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Long-term follow-up on breast cancer patients transplanted with ex vivo expanded peripheral blood CD34+ cells

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Long-term follow-up on breast cancer patients transplanted with ex vivo expanded peripheral blood CD34+ cells
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Candia, Julie Ruth
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Subjects / Keywords:
Breast -- Cancer -- Treatment ( lcsh )
Breast -- Cancer -- Patients -- Rehabilitation ( lcsh )
Hematopoietic stem cells -- Transplantation ( lcsh )
Breast -- Cancer -- Patients -- Rehabilitation ( fast )
Breast -- Cancer -- Treatment ( fast )
Hematopoietic stem cells -- Transplantation ( fast )
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bibliography ( marcgt )
theses ( marcgt )
non-fiction ( marcgt )

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Includes bibliographical references (leaves 133-139).
Statement of Responsibility:
by Julie Ruth Candia.

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University of Florida
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ocm47848338
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LD1190.L44 2001 .C36 ( lcc )

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Full Text
LONG-TERM FOLLOW-UP ON BREAST CANCER PATIENTS
TRANSPLANTED WITH EX VIVO EXPANDED
PERIPHERAL BLOOD CD34+ CELLS
by
Julie Ruth Candia
B.S., Colorado State University, 1988
A thesis submitted to the
University of Colorado at Denver
in partial fulfillment
of the requirements for the degree of
Master of Basic Science
2001


2001 by Julie Ruth Candia
All rights reserved.


This thesis for the Master of Basic Science
degree by
Julie Ruth Candia
has been approved
by
Ian KTfoicNiece
j/30101
Date


Candia, Julie Ruth (M.B.S., Applied Science)
Long-term Follow-up on Breast Cancer Patients Transplanted with Ex Vivo
Expanded Peripheral Blood CD34+ Cells
Thesis directed by Ian K. McNiece, PhD
ABSTRACT
Patients with various stages of breast cancer were administered growth
factor(s) and underwent leukapheresis to collect peripheral blood
progenitor/stem cells for transfusion following high-dose chemotherapy.
Transplantation is required to establish hematopoiesis. The patients in this
study were consented and enrolled consecutively into two cohorts. Cohort 1
consisted of 10 patients who were transfused with both CD34 antigen
(CD34+) selected cells which were expanded ex vivo and with unexpanded
CD34+ selected cells. The first cohort of patients established the safety of
administering expanded CD34+ cells. Cohort 2 consisted of 11 patients who
were transfused only with the CD34 selected cells that were expanded.
Short-term results indicate that all patients in both cohorts demonstrated
rapid engraftment of neutrophils posttransplant, and that neutrophil
engraftment occurs faster with expanded CD34+ cells than unexpanded cells.
IV


The long-term engrafitment kinetics were investigated in the present study by
measuring peripheral blood counts. The median absolute neutrophil count
for Cohort 2 at long-term follow-up was 3.8 X 10A3/wL, and the median
platelet count was 178 X 10A3/mmA3. These results indicate that CD34+
expanded cells can provide durable long-term engrafitment.
This abstract accurately represents the content of the candidates thesis. I
recommend its publication.
Signed
Ian Kf^/IcNiece
v


DEDICATION
I dedicate this thesis to:
Candace B. Malet, RN, BSN (University of Colorado Health Sciences Center),
for her unfaltering understanding, patience, support, and friendship.
My family (Vina Candia, Robert Gonzalez; Julian, Jackie, and Sarah Candia) for
their love and support, and for tolerating hearing the word thesis for two years.


ACKNOWLEDGEMENTS
Dr. Ian K. McNiece (University of Colorado Health Sciences Center), my
advisor, for his patience, understanding, guidance, and knowledge.
Dr. Elizabeth J. Shpall and the Bone Marrow Transplant Program Research
Team for giving me the opportunity to participate in research at the University of
Colorado Health Sciences Center.
Dr. Zenas Hartvigson for his genuine guidance, support, and enthusiasm in the
Master of Basic Science Program at the University of Colorado at Denver.
Dr. William Briggs for his patience and participation as a committee member at
the University of Colorado at Denver.
Dr. Anna Baron and Chan Zeng at the University of Colorado Health Sciences
Center for their assistance with statistical analysis.
Tom Porterfield for his friendship, support, and patience.
Glenda Robles for her lasting friendship, understanding, and support.
Raymond Kingery for his computer program assistance
Julian Candia for his computer program assistance; and for having a cute little
daughter, Sarah, to distract me from my studies, and to make me laugh and
smile.


CONTENTS
Figures.......................................................... xiii
Tables .......................................................... xiv
CHAPTER
1. INTRODUCTION .............................................. 1
Problem Statement ....................................... 1
Purpose of the Study .....................................2
2. HEMATOPOIESIS ..............................................4
Hematopoietic Progenitor and Stem Cells ..................4
Characteristics of Mature Peripheral Blood Cells .........8
Bone Marrow Structure and Function...................... 10
Granulocytopoiesis Maturation Sequence ................. 16
Myeloblast .......................................... 17
Promyelocyte ........................................ 17
Myelocyte............................................ 18
Metamyelocyte........................................ 18
Neutrophilic Band ................................... 18
Segmented Neutrophil
19


3. HISTORY OF PERIPHERAL BLOOD PROGENITOR/STEM
CELL TRANSPLANTATION ..................................21
Cellular Transplants ...............................21
Early Animal Models ................................22
Hematopoietic Stem Cells in Human Peripheral Blood .24
Attempts to Quantitate and Identify the
Progenitor/Stem Cell ...............................26
CD34+ Antigen ......................................27
Optimization of Peripheral Blood
Progenitor/Stem Cell Collection ....................27
4. CD34+CELLS ...........................................30
Discovery of the CD34 Antigen ......................30
5. GRANULOCYTE-COLONY STIMULATING FACTOR ................33
Function ...........................................33
Structure ..........................................34
Mobilization of Progenitor/Stem Cells ..............34
6. EX VIVO EXPANSION AND SELECTION OF HEMATOPOIETIC
PROGENITOR/STEM CELLS .................................37
Ex Vivo Expansion and Cytokines ....................37
Reduced Tumor Cell Content .........................39
Decreased Neutropenia ..............................41
7. HIGH-DOSE CHEMOTHERAPY................................43
IX


Purpose
43
Characteristics of Oncology Drugs ..........................44
Cyclophosphamide (Cytoxan) ..............................44
Cisplatin (Platinol) ....................................45
Carmustine (BCNU) .......................................45
Docetaxel (Taxotere) ....................................46
Carboplatin (Paraplatin).................................46
Melphalan ...............................................47
Paclitaxel (Taxol).......................................47
8. BREAST CANCER ................................................49
Types of Breast Cancer .....................................49
Breast Cancer Risk..........................................51
9. METHODS ......................................................53
Patients ...................................................53
Leukapheresis...............................................55
Materials and Supplies ..................................55
Procedure ...............................................57
CD34+ Selection.............................................59
Materials and Supplies ..................................59
Procedure ...............................................59
x


Ex Vivo Expansion ........................................60
Materials and Supplies ................................60
Procedure .............................................60
High-Dose Chemotherapy ...................................61
Materials and Supplies ................................62
Procedure .............................................62
Peripheral Progenitor/Stem Cell Reinfusion ..............63
Materials and Procedure ...............................64
Complications .........................................64
10. RESULTS....................................................66
Long-term Follow-up on Engraftment.......................66
Disease-Free Survival ...................................69
Overall Survival ........................................70
Patient Health Status ...................................71
11. DISCUSSION ................................................86
Results on Early Neutrophil Engraftment .................86
Platelet Recovery Time ..................................87
CD34+ Counts ............................................88
Tumor Content ...........................................89
Long-term Engraftment....................................90
xi


12. CONCLUSION
93
APPENDIX ........................................................95
A. Appendix A: Follow-up Peripheral Blood Counts
for Cohorts 1 and 2 ...................................95
B. Appendix B: Thesis Defense Slide Presentation ....... 126
REFERENCES .................................................... 133
xii


FIGURES
Figure
2.1 Schematic View of Hematopoiesis .......................................5
2.2 Scheme of Hematopoietic Cell Lines ....................................7
2.3 Fractal Pattern of Bone Marrow ...................................... 12
2.4 Graphic Presentation of Bone Marrow Tissue .......................... 14
2.5 Granulocytopoiesis ...................................................20
4.1 Schematic of Cell Surface Antigens ...................................32
9.1 Methods Schema for Cohort Patients .................................. 54
10.1.1- 10.1.5 Cohort 1: Absolute Neutrophil Count vs Date ........... 75-77
10.2.1- 10.2.6 Cohort 2: Absolute Neutrophil Count vs Date ........... 78-80
10.3.1- 10.3.2 Cohort 1: Platelet Count vs Date ..........................81
10.4.1- 10.4.2 Cohort 2: Platelet Count vs Date ..........................82
10.5.1- 10.5.2 Graphic Overlay of White Blood Cell Count (WBC) vs Days
Post-Peripheral Blood Progenitor Cell Transplant
for Cohort 1 and 2 ........................................83
10.6.1- 10.6.2 Graphic Overlay of Absolute Neutrophil Count (ANC) vs Days
Post-Peripheral Blood Progenitor Cell Transplant
for Cohort 1 and Cohort 2 ................................84
10.7.1- 10.7.2 Graphic Overlay of Platelet Count (PLT) vs Days
Post-Peripheral Blood Progenitor Cell Transplant for Cohort 1
and Cohort 2...............................................85
xiii


TABLES
Table
10.1 Peripheral Blood Counts at Last Follow-up .........................73
10.2 Mean, Median, and Range for Peripheral Blood
Counts at Last Follow-up .........................................73
10.3 Disease-Free/Overall Survival for Cohorts .........................74
xiv


CHAPTER 1
INTRODUCTION
Problem Statement
Ex vivo expanded progenitor cells used for hematopoietic support provide rapid
engraftment of neutrophils in breast cancer patients receiving high-dose
chemotherapy, as demonstrated by researchers at the University of Colorado Health
Sciences Center (UCHSC) (McNiece et al., 2000). In particular, specific
progenitor/stem cells called CD34+ cells are specifically and magnetically selected
from other hematopoietic cells, and subsequently expanded in culture (McNiece et
al., 1999). Richel et al. (2000) have shown that CD34+ cells isolated using
magnetically activated cell sorting techniques provide safe and rapid engraftment in
breast cancer patients. In addition, recent studies suggest that expansion and/or
selection of CD34+ progenitor cells significantly decreases, and may even eliminate,
tumor cells in the final product to be infused for transplantation (Voso et al., 1999;
Lundell, Vredenburgh, Tyer, DeSombre, & Smith, 1998). Studies have shown that
there is consistently a significant loss in the number of CD34+ progenitor/stem cells
to be infused using only CD34+ selection techniques (Richel et al., 2000;
Martin-Henao et al., 2000). However, ex vivo expansion following CD34+ selection


ameliorates the loss of CD34+ selected cells. Thus, ex vivo expansion accomplishes
several important functions including the possibility of increasing the number of
engrafting cells to be infused, and decreasing, or possibly eliminating, contamination
of the transplant product with tumor cells. Expanded cells have the potential of
reducing the period that patients are thrombocytopenic (experience a low platelet
count), which can lead to bleeding complications. In addition, the use of CD34+
expanded cells can shorten the time of neutropenia, and decrease the amount of time
that the patient is immunocompromised following high-dose transplant therapy.
Thus, the possibility of using expanded progenitor/stem cells for transplantation
offers substantial benefits for the patient.
Purpose of the Study
The purpose of the study is to evaluate the long-term engraftment kinetics of
growth factor-mobilized autologous peripheral blood progenitor stem cells (PBPC)
which have been immunomagnetically CD34 antigen selected and expanded ex vivo,
and are subsequently infused into breast cancer patients following high-dose
chemotherapy. Patients were followed for up to two years after receiving their
PBPC transplant. The rate and duration of sustained hematopoietic reconstitution
produced is measured by peripheral blood counts performed on the patients
posttransplantation. The data that is of particular interest are white blood counts,
absolute neutrophil counts, and platelet counts in the follow-up of hematopoietic
9


engraftment. Long-term follow-up is important in evaluating sustained engraftment
of expanded progenitor/stem cells, and may indicate whether or not the most
primitive stem cells are maintained in expansion culture. Without durable
engraftment, patients may become aplastic and are unable to maintain repopulation
of the blood cells which are essential for life. In addition, the patients disease and
health status are important to the study.


