Identification of a Small Subpopulation of Candidate Leukemia Initiating Cells in the Side Population (SP) of Patients with Acute Myeloid Leukemia

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Identification of a Small Subpopulation of Candidate Leukemia Initiating Cells in the Side Population (SP) of Patients with Acute Myeloid Leukemia
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  Part of it has been adapted from:New Approaches for the Detection of Minimal Residual Disease in Acute Myeloid Leukemia A.van Rhenen * B. Moshaver * G.J. OssenkoppeleGJ. Schuurhuis Current Hematologic Malignancy Reports.  2007;2:111-118. *  Both authors (A.v. Rhenen and B. Moshaver) contributed equally to this manuscript 1 General Introduction  11   1. GENERAL: HEMATOPOIESIS AND LEUKEMIA 1.1 Normal hematopoeisis and hematopoietic stem cells Blood cell formation occurs in the yolk sac in the first weeks of gestation. From 6 weeks until the 7th month of fetal life, hematopoiesis takes place in the liver and spleen, after which hematopoiesis is transferred to the bone marrow, which is the only source of blood cells during adult life under healthy conditions. All blood cells are derived from hematopoietic stem cells (HSC), being defined by its ability to reconstitute hematopoiesis in serial bone marrow transplantations in mice who underwent myeloablative therapy. HSCs maintain hematopoeisis by the capacity of both self-renewal and of differentiation into multilineage committed progenitor cells, either a common myeloid progenitor (CMP) or a common lymphoid progenitor (CLP) (1, 2). These cells then give rise to more differentiated progenitors and ultimately, they will mature towards unilineage committed progenitors for monocytes, granulocytes, erythrocytes, platelets and natural killer cells, B- and T-lymphocytes (3, 4). The process of proliferation and differentiation of HSCs is strictly regulated and balanced in the bone marrow environment, consisting of bone marrow stromal cells (BMSCs), such as macrophages, fibroblasts, adipocytes and endothelial cells, cytokines and the extracellular matrix. The relation of hematopoietic cells with their environment is highly dynamic allowing preservation of stem cells in the so-called “stem cell niche” as well as expansion and differentiation on demand (5, 6). Although HSCs were previously thought to be resting cells within the stem cell niche, recent evidence from mouse experiments show that 8-10% of HSC randomly enter the cell cycle every day, with all HSCs entering the cell cycle in 1-3 months (7). 2. ACUTE MYELOID LEUKEMIA  Acute myeloid leukemia (AML) is a clonal expansion of immature hematopoietic progenitor cells, being caused by somatic mutations causing a disturbance in differentiation. In general, only multiple genetic changes will result in the development of AML. The incidence of AML is 3.7 and 2-3 per 100.000 men en women per year in the United States and Western Europe, respectively (8-10).   The disease affects both children and adults. 1 GENERAL   INTRODUCTION  1213 incorporated (table 1). In addition, information on chromosomal aberrancies provides  information on the prognosis of these patients, thereby allowing patient tailored therapy (18). 2.1 Diagnosis Diagnosis of AML in BM and PB samples of patients is based on morphologic, immunophenotypic, and cytogenetic features. Thereby, AML can be classified according to the French-American-British (FAB) and the World Health Organization (WHO) classifications. 2.1.1 Morphology May-Grunwald-Giemsa (MGG) staining is being used to visualize the morphology of the different type cells in patients specimens. AML is usually characterized by hypercellularity with an accumulation of myeloid blasts in BM or PB with displace ment  of normal erythropoietic and myeloid progenitor cells as well as disappearance of megakaryocytes (11). In the FAB classification, AML is subdivided in 8 types on bases of morphological and cytochemical criteria: AML-M0 to AML-M7 (12, 13) (table 1). 