Chapter one Introduction

Chapter one
Introduction
& review of literature

INTRODUCTION
1.1. Introduction
Acute myeloid leukemia (AML) is a malignant clonal disorder characterized by alterations and low production of healthy hematopoietic cells; these alterations inhibit differentiation of cells and induce proliferation or accumulation of blasts. Blasts replace normal hematopoietic tissue,
triggering the appearance of cytopenias. The accumulation of immature cells begins in the bone marrow, but in most cases quickly builds up in the blood(1).

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The etiology of AML is heterogeneous and complex, but it is widely accepted that both environmental and genetic factors play significant roles in the development of the disease (2).

Acute myeloid leukemia (AML) accounts for about 90% of
acute leukemia in adults, and its incidence increases with age.
Although some clinic advances including new molecule targeting
agents and hematopoietic stem cell transplantation
(HSCT) have been applied over the past three decades, the
five-year survival of adult AML still ranges from 30 to 40%
in total patients and is even lower than 15% in the refractory
and relapsed ones3.
Accurate diagnosis and classification in AML are essential for treatment decisions and assessment of prognosis. Initial assessment requires a careful history, physical exam, complete blood count (CBC) with peripheral blood (PB) smear review, bone marrow (BM) examination, flow cytometry (FC), cytogenetics, and selected molecular genetic analyses(4).

Interleukin 35 (IL 35), is a recently identified heterodimeric cytokine of an IL-12 family consisting of Epstein-Barr virus-induced gene protein 3 (EBI3) and the p35 subunit of IL 12 (7). In contrast to all other known IL 12 family members, which are not expressed by T cells, IL 35 is secreted by Treg cells and contributes to their suppressive activity, rather than acting as an immunostimulatory or proinflammatory manner (5).
Interleukin 10 (IL-10)

Interleukin-10 (IL-10) is predominately secreted by immune cells including macrophages, T lymphocytes, and natural killer (killer) NK cells, and constitutes a major determinant of viral clearance vs. persistent infection . IL-10 which still having unclear role in cancer pathogenesis, some studies showed that IL-10 involved in the development and progression of cancer in humans, as a tumor promoting (promote cancer potentially) has immune-stimulating and immuno suppressive (inhibit cancer potentially) (6).
In this study , we examined IL_35 along with IL_10 in adult AML, as a tumor promoting and tumor inhibiting factors that may regulate tumor susceptibility and development.

1.2. Aim of the study:
– To estimate the level of IL-35 along with IL-10 in the serum collected from group of Iraqi patients with AML before and after chemotherapy.

– To correlate between the level of IL-10 and IL-35 with blast percentage in peripheral blood, bone marrow and with other heamatological parameters.

REVIEW OF LITERATURE
1.3. Acute myeloid leukemia (AML)
1.3.1 Definition:
AML can be identified as a malignancy that are originating from transformed multipotent hematopoitic progenitor cells, it is characterized by clonal proliferation of abnormal blast cells in the marrow and impaired production of normal blood cells causing anaemia; thrombocytopenia; and low, normal, or high white cell count depending on the concentration of leukemic cells in the blood(7).
AML occurs in many morphologic variants, each with characteristic cytologic, genetic, and sometimes clinical features.(8)
the term Leukämie (derived from the Greek meaning “white blood.”) were used for the first time in 1847 by Virchow as a medical entity characterized by too many white blood cells (9,10) .
In 1869 Neumann proposed that white blood cells were made in the BM and not the spleen and suggested the term myeloid, meaning marrow-derived. During the 1970s, ;The morphological appearance and cytochemical stain are culminated in the development of what became known as the FAB classification of AML. In 1997 the World Health Organization (WHO) were proposed a new classification sytem that based on genetic, morphologic and immunophenotypic characteristics of the disease(9).

1.3.2 Epidemiology and etiology:
AML accounts for 80% of acute leukemias in adult and 15% to 20% in children. AML is the most frequent leukemia in neonate. This result in bimodal incidence curve with a peak at less than one year of age of approximately 2 per 100,000 infant, dropping to approximately o.4 cases per 100,000 at age 7 years, and then increasing to 1.0 per 100, 000 by age 25 years. Thereafter, incidence increases exponentially to 20 cases per 100,000 on octogenarians.an exception to the striking change in the incidence with age in adults is found in Acute promyelocytic leukemia (APL) in which the incidence by age does not vary as significantly(7).
In Iraq, according to the Iraqi Cancer Registry 2008 (3), leukemia ranks the third among the ten common cancers in Iraq, constituting 6.77% of all cancers with an annual incidence of 3.01 per 100,000 populations(11).
Most AML cases are sporadic. The identification of the cause of developing AML is usually not possible.
Four exposures have been established as causative factors. These include high dose radiation; higher dose chronic benzene exposure, usually in an industrial setting; treatment of other cancers or sever autoimmune syndromes with alkalyting agents, topoisomerase II inhibitors, or some other cytotoxic drugs; and prolonged tobacco smoking table (1.1) (7).

