Leukemias E-Book

• Herausgeber: John Wiley & Sons
• Kategorie: Fachliteratur
• Sprache: Englisch

Seitenzahl: 1528

Veröffentlichungsjahr: 2013

Beschreibung

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

Leseprobe

Contents

Contributors

Preface

Part 1 Background and Diagnostic

Chapter 1 Stem-cell Biology in Normal and Malignant Hematopoiesis

Introduction

Hematopoietic stem-cell biology in normal hematopoiesis

Conclusions

Acknowledgments

References

Chapter 2 Epidemiology and Etiology of Leukemias

Myelodysplastic syndromes and acute myeloid leukemia

Acute lymphoblastic leukemia

Polycythemia rubra vera

Essential thrombocythemia

Idiopathic myelofibrosis

Chronic myeloid leukemia

Chronic lymphocytic leukemia

References

Chapter 3 Traditional Diagnostic Approaches

Myeloproliferative neoplasms

Chronic myeloid leukemia

Acute leukemias

Acute lymphoblastic leukemia

Chronic lymphocytic leukemia and related lymphoproliferative disorders

References

Chapter 4 Diagnosis of Leukemias: New Diagnostic Modalities and Implications for Classification

Introduction

Technology

Acute myeloid leukemia

Acute lymphoblastic leukemia

Chronic myeloid leukemia

Chronic lymphocytic leukemia

References

Chapter 5 Non-cytogenetic Markers and Their Impact on Prognosis

Introduction

Molecular diagnostics and molecular monitoring of disease

Major subchromosomal mutations in leukemia

Acute lymphoblastic leukemias

Therapeutic targeting of molecular abnormalities

Referencess

Part 2 Myelodysplastic Syndromes

Chapter 6 Myelodysplastic Syndromes: Pathophysiology

Introduction

Cell of origin

Genetic alterations within the cell of origin

Altered bone marrow microenvironment/apoptotic pathways

Fas/Bcl-2

Altered immune surveillance

Altered cellular functions/aberrant differentiation

References

Chapter 7 Myelodysplastic Syndromes: The Role of Cytogenetic and Molecular Abnormalities for Classification and Risk Assignment

Introduction

Diagnosis and classification

Molecular genetic findings

Consequences for treatment decisions in the light of old and new compounds

References

Chapter 8 Myelodysplastic Syndromes

Epidemiology and etiology

Clinical and laboratory features

Ancillary studies

Diagnosis

Prognosis

Therapy

References

Part 3 Acute Myeloid Leukemia

Chapter 9 Presentation and Diagnosis: Novel Molecular Markers and their Role in the Prognosis and Therapy of Acute Myeloid Leukemia

Introduction

Involvement of transcription

Alterations of the MLL gene

Dysregulation of HOX genes

Activation of proliferation

Mutations of oncogenes

Interference with cell cycle and apoptosis

Other molecular markers

Molecular risk stratification in acute myeloid leukemia with normal karyotype

Planning of molecular diagnostics in acute myeloid leukemia

Origin and sequence of molecular mutations in acute myeloid leukemia

Gene expression profiling

Conclusions

References

Chapter 10 Induction Therapy of Acute Myeloid Leukemia

Pre-treatment evaluation

Choice of anthracycline

Dose of anthracycline

Dose of cytarabine

Double induction and timed-sequential therapy

Addition of other agents to standard therapy

Priming and growth-factor support

Monoclonal antibodies

FLT3 inhibitors

Alternative strategies

Conclusion

References

Chapter 11 Salvage Therapy for Acute Myeloid Leukemia: Current Strategies and Emerging Therapies

Introduction

Cell cycle-directed therapies

Signal transduction-directed therapies

VEGF-directed therapies

Protein metabolism-directed therapies

Epigenetic therapies

Immune-targeted therapies

Conclusion

References

Chapter 12 Hematopoietic Stem-cell Transplantation for Acute Myelogenous Leukemia

Introduction

Pre-transplant regimens

Myeloablative preparative regimens

Transplants for acute myeloid leukemia in complete remission 1

Autologous transplantation

Transplantation for acute myeloid leukemia in primary induction failure or beyond complete remission 1

Therapy following transplant failure

Aging and allogeneic transplantation for acute myeloid leukemia

Patient selection and results of transplantation

Future directions

Conclusion

References

Part 4 Acute Promyelocytic Leukemia

Chapter 13 The Pathophysiology of Acute Promyelocytic Leukemia

Introduction

Molecular architecture of the t(15;17)

Structure and function of PML and RARα

Other biologic features of APL and their impact on therapy

Conclusions and future perspectives

References

Chapter 14 Acute Promyelocytic Leukemia: Manifestations and Therapy

Introduction

Clinical and biologic characteristics of acute promyelocytic leukemia

First-line treatment of acute promyelocytic leukemia

Treatment of relapsing acute promyelocytic leukemia

Consolidation/maintenance treatment

Treatment in elderly patients and in children

Conclusion

References

Part 5 Acute Lymphoblastic Leukemia

Chapter 15 Acute Lymphoblastic Leukemia: Presentation, Diagnosis and Classification

Introduction

Epidemiology

Presentation

Diagnosis

B-ALL

T-ALL

Differential diagnoses

Cytogenetic and molecular characterization

Cytogenetic and molecular lesions in B-ALL

Cytogenetic and molecular lesions in T-ALL

Conclusion

References

Chapter 16 Induction Therapy in Adult Acute Lymphoblastic Leukemia

Introduction

Historical considerations

Antileukemic drugs used in induction schedules

Remission induction

Subset-specific approaches

Supportive care

Complications during induction

Evaluation of induction therapy

Prognostic factors

References

Chapter 17 Salvage Therapy of Adult Acute Lymphoblastic Leukemia

Introduction

Definitions of refractory and relapsed disease, including minimal residual disease response evaluation

Treatment principles

Outcome after relapse

Prospective studies of salvage therapy

Stem-cell transplant after relapse

Management of specific situations

Prognostic factors for outcome of relapse

New drugs

Nucleoside analogs or related drugs

Liposomal drugs

Monoclonal antibodies

Molecular therapies

Summary and conclusions

References

Chapter 18 Philadelphia Chromosome-positive Acute Lymphoblastic Leukemia: Current Treatment Status and Perspectives

