Chung, Stephen S.a,b
Chromosomal structural variations have been well established as diagnostic and prognostic markers in acute myeloid leukemia (AML) [1,2], consistent with the concept that acquired somatic genetic abnormalities drive disease pathogenesis. However, about half of AMLs are associated with a normal karyotype [2–4], and studies using high-resolution techniques such as comparative genomic hybridization or single-nucleotide polymorphism arrays have found many AMLs to lack clear structural abnormalities [5–7]. The identification of genes harboring recurrent somatic mutations in AML has allowed for improved insight into the pathogenesis of these AMLs without obvious structural abnormalities [8,9]. Importantly, a subset of these genes including fms-like tyrosine kinase 3 (FLT3), nucleophosmin (NPM1), CCAAT/enhancer binding protein alpha (CEBPalpha), and mast/stem cell growth factor receptor (KIT) has allowed for refinement of prognostic models in AML, particularly with regards to selection of patients for optimal postremission therapies such as consolidation chemotherapy or allogeneic transplantation (Table 1[10▪▪,11–13,14▪,15–18,19▪,20–25,26▪,27,28▪▪,29▪▪,30▪,31▪]). Although recently identified mutated genes promise to further improve prognostic models, there remains a large gap between our knowledge of genetic abnormalities that drive disease pathogenesis/prognosis and our ability to exploit them therapeutically. New clinical trials supported by strong preclinical evidence appear poised to finally bridge this gap.
Cytogenetic abnormalities at diagnosis are highly predictive of response to induction chemotherapy, relapse risk, and overall survival (OS) . Patients with core binding factor (CBF)-type leukemias harboring inv(16) or t(8;21) generally have a favorable prognosis, whereas patients with a complex karyotype, monosomies of chromosomes 5/7, mixed-lineage leukemia (MLL)-rearrangements, inv(3), and t(6;9), among other abnormalities, tend to have a much worse prognosis. Postremission therapy with allogeneic transplantation appears to confer the most benefit to patients with unfavorable-risk cytogenetics . More recent studies have associated a monosomal karyotype with a dismal prognosis [12,13]. Of note, P53 alterations are highly associated with a monosomal karyotype and may in fact account for the poor prognostic import of this karyotype [14▪].
MOLECULAR GENETICS IN ACUTE MYELOID LEUKEMIA: EXISTING STANDARD OF CARE
Given the clear heterogeneity in the clinical behavior and outcomes that characterize AML without chromosomal abnormalities [otherwise referred to as cytogenetically normal AML (CN-AML)], candidate gene sequencing approaches have been applied toward identifying somatic mutations with prognostic import. Early efforts identified MLL partial tandem duplications (PTDs)  and FLT3 internal tandem duplications (ITDs)  to be associated with worse relapse-free survival and OS [15–17,34–36]. Conversely, a favorable outcome has been observed in CN-AML with mutated CEBPalpha[37,38] or NPM1[39–42] in the absence of FLT3-ITD. A landmark study of CN-AML patients less than 60 years of age examined in parallel the frequencies and interactions between these mutations, and their influence on outcomes after postremission therapy with allogeneic transplantation or chemotherapy/autologous transplantation . A favorable outcome was observed for those patients harboring NPM1 mutations and lacking FLT3-ITD, and allogeneic transplantation did not appear to improve the outcome of this subgroup. This has led to a change in clinical practice, such that patients with mutated NPM1 and wild-type FLT3 have been placed in the favorable-risk genetic subgroup by the European LeukemiaNet  and are generally not recommended to undergo allogeneic transplantation. However, it is important to note that this analysis was based on transplantation of just 38 patients with this genotype, and more recent analyses have demonstrated heterogeneity within this group of patients [10▪▪], as will be discussed later. In this analysis, there were too few patients with CEBPalpha mutations to determine the impact of allogeneic transplantation. Subsequent studies have suggested that the favorable prognostic import of CEBPalpha is limited to bi-allelic mutations .
