Improved understanding of the molecular basis of AML was incorporated into the 2008 revision of the World Health Organization (WHO) Classification of Tumours of Haematopoietic and Lymphoid Tissues [1, 2], which necessitates application of a range of laboratory tests including cytogenetics and molecular testing to complement morphological and immunophenotypic assessment for disease diagnosis and categorization. The WHO classification is organized in a hierarchical fashion, with the first group being AML with particular balanced translocations or inversions (and their molecular counterparts), which are defined as “AML with recurrent genetic abnormalities” (Table 40.2). These include t(15;17)(q22;q12~21), the diagnostic hallmark of acute promyelocytic leukemia (APL), which accounts for approximately 12 % of AML cases. Cloning of the translocation breakpoints in the early 1990s [3–5] showed that t(15;17) leads to fusion of the gene that encodes the myeloid transcription factor, retinoic acid receptor alpha (RARA), with a previously unknown gene designated PML (for promyelocytic leukemia), which has subsequently been found to be involved in growth suppression and regulation of apoptosis [6]. While the vast majority of APL cases have an underlying PML–RARA fusion, in approximately 1–2 % RARA is fused to an alternative partner [7] and classified as “AML with a variant RARA translocation.” These rare subtypes of APL include involvement of ZBTB16 (PLZF), NPM1, NUMA, FIP1L1, and BCOR, as a result of the t(11;17)(q23;q21), t(5;17)(q35;q21), t(11;17)(q13;q21), t(4;17)(q12;q21), and t(X;17)(p11;q21), respectively, and PRKAR1A and STAT5B in rearrangements involving 17q [8–14]. The nature of the fusion partner has an important bearing on disease biology, particularly the response to molecularly targeted therapies, i.e., all transretinoic acid (ATRA) and arsenic trioxide (ATO) [15]. ATRA targets the ligand-binding domain of the RARα moiety in the C-terminal region of the fusion proteins (as well as wild-type RARα). Sensitivity to ATRA has been documented in APL subtypes involving PML, NPM1, NUMA, and FIP1L1 [15], whereas PLZF-RARα and STAT5B-RARα have both been associated with primary resistance to retinoids and a poorer prognosis [14, 16, 17]. To date, sensitivity to ATO has only been demonstrated in PML–RARA-positive APL, reflecting the capacity of ATO to bind directly to the PML moiety of the fusion protein inducing its degradation via the proteasome [18]. Therefore, ATO should not be used for the treatment of APL as part of front-line therapy or in the context of suspected relapse, unless positive for PML–RARA.

Approximately 10 % of patients with AML are classified as having core-binding factor (CBF) leukemia with balanced chromosomal rearrangements that disrupt genes that encode components of the heterodimeric transcription factor complex, comprising RUNX1 (AML1, CBFα) and CBFβ, which plays a critical role in hematopoiesis [19]. The CBFα subunit is targeted by the t(8;21)(q22;q22), which fuses RUNX1 (AML1, CBFA2) to the gene encoding the RUNX1T1 (formerly ETO for eight twenty one) transcriptional repressor, thereby potentially silencing RUNX1 target genes [19, 20]. The β-subunit is targeted by the inv(16)(p13.1q22) or the less common t(16;16)(p13.1;q22), in which CBFB is fused to the gene encoding myosin heavy chain (MYH11). RUNX1 is a recurrent translocation target in non-CBF acute leukemias, with fusions to ETV6 (TEL) as a result of the cytogenetically cryptic t(12;21)(p13;q22) in pediatric acute lymphoblastic leukemia (ALL), or to a range of partners in AML [21, 22], including MECOM (EVI1/MDS1) as a result of t(3;21)(q26;q22) in “AML with myelodysplasia-related changes” (Table 40.2). RUNX1 also has been implicated in therapy-related leukemias arising following exposure to drugs targeting topoisomerase II [23, 24].

The MLL (for myeloid/lymphoid or mixed-lineage leukemia, at present named KMT2A) gene located at 11q23 is a further recurrent translocation target in acute leukemia, with almost 80 partner genes now characterized [25]. MLL is an epigenetic regulator that plays a critical role in hematopoiesis, modulating HOX gene expression [26]. The most common MLL translocation observed in AML is the t(9;11)(p22;q23), which occurs in approximately 2 % of cases and leads to fusion of MLL with MLLT3 (formerly known as AF9). AML with t(9;11)(p22;q23) has been associated with a relatively favorable outcome in some pediatric [27] and adult [28] AML studies and is distinguished as a separate entity in the 2008 WHO classification. Apart from the t(9;11), the most frequent other MLL translocations observed in AML are t(6;11)(q27;q23) involving MLLT4 (AF6), t(11;19)(q23;p13.3) involving MLLT1 (ENL), t(11;19)(q23;p13.1) involving ELL, and complex rearrangements between 10p12 and 11q23 (e.g., a reciprocal translocation and an inversion of an 11q segment translocated to 10p12 or inverted insertion of an 11q segment into 10p12 or a 10p segment into 11q23), in which the fusion partner is MLLT10 (AF10) [29, 30]. While translocations involving MLL are observed in de novo leukemia and account for the majority of leukemias presenting in infancy, the locus is also a recurrent translocation target in therapy-related leukemias [31], particularly those arising following exposure to the epipodophyllotoxin class of topoisomerase II inhibitors [32]. Such cases are classified within the WHO as “therapy-related AML.”

