AML

Question 1

4 considerations when designing trials for MTT. Developing Molecularly Targeted Therapy for Childhood Leukemia: FLT3 Inhibitors. C4K, Patrick Brown, 2010.

Answer 1

Must use novel clinical trial designs that are driven by translational laboratory science „

1. MTD-based phase I design inappropriate – should be should be based on biologic endpoint (target inhibition), not maximum tolerable dose. „
2. Should base combination studies on preclinical data of how agent best combines with other therapies
3. Develop predictors of response for use in subsequent trials (e.g., ex vivo sensitivity + target inhibition = predicted CR)

„4. Assess effect of MTT on cancer stem cells

Question 2

FLT3. When was it discovered? Where is it expressed? Where is FL expressed? When/who first described activating mutations in AML? Knockout mouse phenotype? Developing Molecularly Targeted Therapy for Childhood Leukemia: FLT3 Inhibitors. C4K, Patrick Brown, 2010.

Answer 2

• Cloned in 1992 from a stem cell cDNA library by Donald Small at Hopkins „- Expressed in hematopoietic precursors and dendritic cells - Expressed on the blasts of most AML and ALL cells „ - Ligand (FL) is ubiquitously expressed - FLT3 activating mutations first reported in AML in 1996 (Nakao, et al) „- Mutations are weakly transforming „- Myeloproliferative disease in mouse models

Question 3

What is FLT3’s CD number? What does FLT3 stand for? Give two alternative names for FLT3?

Answer 3

Cluster of differentiation antigen 135 (CD135) also known as Fms-like tyrosine kinase 3 (FLT-3), receptor-type tyrosine-protein kinase FLT3, or fetal liver kinase-2 (Flk2) is a protein that in humans is encoded by the FLT3 gene.

Question 4

What is FLT3? What is its ligand? Where is it expressed?

Answer 4

• FLT3 (CD135) is a cytokine receptor which belongs to the receptor tyrosine kinase class III. - FLT3 (CD135) is the receptor for the cytokine Flt3 ligand (FLT3L). - It is expressed on the surface of many hematopoietic progenitor cells. Signalling of FLT3 is important for the normal development of haematopoietic stem cells and progenitor cells. - The FLT3 gene is one of the most frequently mutated genes in acute myeloid leukemia (AML). Besides, high levels of wild-type FLT3 have been reported for blast cells of some AML patients without FLT3 mutations. These high levels may be associated with worse prognosis.

Question 5

Describe the structure of FLT3

Answer 5

FLT3 is composed of five extracellular immunoglobulin-like domains, a transmembrane domain, a juxtamembrane domain and a tyrosine-kinase domain consisting of 2 lobes that are connected by a tyrosine-kinase insert. Cytoplasmic FLT3 undergoes glycosylation, which promotes localization of the receptor to the membrane.

Question 6

Describe initial signalling events when FLT3 is bound by FLT3L. Name two cytokines which appear to down modulate FLT3 activity?

Answer 6

CD135 is a Class III receptor tyrosine kinase. When this receptor binds to FLT3L a ternary complex is formed in which two FLT3 molecules are bridged by one (homodimeric) FLT3L. The formation of such complex brings the two intracellular domains in close proximity to each other, eliciting initial trans-phosphorylation of each kinase domain. This initial phosphorylation event further activates the intrinsic tyrosine kinase activity, which in turn phosphorylates and activates signal transduction molecules that propagate the signal in the cell. Signaling through CD135 plays a role in cell survival, proliferation, and differentiation. Two cytokines that down modulate FLT3 activity (& block FLT3-induced hematopoietic activity) are: - TNF-Alpha (Tumor necrosis factor-alpha) - TGF-Beta (Transforming growth factor-beta) TGF-Beta especially, decreases FLT3 protein levels and reverses the FLT3L-induced decrease in the time that hematopoietic progenitors spend in the G1-phase of the cell cycle

Question 7

Describe two common FLT 3 mutations in acute leukaemia

Answer 7

FLT3 mutations in acute leukaemia

1. FLT3 internal tandem duplication (FLT3/ITD)
   
   • In-frame insertions into juxtamembrane domain.
   • Constitutively activates the tyrosine kinase function of the receptor
   • 23%/13% of AML (adults/kids); < 1% of ALL
   • Strong negative prognostic factor
2. FLT3 kinase domain mutations
   
   • Most commonly at D835
   • Constitutively activates the tyrosine kinase function of the receptor
   • 7% of AML; rare in ALL, except hyperdiploid and MLL-r (5-15%)
   • Neutral prognostic factor

Question 8

What percentage of infants and older children with ALL have an MLL rearrangement? Prognostic significance?

Answer 8

80% infants and 5% older children with
ALL are MLL-r.

FLT3 constitutively activated by mutation
and/or wt overexpression/autocrine
activation in MLL-R and hyperdiploid ALL.
􀂄􀂄 MLL-R is associated with a worse prognosis.

Question 9

Developing Molecularly Targeted Therapy for Childhood Leukemia: FLT3 Inhibitors. C4K, Patrick Brown, 2010.

Key Points

Answer 9

1. How to think about designing trials of MTT.
   
   • Need to think about how MTT will interact with standard chemotherapy or other MTTs. Notably, if MTT works by shifting cancer cells in to G0, and chemo works best on actively dividing cells, could theoretically antogonise chemo by giving MTT first, should give it after chemo, and can test this hypothesis in lab during trial design.
2. Value of reading far afield! 1950s penicillin paper - giving patients penicillin, taking plasma samples, seeing if plasms would inhibit growth of patient bacteria in vitro. Applied to finding inhibitory dose of FLT3 inhibitors - the Plasma Inhibitory Assay (PIA).
3. PIA: Patients take CEP-701 (lestaurtinib), whole blood taken at different time points, plasma extracted, FLT3/ITD AML cell line subjected to patient plasma, western blot done for phosphorylated FLT3.
4. FLT3/ITD+ childhood AML and MLL-R infant ALL both have dismal prognosis despite maximally intensified standard therapy, and FLT3 is constitutively activated in both. Inhibiting FLT3 signaling selectively kills these leukaemic cells in vitro and in vivo, but will need to be combined with chemotherapy in syngergic sequences.

Question 10

Mutations and Treatment Outcome in Cytogenetically Normal Acute Myeloid Leukemia

Richard F. Schlenk, …. Hartmut Döhner for the German–Austrian Acute Myeloid Leukemia Study Group

N Engl J Med 2008; 358:1909-1918May 1, 2008DOI: 10.1056/NEJMoa074306

Abstract

Answer 10

Background

Mutations occur in several genes in cytogenetically normal acute myeloid leukemia (AML) cells: the nucleophosmin gene (NPM1), the fms-related tyrosine kinase 3 gene (FLT3), the CCAAT/enhancer binding protein α gene (CEPBA), the myeloid–lymphoid or mixed-lineage leukemia gene (MLL), and the neuroblastoma RAS viral oncogene homolog (NRAS). We evaluated the associations of these mutations with clinical outcomes in patients.

Methods

We compared the mutational status of the NPM1, FLT3, CEBPA, MLL, and NRAS genes in leukemia cells with the clinical outcome in 872 adults younger than 60 years of age with cytogenetically normal AML. Patients had been entered into one of four trials of therapy for AML. In each study, patients with an HLA-matched related donor were assigned to undergo stem-cell transplantation.

Results

A total of 53% of patients had NPM1 mutations, 31% had FLT3 internal tandem duplications (ITDs), 11% had FLT3 tyrosine kinase–domain mutations, 13% had CEBPA mutations, 7% had MLL partial tandem duplications (PTDs), and 13% had NRAS mutations. The overall complete-remission rate was 77%. The genotype of mutant NPM1 without FLT3-ITD, the mutant CEBPA genotype, and younger age were each significantly associated with complete remission. Of the 663 patients who received postremission therapy, 150 underwent hematopoietic stem-cell transplantation from an HLA-matched related donor. Significant associations were found between the risk of relapse or the risk of death during complete remission and the leukemia genotype of mutant NPM1 without FLT3-ITD (hazard ratio, 0.44; 95% confidence interval [CI], 0.32 to 0.61), the mutant CEBPA genotype (hazard ratio, 0.48; 95% CI, 0.30 to 0.75), and the MLL-PTD genotype (hazard ratio, 1.56; 95% CI, 1.00 to 2.43), as well as receipt of a transplant from an HLA-matched related donor (hazard ratio, 0.60; 95% CI, 0.44 to 0.82). The benefit of the transplant was limited to the subgroup of patients with the prognostically adverse genotype FLT3-ITD or the genotype consisting of wild-type NPM1 and CEBPA without FLT3-ITD. Conclusions Genotypes defined by the mutational status of NPM1, FLT3, CEBPA, and MLL are associated with the outcome of treatment for patients with cytogenetically normal AML.

Question 11

MicroRNA Expression in Cytogenetically Normal Acute Myeloid Leukemia

Guido Marcucci, M.D…..and Clara D. Bloomfield, M.D.

N Engl J Med 2008; 358:1919-1928May 1, 2008 DOI: 10.1056/NEJMoa074256

Abstract

Answer 11

Background

A role of microRNAs in cancer has recently been recognized. However, little is known about the role of microRNAs in acute myeloid leukemia (AML).

Methods

Using microRNA expression profiling, we studied samples of leukemia cells from adults under the age of 60 years who had cytogenetically normal AML and high-risk molecular features — that is, an internal tandem duplication in the fms-related tyrosine kinase 3 gene (FLT3–ITD), a wild-type nucleophosmin (NPM1), or both. A microRNA signature that was associated with event-free survival was derived from a training group of 64 patients and tested in a validation group of 55 patients. For the latter, a microRNA compound covariate predictor (called a microRNA summary value) was computed on the basis of weighted levels of the microRNAs forming the outcome signature.

Results

Of 305 microRNA probes, 12 (including 5 representing microRNA-181 family members) were associated with event-free survival in the training group (P<0.005). In the validation group, the microRNA summary value was inversely associated with event-free survival (P=0.03). In multivariable analysis, the microRNA summary value remained associated with event-free survival (P=0.04) after adjustment for the allelic ratio of FLT3-ITD to wild-type FLT3 and for the white-cell count. Using results of gene-expression microarray analysis, we found that expression levels of the microRNA-181 family were inversely correlated with expression levels of predicted target genes encoding proteins involved in pathways of innate immunity mediated by toll-like receptors and interleukin-1β.

Conclusions

A microRNA signature in molecularly defined, high-risk, cytogenetically normal AML is associated with the clinical outcome and with target genes encoding proteins involved in specific innate-immunity pathways

Question 12

CORRESPONDENCE

MicroRNA in Acute Myeloid Leukemia (the Marcucci article)

N Engl J Med 2008; 359:653-654August 7, 2008DOI: 10.1056/NEJMc081231

Answer 12

To the Editor:

Marcucci et al. (May 1 issue)1 highlight a possible association between the microRNA-181 family and innate-immunity genes. Of the 452 genes that correlated strongly with their microRNA summary value, 32 were predicted to be targets of the microRNA-181 family, according to the TargetScan database.2 Although some of these 32 genes indeed contribute to innate immunity, we write to underline the risk of using only one algorithm to predict microRNA targets and recommend the use of multiple algorithms to consolidate predictions. Thus, reanalysis of the published data with an alternative algorithm, miRanda,3 predicted 50 target genes for the microRNA-181 family, only 10 of which overlapped with the TargetScan predictions. Using DAVID,4 we found that these 50 genes were not statistically enriched for the gene-ontology category of “immunity” but rather for “protein binding” and “regulation of apoptosis.” Had the authors used miRanda instead of TargetScan, their conclusions regarding microRNA-181 target genes would have been different. In highlighting this discrepancy between algorithms, we wish to emphasize the need for greater circumspection in their application.

William J. Ritchie, Ph.D.
Stephane Flamant, Ph.D.
Centenary Institute, Sydney, NSW 2050, Australia

John E.J. Rasko, M.B., B.S., Ph.D.
Royal Prince Alfred Hospital, Camperdown, VIC 2050, Australia
j.rasko@centenary.usyd.edu.au

The authors reply: In our article, we showed that the summary value of the microRNA expression signature correlated with expression of 452 genes. Sixteen gene-ontology terms for biologic processes had at least half their members included among these 452 genes. Of the 16 terms, 15 included members participating in innate immunity. This finding did not depend on the prediction of microRNA targets. We used TargetScan to predict which of the 452 genes were putative targets of microRNA-181, the most represented microRNA family in the signature. We noted that seven target genes (all negatively correlated with microRNA-181 expression) were related to innate immunity. The miRanda algorithm also predicts that four of these genes will be microRNA-181 targets, suggesting some similarity in the results obtained from TargetScan and miRanda. Although we agree that algorithms for predicting microRNA targets should be applied carefully, we believe that the optimal approach for definitively linking microRNAs to the regulation of specific genes is experimental validation at the bench.

