Clonal Origin of Myeloid Leukemia Cells

Normal myelopoiesis is a complex differentiation program whereby primitive hematopoietic stem cells (HSCs) develop along a multistep pathway into fully differentiated, functionally active circulating blood cells. This exquisitely controlled process is regulated by the intricate interactions among the expression levels of various transcription factors, growth factors and their receptors, cytokines, enzymes, and still unidentified novel molecules. A series of sequential genetic abnormalities perturbs this normal developmental progression and leads to AML. Although the relationships between specific morphologic subtypes of AML and their specific recurring genetic abnormalities have provided some insight into the mechanisms of leukemogenesis, understanding of the process whereby these fusion gene products interact with normal signal transduction pathways to subvert hematopoietic cell development is incomplete.

Several strong lines of evidence support the hypothesis that AML progresses from a single transformed hematopoietic stem or progenitor cell. More than 30 years ago, studies were pioneered by Beutler and colleagues and Fialkow using X inactivation patterns in female patients to establish the clonal origin of human malignancies, including leukemia. By showing the presence of a single glucose-6-phosphate dehydrogenase (G6PD) isoenzyme in the leukemic myeloblasts of heterozygous females, the clonal origin of myeloid leukemia cells was demonstrated. Females who are heterozygous at the G6PD locus express two isoforms of the enzyme. Approximately half of the cells in normal somatic tissue have randomly inactivated one of the X chromosomes, and approximately half of the cells should therefore express each isoform. Unlike normal somatic cells, AML cells of female patients heterozygous for G6PD expressed only one G6PD isoform, indicating cells of clonal origin. In the 1980s, Vogelstein and coworkers developed a strategy using X chromosome–linked DNA restriction length fragment polymorphisms to determine the clonal origin of human tumors. They established that maturing granulocytic cells arise from the malignant clone in patients with AML. Later, X chromosome inactivation to determine clonality in malignancies was carried out using the polymerase chain reaction (PCR) assay to distinguish X-linked polymorphic genes.

Transformation of Stem Cells with Self-Renewal Capacity

These earlier studies, and those performed by Bonnet and Dick and associates in the 1990s, led to the insight that leukemia cell populations form a hierarchy in which leukemia stem cells or leukemia-initiating cells (LICs) are responsible for their own self-renewal and for the generation of the more differentiated progeny within the leukemic clone. This theory was in contrast to the stochastic model, which stated that malignant properties within cancer cell populations follow Gaussian distributions, resulting in the random or stochastic probability that individual cancer cells within the population are able to initiate malignant progression. Whereas AML was the first human cancer in which cancer stem cells were identified, the case has now been made in breast cancer, brain tumors, and colon cancer, among others. In general, AML stem cells are believed to arise through the transformation of HSCs, which then retain the capacity for self-renewal, or through transformation of more differentiated hematopoietic progenitor cells that acquire the ability to self-renew by virtue of the particular transforming mutations, such as the formation of an MLL fusion gene.

AML is a heterogeneous disease in its clinical manifestations, response to therapy, and molecular genetics; thus insight into the cell of origin would have important ramifications regarding diagnosis and treatment. Moreover, refractory disease and relapse are often attributed to the presence of an LIC population that is resistant to chemotherapy; thus the ability to identify and target this subpopulation may be key to improving overall disease outcome. In the 1990s, Dick’s group produced a series of convincing papers that strongly implicated the primitive, pluripotent HSC as the LIC in some types of AML. “Stemness,” or the capacity to function as an LIC, includes self-renewal, proliferation, and differentiation and is tested in vitro by replating limiting dilution studies and in vivo by serial transplantation assays with the ability to regenerate a clonal leukemia in secondary and subsequent recipient animals. The HSC, with its inherent gene programs ensuring self-renewal, proliferation, and survival appears to be an ideal target to become sabotaged through a series of genetic events, leading ultimately to a fully transformed AML LIC that is capable of producing a clonal population of leukemia cells. According to this argument, different AML phenotypes often occur as a result of the gene(s) that are altered, leading to differentiation arrest within the progeny of the LIC, but not necessarily reflecting the degree of the commitment of the initially transformed cell. Support for this hypothesis initially came from several clonality studies in leukemic cells from patients with AML, which demonstrated multiple-lineage involvement in a high proportion of cases, and implicated a multipotent HSC as the cell of origin. More supportive evidence has come from studies looking at cytogenetic markers and characteristic cell surface antigen expression patterns. Pluripotent HSCs express CD34 but do not express CD38 or HLA-DR, whereas more committed myeloid progenitor cells are CD34+, CD38+, and HLA-DR+. Using fluorescence-activated cell sorting (FACS) of leukemic cell populations, the same two subpopulations, CD34+/CD38− and CD34+/CD38+, were isolated from the bone marrow of patients with different AML subtypes. The two subpopulations were then evaluated using fluorescence in situ hybridization (FISH) to determine the presence of cytogenetic abnormalities. The studies detected the same characteristic cytogenetic abnormalities in the CD34+/CD38− stem cell fraction as in the original leukemic bone marrow samples, implying origin in a very early HSC compartment.

Further evidence supporting the stem cell origin model of AML was derived from transplantation experiments in which purified human AML cells were transplanted into mice with severe combined immunodeficiency (SCID). These experiments defined a SCID mouse leukemia–initiating cell (SL-IC) in the bone marrow of patients with AML and showed that these SL-ICs are CD34+/CD38− and that their engraftment produces large numbers of colony-forming progenitors. The CD34+/CD38+ and CD34− fractions did not engraft. Similar results were obtained through transplantation studies in a modified SCID mouse, the nonobese diabetic mouse with SCID (NOD/SCID), with cells from patients with AML. These studies identified and analyzed AML stem cells from samples of different FAB subtypes on the basis of their ability to initiate human AML after transplantation in NOD/SCID mice. These SL-ICs were able to proliferate and differentiate after transplantation, producing disease in the mice identical to that in the donor, as well as being able to renew themselves, reestablishing AML in secondary recipients, demonstrating that SL-ICs are capable of self-renewal. Again, the SL-ICs were found to reside in the CD34+/CD38− fraction and not in the CD34+/CD38+ or CD34− fractions. The SL-IC phenotype was consistent regardless of the FAB subtype (e.g., M1, M2, M4, M5). As few as 2 • 10 ⁴ CD34+ cells were able to initiate the leukemic clone in recipient mice, whereas 100 times as many CD34− cells failed to engraft. Cells with the CD34 surface antigen were further fractionated on the basis of CD38 expression. Only the CD34+/CD38− fraction contained SL-ICs.

Whereas the transformation of the HSC provides an attractive mechanism to generate hierarchical myeloid leukemia cell populations, several studies have given new credence to a second mechanism through which committed progenitor cells along the pathway of myeloid differentiation are vulnerable to transforming mutations that confer self-renewal properties to progenitor cells that inherently lack this ability. These transformation events create an LIC capable of generating a population of aberrant cells blocked at the differentiation state at which the initial transforming event occurred. Several translocations involving the MLL gene have been shown to initiate self-renewal in myeloid progenitor cells and to result in an AML phenotype. A head-to-head comparison was conducted by retrovirally transducing the human AML-associated MLL-ENL fusion gene into murine HSC, common myeloid progenitors (CMPs), and granulocytic-monocytic–restricted progenitors (GMPs) and evaluating the relative transforming ability and resultant phenotype in each cell type. AML developed in mice transplanted with any of the three transduced cell populations and, in each case, displayed an identical myelomonocytic phenotype as assessed by morphology and flow cytometry. Similarly, the MOZ-TIF2 fusion gene was able to confer self-renewal properties to committed myeloid progenitor cells and resulted in AML when transplanted into irradiated mice. Although the fusion genes MLL-ENL and MOZ-TIF2 appear to impart leukemogenic capabilities to committed progenitor cells convincingly, other fusion genes such as BCR-ABL can induce leukemic transformation only when expressed in an HSC but not a more mature myeloid cell. Similarly, the potent oncogenic combination of Hoxa9 and Meis1 only resulted in AML when transduced into HSCs but not in more committed myeloid progenitors. In this case, expression of β-catenin was found to be the necessary factor for oncogenesis. Present in HSCs but absent in more committed progenitors, the addition of β-catenin to progenitors transduced with Hoxa9 and Meis1 was sufficient to lead to AML. These findings point to the differing transforming abilities of different oncogenes that are likely cell-type and cell-context specific. Hence both cell-of-origin hypotheses appear to be correct, depending on the specific oncogene or tumor suppressor that provides the initiating genetic lesion. Increased proliferation, survival, and self-renewal are all necessary attributes of a leukemia stem cell. Some genetic abnormalities, such as MLL-ENL and MOZ-TIF2 , may be capable of inducing self-renewal and thus are able to transform a more differentiated myeloid cell into an LIC. By contrast, other oncogene fusions, such as BCR-ABL , or oncogenic transcription factor genes, such as Hoxa9 , do not possess self-renewal capabilities and thus require a cell that inherently has this characteristic, namely, the HSC.

More recently, challenges to the cancer stem cell hypothesis have arisen out of a number of studies, predominantly in solid tumors such as melanoma, where the frequency of cells capable of giving rise to a new tumor in an immunocompromised host is of sufficient frequency to call into question whether the LIC represents a unique subpopulation or the original stochastic model of cancer progression holds true. These studies have again called into question previously accepted tenets. CD34+/CD38+ populations, in addition to the CD34+/CD38−, have also been found to contain LICs when injected into more immune-deficient hosts that lack key components of the innate immune system. These studies and additional syngeneic and cogenic transplant studies in mice as well as zebrafish highlight the critical context of the recipient animal in determining the LIC. The heterogeneity of LICs in AML is further demonstrated by an increasing number of cell surface markers in addition to CD34 and CD38, including CD123, TIM3, and CD47. Additionally, a number of studies have attempted to identify a specific LIC genetic signature, which has been found at least in some cases to independently predict prognosis. In this case, the LIC signature was reminiscent of the normal HSC, hearkening back to the original predictions of Dick’s group highlighting the HSC as a putative LIC cell of origin. However, although these findings are provocative, a consistent cadre of LIC genes has not yet been established. Clonality studies demonstrating the progressive acquisition of mutations in a given cell have also been put forward as an alternative to the cancer stem cell hypothesis. Although seemingly at odds, these different concepts can be reconciled into a single integrated framework. Myeloid (and lymphoid) leukemia-initiating events may occur in both HSCs and committed progenitor cells, and each leukemia arises from a combination of multiple genetic abnormalities. Secondary events subsequently are acquired in different subpopulations, generating unique clones with varying malignant potential depending on the genetic lesion and the self-renewal capacity of the cell in which these abnormalities occur. The number of genetic lesions necessary to cause frank AML is being defined based on next-generation sequencing studies recently published by the group at Washington University in St. Louis as well as other centers. The evolution of particular clones appears to determine the eventual phenotype of the AML cells documented at diagnosis ( Fig. 51-1 ).

Integration of the leukemic initiating cell (LIC) and clonal evolution models of acute myelogenous leukemia pathogenesis.

(Modified from Dick JE: Stem cell concepts renew cancer research. Blood 112:4793–4807, 2008.)

These new discoveries have added additional complexity to our understanding of the contribution of LICs to AML pathogenesis by demonstrating that the heterogeneity inherent in this disease extends to the AML stem cells. However, the identification of new LIC surface markers, genetic signatures, and gene-specific mutations hold out promise that LIC-targeted therapies will be realized in the coming years.

