acute myeloid leukemia
Gomes e Silva, F.P.
Citation
Gomes e Silva, F. P. (2009, March 3). Genome wide characterization of minimally differentiated acute myeloid leukemia. Retrieved from https://hdl.handle.net/1887/13569
Version: Corrected Publisher’s Version
License: Licence agreement concerning inclusion of doctoral thesis in the Institutional Repository of the University of Leiden
Downloaded from: https://hdl.handle.net/1887/13569
Note: To cite this publication please use the final published version (if applicable).
1
General introduction
1.1 Leukemia: historical note
The identification of leukemia as a systemic disease is relatively recent. Until approximately 160 years ago, patients with leukemia were commonly diagnosed with infections, anemia, dropsy or other conditions with variable etiologies.1 The first report describing the illness is attributed to Velpeau in 1827,2 and it was followed 20 years later by separate reports from Virchow, Craigie, Bennett, Barth and Donné.3-7 Most of the reports described the high numbers of white blood cells present in the blood as purulent matter. Donné, who popularized microscopic techniques for clinical practice, was one of the first to recognize that the white cells in the blood of these patients were of a different nature than pus.7 However, it was Virchow, the founder of the cellular pathology school in Berlin, who was the first to identify leukemia as a specific and distinct entity and the first to use the term leukemia (Greek leukos,
“white”; haima, “blood”).3;8 Subsequently, Virchow reported a distinction between splenic and lymphatic leukemia, each of which was associated with particular types of white blood cells. This division is broadly equivalent to myeloid and lymphoid leukemia. In 1868 the origin of the cells responsible for the so-called splenic leukemia was traced to the bone marrow.9
Shortly after the establishment of leukemia as a distinct disease the first drugs used in leukemia therapy were discovered. As it would happen with many drugs after, their discovery owes much to a combination of chance and empiricism. By the beginning of the XX century X-rays were used for therapy. But only in 1930 the first case of a successful treatment of acute leukemia was reported, in a patient who would live to the age of 102, 52 years after therapy.10
During the second half of the XX century the study of leukemia saw great progress.
Developments in the areas of cytogenetics, genetics, molecular biology and immunology have fomented the design of new drugs, resulted in the development of bone marrow therapy, provided new targets for treatment, allowed the definition of new subsets of leukemia and permitted the identification of residual disease.
1.2 Hematopoiesis
Blood is a connective tissue composed of plasma and different types of cells. Each cell type has its function. Erythrocytes are responsible for the transport of oxygen and carbon dioxide. Platelets participate in hemostasis (coagulation) and in the inflammation response.
Granulocytes, monocytes and lymphocytes play different roles in the immune response.
Nevertheless, how different these cells might be, it is currently accepted that they share a same progenitor: the hematopoietic stem cell (HSC).
The process known as hematopoiesis starts with the HSCs, including their self-renewal, goes through the commitment of a HSC to a specific lineage and ends in differentiation into a particular blood cell type (Figure 1).
Figure 1. Hematopoietic cell differentiation. Hematopoietic stem cells are both able to self-replicate and to give origin to lineage committed progenitor cells. The differentiation into mature blood cells occurs through a series of stages each of which further restricts the lineage choice. Maturation occurs in either the bone marrow, for most of the myeloid lineage cells, or the lymphoid organ, for the lymphoid lineage cells. Detailed maturation stages of the committed progenitor cells are not represented. With each new commitment step there is a considerable cell amplification, which is not depicted in the diagram. (Adapted from Bondurant and Koury, 2004).11
1.2.1 The hematopoietic system origins
Unlike other body systems, the ontogeny of the hematopoietic system goes through different phases. Two sorts of hematopoiesis occur, primitive and definitive, and the site of hematopoiesis changes several times during the neonatal period.
Primitive hematopoiesis is transient, lasting only till the eighth week of the embryo development. It arises in the blood islands of the extra-embryonic yolk sac. Primitive cells consist mainly of erythroid cells that express embryonic hemoglobin.
Definitive hematopoiesis takes place in the aortic-gonadal-mesonephros region of the embryo.
Definitive hematopoietic cells consist of all blood cell types. The origin of HSCs that give rise to definitive hematopoiesis is unclear, though there is evidence that they originate in specialized endothelial cells in the dorsal aorta.12;13 During later phases of fetal development, HSCs migrate to the bone marrow (BM), which becomes the exclusive site of postnatal hematopoiesis through the lifespan of normal individuals. Whether the HSC that give rise to
primitive and definitive hematopoiesis has the same origin remains controversial.13 1.2.2 The hematopoietic stem cell
HSCs are characterized by their capacity to self-renew and to differentiate into all blood cell lineages. There is approximately 1 HSC per 1 105 BM cells. Most HSCs are quiescent, only a fraction goes into the cell cycle either to proliferate and give rise to different lineage progenitors or to replenish the HSC pool. Several studies in mice have demonstrated that, after transplantation, some HSC contribute to hematopoiesis through the lifespan of the host, others contribute and then become extinct and finally, some may remain quiescent for a period before they contribute.14-16
In an attempt to define HSCs several surface markers were proposed as characteristic for these cells. Nevertheless, there is uncertainty in this area. Until recently it was accepted that HSCs were characterized by the expression of CD34, CD59, CD117 and CD90 and absence of lineage specific markers and CD38.17;18 However, more recent evidence shows that some HSCs do not express CD34, casting doubt over which are the characteristic surface markers of the HSCs.18-20
Closely related to the ability of the HSCs to differentiate into all the blood cell lineages is the question of stem cell plasticity. Plasticity can be defined as the capacity of tissue-specific adult stem cells to generate the differentiated cell types of another tissue. Though research has produced data indicating that HSC were able to differentiate into cells of different tissues,21 the criteria for these experiments might have been not stringent enough. Some evidence suggests that, in vivo, adult stem cells are tissue specific.11 Still it is possible that in particular conditions adult stem cells can be induced to dedifferentiate or that there are adult stem cells more primitive than HSCs that can give rise to HSCs and other stem cells.11
1.2.2.1 Regulation of the hematopoietic stem cell fate
HSCs division can be symmetric, where two identical HSCs are produced, or asymmetric, where one HSCs remains as such and the remaining cell undergoes differentiation or apoptosis.
When a HSC begins differentiating it gives rise to committed hematopoietic progenitor cells.
This process is irreversible. In turn early committed progenitors can increase their numbers by cell division and can give rise to representatives of several, but not all, blood lineages.
Different committed progenitors give rise to specific blood lineages according with their early stage of differentiation. The descendents of these multi-lineages progenitor cells will ultimately be restricted to a more mature, single-lineage committed progenitor cell that gives rise to terminally differentiated cells. The number of cell divisions allowed in each stage of the process is unknown. For some lineages the number of cell divisions that cells in a more mature stage of development can undergo seems to be more limited than that of their progenitors: the further along the differentiation pathway a cell is, the less cell divisions it can undergo. However there are exceptions to this rule: for example lymphocytes, based on in vitro studies, might have vast or unlimited potential to proliferate.22 Either way, at the final stage of hematopoiesis, terminally differentiated cells have become the most prevalent cells of the hematopoietic system (Figure 1).