DISCOVERY OF NOVEL DISEASE ALLELES IN ACUTE MYELOID LEUKEMIA
Recent efforts have used systematic approaches to identify new recurrent disease alleles in AML. Ley et al. sequenced the whole genome of a patient with CN-AML, representing the first time the entire genome of a malignancy had been sequenced. NPM1 and FLT3-ITD abnormalities were found, as well as eight other somatic mutations of as yet unclear significance. Identification of multiple mutations in AML poses the dilemma of determining which drive disease pathogenesis and impact clinical outcomes. One method of inferring pathologic significance is by identifying recurrently mutated genes. In that vein, whole genome sequencing studies followed by directed sequencing of candidate alleles have identified recurrent mutations in genes such as isocitrate dehydrogenase 1 (IDH1)  and DNA methyltransferase 3A (DNMT3A) [47,48]. Recent candidate gene sequencing approaches have further identified novel recurrent somatic mutations, including abnormalities in epigenetic modifiers such as addition of sex combs-like 1 (ASXL1)[10▪▪,49] and tet methylcytosine dioxygenase 2 (TET2) , which are present in 5–15% and 10% of AML cases, respectively. Metabolic profiling of AML has also helped to identify IDH2 mutations as frequent abnormalities in addition to IDH1, with the two mutations collectively occurring in 15–30% of AML. In elegant studies by Welch et al.[52▪▪], whole genome sequencing of 12 French American British (FAB) Classification M1 AML cases (in which the ‘driver’ lesion is unknown) and 12 FAB M3 acute promyelocytic leukemia cases [in which the ‘driver’ lesion is presumed to be t(15;17)] was used to infer potential driver versus cooperating mutations in FAB M1 AML cases. Intriguingly, the use of normal bone marrow controls encompassing a spectrum of ages demonstrated that many mutations identified by whole genome sequencing are ‘normal’ age-related lesions of unclear pathologic significance. NPM1, DNMT3A, and IDH1 were identified as driver mutations, and FLT3-ITD was predicted to be a cooperating mutation, consistent with the high concordance of NPM1 mutations and high discordance of FLT3-ITD mutations between diagnosis and relapse [53–55]. Future studies incorporating similar principles promise to identify the most relevant mutated genes in AML.
NEW AND EVOLVING PROGNOSTIC MODELS BASED ON MOLECULAR GENETICS
The majority of existing studies correlating molecular genetic abnormalities with clinical outcome examined single genes in isolation. By failing to assess for the co-occurrence and interaction between different mutated genes, these studies may not be adequate to determine the independent contributions of mutated genes in the context of complex genotypes. To address this issue, Patel et al.[10▪▪] performed high-throughput re-sequencing of 18 genes previously identified to be recurrently mutated in AML (FLT3, NPM1, CEBPalpha, KIT, IDH1, IDH2, DNMT3A, PHF6, WT1, TP53, RUNX1, EZH2, PTEN, HRAS, KRAS, NRAS, TET2, and ASXL1) in 398 patients enrolled on the Eastern Cooperative Oncology Group E1900 clinical trial. This was a large, uniformly treated, and well annotated cohort of patients randomized to induction chemotherapy incorporating standard or intensified dose daunorubicin (DNR), allowing for a robust correlation of mutational profile with survival and response to chemotherapy. Mutations associated with clinical outcome across the entire cohort of patients included IDH2 mutations (specifically R140), which were associated with an improved OS, and ASXL1 and PHF6 mutations, which were associated with worse OS. Perhaps most importantly, molecular profiling allowed for refinement of prognostication in intermediate-risk CN-AML. For example, in patients considered to have favorable-risk CN-AML (i.e., those harboring an NPM1 mutation and wild-type FLT3), the presence of a concurrent IDH mutation conferred a very favorable outcome, better even than that of patients with CBF AML. Conversely, those lacking IDH mutations had substantially worse outcomes, representing a group of patients who may be receiving suboptimal postremission therapy strategies under current standard practice. Amongst intermediate-risk patients with wild-type FLT3, the presence of TET2, ASXL1, PHF6, or MLL-PTD mutations was associated with a much worse survival, and amongst intermediate-risk patients positive for FLT3-ITD, the presence of TET2, DNMT3A, MLL-PTD, or trisomy 8 abnormalities was associated with a much worse survival. Interestingly, those patients positive for FLT3-ITD but lacking the aforementioned adverse-risk mutations had a more favorable outcome that was comparable to those with both FLT3-ITD and CEBPalpha mutations. This implies that a significant component of the adverse prognostic import of FLT3-ITD mutations may be conferred by high-risk co-occurring lesions, perhaps explaining in part the limited efficacy of FLT3-targeted therapies to date.
