Mutations in NPM1 Gene

NPM1 mutations (duplication and insertion) are the most frequent AML-related mutation occurred in adults and found in one third of all adult cases of AML (~30%) (Figures 2 and 3). NPM1 mutations are enriched in CN-AML (about 45–64% of CN-AML cases) (Figure 3A), wherein exhibit good response to conventional induction chemotherapy (based on a combination of anthracycline and cytarabine) and favorable outcomes are achieving in CN-AML subtype without the FLT3-ITD mutation (high CR rate ~ 85%, EFS ~ 50–60%, OS rates ~ 50%).11,16,18

In pediatric AML, however, findings indicate that NPM1 mutations generally confer an independent favorable prognostic impact despite FLT3-ITD mutations. In addition, pediatric AML patients with both NPM1 and FLT3-ITD mutations appear to have favorable prognoses and may not need hematopoietic stem-cell transplantations. Incidence of NPM1 mutations cannot be found below the age of 3 years, but above this age increases with age. NPM1 mutations occur frequently in adult CN-AML and confer favorable outcome. The studies on CN-AML in adults have found that the prognosis of NPM1 mutations positive for FLT3-ITD is unfavorable. The European LeukemiaNet (ELN) scheme proposes that NPM1 mutations with FLT3-ITD allele ratio (AR) <0.5 (low AR) has a favorable prognosis, and allogeneic hematopoietic stem-cell transplant (HSCT) in the first complete remission (CR1) period is not actively recommended.22–24

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Then in adults, CN-AML with isolated NPM1 mutations (ie the NPM1+FLT3-subgroup) exhibits a good response to chemotherapy, relapse-free survival (RFS), and improved OS. This favorable effect is however lost in the presence of an FLT3-ITD. The survival of double-positive subtype NPM1+/FLT3+ will be similar to the NPM1-/FLT3-ITD+ subtype. NPM1+/FLT3+ mutations show an intermediate prognosis where there is no survival difference between NPM1+/FLT3+ and NPM1-/FLT3+. Postulating, treatment of NPM1+/FLT3+ patients with recently developed FLT3 inhibitors would be effective by converting NPM1+/FLT3+ to a more chemotherapy-sensitive status (Table 4).11,16,18

NPM1 mutation is the only detectable genetic mutation in approximately 28% of CN-AML cases, whereas in the majority of cases, additional mutations exist in class I genes such as FLT3, NRAS and WT1, (approximately in 40%, 21% and 17.5% of cases, respectively) (Figure 3B).2,11 Additionally, NPM1 mutation can also coincide with secondary chromosomal abnormalities, such as trisomy 8 and 4 and del(9q).

Generally, there are two prognoses for the outcome of NPM1 mutation without FLT3-ITD; 1) patients do not necessarily benefit from allogeneic stem-cell transplant following conventional induction chemotherapy (based on anthracycline and cytarabine), and 2) elderly patients might benefit from combination of ATRA to their chemotherapy regimen.1,3,5

At the molecular level, the nucleophosmin 1 (NPM1) gene encodes a multi-functional chaperone protein shuttling between nucleus and cytoplasm. NPM1 seems to be mainly involved in ribosomal protein assembly and biogenesis, stabilization of the tumor-suppressor p19ARF and p53 pathway, DNA-repair process, genomic stability, and finally regulation of DNA transcription through modulation of chromatin structure. Any mutation that disrupts NPM1 normal function as a tumor suppressor would lead to malignant transformation. Herein, NPM1 mutations prevent proper folding and lead to loss-of-function. Moreover, NPM1 single allele mutations perturb the function of WT protein through cytoplasmic localization of both mutant and WT protein.8,18

Other concordant conclusions are that NPM1 mutations are mostly present in adult AML with more preference in females, associated with increased blood leukocyte count, myelomonocytic phenotype and low CD34 expression. This highly prevalent mutation provides a suitable marker for monitoring minimal residual disease (MRD) of AML.11,16 QRT-PCR is an implement to quantify mutated transcripts and to evaluate the presence of MDR. Herein, the persistence of mutated transcripts in blood of patients would associate with a greater risk of relapse after chemotherapy cycle, for example, NPM1-mutated transcripts detected in the blood of 15% of patients after the second chemotherapy cycle, associates with a greater risk of relapse after 3 years of follow-up as well as a lower rate of survival.3,5

