Patients with AML and a normal karyotype who present between the ages of 16 and 60 years carry an intermediate prognosis. However, cytogenetically normal AML is highly heterogeneous at the molecular level, both mutations and overexpression of single genes have been identified, and their complex interactions are frequently utilized to provide more accurate risk stratification schemas. Within the normal cytogenetic category, 45% to 62% of patients with AML have nucleophosmin 1 (NPM1) mutations, 25% to 35% have FLT3 mutations, 5% to 10% have MLL tandem duplications, and 8% to 15% have CEBPA mutations. The prognosis of patients with a normal karyotype differs depending on the presence of each of these mutations (Table 1). Patients with NPM1 mutations alone have a favorable prognosis, with 60% of patients living longer than 11 years. Even in patients age 60 to 74, the presence of an NPM1 mutation is an independent predictor of a more favorable outcome. More than 20 different mutations have been described in the C-terminal portion of the protein that lead to loss of tryptophan residues and generation of a nuclear export signal that acts in concert to cause delocalization of NPM1 from the nucleus to the cytoplasm. Because the functional integrity of NPM1 is dependent on its ability to shuttle between the nucleus and cytoplasm, this ability is severely compromised in NPM1 mutated AML. NPM1-mutated AML is associated with distinctive biologic and clinical features including older age, female predominance, multi-lineage involvement, extramedullary disease, high blast percentages and increased white blood cell counts, and lack of CD34 expression. As an isolated molecular abnormality, NPM1-mutated AML is associated with a highly favorable prognosis, with complete remission (CR) rates with modern chemotherapy being between 70% and 80%. The most common mutation (type A, accounting for 75% of all mutations) generates an aberrant extra nuclear export signal. Although NPM1 mutations are heterozygous, hetero/homodimerization with wild-type NPM1 results in cytoplasmic mislocalization of both mutant and wild-type protein. This alteration in subcellular location perturbs normal NPM1 function, including the mislocalization and stabilization of critical proteins such as the TP53 regulator p14ARF. This process also generates a distinct transcriptional signature. NPM1 mutations are considered to be early events in the pathogenesis of AML. In childhood AML with normal cytogenetics, NPM1 mutations are relatively uncommon, occurring at a frequency of 8% of cases.
Table1. Gene Mutations in Patients With Acute Myeloid Leukemia and a Normal Karyotype
In contrast, the presence of FLT3 and MLL mutations are associated with an adverse prognosis, and coexistence of FLT3 and NPM1 does not improve the prognosis. Internal tandem duplications (ITDs) of the FLT3 gene confer an increased risk for relapse and death when compared with patients without FLT3-ITD. Three types of FLT3 gene changes are present, ITD of the juxtamembrane domain-coding sequence, a point mutation within the activation loop domain, and the copy-neutral loss of heterozygosity. Although both mutations lead to the constitutive activation of the receptor, only presence of FLT3-ITD with high allelic ratio has been associated with an inferior outcome in young patients. This mutation initially develops as a het erozygous mutation and over time the FLT3-IDT blasts acquire copy neutral loss of heterozygosity resulting in homozygous mutated allele and high FLT3-ITD allelic ratio. OS of patients with FLT3-ITD differs significantly when categorized by the 2017 ELN risk stratification criteria. Therefore, at diagnosis of AML a complete work-up according to ELN recommendations, including assessment of ITD allelic burden is mandatory to select patients eligible for intensive therapy
Intact functioning of the CCAAT/enhancer binding protein (CEBPA) gene is essential for normal granulocytic differentiation. It is a lineage-specific transcription factor that is required for the formation of committed myeloid progenitors from multipotent pro genitor cells. CEBPA executes this function by coupling the direct transcriptional activation of myeloid-specific genes with the arrest of cell proliferation. CEBPA is an intron-less gene with mRNA that can be translated into two different AUG codons to give rise to two dis tinct isoforms, p42 and p30. Only the p42 isoform of CEBPA can promote proliferation arrest. CEBPA mutations, leading to arrest of differentiation, are seen in 5% to 10% of de novo AML and in up to 15% of normal karyotype AML. Only mutations affecting both alleles, double mutations, confer superior survival as compared to single mutations. Concomitant GATA1 and WT1 mutations are seen more often in double mutants while FLT3-ITD, NPM1, ASXL1, and RUNX1 mutations are more commonly associated with single mutations, possibly contributing to their poorer prognosis.
A rare but specific subset of adult AML can be defined by the cytogenetically cryptic NUP98-NSD1 fusion gene that involves the nucleoporin gene 98 (NUP98) on chromosome1, 1p15, and the non homeobox gene NSD1 present on chromosomal band 5q35. NUP98 encodes a 98-kDa protein of the nuclear pore complex, which is known to fuse with at least 21 different fusion gene partners in chromosomal rearrangements in various hematologic malignancies. NSD1 is thought to function both as a transcriptional co-activator and a co repressor. Among 293 pediatric and 808 adult cytogenetically normal cases of AML the NUP98-NSD1 fusion gene was described in 16.1% of pediatric and 2.3% of adult AML cases. Apparently NUP98-NSD1 has been shown to correlate with FLT3-ITD, which is associated with increased blast cell percentages.
TET2 proteins are necessary for the conversion of 5-methylcytosine to 5-hydromethylcytosine and play a role in demethylation processes within the cell. TET2 mutations have been reported in 10% to 23% of patients with AML with a normal karyotype. The impact of TET2 mutations on the prognosis and outcome remains poorly defined.
DNMT3A mutations, which affect the DNA methyltransferase enzymes and subsequent epigenetic modulation, have been described in about 27% to 33% of normal karyotype AML and are associated with a worse OS (see earlier section on clonal origin and early mutations).
Currently, a newly diagnosed patient with AML and a normal karyotype should be checked for common mutations such as FLT3, NPM1, CEBPA, RUNX1, MLL-PTD, or EVI1, TET2, and DNMT3A, which may have prognostic and possibly therapeutic implications (Table 2).
Table2. The European Leukemia Net 2017 Risk Stratification of Acute Myeloid Leukemia
Integration of NGS and high-density SNP array in a study of 252 patients with de novo AML and normal karyotype showed co-occurrence of mutations and cryptic cytogenetic abnormalities in 58% of which 49% harbored copy-number abnormalities or copy neutral loss of heterozygosity (CN-LOH).7 Of the 282 cryptic acquired abnormalities, genomic losses were more frequent than gains or CH-LOH (54%, 27%, and 19%, respectively). Losses were distributed virtually across all chromosomes, differently from CN-LOH and gains. Most important, the majority of lesions were not recurrent with the exception of those located on 13q. Losses and CN-LOH were concentrated in chromosomes 1, 2, 5q, 7, 11q, and 13q. Due to their location, and general implications of losses and CN-LOH, these genomic cryptic abnormalities could be acting as a second hit in the leukemogenesis process due to the loss of the wild-type alleles. Initial results are consistent that the presence of two or more genomic lesions has a negative impact on patient outcomes.