CHAPTER 2
HEMATOPOIESIS
The dynamic process of blood cell production and development is called
hematopoiesis. Mature blood cells, such as leukocytes (white cells), erythrocytes
(red cells), and thrombocytes (platelets) must be maintained at adequate levels in the
body, and are very important to an individuals health. Leukocytes are comprised of
granulocytes, lymphocytes, and monocytes. Granulocytes are important in fighting
infections, lymphocytes play an important role in the immune response, and
monocytes engulf debris and pathogens. Erythrocytes provide a variety of
important functions in the body, such as oxygen transport and excretion of carbon
dioxide, and delivery of nutrients to other cells. Thrombocytes are important in the
mechanism of blood clotting termed hemostasis (Bell & Hughes, 1997).
Hematopoietic Progenitor and Stem Cells
All of the above cells are derived from undifferentiated somatic cells called
hematopoietic stem cells, which are morphologically indistinct. Pluripotent stem
cells are not only able to self-replicate, but they can also differentiate into either the
multipotential (myeloid) stem cell or the lymphoid stem cell. The lymphoid stem cell
line only produces the T and B lymphocytes. The multipotential (myeloid) stem cell
4


is capable of giving rise to several different cell lines. Multipotential cells which
become committed to a particular cell line are called progenitor cells. These various
blood cells lines are granulocytes, erythrocytes, monocytes, and megakaryocytes.
Each cell line has morphologically distinct characteristics and functions (Bell &
Hughes, 1997; Hoffman, 1995). Stem cells and progenitor cells express an
identifying antigen on the cell surface known as the CD34 antigen. These cells are
termed CD34+ cells (see Figure 2.1).
Figure 2.1 Schematic view of hematopoiesis.
Hematopoiesis
Stem Cells
Long-term
Progenitor Cells] Short-term
Precursor Cells
Mature Cells
5


Committed progenitors can only proliferate and mature along a single pathway
(Naeim, 1998). A committed progenitor can be grown in culture to form colonies of
cells called colony-forming cell (CFC) or colony-forming units (CFU). In vitro
growth of committed progenitors requires specific colony-stimulating factors.
CFU-GM is the progenitor cell for neutrophilic granulocytes and monocytes. The
CFU-GM becomes even more specific and becomes either a CFU-G, which
differentiates into a neutrophil, or a CFU-M, which differentiates into a monocyte.
CFU-Eo originate from the multipotential myeloid cell line and differentiate along
the eosinophilic pathway. CFU-Bas originate from the myeloid cell line to become
basophils. Erythrocytes (red blood cells) will be produced from burst-forming
unit-erythroid (BFU-E). Megakaryocytes will grow from cells which have
committed to the CFU-Meg line and will eventually become mature thrombocytes
(platelets) (Naeim, 1998) (see Figure 2.2).
6


1130 ES
Figure 2.2 Schema of hematopoietic cell lines: showing
development of different cell lines into mature cells. Schema
does not detail complete maturation sequence.
7


Studies propose a stochastic model for self-replication and differentiation of
hematopoietic cells. This model is based on differentiation as a random event and
the ability of the stem cell to choose from several options at the time of
commitment. A slight change in initial conditions may result in thousands of mature
cells formed from one stem cell. Stem cells are influenced towards self-replication
or differentiation based on complex interactions with extrinsic and intrinsic factors
(Naeim, 1998).
Characteristics of Mature Peripheral Blood Cells
Granulocytes consist of cells called neutrophils, eosinophils, and basophils.
These cells are named for their characteristic granules found in the cytoplasm.
These granules and other morphologic characteristics become evident when the cell
is stained with Wrights stain. The granulocytes are very similar in size,
approximately 14 micrometers (m). (Bell & Hughes, 1997). However, each type of
granulocyte is unique and can easily be distinguished under the microscope after
staining.
Neutrophils are the most numerous cell in the peripheral blood, comprising
50-70% of the total white cells. They have a very important function in fighting
bacterial infections. The cytoplasm of these cells stains light pink with many tiny,
pink granules The nucleus is usually segmented, and stains dark purple with
Wrights stain. Eosinophils function in allergic reactions. The granules of this cell
8


are large and round, and they stain a very distinct bright orange. The nucleus is
bilobed and stains dark purple. Basophils fight parasitic infections. They have very
large, coarse granules, which stain very dark purple to black. Basophils and
eosinophils comprise the smallest percentage of cells in normal adult peripheral
blood (0-6% total) (Bell & Hughes, 1997).
Normal adult blood contains 20% to 40% lymphocytes and is the next most
numerous cell. Lymphocytes consists of T and B immune cells; both are derived
from a common lymphoid progenitor cell. There are two different types of T cells,
Helper T cells and Cytotoxic T cells. When activated, Helper T cells produce
polypeptides which help to activate B cells and Cytotoxic T cells. B cells are
responsible for producing the five different antibodies (IgM, IgG, IgA, IgD, IgE). T
and B cells are difficult to distinguish morphologically, but are easily distinguished
by cell surface markers. Lymphocytes are smaller than the granulocytes, usually 7 to
10 wm The dense, round nucleus is large compared to the cytoplasm and stains dark
purple. The cytoplasm usually has round margins and stains a fairly dark blue.
However, lymphocytes that are reacting to appropriate antigenic stimuli will cause
the cell to enlarge and the nucleus to become irregularly shaped and less dense (Bell
& Hughes, 1997).
Monocytes comprise about 2% to 9% of the leukocytes in normal adult blood.
Monocytes are known for their ability to phagocytize various debris, such as cell
9


fragments, bacteria, or fungi. The monocyte is larger than a neutrophil, ranging in
size from 15 to 18 wm. The abundant cytoplasm stains grayish-blue and may be
irregular-shaped. Also, the cytoplasm contains numerous small reddish granules.
The nucleus is fairly large and is usually kidney-shaped and deeply folded. The
distinctive monocyte nucleus resembles a brain because of the indentations or
convolutions (Bell & Hughes, 1997). Monocytes also migrate from the bloodstream
to the tissues and are then called macrophages. Macrophages perform the same
functions in the tissues as in the bloodstream.
Thrombocytes (platelets) are the smallest cells in the peripheral blood, varying in
size from 1 to 4 wm. However, this is the mature form of the cell; the thrombocyte
descends from the largest hematopoietic cell in the bone marrow called the
megakaryocyte. When a megakaryocyte has completed maturation., the cytoplasm
fragments to form several thousand individual thrombocytes (or platelets). The
platelet has a very important role in the blood clotting cascade for preventing
bleeding with injury to vessels. Platelets do not have a nucleus since they are
cytoplasmic fragments. Platelets stain purple-blue and have small reddish granules
(Bell & Hughes, 1997). They resemble tiny little cotton balls.
Bone Marrow Structure and Function
All of the various cell lines follow a sequence of maturation. Maturation and
production of hematopoietic cells in the adult begins in the bone marrow, called
10


medullary hematopoiesis. Medullary hematopoiesis is usually a steady state process.
In the adult, hematopoietic cells are produced in the sternum, ribs, pelvis, vertebrae,
and skull (Bell & Hughes, 1997). Extramedullary hematopoiesis occurs in the
spleen, liver, thymus, and lymph nodes, usually in response to a demand for
increased blood cells. Hematopoietic stem cells in the bone marrow respond to a
variety of regulatory cytokines that cause them to become committed progenitor
cells. The progenitor cell will become committed to one particular cell line, which
has morphologically distinct maturation characteristics. The granulocytic cell line
will be used later to illustrate the maturation process (Bell & Hughes, 1997).
Bone marrow (BM) is derived from mesenchymal tissue (Naeim, 1998) which is
a diffuse network of tissue derived from the embryonic mesoderm (Anderson, K., &
Andeson, L., 1998). The spatial organization of the bone marrow is a fractal
geometry which consists of many spaces and surfaces that wrinkle, branch, and fold


Figure 23 Fractal pattern of bone marrow: shows
pattern of cell distribution in the bone marrow.
12


The hematopoietic component supported by the microenvironment of the bone
marrow constantly cycles through proliferation and differentiation. The
microenvironment is composed of stromal cells which include endothelial cells,
macrophages, fibroblasts, adipocytes, osteoblasts, and osteoclasts (Naeim, 1998;
Link, 2000). The stromal cells are distributed randomly and unevenly to form the
extracellular matrix (ECM) of the bone marrow. Hematopoietic cells are supported
by a fine reticulin mesh which is made up of subtances produced by the stromal
cells, such as collagen, fibronectin, laminin, thrombospondin, hemonectin,
proteoglycans, glycoproteins, and glycosaminoglycans (Naeim, 1998; Link 2000)
(see Figure 2.4).


