Tài liệu HEMATOPOIETIC GROWTH FACTORS IN ONCOLOGY BASIC SCIENCE AND CLINICAL THERAPEUTICS pptx

Edited by
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Graham J. Lieschke, MBBS, PhD, FRACP
CANCER DRUG DISCOVERY AND DEVELOPMENT
CANCER DRUG DISCOVERY AND DEVELOPMENT
Hematopoietic
Growth Factors
in Oncology
Basic Science and Clinical
Therapeutics
Hematopoietic
Growth Factors
in Oncology
Basic Science and Clinical
Therapeutics
Edited by
George Morstyn, MBBS, PhD,FRACP
MaryAnn Foote, PhD
Graham J. Lieschke, MBBS, PhD, FRACP
H
EMATOPOIETIC
G
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ACTORS IN
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ISCOVERY AND
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HUMANA PRESS
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Edited by
GEORGE MORSTYN, MBBS, PhD, FRACP
Amgen, Australia, Pty. Ltd.
Monash University, Victoria, Australia
MARYANN FOOTE, PhD
Amgen Inc., Thousand Oaks, CA
GRAHAM J. LIESCHKE, MBBS, PhD, FRACP
Ludwig Institute for Cancer Research, Royal Melbourne Hospital
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Hematopoietic growth factors in oncology basic science and clinical therapeutics / edited by George Morstyn, MaryAnn Foote,
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Foote, MaryAnn. III. Lieschke, Graham J. IV. Series.
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2003017466
v
PREFACE
Several hematopoietic growth factors (HGFs) have achieved widespread clinical
application. In the United States alone, more than US $5 billion per year of the health care
budget is spent on these factors. The first patients were treated with recombinant human
erythropoietin (rHuEPO, epoetin alfa, Epogen
®
) in 1985 and the first patients received
recombinant methionyl human granulocyte colony-stimulating factor (r-metHuG-CSF,
filgrastim, Neupogen
®
) or recombinant human granulocyte-macrophage colony-
stimulating factor (rHuGM-CSF, sargramostim, Leukine
®
or Prokine
®
) in 1986. The first
agent promoting platelet recovery was formally approved in 1997 (recombinant human
interleukin-11 [rHuIL-11], oprelvekin, Neumega
®
). In 2002, sustained-duration
derivative r-metHuG-CSF (pegfilgrastim, Neulasta
®
) was formally approved for clinical
use. Likewise in 2002, a new erythropoietic protein (darbepoetin alfa, Aranesp

®
) with a
longer serum half-life and increased biologic activity compared with rHuEPO was
formally approved for clinical use. Pharmaceutical forms of several other agents have
been assessed in clinical studies but are yet to find a widespread clinical utility or niche
(e.g., stem cell factor, thrombopoietin, interleukin-3, colony-stimulating factor-1
[macrophage colony-stimulating factor]). The efficacy of the marketed agents to
ameliorate the complications of cancer and the side effects of chemotherapy has led to
their broad clinical application; however, their cost has led to efforts to ensure that their
use is focused onto clinically appropriate indications. Hematopoietic Growth Factors in
Oncology: Basic Science and Clinical Therapeutics is a further contribution to this
endeavor.
HGFs are produced in the bone marrow, kidney, brain, and fetal liver by a wide variety
of cells, and they exhibit exquisite selectivity of action dependent on the expression of
specific receptors by target cells. The factors stimulate proliferation and differentiation,
have antiapoptotic effects, and enhance the function of mature cells.
Hematopoietic Growth Factors in Oncology: Basic Science and Clinical Therapeutics
introduces the molecular basis for the activity of HGFs and discusses their specific role
in the treatment of various malignancies. The clinical application of these agents continues
to expand because of their benefits and relative lack of side effects. Chemotherapy
remains a mainstay of cancer treatment despite the introduction of newer therapeutic
approaches, and so there remains a need to optimize chemotherapy-related supportive
care. In the chapters presented from a systematic oncology perspective, we hope to help
oncologists treating patients with particular tumor types to make informed evidence-
based decisions about adjunctive HGF therapy within disease-focused treatment
regimens. The volume also describes progress in various areas of basic science that may
lead to further advances in hemopoietic cell regulation. There are also sections on the
utility of growth factors in infectious disease settings such as AIDS.
Some notes about the preparation of the book are in order. Because of the nature of
scientific inquiry, the editors have allowed overlap in chapter topics and varying opinions.

We encouraged the authors to be comprehensive regarding the available HGFs, and we
actively sought chapters covering the currently available agents. The opinions expressed
vi Preface
are not necessarily the opinions of the editors or the publisher. Great care has been taken
to ensure the integrity of the references and drug doses, but the package inserts of any drug
should always be consulted before administration.
Readers will realize that many scientists and clinicians worldwide have worked and
continue to work in the fields of basic and applied research of HGFs. We would, however,
like to recognize one of our colleagues, Dr. Dora M. Menchaca. Dora joined Amgen in
July 1991 as a clinical manager and was a close colleague of MaryAnn Foote and George
Morstyn. She was involved in the design and conduct of many clinical trials, including
the use of filgrastim in the setting of acute myeloid leukemia and myelodysplastic
syndromes; the use of stem cell factor in many clinical settings; the use of megakaryocyte
growth and development factor for the treatment of thrombocytopenia and for harvesting
peripheral blood progenitor cells; and several other molecules. Dora was an advocate for
patients enrolled in clinical trials and worked diligently to help get new therapeutic
molecules registered and marketed to help patients worldwide. Dora was returning on an
early morning flight after a meeting with the FDA and was on American Airlines flight
77 that was hijacked and crashed into the US Pentagon on September 11, 2001. We still
mourn the loss of this dedicated scientist and continue to miss her enthusiasm, her
intelligence, her warm and caring personality, and her infectious smile and laughing eyes.
We dedicate this book to Dora.
George Morstyn,
MBBS, PhD, FRACP
MaryAnn Foote, PhD
Graham J. Lieschke, MBBS, PhD, FRACP
vii
CONTENTS
Preface v
Contributors ix

