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The Oncologist, Vol. 12, No. 3, 341-355, March 2007; doi:10.1634/theoncologist.12-3-341
© 2007 AlphaMed Press

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 Prognostic Factors and Risk-Based Therapy in Pediatric Acute Myeloid Leukemia
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Pediatric Oncology

Prognostic Factors and Risk-Based Therapy in Pediatric Acute Myeloid Leukemia

Soheil Meshinchia, Robert J. Arcecib

aFred Hutchinson Cancer Research Center, University of Washington, Department of Pediatrics, Division of Clinical Research, Seattle, Washington, USA; bPediatric Oncology, Sidney Kimmel Comprehensive Cancer Center at Johns Hopkins, Baltimore, Maryland, USA

Correspondence: Robert J. Arceci, M.D., Ph.D., Pediatric Oncology, Sidney Kimmel Comprehensive Cancer Center at Johns Hopkins, Bunting-Blaustein Cancer Research Building, 1650 Orleans Street, Room 2M51, Baltimore, Maryland 21231, USA. Telephone: 410-502-7519; Fax: 410-502-7223; email: arcecro{at}jhmi.edu

Received July 19, 2006; accepted for publication January 17, 2007.


   LEARNING OBJECTIVES
Top
 Learning Objectives
Abstract
 Introduction
 Prognostic Factors
 Conclusions
 Disclosure of Potential...
 References
 
After completing this course, the reader will be able to:

  1. Identify specific favorable and high-risk factors in pediatric AML.
  2. Discuss the rationale and indications for the use of stem cell transplantation in pediatric AML.
  3. Describe new technologies and emerging molecular prognostic markers in pediatric AML.

Access and take the CME test online and receive 1 AMA PRA Category 1 CreditTM at CME.TheOncologist.com


   ABSTRACT
Top
 Learning Objectives
 Abstract
Introduction
 Prognostic Factors
 Conclusions
 Disclosure of Potential...
 References
 
Acute myeloid leukemia (AML) has posed significant therapeutic challenges to pediatric oncologists. Despite intensive therapy, half of the children with AML relapse and die from their disease. Efforts to identify risk factors in AML are directed toward defining populations who may benefit from alternative therapies. Patients at lower risk for relapse may benefit from treatment de-escalation, sparing them adverse side effects. Management of high-risk patients may prove more difficult, as the nearly myeloablative nature of AML therapy leaves little room for therapy escalation short of stem cell transplantation. This review evaluates prognostic factors in pediatric AML and discusses the feasibility of using these factors in risk-adapted therapy regimens.

Disclosure of potential conflicts of interest is found at the end of this article.


   INTRODUCTION
Top
 Learning Objectives
 Abstract
 Introduction
Prognostic Factors
 Conclusions
 Disclosure of Potential...
 References
 
Acute myeloid leukemia (AML) accounts for one fourth of the acute leukemias in children, but it is responsible for more than half of the leukemic deaths in this patient population. In contrast to the tremendous success in the treatment of acute lymphocytic leukemia (ALL) in the last three decades, resulting in a >80% cure rate, improvements in AML therapy have been limited, and only about half of the patients with AML are cured of their disease. Risk-adapted therapy has been the cornerstone of ALL therapy. High-risk patients are identified and their treatment "augmented," whereas therapy for the lower-risk patients can be de-escalated. Such a risk-based approach has led to >90% event-free survival in low-risk patients and significant improvements in outcome in traditionally high-risk patients. The underlying reason for the success of this approach in ALL is that standard ALL induction and consolidation are amenable to additional intensification without causing significant morbidity. In AML, the leukemic stem cell is inherently drug resistant, and improvements in outcome have been generally associated with escalation of induction therapies to maximally tolerated doses in conjunction with intensive supportive care measures.

Limited biological stratification of AML has been successful in developing highly effective therapies, including all-trans retinoic acid (ATRA) and arsenic trioxide for patients with acute promyelocytic leukemia (APML) with the t(15;17) translocation resulting in expression of the PML/retinoic acid receptor alpha (RAR-{alpha}) fusion product. Similarly, the acute megakaryocytic leukemia (AMKL) of young children with Down syndrome shows a favorable response to standard intensive therapies.

Although prognostic factors have also been identified in pediatric AML, they have not been tested and used to the fullest extent. A major reason for the lack of use of prognostic markers has been, in part, the extreme intensity of AML therapy, with patients receiving near myeloablative chemotherapy and sometimes stem cell transplantation (SCT) for those with matched family donors.

Intensive AML induction therapy leaves little room for treatment intensification short of unrelated donor (URD) SCT. Furthermore, postrelapse treatment outcomes remain unsatisfactory despite intensive reinduction attempts and URD SCT. Because URD SCT is associated with significant long- and short-term toxicities, such intensive therapy is currently reserved for patients with relapsed disease or primary induction failure. In considering the use of URD SCT in the high-risk population, one must weigh the toxicity of the transplantation against the risk for relapse and establish an outcome threshold where patients whose outcome is anticipated to fall below that threshold are referred for URD SCT. Identification of additional patient groups at extremely high risk for relapse may justify the use of URD SCT in this patient population.

Alternatively, favorable prognostic markers may identify patients who can then be spared myeloablative therapy. The outcome for patients with "favorable" cytogenetic markers remains in the 60% range with current therapies, which is not high enough to justify therapy de-escalation. Thus, while prognostic factors have been identified in AML, there is still a great deal of controversy as to how best to use them for treatment stratification in most instances.