Figure 2.4 Graphic presentation of bone marrow tissue:
(A) arteriole (B) endothelial cells (C) central sinus
(E) areas of erythropoiesis (F) fat tissue (G) areas of
granulocytopoiesis (H) nutrient histiocyte (I) lymphocyte
(J) plasma cell (K) megakaryocyte (L) supportive
adventitial-reticulum cells.
14


The stromal substances which comprise the ECM are believed to regulate
hematopoietic activity via specific cell surface receptors called adhesion molecules
(Link 2000). Thus, the ECM is responsible for facilitating cell-cell interactions and
binding growth factors to interact with hematopoietic cells. In addition, the ECM is
probably involved in the migration of hematopoietic cells (Naeim, 1998; Link 2000).
Adhesion molecules on the surface of cell membranes of stromal cells also have a
role in proliferation, activation, and homing of hematopoietic cells to the bone
marrow microenvironment after intravenous infusion. Link (2000) refers to studies
that indicate that lectins on the membrane surface of hematopoietic progenitor cells
may recognize galactose and mannose in the bone marrow microenvironment, and
this may be the molecular basis for recognition. The very late antigen-4 (VLA-4) is
an integrin thought to be important in homing and is expressed on hematopoietic
progenitor/stem cells. Link (2000) also refers to studies in which antibodies to
VLA-4 inhibit adhesion of CD34+ cells to bone marrow stromal cells. Inhibition of
adhesion in the bone marrow leads to mobilization of CD34+ cells in the peripheral
circulation.
Hematopoiesis is restricted to the extravascular spaces of the bone marrow.
However, hematopoietic cells are interspersed among the thin-walled venous sinuses
within the bone marrow. Endothelial cells line the venous sinuses (Naeim, 1998;
Link, 2000). Migration of hematopoietic cells into the intravascular space probably
15


occurs near interendothelial cell junctions and regions where the endothelial-cell
basement membrane ECM is thinned or absent Link, 2000, p. 25).
Granulocvtopoiesis Maturation Sequence
Production of the neutrophil starts in the bone marrow from precursors
committed to the myeloid cell line. The neutrophilic cell starts as a very immature
cell called a myeloblast and progresses in a continuum of cell maturation to the most
mature cell called a segmented neutrophil. Several notable cytologic characteristics
signify the progressive stages of the maturation sequence (Bell & Hughes, 1997) (see
Figure 2.5):
Generally the diameter of the immature cell is greater than the mature cell. The
overall size of the cell becomes smaller as the cell matures.
The immature cell will has a high nucleus to cytoplasm (N:C) ratio; thus the
nucleus is much more prominent than the cytoplasm. The nucleus becomes
smaller as the cell matures, and the shape of the nucleus changes.
The nucleus also shows other obvious changes as the cell matures. The
immature nuclear chromatin is very fine, but as the nucleus matures it becomes
more dense. Nucleoli (contains deoxyribonucleic acid [DNA] templates for
ribosomal ribonucleic acid [RNA]; enlarged during protein synthesis) are visible
in immature cells, but not in more mature cells.
16


The previously mentioned cell characteristics are visible with staining, as are
other characteristics, such as changes in granulation, and the intensity and color
produced with Wrights stain.
Myeloblast
The myeloblast has a round nucleus which stains predominately red and contains
one or more nucleoli. The chromatin is very delicate and fine resembling a lacy
pattern. The slight to moderate cytoplasm is nongranular and stains blue, but is
darker at the periphery than next to the nucleus. The lighter area adjacent to the
nucleus is due to the Golgi body (Bell & Hughes, 1997).
Promyelocyte
Development of large primary granules identifies the cell as a promyelocyte.
These early granules may cover the entire cell and stain dark blue or reddish blue.
The granules are usually round, but can vary in shape. Like the myeloblast, the
nucleus is usually round and still occupies a large volume of the cell. However, the
chromatin pattern is slightly more coarse, and nucleoli may be visible but may not be
as distinct as in the myeloblast. The nucleus has less red and stains more bluish.
The cytoplasm is still blue with a lighter area adjacent to the nucleus (Bell &
Hughes, 1997).
17


Myelocyte
The initial presence of secondary lysosomal granules is referred to as the dawn to
neutrophilia and the cell is classified as a myelocyte. The smaller secondary
granules stain reddish or pinkish; thus as the cell ages the cytoplasm becomes less
bluish and more pinkish. The nucleus may be round or oval, or flattened on one
side. The chromatin continues to thicken and nucleoli are either absent or indistinct.
Also, the cytoplasm becomes more abundant than the promyelocyte (Bell & Hughes,
1997).
Metamyelocyte
When the nucleus becomes indented or bean-shaped, the cell is classified as a
metamyelocyte. The nuclear chromatin is noticeably clumped and the purple
nucleus becomes smaller. The cytoplasm stains more pinkish because of the
predominance of small, pinkish secondary granules (Bell & Hughes, 1997).
Neutrophilic Band
The nuclear indentation becomes more marked as the cell continues to mature.
Often the now coarser, dark purple nucleus is shaped like a horseshoe, and shows
signs of degeneration. The cytoplasm is filled with small, pink secondary granules
18


and maybe an occasional dark primary granule (Bell & Hughes, 1997). These
defining characteristics now classify the cell as a band.
Segmented Neutrophil
The mature neutrophil has a nucleus which is separated into lobes which are
connected by a narrow filament. The nuclear chromatin is very clumped and appears
dark purple. The cytoplasm stains a light pink and contains many small, pink
secondary granules. Few primary granules may be present (Bell & Hughes, 1997).
Neutrophils are stored in the bone marrow and are released into the peripheral blood
for approximately 7 to 10 hours. At this time the cells exit the blood stream and enter
tissues and are subsequently replaced by other cells from the marrow.
I
19


Figure 2.5 Granulocytopoiesis. Stages of neutrophilic
granulocytopoiesis illustrate sequence starting from an
immature precursor, the myeloblast, to the mature
segmented neutrophil.
20


CHAPTER 3
HISTORY OF PERIPHERAL BLOOD PROGENITOR/STEM CELL
TRANSPLANTATION
Cellular Transplants
Exciting research in several different clinical disciplines over the last 50 years has
led to the development of more than 20,000 human cellular transplantations being
performed annually. Cellular transplants include hematopoietic cells from the bone
marrow, peripheral blood, or cord blood. The focus of this paper is on
hematopoietic progenitor stem cells mobilized into the bloodstream, which are
called peripheral blood progenitor stem cells (PBPC). PBPC is generally used as an
abbreviation for peripheral blood progenitor cells, and encompasses a transplant
product that contains both progenitor and stem cells. Initially, cellular transplants
were only carried out on patients with terminal diseases. However, today
transplantation may be used preferentially in treating patients in the early stages of
disease. Cellular transplants have been used to treat patients with a variety of
hematologic and oncologic malignancies such as, acute and chronic leukemias,
non-Hodgkins and Hodgkins lymphomas, Multiple Myeloma, and a variety of solid
tumors (Thomas, 1999; Mangan, 1995).
21


In 1909, Alexander Maximow, a Russian from St. Petersburg, gave a lecture on
the possible existence of small, easily transportable cells, which were part of the
white blood cell population in the bloodstream, and were capable of regaining their
full pluripotentiality (able to differentiate into all hematopoietic lineages). His was
the first publication to suggest the existence of hematopoietic stem cells in the
bloodstream. Forty years later, research begin in several areas which contributed to
our knowledge of the physiology of the stem cell (Korbling & Fliedner, 1996).
Early Animal Models
Studies after World War II began to unravel the mysterious nature of the stem
cell. In 1949, pivotal experiments showed that by shielding the spleen of mice
during lethal total body irradiation permitted their survival. Thus, the theory of
stem cell migration was demonstrated by showing that hematopoietic cells in the
spleen were capable of circulating and repopulating or engrafting the ablated bone
marrow (Jacobson, Marks, Robson, et al., 1949).
In addition, continuing research supported the existence of stem cells in the
circulating blood, and their ability to restore hematopoiesis in several animal
models. Radiolabeling cell techniques provided additional evidence that stem cells
were permanent residents of the circulating blood (Bond, Fliedner, & Archambeau,
1965). Previously, it was believed that these immature cells were only found in
the peripheral blood under pathological conditions. In the 1960s, studies in canines
22


provided even more evidence for the peripheral circulating stem cell. Canines were
also used in clinical PBPC transplantation studies. Successful autologous
transplantation was performed after lethally irradiating dogs and subsequently giving
them their own PBPC back. The canine autologous stem cells were collected, and
either set aside or cryopreserved before the stored cells were infused intravenously
(Cavins, Scheer, Thomas, & Ferrebee, 1962). Of note, autologous human bone
marrow transplants were already being performed in the 1950s (Kumick et al.,
1958). A patient with malignant lymphoma received an autologous bone marrow
transplant in 1959, following treatment with high-dose nitrogen mustard, and
remained in remission for 21 years (Haurani, 1997). The first peripheral blood
transplant was not performed until the 1970s (Thomas, 1999; Mangan, 1995;
Korbling & Fliedner, 1996).
The canine model contributed to other important observations in hematopoietic
stem cell studies which are applicable to human treatment today. Dogs that were
given supralethal irradiation and allogeneic (cells transplanted into a recipient from
a donor) transplants experienced various problems in transplantation, such as failure
to engraft and engraftment with Graft Versus Host Disease (GVHD) (immune
reaction of the engrafted lymphoid cells against the tissues of the host) (Thomas et
al., 1962). Also, studies showed that dogs could be successfully engrafted using
chemotherapy only with no prior irradiation (Storb et al., 1969).
23


Hematopoietic Stem Cells in Human Peripheral Blood
E. J. Friereich was involved in experiments in the early 60s which showed that
bone marrow engraftment occurred in patients with myelosuppression after being
transfused with peripheral blood leukocytes from patients with chronic myelogenous
leukemia. McCredie, Hersh, & Freireich (1971) were also involved in previous
experiments which showed that engraftment occurred when peripheral blood
leukocytes were collected with a cell separator from donors, and transfused into
patients who were myelosuppressed with chemotherapy. McCredie et al. (1971)
took note of these experiments and the earlier experiments with the lethally
irradiated and normal dogs. In light of these observations, they proposed that stem
cells exist in the peripheral blood are capable of repopulating the bone marrow.
McCredie et al. (1971) collected white blood cells (leukocytes) from normal
donors by three different techniques. They collected bone marrow leukocytes
(BML), peripheral blood leukocytes (PBL), and peripheral blood leukocytes
collected by a blood-cell separator, or leukapheresis (LL). The three different
leukocyte collections were cultured and observed for colony formation.
Colony-forming cells (CFC) were grown from all three types of collections. CFC
grew more regularly in the BML than the PBL or LL. CFC were higher in the LL
culture than the PBL culture. This observation indicated that CFC are more
24


concentrated with the leukapheresis procedure than a peripheral blood draw.
Furthermore, the majority of circulating leukocytes collected by leukapheresis are
large mononuclear cells. Thus, the investigators suggested that the CFC (i.e.
progenitor cells) must be present in this cell fraction. Furthermore, they proposed
that peripheral blood collections may be as efficient as bone marrow in
reconstitution of granulocytic cells in patients that are myelosuppressed (McCredie
et al 1971).
Barr, Whang-Peng, & Perry (1975) may have been the first to clearly demonstrate
the presence of hemopoietic stem cells in human peripheral blood. They harvested
mononuclear cells from the peripheral blood of normal donors. Since hemopoietic
cells closely resemble lymphocytes, the collection of mononuclear cells were
separated from thymus-dependent lymphocytes (T cells) by rosette formation with
sheep erythrocytes. The mononuclear cell fraction, which failed to exhibit resetting,
was further separated based on size. A monocyte concentrate was obtained which
consisted mainly of monocytes, and a smaller portion of basophils and lymphoid
cells. Monocytes, bursa-equivalent lymphocytes (B cells), and basophils were
removed from this concentrate using immunoglobulin G and complement
complexes. This caused resetting formation of these cells.
Cells that exhibited resetting characteristics failed to grow in culture, and only
contained the original cells (i.e. lymphocytes, monocytes, and basophils). Residual
25


