**2. Molecular basis and gene expression in acute myeloid leukemia**

#### **2.1. Mutations related to pathogenesis**

Mutations in AML are different according to the age of patient. Balanced translocations are common in children and adolescents, while in elderly complex karyotypes are common [11]. To date, more than 80 mutations are identified. These rearrangements act as driver mutations to initiate leukemic phase. Chromosomal derangement leads to disruption of transcription factor that controls directly hematopoiesis. Example is RARα that is formed by realignment of 17q21 [12]. Besides fusion genes other chromosomal abnormalities are also important in pathogenesis of AML. These are divided into three groups. (1) mutations abnormally controlled transcription factors that are helpful in hematopoieses, (2) mutations related to certain receptors, i.e., tyrosine kinase receptors, and (3) mutations involving the gene that encodes nucleophosmin [13].

#### **2.2. Abrasions in gene associated with transcription**

Hematopoiesis is regulated by various transcriptions factors which encodes with genes. These genes can get mutation which leads to inactivated or dominated in regulation of hematopoietic functions. Indeed, AML1mutations are detected in up to 25% of M0 cases and are frequently biallelic [14]. The CEBPA, which plays an important role in granulopoiesis, also is a relatively common target in AML, being potentially deregulated by the AML1-ETO and promyelocytic leukemia-retinoic acid receptor alpha (PML-RARα) oncoproteins. Furthermore, mutations involving CEBPA are present in approximately 10% of cases of AML [15].

#### **2.3. Karyotypic abnormalities in AML**

Novel molecular markers of prognostic and more importantly of predictive significance have been identified in different leukemias. The link between the leukemogenic importance of these markers and their role as potential targets for novel drugs has started to contribute to the stepwise replacement of risk adapted by treatment strategies, e.g., imatinib in chronic myeloid leukemia (CML) and all-trans-retinoic acid (ATRA) in acute promyelocytic leukemia (APL).

Over the past few decades, it has become clear that a significant proportion of cases of AML are characterized by at least one of a varieties of recurrent chromosomal abnormalities, e.g., t (8;21), t(15;17), etc., or by the presence of prognostic markers, e.g., FMS-like tyrosine kinase 3 (FLT3), multilineage leukemia (MLL), nucleophosmin 1 (NPM1) and CCAAT/enhancerbinding protein-α (CEBPA). A key challenge for the future is to use information gained from cytogenetic analysis in conjunction with molecular diagnosis and gene expression profiling to achieve greater consensus in the risk group assignment of AML, and risk adapted therapy. FLT 3 is widely known as FLT3 is a class III tyrosine kinase receptor [2]. Its structure consists of an extracellular ligand-binding domain, a single transmembrane domain, and a cytoplasmic region consisting of juxtamembrane domain and kinase domain interrupted by kinase insert [2, 3]. FLT3 is normally expressed on hematopoietic progenitor stem cells (HPSCs) where it plays an important role in survival and proliferation of stem cells. Its expression is

FLT3 mutations have been detected in one-third of AML patients and a small number of ALL patients [2].The mutations most commonly involve internal tandem duplications (ITDs) of the juxtamembrane domain (JM) (detected in 23% of AML patients and <1% of ALL patients) and point mutations in the activation loop of tyrosine kinase domain [2–4]. It has been detected in

Prevalence of FLT3-ITD is according to age, i.e., rare in infants. FLT3 positivity was reported to be 5–10% in patients younger than 10 years, but it rose to 35% in middle-aged patients. Patients with this mutation usually present with increased white blood counts (WBC) and have normal cytogenetics [6]. In literature, there is no marked difference in complete response (CR) between FLT3 positive and FLT 3 negative patients, but relapse rate and overall survival(OS) are lower in FLT3-ITD-positive patients specially in younger than 60 years [7].

Further studies have confirmed that FLT3-ITD is not only inversely correlated with relapse but also associated with decreased overall survival [8–10]. In Kottaridis study, the prevalence of FLT3ITD was 27%. In same study, this mutation was strongly associated with hyperleukocytosis and normal cytogenetics. In literature, on multivariate analysis this mutation has the

Mutations in AML are different according to the age of patient. Balanced translocations are common in children and adolescents, while in elderly complex karyotypes are common [11]. To date, more than 80 mutations are identified. These rearrangements act as driver mutations to

**2. Molecular basis and gene expression in acute myeloid leukemia**

lost with HPSC differentiation [2].

66 Myeloid Leukemia

7–12% of AML patients and about 3% of ALL patients [4, 5].

strongest correlation with decrease DFS [10].

**2.1. Mutations related to pathogenesis**

Evaluation of karyotypic abnormalities has prognostic and therapeutic implications. Detection of t(15:17) by cytogenetic analysis shows favorable treatment response by ATRA. Similarly, t(9:22) is not responded to conventional chemotherapy but showed good results to imatinib. Approximately 60% of newly diagnosed cases with less than 20% blasts in the bone marrow have abnormal karyotype. Prognostically, t(15:17),inv(16), and t(8:21) showed relatively better prognosis in comparison to monosomies 5 or 7 [16].

#### **2.4. Chromosomal aberration detection by FISH**

The absence of fusion genes in good prognostic group necessitates evaluation by FISH to accurately define prognosis. This can be exemplified by complex pattern of losses and loss of chromosome 5 or 7. Other than FISH, southern blot analysis can be used for chromosomal abnormality evaluation. However, it is more laborious than real-time PCR but more informative in certain circumstances, i.e., MLL gene rearrangement [17]. This approach can be used in the future for minimal residual disease assessment in AML.

#### **2.5. Predictive mutations in leukemogenesis**

The most notable is FLT3-ITD, which is associated with decreased duration of remission. However, its impact on survival after post bone marrow transplant in the first CR is not clearly documented [18]. The presence of EVI1 in the absence of chromosome 3q abnormalities depicts poor survival. Mutations that show good response to conventional chemotherapy and do not need bone marrow transplant in the first CR are the presence of NPM1 or CEBPA mutation without concomitant FLT3-ITD mutation [19]. In the era of molecular medicine, new tests at molecular levels are being under process. New molecularly targeted agents are underway on the basis of these special tests. Examples of these are FLT3 inhibitors that can modify outcome of poor prognostic group.

#### **2.6. Mutations in AML**

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

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

Genomics of Acute Myeloid Leukemia http://dx.doi.org/10.5772/intechopen.72757 69

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

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

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

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

marrow hyperplasia, splenomegaly, hepatomegaly, and enlarged lymph nodes [39].

domain [38].

unknown [27].

**3.3. FLT3 ligand(FL)**

uncontrolled FL secretion [37].

**3.5. FLT3 receptor signaling**

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

**3.2. FLT3 receptor expression**

#### **2.7. Prognosis of AML**

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 decisions on allogeneic transplantation [9, 30].

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

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].