Question 13

Prognostic Relevance of Integrated Genetic Profiling in Acute Myeloid Leukemia

Jay P. Patel, ….. and Ross L. Levine, M.D. (Memorial Sloan Kettering)

N Engl J Med 2012; 366:1079-1089March 22, 2012DOI: 10.1056/NEJMoa1112304

Abstract

Answer 13

Background

Acute myeloid leukemia (AML) is a heterogeneous disease with respect to presentation and clinical outcome. The prognostic value of recently identified somatic mutations has not been systematically evaluated in a phase 3 trial of treatment for AML. Methods

We performed a mutational analysis of 18 genes in 398 patients younger than 60 years of age who had AML and who were randomly assigned to receive induction therapy with high-dose or standard-dose daunorubicin. We validated our prognostic findings in an independent set of 104 patients.

Results

We identified at least one somatic alteration in 97.3% of the patients. We found that internal tandem duplication in FLT3 (FLT3-ITD), partial tandem duplication in MLL (MLL-PTD), and mutations in ASXL1 and PHF6 were associated with reduced overall survival (P=0.001 for FLT3-ITD, P=0.009 for MLL-PTD, P=0.05 for ASXL1, and P=0.006 for PHF6); CEBPA and IDH2 mutations were associated with improved overall survival (P=0.05 for CEBPA and P=0.01 for IDH2). The favorable effect of NPM1 mutations was restricted to patients with co-occurring NPM1 and IDH1 or IDH2 mutations. We identified genetic predictors of outcome that improved risk stratification among patients with AML, independently of age, white-cell count, induction dose, and post-remission therapy, and validated the significance of these predictors in an independent cohort. High-dose daunorubicin, as compared with standarddose daunorubicin, improved the rate of survival among patients with DNMT3A or NPM1 mutations or MLL translocations (P=0.001) but not among patients with wild-type DNMT3A, NPM1, and MLL (P=0.67). Conclusions We found that DNMT3A and NPM1 mutations and MLL translocations predicted an improved outcome with high-dose induction chemotherapy in patients with AML. These findings suggest that mutational profiling could potentially be used for risk stratification and to inform prognostic and therapeutic decisions regarding patients with AML. (Funded by the National Cancer Institute and others.)

Nature Commentary: Previous studies in small, single-
institution cohorts of patients with
AML have shown specific mutations to
be associated with different outcomes.
However, these data have not been
validated in larger populations treated
homogenously. In the second study,
Ross Levine (Patel et al) and colleagues performed
mutational analysis using high-
throughput screening to assess 18 genes
from 398 patients with AML who were
part of the ECOG E1900 phase III trial.
Patients were randomly assigned to receive either standard-dose or high-
dose daunorubicin treatment. The
prognostic findings from this analysis
were validated in an independent cohort
of 104 patients. This study showed that
mutational analysis of a large set of genetic
alterations can be used in the clinic to
retrospectively classify patients with
AML into more-precise subgroups with
a favourable-risk, intermediate-risk or
unfavourable-risk profile.
Levine summarizes the main findings,
“we made two important observations.
First we found that a set of mutations were
associated with improved, or adverse,
outcome in AML. Previous studies
showed that many of the individual
mutations were of prognostic value, but
we developed a classifier that showed
which mutations, and combinations of
mutations, had independent prognostic
value. ” Importantly, Levine’s team
showed that patients with three specific
mutations in DNMT3A, MLL, and NPM1,
had improved outcome with high-dose
daunorubicin chemotherapy. Levine and
his team are planning to develop tests
to implement this approach in the clinic and to validate other genetic classifiers in
additional patient cohorts.

Question 14

Prognostic Relevance of Integrated Genetic Profiling in Acute Myeloid Leukemia

Patel et al NEJM 2012

Which four somatic mutations were associated with reduced overall survival?

Answer 14

1. Internal tandem duplication in FLT3 (FLT3-ITD)
2. Partial tandem duplication in MLL (MLL-PTD)
3. Mutations in ASXL1
4. Mutations in PHF6

Question 15

Prognostic Relevance of Integrated Genetic Profiling in Acute Myeloid Leukemia

Patel et al NEJM 2012

Which somatic mutations were associated with improved overall survival?

Answer 15

1. CEBPA
2. IDH2
3. The favourable effect of NPM1 mutations was restricted to patients with co-occurring NPM1 and IDH1 or IDH2 mutations.

Question 16

Prognostic Relevance of Integrated Genetic Profiling in Acute Myeloid Leukemia

Patel et al NEJM 2012

Which mutations predicted benefit from high dose daunorubicin?

Answer 16

High-dose daunorubicin, as compared with standard-dose daunorubicin, improved the rate of survival among patients with

1. DNMT3A mutations
2. NPM1 mutations
3. MLL translocations

Question 17

Which mutatations were screened for in this cohort of NK AML? Roughly which percentage of patients had each?

Mutations and Treatment Outcome in Cytogenetically Normal Acute Myeloid Leukemia

Richard F. Schlenk, …. Hartmut Döhner for the German–Austrian Acute Myeloid Leukemia Study Group

N Engl J Med 2008; 358:1909-1918May 1, 2008DOI: 10.1056/NEJMoa074306

Answer 17

NPM1, FLT3, CEPBA, MLL, NRAS.

Around half had mutated NPM1, FLT3-ITD 1/3, FLT3 TKD mutation 11%, CEPBA 13%, MLL PTDs 7%, NRAS mutations 13%.

Question 18

Which mutations were significantly associated with complete remission?

Which significant associations were found between genotype and risk of relapse or death in patients who received postremission therapy?

Which patient groups benefited from transplant?

Mutations and Treatment Outcome in Cytogenetically Normal Acute Myeloid Leukemia

Richard F. Schlenk, …. Hartmut Döhner for the German–Austrian Acute Myeloid Leukemia Study Group

N Engl J Med 2008; 358:1909-1918May 1, 2008DOI: 10.1056/NEJMoa074306

Answer 18

1. Associated with complete remission:
   
   1. NPM1 mutation without FLT3 ITD
   2. CEBPA mutation
   3. Younger age
2. Associated with risk of relapse or death during complete remission:
   
   1. NPM1 without FLT3 ITD (hazard ration 0.44)
   2. CEBPA mutant (HR 0.48)
   3. MLL-PTD genotype (HR 1.56)
   4. Receipt of HLA matched transplant (HR 0.60)
3. Benefit of BMT restricted to
   
   1. FLT3-ITD genotype
   2. Genotype of wild type NPM1 and CEBPA without FLT3

Question 19

What percentage of adult vs paed AMLs have FLT3 ITD mutations?

Answer 19

Around 23% of adult AMLs have FLT3-ITD mutations (though about half of NK-AMLs are FLT3-ITD mutants). About 13% of paed AMLs have FLT3-ITD genotype.

Question 20

Inositol polyphosphate 4-phosphatase II (INPP4B) is associated with chemoresistance and poor outcome in AML.

Blood. 2015 Apr 30;125(18):2815-24. doi: 10.1182/blood-2014-09-603555. Epub 2015 Mar 3.

Rijal S …Wei AH. (Melbourne)

ABSTRACT

Answer 20

Abstract

Phosphoinositide signaling regulates diverse cellular functions. Phosphoinositide-3 kinase (PI3K) generates PtdIns(3,4,5)P3 and PtdIns(3,4)P2, leading to the activation of proliferative and anti-apoptotic signaling pathways. Termination of phosphoinositide signaling requires hydrolysis of inositol ring phosphate groups through the actions of PtdIns(3,4,5)P3 3-phosphatase (PTEN), PtdIns(3,4,5)P3 5-phosphatases (eg, SHIP), and PtdIns(3,4)P2 4-phosphatases (eg, INPP4B). The biological relevance of most of these phosphoinositide phosphatases in acute myeloid leukemia (AML) remains poorly understood. Mass spectrometry-based gene expression profiling of 3-, 4- and 5-phosphatases in human AML revealed significant overexpression of INPP4B. Analysis of an expanded panel of 205 AML cases at diagnosis revealed INPP4B overexpression in association with reduced responses to chemotherapy, early relapse, and poor overall survival, independent of other risk factors. Ectopic overexpression of INPP4B conferred leukemic resistance to cytosine arabinoside (ara-C), daunorubicin, and etoposide. Expression of a phosphatase inert variant (INPP4B C842A) failed to abrogate resistance of AML cells to chemotherapy in vitro or in vivo. In contrast, targeted suppression of endogenously overexpressed INPP4B by RNA interference sensitized AML cell lines and primary AML to chemotherapy. These findings demonstrate a previously unsuspected and clinically relevant role for INPP4B gain of function as a mediator of chemoresistance and poor survival outcome in AML independent of its phosphoinositide phosphatase function.

Comment by Recher in Blood. 2015 , DOI: 10.1182/blood-2015-03-633669, PMID: 25931577:

How does INPP4B drive chemoresistance in AML? The feld is open. Because INPP4B reduces the activity of drugs with various mechanisms of action, it is likely that it could be involved in apoptotic response rather than in drug-specifc metabolisms. INPP4B overexpression in leukemic cells did not impact the phosphorylation of AKT or the expression of antiapoptotic members of the Bcl-2 family. Thus, critical downstream targets of INPP4B remain to be determined. The finding that
INPP4B could act independently of its phosphatase activity raises several clues to investigate how cells resist to chemotherapy. Phosphoinositide enzymes have both a catalytic and a molecular adapter activity that are crucial to organizing multimolecular complexes.
As discussed by the authors, INPP4B contains an N-terminal C2-lipid binding domain, which interacts with membranes. It also contains a Nervy homology 2 domain known to mediate oligomerization (ie, AML1-
ETO oligomerization) or protein-protein interaction).8 Thus, the findings of Rijal et al pave the way to perform further molecular studies of the INPP4B interactome in order to identify new therapeutic targets aimed at
unlocking chemoresistance in AML.

Question 21

Name three mechanisms of constitutive PI3K pathway activation in AML.

Answer 21

Most AML display a constitutive activation of the PI3K pathway that contributes to cell proliferation and
chemoresistance. Several mechanisms converge to activate the PI3K pathway in AML, including

• mutated receptor tyrosine kinases (ie, FLT3-ITD),
• growth factors (ie, insulin-like growth factor 1), or
• microenvironment players (ie, SDF1a/ CXCR4 or fibronectin/VLA-4 modules).

Park S, Chapuis N, Tamburini J, et al. Role of the
PI3K/AKT and mTOR signaling pathways in acute
myeloid leukemia. Haematologica. 2010;95(5):819-828.

Question 22

What is phosphorylation of protein kinase B (AKT)
on serine 473 a surrogate marker of?

Answer 22

The phosphorylation of protein kinase B (AKT) on serine 473 is used as a surrogate marker of PI3K
activation.

Question 23

IL8-CXCR2 pathway inhibition as a therapeutic strategy against MDS and AML stem cells

Carolina Schinke….Amit Verma (Albert Einstem med college NY). Blood 2015

Comment in

Targets of opportunity for precision medicine.

Yogen Saunthararajah

Answer 23

Key Points

• IL8-CXCR2 is overexpressed in purified stem cells from AML and MDS, and CXCR2 expression is associated with worse prognosis.
• Inhibition of CXCR2 by genetic and pharmacologic means leads to decreased viability in AML/MDS stem cells and in vitro and in vivo models.

Abstract

Acute myeloid leukemia (AML) and myelodysplastic syndromes (MDS) are associated with disease-initiating stem cells that are not eliminated by conventional therapies. Novel therapeutic targets against preleukemic stem cells need to be identified for potentially curative strategies. We conducted parallel transcriptional analysis of highly fractionated stem and progenitor populations in MDS, AML, and control samples and found interleukin 8 (IL8) to be consistently overexpressed in patient samples. The receptor for IL8, CXCR2, was also significantly increased in MDS CD34+ cells from a large clinical cohort and was predictive of increased transfusion dependence. High CXCR2 expression was also an adverse prognostic factor in The Cancer Genome Atlas AML cohort, further pointing to the critical role of the IL8-CXCR2 axis in AML/MDS. Functionally, CXCR2 inhibition by knockdown and pharmacologic approaches led to a significant reduction in proliferation in several leukemic cell lines and primary MDS/AML samples via induction of G0/G1 cell cycle arrest. Importantly, inhibition of CXCR2 selectively inhibited immature hematopoietic stem cells from MDS/AML samples without an effect on healthy controls. CXCR2 knockdown also impaired leukemic growth in vivo. Together, these studies demonstrate that the IL8 receptor CXCR2 is an adverse prognostic factor in MDS/AML and is a potential therapeutic target against immature leukemic stem cell–enriched cell fractions in MDS and AML.

