**3. Structural and functional characteristics of Fms-like tyrosine kinase 3**

#### **3.1. FLT3 receptor**

Transmembrane FLT3 receptor is a member of type III receptor tyrosine kinase (RTK) family. Other receptors included in this group are KIT, c-FMS, and platelet-derived growth factor receptor (PDGFR) [34–37]. These receptors keep control in normal maturation and proliferation of hematopoietic precursor cells. The FLT3 is approximately 1000 kilobases in length and consists of 24 exons situated on chromosomes 14 and 15. This gene encodes a 993-amino-acid protein that is observed as a major 140 kDa band and a minor 160 kDa band because of N-linked glycosylation, and a 130 kDa band when unglycosylated and not membrane bound. The FLT3 receptor has an extracellular domain, one transmembrane region and two cytoplasmic kinase domains. Extracellular domain comprises of five immunoglobulin-like domains, while transmembrane region has a cytoplasmic juxtamembrane (JM) domain and cytoplasmic kinase is linked by an intracellular kinase domain [38].

### **3.2. FLT3 receptor expression**

**2.6. Mutations in AML**

68 Myeloid Leukemia

**2.7. Prognosis of AML**

**kinase 3**

**3.1. FLT3 receptor**

decisions on allogeneic transplantation [9, 30].

In previous few decades, there is a great insight in biology of this disease that leads to riskadapted treatment approaches. With better understanding of the disease, it is evident that AML is heterogeneous at the molecular level. Around 45% of de novo AML patients belong to normal cytogenetics [20–25]. Recently, molecular dissection of this group identified better prognostication. These molecular alterations include internal tandem duplication of FLT3,

In AML risk stratification, there are various clinical and biological factors relevant with treatment outcome [29]. These risk factors included are age, performance status, leucocyte counts, platelets counts, lactate dehydrogenase, drug resistance, immunophenotyping, cytogenetics, molecular genetics, epigenetics, micro RNA and so on. In various literature, these factors shows significant role to identify the potential role for treatment outcome in AML; for example, various mutations have targeted agents (e.g., ATRA and arsenic trioxide in PML-RARAα acute promyelocytic leukemia or FLT3 inhibitors in AML with FLT3 mutations), informing

These clinical and biological analyses classified the AML into three risk stratification groups: favorable, intermediate, and unfavorable. Current updates reclassified into further subgroups after more markers includes with the deep molecular analysis like next-generation sequencing (NGS) of the whole genome of AML patients [5, 31, 32]. There are multiple large cohort done previously and currently as well in normal karyotypes of AML in which significance of mutations like FLT3, NPM1, and CEBPA has been subclassified into subgroups according to their

When taking into account immunophenotyping, human leukocyte antigen (HLA)-DR and CD14 expression are associated with the elderly, highest WBC count, and unfavorable-risk cytogenetics; CD4, CD7, and CD11b expressions are correlated with the highest WBC count and unfavorable-risk cytogenetics; CD22, CD34, CD123, and terminal deoxynucleotidyl transferase (TdT) expressions are correlated with unfavorable-risk cytogenetics; CD19 is associated with children and favorable-risk cytogenetics; and myeloperoxidase (MPO) and glycophorin A (gly-A) expressions are associated with lower WBC count and favorable-risk cytogenetics [33].

Transmembrane FLT3 receptor is a member of type III receptor tyrosine kinase (RTK) family. Other receptors included in this group are KIT, c-FMS, and platelet-derived growth factor receptor (PDGFR) [34–37]. These receptors keep control in normal maturation and proliferation of hematopoietic precursor cells. The FLT3 is approximately 1000 kilobases in length and consists of 24 exons situated on chromosomes 14 and 15. This gene encodes

presence and absence for different treatment outcomes and survival rates [33].

**3. Structural and functional characteristics of Fms-like tyrosine** 

partial tandem duplication of MLL gene, and mutations of CEBPA [26–28].