The remaining subtypes of AML distinguished as separate disease entities on the basis of cytogenetics are those characterized by the t(6;9)(p23;q34), inv(3)(q21q26.2) or t(3;3)(q21;q26.2), and t(1;22)(p13;q13). The t(6;9)(p23;q34) is found in approximately 1 % of AML and leads to fusion of DEK at 6p23 with NUP214 (CAN) at 9q34, which encodes a component of the nuclear pore complex [33, 34]. The inv(3) or t(3;3) occurs in a similar proportion of AML cases and is associated with upregulation of the zinc finger transcription factor MECOM (EVI1), which is involved in normal hematopoiesis. AML with inv(3) or t(3;3) may present de novo, or secondary to prior myelodysplastic syndrome (MDS), and is characterized by a normal or elevated presenting platelet count and abnormal megakaryopoiesis with micromegakaryocytes in the bone marrow (BM) [35]. MECOM has long been implicated in leukemogenesis, having been identified as a recurrent integration target in murine retroviral mutagenesis screens [36, 37]; further evidence has been provided by characterization of MDS associated with insertional activation of MECOM occurring as a complication of gene therapy for chronic granulomatous disease [38]. Interestingly, these have provided important insights into potential mechanisms underlying acquisition of additional cytogenetic abnormalities in AML, with both cases showing monosomy 7, which is well recognized as a frequent secondary abnormality in cases with inv(3) or t(3;3). Forced overexpression of MECOM was found to disrupt normal centrosome duplication, suggesting that activation of MECOM as a result of retroviral insertion or chromosomal translocation leads to genomic instability, giving rise to acquisition of additional changes such as monosomy 7 involved in progression to MDS and AML.

AML with t(1;22)(p13;q13) is extremely rare (only approximately 40 cases reported worldwide [39]) and is associated with acute megakaryoblastic leukemia occurring in infants and young children (<3 years of age), particularly those without Down syndrome. The translocation fuses RBM15 (for RNA-binding motif protein 15, also known as OTT) with MKL1 (for MegaKaryoblastic Leukemia [Translocation] 1, or MAL) [40, 41], which is involved in normal megakaryocyte maturation [42].

The FLT3 tyrosine kinase has been an intense focus for research. Groups working in Japan in the late 1990s identified two major classes of receptor-activating mutations of FLT3 that, cumulatively, are found in approximately 30 % of AML patients [127, 128]. In normal hematopoiesis, the membrane-spanning FLT3 receptor is expressed on early progenitor cells including CD34+ hematopoietic stem cells and plays an important role in proliferation, survival, and differentiation. High levels of abnormal FLT3 expression are seen in 70–100 % of AML [129].

FLT3 internal tandem duplication mutations (FLT3-ITDs) are in-frame palindromic duplications of exons 14 and 15 of the FLT3 gene, between three and 400 bp in length, that disrupt the autoinhibitory function of the FLT3 juxtamembrane domain, leading to activation of signaling pathways downstream of FLT3. Pooled data show an overall FLT3-ITD incidence of 23 % in newly diagnosed AML, with a lower incidence in children [130]. Although ITD length has previously been thought not to influence prognosis, some longer ITDs, which integrate within the first tyrosine kinase domain of the receptor, are associated with lower complete remission (CR) rate and poor relapse-free survival [131]. FLT3-ITDs are accompanied by NPM1 mutations in a significant proportion of CN-AML patients (see below) [58]. FLT3-ITDs are predominantly seen in CN-AML, being associated with proliferative disease with a high presenting white blood cell (WBC) count and poor prognosis in terms of significantly increased relapse rate and decreased survival in both adults and children [48, 132]. FLT3-ITD mutations at diagnosis are highly heterogeneous with respect to FLT3-ITD allelic ratio, ITD length, and cooperating partner mutations. Patients with a high “ITD mutant-to-wild-type FLT3 allelic ratio,” sometimes due to mitotic recombination leading to partial uniparental disomy, have an especially poor prognosis and usually relapse within 12 months of initial treatment [57]. Relapsed FLT3-ITD-mutated AML carries a particularly dismal prognosis; blasts in this setting are highly dependent on FLT3 kinase signaling, carrying a particularly high FLT3-mutant allelic burden with the wild-type FLT3 allele often virtually absent [133].