Comment: Targets of opportunity for precision medicine

The intent driving the idea of “precision medicine,” or the individualization of oncotherapy based on case-by-case cancer genetics, is selective suppression of malignant clones while sparing the normal stem cells needed for health and life—stated simply,
a better therapeutic index. Schinke et al identified higher expression of CXCR2 in MDS/AML stem cells than in normal HSCs and demonstrated preclinical therapeutic proof of principle, affirming that molecular targets offering a good therapeutic index are not
restricted to mutated oncoproteins. In fact, CXCR2 and othernononcogene cancer molecular addictions could have advantages as targets for therapy, such as near-term drugability and broad applicability to a
histologic diagnosis. Per the latter, high CXCR2 expression is a characteristic of some morphologic subtypes of AML despite different underlying driver
mutations. Stated another way, there is convergent neoplastic evolution, from disparate genetic origins toward expansion of myeloblasts bearing gross molecular and morphologic similarities. Why do multiple
roads, cobbled by Darwinian selective pressures working for years, lead to similar molecular-morphologic destinations that are distinct from normal HSCs? Here are some clues: “first hits” in the multihit process of
leukemogenesis (eg, RUNX1 or DNMT3A mutations) originate in the germline or HSCs (cells of origin) and, accordingly, can be detected in the germline or HSCs. Subsequent evolution into AML-initiating cells (AML stem cells), however, occurs in lineage-committed daughter cells of these cells of origin, most cleanly documented by the detection of secondary mutations needed for transformation into AML (eg, FLT3 or NPM1
mutations) only in committed progenitors
and not in HSCs.3-5 In fact, first hits such as
RUNX1 loss-of-function have been shown
to manifest their most dramatic phenotypic
effects not in HSCs but in committed daughter cells (progenitors) by impeding the
differentiation (maturation) that is the natural
control on the cell growth and division of
this intrinsically MYC-enriched, exponentially
expanding compartment.6 In short,
transformation into MDS/AML is in a cellular
context biologically distinct from that of
HSCs,7,8 and CXCR2 and a number of other
molecular targets that have been found to
discriminate between MDS/AML stem cells
and HSCs8 are opportunistic targets, reecting
this compartment shift from stem cells to
progenitors that occurs in neoplastic evolution. The wealth of discriminating
molecular targets so created is great news,
because there could be many reasons, such
as lack of drugability without off-target effects
or importance to some other normal function, that could undermine the therapeutic index of
any one target. This target-rich environment,
not restricted to mutated oncoproteins, offers
real hope for breakthroughs (or at least
improvements in safety and effectiveness) in
MDS/AML therapy.

Question 24

The transcriptomic landscape and directed chemical interrogation of MLL-rearranged acute myeloid leukemias.

Nature genetics. 2015 , DOI: 10.1038/ng.3371, PMID: 26237430

Vincent-Philippe P Lavallée,… Guy Sauvageau

Answer 24

ABSTRACT

Using next-generation sequencing of primary acute myeloid leukemia (AML) specimens, we identified to our knowledge the first unifying genetic network common to the two subgroups of KMT2A (MLL)-rearranged leukemia, namely having MLL fusions or partial tandem duplications. Within this network, we experimentally confirmed upregulation of the gene with the most subtype-specific increase in expression, LOC100289656, and identified cryptic MLL fusions, including a new MLL-ENAH fusion. We also identified a subset of MLL fusion specimens carrying mutations in SPI1 accompanied by inactivation of its transcriptional network, as well as frequent RAS pathway mutations, which sensitized the leukemias to synthetic lethal interactions between MEK and receptor tyrosine kinase inhibitors. This transcriptomics-based characterization and chemical interrogation of human MLL-rearranged AML was a valuable approach for identifying complementary features that define this disease.

Question 25

What does MLL stand for and what is an alternative name for this gene?

Where is it?

What does it encode?

Whate percentage of adult AMLs have an MLL fusion?

What are the four commonest MLL fusions in MLL-F AMLs and what is their prognostic significance?

Answer 25

• mixed-lineage leukemia; also known as KMT2A
• on chromosome band 11q23
• encodes a histone H3 lysine 4 (H3K4) methyltransferase.
• KMT2A/MLL is fused to numerous partners in 5−10% of adult AMLs (MLL-F AMLs)
• The most common MLL translocations found in AML are
  
  • t(9;11)(p22;q23) resulting in MLL fused to MLLT3 (also known as AF9)
  • t(6;11)(q27;q23) resulting in MLL fused to MLLT4 (also known as AF6)
  • t(11;19)(q23;p13.3) resulting in MLL fused to MLLT1 (also known as ENL)
  • t(11;19)(q23;p13.1) resulting in MLL fused to ELL2.
• With the exception of MLL-MLLT3 AML, most MLL-F AML subtypes are a/w an adverse clinical outcome.

Question 26

Depletion of the chromatin remodeler CHD4 sensitizes AML blasts to genotoxic agents and reduces tumor formation.
Blood. 2015 Aug 11. pii: blood-2015-03-631606. [Epub ahead of print]

Sperlazza J….Ginder GD.

Answer 26

Abstract

Chromodomain Helicase DNA-binding protein 4 (CHD4) is an ATPase that alters the phasing of nucleosomes on DNA and has recently been implicated in DNA double stranded break (DSB) repair. Here, we show that depletion of CHD4 in Acute Myeloid Leukemia (AML) blasts induces a global relaxation of chromatin that renders cells more susceptible to DSB formation, while concurrently impeding their repair. Furthermore, CHD4 depletion renders AML blasts more sensitive both in vitro and in vivo to genotoxic agents used in clinical therapy: daunorubicin (DNR) and cytarabine (ara-C). Sensitization to DNR and ara-C is mediated in part by activation of the ATM pathway, which is preliminarily activated by a Tip60-dependent mechanism in response to chromatin relaxation and further activated by genotoxic-agent induced DSBs. This sensitization preferentially affects AML cells, as CHD4 depletion in normal CD34+ hematopoetic progenitors does not increase their susceptibility to DNR or ara-C. Unexpectedly, we found that CHD4 is necessary for maintaining the tumor forming behavior of AML cells, as CHD4 depletion severely restricted the ability of AML cells to form xenografts in mice and colonies in soft agar. Taken together, these results provide evidence for CHD4 as a novel therapeutic target whose inhibition has the potential to enhance the effectiveness of genotoxic agents used in AML therapy.

Question 27

Antibodies targeting human IL1RAP (IL1R3) show therapeutic effects in xenograft models of acute myeloid leukemia.
Proc Natl Acad Sci U S A. 2015 Aug 10. pii: 201422749.

Ågerstam H…. Fioretos T (Lund University, Sweden)

Answer 27

Abstract

Acute myeloid leukemia (AML) is associated with a poor survival rate, and there is an urgent need for novel and more efficient therapies, ideally targeting AML stem cells that are essential for maintaining the disease. The interleukin 1 receptor accessory protein (IL1RAP; IL1R3) is expressed on candidate leukemic stem cells in the majority of AML patients, but not on normal hematopoietic stem cells. We show here that monoclonal antibodies targeting IL1RAP have strong antileukemic effects in xenograft models of human AML. We demonstrate that effector-cell-mediated killing is essential for the observed therapeutic effects and that natural killer cells constitute a critical human effector cell type. Because IL-1 signaling is important for the growth of AML cells, we generated an IL1RAP-targeting antibody capable of blocking IL-1 signaling and show that this antibody suppresses the proliferation of primary human AML cells. Hence, IL1RAP can be efficiently targeted with an anti-IL1RAP antibody capable of both achieving antibody-dependent cellular cytotoxicity and blocking of IL-1 signaling as modes of action. Collectively, these results provide important evidence in support of IL1RAP as a target for antibody-based treatment of AML.

Question 28

Outcomes of Nonmyeloablative HLA-Haploidentical Blood or Marrow Transplantation With High-Dose Post-Transplantation Cyclophosphamide in Older Adults.

J Clin Oncol. 2015 Aug 10. pii: JCO.2014.60.4777.

Kasamon YL… Jones RJ.

Answer 28

Abstract

PURPOSE:

Recent advances in nonmyeloablative (NMA), related HLA-haploidentical blood or marrow transplantation (haplo-BMT) have expanded the donor pool. This study evaluated the effect of age on NMA haplo-BMT outcomes in patients age 50 to 75 years.

PATIENTS AND METHODS:

A retrospective analysis was performed of 271 consecutive patients with hematologic malignancies, age 50 to 75 years, who received NMA, T-cell-replete haplo-BMT with high-dose post-transplantation cyclophosphamide.

RESULTS:

The median age was 61 years, with 115 patients (42%) age 50 to 59, 129 (48%) age 60 to 69, and 27 (10%) age 70 to 75 years. Overall, 84% of patients had intermediate- or high-/very high-risk disease. The 6-month probabilities of grade 3 or 4 acute graft-versus-host disease (GVHD) and nonrelapse mortality (NRM) were 3% and 8%, respectively. Patients in their 50s, 60s, and 70s had 6-month NRM probabilities of 8%, 9%, and 7%, respectively (P = .20). With a median follow-up of 4 years, corresponding 3-year progression-free survival probabilities were 39%, 35%, and 33% (P = .65), and corresponding 3-year overall survival probabilities were 48%, 45%, and 44% (P = .66). Three-year progression-free survival probabilities were 40% in acute myeloid leukemia (n = 65), 39% in aggressive non-Hodgkin lymphoma (n = 83), and 37% in indolent or mantle-cell lymphoma (n = 65). Older patient age was associated with a significantly higher risk of grade 2 to 4 acute GVHD but not grade 3 to 4 acute or chronic GVHD. No statistically significant associations were found between older age (relative to age 50 to 59 years or as a continuous variable) and NRM, relapse, or survival.

CONCLUSION:

NMA haplo-BMT with post-transplantation cyclophosphamide has encouraging safety and survival outcomes in patients age 50 to 75 years. In patients otherwise fit for BMT, the results support consideration of this approach despite advanced age.

Question 29

High rate of hematological responses to sorafenib in FLT3-ITD acute myeloid leukemia relapsed after allogeneic hematopoietic stem cell transplantation.
Eur J Haematol. 2015 Aug 11. doi: 10.1111/ejh.12647.

Freitas…Ciceri F

Answer 29

Abstract

Relapse represents the most significant cause of failure of allogeneic hematopoietic stem cell transplantation (HSCT) for Flt3-ITD positive acute myeloid leukemia (AML), and available therapies are largely unsatisfactory. In the present study, we retrospectively collected data on the off-label use of the tyrosine-kinase inhibitor sorafenib, either alone or in association with hypomethylating agents and adoptive immunotherapy, in 13 patients with post-transplantation Flt3-ITD positive AML relapses. Hematologic response was documented in 12/13 patients (92%), and 5/13 (38%) achieved complete bone marrow remission. Treatment was overall manageable in the outpatient setting, although all patients experienced significant adverse events, especially severe cytopenias (requiring a donor stem cell boost in 5 patients) and typical hand-foot syndrome. None of the patients developed graft-versus-host disease following sorafenib alone, whereas this was frequently observed when this was given in association to donor T cell infusions. Six patients are alive and in remission at the last follow-up, and 4 could be bridged to a second allogeneic HSCT, configuring a 65±14% overall survival at 100 days from relapse. Taken together, our data suggest that sorafenib might represent a valid treatment option for patients with Flt3-ITD positive post-transplantation relapses, manageable also in combination with other therapeutic strategies.

Question 30

Genomic and Epigenomic Landscapes of Adult De Novo Acute Myeloid Leukemia

The Cancer Genome Atlas Research Network

N Engl J Med 2013; 368:2059-2074May 30, 2013DOI: 10.1056/NEJMoa1301689

Answer 30

Abstract

BACKGROUND:

Many mutations that contribute to the pathogenesis of acute myeloid leukemia (AML) are undefined. The relationships between patterns of mutations and epigenetic phenotypes are not yet clear.

METHODS:

We analyzed the genomes of 200 clinically annotated adult cases of de novo AML, using either whole-genome sequencing (50 cases) or whole-exome sequencing (150 cases), along with RNA and microRNA sequencing and DNA-methylation analysis.

RESULTS:

AML genomes have fewer mutations than most other adult cancers, with an average of only 13 mutations found in genes. Of these, an average of 5 are in genes that are recurrently mutated in AML. A total of 23 genes were significantly mutated, and another 237 were mutated in two or more samples. Nearly all samples had at least 1 nonsynonymous mutation in one of nine categories of genes that are almost certainly relevant for pathogenesis, including transcription-factor fusions (18% of cases), the gene encoding nucleophosmin (NPM1) (27%), tumor-suppressor genes (16%), DNA-methylation-related genes (44%), signaling genes (59%), chromatin-modifying genes (30%), myeloid transcription-factor genes (22%), cohesin-complex genes (13%), and spliceosome-complex genes (14%). Patterns of cooperation and mutual exclusivity suggested strong biologic relationships among several of the genes and categories.

CONCLUSIONS:

We identified at least one potential driver mutation in nearly all AML samples and found that a complex interplay of genetic events contributes to AML pathogenesis in individual patients. The databases from this study are widely available to serve as a foundation for further investigations of AML pathogenesis, classification, and risk stratification. (Funded by the National Institutes of Health.).

Question 31

HLA-C–Dependent Prevention of Leukemia Relapse by Donor Activating KIR2DS1

Jeffrey M. Venstrom, M.D…..and Katharine C. Hsu, M.D., Ph.D.