Table (1.1) risk factor for AML.
Genetic Disorders:
• Down syndrome
• Klinefelter syndrome
• Patau syndrome
• Ataxia-telangiectasia
• Shwachman syndrome
• Kostmann syndrome
• Neurofibromatosis
• Fanconi anaemia
• Familial platelet disorder syndrome
• Li-Fraumeni syndrome
Acquired Disorders Myeloproliferative disorders
• Myelodysplastic syndrome (MDS)
• Aplastic anaemia • Paroxysmal nocturnal haemoglobinuria (PNH)
• Multiple myeloma
Physical and Chemical Exposure:
• Benzene
• Cigarettes smoking
• Embalming fluid (ethylene oxide)
• Pesticides

Radiation exposure:
• Non-therapeutic • Therapeutic radiation
Chemotherapy:
• Alkylating agents
• Topoisomerase-II inhibitors
• Anthracyclines
• Taxanes

1.3.3 Pathogenesis:
AML results from a sequence of mutations in a multipotential hematopoitic cells or, a more differentiated, lineage restricted progenitor cell. These mutations causing disruption of differentiation of stem cell and maturation of unilineage progenitor, regulation of proliferation, and of cell survival (apoptosis) in varying combination. This complexity results in many phenotype. AML cases either result from clonal evolution of chronic myeloid disorder such as polycythemia vera, primary myelofibrosis, essential thrombocythemia, and chronic myeloid leukemia, or may arise de novo as an acquired cytogenetic changes, including translocation, inversions, and deletions. These changes lead to mutation of proto-oncogens and the formation of oncogens. (7).

There are different types of mutations which appear to culminate in the pathogenesis of AML:(12)
• Class I comprises mutations which activate signal transduction pathways resulting in enhanced proliferation and/or survival of leukaemic progenitor cells such as mutations leading to activation of the receptor tyrosine kinase Fms-like tyrosine kinase 3 (FLT3) or the RAS signalling pathway.
• Class II comprises mutations that affect transcription factors or components of the transcriptional co-activation complex, resulting in impaired differentiation and/or aberrant acquisition of self-renewal properties by haemopoietic progenitors, such as the recurring gene fusions resulting from t(8;21), inv(16)/t(16;16), t(15;17), as well as mutations in CCAAT-enhancer-binding protein (CEBPA) and mixed lineage leukaemia (MLL) genes.

1.3.4 Cytogenetic and molecular genetics:

Cytogenetic abnormalities can be recognized in approximately 60% of newly diagnosed AML patients. Most of these cases are associated with balanced chromosomal translocations that often result in gene rearrangement.(13)
One of these is the t(8;21)(q22;q22) translocation which produces RUNX1-RUNX1T1 fusion product that enhances self-renewal of haemopoietic stem cells and blocks haemopoietic differentiation. The inv(16)(p13q22) or t(16;16)(p13;q22) produces the leukaemogenic CBF?-MYH11 fusion gene which blocks differentiation of haematopoietic stem cells by inhibiting the function of Runt-related transcription factor 1 (RUNX1). Acute promyelcytic leukaemia cells usually have t(15;17)(q22;q11-21) producing PML-RARA fusion products which also behave as a transcriptional repressor.(14)
Overall survival, remission rate, and relapse can be assessed by cytogenetic which act as an important prognostic factor.(15) Besides cytogenetic abnormalities, genetic mutations are frequent in AML patients, including mutations of FLT3 occur in two major mutant forms, an internal tandem duplication (ITD) or a point mutation in the tyrosine kinase domain (TKD) in40% and 6% of AML patient with normal karyotype, respectively. Activating mutations at codon 12, 13 or 61 of the NRAS occur in 10% of AML patients. About 15% of AML patients have inactivating mutations of CEBPA. Nucleophosmin1 (NPM1) is mutated in 50% of AML patients with normal karyotype. This protein has an important role in ribosome biogenesis, including nuclear export of ribosomal proteins, mutant NPM1 has more favorable outcomes and it is most frequently mutated gene in AML.(16)