Part I. Basic Research
1 Introduction to Hematopoietic Growth Factors: A General Overview 3
George Morstyn and MaryAnn Foote
2Animal Models of Hematopoietic Growth Factor Perturbations
in Physiology and Pathology 11
Graham J. Lieschke
3 The Jak/Stat Pathway of Cytokine Signaling 45
Ben A. Croker and Nicos A. Nicola
4Small-Molecule and Peptide Agonists: A Literature Review 65
Ellen G. Laber and C. Glenn Begley
Part II. Hematopoietic Growth Factors
5Granulocyte Colony-Stimulating Factor 83
Graham Molineux
6 Erythropoietic Factors: Clinical Pharmacology and Pharmacokinetics 97
Steven Elliott, Anne C. Heatherington, and MaryAnn Foote
7 Thrombopoietin Factors 125
David J. Kuter
8 Stem Cell Factor and Its Receptor, c-Kit 153
Keith E. Langley
9Hematopoietic Growth Factors: Preclinical Studies of Myeloid
and Immune Reconstitution 185
Ann M. Farese and Thomas J. MacVittie
Part III. Use of Hematopoietic Growth Factors in Oncology
10 Commentary on the ASCO and ESMO Evidence-Based Clinical Practice
Guidelines for the Use of Hematopoietic Colony-Stimulating Factors 211
Richard M. Fox
11 Neutropenia and the Problem of Fever and Infection
in Patients With Cancer 219
David C. Dale
12 Thrombocytopenia and Platelet Transfusions in Patients With Cancer 235

Lawrence T. Goodnough
viii Contents
13 Hematopoietic Growth Factors in Lung Cancer 249
Johan F. Vansteenkiste and Christophe A. Dooms
14 Role of Hematopoietic Growth Factors As Adjuncts
to the Treatment of Hodgkin’s and Non-Hodgkin’s Lymphomas 275
Marcie R. Tomblyn and Jane N. Winter
15 Use of Granulocyte Growth Factors in Breast Cancer 285
Eric D. Mininberg and Frankie Ann Holmes
16 Role of Cytokines in the Management
of Chronic Lymphocytic Leukemia 311
Carol Ann Long
17 Hematopoietic Growth Factor Therapy
for Myelodysplastic Syndromes and Aplastic Anemia 333
Jason Gotlib and Peter L. Greenberg
18 Use of Hematopoietic Growth Factors in AIDS-Related Malignancies 357
MaryAnn Foote
Part IV. Safety and Economic Implications
19 The Safety of Hematopoietic Growth Factors 375
Roy E. Smith and Barbara C. Good
20 Long-Term Safety of Filgrastim in Chronic Neutropenias 395
Karl Welte
21 Economics of Hematopoietic Growth Factors 409
Gary H. Lyman and Nicole M. Kuderer
Part V. Future Directions
22 Potential for Hematopoietic Growth Factor Antagonists in Oncology 447
Hayley S. Ramshaw, Timothy R. Hercus, Ian N. Olver,
and Angel F. Lopez
Acronyms and Selected Abbreviations 467
Index 475

CONTRIBUTORS
ix
C. GLENN BEGLEY, MBBS, PhD, FRACP, FRCPath, FRCPA • Senior Director, Basic Research
in Hematology, Amgen Inc., Thousand Oaks, CA
B
EN A. CROKER, BSC • Cancer and Hematology Division, The Walter and Eliza Hall
Institute of Medical Research, Victoria, Australia
D
AVID C. DALE, MD • Professor, Department of Medicine, University of Washington,
Seattle, WA
C
HRISTOPHE A. DOOMS, MD • Respiratory Oncology Unit (Pulmonology), University
Hospital Gasthuisberg, Leuven, Belgium
S
TEVEN ELLIOTT, PhD • Fellow, Hematology Department, Amgen Inc., Thousand Oaks,
CA
A
NN M. FARESE, MS, MT (ASCP) • Greenebaum Cancer Center, University of Maryland,
Baltimore, Maryland
M
ARYANN FOOTE, PhD • Director, Medical Writing, Amgen Inc., Thousand Oaks, CA
R
ICHARD M. FOX, MB, PhD, FRACP • Department of Medical Oncology, Royal Melbourne
Hospital, Melbourne, Australia
B
ARBARA C. GOOD, PhD • Director, Scientific Publications, National Surgical Adjuvant
Breast and Bowel Project, Pittsburgh, PA
L
AWRENCE T. GOODNOUGH, MD • Professor, Departments of Medicine and Pathology
and Immunology, Washington University School of Medicine, St. Louis, MO