   PROGNOSTIC FACTORS
Top
 Learning Objectives
 Abstract
 Introduction
 Prognostic Factors
Conclusions
 Disclosure of Potential...
 References
 
Overview
Prognostic factors include host factors, response to therapy, as well as disease characteristics. These factors are generally interdependent, the sum of which ultimately determines disease response and patient outcome. In addition, prognostic factors may change as treatment changes, thus necessitating the evaluation of all established and putative prognostic markers within the framework of a defined therapy. This article reviews the established prognostic factors and discusses novel prognostic markers that are emerging as significant predictors of disease outcome in pediatric AML.

Host Factors
Host factors, such as gender, age, race, and constitutional abnormalities, have been associated with outcome in pediatric patients with AML. For example, while female patients may do slightly better than male patients, this association has not been strong enough to include in therapeutic stratification. Similarly, different reports have indicated varying outcomes for infants with AML, but most current studies treat infants in a fashion similar to older children, with equal or slightly better outcomes [1, 2]. However, racial variation does seem to influence clinical outcome, as non-Caucasian patients appear to have a significantly worse outcome than Caucasians despite the same chemotherapy. In a recent study of nearly 1,600 children treated in the Children's Cancer Group (CCG) 2891 and 2961 trials, Aplenc et al. [3] demonstrated that African-American and Hispanic patients had an overall survival rate of approximately 35%, compared with a survival rate of 48% for Caucasian patients. Constitutional trisomy 21 has been shown to impact the outcome of children with AML. AML patients with Down syndrome, particularly those under 2 years of age, appear to be extremely sensitive to cytotoxic therapy, as they show greater toxicity as well as a better outcome with less-intensive therapy, with a remission rate of 90% and an overall survival rate of 80%. These patients are now treated with intensity-reduced, alternative approaches [4–7].

Variance from an ideal body weight in AML patients at the time of diagnosis has recently been shown to impact clinical outcome. Lange et al. [8] looked at the survival rates of children with AML who were either underweight (≤10th percentile) or overweight (>95th percentile) at diagnosis and compared their clinical outcome with that of the remainder of the patients in the study. In comparison with the middleweight patients, underweight or overweight patients had a nearly twofold higher risk for mortality. They demonstrated that excess treatment-related mortality led to worse survival in both underweight and overweight patients [8].

Additional host factors are being studied that might influence response to therapy and/or toxicity in terms of pharmacologic metabolism of drugs. For example, inherited alterations in the detoxification enzyme, glutathione S-transferase theta (GST-{theta}), that result in a null phenotype are associated with a significantly shorter survival duration as a result of excess toxicity [9]. This association of toxicity with GST genotype is likely associated with the specific drug combinations used in different AML therapy platforms.

Response to Therapy
Response to therapy has been an important predictor of clinical outcome in leukemias. Historically, response to therapy has been measured by the morphologic presence of disease at defined periods after the start of induction therapy. In addition, the presence of disease below the level of morphologic detection has been evaluated. We review induction failure as well as the presence of minimal residual disease (MRD) as a means of identifying high-risk patients.

Primary Induction Failure
Studies evaluating the morphologic presence of disease have shown that such patients have a dismal outcome even if they are reinduced into remission [2, 10]. The Medical Research Council (MRC) studies methodically evaluated the role of response to therapy as part of the MRC 10 AML trials, in which they demonstrated that patients with >15% marrow blasts prior to the start of the second induction course had a significantly worse outcome than those with <15% disease [2, 11]. They established this clinical cutoff by demonstrating that patients with partial remission (5%–15% blasts) at the end of induction had a survival rate rather similar to those with <5% blasts, whereas the survival for those with 15%–20% blasts was poor and mirrored the outcome for those with >20% marrow blasts. Based on these findings, the MRC used the threshold of 15% to define refractory disease. Many clinical trials, including the Children's Oncology Group (COG) pediatric AML trials, are now using the threshold of 15% to define primary induction failure (PIF). AML patients with PIF have an extraordinarily poor prognosis, and current COG approaches are studying URD SCT in this very high-risk group.

MRD
Because morphologic disease response has been shown to be such a powerful prognostic factor, the role of disease persistence below detection at the morphologic level (MRD) has been evaluated as a prognostic factor in AML.

More than 80% of pediatric patients with AML who undergo induction therapy achieve complete remission (CR), as assessed by morphologic evaluation of the marrow at the end of induction therapy. However, nearly half of these patients are destined for relapse and poor outcome. Identification of occult disease in patients in morphologic remission may identify patients at high risk for impending relapse. Appropriate intervention in this group of patients could potentially prevent morphologic relapse and be more effective. Despite its potential in risk management in AML, the clinical utility of MRD, which represents an in vivo measure of response to therapy, is related to several factors. First, MRD should have general applicability and be able to identify a significant proportion of patients at risk for relapse. Second, there should be adequate time from the detection of MRD to morphologic relapse to allow for intervention. And, most importantly, therapy of MRD must lead to a better outcome, otherwise the detection of MRD is clinically meaningless.