Introduction

The molecular biology of the philadelphia chromosome

Treatment

Conclusion

References

Chapter 19 Acute Lymphoblastic Leukemia in Adolescents and Young Adults

Introduction

Incidence of acute lymphoblastic leukemia in adolescents and young adults

Therapeutic approach to adolescents and young adults with acute lymphoblastic leukemia

Conclusions

References

Chapter 20 The Role of Hematopoietic Stem-cell Transplantation in Adults with Acute Lymphoblastic Leukemia

Abstract

General principles of acute lymphoblastic leukemia therapy

Factors influencing transplant outcome

Immunomodulation post transplant

Conclusion

References

Part 6 Chronic Myeloid Leukemia

Chapter 21 Pathophysiology of Chronic Myeloid Leukemia

Introduction

The molecular biology of chronic myeloid leukemia

Pathogenetic consequences of Bcr–Abl

The clinical evolution of chronic myeloid leukemia

Challenges for the future

References

Chapter 22 Therapy of Newly Diagnosed and Chronic-phase Chronic Myeloid Leukemia

Introduction

Chronic myeloid leukemia pathophysiology

Use of tyrosine kinase inhibitors for the treatment of chronic myeloid leukemia

Prognostic factors

Conclusions

Acknowledgments and disclosures

References

Chapter 23 Therapy of Advanced-stage and Resistant Chronic Myeloid Leukemia

Introduction

How to monitor newly diagnosed patients with chronic-phase chronic myeloid leukemia receiving imatinib as a first-line therapy

Imatinib resistance

Therapy for imatinib-resistant patients with chronic-phase chronic myeloid leukemia

How to choose the tyrosine kinase inhibitor for imatinib-failure patients with chronic-phase chronic myeloid leukemia

How to monitor patients on second-generation tyrosine kinase inhibitors

Advanced-stage chronic myeloid leukemia

Recommendations for patients with de novo advanced-phase disease and blast crisis

Conclusion

References

Part 7 Chronic Lymphocytic Leukemia

Chapter 24 Chronic Lymphocytic Leukemia: Pathophysiology, Diagnosis and Manifestations, and Prognostic Markers

Introduction

Pathophysiology

Morphology

Histology

Immunophenotyping

Differential diagnosis

Work-up and staging

Radiological assessment

Prognosis

Clinical stage according to Binet and Rai

Markers of tumor burden

IGHV mutational status and surrogate markers

Genomic aberrations and chronic lymphocytic leukemia

References

Chapter 25 Therapy of Chronic Lymphocytic Leukemia: Front-line and Salvage

Introduction

Indications for front-line therapy

Choice of front-line therapy

Indications for salvage therapy

Choice of salvage therapy

Perspective

References

Chapter 26 The Role of Stem-cell Transplantation in Chronic Lymphocytic Leukemia

Introduction

Patient selection for stem-cell transplantation

Management strategies in chronic lymphocytic leukemia

Role of transplantation in chronic lymphocytic leukemia

Conclusions

References

Chapter 27 Special Situations: Management of Elderly Patients and Chronic Lymphocytic Leukemia in Transformation

Management of chronic lymphocytic leukemia in transformation

Management of chronic lymphocytic leukemia in elderly patients

Conclusions

References

Part 8 Other Leukemic Disorder

Chapter 28 Hairy Cell Leukemia

Introduction

Pathology and diagnosis

Treatment of hairy cell leukemia

Conclusions

References

Chapter 29 Therapeutic Approaches to the Mature T-cell Lymphoproliferative Leukemias

Introduction

T-cell prolymphocytic leukemia

T-cell large granular lymphocyte leukemia

Natural killer-cell leukemias

Adult T-cell leukemia/lymphoma

Conclusion

References

Chapter 30 Philadelphia Chromosome-negative Myeloproliferative Neoplasms

Myeloproliferative neoplasms

Essential thrombocythemia and polycythemia vera

Myelofibrosis

Hypereosinophilic syndrome and chronic eosinophilic leukemia

Systemic mast cell disease

References

Part 9 General Treatment Principles and Clinical Developments

Chapter 31 Management of Emergencies in Leukemias

Introduction

Hyperleukocytosis and leukostasis

Metabolic complications

Mediastinal mass/superior vena cava syndrome

Management of bleeding complications in leukemias

Management of acute promyelocytic leukemia-associated coagulopathy

Neurologic complications

Other organ involvement

References

Chapter 32 Management of Infectious Complications in Patients with Leukemia

Introduction

Risk and epidemiology of infections in patients with leukemia

Common clinical infectious syndromes

Infections in special hosts

Management of infection

Non-neutropenic patients

Acknowledgments

References

Chapter 33 Transfusion Support and Hematopoietic Growth Factors

Principles of blood transfusion

Practice of blood transfusion in leukemia

Clinical uses of hematopoietic growth factors in leukemia

Conclusions

Acknowledgments

References

Chapter 34 New Designs for Clinical Trials: Acute Myeloid Leukemia as an Example

Introduction

Bayesian approach

Simon two-stage design

References

Index

Color plate

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ISBN: 978-1-4051-8235-5

A catalogue record for this book is available from the British Library.

Library of Congress Cataloging-in-Publication Data

Leukemias: principles and practice of therapy/edited by Stefan Faderl, Hagop Kantarjian.

p.; cm.

Includes bibliographical references and index.

ISBN 978–1-4051-8235-5 (alk. paper)

1. Leukemia. I. Faderl, S. H. (Stefan H.) II. Kantarjian, Hagop, 1952- [DNLM: 1. Leukemia--diagnosis. 2. Leukemia--therapy. WH 250 L65597 2011]

RC643.L433 2011

616.99'419-dc22

2010024070

Set in 9.25/12pt Palatino by Toppan Best-set Premedia Limited

Contributors

LionelAdes

Service d’hematologie clinique

Hopital Avicenne (Assistance Publique-Hopitaux de Paris)

and Paris 13 University

Bobigny, France

Gheath Alatrash do, phD

Assistant Professor of Medicine

Department of Stem Cell Transplantation and Cellular Therapy

Division of Cancer Medicine

University of Texas

MD Anderson Cancer Center

Houston, Texas, USA

Maher Albitar

Quest Diagnostics Nichols Institute

San Juan Capistrano

California, USA

Elias Anaissie MD

Professor

Deputy Chair and Head, Division of Supportive Care

Myeloma Institute for Research and Therapy

University of Arkansas for Medical Sciences

Little Rock, Arkansas, USA

John Anastasi MD

Section of Hematopathology

Department of Pathology

University of Chicago

Chicago, Illinois, USA

Ulrike Bacher

Department of Stem Cell Transplantation

University of Hamburg

Hamburg, Germany

David Bowen

Consultant Haematologist and Honorary Professor of Myeloid Leukaemia Studies

St. James’s Institute of Oncology

Leeds Teaching Hospitals

Leeds, UK

Carlos Bueso-Ramos MD, phD

Professor

Department of Hematopathology

University of Texas

MD Anderson Cancer Center

Houston, Texas, USA

Daniel J DeAngelo MD, phD

Associate Professor of Medicine

Harvard Medical School;