Figure 2. Simplified diagram of the different stages of hematopoietic development and associated transcription factors. HSC – hematopoietic stem cell; CHC – committed hematopoietic cell; CLP – committed lymphoid progenitor; BCP – B-cell progenitor; TNK – T-cell and natural killer cell precursor;
TCP – T-cell progenitor; NKP – natural killer cell progenitor; CMP – committed myeloid progenitor; GM – granulocyte-monocyte progenitor cell; MP – monodendritic progenitor; NP – neutrophil progenitor;
EoP – eosinophil progenitor; BP – basophil progenitor; EMk – Erythro-megakaryocyte progenitor; EP - erythrocyte progenitor and MkP – megakaryocyte progenitor. Adapted from Kaushansky, 2006.30 Both the molecular mechanisms that lead to the commitment of HSCs to differentiation and the molecular mechanism that guide the cells through the differentiation process are not fully understood. There are two diverging models for the commitment of HSCs to differentiation.
The first to arise, the stochastic model, proposes that HSCs randomly commit to self-renew or differentiate.23 In this model the changes in gene expression necessary for differentiation are probabilistic and unaffected by the cellular environment. The second model, the instructive model, supports the concept that the microenvironment in which the HSCs reside determines the fate of the cell. Most likely the cells would respond to cell-cell, cell-extracellular matrix or cell-soluble ligands signals. These interactions start a cascade of events that would lead to a certain stage of differentiation. In turn, further signals would direct the differentiation process towards a final stage. Conclusive prove for either model is lacking. Though several experiments
point in one or the other direction, the results are subject to different interpretations.24-26 In any case it is now generally accepted that subsequent lineage differentiation is cued by external signals, where hematopoietic growth factors play a major role.24-26
Independently of how the HSCs fate is regulated, molecular signatures for differentiation are known. Some transcription factors are expressed in early stages of differentiation, while others seem to be restricted to later stages and specific blood lineages (Figure 2). Among the first, the most prominent are some of the homeobox (HOX) family members.27 Genes like Ikaros, RUNX1, PU.1, GATA1, GATA3 and CEBPA are ascribed to more lineages restricted roles.28;29 For a review on the subject see Laiosa et al., 2006.29
1.3 Leukemia, general mechanisms
Leukemia is believed to be the result of deregulation of normal hematopoiesis. Leukemic cells, or at least part of the leukemic cell population, have an unlimited cell division potential.
Leukemic cells express markers seen in particular stages of differentiation, though they can co-express markers related to different stages and even different lineages. Even if there is no direct correspondence between the leukemia cells and a normal differentiating hematopoietic cell lineage, the expression of common markers might indicate that these two groups of cells share the same early commitment to a blood lineage, an important point in understanding leukemogenesis.
1.3.1 Molecular mechanisms of leukemogenesis
Central to the molecular pathogenesis of acute leukemia is the concept that leukemia consists of blasts whose differentiation has been arrested. Within the models of leukemogenesis proposed, some describe the differentiation arrest as a secondary event that results either from the disruption of cell cycle control or of later events not necessary for leukemogenesis itself.
Others consider the block in differentiation as a central factor for the leukemogenic process, together with the disruption of cell cycle control.31 This last view seems to be the most widely accepted. Again, there are different views on how the block of differentiation and cell cycle control is achieved. One possibility is that the mutation of a single gene can affect both processes. Many growth factor receptors are involved in proliferation and survival via the signaling network. It has been shown that growth factor receptors usually found mutated in leukemia, such as FLT3 and G-CSFR, play also important roles in myeloid differentiation.32 Conversely, transcription factors involved in differentiation, such as CEBPA, can also play a part in proliferation and survival, either by altering the expression of genes related to apoptosis and cell cycle, or by direct interaction with such genes.32 Although the theory that mutation of a single gene can affect several pathways and result in leukemia should not be dismissed, there is no actual prove that this happens. On the other hand there are genetic data, backed by animal models, which support a multistep model of leukemogenesis. As the name suggests, this model defends that acute leukemia is the result of multiple mutations. Animal models have shown that expression of acute myeloid leukemia-associated fusion proteins in mice, like AML1-ETO and CBFB-MYH11, is not sufficient to induce leukemia.33;34 Only after exposure to mutagenic agents do these mice developed leukemia, implying that mutation of
other critical genes is necessary.34 More evidence can be found in humans. RUNX1 associated myelodysplastic disorder (RUNX1/MDS) is a clonal disorder characterized by ineffective hematopoiesis, which can lead to either fatal cytopenias or AML. MDS evolution to AML has been associated with further mutation of proliferation associated genes like FLT3.35;36 Germ line mutationsof RUNX1 have been shown to occur in an autosomal dominantdisorder, familial platelet disorder (FDP). FDP affected individuals have propensity to develop AML but only after acquisition of further mutations.37 Equally important is the evidence showing that cells with t(12;21), associated with ETV6-RUNX1 fusion, were found present during uterus development in embryos, but development of leukemia only occurs later in life.38
Figure 3. “Two-hit” leukemia model.40 This model hypothesizes that a minimum of two mutations is necessary for development of leukemia. One class of mutations confers a proliferative or cell survival advantage while the second class serves primarily to impair hematopoietic differentiation and apoptosis.
In a compound model for the pathology of acute myeloid leukemia several aspects have to be considered covering inappropriate proliferation in the absence of normal growth stimulus, indefinite self-renewal in a manner analogous to a stem cells, escape from programmed cell death, inhibition of differentiation, aberrant cell cycle checkpoint, genomic instability and multi-organ dissemination of leukemic cells.39 These features can have a direct or indirect role on leukemogenesis. They can also have different importance for each leukemia case and
be the result of a few or many genetic lesions.
A simplified version of the multistep model is a cooperative model which focuses on mutation in two separate classes of genes resulting in leukemia. Class I mutations confer a proliferative advantage to the cells while class II mutations interfere with differentiation, and ultimately, apoptosis of these cells (Figure 3). This model also predicts that a minimum of one mutation in each class would be required for the development of acute leukemia.40;41
1.3.2 The primordial leukemic stem cell
Recent studies have shown that, as with normal hematopoiesis, there is a hierarchical structure among the leukemic population, including a fraction with self-renewal properties:
the leukemic stem cells (LSC). Population of cells able to self-renew were found among the leukemic cells derived from different AML subtypes.42 These LSCs populations are not functionally homogeneous but are comprised of different layers (strata) like the normal HSCs populations. Within these strata there are cells that divide at different rates, including cells that remain quiescent.43;44 The ability of LSCs to control self-renewal suggests that the leukemogenic process does not abolish all the pathways normally regulated in HSCs, though the rate of self-renewal of LSCs is higher than normal HSCs.43
Because normal HSCs and LSCs share the ability to self-renew, which in turn increases the chance of these cells to accumulate mutations, it has been postulated that LSCs derive from HSCs (Figure 4). Evidence of HSCs as origin for LSCs has its base in studies that show that both cell types are CD34+CD38-.45 Nevertheless, this does not exclude the possibility that more mature progenitors cannot reacquire the CD34+CD38- phenotype by reacquiring the self-renewing properties. Analyses of leukemia associated genes in mouse have shown that more committed progenitor cells can be used to generate mouse models of human leukemia supporting the idea that LSCs developed from more committed progenitors cells that reacquired the ability to self-renew by mutational events (Figure 4).46;47 Furthermore, in a study of chronic myeloid leukemia, myeloid progenitor populations were found to have increased proliferative and self-renewing capacity following events that resulted in increased expression of the β-catenin pathway and progression to blast crisis.48 It seems possible that acute leukemia might arise from both HSCs and committed progenitors depending on the mutations involved. It is also feasible that committed progenitors will require more mutations to revert to a more HSC comparable phenotype.