Class I Mutations

Among the earliest examples of class I mutations, RAS mutations were described in the early 1980s. There are three functional RAS gene family members— NRAS, KRAS, and HRAS —that all act as guanosine nucleotide phosphate (GTP)-binding proteins. Mutations of NRAS are the most prominent RAS mutations in AML and have been found in as many as 30% of adult patients with AML. Mutations, most frequently in codons 12, 13, and 61, result in the retention of the RAS protein in its active GTP-bound state and lead to the constitutive activation of downstream effector proteins, which causes the transcriptional activation of various target genes that direct cellular differentiation, proliferation, and survival ( Fig. 51-3 ). RAS proteins may be alternatively activated by mutations in genes encoding a number of regulatory factors. Activating point mutations in the SHP-2/PTPN11 phosphatase provide a stimulatory signal through guanine nucleotide exchange factors, such as “Son of Sevenless” (SOS), resulting in increased RAS pathway signaling, as do inactivating mutations of neurofibromin-1 (NF1) , which codes for a GTPase-activating protein (GAP) of the same name. NF1, normally functions, at least in part, by increasing the GTPase activity of RAS, thereby inactivating RAS-GTP by converting it into the inactive RAS-GDP form. These three types of mutations function independently to activate RAS signaling and lead to disturbances in hematopoietic cell differentiation and proliferation that may ultimately contribute to the development of MDS, myeloproliferative disease (MPD), and AML (see Fig. 51-3 ). Mutations in NF1 and PTPN11 have been particularly associated with JMML (see later).

Constitutive activation of the RAS pathway is central to myeloid leukemogenesis.

RAS pathway signaling may be constitutively activated via a number of mutually exclusive mechanisms (horizontal red arrows). These include the following: mutations in the GM-CSF or FLT3 surface receptors, resulting in ligand independent activation (found primarily in de novo AML); activating mutations in JAK2 (found in PV, ET, and PM) or SHP-2, c-CBL (found in JMML); activating mutations in RAS itself (found in all types of AML); or inactivating mutations in neurofibromin (found in JMML). PV, polycythemia vera; ET, essential thrombocytosis; PM, primary myelofibrosis; FLT3, fms-like tyrosine kinase 3; GM-CSF, granulocyte-macrophage colony-stimulating factor; JAK2, Janus 2 kinase; JMML, juvenile myelomonocytic leukemia.

(Modified from Loh ML, Vattikuti S, Schubbert S, et al: Mutations in PTPN11 implicate the SHP-2 phosphatase in leukemogenesis. Blood 103:2325–2331, 2004; and Proytcheva M: Juvenile myelomonocytic leukemia. Semin Diagn Pathol 28:298–303, 2011.)

There has been significant disagreement regarding the prognostic implications of RAS mutations in MDS and AML. Several studies have demonstrated that RAS mutations in patients with MDS confer a poor prognosis and increase the risk for progression to acute leukemia. Other studies have reported no difference in survival in AML patients who carried RAS mutations compared with patients without RAS mutations, whereas some have suggested improved survival in patients with AML and NRAS mutations. A large study of more than 2500 patients with AML demonstrated no difference in prognosis in patients with or without RAS mutations. This study also demonstrated an association between RAS mutations and a leukemia karyotype containing inv(16), a class II mutation resulting in the CBFβ-MYH11 fusion protein and altered function of the core-binding factor (CBF) transcriptional complex. A recent report from the COG examining 825 pediatric AML samples treated on two recent trials identified NRAS mutations in 10% of patients predominantly in codons 12 and 13, with no mutations in codon 61. Interestingly, most of these mutations were associated with good prognosis NPM1 mutations (see later) and not CBF alterations. However, multivariate analysis confirmed a lack of independent impact of NRAS mutations on prognosis.

Another group of class I mutations involves constitutive activation of receptor or cytoplasmic-nuclear tyrosine kinases. The BCR-ABL translocation that is the sine qua non of CML is an example of a class I mutation causing the activation of the ABL tyrosine kinase. Similarly, receptor tyrosine kinase mutations activate the human protooncogenes KIT and FLT3 in AML. The KIT protein is activated by the binding of its ligand stem cell factor and is critical for the development and growth of mast cells, melanocytes, HSCs, and the interstitial cells of Cajal. Mutations in KIT allow ligand-independent activation of KIT and confer factor-free growth and tumorigenicity in hematopoietic cell lines. Blasts from patients with AML express KIT on their cell surfaces in most cases. Deletional and insertional mutations of KIT have been identified in the blast cells of patients with AML, often in association with the CBF abnormalities inv(16) or t(8;21).

The FLT3 gene encodes a class III fms-like receptor tyrosine kinase expressed in early hematopoietic progenitors. FLT3 mutations occur in two varieties, internal tandem duplications (ITDs) in the juxtamembrane region and activation loop mutations (ALMs), primarily at codons 835 and 836. The FLT3 -ITD is one of the most frequent abnormalities in adult AML, documented in 25% to 30% of patients, and generally confers a poor prognosis. By contrast, ALMs occur in only 10% of adult patients and have not been associated with inferior survival rates. FLT3 -ITDs have been observed in 5% to 16% of pediatric AML cases and ALMs in 2% to 5% of cases. Paralleling the adult studies, FLT3 -ITDs, but not ALMs, were associated with a poor prognosis in children. FLT3 -ITDs result in the constitutive activation of FLT3 and cause interleukin-3–independent growth in Ba/F3 and 32D cells. This activation, in turn, results in the signaling of multiple downstream pathways, including the aforementioned RAS pathway, and the inhibition of caspase-mediated apoptosis through the stimulation of phosphatidylinositol 3′-kinase (PI3K) and AKT (see Fig. 51-3 ). However, mice transplanted with FLT3-transformed hematopoietic cells develop a myeloproliferative disorder characterized by splenomegaly and leukocytosis but they do not develop AML. FLT3 -ITDs often occur in conjunction with other mutations, including NPM1 (see later) and WT1 (see later).

Class II Mutations

In class II mutations, the molecular defect appears to be at the level of transcriptional activation, whereby transcriptional repressors cause a dominant negative inhibition of normal hematopoietic cell differentiation. AML can be subtyped based on the type of class II mutations in the blast cells of patients. The type 1 subset of class II mutations occurs in patients with de novo AML. These are typical chromosomal translocations resulting in chimeric oncoproteins that in some cases may cause the inhibition of differentiation. The type 2 subset of class II mutations usually are found in patients with AML arising after a prodrome of MDS and manifest more commonly in older adults or in patients who have received previous chemotherapy. Cytogenetic studies of the blast cell populations in these patients have complex karyotypes, lacking translocations but harboring deletions such as 5q−, monosomy 7, and 20q−.

Type 1 Mutations

Translocations Involving Core-Binding Factor Complex.

Several of the translocations that have been identified in adult and childhood de novo AML involve the CBF complex. The CBF complex is the most frequent target of chromosomal translocations in the human leukemias. The CBF regulatory complex consists of a DNA-binding subunit, RUNX1 (also called CBFα or AML1) and CBFβ, a subunit that does not bind DNA independently but heterodimerizes with RUNX1 or one of its closely related family members. Chromosomal translocations that modify the CBF complex in AML include the following: t(8;21)(q22;q22), which generates the AML1-ETO fusion protein, more recently referred to as RUNX1-RUNX1T1 ( Fig. 51-4 ); t(3;21)(q26;q22), which gives rise to AML1-EVI1; and inv(16)(p13;q22), which fuses the CBFβ to smooth muscle myosin heavy chain (SMMHC), resulting in the CBFB-MYH11 fusion gene. The AML1 gene was inactivated in the germline of mice and was shown to be essential for definitive hematopoiesis of all cell lineages. Homozygous animals die early in embryogenesis of central nervous system (CNS) hemorrhage, but they have normal morphogenesis and yolk sac hematopoiesis lineages. Inactivation of the CBFβ gene in the mouse has shown a similar phenotype in the homozygous null mice. These experiments have demonstrated that the AML1/CBFα complex is essential for normal hematopoiesis and that chromosomal rearrangements involving this complex may interfere with its regulatory function in ways that lead to disruption of cellular differentiation, and eventually malignant transformation.

Consequences of the t(8;21) AML1-ETO (RUNX1-RUNX1T1) translocation.

A, Karyotype demonstrating the reciprocal t(8;21)(q22;q22) translocation in an affected patient. B, Interphase fluorescence in situ hybridization (FISH) using the AML1/ETO dual-color, dual-fusion Vysis probe demonstrates the novel AML1-ETO fusion by the presence of linked green AML1 and red ETO signals. C, The normal AML1-CBFβ transcription factor complex. D, The molecular consequences of the AML1-ETO fusion; namely, transcription repression may occur in contexts in which the normal AML1/RUNX1-CBFβ acts as to transactivate gene expression.

(Courtesy of Dr. Barbara Morash, Cytogenetics, IWK Health Centre, Halifax, Nova Scotia, Canada; D modified from Downing JR: The core-binding factor leukemias: lessons learned from murine models. Curr Opin Genet Dev 13:48–54, 2003.)

Evidence primarily from knock-in studies in mice has supported the hypothesis that the AML1-ETO fusion protein functions as a dominant negative inhibitor of wild-type functions. In these experiments, the AML1-ETO fusion gene is “knocked in” to the wild-type AML1 locus. The phenotype of these mice is identical to the AML1 knockout; the mice die early in embryogenesis from CNS hemorrhage and lack any evidence of definitive hematopoiesis, although the rare cells that survive and express AML1-ETO have an increased capacity for self-renewal. Biochemical experiments have shown that the ETO portion of the AML1-ETO chimeric protein recruits nuclear corepressor complexes to the CBF promoters, resulting in transcriptional inhibition of target genes normally activated by the AML1/CBFβ heterodimeric complex (see Fig. 51-4D ). Subsequent studies using inducible murine model systems to bypass the embryonic lethality of AML1-ETO overexpression have demonstrated that overexpression of this transgene at later developmental time points confers enhanced self-renewal capacity to bone marrow progenitors, as demonstrated by serial culture experiments. Importantly, the mice do not develop AML but appear to be predisposed to leukemogenesis. Thus after treatment with N -ethyl- N -nitrosourea (ENU), the mice developed rapid onset of AML compared with similarly exposed wild-type mice. Moreover, as noted, there is an association in myeloid leukemia of AML1-ETO translocations and mutant tyrosine kinases, such as KIT. Interestingly, AML1-ETO transcripts may persist for a long time in patients with AML1-ETO –expressing AML in sustained remission.

Translocations Involving the Retinoic Acid Receptor-α.

Reciprocal chromosomal translocations involving the retinoic acid receptor-α (RARA) gene locus on chromosome 17 are the defining molecular features of acute promyelocytic leukemia (APL). Before the RARA translocations were described, retinoic acid was known to induce myeloid differentiation in vitro and therapy with all- trans -retinoic acid (ATRA) in patients with APL was shown to induce complete remission (CR). The intense investigation of the RARA gene triggered by these observations culminated in identification of the promyelocytic leukemia PML-RARA translocation breakpoint, t(15;17)(q22;q11), reported simultaneously by several groups. Although most APL cases have the associated t(15;17), other reciprocal translocations involving the RARA gene have been identified in which the gene is fused to other gene partners. The most common of these include the promyelocytic leukemia zinc finger (PLZF) gene on chromosome 11 in t(11;17)(p13;q11) and the nucleophosmin gene (NPM) on chromosome 5 in t(5;17)(q31;q11).

Normally RARA functions as a ligand-dependent activator of transcription. It forms a heterodimer with the related protein, retinoid X receptor (RXR), and binds through its zinc finger domain to a specific promoter sequence; in the presence of the retinoic acid ligand, the RARA-RXR heterodimer activates transcription of retinoic acid–responsive genes. This process is accomplished in concert with a coactivator complex that includes proteins such as p300 and pCAF. In the absence of retinoic acid, a corepressor complex is recruited, composed of N-CoR (nuclear receptor corepressor) or SMRT (silencing mediator of retinoid and thyroid receptors), which binds to another protein called Sin3 and then to HDAC1. HDAC1 is a histone deacetylase protein, which epigenetically alters histones to keep DNA in an untranscribable form. The expression of retinoic acid–responsive genes is essential for normal myeloid development.