The results from the E1900 study were validated in an independent cohort of patients treated on the same study, but further validation in other patient cohorts will be needed to confirm these findings for their general applicability. For example, several other studies have found IDH1/IDH2 to confer a worse prognosis [21,56,57] in the cytogenetically normal mutated NPM1 wild-type FLT3 subgroup, whereas a recent study by the Medical Research Council identified a favorable prognosis conferred specifically by the IDH2 R140 mutation in a cohort of young patients who received relatively aggressive therapy (induction therapy followed by autologous or allogeneic transplantation) . Therefore, patient age, as well as intensity and uniformity of treatment, may underlie the inconsistent results between trials thus far.
Molecular genetic studies have mostly been performed in patients younger than 60–65 years, and given that the median age of AML patients at presentation is 69, validation of these studies in older patients will be critical to determining their widespread applicability. Given the evidence that the favorable prognostic import of t(8;21) and inv(16) extends to elderly patients harboring these abnormalities , molecular genetic abnormalities may extend their predictive capability into this age group as well. Indeed, a recent analysis of 428 older patients with AML demonstrated that ASXL1 mutations confer a poor prognosis . In the myelodysplastic syndromes, a recent comprehensive mutational analysis of 111 genes in 738 patients demonstrated that the presence of ‘driver’ mutations predicted prognosis to a degree comparable to well validated clinical variables . A similar principle may hold in AML with continued integrated analysis of a larger number of relevant disease alleles, such that molecular genetics may largely supplant time-honored clinical prognostic indicators such as white blood cell count and age.
MOLECULAR GENETICS FURTHER REFINE DISTINCT CYTOGENETIC SUBGROUPS
Somatic mutations may modify the prognostic impact of structural chromosomal abnormalities, such as with P53 mutations and a monosomal karyotype, as discussed above [14▪]. The best example of such an interaction that is clinically relevant is the close association between CBF abnormalities [e.g., t(8;21) and inv(16)] and KIT mutations , as reported by Paschka et al.. Although this initial study reported that KIT mutations (both exon 17 and exon 8) are associated with a worse OS in inv(16)-associated AML and an increased risk for relapse (but no survival difference) in t(8;21) AML, the sample size of inv(16) cases was small. Multiple recent studies have confirmed the poor prognostic import of KIT mutations in t(8;21)-associated AML but failed to confirm any prognostic significance for KIT mutations in inv(16)-associated AML [10▪▪,60–63]. Thus, although it has become standard practice to recommend allogeneic transplantation for patients with CBF AML and KIT mutations, this may only apply to patients with t(8;21). Nevertheless, even in patients with CBF AML without KIT mutations, the long-term OS is only 55–60%. Similar to the ‘favorable-risk’ cytogenetically normal mutated NPM1 wild-type FLT3 subgroup, this group of patients may be heterogeneous, and future studies may identify additional genetic lesions that impact on prognosis.
MOLECULAR GENETICS AS DETERMINANTS OF RESPONSE TO THERAPY
Certain adverse-risk genetic subtypes of AML tend to be associated with robust responses to chemotherapy but increased rates of relapse. This may influence the timing of clinical interventions to overcome adverse risk. For example, FLT3-ITD positive AML tends to respond robustly to induction chemotherapy but is characterized by high rates of relapse [15–18]. Thus, standard induction chemotherapy may be an excellent upfront therapeutic modality for this and other adverse-risk mutations with similar features (e.g., CBF AML with KIT mutations ). Furthermore, FLT3 ligand levels may rise significantly during chemotherapy-induced aplasia, potentially contributing towards resistance to FLT3 inhibitors and relapse [24,25,64], and thus prompt allogeneic transplantation may be of particular importance.