1.3.5 Clinical Features:
Signs and symptoms
Acute myeloid leukaemia was found to be a highly variable disease that presented with non-specific signs and symptoms. The presenting signs and symptoms of AML are related to proliferation of leukemic cells or bone marrow failure .17
Pallor, fatigue, and dyspnea indicate the development of anaemia.19 Common presenting feature is fever occur in about 20% of patients; as a result of infection due to neutropenia or result from leukaemia itself.
common initial manifestations are petechiae, ecchymosis, gums bleeding, epistaxis, and bleeding after trivial trauma. (17,18)
Hyper leukocytosis is present in 10 to 20 percent of patients with newly diagnosed (AML). It is more common in patients with myelomonocytic (FAB-M4) leukemia, monocytic (FAB-M5) leukemia, or the microgranular variant of acute promyelocytic leukemia (FAB-M3)(19,20).
Symptoms of leukostasis occur less frequently and typically affect patients with white blood cell (WBC) counts over 100 x 109/L(21).
lymphadenopathy or organomegaly may occur, but are unusual features.
Disseminated intravascular coagulation, due to consumption of platelets and clotting factors, is a common presenting feature of acute promyelocytic leukaemia. Gum hypertrophy and skin lesion are seen in the monocytic variants.
unlike in acute lymphoblastic leukaemia, cerebrospinal infiltration is not usual. (13).
Bone pain occur in 25% of cases and it is an important consideration in the treatment of the monocytic type of AML.(22)

• granulocytic sarcoma (MS), is defined as the occurrence of one or multiple tumour masses composed of immature myeloid cells presenting at an extramedullary site; occurs most frequently in the skin, bone or lymph node. It may develop before AML by months or years or occur concurrently.23

1.3.6 Classification
1.6.1 French-American-British classification (FAB)

The classification of acute leukemia has evolved initially in the past few decades. The FAB classification was based on morphologic features of blast cell population on Romanovsky-stained bone marrow aspirate smears and the result of cytochemical studies.24
This classification did not depend on morphologic features of non blast cells in AML particularly whether dysplastic features is present or not. The FAB classification determine the 30% bone marrow blast cell cutoff. (25)

This classification divides AML into 8 subtypes (M0 to M7). Although AML blasts evolve from common myeloid precursors, the 8 subtypes differ in degree of maturation as elucidated in the table (1-2). (26)

Table (1-2) FAB classification 26

subtype
M0 AML with no evidence of differentiation
M1 Myeloblastic leukaemia with little maturation
M2 Myeloblastic leukaemia with maturation
M3 Acute promyelocytic leukemia
? Hypergranular variant
? Microgranular variant
M4 Acute myelomonocytic leukaemia
? M4Eo: with dysplastic marrow eosinophils
M5 Acute monoblastic leukaemia
? M5a: acute monoblastic leukaemia
? M5b: acute monocytic leukaemia
M6 Erythroleukaemia
? AML with erythroid dysplasia
? Erythroleukaemia
M7 Acute megakaryoblastic leukaemia

1.6.2. World Health Organization (WHO) classification

In 2001, the World Health Organization (WHO) introduced a new classification system followed by a revised version in 2008. Later in 2016 a new revised version was released, the WHO classification of AML distinguishes itself by incorporating genetic information with morphology, immunophenotype and clinical presentation to define six major disease entities: (Table 1)(27)

In the WHO system, the cut-off blast cells percentage for making a diagnosis of AML was lowered to 20% in peripheral blood or bone marrow; however, clonal, recurring cytogenetic abnormalities,(28)
t(8; 21)(q22; q22), t (16; 16)(p13; q22), inv (16)(p13; q22), or t (15; 17)(q22; q12), should be considered AML regardless of blast cells percentage(29).