Mutations in FLT3 Gene

FLT3 (FMS-like tyrosine kinase) is a member of class III of receptor tyrosine kinases (RTK, the same family of FMS, KIT, PDGFR-α/β), whereby ligand binding signals intracellular pro-proliferative pathways. FLT3 mutations are the most common RTK mutation in AML (~30% of all cases). The most frequent FLT3 mutations (~25%) are the internal tandem duplication (ITD) within the cytoplasmic-juxtamembrane (JM) region (FLT3-ITD). Less frequent (~7%) are point mutations in the activation loop of the tyrosine kinase domain (FLT3-TKD mutation), such as the D835Y mutation. FLT3-ITD usually occurs in CN-AML wherein is associated with a dismal outcome (Figure 3) (Table 4). Both types of mutations are classified as class I mutations which provide cells constitutive-proliferative and survival advantages. FLT3 mutations result in ligand-independent kinase activation of FLT3 and its downstream signaling transducers STAT5, RAS/MAPK, PI3K, src homologous and collagen gene (SHC), and cytoplasmic tyrosine phosphatase SH2 domains (SHP2).5,6,8

Clinically, FLT3-ITD patients represent high blast counts and normal cytogenetics. The t(15;17) may coincide FLT3-ITD but rarely co-occurs in AML with complex karyotype or CBF-leukemia (CBF-MYH11 and AML1-ETO). The FLT3-ITD presence is a negative risk factor for OS, EFS and prognoses (Tables 3 and 4).10,11,16

Patients who are homozygous for FLT3-ITD or lose wild-type FLT3 allele would experience inferior outcomes or poor prognosis. By using DNA fragment analysis (PCR products sized by capillary electrophoresis which detecting ITD abnormally large amplicons), labs can quantify the relative level of the mutant allele and are able to define a cut-off value that distinguishes between prognostic subgroups.2,5

Unfavorable prognosis and inferior outcome depend, especially, on a high ratio of the expression of FLT3 mutant allele to FLT3 wild-type allele (the allelic ratio >0.78). The high level of the mutant allele (some studies, however, mentioned a mutant allele/wt allele ratio >0.5), is likely of importance which leads to shorter CR duration, death-free survival (DFS) and OS.2,15,8

Expression levels of FLT3 (CD135) are associated with certain subtypes of AML; with a minimum level in FAB M3 subtype and a maximum level in FAB M5 subtype.3,8 Small molecule tyrosine kinase inhibitors are remarkable strategies to inhibit FLT3 signaling and to specifically kill leukemic cells.4,8 From clinical trials, multi-targeted tyrosine kinase inhibitors showed a great benefit to the therapeutic anti-leukemia activity. Nevertheless, none of the designed inhibitors would lead to sustained therapeutic response on their own while in combination with other therapeutics make a preferred strategy for the treatment of AML patients. Some RTK inhibitors include midostaurin, lestaurtinib (CEP701) and sorafenib.3

Mutations in CEBPA

CEBPA encodes CCAAT-enhancer-binding protein-α (CEBP-α), a member of transcription factors with leucine-zipper motifs. Members of basic leucine-zipper (bZIP) transcription factors consist of C-terminal DNA-binding (basic region) and dimerization (leucine zipper) motifs and two less-conserved N-terminal transactivation domains.5,8 CEBP-α is a master transcription factor with a primary role in myeloid differentiation. About 10% of AML patients experience CEBPA mutations which mostly coincide with intermediate-risk karyotypes, and associate with a good prognosis.11,18 About 7-15% of CN-AML patients experience CEBPA mutations (Figure 3A) (Table 4), the majority of which being FAB-M2 subtype. CEBPA double-mutations show favorable outcomes (longer remission duration and higher OS ~ 8 years), and better prognostic impact (especially in patients aged 16–60 years).5,8 Importantly, aberrations such as FLT3 mutations and MLL-PTD rearrangement have no significant influence on the prognosis outcome of CEBPA double-mutation. This benefit is lost in the presence of CEBPA wild-type allele or CEBPA single-mutation which is an independent adverse prognostic marker affecting remission duration and OS rates.11,16 The coincidence of CEBPA double-mutation with FLT3-ITD mutation is rare, where CEBPA mutation is mutually exclusive with NPM1 mutation.3,11,18 At the molecular level, CEBPA has a single exon mapped to band 19q 13.1, with tumor-suppressor activity. CEBP-α induces a number of genes involved in granulocytic specific differentiation. Consecutive up-regulation of CEBPA gives rise to granulocytic differentiation, while its further conditional expression triggers neutrophil differentiation. CEBPA mutations result in loss-of-function trait contributing to an early block of granulocyte maturation. The t(8;21) is an alternative mechanism for CEBPA inactivation wherein the AML1-ETO fusion protein suppresses CEBPA transcription and hence blocks granulocyte differentiation.2,16,17

