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Emerging Strategies for High-Risk and Relapsed/Refractory Acute Myeloid Leukemia: Novel Agents and Approaches Currently in Clinical Trials

Blood Reviews


High-risk acute myeloid leukemia (AML) is defined by clinical and biologic features that predict for poor response to induction chemotherapy and high risk of relapse. Despite even the most aggressive and well-developed strategies for care, most patients succumb to the disease. No currently available treatment has demonstrated consistent efficacy in terms of remission induction or long-term survival. This review will highlight some of the emerging strategies to treat high-risk AML with an emphasis on clinical trials of novel strategies currently enrolling patients. Targeted molecular therapies, novel cytotoxics, and immune-based therapies are under investigation for the management of high-risk AML. Some of the agents covered include tyrosine kinase inhibitors targeted to AML specific oncoproteins, nanoparticle formulations of existing drugs, nucleoside analogues, monoclonal antibodies, chimeric antigen receptors, bispecific T-cell engaging antibodies, and vaccines. As our understanding of the biology of AML has improved, targeted therapy for AML has emerged, offering to change not only response rate, but also the nature of response. Differentiation, rather than necrosis or apoptosis, is often seen in response to targeted agents and may be seen more frequently in the future. Interventions that might be more widely used in the near future include FLT3 inhibitors and nanoparticle formulations of drugs already known to have activity in the disease. Longer term, immune therapy holds significant promise.

Keywords: High Risk AML, FLT3, Sorafenib, Midostaurin, Quizartinib, Volasertib, IDH, CPX-351, Sapacitabine, Vosaroxin, Gemtuzumab Ozogamicin, Bispecific T-cell Engaging Antibodies.


Clinically and biologically, acute myeloid leukemia (AML) in adults is a heterogeneous disease, characterized by unique risk profiles. High-risk AML clusters in older adults and is biologically and clinically characterized by a number of factors ( Table 1 ). At diagnosis, if a patient has adverse molecular or cytogenetic features, an antecedent hematological disorder (typically myelodysplastic syndrome or myeloproliferative neoplasms), prior cytotoxic chemotherapy (therapy-related AML), or extramedullary disease (eg. CNS disease, myeloid sarcoma), they would conventionally be considered high-risk. Advancing age may not truly qualify as high-risk: while the biology in this group is usually more aggressive, high-risk could also include inability to tolerate standard therapy. The prognosis of most patients older than 70 years of age with AML is often poor (with the notable exception of acute promyelocytic leukemia) with intensive chemotherapy; among the elderly, not only is it common to see complex cytogenetics, it is common to see adverse cytogenetic and molecular features. The 8-week mortality exceeds 30% and the median survival is less than 6 months. As with any other malignancy, performance status and comorbid conditions have a significant impact on survival rates. Given that, physiological age is probably more important than chronological age. Moreover, certain patients initially considered favorable- or intermediate-risk might later declare themselves as high-risk if they have AML refractory to two cycles of induction therapy, a short duration of remission, or relapse following an allogeneic hematopoietic cell transplant (HCT). 1–7

Table 1 Risk Assessment for Patients with AML.

Risk Cytogenetic Features Molecular Features Clinical Features
Favorable t(8:21), inv (16) or t(16:16) Mutated CEBPA or NPM1 N/A
Intermediate Normal or trisomy 8 FLT3-ITD, mutation in KIT, TET2, MLL-PTD, DNMT3A, ASXL1, or PHF6 N/A
High-Risk − 5/-7, 11q23, 20q-, 3 or more N/A Prior cytotoxic chemotherapy (therapy-related AML), extramedullary disease (eg. CNS, myeloid sarcoma), antecedant hematologic disorder (MDS, MPN), relapse after allogeneic HCT, or refractory to 2 cycles of 7 + 3 induction

Despite decades of research and clinical trials, high-risk AML is still associated with a bleak long-term outcome. Unfortunately, there has been no regulatory pathway to approval of novel agents based on adverse disease biology or clinical features that predict for adverse outcome.8–10 Clinical trials typically do not rigorously and prospectively risk-stratify patients with AML, although lately there has been an attempt to define more homogenous populations for the purpose of drug-approval. Conclusions about the response to therapy with regards to risk must then be made retrospectively, frequently in the setting of a variety of consolidation strategies. In the therapeutic management of high-risk AML, practitioners still have a very limited portfolio. For patients who can tolerate it, the only therapy that offers a chance at long-term disease-free survival is still standard induction with cytarabine/anthracycline-based therapy (hereafter referred to as 7 + 3) and consolidation with high-dose cytarabine or allogeneic HCT. Allogeneic HCT has been the only strategy that seems to improve the otherwise dismal outcome after conventional induction and consolidation chemotherapy.5 Unfortunately, those who might benefit most from an allogeneic HCT are also those most likely to be ineligible for transplant either due to failure of induction chemotherapy or early relapse.11 Given that traditional therapies have proven largely ineffective for this group of patients, clinical trials constitute a preferred management pathway. This review will highlight some of the emerging strategies to treat high-risk AML.

Targeted Molecular Therapy with FLT3 Inhibitors

Although there are many possible targets in AML, few have been exploited therapeutically in a clinically significant way. In about 20% of AML samples, internal tandem duplication (ITD) mutations in FLT3 are detected and are associated with inferior outcome.12 An additional 5-10% of patients with AML harbor a tyrosine kinase domain (TKD) constitutively activating point mutation in FLT3, commonly at the activation loop residue D835, though this is less prognostic than the ITD form.13,14 Given the success of kinase inhibitors in other diseases, FLT3 has been a target of choice for years, though early FLT3 inhibitors showed disappointing results. This was thought to be largely attributable to lack of potency, selectivity, and favorable pharmacokinetic properties.15–17 Newer agents may be more auspicious. For an overview, see Table 2 .

