Leukemias

Principles and Practice of Therapy

Stefan Faderl, Hagop Kantarjian, Hagop Kantarjian, Stefan Faderl, Hagop Kantarjian, Stefan Faderl

• Publisher: Wiley-Blackwell
• Year: 2011
• Print ISBN: 9781405182355
• eBook ISBN: 9781444347814
• Edition: 1
• Topic: Medicine
• Subtopic: Hematology

Edited by experts from one of the world's largest leukemia centers, this book provides information on the biology of the variety of leukemic disorders, up-to-date diagnostic testing and many new developments in therapy. Chapters covering new treatments present an outlook for the future and explain the rationale for ongoing clinical trials.

Topics include:

• Targeted therapy, e.g. tyrosine kinase inhibitors (Flt3, Aurora kinase inhibitors, kit inhibitors, BCR-ABL inhibitors)
• Ras inhibitors
• Epigenetic therapy (hypomethylaters and histone deacetylase inhibitors)
• Lenalidomide analogs
• New chemotherapy drugs, e.g. clofarabine, cloretazine, sapacitabine, forodesine
• Combinations of chemotherapy with kinase inhibitors (e.g. ALL induction protocols in combination with dasatinib or imatinib)
• New monoclonal antibodies (lumiliximab, humaxCD20, anti-CD40)
• Thrombopoietic agents

Leukemias: Principles and Practice of Therapy

• Includes practical information to guide you in challenging situations, such as treatment of elderly patients, pregnancy, relapsed and refractory disease
• Incorporates chapters on supportive care and pharmacologic information about the most frequently used drugs in this area

Part 1

Background and Diagnostic

Chapter 1

Stem-cell Biology in Normal and Malignant Hematopoiesis

Amer Zeidan¹ and Meir Wetzler²

¹ Division of Hospital Medicine, General Medicine Unit, Rochester General Hospital, Rochester, New York, USA

²Department of Medicine, Roswell Park Cancer Institute, Buffalo, New York, USA

Introduction

Hematopoiesis is the highly orchestrated process of blood cell production that maintains homeostasis by reproducing billions of white blood cells (WBCs), red blood cells (RBCs), and platelets on a daily basis [1].

Hematopoietic stem cells (HSCs) represent the small population of long-lived, quiescent, undifferentiated, pluripotent cells which are characterized by the capacity of self-renewal, exceptional proliferation potential, resistance to apoptosis, and the ability of multilineage differentiation into all blood cell types mediated by the production of several lineage-committed progenitors [1–5].

The central role of leukemia stem cells (LSCs) in the pathogenesis of some forms of leukemia has become well recognized over the last two decades. LSCs share many basic characteristics with HSCs, including quiescence, self-renewal, extensive proliferative capacity, and the ability to give rise to differentiated progeny in a hierarchical pattern [6–12]. Some scientists even view leukemia as a newly formed, abnormal hematopoietic tissue initiated by a few LSCs that undergo an aberrant and poorly regulated process of organogenesis analogous to that of the normal HSCs [13].

Many researchers believe that the persistence of LSCs, which are resistant to most of the traditional chemothera-peutic agents that kill the bulk of the leukemic cell populations, is a major cause of leukemia relapse after “successful” remission induction. Subsequently, designing effective therapeutic modalities that specifically target the LSCs is likely to reduce the incidence of relapse, and possibly even lead to a cure. As discussed below, the ongoing efforts to develop “magic bullets” targeting the LSCs will continue to face significant challenges because of their similarities to normal HSCs [14]. It is very important to further delineate the differences between normal HSCs and LSCs in order to design novel therapeutic modalities that offer maximal cytotoxicity to LSCs while sparing the normal HSCs [4,14].

In this chapter, we will briefly review the basic principles of the biology of HSCs and LSCs and examine the major scientific advances in this field. We will also discuss some of the ongoing efforts to utilize this growing knowledge for the purpose of developing targeted therapies directed against LSCs that could reduce the frequency of leukemia relapse.