Molecular MRD
The majority of the data on the detection of MRD in AML has been generated using polymerase chain reaction (PCR)-based methods in which detection of unique fusion genes has been correlated with morphologic relapse [12–15]. In these studies reverse transcription (RT)-PCR was used to detect MRD in patients with specific cytogenetic abnormalities [acute myeloid leukemia with t(8;21), inv(16), or t(15;17)] and correlate the presence of occult disease with morphologic relapse. The only AML subtype for which utility of MRD has been conclusively demonstrated to be of clinical utility is APML characterized by the t(15;17) fusion product PML/RAR-{alpha}. In APML, detection of persistent t(15;17) fusion product is significantly associated with a high risk for relapse, and early therapeutic intervention, prior to morphologic relapse, has been shown to improve outcome [16, 17]. In contrast, the t(8;21) translocation-generated fusion product may not only be present in the general population [18], but may remain positive by PCR for many years in patients with AML in morphologic remission [19]. Thus, the mere detection of an abnormal transcript may not be clinically meaningful. More recent studies, using real-time quantitative PCR have proven to be more important in the identification of clinically relevant MRD. Schnittger et al. [20] demonstrated that the expression level of the abnormal transcript for t(8;21) and inv(16) at the time of diagnosis, as well as the rate of decline in the transcript, may predict clinical outcome. In addition, patients with increasing transcript levels are at extremely high risk for relapse. The question of whether therapeutic intervention in the context of molecular MRD in core binding factor (CBF) leukemias improves clinical outcome needs be addressed.

Immunophenotypic MRD
Leukemic blasts usually express aberrant surface antigen patterns that differ from the pattern observed in normal progenitors. This difference has been exploited to develop flow cytometric–based MRD assays with which the presence of one cell with a leukemic immunophenotype can be detected in 1,000–10,000 normal nucleated cells [21]. The advantage of flow cytometry over PCR-based technology is that it is applicable to most patients with AML. Recent studies have evaluated the utility of multidimensional flow cytometry to detect disease presence in patients in morphologic remission and correlated the presence of MRD with clinical outcome. In an evaluation of 126 adult AML patients in clinical remission, San Miguel et al. [22] used flow cytometry to determine the presence of MRD. They demonstrated that patients with occult disease detected by flow cytometry had a significantly greater risk for relapse than patients without occult leukemia. In a CCG study of 252 pediatric patients with AML in morphologic remission, Sievers et al. [23] established flow cytometric detection of MRD as a viable means of identifying patients at high risk for relapse. In that study, 16% of the patients in CR were identified as having occult disease by flow cytometry. These patients had a fivefold higher risk for relapse than the MRD-negative patients, with a relapse-free survival rate from remission of 35%, compared with 65% for the MRD-negative patients. In a multivariate analysis, flow cytometric detection of MRD showed the strongest correlation with relapse-free survival. That study thus demonstrated that flow cytometry can be used to screen for occult disease in pediatric AML, and that patients with MRD are at high risk for relapse. More importantly, in that study, the median time to relapse for the MRD-positive population was 173 days, more than adequate for intervention. In contrast, results from a Berlin-Frankfurt-Muenster (BFM) report have shown that MRD detection did not provide prognostic information additional to that of the more traditional risk factors [24]. The question of how to optimally manage such MRD-positive patients, however, has not been resolved. There are currently no data to suggest whether intervention in MRD-positive patients would alter their overall clinical outcome.

The utility of flow-based MRD is being prospectively evaluated as part of clinical trials being conducted through a St. Jude Children's Research Hospital consortium and by COG. In ALL, a reduction in MRD has been shown to generally parallel other risk factors [25]. However, the genetic context of MRD may be critical, as a significant fraction of ALL patients with favorable trisomies have MRD at the end of induction. The potentially very important role of the genetic context of MRD in AML is under current investigation in the COG-AAML03P1 trial.

Disease Characteristics
Historically, characteristics believed to be inherent to leukemia have included factors such as diagnostic WBC, morphologic classification (French American British [FAB] subtype), and biological characteristics such as cytogenetics. More recently, with the advances in molecular diagnostics and genomic and proteomic profiling, disease classification has expanded significantly. Diagnostic WBC has been demonstrated to be a prognostic factor in AML. A WBC <20,000 cells/ml3 has been associated with a better prognosis, and a WBC of >100,000 cells/ml3 has been linked to an unfavorable outcome [26]. Diagnostic WBC has been shown to be a continuous variable for outcome, as an increase in the WBC is associated with an incremental decline in outcome. Such a continuous variable has thus far been difficult to incorporate into risk stratification strategies in AML, as many molecular events that mediate myeloid leukemogenesis lead to leukocytosis (i.e., FLT3/internal tandem duplication [FLT3/ITD]). Thus, identification of the underlying biological mechanisms responsible for leukemic proliferation and survival characteristics leading to leukocytosis should provide a more definitive means of assessing the risk for treatment failure.

Historically, AML has been classified based on morphologic appearance using the FAB classification. Because of the subjective nature of such a classification and lack of uniformity or correlation with underlying biology, the World Health Organization (WHO) recently developed a system for comprehensive AML classification based on cytogenetics, disease biology, and clinical history (Table 1). Scrutiny of the FAB subtypes (e.g., FAB M6 and M7) that have traditionally been associated with poor outcome has revealed that high-risk cytogenetics are significantly overrepresented in those with FAB M6/M7, and that the prognostic significance of these subtypes may be a result of the predominance of cytogenetic groups associated with poor outcome. The WHO classification schema relies mainly on recurrent cytogenetic alterations and clinical history for AML classification [27].