J
ASON GOTLIB, MD • Clinical Research Fellow, Hematology Division, Stanford
University Medical Center, Stanford, CA
P
ETER L. GREENBERG, MD • Professor, Department of Medicine, Stanford University
Medical Center, Stanford, CA; Head, Hematology, VA Palo Alto Health Care
System, Palo Alto, CA
A
NNE C. HEATHERINGTON, PhD • Research Scientist, Department of Pharmacokinetics
and Drug Metabolism, Amgen Inc., Thousand Oaks, CA
T
IMOTHY R. HERCUS, PhD • Cytokine Receptor Laboratory, Hanson Institute, Adelaide,
Australia
F
RANKIE ANN HOLMES, MD, FACP • US Oncology; Texas Oncology, Houston, TX
N
ICOLE M. KUDERER, MD • James P. Wilmot Cancer Center, University of Rochester
Medical Center, Rochester, NY
D
AVID J. KUTER, MD, DPhil • Chief of Hematology, Massachusetts General Hospital,
and Associate Professor of Medicine, Harvard Medical School, Boston, MA
E
LLEN G. LABER, PhD • Senior Medical Writer, Medical Writing, Amgen Inc., Thousand
Oaks, CA
x Contributors
KEITH E. LANGLEY, PhD • Principal Medical Writer, Medical Writing, Amgen Inc.,
Thousand Oaks, CA
G
RAHAM J. LIESCHKE, MBBS, PhD, FRACP • Assistant Member and Laboratory Head,
Cytokine Biology Laboratory, Ludwig Institute for Cancer Research, Melbourne

Tumour Biology Branch, Parkville, Victoria, Australia; Clinical Hematologist,
Department of Clinical Hematology and Medical Oncology, The Royal Melbourne
Hospital, Parkville, Victoria, Australia
C
AROL ANN LONG, PhD • Newbury Park, CA
A
NGEL F. LOPEZ, MD, PhD • Cytokine Receptor Laboratory, Hanson Institute, Adelaide,
Australia
G
ARY H. LYMAN, MD, MPH, FRCP • James P. Wilmot Cancer Center, University
of Rochester Medical Center, Rochester, NY
T
HOMAS J. MACVITTIE, PhD • Greenebaum Cancer Center, University of Maryland,
Baltimore, MD
E
RIC D. MININBERG, MD • MD Anderson Cancer Center, University of Texas,
Houston, TX
G
RAHAM MOLINEUX, PhD • Associate Director, Hematology Department, Amgen Inc.,
Thousand Oaks, CA
G
EORGE MORSTYN, MBBS, PhD, FRACP • Special Advisor, Development, Amgen Inc.,
Thousand Oaks, CA; Department of Microbiology, Monash University, Clayton,
Victoria, Australia
N
ICOS A. NICOLA, PhD • Professor, Molecular Hematology, and Assistant Director,
The Walter and Eliza Hall Institute of Medical Research, Parkville, Victoria,
Australia
I
AN N. OLVER, MD, PhD • Clinical Director, Royal Adelaide Hospital Cancer Center,

Adelaide, Australia
H
AYLEY S. RAMSHAW, PhD • Cytokine Receptor Laboratory, Hanson Institute,
Adelaide, Australia
R
OY E. SMITH, MD • Director, Medical Affairs and Medical Oversight, National
Surgical Adjuvant Breast and Bowel Project, Pittsburgh, PA
M
ARCIE R. TOMBLYN, MD • Fellow, Division of Hematology/Oncology, Feinberg
School of Medicine, Northwestern University; Robert H. Lurie Comprehensive
Cancer Center, Chicago, IL
J
OHAN F. VANSTEENKISTE, MD, PhD • Respiratory Oncology Unit (Pulmonology),
University Hospital Gasthuisberg, Leuven, Belgium
K
ARL WELTE, MD, PhD • Professor of Pediatrics, Hannover Medical School; Head,
Department of Pediatric Hematology and Oncology, Children Hospital,
Hannover, Germany
J
ANE N. WINTER, MD • Professor of Medicine, Division of Hematology/Oncology,
Feinberg School of Medicine, Northwestern University; Robert H. Lurie
Comprehensive Cancer Center, Chicago, IL
I
B
ASIC
R
ESEARCH

1. INTRODUCTION
A complex, inter-related, and multistep process called hematopoiesis controls the

production and development of specific bone marrow cells from immature precursor
cells to functional mature blood cells. The earliest cells are stem cells and are multipo-
tential and able to self-renew. Up to 10
11
blood cells are produced in an adult human
each day. The proliferation of precursor cells, the commitment to one lineage, the mat-
uration of these cells into mature cells, and the survival of hematopoietic cells require
the presence of specific growth factors, which act individually and in various combina-
tions in complex feedback mechanisms. The hematopoietic growth factors (HGFs)
stimulate cell division, differentiation, maturation, and survival, convert the dividing
cells into a population of terminally differentiated functional cells (Fig. 1), and in some
cases also activate their mature functions (1–4). Because the literature concerning
every aspect of HGF discovery, cloning, function, and clinical use is burgeoning, in this
chapter, we mention only a few of the most significant works and cite general refer-
ences where possible.
These factors are important for both maintaining the steady state and mediating
responses to infection. More than 20 HGFs have been identified. The properties of some
are described in Table 1. The structure and function of these growth factors have been
characterized and the gene that encodes for each factor identified and cloned. Several
HGFs are commercially available as recombinant human forms, and they have utility in
3
From: Cancer Drug Discovery and Development
Hematopoietic Growth Factors in Oncology: Basic Science and Clinical Therapeutics
Edited by: G. Morstyn, M. A. Foote, and G. J. Lieschke © Humana Press Inc., Totowa, NJ
1
Introduction to Hematopoietic
Growth Factors
A General Overview
George Morstyn, MBBS,
P

h
D
, FRACP
and MaryAnn Foote,
P
h
D
C
ONTENTS
I
NTRODUCTION
D
ISCOVERY OF
H
EMATOPOIETIC
G
ROWTH
F
ACTORS
C
LINICAL
D
EVELOPMENT OF
H
EMATOPOIETIC
G
ROWTH
F
ACTORS
F