Figure 4. Leukemic cell origin. The central path represents the normal hematopoietic development.
Hematopoietic stem cells (HSC), which are able to self-renew, give rise to progenitor cells that differentiate into mature blood cells. In the top panel acquired mutations disrupt the normal hematopoietic development leading to a pool of leukemia stem cells (LSC) that are also able to self-renew. The process ends with the formation of leukemic blasts. Alternatively, as depicted in the lower panel, the origin of the leukemic blasts can be leukemic cells derived from mutated progenitor cells. These leukemic cells must reactivate the ability to self-renew in order to become LSC. Adapted from Bonnet, 2005.45
1.4 Clonal hematopoietic disorders and classification
The classification of acute leukemia has been the focus of hematologists since the beginning of the XX century. A wide variety of blood related syndromes or diseases result from abnormal hematopoiesis. The first criterion for the classification of these disorders is the division into two major groups: myeloid or lymphoid. This distinction is based on the retained differentiation exhibited by affected cells. Table 1 presents a summary of neoplastic myeloid disorders based on the latest recommendations of the World Health Organization (WHO).49 In addition the WHO also provides a detailed classification of lymphoid neoplasms which consists of four major groups: precursor lymphoid neoplasms, mature B-cell neoplasms, mature T- and NK-cell neoplasms and Hodgkin’s lymphoma.
Table 1. Revised 2008 WHO classification of myeloid neoplasms Myelodysplastic syndromes (MDS)
Refractory cytopenia with unilineage dysplasia (RCUD) Refractory anemia (RA)
Refractory neutropenia (RN) Refractory thrombocytopenia (RT)
Refractory anemia with ring sideroblasts (RARS)
Refractory cytopenia with multilineage dysplasia (RCMD) Refractory anemia with excess blasts (RAEB)
Myelodysplastic syndrome associated with isolated del(5q) Myelodysplastic syndrome, unclassifiable (MDS, U) Childhood myelodysplastic syndrome
Refractory cytopenia of childhood (RCC) - provisional Myelodysplastic / myeloproliferative neoplasms (MDS/MPN) Chronic myelomonocytic leukemia (CMML)
Atypical chronic myeloid leukemia, BCR-ABL1 negative (aCML) Juvenile myelomonocytic leukemia (JMML)
Myelodysplastic/myeloproliferative neoplasm, unclassifiable MDS/MPN, U) Refractory anemia with ring sideroblasts and thrombocytosis (RARS-T) - provisional
Myeloproliferative neoplasms (MPN)
Chronic myelogenous leukemia, BCR/ABL positive (CML) Chronic neutrophilic leukemia (CNL)
Polycythemia vera (PV) Primary myelofibrosis (PMF) Essential thrombocythemia (ET)
Chronic eosinophilic leukemia, not otherwise specified (CEL, NOS) Mastocytosis
Myeloproliferative neoplasm, unclassifiable (MPN, U)
Myeloid and lymphoid neoplasms with eosinophilia and abnormalities of PDGFRA, PDGFRB or FGFR1
Myeloid and lymphoid neoplasms with PDGFRA rearrangement Myeloid neoplasm with PDGFRB rearrangement
Myeloid and lymphoid neoplasms with FGFR1 rearrangement Acute myeloid leukemia and related precursor neoplamsa Acute leukemia of ambiguous lineage
a – See Table 3 for complete information. Adapted from Swerdlow et al., 2008.49
The diversity of clonal blood disorders demands a correct classification, as more specific treatments arise for each disease subgroup.
1.4.1 Acute leukemia classification
One of the most important efforts to standardize the classification of acute leukemia was the French/American/British (FAB) classification, based on morphologic data.50 This classification proposed in 1976, although revised since, was found with time to have little predictive power for treatment outcome. Although it lost its value for clinical classification, it remains a useful descriptor for leukemia morphology. FAB classification of AML consists of
8 subtypes (Table 2). Subtypes M1, M2 and M4 are the largest groups comprising respectively 17, 30 and 25 percent of all cases. Subtypes M3 and M5 both account for about 10 percent of cases, while M0, M6 and M7 less than 5 percent each.51
Table 2. FAB classification of AML Lineage Abbreviated
name
FAB
Subtype Common name
Myelogenous
AML M0 Myeloblastic without cytological maturation M1 Myeloblastic with minimal maturation M2 Myeloblastic with significant maturation
APL M3 Acute promyelocytic leukemia
M3 variant APL unusual hypogranular form Myelogeneous and
monocytic
AMML M4 Acute myelomonocytic leukemia
M4eo M4 with eosinophilic maturation M4baso M4 with basophilic maturation
Monocytic
AMoL M5a Acute monoblastic leukemia (poorly differentiated)
M5b Acute monoblastic leukemia (more differentiated)
Erythroid/myeloid AEL M6 Acute erythroid leukemia
Megakaryoblastic M7 Acute megakaryoblastic leukemia
Mixed lineage AMLL Acute mixed lineage leukemia
Progenitor cells AUL Acute undifferentiated leukemia
Adapted from Goasguen et al., 1996.51
The FAB classification of acute lymphoid leukemia (ALL) consists of three subtypes: from L1 to L3. The subtypes were defined according with cell size and nucleus morphology and number. L1 and L2 have no current clinical significance. L3 corresponds to the leukemia counterpart of Burkitt lymphoma.
In 2001 the WHO published a worldwide consensus classification of hematopoietic and lymphoid neoplasms which was updated in 2008.49;52 The WHO classification evolved to include not only morphology, but also clinical, immunophenotypic and cytogenetic data. In this classification ALL subtypes have been divided according to similar biologic and genetic rather than clinical characteristics of the disease. The WHO classification of AML relies on seven major categories summarized in Table 3. Though this classification is more refined, the fourth category, AML not otherwise categorized, still falls back to a modified FAB classification to characterize cases that do not fulfill criteria for the other groups.
Table 3. WHO classification of acute myeloid leukemia and related precursor neoplasms Acute myeloid leukemia 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); CBFβ-MYH11 Acute promyelocytic leukemia (APL) with t(15;17)(q22;q12); PML/RARA AML with t(9;11)(p22;q23); MLLT3-MLL
AML with t(6;9)(p23;q34); DEK-NUP214
AML with inv(3)(p21q26.2) or t(3;3)(p21;q26.2); RPN1-EVI1 AML (megakaryoblastic) with t(1;22)(p13;q13); RBM15-MKL1 AML with mutated NPM1 - provisional
AML with mutated CEBPA - provisional
Acute myeloid leukemia with myelodysplasia-related changes Therapy-related myeloid neoplasms
Acute myeloid leukemia not otherwise categorized AML with minimal differentiation AML without maturation AML with maturation
Acute myelomonocytic leukemia Acute monoblastic/monocytic leukemia Acute erythroid leukemia
Pure erythroid leukemia
Erythroleukemia, erythroid/myeloid Acute megakaryoblastic leukemia Acute basophilic leukemia
Acute panmyelosis with myelofibrosis Myeloid sarcoma
Myeloid proliferation related to Down syndrome Transient abnormal myelopoiesis
Myeloid leukemia associated with Down syndrome Blastic plasmacytoid dendritic cell neoplasms
1.5 Acute myeloid leukemia
AML is characterized by the proliferation of blasts showing little differentiation into a population that can consist of an excess of 10 billion cells. As result, normal hematopoiesis is inhibited and normal blood cell levels fall. The limited number of red blood cells results in anemia with associated weakness, exertional limitations and pallor. The thrombocytopenia, low levels of platelets, results in hemorrhage. The decrease in neutrophil and monocyte levels lead to poor wound healing and infections. Severe infections can arise if the leukemia is left untreated.53 Fever, weight loss and malaise can occur as early manifestations of AML.