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  		Table 1. WHO classification of acute myeloid leukemia

 

Cytogenetics
Diagnostic cytogenetics is widely recognized as one of the most significant prognostic factors in AML. Informative cytogenetics are usually available in 70%–80% of pediatric patients with AML, and clonal abnormalities are demonstrated in nearly 80% of those with informative cytogenetics [28–30]. The prognostic significance of karyotypic abnormalities has been evaluated retrospectively in several trials, and specific favorable and unfavorable subgroups of AML have been identified. We discuss specific favorable and unfavorable cytogenetic markers and their clinical impact across several pediatric trials.

Favorable, Cytogenetics. t(8;21) and inv(16) are two of the most commonly identified translocations in pediatric AML and are at the core of the WHO classification system. Together, these leukemias are often referred to as CBF leukemias because the AML1/ETO fusion produced by the t(8;21) and the CBF-{alpha}–MYH11 fusion produced by the inv(16) both disrupt CBF [31, 32]. Clinical trials conducted by the Cancer and Leukemia Group B (CALGB) in adults indicated that patients with CBF AML had superior outcomes when compared with patients with all other types of AML, and had a particularly good outcome when treated with high-dose cytarabine [33]. The presence of t(8;21) or inv(16) has also been associated with longer survival in pediatric patients [34]; however, available data suggest that there may be a difference in outcome between patients with t(8;21) and inv(16) in different clinical trials. MRC AML clinical trials of children and adults demonstrated that 17% of the pediatric patients had CBF leukemia (12% with t(8;21) and 5% with inv(16)) [1, 35]. Patients with t(8;21) and inv(16) had significantly better remission induction and overall survival than patients with the normal karyotype. However, this difference was less marked in the pediatric population, as shown in the MRC 10 trial, in which the relapse-free survival rate from remission for the "good risk" CBF group was similar to that of the patients in the "standard risk" group [36].Go


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  		Table 2. Potential risk factors for pediatric acute myeloid leukemia

 
The prognostic significance of CBF leukemias has been evaluated in both Pediatric Oncology Group (POG) and CCG clinical trials, with somewhat different results. The clinical significance of cytogenetic abnormalities in 478 children with pediatric AML was evaluated in the POG 8821 study [28]. Patients with t(8;21) and inv(16) had a significantly better remission induction rate than patients with the normal karyotype. However, the postremission outcome was different between patients with inv(16) and those with t(8;21). Patients with inv(16) exhibited an overall survival rate of 75%, compared with 54% for patients with the normal karyotype. In contrast, patients with t(8;21) had a higher relapse rate, which was not different from that of patients with the normal karyotype. However, the salvage rate for patients with t(8;21) was significantly higher than for those with the normal karyotype, leading to longer overall survival. The recent CCG 2961 study further demonstrated that, among 98 patients in first CR (CR1) with t(8;21) or inv(16), outcomes in those without and with a donor were not different (disease-free survival rate, 61% versus 72%; p = .49 and overall survival rate, 78% versus 77%; p = .85) [8]. Such data have given support to withholding SCT in patients with CBF AML in CR1, and to reserving transplantation in case of relapse.

APML is currently the most curable form of AML, with cure rates of 70%–90% in children and adults [37]. The underlying t(15;17) translocation in APML, which leads to formation of the PML/RAR-{alpha} transcript, causes maturational arrest in the promyelocyte stage. This maturation arrest can be overcome with pharmacologic doses of ATRA. Because of its sensitivity to differentiation therapy with ATRA, APML is treated differently from other AML subtypes, with excellent outcomes [38–40].

Mixed lineage leukemia (MLL) rearrangements have been implicated in myeloid leukemogenesis, and their clinical significance has been evaluated in numerous trials [29, 41, 42]. Although in earlier studies the presence of 11q23 abnormalities was associated with an unfavorable outcome, more contemporary studies have not shown such a prognostic significance [29, 35]. Evaluation of 42 patients with 11q23 treated in St. Jude Children's Research Hospital trials in 1980–1997 showed no overall difference in the outcome of patients with or without 11q23. However, within the 11q23 population, patients with t(9;11) had a significantly better overall and event-free survival rate than patients with the normal karyotype or other 11q23 abnormalities [30, 43]. However, larger MRC, CCG, or POG studies have not demonstrated similar findings in patients with t(9;11) leukemias [28, 35]. Further evaluation is needed regarding this cytogenetic subtype.

Unfavorable Cytogenetics
Karyotypes associated with poor outcome have been identified in a small proportion of pediatric patients with AML. Abnormalities of chromosome 5 and 7 have been associated with poor outcome in pediatric AML [2, 28, 35]. The MRC 10 study, which evaluated adult and pediatric patients for risk groups, initially identified chromosome 3 abnormalities as a high-risk karyotype [35]. Subsequent evaluation of pediatric patients demonstrated that while -7 and del/-5 are significant predictors of poor outcome in pediatric AML, in contrast to adult AML, an abnormal chromosome 3 does not carry prognostic significance in children [44]. The prevalence of these abnormalities is in the range of 2%–4%, and collectively they account for <10% of patients at high risk for a poor outcome. These patients have a lower rate of remission induction and a worse overall outcome. MRC studies have confirmed these abnormalities as markers of poor outcome, with a remission induction rate of approximately 50%, a high relapse rate, and an overall survival rate of <20% [2, 35, 36, 44]. Other uncommon, recurrent cytogenetic abnormalities, such as t(6;9), have been associated with poor outcome. However, because of its low prevalence and significant association with other molecular abnormalities (i.e., FLT3/ITD), the true prognostic significance of t(6;9) has not been clearly established in children.