Table 2 Activity of FLT3 Inhibitors in FLT3-ITD AML.

Agent Single Agent Activity Response Duration Resistance Mechanism Differentiation Seen?
Sorafenib ORR 92%

(N = 12/13 with 6 CRi, 6 nCRi)
Median 72 days Possibly expression of ALDH1A1, JAK3, and MMP15. TKD mutation at D835. Yes
Quizartinib CRc 48%

(N = 92/191)
Median 79 and 89 days in two cohorts Mutated C/EBPα or TKD mutation at F691 or D835 Yes, with differentiation-like syndrome
Midostaurin 2% PR

(N = 1/35)
60 days Unknown Not reported
Ponatinib 30% ORR

(N = 3/10)
3-6 months Unknown, though the only responders with FLT3 inhibitor-naive Not reported


Off-label use of sorafenib may offer some benefit in relapsed/refractory FLT3-ITD AML. However, the use of sorafenib during induction and consolidation, especially for elderly patients with AML, has not been as encouraging, and even shows toxicity. In one trial, elderly patients (median age 68) received 7 + 3 and up to two cycles of intermediate-dose cytarabine consolidation.18 201 patients were randomized 1:1 to receive either sorafenib or placebo between the chemotherapy cycles and for up to 1 year after the beginning of therapy. Not only did sorafenib fail to improve EFS or OS, regardless of subgroup (including those with FLT3 ITD), there was higher treatment-related mortality and lower CR rates. Due to higher toxicity, fewer patients received consolidation. In another study, a phase II trial was performed with 43 patients, 93% of whom had leukemia characterized by FLT3-ITD, median age of 64, monthly cycles of 5-azacytidine was given for 7 days with continuous sorafenib.19 The response rate was 46%, including 10 (27%) complete response with incomplete count recovery (CRi), 6 (16%) complete responses (CR), and 1 (3%) partial response. The median duration of response was 2.3 months with a wide range: 1–14.3 months. The median number of cycles required to achieve CR/CRi was two. Another study in 13 younger patients (median age 47) with relapsed/refractory FLT3-ITD AML treated with sorafenib, 12 showed clearance or near clearance of bone marrow myeloblasts after 27 (range 21–84) days with evidence of leukemia differentiation.20 The sorafenib response was lost in most patients after 72 (range 54–287) days but the FLT3 and downstream effectors remained suppressed. Resistant cells expressed several genes including ALDH1A1, JAK3, and MMP15, whose functions were unknown in AML and both ITD and TKD at D835 were identified in leukemia initiating cells (LICs) from samples prior to and after sorafenib treatment. This suggests that there may be preexisting LICs bearing both FLT3-ITD and TKD mutations that were selected out and expanded during treatment. In summary, sorafenib appears to provide a useful option for treatment of relapsed/refractory AML patients but has not yet been shown to be a good choice when incorporated into induction and consolidation in older patients. However, a large prospective study is needed to confirm the results from the small observational studies.


Quizartinib (AC220) was arguably the first FLT3 inhibitor to achieve a meaningful single-agent activity with a composite complete remission (CR) rate of approximately 50% in a phase II study in patients with relapsed/refractory FLT3-ITD AML.21,22 Interestingly, in 13 of 14 FLT3-ITD AML patients treated with quizartinib, terminal myeloid differentiation of BM blasts was observed in association with a clinical differentiation syndrome.23 In vitro, primary blasts cocultured with human BM stroma, FLT3 inhibition with quizartinib induced cell-cycle arrest and differentiation rather than apoptosis.23 In an as yet unreported multicenter, international phase 2 study (accrual completed and interim clinical results presented in abstract form) in adults with relapsed/refractory AML, quizartinib was administered as a single agent.23 Final results remain to be seen, but an interesting laboratory correlative study of patients in this trial emerged. In an analysis of only patients with normal karyotype and FLT3/ITD mutations included, 14 were evaluable. Treatment with quizartinib was associated with rapid peripheral clearance of circulating blasts. Interestingly, although neutrophil counts tended to be remarkably low prior to treatment, a surge of peripheral neutrophils after several weeks of therapy that usually peaked around day 40 was observed in 13 of the 14 study subjects. In parallel with this neutrophil surge, several patients developed fever and inflammatory infiltrates in the lungs, soft tissue, or skin without an infectious source found. Skin biopsy of lesions, when present, showed neutrophilic lobular panniculitis, consistent with Sweet syndrome. Treatment with corticosteroids provided rapid relief. The similarity of the clinical and laboratory finding to retinoic acid differentiation syndrome in acute promyelocytic leukemia (APL) cannot be ignored. Experiments indicated that the maturing myeloid elements were derived directly from leukemia precursors, and the cells retained their ITD allele. This difference in the response of blasts in the peripheral blood (cytotoxicity) compared with the bone marrow (differentiation) highlights the influence of the marrow microenvironment on the outcome of leukemia cells. There was one patient (out of 14) who failed to display this pattern of differentiation with quizartinib. This patient harbored a C/EBPα b-ZIP mutation. Another patient in this series developed clinical resistance to quizartinib which was associated with a new C/EBPα trans-activation domain mutation. This suggests that differentiation of leukemia cells in response to a FLT3 inhibitor is dependent upon a functional C/EBPα.23 Since use of this drug can also be associated with an incomplete recovery of blood counts despite bone marrow blast clearance, the distinct difference from chemotherapy treatment response questions response criteria used for evaluation of quizartinib and underscores the need for good overall survival data. In addition, in another study, of eight patients with FLT3-ITD treated with quizartinib who initially had a bone marrow response, all had evolution of TKD mutations, confirming that relapses were mediated by the reactivation of FLT3 kinase activity.24 Mutations occurring at the F691 and D835 are common causes of resistance to quizartinib and sorafinib and might change the way the drug binds and/or shift kinases into an autoactivated conformation.20,24,25 Since clinical data are limited, it’s not clear if these are equally valid therapeutic targets. On a phase I study, ponatinib, an ABL/FLT3 inhibitor that was recently FDA-approved for the treatment of TKI-resistant CML, was able to induce a CR in 2 of 7 patients with FLT3-ITD AML who had not previously been treated with a TKI.26 It may have activity against quizartinib-resistant FLT3 mutations at F691.27 The side effect profile of ponatinib (serious arterial thrombotic events) makes it a less attractive drug and other inhibitors with in vitro activity against FLT3 TKD mutations are currently undergoing clinical trials. PLX3397 has potent activity against the FLT3 F691L mutant.28 Crenolanib has activity against FLT3-ITD mutants, FLT3/D835 point mutations and, at least in vitro, can overcome the resistance to quizartinib and sorafenib.29 Another potential advantage of crenolanib is its reduced inhibition of c-Kit compared with quizartinib. G-749 is a novel FLT3 inhibitor that has potent and sustained in vitro inhibition of the FLT3 wild type and FLT3-ITD, FLT3-D835Y, FLT3-ITD/N676D, and FLT3-ITD/F691L.30 It has antileukemia activity in human AML bone marrow blasts regardless of FLT3 mutation and shows activity in animal models. Since patients with FLT3-ITD AML who would otherwise be eligible for an allogeneic HCT often relapse prior to the procedure, the best use of kinase inhibitor may prove to be to maintaining remission as a bridge to transplant.