(Table 1) World Health Organization (WHO) classification

AML with recurrent genetic abnormalities
AML with t(8;21)(q22;q22), RUNX1–RUNX1T1
AML with inv(16)(p13.1q22) or t(16;16)(p13.1;q22), CBF?–MYH11
Acute promyelocytic leukaemia with t(15;17)(q22;q11-12), PML–RARA
AML with t (9; 11)(p22; q23), MLLT3–MLL
AML with t (6; 9)(p23; q34), DEK–NUP214
AML with inv(3)(q21q26.2) or t(3;3)(q21;q26.2), RPN1–EVI1
AML (megakaryoblastic) with t(1;22)(p13;q13), RBM15–MKL1
Provisional entity: AML with mutated NPM1
Provisional entity: AML with mutated CEBPA

AML with myelodysplasia – related changes
Therapy- related myeloid neoplasms
AML, not otherwise categorized
AML with minimal differentiation
AML without maturation
AML with maturation
Acute myelomonocytic leukaemia
Acute monoblastic/monocytic leukaemia
Acute erythroid leukaemia
– Pure erythroid leukaemia
– Erythroleukaemia, erythroid/myeloid
Acute megakaryoblastic leukaemia
Acute basophilic leukaemia
Acute panmyelosis with myelofibrosis (acute myelofibrosis; acute myelosclerosis)
Myeloid sarcoma (extramedullary myeloid tumor; granulocytic sarcoma; chloroma)
Blastic plasmacytoid dendritic cell neoplasm
Acute leukemia’s of ambiguous lineage
Acute leukaemias of ambiguous lineage
Acute undifferentiated leukaemia
Mixed phenotype acute leukaemia (MPAL) with t (9; 22)(q34.1; q11.2); BCR-ABL1
MPAL with t (v; 11q23.3); KMT2A rearranged
MPAL, B/myeloid, NOS
MPAL, T/myeloid, NOS

1.6.3 European Group for the immunological classification of leukaemia (EGIL)
The immunological classification of acute leukemias are suggested by an established European group designated EGIL. The main aims of EGIL are to establish guidelines for the characterization of acute leukemias based on marker expression and provide basis for the diagnosis of the different types of these hemopoietic malignancies which should be valuable for future clinical and laboratory investigations. This classification has suggested that acute leukaemia can be classified as myeloid, B lineage, T lineage, or biphenotypic. It also suggests criteria for distinguishing biphenotypic leukaemia from AML with aberrant expression of lymphoid antigens, and from ALL with aberrant expression of myeloid antigens. (30)
However, a purely immunological classification has the disadvantage that discrete entities may fall into one of two categories; for example some cases of AML of FAB M2 subtype associated with t (8;21)(q22;q22) would be classified as “AML of myelomonocytic lineage”, while others would be classified as “AML with lymphoid antigen expression,” depending on whether or not a case showed aberrant expression of CD19. In addition, rare cases of acute leukaemia have been described which were clearly myeloid when assessed by cytology and cytochemistry but which did not express any of the commonly investigated myeloid antigens. The consensus considers a 20% minimum threshold to define a positive reaction of blast cells to a given monoclonal antibody.(24)

1.3.7 Diagnosis

the diagnosis of AML is important for treatment decisions and assessment of prognosis. Once clinical suspicion is started, a precise history ( medical history and family history), physical exam, complete blood count (CBC) with blood film, BM examination, flow cytometric analysis, cytogenetic, and molecular genetic analyses should performed for accurate diagnosis.(31)

1.3.7.1 CBC and Blood Film
Anaemia and thrombocytopenia are nearly always present. Half the patient with AML have a platelet count less than 50*109/l. The first clue for the diagnosis of AML is a defective result of the total leucocytes count which is below 5.0 *109/l in about one half of the patients, and the absolute neutrophil count is less than 1.0*109/l in more than one half of the patients at diagnosis(18).
high leucocyte count (100 x 109/L) may be present in 5-30% of patients. AML may also present with a normal leucocyte count. Eosinophilia and basophilia may be present in some subtypes of AML (32).
Blood film examination is based on identification of leukemic blast cells in the peripheral blood smear. Auer rods are considered as a pathogenic feature specific for AML (33).
Presumptive diagnosis of AML can be made by examining the PB smear where leukaemic blasts are circulating in PB, but definitive diagnosis is made by examination of the aspirate or biopsy of BM. In some cases, if the condition of the patient does not allow the puncture of BM or biopsy and if there is evidence of 20% blasts in PB, the diagnosis can be made.(24)

1.3.7.2 Bone marrow examination

the presence of a minimum of 20% blasts in the BM is required or diagnosis of AML with exception for AML with t(15;17), t(8;21), inv(16) or t(16;16), so bone marrow aspirate are strongly recommended as routine diagnostic test for suspected AML cases. The term Blast include myeloblasts, monoblasts, and megakarioblasts, monoblasts and promonocytes, but not abnormal monocytes. Erythroblasts are not considered as blasts except in the rare cases of erythroleukaemia.34

