells expressing the KIR2DS1-activating receptor efficiently kill T-cell blasts anddendritic cells: implications in haploidentical HSCT. Blood 2011;117:4284–92.

[57] Biassoni R, Cantoni C, Pende D, Sivori S, Parolini S, Vitale M, et al. Human naturalkiller cell receptors and co-receptors. Immunol Rev 2001;181:203–14.

[58] Stewart CA, Laugier-Anfossi F, Vely F, Saulquin X, Riedmuller J, Tisserant A, et al. Rec-ognition of peptide-MHC class I complexes by activating killer immunoglobulin-likereceptors. Proc Natl Acad Sci U S A 2005;102:13224–9.

[59] Bauer K, Rancea M, Roloff V, Elter T, Hallek M, Engert A, et al. Rituximab,ofatumumab and other monoclonal anti-CD20 antibodies for chronic lympho-cytic leukaemia. Cochrane Database Syst Rev 2012;11:CD008079.

[60] Cang S, Mukhi N, Wang K, Liu D. Novel CD20monoclonal antibodies for lymphomatherapy. J Hematol Oncol 2012;5:64.

[61] Kantarjian H, Thomas D, Wayne AS, O'Brien S. Monoclonal antibody-based thera-pies: a new dawn in the treatment of acute lymphoblastic leukemia. J Clin Oncol2012;30:3876–83.

[62] Cheson BD. Monoclonal antibody therapy of chronic lymphocytic leukaemia.Best Pract Res Clin Haematol 2010;23:133–43.

[63] Feldman EJ, Brandwein J, Stone R, Kalaycio M, Moore J, O'Connor J, et al. Phase IIIrandomized multicenter study of a humanized anti-CD33 monoclonal antibody,lintuzumab, in combination with chemotherapy, versus chemotherapy alone inpatients with refractory or first-relapsed acute myeloid leukemia. J Clin Oncol2005;23:4110–6.

[64] Walter RB, Appelbaum FR, Estey EH, Bernstein ID. Acute myeloid leukemia stemcells and CD33-targeted immunotherapy. Blood 2012;119:6198–208.

[65] Bross PF, Beitz J, Chen G, Chen XH, Duffy E, Kieffer L, et al. Approval summary:gemtuzumab ozogamicin in relapsed acute myeloid leukemia. Clin Cancer Res2001;7:1490–6.

[66] McKoy JM, Angelotta C, Bennett CL, Tallman MS, Wadleigh M, Evens AM, et al.Gemtuzumab ozogamicin-associated sinusoidal obstructive syndrome (SOS):an overview from the research on adverse drug events and reports (RADAR)project. Leuk Res 2007;31:599–604.

[67] Kopecky KJ, Stuart RK, Larson RA, Nevill TJ, Stenke S, Slovak ML, et al. PreliminaryResults of Southwest Oncology Group Study S0106: An International IntergroupPhase 3 Randomized Trial Comparing the Addition of Gemtuzumab Ozogamicinto Standard Induction Therapy Versus Standard Induction Therapy Followed by aSecond Randomization to Post-Consolidation Gemtuzumab Ozogamicin VersusNo Additional Therapy for Previously Untreated Acute Myeloid Leukemia.Blood 2009;114 [Abstract 790].

[68] Castaigne S, Pautas C, Terre C, Raffoux E, Bordessoule D, Bastie JN, et al. Effect ofgemtuzumab ozogamicin on survival of adult patients with de-novo acutemyeloid leukaemia (ALFA-0701): a randomised, open-label, phase 3 study. Lancet2012;379:1508–16.

[69] Estey EH, Giles FJ, Beran M, O'Brien S, Pierce SA, Faderl SH, et al. Experience withgemtuzumab ozogamycin (“mylotarg”) and all-trans retinoic acid in untreatedacute promyelocytic leukemia. Blood 2002;99:4222–4.

[70] Lo-Coco F, Cimino G, Breccia M, Noguera NI, Diverio D, Finolezzi E, et al.Gemtuzumab ozogamicin (Mylotarg) as a single agent for molecularly relapsedacute promyelocytic leukemia. Blood 2004;104:1995–9.

[71] Eppert K, Takenaka K, Lechman ER, Waldron L, Nilsson B, van Galen P, et al. Stemcell gene expression programs influence clinical outcome in human leukemia.Nat Med 2011;17:1086–93.