Clinical Director, Adult Leukemia Program

Department of Medical Oncology

Dana-Farber Cancer Institute

Boston, Massachusetts, USA

Claire Dearden MD, frcp, frcpath

Consultant Haematologist

Royal Marsden NHS Foundation Trust

Surrey, UK

Marcosde Lima, MD

Associate Professor of Medicine

Department of Stem Cell Transplantation and Cellular Therapy

University of Texas

MD Anderson Cancer Center

Houston, Texas, USA

Hartmut Döhner MD

Department of Internal Medicine III

University of Ulm Ulm,

Germany

BarbaraEichhorst, MD

Specialist for Hematology and Oncology

Department I of Internal Medicine

University Clinic of Cologne

Cologne, Germany

Elihu Estey

Clinical Research Department

Fred Hutchinson Cancer Research Center

Division of Hematology

University of Washington School of Medicine

Seattle, Washington, USA

Pierre Fenaux MD, phD

Service d’hematologie clinique

Hopital Avicenne (Assistance Publique-Hopitaux de Paris)

and Paris 13 University

Bobigny, France

Alessandra Ferrajoli MD

Associate Professor of Medicine

Department of Leukemia

University of Texas

MD Anderson Cancer Center

Houston, Texas, USA

Andrew Fletcher mbchb, mrcpch

Department of Pediatric Hematology

St. James ’ s University Hospital

Leeds, UK

Olga Frankfurt MD

Assistant Professor of Medicine

Northwestern University Feinberg School of Medicine

Robert H. Lurie Comprehensive Cancer Center

Chicago, Illinois, USA

Guillermo Garcia-Manero MD

Associate Professor of Medicine

Chief, Section of Myelodysplastic Syndromes

Department of Leukemia

University of Texas

MD Anderson Cancer Center

Houston, Texas, USA

Ulrich Germing MD, phD

Department of Hematology, Oncology and Clinical Immunology

Heinrich-Heine-University

Dusseldorf, Germany

Valentin Goede MD

Resident

Department I of Internal Medicine

University Clinic of Cologne

Cologne, Germany

Nicola Gökbuget MD

Head of Study Center

Goethe University Hospital

Department of Medicine II, Hematology/Oncology

Frankfurt, Germany

Steven D Gore MD

Professor of Oncology

Hematologic Malignancies

Sidney Kimmel Comprehensive Cancer Center

Johns Hopkins

Baltimore, Maryland, USA

John G Gribben MD, DSc, FRCP, FRCPath, FMedSci

Professor

Institute of Cancer

Bart’s and the London School of Medicine

London, UK

Sandeep Gurbuxani mbbs, phD

Section of Hematopathology

Department of Pathology

University of Chicago

Chicago, Illinois, USA

Claudia Haferlach

MLL Munich Leukemia Laboratory

Munich, Germany

Torsten Haferlach

MLL Munich Leukemia Laboratory

Munich, Germany

Simon Hallam ma, mb, BChir, mrcp

Institute of Cancer

Bart’s and the London School of Medicine

London, UK

Michael Hallek MD

Director

Department I of Internal Medicine

University Clinic of Cologne

Cologne, Germany

Syed Khizer Hasan phD

Department of Biopathology

University Tor Vergata

Rome, Italy

Devendra Hiwase MD

Consultant Haematologist

Haematology Division

SA Pathology

Adelaide, Australia

Andreas Hochhaus MD

Professor of Internal Medicine, Hematology and Oncology

Universitatsklinikum Jena, Klinik für Innere Medizin II

Abteilung Hamatolgie und Internistische Onkologie

Jena, Germany

Dieter Hoelzer MD

Professor of Internal Medicine

Onkologikum am Museumsufer

Frankfurt, Germany

Timothy Hughes MD

Professor of Haematology

Haematology Division

SA Pathology, Adelaide, Australia

JudithE Karp md

Professor of Oncology and Medicine

Director, Leukemia Program

Sidney Kimmel Comprehensive Cancer Center

Johns Hopkins University

Baltimore, Maryland, USA

Partow Kebriaei MD

Associate Professor of Medicine

University of Texas

MD Anderson Cancer Center

Houston, Texas, USA

Wolfgang Kern, MD

MLL Munich Leukemia Laboratory

Munich, Germany

Hartmut Koeppen MD, phD

Staff Pathologist

Department of Pathology

Genentech, Inc

South San Francisco, California, USA

Sergej Konoplev MD, phD

Department of Hematopathology

University of Texas

MD Anderson Cancer Center

Houston, Texas, USA

Francesco Lo-Coco MD

Department of Biopathology

University Tor Vergata

Rome, Italy

Junia VMelo MD, phD, frcpath

Professor of Haematology

University of Adelaide

Centre for Cancer Biology—SA Pathology

Adelaide, SA, Australia

Ruben Mesa MD

Professor of Medicine

Director, Acute and Chronic Leukemias Program

Mayo Clinic

Scottsdale, Arizona, USA

Bijay Nair MD, mph

Assistant Professor of Medicine

Myeloma Institute for Research and Therapy

University of Arkansas for Medical Sciences

Little Rock, Arkansas, USA

Marcio Nucci MD

University Hospital

Universidade Federal do Rio de Janeiro

Rio de Janeiro, Brazil;