N Engl J Med 2012; 367:805-816August 30, 2012DOI: 10.1056/NEJMoa1200503

Answer 31

Abstract

BACKGROUND:

Of the cancers treated with allogeneic hematopoietic stem-cell transplantation (HSCT), acute myeloid leukemia (AML) is most sensitive to natural killer (NK)-cell reactivity. The activating killer-cell immunoglobulin-like receptor (KIR) 2DS1 has ligand specificity for HLA-C2 antigens and activates NK cells in an HLA-dependent manner. Donor-derived NK reactivity controlled by KIR2DS1 and HLA could have beneficial effects in patients with AML who undergo allogeneic HSCT.

METHODS:

We assessed clinical data, HLA genotyping results, and donor cell lines or genomic DNA for 1277 patients with AML who had received hematopoietic stem-cell transplants from unrelated donors matched for HLA-A, B, C, DR, and DQ or with a single mismatch. We performed donor KIR genotyping and evaluated the clinical effect of donor KIR genotype and donor and recipient HLA genotypes.

RESULTS:

Patients with AML who received allografts from donors who were positive for KIR2DS1 had a lower rate of relapse than those with allografts from donors who were negative for KIR2DS1 (26.5% vs. 32.5%; hazard ratio, 0.76; 95% confidence interval [CI], 0.61 to 0.96; P=0.02). Of allografts from donors with KIR2DS1, those from donors who were homozygous or heterozygous for HLA-C1 antigens could mediate this antileukemic effect, whereas those from donors who were homozygous for HLA-C2 did not provide any advantage (24.9% with homozygosity or heterozygosity for HLA-C1 vs. 37.3% with homozygosity for HLA-C2; hazard ratio, 0.46; 95% CI, 0.28 to 0.75; P=0.002). Recipients of KIR2DS1-positive allografts mismatched for a single HLA-C locus had a lower relapse rate than recipients of KIR2DS1-negative allografts with a mismatch at the same locus (17.1% vs. 35.6%; hazard ratio, 0.40; 95% CI, 0.20 to 0.78; P=0.007). KIR3DS1, in positive genetic linkage disequilibrium with KIR2DS1, had no effect on leukemia relapse but was associated with decreased mortality (60.1%, vs. 66.9% without KIR3DS1; hazard ratio, 0.83; 95% CI, 0.71 to 0.96; P=0.01).

CONCLUSIONS:

Activating KIR genes from donors were associated with distinct outcomes of allogeneic HSCT for AML. Donor KIR2DS1 appeared to provide protection against relapse in an HLA-C-dependent manner, and donor KIR3DS1 was associated with reduced mortality. (Funded by the National Institutes of Health and others.).

Question 32

Clonal Architecture of Secondary Acute Myeloid Leukemia

Matthew J. Walter, M.D…..Graubert, M.D.

N Engl J Med 2012; 366:1090-1098March 22, 2012DOI: 10.1056/NEJMoa1106968

Answer 32

Abstract

BACKGROUND:

The myelodysplastic syndromes are a group of hematologic disorders that often evolve into secondary acute myeloid leukemia (AML). The genetic changes that underlie progression from the myelodysplastic syndromes to secondary AML are not well understood.

METHODS:

We performed whole-genome sequencing of seven paired samples of skin and bone marrow in seven subjects with secondary AML to identify somatic mutations specific to secondary AML. We then genotyped a bone marrow sample obtained during the antecedent myelodysplastic-syndrome stage from each subject to determine the presence or absence of the specific somatic mutations. We identified recurrent mutations in coding genes and defined the clonal architecture of each pair of samples from the myelodysplastic-syndrome stage and the secondary-AML stage, using the allele burden of hundreds of mutations.

RESULTS:

Approximately 85% of bone marrow cells were clonal in the myelodysplastic-syndrome and secondary-AML samples, regardless of the myeloblast count. The secondary-AML samples contained mutations in 11 recurrently mutated genes, including 4 genes that have not been previously implicated in the myelodysplastic syndromes or AML. In every case, progression to acute leukemia was defined by the persistence of an antecedent founding clone containing 182 to 660 somatic mutations and the outgrowth or emergence of at least one subclone, harboring dozens to hundreds of new mutations. All founding clones and subclones contained at least one mutation in a coding gene.

CONCLUSIONS:

Nearly all the bone marrow cells in patients with myelodysplastic syndromes and secondary AML are clonally derived. Genetic evolution of secondary AML is a dynamic process shaped by multiple cycles of mutation acquisition and clonal selection. Recurrent gene mutations are found in both founding clones and daughter subclones.

Nature commentary:

Previous work had indicated that the
size of the MDS clone was larger than
that reflected by the myeloblast count,
but what was surprising was that most of
the cells in the bone marrow of patients
with MDS were clonal—that is, they had
expanded from a single cell that contained
a distinct group of mutations. Moreover,
the MDS clone was not significantly larger
when the patients’ disease had progressed
to AML. Furthermore, in all cases that
were assessed, new clonal populations
emerged, either when patients still had
MDS or when they progressed to AML. In
all cases, the clones present in AML could
be tracked back to the original ‘founding’
clone present in MDS. “Our results
suggest that MDS and secondary AML
are both highly clonal haematopoietic
malignancies, and that evolution to AML
is characterized by the emergence of clonal
populations of cells that are derived from
a founding clone that is present in MDS, ”
explains Graubert. His team are planning
to determine which genetic changes are
driving outgrowths of new clones.

Question 33

Randomised Introduction of 2-CDA as Intensification during Consolidation for Children with High-risk AML–results from Study AML-BFM 2004.
Klin Padiatr. 2015 May;227(3):116-22. doi: 10.1055/s-0035-1548816. Epub 2015 May 18.

Creutzig U…Reinhardt D

Answer 33

Abstract

BACKGROUND:

The outcome in children and adolescents with high-risk (HR) acute myeloid leukemia (AML) is still unsatisfactory. Therefore, in study AML-BFM 2004 we aimed to improve outcome of HR-patients by adding moderately dosed 2-Chloro-2-Deoxyadenosine (2-CDA = cladribine) to the respective consolidation treatment backbone without increasing toxicity. The aim was to improve prognosis especially in FAB M4/M5/MLL patients, who represent the largest subgroup of HR patients.

PATIENTS AND METHODS:

In total, 343 children and adolescents with HR-AML were randomized to receive or not 2-CDA (6 mg/m²/d, days 1, 3) in combination with cytarabine/idarubicine (AI=500 mg/m² cytarabine 5 days continuous infusion plus 7 mg/m²/d idarubicin, days 3 and 5).

RESULTS:

RESULTS for patients of the AI/2-CDA arm (n=168) vs. the AI-arm (n=175) were similar: 5-year overall survival 68±4 vs. 72±4%, plogrank=0.38, event-free survival 53±4 vs. 49±4%, plogrank=0.77; cumulative incidence of relapse at 5 years: 35±4 vs. 37±4%, p(Gray)=0.89. RESULTS in patients with MLL rearrangement or FAB M4/M5 were also similar in the treatment groups. In addition, toxicities did not differ between the two arms.

CONCLUSION:

We conclude that additional, moderate dose 2-CDA does not improve prognosis in HR-patients when given during consolidation treatment. Its effect might be too low in this multidrug regimen, where the strongest effects are achieved during induction, or the chosen dose of 2-CDA might have been too low.

Question 34

Randomized trial comparing liposomal daunorubicin with idarubicin as induction for pediatric acute myeloid leukemia: results from Study AML-BFM 2004

Ursula Creutzig….Dirk Reinhardt

Blood. 2013 Jul 4;122(1):37-43. doi: 10.1182/blood-2013-02-484097.

Answer 34

Key Points

AML induction with liposomal daunorubicin (80 mg/m2 per day for 3 days) shows antileukemic activity comparable to idarubicin (12 mg/m2 per day for 3 days).

Liposomal daunorubicin promises to be more active in the t(8;21) subgroup and causes less treatment-related toxicity.

Abstract

Outcomes of patients with acute myeloid leukemia (AML) improve significantly by intensification of induction. To further intensify anthracycline dosage without increasing cardiotoxicity, we compared potentially less cardiotoxic liposomal daunorubicin (L-DNR) to idarubicin at a higher-than-equivalent dose (80 vs 12 mg/m(2) per day for 3 days) during induction. In the multicenter therapy-optimization trial AML-BFM 2004, 521 of 611 pediatric patients (85%) were randomly assigned to L-DNR or idarubicin induction. Five-year results in both treatment arms were similar (overall survival 76% ± 3% [L-DNR] vs 75% ± 3% [idarubicin], Plogrank = .65; event-free survival [EFS] 59% ± 3% vs 53% ± 3%, Plogrank = .25; cumulative incidence of relapse 29% ± 3% vs 31% ± 3%, P(Gray) = .75), as were EFS results for standard (72% ± 5% vs 68% ± 5%, Plogrank = .47) and high-risk (51% ± 4% vs 46% ± 4%, Plogrank = .45) patients. L-DNR resulted in significantly better probability of EFS in patients with t(8;21). Overall, treatment-related mortality was lower with L-DNR than idarubicin (2/257 vs 10/264 patients, P = .04). Grade 3/4 cardiotoxicity was rare after induction (4 L-DNR vs 5 idarubicin). Only 1 L-DNR and 3 idarubicin patients presented with subclinical or mild cardiomyopathy during follow-up. In conclusion, at the given dose, L-DNR has overall antileukemic activity comparable to idarubicin, promises to be more active in subgroups, and causes less treatment-related mortality. This trial was registered at www.clinicaltrials.gov as #NCT00111345.

Question 35

Favorable outcome in infants with AML after intensive first- and second-line treatment: an AML-BFM study group report

U Creutzig… D Reinhardt

Leukemia. 2012 Apr;26(4):654-61. doi: 10.1038/leu.2011.267. Epub 2011 Oct 4.

Answer 35

Infants <1 year of age have a high prevalence of prognostically unfavorable leukemias and a presumed susceptibility to treatment-related toxicities. A total of 125 infants with acute myeloid leukemia (AML) were treated in studies AML-BFM-98 (n=59) and -2004 (n=66). Treatment regimens of both studies were comparable, consisting of intensive induction followed by four courses (mainly high-dose cytarabine and anthracyclines). Allogeneic-hematopoietic stem-cell-transplantation (allo-HSCT) in 1st remission was optional for high-risk (HR) patients. Most infants (120/125=96%) were HR patients according to morphological, cytogenetic/molecular genetic and response criteria. Five-year overall survival was 66±4%, and improved from 61±6% in study-98 to 75±6% in study-2004 (Plogrank 0.14) and event-free survival rates were 44±6% and 51±6% (Plogrank 0.66), respectively. Results in HR infants were similar to those of older HR children (1–<2- or 2–<10-year olds, Plogrank 0.90 for survival). Survival rates of HSCT in 1st remission, initial partial response and after relapse were high (13/14, 2/8 and 20/30 patients, respectively). The latter contributes to excellent 5-year survival after relapse (50±8%). Despite more severe infections and pulmonary toxicities in infants, treatment-related death rate was identical to that of older children (3%). Our data indicate that intensive frontline and relapse AML treatment is feasible in infants, toxicities are manageable, and outcome is favorable.

Question 36

Favourable outcome of patients with childhood acute promyelocytic leukaemia after treatment with reduced cumulative anthracycline doses.
Br J Haematol. 2010 May;149(3):399-409. doi: 10.1111/j.1365-2141.2010.08107.x. Epub 2010 Mar 8.

Creutzig U…Reinhardt D.

Answer 36

Abstract

Acute promyelocytic leukaemia (APL) treatment often includes high cumulative doses of anthracyclines, which can cause long-term cardiotoxicity. Here, we report the favourable outcome in 81 paediatric APL patients treated according to the consecutive acute myeloid leukaemia-Berlin/Frankfurt/Muenster (AML-BFM) trials -93/-98/-2004 with an anthracycline-cytarabine regimen in combination with all-trans-retinoid acid (ATRA). Outcomes achieved by treatment with a reduced cumulative anthracycline dose (350 mg/m(2)) were comparable to those reported for studies with higher doses. Five-year overall survival of the total cohort was 89 +/- 4% and event-free survival (pEFS) was 73 +/- 6%. Overall survival was similar when comparing AML-BFM trial periods (trial 93: 88 +/- 8%, 98: 85 +/- 7% and 2004: 94 +/- 8%, P((logrank)) = 0.63). Seventy-five (93%) patients achieved complete remission. Most fatal events occurred during the first 6 weeks of treatment. Long-term cardiotoxicity was observed in one patient. Two patients suffered from secondary haematological malignancies. Salvage treatment was effective in 7/9 patients (78%) with relapsed APL, who now are long-term survivors after second line combination treatment with arsenic trioxide (4/7 patients) and stem cell transplantation (5/7 patients). Our results demonstrate that - combined with ATRA - a lower cumulative anthracycline dose can be used safely to maintain high cure rates and promote the reduction of long-term sequelae, such as cardiotoxicity in APL patients.