The enumeration of blasts in the bone marrow is crucial in the diagnosis of acute leukaemia and the definition of a blast cell is therefore important. Myeloblasts were defined in terms of several nuclear characteristics, including a high nuclear/cytoplasmic ratio, easily visible nucleoli and usually, but not invariably, fine nuclear chromatin. Nuclear shape is variable. Cytoplasmic characteristics include variable cytoplasmic basophilia; there may or may not be granules or Auer rods but no Golgi zone is detected. The exception to this last observation may be seen in cases of AML with t(8;21) where there may be blast cells with a small distinct Golgi, with or without an Auer rod, but with no other features of a promyelocyte.35

1.3.7.3 Cytochemistry
The cytochemistry of blast cells as demonstrated by staining techniques is useful in the diagnosis of the acute leukemia.
The principal uses of cytochemistry in AML are:36

1. To recognize the blast cells in acute leukaemia as myeloid lineage.
2. To demonstrate myeloperoxidase (MPO) or non-specific esterases (NSEs) and thus contribute to a diagnosis of mixed-phenotype acute leukaemia.
3. To identify granulocytic and monocytic components in AML.
4. To identify unusual lineages occasionally involved in AML (e.g. basophils and mast cells).

Myeloperoxidase (MPO) or Sudan black B (SBB) are the cytochemical stains that are advised for granulocytic differentiation and non- specific esterase (NSE) stain such as @ – Naphthyl acetate esterase (ANAE) for monocytic differentiation.
A Naphthol AS-D chloroacetate esterase (chloroacetate esterase, CAE) stain is of granulocytic spesifity but less benefit than MPO/SBB for Auer rods identification (37).
Control blood or marrow slides should always be stained in parallel to ensure the quality of the staining(36).
These stains can be performed on peripheral blood, bone marrow aspirate smears, fresh or archived touch imprint slide. Less mature or minimally differentiated AML subtypes can be distinguished by Myeloperoxidase, Sudan Black B, and Specific Esterases staining of primary granules of myeloid cells(38).
Less than 3% of blasts are demonstrated with positive granules in AML with minimal differentiation, whereas intensely positive field will appear in the majority of blasts seen in AML.
Monocytes are seem to have a faint dusty staining, and should be interpritated as appositive result, especially with Suddan black(38).
Beside monocytes and histiocytes, ?-naphthyl acetate esterase is also positive in megakaryocytes and platelets(39).
About 20% of monocytic leukemias can be negative esterase(38).
Periodic acid schiff oxidises glycol groups to produce stable dialdehydes. These dialdehydes give a red reaction product when exposed to Schiff reagent (leucobasic fuchsin).
Positive reactions occur with carbohydrates, principally glycogen. In haemopoietic cells, the main source of positive reactions is glycogen.
A positive PAS stain in erythroblasts is a common finding in erythroleukaemia where it has a coarsely granular pattern in cells of early stage and a finely granular pattern in cells of later stage. PAS staining of the periphery of the cytoplasm, especially in cytoplasmic protrusion, is characteristic for megakaryocytes and megakaryoblasts, also shows block positivity in B lymphocytes more than T(36).

1.3.7.4 Flow cytometric immunophenotyping

Flow cytometry plays an indispensible role in the diagnosis and subclassification of acute myeloid leukemia (AML). Using a multiparametric approach, flow cytometry immunophenotyping has the advantage of efficiency with high sensitivity(40).
Acute leukemia (AL) displays characteristic patterns of antigen expression, which facilitate their identification and proper classification.Distinction between lymphoid and myeloid leukemia, most often made by flowcytometry, is crucially important. Acute leukemias reflect the pattern of antigen acquisition seen in normal hematopoietic differentiation, yet invariably demonstrate distinct aberrant immunophenotypic features. Detailed understanding of these phenotypic patterns of differentiation, particularly in myeloid leukemia, allows for more precise classification of leukemia than does morphology alone. Multiparameter flowcytometry is a useful adjunct to morphology and cytochemistry and it is an invaluable tool in the diagnosis of AL. Flowcytometry of leukemic cells plays essential role in identification of leukemia cell line, maturation stage and detection of residual disease(41).