[72] Majeti R. Monoclonal antibody therapy directed against human acute myeloidleukemia stem cells. Oncogene 2011;30:1009–19.

[73] Jin L, Hope KJ, Zhai Q, Smadja-Joffe F, Dick JE. Targeting of CD44 eradicateshuman acute myeloid leukemic stem cells. Nat Med 2006;12:1167–74.

[74] Majeti R, Chao MP, Alizadeh AA, Pang WW, Jaiswal S, Gibbs Jr KD, et al. CD47 isan adverse prognostic factor and therapeutic antibody target on human acutemyeloid leukemia stem cells. Cell 2009;138:286–99.

[75] Jin L, Lee EM, Ramshaw HS, Busfield SJ, Peoppl AG, Wilkinson L, et al. Monoclonalantibody-mediated targeting of CD123, IL-3 receptor alpha chain, eliminateshuman acute myeloid leukemic stem cells. Cell Stem Cell 2009;5:31–42.

[76] Zhang L, Gajewski TF, Kline J. PD-1/PD-L1 interactions inhibit antitumor immuneresponses in a murine acute myeloid leukemia model. Blood 2009;114:1545–52.

[77] Brahmer JR, Tykodi SS, Chow LQ, Hwu WJ, Topalian SL, Hwu P, et al. Safety andactivity of anti-PD-L1 antibody in patients with advanced cancer. N Engl J Med2012;366:2455–65.

[78] Topalian SL, Hodi FS, Brahmer JR, Gettinger SN, Smith DC, McDermott DF, et al.Safety, activity, and immune correlates of anti-PD-1 antibody in cancer. N EnglJ Med 2012;366:2443–54.

[79] WingK, Onishi Y, Prieto-Martin P, Yamaguchi T,MiyaraM, Fehervari Z, et al. CTLA-4control over Foxp3+ regulatory T cell function. Science 2008;322:271–5.

[80] Qureshi OS, Zheng Y, Nakamura K, Attridge K, Manzotti C, Schmidt EM, et al.Trans-endocytosis of CD80 and CD86: a molecular basis for the cell-extrinsicfunction of CTLA-4. Science 2011;332:600–3.

[81] Hodi FS, O'Day SJ, McDermott DF, Weber RW, Sosman JA, Haanen JB, et al.Improved survival with ipilimumab in patients with metastatic melanoma.N Engl J Med 2010;363:711–23.

[82] Lipson EJ, Drake CG. Ipilimumab: an anti-CTLA-4 antibody for metastatic melanoma.Clin Cancer Res 2011;17:6958–62.

[83] Szczepanski MJ, Szajnik M, Czystowska M, Mandapathil M, Strauss L, Welsh A,et al. Increased frequency and suppression by regulatory T cells in patientswith acute myelogenous leukemia. Clin Cancer Res 2009;15:3325–32.

[84] Shenghui Z, Yixiang H, Jianbo W, Kang Y, Laixi B, Yan Z, et al. Elevated frequen-cies of CD4(+) CD25(+) CD127lo regulatory T cells is associated to poor prog-nosis in patients with acute myeloid leukemia. Int J Cancer 2011;129:1373–81.

[85] Vey N, Bourhis JH, Boissel N, Bordessoule D, Prebet T, Charbonnier A, et al.A phase 1 trial of the anti-inhibitory KIR mAb IPH2101 for AML in completeremission. Blood 2012;120:4317–23.

[86] Zhang Y, Patel S, Abdelouahab H, Wittner M, Willekens C, Shen S, et al. CXCR4 in-hibitors selectively eliminate CXCR4-expressing human acute myeloid leukemiacells in NOG mouse model. Cell Death Dis 2012;3:e396.

[87] Peled A, Tavor S. Role of CXCR4 in the pathogenesis of acute myeloid leukemia.Theranostics 2013;3:34–9.

[88] Uy GL, Rettig MP, Motabi IH, McFarland K, Trinkaus KM, Hladnik LM, et al. Aphase 1/2 study of chemosensitization with the CXCR4 antagonist plerixafor inrelapsed or refractory acute myeloid leukemia. Blood 2012;119:3917–24.

[89] Beider K, Begin M, Abraham M, Wald H, Weiss ID, Wald O, et al. CXCR4 antagonist4F-benzoyl-TN14003 inhibits leukemia and multiple myeloma tumor growth. ExpHematol 2011;39:282–92.