Myeloma Institute for Research and Therapy

University of Arkansas for Medical Sciences

Little Rock, Arkansas, USA

Nnenna Osuji

Honorary Consultant, Section of Haemato-Oncology

Royal Mardsen NHS Foundation Trust/Institute of Cancer

Research

Surrey, UK

Keith W Pratz MD

Assistant Professor of Oncology

Sidney Kimmel Comprehensive Cancer Center

Johns Hopkins University

Baltimore, Maryland, USA

Jerald Radich MD

Clinical Research Division

Fred Hutchinson Cancer Research Center

Seattle, Washington, USA

Farhad Ravandi MD

Associate Professor of Medicine

Department of Leukemia

University of Texas

MD Anderson Cancer Center

Houston, Texas, USA

PaoloRebulla MD

Director, Center of Transfusion Medicine

Cellular Therapy and Cryobiology

Foundation ’Ca’ Granda Ospedale Maggiore Policlinico’

Milano, Italy

MichaelRyttingMD

Assistant Professor- Pediatrics and Leukemia

University of Texas

MD Anderson Cancer Center

Houston, Texas, USA

OlgaSala-TorraMD

Clinical Research Division

Fred Hutchinson Cancer Research Center

Seattle, Washington, USA

Eric Schafer MD

Instructor in Pediatrics and Oncology

Sidney Kimmel Comprehensive Cancer Center

Johns Hopkins University

Baltimore, Maryland, USA

Susanna Schnittger

MLL Munich Leukemia Laboratory

Munich, Germany

Donald Small MD, phD

Kyle Haydock Professor of Oncology

Professor of Oncology, Pediatrics, Cellular and Molecular

Medicine

Director, Pediatric Oncology

Sidney Kimmel Comprehensive Cancer Center

Johns Hopkins University

Baltimore, Maryland, USA

William Stevenson mbbs, phD

Hematologist

Royal North Shore Hospital

Sidney, Australia

Stephan Stilgenbauer MD

Department of Internal Medicine III

University of Ulm Ulm,

Germany

Sylvain Thépot

Service d’hematologie clinique

Hopital Avicenne (Assistance Publique-Hopitaux de Paris)

and Paris 13 University

Bobigny, France

Xavier Thomas MD, phD

Leukemia Unit

Department of Hematology

Edouard Herriot Hospital

Lyon, France

Wei-Gang Tong MD, phD

Clinical Fellow

Department of Leukemia

University of Texas

MD Anderson Cancer Center

Houston, Texas, USA

Srdan Verstovsek MD, phD

Associate Professor

University of Texas

MD Anderson Cancer Center

Houston, Texas, USA

Martha Wadleigh, MD

Assistant Professor of Medicine

Harvard Medical School

Department of Medical Oncology

Dana-Farber Cancer Institute

Boston, MA

Erica D. Warlick, MD

Assistant Professor

Division of Hematology, Oncology, and Transplantation

Department of Medicine University of Minnesota

Minneapolis, Minnesota, USA

MeirWetzler MD, facp

Professor of Medicine

Chief, Leukemia Section

Department of Medicine

Roswell Park Cancer Institute

Buffalo, New York, USA

Annika Whittle

Specialist Registrar

Department of Hematology

St. James’s Institute of Oncology

Leeds, UK

Agnes S M Yong mb BCh, mrcp (uk), phD, FRCPath

Senior Research Fellow

Hematology Branch

National Institutes of Health

Bethesda, Maryland, USA

Amer Zeidan MD

Academic Hospitalis

Division of Hospital Medicine

General Medicine Unit

Rochester General Hospital

Rochester, New York, USA;

Sidney Kimmel Comprehensive Cancer Center at Johns Hopkins

Baltimore, Maryland, USA

Thorsten Zenz MD

Department of Internal Medicine III

University of Ulm

Ulm, Germany

Preface

Progress in the definition and therapy of hematologic malignancies has been at the forefront of a therapeutic revolution in cancer medicine. The introduction of imatinib mesylate in the management of patients with Philadelphia chromosome-FI positive chronic myeloid leukemia only 10 years ago has sparked a concerted effort into targeted therapies that extend, by now, far beyond the initial BCR-ABL kinase. Exciting developments in unraveling the biology of leukemic disorders coincide with the availability of an array of drugs never before realized. New prognostic markers, which are being tested in clinical trials, are helping to fine-tune the treatment approaches in many different leukemic disorders. Markers predictive of response to specific therapies are integrated into clinical algorithms, as, for example, the finding of 17p abnormalities in patients with chronic lymphocytic leukemias has demonstrated. As so often is the case, the ever increasing body of knowledge also raises many more new questions. Hence the practicing physician and academician is constantly faced with new challenges and confronted with the frequently daunting task to reconcile the major developments of a specialty that is becoming both more spread out and specialized “within.” This book, dedicated exclusively to leukemic disorders, aims to discuss and summarize new pathophysiologic concepts, therapeutic approaches, developments of supportive care, and future investigational strategies. Experts in their respective fields from all over the world have been invited to share their unique insights, vision, and expertise. To compile a book like this is always a trying task as it will struggle with the same challenge as every one of us faces, that is to keep up with all the new information in this incredibly dynamic field, even as it is going through the production process. It is the wish of the editors that this book will be instructive and helpful to its hopefully many readers.

Stefan Fade

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 plasticity

A fascinating aspect of the biology of HSCs is their potential plasticity. The term plasticity refers to the ability of organ-specific stem cells to recover their ability to differentiate into cells of other lineages, either in vitro or after transplantation in vivo [1,66,67]. There have been a number of reports that documented HSCs differentiating into non-hematopoietic cells including myocardium, muscle cells, neurons, and hepatocytes [68–71]. A detailed discussion of this phenomenon is beyond the scope of this review, but the interested reader can refer to some of the many reports published on this subject [1,66–71].

Leukemic stem cells

Two interesting observations suggested the presence of LSCs. First, despite the aggressive course of acute myeloid leukemia (AML), it has long been recognized that most leukemic blasts have limited in vivo proliferative capacity and that only a minority of these blasts are capable of forming colonies in vitro [72,73]. Second, the leukemic blasts in any individual, despite their morphologic homogeneity, are characterized by significant intercell biologic heterogeneity [74]. While it has long been assumed that stochastic variations related to the genomic instability of the tumor cells are largely responsible for this phenomenon, it has become increasingly evident that intrinsic development processes such as those normally found within stem-cell-based hierarchies also play a significant role [12]. Mounting evidence over the last two decades suggested that the leukemic clone is maintained by a rare population of stem cells—the LSCs, also known as the leukemia-initiating cells (LICs) [75,76]. Some researchers even viewed 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 normal HSCs, but the differentiation from the LSC population gives rise to “blast” cells, which represent arrested or aberrant stages of myeloid development [11,13].