Much evidence supports the hypothesis that the PML-RARA fusion protein functions as a dominant negative inhibitor of the PML protein and RXR. PML proteins are normally located in macromolecular nuclear organelles, called PML oncogenic domains (PODs). The PML-RARA fusion protein disrupts the PODs, causing normal PML, RXR, and other nuclear proteins to disperse in an abnormal pattern. ATRA and arsenic trioxide (As ₂ O ₃ ) have shown activity in APL blast cells and in patients with APL, and studies of these drugs have afforded important insights into the mechanisms of leukemogenesis in APL. ATRA and As ₂ O ₃ appear to act through different biochemical pathways, and promyelocytes resistant to ATRA are often sensitive to treatment with As ₂ O ₃ . Both drugs induce degradation of the PML-RARA fusion protein but through different mechanisms. Retinoic acid binding to wild-type retinoic acid receptors (RARs) in the cell nucleus causes degradation of the PML-RARA protein through the ubiquitin-proteosome and caspase systems, thus allowing for the terminal differentiation of leukemic promyelocytes. This binding also results in derepression whereby N-CoR is dissociated from the PML-RARA fusion protein and, subsequently, the nuclear coactivator complex is recruited to reverse histone deacetylase-mediated repression. PODs are relocated back to the normal nuclear pattern. By contrast, As ₂ O ₃ appears to target the PML portion of the PML-RARA fusion protein preferentially and causes its degradation by inducing apoptosis, potentially through downregulation of the antiapoptotic factor BCL2. The activity of these agents has led to the inferences that fusion proteins may interfere with normal myeloid cell development in several ways—through inhibitory effects on assembly of the PODs that contain PML or through dominant inhibitory effects on transcriptional targets of dimeric complexes with normal retinoid receptors, leading to arrest of differentiation at the promyelocyte stage.

Studies using transgenic mice have shown that the PML-RARA fusion product is involved in the development of leukemia. Transgenic mice generated with the PML-RARA fusion protein specifically expressed in the myeloid-promyelocytic lineage develop a myelodysplastic-like disorder during the first year of life. A subset of these mice then develops a form of acute leukemia that closely mimics human APL and responds to ATRA. Given the relatively long latency period and the development of leukemia in only a fraction of the mice, it is likely that the PML-RARA translocation is insufficient by itself to cause APL and that second mutations are necessary for leukemic transformation.

Mixed-Lineage Leukemia Gene Translocations.

Transcriptional coactivators and corepressors have been implicated in leukemogenesis. An example is the myeloid-lymphoid or MLL protein, a large protein containing transcriptional activation and repression domains, which is believed to act epigenetically to modulate gene expression through its effects on chromatin structure and configuration. Wild-type MLL is required for normal hematopoiesis and HSC development. Mice deficient in MLL die on embryonic day 10.5 and have numerous skeletal, neural, and hematologic deficits. Heterozygous mice have defects in embryonic segmentation and yolk sac hematopoiesis, and adults demonstrate a mild anemia and thrombocytopenia. Chromosomal translocations of the MLL gene on chromosome segment 11q23 have been identified in human AML and ALL and confer a poor prognosis. Many gene partners have been identified that are involved in these translocations, including AF4, AF9, ENL, AF6, ELL , and AF10 . In most cases, the fusion proteins incorporate the 5′ end of MLL and the 3′ end of the partner. Despite the number of different fusion partners, leukemias with MLL translocations tend to possess a distinct genetic signature compared with AML and ALL samples lacking an MLL translocation. This genetic signature includes the upregulation of a number of specific HOX family genes, including HOXA9, HOXA7, and MEIS1 . Wild-type MLL is known to play a major role in HOX gene transcriptional regulation. In turn, HOX genes play critical roles in embryonic development and normal hematopoiesis. A number of studies have clearly demonstrated that overexpression of specific major HOX genes in mouse models and human bone marrow samples, whether by involvement in reciprocal translocations or by gene upregulation, is linked to the transformation of malignant HSCs in AML. The mechanism whereby MLL fusion proteins upregulate HOX gene expression may differ, depending on whether the fusion partner encodes a nuclear partner with direct transcriptional activity or a cytoplasmic protein that results in MLL protein dimerization and subsequent transcriptional activation.

Transcriptional control may be mediated through the modification of histone proteins, which function to maintain chromatin structure. Recently, the DOTL1 complex was identified as a critical factor in MLL-induced leukemogenesis. DOT1L is a histone-3-lysine-79 (H3K79) methyltransferase, which was previously believed to play a fairly ubiquitous role in methylating gene targets in concert with transcription. However, Zhang and Armstrong’s group definitively demonstrated through a series of elegant in vitro and in vivo experiments a specificity for DOT1L in the epigenetic regulation of MLL-translocated downstream targets. When DOT1L was knocked out or absent from MLL-AF9 –transformed cell lines or transgenic mice, leukemia was prevented. The MLL gene is rearranged in up to 20% of pediatric cases of AML. Translocations of MLL are the single most common genetic alteration in infants with acute leukemia, regardless of phenotype, and account for approximately 70% of all cases of AML and ALL in infants. In pediatric and adult cases of AML, the presence of the 11q23 translocation is generally associated with an unfavorable prognosis.

Although less common than MLL translocations, the MLL gene can be altered by partial tandem duplications (PTDs) of particular exons. These PTDs occur in adult and pediatric AMLs with a normal karyotype, as well as those with trisomy 11. Similar to MLL fusions, the MLL -PTD tends to be associated with a poor prognosis and often with the presence of an FLT3 mutation. The mechanisms underlying MLL-PTD leukemogenesis have not yet been elucidated but appear to be different than those of MLL fusions. Interestingly, MLL -PTD appears to repress wild-type MLL expression, which may be an important feature underlying the leukemogenic potential of this abnormality.

French-American-British Classification

In 1976, the FAB group proposed a system of classification of AML based on morphologic and cytochemical features (see Table 51-1 ). The system divides AML into seven subtypes, M1 through M7. An M0 subtype was later added to describe undifferentiated leukemia and, since 1976, immunophenotypic data have also been included. In general, AML can be differentiated from ALL based on morphologic features and cytochemical stains. Cells of myeloid origin should stain with myeloperoxidase and SBB. Cells of monocytic derivation usually stain with NSE. AML blasts usually do not have PAS activity, except for erythroblasts in AML-M6 and eosinophils of the M4Eo subtype.

World Health Organization Classification

The discovery of cytogenetic and molecular genetic abnormalities in malignant disease has had a significant impact on our understanding of malignant transformation. In myeloid malignancies, some genetic abnormalities have prognostic significance whereas others appear to define specific disease subtypes. The WHO has proposed a classification system for neoplastic diseases of the hematopoietic and lymphoid tissues that includes a classification for AML (see Box 51-2 and Fig. 51-5 ). This classification uses the traditional FAB-type morphologic categories of disease and includes additional entities such as immunophenotype, molecular genetic, and clinical characteristics that make the system more clinically relevant for diagnosis, prognosis, and treatment. The WHO classification system divides myeloid diseases into several major subtypes—myeloproliferative neoplasms; myeloid neoplasm associated with eosinophilia and abnormalities of PDGFRA , PDGFRB , or FGFR1 ; myelodysplastic-myeloproliferative neoplasms; MDS, AML and related neoplasms; and acute leukemias of ambiguous lineage. The 2008 revision of the WHO classification has expanded the category of AML to include AML with recurrent cytogenetic translocations, AML with myelodysplasia-related changes, therapy-related myeloid neoplasms, AML not otherwise specified, myeloid sarcoma, myeloid proliferations related to Down syndrome, and blastic plasmacytoid dendritic cell neoplasms.

Within the subtype containing recurrent cytogenetic translocations, seven additional categories have been described:

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  AML with t(8;21)(q22;q22); AML1-ETO ; RUNX1-RUNX1T1

  
  
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  AML with inv(16)(p13.1q22) or t(16;16)(p13.1;q22); CBFB-MYH11

  
  
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  APL with t(15;17)(q22;q12); PML-RARA

  
  
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  AML with t(9;11)(p22;q23); MLLT3-MLL

  
  
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  AML with t(6;9)(p23;q34); DEK-NUP214

  
  
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  AML with inv(3)(q21q26.2) or t(3;3)(q21;q26.2); RPN1-EVI1

  
  
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  AML (megakaryoblastic) with t(1;22)(p13;q13); RBM15-MKL1

AML with t(6;9)(p23;q34) and AML with inv(3)(q21q26.2) is relatively uncommon in children. Myeloid sarcomas, formerly called chloromas, may precede hematopoietic evidence of disease and so are now classified as a separate entity. The myeloid diseases of Down syndrome were incorporated into the revised WHO classification in 2008 and are described later in this chapter. Plasmacytoid dendritic cell neoplasms were formerly referred to as blastic NK-cell lymphomas, but the nomenclature has changed to reflect the origin of this myeloid malignancy in this subset of dendritic cells. This rare disease can manifest in children with skin lesions, adenopathy, and ultimately bone marrow involvement. Blast cells characteristically express CD4, CD43, CD56, and CD123 without expression of CD34 or CD117.

The FAB standard used to define AML had been 30% replacement of the bone marrow by the blast cells, but later studies have shown that patients with 20% to 30% blasts (previously classified as refractory anemia with excess blasts in transformation [RAEB-T]) have a prognosis similar to that of patients with more than 30% blasts. The blast count for the diagnosis of AML in the WHO classification was therefore changed to 20%, and the category of RAEB-T was eliminated.

Certain specific and recurrent cytogenetic abnormalities are common in MDS, in alkylating agent–related AML, and in de novo AML with a poor prognosis. These abnormalities include 3q−, −5, 5q−, −7, 7q−, +8, +9, 11q−, 12p−, −8, −19, 20q−, +21, t(1;7), t(2;11), and complex karyotypes. In the classification scheme, these cytogenetic abnormalities were considered to indicate a poor prognosis. Prior therapy with topoisomerase II inhibitors (i.e., epipodophyllotoxins and doxorubicin) was also associated with a poor prognosis. Typically, patients in whom AML develops after exposure to these agents have translocations involving 11q23 ( MLL ). The WHO classification includes these patients in the poor-prognosis category but distinguishes them from having alkylating agent–related secondary leukemia.

Patients may have MDS with unilineage or multilineage dysplasia, and these are reflected as distinct diseases in the most recent iteration of the WHO classification. Dysplasia is defined as being observed in greater than or equal to 10% of the cells of a given myeloid lineage. Children with 2% to 19% peripheral blasts or 5% to 19% bone marrow blasts can be classified according to the same criteria as adult MDS, but a new category of refractory cytopenia of childhood has been introduced to define children with persistent cytopenias and dysplasia of two or more lineages but less than 2% peripheral blasts or less than 5% bone marrow blasts.

Remission Induction

During the past 40 years, survival rates for patients with AML have risen from less than 10% to more than 50% of patients because of intensification of chemotherapy, use of hematopoietic stem cell transplantation (HSCT), and improved supportive care. The first phase of chemotherapy is termed induction and consists of two to four cycles of intensive chemotherapy with the goal of inducing remission. Morphologic remission is defined as the presence of fewer than 5% blasts visible on bone marrow aspirate obtained after recovery of counts. Patients may also achieve cytogenetic and FISH-negative remission, as defined by the absence of a previously detected leukemic clone–associated cytogenetic abnormality in the bone marrow cells. Remission is also now being defined by the absence of MRD, assessed by multiparameter flow cytometry that is designed to detect as few as 0.1% leukemic blasts in the marrow. The inclusion of MRD evaluation has not only identified children at risk for relapse who appeared to be in morphologic remission but has also revealed false-positive recovering marrows that had been deemed refractory disease.