A further 7–10 % of AML patients have mutations involving the activation loop within the tyrosine kinase domain of the FLT3 receptor (FLT3-TKDs), usually single base substitutions (most frequently D835Y) or small deletions. Although similarly FLT3 activating and associated with high presenting WBC levels, prognostic implications of FLT3-TKDs are less clear; patient series describe conflicting adverse, intermediate, and even favorable prognostic associations [51, 56, 134]. In contrast to FLT3-ITD mutations, FLT3-TKD mutations appear to frequently be “late genetic hits,” are often lost at relapse, and have subtly different effects than ITDs on FLT3 downstream signaling [135].

Whether or not allogeneic stem cell transplantation (SCT) in first CR improves outcomes in FLT3-ITD AML patients remains controversial [136], although the presence of the mutation in the context of wild-type NPM1 is considered an indication for SCT in the consensus document by the European LeukemiaNet [137]. The high incidence and clear deleterious prognostic impact of FLT3-ITD mutations, however, coupled with the tangible clinical gains achieved through targeting dysregulated tyrosine kinase activity in other malignancies, such as chronic myeloid leukemia (CML) and breast cancer, have provided a strong rationale for the development of FLT3-targeted therapy in AML. Several multi-kinase inhibitors with FLT3-inhibitory activity, the most developed of which being midostaurin (PKC412), lestaurtinib (CEP701), and sorafenib, have been assessed in combination with chemotherapy in international phase III clinical trials, with results currently awaited. Newer and more potent FLT3-inhibitory molecules, most notably quizartinib (AC220) and ASP2215, are in earlier stages of clinical development [138].

Other molecular abnormalities that have been consistently shown to confer a poorer prognosis include mutations in the RUNX1 gene [96–98, 139] and partial tandem duplications of the MLL gene (MLL-PTD) [52, 90–92], although recent data indicate that intensive consolidation therapy that includes autologous transplant in first CR may improve the outcome of CN-AML patients with the latter rearrangement [94].

Major steps forward in understanding the molecular pathogenesis of AML were the discoveries of mutations in the genes encoding CCAAT/enhancer-binding protein-α (CEBPA) and nucleophosmin (NPM1) that serve to identify subsets of patients with relatively favorable prognosis [63, 140] and which were recognized as provisional disease entities in the 2008 WHO classification [1]. Mutations in CEBPA, which encodes a myeloid transcription factor, were first described in 2001 and occur in approximately 10 % of cytogenically normal AML (CN-AML) [141]. Mutations cluster in both the amino- and carboxy-terminal regions, with the former leading to expression of a truncated 30 kDa isoform of CEBPA (p30) and loss of the 42 kDa full-length protein (p42) [142]. Carboxy-terminal mutations affect regions involved in mediating dimerization and DNA binding. Interestingly, in the majority of patients with CEBPA mutations, both alleles are involved, combining an upstream mutation in one allele with a downstream mutation in the other [141, 142]. The analysis of patient samples has shown that CEBPA mutations can be inherited, with progression to AML in later life being associated with acquisition of additional mutations, which can include involvement of the other CEBPA allele [143, 144]. Significant insights into the biology of CEBPA mutations have been provided by murine models, which have shown how loss of p42 expression (mimicking biallelic N-terminal CEBPA mutations), or compound heterozygous mutations affecting amino- and carboxy-terminal regions in combination, affects hematopoiesis and gives rise to AML [145, 146]. While early studies reported that CEBPA mutation predicts a relatively favorable outcome in AML [52, 147–149], subsequent studies have shown that the more favorable prognosis is seen in the subset of patients with biallelic mutations, especially those who lack FLT3-ITD [59, 60, 150–152].

Mutations in NPM1, discovered by Brunangelo Falini and colleagues in 2005 [63], represent the most common molecular lesion identified in AML to date, occurring in a third of cases, including 50–60 % of those with CN-AML. Over 30 different mutations have been described, which involve the C-terminal region of the protein. These lead to loss of tryptophan residues and generation of a nuclear export signal resulting in delocalization of nucleophosmin from the nucleoli to the cytoplasm [153]. NPM1 mutation is considered, an AML-defining lesion, being stable in the vast majority of cases over the disease course [154, 155]. Indeed, NPM1 mutation has been shown to enhance self-renewal of hematopoietic progenitors, associated with expanded myelopoiesis leading to the development of AML in a murine model [156]. Patients with AML harboring an NPM1 mutation in the absence of a FLT3-ITD have a relatively favorable prognosis [64–66]. NPM1 mutations also have been shown to be strong, independent predictors of better outcome in older patients, especially those aged 70 years and older [68]. Whereas most studies have focused on CN-AML [157], patients with AML with cytogenetic abnormalities that would be considered to have a standard risk but who harbor an NPM1 mutation also have a better outcome in the absence of FLT3-ITD; however, those with FLT3-ITD in the absence of the protective effect of an NPM1 mutation have a very poor prognosis [43, 158]. Whether the combined FLT3-ITD/NPM1-mutated genotype is itself associated with an intermediate or adverse outcome remains open to debate.