The diagnosis is made by identification and count of leukemic blast cells in the blood or bone marrow. The non lymphoid derivation of AML can be determined by the presence of Auer rods or by Sudan black, myeloperoxidase (MPO), chloroacetate esterase, or non specific esterase on cytochemical stains.54 In addition to morphology and cytochemistry, flow cytometry can be used to study the expression of myeloid lineage specific surface markers.
Based on morphologic and immunophenotypic data the leukemia can be further classified to
a specific myeloid lineage. Table 4 presents the immunologic phenotypes of AML.
Table 4. Immunologic phenotypes of AML
Lineage Usually positive for
Myeloblastic CD11, CD13, CD15, CD33, CD117, HLA-DR Myelomonocytic CD11, CD13, CD14, CD15, CD32, CD33, HLA-DR
Erythroblastic Glycophorin, spectrin, ABH antigens, carbonic anhydrase I, HLA-DR
Promyelocytic CD11, CD13, CD15, CD33
Monocytic CD11, CD13, CD14, CD33, HLA-DR
Megakaryoblastic CD34, CD41, CD42, CD61, von Willebrand factor
1.5.1 Cytogenetic characterization of acute myeloid leukemia
Acquired chromosome abnormalities are frequently found in leukemic blast cells from AML patients. In many cases they are specific cytogenetic abnormalities closely or uniquely associated with morphological and clinical distinct types of leukemia. Furthermore these abnormalities correlate with prognosis.55
The incidence of abnormal karyotypes varies between 53 and 78 percent in adults, and between 68 and 85 percent in children.56;57 The reason for the discrepancy between adults and children is unknown. Approximately 55 percent of the AML cases show a single cytogenetic abnormality, 15 to 20 percent involving a chromosome number change.58 The most common balanced chromosomal abnormalities in AML include t(8;21)(q22;q22), t(15;17)(q22;q12), inv(16)(p13q22) or t(16;16)(p13;q22), 11q23 abnormalities/MLL rearrangements. These abnormalities are found in 15% to 25% of all AML cases and are combined in the first step of the new WHO classification system (Table 3).56;57 Table 5 summarizes the most common recurring chromosome aberrations in AML and their associated clinical features and prognosis.
Table 5. Recurring chromosome aberrations in AML
Rearrangement Genes involved Hematologic clinical features Prognosis
t(1;3)(p36;q21) Preceded by MDS, M1, M4 Poor
t(1;7)(q10;q10) Preceded by MDS, M1, M4, genotoxic
exposure Poor
t(1;11)(p32;q23) AF1p, MLL M0, M5 Poor
t(1;11)(q21;q23) AF1q, MLL M4, M5, infants Poor
t(1;22)(p13;q13) M7 Poor
inv(3)(q21;q26);
t(3;3)(q21;q26)
EVI1, RPN1 Preceded by MDS, M1, M4, M6 Poor t(3;5)(q25;q35) MLF1, NPM1 M6, Sweet syndrome Intermediate/poor t(3;21)(q26;q22) EVI1, MDS or
EAP; RUNX1
No FAB preference, genotoxic
exposure Poor
+4 M1, M2, M4; subcutaneous tumors Poor
-5/del(5q) No FAB preference, genotoxic
exposure
Poor
t(5;17)(q35;q11) NPM1, RARA M3 Poor
t(6;9)(p23;q34) DEK, CAN Preceded by MDS; M2 and M4 Poor
t(6;11)(q27;q23) AF6, MLL M4 and M5, localized infections Poor t(7;11)(p15;p15) HOXA9, NuP98 M2 with Auer rods Intermediate
-7/del(7q) No FAB preference, genotoxic
exposure
Poor
+8 M2, M4, and M5; preceded by MDS Intermediate/poor
t(8;16)(p11;q13) MOZ, CBP M5 Poor
t(8;21)(q22;q22) ETO, RUNX1 M2 with Auer rods Good
t(9;11)(p21- 22;q23)
AF9, MLL M5 Intermediate
t(9;22)(q34;q11) ABL, BCR M1 and M2; biphenotypic rare Poor t(10;11)(p11-
15;q13-23) AF10, MLL M5 Poor
+11 MLL M1, M2 Poor
inv(11)(p15;q22) NUP98, DDX10
No FAB preference, genotoxic exposure
t(11;16)(q23;p13) MLL, CBP M4, M5, infants Poor
t(11;17)(q23,q25) MLL, AF17 M2, M4, and M5 Poor
t(11;17)(q23;q11) PLZF, RARA M3 Intermediate
t(11;19)(q23;p13) MLL, ENL, ELL M4 and M5; biphenotypic Poor t/del(11q23) MLL M5, biphenotypic, genotoxic exposure Poor
t/del(12p) No FAB preference, genotoxic
exposure
Poor
i(12)(p10) Concurrent germ cell tumors Poor
t(12;22)(p13;q11) TEL, MN1 Preceded by MDS, M1, M4, M7 Poor
+13 Poor
t(15;17)(q22;q11) PML, RARA M3, M3v Good
inv(16)(p13;q22), t(16;16)(p13;q22), del(16)(q22)
MYH11, CBFβ M4Eo Good
t(16;22)(p11;q22) FUS, ERG No FAB preference Poor
i(17)(q10) Preceded by MDS, no FAB preference Poor
del(20q) No FAB preference Poor
+21 No FAB preference Intermediate
+22 M4 Intermediate
Adapted from Greer et al., 2004 .58
1.5.2 Epidemiology and etiology of acute myeloid leukemia
AML incidence varies with population, gender and age. Accordingly to the GLOBOCAN 2002 database, the world overall leukemia age-standardized incidence rate per 100,000 persons per year is 5.8 for males and 4.1 for females. These values are lower in less developed regions, 4.4 for males and 3.2 for females, than in developed regions, 9.1 for males and 5.9 for females. The world overall leukemia age-standardized mortality rate averages 3.7 per 100,000 persons.59 Although AML is the predominant form of neonatal leukemia it only accounts for less than 15 percent of leukemia cases during childhood and 15 to 30 percent during adolescence.53 In adults AML accounts for 80 to 90 percent of cases of acute leukemia.58
Three major factor groups can predispose to the development of AML: environmental factors, inherited conditions and acquired diseases. Among the environment factors benzene, ionizing radiation and therapy related drugs (alkylating agents and topoisomerase II inhibitors) are established causal agents. Benzene has been shown to increase AML rates in factory workers 4 to 7 times.60 The relation of ionizing radiation to AML has been established early after its discovery at the beginning of the XX century. Medical uses, atomic bomb exposure and nuclear facilities accidents and exposure have shown the power of radiation as a leukemogenic agent.58 Therapy related drugs increase the chance of leukemia by inducing DNA damage and chromosomal abnormalities.61 Cigarette smoking has also been correlated to AML.58
Genetic factors also play a role in AML. A number of inherited conditions contribute to an increased risk of AML. The genes affected in these conditions usually play major roles in one or more of the following functions: DNA repair and chromosomal stability (e.g. Bloom syndrome), hematopoiesis (e.g. FPD) or tumor suppression (neurofibromatosis). Table 6 presents some of the inherited and acquired conditions that have an increased risk for AML.