Induction chemotherapy should be initiated as soon as the diagnosis of AML is confirmed, preferably by morphologic, immunophenotypic, and cytogenetic studies of the bone marrow. In some cases, particularly in patients presenting with hyperleukocytosis, the patient’s condition may be too unstable for bone marrow evaluation, in which case the diagnosis can often be confirmed on studies of the peripheral blood. Induction chemotherapy regimens consist of high-dose myelosuppressive cytotoxic agents that result in prolonged pancytopenia. The period of bone marrow hypoplasia generally lasts from 21 to 30 days from the initiation of therapy but may last considerably longer. During this period, patients have a high risk for life-threatening infection and bleeding. Aggressive supportive care is necessary (see later).

Postremission Therapy

Several randomized studies have demonstrated that without postremission therapy almost all patients with newly diagnosed AML suffer a relapse within 2 years. The strategies for postremission therapy include autologous HSCT, allogeneic HSCT, or continued courses of chemotherapy. The CCG trials have shown no benefit to autologous HSCT over continued cycles of chemotherapy so, although that strategy continues to be used in some European trials, autologous HSCT is not included in current U.S. trials. Most studies have shown a lower relapse rate for AML patients treated with matched related donor HSCT versus chemotherapy, but the OS is often no better because of the significant morbidity and mortality of HSCT. For this reason, until the most recent trials, unrelated donor HSCT was believed to be too morbid for patients in first remission and thus trials used a genetic randomization, wherein all patients with a matched related donor proceeded to HSCT and all others proceeded with further cycles of chemotherapy.

Based on the success of the MRC trials with chemotherapy alone using a combination of cytogenetic characteristics and response to therapy, the COG and SJCRH adopted risk-based criteria for HSCT. HSCT, even from a matched related donor, is not recommended for patients with low-risk disease. This was previously defined by COG as the presence of t(8;21) or inv(16) in the leukemic clone at diagnosis, but more recently the presence of a CEBPA or NPM1 mutation has been added to this definition, based on the data revealing the better outcome for these patients. The COG has classified high-risk disease as monosomy 7, −5/5q−, FLT3 -ITD with high allelic ratio, MRD positive after induction chemotherapy, or more than 15% blasts in the marrow after one course of induction chemotherapy. * The incorporation of MRD after induction has enabled the COG to relegate normal karyotype patients to high- and low-risk groups, thereby dichotomizing patients and eliminating the intermediate-risk category. Therefore on the current AAML1031 protocol, high-risk patients will receive best available donor HSCT for consolidation, whereas good-risk patients will not go to transplant even if a sibling donor match is available. With recent advances in supportive care and improved therapy to prevent and treat graft-versus-host disease (GVHD), outcomes have improved for matched related and matched unrelated donor HSCT. Therefore for patients with high-risk disease, both trials recommend HSCT in first CR with an unrelated donor if no matched family donor is identified. The optimal number of cycles of chemotherapy before HSCT to balance minimizing toxicity while minimizing MRD is variable, with the MRC10 trial using four cycles, the current SJCRH trial including two to three cycles, and the COG trial using three cycles before HSCT.

* References .

† References .

Supportive Therapy at Diagnosis and during Therapy

The diagnosis and treatment of childhood AML require a multidisciplinary approach involving pediatric oncologists, hematopathologists, pediatric surgeons, infectious disease specialists, pediatric intensivists, and pediatric nurses. While studies and procedures are initiated to make the diagnosis, special care must be taken to prevent and treat the life-threatening consequences of leukemia and accompanying pancytopenia. The most serious complications of leukemia at diagnosis are infection and bleeding with pancytopenia, tumor lysis syndrome, and leukostasis. The most common causes of death once remission is achieved are relapse, infection, hemorrhage, and cardiac events. The improvement in OS of patients with AML over the past several decades is attributable as much to better supportive care efforts as to more effective treatment regimens.

Extramedullary Disease

Between 20% and 40% of children with AML have evidence of extramedullary disease at the time of diagnosis. The most common manifestations of extramedullary disease are skin infiltrates, soft tissue or bone involvement, gingival infiltration, and CNS involvement. Some patients, particularly infants, may have isolated extramedullary leukemia without other evidence of disease, but bone marrow infiltration invariably occurs in these patients without systemic therapy.

Demographic Risk Factors

Age older than 10 years was historically reported to carry a worse prognosis in clinical trials at SJRCH, as well as from the BFM and Japanese groups. However, a recent report from the SJCRH AML02 study demonstrated comparable EFS and OS in children as old as age 21 years as in their younger counterparts, although these older children had higher toxic death rates predominantly of infectious etiology. Race and ethnicity play a role in prognosis as well. Patients on CCG2891 who received chemotherapy alone had an OS of 48% for white children versus only 37% for Hispanic children ( P = .016) and 34% for black children ( P = .007). On the SJCRH AML97, there was a trend toward inferior outcomes for black compared with white children (hazard ratio, 1.61; 95% confidence interval, 0.76 to 3.42; P = .22 with 5-year survival rates of 55.6% vs. 27.3%). In these trials, there was no difference in other prognostic variables between the groups and these patients were hospitalized and received intravenous chemotherapy, received the same supportive care, and had the same compliance rates. Therefore, the observed differences may be attributable to pharmacogenomic differences that affect response to and toxicity from chemotherapy. Patients who are overweight (body mass index ≥95th percentile) or underweight (≤10th percentile) also carry a worse prognosis and are less likely to survive and more likely to suffer treatment-related mortality.

Cytogenetic Factors

Results of several large multicenter trials have confirmed the correlation between certain cytogenetic abnormalities and clinical outcome and have shown that karyotype in AML represents an independent prognostic variable for attainment of complete response and OS. In children and adults, the highest complete response rates and longest survival times have been linked to association with t(8;21) and inv(16) or t(16;16). These translocations result in modifications to the core-binding complex, a group of proteins that appear to play a vital role in hematopoiesis. AML cells with inv(16) have increased incorporation of cytarabine into nuclear DNA and are more susceptible to cytarabine chemotherapy in vitro. Patients with APL and t(15;17) previously represented a poor-risk group, but, since the advent of ATRA and As ₂ O ₃ therapy (see later), now represent a group with a favorable prognosis. Other cytogenetic aberrations have been associated with an unfavorable outcome. These include monosomy 5, del(5q), monosomy 7, abnormalities of 3q, aberrations of 12p, and complex cytogenetic abnormalities. For pediatric patients with monosomy 7, several studies have shown better OS when HSCT is used as consolidation therapy as opposed to conventional chemotherapy. Patients with normal karyotypes have traditionally been grouped together in an intermediate-prognosis group, but now mutations such as the FLT3 -ITD (poor prognosis) and NPM1 and CEBPA (good prognosis) have been demonstrated to alter prognosis sufficiently to influence the risk assignment of these patients at diagnosis on COG, SJCRH, BFM, and MRC trials. Abnormal karyotypes linked with an intermediate prognosis include del(7q), 11q23 translocations, +21, +8, +22, and del(9q). Other karyotypes such as t(6;9)(p23;q34), t(8;16)(p11;p13), and t(16;21)(q24;q22) are uncommon but appear associated with a poorer prognosis. While more studies with large numbers of patients undergoing cytogenetic evaluation are completed, certain groups of karyotype aberrations are showing heterogeneity in their prognostic significance. For example, all 11q23 translocations were previously grouped together in the intermediate- to poor-prognosis category. However, studies have shown that the 11q23 aberration may be subdivided into groups of lesser and greater prognostic risk. Patients with the t(9;11)(p22;q23) have been found in some studies to have a longer EFS and OS than patients with other 11q23 translocations. However, this was not found to be true in a recent review of outcome on MRC10 and 12 trials. As new therapeutic agents are developed that target the specific molecular aberrations identified by cytogenetic analysis, accurate detection of these abnormalities becomes even more important.

Molecular Genetic Factors

Analysis of molecular genetics is emerging as an invaluable tool in prognostic determination for patients with AML, as well as for providing insight into the pathogenesis of AML and elucidating possibilities for targeted therapy. RAS mutations have been identified in pediatric AML patients, with NRAS mutations in 10% to 15% and KRAS mutations in 3% of patients. HRAS mutations are rare in AML. In both adults and children these mutations are associated with favorable-risk AML. In adults, the association is with CBF leukemias and, in particular, inv(16). Although some pediatric studies have also found a link between CBF AML and NRAS mutations, others have found a distinct absence of NRAS with inv(16) and a unique correlation of NRAS and NPM mutations not seen in adult AML. Independently, however, NRAS mutations have no significant impact on prognosis. Patients had similar 5-year EFS and OS, although patients with NRAS mutations were associated with increased treatment-related mortality on one study. KIT mutations have been identified in 11.3% of pediatric AML patients, predominantly in the CBF leukemias, ranging from 17% to 41% of patients. The role of KIT mutations in prognosis has been unclear: in some series, the KIT mutations do not seem to have prognostic significance, in others, KIT mutations have been shown to confer worse prognosis in patients with t(8;21) but not in patients with inv(16) and, in still other trials, KIT mutations confer a worse prognosis in all subsets of CBF leukemias. However, a recent review of more than 200 children with CBF AML enrolled on four COG AML trials concluded that KIT mutations do not alter prognosis. Owing to their good outcome with chemotherapy alone, patients with CBF AML are currently nonrandomly assigned to chemotherapy without HSCT in CR1 even if a matched family donor is available. Based on these recent data, on current COG trials the presence of a KIT mutation does not alter risk stratification. This is in contrast to adult data which show that KIT mutations appear to confer an inferior outcome. These prognostic differences in adult versus pediatric CBF AML may reflect more aggressive therapy in children and, potentially, an earlier cell of origin in the adult disease that contributes to increased resistance to therapy.

As noted, FLT3 mutations include ITDs in the juxtamembrane domain and point mutations in the activation loop (ALM). FLT3 -ITDs have a worse prognosis, particularly when present in a high allelic ratio (>0.4), whereas FLT3 -ALMs seem to have no impact on prognosis. Partial tandem duplications of the MLL gene have also been reported in 13% of pediatric AML patients and are associated with a worse prognosis in adult and pediatric patients. Frameshift mutations in NPM1 lead to cytoplasmic localization of nucleophosmin and have been identified in 6% to 8% of all children with AML and 25% to 30% of pediatric AML patients with normal cytogenetics. When identified as an isolated mutation in adults and children, it is associated with improved prognosis, but when found in conjunction with an FLT3 -ITD, the poor prognostic impact of the FLT -ITD prevails. With contemporary chemotherapy, children with AML with a normal karyotype and isolated NPM1 mutation have outcomes in keeping with CBF leukemia with OS greater than 80%; thus these children have been assigned to the low-risk arm on the current COG trial (R. Aplenc, personal communication, June 2011). PTPN11 mutations are found in only 4% of pediatric patients with AML (4%), and 18% to 36% of the PTPN11 mutations are seen in M5 AML. In pediatrics, this mutation does not appear to have prognostic importance. The Wilms tumor gene (WT1) is aberrantly expressed in AML; in adults, high expression levels of WT1 at diagnosis have been associated with a poor prognosis in many studies. In pediatric patients, however, the available data have been contradictory. One small study of 47 patients has shown that low levels of WT1 at diagnosis are associated with good outcome, whereas another small study of 41 patients has shown that high levels of WT1 are more commonly seen in association with the t(8;21) and inv(16) favorable prognosis cytogenetic findings, and therefore associated with a better prognosis. Hollink and colleagues found an association between WT1 mutations and an inferior outcome. More recently, a large study performed by the COG identifying 70 patients with WT1 mutations found that although there was significant overlap of the WT1 mutation with either CBF or FLT3 -ITD, independently, WT1 mutations did not influence prognosis. Interestingly, 28% of pediatric AML patients harbor a minor single nucleotide polymorphism (SNP) in exon 7 of WT1 (rs16754) that was found to result in an improved 5-year OS (62% vs. 44%) for patients treated on CCG 2961 compared with those with leukemic expression of the major WT1 allele. Levels of expression of wild-type WT1 may also influence prognosis. However, at present, WT1 mutational status, presence of the minor SNP, and wild-type expression levels continue to be studied but are not being used to make treatment decisions. In addition to its role as a partner in the t(8;21) translocation, RUNX1 may also be mutated in approximately 5% of pediatric AML patients, specifically with point mutations in exons 3 and 8. Although typically associated with AML secondary to MDS, in de novo AML these patients had less organomegaly, a higher incidence of chloromas, and a trend toward improved outcome with modern intensive AML therapy. However, RUNX1 mutations are not currently used for risk stratification.