FLT3 is present in precursors of lymphoid and myeloid cells. These progenitor cells are converted into granulocyte, monocyte, B cell, and T cell, but in comparison to their counterpart, cells are unable to produce erythroid and megakaryocyte cells. FLT3 is also expressed in other tissues like the placenta, gonads, and brain, but its significance in these areas is unknown [27].

## **3.3. FLT3 ligand(FL)**

FLT3 regulates early hematopoiesis by stimulating the FLT3 signal transduction pathway. mRNA of FLT3 is identified in hematopoietic as well as non-hematopoietic tissues. But identification of membrane-bound and soluble isoform is restricted to bone marrow s T lymphocytes and stromal fibroblasts. This protein in non-hematopoietic cells acts similarly as cells expresses FLT3 receptor shows FLT3 has autocrine and paracrine signaling mechanisms. It is identified during resting phase, but it is detected in serum at lower concentration. Under controlled circumstances release of FL is at a lower level to avoid hyperstimulation of progenitor hematopoietic cells. Current research depicts that one pathway for leukemia development is uncontrolled FL secretion [37].

#### **3.4. Synergy of FL with other cytokines**

FL needs cytokines for its action and proliferation. Interleukin-3 (IL-3), granulocyte colony-stimulating factor (G-CSF), colony-stimulating factor-1 (CSF-1), and granulocyte macrophage colonystimulating factor (GM-CSF) are growth factors that help in FL-mediated signal transduction [27]. However, working in conjunction with cytokines, FL induces expansion of hematopoietic progenitor cell [38]. In vivo analysis of FL function further supports its vital role in maintenance and proliferation during early hematopoiesis. Blocking of FL in mice decreased myeloid progenitor cells, whereas stimulation of FL revealed transient HSC proliferation evidenced by bone marrow hyperplasia, splenomegaly, hepatomegaly, and enlarged lymph nodes [39].

### **3.5. FLT3 receptor signaling**

After binding of FL to FLT3 receptor there is a formation of homodimer in plasma membrane. The dimer joins cytoplasmic domains and consequently phosphorylation of tyrosine residues, likely Tyr-589 and Tyr-591, on the JM domain [40]. This combination leads to conformational change at receptor sites to initiate autophosphorylation and leads to downstream signaling cascade which involves activation of cytoplasmic molecules that control pathways of apoptosis, proliferation, and differentiation (**Figure 1**). FLT3 receptor sends signals to the p85 subunit

pathophysiology of leukemia. Mutations and expression levels of FLT3 distinguish a dis-

Genomics of Acute Myeloid Leukemia http://dx.doi.org/10.5772/intechopen.72757 71

These studies reveal that FLT3 mutations are mainly identified in AML and more infrequent in secondary AML developed from myelodysplastic syndrome (MDS) or therapy-related AML than de novo AML. This mutation is also found in ALL and MDS, but never in chronic myeloid leukemia, chronic lymphoid leukemia, multiple myeloma, malignant lymphoma, or myeloproliferative disorders (MPD) [50]. Frequency of this mutation in MDS is reported to be 3%, but it increased to 15% in advanced stage and after AML conversion [46]. The incidence of FLT3 mutations is directly proportional to the age of patients with AML. In elderly patients, FLT3-ITD has been found in approximately 25% of this population; among them 31.4% were

FLT3 mutations are detected in 25% of AML patients, revealing that the FLT3 gene is the target most frequently mutated. FLT3 is also implicated in the pathogenesis in few patients of ALL, such as those with MLL gene rearrangement or hyperdiploidy. As FLT3 mutation is closely associated with poor prognosis, routine testing of this mutation is recommended to stratify the patients into distinct risk groups. However, optimal treatment strategy according to this mutation is under evaluation because it remains uncertain that high-dose chemotherapy and/ or myeloablative therapy followed by hematopoietic stem cell transplantation will change the prognosis of FLT3-mutated patients. Mutated or overexpressed FLT3 is an important molecular target in the treatment of leukemia and several potent inhibitors in clinical phase 1 and 2 trials are underway [52–55]. At present, the clinical efficacy of FLT3 inhibitors seems unimpressive, and problems related to adverse effects and pharmacokinetics are observed. Further studies are required to establish the role of FLT3 inhibitors in the treatment of leukemia.