For a more complete review on the subject consult Greer et al., 2004.58 Table 6. Inherited and acquired conditions predisposing to AML
Congenital defects Marrow failure syndromes Down Syndrome Fanconi anemia and related disorders Bloom Syndrome Dyskeratosis congenital
Monosomy 7 syndrome Shwachman-Diamond syndrome Klinefelter syndrome Amegakaryocytic thrombocytopenia Turner syndrome Blackfan-Diamond syndrome Neurofibromatosis Kostmann agranulocytosis Congenital dysmorphic syndrome Familial aplastic anemia WT syndrome Familial platelet disorder
Ataxia-pancytopenia
Adapted from Greer et al. 2004 and Liesveld and Lichtman, 2006.53;58
As mentioned before AML can also develop from the progression of other hematopoietic clonal disorders such as chronic myelogenous leukemia, idiophatic myelofibrosis, primary thrombocythemia, polycythemia vera, clonal cytopenias or aplastic anemia, for example. The evolution can occur spontaneously or be enhanced by therapy.
1.5.3 Frequently mutated genes in acute myeloid leukemia
Some genes have been implied in AML. The function of these genes falls within several categories. Central to the current models of leukemogenesis are two major groups of genes found mutated: the ones related to differentiation and apoptosis, which are in most part transcription factors, and the ones related to a proliferative advantages that are usually implied in signal transduction or cell cycle control. A third category of genes comprises those genes, generally known as caretaker, whose inactivation leads to genetic instability that only indirectly contributes to the leukemia (or any type of tumor) by increasing the rate of mutation.
1.5.3.1 Signal transduction
In normal myeloid cells the choice between proliferation, differentiation or apoptosis depends to a large extent on external signals.62 On the other hand, the response to the external stimulus will depend on the cellular context. Once the machinery is present that allows the transduction of a signal, mutation in one of the elements that compose the transduction pathway can wrongly induce signaling.
There are two major classes of signal receptors. Receptor tyrosine kinases (RTK) that have intrinsic tyrosine kinase activity and cytokine receptors which need a subunit to transmit the signal. Activation of either receptors results in the activation of several intracellular signaling cascades that exhibit a certain degree of overlap and induce changes important for proliferation and survival. Among these pathways are the RAS-MAPK, the PI3K/AKT and the JAK-STAT pathways (Figure 5). Among the signaling transduction pathways elements, FLT3, KIT, RAS, PTPN11, NF1 and JAK2 are found mutated in AML.
FLT3
FLT3 is a class III receptor tyrosine kinase expressed in early multipotent progenitor cells but not in HSCs. FLT3 expression seems to be associated with a decrease of self-renewal capacity during haematopoiesis.63 Two main types of FLT3 mutation occur and result in constitutive activation. Internal tandem duplication (ITD) within the juxtamembrane domain of FLT3 occurs in about 25 percent of AML patients. The second type of mutations, usually base substitutions, occur within the activation loop of the second kinase domain in approximately 10 percent of AML patients.40
KIT
KIT is also a class III receptor tyrosine kinase and shows high homology with FLT3. In AML, KIT mutation is associated with core binding factor (CBF) leukemia. Kit exon 8 mutations coding for the extracelular domain and mutation at codon 816 in the activation loop of the catalytic domain are detected in approximately 20 to 30 percent of patients with t(8;21) or inv(16)/t(16;16) but are uncommon in other types of AML.64
Figure 5. Overview of the RAS-MAPK, PI3K/AKT and JAK-STAT signalling pathways. Activation of a cytokine receptor by a ligand, results in activation of JAK, recruitment and activation of Stat signaling proteins and phosphorylation and activation of downstream pathways including the PI3K and RAS- MAPK signaling pathway. In a similar way binding of the ligand by a receptor tyrosine kinase leads to its activation and recruitment of Grb2 and SOS which in turn activate the RAS-MAPK pathway.
Activation of the PI3K/AKT signaling pathway by a receptor tyrosine kinase is also possible. PTPN11 is known to be also involved in the activation of the RAS-MAPK signaling pathway, whereas NF1 has an opposite role. Though these signaling pathways can play different functions, in AML they are implied in the activation of genes necessary for proliferation and survival. The figure is based on the different information available in this section.
RAS
KRAS, NRAS and HRAS are part of a superfamily of regulatoryGTP hydrolases. Constitutive activation of NRAS and KRAS by codons 11, 12 and 61 mutation occurs in 20 to 30 percent of AML patients and are not related to any particular FAB subtype.65;66HRAS mutation is very rare in AML.67
PTPN11
PTPN11 (SHP2) is ancomponent of signaling pathways and is implied in the activation of the RAS-MAPK and PI3K pathways.68 PTPN11 mutation is found in 35 percent of sporadic
juvenile myelomonocytic leukemia cases and in 5 percent of childhood AML but shows low incidence in adult AML.68;69
JAK2
Janus kinase family of tyrosine kinases are involved in the downstream signaling of cytokines.
Activated JAKs phosphorylate STAT proteins that migrate to the nucleus where they regulate gene transcription.70 JAK2 V617F activating mutation was first reported in 2005. The highest incidence of JAK2 mutations reported to this date is 18 percent for AML-M7, but overall incidence in AML is below 5 percent.71
NF1
NF1 is a tumor suppressor gene involved in the negative regulation of the signaling pathway.
It acts by inactivating RAS.72 Loss of NF1 results in increased Ras-mediated signaling in response to stimuli leading to proliferation and increased cell survival of blood progenitor cells.73 Germline mutations of NF1 are found in type I, an autosomal dominant disorder. The risk of malignant myeloid disorders in young children with this neurofibromatosis is 200 to 500 times the normal risk.74
1.5.3.2 Differentiation and apoptosis
As mentioned before hematopoiesis is characterized by the expression of different transcription factors at different stages that direct the differentiation of hematopoietic progenitors into specific lineages (Figure 2). The inhibition of this process resulting in a differentiation block is a hallmark of leukemia and in AML has been associated with the disruption of certain transcriptions factors such as RUNX1, CBFβ, CEBPA, RARA, MLL and possibly SPI1 and NPM1.