2.1.2 Immunophenotyping Immunophenotype analysis has a central role in distinguishing between minimally differentiated AML and acute lymphoblastic leukemia (ALL). Immunophenotyping performed by flow cytometry concerns the protein expression on the surface and in the cytoplasm of the blast population. Myeloid immature cells are characterized by low side-scatter (SSC), low expression of CD45 (leukocyte marker), expression of CD34 and/or CD117, and the expression of lineage specific markers CD13, CD33 and myeloperoxidase (MPO). Multiple markers analysis helps to identify phenotypes associated with particular cytogenetic aberrations, or may help to identify immunophenotypic abnormalities that are useful in monitoring patients for the presence of minimal residual disease (MRD) (14, 15). 2.1.3 Cytogenetic aberrations Cytogenetic studies are important for the sub-classification of hematopoietic malignancies. Approximately 50% of patients with de novo  AML show chromosomal aberrancies. Some of these rearrangements coincide largely or completely with certain FAB classes: t(8;21) (q22;q22) for FAB M2 (AML1/ETO), t(15;17)(q22;q12) for FAB M3 (PML/RAR α ), inversion(16) (p13;q22) or t(16;16) for FAB M4 (CBF β  / MYH11), while for monocytic leukemia 11q23 (MLL) translocation is frequently involved (16, 17) (table 1). However, most of the chromosomal abnormalities add to the FAB-classification. Therefore, the WHO has recently developed a new classification of AML in which information on chromosome analysis is now being CHAPTER   1 1 GENERAL   INTRODUCTION Table 1  The WHO 2008 classification system for AML WHO classification ≥ 20% blastsFAB classification ≥ 30% blasts AML with recurrent cytogenetic translocations  AML t(8;21)(q22;q22) [AML1/ETO] Particularly in M2 AML t(15;17)(q22;q11-12) [PML/RAR ] M3 and variants AML inv(16)(p13.1q22) or t(16;16)(p13;q22) [CBF /MYH11]  AML t(9;11)(p22;q12) [MLLT3-MLL]  AML t(6;9)(p23;q34) [DEK-NUP214]  AML inv(3)(q21q26.2) or t(3;3)(q21;q26.2) [RPN1-EVI1]  AML (megakaryoblastic) t(1;22)(p13;q13) [RMB15-MKL1]  AML with mutated NPM1 AML with mutated CEBPAM4eoM7  AML with MDS-related changes Therapy-related myeloid neoplasms  AML not otherwise specified  AML with minimally differentiationM0 AML without maturationM1 AML with maturationM2 AML myelomonocyticM4 AML monoblastic and monocyticM5 AML erythroidM6 AML megakaryoblasticM7 AML basophilic  AML panmyelosis with myelofibrosis  AML:  acute myeloid leukemia FAB:  French-American-British MDS:  myelodysplastic syndrome  1415 in first remission for intermediate and/or high-risk patients with an HLA-matched sibling or a matched-unrelated donor. Patients generally receive immuno-suppressive chemotherapy in combination with total body irradiation (TBI).  Although this strategy carries a high risk on short-term mortality and long-term morbidity as a result of graft versus host disease (GVHD), studies have been reported an improved overall survival for the patients with poor/high risk cytogenetics who underwent an allogeneic hematopoietic stem cell transplantation (HSCT), compared with those without a donor (22, 23). The reason for that probably lies in the immune graft versus leukemia (GVL) effect, as donor lymphocytes recognize the recipient leukemic cells as foreign with subsequent elimination of leukemic cells. This principle is also being applied in allogeneic transplanted patients with a relapse, in which the infusion of donor T-lymphocyte infusions (DLI) might lead to a second complete remission (24, 25).  Although about 80% of adult patients (<60 years old) achieve complete remission after above described intensive treatment strategies, only 30% to 40% of patients are alive after 5 years after diagnosis, mainly being the result of relapse of the disease. For elderly patients the prognosis is even worse (26). A relapse of the disease is now thought to be caused by the outgrowth of MRD. This concept has been supported by the finding that the detection of MRD after completion of therapy predicts relapse (27). Therefore, MRD detection after induction chemotherapy, might help to decide on which type of consolidation therapy. Currently, this decision is mainly based on molecular classification of AML as well as on the comorbidity score. However, it would be ideal to decide on the basis of the response of the disease to induction chemotherapy in order to improve efficacy where needed and to circumvent side effects in patients with a low risk for relapse. 