CEBPA mutations have been found to be an independent prognostic marker conferring a good outcome in both pediatric and adult AML and, similar to that seen for CBF AML, at least in children. Interestingly, pediatric patients with AML and CEBPA mutations tend to be older (age >10 years) and predominantly have a normal karyotype with an absence of other good- or poor-risk molecular abnormalities. Furthermore, patients with CEBPA mutations did not benefit from HSCT in first remission. Thus, in current studies, presence of a CEBPA mutation classifies a patient as low risk. Recent next-generation sequencing–based studies have revealed frequent mutations in the DNA methyltransferase gene, DNMT3A, the gene coding for the demethylating enzyme, TET2, and the genes IDH1 and IDH2 , which code for proteins in the Krebs cycle. DNMT3A mutations are generally associated with a poor outcome and contribute to a worse prognosis when combined with FLT3 ITD. TET2 mutations are similarly associated with a poor prognosis, with or without corresponding FLT3 abnormalities. By contrast, IDH1/IDH2 mutations appear to be more positive prognostic features. However, these abnormalities are generally rare in pediatric AML ; thus their impact cannot be easily determined, and at this time they have been excluded from any current pediatric AML risk stratification schemas.

Prognosis in Relapsed or Refractory Disease

A durable remission is less likely to be achieved and maintained in patients with relapsed or refractory AML than in patients with newly diagnosed AML, but the salvage rate in these patients has improved in recent years. The single most important prognostic indicator after relapse is duration of first remission, with less than 1 year to relapse carrying an OS rate of 0% to 21% and longer than 1 year to relapse carrying an OS rate of 33% to 62%. Other characteristics that have been associated with better prognosis after relapse are male gender, good-risk cytogenetic features of t(8;21) and inv(16), chemotherapy alone without HSCT as consolidation during first CR, and the use of HSCT as part of relapse therapy. The characteristics associated with worse prognosis after relapse are early recurrence, M5 or M7 morphology, poor-risk cytogenetics, and HSCT during first CR.

Conventional Therapy for Relapsed or Refractory Disease

There is no accepted standard therapy for adult or pediatric patients with relapsed or refractory AML. However, several researchers have demonstrated activity of high-dose cytarabine in patients with relapsed disease, even in those who had been exposed to cytarabine during prior treatment. High-dose cytarabine has been used in combination with other agents, such as mitoxantrone, asparaginase, etoposide, fludarabine, cladribine, and clofarabine. The combination of cytarabine and mitoxantrone was particularly effective as reinduction with a remission rate of 76%, but now that this regimen has been incorporated into the current standard first-line regimens, its use as a reinduction regimen may provide too much cumulative anthracycline for many patients. Fludarabine, cladribine, and clofarabine are all deoxyadenosine analogues that have been used for the treatment of AML. Trials using high-dose cytarabine in combination with fludarabine and G-CSF (i.e., the FLAG regimen) have demonstrated a 70% remission rate in patients with relapsed AML, and this regimen is frequently used by many centers for patients who have experienced a relapse of their disease. The addition of idarubicin to the FLAG regimen has resulted in longer-lasting remissions in some studies, but it has also been associated with more significant toxicity and for this reason is not frequently incorporated. The combination of cytarabine and cladribine has been successful as a relapse regimen in some trials for adult patients and pediatric patients with de novo AML, particularly those with the M5 subtype. However, this combination seems to be of little benefit in pediatric patients with relapsed or refractory AML. Clofarabine was found to be effective as a single agent in relapsed ALL and AML and was subsequently approved by the FDA for use in relapsed ALL. Clofarabine has been shown to be safe and effective in combination with cytarabine in regimens for adult patients, and this combination has been tested for pediatric patients with relapsed or refractory AML. The recently closed COG study AAML0523 examined the combination of clofarabine and cytarabine for relapsed AML and ALL and found that a dose of 52 mg/m ² of clofarabine was tolerated by patients with relapsed AML. Of the 46 patients who were evaluable, only 21% responded as evaluated by complete response or complete response with incomplete platelet recovery, which did not meet the predetermined statistical threshold for efficacy in overall response rate. Based on these findings, this regimen has somewhat fallen out of favor compared with FLAG. However, as a result of compliance issues and failure to recognize complete response with incomplete count recovery as evidence of response, results from this study may in fact underestimate the efficacy of this combination therapy; thus clofarabine and cytarabine for relapsed AML as a bridge to HSCT (see later) may still be worthy of consideration. For patients who cannot tolerate high-dose cytarabine or further anthracyclines, one alternative regimen that has been used at the Memorial Sloan-Kettering Cancer Center is topotecan, vinorelbine, thiotepa, dexamethasone, and gemcitabine (TVTG), with 5 of 9 AML patients responding to this therapy.

Long-term remission for patients with relapsed or refractory disease requires consolidation therapy with allogeneic HSCT. Traditionally, this has included matched family members or matched unrelated donors, but the more recent use of alternative HSC sources, such as banked umbilical cord blood or haploidentical family donors, has increased the number of patients with a potential marrow source. A recent trend has been in using non–total-body irradiation (TBI)–based conditioning regimens for AML in first remission due to equal efficacy and less toxicity, but TBI may still be used in the conditioning of patients with relapsed AML. It is possible to cure patients who have persistent, refractory disease at the time of allogeneic HSCT, particularly if they have less than 20% blasts in the marrow. Those patients with shorter remissions and higher blast percentages in their marrow have traditionally been associated with only a 25% chance of cure, whereas for patients who have had a long first remission and proceed to HSCT in a second CR the OS rates are much better and may be as high as 40% to 62%. However, although lower levels of MRD were associated with improved overall outcomes, investigators at the SJCRH found that with contemporary risk-adapted therapeutic regimens, allogeneic HSCT may be curative for patients with AML who continue to be MRD positive or even have persistent marrow blasts, with 5-year OS of 67% and 58%, respectively. This analysis included patients with relapsed AML. These findings are in contrast to the impact of residual disease in ALL, which may be attributed at least in part to the graft-versus-leukemia (GVL) in AML. This effect is difficult to measure directly but has been indirectly inferred from the presence of GVHD. The presence of acute and chronic GVHD has been associated with lower relapse rates in AML. Donor lymphocyte infusions have been used successfully to induce GVL effect in patients with CML who experience relapse after allogeneic HSCT, but this strategy is much less effective in patients with AML who experience relapse after allogeneic HSCT.

There can be GVL effect without GVHD, however, as evidenced by lower relapse rates in patients receiving HSCT from a matched sibling versus an identical twin, even in the absence of GVHD. There is much interest in maximizing the GVL effect, and recent studies have concentrated on the role of alloreactive natural killer (NK) cells in this process. Donors who are mismatched at the KIR ligand produce NK cells that recognize and destroy host AML cells in response to the lack of expression of a self class I ligand on the AML cells. Deliberate KIR mismatching through haploidentical HSCT has been shown in small studies to decrease the relapse rate without increasing GVHD, therefore resulting in improved long-term survival. This strategy is being used in clinical trials for patients with high-risk de novo AML or relapsed and refractory disease and is currently incorporated into ongoing pediatric trials of the COG and SJCRH. The feasibility of engrafting haploidentical NK cells in children with AML in remission was recently demonstrated.

Novel Agents and Targeted Therapy

Conventional therapy for AML is predominantly a “shotgun” approach in which leukemia cells, as well as normal hematopoietic stem and progenitor cells and many other normal tissues, are injured, with the hope that the normal cells will recover faster and more completely than the leukemia cells. As discussed earlier, this approach has not resulted in superior cure rates in AML and the intensity has been increased to the point that most patients suffer toxicity and treatment-related morbidity; a substantial fraction of patients suffer treatment-related mortality. While the molecular pathogenesis of AML has been elucidated, opportunities have appeared for the development of novel and targeted agents that preferentially destroy leukemia cells, with far less significant effects on normal hematopoietic cells and other normal tissues. However, preclinical and adult clinical data, not to mention drug availability, are in rapid flux, given the growing interest in developing novel targeted therapeutics. Thus current therapeutic platforms for relapsed AML are moving to the model of having a standardized backbone to which novel agents can be rapidly inserted to be sufficiently nimble to adapt to the availability and promise of emerging drugs.

All- Trans -Retinoic Acid

With the introduction of ATRA, the treatment and outcome of APL have changed dramatically. Although previously considered primarily a differentiation agent, a number of recent studies in PML-RARA expressing mice and cell lines containing the alternative PLZF-RARA fusion have uncoupled differentiation and leukemic cell death, suggesting that exclusive acceptance of a differentiation paradigm belies ATRA’s true mechanism of action. In keeping with these recent findings, while even low-dose ATRA induces differentiation in APL blasts in vivo and in vitro and induces remission in up to 90% of patients with newly diagnosed APL, these affects are transient without additional chemotherapy or As ₂ O ₃ (see later). Although ATRA therapy results in a lifting of the repression complex that inhibits APL blast differentiation, higher doses, As ₂ O ₃ , or chemotherapy is needed to degrade the PML-RARA fusion protein and halt the self-renewal process, ultimately eliminating the malignant clone. Elimination of PML-RARA may further result in more durable cures by targeting the LIC population in APL in which the initial t(15;17) occurred, as well as restoring the normal function of the intracellular PML bodies disrupted by the presence of the fusion protein. Thus recent strategies combine ATRA with conventional chemotherapy, resulting in improved prognosis for patients with APL compared with chemotherapy alone. A long-term analysis has demonstrated significantly higher actuarial EFS rates, lower relapse rates, and improved OS in the group treated with both ATRA and chemotherapy. The addition of ATRA to the chemotherapy regimen has been particularly effective for pediatric patients. The APL93 trial used ATRA in combination with daunorubicin and cytarabine in induction, and the children in this trial had a complete remission rate of 97%. The PETHEMA and GIMEMA-AIEOPAIDA groups have completed pediatric trials using ATRA and idarubicin as induction chemotherapy, with reported CR rates of 92% to 96%. As such, this lower dose has become the standard for current North American APL studies in children. Current recommendations are to initiate ATRA therapy early, at the time when a diagnosis of APL first is suspected, to correct and prevent devastating consequences from an evolving coagulopathy.

One potentially life-threatening complication of ATRA therapy is the retinoic acid syndrome. This syndrome occurs in approximately 25% of patients and is characterized by fever, respiratory distress, pleural or pericardial effusions, edema, hypotension, and, in some cases, renal failure. In most patients, the symptoms are preceded by an increasing WBC count. Retinoic acid syndrome can be life threatening, and the mortality rate ranges from 8% to 15% in different reports. The syndrome is also associated with a higher risk for bone marrow and extramedullary relapse. The use of chemotherapy concurrently with ATRA therapy during induction, particularly in patients with high presenting WBC counts, results in a significantly lower incidence of fatal retinoic acid syndrome ; and given the dose dependence, ATRA syndrome now occurs less frequently with the use of 25 mg/m ² as the standard therapeutic dosing (see later). The use of high-dose corticosteroids at the first sign of symptoms has also been shown to be effective in preventing ATRA syndrome and reducing its mortality rate. Another serious complication of ATRA therapy is pseudotumor cerebri, presenting as headache and papilledema. True pseudotumor cerebri is observed in up to 16% of children, and an additional 39% of children develop headaches without other signs of increased intracranial pressure. Pseudotumor cerebri may require treatment with diuretics such as acetazolamide or serial lumbar punctures to reduce intracranial pressure; and, in some cases, discontinuation or dose reduction of ATRA is required. Close follow-up with an ophthalmologist is advised because these patients may suffer visual loss, which in rare cases may be irreversible. Because of the high rate of ATRA-related side effects, the AML-BFM study group studied dose reduction of ATRA during induction from 45 to 25 mg/m ² /day. They found that this dose was still effective, although in pediatric patients there was still a very high rate of CNS side effects, with 57% of children exhibiting headache, increased intracranial pressure, or frank pseudotumor cerebri. The CALGB trial C9710 included pediatric patients and used the higher dose of ATRA, but recent data demonstrated no additional benefit and only increased toxicity compared with 25 mg/m ² dosing. Therefore this lower dose was incorporated into the recently closed first exclusively pediatric COG APL study, AAML0631, and this dose is now considered the standard of care in pediatric APL.