Midostaurin (PKC412; N-benzoylstaurosporin) is a multitargeted tyrosine kinase inhibitor and has shown activity in patients with AML with FLT3 mutations. One phase II trial in patients with AML or MDS with either wild-type (n = 60) or mutated (n = 35) FLT3 were given oral midostaurin.31 The rate of bone marrow response (reduction in peripheral blood or bone marrow blasts by ≥ 50%) was 71% in patients with FLT3-mutant and 42% in patients with FLT3 wild-type. One partial response (PR) occurred in a patient with FLT3-mutated AML. As midostaurin can inhibit multiple enzyme pathways presumptively involved in the control of cell proliferation, including c-kit, platelet-derived growth factor receptor, and protein kinase C, there were concerns that this drug might be toxic, however, midostaurin was generally well-tolerated. There is a phase III randomized, double-blind, placebo controlled study of induction and consolidation with midostaurin or placebo in patients < 60 years with newly diagnosed FLT3 mutated AML, that is ongoing, but not recruiting participants (NCT00651261).

A phase I study of midostaurin combined either sequential or concurrent decitabine (DAC).32 Eight patients were over age 60 with newly diagnosed with AML and 8 were 18 years or older with relapsed AML. Only 2 of 16 patients (13%) had FLT3-ITD and no patient had KIT mutations. Based on an intent-to-treat analysis, 57% of the patients achieved stable disease or better while enrolled in the trial; 25% had a complete hematologic response. Currently enrolling patients is a phase II study of decitabine with midostaurin for patients older than 60 with newly diagnosed FLT3-ITD/TKD mutated AML (NCT01846624). Decitabine is given days 1–10 and midostaurin on days 11–28. Similarly, there is an ongoing phase I/II trial comparing MS in combination with azacitidine for patients of any age with AML who are not considered candidates for standard induction chemotherapy or have poor risk AML (t-AML, secondary AML, adverse cytogenetics or complex karyotype), or any subjects over 70 years of age with untreated AML (NCT01093573). Midostaurin plus bortezomib is also being studied in a phase I trial (NCT01174888) for patients with elapsed/refractory AML in combination with mitoxantrone, etoposide, and cytarabine.


Polo-like kinases (Plks) are a family of conserved serine/threonine kinases that have important effects on cell-cycle regulation and progression through multiple stages. Plks participate in centrosome maturation, bipolar spindle formation, chromosome segregation, activation of CDK/cyclin complexes during M-phase of the cell cycle, and cytokinesis.33 Volasertib is a selective and potent Plk inhibitor and has recently received “Breakthrough Therapy” and “Orphan Drug” designation by the US FDA and European Commission for use in AML. Orphan Drug Designation is a status given to drugs under investigation to treat a rare disease that has limited treatment options, without changing the regulatory requirements needed for approval. These statuses were given due to encouraging results in a phase II study in patients with previously untreated AML ineligible for intensive therapy.34 This trial compared volasertib in combination with low-dose cytarabine (LDAC) versus LDAC alone for patients age > 65 who were ineligible for intensive remission induction therapy. Objective responses were observed in 31% of patients (13 of 42 patients) treated with the combination of volasertib plus LDAC compared to 13% of the patients treated with LDAC alone (p = 0.0523). A trend for OS benefit (8.0 months compared to 5.2 months, p = 0.996) was observed. There was no excessive or unmanageable toxicity reported. A phase III trial using the same eligibility criteria and comparison strategy is now underway (NCT01721876).