In the EGIL classification, AMLs are defined immunologically by the expression of 2 or more of the following myeloid Markers: MPO, CD13, CD33 and CD117.( 42)
recurrent genotypic abnormalities are associated with some phenotypes in AML. For example AML with t(8; 21)(q22;q22) is associated with aberrant expression of CD19, CD56, and sometimes terminal deoxynucleotidyl transferase (TdT). The phenotype of Acute promylocytic luekaemia with t(15;17)(q22;q12) are : CD34 negative or only partially positive, HLA-DR negative or only partially positive, CD11b negative, CD13 heterogeneous, CD117 positive, CD33 positive (homogeneous bright staining), and CD15 negative or weak intensity staining.
A similar CD34, HLA-DR phenotype has been described in a subset of AML with cup-shaped nuclear invaginations, normal cytogenetics, and an apparent association with FLT3-ITD.(43)
However, although flow cytometric immunophenotypic studies may be used as a screening tool, they lack specificity and sensitivity for the detection of genotypic abnormalities.(44)

Table (1-3 )
Expression of cell-surface and cytoplasmic markers for the diagnosis of AML and MPAL(34,45)

Expression of cell-surface and cytoplasmic markers
Diagnosis of AML
Precursors CD34, CD117, CD33, CD13, HLA-DR
Granulocytic markers CD65, cytoplasmic MPO
Monocytic markers CD14, CD36, CD64
Erythroid markers CD235a (glycophorin A), CD36
Megakaryocytic markers CD41 (glycoprotein IIb/IIIa),
CD61 (glycoprotein IIIa)

Diagnosis of MPAL
Myeloid lineage MPO (flow cytometry, immunohistochemistry, or
cytochemistry) or monocytic differentiation (at
least 2 of the following: NSE
cytochemistry, CD11c, CD14, CD64, lysozyme)

T-lineage Strong cytoplasmic CD3 (with antibodies to CD3
? chain) or surface CD3
B-lineage Strong CD19 with at least 1 of the following
strongly expressed: cytoplasmic CD79a, cCD22,
or CD10 or weak CD19 with at least 2 of the
following strongly expressed: CD79a, cCD22
or CD10

Megakaryocytic markers CD41 (glycoprotein IIb/IIIa),
CD61 (glycoprotein IIIa)

Diagnosis of MPAL
Myeloid lineage MPO (flow cytometry, immunohistochemistry, or
cytochemistry) or monocytic differentiation (at
least 2 of the following: NSE
cytochemistry, CD11c, CD14, CD64, lysozyme)

T-lineage Strong cytoplasmic CD3 (with antibodies to CD3
? chain) or surface CD3
B-lineage Strong CD19 with at least 1 of the following
strongly expressed: cytoplasmic CD79a, cCD22,
or CD10 or weak CD19 with at least 2 of the
following strongly expressed: CD79a, cCD22
or CD10

1.8.4 Cytogenetic Diagnosis
Cytogenetic is widely recognized as one of the most important prognostic determinants in AML. Overt cytogenetic abnormalities are present in about half to three-fourths of patients. Abnormalities such as t(8;21), t(15;17), inv16, and translocations involving 11q are the most common abnormalities(46).
The WHO recommends that cases with at(8;21)(q22;q22), inv(16)(p13.1q22), t(16;16)(p13.1;q22), or t(15;17)(q22;q12) abnormality be considered AML with that specific recurrent genetic abnormality(47).
Somatically acquired mutations have been identified in several genes for example NPM1, FLT3, CEBPA, MLL, NRAS, WT1, KIT, and RUNX1 (34).

Table (1.4) cytogenetic risk group(48).

1.9. Treatment

General AML management approach still relies largely on intensive chemotherapy and allogeneic hematopoietic stem cell transplantation (HSCT), at least in younger patients who can tolerate such intensive treatments.
In contrast, minimal advances have been made for patients unable to tolerate intensive treatment, mostly representing older patients(49).

Two treatment phases of adult AML are (50):

1- Front-line induction of remession therapy

Is referred to as applying chemotherapy to get ride of leukemic cells and Improve marrow function by inducing complete remission (CR).
CR is defined as less than 5% blast cells in a cellular marrow durable for at least 28 days with a peripheral neutrophil count of 1.5 × 109/L and platelet count above 100 × 109/L, and absence of extramedullary disease.
In some cases, these criteria may be met, but the morphology is dysplastic.
Induction therapy with cytarabine and an anthracycline remains a standard of care in AML. The standard combination is the 7+3, with a 7-day continuous infusion of cytarabine at the dosage of 100 or 200mg/m2 per day on days 1 to 7 and daunorubicin at 60 mg/m2 per day on days 1 to 3.