[90] Kuhne MR, Mulvey T, Belanger B, Chen S, Pan C, Chong C, et al.BMS-936564/MDX-1338: a fully human anti-CXCR4 antibody induces apoptosisin vitro and shows antitumor activity in vivo in hematologic malignancies. ClinCancer Res 2013;19:357–66.

[91] Tonks A, Hills R, White P, Rosie B, Mills KI, Burnett AK, et al. CD200 as a prognosticfactor in acute myeloid leukaemia. Leukemia 2007;21:566–8.

[92] Kretz-Rommel A, Qin F, Dakappagari N, Cofiell R, Faas SJ, Bowdish KS. Blockadeof CD200 in the presence or absence of antibody effector function: implicationsfor anti-CD200 therapy. J Immunol 2008;180:699–705.

[93] Wei G, Ni W, Chiao JW, Cai Z, Huang H, Liu D. A meta-analysis of CAG (cytarabine,aclarubicin, G-CSF) regimen for the treatment of 1029 patients with acute myeloidleukemia and myelodysplastic syndrome. J Hematol Oncol 2011;4:46.

[94] Foon KA, Smalley RV, Riggs CW, Gale RP. The role of immunotherapy in acutemyelogenous leukemia. Arch Intern Med 1983;143:1726–31.

216 A. Martner et al. / Blood Reviews 27 (2013) 209–216

[95] Anguille S, Lion E, Willemen Y, Van Tendeloo VF, Berneman ZN, Smits EL.Interferon-alpha in acute myeloid leukemia: an old drug revisited. Leukemia2011;25:739–48.

[96] Goldstone AH, Burnett AK, Wheatley K, Smith AG, Hutchinson RM, Clark RE.Attempts to improve treatment outcomes in acute myeloid leukemia (AML) inolder patients: the results of the United Kingdom Medical Research CouncilAML11 trial. Blood 2001;98:1302–11.

[97] Simonsson B, Totterman T, Hokland P, Lauria F, Carella AM, Fernandez MN, et al.Roquinimex (Linomide) vs placebo in AML after autologous bone marrow trans-plantation. Bone Marrow Transplant 2000;25:1121–7.

[98] Foa R, Meloni G, Guarini A, Vignetti M, Marchis D, Tosti S, et al. Interleukin 2(IL2) in the management of acute myeloid leukemia: clinical and biological find-ings. Leukemia 1992;6(Suppl. 3):115S–6S.

[99] Baer MR, George SL, Caligiuri MA, Sanford BL, Bothun SM, Mrozek K, et al.Low-dose interleukin-2 immunotherapy does not improve outcome of patientsage 60 years and older with acute myeloid leukemia in first complete remission:Cancer and Leukemia Group B Study 9720. J Clin Oncol 2008;26:4934–9.

[100] BlaiseD, AttalM, Reiffers J,MichalletM, Bellanger C, Pico JL, et al. Randomized studyof recombinant interleukin-2 after autologous bone marrow transplantation foracute leukemia in first complete remission. Eur Cytokine Netw 2000;11:91–8.

[101] Kolitz JE, Hars V, DeAngelo DJ, Allen SL, Shea TC, Vij R, et al. Phase III trial ofimmunotherapy with recombinant interleukin-2 (rIL-2) versus observation inpatients b 60 years with acute myeloid leukemia (AML) in first remission (CR1):preliminary results fromCancer and Leukemia GroupB (CALGB)19808. ASHAnnualMeeting Abstracts, 110; 2007. p. 157.

[102] Lange BJ, Smith FO, Feusner J, Barnard DR, Dinndorf P, Feig S, et al. Outcomes inCCG-2961, a children's oncology group phase 3 trial for untreated pediatric acutemyeloid leukemia: a report from the children's oncology group. Blood 2008;111:1044–53.

[103] Lotzova E, Savary CA, Herberman RB. Induction of NK cell activity against freshhuman leukemia in culture with interleukin 2. J Immunol 1987;138:2718–27.

[104] Pautas C,Merabet F, Thomas X, Raffoux E, Gardin C, CormS, et al. Randomized studyof intensified anthracycline doses for induction and recombinant interleukin-2 formaintenance in patients with acute myeloid leukemia age 50 to 70 years: resultsof the ALFA-9801 study. J Clin Oncol 2010;28:808–14.