Core binding factors
The CBF transcription complex consists of the DNA binding RUNX1 (AML1) subunit and the CBFB subunit, which increases the DNA binding affinity of the complex. RUNX1 has been implicated in the developmental specification of HSC fate and lineage.75 Mutation of the CBF members is frequently found in AML either by the involvement of its members in translocations (Table 5) or by inactivating mutations in RUNX1. The most frequent translocation in AML is the t(8;21)(ETO/RUNX1), associated with AML-M2 subtype, found in 10 to 15 percent of cases.76 Inv(16) and the less common t(16;16), both involving CBFB, are often associated with AML-M4Eo and occurs in 5 percent of AML cases.57 RUNX1 point mutations are usually found in the runt domain (DNA binding) and are in most cases biallelic.36 Somatic point mutations occur in up to 35 percent of AML-M0 cases.77 Germ line mutationsof RUNX1 are seen in FPD and predispose to AML.37
CEBPA
CEBPA expression is essential for the granulocytic differentiation of common myeloid progenitors.78CEBPA mutations either disrupt the ability of the protein to bind DNA and other proteins, in which case they are frequently biallelic, or result in a dominant negative truncated form.79 Mutation occurs in 10 percent of reported AML cases with predominance in M1 and M2 subtypes.79 The morphologic characteristics of M1 and M2 subtype (granulocytic features) fit with the role of CEBPA in hematopoiesis.
RARA
RARA is an all-trans retinoicacid (ATRA) receptor. In the hematopoietic system, ATRA has been shown to inhibit growth, induce differentiationof myelomonocytic progenitor cells, and to enhance self-renewalof more immature multipotent stem cells.80 Promyelocytic leukemia (AML-M3), accounting for 10 percent of AMLs, is characterized by translocations involving RARA. The most frequent translocation partner is PML. Other translocations involve NPM1 and PLZF (Table 5).81
MLL
MLL is required for the proper maintenance of HOX gene expression during development and hematopoiesis. The exact regulatory mechanism of HOX gene expression by MLL is poorly understood, but it is believed that MLL functions at the level of chromatin organization.82 MLL rearrangements include non-constitutional or acquired deletions, duplications, inversions and reciprocal translocations at 11q23. MLL is involved in 5 percent of AML patients, but has much higher incidence in pediatric AML.83;84 More recently, partial tandem duplications have also been detected and are present in the majority of patients with trisomy 11 and in 5 percent of other AML.85;86
SPI1
SPI1 (Pu.1) is an ETS-family transcription factor expressed in hematopoietictissues, and it is fundamental for normal myeloid and lymphoid lineage differentiation.87 Heterozygous mutations in SPI1 have been detected in one study in 7 percent of AML patients. The incidence in the M0 subtype was 3 mutations in 13 patients (23 percent) but could also be detected in the M1, M4, M5 and M6 FAB subtypes.88
NPM1
Heterozygous mutations of the nucleophosmin gene (NPM1) have been reported in 35 percent of adult and 6.5 percent of pediatric patients.89;90 In adults NPM1 mutations are present in about 50 percent of all AML cases with normal karyotype.91 NMP1 is a nucleolar phosphoprotein that shuttles between the nucleus and cytoplasm. It is involved in several processes including ribosome biogenesis, control of centrosomal duplication during the cell cycle, regulation
of DNA transcription via chromatin remodeling and regulation of function and stability of several important nuclear proteins such as ARF and MDM2.92 NPM1 mutations reported consist of small insertions or deletions at exon 12 resulting in disruption of the NPM nucleolar localization signal.91 The exact mechanism by which NPM1 is involved in leukemogenesis is not known. NPM1 mutations are associated with a good prognosis.89;91
1.5.3.3 Caretaker genes
Defective mismatch repair leading to genomic instability plays a minor role in AML when compared with solid tumors.93 Nevertheless, there have been reports associating deficiency of MLH1, a gene involved in mismatch repair, with acute leukemia.94 On the other hand, genomic instability associated with chromosomal aberrations is more common. A complex aberrant karyotype, found in 10 to 20 percent of AMLs, is frequently associated with deletion or mutation of TP53, an important gene for genomic stability.95 Another example is Fanconi anemia (FA) a disorder associated with high risk of AML.96 Genes mutated in FA, such as BRCA2, play important roles or interact with proteins involved in double-strand break repair and/or homologous recombination.97 The AML risk associated with FA might be related with chromosome maintenance, though direct role on hematopoiesis cannot be dismissed.98;99 1.6 Minimally differentiated acute myeloid leukemia: AML-M0
Minimally differentiated acute myeloid leukemia, FAB M0 subtype, accounts for less than 5 percent of all AMLs.100 In the WHO classification AML-M0 has become, in its essence, part of the minimally differentiated AML subcategory. AML-M0 cannot be diagnosed on morphologic or cytochemistry grounds alone, as they can resemble lymphoblasts and lack Auer rods. They are negative for myeloperoxidase (MPO) and Sudan Black B reaction, express at least one myeloid antigen (CD13, CD33, CD15) and are negative for lymphoid antigens.100;101 Nevertheless, expression of some lymphoid associated antigens (CD2, CD7 and CD19) and TdT can be found but do not preclude the diagnosis.102-105 AML-M0 frequently expresses CD34 and HLA-DR associated with early progenitor cells102;103;106. Incidence of cytogenetic abnormalities is more frequent in AML-M0 (71 to 81 percent) than in other subtypes. Complex aberrant karyotypes are detected in about 20 percent of cases, and unbalanced chromosome changes involving –5 or 5q, -7 or 7q, +8 or +13 are frequent.103;104;107
As previously mentioned, RUNX1 point mutations are associated with AML-M0.36 FLT3 mutation can be detected in about 20 percent of cases.108;109SPI1 might be an important factor in AML-M0 as mutation was detected in 23 percent of AML-M0 cases in one study.88 Prognosis of AML-M0 is poor compared with other de novo AML.104;106;110
Reference list
Henderson ES. History of Leukemia. In: Henderson E.S., Lister T.A., Greaves M.F., eds.
1.
Leukemia.: W.B. Saunders Company; 1996:1-7.
Velpeau A. Sur la resorption du puseat sur l’altération du sang dans les maladies clinique de 2.
persection nenemant. Premier observation. Rev Med 1827;2:216.
Virchow RLK. Weisses Blut. N.Notiz.Geb.Natur.u Heilk 1845;36:151-156.
3.
Craigie D. Case of disease of the spleen in which death took place in consequence of the presence 4.
of purulent matter in the blood. Edinb.Med.Surg.J. 1845;64:400-413.
Bennett JH. Case of hypertrophy of the spleen and liver, in which death took place from suppuration 5.
of the blood. Edinb.Med.Surg.J. 1845;64:413-423.
Barth R. Alterations du sang remarquable par le prédominance des globules blanc ou maqueux 6.
hypertrophie considérable de la rate. Bull.Soc.Méd.Hop. 1856;3:39.
Donné A. Cours de microscopie complémentaire des études médicales. Paris: J.B. Baillière;
7.
1844.
Virchow RLK. Weisses Blut und Milztumoren. Med.Z. 1846;15:157.
8.
Neumann E. Ein Fall von Leukämie mit Erkrankung des Knochenmarkes. Arch.Heilk 1870;11:1- 9.
14.
Gloor W. Ein Fall von geheiltes Myeloblastenleukämie. Munchn Med.Wochenschr 1930;77:1096- 10.
1098.
Bondurant MC, Koury MJ. Origin and Development of Blood Cells. In: Greer PG, Foerster J, 11.