Question 37

Second induction with high-dose cytarabine and mitoxantrone: different impact on pediatric AML patients with t(8;21) and with inv(16).
Blood. 2011 Nov 17;118(20):5409-15. doi: 10.1182/blood-2011-07-364661. Epub 2011 Sep 26.

Creutzig U… Reinhardt D.

Answer 37

Abstract

Patients with core binding factor acute myeloid leukemia (CBF-AML) benefit from more intensive chemotherapy, but whether both the t(8;21) and inv(16)/t (16;16) subtypes requires intensification remained to be determined. In the 2 successive studies (AML-BFM-1998 and AML-BFM-2004), 220 CBF-AML patients were treated using the same chemotherapy backbone, whereby reinduction with high-dose cytarabine and mitoxantrone (HAM) was scheduled for these cohorts only in study AML-BFM-1998 but not in AML-BFM-2004 against the background to minimize overtreatment. Five-year overall survival (OS) and event-free survival (EFS) were significantly higher and the cumulative incidence of relapse (CIR) lower in t(8;21) patients treated with HAM (n = 78) compared with without HAM (n = 53): OS 92% ± 3% versus 80% ± 6%, p(logrank)0.047, EFS 84% ± 4% versus 59% ± 7%, p(logrank)0.001, and CIR 14% ± 4% versus 34% ± 7%, p((gray))0.006. These differences were not seen for inv(16) (n = 43 and 46, respectively): OS 93% ± 4% versus 94% ± 4%, EFS 75% ± 7% versus 71% ± 9% and CIR 15% ± 6% versus 23% ± 8% (not significant). The subtype t(8;21), but not inv(16), was an independent predictor of worse outcome without HAM reinduction. Based on our data, a 5-year OS of > 90% can be expected for CBF-AML, when stratifying t(8;21), but not inv(16), patients to high-risk chemotherapy, including HAM reinduction.

Question 38

Early deaths and treatment-related mortality in children undergoing therapy for acute myeloid leukemia: analysis of the multicenter clinical trials AML-BFM 93 and AML-BFM 98.
J Clin Oncol. 2004 Nov 1;22(21):4384-93.

Creutzig U… Lehrnbecher T.

Answer 38

Abstract

PURPOSE:

The rates of early death (ED) and treatment-related mortality (TRM) are unacceptably high in children undergoing intensive chemotherapy for acute myeloid leukemia (AML). Better strategies of supportive care might help to improve overall survival in these children.

PATIENTS AND METHODS:

In a retrospective study, we analyzed incidence, clinical features, and risk factors for lethal complications of 901 children enrolled onto the multicenter trials Acute Myeloid Leukemia-Berlin-Frankfurt-Muenster (AML-BFM) 93 and AML-BFM 98.

RESULTS:

One hundred four patients (11.5%) enrolled onto the clinical trials AML-BFM 93 and AML-BFM 98 died shortly after diagnosis or as a result of treatment-related complications. Thirty-two patients (3.5%) died before (six patients) or during (26 patients) the first 14 days of treatment, mainly as a result of bleeding or leukostasis. Low performance status, hyperleukocytosis, and French-American-British type M5 were the main risk factors for a lethal event before day 15. After day 15, the predominant causes of death were complications caused by infections, particularly bacterial and fungal infections. The incidence of lethal infections was highest during induction therapy and decreased thereafter. When comparing both clinical trials, significantly fewer patients died within the first 6 weeks in AML-BFM 98 than in AML-BFM 93 (14 [3.5%] of 430 patients v 35 [7.4%] of 471 patients; P = .01).

CONCLUSION:

To reduce the high incidence of ED and TRM in children with AML, early diagnosis and adequate treatment of complications are needed. Children with AML should be treated in specialized pediatric cancer centers only. Prophylactic and therapeutic regimens for better treatment management of bleeding disorders and infectious complications have to be assessed in future trials to ultimately improve overall survival in children with AML.

Question 39

The hidden genomic landscape of acute myeloid leukemia: subclonal structure revealed by undetected mutations.
Blood. 2015 Jan 22;125(4):600-5. doi: 10.1182/blood-2014-05-576157. Epub 2014 Dec 12.

Bodini M… Riva L

Answer 39

Abstract

The analyses carried out using 2 different bioinformatics pipelines (SomaticSniper and MuTect) on the same set of genomic data from 133 acute myeloid leukemia (AML) patients, sequenced inside the Cancer Genome Atlas project, gave discrepant results. We subsequently tested these 2 variant-calling pipelines on 20 leukemia samples from our series (19 primary AMLs and 1 secondary AML). By validating many of the predicted somatic variants (variant allele frequencies ranging from 100% to 5%), we observed significantly different calling efficiencies. In particular, despite relatively high specificity, sensitivity was poor in both pipelines resulting in a high rate of false negatives. Our findings raise the possibility that landscapes of AML genomes might be more complex than previously reported and characterized by the presence of hundreds of genes mutated at low variant allele frequency, suggesting that the application of genome sequencing to the clinic requires a careful and critical evaluation. We think that improvements in technology and workflow standardization, through the generation of clear experimental and bioinformatics guidelines, are fundamental to translate the use of next-generation sequencing from research to the clinic and to transform genomic information into better diagnosis and outcomes for the patient.

Question 40

Mouse models of NPM1-mutated acute myeloid leukemia: biological and clinical implications.
Leukemia. 2015 Feb;29(2):269-78. doi: 10.1038/leu.2014.257. Epub 2014 Sep 2.

Sportoletti P1, Varasano E1, Rossi R1, Mupo A2, Tiacci E1, Vassiliou G2, Martelli MP1, Falini B1.

Answer 40

Abstract

Acute myeloid leukemia (AML) carrying nucleophosmin (NPM1) mutations displays distinct biological and clinical features that led to its inclusion as a provisional disease entity in the 2008 World Health Organization (WHO) classification of myeloid neoplasms. Studies of the molecular mechanisms underlying the pathogenesis of NPM1-mutated AML have benefited greatly from several mouse models of this leukemia developed over the past few years. Immunocompromised mice xenografted with NPM1-mutated AML served as the first valuable tool for defining the biology of the disease in vivo. Subsequently, genetically engineered mouse models of the NPM1 mutation, including transgenic and knock-in alleles, allowed the generation of mice with a constant genotype and a reproducible phenotype. These models have been critical for investigating the nature of the molecular effects of these mutations, defining the function of leukemic stem cells in NPM1-mutated AML, identifying chemoresistant preleukemic hemopoietic stem cells and unraveling the key molecular events that cooperate with NPM1 mutations to induce AML in vivo. Moreover, they can serve as a platform for the discovery and validation of new antileukemic drugs in vivo. Advances derived from the analysis of these mouse models promise to greatly accelerate the development of new molecularly targeted therapies for patients with NPM1-mutated AML.

Question 41

3 ways of classifying AML

Answer 41

Acute myeloid leukemia (AML) is composed of a heterogeneous
group of diseases that can be classied by morphology, lineage, and genetics.

Question 42

What is RUNX1-RUNX1T1 synonymous with (older name of this fusion transcript)?

Answer 42

RUNX1-RUNX1T1 formerly known as AML1/ETO

Question 43

How I treat pediatric acute myeloid leukemia.

Rubnitz JE1.

Blood. 2012 Jun 21;119(25):5980-8. doi: 10.1182/blood-2012-02-392506. Epub 2012 May 7.

Answer 43

Abstract

Acute myeloid leukemia is a heterogeneous disease that accounts for approximately 20% of acute leukemias in children and adolescents. Despite the lack of targeted therapy for most subtypes and a dearth of new agents, survival rates have reached approximately 60% for children treated on clinical trials in developed countries. Most of the advances have been accomplished by better risk classification, the implementation of excellent supportive care measures, adaptation of therapy on the basis of each patient’s response to therapy, and improvements in allogeneic hematopoietic stem cell transplantation. However, it is unlikely that further gains can be made through these measures alone. In this regard, high-resolution, genome-wide analyses have led to greater understanding of the pathogenesis of this disease and the identification of molecular abnormalities that are potential targets of new therapies. The development of molecularly targeted agents, some of which are already in clinical trials, holds great promise for the future.

Question 44

Mouse models for core binding factor leukemia.

Leukemia (13 July 2015)

D W L Chin… M Osato

Answer 44

Abstract

RUNX1 and CBFB are among the most frequently mutated genes in human leukemias. Genetic alterations such as chromosomal translocations, copy number variations and point mutations have been widely reported to result in the malfunction of RUNX transcription factors. Leukemias arising from such alterations in RUNX family genes are collectively termed core binding factor (CBF) leukemias. Although adult CBF leukemias generally are considered a favorable risk group as compared with other forms of acute myeloid leukemia, the 5-year survival rate remains low. An improved understanding of the molecular mechanism for CBF leukemia is imperative to uncover novel treatment options. Over the years, retroviral transduction-transplantation assays and transgenic, knockin and knockout mouse models alone or in combination with mutagenesis have been used to study the roles of RUNX alterations in leukemogenesis. Although successful in inducing leukemia, the existing assays and models possess many inherent limitations. A CBF leukemia model which induces leukemia with complete penetrance and short latency would be ideal as a platform for drug discovery. Here, we summarize the currently available mouse models which have been utilized to study CBF leukemias, discuss the advantages and limitations of individual experimental systems, and propose suggestions for improvements of mouse models.

Question 45

Secondary mutations as mediators of resistance to targeted therapy in leukemia

Naval Daver…Hagop Kantarjian

Blood. 2015 May 21;125(21):3236-45.

Answer 45

Abstract

The advent of small molecule-based targeted therapy has improved the treatment of both acute and chronic leukemias. Resistance to small molecule inhibitors has emerged as a common theme. The most frequent mode of acquired resistance is the acquisition of point mutations in the kinase domain. FLT3 inhibitors have improved response rates in FLT3-mutated acute myeloid leukemia (AML). The occurrence of the ATP-binding site and activation loop mutations confers varying degrees of resistance to the individual FLT3 inhibitors. Second-generation FLT3 inhibitors such as crenolanib may overcome the resistance of these mutations. Furthermore, nonmutational mechanisms of resistance such as prosurvival pathways and bone marrow signaling may be upregulated in FLT3 inhibitor-resistant AML with secondary kinase domain mutations. More recently, point mutations conferring resistance to the Bruton tyrosine kinase inhibitor ibrutinib in chronic lymphocytic leukemia, arsenic trioxide in acute promyelocytic leukemia, and the BH3-mimetic ABT199 in lymphoma have been identified. In chronic myeloid leukemia, the emergence of tyrosine kinase domain mutations has historically been the dominant mechanism of resistance. The early identification of secondary point mutations and their downstream effects along with the development of second- or third-generation inhibitors and rationally designed small molecule combinations are potential strategies to overcome mutation-mediated resistance.

Question 46

Leukemia-Associated Somatic Mutations Drive
Distinct Patterns of Age-Related Clonal Hemopoiesis

McKerrell T1, Park N2, Moreno T3, Grove CS1, Ponstingl H1, Stephens J4; Understanding Society Scientific Group, Crawley C5, Craig J5, Scott MA5, Hodkinson C6, Baxter J6, Rad R7, Forsyth DR8, Quail MA2, Zeggini E9, Ouwehand W10, Varela I3, Vassiliou GS11.

Cell Rep. 2015 Mar 3;10(8):1239-45.

Answer 46

In Brief: McKerrell et al. employ ultra-deep
sequencing to show that age-related
clonal hemopoiesis is much more
common than previously realized. They
find that clonal hemopoiesis, driven by
mutations in spliceosome genes SF3B1
and SRSF2, was noted exclusively in
individuals aged 70 years or older and
that NPM1 mutations are not seen in
association with this phenomenon,
endorsing their close association with
leukemogenesis.

Highlights

• Clonal hemopoiesis is an almost inevitable consequence of aging in humans
• Spliceosome gene mutations drove clonal hemopoiesis only in persons aged >70 years
• NPM1 mutations behave as gatekeepers for leukemogenesis

SUMMARY

Clonal hemopoiesis driven by leukemia-associated
gene mutations can occur without evidence of a blood
disorder. To investigate this phenomenon, we interro-
gated 15 mutation hot spots in blood DNA from 4,219
individuals using ultra-deep sequencing. Using only
the hot spots studied, we identified clonal hemopoie-
sis in 0.8% of individuals under 60, rising to 19.5% of
those >90 years, thus predicting that clonal hemopoi-
esis is much more prevalent than previously realized.
DNMT3A-R882 mutations were most common and,
although their prevalence increased with age, were
found in individuals as young as 25 years. By contrast,
mutations affecting spliceosome genes SF3B1 and SRSF2, closely associated with the myelodysplastic
syndromes, were identified only in those aged >70
years, with several individuals harboring more than
one such mutation. This indicates that spliceosome
gene mutations drive clonal expansion under selection
pressures particular to the aging hemopoietic system
and explains the high incidence of clonal disorders
associated with these mutations in advanced old age.