UTURE
D
IRECTIONS
R
EFERENCES
clinical practice. These factors include the recombinant forms of two myeloid hematopoi-
etic growth factors, granulocyte colony-stimulating factor (G-CSF) and granulocyte-
macrophage colony-stimulating factor (GM-CSF); erythropoietin (EPO), the red cell
factor; stem cell factor (SCF), an early-acting HGF; and thrombopoietin (TPO) and
interleukin-11 (IL-11), platelet factors. T lymphocytes, monocytes/macrophages, fibrob-
lasts, and endothelial cells are the important cellular sources of most HGFs, excluding
EPO and TPO (5,6). EPO is produced primarily by the adult kidney (7–9), and TPO is
produced in the liver and in the kidney (10–12).
G-CSF (recombinant products: filgrastim, lenograstim, pegfilgrastim) maintains neu-
trophil production during steady-state conditions and increases production of neutrophils
during acute situations, such as infections (13). Recombinant human G-CSF (rHuG-CSF)
reduces neutrophil maturation time from 5 d to 1 d, leading to the rapid release of mature
neutrophils from the bone marrow into the blood (14). rHuG-CSF also increases the cir-
culating half-life of neutrophils and enhances chemotaxis and superoxide production (15).
Pegfilgrastim is a sustained-duration formulation of rHuG-CSF that has been developed
by covalent attachment of a polyethylene glycol molecule to the filgrastim molecule (16).
GM-CSF (recombinant products: molgramostim, sargramostim) is locally active and
remains at the site of infection to recruit and activate neutrophils (13). Like G-CSF,
4Part I / Basic Research
Fig. 1. Hematopoietic tree. EPO, erythropoietin; G-CSF, granulocyte colony-stimulating factor;
GM-CSF, granulocyte-macrophage colony-stimulating factor; mGDF, megakaryocyte growth and
development factor; SCF, stem cell factor; TPO, thrombopoietin. (Courtesy of Amgen, Thousand
Oaks, CA.)
GM-CSF and rHuGM-CSFs stimulate the proliferation, differentiation, and activation
of mature neutrophils and enhance superoxide production, phagocytosis, and intracel-

lular killing (17–19). GM-CSF and rHuGM-CSF, unlike G-CSF, stimulate the prolifer-
ation, differentiation, and activation of mature monocytes/macrophages (18).
Erythropoietic factors (recombinant products: epoetin alfa, epoetin beta, darbepoetin
alfa) increase red blood cell counts by causing committed erythroid progenitor cells to
proliferate and differentiate into normoblasts, nucleated precursors in the erythropoietic
lineage (20–22). Tissue hypoxia resulting from anemia induces the kidney to increase its
production of EPO by a magnitude of a 100-fold or more. EPO stimulates the production
of erythroid precursor cells and therefore increases the red blood cell content and oxy-
gen-carrying capacity of blood. Anemia in patients with cancer can be owing to direct or
indirect effects of the malignancy on the marrow, or as a complication of myelotoxic
chemotherapy or radiotherapy. The onset is often insidious, and some of the clinical
effects of anemia have in the past been wrongly attributed to the underlying malignancy.
Darbepoetin alfa is another erythropoietic factor that has an extended half-life owing to
its increased number of sialic acid-containing carbohydrate molecules (20–21).
Chapter 1 / Introduction 5
Table 1
Hematopoietic Growth Factors and Their Activities
Factor MW (kDa) Abbreviation Target cell Actions
Erythropoietin 34–39 EPO Erythoid progenitors Increase red blood
(BFU-E, CFU-E) count
Granulocyte colony- 18 G-CSF Granulocyte progenitors; Increase ANC
stimulating factor mature neutrophils
(G-CFC)
Granulocyte- 14–35 GM-CSF Granulocyte, macrophage Increase neutrophil,
macrophage colony- progenitors (GM-CFC) eosinophil, and
stimulating factor eosinophil progenitors monocyte count
Interleukin-3 28 IL-3 Multipotential Increase
progenitor cells hematopoietic and
lymphoid cells
Interleukin-5 40–50 IL-5 Eosinophil progenitor Increase eosinophils

cells
Interleukin-7 25 IL-7 Early B and T cells Stimulate B
and T cells
Interleukin-11 23 IL 11 Early hemopoietic Increase platelet
progenitors, count
megakaryocytes
Monocyte colony- 40–70 M-CSF Monocyte progenitor Increase monocytes;
stimulating factor cells but decrease in
platelet count
Thrombopoietin 35 TPO Stem cells, Increase platelet
megakaryocyte and count
erythroid progenitors
A
BBREVIATIONS
:ANC, absolute neutrophil count; BFU-E, blast-forming unit-erythroid; CFU-E, colony-
forming unit-erythroid; G-CFC, granulocyte colony-forming cell; GM-CFC, granulocyte-macrophage
colony-forming cells.
SCF (recombinant product: ancestim) is an early-acting hematopoietic growth factor
that stimulates the proliferation of primitive hematopoietic and nonhematopoietic cells
(2,23). In vitro, SCF has minimal effect on hematopoietic progenitor cells, but it syner-
gistically increases the activity of other HGFs, such as G-CSF, GM-CSF, and EPO.
SCF and recombinant human (rHu)SCF stimulate generation of dendritic cells in vitro
and mast cells in vivo, and rHuSCF has been used in combination with rHuG-CSF to
increase progenitor cell mobilization (24).
Thrombopoietic factors (recombinant products: rHuTPO, pegylated megakaryocyte
growth and development factor [PEG-rHuMGDF], and rHuIL-11 [oprelvekin]) stimu-
late the production of megakaryocyte precursors, megakaryocytes, and platelets
(10,25,26). IL-11 has many effects on multiple tissues and can interact with IL-3, TPO,
or SCF. Endogeous TPO values are increased in patients with thrombocytopenia; it is
very effective at increasing the platelet count. TPO is thought to be the major regulator