Lukens JN, Rodgers GM, Paraskevas F, Glader B, eds. Wintrobe’s Clinical Hematology.: Lippincott Williams & Wilkins; 2004:170-193.
Tavian M, Coulombel L, Luton D, Clemente HS, Dieterlen-Lievre F, Peault B. Aorta-associated 12.
CD34+ hematopoietic cells in the early human embryo. Blood 1996;87:67-72.
Galloway JL, Zon LI. Ontogeny of hematopoiesis: examining the emergence of hematopoietic 13.
cells in the vertebrate embryo. Curr.Top.Dev.Biol. 2003;53:139-158.
Jordan CT, Lemischka IR. Clonal and systemic analysis of long-term hematopoiesis in the mouse.
14.
Genes Dev. 1990;4:220-232.
Capel B, Hawley RG, Mintz B. Long- and short-lived murine hematopoietic stem cell clones 15.
individually identified with retroviral integration markers. Blood 1990;75:2267-2270.
Keller G, Snodgrass R. Life span of multipotential hematopoietic stem cells in vivo. J.Exp.Med 16.
1990;171:1407-1418.
Berenson RJ, Andrews RG, Bensinger WI, Kalamasz D, Knitter G, Buckner CD, Bernstein ID.
17.
Antigen CD34+ marrow cells engraft lethally irradiated baboons. J.Clin.Invest 1988;81:951-955.
Weissman IL, Shizuru JA. The origins of the identification and isolation of hematopoietic stem 18.
cells, and their capability to induce donor-specific transplantation tolerance and treat autoimmune diseases. Blood 2008;112:3543-3553.
Ogawa M. Changing phenotypes of hematopoietic stem cells. Exp.Hematol. 2002;30:3-6.
19.
Zanjani ED, Almeida-Porada G, Livingston AG, Flake AW, Ogawa M. Human bone marrow. Exp.
20.
Hematol. 1998;26:353-360.
Zubair AC, Silberstein L, Ritz J. Adult hematopoietic stem cell plasticity. Transfusion 2002;42:1096- 21.
1101.
Kimoto M, Fathman CG. Antigen-reactive T cell clones. I. Transcomplementing hybrid I-A-region 22.
gene products function effectively in antigen presentation. J.Exp.Med 1980;152:759-770.
Till JE, Mcculloch EA, Siminovitch L. A stochastic model of stem cell proliferation, based on the 23.
growth of spleen colony-forming cells. Proc.Natl.Acad.Sci.U S.A 1964;51:29-36.
Metcalf D. Lineage commitment and maturation in hematopoietic cells: the case for extrinsic 24.
regulation. Blood 1998;92:345-347.
Enver T, Heyworth CM, Dexter TM. Do stem cells play dice? Blood 1998;92:348-351.
25.
Robb L. Cytokine receptors and hematopoietic differentiation. Oncogene 2007;26:6715-6723.
26.
Payne KJ, Crooks GM. Human hematopoietic lineage commitment. Immunol.Rev 2002;187:48- 27.
64.
Zhang DE, Hohaus S, Voso MT, Chen HM, Smith LT, Hetherington CJ, Tenen DG. Function 28.
of PU.1 (Spi-1), C/EBP, and AML1 in early myelopoiesis: regulation of multiple myeloid CSF receptor promoters. Curr.Top.Microbiol.Immunol. 1996;211:137-147.
Laiosa CV, Stadtfeld M, Graf T. Determinants of lymphoid-myeloid lineage diversification. Annu.
29.
Rev Immunol. 2006;24:705-738.
Kaushansky K. Hematopoietic stem cells, progenitors and cytokines. In: Lichtman MA, Beutler 30.
E, Kipps TJ, Seligsohn U, Kaushansky K, Prchal JT, eds. William’s Hematology.: McGraw-Hill;
2006:201-220.
Tenen DG. Disruption of differentiation in human cancer: AML shows the way. Nat.Rev Cancer 31.
2003;3:89-101.
Steffen B, Muller-Tidow C, Schwable J, Berdel WE, Serve H. The molecular pathogenesis of 32.
acute myeloid leukemia. Crit Rev Oncol.Hematol. 2005;56:195-221.
Castilla LH, Garrett L, Adya N, Orlic D, Dutra A, Anderson S, Owens J, Eckhaus M, Bodine D, 33.
Liu PP. The fusion gene Cbfb-MYH11 blocks myeloid differentiation and predisposes mice to acute myelomonocytic leukaemia. Nat.Genet. 1999;23:144-146.
Higuchi M, O’Brien D, Kumaravelu P, Lenny N, Yeoh EJ, Downing JR. Expression of a conditional 34.
AML1-ETO oncogene bypasses embryonic lethality and establishes a murine model of human t(8;21) acute myeloid leukemia. Cancer Cell 2002;1:63-74.
Horiike S, Yokota S, Nakao M, Iwai T, Sasai Y, Kaneko H, Taniwaki M, Kashima K, Fujii H, 35.
Abe T, Misawa S. Tandem duplications of the FLT3 receptor gene are associated with leukemic transformation of myelodysplasia. Leukemia 1997;11:1442-1446.
Osato M. Point mutations in the RUNX1/AML1 gene: another actor in RUNX leukemia. Oncogene 36.
2004;23:4284-4296.
Song WJ, Sullivan MG, Legare RD, Hutchings S, Tan X, Kufrin D, Ratajczak J, Resende IC, 37.
Haworth C, Hock R, Loh M, Felix C, Roy DC, Busque L, Kurnit D, Willman C, Gewirtz AM, Speck NA, Bushweller JH, Li FP, Gardiner K, Poncz M, Maris JM, Gilliland DG. Haploinsufficiency of CBFA2 causes familial thrombocytopenia with propensity to develop acute myelogenous leukaemia. Nat.Genet. 1999;23:166-175.
Wiemels JL, Cazzaniga G, Daniotti M, Eden OB, Addison GM, Masera G, Saha V, Biondi A, 38.
Greaves MF. Prenatal origin of acute lymphoblastic leukaemia in children. Lancet 1999;354:1499- 1503.
Licht JD, Sternberg DW. The molecular pathology of acute myeloid leukemia. Hematology.
39.
Am.Soc.Hematol.Educ.Program. 2005137-142.
Gilliland DG, Griffin JD. The roles of FLT3 in hematopoiesis and leukemia. Blood 2002;100:1532- 40.
1542.
Dash A, Gilliland DG. Molecular genetics of acute myeloid leukaemia. Best.Pract.Res.Clin.
41.
Haematol. 2001;14:49-64.
Bonnet D, Dick JE. Human acute myeloid leukemia is organized as a hierarchy that originates 42.
from a primitive hematopoietic cell. Nat.Med 1997;3:730-737.
Hope KJ, Jin L, Dick JE. Acute myeloid leukemia originates from a hierarchy of leukemic stem 43.
cell classes that differ in self-renewal capacity. Nat.Immunol. 2004;5:738-743.
Guan Y, Hogge DE. Proliferative status of primitive hematopoietic progenitors from patients with 44.
acute myelogenous leukemia (AML). Leukemia 2000;14:2135-2141.
Bonnet D. Normal and leukaemic stem cells. Br.J.Haematol. 2005;130:469-479.