The seminal work by John Dick, laboratory in 1994 [75] provided the first proof for the presence of LSCs by isolation of a population of CD34+/CD38−cells from patients with AML followed by engraftment of these cells into SCID mice. This resulted in a pattern of dissemination and leukemic cell morphology similar to that seen in the original patients. Work from the same laboratory later demonstrated that fewer CD34+/CD38−LSCs were needed to induce AML in NOD/SCID mice than in SCID mice [9]. The authors concluded that the LSCs possess the differentiative and proliferative capacities and the potential for self-renewal seen in HSCs, and that LSCs were able to differentiate, n vivo into leukemic blasts, indicating that the leukemic clone is organized as a hierarchy [9]. Subsequently, the presence of LSCs has been demonstrated in chronic myeloid leukemia (CML), and also some forms of acute lymphoblastic leukemia (ALL) (see below for more detailed discussion).

LSCs, which constitute a minority of the tumor bulk, are functionally defined on the basis of their ability to transfer leukemia into an immunodeficient recipient animal [14]. LSCs share many of the basic characteristics with normal HSCs including quiescence, self-renewal, extensive proliferative capacity, and the ability to give rise to differentiated progeny in a hierarchical pattern [6–12] (Table 1.1). It was shown that LSCs are not functionally homogeneous but, similar to the normal HSC compartment, comprise distinct hierarchically arranged LSC classes [77]. In a xenotransplantation model, some LSCs emerged only in recipients of serial transplantation, indicating that they divided rarely and underwent self-renewal rather than commitment after cell division within primary recipients. On the other hand, other LSCs gave only short-term leukemic repopulation in secondary recipients. This led to the conclusion that the distinct LSC fates were derived from heterogeneous self-renewal potential, which is analogous to the hierarchy observed in normal HSCs with long and short repopulation potentials [25,77].

Most of the chemotherapeutic agents traditionally used to treat leukemias are cell-cycle active agents, which primarily target dividing cells. These agents are highly unlikely to eradicate the quiescent LSCs [8,78,79]. In addition, the LSCs seem to be biologically distinct from their more differentiated progeny, with specific cellular and molecular mechanisms that control their behavior; these mechanisms are quite different from those controlling the more mature leukemic blasts [11]. Therefore, it seems plausible that the agents acting against the more mature blasts will not be as efficient in eradicating the LSCs, possibly significantly contributing to treatment failure and future relapse [11]. In addition, it is highly likely that LSCs, similar to their normal HSC counterparts, possess efflux pumps, such as the P-gp, which confer resistance to traditional chemotherapy by quickly removing the cytotoxic agents from the cells [43,44,80]

Table 1.1 Common characteristics of hematopoietic stem cells and leukemia stem cells.

Quiescence Self-renewal Extensive proliferative capacity Ability to give rise to a differentiated progeny Expression of efflux proteins Survival in a “niche” Resistance to apoptosis CD34+/CD38−/CD71−/HLA-DR−immunophenotype Comprising hierarchy-arranged classesBmi-1 expression

Where does the leukemic stem cell come from?

The experimental data from John Dick's laboratory [9,75] led the authors to conclude that, given the homogeneous CD34+/CD38− immunophenotype of LSCs and their ability for hierarchical differentiation, proliferation, and self-renewal, leukemic transformation occurred at the level of the normal HSCs rather than the committed progenitor cells. Later, this postulation that LSCs necessarily arise from aberrant HSCs [9,25,81] was challenged. Alternatively, it was suggested that LSCs could arise from more committed progenitors caused by mutations and/or selective expression of genes that enhance their otherwise limited self-renewal capabilities [13,82–86]. In fact, several groups [83–86] reported the ability to induce transformation of committed progenitor cells to LSCs by transducing these cells with the oncogenic fusion genes. Cozzio et al. [83] reported similar latencies of developing AML in recipient mice when transducing the leuke-mogenic chimeric gene MLL-ENL (MLL, also known as ALL1, mixed-lineage-leukemia, and ENL, 11–19-lysine-rich-[eukemia) into either HSCs or myeloid progenitors with granulocyte-macrophage differentiation potential (GMPs). Similarly, Krivtsov et al. [84] demonstrated that the oncogenic fusion protein MLL-AF9 (ALL1 fused gene from chromosome 9) can transform GMPs. These findings established the ability of transient repopulating progenitors to initiate myeloid leukemias in response to the MLL oncogene, thus supporting the existence of an LSC that overlaps with the multipotent HSC [83]. This led some researchers to propose that the nature of the LSC may vary depending upon the particular stage in normal hematopoiesis during which the insult occurred, resulting in significant pathogenetic and therapeutic differences of the leukemic phenotypes [10,12,74,77].

In addition, Huntly et al. [85] showed that while CMPs that were transduced with the oncogene MYST histone acetyltransferase (monocytic leukemia) 3-transcriptional intermediary factor 2 (MOZ-TIF2) resulted in an AML in vivo that could be serially transplanted, BCR–ABL trans-duction into CMPs conferred none of these properties. These data demonstrated that some, but not all, leukemia oncogenes can transfer properties of LSCs to HPCs destined to undergo apoptotic cell death. Thereafter, Stubbs et al. [86] showed that GMPs transduced with MLL-AF9 and FMS-like tyrosine kinase-internal tandem duplication (FLT3-ITD) mutation cooperated to produce a more aggressive AML when compared with AML induced by MLL-AF9 alone. These observations supported the theory of multistep nature of leukemogenesis where the initial genetic event (first hit) often leads to the expression of chimeric oncogenes (e.g. MLL-AF9) encoded by recurrent chromosomal translocations, while subsequent mutations (second hit) may activate specific signaling pathways (e.g. FLT3-ITD) [86]. It is important to note that all of the data on the progenitor origin of LSCs come from mouse models.

The terms “LSC” and “cancer stem cell” have caused significant confusion in the literature, partly because they implied that these cells originate from a normal stem cell. Recently, a panel of the American Association for Cancer Research agreed that the term “cancer stem cell” in general does not refer to the cell of origin of the cancer [87,88]. Rather, this term encompasses the notion that the cell type that sustains the growth of many cancers possesses stem-cell properties and lies at the pinnacle of a neoplastic hierarchy, giving rise to a “differentiated” progeny that lacks these same properties [87,88].