Conversely, older patients with a complex karyotype/monosomal karyotype, as well as other high-risk cytogenetic subgroups such as inv(3), encounter high rates of primary induction failure [3,4,11]. Prioritizing these patients for clinical trials should be of utmost importance. For non-clinical trial based therapy, one might consider the use of hypomethylating agents (HMAs) currently designated for patients ‘not fit’ for intensive induction chemotherapy. HMAs appear to have efficacy independent of cytogenetic risk, and therefore in patients with a complex karyotype they may confer response rates comparable to standard induction chemotherapy (20–50% complete remission) with much less toxicity [65,66]. It remains to be seen whether HMAs will maintain this breadth of efficacy across different molecular genetic subgroups. In fact, mutations in epigenetic modifiers such as DNMT3A and TET2 have been described to predict for response to HMAs [67,68], and if validated these may represent useful biomarkers to select patients for these agents.
The E1900 clinical trial found an OS benefit to DNR intensification when given as part of standard induction chemotherapy to patients younger than 60 years of age. A post-hoc analysis of molecular genetic subgroups in this cohort found that this benefit was restricted to patients with MLL-rearrangements or mutated NPM1 or DNMT3A. These results await prospective validation, but they raise the possibility that a substantial proportion of patients may not benefit from (and thus might be spared the toxicity of) DNR intensification. As molecular genetic abnormalities such as these influence upfront treatment decisions, it will become increasingly important to develop expedient and clinically tractable methods to obtain this information in a timely manner.
Inhibition of FLT3 in patients with relapsed/refractory AML with the tyrosine kinase inhibitor (TKI) quizartinib (AC220) has recently been shown to induce a relatively high rate of response, particularly in patients positive for FLT3-ITD (53% complete remission or partial remission, versus only 14% in FLT3 wild-type patients) [19▪]. On the basis of these results, prospective trials incorporating quizartinib into upfront intensive induction chemotherapy are ongoing. Sorafenib, another TKI targeting FLT3, has shown some benefit as upfront therapy in combination with 5-azacytidine [69▪] but not standard induction chemotherapy [70▪]. Unfortunately, rapid and frequent resistance to FLT3-targeted therapies has been observed, even after allogeneic transplantation [71,72,73▪▪], and this is likely attributable to the development of drug resistant mutations [74▪] and/or the presence of other high-risk co-occurring genetic lesions. Other rationally designed therapeutics targeting molecular genetic abnormalities currently entering early phase clinical trials include DOT1-like (DOT1L) histone methyltransferase inhibitors for MLL-rearranged AML [27,28▪▪], IDH inhibitors for IDH-mutated AML [29▪▪,30▪], and inhibitors of the nuclear export protein chromosome region maintenance 1 (CRM-1) for NPM1-mutated AML [31▪].
The past several years have witnessed significant advances in our understanding of how molecular genetics impact on clinical outcome, with a particular influence on the selection of optimal postremission strategies. Further stratification of ‘favorable-risk’ AML subgroups may help to identify which patients are truly most likely to do well with chemotherapy-based postremission therapy. Although the identification of the highest risk patients within already adverse-risk subtypes of AML may not necessarily influence the existing recommendations for allogeneic transplantation, the complement of mutations whose adverse risk can be overcome by transplantation remains to be determined. These patients who have poor outcomes with any standard therapies should be prioritized for enrollment on clinical trials incorporating innovative new therapies. We will undoubtedly continue to discover new molecular genetic abnormalities and potential ‘druggable’ targets through comprehensive efforts such as the Cancer Genome Atlas [75▪▪]. As the accumulation of molecular genetic data appears to ‘split’ AML into the heterogeneous disorder it has always intuitively appeared to be, common biologic features between different subtypes of AML may in fact allow us to ‘lump’ certain subtypes of AML together. This may allow for identification of therapies with the potential to benefit a larger swath of the spectrum of AML patients. Together, these advances promise to bring our now vast knowledge of the molecular genetics of AML to bear on clinical decision-making and novel therapeutics.
S.S.C is supported by a Young Investigator Award from the Conquer Cancer Foundation of the American Society of Clinical Oncology and a U.S. Department of Defense Postdoctoral Fellow Award in Bone Marrow Failure Research (BM120096).
Conflicts of interest
There are no conflicts of interest.
REFERENCES AND RECOMMENDED READING
Papers of particular interest, published within the annual period of review, have been highlighted as:
- ▪ of special interest
- ▪▪ of outstanding interest
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65. Cashen AF, Schiller GJ, O’Donnell MR, DiPersio JF. Multicenter, phase II study of decitabine for the first-line treatment of older patients with acute myeloid leukemia. J Clin Oncol. 2010; 28:556–561.