of platelet production.
2. DISCOVERY OF HEMATOPOIETIC GROWTH FACTORS
The study of hematopoiesis was greatly facilitated in the mid-1960s when tech-
niques for studying hematopoietic stem cells and progenitor cells in vivo (27) and in
clonal culture (28,29) were developed. It was clear that the proliferation and devel-
opment of these cells was dependent on growth factors. In cultures, these growth
factors were provided by serum, conditioned medium, or cell underlayers. The
growth factors present in these sources were called colony-stimulating factors
(CSFs) (30).
In the 1970s and early 1980s, many of the growth factors were purified. It was rec-
ognized that several growth factors acted on the granulocyte lineage, including G-CSF,
GM-CSF, and IL-3. At the time, it was a great challenge to achieve purity because
these factors were present at very low concentrations. By the mid-1980s, it was appar-
ent that the criterion for purity was when a single protein sequence could be obtained
from the pure preparation and the gene encoding this sequence could be cloned and
expressed to produce the same protein.
Once recombinant human forms of HGF were produced by recombinant DNA tech-
nology in large amounts, the focus shifted to studying the pharmacology and clinical
effects. The focus of laboratory research changed from identifying additional growth
factors to studying their mode of action. Site-directed mutagenesis and other tech-
niques allowed the structure of the growth factors, their binding to receptors, and their
intracellular signaling to be defined in detail. The study of HGFs in vivo was greatly
facilitated by gene knockout as well as by gene overexpression studies. These areas are
reviewed in later chapters of this book.
When the clinical development of recombinant human forms of HGF was initiated, a
common belief was that since they were natural regulators of hematopoiesis, they
would be well tolerated. Laboratory studies indicated redundancy in the effects of these
HGF and also showed that their maximal effects were produced when they were used
in combination with each other. Clinical studies appeared to indicate that the factors
most selective on one cell lineage (such as recombinant forms of G-CSF, EPO, and

TPO) were better tolerated than broadly acting factors. Combinations of recombinant
growth factors in the clinic were not extensively tested, but combinations of rHuG-CSF
and rHuEPO, rHuG-CSF and rHuGM-CSF, and rHuSCF and rHuG-CSF have been
6Part I / Basic Research
studied, with beneficial effects reported in some cases, for example, rHuG-CSF and
rHuSCF for progenitor cell mobilization (31).
Other chapters in this volume review the pharmacology of HGF in normal and spe-
cial populations, as well as in relation to some of the most common cancers. The use of
HGF in the oncology sector revolutionized the treatment of patients. Further work
should offer more innovative methods for the treatment of patients with cancer.
3. CLINICAL DEVELOPMENT OF HEMATOPOIETIC
GROWTH FACTORS
The clinical development of recombinant forms of HGF were directed by an extensive
understanding of the biologic effects of these factors. The human gene encoding EPO
was cloned in 1983 (22), and clinical development of epoetin alfa began soon after. Ini-
tial studies were focused on patients with an endogenous EPO deficiency, such as
patients with severe chronic renal failure receiving dialysis. The effects of epoetin alfa
were apparent in the first dose levels with an increase in hemoglobin concentration and
hematocrit. A reduction in the requirement for red blood cell transfusions was ultimately
proved in the pivotal phase 3 trial. Further studies focused on defining a safe rate of rise
in hemoglobin and an appropriate target; however, a conservative target rather than nor-
malization of hematocrit was initially approved in the dialysis setting. In patients with
underlying heart disease, the safety and benefits of correction to a normal hematocrit are
still under investigation almost 20 years after the initiation of clinical studies (32,33).
For the development of rHuEPO in the setting of cancer, a major challenge was to
recognize the benefits of maintaining hematocrits at higher volumes than had been the
previous practice when only blood transfusions were available. A second challenge was
to obtain sufficient information on the reduction in the need for transfusions and
improvement in quality of life to justify the cost of therapy with rHuEPO. Recent
guidelines developed by the American Society of Clinical Oncology (ASCO) and the

American Society of Hematology (ASH) address the optimum use of rHuEPO. They
do not yet evaluate the impact of darbepoetin alfa on this field.
Another set of blood factors studied were the factors stimulating the platelet lineage.
The development of factors stimulating platelets was impaired by several observations.
The first was that the factors available, rHuIL-11 and rHuTPO, act on increasing the
number and ploidy of megakaryoctes but do not stimulate platelet shedding. Therefore,
the increase in platelet counts is slow. Second, IL-11 was pleiotropic and was associ-
ated with significant adverse events. The recombinant thrombopoietins (rHuTPO,
PEG-rHuMGDF) tested in the clinic induced antibodies that inhibited their own activ-
ity and the activity of TPO, leading to prolonged thrombocytopenia.
HGFs such as SCF and flt3 ligand were also tested for activity on multipotential
stem cells. rHuSCF enhanced progenitor cell mobilization induced by rHuG-CSF. The
problems of severe stem cell deficiency states such as aplastic anemia remain unsolved,
and rHuSCF was associated with side effects related to mast cell activation. Neverthe-
less, rHuSCF received marketing approval in Australia, New Zealand, and Canada.
4. FUTURE DIRECTIONS
A number of chapters in this volume focus on current research that could lead to
future clinical applications. Croker and Nicola review the pathways of cytokine signal-
ing. These pathways when aberrant could be involved in oncogenesis, and thus an
Chapter 1 / Introduction 7
understanding of signaling may lead to new targets for the development of therapeu-
tics. It is also possible that for some applications, targeting of the intracellular signaling
pathway will lead to more selective and orally active stimulants of hematopoietic cells.
It may be possible to stimulate selectively early cells, mature cells, or mature cell func-
tion. Identifying more effective ways of reconstituting the marrow of patients with
severe aplastic anemia or other forms of aplasia or dysplasia, would also be an impor-
tant clinical objective. The development of antagonists for the treatment of inflamma-
tory states or some types of leukemia may prove valuable.
An important area for future development arises from structure and function analy-
ses of HGF. For example, at one time, it was not considered possible to modify the pro-