[105] Berry SM, Broglio KR, Berry DA. Addressing the incremental benefit of histaminedihydrochloridewhen added to interleukin-2 in treating acutemyeloid leukemia: aBayesian meta-analysis. Cancer Invest 2011;29:293–9.

[106] Buyse M, Squifflet P, Lucchesi KJ, Brune ML, Castaigne S, Rowe JM. Assessment ofthe consistency and robustness of results from a multicenter trial of remissionmaintenance therapy for acute myeloid leukemia. Trials 2011;12:86.

[107] HanssonM, Asea A, ErssonU, Hermodsson S, Hellstrand K. Induction of apoptosis inNK cells by monocyte-derived reactive oxygen metabolites. J Immunol 1996;156:42–7.

[108] Thoren FB, Romero AI, Hellstrand K. Oxygen radicals induce poly(ADP-ribose)polymerase-dependent cell death in cytotoxic lymphocytes. J Immunol 2006;176:7301–7.

[109] Kono K, Salazar-Onfray F, Petersson M, Hansson J, Masucci G, Wasserman K, et al.Hydrogen peroxide secreted by tumor-derived macrophages down-modulatessignal-transducing zeta molecules and inhibits tumor-specific T cell-and naturalkiller cell-mediated cytotoxicity. Eur J Immunol 1996;26:1308–13.

[110] Hellstrand K, Hermodsson S. Cell-to-cell mediated inhibition of natural killer cellproliferation by monocytes and its regulation by histamine H2-receptors. Scand JImmunol 1991;34:741–52.

[111] Mellqvist UH, Hansson M, Brune M, Dahlgren C, Hermodsson S, Hellstrand K.Natural killer cell dysfunction and apoptosis induced by chronic myelogenous leuke-mia cells: role of reactive oxygen species and regulation by histamine. Blood 2000;96:1961–8.

[112] Romero AI, Thoren FB, Brune M, Hellstrand K. NKp46 and NKG2D receptorexpression in NK cells with CD56dim and CD56bright phenotype: regulationby histamine and reactive oxygen species. Br J Haematol 2006;132:91–8.

[113] Martner A, Thoren FB, Aurelius J, Soderholm J, Brune M, Hellstrand K. Immuno-therapy with histamine dihydrochloride for the prevention of relapse in acutemyeloid leukemia. Expert Rev Hematol 2010;3:381–91.

[114] Romero AI, Thoren FB, Aurelius J, Askarieh G, Brune M, Hellstrand K. Post-consolidation immunotherapy with histamine dihydrochloride and interleukin-2in AML. Scand J Immunol 2009;70:194–205.

[115] Brune M, Castaigne S, Catalano J, Gehlsen K, Ho AD, Hofmann WK, et al.Improved leukemia-free survival after postconsolidation immunotherapy with

histamine dihydrochloride and interleukin-2 in acute myeloid leukemia: resultsof a randomized phase 3 trial. Blood 2006;108:88–96.

[116] Thoren FB, Romero AI, Brune M, Hellstrand K. Histamine dihydrochloride andlow-dose interleukin-2 as post-consolidation immunotherapy in acute myeloidleukemia. Expert Opin Biol Ther 2009;9:1217–23.

[117] Aurelius J, Martner A, Brune M, Palmqvist L, Hansson M, Hellstrand K, et al. Re-mission maintenance in acute myeloid leukemia: impact of functional histamineH2 receptors expressed by leukemic cells. Haematologica 2012;97:1904–8.

[118] Sakaguchi S. Regulatory T, cells: key controllers of immunologic self-tolerance.Cell 2000;101:455–8.

[119] Long SA, Buckner JH, Greenbaum CJ. IL-2 therapy in type 1 diabetes: “trials” andtribulations. Clin Immunol 2013 (epub ahead of print).

[120] Liao W, Lin JX, Leonard WJ. Interleukin-2 at the crossroads of effector responses,tolerance, and immunotherapy. Immunity 2013;38:13–25.

[121] Beyer M. Interleukin-2 treatment of tumor patients can expand regulatoryT cells. Oncoimmunology 2012;1:1181–2.

[122] Levin AM, Bates DL, Ring AM, Krieg C, Lin JT, Su L, et al. Exploiting a natural conforma-tional switch to engineer an interleukin-2 ‘superkine’. Nature 2012;484:529–33.