FLT3 receptor is expressed in the vast majority of human B-lineage acute lymphocytic leukemias (ALL) and most myeloid leukemias in different types of AML (M0–M7) [56–58]. A smaller proportion of T-cell-related leukemias also possess (less than 30%) FLT3 receptor [50, 59–62]. Further analyses revealed that the mean mRNA expression level of FLT3 is higher in leukemic cells than in normal progenitor cells, but percentage is different in various samples of AML patients. Nearly, all primary AML patients and other immature leukemic- cells demonstrate FL and FLT3 co-expression [50, 59–61]. Thus, there is recognized evidence that FL

FLT3-activating mutations represent the most frequent identified genetic mutation in de novo AML (−30%) and rarely in myelodysplastic syndromes (−5%). FLT3 genetic aberration is never observed in CML, bi phenotypic ALL, chronic lymphoid leukemia, non-Hodgkin's lymphoma,

In 1996, the first type of FLT3 mutation was discovered as a duplication of gene (internal tandem duplication [ITD]) within the JM domain of FLT3 programmed by exons 14 and 15.

causes autocrine effect in AML, which leads to development of leukemia.

over 55 years of age [51]. In contrast, only 10% pediatric group had this mutation.

**3.7. Clinical prevalence and characteristics of FLT3 mutations**

ease entity of leukemia.

**3.8. Future perspectives**

**3.9. Genomic alteration of FLT3 in AML**

or multiple myeloma patients.

**Figure 1.** Signal transduction pathways downstream of FMS-like tyrosine kinase-3 receptor activation. Abbreviations: CEBPA, CCAAT/enhancer-binding protein alpha; ERK, extracellular signal-regulated kinase; MAP, mitogen-activated protein; PI3-kinase, phosphatidylinositol 3-kinase. Adapted from STEMCELLS 2006;24:1174–1184. www.StemCells.com

of phosphatidylinositol 3-kinase and to the adaptor protein growth factor receptor-bound protein 2, phospholipase C\_1 and a GTPase-activating protein of the Ras proliferation pathway [40, 41]. Normally, FLT3 activates extracellular kinase 1/2 but weaker phosphorylation of STAT5, which is a key target during deregulated signaling [23].

#### **3.6. Significant clinical mutation in FLT3 receptors**

FL-FLT3 interaction controls survival, proliferation, and differentiation of stem cells. FLT3 is also expressed on the surface of a high proportion of AML and B-lineage ALL cells [25, 42–45]. In FLT3-expressing leukemia cells, FL-stimulation leads to increased survival of leukemic cells [46]. In 1996, a unique mutation of the FLT3 gene was first identified in AML cells. This mutation (FLT3-ITD) is produced when a fragment portion of the JM domaincoding sequence is multiplied in a direct head-to-tail orientation [47]. There are two types of mutations of FLT3 receptor, i.e., FLT3/ITD and FLT3/KDM that occur in 15–35 and 5–10%, respectively. Mutations of the FLT3 gene are, therefore, the most frequent genetic alterations so far reported as having involvement in AM which should be deleted or written somewhere else [8, 9, 33, 48, 49]. In comparison to AML, FLT3-ITD is far less common in patients with ALL, but FLT3/KDM is recurrently found in patients with ALL, especially in those harboring an MLL gene rearrangement or hyperdiploidy [7]. It is observed that ALL cells, which strongly express FLT3 but do not carry FLT3 mutations, have the same sensitivity to a potent FLT3 inhibitor as leukemia cells with FLT3 mutations [10]. Recently, high concentration of FLT3 transcripts in AML patients without FLT3 mutations is associated with a poor prognosis [6]. These studies indicate that FLT3 is greatly involved in the pathophysiology of leukemia. Mutations and expression levels of FLT3 distinguish a disease entity of leukemia.