tein backbone of the HGF and thereby improve their pharmacologic properties,
because of the risk of a loss of efficacy or the induction of immunogenicity. The recent
regulatory approval of darbepoetin alfa, however, shows that changing the amino acid
sequence of rHuEPO to produce a hyperglycosylated molecule results in prolonged
half-life and maintained efficacy without inducing neutralizing antibodies. It is likely
that further modifications will be explored.
We are currently able to stimulate the neutrophil and erythroid lineage effectively;
however, Kuter reviews the data demonstrating that although recombinant TPO is
effective in preclinical and clinical studies to increase platelet counts, it does not work
rapidly enough to prevent platelet transfusions now that the trigger for these is as low
as 10 × 10
9
/L. In addition, the immunogenicity of the first-generation molecules needs
to be overcome. Much work needs to be done to define better both the clinical need,
and the optimal properties of a platelet stimulant.
Although in retrospect the clinical success of recombinant human HGF may seem to
have been easily achieved, the chapter by Farese and MacVittie describes preclinical
studies of chimeric growth factor receptor agonists that have not transitioned success-
fully to the clinic.
New anticancer agents are continually being developed, and these need to be inte-
grated with current chemotherapy and radiotherapy regimens and hematologic support.
A major area of investigation discussed by Fox and by Lyman and Kuderer is the effect
on cost of introducing new agents. A positive development has been the objective review
of data and the production of treatment guidelines by societies such as ASCO and ASH.
Although such guidelines need to be updated, they have become an objective standard by
which to define therapy for individual patients. The field of HGF has developed in 40
years from an in vitro cell culture phenomenon to an established field providing benefit
to patients. The specificity of the late-acting factors and the ability to measure blood cell
counts as surrogate endpoints greatly facilitated dose finding and clinical development.
Science now moves more quickly, and there is an expectation that basic discoveries

can be applied clinically in 2–3 years. Perhaps an understanding of how the biologic
effects of HGF were applied clinically will be useful for the successful development of
other areas of translational medicine.
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8Part I / Basic Research
3. Lieschke GJ, Grail D, Hodgson G, et al. Mice lacking granolocyte colony-stimulating factor have
chronic neutropenia, granulocyte and macrophage progenitor cell deficiency, and impaired neutrophil
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4. Du XX, Williams DA. Interleukin-11: review of molecular, cell biology, and clinical use. Blood 1997;
89:3897–3908.
5. Vellenga E, Rambaldi A, Ernst TJ, Ostapovicz D, Griffin JD. Independent regulation of M-CSF and G-
CSF gene expression in human monocytes. Blood 1988; 71:1529–1532.
6. Groopman JE, Molina JM, Scadden DT. Hematopoietic growth factors. Biology and clinical applica-
tions. N Engl J Med 1989; 321:1449–1459.
7. Jacobson LO, Goldwasser E, Fried W, et al. Role of the kidney in erythropoiesis. Nature 1957;
179:633–634.
8. Mirand EA, Prentice TC. Presence of plasma erythropoietin in hypoxic rats with and without kidneys
or spleen. Proc Soc Exp Biol Med 1957; 96:49–51.
9. Erslev AJ. Erythropoietin. N Engl J Med 1991; 324:1339–1344.
10. Bartley TD, Bogenberger J, Hunt P, et al. Identification and cloning of a megakaryocyte growth and
development factor that is a ligand for the cytokine receptor Mpl. Cell 1994; 77:1117–1124.
11. de Sauvage FJ, Hass PE, Spencer SD, et al. Stimulation of megakaryocytopoiesis and thrombopoiesis
by the c-Mpl ligand. Nature 1994; 369:533–538.
12. Foster DC, Sprecher CA, Grant FJ, et al. Human thrombopoietin: gene structure, cDNA sequence,
expression, and chromosomal localization. Proc Natl Acad Sci USA 1994; 91:13023–13027.