cells, which failed to rosette, were cultured in vivo. However, these cells
proliferated and differentiated into erythrocytic, granulocytic, and megakaryocytic
progeny, thus demonstrating true pluripotentiality of a small fraction of cells in
human peripheral blood (Barr, Whang-Peng, & Perry, 1975).
Later studies performed with long-term cultures of normal human peripheral
blood showed that primitive hematopoietic cells, which are closely related to stem
cells, are present in the circulation, although at very low levels. Furthermore, these
cells are indistinguishable in their properties of differentiation and proliferation from
primitive cells in normal bone marrow (Udomsakdi et al., 1992).
Attempts to Quantify and Identify the Progenitor/Stem Cell
The case for the circulating stem cell, capable of protecting lethally irradiated
animals and differentiating into all hematopoietic cell lines, was definitely strong
after monumental research in the 1950s and 1960s. The next step was to be able to
identify and quantify the progenitor/stem cell in the blood. This would not be an
easy feat because the progenitor/stem cell is a morphologically indistinct cell,
appearing as a medium-sized mononuclear lymphoid-looking cell (Mangan, 1995).
The first quantitative experiments used colony forming units (CFUs) for
enumeration. Today, CFUs are known to be characteristic of the progenitor cell, not
the stem cell. Scientists discovered that colonies formed on the spleen of mice
which had been irradiated and subsequently injected with bone marrow. Eventually,
26


an estimate of human progenitor/stem cells was obtained by culturing blood or
marrow white blood cells, specifically mononuclear cells (Till & McCulloch, 1963).
In vitro assays improved by using LTC (long-term cultures), which are indicative of
stem cells (Udomaski et al., 1992). LTC further improved the approximation of
progenitor/stem cells in the peripheral blood However, these culturing techniques
were time consuming, and thus were impractical for routine identification and
quantitation of peripheral blood progenitor/stem cells (Mangan, 1995).
C34 + Antigen
In 1984, an antigen expressed on all types of progenitor and stem cells was
discovered and termed the CD34 antigen. Cells expressing the CD34 antigen are
referred to as CD34 positive cells (CD34+). This discovery enabled more rapid
identification of the circulating progenitor/stem cells. Monoclonal antibodies and
flow cytometry were used to identify subsets of the CD34+ cells, either the
committed progenitor cell group (CD38+) or the piuripotential cell group (CD38-).
Again, these cells commonly express the CD34 antigen, but also express different
antigens that further differentiate these cells (Civin et al., 1984).
Optimization of Peripheral Blood Progenitor/Stem Cell Collection
The discovery of the CD34+ antigen helped in the enumeration of PBPC under
steady state conditions; however, researchers discovered that less than 1% of all the
27


mononuclear cells in the bloodstream were CD34+, and of the CD34+ cells less than
0.1% were actual stem cells (Sienna, Pregni, Brando, et al., 1991). The feasibility
of collecting PBPC under steady state conditions was indeed questionable.
In the 1970s blood separation devices called apheresis machines were being used
in most blood banks for collecting platelets and white blood cells. Cells are
separated based on cell density and collected by centrifugation. The apheresis
technique was adapted for the collection of PBPC. Central venous catheters, and the
automation of the apheresis instrument allowed convenient access to large volumes
of peripheral blood processing. Nevertheless, this meant that large volumes of blood
had to be processed in the laboratory. This cumbersome method was not
cost-effective, and was potentially toxic because of the large volume of cells
cryopreserved with chemicals and subsequently infused. Therefore, under steady
state conditions, many apheresis procedures were required for the collection of an
adequate number of PBPC (Mangan, 1995). Studies conducted in the 1970s and
early 80s showed that stem cells were regulated by numerous growth factors. The
factors are named for the cell line that they effect, such as granulocyte-colony
stimulating factor (GCSF), which stimulates the production of a particular a group of
white cells called granulocytes. Clinical use of this growth factor in the early 80s
demonstrated that it was capable of causing the bone marrow to release
progenitor/stem cells into the peripheral blood, a process termed mobilization. In
28


addition, in 1976, progenitor/stem cells were found to be increased in the peripheral
blood after the administration of chemotherapy. This rebound recovery of cells in
the bone marrow subsequently caused the release of progenitor/stem cells into the
circulation (Richman, Weiner, & Yankee, 1977). Further research showed that
chemotherapy followed by administration of growth factors resulted in even greater
numbers of progenitor/stem cells in the peripheral blood (Pettingill, Testa, Swindill,
et al., 1993).
With recent scientific developments in the last 20 years, autologous PBPC
transplants have practically replaced bone marrow transplants. Mobilized peripheral
blood transplants offer several advantages over bone marrow transplants including
more rapid recovery of circulating granulocytes and platelets. Peripheral blood
collections via apheresis do not require sedation, and the procedure is not as invasive
as bone marrow harvest surgery. Furthermore, a BM harvest requires general
anesthesia with more complications than apheresis, including death.
29


CHAPTER 4
CD34+ CELLS
Discovery of the CD34+ Antigen
Civin et al. (1984) was one of the first to describe the antigenic analysis of
hematopoietic progenitor cells Civin used mice to produce a monoclonal antibody
(anti-My-10) against the immature human myeloid leukemic cell line (KG-la).
KG-la, are immature, undifferentiated white cells, were introduced into mice to
stimulate the production of antibodies to cell surface antigens. Analysis of
anti-My-10 showed the antibody to belong to the IgG class (Civin et al., 1984).
Indirect immunofluorescence was used to measure binding of the antibody to the
KG-la cell line as well as mature granulocytes from the peripheral blood of normal
donors. Among other cells tested against the antibody were lymphocytes,
monocytes, and marrow cells from normal donors. Results of the experiment
revealed that the KG-la cells were as fluorescent as the positive control, thus the cell
line contains large amounts of the My-10 antigen. The granulocytes, monocytes,
and lymphocytes from the peripheral blood were not labeled by the antibody.
Although progenitor and stem cells are known to circulate in the peripheral blood in
extremely small numbers, they were not detectable as a subpopulation of the
30


peripheral blood by immunofluorescence in this experiment. However, a small
percent (1.3%) was detectable as My-10 antigen marrow cells (Civin et al., 1984).
Morphologic and cytochemical analysis of the positive normal human marrow
cells showed that they were predominately very immature cells, such as myeloblasts
and monoblasts. Expression of the antigen decreased as the myeloid cell line
matured because only rare more mature cells were found to be My-10-positive
(Civin et al., 1984).
The My-10 antigen was radiolabeled, and immunoprecipitated from the
surface of the KG-la cells with the anti-My-10 antibody. SDS polyacrylamide gel
electrophoresis and autoradiography was performed on the antigen; a protein with a
molecular radius of about 115 kilodaltons (KD) was revealed, the (',1)34 antigen
(Civin et al., 1984) (see Figure 4.1). The authors definitely saw the potential for
further hematopoietic research and the isolation of an immature hematopoietic cell
population for human bone marrow transplantation.
31


Figure 4.1 Schematic of cell surface antigens.
Presentation includes some of the significant cell
surface antigens on stem, progenitor, and precursor
cells.
Stem Cell
cd34+, cd33- cd38-, ^cd45ro+, cd45ra-
Myeloid Progenitor
cd34+, cd33+^8-
CFU-M, cd34+
cd33+, 13
ymphoid Progenitor
T-cell
Monoblast
cd33-^d38+ cd33+,"
cdl4
Monocyte
Progenitors^
cd34+,'lcdl0+ cd34+,^Cd45ra+
cd7+, cd33-
T-cell
32


CHAPTER 5
GRANULOCYTE-COLONY STIMULATING FACTOR
Function
Granulocyte-colony stimulating factor (GCSF) is a hematopoietic growth factor
(cytokine) which regulates the proliferation (production) and differentiation
(maturation) of neutrophils. Neutrophils comprise approximately 70% of the
leukocytes in the circulation. They have a very important role in providing
protection from bacterial pathogens. However, these cells have a short half-life; thus
neutrophils must be continuously replenished from pluripotent stem cells in the bone
marrow (Nagata, 1994). Morstyn et al. (1998) state that native human GCSF is
produced by stromal cells, endothelial cells, fibroblasts, and monocytes (p. 424).
Bagby and Heinrich (1999) list the following functions of GCSF (p. 3 4):
Stimulates growth of progenitor cells committed to the neutrophil lineage
Stimulates neutrophil maturation of certain leukemic cells
Activates phagocytic function of mature neutrophils
Stimulates quiescent pluripotent hematopoietic progenitor cells to enter Gl-S
Stimulates mobilization of stem cells and progenitors from hematopoietic niches
into peripheral blood
33


Maintenance of steady-state neutrophil numbers
Structure
GCSF protein is encoded on the long arm of chromosome 17 and has core
molecular weight of 18.6 kilodaltons. Human GCSF exists in two forms, a form
with 177 amino acids and another form with 174 amino acids, which may be more
active. Native human GCSF is glycosylated and the sugar chain is attached to the
C-D loop at the threonine 134 position. Glycosylation does not appear to be
important for the biological function of the molecule, but may play a role in
protecting the GCSF protein from proteolytic degradation. Further, filgrastim
(rHuG-CSF) is a non-glycosylated commercial hematopoietic growth factor and was
the GCSF used in this study (Morstyn et al., 1998).
As a member of the a superfamily of cytokines, GCSF has a protein structure
consisting of a four alpha-helical bundle; the helices are labeled A-D starting at the
N-terminus. Also, a short helix exists in the A-B loop (Layton et al., 1999).
Furthermore, human GCSF contains four disulfide bonds which are essential for
proper folding and biological activity of the molecule (Nagata, 1994).
Mobilization of Progenitor/Stem Cells
Hematopoietic cytokines, such as GCSF, GM-CSF, stem cell factor (SCF),
Interleukin 1 (IL-1), IL-7, IL-8,1L-11, and IL-12 are capable of mobilizing
34