The presence of complex cytogenetics has been associated with worse outcome in AML. Some classification systems define complex karyotype as the presence of five or more abnormalities [35, 45], whereas others use three or more abnormalities [46–48]. A majority of the data on the prognostic significance of complex cytogenetics has been derived from adult studies, with scant published data on pediatric patients. Available data suggest that complex cytogenetics are highly associated with specific high-risk karyotypes, where a significant proportion of those with defined complex cytogenetics have chromosome 5 or 7 abnormalities [49]. In the Farag et al. [49] study cohort, of the 94 patients with at least five abnormalities, 75 had -5/5q- and 48 had -7/7q- abnormalities, with no patients with CBF AML. Other studies have demonstrated that the favorable outcome of those with CBF AML is not diminished in the presence of complex cytogenetics [50], supporting the notion that the presence of specific translocations, and not the number of translocations, may define clinical outcome. This is a particularly important distinction to make in pediatric patients, because the prevalence of complex karyotypes is lower and the prevalence of favorable cytogenetics is higher than in adult patients. As those with complex cytogenetics are likely to be enriched with high-risk karyotypes (-5/del5q or -7/del7q), it is not clear whether the presence of multiple abnormalities in the absence of specific high-risk karyotypes has prognostic significance. Recent evaluation of CCG 2961 data failed to demonstrate prognostic significance for the presence of at least five karyotypic abnormalities in those with standard risk AML (T. Alonzo, unpublished data).

Larger numbers of patients with subtypes of AML characterized by specific chromosomal alterations and treated with uniform approaches may be required to prove smaller but still relevant outcome differences. Importantly, effective targeting of fusion proteins resulting from chromosomal translocations and/or downstream consequences of such abnormalities may eventually eliminate the question of their prognostic significance using conventional therapy.

Multidrug Resistance
Therapeutic resistance is a major obstacle in the treatment of AML. Such resistance has been associated with rapid drug efflux mediated by the multidrug resistance gene 1 (MDR1) encoding P-glycoprotein (Pgp) as well as expression of other proteins conferring MDR, such as the MDR-associated protein 1 (MRP1) and lung resistance protein (LRP). It is expected that expression levels of genes that mediate drug resistance may correlate with response to chemotherapy and clinical outcome. Numerous studies have evaluated the prognostic significance of expression of MDR genes with varying conclusions [51–54]. Early studies demonstrated significant prognostic significance to the expression of MDR genes [55–57], wherein those whose leukemic blasts had a high expression level of MDR genes were more resistant to chemotherapy and had a worse survival. However, more comprehensive evaluation of the prognostic significance of MDR expression in the context of contemporary, intensive chemotherapy protocols failed to demonstrate independent prognostic significance to MDR expression when evaluated in the context of other adverse prognostic factors such as cytogenetics [53]. These studies have demonstrated that MDR genes are highly expressed in older patients and those with high-risk cytogenetics, thus, not providing additional, clinically useful prognostic information. Evaluation of MDR genes in pediatric patients also failed to demonstrate prognostic significance [52]. Sievers et al. [52] demonstrated a prevalence of 13% for the expression of PgP in a group of 130 pediatric AML patients treated in the CCG 2891 trial. However, the clinical outcomes of those with and without PgP expression were not different. Additional pediatric studies have demonstrated that MDR-1 expression is not higher overall in patients with relapsed AML [58]. Although MDR expression may not be an independent prognostic factor, it may be a useful therapeutic target in the management of AML. Several agents have been shown to impair the function of proteins encoded by MDR genes, which may potentially sensitize the cells to the therapeutic effects of the specific chemotherapy agents [59–61]. In combination with conventional chemotherapy, such agents may augment response to chemotherapy and improve survival.

Molecular Abnormalities

Receptor Tyrosine Kinase Mutations
Receptor tyrosine kinases (RTKs) and their downstream effectors (RAS, Janus kinase/signal transducer and activator of transcription [JAK/STAT]) have emerged as significant components in the pathogenesis of a variety of cancers [62, 63]. Whether it is the constitutive activation of the receptor by an intrinsic receptor mutation (FLT3, c-KIT, and c-Fms mutations) [64–66], the autocrine/paracrine stimulation of the receptor by a ligand secreting tumor (vascular endothelial growth factor [VEGF] receptor) [67, 68], or the activation of the downstream effectors (e.g., RAS) [69–71], such activating events directly contribute to disease pathogenesis, progression, and resistance to chemotherapy. Mutations of the FLT3 and c-KIT receptor genes are the most common RTK mutations in AML and are addressed specifically below.

FLT3 Mutations. Mutations in the FLT3 receptor gene have been demonstrated to be the most common genetic alteration in AML thus far identified. FLT3/ITD as well as the FLT3 activation loop mutation (FLT3/ALM) lead to constitutive activation of the receptor kinase (Fig. 1) [72–74]. Furthermore, these mutations have been implicated in rapid disease progression and resistance to conventional therapy [72, 75–77]. FLT3/ITD has a prevalence of 12%–15% in pediatric patients [75, 78], 20%–25% in young adults [77], and nearly 35% in the older adult population [79].


Figure 1
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  		Figure 1. FLT3 receptor structure. The FLT3 receptor is composed of an immunoglobulin-like extracellular domain (EC), a transmembrane domain (TM), a juxtamembrane domain (JM), and two tyrosine kinase (TK) domains. Mutations of the FLT3 receptor gene have been identified in the JM domain (FLT3/ITD) and in the TK domain (FLT3/ALM).