45.
Brown D, Kogan S, Lagasse E, Weissman I, Alcalay M, Pelicci PG, Atwater S, Bishop JM. A 46.
PMLRARalpha transgene initiates murine acute promyelocytic leukemia. Proc.Natl.Acad.Sci.U S.A 1997;94:2551-2556.
Jaiswal S, Traver D, Miyamoto T, Akashi K, Lagasse E, Weissman IL. Expression of BCR/
47.
ABL and BCL-2 in myeloid progenitors leads to myeloid leukemias. Proc.Natl.Acad.Sci.U S.A 2003;100:10002-10007.
Jamieson CH, Ailles LE, Dylla SJ, Muijtjens M, Jones C, Zehnder JL, Gotlib J, Li K, Manz 48.
MG, Keating A, Sawyers CL, Weissman IL. Granulocyte-macrophage progenitors as candidate leukemic stem cells in blast-crisis CML. N.Engl.J.Med 2004;351:657-667.
Swerdlow SH, Campo E, Harris NL, Jaffe ES, Pireli SA, Stein H, Thiele J, Vardiman JW. WHO 49.
classification of tumours of haematopoietic and lymphoid tissues. Lyon: IARC; 2008.
Bennett JM, Catovsky D, Daniel MT, Flandrin G, Galton DA, Gralnick HR, Sultan C. Proposals 50.
for the classification of the acute leukaemias. French-American-British (FAB) co-operative group.
Br.J.Haematol. 1976;33:451-458.
Goasguen JE, Bennett JM, Henderson ES. Biologic Diagnosis of Leukemias. In: Henderson E.S., 51.
Lister T.A., Greaves M.F., eds. Leukemia.: W.B. Saunders Company; 1996:8-33.
Jaffe ES, Harris NL, Stein H, Vardiman JW. Pathology and Genetics of Tumours of Haematopoietic 52.
and Lymphoid Tissues. Lyon: IARC Press; 2001.
Liesveld JL, Lichtman MA. Acute myelogenous leukemia. In: Lichtman MA, Beutler E, Kipps TJ, 53.
Seligsohn U, Kaushansky K, Prchal JT, eds. William’s Hematology.: McGraw-Hill; 2006:1183.
Cheson BD, Cassileth PA, Head DR, Schiffer CA, Bennett JM, Bloomfield CD, Brunning R, Gale 54.
RP, Grever MR, Keating MJ, . Report of the National Cancer Institute-sponsored workshop on definitions of diagnosis and response in acute myeloid leukemia. J.Clin.Oncol. 1990;8:813-819.
Mrozek K, Heinonen K, Bloomfield CD. Prognostic value of cytogenetic findings in adults with 55.
acute myeloid leukemia. Int.J.Hematol. 2000;72:261-271.
Haferlach T, Schnittger S, Kern W, Hiddemann W, Schoch C. Genetic classification of acute 56.
myeloid leukemia (AML). Ann.Hematol. 2004;83 Suppl 1:S97-100.
Mrozek K, Heerema NA, Bloomfield CD. Cytogenetics in acute leukemia. Blood Rev 2004;18:115- 57.
136.
Greer PG, Baer MR, Kirshner JR. Acute Myeloid Leukemia in Adults. In: Greer PG, Foerster 58.
J, Lukens JN, Rodgers GM, Paraskevas F, Glader B, eds. Wintrobe’s Clinical Hematology.:
Lippincott Williams & Wilkins; 2004:2097-2139.
Ferlay, J., Bray, F., Pisani, P., and Parkin, D. M. GLOBOCAN 2002: Cancer Incidence, Mortality 59.
and Prevalence Worldwide. http://www.dep.iarc.fr/ IARC CancerBase No. 5(version 2.0). 2004.
IARCPress.
Yin SN, Li GL, Tain FD, Fu ZI, Jin C, Chen YJ, Luo SJ, Ye PZ, Zhang JZ, Wang GC, . Leukaemia 60.
in benzene workers: a retrospective cohort study. Br.J.Ind.Med 1987;44:124-128.
Karp JE, Smith MA. The molecular pathogenesis of treatment-induced (secondary) leukemias:
61.
foundations for treatment and prevention. Semin.Oncol. 1997;24:103-113.
Shaheen M, Broxmeyer HE. The Humoral Regulation of Hematopoiesis. In: Hoffman R, Benz EJ, 62.
Shattil SJ et al., eds. Hematology: Basic Principles and Practice.: Elsevier; 2005:233.
Adolfsson J, Borge OJ, Bryder D, Theilgaard-Monch K, Astrand-Grundstrom I, Sitnicka E, Sasaki 63.
Y, Jacobsen SE. Upregulation of Flt3 expression within the bone marrow Lin(-)Sca1(+)c-kit(+) stem cell compartment is accompanied by loss of self-renewal capacity. Immunity. 2001;15:659- 669.
Care RS, Valk PJ, Goodeve AC, Abu-Duhier FM, Geertsma-Kleinekoort WM, Wilson GA, Gari 64.
MA, Peake IR, Lowenberg B, Reilly JT. Incidence and prognosis of c-KIT and FLT3 mutations in core binding factor (CBF) acute myeloid leukaemias. Br.J.Haematol. 2003;121:775-777.
Byrne JL, Marshall CJ. The molecular pathophysiology of myeloid leukaemias: Ras revisited.
65.
Br.J.Haematol. 1998;100:256-264.
Reuter CW, Morgan MA, Bergmann L. Targeting the Ras signaling pathway: a rational, mechanism- 66.
based treatment for hematologic malignancies? Blood 2000;96:1655-1669.
Janssen JW, Steenvoorden AC, Lyons J, Anger B, Bohlke JU, Bos JL, Seliger H, Bartram CR. RAS 67.
gene mutations in acute and chronic myelocytic leukemias, chronic myeloproliferative disorders, and myelodysplastic syndromes. Proc.Natl.Acad.Sci.U S.A 1987;84:9228-9232.
Bentires-Alj M, Paez JG, David FS, Keilhack H, Halmos B, Naoki K, Maris JM, Richardson 68.
A, Bardelli A, Sugarbaker DJ, Richards WG, Du J, Girard L, Minna JD, Loh ML, Fisher DE, Velculescu VE, Vogelstein B, Meyerson M, Sellers WR, Neel BG. Activating mutations of the noonan syndrome-associated SHP2/PTPN11 gene in human solid tumors and adult acute myelogenous leukemia. Cancer Res. 2004;64:8816-8820.
Tartaglia M, Gelb BD. Germ-line and somatic PTPN11 mutations in human disease. Eur.J.Med 69.
Genet. 2005;48:81-96.
Valentino L, Pierre J. JAK/STAT signal transduction: regulators and implication in hematological 70.
malignancies. Biochem.Pharmacol. 2006;71:713-721.
Tefferi A, Pardanani A. Mutation screening for JAK2V617F: when to order the test and how to 71.
interpret the results. Leuk.Res. 2006;30:739-744.
DeClue JE, Papageorge AG, Fletcher JA, Diehl SR, Ratner N, Vass WC, Lowy DR. Abnormal 72.
regulation of mammalian p21ras contributes to malignant tumor growth in von Recklinghausen (type 1) neurofibromatosis. Cell 1992;69:265-273.