Arsenic Trioxide

Arsenic trioxide (As ₂ O ₃ ,) has been used for decades in China as treatment for leukemia. The precise mechanism of action is actively being studied, but As ₂ O ₃ appears to induce both differentiation and apoptosis of APL blasts and to degrade the PML-RARA fusion protein while not affecting transcriptional regulation per se. As ₂ O ₃ appears to induce nonterminal differentiation followed by apoptosis via the caspase pathway. When As ₂ O ₃ was used as a single agent in patients with APL, remission rates of 65% to 85% were reported and the 10-year survival was as high as 30%. Later studies done in China and the United States showed favorable results, with moderate toxicity, using As ₂ O ₃ in patients with relapsed APL who had previously received ATRA therapy. Several studies have shown promising results using As ₂ O ₃ in remission induction and consolidation therapy for patients with newly diagnosed APL. One small series of 11 children with newly diagnosed APL treated with As ₂ O ₃ as a single agent showed mild toxicity, including leukocytosis, skin changes, and mild neuropathy. CR was induced in 91% of these patients, with 1 patient dying of cerebral hemorrhage during induction. With a relatively short median follow-up of 30 months, the DFS was 81% with only 1 relapse; the patient who experienced relapsed achieved a second CR, and thus the OS rate remains 91%. On the U.S. phase I pediatric study of As ₂ O ₃ for relapsed leukemia, 13 patients with relapsed APL received As ₂ O ₃ intravenously daily at 0.15 mg/kg/dose for 5 days per week for 4 weeks with a 2-week break with maximum of 70 doses. A CR rate of 85% was observed after a median of 20 doses. Dose-limiting toxicities included rare vascular leak, anorexia, neuropathic pain, and QTc prolongation (see later).

ATRA and As ₂ O ₃ have been used safely together as induction therapy in children and adults. The drugs may have a synergistic effect when used together; one study showed a shorter median time to CR in the group receiving the combination (40.5 days for ATRA alone, 31 days for As ₂ O ₃ alone, 25.5 days for ATRA with As ₂ O ₃ ). The combination also provided longer duration of remission in this study. Because of these promising data, several groups have included As ₂ O ₃ as part of induction or consolidation therapy for newly diagnosed pediatric patients with APL, with the hope of reducing the total cumulative dose of anthracycline exposure. As ₂ O ₃ was used in a randomized fashion during consolidation in the CALGB 9710 trial that included pediatric patients through the COG and demonstrated a dramatic improvement in outcome. Patients 15 years of age and older who had the addition of As ₂ O ₃ after a common ATRA and chemotherapy induction demonstrated significantly improved 3-year EFS (77% vs. 59%) and OS (86% vs. 77%) compared with patients who received ATRA and chemotherapy alone. These results led to the nonrandom incorporation of As ₂ O ₃ into the successor COG study AAML0631, such that all patients nonrandomly received As ₂ O ₃ in a dose of 0.15 mg/m ² for 5 days a week for five courses during consolidation therapy.

As ₂ O ₃ therapy is generally well tolerated, often with only minimal and reversible toxicity. However, care must be taken with its use, because it can produce a prolonged QTc interval, which may be asymptomatic but can progress to torsades de pointes and fatal cardiac arrhythmia. In one retrospective analysis, the rate of arrhythmia was significantly higher in African American patients (3 of 4) versus non–African-American patients (1 of 73). QTc intervals should be followed closely by weekly electrocardiography during As ₂ O ₃ treatment, and hypokalemia and hypomagnesemia should be meticulously avoided. Because of the induction of cellular differentiation, As ₂ O ₃ commonly results in a condition similar to retinoic acid syndrome, most often characterized by fluid retention and pleural and pericardial effusions. This may be successfully treated in the same way as ATRA-induced retinoic acid syndrome. As ₂ O ₃ can also cause a significant polyneuropathy, particularly when given in repeated courses. Less dangerous side effects include skin changes with rash, hyperpigmentation, keratosis, and transient liver function test abnormalities. In a COG phase I study of As ₂ O ₃ in children with refractory or relapsed acute leukemia, dose-limiting toxicities included prolonged QTc, pneumonitis, and neuropathic pain, whereas non–dose-limiting toxicities included elevated liver function test results, nausea, vomiting, abdominal pain, constipation, electrolyte changes, hyperglycemia, dermatitis, and headache.

Owing to their synergistic effects, ATRA and As ₂ O ₃ were both used upfront on the AAML0631 study and were generally well tolerated. Although used together with chemotherapy on this recently completed trial, the efficacy of this combination and recent adult studies demonstrating the ability of this combination therapy to lead to sustained remission without additional cytotoxic chemotherapy are likely to lead to future trials in which additional chemotherapy is significantly reduced or even eliminated to attain a first CR at least in standard patients. Most striking was the recent APL0406 study, which randomized adult APL patients to ATRA and As ₂ O ₃ or ATRA and idarubicin in induction and found a 2-year EFS of 97% versus 86.7% in favor of the ATRA/As ₂ O ₃ trial arm. Thus the addition of anthracyclines and cytarabine may be reserved for APL patients designated high risk at diagnosis based on WBC count, those with more resistant disease (PML-RARA positive post-consolidation [see later]), or first recurrence.

Cytarabine

Although cytarabine is the backbone of induction chemotherapy for other subtypes of AML, its role in APL has been less clear. The PETHEMA group completed a pediatric trial using ATRA and idarubicin as induction chemotherapy without cytarabine, and the CR rate was 92%. The GIMEMA-AIEOPAIDA protocol also used ATRA and idarubicin during induction without cytarabine and showed a 96% CR rate in children. However, this question was subsequently studied in a randomized clinical trial in which patients younger than 60 years and with a presenting WBC count less than 10,000/µL were randomly assigned in induction to ATRA and daunorubicin, with or without cytarabine. CR rates were comparably high for the two groups—99% with cytarabine and 94% without; however, in the cytarabine arm, the relapse rate was significantly lower (4.7% vs. 15.9%; P = .011), the EFS higher (93.3% vs. 77.2%; P =.0021), and the OS higher (97.9% vs. 89.6%; P = .0066). Of note, the current trials all use high cumulative anthracycline doses to achieve excellent OS rates but there is concern about long-term cardiac toxicity, particularly in pediatric patients. Considering the improved results with the addition of cytarabine to the induction and consolidation regimens, several groups have been studying the use of more cytarabine with less anthracycline in pediatric patients with APL. On AAML0631, 6 g/m ² of cytarabine was given during the second consolidation cycle for all patients, with an additional 6 g/m ² of cytarabine for high-risk patients. The cumulative anthracycline dose was decreased from 655 mg/m ² on prior trials to 355 mg/m ² for standard-risk patients and 405 mg/m ² for high-risk patients.

Maintenance Therapy

Another difference between APL and other subtypes of AML historically has been an identified benefit from maintenance therapy. The APL93 trial randomized pediatric patients after consolidation to an arm with no further therapy or to arms with 2 years of intermittent ATRA, continuous chemotherapy with 6-mercaptopurine and methotrexate, or both ATRA and chemotherapy. Although 23% of pediatric patients in this trial experienced relapse, none of the patients who received both chemotherapy and ATRA during maintenance therapy did so. In this trial, all patients with relapsed APL achieved a second CR. The 5-year EFS was 71%, and OS was 90%. The PETHEMA group also used ATRA plus 6-mercaptopurine and methotrexate during 2 years of maintenance therapy for pediatric patients and demonstrated a DFS of 82% and an OS of 87%. Since these earlier studies, all recent trials for APL have included a maintenance phase of therapy. To be congruent with parallel European studies, the recently completed AAML0631 study included 2 years of maintenance therapy with ATRA, 6-mercaptopurine, and methotrexate, whereas the CALGB study only included a single year of maintenance with the same agents. However, the future of maintenance therapy in APL is uncertain. The need for chemotherapy versus ATRA alone in maintenance therapy has been raised. Preliminary results of upfront ATRA and As ₂ O ₃ in both Italian and M.D. Anderson Cancer Center adult trials without inclusion of maintenance therapy have prompted consideration of elimination of maintenance therapy altogether. Inclusion of maintenance therapy and whether any chemotherapy is necessary will be key therapeutic goals explored in upcoming trials.

Targeted Therapy

With the use of ATRA and As ₂ O ₃ , targeted therapy for APL has become a reality. Other targeted agents are also being used successfully in APL. GMTZ (see earlier) is a recombinant, humanized, anti-CD33 monoclonal antibody linked to the tumor antibiotic calicheamicin. APL cells express very high levels of CD33, making them an attractive target for GMTZ. GMTZ has been used successfully as a single agent to induce remission in patients with multiply relapsed APL. In a small series of 12 patients, GMTZ was used safely in combination with ATRA with or without idarubicin, depending on the extent of MRD, as induction therapy for patients with de novo APL with a CR rate of 84%. Some groups are now studying the use of GMTZ as induction therapy in larger numbers of patients. Although not currently used initially for children with APL, GMTZ may be included for the management of high-risk patients and standard-risk patients who have a rise in their WBC count over 10,000/µL during induction therapy or, alternatively, as a strategy to induce remission after relapse in patients who have already received ATRA, As ₂ O ₃ , and cytotoxic chemotherapy.

As discussed earlier, mutations in FLT3 have been identified in up to 43% of patients with APL, making the use of FLT3 inhibitors an attractive option. Most FLT3 inhibitors are oral agents with little toxicity. In one mouse model of APL with the FLT3 mutation, treatment with doxorubicin alone had no impact on survival, but treatment with the FLT3 inhibitor SU11657, with or without doxorubicin, resulted in prolonged survival. In another mouse model, treatment with ATRA and SU11657 resulted in the rapid regression of APL. There is considerable interest in using FLT3 inhibitors in combination with ATRA and other targeted agents to improve therapy for patients with APL.

Hematopoietic Stem Cell Transplantation

Because the combination of chemotherapy and ATRA, and now ATRA and As ₂ O ₃ , results in such high cure rates for APL, HSCT is not recommended for patients in first CR, even with a matched sibling donor. There is evidence for prolonged remission without HSCT, even after relapse for patients treated with agents such as ATRA, As ₂ O ₃ , and GMTZ, especially when combined with chemotherapy. Both autologous and allogeneic HSCT have been used successfully in patients with relapsed APL. For autologous HSCT to be successful, peripheral blood stem cells or bone marrow cells must be harvested once the patient has achieved negative MRD by PCR assay. The GVL effect appears to play a role in the allogeneic HSCT treatment of APL, as evidenced by the fact that relapse rates are significantly lower after allogeneic HSCT compared with autologous HSCT and that patients who suffer a molecular relapse after allogeneic HSCT may achieve remission after the withdrawal of immunosuppression. However, despite the lower relapse rate, because of significantly higher transplantation-related mortality (TRM) after allogeneic HSCT, OS in adults is higher after autologous HSCT than after allogeneic HSCT (postallogeneic HSCT: DFS, 92.3%; EFS, 52.2%; OS, 51.8%; TRM, 39%; and postautologous HSCT with negative preharvest PCR: DFS, 87.3%; EFS, 76.5%; OS, 75.3%; TRM, 6%). In pediatric patients, allogeneic HSCT is much better tolerated. A retrospective analysis demonstrated no difference in OS after allogeneic HSCT versus autologous HSCT, although the allogeneic HSCT group had a lower relapse rate and higher TRM (postallogeneic HSCT: RR, 10%; EFS, 71%; OS, 76%; TRM, 19%; and postautologous HSCT: RR, 27%; EFS, 73%; OS, 82%; TRM, 0%). Given these data, in adults, autologous HSCT is usually the preferred choice versus allogeneic HSCT for patients in second CR, whereas in children, allogeneic HSCT is often the preferred choice, particularly if a fully matched sibling donor is available.