hematopoietic cells into the circulation. Chemotherapy also causes rebound
mobilization of hematopoietic cells into the circulation (Link, 2000). For example,
GCSF is capable of inducing a 20-fold increase in peripheral blood neutrophils and a
100-fold increase in peripheral blood hematopoietic cells. SCF causes a 24-fold
increase in peripheral blood neutrophils and a 20-fold increase in peripheral blood
hematopoietic cells. Another comparison shows that IL-1 causes a 5-fold and
30-fold increase, respectively. Mobilization with chemotherapy alone produces a
8-fold and 80-fold increase, respectively (Link, 2000). GCSF is most commonly
used mobilization agent because this cytokine is potent and lacks serious toxic
effects (Link, 2000). The fact that hematopoietic cytokines with distinct biologic
activities can mobilize hematopoietic cells leans towards a common mechanism.
Link (2000) describes the following common characteristics of cytokine
mobilization with few exceptions existing:
Increased circulating hematopoietic cells
Broad spectrum mobilization of hemapoietic cells including primitive pluripotent
to committed progenitors of different cell lines
Comobilization of neutrophils with hematopoietic cells
Cytokine mobilization of hematopoietic cells into the circulation may occur by
phenotypic changes in hematopoietic cells or by changes in the actual BM
microenvironment. Evidence exists for both scenarios. Studies have indicated
35


differences in the phenotypic characteristics of mobilized hematopoietic cells
compared to hematopoietic cells found in the steady-state environment of the BM.
However, experiments with the 1L-8 chemokine indicate that changes in the BM
microenvironment lead to mobilization (Link, 2000). GCSF-R (GCSF receptor)
deficient mice are defective in granulopoiesis leading to neutropenia. In addition,
the GCSF-R has been shown to act synergistically with other agents. For example,
GCSF-R is necessary for hematopoietic cell mobilization in mice treated with
chemotherapy, specifically cyclophosphamide; GCSF-R-deficient mice are deficient
in circulating hematopoietic cells. The GCSF-R is also required for mobilization of
hematopoietic cells from the BM into the circulation by IL-8. Although, other
cytokines do not require the presence of the GCSF-R for mobilization. The author
believes that GCSF-R-dependent signals are produced by hematopoietic cells in
response to stimuli (such as cytokines), which in turn cause changes in the BM
microenvironment, and thus lead to hematopoietic cell mobilization. Therefore, the
model favors BM microenvironment changes in mobilization, and places less
importance on the cells themselves (Link, 2000). Furthermore, evidence suggests
that VLA-4 adhesion molecules are down-regulated in the BM and allow release of
cells into the peripheral circulation.
36


CHAPTER 6
EX VIVO EXPANSION AND SELECTION OF HEMATOPOIETIC
PROGENITOR/STEM CELLS
Ex vivo expansion/selection of hematopoietic progenitor/stem cells from purified
CD34+ cells offer several benefits in transplantation (Brugger, Scheding, Ziegler,
Buring, & Kanz, 2000; Andrews, Briddell, Hill, Gough, & McNiece, 1999):
Only a small volume of peripheral blood collection is required
Reduced tumor cell load in reinfusion product
Rapid engraftment leading to decreased duration and severity of neutropenia
Ability to perform repeated clinical applications as with multiple high-dose
chemotherapy
The possibility of increasing stem cells in allogenic cases where human
leukocyte antigen is mismatched, allowing decreased graft rejection
Ex Vivo Expansion and Cytokines
Brugger et al. (2000) state that optimal hematopoietic growth factor conditions
have not yet been elucidated in progenitor/stem cell ex vivo expansion. SCF is
thought to be an important growth survival factor for hematopoietic progenitor/stem
cells. Some cytokines, such as IL-1, IL-3, and IL-6, act in the early stages of
37


expansion. On the other hand, GCSF, GM-CSF, and erythropoietin (EPO) are
late-acting growth factors.
The Brugger team (2000) found from previous experiments that a combination of
SCF, IL-1, IL-3, IL-6, and EPO in a stroma-free liquid culture was capable of
expanding CD34+ cells collected from the peripheral blood. In this system,
progenitor cells were expanded well, but more primitive progenitor and stem cells,
identified by LTC-IC (long-term culture-initiating cells), were not expanded;
nevertheless these cells were maintained in culture. However, other studies indicate
that a cytokine combination of SCF, FL, thrombopoietin (TPO), and IL-3 greatly
improve generation of more primitive (noncommitted) progenitor/stem cells. TPO
not only effects primitive progenitor/stem cell generation, but also enhances the
megakaryocytic cell line responsible for replenishing platelets. GCSF, GM-CSF, and
EPO appear to play an important role in expansion of committed progenitor cells
(Brugger et al., 2000).
Brugger et al. (2000) deem that controversy still exists in the ex vivo expansion of
hematopoietic progenitor/stem cells, mainly the ability of the transplanted cells to
home and provide terminally differentiated cells in vivo, especially in patients
receiving myeloablative therapy. Committed progenitors are probably responsible
for the rapid kinetics seen in the recovery of peripheral blood cells. However,
Brugger et al. (2000) propose that stem cells may be spent from growth factor
38


addition in culture. Albella, Segovia, Guenechea, Pragnell, & Bueren (1999)
invested combinations of cytokines that prevent differentiation of stem cells and
enhance self-renewal in the stem cell compartment. They used mouse BM expanded
ex vivo with the SCF and IL-11 (in combination with or without other factors known
as Flt3 ligand and macrophage inflammatory protein-1 alpha) cytokines. The results
showed significant increase in both short-term and mid-term murine hematopoietic
repopulating ability, 40 days and 180 days posttransplantation, respectively. The
authors conclude that stem cells are not expanded in culture; however, stem cells are
maintained in culture, and thus not differentiated. Though, the maintenance of
long-term repopulating ability in the mouse model was observed one year following
transplantation.
Reduced Tumor Cell Content
Tumor cell content in ex vivo expanded cells may be reduced because less
volume of blood is required. In addition, culture conditions which favor expansion
of normal hematopoietic progenitor/stem cells may inhibit propagation of malignant
cells (called passive purging) (Brugger et al., 2000; Carlos et al., 1999). Lundell,
Vredenburgh, Tyer, DeSombre, & Smith (1998) refer to previous studies which
indicate decreased survival in breast cancer patients receiving BM or PBSC with
detectable tumor cells. Furthermore, other studies noted indicate that reduction in
the number of tumor cells infused in the transplant product may delay disease
39


relapse in these patients. However, reinfused tumor cells must be distinguished from
the possibility of residual tumor cells.
Lundell et al. (1998) expanded tumor cell positive BM from stage IV breast
cancer patients, and subsequently immunostained the expanded cells for
identification of tumor cells (immunostaining can detect one tumor in 10A6 normal
BM cells). Pre-expansion samples contained 6-2128 tumor cells in 5 x 10A6
nucleated cells evaluated. Post-expansion samples revealed no tumor cells in four of
seven different samples. The other three samples had a decrease of 44 to two, 40 to
two, and 2128 to four per 5 x 10A6 nucleated cells examined. This experiment
demonstrates passive purging resulting in tumor cell reduction of one to four logs.
Carlos et al. (1999) also demonstrated significant decreased tumor cell content after
ex vivo expansion of BM cells from breast cancer patients with detectable tumor cell
contamination prior to expansion. Additionally, studies have addressed whether
concomitant clonogenic growth of tumor cells occurs with expansion of CD34+ cells
under the influence of cytokines. Brugger et al. (2000) conclude that tumor cells do
not expand significantly in cytokine-induced cultures for CD34+ cells. Besides,
these researchers found that no tumor cells were detected in previous tumor-positive
samples after CD34+ cell selection, or after expansion of these CD34+ cells.
40


Decreased Neutropenia
Studies on the use ex vivo expanded CD34+ PBPC cells plus posttransplant
growth factor have been shown to significantly decrease neutropenia in lethally
irradiated primate models (Andrews, Briddell, Hill, Gough, & McNiece, 1999).
Reiffers et al. (1999) found similar results in myeloma patients treated with or
without irradiation and high-dose chemotherapy, and transplanted with CD34+ ex
vivo expanded cells. McNiece et al. (2000) have also demonstrated decreased time
of neutropenia in breast cancer patients transplanted with CD34+ expanded cells.
Abrogation of neutropenia in these studies is probably due to the high numbers of
committed progenitor cells (Andrew et al., 1999; Reiffers et al., 1999). Thus, some
studies are aimed at determining how to specifically generate granulopoietic
postprogenitor cells (GPPC) that would be additionally transplanted with
progenitor/stem cells to help abrogate neutropenia (Stefan et al., 2000).
Nevertheless, treatment protocols that require high-dose myeloablative
chemotherapy would benefit from ex vivo expanded cells because of the morbidity
associated with infection during the induced neutropenic phase.
Andrews et al. (1999) demonstrated that juvenile baboons transplanted with
expanded CD34+ PBPC and subsequently given GCSF and megakaryocyte growth
and development factor (MGDF), significantly shortened the time of neutropenia.
41


This shortened time of neutropenia was much more considerable when compared to
baboons who received unmanipulated cells of the same type along with growth
factor, or in animals that received only expanded cells, but no growth factor.
However, animals receiving either unmanipulated cells or expanded cells
demonstrated similar duration of neutropenia without the administration of growth
factor. Notably, the scientists found that thrombocytopenia seemed to be prolong in
animals that received expanded cells compared to animals receiving unmanipulated
cells. Though, this is somewhat puzzling since megakaryocytes (platelet precursors)
are also expanded in culture. Furthermore, the majority of animals receiving
expanded cells maintained sufficient peripheral blood counts for six months
posttransplant, and these findings may imply that stem cells were maintained in
culture (Adrews et al., 1999).
42


CHAPTER 7
HIGH-DOSE CHEMOTHERAPY
Purpose
Chemotherapy drugs interfere with cell replication and division, and can act at
different phases in the cell cycle. However, these drugs are non-discriminating and
they also effect normal cells which divide rapidly, such as bone marrow,
gastrointestinal mucosa, gonads, and hair follicles (Wilkes et al., 1999).
High-dose chemotherapy (HDT) is the use of chemotherapeutic agents to cause
myeloablation of the bone marrow for the treatment of cancer and other diseases.
Thus, the goal of high-dose chemotherapy is to effectively eradicate tumors, and/or
to allow the immune system to fight residual amounts of tumor (Colvin & Petros,
2000). Since the bone marrow is myeloablated, peripheral blood progenitor/stem cell
or bone marrow transplants are required to reestablish hematopoiesis. HDT
regimens usually include a minimum of two agents which differ in action. Use of
HDT regimens may serve several important functions: 1) to improve tumor toxicity
by synergistic activity, 2) to decrease drug toxicity, 2) to prevent tumor drug
resistance (Colvin & Petros, 2000).
43