IDH Inhibitors

IDH mutations occur in about 20% of human AML, and more frequently in cytogenetically normal AML.35 The normal function of isocitrate dehydrogenases is to convert isocitrate into α-ketoglutarate (α-KG) and carbon dioxide as part of the krebs cycle. Three isofroms exist (IDH1, IDH2, IDH3) but only IDH1 and IDH2 have been implicated in leukemogenesis. To date, all reported IDH1 or IDH2 mutations are heterozygous, which argued that a gain of function is likely. However, the paradigm of most enzymatic mutations associated with cancer has traditionally thought of as either a gain of function of the native enzymatic process producing a constitutively active protein or catalytic inactivation. IDH1 and IDH2 mutations are unique in that the product is an enzyme with novel activity.35 The mutant product of this neomorphic (new function) enzyme is ability to convert α-KG to 2-hydroxyglutarate (2HG). In this setting, the 2HG levels are consistently 10–100 fold elevated. As with most other putative oncogenes in AML, IDH mutations by themselves are not sufficient to induce AML but can cooperate with others to cause it. Though the details of the role of IDH enzymes and 2HG as an oncometabolite and in leukemogenesis are still being investigated, one likely explanation is that it induces histone- and DNA-hypermethylation through inhibition of epigenetic regulators resulting in a block of differentiation. Global DNA hypermethylation with a specific signature is seen in AML with mutant IDH1/2 and TET2 loss-of-function mutations are associated with similar epigenetic defects.36 Forced expression of mutant IDH impairs TET2 catalytic function, suggesting that 2HG might inhibit the function of TET2; this was later confirmed biochemically.37 Since Hox genes frequently have increased expression associated with IDH-mutated AML, HoxA9 and mutant IDH1 were retrovirally transduced in a murine bone marrow model and a myeloproliferative phenotype was seen with increased cell cycling, decreased CDK gene expression, and increase mitogen-activated protein kinase activity.39 The obvious next step was to attempt to inhibit the neopmorphic enzymatic activity of IDH1/2, especially since preclinical data suggested that doing so induced differentiation in vitro.39 There is now a phase I study using a novel drug, AG-221, an oral, potent, reversible, and selective inhibitor of mutated IDH2, in patients with hematologic malignancies that harbor IDH2 mutations (NCT01915498). Data are still quite early and have not been published yet, though a presentation of some early results look promising. So far therapy has been well tolerated and 6 of 10 patients have had objective responses, including 2 CRs. The striking aspect of the responses is the recapitulation of the preclinical response: marked differentiation of myeloblasts into mature forms.40

All-trans retinoic acid (ATRA)

ATRA has historically only been used in acute promyleocytic leukemia (APL) and is a potent differentiative agent as recently reviewed by Watts and Tallman in this journal.41 However, ATRA can also down-regulate bcl-2 expression and increase AML sensitivity to cytarabine in vitro when tested on non-APL AML cells.42 A phase III study of 242 patients older than 60 with AML randomly assigned patients to ATRA beginning on day + 3 after the initiation of chemotherapy or no ATRA in combination with induction and first consolidation therapy.43 There was a difference in the rates of CR favoring ATRA (52 vs 39%; P = 0.05). From that, the 61 patients who achieved CR were randomly assigned 1:1 to second intense consolidation or 1-year oral maintenance therapy. OS after second randomization was significantly better for patients assigned to intensive consolidation therapy (P < 0.001). The addition of ATRA to induction and consolidation therapy may improve CR rate, EFS and OS in elderly patients with AML. Another phase III study is enrolling to test a more “age-adapted” combination with less intensity: LDAC and etoposide with or without ATRA in patients > 60 with AML who are not eligible for intensive therapy and also have the NPM1 mutation (NCT01237808).

One of the unfortunate shortcomings of a strategy of combined cytotoxic and differentiative agents is that if non-APL leukemia cells respond to ATRA, they do so only at a much higher concentration of ATRA. There is now interest in sensitizing leukemia cells to ATRA with combination therapy. Inhibition of glycogen synthase kinase 3 (GSK3) enhances ATRA-mediated AML differentiation and growth inhibition.44 GSK3 mediated phosphorylation regulates the expression and transcriptional activity of the receptor for ATRA, the retinoic acid receptor. Hopefully this combination will lower the effective concentration of ATRA required for differentiation of leukemia cells to therapeutic ranges and improve clinical responses.

Novel Cytotoxics

Many elderly patients with AML present challenges in that due to comorbid conditions, intensive standard therapy isn’t well tolerated or might even be completely contraindicated. In addition, they have a high rate of complex cytogenetics, making targeted therapy less optimal. For this group, or any other patient with high-risk AML in whom intensive treatment is less desirable, novel cytotoxic therapy should be especially considered.


CPX-351 is a liposomal formulation of cytarabine and daunorubicin in a 5:1 molar ratio. Liposomal encapsulation can reduce the toxicity and decrease the required effective drug doses while maintaining efficacy via controlled-release effect. There are preclinical data to suggest that maintaining this drug ratio might be more effective in killing AML cells by avoiding the potential for less potency, and sometimes even antagonistic effects, at different drug ratios.45 CPX-351 has so far shown reduced non-hematologic toxicities such as hair loss, gastrointestinal side effects, and hepatic toxicity, while retaining hematopoietic cytotoxicity.46 One hundred twenty-six newly diagnosed, treatment naïve, older AML patients were randomized 2:1 to CPX-351 or 7 + 3 induction in a phase II study.47 CPX-351 produced higher response (CR + CRi) rates (66.7%vs.51.2%, P = 0.07), without any difference seen in EFS and OS for the group. A planned retrospective analysis of the secondary AML subgroup showed an improved response rate (57.6% vs.31.6%, P = 0.06), EFS (HR = 0.59, P = 0.08) and OS (HR = 0.46, P = 0.01). Cytopenias were slightly longer with CPX-351 with more grade 3–4 infections. However, no increase in infection-related deaths (3.5%vs.7.3%) or 60-day mortality (4.7% vs.14.6%) was seen. The current phase III randomized study compares CPX-351 with conventional 7 + 3 regimen in newly diagnosed AML patients aged 60–75 with AML characterized by high-risk features, enriched for secondary AML (NCT01696084).