Pathobiology

Down syndrome, characterized by trisomy 21, is the most common congenital chromosomal abnormality, occurring in an estimated 1 in 600 to 1000 live births. Up to 10% of infants with Down syndrome are born with a transient myeloproliferative disorder (TMD) (also referred to as transient leukemia [TL] or transient abnormal myelopoiesis [TAM]). TMD is characterized by the presence of immature megakaryoblasts in the liver, bone marrow, and peripheral blood. The disorder usually spontaneously regresses within 3 months. By morphology, TMD blasts are identical to AMKL blasts and, even though in most cases they regress, up to 30% of Down syndrome infants with TMD subsequently develop AMKL within 3 years. Studies have shown that TMD blasts are clonal and that the same clone subsequently evolves into AMKL in a subset of patients.

In 2002, Wechsler and colleagues first reported mutations in exon 2 of GATA1 in Down syndrome–associated AMKL. Soon thereafter, several groups reported that GATA1 mutations were detectable in almost all patients with Down syndrome–associated TMD. Interestingly, GATA1 mutations have also been detected in approximately 10% of Down syndrome patients who have never been diagnosed with a hematologic abnormality. It is not clear whether these patients have a history of subclinical TMD that regressed without detection or if the mutation simply did not result in the TMD phenotype in these cases. Normal GATA1 plays a vital role in the maturation of erythroid cells and megakaryocytes. The GATA1 mutations in Down syndrome TMD and AMKL result in GATA1s, a truncated protein lacking the amino-terminal activation domain. GATA1s appears to be a normal isoform of GATA1 and has been detected in human hematopoietic cell lines and mouse fetal liver. However, because GATA1 is an X-linked gene, only the truncated GATA1s is expressed in Down syndrome TMD and AMKL blasts. It has been hypothesized that the presence of a trisomy 21 in conjunction with a GATA1s allows abnormal proliferation of megakaryoblasts, but only in the fetal hematopoietic system (i.e., the fetal liver); thus TMD resolves with the loss of fetal hematopoiesis. Although the combination of trisomy 21 and a GATA1 mutation appears sufficient to result in TMD, this is not sufficient to result in frank leukemia, and only if additional mutations are accumulated over time will the disease progress to frank AMKL.

Wilms tumor gene (WT1) levels detected by PCR assay are elevated in patients with TMD. Levels normalized in four of five patients studied who had regression of TMD, with no further sequelae, but did not normalize in the fifth, who went on to develop AMKL at 11 months of age, making this an attractive marker for MRD and prognosis. Both gain- and loss-of-function mutations in Janus kinase 3 (JAK3) have been identified in a subset of patients with TMD, although the prognostic relevance of this is not yet known.

Clinical Presentation

Because Down syndrome infants with TMD are often asymptomatic, the incidence of 10% may be an underestimate, because many patients may have TMD that spontaneously regresses, without ever being detected. For this reason, many physicians obtain a routine screening complete blood cell (CBC) count at birth for all infants with known or suspected Down syndrome. However, some infants with Down syndrome have a dramatic presentation of TMD at or shortly after birth, with hydrops fetalis, pleural and pericardial effusions, ascites, and massive hepatosplenomegaly. These patients may rapidly progress to liver fibrosis or multisystem organ failure, both of which are most often fatal. In the Pediatric Oncology Group (POG) trial 9481, 48 children with TMD were followed prospectively; 19% developed life-threatening disease with hepatic fibrosis or cardiopulmonary failure, with an overall mortality at 3 months of age of 10.4%. Another retrospective analysis from Japan has shown that 22.9% of patients with TMD die before the age of 6 months, and the main causes of death were hepatic or cardiopulmonary failure. In this study, early gestational age, WBC count greater than 100,000/µL, high percentage of peripheral blasts, elevated aspartate aminotransferase (AST), elevated direct bilirubin, and low Apgar score were significantly associated with poor survival. They noted that only 7.7% of term Down syndrome infants with a WBC count less than 100,000/µL died, whereas 54.5% of preterm infants with a WBC count higher than 100,000/µL died, suggesting that risk stratification may be possible to determine high-risk subgroups of patients who require earlier and more aggressive therapy.

Treatment

Because most patients with TMD will exhibit spontaneous regression, treatment often consists of supportive care only. Many groups, including COG protocols 2971 and 08B1, have recommended supportive care only for TMD patients unless they exhibit signs or symptoms of life-threatening disease, including hyperviscosity, with a total blast count higher than 100,000/µL, organomegaly causing respiratory compromise, congestive heart failure not caused by a congenital heart defect, hydrops fetalis, life-threatening hepatic dysfunction, defined as significant (grade 4 level toxicity) DIC, hyperbilirubinemia, ascites, or transaminitis. For patients who require therapy, the choices include leukapheresis, exchange transfusion, or low-dose cytarabine. Low-dose cytarabine has been effective when used early in critically ill patients. In patients with hyperleukocytosis or who require therapy with cytarabine, treatment to prevent the consequences of rapid tumor lysis (see earlier) should be promptly initiated.

Pathobiology

Children with Down syndrome have a markedly increased risk for leukemia during the first 10 years of life. Acute leukemia is 10 to 20 times more common in children with Down syndrome than in children in the general population, and leukemia in Down syndrome patients younger than 4 years is most commonly AML (specifically AMKL), whereas leukemia in older children with Down syndrome is most commonly ALL. The ratio of ALL to AML is approximately 1 : 1 in children with Down syndrome, but in the first 4 years of life, AML is 100 times more common than ALL. Although AMKL is a rare subtype of AML in the general population, it is the predominant form of AML in children with Down syndrome, making Down syndrome patients 500 times more likely than other children to develop AMKL. Unlike other children with AML, AMKL in children with Down syndrome often manifests after a period of myelodysplasia, usually characterized by months of thrombocytopenia and later by anemia. Most cases of AMKL in Down syndrome patients are preceded by TMD (see earlier). Several studies have confirmed that when there is a proven antecedent TMD, the same TMD clone subsequently evolves into AMKL.

Also characteristic of AMKL in Down syndrome are GATA1 mutations (see earlier). Because GATA1 encodes a transcription factor essential for megakaryocyte development, samples from patients with AMKL were studied for evidence of GATA1 mutations. Mutations in exon 2 of GATA1 were detected in AMKL blasts from Down syndrome patients but not in other subtypes of AML in Down syndrome patients or in AMKL blasts from non–Down syndrome patients. Several subsequent studies have confirmed GATA1 mutations leading to a premature stop codon in almost all cases of Down syndrome–associated AMKL. GATA1 mutations have been identified in the blood spots of three of four Down syndrome patients with AMKL who had not been previously diagnosed with TMD, suggesting that in most cases of AMKL the mutation has been present since birth. Down syndrome patients who develop AML after the age of 4 years do not usually have GATA1- mutated AMKL but have AML more typical of the general pediatric population (non–Down syndrome AML). This disease is not as sensitive to chemotherapy and does not carry the exceptionally good prognosis carried by Down syndrome AMKL (see later).

Additional mutations beyond the GATA1 mutation in the presence of trisomy 21 are necessary to produce AMKL. The good-risk translocations common in other forms of pediatric AML, including t(8;21), t(15;17), inv(16), and t(9;11), are almost never seen in Down syndrome AMKL, although they are more commonly reported in Down syndrome patients who develop AML after the age of 4. Other than trisomy 21, trisomy 8 is the most common cytogenetic abnormality in Down syndrome AMKL. Other translocations commonly reported in AMKL in the absence of Down syndrome, including t(1;22) and t(1;3), are only rarely documented in Down syndrome AMKL. Monosomy 7, a widely accepted marker of pediatric and adult high-risk AML in the absence of Down syndrome, is also seen in 3.5% to 10% of patients with Down syndrome and AML and, although data are limited, it is generally associated with a worse outcome (see later). Approximately 25% of patients have no cytogenetic abnormalities other than the constitutional trisomy 21, although some patients will exhibit tetrasomy 21. As in TMD, overexpression of WT1 and mutations in JAK3 have been reported in Down syndrome AMKL. More recently, next generation sequencing approaches have identified a number of potential somatic mutational drivers that might contribute to the progression of TMD to AMKL including mutations in the cohesin complex; the epigenetic regulator, EZH2; as well as activating lesions in the RAS, WNT, mitogen activated protein kinase (MAPK) and phosphoinositide 3-kinase (PI3K) pathways.

Clinical Presentation

Children with Down syndrome who develop AML present at a younger age than average for childhood AML, with 60% to 70% of patients diagnosed when they are younger than 2 years. Down syndrome patients generally present with lower total WBC counts (median, 7,000 to 10,000/µL) and lower platelet counts (median, 25,000 to 30,000/µL) than other children. Hepatosplenomegaly is more common in Down syndrome patients with AML than in other children, whereas lymphadenopathy and CNS involvement are less common. Down syndrome AMKL is frequently preceded by TMD, which is almost absent in non–Down syndrome patients, or MDS, which is far less common in non–Down syndrome patients. Older Down syndrome patients with AML have presenting signs and symptoms that more closely mirror those of the general population, reflecting the virtual lack of AMKL with GATA1 mutations in patients older than 5 years.

Treatment and Outcome

Many children with Down syndrome have been entered into large clinical trials since 1982, when legislation was passed that prohibits withholding medical intervention from children with disabilities. In the early clinical trials that included patients with Down syndrome and AML, the outcome was poor because of excessive toxicity. The associated congenital abnormalities, increased susceptibility to infection, and altered drug metabolism in patients with Down syndrome made it challenging to find appropriate therapy for this group. However, while general medical care for children with Down syndrome has improved, the outcome for children with AML and Down syndrome has also improved. Multiple studies from around the world have consistently demonstrated that Down syndrome AML patients have a better overall prognosis than children with AML in the general population. A remarkable feature of AML in children with Down syndrome is its extraordinary responsiveness to AML chemotherapy. Compared with non–Down syndrome AML, leukemic blasts are 4.5-fold and 12-fold more sensitive in vitro to cytarabine (median half maximal inhibitory concentration [IC ₅₀ ], 77.5 vs. 350.9 nM) and daunorubicin (median IC _50, 5.8 vs. 71.2 nM), respectively. The increased sensitivity of Down syndrome AML blasts to cytarabine has been explained by a 5.2-fold greater accumulation of the active drug metabolite cytarabine-CTP compared with non–Down syndrome AML blasts. Down syndrome AMKL blasts are significantly more sensitive to other chemotherapy agents, including amsacrine (16-fold), etoposide (20-fold), 6-thioguanine (3-fold), busulfan (5-fold), and vincristine (23-fold). In addition to constitutionally expressed genes encoded on chromosome 21, which modulate drug metabolism in Down syndrome AML blasts, such as cystathionine-β-synthase (CBS), the expression of mutant GATA1 protein itself contributes to increased drug accumulation. In addition, the cytidine deaminase (CDA) levels in Down syndrome AMKL blasts are low, leading also to increased sensitivity to cytarabine. The CDA promoter contains several binding sites for GATA1, suggesting that GATA1 mutations may contribute to the sensitivity of these blasts.