Characteristics of Oncology Drugs
Patients in this study received several different high-dose chemotherapy regimens
based on the stage of their disease. Several regimens used for high-dose
chemotherapy consisted of the following oncology drugs: cyclophosphamide,
cisplatin, BCNU, Taxotere, carboplatin, melphalan, and taxol. Three different
combinations were used: 1) cyclophosphamide, cisplatin, and BCNU, 2) Taxotere,
carboplatin, and melphalan, 3) taxol, cyclophosphamide, and cisplatin. The drug
class, mechanism of action, pharmacokinetics, dosage, and toxicities/side-effects of
each drug will be discussed.
Cyclophosphamide (Cytoxan)
Cyclophosphamide is an alkylating agent that prevents DNA synthesis by causing
crosslinkage in DNA strands. The drug is activated in the liver and by serum
enzymes. The drug
and its metabolites are excreted by the kidneys, and the half-life of the drug is 6-12
hours. The dosage range is from 100-1500 milligrams per meter squared (mg/mA2).
High-dose cyclophosphamide used in this study was 1875 mg/mA2/day intravenous
(IV) x 3. Possible toxicities/side-effects include: infection and bleeding caused by
bone marrow depression; accumulation of drug metabolites in the bladder causing
44


hemorrhagic cystitis; nausea, vomiting, and diarrhea leading to malnutrition or
anorexia; alopecia and changes in skin and nails; amenorrhea, and reproductive
hazards due to mutagenicity and probable tetragenicity; secondary malignancy;
alterations in cardiac function; pulmonary toxicity (Wilkes, Ingwersen, & Burke,
1999).
Cisplatin (Platinol)
Cisplatin is a heavy metal acting similar to an alkylating agent and also inhibits
DNA synthesis by crosslinking, and by denaturing the double helix structure. The
drug is rapidly distributed to the liver and kidneys, and the drug is excreted in the
urine within 24 hours. Dosage ranges from 15-120 mg/mA2. Dosage in this study
was 55 mg/mA2/day IV x3. Possible toxicities/side-effects include: renal damage;
nausea and vomiting, and taste alterations; anaphylaxis (tachycardia, wheezing,
hypotension, facial edema, rash, urticaria, erythema, pruritus); neuropathy; anemia;
infection and bleeding; reproductive hazard; alterations in cardiac function (Wilkes
etal., 1999).
Carmustine (BCNU)
BCNU is classified as a nitrosourea which acts as an alkylating agent, and also
inhibits DNA repair. This drug is rapidly distributed to tissues and is rapidly
metabolized with a half-life of one hour. However, the drugs lipid solubility allows
45


the drug to remain in the cerebrospinal fluid nine times longer. Excretion occurs
mainly by the kidneys. Dosages range from 40-225 mg/mA2, or 450-600 mg/mA2
with transplants. The BCNU dose used in this study was 600 mg/mA2/day IV x 1.
Possible toxicities/side-effects are: infection and bleeding; pulmonary toxicity;
nausea, vomiting, and reversible liver dysfunction; irritation causing venospasms and
skin flushing; kidney damage; reproductive hazards; ocular toxicity causing damage
to optic nerve, retinal hemorrhage, and neuroretinitis (Wilkes et al., 1999).
Docetaxel (Taxotere)
Docetaxel, a mitotic spindle poison, acts by arresting cell division in metaphase
by enhancing microtubule assembly and inhibiting depolymerization. The drug is
metabolized by an isoenzyme, and is highly protein-bound in the plasma. Excretion
of the drug occurs mainly by the intestines with waste. The dosage range is 60-100
mg/mA2, and the dosage administered in this study was 200-300 mg/mA2 x 1.
Possible toxicities/side-effects include: anaphylaxis; infection and bleeding; fluid
retention causing edema, pleural effusion, and ascites; alopecia and skin rash;
neuropathy; nausea, vomiting, diarrhea, and stomatitis (Wilkes et al., 1999).
Carboplatin (Paraplatin)
This alkylating agent is similar to cisplatin and acts by causing intrastrand and
interstrand DNA crosslinking. The majority of carboplatin is excreted in the urine
46


with a half-life of about 100 minutes. The dosage range is 300-360 mg/mA2, and the
dosage used in this study was 1000 mg/mA2/day x 3. Possible toxicities/side-effects
include: infection and bleeding; nephrotoxicity; nausea, vomiting, diarrhea,
stomatitis, and reversible liver dysfunction; neurologic dysfunction; reproductive
hazards; anaphylaxis (Wilkes et al., 1999).
Melphalan
This alkylating agent is a derivative of a nitrogen mustard which prevents cell
replication via DNA crosslinking and breakage. Melphalan has variable
bioavailability. The drug is excreted by both the intestines and the kidneys with a
half-life of about two hours. Dosages ranges for bone marrow transplantation is
usually 50-60 mg/mA2, but the dosage administered in this study was 150
mg/mA2/day x 3. Possible toxicities/side effects are: infection and bleeding; nausea
and vomiting; anaphylaxis causing cardiac problems; reproductive hazards;
secondary malignancy; pulmonary toxicity; alopecia, skin rash, and urticaria (Wilkes
et al., 1999).
Paclitaxel (Taxol)
Paclitaxel causes cell death by the same mechanism of action as docetaxel. This
drug is highly protein-bound in the plasma and is metabolized in the liver. Drug
metabolites are excreted mainly in the bile. Dosage varies from 135-250 mg/mA2.
47


The dosage used in this study was 725 mg/mA2/day x 1. Possible
toxicities/side-effects are as follows: anaphylaxis; bleeding and infection; sensory
neuropathy; alopecia; nausea, vomiting, diarrhea, stomatitis, and liver toxicity
(Wilkes etal., 1999).
48


CHAPTER 8
BREAST CANCER
Types of Breast Cancer
Breast cancer occurs when a malignant tumor develops from previously normal
breast tissue. The female breast is comprised of four main tissue components:
lobules which are the milk-producing glands; ducts through which milk passes to the
nipple; stroma which consists of fatty tissue and ligaments; lymphphatic vessels
which lead to the axillary lymph nodes (American Cancer Society [ACS], 1999).
There are many different types of breast cancers effecting different cells and
tissues of the breast; thus, the types of breast cancers vary in their prognosis and
treatment options. Adenocarcinoma starts in the glandular tissue and includes
ductal carcinomas and lobular carcinomas. Most breast cancers are
adenocarcinomas because they originate in the glandular tissue. Ductal carcinoma
in situ (DCIS) is a form of noninvasive breast cancer where cancer cells are present
inside the ducts, but have not spread to other breast tissue. DCIS can be
subclassified according to grade and type. Grade distinguishes the severity of the
cancer cells detected under the microscope. Comedocarcinoma describes DCIS with
areas of dead or degenerating tumor cells. In situ breast cancer indicates an early
49


stage of cancer where the tumor is confined only to the area where the cancer began.
Infiltrating ductal carcinoma (IDC) began in the duct but has passed through the
duct wall and invaded fatty tissue. Metastasis is now possible through the lymphatic
and circulatory system. This type of cancer is responsible for approximately 80% of
breast cancers. Infiltrating lobular carcinoma (1LC) starts in the milk-producing
gland and has the potential to metastasize. Lobular carcinoma refers to a special
type of invasive ductal or lobular carcinoma with a better prognosis than usual ILC
or IDC. Inflammatory breast cancer occurs when cancer cells block lymphatic
vessels in the skin over the breast. The blockage causes the breast skin to become
red and warm with a thick, pitted appearance. This type of invasive breast cancer is
rare, but it is aggressive. Lobular carcinoma in situ (LCIS) begins in the breast
gland but does not penetrate the lobule wall. This type is a neoplasia rather than
cancer, and is usually not invasive. However, women with this classification may
have an increased risk for invasive breast cancer. Medullary carcinoma of the breast
is invasive, but with a clear delineation between tumor tissue and normal tissue.
This type of invasive breast cancer has a good prognosis, but accounts for only 5%
of breast cancers. Mucinous carcinoma is a rare type of invasive breast cancer
consisting of mucus-producing cancer cells. Paget's disease of the nipple originates
in the duct and metastasizes to the nipple and areola skin. This rare type of breast
cancer can cause the skin of the nipple and areola to become crusty, scaly, and red,
50


with areas of bleeding. Phyllodes tumor is a very rare type of tumor that forms from
connective breast tissue. These type of breast tumors are usually benign and rarely
become malignant (ACS, 1999).
Breast Cancer Risk
According to the American Cancer Society the following are risk factors for
breast cancer that cannot be changed by the individual: female, aging, genetics,
family history of breast cancer, personal history of breast cancer, race, previous
breast biopsy, previous breast irradiation, and early menarche or late menopause
(ACS, 1999).
Breast cancer is about 100 times more common in women than men.
Seventy-seven percent of women with breast cancer are over 50 years when
diagnosed.
About 50% of women with inherited mutations of the BRCA1 or BRCA2 will get
breast cancer by age 70. Inherited mutations of p53 tumor suppressor gene also
increases a womens risk of developing several types of cancers, including breast
cancer. A womans risk is doubled if she has a first-degree relative (mother,
daughter, sister) with breast cancer. Women with cancer in one breast have an
increased risk for new cancer developing in the opposite breast. Caucasian women
have a slightly greater risk than African-American women, but African-Americans
are more likely to die. Asian and Hispanic women have a lower risk (ACS, 1999).
51


The ACS reports that women with a previous breast biopsy have increased risk
for breast cancer, but the degree of risk depends on the classification or diagnosis of
the breast biopsy. Parker et al. (1999) found that 82% of women in a study with
known breast cancer had undergone a previous breast biopsy or fine-needle
aspiration which was initially negative for cancer. Also, women who received chest
radiation at a young age as treatment for other cancers have a significant increased
risk for breast cancer (ACS, 1999).
The ACS also states that early menarche (before age 12) and late menopause
(after age 50) contribute a slightly higher risk for breast cancer.
Health Net conducted a recent survey study to determine risk factors for breast
cancer in younger women. A Breast Health Assessment survey was sent to women
from age 34 to 49 years. Risk factors were compared for women with known breast
cancer and the general population. The study concluded that breast cancer risk was
increased by age, first-degree relative with breast cancer, and previous breast biopsy
for cysts. No statistical difference between the two groups was related to menarche,
nulliparity, first pregnancy after age 30, or breast feeding (Parker et al., 1999).
52


CHAPTER 9
METHODS
Patients
The study was approved by the Combined Multiple Institutional Review Board at
the University of Colorado Health Sciences Center. The study group consisted of 21
women with advanced breast cancer ranging from stage II to IV disease. The
subjects were consecutively assigned to either Cohort 1 (N=10) or Cohort 2 (N=l 1).
The subjects in Cohort 1 and Cohort 2 both underwent leukapheresis over a period of
five days to collect peripheral blood progenitor/stem cells which had been mobilized
from the bone marrow with GCSF (Neupogen). Cohort 1 patients received both ex
vivo expanded CD34+ selected cells and CD34+ selected unexpanded cells. Cohort
2 received only ex vivo expanded CD34+ selected cells as the sole hematopoietic
support. Both groups received various regimens of high-dose chemotherapy before
being infused with the CD34+ selected cells. Cohorts 1 and 2 also received
recombinant human GCSF posttransplantation (Figure 9.1)
53


Figure 9.1 Methods schema for cohort patients. (A) Cohort 1: CD34+ selected and
CD34+ ex vivo expansion infusion. (B) Cohort 2: CD34+ ex vivo expansion
infusion (Reprinted with permission of McNiece et al., 2000).
A
B_____________
Mobilization 1
54