Nucleoside analogues (eg. cladribine, clofarabine, cytarabine, azacitidine, decitabine) are a major class of agents used in AML. Oral sapacitabine is a prodrug of CNDAC (2′-C-cyano-2′-deoxy-1-β-D-arabino-pentofuranosylcytosine), a nucleoside analog which induces single-strand breaks (SSBs) when it incorporates into DNA. When the DNA replicates, unrepaired SSBs are converted into double-strand breaks (DSBs). Importantly, CNDAC-induced SSBs can be repaired by the transcription-coupled nucleotide excision repair pathway but DSBs are mainly repaired through homologous recombination (HR).48 The dependency of the HR pathway for repair of CNDAC-induced DSBs is unique for a nucleoside analog in general and distinctly different from repair mechanisms used for cytarabine-induced DNA damage, thus sapacitabine may have clinical applications different from or complementary to cytarabine. Sapacitabine was tested in a randomized, phase 2 study with patients older than 70 with AML that were either treatment-naive or in first relapse.49 105 patients were randomized to receive one of three schedules of oral sapacitabine on a 28 day cycle, 200 mg twice a day for 7 days (group A); 300 mg twice a day for 7 days (group B); and 400 mg twice a day for 3 days each week for 2 weeks (group C). The 1-year overall survival was 35% in group A, 10% in group B, and 30% in group C. 14 (13%) of 105 patients died within 30 days and 27 (26%) died within 60 days. The most common grade 3–4 adverse events were cytopenias, febrile neutropenia, and pneumonia. Seven deaths were thought to be probably or possibly related to the drug. Sapacitabine seems active and tolerable in elderly patients with AML and in the future is being combined with other low-intensity therapies in elderly patients with AML.


In a substantial portion of AML patients, there is a deficient uptake of cytarabine, often explained by lack of a transport protein (hENT1) in the leukemia cell membrane. Elacytarabine is the elaidic acid ester derivative of cytarabine whose uptake that is independent of this transport protein.50 A phase III trial was done that randomized patients with AML > 65 years with adverse cytogenetics or multiply relapsed disease to either elacytarabine or clinician’s choice of 7 control treatments. There was no benefit to survival or response rate seen in the phase III trial. Reasons for lack of benefit seen are speculative, though could be related to patient selection. Perhaps evaluation of patients based on hENT1 expression might be expected to yield more fruitful results. It could be that the drug doesn’t have enough measurable clinical activity above standard cytarabine.51


Formerly known as SNS-595/AG-7352/voreloxin, vosaroxin is a first-in-class quinolone derivative that inhibits topoisomerase II and has a similar mechanism of action to anthracyclines. It has cytotoxicity against a broad range of aggressive and multidrug resistant (MDR) tumors.52 This is attributed largely to its high permeability and lack of interaction with and susceptibility to the MDR P-glycoprotein pump, unlike most other topoisomerase II inhibitors. Moreover, it also avoids the p53 resistance pathway by activation of caspase-3, a key mediator of apoptosis independent of p53.53 In a phase I study, 73 older adult patients (85% of whom had AML and most had relapsed/refractory disease) were treated and 5 patients had a CR/Cri with a median duration of 3.1 months.54 The safety profile appears acceptable.

L-Asparaginase Encapsulated in Red Blood Cells

In acute lymphoblastic leukemia, L-asparaginase (L-Aspa) has proven efficacy, though its use in adults is hampered by frequent toxicity (usually hepatic), often prohibitory in the elderly. The rational use of L-Aspa is based on asparagine synthetase (ASNS) deficiency in leukemic cells; by depleting plasma asparagine, it starves the leukemia cells and impedes protein synthesis. In AML, promising results have been obtained in clinical trials with an improvement of complete remission rates from 18% to 54% in refractory patients younger than 60 and from 0% to 31% in refractory patients older than 60.55 AML cells from 71% of AML patients were found to be sensitive to L-Aspa in vitro.56 Despite this, L-Aspa is not typically used in the treatment of AML, mainly because of the commonly observed adverse effects. To circumvent the issues of toxicity, investigators have used a form of L-Asparaginase encapsulated in red blood cells called GRASPA. This allows systemic depletion of L-Aspa while at least partially shrouding the enzyme from the liver and circulating proteolytic enzymes, thus (hopefully) minimizing toxicity. When GRASPA was tested on patients older than age 55 (median age 67) with newly diagnosed Philadelphia chromosome negative acute lymphblastic leukemia, it had a better safety profile than the unencapsulated counterpart, allowing use even in elderly patients.57 A clinical study is currently recruiting patients with AML unfit for intensive chemotherapy in order to evaluate GRASPA’s efficacy in combination with low-dose cytarabine (NCT01810705). The investigators also plan to analyze the relationship between L-Aspa sensitivity and clinical response with ASNS expression in primary tumor cells at diagnosis.

Immune Therapy

With the recent successes of immune-based therapies in lymphoid malignancies it is expected that myeloid malignancies might similarly benefit. No known leukemia-specific cell surface antigens exist; all are shared in some way with their normal counterparts or other normal cells. One side effect of using an antigen common to normal and malignant lymphoid cells is depletion of a normal lymphoid subset. Yet while depleting a patient’s B cell pool long-term (along with a B cell derived malignancy) has acceptable therapeutic consequences, using a pan myeloid target and potentially causing depletion of myeloid cells long-term would clearly not be as forgiving.