Table 1. Mutation Hot Spots Interrogated in This Study
Gene Target Codon
DNMT3A R882
JAK2 V617
NPM1 L287
SRSF2 P95
SF3B1 K666
SF3B1 K700
IDH1 R132
IDH2 R140
IDH2 R172
KRAS G12
NRAS G12
NRAS Q61
KIT D816
FLT3 D835
FLT3 N676

Question 47

History of paed AML therapy.

Approach to treatment in the 60s? Doses used? CR rates?

Approach to rx in the 70s? Doses?

Which approaches have been tried to improve induction and results?

Approaches to post remission therapy and results?

Role of allogeneic SCT?

C4K Rubnitz 2011

Answer 47

EARLY AML INDUCTION THERAPY

• 1960s: development of cytarabine (AraC) and daunorubicin (DNR)
  
  • AraC: 100-200 mg/m2/day x 5-7 days
  • DNR: 60 mg/m2/day x 3-7 days
  • Each produced CR rates about 40%
• 1970s: development of “3 + 7” regimen
  
  • DNR 45-60 mg/m2/day x 3 days
  • AraC 100-200 mg/m2/day x 7 days
  • Still considered standard of care

ATTEMPTS TO IMPROVE INDUCTION

• Choice of anthracycline
  
  • DNR vs. Idarubicin vs. Mitoxantrone
• Intensification of therapy
  
  • Dose (SD-AraC vs. HD-AraC)
  • Timing (standard vs. intensive)
• Addition of other agents
  
  • Etoposide vs. Thioguanine
• Results: almost all randomized trials showed no difference between arms

POST-REMISSION THERAPY

• ECOG study in 1980s (chemo vs no chemo) demonstrated that postremission therapy is beneficial
• Benefit of intensive chemotherapy confirmed by adult and pediatric groups
• Maintenance therapy: worse outcome for patients who received MT (harder to salvage post relapse)
• Additional courses of intensification: 4 courses equal to 5 courses
• Autologous SCT: no benefit
• Allogeneic SCT

ROLE OF ALLOGENEIC SCT

• Reduces risk of relapse, but…
  
  • Higher TRM and late effects
  • Higher costs
  • Lower salvage rates
  • Most studies do not show survival benefit
• Important issues
  
  • Donor selection
  • Patient selection: who should receive SCT in CR1?

Question 48

Genetic features with proven, probably and uncertain prognostic implications in paed AML as of 2012

How I treat pediatric acute myeloid leukemia Jeffrey E. Rubnitz Blood 2012

Answer 48

PROVEN

Favourable:

1. t(8;21)(q22;q22)/RUNX1-RUNX1T1 (15%)
2. inv(16)(p13.1;q22)/CBFß-MYH11 and t(16;16)(p13.1;q22)/CBFß-MYH11 (10%)

Unfavourable:

1. FLT3-ITD (12%): poor prognosis, especially in cases with a high ratio of mutant to wild-type allele;
   may benefit from HSCT or treatment with FLT3 inhibitors
2. -7 (1%): Poor prognosis

PROBABLE

1. 11q23; MLL rearrangements (20%)
   
   1. t(9;11)(p12;q23)/MLL-AF9 (8%): favorable prognosis in some studies
   2. t(1;11)(q21;q23)/MLL-AF1q (1%): favorable prognosis
   3. t(6;11)(q27;q23)/MLL-AF6 (1%): poor prognosis
   4. t(10;11)(p12;q23)/MLL-AF10 (1%): poor prognosis
   5. Others (9%): intermediate prognosis
2. t(1;22)(p13;q13)/RBM15-MKL1 (1%): only observed in megakaryoblastic leukemia, probably a/w favorable prognosis
3. NPM1 mutations (8%): seen in 20% of cases with normal karyotype; favorable prognosis, except in cases with FLT3-ITD
4. CEBPA mutations (5%): Seen in 17% of cases with normal karyotype; favorable prognosis, except in cases with FLT3-ITD; favorable prognosis probably limited to cases with biallelic mutations
5. t(6;9)(p23;q34)/DEK-NUP214 (1%): poor prognosis
6. t(8;16)(p11;p13)/MYST3-CREBBP (1%): poor prognosis
7. t(16;21)(q24;q22)/RUNX1-CBFA2T3 (1%): poor prognosis

Genetic features with unknown prognostic implications

1. WT1
   
   1. Mutation (10%): unknown
   2. SNP rs16754 (25%): May be associated with favorable outcome
2. IDH1 and IDH2
   
   1. Mutation (4%): unknown
   2. IDH1 SNP rs11554137 (10%): unknown
3. RUNX1 mutation (rare): unknown
4. TET2 mutation (rare): inknown
5. DNMT3A mutation (rare): unknown

Question 49

Risk classification of patients with paed AML (How I treat pediatric AML, Rubnitz, Blood 2012)

Answer 49

Risk classification scheme based on features at diagnosis and the presence of MRD.

Low risk

1. Patients with t(8;21), inv(16), or t(16;16) regardless of other genetic alterations
2. Patients with NPM1 mutations or biallelic CEBPA mutations are provisional LR, except in the presence of FLT3-ITD.
3. Provisional LR patients are moved to the intermediate-risk group if they are MRD-positive after one course of induction therapy

High Risk

1. t(6;9)
2. t(8;16)
3. t(16;21)
4. -5
5. -7
6. Megakaryoblastic (M7) without t(1;22)
7. Treatment-related AML
8. MDS-related AML
9. Flt3-ITD high AR

HR patients include those with any of the features above, regardless of response to therapy.

Patients who lack LR and HR features are provisionally classified as intermediate risk but are moved to the HR group if they have a poor response to therapy as assessed by MRD.

Question 50

NK cell role in AML treatment (C4K Rubnitz 2012):

• When were NK cells first described?
• NK cells comprise what % of lymphocytes?
• How are NK cells phenotypically defined?
• Rationale for NK cell therapy in AML?
• Four roles for donor NK cells in SCT?
• Effects of KIR mismatch in haploidentical SCT?
• Effects of KIR mismatch in URD SCT?
• Role of NK cells in the non SCT setting?

Answer 50

• Described in 1975 as capable of in vitro cytotoxicity against leukemic cells without prior exposure
• 5-15% of lymphocytes
• Phenotypically defined
  
  • Expression of CD56
  • Lack of CD3 and TCR

REGULATION OF NK CYOTOXICITY

• Cytotoxicity regulated by activating and inhibitory mechanisms
  
  • Killer immunoglobulin-like receptors (KIRs) include activating and inhibitory molecules
  • Inhibitory KIRs recognize & ligate specific HLA class I molecules on target cells and inhibit NK functions
  • Inhibition dominates over activation

RATIONALE FOR NK CELL THERAPY IN AML

• Cytotoxicity does not require prior sensitization
• NK cells do not cause GVHD
• NK cells do not permanently engraft
• Anti-malignancy effect vs. AML
• Optimal donor can be chosen by appropriate receptor-ligand mismatch
• Large-scale NK cell isolation is feasible

ROLES OF DONOR NK CELLS IN SCT

• Reduce relapse
  
  • Direct cytotoxicity of donor NK cells against patient AML cells
  • Effective if the HLA class I epitope for the donor’s inhibitory KIR is absent from the recipient tumor cells
• Reduce GVHD
  
  • Donor NK cells may kill host dendritic cells
• Reduce Infection: enhanced immune reconstitution
• Reduce Rejection

EFFECTS OF KIR MISMATCH IN HAPLOIDENTICAL SCT

• Landmark study was Ruggeri et al, Science 2002
  
  • Haploidentical SCT performed in 57 patients with high-risk AML
  • KIR ligand incompatibility completely protected against rejection, GVHD, and AML relapse
• Confirmatory study (Blood 2007)
  
  • NK Alloreactive Donor
    
    • AML in CR -> 67% EFS
    • AML in relapse -> 34% EFS
  • Non-NK Alloreactive
    
    • AML in CR -> 18% EFS
    • AML in relapse -> 6% EFS

EFFECTS OF KIR MISMATCH IN URD SCT

• Giebel et al, Blood 2003: Unrelated donor SCT performed in 87 patients with AML
  
  • KIR mismatch (n=13): 100% DFS, 100% OS, 0% RR, 0% TRM
  • No KIR mismatch (n=74): 37% DFS, 45% OS, 18% RR, 45% TRM

ROLE OF NK CELLS IN THE NON SCT SETTING

Miller et al, Blood 2005

• First showed that NK cells, infused in a non-stem cell transplant setting, could expand in vivo and exert a potent anti-cancer effect
  
  • 19 adult high-risk AML patients received fludarabine, cyclophosphamide, IL-2, and an infusion of 2 x 10⁷/kg CD3-depleted NK cell product (~40% NK cells)
  • 8/15 patients had ≥ 1% engraftment at day 7 or later
  • The number of circulating NK cells was significantly greater in the patients who achieved remission
  • Evidence for anti-leukemic effects of KIR-mismatched NK cells:
    
    • 5 of 19 patients overall achieved CR
    • 3 of 4 mismatched patients achieved CR

NKAML PROTOCOL

Hypothesis: Haploidentical NK cell therapy will provide a safe alternative mode of consolidation therapy and will reduce the risk of relapse by eliminating residual leukemic blasts in patients with AML in first remission.

Aims:

• To study the engraftment, phenotype, and function of transplanted haploidentical NK cells
• To assess the feasibility and toxicity of NK cell transplantation in the non-SCT setting

Patients and Methods

• 10 patients with AML in first remission
• Regimen
  
  • Cyclophosphamide: 60 mg/kg on day -7
  • Fludarabine: 25 mg/m2/day on days -6 thru -2
  • Interleukin-2: 1 million units/m2 sc QOD X 6
  • NK cell products: Purified by CD3 depletion, CD56 selection. Infused on day 0.

Toxicity

• Non-hematologic toxicity
  
  • 1 swelling and erythema at IL-2 site
  • 1 viral respiratory infection
  • 1 episode of fever and neutropenia
• Hematologic toxicity: Median time to ANC > 500 12 days
• Hospitalizations: Median length 2 days (0-3 days)

Cell Products

• NK cells: median 29 x 106/kg, range 5 to 81 x 10⁶/kg
• B cell contamination: median 0.097 x 10⁶/kg
• T cell contamination: no detectable T cells in 9 cases; 1 x 103/kg in 1 case (10-fold lower than needed for GVHD)

Engraftment

• Length of NK cell engraftment
  
  • Median: 10 days
  • Range: 2 to 189 days
• NK chimerism
  
  • Median peak: 7% donor
  • Range of peaks: 1% to 30% donor

Expansion of NK cells

• Observed in all 9 cases with KIR-mismatched donor
• Median peak expansion at day 14 (highly variable between patients)

Outcomes and Conclusions

• With a median follow-up time of >1000 days, all patients remain in remission
  
  • 2-year EFS 100%, 95% confidence interval, 69% - 100%
  • No long term toxicity
• Immunosuppression with cyclo and fludarabine, followed by the infusion of KIR mismatched NK cells, is safe and feasible in patients with AML in first remission

Question 51

Dactinomycin in NPM1-Mutated Acute Myeloid Leukemia

Falini et al. N Engl J Med 2015; 373:1180-1182

Answer 51

To the Editor:

NPM1-mutated acute myeloid leukemia (AML) is a distinct leukemia entity that accounts for one third of cases of AML in adults.1 NPM1 is a crucial protein for normal nucleolar integrity and function. We hypothesized that the nucleolus of NPM1-mutated AML cells might be vulnerable to drugs that trigger a nucleolar stress response because it contains a low level of nonmutant NPM1 (owing to haploinsufficiency and cytoplasmic retention of nonmutant NPM1 by the NPM1 mutant1). 2 Moreover, the p53-mediated nucleolar stress response is retained in NPM1-mutated AML because NPM1-mutated AML cells lack p53 mutations or deletions. 3

Among potentially active drugs, we focused on dactinomycin because it induces nucleolar stress by interfering with ribosome biogenesis through inhibition of RNA polymerase I. 4 Dactinomycin is active in Wilms’ tumor and some other tumors, but we could find no study on dactinomycin use in AML.

Therefore, we used it to treat a 60-year-old patient with NPM1-mutated AML without FLT3 internal tandem duplication mutations. Because dactinomycin lacks cardiotoxicity, we selected a patient for whom intensive chemotherapy was not appropriate owing to a low left ventricular ejection fraction (35%). Leukemia had progressed after an initial single cycle of azacitidine. Therefore, with the patient’s consent, we administered dactinomycin as an off-label single agent (approved by the institutional review board at Perugia Hospital), at a dose of 12.5 μg per kilogram of body weight per day for 5 consecutive days, as recommended for low-risk gestational trophoblastic tumors. 5 Morphologic and immunohistochemical complete remission was achieved after two cycles of therapy, and we administered four more cycles for a total of six, at intervals of 3 to 4 weeks. A quantitative reverse-transcriptase–polymerase-chain-reaction assay for mutant copies of NPM1 showed a molecular complete remission after the fourth cycle (Figure 1G). The patient has now had a morphologic and molecular complete remission lasting 14 months. Adverse events included febrile neutropenia and thrombocytopenia of grade 4 during cycles 1 and 2, grade 2 oral mucositis during cycles 1 through 3, and superficial skin erosions during cycle 2. Cycles 3 through 6 were administered on an outpatient basis, with no need for blood transfusions.