Leukemic stem cells in BCR–ABL-positive leukemia (CML, AML, and ALL)

In CML the mature leukemic cells and their progenitors are morphologically indistinguishable from their normal counterparts, and the distinction requires proof of the presence or absence of the Philadelphia (Ph) chromosome or the Bcr–Abl transcript in these cells [89]. The Ph chromosome is the cytogenetic hallmark of CML, and it results from the reciprocal translocation between the long arms of chromosomes 9 and 22 that leads to the formation of the chimeric oncogenic tyrosine kinase BCR–ABL, which is central to the pathogenesis of the disease [90]. The Ph chromosome has been shown to be present in nearly all hematopoietic lineages, indicating that the cell of origin of CML has a multilineage differentiation potential [91]. Researchers were able to transplant CD34+ malignant primitive cells from patients with CML into immunode-ficient SCID and NOD/SCID mice, which subsequently were able to proliferate and produce mature Ph+ progeny with kinetics that recapitulated the phase of the donor's disease, and thus providing an i n vivo model for CML biology [92–94].

Holyoake et al. [95] provided the first direct and definitive evidence of the presence of a reversibly quiescent subpopulation of BCR–ABL+/CD34+ leukemic cells that exhibit both in vivo and in vitro stem-cell properties in patients with CML. In a later study, the same group [95,96] demonstrated that those stem cells were leukemic (BCR–ABL+), expressed the immunophenotype CD34+/CD38−/CD45RA−/CD71−, and could spontaneously exit G0 state to enter a continuously cytokine-independent proliferating state to produce a BCR–ABL+ progeny.

The development of imatinib mesylate (IM) (Gleevec®, Novartis, Basel, Switzerland), a tyrosine kinase inhibitor (TKI) that targets BCR–ABL, represented a major advance in the treatment of CML, and is now widely accepted as the standard of care first-line treatment of chronic phase CML (CP-CML) [97]. Despite its impressive results, a significant minority of patients with CP-CML and a majority of the patients with the accelerated phase (AP-CML) and blastic phase (BP-CML) either do not respond (primary resistance) or lose their initial response (secondary resistance) to imatinib [98]. Even among imatinib responders, only a few patients achieve complete molecular remission, that is the complete disappearance of BCR–ABL transcripts in highly sensitive reverse transcriptase polymerase chain reaction assays [90,99]. There is accumulating evidence that CML LSCs are not eradicated by IM in vivo, with patients in complete cytogenetic response (CCR) still demonstrating Ph+ CD34+ cells, CFUs, and LTC-ICs [100]. This indicates that the disease is not eradicated in the vast majority of patients, which is concerning for the possibility of future relapse. Based on these observations, it has been proposed that the successful targeting of the quiescent CML LSC population might be the “holy grail” for achieving cure for this disease.

The most common mechanism of imatinib resistance in CML is the development of mutations in ABL [90,101,102]. An important, recent addition to the arsenal against IM-resistant CML, and Ph leukemias in general, was the development of nilotinib (Tasigna®, Novartis, Basel, Switzerland) and dasatinib (Sprycel®, Bristol-Myers Squibb, New York). Nilotinib and dasatinib are second-generation TKIs with greater potency of BCR–ABL inhibition than imatinib. Both nilotinib and dasatinib were demonstrated to be clinically effective in patients with different phases of imatinib-resistant CML, and both were capable of inhibiting the majority of kinase mutations in imatinib-resistant CML [103–107]. However, none of the TKIs in clinical use for CML target the CML LSC [108–111].

Several possible mechanisms were proposed to explain the resistance of CML LSCs to TKIs in addition to mutations. One such mechanism, which is unique to CML, is the amplification of the BCR–ABL transcript and BCR–ABL protein [111,112]. Other mechanisms include increased expression of interleukin (IL) 3 receptor, granulocyte colony-stimulating factor receptor, MDR1, and suppressed expression of organic cation transporter 1 (an influx transporter important for imatinib uptake) [112]. It is not clear whether one of these mechanisms is more important than the others or whether they act in concert.

BMS-214662 is a very promising farnesyl transferase inhibitor (FTI) that has been shown to induce selective apoptosis of CML LSCs in vitro, both as a single agent and in combination with imatinib or dasatinib, with little effect on normal HSCs [113,114]. BMS-214662 potently induced apoptosis of both proliferating and quiescent CML stem/progenitor cells with <1% recovery of Ph+ LTC-Cs whether harboring wild-type or mutant BCR–ABL [114]. Its mechanism of action involves induction of apoptosis through activation of caspases 3 and 8, inhibition of the mitogen activated protein kinase pathway, the inhibitor of apoptosis protein 1, nuclear factor (NF) κB, and the inducible nitric oxide synthase, in addition to the traditional mechanism of action of FTIs, which involves RAS inhibition, 113,114]. This agent offers the potential for eradication of CP~CML, and a clinical trial is forthcoming [113,114].

The LIC in Ph ALL (for further discussion of LSCs in ALL please see below) seems to be considerably more differentiated than an HSC since the phenotype is almost exclusively of B lineage. Castor et al. [115] showed that p210BCR–ABL cells originated from HSCs, whereas p190BCR–ABL cells originated from B-cell progenitors. Their data suggested that p210BCR–ABL and p190BCR–ABL represent largely distinct biologic and clinical entities.

Leukemic stem cells in acute myeloid leukemia

The primitive AML leukemic subpopulation capable of leukemic repopulation into NOD/SCID mice has a distinct immunophenotype that is quite similar across the different AML subtypes except acute promyelocytic leukemia, in which direct assessment of the frequency and immunophenotype of LSCs has not been achieved [9,11,76,116]. While heterogeneity exists between individual patients, most AML LSCs share the CD34+/CD38−/CD71−/HLA-DR−phenotype with normal HSCs (Table 1.1) [9,76]. Nevertheless, several cell-surface antigens were reported to have differential expression between LSCs and normal HSCs (Table 1.2). AML LSCs, for example, differ from normal HSCs by the lack of Thy-1 (CD90) and c-Kit (CD117) expression, and by the expression of IL-3 receptor a chain (CD123) and the novel antigen C-type lectin-like molecule 1 (CLL-1) [117–121]. Additionally, most AML LSCs express CD33, but some normal HSCs may express this antigen as well [81]. Finally, CD44 was reported to be overexpressed in both AML and CML LSCs in comparison with normal HSCs [122,123]. In summary, while AML LSCs share some characteristics with HSCs, they differ from HSCs by others, suggesting that these differences should play a role in targeting the LSCs while sparing the non-malignant HSCs.