Gemtuzumab Ozogamicin

Immune therapy agents in AML began with gemtuzumab ozogamicin (GO). GO is a humanized anti-CD33 monoclonal antibody conjugated with calicheamicin, a potent antitumor anthracycline antibiotic. GO is an active therapeutic agent but is no longer available in the United States or Europe. In 2000, GO was granted accelerated approval by the US Food and Drug Administration (FDA) based on promising phase II data in older adults with relapsed AML.58 Many felt that GO might also improve outcomes in newly diagnosed patients. Several phase III studies were designed to test GO in the upfront setting. The results of a randomized study by the Southwest Oncology Group (SWOG) led to the voluntary withdrawal of GO in 2010 when it failed to show improved efficacy and toxicity appeared excessive.59 One explanation for this this lack of improved efficacy with GO was that the dose of daunorubicin in the study arm was only 45 mg/m2 compared with 60 mg/m2 in the standard arm. Increased survival has been seen with dose intensifying anthracyclines in induction, especially in the young. Since the efficacy was similar this could suggest that the GO might have made up for the disadvantage of the lower anthracycline dose. It is also important to note that the induction mortality of 5% in the GO group was considered typical, while the 1% in the control group seemed unusually low. Since that trial, four additional randomized studies have been completed that, taken together, argue that GO might be a very useful drug with acceptable toxicity. These studies include the British AML 15 trial, British AML 16 study, Acute Leukemia French Association 0701 trial, and French Groupe Ouest Est d'Etude des Leucémies Aiguës et Autres Maladies du Sang AML 2006 trial.60–63 Typically GO was added into the induction and continued in the consolidation and compared to patients randomized to the identical induction and consolidation without GO. Patients getting GO consistently showed improved relapse rate, EFS and OS, with a bias toward patients with favorable cytogenetics deriving the bulk of the benefit. Elderly patients and those with high-risk features did not sustain any benefit of added GO. There were typically no differences in the initial response rate, CR rate, and the treatment-related mortality was similar, without any increase in toxicity with GO. Although now largely considered to be an “active” drug in AML, particularly in the lower- and intermediate-risk groups, it is likely that some degree of controversy will continue with regard to its therapeutic benefit, thus making widespread re-approval of GO in the coming year unlikely.

Other Monocolonal Antibodies

Lintuzumab (SGN-33; HuM195) is a another humanized monoclonal antibody against CD33 and it failed to show a survival benefit in a phase IIb trial evaluating whether addition of lintuzumab to low-dose cytarabine would increase overall survival in adults > 60 years with untreated AML.64 Some of the pitfalls of GO and lintuzumab might prove to be ameliorated with SGN-CD33A, a humanized anti-CD33 antibody with engineered cysteines conjugated to a highly potent, synthetic DNA cross-linking pyrrolobenzodiazepine dimer via a protease-cleavable linker.65 Although data with GO demonstrate target validity and activity in some patients with AML, broader therapeutic benefit might be limited by heterogeneous drug conjugation (approximately 50% of the anti-CD33 mAb molecules are unconjugated in clinical-grade GO), linker instability, and cellular efflux of the toxic GO payload, calicheamicin-γ1. These have been consistently identified as reasons for GO resistance or failure. Expression of transporter proteins has been associated with resistance to GO which is a serious concern given that MDR expression is common in AML.66 The novel linker used with SGN-CD33A enables uniform drug loading of antibodies and uses a toxic moiety that maintains antitumor activity despite the MDR phenotype. In preclinical testing using AML cell lines, primary AML cells in vitro, and in xenotransplantation studies in mice, SGN-CD33A is more potent than GO. And unlike GO, antileukemia activity is observed with SGN-CD33A in AML models with the MDR. A phase I trial is currently enrolling (NCT01902329). Rather than conjugation to a cytotoxic, another model is radio-immunoconjugatation of CD33 directed antibodies. Lintuzumab has been conjugated to the α-particle-emitting radionuclide bismuth-213 ((213)Bi) in a phase I/II trial that included 31 patients with newly diagnosed (n = 13) or relapsed/refractory (n = 18) AML (median age, 67 years). They were treated with cytarabine (200 mg/m (2) /d) for 5 days followed by (213)Bi-lintuzumab. The maximum tolerated dose was largely determined by myelosuppression lasting > 35 days. Reductions in bone marrow blasts were seen across all doses with a 6 month median duration of response.67 Unfortunately, the use of the isotope (213)Bi is limited by its short 46-min half-life. To circumvent that problem, Actinium-225 conjugated lintuzumab yields 4 α-emitting isotopes with a ten day half-life - more clinically translatable. Clinical benefit so far is modest; in the 12 evaluable treated patients with relapsed/refractory AML, peripheral blood blasts were cleared in 7 and one patient death possibly related to myelosuppression, the expected dose limiting toxicity. Six patients had bone marrow blast reductions of > 33% at 4 weeks.68

The number of other AML surface antigens targeted by monocolonal antibodies (mAbs) in clinical trials is small. A mAb against CD123 mAb (CSL360) was used in a Phase I clinical trial (NCT00401739) for 26 patients with relapsed/refractory or high-risk AML.69 No treatment-related toxicities were observed other than mild infusion reactions and one infection, and one complete remission was reported. Now a new Phase I clinical trial (NCT01632852) with CSL362, a different chimeric anti-CD123 mAb, is recruiting patients with CD123 + high-risk AML who are in complete remission.