 
Initial studies correlated the presence of FLT3/ITD with significant leukocytosis and demonstrated a strong prognostic significance for the presence of FLT3/ITD, in which patients that harbored this mutation had an extremely poor outcome compared with the patients without FLT3/ITD [75, 77]. In more recent studies using contemporary chemotherapy, the prognostic significance of FLT3/ITD has been less dramatic, with an overall survival rate of approximately 30% for the FLT3/ITD population compared with 45% for patients without FLT3/ITD [78, 80–82]. However, these studies also identified a subclass of patients with FLT3/ITD in whom the ratio of ITD to wild-type allele (ITD-allelic ratio [AR]) correlates with clinical outcome. Several studies have now shown that the proportion of the ITD to wild-type PCR product varies significantly from patient to patient, and this difference may have clinical implications. For example, some patients have a higher predominance of mutant ITD product (high ITD-AR), whereas others have a higher proportion of normal product (low ITD-AR). The ITD-AR has been used to identify patients with FLT3/ITD at a higher risk for relapse and poor outcome in a number of clinical trials [81, 83, 84]. Thiede et al. [81] used the ITD-AR to define relapse risk. Patients with an ITD-AR less than the median (≤0.78) had an overall survival rate of nearly 60%, compared with an overall survival rate of 0% in patients with an ITD-AR >0.78 (p = .006). They also suggested that the ITD-AR may be a continuous variable, because changing the ITD AR cutoff altered the clinical outcome. We confirmed the utility of the ITD-AR in a pediatric population treated on samples from AML BFM studies. Patients with a high ITD-AR (greater than the median of 0.69) had a poor outcome, whereas the outcome for those with a low ITD-AR (≤0.69) was not different from that of patients without FLT3/ITD [83]. More recent evaluation of over 630 patient samples from the CCG 2961 study established an ITD-AR threshold of 0.4 as a clinically meaningful threshold that accurately distinguishes FLT3/ITD-positive patients that are at high risk for relapse from those who are expected to do well. In that study, those with FLT3/ITD with a high ITD-AR (>0.4) had a relapse rate of nearly 80% and a survival rate of <20%, compared with the patients with a low ITD-AR, or no FLT3/ITD (Fig. 2) [80]. This threshold was further validated in a European cohort, demonstrating that an ITD-AR of 0.4 can be used more broadly for risk identification in FLT3/ITD-positive patients.


Figure 2
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  		Figure 2. Prognostic significance of FLT3 mutations in pediatric AML: progression-free survival from study entry for patients with FLT3/ITD with a high or low ITD-AR, FLT3/ALM, and FLT3/WT is demonstrated. The only population with poor outcome are those with FLT3/ITD with a high ITD-AR (>0.4). Abbreviations: ALM, activation loop mutation; AR, allelic ratio; ITD, internal tandem duplication; WT, wild type.

 
Recent identification of an additional activating mutation of the FLT3 gene has further increased the interest in FLT3 as a prognostic marker. This mutation is a missense mutation in the activation loop domain of the FLT3 gene (FLT3/ALM) at codons 835/836, which leads to the constitutive activation of the kinase domain [74]. These mutations have been identified in approximately 7% of adult and pediatric patients with AML. Despite being an activating mutation, FLT3/ALM does not appear to have prognostic significance in AML [74, 81, 85]. We have demonstrated that FLT3/ALM is present in pediatric AML with the same frequency as reported in the adult population [78, 85] and that FLT3/ALM-positive patients have a lower diagnostic WBC, higher remission induction rate, and better overall survival than patients with FLT3/ITD (Fig. 2). It is unclear why these two types of activating mutation involving the same receptor are associated with different AML phenotypes and patient outcome.

c-KIT Mutations. Activating mutations in the c-KIT receptor gene involve mutations in the juxtamembrane or the kinase domains of the receptor gene and lead to constitutive activation of the c-KIT receptor. Activating mutations of the c-KIT receptor gene have been identified in 2%–15% of myeloid malignancies [66, 86–90], including mastocytosis, myelodysplastic syndrome (MDS) and AML. More recently, c-KIT mutations have been demonstrated to be quite prevalent in CBF leukemias, with a prevalence of 40% in patients with t(8;21) or inv(16) AML [90]. Although initial studies did not demonstrate a prognostic significance for c-KIT mutations, separate evaluation of D816 mutations suggested that, in patients with CBF leukemia, those with D816 mutations have a significantly higher relapse risk than those without mutations [91–93]. More recent studies have provided additional data on the prognostic significance of KIT mutations in those with CBF, where relapse risk in those with KIT mutation was >60%, compared with <30% for those without mutations [94]. The prevalence of KIT mutations and their prognostic significance in pediatric AML remain to be established, although a report from Shimada et al. [93], in a relatively small number of children with CBF ALM, suggests an association between activating c-KIT mutations and poor outcome in children.

Small molecule inhibitors targeting RTK receptors have shown significant efficacy in vitro and in animal models; however, their use as single-agent therapy in relapsed AML has not resulted in significant responses [95–98]. Trials evaluating the utility of FLT3 inhibitors in combination with conventional chemotherapy in both relapsed AML as well as de novo AML are ongoing [95, 99–101]. Early data are promising, and it appears that FLT3 inhibitors may augment response to chemotherapy in those with FLT3 mutations.