#### **3.7. Clinical prevalence and characteristics of FLT3 mutations**

These studies reveal that FLT3 mutations are mainly identified in AML and more infrequent in secondary AML developed from myelodysplastic syndrome (MDS) or therapy-related AML than de novo AML. This mutation is also found in ALL and MDS, but never in chronic myeloid leukemia, chronic lymphoid leukemia, multiple myeloma, malignant lymphoma, or myeloproliferative disorders (MPD) [50]. Frequency of this mutation in MDS is reported to be 3%, but it increased to 15% in advanced stage and after AML conversion [46]. The incidence of FLT3 mutations is directly proportional to the age of patients with AML. In elderly patients, FLT3-ITD has been found in approximately 25% of this population; among them 31.4% were over 55 years of age [51]. In contrast, only 10% pediatric group had this mutation.

#### **3.8. Future perspectives**

of phosphatidylinositol 3-kinase and to the adaptor protein growth factor receptor-bound protein 2, phospholipase C\_1 and a GTPase-activating protein of the Ras proliferation pathway [40, 41]. Normally, FLT3 activates extracellular kinase 1/2 but weaker phosphorylation of

**Figure 1.** Signal transduction pathways downstream of FMS-like tyrosine kinase-3 receptor activation. Abbreviations: CEBPA, CCAAT/enhancer-binding protein alpha; ERK, extracellular signal-regulated kinase; MAP, mitogen-activated protein; PI3-kinase, phosphatidylinositol 3-kinase. Adapted from STEMCELLS 2006;24:1174–1184. www.StemCells.com

FL-FLT3 interaction controls survival, proliferation, and differentiation of stem cells. FLT3 is also expressed on the surface of a high proportion of AML and B-lineage ALL cells [25, 42–45]. In FLT3-expressing leukemia cells, FL-stimulation leads to increased survival of leukemic cells [46]. In 1996, a unique mutation of the FLT3 gene was first identified in AML cells. This mutation (FLT3-ITD) is produced when a fragment portion of the JM domaincoding sequence is multiplied in a direct head-to-tail orientation [47]. There are two types of mutations of FLT3 receptor, i.e., FLT3/ITD and FLT3/KDM that occur in 15–35 and 5–10%, respectively. Mutations of the FLT3 gene are, therefore, the most frequent genetic alterations so far reported as having involvement in AM which should be deleted or written somewhere else [8, 9, 33, 48, 49]. In comparison to AML, FLT3-ITD is far less common in patients with ALL, but FLT3/KDM is recurrently found in patients with ALL, especially in those harboring an MLL gene rearrangement or hyperdiploidy [7]. It is observed that ALL cells, which strongly express FLT3 but do not carry FLT3 mutations, have the same sensitivity to a potent FLT3 inhibitor as leukemia cells with FLT3 mutations [10]. Recently, high concentration of FLT3 transcripts in AML patients without FLT3 mutations is associated with a poor prognosis [6]. These studies indicate that FLT3 is greatly involved in the

STAT5, which is a key target during deregulated signaling [23].

**3.6. Significant clinical mutation in FLT3 receptors**

70 Myeloid Leukemia

FLT3 mutations are detected in 25% of AML patients, revealing that the FLT3 gene is the target most frequently mutated. FLT3 is also implicated in the pathogenesis in few patients of ALL, such as those with MLL gene rearrangement or hyperdiploidy. As FLT3 mutation is closely associated with poor prognosis, routine testing of this mutation is recommended to stratify the patients into distinct risk groups. However, optimal treatment strategy according to this mutation is under evaluation because it remains uncertain that high-dose chemotherapy and/ or myeloablative therapy followed by hematopoietic stem cell transplantation will change the prognosis of FLT3-mutated patients. Mutated or overexpressed FLT3 is an important molecular target in the treatment of leukemia and several potent inhibitors in clinical phase 1 and 2 trials are underway [52–55]. At present, the clinical efficacy of FLT3 inhibitors seems unimpressive, and problems related to adverse effects and pharmacokinetics are observed. Further studies are required to establish the role of FLT3 inhibitors in the treatment of leukemia.