With regards to HCT conditioning regimens, it seems that any further increase in total body irradiation or chemotherapy above that administered in traditional regimens decreased relapse rates at the expense of increased life-threatening toxicity, ultimately with no change in overall survival. One way to increase radiation while sparing non-hematologic tissues is radioimmunotherapy. In a trial of 58 patients (median age 63) with high-risk AML or advanced MDS, a reduced-intensity approach of fludarabine and 2 Gy TBI was augmented by escalating doses of 131I anti-CD45 antibodies. The CR rate was 100% with engraftment failures. Nonrelapse day 100 mortaility rate was 12%. At the maximum tolerated dose, median OS and DFS were 206 and 189 days, respectively. Estimated 1-year survival of 41%, which is encouraging since 86% had refractory disease or were in florid relapse at the time of treatment and these results are similar when compared to lower-risk AML patients transplanted using a regimen of fludarabine and TBI alone.70

Targeting CD47

AML is initiated and maintained by a subset of self-renewing leukemia stem cells (LSC) that also have increased CD47 expression. CD47 is a widely expressed transmembrane protein and its natural function is as a ligand for signal regulatory protein alpha (SIRPα), which is expressed on phagocytic cells. Upon interaction with SIRPα, activation initiates a signal transduction cascade resulting in inhibition of phagocytosis. Preclinical data suggest that CD47 overexpression contributes to pathogenesis by inhibiting LSC phagocytosis and is associated with a worse overall survival in adult AML patients.71 Blocking monoclonal antibodies directed against CD47 can disrupt the CD47-SIRPα interaction and preferentially enabled phagocytosis of AML LSCs; they also inhibited their engraftment in vivo in mice.72 Mouse CD34(+)CD38(−) AML LSCs were separated and transplanted into NOD/SCID mice, then anti-human CD47 antibody, alone or combined with Ara-C was used systemically. The addition of anti CD47 increased survival time from 7 to 14 days. In an in vitro culture, the phagocytic index in the culture medium containing anti-CD47 monoclonal antibody was significantly higher than in the culture medium containing anti-CD47 monoclonal antibody (76.9% ± 12.2% vs 7.60% ± 2.4%; P < 0.05).

Bispecific T-Cell Engaging (BiTE) Antibodies

BiTEs have proven to be a simple yet elegant new way to harness the immune system and have yielded meaningful results in acute lymphoblastic leukemia. BiTE antibodies are genetically engineered single-chain antibodies, without a constant region, that use a linker combining two variable regions of a normal antibody with different specificities. The goal is close approximation of cells expressing the antigens. Blinatumomab is a BiTE that uses T-cell-specific CD3 and B-cell-specific CD19 region and has been shown to initiate a T-cell cytotoxic response against CD19 expressing cells.73,74 BiTEs can stimulate a polyclonal T-cell response because they do not require T-cell specificity to the tumor or rely on MHC molecules. Blinatumomab’s success in B cell ALL has stimulated interest in a BiTE for AML. AMG 330 is a CD33/CD3-BiTE antibody engineered to target AML that has potent in vitro activity. In the presence of T-cells, AMG 330 was highly active against human and primary AML cells lines in a dose- and effector- to- target cell ratio-dependent manner.75 The activity was not affected by common CD33 single-nucleotide polymorphisms or expression of the ATP-binding cassette transporter proteins, P-glycoprotein or breast cancer resistance protein. AMG 330 did not reduce surface CD33 expression, a phenomenon often seen with bivalent CD33 antibodies.76 In cultures that supported the growth of primary AML blasts, AMG 330 recruited and expanded CD3(+)/CD45RA(−)/CCR7(+) memory T cells within patient samples. Even at low effector-to-target ratios, the majority of the samples showed that the recruited T cells completely or substantially lysed autologous blasts. In vivo activity of this agent will be assessed in the future. Whether this agent can maintain activity against the AML cells without causing prolonged cytopenias is the question, since CD33 is expressed on normal early myeloid cells. BiTEs for other AML targets might also be seen in the future.

Chimeric Antigen Receptors

The success of allogeneic HCT in producing long-term leukemia-free survival relies, in part, on the graft vs. leukemia (GvL) effect. This is especially true in the context of non-myeloablative HCTs. Further evidence of an immune-mediated killing of AML by donor cells is the effect of salvage therapy with donor lymphocyte infusions (DLIs) which can be therapeutic in up to 40% of AML patients who relapsed after HCT.77 However, the attendant risk of serious, life-threatening graft vs. host disease (GVHD), risk of conditioning, and the lack of a suitable donor limit the applicability and clinical usefulness of these therapies.3,11 In the future, T-cells might be altered to allow the development of a specific T-cell based immunotherapy. Chimeric antigen receptors (CARs) are synthetic molecules consisting of an extracellular antigen-binding domain (typically derived from variable heavy and light chains of a monoclonal antibody) joined via a spacer region to intracellular signaling domains. Later versions of CARs contain dual intracellular signaling domains: the endodomain from a T-cell costimulatory molecule such as CD28, 4-1BB, or OX-40, and the intracellular domain of CD3ζ.78 The major histocompatibility complex is not necessary for CAR-expressing T cells to target their cell surface antigens, become activated, and induce an immune response. The interleukin-3 receptor α chain (IL3RA, also known as CD123) is overexpressed on AML cells as compared to normal bone marrow.79 There are now many groups researching CD123-targeting monoclonal antibodies and recombinant immunotoxins which have shown some preclinical activity.80,81 Phase I trials have reported antileukemia responses in some patients. CAR T-cells targeting CD123 might also be promising, though since CD123 is expressed on common myeloid progenitor (CMP) cells, long term persistence of the T-cells would be potentially lethal due to prolonged cytopenias.82 Enabling a suicide gene into the CAR T-cells or other methods to control the response might be warranted. In one preclinical study, all chemotherapy-resistant AML samples were variably susceptible to CAR T-cell mediated killing, independent of chromosomal abnormalities.83,84 Resistance to chemotherapy does not necessarily confer immunologic resistance. Planning of a phase I clinical trial to treat patients with relapsed or refractory AMLs using CD123 CAR T cells is underway.