Leukapheresis
The COBE Spectra Apheresis System version 6 was used to collect
mononuclear cell-rich peripheral blood progenitor/stem cells. The COBE Spectra
collects cells in the same manner as centrifugation where heavier cells, such as red
cells, fall to the bottom and lighter cells settle on the top layer known as the buffy
coat. The COBE Spectra only has the capability to collect the buffy coat layer and
cannot morphologically distinguish cell types. Blood is continuously pumped
through the machine as the PBPC are removed and the remainder of the blood is
returned to the patient.
Materials and Supplies
Materials and supplies used in the procedure consist of the following. COBE
Spectra disposable Auto PBPC blood tubing set #777-006-100; COBE
SpectraTHERM disposable blood warmer set #777-000-200; Anticoagulant
Citrate Dextrose to prevent clotting of the blood in the apheresis machine; 0.9%
normal saline used to initially prime the PBPC and blood warmer tubing; Baxter
Fenwal sampling Site Coupler #4C2405 to gain sterile access to final product bag;
sterile drapes, Betadine, alcohol wipes, and latex exam gloves to maintain sterility of
the catheter site and product throughout the apheresis procedure. Blood collection
55


tubes, sterile needles, and syringes to collect required peripheral blood specimens,
and to collect a sample from the final apheresis product. Equipment used in the
procedure, in addition to the COBE Spectra, includes the SpectraTHERM used
to warm blood as it is returned to the auto donor, and the Sebra Tube Sealer to
close off all tubing at the conclusion of the procedure, and to seal and detach the
product from the rest of the apheresis tubing set.
All autologous patients for the autologous apheresis procedure have orders signed
by the Bone Marrow Transplant attending physician. Subjects also signed an
informed consent for the leukapheresis procedure, and for high-dose chemotherapy.
Accordingly, all patients in the study signed an informed consent for participation in
this particular study, and the study was previously approved by the International
Review Board at the UCHSC.
In addition, subjects must complete specific requirements before starting
apheresis. All subjects were required to complete the same standards as all
autologous apheresis patients. The following was required for all subjects in the
study: health and physical exam; height and weight; comprehensive infectious
disease panel; ABO blood group and Rh type; pregnancy assessment on all females
of childbearing potential; completed HIV questionnaire, central line placement;
GCSF mobilization four days before the start of apheresis.
56


Procedure
The leukapheresis procedure normally takes four to six hours to complete. Cells
are usually collected on five consecutive days. The COBE Spectra is loaded with
the PBPC tubing set and primed in accordance with the COBE Operational
Manual-Section 3. Once the machine has been primed with normal saline, alarm
tests will performed to insure proper loading and functioning of the machine. Also,
the blood warmer tubing is inserted on the SpectraTHERM and connected to the
PBPC tubing. Data must be entered into the COBE Spectra computer to calculate
the volume of product that each individual patient will collect in the product bag.
Data required for the procedure includes: height and weight, sex, white count, the
percent of mononuclear cells (the total monocytes, lymphocytes, myelocytes,
metamyelocytes, promyelocytes, and blasts), and hematocrit. The total blood
volume (TBV) of each individual patient to be processed is based on sex, height and
weight. The patients TBV is processed a total of four times through the instrument.
Vital signs are taken before and after apheresis. The patient is monitored closely
during the procedure for any adverse reactions that may occur. The most common
reaction is citrate sensitivity due to the anticoagulant used during the procedure. The
anticoagulant works by binding ionized calcium in the bloodstream. Calcium is a
necessary component in hemostasis, thus removal of calcium prevents blood from
57


clotting in the instrument tubing and prevents clotting of the final product containing
the progenitor/stem cells. The anticoagulant is eventually metabolized when the
processed blood in returned to the patient. However, during apheresis, the patient
may experience a sensation of tingling or numbness in varying degrees, and in
several areas of the body. This occurs as the calcium levels drop in the bloodstream
and the body begins to remove blood from the muscles, which require calcium for
muscle contraction. The tingling sensation is usually located around the mouth,
hands, feet, arms, and legs. Some degree of tingling, or citrate sensitivity, is
expected and patients are given calcium supplements prophylactically at the
beginning of the procedure and as needed thereafter. Ionized calcium peripheral
blood levels are monitored if symptoms are more severe, and intravenous calcium
may be required to treat hypocalcemia. Patients may experience other adverse
reactions, but most of these reactions are less common: muscle tetany, hypotension,
blurred vision, nausea, and vasovagal syncope. The patient receives GCSF while
undergoing the apheresis procedure to mobilize progenitor/stem cells. GCSF also
has several side-effects, most commonly bone pain and headache.
58


CD34+ Selection
Materials and Supplies
Materials and supplies used in CD34+ selection process include the following:
Isolex 3001 Magnetic Cell Separator; Isolex Stem Cell Reagent Kit, and other
necessary laboratory reagents; Isolex 3001 Magnetic Cell Separator Disposable
Set; MPC-1 Dynal Magnetic Particle Concentrator; Coulter Z1 Cell Counter;
Sebra Tube Sealer; other minor laboratory supplies.
Procedure
The Isolex 3001 Cell Selection System is used to immunomagnetically select
CD34+ cells from other cell populations contained within the apheresis collection
product. First, the cells are washed and concentrated. Second, the concentrated
cells are incubated with murine antihuman CD34+ monoclonal antibody, and
subsequently washed to remove any excess antibody. Third, the sensitized cells are
incubated with tiny paramagnetic beads which are coated with another antibody, and
the beads form rosettes containing the CD34+ cells when exposed to a primary
magnet. Finally, the bead/cell rosettes are incubated with a stem cell releasing agent,
and the cells are washed.
59


Ex Vivo Expansion
Materials and Supplies
Materials and supplies included an ex-vivo cell processing bag, defined medium,
and several cytokines: Stem Cell Factor (SCF), Granulocyte Stimulating Factor
(GCSF), Megakaryocyte Growth and Differentiation Factor (MGDF) (Amgen,
Inc.).
Procedure
Cohort 1 received unexpanded CD34+ selected cells that were frozen at -180
degrees Celsius in liquid nitrogen until the product was released for infusion or
expansion (10% dimethyl sulfoxide [DMSO] was used as a cryopreservant). Cohort
1 patients were also infused with CD34+ cells that were frozen, thawed, and then
expanded in culture. Cohort 2 patients received only CD34+ cells that were frozen,
thawed, and then ex vivo expanded. Unmanipulated PBPC fractions were frozen as
back-up cells for both cohorts.
Prior to expansion, a cell count was performed to determine the number of cells
available following CD34+ selection. The cell culture procedure was performed
using sterile technique under laminar air flow hood. Nutrient media was transferred
to the cell processing bag. The cytokines were reconstituted according to
60


manufacturers instructions. The volume of each cytokine to be added to the culture
was calculated using the culture volume and cytokine concentration. Cells were
injected into the processing bag, and the culture bag was placed in a 37 degrees
Celsius humidified incubator with five percent carbon dioxide for ten days. The
cultures were checked daily for unusual turbidity and media indicator color change.
Also, cultured cells were tested for sterility, including mycoplasma detection.
After ten days an aliquot of cultured cells is removed for viability testing and a
cell count is performed. The cultured cells were processed using the COBE 2991.
After washing the cells, a cell count, viability, and sterility testing is performed on
the supernatant. The final product is only released for infusion if sterility testing is
negative, visual inspection is acceptable, and the viability is greater than 70%.
High-Dose Chemotherapy
Several patient criteria are required before the administration of high-dose
chemotherapy. Chemotherapy dose is based on the patients body surface area
(BSA). Body surface area is calculated using the individual patients height and
weight. The patients BSA and the chemotherapy drug dosage /mA2 is recalculated
prior to infusion. In addition, several tests are required prior to the administration of
chemotherapy: serum creatinine; creatinine clearance; electrocardiogram (EKG);
liver function studies.
61


Consent forms are signed by the attending physician, the patient and a witness.
Chemotherapy orders must be signed by the attending physician, and nurse
practitioner (NP) or fellow. Protocol eligibility and cryopreservation forms are
signed by the attending. Also, the patient must be well hydrated the previous six
hours to chemotherapy, and the bladder must be irrigated for the previous four hours
if applicable. Urine output should be at least 200 cc/hr for three hours. Intravenous
(IV) access must be available other than the central line catheter that is used for
chemotherapy administration, usually a peripheral IV or arterial line. This access is
used to draw blood samples for pharmacokinetics.
Materials and Supplies
Materials and supplies include the following: pumps for bladder irrigation and
cc/cc replacement; pumps for infusion of chemotherapy agents; cardiac monitor;
chemotherapy gloves and gown; disposable underpad; alcohol wipes, gauze, needles,
syringes, tubing with Luer-lock connectors; normal saline for flush solution.
Procedure
Permanent venous catheters and central lines are preferred for the administration
of chemotherapy. If a venous catheter or central line is not available, a peripheral IV
must be inserted with careful consideration given to the site of insertion
(i.e. avoiding areas with hematomas, inflammation, sclerosing, phlebitis, etc ).
62


Chemotherapy drugs that are vesicants (agents that cause blistering or tissue
necrosis) must be infused through a central line.
Chemotherapy infusion is most often performed using a central line catheter. The
line is permanently placed through the patients chest and is tunneled into the
superior vena cava. Tubes are held in place by suture on the outside of the patients
chest. The tubes (lines) on the outside of the chest have caps and clamps for access
to venous circulation. Blood return is checked prior to chemotherapy infusion to
insure proper line placement. A separate tubing set is filled with normal saline, and
this tubing is connected to a bag containing the chemotherapy drug which is in fluid
form. Aseptic technique is used to attach the tubing and bag to the central line, or
peripheral line if applicable. The infusion is usually continuous and a IV pump is
used to provide consistent administration of the drug. The patient is monitored
closely during the infusion for signs of hypersensitivity and anaphylaxis. At
completion of the infusion, the line is flushed with 10 cc of sterile solution. The
patient is monitored periodically thereafter for signs of extravasation (leakage or
infiltration of a vesicant chemotherapy agent into local tissue).
Peripheral Blood Prouenitor/Stem Cell Reinfusion
The peripheral stem/progenitor cells are infused through the patients central line
into the venous circulation and eventually find their way back to the bone marrow
63


where they engraft. The patients receive their cells back after receiving six to eight
days of high-dose chemotherapy.
Materials and Procedure
The patient is hydrated and medicated with Tylenol, Benadryl, and other
medications if necessary. A cardiac monitor is used to prior to infusion and for two
hours after the infusion is complete. Free flowing normal saline (NS) is attached to
the patients central line with intravenous (IV) tubing. The previously prepared cells
are contained in a syringe and are simply pushed into a port in the free flowing NS
tubing at a rate of 1 cc every 10 seconds. At completion of the infusion the central
line is flushed with 10-20 cc NS to completely flush the residual cells into the blood
stream.
Complications
The patient is monitored for signs of any adverse reactions during and after the
PBPC infusion. Symptoms of adverse reactions include the following: hives,
temperature >38.3 degrees Celsius, chills, dyspnea, cough, bronchospasm,
hyper/hypotension, bradycardia, chest pain, back or flank pain, hematuria.
The above signs of adverse reactions can be attributed to several factors directly
related to the infusion product. Bacterial contamination can occur via normal skin
flora or waterborne flora, such as coagulase negative Staphylococcus and
64