Hematopoietic stem-cell biology in normal hematopoiesis

The HSC is the best-defined somatic stem cell to date [15]. The experimental data support the presence of an HSC compartment that is arranged as a continuum with unidirectional, irreversible progression of cells with decreasing capacities for self-renewal, increasing likelihood for differentiation, and increasing proliferative activity [16]. HSCs generate all the multiple hematopoietic lineages for the entire lifespan through a successive series of intermediate progenitors, known as colony-forming units (CFUs) or colony-forming cells (CFCs) [4,5]. As these intermediate progenitors continue to mature, they become more restricted in terms of the number and type of lineages that they can generate and exhibit a reduced self-renewal capacity [4,5]. Researchers have demonstrated the presence of a common lymphoid progenitor (CLP) and a common myeloid progenitor (CMP), which possibly reflects the earliest branching points between the lymphoid and myeloid lineages [17,18].

Studies on murine hematopoietic stem cells

Studies in mice have contributed considerably to our current understanding of HSC biology. Initially, it was demonstrated that bone marrow cells injected into lethally irradiated recipient mice re-established hematopoiesis [19]. Later, it was shown that the first step in this engraftment was the formation of multilineage colonies in the spleen within 10 days of the injection [20]. Each of these spleen multilineage colonies actually arose from a single pluripotent stem cell, the spleen colony-forming unit (CFU-S) [21]. Those spleen colonies containing the CFU-S were capable of giving rise to new colonies in secondary recipients [22]. Subsequent studies demonstrated that the CFU-S actually consists of a heterogeneous population of more advanced progenitor cells that are distinct from the more primitive and more highly renewing HSCs, and that CFU-S are not capable of long-term multilineage hematopoietic reconstitution in vivo [23].

Jones et al. [24] showed that serial bone marrow transplantations, which eventually failed to reconstitute lethally irradiated mice, dissociated two phases of engraftment. The first unsustained phase was maintained with repeated serial transfer and appeared to be produced by committed progenitors, like granulocyte-macrophage colony-forming units (CFU-GM) and the CFU-S. The second sustained phase was eventually lost with repeated serial transfer, apparently due to decreasing numbers of pluripotent HSCs. Prolonging the time interval between serial transfers reestablished the ability of the serially transplanted marrow to reconstitute hematopoiesis [24], suggesting that the HSCs needed more time to allow long-term engraftment. Thereafter, Morrison et al. concluded that marrow reconstitution in mice was deterministic, not stochastic [25].

Studies on human hematopoietic stem cells

Owing to the clear limitations of experimenting on humans, most of our current knowledge about human HSCs was obtained indirectly from in vitro studies and xenotransplantation of human cells into immunodeficient animals [1]. Despite the presence of important differences, evidence suggests that the human HSC compartment, although not completely defined, parallels that of the murine counterpart, with a heterogeneous population of primitive cells with varying capacities for differentiation, proliferation, and self-renewal [1,4].

The in vitro culture assays [26–29] can evaluate some of the important characteristics of HSCs such as pluripotency and proliferative potential, but cannot accurately measure the bona fide properties of HSCs: the sustained and complete hematopoietic repopulating ability, and the maximal differentiating ability [5,30]. The severe combined immune-deficient (SCID) mice, which lack adaptive immunity, and the non-obese diabetic SCID (NOD/SCID) mice, which lack both innate and adaptive immunity, offered a more accurate reflection of the human HSC function than the in vitro culture assays [14,31–33]. The accuracy of these i n vivo repopulation assays has been further improved by co-injection of distinguishable reference cells [5].