Table 1.2 Key features that distinguish acute myeloid leukemia leukemic stem cells from normal, non-malignant hematopoietic stem cells.

NF-κB upregulation PI3K upregualtion PTEN depletion Lack of expression of c-Kit (CD117), Thy-1 (CD90) Expression of IL-3 receptor alpha chain (CD123), C-type lectin-like molecule-1 (CLL-1), and VLA-4 Possible overexpression of CD44 and CD33

Leukemic stem cells in acute lymphoblastic leukemia

The presence of an LSC in ALL is a controversial subject. Similar to AML, ALL is a heterogeneous disease with clinically and genetically different subtypes. Rearrangements of the T-cell receptor (TcR) or the immunoglobulin heavy chain (IgH) genes support the theory that T and B-lineage ALL originate in cells already committed to the T or B-cell lineages [124–126].

The development of ALL from a committed B-cell progenitor was suggested by Castor et al. [115], who demonstrated that primary ETV6-R UNX1 (previously TEL-AML1; t[12;21][p13;q22]) fusions and subsequent leukemic transformations were targeted to committed B-cell progenitors. Similarly, Kong et al. [127] used a novel in vivo xenotransplantation model in which purified CD34+/CD38+/CD19+, CD34+/CD38−/CD19+, andCD34+/CD38−/CD19−cells from pediatric patients with B-ALL were injected into sublethally irradiated newborn NOD/SCID/IL2ry(null) mice. The authors found that both CD34t/CD38+/CD19+ and CD34+/CD38−/CD19+ cells initiate BtALL in primary recipients, whereas the recipients of CD34+/CD38−/CD10−/CD19−cells showed normal human hematopoietic repopulation. It was noted that the extent of leukemic infiltration into the spleen, liver, and kidney was similar between the recipients transplanted with CD34+/CD38+/CD19+ cells and those transplanted with CD34+/CD38−/CD19+ cells. In addition, transplantation of CD34t /CD38+/CD19+ cells resulted in the development of BtALL in secondary recipients, demonstrating self-renewal capacity. The authors concluded that the identification of CD34+CD38+CD19+ self-renewing B-ALL cells proposes a hierarchy of LICs distinct from that of AML.

Over the last few years there have been several reports indicating that in some ALL subtypes, the leukemic blasts may arise from a more phenotypically primitive HSC rather than a lymphoid-lineage committed progenitor. For example, cytogenetically aberrant cells have been shown to be present in the CD34+/CD38−/CD33−/CD19−bone marrow compartment in children with Btcell precursor-ALL (BCP-ALL) indicating that ALL blasts in some patients may evolve from a precursor compartment [128]. Similarly, Cox et al. [129,130] have demonstrated the presence of cells capable of long-term proliferation in the CD34+/CD10−/CD19−subfraction of BCPtALL samples, and in the CD34+/CD4−and CD34+/CD7−subfractions of T-ALL pediatric samples. This suggested that a more primitive phenotype was the target for leukemic transformation in these cases. Finally, le Viseur et al. [131] showed that in pediatric ALL, blasts at different stages of immu-nophenotypic maturation have stem-cell properties. The investigators transplanted human leukemic bone marrow into NOD/SCID mice, and found that blasts representative of all of the different maturational stages (CD34,/CD19−, CD34+/CD19+, and CD34−/CD19+) were able to reconstitute and re-establish the complete leukemic phenotype in vivo, This represents the potential malleability or plasticity of LSCs, that is the ability of more differentiated leukemia cells to reacquire the LSC characteristics [132]. It is clear that the ALL story is not as straightforward as the AML story.

Targeting the leukemic stem cell

Many genetic mutations, molecular aberrations, and signaling pathway disruptions have been reported to drive leukemogenesis in AML, but little is known on how these abnormalities affect the LSCs [11]. Given the many similarities between LSCs and HSCs, and the central role that LSCs play in leukemia maintenance, studies have focused on identifying pathways of proliferation, self-renewal, and survival that are differentially active in LSCs rather than HSCs. The clear goal is to introduce drugs that are capable of selective targeting pathways that maintain LSCs while sparing normal HSCs (see Table 1.3 for a summary of the targeted approaches discussed below).

The importance of minimal residual disease (MRD) in causing relapse after achieving complete remission (CR) in leukemia is well established [133]. For example, van Rhenen et al. recently showed that phenotypically defined LSCs could be detected in patients with AML who achieved CR, and that the frequency of these LSCs at diagnosis correlates with MRD after chemotherapy and with survival [120,121]. These observations suggest that MRD can be detected at the stem-cell level, which may allow for development of a therapy targeted at these residual cells.

Table 1.3 Agents that target leukemia stem cells and spare the hematopoietic stem cells.

Target Agent NF-κB MG-132   Bortezomib   DMAPT   TDZD-8 PI3K Rapamycin   ET-18-OCH Farnesyl transferase BMS-214662 Mechanism unknown Hyperthermia Niche AMD3100   Anti-CD44

Targeting leukemic stem-cell survival pathways

The identification of survival pathways that are preferentially overexpressed in LSCs suggest that differential activation of apoptosis mechanisms in LSCs should be possible, and strategies specifically modulating these pathways are likely to be effective in eradication of LSCs [8,12,79,134–136].

Among the more characterized dysfunctional signal transduction pathways that control cell survival in LSCs are NF-κB and phosphatidyl-inositide-3 kinase (PI3K). While unstimulated normal HSCs do not express NF-κB, AML LSCs exhibit readily detectable NF-κB levels [8]. NF-κB is a transcription factor that often has anti-apoptotic effects which render survival advantage to malignant cells [137,138]. Taking advantage of NF-κB, Guzman et al. [8] demonstrated that inhibiting NF-κB with the proteasome inhibitor MG-132 contributed to the rapid induction of death of LSCs, whereas normal HSCs were minimally affected, if at all. The agents targeting NF-κB would be of particular appeal since they are expected to have a quite favorable therapeutic index given the almost undetectable levels of NF-κB in normal HSCs [8,11]. The same group [79] combined MG-132 and the anthracycline idarubicin; this combination induced a rapid and extensive apoptosis of the LSC population while leaving normal HSCs viable. Molecular genetic studies demonstrated that inhibition of NF-κB and activation of p53-regulated genes contributed to LSC apoptosis [79].