The mechanism of action of dactinomycin in AML remains to be clarified. The progressive decrease in NPM1 mutant transcripts (until the achievement of negativity for minimal residual disease) is reminiscent of dactinomycin-induced normalization of serum levels of human chorionic gonadotropin in patients with gestational trophoblastic tumors. 5 We treated six additional patients with refractory or relapsed NPM1-mutated AML with cycles of dactinomycin at a dose of 15.0 μg per kilogram per day for 5 consecutive days. A hematologic complete remission was achieved in two patients: one (74 years of age) had not had a response to azacitidine, and another (72 years of age) had had a relapse after multiple lines of chemotherapy. Future studies are warranted to investigate dactinomycin in NPM1-mutated AML and other AML genotypes.

Question 52

Acute Myeloid Leukemia

Hartmut Döhner, M.D., Daniel J. Weisdorf, M.D., and Clara D. Bloomfield, M.D.

N Engl J Med 2015; 373:1136-1152

• 35-40% adults <60 cured
• 5-15% >60yrs cured
• Adults unable to tolerate intensive chemo -> median survival 5-10 months
• Cytogenetic heterogeneity 30yrs, molecular heterogeneity 15yrs. Well accepted prognostic importance, nascent therapeutic importance.

DISEASE CLASSIFICATION

• Classification based on morphology, flow cytometry, conventiional cytogenetic testing for chromosomal abnormalities, screening for selected molecular genetic lesions.
• WHO 2008 classification: AML with recurrent genetic abnormalities, AML with myelodysplasia-related changes, therapy-related AML, and AML not otherwise specified

Answer 52

Acute myeloid leukemia (AML) is a form of cancer that is characterized by infiltration of the bone marrow, blood, and other tissues by proliferative, clonal, abnormally differentiated, and occasionally poorly differentiated cells of the hematopoietic system. Although it was incurable 50 years ago, AML is now cured in 35 to 40% of adult patients who are 60 years of age or younger and in 5 to 15% of patients who are older than 60 years of age.1 The outcome in older patients who are unable to receive intensive chemotherapy without unacceptable side effects remains dismal, with a median survival of only 5 to 10 months.

Although the cytogenetic heterogeneity of AML has been recognized for more than 30 years, the enormous molecular heterogeneity of the disease has become increasingly apparent over the past 15 years. The prognostic importance of this biologic heterogeneity is well accepted, but translation of this new information into improved therapy is just beginning. In this article, we describe recent advances in the disease classification, understanding of the genomic landscape, identification of prognostic factors, current treatment, and new therapies under investigation in types of adult AML other than acute promyelocytic leukemia.

DISEASE CLASSIFICATION

Morphologic assessment of bone marrow specimens and blood smears, analysis of the expression of cell-surface or cytoplasmic markers by means of flow cytometry, identification of chromosomal findings by means of conventional cytogenetic testing, and, more recently, screening for selected molecular genetic lesions are the diagnostic procedures used to classify AML. AML is classified according to the World Health Organization (WHO) Classification of Tumours of Haematopoietic and Lymphoid Tissues,2 which was last updated in 2008. The major categories of the current classification include AML with recurrent genetic abnormalities, AML with myelodysplasia-related changes, therapy-related AML, and AML not otherwise specified.

A revision of the WHO classification is under way. Changes to the section on AML with recurrent genetic abnormalities are being discussed. First, the molecular basis of inv(3)(q21q26.2) or t(3;3)(q21;q26.2) has been revisited,3 so that the revision shows rearrangement of a GATA2 oncogenic enhancer element, rather than of the RPN1 gene, in band 3q21 with the MECOM (EVI) gene in band 3q26.2. Second, the provisional entities “AML with NPM1 mutation” and “AML with CEBPA mutation” will become entities; “AML with CEBPA mutation” will be restricted to patients with AML in whom there is a biallelic (and not a monoallelic) mutation, because only that form of AML defines a clinicopathologic entity that is associated with a favorable prognosis.4 Finally, “AML with RUNX1 mutation”5,6 and “AML with BCR-ABL1” gene fusion7 are being considered as provisional entities on the basis of their characteristic clinicopathologic features, and in the section on AML with BCR-ABL1 gene fusion, the need for including the use of tyrosine kinase inhibitor therapy is being discussed.

A section on familial myeloid neoplasms, which reflects the increasing recognition of familial syndromes, is also under development.8 Inherited forms of myeloid neoplasms have been associated with germline mutations in at least 10 genes, 8-10 — ANKRD26, CEBPA, DDX41, ETV6, GATA2, RUNX1, SRP72, TERC, TERT, and TP53. For additional families carrying mutations associated with familial syndromes to be detected, it is important that physicians take detailed patient family histories, including data on cancer and bleeding problems. Awareness of inherited syndromes is clinically relevant, since these patients may require unique care, and family members should be screened, especially if allogeneic donor hematopoietic-cell transplantation is considered.

GENOMIC LANDSCAPE

Emerging data gleaned with the use of new genomic techniques — in particular, next-generation sequencing — are providing an unprecedented view of the spectrum and frequency of mutations, their distinct patterns of cooperativity and mutual exclusivity, their subclonal architecture, the clonal evolution during the disease course, and the epigenetic landscape of the disease.

The Cancer Genome Atlas Research Network analyzed the genomes of 200 patients with AML (50 with the use of whole-genome sequencing and 150 with the use of whole-exome sequencing, along with RNA and microRNA sequencing and DNA-methylation analysis).11 Genes that were significantly mutated in AML were organized into several functional categories.

Data are lacking from studies involving larger patient cohorts to elucidate the complex interplay of these genetic lesions in individual patients with AML.

Studies have shown that most cases of AML are characterized by clonal heterogeneity at the time of diagnosis, with the presence of both a founding clone and at least one subclone.11 Various patterns of dynamic clonal evolution that occur at relapse probably contribute to resistance to therapy.12

Other important findings revealed by next-generation sequencing studies relate to the pattern of mutation acquisition and the existence of preleukemic stem cells. Data from clonal evolution studies provide support for a model in which genes that are commonly involved in epigenetic regulation (i.e., DNMT3A, ASXL1, IDH2, and TET2) are present in preleukemic hematopoietic stem cells and occur early in the evolution of AML.13-15 Such ancestral preleukemic stem cells are capable of multilineage differentiation, can survive chemotherapy, and can expand during remission, eventually leading to relapse. Recent studies show that clonal hematopoiesis with somatic mutations, commonly involving the same genes (DNMT3A, TET2, and ASXL1), increases as people age and is associated with an increased risk of hematologic cancer and death.16-18 In absolute value, this risk is relatively low, and currently it has no clinical consequences.

The mutational pattern may be indicative of AML ontogeny (that is, whether the AML was newly diagnosed as primary disease or as a secondary disorder after an antecedent myeloid disorder such as a myelodysplastic syndrome). In a recent study, the presence of mutations in SRSF2, SF3B1, U2AF1, ZRSR2, ASXL1, EZH2, BCOR, or STAG2 defined a distinct genetic subtype of AML that shares clinicopathologic features with clinically confirmed secondary AML. 19

PROGNOSTIC CLASSIFICATION FACTORS

Prognostic factors can be subdivided into those that are related to the patient and those that are related to the disease. Patient-associated factors (e.g., increasing age, coexisting conditions, and poor performance status) commonly predict treatment-related early death, whereas disease-related factors (e.g., white-cell count, prior myelodysplastic syndrome or cytotoxic therapy for another disorder, and leukemic-cell genetic changes) predict resistance to current standard therapy. Because of marked improvements in supportive care in many older patients, the risk of treatment-related death is considerably lower than the risk that the disease will prove to be resistant to treatment. Indeed, treatment-related mortality appears to have decreased substantially in recent years. 20

The evaluation of molecular genetic lesions as prognostic and predictive markers is an active research area. Currently, three molecular markers (NPM1 and CEBPA mutations and FLT3 internal tandem duplications) are used in clinical practice, as reflected in the European Leukemia Net (ELN) recommendations.

It is expected that additional markers (e.g., RUNX1, ASXL1, and TP53) that have consistently been associated with an inferior outcome will soon be included in these recommendations. The prognostic importance of other mutated genes (e.g., DNMT3A, IDH1, IDH2) is less clear.

Despite the introduction of genetic testing at the initial diagnostic workup, the ability of clinicians to forecast resistance to treatment remains limited. 23 The monitoring of minimal residual disease by means of a quantitative reverse-transcriptase–polymerase-chain-reaction (RT-PCR) assay that detects leukemia-specific genetic targets or by means of multiparameter flow cytometry that identifies leukemia-associated aberrant phenotypes is another powerful tool to predict outcome. 24 The monitoring of minimal residual disease in core-binding factor AML and AML with the NPM1 mutation is already integrated into clinical trials, and it allows for preemptive intervention when minimal residual disease is persistent or recurrent. Such monitoring probably will become the standard of care in many patients with AML.

CURRENT THERAPY

The general therapeutic strategy in patients with AML has not changed substantially in more than 30 years.1 Initial assessment determines whether a patient is eligible for intensive induction chemotherapy. If complete remission is achieved after intensive therapy, appropriate postremission therapy is essential.

Induction Therapy

Continuous-infusion cytarabine with an anthracycline remains the mainstay of induction therapy.

Higher doses of daunorubicin than the doses that are currently used are being studied. In the United Kingdom National Cancer Research Institute (NCRI) AML17 trial, 1206 adults, most of whom were younger than 60 years of age, were randomly assigned to first induction therapy with daunorubicin at a dose of either 60 mg per square meter of body-surface area or 90 mg per square meter; no significant difference was shown with respect to the rate of complete response or the rate of overall survival.25 Confirmatory studies may be needed, since the effect of the dose of daunorubicin may be related to the amount of additional anthracycline therapy used.

A complete response is achieved in 60 to 85% of adults who are 60 years of age or younger. In patients who are older than 60 years of age, complete response rates are inferior (40 to 60%). Older age per se should not be a reason to withhold intensive therapy; however, weighing disease-related and patient-related prognostic factors against treatment intensity is crucial. For example, older patients more often have adverse cytogenetic abnormalities, clinically significant coexisting conditions, or both. Such patients are less likely to benefit from standard induction therapy and are candidates for investigational therapy.

No other induction regimen has been shown convincingly to be superior, with one possible exception: the addition of gemtuzumab ozogamicin, a humanized anti-CD33 monoclonal antibody conjugated with the cytotoxic agent calicheamicin. A recent meta-analysis of five randomized trials showed that although adding gemtuzumab ozogamicin to induction therapy did not increase response rates, it reduced the risk of relapse and improved survival among younger and older adults with favorable-risk and intermediate-risk (but not adverse-risk) cytogenetic findings. 26

Consolidation Therapy

Standard postremission strategies include conventional chemotherapy as well as hematopoietic-cell transplantation. Whether allogeneic transplantation is recommended depends mainly on the leukemic genetic-risk profile, scores on established scales that predict the risk of treatment-related death, and specific transplantation-associated factors in the patient.1,27-29

Consolidation with Intensive Chemotherapy

In adults who are 60 years of age or younger, an increasingly preferred regimen is 2 to 4 cycles of intermediate-dose cytarabine (Table 3). The most appropriate dose and number of cycles remain open issues; however, compelling data indicate that doses of 2000 to 3000 mg per square meter are above the plateau of the maximal therapeutic effect. 30 Consolidation therapy with intermediate-dose cytarabine is generally administered in patients with leukemic cells that have a more favorable ELN genetic risk profile, and cure rates among these patients is 60 to 70%. In clinical trials, monitoring of minimal residual disease with the use of a quantitative RT-PCR assay can guide postremission therapy in these patients, and preemptive salvage therapy, including allogeneic hematopoietic-cell transplantation, may be performed when there is molecular detection of persistent or relapsed AML.

Prospective randomized trials comparing single-agent higher doses of cytarabine with multiagent postremission therapy in adult patients who are 60 years of age or younger generally have not shown a significant difference in survival. 31 There is sparse evidence that combination therapy may be superior in patients with adverse-risk cytogenetic findings.32 Autologous hematopoietic-cell transplantation generally does not improve the outcome, but it may still be considered as alternative consolidation therapy in selected patients.33

The outcomes in patients who are older than 60 years of age remain highly unsatisfactory. Randomized trials have compared more intensive consolidation chemotherapy with less intensive consolidation chemotherapy, but the results have been inconclusive.31 Currently, it is generally recommended that patients with a favorable-risk ELN genetic profile and good performance status should receive repetitive cycles of an intermediate-dose cytarabine-based regimen (Table 3). Patients with an unfavorable genetic risk, clinically significant coexisting conditions, or both are unlikely to benefit from such therapy. Although patients with intermediate-risk genetic factors may fare better, the outcome also remains poor, with cure rates of only 10 to 15%. Given such dismal results, these patients should be offered investigational treatment that may include new maintenance therapies.