Characteristics of hematopoietic stem cells

In contrast to the morphologically well-defined committed precursors and mature cells, the HSCs are morphologically indistinguishable from the hematopoietic progenitor cells (HPCs). On the other hand, the HSCs can be phenotypically distinguished from the HPCs through multiparameter flow cytometry. The most commonly used surface antigen to enrich for HSCs is cluster designation CD34. Unlike their murine counterparts that are usually negative for the murine homolog of CD34 (mCD34⁻), primitive human HSCs are usually CD34⁺ [34]. Terstappen et al. [35] demonstrated that 1% of the CD34⁺ cells did not express the CD38 antigen. The CD34⁺/CD38⁻cells were homogeneous and lacked lineage-commitment specific markers (Lin⁻), in agreement with what is expected from putative pluripotent HSCs. In contrast, the CD34⁺/CD38⁺ cells were heterogeneous and contained myeloblasts and erythroblasts, as well as lym-phoblasts, suggesting an upregulation of CD38 antigen upon differentiation of the CD34⁺/CD38⁻cells [35]. Later, the CD34⁺/CD38⁻cell subset was shown to generate long-term, multilineage human hematopoiesis in the human-fetal sheep in vivo model. In contrast, the CD34⁺/CD38⁺ cells generated only short-term human hematopoiesis, suggesting again that the CD34⁺/CD38⁺ cell population contained relatively early multipotent HPCs, but not HSCs [36]. This work proved that the CD34-/CD38⁻cell population has a high capacity for long-term multilineage hematopoietic engraftment, indicating the presence of stem cells in this minor adult human marrow cell subset [36].

In addition, HSCs were found to typically express high levels of stem-cell antigen 1 (SCA-1) and permeability glycoprotein (P-gp), a multidrug efflux transporter located in the plasma membrane and encoded by the multidrug resistance 1 gene (MDR1) [37,38]. On the other hand, HSCs typically have either absent or low levels of expression of Thy-1.1, CD33, CD71, CD10, CD45RA, and HLA-DR, while the more mature progenitors express one or more of these markers [1,15,35,39]. Finally, Gunji et al. [40] showed that the CD34⁺ cell fraction that exhibited low expression of the c-Kit proto-oncogene protein (c-kit-low) contained CD34⁺/CD38⁻cells that are considered to be the more primitive hematopoietic cells. In comparison, the CD34⁺/c-Kit-high cell fraction contained many granulocyte-macrophage -committed progenitor cells. Osawa et al. [34] showed that injecting a single murine pluripotent HSC (characterized by the phenotype mCD34[lo/⁻], c-Kit⁺, SCA1⁺, lineage markers negative [Lin⁻]) resulted in long-term reconstitution of the hematopoietic system. These data suggest that all primitive cells are c-Kit⁺, but HSCs have lower expression of c-Kit than the less primitive progenitors.

The dogma that all HSCs express CD34 has been challenged recently by studies suggesting the existence of an unrecognized population of HSCs that lack the CD34 surface marker and are characterized by their ability to efflux the Hoechst dye [41,42]. These cells were referred to as “side population” (SP) cells [43,44]. These SP cells were found to be highly enriched for long-term culture-initiating cells (LTC-ICs), an indicator of primitive hematopoietic cells [42]. Similarly, Zanjani et al. [45] demonstrated that the CD34⁻/Lin⁻ fraction of the normal human bone marrow contained cells which were capable of engraftment and differentiation into CD34⁺ progenitors and multiple hematopoietic lineages in primary and secondary hosts.

It is evident from the above discussion that we are still facing significant challenges that limit our ability to accurately identify and isolate HSCs. A major goal of future investigations is to determine whether novel markers or marker combinations exist that will allow HSCs to be prospectively identified and isolated from any source [39].

Hematopoietic stem cells and self-renewal

Hematopoiesis encompasses a complex interaction between the HSCs and their microenvironment, which plays a critical role in the maintenance of HSCs. This complex interaction determines whether the HSCs, HPCs, and mature blood cells remain quiescent, proliferate, differentiate, self-renew, or undergo apoptosis [1,46]. While the majority of HSCs are quiescent in the G₀ phase in steady-state bone marrow, many of the stem cells are actually cycling regularly, although slowly, to maintain a constant flow of short-lived HPCs that can generate enough cells to replace those that are constantly lost during normal turnover [15,47].