Parthenolide (PTL), a recently described agent, was found to preferentially induce robust apoptosis in primary human AML cells, AML progenitor cells, and AML stem-cell populations, while sparing normal HSCs [135]. The molecular mechanism of PTL-mediated apoptosis was shown to be strongly associated with NF-κB inhibition and p53 activation [135]. However, PTL has relatively poor pharmacologic properties that limit its potential clinical use [10,136]. Consequently, the investigators [136] developed an oral analog of PTL, dimethylamino-parthenolide (DMAPT), which was demonstrated to induce rapid death of primary human LSCs from both myeloid and lymphoid leukemias, combined with high cytotoxicity to bulk leukemic cell populations. Another exciting, recently described agent is TDZD-8 (4-benzyl-2-methyl-1,2,4-thiadiazolid-ine-3,5-dione), a molecule that belongs to a family of compounds with glycogen synthase kinase-3β and NF-κB inhibitory activity [10,139]. TDZD-8 selectively induces the death of primary myeloid LSCs and leukemia progenitor cells without causing significant harm to the normal HSCs [139]. In xenotransplantation assays, TDZD-8 inhibited the engraftment of AML LSCs, but did not significantly inhibit the engraftment of normal HSCs [139]. In addition to killing the myeloid LSCs, this agent exhibited potent cytotoxic activity against the bulk blast populations from both lymphoid and myeloid malignancies [139].

Several groups have studied the targeting the PI3K pathway [140–142]. Xu et al. [140] reported that the PI3K pathway was constitutively activated in AML cells. The authors also demonstrated that inhibition of either of two important downstream mediator proteins of PI3K, namely Akt/protein kinase B (PΚB) or the mammalian target of rapamycin (mTOR), leads to decreased survival of the malignant cells [140]. The same group [141] demonstrated that the combination of rapamycin, an mTOR inhibitor, and etoposide resulted in significant cytotoxicty against AML blasts and reduced LSC survival. Finally, Wierenga et al. [142] showed that LSCs have an increased hyperther-mic sensitivity compared with their normal counterparts and that this difference can be further increased in combination with ET-18-OCH, another known PI3K inhibitor.

Phosphatase and tensin homolog deleted on chromosome 10 (PTEN) was first described as a tumor suppressor located on chromosome 10q23, and was found to be an important suppressor of the PI3K/Akt/mTOR pathway [143–146]. Yilmaz et al. [147] demonstrated that conditionally deleting the PTEN gene in mice led to a myeloproliferative disease (MPD) within days that evolved into transplantable leukemias within weeks. PTEN gene deletion led to HSC depletion, preventing these cells from stably reconstituting irradiated mice, indicating that, in contrast to LSCs, normal HSCs were unable to maintain themselves without PTEN [147]. These effects were mostly mediated by mTOR as they were inhibited by rapamycin, which not only depleted LSCs in mice with established leukemia, but also restored normal HSC function [147].

Based on these observations, Jordan and Guzman [12] proposed a model in which the preferential induction of apoptosis in the LSC population, while sparing the normal HSC population, is achieved by a combination of specific types of cellular stress (e.g. hyperthermia or genotoxic stress caused by idarubicin) and inhibition of survival signals (by NF-κB or PI3K inhibitors).

Targeting leukemic stem cell surface antigens and microenvironment

Another appealing mechanism in the attempts to eradicate the LSCs is the use of monoclonal antibodies directed against some of the surface antigens that are differentially expressed on LSCs. CD44 is a transmembrane glycoprotein that has been implicated in cell homing and migration, and was demonstrated to be expressed on the leukemic blasts from most patients with AML [148]. Recent reports have identified the CD44 receptor as a necessary factor in hematopoietic mobilization/homing of both AML and CML LSCs [125,149]. Anti-CD44 monoclonal antibodies were found to reverse myeloid differentiation blockage in monocytic and non-monocytic AML and to induce apoptosis of AML blasts, 148]. Later, Jin et al. [122] used the same monoclonal antibody (H90 anti-CD44) in NOD/SCID mice transplanted with human AML. This resulted in marked reduction of leukemic repopulation and in absence of leukemia in serially transplanted mice, indicating that AML LSCs were directly targeted [122]. In addition, these findings indicated that homing is crucial for AML LSCs to maintain their stem-cell functions, suggesting that the leukemogenic process does not completely abrogate niche dependence for AML LSCs [122]. The authors [122] pointed out that AML LSCs were more sensitive to anti-CD44-induced eradication than HSCs, probably as a result of the greater abundance of CD44 on the surface of LSCs as compared with normal HSCs.

SDF-1, produced by stromal cells in the bone marrow niche, and its receptor, CXCR4, expressed on normal HSCs, both play an important role in the survival, homing, and engraftment of human HSCs and their retention in the bone marrow niche [150,151]. Similarly, SDF-1-CXCR4 interactions were found to participate in the migration, repopulation, survival, and development of AML LSCs in the bone marrow, therefore neutralizing the SDF-1-CXCR4 axis can act as a potential treatment for AML [152]. Some experimental data suggested that AML LSCs are more sensitive to anti-CXCR4 treatment than normal human HSCs [152]. AMD3100, a selective antagonist of SDF-1 by binding to its receptor CXCR4, was found to inhibit the transmigration of AML blasts, the outgrowth of leukemic CFUs, and the ability of SDF-1 to induce engraftment of AML cells [152,153]. Future studies will determine the selectivity of AMD3100 to LSCs compared with normal HSCs.

Another important surface antigen, CD33, is expressed on normal immature cells of the myeloid lineage, on many AML blasts, and LSCs, regardless of the AML subtype [81]. Gemtuzumab ozogamicin (Mylotarg®, Wyeth, Maidenhead, UK), an anti-CD33 antibody conjugated with calicheamicin with proven activity against AML blasts, has been postulated to cause some direct killing of CD33+, AML LSCs, possibly explaining its efficacy in some AML cases where the majority of the blasts were CD33−, 81,154]. However, despite relatively good efficacy and certain specificity for LSCs compared with normal HSCs, the drug does not work uniformly in all patients and has significant side-effects [154]. The occurrence of prolonged cytopenias in some patients treated with Gemtucumab suggested that CD33 might be expressed on some normal HSCs [81].