Novel Molecular Markers
Investigations have been conducted to identify novel and more predictive markers of high-risk disease in AML in order to help in risk-based therapy. Such efforts have led to the identification of potential prognostic markers as well as the refinement and reassessment of a number of historic markers. New technologies and molecular tools have enabled investigators to evaluate biologically relevant markers for their role in disease response. Mutations in genes regulating critical pathways in hematopoiesis have been identified and their prognostic significance is under study. Novel mutations in the CCAAT/enhancer binding protein-alpha (CEBP-{alpha}) and nucleophosmin (NPM) genes have been identified in AML, which may have clinical implications. The presence of CEBP-{alpha} mutations, which modulate granulocytic differentiation and lead to maturational arrest, have been identified in nearly 10% of adult AML patients, and their expression has been associated with favorable outcome [102, 103]. The prevalence of CEBP-{alpha} mutations in pediatric AML is somewhat lower, and its clinical significance in children has not been clearly defined [104]. NPM, a nucleocytoplasmic shuttling protein with prominent nucleolar localization, regulates the ADP-Ribosylation Factor (ARF)-p53 tumor-suppressor pathway. Mutations in the NPM gene have been reported in AML that lead to the abnormal cytoplasmic localization of the affected protein [105]. NPM mutations have been reported in 30%–50% of adult AML patients [106], with a prevalence of approximately 10% in children [107]. Evaluation of the prognostic significance of NPM mutations suggests that the presence of NPM mutations correlates with favorable outcome in adult AML patients with the normal karyotype without FLT3/ITD [106, 108]. However, evaluation of the prevalence and prognostic significance of NPM mutations in children with AML treated in the CCG 2961 trial failed to demonstrate any prognostic significance for this mutation in pediatric AML [109].

In addition to such function-altering mutations, regulation of the expression level of various transcription factors may have biologic and prognostic significance. The expression level of the Wilms' tumor gene (WT1) has been implicated in the pathogenesis and prognosis of AML. Although the WT1 expression level at the time of diagnosis has been correlated with clinical outcome [110], such findings have not been uniformly observed [111]. However, more recently, it was demonstrated that patients with a high WT1 expression level at the end of induction had a worse clinical outcome, suggesting its utility as an MRD marker at the time of clinical remission [112]. Telomerase activity has been implicated in leukemogenesis, and there are data to suggest that telomerase activity may have prognostic significance in pediatric AML [113]. BAALC (brain and acute leukemia, cytoplasmic) is a gene whose elevated expression level has recently been associated with adverse outcome in adults with AML [114]. AF1q, an MLL fusion partner whose expression regulates hematopoietic differentiation, is differentially expressed in AML, and a high expression level of this gene has been shown to be associated with an undifferentiated phenotype and worse outcome [115, 116]. VEGF ligand expression has been shown to be elevated in leukemias, and early data suggest that high VEGF ligand expression (and subsequent autocrine/paracrine stimulation) may be associated with poor outcome [73]. Substantiation of all novel prognostic markers should be done in large, multicenter trials, and preferably analyzed prospectively, prior to their use in therapeutic planning and stratification. In addition, there is a growing need for evaluation of all putative prognostic markers in the same patient population in order to delineate overlap and possible interaction with prognostic factors, as well as with the type of treatment used.

Genomics and Proteomics in Risk Assessment
New technologies allowing the determination of gene- and protein-expression profiles have opened up an important era in refining the diagnostic subtyping of AML, identification of new prognostic factors, and drug development. DNA microarray analysis has allowed disease classification based on gene-expression profiling [117]. This technology has recently been successfully applied to predict outcome in adult malignancies [118–123]. Genomic classification of relapse risk is being applied to pediatric AML and early data are encouraging [124]. Lacayo et al. [125] used DNA microarray technology to evaluate relapse risk in a cohort of patients with pediatric AML with FLT3 mutations. They identified an expression profile that identified patients with FLT3 mutations and were further able to determine high-risk and low-risk subpopulations among the patients with FLT3 mutations. Furthermore, they were able to validate their microarray findings using quantitative RT-PCR, wherein they assigned relapse risk using the expression level of two genes previously identified by microarray profiling. Yagi et al. [124] used gene-expression profiling to evaluate diagnostic marrow specimens from 54 pediatric patients with AML. They identified 35 genes whose expression pattern correlated with clinical outcome. More recent studies in adult AML used microarrays to identify specific expression profiles that correlated with disease response and clinical outcome [126, 127]. Such studies have demonstrated that the clustering was primarily driven by the presence of chromosomal alterations. This finding highlights the significant impact of the underlying cytogenetic characteristics of the leukemia and their profound prognostic significance. Larger studies using gene-expression profiling for prognostic determination from pediatric cooperative group studies are required to establish the role of genomic profiling in risk identification in pediatric AML.

Future application of this technology may not only allow for prognostic determination in AML, but may identify specific therapeutic targets. With the elucidation of the human genome sequence and emerging data on epigenetic changes, the field of molecular medicine is also moving toward exploring the utility of the proteome. Proteomics allows for the identification of a protein-expression pattern just as genomics uses a gene-expression pattern. Although this field is in its early stages and data on clinical application are scarce, it remains a promising frontier in diagnostics and prognostics in AML.