Allogeneic Hematopoietic-Cell Transplantation

Postremission therapy with allogeneic hematopoietic-cell transplantation provides the strongest antineoplastic therapy because of pretransplantation cytoreductive conditioning and the immunologic antileukemic graft-versus-leukemia effect.29 Allogeneic hematopoietic-cell transplantation is reserved for patients who are unlikely to have extended complete remission with conventional approaches other than transplantation (Table 3).27,28,34-38

Addressing selection bias in trials of various treatments requires adjustment for comparative eligibility and time-to-treatment effects. Such adjustments have been used in multicenter network trials in which therapies that include hematopoietic-cell transplantation are compared with those that do not include hematopoietic-cell transplantation. Examples of such trials are the U.S. Blood and Marrow Transplant Clinical Trials Network (BMT CTN) and the U.K. NCRI trials.

Transplantation Techniques

Chemoradiotherapy conditioning is chosen because of its antileukemia potency plus sufficient immunosuppression to permit engraftment. Nonmyeloablative or reduced-intensity conditioning must be sufficiently immunosuppressive to prevent rejection of the donor graft. Agents that do not cause adverse effects beyond any limiting myelosuppression are best-suited for hematopoietic-cell transplantation, since bone marrow toxicity is no longer dose-limiting. Fludarabine plus cyclophosphamide or other alkylating agents (such as busulfan and melphalan) and total-body irradiation are often used. Older patients and those with coexisting conditions often receive reduced-intensity conditioning; however, too little conditioning can increase the risk of relapse.

One prospective comparison (though underpowered) between fully myeloablative and reduced-intensity conditioning hematopoietic-cell transplantation has been reported,39 and another prospective trial, BMT CTN 0901 (ClinicalTrials.gov number, NCT01339910), has completed enrollment.

Donor Graft and Cell Source Options

An HLA-matched graft is generally preferred. Grafts from HLA-matched siblings are most often used, but since the late 1980s, grafts from HLA-matched volunteer adult unrelated donors have yielded nearly equivalent outcomes. However, these donors are not identified as often for patients with minority racial or ethnic backgrounds. 40 HLA heterogeneity, particularly in black or mixed-race populations, limits the identification of allele-matched unrelated donors, even in large worldwide networks (which include >20 million potential donors). Closely matched units of umbilical-cord blood and grafts from partially matched family donors provide graft alternatives. 41-45

Hematopoietic-cell transplant grafts are large-volume marrow aspirates (harvests). For adults, but not children, filgrastim-mobilized peripheral-blood stem cells have largely replaced marrow. Numerous randomized trials have shown no overall survival advantage associated with the use of peripheral-blood stem cells, and one large trial involving unrelated donors showed similar rates of survival but higher rates of chronic graft-versus-host disease (GVHD) with grafts of mobilized peripheral-blood stem-cells. 46

Complications of Allotransplantation

Early in the period after hematopoietic-cell transplantation, the risks of mucositis, veno-occlusive disease, interstitial pneumonitis, and infection predominate. 47 Acute and later chronic GVHD are major hazards that are not related to relapse. Excessive immunosuppression to limit GVHD can magnify the risks of opportunistic infection (e.g., reactivation of Epstein–Barr virus infection and lymphoproliferative disease) and recurrence of leukemia. 48 Acute or chronic GVHD may augment graft-versus-leukemia protection against relapse of leukemia, but more severe GVHD does not enhance antitumor effects.

A relapse of AML is the major complication. Factors dictating the risk of relapse are the biologic characteristics of the AML and the degree of detectable residual leukemia. High-risk cytogenetic and molecular subgroups, therapy-related AML, AML after a myelodysplastic syndrome or myeloproliferative neoplasms, or hematopoietic-cell transplantation after the first complete remission all increase the risk of relapse. Pretransplantation consolidation therapy may not reduce the risk of relapse, but minimal residual disease that is detectable before transplantation may increase the risk. 49

Relapse after Transplantation

The relapse of leukemia, particularly early after transplantation, is challenging to manage. 50 Some patients receive reinduction, either alone or supplemented with additional donor lymphocytes that were not immunologically tolerant to the recipient, in order to augment graft-versus-leukemia effects. Reinduction can yield durable remissions in a selected 20 to 30% of patients.

The rate of survival after relapse is poor, except among patients with a relapse 1 or more years after transplantation. For a minority of patients, a second allotransplantation during remission can extend leukemia-free survival.

New Approaches to Improving Outcomes of Transplantation

Limiting the risk of relapse and reducing the effects of GVHD in the peritransplantation period are both essential. New investigational antileukemic approaches include post-transplantation maintenance therapy (e.g., azacitidine) 51 and specific mutation inhibitors (e.g., the FLT3-tyrosine kinase inhibitor). Targeted therapy with immunotoxins (such as gemtuzumab ozogamicin), targeted radioantibody therapy, and total marrow irradiation 52 to augment pretransplantation myeloablation have been explored. Cytomegalovirus reactivation can induce sustained antileukemic activity, but its mechanism and the method for inducing it are uncertain. 53 Similarly, in spite of retrospective observations, data are lacking to better define the mechanism by which the donor killer-cell immunoglobulin-like receptor (KIR) genotype can limit post-transplantation relapse of AML. 54 Supplementation of post-transplantation treatment with antileukemia antibodies, synthetic bispecific T-cell engagers, or vaccines targeting leukemia-associated WT1 or PR1 antigens are under study.55

Treatment for Patients Who Are Ineligible for Intensive Therapy

The treatment of older or frail patients with AML includes best supportive care (including hydroxyurea), low-dose cytarabine, and, more recently, the hypomethylating agents decitabine and azacitidine (Table 3). Currently, no widely accepted algorithm provides treatment guidelines for older patients who cannot receive intensive chemotherapy. In clinical practice, the patient’s age, general health, and specific coexisting conditions, as well as the disease features, the patient’s wishes (and those of the patient’s relatives), and the physician’s attitude and interest all influence decision making.

Low-dose cytarabine induces responses in 15 to 20% of patients, but median survival is only 5 to 6 months. Systematic attempts to improve on this outcome (e.g., with the “pick a winner” program 56 of the Medical Research Council–NCRI AML Group, which involves serial testing of investigational compounds with low-dose cytarabine) have so far failed.

The hypomethylating agents may have promise. Both decitabine and azacitidine have been studied in phase 3 trials. 57,58 In an unplanned survival analysis, the use of decitabine, as compared with treatment chosen by the patient and physician (usually low-dose cytarabine), was associated with a survival advantage (median, 7.7 months vs. 5.0 months). 57 On the basis of this increase in survival, the European Medicines Agency, but not the U.S. Food and Drug Administration, granted approval for the use of decitabine for the treatment of older patients with AML. The AZA-AML-001 trial compared azacitidine with three conventional care regimens (i.e., low-dose cytarabine, intensive chemotherapy, or supportive care only). 58 The median survival was longer with azacitidine than with the conventional care regimens (10.4 months vs. 6.5 months), but the between-group difference was not significant.

Treatment of Relapsed and Primary Refractory AML

Disease recurrence occurs in most patients with AML within 3 years after diagnosis. A short duration of remission (i.e., <6 months), adverse genetic factors, prior allogeneic transplantation, older age, and poor general health status are major determinants of outcome after relapse. At relapse, the major question is whether a patient is physically able or unable to receive intensive salvage therapy. The decision-making process should always be in keeping with the patient’s goals.

Data are scarce with respect to controlled trials involving patients with relapsed or primary refractory AML. 59 Commonly used intensive salvage regimens aim at achieving a complete remission so that the patient can subsequently undergo allogeneic hematopoietic-cell transplantation (Table 3). Usually, the only treatment options for patients who are physically unable to receive intensive salvage therapy are low-intensity therapy or best supportive care. Given the poor outcome after these conventional care regimens, patients who are physically unable, as well as those who are physically able, to receive intensive salvage therapy should have the option of declining treatment or, if they wish, receiving new investigational therapies.

NEW THERAPIES

New compounds in the treatment of AML target a variety of cellular processes such as signaling through tyrosine kinases or other pathways, epigenetic regulation of DNA and chromatin, nuclear export of proteins, and antigens that are expressed on hematopoietic cells or, more specifically, on leukemic stem cells by antibody-based therapy.

The frequent occurrence of mutations in receptor tyrosine kinase genes (FLT3 and KIT) has generated interest in the development of tyrosine kinase inhibitors. Results with first-generation FLT3 inhibitors so far have been disappointing. 69 When used as single agents, these inhibitors lead to only transient reductions in blast counts. Other drawbacks include toxicity due to their nonselectivity for FLT3 and the development of FLT3 resistance mutations. The results of a recent randomized trial of sorafenib that involved 267 younger adult patients irrespective of their FLT3 mutational status suggested a beneficial effect of the kinase inhibitor on event-free survival but no significant effect on overall survival. 63 A trial evaluating the use of standard chemotherapy with or without midostaurin as front-line therapy in 717 patients with FLT3 mutations is under way (NCT00651261). Initial data from studies of second-generation FLT3 inhibitors suggest higher potency, but phase 3 trials have only started.

The presence of frequent mutations in genes involved in DNA methylation and chromatin modification, as well as the identification of new epigenetic targets by global proteomic approaches and functional screens, have informed another exciting and rapidly expanding therapeutic area — the development of new epigenetic therapies. 70,71 One promising new targeted approach is the inhibition of the mutant metabolic enzymes IDH1 and IDH2, which are frequently mutated in AML. 73 AG-120 and AG-221 are oral inhibitors of IDH1 and IDH2, respectively. In phase 1 trials, they have shown encouraging activity by triggering terminal differentiation of leukemic blasts in AML with IDH mutations. 61

Besides addressing mutant proteins directly, investigators have shown increasing interest in targeting mutation-specific dependencies. For example, by using a functional-genomics screen, Chan et al 74 showed that survival of IDH1-mutated and IDH2-mutated cells was highly dependent on antiapoptotic B-cell CLL–lymphoma 2 protein (BCL2) expression. Consistent with this finding, IDH1-mutated and IDH2-mutated AML cells were more sensitive to the BCL2 inhibitor venetoclax (also called ABT-199 or GDC-0199); this provided the basis for combinatorial therapy. Another example is the identification of BRD4, a member of the bromodomain and extraterminal (BET) family of bromodomain epigenetic readers, as a potential therapeutic target in AML70; BET bromodomain inhibitors such as OTX015 are in clinical development. 62

SGI-110, a second-generation hypomethylating agent, is a dinucleotide of decitabine and deoxyguanosine that increases the in vivo exposure of decitabine by protecting it from inactivation by cytidine deaminase. 60 A phase 3 trial of this compound in older patients who are not candidates for intensive therapy is under way.

Inhibition of chromosome region maintenance 1 (CRM1), the major nuclear export receptor, is another promising approach. High expression of CRM1 is associated with short survival in AML. 75 A pivotal study in which selinexor, a new CRM1 inhibitor, 64 is being compared with specified investigator choices in older patients with relapsed or refractory AML is ongoing (NCT02088541).

New formulations of classic cytotoxic agents are also being developed. Vosaroxin, a new anticancer quinolone derivative, inhibits topoisomerase II. A pivotal study evaluated intermediate-dose cytarabine with or without vosaroxin in 711 patients with relapsed or refractory AML. Although the primary end point was not reached in the trial, in a prespecified subgroup analysis, a significant survival benefit was seen among patients 60 years of age or older who received cytarabine with vosaroxin (7.1 months vs. 5.0 months). 66 CPX-351 is a liposomal formulation of cytarabine and daunorubicin packaged at a 5:1 molar ratio within liposomes that are 100 nm in diameter. Results from a phase 2 study 67 suggest a clinical benefit, especially among patients with secondary AML; a pivotal phase 3 trial is under way.

Finally, antibody therapy for AML is undergoing a renaissance. 72 Current activities focus on the development of new monoclonal antibodies targeting CD33, either with the use of antibody–drug conjugates or bispecific antibodies (anti-CD33 and CD3). Another strategy aims at targeting antigens such as CD123, the transmembrane alpha chain of the interleukin-3 receptor, that are preferentially expressed on leukemic stem cells. CD123 is currently also under investigation as a target for chimeric antigen receptor T-cell–engineered cellular therapy. 76 Another interesting target for chimeric antigen receptor T cells is the expression of folate receptor β. 77

Exciting developments in our understanding of the molecular pathogenesis of AML have not yet been translated into clinical practice. New compounds hold promise to improve treatment outcomes; however, it is unlikely that any of these compounds, when used as single agents, will cure the disease. A major challenge will be to identify predictors for a response to specific agents, which will allow for the rational design of combinatorial therapies.