66. Blum W, Garzon R, Klisovic RB, et al. Clinical response and miR-29b predictive significance in older AML patients treated with a 10-day schedule of decitabine. Proc Natl Acad Sci U S A. 2010; 107:7473–7478.
67. Metzeler KH, Walker A, Geyer S, et al. DNMT3A mutations and response to the hypomethylating agent decitabine in acute myeloid leukemia. Leukemia. 2012; 26:1106–1107.
68. Itzykson R, Kosmider O, Cluzeau T, et al. Impact of TET2 mutations on response rate to azacitidine in myelodysplastic syndromes and low blast count acute myeloid leukemias. Leukemia. 2011; 25:1147–1152.
69▪. Ravandi F, Alattar ML, Grunwald MR, et al. Phase 2 study of azacytidine plus sorafenib in patients with acute myeloid leukemia and FLT-3 internal tandem duplication mutation. Blood. 2013; 121:4655–4662.
This phase I/II study combined the FLT3 inhibitor sorafenib with 5-azacytidine in patients with relapsed FLT3-ITD positive AML. The combination was well tolerated, and the overall response rate was 46%.
70▪. Serve H, Krug U, Wagner R, et al. Sorafenib in combination with intensive chemotherapy in elderly patients with acute myeloid leukemia: results from a randomized, placebo-controlled trial. J Clin Oncol. 2013; 31:3110–3118.
This trial randomized elderly patients with AML to standard induction chemotherapy with or without sorafenib irrespective of FLT3 mutational status. The addition of sorafenib did not significantly improve EFS or OS and was associated with significant additional toxicity.
71. Leung AY, Man CH, Kwong YL. FLT3 inhibition: a moving and evolving target in acute myeloid leukaemia. Leukemia. 2013; 27:260–268.
72. Sharma M, Ravandi F, Bayraktar UD, et al. Treatment of FLT3-ITD-positive acute myeloid leukemia relapsing after allogeneic stem cell transplantation with sorafenib. Biol Blood Marrow Transplant. 2011; 17:1874–1877.
73▪▪. Man CH, Fung TK, Ho C, et al. Sorafenib treatment of FLT3-ITD(+) acute myeloid leukemia: favorable initial outcome and mechanisms of subsequent nonresponsiveness associated with the emergence of a D835 mutation. Blood. 2012; 119:5133–5143.
In this study, therapy of 13 patients with relapsed FLT3-ITD-positive AML led to clearance of blasts from the bone marrow in 12 patients at a median of 27 days. However, sorafenib response was lost in nearly all patients at a median of 72 days, including patients who had proceeded to allogeneic transplantation. This was accompanied by the outgrowth of sorafenib-resistant FLT3-TKD harboring subclones, suggesting that this TKI resistant mutation may be responsible for the eventual failure of most existing FLT3-targeted therapies.
74▪. Smith CC, Wang Q, Chin CS, et al. Validation of ITD mutations in FLT3 as a therapeutic target in human acute myeloid leukaemia. Nature. 2012; 485:260–263.
This protein structural study identified point mutations in FLT3 that appear to confer resistance to the FLT3 inhibitor AC220, helping to guide the future development of improved FLT3 inhibitors.
75▪▪. Genomic and epigenomic landscapes of adult de novo acute myeloid leukemia. N Engl J Med. 2013; 368:2059–2074.
In this large collaborative effort by The Cancer Genome Atlas, 200 AML samples were comprehensively analyzed using a number of genomic platforms, including conventional karyotyping, exome sequencing, gene expression profiling, RNA sequencing, and epigenomic profiling. These studies confirmed previously identified genetic lesions in AML, extended our knowledge about their frequency and co-occurrence, and identified novel recurrent genetic lesions. Like previous studies, mutations in signaling molecules, transcription factors, and genes regulating methylation were identified. In addition, mutations affecting cohesin, a chromatin modifier, and the spliceosome, a complex that regulates the processing and maturation of RNA, were identified. This study did not attempt to identify clinically relevant disease subgroups but rather provides a plethora of data from which such clinically useful information may be mined in the future.