13. Cebon J, Layton JE, Maher D, Morstyn G. Endogenous haemopoietic growth factors in neutropenia
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14. Lord BI, Bronchud MH, Owens S, et al. The kinetics of human granulopoiesis following treatment
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15. Welte K, Gabrilove J, Bronchud MH, Platzer E, Morstyn G. Filgrastim (r-met Hu G-CSF): the first 10
years. Blood 1996; 88:1907–1929.
16. Molineux G, Kinstler O, Briddell B, et al. A new form of filgrastim with sustained duration in vivo and
enhanced ability to mobilize PBPC in both mice and humans. Exp Hematol 1999; 27:1724–1734.
17. Nemunaitis J, Rabinowe SN, Singer JW, et al. Recombinant granulocyte-macrophage colony-stimulat-
ing factor after autologous bone marrow transplantation for lymphoid cancer. N Engl J Med 1991;
324:1773–1778.
18. Armitage JO. Emerging applications of recombinant human granulocyte-macrophage colony stimulat-
ing factor. Blood 1998; 92:4491–4508.
19. Angel JB, High K, Rhame F, et al. Phase III study of granulocyte-macrophage colony-stimulating fac-
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14:387–395.
20. Egrie JC, Dwyer E, Browne JK, Hitz A, Lykos MA. Darbepoetin alfa has a longer circulating half-life
and greater in vivo potency than recombinant human erythropoietin. Exp Hematol 2003; 31:290–299.
21. Elliott S, Lorenzini T, Asher S, Aoki K, et al. Enhancement of therapeutic protein in vivo activities
through glycoengineering. Nat Biotechnol 2003; 21:414–421.
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Acad Sci USA 1985; 82:7580–7584.
23. Martin FH, Suggs SV, Langley KE, et al. Primary structure and functional expression of rat and human
stem cell factor DNAs. Cell 1990; 63:203–211.
24. Broudy VC. Stem cell factor and hematopoiesis. Blood 1997; 90:1345–1364.
25. Lok S, Kaushansky K, Holly RD, et al. Cloning and expression of murine thrombopoietin CDNA and
stimulation of platelet production in vivo. Nature 1994; 369:565–568.
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Chapter 1 / Introduction 9
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339:584–590.
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10 Part I / Basic Research
11
From: Cancer Drug Discovery and Development
Hematopoietic Growth Factors in Oncology: Basic Science and Clinical Therapeutics
Edited by: G. Morstyn, M. A. Foote, and G. J. Lieschke © Humana Press Inc., Totowa, NJ
1. INTRODUCTION
The clinical use of hematopoietic growth factors (HGFs) is built on nearly 20 years
of in vitro studies followed by preclinical animal studies. These laboratory and animal
studies, undertaken before first use in humans, provided the basis for expectations of
what the biologic effects in humans would be.

Reflecting the available technologies, the initial animal studies primarily evaluated
the in vivo effects of factor excess after administration of factors to various animal
species and included transgenic models, particularly when the supply of factor itself
was limiting or issues of chronic factor exposure were to be addressed. With the devel-
opment of genetic technologies to disrupt genes in mice selectively, animal models of
2
Animal Models of Hematopoietic
Growth Factor Perturbations
in Physiology and Pathology
Graham J. Lieschke, MBBS, PhD, FRACP
C
ONTENTS
I
NTRODUCTION
A
NIMAL
M
ODELS OF
H
EMATOPOIETIC
G
ROWTH
F
ACTOR
D
EFICIENCY
A
NIMAL
M
ODELS OF

H
EMATOPOIETIC
G
ROWTH
F
ACTOR
E
XCESS
A
NIMAL
M
ODELS OF
H
EMATOPOIETIC
G
ROWTH
F
ACTOR
A
DMINISTRATION
A
FTER
C
HEMOTHERAPY OR
R
ADIOTHERAPY
A
NIMAL
M
ODELS

E
VALUATING
H
EMATOPOIETIC
G
ROWTH
F
ACTOR
S
IGNALING IN
P
ATHOLOGIC
P
ROCESSES
C
ONCLUSIONS
A
CKNOWLEDGMENTS
R
EFERENCES
factor deficiency were developed in the 1990s. These models were particularly useful
for defining the indispensable and physiologic roles of factors and their multicompo-
nent receptors. Increasing sophistication of the technologies for transgenesis and tar-
geted gene modification enabled generation of animal models with inducible and
tissue-specific genetic modifications that included not only gene disruptions but also
truncations, point mutations, and gene replacement. Animal models incorporating
these latter changes were usually generated to test hypotheses regarding the role of spe-
cific lesions in gene function or disease pathogenesis. This range of approaches collec-
tively contributes to the preclinical evaluation of new biologic agents or to the
modeling of particular disease processes so that pathogenic mechanisms can be better

understood and therapeutic strategies can be assessed.
This chapter presents a descriptive overview of animal models of perturbed amounts
of HGF, with a particular emphasis on genetic models, and focuses on those factors in
clinical use: erythropoietin (EPO), granulocyte colony-stimulating factor (G-CSF),
granulocyte-macrophage colony-stimulating factor (GM-CSF), and interleukin (IL)-
11. Since diseases are often acquired and not infrequently present somatic rather than
germline genetic lesions, animal models with acquired rather than congenital perturba-
tions of HGF concentrations and signaling are also described.
2. ANIMAL MODELS OF HEMATOPOIETIC GROWTH
FACTOR DEFICIENCY
Early models of induced factor deficiency relied on immunologic mechanisms to
neutralize factor activity. The ability to disrupt individual genes selectively by gene tar-
geting provided a powerful method of generating mice with deficiencies of either
selected ligands, receptors, or downstream-signaling molecules. Such engineered defi-
ciencies have usually been designed to be absolute and are life-long, providing insight
into the cumulative effects of nonredundant roles of the absent gene product. This
genetic approach has been pre-eminent in defining the essential physiologic role of var-
ious factors. However, animal models with less-than-total factor deficiency have been
generated using other methodologies, both before the gene targeting era and more
recently. These models offer several advantages: although they may not result in
absolute factor deficiency, they offer flexibilities including inducibility, reversibility,
and nonlethality. A new approach, not yet applied to studying HGF, is the use of RNA
interference (1). Various experimental approaches to factor ablation are listed in Table
1 with some comparative relative advantages and disadvantages.
2.1. Spontaneously Arising Mutants
With Hematopoietic Factor Deficiency
The first durable models of HGF deficiency resulted fortuitously from sponta-
neously arising or induced mutations in the genes encoding growth factors or their
receptors. The two examples of this are mutants deficient in stem cell factor (SCF) and
colony-stimulating factor-1 (CSF-1; also known as macrophage colony-stimulating