2- Consolidation therapy
Clinical experience has demonstrated that further intensive post remission treatment is required to ‘consolidate’ CR. This is delivered at the same intensity as induction in order to achieve further cytoreduction. two or three intensive consolidation courses are generally used in younger patients, and stem cell transplantation may be included. Where intensive induction and consolidation can be given, for example in younger patients, maintenance chemotherapy is not required.
For younger patients not undergoing HSCT, administration of several High dose –cytrabine HiDAC consolidation courses using cytarabine twice daily at a 3 g/m2 dose on days 1, 3, and 5 has been a widely used option.

Allogeneic HSCT

One of the most important treatment decisions in AML is to estimate the
benefit/risk associated with allogeneic HSCT in first remission for a
given patient.
Transplantation offers the best means of preventing AML
recurrence, but remains associated with higher treatment-related morbidity
and mortality (TRM), especially in older patients. In patients with
favorable-risk AML, the relapse risk may be low enough and the
salvage rate high enough to postpone HSCT to second remission.

General treatment approach for acute myeloid leukaemia(51):
Fit patients (30,000/?L) who are unlikely to respond to low-intensity therapy, consider therapies as in younger patients; however, tolerance and response may be poor

For Acute Promyelocytic Leukaemia patient (53):
The major component of induction therapy is all-trans retinoic acid (ATRA), which is commonly combined with other agents. The combination of arsenic trioxide with ATRA may be considered for patients who are not candidates for anthracycline-based therapy.
Several regimens are commonly used for induction therapy, including the European APL regimen, the Programa para el Tratamiento de Hemopatias Malignas (PETHEMA) regimen, and, in specific populations, combination therapy with arsenic trioxide.

1.10 Prognosis
The outcome for an individual patient with AML will depend on a number of factors including age and WBC at presentation (Table 1-5). However; the genetic abnormalities in the tumour are the most important determinant.
Complete remission is defined as less than 5% blasts in BM without Auer rods, neutrophil count greater than 1.0 x 109/L, platelet count greater than 100 x 109/L, independence of red cell transfusions and no extramedullary disease (54).
Table (1-5) Prognostic factors in acute myeloid leukaemia (54):
Cytogenetics t(15;17)
t(8;21)
inv(16) Normal
Other non complex change Deletions of chromosome 5 or 7, TP53 mutated
Complex rearrangements (;3 unrelated
abnormalities)
Molecular genetics NPM mutation
CEBPA mutation Wild type FLT3 –ITD
Bone marrow response
to remission induction 20% blasts after first course
Age child 60 years
Performance status Good Bad
Co?morbidities absent Present
WBC 100 × 109/L
Secondary leukaemia absent Present, e.g. to previous chemotherapy or
marrow disease
Minimal residual
disease in remission Absent Present (;0.1% of cells)

1.11 Immunopathological events in AML

The leukemias represent unique models to assess the impact of cancer on the host immune system as the cancer cells and immune cells originate from the same hematopoietic tissue and are in close proximity in peripheral blood and bone marrow(55).
Acute myeloid leukemia (AML) is characterized by rapid expansion of immature myeloid cells in bone marrow and peripheral blood. In AML, the malignant clone is apparently controlled by cellular immunity, including natural killer (NK) cells and subsets of cytotoxic ( CD8+) T cells.
several other immunosuppressive mechanisms important to the course of disease have been described including immunosuppression exerted by NOX2-derived reactive oxygen species (ROS) released from myeloid cells (56).

Treg cells, a subpopulation of CD4+ T cells, inhibit the immune response by influencing the activity of other cell types. Typically, Treg cells are classified into naturally occurring CD4+CD25+ Treg cells, interleukin 10 (IL 10) secreting Treg cells, and transforming growth factor ? (TGF ?) secreting Treg cells based on the types of cytokines they produce, the immunosuppressive function of Tregs contributes to leukemia progression(57).

Recent studies have demonstrated that tumor immune escape mechanism governed by CD4+CD25+ regulatory T cells (Tregs) is a key factor in the tumorigenesis of AML. Especially, inhibitory cytokines TGF-b and IL-10 have been implicated as an important mediator contributing to immunosuppressive functions of Tregs, and are involved in the pathophysiological process of AML. However, blocking TGF-b and IL-10 does not absolutely abrogate the suppressive activity of Tregs,
suggesting some other mechanisms may be responsible for
Tregs-mediating suppression(58).