#### **3.9. Genomic alteration of FLT3 in AML**

FLT3 receptor is expressed in the vast majority of human B-lineage acute lymphocytic leukemias (ALL) and most myeloid leukemias in different types of AML (M0–M7) [56–58]. A smaller proportion of T-cell-related leukemias also possess (less than 30%) FLT3 receptor [50, 59–62]. Further analyses revealed that the mean mRNA expression level of FLT3 is higher in leukemic cells than in normal progenitor cells, but percentage is different in various samples of AML patients. Nearly, all primary AML patients and other immature leukemic- cells demonstrate FL and FLT3 co-expression [50, 59–61]. Thus, there is recognized evidence that FL causes autocrine effect in AML, which leads to development of leukemia.

FLT3-activating mutations represent the most frequent identified genetic mutation in de novo AML (−30%) and rarely in myelodysplastic syndromes (−5%). FLT3 genetic aberration is never observed in CML, bi phenotypic ALL, chronic lymphoid leukemia, non-Hodgkin's lymphoma, or multiple myeloma patients.

In 1996, the first type of FLT3 mutation was discovered as a duplication of gene (internal tandem duplication [ITD]) within the JM domain of FLT3 programmed by exons 14 and 15. The duplicated areas are inconsistent in size (between 3 and 400 bp) and also in location, but the resulting product is always rich in tyrosine residues. It is hypothesized that modification of the length of the JM domain, rather than increase of tyrosine chain, causes enhancement of function of FLT3. Furthermore, an activating point mutation has been identified in the JM domain in immature leukemia cell. Mono Mac 1 and Mono Mac6 causes substitution of valine by alanine at position valine 592 (V592A) [62], but this mutation has not yet been reported in primary AML blasts.

whether FLT3-TKD receptors phosphorylate the same receptor and signal similarly to FLT3- ITD receptors. It is well known that FLT3-D835 mutations are not associated with a significant decrease on the overall survival. FLT3-TKD mutations are also less tumorigenic than FLT3- ITD mutations, even hypothesized that FLT3- D835Y receptors show a higher level of tyrosine

Genomics of Acute Myeloid Leukemia http://dx.doi.org/10.5772/intechopen.72757 73

**4. Co-expression of other mutant genes with FLT 3/ITD & their** 

**4.1. Prognostic and predictive value of the NPM1, FLT3, and CEBPA genotypes**

Actually, no data is available for specific chemotherapy for NPM1mut [74].

NPM1 mutation or combined genotype NPM1mut/FLT3-ITDneg is reported as a favorable prognostic marker for attainment of a complete remission after induction therapy [70–73].

In a more recent study, NPM1 was shown to act as a co-repressor in retinoic acid-associated transcriptional regulation in a manner such that during retinoic acid-induced cellular differentiation, activating protein transcription factor 2 (AP2) recruits NPM1 to the promoter of certain retinoic acid-responsive genes. The German-Austrian AML Study Group (AMLSG) reported favorable effect of ATRA if given with conventional chemotherapy on complete remission rate, event-free survival, and OS in elderly patients with (non- APL) AML1 [50]. This study finding was coinciding with retrospective data in which beneficial effect of ATRA was restricted to NPM1mut/FLT3-ITDneg patients [75]. So, the genotype NPM1mut/FLT3- ITDneg appears as a predictive genotypic marker for the valuable effect of ATRA in non-