Self-renewal is the ability of a stem cell to divide, yielding one daughter cell that can differentiate and another that maintains the pluripotent stem-cell function [14,15,48,49]. There are two hypothetical mechanisms by which asymmetric cell division might be achieved: divisional asymmetry and environmental asymmetry [2]. In divisional asymmetry, specific cell -fate determinants redistribute unequally before the onset of or during cell division [2,15]. As a result, only one daughter cell receives those determinants and therefore retains the HSC fate, while the other daughter cell proceeds to differentiation. In environmental asymmetry, a stem cell would first undergo a symmetric division, producing two identical daughter cells [15]. However, only one cell remains in the HSC niche (see below) and conserves its HSC fate, while the other cell enters a different microenvironment and subsequently produces signals initiating differentiation instead of preserving its stem-cell phenotype [2,15].

The longevity of the HSCs is another area of active research. Morrison et al. [50] showed that HSCs from old mice were only one-quarter as efficient at homing to and engrafting the bone marrow of irradiated recipients in comparison with HSCs from young mice, suggesting that the self-renewal capacity of HSCs is not infinite. Two of the proposed theories to explain this phenomenon are the progressive telomeric DNA shortening and the accumulation of DNA damage leading to stem-cell exhaustion [51,52].

Despite recent advances in our understanding of the complex molecular mechanisms that underlie the process of HSC self-renewal, there are still many aspects of this process that require further elucidation. Detailed discussion of the proposed molecular regulatory mechanisms controlling self-renewal in normal HSCs is beyond the scope of this review, but it is important to note that a large number of transcription factors, proteins, and signaling pathways have been implicated in the regulation of this process (reviewed in refs 2,5,38).

Hematopoietic stem cells and the microenvironment

The mechanisms of bone and blood formation have traditionally been viewed as distinct unrelated processes, but compelling evidence suggests that they are intertwined [53]. HSCs reside in the bone marrow, close to the endosteal surfaces of the trabecular bone in what is commonly referred to as “the niche” [54]. A stem-cell niche can be defined as a spatial structure in which HSCs are housed for an indefinite period of time and are maintained by allowing progeny production through self-renewal in the absence of differentiation [15,54,55]. There is accumulating evidence indicating that the stromal cells in the niche, especially the endosteal osteoblasts, play a major role in regulating the HSC maintenance, proliferation, and maturation [53,56–58]. Although the osteoblast is one of the main cellular elements of the HSC niche, the exact nature of the factors produced by the osteoblast that participate in the regulatory microenvironment for HSCs are known in only limited detail [15,48,53].

Several cell-surface receptors were implicated in controlling the localization of HSCs to the endosteal niche. One example is the calcium-sensing receptor (CaR) [15,49]. A unique feature of the bone that may contribute to the HSC homing might be the high concentration of calcium ions at the HSC-enriched endosteal surface [48]. It was shown that CaR-deficient HSCs from murine fetal liver failed to engraft in the bone marrow [49]. In addition, these cells were highly defective in localizing anatomically to the endosteal niche following transfer to lethally irradiated wild-type recipients, indicating the importance of CaR in homing of HSCs to the bone marrow niche [49]. Several other cell-surface receptors were described to be involved in the localization of the HSCs to the niche; one additional example, chemokine (C-X-C motif) receptor 4 (CXCR-4), and its ligand, the stromal-cell derived factor 1 (SDF-1), will be discussed later in the chapter.

While the majority of HSCs and HPCs are located in the bone marrow, a significant minority of them that play an important role in the establishment and functioning of the hematopoietic system are found in the peripheral blood under steady-state conditions [60,61]. The realization of the presence of large numbers of HSCs and HPCs in the umbilical cord blood and the ability to mobilize these cells into the circulation with chemotherapy and hematopoietic growth factors led to very significant advances in the fields of stem-cell biology, transplantation, and gene therapies [62–65].

Hematopoietic stem-cell plasti...

Table of contents

• Cover
• Title page
• Copyright
• Contributors
• Preface
• Part 1: Background and Diagnostic
• Part 2: Myelodysplastic Syndromes
• Part 3: Acute Myeloid Leukemia
• Part 4: Acute Promyelocytic Leukemia
• Part 5: Acute Lymphoblastic Leukemia
• Part 6: Chronic Myeloid Leukemia
• Part 7: Chronic Lymphocytic Leukemia
• Part 8: Other Leukemic Disorder
• Part 9: General Treatment Principles and Clinical Developments
• Index
• Color plate