2.3 Minimal residual disease There are different hypotheses in order to explain the persistence of a minimal number of leukemic cells after the completion of chemotherapy treatment. The primary reason for treatment failure in AML is cellular drug resistance, which can be either caused by drug efflux, by inherent expression of apoptosis-resistance proteins or by micro-environment induced expression of apoptosis resistance proteins. Resistance mediated by increased drug efflux in AML occurs via high expression of P-glycoprotein (Pgp/ABCB1), multidrug resistance–associated protein-1 (MRP1/  ABCC1), lung resistance-related protein (LRP) and the breast cancer- resistant protein (BCRP/ABCG2). Especially, high expression of Pgp and BCRP has been 2.2 Therapy  AML is a clinically heterogeneous disease, depending on the localisation of proliferating cells; BM, PB or in other tissues. BM localisation leads to suppression of normal hematopoiesis and thereby to anemia, trombocytopenia and neutropenia. An increase in leukemic cells in PB can increase viscosity, thereby giving rise to a hyperviscosity syndrome being the result of a compromised circulation in the central nervous system and in the pulmonary vasculature, leading to dizziness, stupor, visual disturbances and dyspnoe. Local proliferation of leukemic cells, called chloromas results in specific complaints depending on the localisation. However, even in case the patient presents without symptoms, acute therapeutic intervention is required to prevent the life-threatening effects of hyperviscosity and of suppression of normal hematopoiesis. The cornerstone of the therapy is intensive chemotherapy in order to eradicate leukemic cells. This will be followed by consolidation therapy to prevent relapses of the disease. The type of consolidation treatment highly depends on the molecular subtyping of AML, either a third course of chemotherapy, an autologous stem cell transplantation or an allogeneic transplantation is given (19). 2.2.1 Chemotherapy as consolidation in good risk AML  After two chemotherapy courses, consisting of anthracyclines (daunorubicine or idarubicine) and cytarabine-arabinoside (ARA-C), a third course of consolidation chemotherapy might be given without the need for a stem cell transplantation. Post-remission dose-intensive chemotherapy has indeed been found to prolong the duration of remission (20). This approach generally accounts for patients with good risk AML, defined as AML with a t(8;21), an inversion 16 or a t(15;17). 2.2.2 Autologous hematopoietic stem cell transplantation as consolidation in standard risk AML Harvesting peripheral normal stem cells after having reached a complete remission allows dose intensification of consolidation therapy. Myeloablative therapy followed by a peripheral stem cell transplantation is now generally being performed in patients with standard risk AML, not being able to undergo an allogeneic stem cell transplantations (21). 2.2.3 Allogeneic hematopoietic stem cell transplantation as consolidation in standard and high risk AML  Allogeneic BM or PB transplantation is an established procedure for consolidation CHAPTER   1 1 GENERAL   INTRODUCTION  1617 upon inhibition of -catenin, one of the mediating proteins in Wnt signaling (43). In view of the important role of bone marrow microenvironment-induced chemo - therapy resistance and maintenance of malignant stem cells, it is important to elucidate the responsible mechanisms. Moreover, it is tempting to hypothesize that exposure of BMSCs to chemotherapy affects its leukemic supportive effect, however this has never been addressed. This is of importance taking into account the known hazardous effect of chemotherapeutics to BMSCs (44). Hopefully, this will lead to potential new therapeutic targets in AML. 2.3.2 MRD detection In patients with acute leukemia, detection and quantification of malignant cells after chemotherapy in remission BM provides powerful prognostic information for the identification of patient risk categories. The clinical importance of MRD detection and quantification in childhood ALL is already established and led to a risk-adapted approach in pediatric ALL trials (45, 46). A number of studies have reported on the clinical value of MRD detection in childhood AML (47, 48) and adult AML (27, 49, 50). The obtained knowledge is expected to facilitate the early detection of impending relapse, may result in risk-adapted therapies, and may offer a short-term endpoint to assess the effectiveness of new targeted therapies. 