FLT3-ITD has been reported consistently as an unfavorable prognostic marker for RFS and OS. Whether other molecular markers, in particular NPM1mut, add to prognostication in FLT3-ITDpos, AML is unclear [76, 77]. It is reported in some studies that genotype NPM1mut/ FLT3-ITDpos shows more favorable prognosis compared with the genotype NPM1WT/FLT3- ITDpos; however, confirmation by other studies are due now [56]. More recent data give insight that outcome is also related to the concentration of the mutant allele and not just its mere presence [77]. However, if NPM1 mutation status was included to the prognostic model, the mutant wild-type ratio of FLT3-ITD was not an important prognostic factor. Currently in randomized multicenter phase III trial [Cancer and Leukemia Group B (CALGB) 10603; clinicaltrials.gov, NCT00651261] wild-type ratio (high versus low) is applied for midostaurin

CEBPA mutations are another genetic abnormality that consistently associated with good prognosis, either in the subset of patients with intermediate-risk cytogenetics or with normal karyotypes [74]. In the context of other molecular markers, the mutated CEBPA alone retained its prognostic significance for RFS and OS; additional mutations did not affect outcome in the CEBPA mut subgroup. This needs validation. Actually, even in the largest cohort of patients analyzed so far in CN-AML, the sample size in the CEBPA mut subgroup was too

kinase activity than FLT3-ITD receptors.

(PKC412) in young adult AML patients.

**predictive value**

APL AML.

A different group of genetic makeup has been described in the activation loop within the second tyrosine kinase domain of the FLT3 receptor that normally inhibits the binding of adenosine triphosphate and substrate to the kinase domain when the receptor is in a dormant state. Frequently, these involve mutations at aspartic acid 835 (D835) or isoleucine 836 (1836) in exon 20.

### **3.10. FLT3-ITD**

FLT3-ITD mutations are present in 20–25% of adult patients with AML and correlate with higher WBC count at diagnosis, increased relapse risk, and poor prognosis*.* Moreover, simultaneous loss of the wild-type FLT3 allele is associated with significantly inferior outcome and decreased overall survival in FLT3-ITD patients*.* In pediatric AML patients, FLT3-ITD is detected in 11–16% of cases and linked to worse prognosis [30, 63–65]*.* FLT3-ITD mutations more frequently occur in acute prolymphocytic leukemia (30–39%) containing t(15;17), which produces the PML-RARα oncogene. FLT3-ITD receptors are characterized by ligand-independent receptor dimerization and phosphorylation, but the accurate mechanism of this mutation binding and how it causes constitutive kinase activity has not yet been fully elucidated. Both lengthening as well as shortening of the JM domain result in activation of FLT3 receptor [65]*.*

Structural analysis of the EphB2 RTK, it described that JM domain takes up a z-helical conformation, which inhibits the activation of kinase and also self-dimerization. FLT3-ITD would affect the hindrance of kinase domain and, with the help of JM domain, would produce auto phosphorylation. In further studies of HSCT bone marrow cells in mice which done by retro virally transduced with FLT3-ITD, which produced oligo clonal band of MPD due to oncogenic potential of FLT3 ITD mutation [66].

#### **3.11. TKD mutations**

Activating TKD mutations are noticed in 7–14% of adults with AML, but D835 has no significant correlation with poor outcome. In pediatric group this mutation was observed 3–8%. Clustering algorithms has identified that childhood acute leukemias carrying rearrangements of MLL gene on chromosome 11q23 show overexpression of wild-type FLT3 mRNA easily distinguishing them from conventional pre-B ALL and AML. Interestingly, FLT3-TKD deletions involving codons D835 and I836 were identified more frequently with MLL gene [67, 68]. Patient with these activating FLT3-TKD alterations expresses higher levels of FLT3 transcripts [69].

Like FLT3-ITD receptors, FLT3-TKD mutations stimulate ligand-independent receptor activation and promote growth factor independence in 32D cells. However, it is under evaluation whether FLT3-TKD receptors phosphorylate the same receptor and signal similarly to FLT3- ITD receptors. It is well known that FLT3-D835 mutations are not associated with a significant decrease on the overall survival. FLT3-TKD mutations are also less tumorigenic than FLT3- ITD mutations, even hypothesized that FLT3- D835Y receptors show a higher level of tyrosine kinase activity than FLT3-ITD receptors.