Targeting WT1

Wilms’ tumor 1 (WT1, a zinc finger transcription factor) is overexpressed in 73–100% of AML patients, is a marker for worse outcome, and is actually expressed in both LSCs and their more mature counterparts.85,86 WT1 expression has also been detected in normal hematopoietic stem cells, albeit at much lower levels.87 Anti-WT1 cytotoxic T-cell clones that were able to eliminate CML cells in vitro did not inhibit colony formation of normal CD34 + cells, possibly because their expression is below the limit necessary for cell killing.88 In a Phase I/II clinical trial (NCT00153582, NCT00834002) testing peptide and dendritic cell-based vaccinations, no toxicity related to WT expression in other tissues was seen.89,90 Multiple Phase I/II studies (NCT01640301, NCT01621724) involving the adoptive transfer of donor or autologous derived T cells transduced with a WT1-specific TCR in patients with AML are ongoing. There was one study that suggested a short lived response with WT1 vaccination.91

Concluding Remarks

There are no currently acceptable conventional treatments for high-risk AML. Large, well-designed clinical trials of novel agents are the only way to make progress in this otherwise leathal disease, since traditional cytotoxic agents are inadequate. HCT has been the only modality that seems to improve the expected dismal future that awaits patients with high-risk AML, but the therapeutic impact on a limited population, and continued treatment-related toxicity demonstrate its shortcomings. It is the opinion of the authors that all patients with high-risk AML be evaluated and considered for an allogeneic HCT in first remission the context of their physiologic age, pretransplant conditioning regimen, donor availability and type, social situation, and other variables. The interpretation of the data regarding the benefit of transplant for high-risk AML is complicated and problematic given there is not currently a prospective randomized trial comparing transplant to other strategies (all other things being equal), and probably never will be. Encouragingly, even when transplant is done in the context of active disease, it seems more effective than salvage chemotherapy.92 However, for a discussion on the complexities of interpreting the data on transplantation outcomes the reader is referred to the final two references in this review.93,94

At present, there are no approved alternatives available to improve the results of consolidation therapy for patients with high-risk disease and no drugs in development that target postremission management as a pathway to regulatory approval. Ideally, if other therapy enables similar or better long term leukemia-free survival, HCT might be required for fewer patients, thus avoiding the attendant toxicities. All patients who have high-risk AML and are eligible for a clinical study should be encouraged to participate since therapy available outside of an experimental setting is usually inadequate. Yet all too often, patients with high-risk AML have refractory disease or relapse too soon to be considered for allogeneic HCT or other consolidation strategies. In this situation, salvage therapy frequently yields disappointing results and experimental therapy is advised. For those who do attain a remission, it is often short lived. This could be an ideal place for certain targeted therapies, with a goal of remission maintenance.

Though the outcomes for high-risk AML have changed little in the past few decades, it is highly likely that the outpouring of new knowledge and rationally directed therapy in the recent few years will change this for the better. Today, for investigators, clinicians, and (most importantly) patients, there are far more choices for experimental therapy than ever before. Many of these agents are rationally designed and have encouraging preliminary results. As the biology of AML is more fully elaborated, new targets emerge. The most achievable early advance in the treatment ofAML will likely be targeted therapy, although targets may change in the course of treatment of an individual patient and “moving target” therapy might be more appropriate. Another modality expected to have potential for modest improvement is nanoparticle formulations. While repackaging an old drug or combination of drugs may not lead to dramatic results, it may be a step forward. Long term, immunotherapy holds promise, but its use is hampered by shared antigens on normal myeloid stem cells and potential for fatal prolonged cytopenias. While still in its infancy, targeting CD47 might prove quite beneficial if in vitro and animal results are confirmed in human trials. For decades, the goal has been to eradicate AML by direct cytotoxicity, but newer agents that target specific molecular derangements show more and more that differentiation of myeloblasts, rather than direct apoptosis or necrosis, is therapeutically appealing. Because of this, evaluating response rate as well as response type will be essential in clinical trials with newer agents in AML, and will need to be considered for regulatory approval. Optimistically, differentiative therapy is mirroring the early days of the treatment of APL, a once highly lethal disease that is now effectively treated with a “vitamin and mineral”. We look forward to the day when a similar sentiment can be said of high-risk AML.

Practice Points

• Look for clinical trials for the subgroups of patients with high-risk AML

• Consider allogeneic transplant as soon as the diagnosis of high-risk AML is made

Research Agenda

• Laboratory tests required to characterize high-risk acute myeloid leukemia

• Clinical value of novel therapeutics in management

• Role of allogeneic transplantation in the management of high-risk AML

Conflict of Interest

Dr. Joshua Sasine has no conflict of interests to disclose. Dr. Gary Schiller discloses funding from the following: Sunesis Pharmaceutical, Amgen, Celator Pharmaceutical, Boehringer-Ingelheim, Genzyme, Astellas, Novartis.


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UCLA Department of Medicine, Division of Hematology and Oncology, Room 42–121 Center for Health Sciences, David Geffen School of Medicine at UCLA, Los Angeles, CA 90095, USA

lowast Corresponding author. Tel.: + 1 310 825 5513.