   CONCLUSIONS
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 Learning Objectives
 Abstract
 Introduction
 Prognostic Factors
 Conclusions
Disclosure of Potential...
 References
 
Despite significant efforts toward improving the clinical outcome of pediatric AML patients, much progress is still needed. A contributing factor to the relatively slow progress may be that, despite significant heterogeneity in AML, we have historically treated AML uniformly. The idea of identifying subpopulations within AML for treatment stratification is likely to play an increasingly important role in future therapeutic strategies. APML has been proven to be a unique subgroup of AML with very specific therapy requirements in both children and adults. Other data are also emerging that particular morphologic or cytogenetic subgroups may respond differently to specific therapies [43, 128, 129]. The St. Jude Children's Research Hospital AML consortium is putting this hypothesis to test in an ongoing multicenter AML trial in which patients with specific FAB, molecular, or cytogenetic subtypes are treated differently, although such approaches may be compromised in their ability to reach firm conclusions without prospective randomization. In addition, many risk factors have not been generally acted upon because of limited therapeutic options, lack of prospectively acquired data, and significant nonrelapse mortality, which has obscured appropriate statistical analysis of potential risk factors.

At least three areas are emerging that have great potential to help identify high-risk patients. Cytogenetic markers associated with poor outcome appear to maintain significance across nearly all trials tested. Abnormalities of chromosome 5 and 7, which collectively account for 7%–10% of pediatric AML cases, portend poor outcome. Unfortunately, nearly half of these patients fail to achieve a CR, thus limiting possible postremission interventions. The presence of FLT3/ITD and MRD by flow cytometry have received significant attention for identification of high-risk patients. FLT3/ITD, which is primarily seen in patients with the normal karyotype, can be used to identify nearly 15% of pediatric patients with AML, and use of the ITD-AR may further delineate risk status. Flow cytometric evaluation of MRD appears to be able to identify an additional 15%–20% of patients with a high risk for relapse. Thus collectively, cytogenetics, FLT3/ITD, and MRD can identify approximately one third of the AML patients with a high risk for relapse. These markers will be prospectively evaluated for validation as part of an ongoing COG AML trial.

Options to intervene in such high-risk patients must be carefully evaluated. Targeted therapies are being developed for patients with FLT3 mutations, but their clinical applications for newly diagnosed patients will require evaluation in clinical trials, and patients with MRD or poor cytogenetic markers may not have readily available alternatives. At this time, the standard of care for patients who relapse is intensive chemotherapy remission reinduction prior to allogeneic SCT. If a patient group that is determined to be at an extremely high risk for relapse can be identified, it can be argued that such patients need to be considered for hematopoietic stem cell transplantation (HSCT) prior to relapse. Unfortunately, despite the routine utility of SCT in relapsed AML, its role in the treatment of high-risk patients has not been clearly established. The question of treatment options for high-risk patients needs to be addressed within the context of multi-institutional trials; and whether such high-risk patients are to be quickly transplanted or randomized to HSCT versus intensive chemotherapy with or without targeted agents needs be addressed. Given the nature of AML therapy, HSCT may be the only available short-term option for therapy intensification in high-risk patients, and because most patients do not have matched family donors for transplantation, the use of matched URD transplantation needs to be considered in patients without family donors. Given that HSCT, especially from a URD, carries significant short- and long-term toxicities, its utility in high-risk patients must therefore be carefully examined. However, if patients at high risk for relapse do not receive an HSCT during CR1, there is a high chance that they will relapse and will need a transplant as therapy after relapse if they achieve a second CR. Thus, the option for these patients may not be whether they should receive an HSCT, but whether they should be transplanted in first or second CR (if a second CR is achievable).

Similarly, for patients with prognostic features placing them in a good risk category, the use of HSCT from matched family donors remains controversial [130–132]. Several cooperative groups, including the MRC and BFM, have concluded that patients with good-risk AML can be effectively treated with only chemotherapy and that allogeneic HSCT should be reserved for patients who relapse [130, 132]. This type of approach depends on the ability to reinduce a remission as well as the effectiveness of HSCT in this group of patients. North American studies have demonstrated that the best relapse-free and overall survival for pediatric patients with AML is achieved in those receiving family donor HSCT in CR1, except for patients with inv(16) [131, 133]. Because HSCT may not have the same effectiveness in all groups of newly diagnosed or relapsed patients, as well as uncertainties regarding the long-term outcomes for relapsed patients following different initial therapies, questions regarding the application of HSCT should best be determined through prospective clinical trials.

As clinicians caring for children with AML, our most important objective is to improve the outcome with the least toxicity. Managing patients who are at extremely high risk for relapse is difficult, but the reality is that one may have to choose extremely intensive therapy to overcome disease resistance in at least the near future. Prognostic markers for relapse should also be prospectively studied and validated in large multi-institutional trials. Once such markers are validated, they must be acted upon, and a relapse threshold and survival after relapse must be established; patients identified with a particular marker(s) that would put them below an accepted threshold would be promptly referred for HSCT with the hope that this would improve outcome. Future work should be directed not only toward identifying prognostic factors, but also toward therapeutically exploiting those factors. The development of specifically targeted therapies that will both cytoreduce the leukemic burden and also eliminate or control the leukemic stem cell population is likely to be critical for achieving improved outcomes for patients with AML. As these more effective therapies are developed to target one or more of the critical genetic changes observed in specific subtypes of AML, the role of SCT will hopefully decrease. This hope should be applicable for children and adults with both high-risk as well as good-risk AML.


   DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
Top
 Learning Objectives
 Abstract
 Introduction
 Prognostic Factors
 Conclusions
 Disclosure of Potential...
References
 
R.J.A. has acted as a consultant for Genzyme and Novartis.


   REFERENCES
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 Learning Objectives
 Abstract
 Introduction
 Prognostic Factors
 Conclusions
 Disclosure of Potential...
 References
 

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