2.3.3 Different approaches in MRD detection If the leukemic cell at diagnosis carries an antigenic or molecular marker that distinguishes it from its normal counterpart, this marker can be used after chemotherapy to detect residual malignant cells in BM. This allows the application of two sensitive methods to identify MRD in both ALL and AML: polymerase chain reaction (PCR)–based and multiparameter flow cytometry (MFC) techniques (51). Each method has its advantages and disadvantages which will be discussed in more detail for AML. 2.3.3.1 PCR detection Real-time quantitative reverse transcriptase PCR (qRT-PCR) is the most sensitive molecular MRD detection method (cell frequency of 10 –4  to 10 –5/-6 ) used in recent years in AML (52, 53). It permits absolute quantification of target DNA or mRNA, in contrast to end-point quantification of the more classic PCR method. This technique is based on genetic aberrations such as mutations and fusion genes, occurring in subgroups of AML. The most common rearrangements producing fusion genes in AML are t(8;21), t(15;17), and inv(16)/t(16;16), which occur in a found to predict a poor prognosis (28-30). Moreover, patients with a multidrug resistant profile at diagnosis have higher MRD frequencies after chemotherapy than patients with a more sensitive phenotype (29). High expression of the anti-apoptotic protein Bcl-2 and/or low expression of the pro-apoptotic protein Bax at diagnosis, have been shown to predict poor survival in AML (31). This apoptotic profile is either an inherent characteristic of leukemic cells, but can also be induced by the bone marrow microenvironment. Again, an apoptosis-resistant profile is correlated with higher levels of MRD after chemo- therapy (32). 2.3.1 The role of the bone marrow microenvironment in MRD Recently the bone marrow microenvironment has been proposed to be a key player in drug resistance in AML (33). Inhibition of chemotherapy-induced apoptosis is generally considered to underlie protection by BMSCs (34). However, both promotion of proliferation and inhibition of spontaneous apoptosis might add to the observed maintenance of leukemic cells by BMSCs (35- 38). The latter is of particular interest in view of the reported prognostic significance of spontaneous apoptosis in AML (39). Moreover, in the situation of MRD, after the completion of chemotherapeutic treatments, the process of spontaneous apoptosis is likely to be of importance in the prevention of the outgrowth to relapse (40).The underlying mechanism in protection from apoptosis, is a reciprocal signaling dialogue between tumor cells and the surrounding microenvironment (37). For example, it has been shown that very late antigen-4 (VLA-4)-positive leukemic cells acquire resistance drug-induced apoptosis through the phosphatidylinositol- 3-kinase (PI-3K)/AKT/Bcl-2 signaling pathway, which is activated by the interaction of VLA-4 and fibronectin (41). Accordingly, VLA-4 expression was found to predict prognosis of AML patients. Moreover, stromal cells have been found to prevent apoptosis of AML cells by up-regulating anti-apoptotic proteins in leukemic blasts (42). In addition, discrete cellular spaces within the bone marrow microenvironment, termed “niches”, are thought to provide the mechanical support and extrinsic molecular factors that maintain stem-ness. Osteoblasts were identified as key cellular components, producing Wnt-proteins, thereby linking the concept of activated self-renewal pathways within stem cells and the concept of an inductive stem cell niche. Indeed, the re-plating efficiency of HSC, transduced with fusion proteins encoded by translocations commonly found in AML, was abrogated CHAPTER   1 1 GENERAL   INTRODUCTION
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