Cytokines are secreted by different types of cells in response to a variety of stimuli such as tissue damage or infection and regulate the immune response and other biologic processes.

In AML patients, cytokines can be produced by both leukemic
blasts and cells of the immune system and the role of cytokines
in the pathogenesis of acute leukemia has not been fully clarified. Thus, aberrant cytokine signaling is a feature of leukemia
that may contribute to proliferation, blast survival, resistance to
chemotherapy and patients’ prognosis (59).

The topic of cytokines effect on the immune system against cancer is remains controversial point (60). two important cytokines are IL-35 and IL-10 which maintains the growth of cancer stem cells.

Interleukin (IL)-35
Interleukin (IL)-35 is a relatively newly discovered member of IL-12 cytokine family that is unique in that it is contain IL-12? chain p35 and IL-27? chain Epstein-Barr virus-induced gene 3 (Ebi3) connected by disulfide bond. IL-35, initially named at the 13th International Congress of Immunology, is the
new focus of cytokines research. IL-35 is similar to other IL-12 family members which are heterodimeric glycoproteins formed with disulfide-linked ? (p19, p28, or p35) and ? (p40 or EBi3) chains. The ?-chain has 4-?-helical bundles, a typical cytokine structure, and the ?-chain is homologous to the soluble cytokine receptor. p35 and p40 combine to form IL-12; p19 and p40 combine to form IL- 23; p28 and Ebi3 combine to form IL-27 . IL-35 is composed of p35 and Ebi3, and it differs from the expression and secretion way of other IL-12 members. In response to
bacteria, bacterial products, or intracellular parasites, IL- 12, IL-23, and IL-27 are secreted by activated antigen presenting cells, including B cells, monocyte, macrophages and dendritic cells. IL-35 was initially reported to be produced by Treg cells and was essential for maximizing
the inhibitory role of Treg cells. Recently studies suggest that regulatory B cells (Breg) also produce IL-35 and rIL-35 fusion proteins can induce Breg cells to secret IL-10 and IL-35(60).

Tregs-derived from adult AML patients produced IL-35 in a stimulation dependent manner. IL-35 promoted AML blasts immune escape by expanding Tregs and inhibiting CD41CD25-effector T cells (Teffs). Furthermore, IL-35 directly promoted the proliferation of AML blasts and reduced the apoptosis of AML blasts. Together, our study demonstrates that IL-35-derived from Tregs promotes the growth of adult AML blasts, suggesting that IL- 35 has an important role in the pathogenesis of AML
(58).
Interleukin-10
Interleukin- 10 (IL- 10) is a 36 kDa homodimeric cytokine that was originally described as ‘cytokine synthesis inhibitory factor’ (CSIF) because of its ability to inhibit the secretion of cytokines from T helper type I (Thl)T-cell clones . IL-10, which is produced by T cells, B cells and macrophages. has different effects on cytokine production and activation in different cell types(61).
Helper T lymphocyte subsets are consisted of Th1 and Th2 subsets and IL-10 are mainly secreted by Th2 subsets. Interleukin-10 (IL-10) has pleiotropic effects in immunoregulation and inflammation and influences many aspects of the immune response . The IL-10 gene is located on chromosome 1. IL-10 has immune-stimulating (promote cancer potentially) and immuno-suppressive (inhibit cancer potentially) and may regulate tumor susceptibility and development . Recent studies showed that IL-10 single nucleotide pleomorphism (SNP) was associated with the non-Hodgkin’s lymphoma . There was no relative research on the relation between IL-10 SNP and myeloid systemic tumor, acute myeloid leukemia specially. (62)

Dual biological functions of IL-10 makes a controversial role in carcinogenesis in humans, as a tumor promoting and tumor inhibiting factor, may regulate tumor susceptibility and development (6).

Although these cytokines were secreted by different subtypes of Treg cells. IL 10 is known to inhibit cytokine production by T cells, and exerts anti-inflammatory and immunosuppressive activities. It inhibits the production of IL 2, IFN? and granulocyte macrophage colony-stimulating factors (GM CSF) as well as the proliferative response of T helper (Th) 1 cells (57).

However, the precise roles of Treg cells secreting cytokines (IL-35 and IL-10) in AML still unknown.

To investigate the role and disregularity of Treg related cytokines in the pathogenesis of AML, we measured the serum concentration of the two Treg-associated cytokines (IL 35, and IL 10) before and after chemotherapy and evaluate their clinical and heamatological relevance.