Mutations in CEBPA have a favorable prognosis and good response to the conventional chemotherapy and are associated with higher CR rate and better RFS and OS, comparable with that of CBF-AML (Tables 1 and 2).2,6,8

BAALC Over-Expression

The brain and acute leukemia, cytoplasmic (BAALC) gene is mapped to band 8q22.3. BAALC expression occurs in neural and hematopoietic stem cells. In HPCs, BAALC is a specific marker for the proliferation stage, as its down-regulation leads to cell differentiation. CD34+ progenitor cells from both normal and abnormal bone marrow express BAALC. Since all subtypes of CD34 + cells express BAALC, it would be present in early progenitor cells, as a novel marker common for the myeloid, lymphoid, and erythroid pathways. As patients with an elevated level of BAALC show a higher rate of primary resistant leukemia, a higher CIR and an inferior OS (3 years), its over-expression is an independent risk factor associated with chemotherapy resistance and unfavorable outcomes. To improve the unfavorable outcome for BAALC high-risk patients, modifying and adjusting induction therapy and intensifying post-remission therapy should be taken into account. Reports suggest that AML patients with a high level of BAALC would benefit from consolidation with allogeneic SCT, while therapy with autologous SCT seems unfavorable. Clinically, high-level BAALC patients exhibit an escalated percentage of blood blasts and immature FAB M0/M1 subtypes, while low BAALC expression associates with monocytic differentiation and FAB M5b subtype and gingival hyperplasia. BAALC over-expression may be accompanied by FLT3-ITD, CEBPA, MLL-PTD, and high ERG over-expression mutations. Importantly, over-expressed BAALC induces drug resistance gene (MDR1) and stem-cell markers (CD133, CD34, KIT).5,15,8

RAS Mutations

RAS (rat sarcoma) oncogenes encode a family of membrane-associated G proteins that regulate signal transduction by binding to a variety of membrane receptors. The RAS G proteins are involved in signal transduction pathways that induce proliferation. The genome contains three functional RAS genes: N-(neuroblastoma), K-(Kirsten), and H-(Harvey) RAS. Each RAS gene (H-RAS, K-ras, N-RAS) contains 4 exons can carry transforming mutations exclusively in codons 12/13/61, which leads to constitutive activation of RAS protein identified in many cancer types.3,8

Accordingly, RAS mutations are the most frequent mutations occurring in CBF-AML patients (45% N-RAS; 13% K-ras) (Figure 2) (Table 2) and N-RAS is prominent (11–30% in all AML) (Figures 2 and 3), especially in those under the 60 years of age. As mentioned, RAS mutations occur with a higher frequency in the favorable risk groups wherein lead to poor outcomes. Screening RAS mutations in AML patients are thereby proposed as a benefit guide for therapeutic decisions. AML patients with RAS mutations showed a good response to post-remission high-dose cytarabine therapy.8,16,18

WT1 Mutations

Wilms tumor (WT1) mutations (insertions or deletions) are detectable in 10% of AML cases, mainly clustered in exons 7 and 9, and show induction failure. WT1 mutations belong to the class I oncogene activating mutations which confer WT1 constitutive activation.2,16

The WT1 gene is mapped to band 11p13 which encodes the transcription factor WT1, an oncogene expressed in various cell types to promoting proliferation, as well as to blocking differentiation. At the molecular level, WT1 mutations mostly occur with FLT3-ITD mutation and are failure to standard induction chemotherapy. WT1 can be a reliable marker for MDR assessment in acute leukemia patients. After induction chemotherapy, the WT1 levels in blood samples of treated patients would allow to distinguish those with continuous CR from those who obtain only an “apparent” CR or who relapse within a few months. The level of WT1 helps identify patients at high risk of relapse soon after induction chemotherapy, those who need post-induction therapy being intensified. Moreover, the expression level of the WT1 gene in normal HPCs is approximately 10 times less than those in leukemic cells.2,8

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