factor [M-CSF]). These models presented prototypes for the models of other factor
deficiencies generated by gene targeting.
The steel (Sl) mutation arose in 1956 (2). In its most severe form, animals homozy-
gous for the original Sl allele die before birth with macrocytic anemia, absent germ cell
12 Part 1 / Basic Research
development, and defective skin pigment cell development (2,3). Heterozygous Sl/+
animals have diluted hair pigment and mild macrocytic anemia and are fertile. Other
alleles were noted that resulted in less severe phenotypes in homozygous animals, e.g.,
Sl
d
(steel-Dickie), for which homozygotes are viable but have severe anemia, sterility,
and a black-eyed/white-coated phenotype. A full list of characterized Sl alleles is found
in Peters et al. (4), and an overview of the major phenotypic subtleties is described in
Russell (5). When in 1990 the ligand for the cellular proto-oncogene c-kit was cloned
by several groups (6–8), it was shown to be the product of the steel locus on mouse
chromosome 10 (7,8). The steel gene product was a previously unknown growth factor
that, among other functions, acts as a hematopoietic CSF in vitro (8,9) and was desig-
nated variously as kit-ligand, steel factor, mast cell growth factor, or stem cell factor.
Chapter 2 / Animal Models 13
Table 1
Comparison of Various Approaches to Impair Hematopoietic Growth Factor Action
or Reduce Factor Production
Method of factor Durability
deficiency or
Degree of
Technical difficulty
impairment
impairment
In vitro In vivo and issues Comments
Neutralization Transient Yes Yes If antibody available, Standard approach to demonstrate

by antibody Incomplete straightforward specificity of factor effects in vitro;
systemic administration may not
achieve neutralization in all
body compartments and local sites
Antisense RNA Transient Yes No Oligonucleotide Specificity must be demonstrated
from transfected Incomplete stability and
construct potential toxicity
Antisense RNA Permanent Yes Yes Similar to other Expression of transgene may be
from stable Incomplete transgenic projects variable in different tissues
transgene leading to variable degrees of
factor impairment
Administration Transient Yes Yes Specific antagonist Antagonism at level of receptor
of antagonist Incomplete must be developed most appropriate to study factor
and validated physiology
Induced innate Transient in No Yes Requires immunogenic No control over induced
autoimmunity long term form of factor immune response, which
to factor Incomplete may be nonspecific
or nonneutralizing
Natural or Permanent No Yes Capricious and Structure of disrupted allele
randomly- Complete unreliable must be characterized; may
induced or incomplete involve several adjacent or
mutation separate loci.
Targeted gene Permanent Yes Yes Difficult multistage Total factor deficiency must still
disruption Complete process requiring be formally proven at the
several mouse protein level
generations
Targeted gene Under Yes Yes Difficult multistage More flexible than germline
modification experimental process requiring gene disruption—can be
or inducible control several mouse controlled in both time
disruption Incomplete generations and anatomical

location
RNA interference Depends on Yes Yes Techniques Not yet applied to growth factor
methodology still under models
Incomplete development in
mammalian systems
Applicable
for use
Mice with spontaneously arising mutations at the dominant spotting W locus have long
been known (10,11); this locus was only relatively recently molecularly characterized
as being the SCF receptor c-kit (12).
The osteopetrosis (op) mutant arose in 1970 and was characterized in 1976 (13). The
mutation was characterized as a base insertion generating a premature stop codon in
the Csfm (M-CSF) gene on mouse chromosome 3 (14). op/op mice have severe
osteopetrosis with disordered bone remodeling and osteoclast deficiency (13,15),
marked but not absolute monocyte and tissue macrophage deficiency (16–21), impaired
female fertility (22), a lactation defect (23), and reduced survival (13). Mice lacking
the CSF-1 receptor were generated by gene targeting that largely replicate the ligand-
deficiency phenotype (24).
A challenge in interpreting the phenotype of naturally occurring mutations is to
know whether the factor deficiency is absolute or partial. This question can be
addressed by combining knowledge of necessary functional domains, gene expression
analysis, and determination of amounts of bioactive and immunoreactive protein. Com-
parison of mice lacking ligand with those lacking the corresponding receptor can be
helpful. Some spontaneous mutations involve deletions, which may potentially encom-
pass several genes, thus potentially confounding the phenotype.
2.2. Erythropoietin
Early studies used serum from rabbits immunized with concentrated EPO-containing
urine to achieve neutralization of endogenous EPO in recipient rabbits (25–27). Pas-
sively immunized rabbits and mice developed anemia. In a more recent study involving
active rather than passive immunization, monkeys treated with a human (Hu)GM-CSF-

EPO fusion moiety developed anti-EPO (but not anti-GM-CSF) antibodies (Ab), with
resultant anemia (28) (Table 2).
14 Part 1 / Basic Research
Table 2
Animal Models of Reduced Eythropoietin Levels or Signaling
Method of reduced Major phenotypic
Animal erythropoietin signaling consequences Reference
Rabbit Passive immunization with serum containing Anemia 26, 27
presumed anti-EPO antibodies
Mice Passive immunization with serum containing Anemia 25
presumed anti-EPO antibodies
Monkey Immunization during GM-CSF EPO hybrid Anemia 28
protein administration resulting in anti-EPO
antibodies crossreacting with simian EPO
Mice Targeted disruption of EPOR gene Death in utero at E13.5 29, 30
Ventricular hypoplasia
Vascular abnormalities
Mice Targeted disruption of EPOR receptor gene Death in utero at E13.5 29, 30
Ventricular hypoplasia
Vascular abnormalities
Haploinsufficiency
A
BBREVIATIONS
:E,embryonic day; EPO, erythropoietin; EPOR, EPO receptor; GM-CSF, granulocyte-
macrophage colony-stimulating factor.