[123] Klein C, Inja W, Nicolini V, Claire D, Freimoser A, Herter S, et al. Noveltumor-targeted, engineered IL-2 variant (IL-2v)-based immunocytokines for im-munotherapy of cancer. AACR Special Conference on Tumor Immunology; 2012(Abstract:55).

[124] Waldmann TA, Lugli E, Roederer M, Perera LP, Smedley JV, Macallister RP, et al.Safety (toxicity), pharmacokinetics, immunogenicity, and impact on elementsof the normal immune system of recombinant human IL-15 in rhesus macaques.Blood 2011;117:4787–95.

[125] Greiner J, Ono Y, Hofmann S, Schmitt A, Mehring E, Gotz M, et al. Mutatedregions of nucleophosmin 1 elicit both CD4(+) and CD8(+) T-cell responsesin patients with acute myeloid leukemia. Blood 2012;120:1282–9.

[126] Maslak PG, Dao T, Krug LM, Chanel S, Korontsvit T, Zakhaleva V, et al. Vaccinationwith synthetic analog peptides derived from WT1 oncoprotein induces T-cellresponses in patients with complete remission from acute myeloid leukemia.Blood 2010;116:171–9.

[127] Van Tendeloo VF, Van de Velde A, Van Driessche A, Cools N, Anguille S, Ladell K,et al. Induction of complete and molecular remissions in acute myeloid leukemiabyWilms' tumor 1 antigen-targeted dendritic cell vaccination. Proc Natl Acad SciU S A 2010;107:13824–9.

[128] Van Driessche A, Berneman ZN, Van Tendeloo VF. Active specific immunotherapytargeting theWilms' tumor protein 1 (WT1) for patientswith hematologicalmalig-nancies and solid tumors: lessons from early clinical trials. Oncologist 2012;17:250–9.

[129] Scheibenbogen C, Letsch A, Thiel E, Schmittel A, Mailaender V, Baerwolf S, et al.CD8 T-cell responses to Wilms tumor gene product WT1 and proteinase 3 inpatients with acute myeloid leukemia. Blood 2002;100:2132–7.

[130] Brentjens RJ, Davila ML, Riviere I, Park J, Wang X, Cowell LG, et al. CD19-targetedT cells rapidly induce molecular remissions in adults with chemotherapy-refractory acute lymphoblastic leukemia. Sci Transl Med 2013;5:177ra38.

[131] McKenna DH, Kadidlo DM, Cooley S, Miller JS. Clinical production and therapeu-tic applications of alloreactive natural killer cells. Methods Mol Biol 2012;882:491–507.

[132] Di Giacomo AM, Biagioli M, Maio M. The emerging toxicity profiles of anti-CTLA-4antibodies across clinical indications. Semin Oncol 2010;37:499–507.

[133] Yan Y, Zhang GX, Gran B, Fallarino F, Yu S, Li H, et al. IDO upregulates regulatoryT cells via tryptophan catabolite and suppresses encephalitogenic T cell responsesin experimental autoimmune encephalomyelitis. J Immunol 2010;185:5953–61.

[134] Curti A, Pandolfi S, Valzasina B, Aluigi M, Isidori A, Ferri E, et al. Modulation oftryptophan catabolism by human leukemic cells results in the conversion ofCD25- into CD25+ T regulatory cells. Blood 2007;109:2871–7.

[135] Buzyn A, Petit F, Ostankovitch M, Figueiredo S, Varet B, Guillet JG, et al.Membrane-bound Fas (Apo-1/CD95) ligand on leukemic cells: a mechanism oftumor immune escape in leukemia patients. Blood 1999;94:3135–40.

[136] Whiteside TL. Immune modulation of T-cell and NK (natural killer) cell activitiesby TEXs (tumour-derived exosomes). Biochem Soc Trans 2013;41:245–51.

[137] Coles SJ, Hills RK, Wang EC, Burnett AK, Man S, Darley RL, et al. Expression ofCD200 on AML blasts directly suppresses memory T-cell function. Leukemia2012;26:2148–51.

[138] Coles SJ, Hills RK, Wang EC, Burnett AK, Man S, Darley RL, et al. Increased CD200expression in acute myeloid leukemia is linked with an increased frequency ofFoxP3+ regulatory T cells. Leukemia 2012;26:2146–8.