Observations of increased in vitro sensitivity of Down syndrome AMKL blasts to cytotoxic drugs have been complemented by pioneering studies in which very low doses of cytarabine, which were administered over a prolonged period of time (10 mg/m ² subcutaneously for 12 hours for 7 days every 2 weeks for 2 years), resulted in durable remissions in as many as 67% of cases (intention to treat analysis ). Although outcomes of the very-low-dose cytarabine regimen eventually did not match those of standard dose cytarabine, the observation highlights a unique degree of drug sensitivity of Down syndrome AMKL blasts to cytarabine that is not encountered in pediatric non–Down syndrome AML. Recently, Japanese investigators showed that high-dose cytarabine could successfully be eliminated from a treatment protocol for Down syndrome AML while maintaining a 4-year EFS of 83%. In parallel, studies maximizing dose intensity for the treatment of AML, although successful in the general pediatric population, did not translate into any improvements in EFS for patients with Down syndrome and AML owing to excessive mortality during induction therapy in this group. In the German collaborative AML-BFM93 protocol, children with Down syndrome fared worse than other patients because of an increased frequency of infectious complications, but none of the patients experienced relapse and EFS was approximately 70%. Therefore, in the subsequent AML-BFM98 protocol, Down syndrome patients received a reduced intensity regimen and the EFS improved dramatically, to almost 90%. In the Nordic Society for Paediatric Haematology and Oncology-Acute Myeloid Leukaemia (NOPHO-AML) trials, the earlier, more intensive NOPHO-AML88 trial showed a high death rate from infectious complications for Down syndrome patients with AML, with an EFS of only 47%, compared with much better outcomes in the later, less-intensive NOPHO-AML93 trial, with an EFS of 85% (76% for those who received full-dose therapy and 92% for those who received reduced-dose therapy). The MRC trials showed similar results with Down syndrome patients receiving the same standard therapy as other patients, resulting in a higher rate of death from toxicity but lower relapse rate when compared with other children. Perhaps the most striking example comes from the CCG 2891 study. In this study, all children were randomly assigned to receive induction chemotherapy with intensive timing (i.e., proceeding with the second cycle, regardless of counts) versus standard timing (i.e., awaiting count recovery before initiating the second cycle). The Down syndrome patients in the intensive timing arm of the study suffered a 32% mortality rate compared with an 11% mortality rate for the remainder of the patients, prompting early closure of that study arm for Down syndrome patients. Standard timing induction for Down syndrome patients resulted in an impressively high CR rate of 95% (2.4% with toxic death and 2.4% with resistant disease). Postremission consolidation with allogeneic HSCT in this trial also resulted in an unacceptably high treatment-related mortality for Down syndrome patients, with only 33% survival at 4 years. In this study, standard timing induction followed by consolidation with chemotherapy was clearly the superior treatment for Down syndrome patients, with a 4-year DFS of 88% compared with a DFS of only 42% for this regimen in the non–Down syndrome patients. Given these collective data, COG trial A2971 used a less intensive approach for Down syndrome patients, with four cycles of standard timing low-dose cytarabine, daunorubicin, and thioguanine, followed by a cycle of high-dose cytarabine with asparaginase and then intrathecal therapy. Five-year outcome data were comparable to those of CCG2891 with OS of 84% and EFS of 79%.

The first toxicity to which DS-AML patients were identified as being particularly susceptible was cardiotoxicity. On the POG9421 AML study in which the total cumulative anthracycline equivalent dose was 535 mg/m ² , 21% of DS children developed congestive heart failure with diminished shortening fractions on echocardiogram requiring chronic diuretics and/or inotropes. Sixty percent of these patients had congenital heart defects, and a third of the patients with cardiac dysfunction died. On both CCG2891 and A2971, the cumulative anthracycline dose was reduced to 350 mg/m ² , but together with the overall reduction in chemotherapy on A2971, grade 3 or higher cardiac toxicity occurred in only 4% of patients during induction and 2% of patients during intensification. The most recent COG Down syndrome AML study, AAML0431, was designed to further reduce the cumulative dose of anthracycline based on A2971 and gave a cumulative dose of only 240 mg/m ² . The study closed in December 2011 and to date no significant cardiac toxicity has been reported (J. Taub, personal communication, June 2014). However, the second cycle of induction chemotherapy during which high-dose cytarabine is given resulted in the greatest number of adverse events, including longest median duration, longest period of myelosuppression, and highest rate of bacterial infections. These toxicities, together with prior data demonstrating the exquisite sensitivity of Down syndrome AMKL blasts to cytarabine, have prompted plans for elimination of high-dose cytarabine for the majority of patients on the upcoming COG Down syndrome AML trial.

Despite the overall favorable outcomes for AML in Down syndrome, 15% to 25% of patients do not achieve long-term survival with current treatment protocols, predominantly owing to relapse or refractory disease. Lack of response to therapy is associated with very poor outcomes with survival rates of 20-26% even after HSCT. Prognostic markers in AML in Down syndrome are few because many of the predictive genetic lesions discussed earlier for non–Down syndrome AML are very infrequent findings in Down syndrome patients. To date, age older than 4 years and monosomy 7 appear the only known high-risk factors, both of which are relatively rare, and the outcome in patients harboring a monosomy 7 is still somewhat controversial. In the BFM-93 trial, 3 of 4 patients older than 4 years lacked the typical AMKL of Down syndrome and only 1 of the 3 survived. In a combined analysis of European trials, 17 of 317 Down syndrome patients diagnosed with AML were older than 4 years. DNA was available from blasts of 10 of these patients and, of these, 3 children younger than 7 years had the characteristic AMKL and 1 additional younger child had M0 AML with a GATA1 mutation. Of the 6 remaining patients without AMKL or a GATA1 mutation, all were 7 years of age or older and 4 experienced relapse. In CCG 2891, children older than 4 years of age ( n = 9) had a significantly lower EFS at 6 years of only 33% ± 31%. On A2971, patients younger than 4 years of age fared significantly better than older children (5 year EFS, 81% ± 7% vs. 33% ± 38%), although only 6 children older than age 4 years were included. An international retrospective study of 451 Down syndrome patients with AML found monosomy 7 was associated with a moderately worse outcome, with a 5-year EFS of 69%. CCG studies 2891 and A2971, as well as a recent Japanese study, found that only 11 of 16 patients with Down syndrome and AML and monosomy 7 were in complete clinical remission. These limited data suggest that older children with Down syndrome and AML, particularly given the absence of GATA1 mutations, have disease more reminiscent of non–Down syndrome AML and should be treated more aggressively. Similarly, the presence of monosomy 7 may contribute to an inferior outcome and justify more intensive therapy for this cohort. However, most children with Down syndrome and AML are younger than age 4 years and lack monosomy 7. For this group, similar to non–Down syndrome AML, MRD after induction may enable the ability to identify high-risk patients. These data will be forthcoming from the recently closed AAML0431 study and will inform future clinical trials.

Morphology

The morphologic features of MDS include bone marrow dysplastic changes involving all three cell lineages (i.e., trilineage dysplasia). A common theme underlying these features is asynchrony between the usual cytoplasmic and nuclear differentiation programs, so that nuclei appear abnormal and less mature than the features of the surrounding cytoplasm. Typical erythrocytic abnormalities include megaloblastoid changes, perinuclear ringlike deposits of iron (i.e., ringed sideroblasts), multinucleation, nuclear budding and fragmentation, and an increased percentage of immature forms. Dysmegakaryopoiesis is characterized by micromegakaryocytes, abnormal nuclei, and abnormal nuclear lobulation. The dysplastic features of the granulocytic cells comprise immature forms, hypogranulation, and the Pelger-Huët–type abnormality. Monocytic dysplasia includes increased numbers of bone marrow monocytes, abnormal granulation with increased azurophilic granules, hemophagocytosis, abnormal nuclei, and giant forms. Bone marrow cellularity is usually normal or increased, and the reticulin concentration is increased in most cases.

Patients with MDS may present initially with a single peripheral blood cytopenia, which usually progresses to pancytopenia with anemia, leukopenia, and thrombocytopenia. The anemia in MDS is caused by ineffective erythropoiesis with low reticulocyte counts and macrocytosis, teardrop cell formation, and moderate poikilocytosis. In addition, megaloblastoid circulating nucleated red blood cells are frequently detected. Peripheral blood dysgranulopoiesis manifests as circulating myeloblasts, progranulocytes, and Pelger-Huët cells. Thus early signs may be a falling hematocrit with increasing mean corpuscular volume or unexplained thrombocytopenia. The peripheral blood may also exhibit increased numbers of monocytes and monoblasts.

Cytogenetics

Cytogenetic abnormalities are common in children with MDS and were reported in 59% of 227 children tested in one study. Chromosomal deletions are the hallmark of MDS and frequently involve the long arms of chromosomes 5, 7, and 20 ( Fig. 51-8 ). Other common cytogenetic abnormalities include trisomy 8, del(17p), +21, inv(1), and t(7;16). These chromosomal abnormalities are accompanied by other complex karyotypic changes in many cases. Other abnormalities that have been reported in pediatric cases of MDS include +6, +9, and +11, whereas deletions of chromosomes 11, 12, and 13 and Y are rare. Common balanced chromosomal translocations found in AML, such as t(8;21), t(15;17), and inv(16), are not usually detected in MDS.

The hallmark of myelodysplastic syndrome (MDS) is the loss of specific chromosomal regions. Deletions of the long arm of chromosomes 5 and 7 (5q−, 7q−) occur in de novo MDS and acute myelogenous leukemia (AML) but much more frequently in therapy-related MDS and therapy-related AML after treatment with alkylating agents. 20q− abnormalities are less common but also occur in both de novo MDS and AML and after prior chemotherapy.

(Data from Heaney ML, Golde DW: Myelodysplasia. N Engl J Med 340:1649–1660, 1999; Hofmann WK, Lubbert M, Hoelzer D, et al: Myelodysplastic syndromes. Hematol J 5:1–8, 2004; Pedersen-Bjergaard J, Andersen MT, Andersen MK: Genetic pathways in the pathogenesis of therapy-related myelodysplasia and acute myeloid leukemia. Hematol Am Soc Hematol Educ Prog 2007:392–397, 2007.)

Classification

In 1982, the FAB cooperative group defined five categories for the adult myelodysplastic and myeloproliferative syndromes—refractory anemia (RA), RA with ringed sideroblasts (RARS), RA with excess blasts (RAEB), RA with excess blasts in transformation (RAEB-T), and chronic myelomonocytic leukemia (CMML). Classification of childhood MDS into defined FAB subcategories is not always straightforward. Many children show overlapping features of MPD and MDS, particularly patients with JMML and infant monosomy 7 syndrome. The classification of pediatric MDS is also complicated by the fact that some children develop MDS in the context of inherited predispositions such as neurofibromatosis type 1, severe congenital neutropenia, and Down syndrome. Using the FAB system of classification, the most common subtypes of MDS in children are RAEB and RAEB-T, whereas RA and RARS are uncommon and CMML is rare.

The WHO proposed a revised classification of MDS in 1997. The WHO system includes the following changes to the FAB criteria: elimination of the RAEB-T subtype through a reduction in the marrow blast count to 20% for the diagnosis of AML and the division of the RAEB subtype into RAEB-I, consisting of 5% to 10% marrow blasts, and RAEB-II, comprising 11% to 20% blasts. Other WHO recommendations include two new categories for patients with refractory cytopenias with multilineage dysplasia and a new category for unclassified MDS. In the new system, CMML is removed completely and reclassified as a myeloproliferative disorder.

A third classification system is used in parallel with the FAB and WHO systems, the IPSS Scoring System. This system assigns a numeric value to each of the number of cytopenias, cytogenetic abnormalities, and percentage of bone marrow blasts, and a higher score is associated with a poor prognosis. The use of the IPSS in children is of somewhat limited value. The only factors found to be predictive were bone marrow blasts greater than 5% and platelet counts greater than 100,000/µL.