Pseudomonas paucimobilis, respectively. The DMSO used for cryopreservation can
cause coughing or a choking sensation, bronchial spasms and dyspnea, and nausea
and vomiting. Also, the DMSO can cause a garlic-like taste in the mouth. However,
not all patients received DMSO preserved cells. Allergic reactions may be attributed
to reagents or antibiotics used in the procedure. Pulmonary edema may result form
circulatory overload of transplanted product and saline infusions. Anticoagulants
contained in the infusion product can cause bleeding due to inactivation of
coagulation proteins and platelets. On the other hand, the product may contain
microaggregates of fibrin, bone, and cell clumps which could lead to thrombotic
problems and possibly cause pulmonary embolism. However, these microaggregates
would more likely be found in bone marrow aspiration products than peripheral
progenitor/stem cell collections.
Furthermore, metabolic possible complications include: hypothermia, citrate
toxicity (affects muscles, including the heart), acidosis, and hyperkalemia (increased
potassium) or hypokalemia (decreased potassium) due to hemolysis during
cryopreservation of the product. Hemolyzed blood may also cause hemoglobinuria,
chills, fever, disseminated intravascular coagulation (DIC), and renal failure.
However, perinephrial blood progenitor/stem cell collections typically have a very
low percentage of red blood cells in the final transplant product. Finally, there is the
critical hematopoietic cell lines that are necessary to sustaining life.
65


CHAPTER 10
RESULTS
Long-term Follow-up on Engraftment
All patients received peripheral blood progenitor transplants between July 1998
and January 1999. Cohort 1 patients were transplanted with a CD34+ expanded and
unexpanded fraction between July 1998 and November 1998. Cohort 2 patients
received only a CD34+ expanded fraction between November 1998 and January
1999. Thus, patients are at various stages posttransplantation. Patients were
followed for the past two years to obtain information on peripheral blood counts to
monitor sustained hematopoietic engraftment.
All follow-up data obtained for white blood counts (WBC), absolute neutrophil
counts (ANC), and platelet counts are presented in Appendix A. Mean WBC for
Cohort 1 patients at last follow-up was 4.8 (range 1.6 to 8.4), and the mean ANC
was 3.0 (range 0.6 to 5.5). Median ANC was 3.2. Mean platelet count for patients
in Cohort 1 at last follow-up was 147, 000 (range 22,000 to 211,000). Patient 3 was
excluded from mean and median calculation due to recent gastrointestinal bleed.
However, she has still maintained a PLT > 20,000/mmA3. Surviving Cohort 2
patients had a mean WBC and ANC of 3.8 (range 2.1 to 4.7) and 2.4 (range 1.2 to
66


3.1), respectively, at last follow-up. Median ANC was 2.4. Cohort 2 had a mean
platelet count of 163,000 (102,000 to 198,000). Blood count results at last follow-up
are presented in Table 10.1. Mean, median, and range for peripheral blood counts
are shown in Table 10.2. The normal reference range for WBC is 4.8 to 10.8 (X
10A3/L/ and the normal range for ANC is 2.0 to 8.1 (X 10A3/wL). The normal
reference range for platelet count is 150,000 to 450,000 (/mmA3) (Harmening, 1997).
Normal ranges for peripheral blood counts will vary depending on methodology
used at different institutions. Patients were considered neutropenic when the ANC
was < 500 per microliter (uL) of peripheral blood. Thrombocytopenia occurred
when patients had a platelet count < 20,000 per millimeter cubed (/mmA3) of
peripheral blood. Patients are already compromised with their disease and prior
chemotherapy. Furthermore, about 60% of patients undergo chest wall radiation
therapy subsequent to their PBPC transplant. Accordingly, patient counts cannot be
evaluated solely on the basis of what is considered normal for the rest of the
population. Therefore, both cohorts have mean counts that are acceptable, and thus
capable of providing protection against infection and bleeding.
The data shows that the majority of patients in both cohorts have maintained
peripheral blood counts at adequate levels over long-term follow-up. However,
Patient 4 in Cohort 1 had problems with pancytopenia (decreased counts in all cell
lines). She had disease recurrence and there is a possibility that bone marrow
67


involvement was preventing normal cells from growing properly. Despite disease,
last follow-up counts were adequate. Further, Patient 8 in Cohort 2 had problems
with slow platelet engraftment and failed to obtain adequate platelet counts. She
was reinfused with back-up cells approximately 5 months after the first transplant.
Figure 10.1.1 to 10.1.5, and Figure 10.2.1 to 10.2.6 represent WBC and ANC for
both cohorts (odd numbered patients chosen for examples) from date of transplant to
date of last known follow-up. Figure 10.3.1 to 10.3.2, and Figure 10.4.1 to 10.4.2
represent examples of platelet counts, for both cohorts, monitored over time. The
first date shown on the graphs is the date of the peripheral blood transplant
commonly referred to as day 0. Initially, patients peripheral blood counts are
dramatically decreased due to high-dose chemotherapy just prior to infusion of cells.
All patients were given GCSF after infusion of cells. The spike early in the graphs
for both cohorts represents response to growth factor and initial engraftment of cells.
Long-term durable engraftment is indicated by a stable leveling off in the graphs and
also demonstrates maintenance of the hematopoietic cell line; thus the results
indicate that the bone marrow can maintain function after myeloablation followed by
progenitor/stem cell rescue. Surviving patients in both cohorts have maintained
peripheral blood counts over time which indicates sustained engraftment of the
hematopoietic cell lines.
68


Figures 10.5.1-10.7.2 represent overlay graphs of all data (WBC, ANC, PLT) for
all patients in Cohort 1 and Cohort 2. All patients are graphed from the same
starting point (day 0) instead of individual transplant date. Patient 3 (Cohort 1) was
excluded from the graphs in Figures 10.5.1 and 10.6.1 for statistical representation of
the majority of the data. Likewise, patients 3 and 4 (Cohort 2) were excluded from
the graphs in Figures 10.5.2 and 10.6.2.
Disease-Free Survival
Medical records were researched to monitor patients disease and survival status.
Patients were monitored for recurring disease by physical examination, tumor
markers, and Computed Tomography (CT scan) of the brain, chest, abdomen, pelvis,
and bone. Data was collected for all 10 patients in Cohort 1, 8 out of 10 patients
remain with no evidence of disease at last follow-up. Thus, the proportion of Cohort
1 patients remaining in remission was 0.80. Patient 3 had metastatic disease to the
liver and brain. Patient 4 had metastasis to the lungs. Disease free survival (DFS) in
months is recorded in Table 10.3. At last known follow-up, Cohort 1 patients with
no evidence of disease have remained in remission from 10 to 28 months.
The proportion of DFS for Cohort 2 was 0.70. Patients 3 did not have recent
documentation on disease status at last follow-up. Seven out of the 10 remaining
patients have no evidence of disease at their individual follow-up, and remain in
remission from 3 to 23 months. Patient 4 had additional chemotherapy for
69


secondary disease to the lungs two months following transplant. She received a stem
cell reinfusion one year later and was well engrafted, but brain metastasis was
discovered five months later. A BM biopsy one month after this revealed
myelodysplastic syndrome (MDS), or preleukemic syndrome. Patient 8 had
metastatic disease to the liver approximately 6 months posttransplant. Patient 11
had slow very slow disease progression in the bone only, at last follow-up. DFS
recorded is based on last written documentation of disease status from follow-up
clinics; some patients may actually be in remission longer than indicated.
Overall Survival
Overall posttransplantation survival (OS), recorded in months, was based on last
known follow-up of peripheral blood counts obtained from patients (Table 10.3).
All Cohort 1 patients were alive at last known follow-up (proportion of overall
survival = 1.0). Based on the last known documentation of peripheral blood counts,
patients range of overall survival (OS) in Cohort 1 is 12 to 28 months.
Proportion of OS for patients in Cohort 2 is 0.82; nine out of the 11 patients were
alive at last follow-up. Patient 4 developed MDS, and Patient 8 had liver metastasis.
Cohort 2 patients received their transplants up to 6 months later than Cohort 1; range
of OS is 3 23 months at last individual patient documentation. OS is based on last
documentation of blood count data from follow-up clinics; thus patients may have a
longer OS than presently indicated.
70


Patient Health Status
Patients experienced some significant changes in health status following
treatment during peripheral blood cell transplant. At last follow-up, the most
common complaints in both cohorts were pulmonary toxicity, peripheral
neuropathy, and post herpetic neuralgia. Pulmonary injury and neuropathies were
caused by drugs used during the high-dose therapy regimen of treatment.
Pulmonary injury manifests in complaints of shortness of breath, and oxygen
desaturation with exertion. Pulmonary toxicity was treated with steroids, such as
prednisone. Neuropathies are most commonly manifested as numbness and tingling
in hands and feet. Neuropathies were treated with a drug called Neurotonin.
Pulmonary toxicity and neuropathies may resolve with treatment; however, the
degree of recovery may not be complete.
Further, some patients complained of fatigue, and musculoskeletal pain. A few
patients also experienced a decrease in short-term memory. Memory loss is
occasionally seen shortly after transplant; however, it is unusual for this to persist in
the long-term. Some patients required treatment for depression. Further, a few
patients experienced vision problems, including blurred vision and ischemic optic
neuritis resulting from chemotherapy drugs. One patient tried to get pregnant
after transplant, but was diagnosed with primary infertility.
71


Furthermore, several patients complained of hot flashes which were secondary
to Tamoxifen therapy in the posttransplant period. Tamoxifen is an antiestrogen
usually used in the palliative treatment of patients with advanced breast cancer
who have estrogen-dependent tumors. On the other hand, a number of patients
were reported as doing very well at last follow-up. One patient had a one year
remission of asthmatic symptoms following transplant. Some patients had no
major complaints at last follow-up, and several patients returned to work part-time
and full-time.
72


Table 10.1 PERIPHERAL BLOOD COUNTS AT LAST FOLLOW-UP
ID COHORT DATE WBC (uL) ANC (uL) PLT mmA3
1 1 11/27/00 8 3.41 211
2 1 12/15/00 4 2.9 156
3 1 12/26/00 8.4 5.5 22
4 1 11/21/00 4.5 3.69 103
5 1 10/06/00 5.4 4.6 140
6 1 03/27/00 4.4 2.93 142
7 1 11/09/00 3.6 1.4 174
8 1 12/21/00 2 1.21 121
9 1 10/31/00 6.5 4.23 145
10 1 11/07/00 1.6 0.6 132
1 2 11/13/00 3.7 1.9 194
2 2 06/01/00 4.2 2.7 102
3 2 03/13/00 3.5 2.1 131
4 2 10/05/00 6.2 5 160
5 2 04/20/00 3.8 2.4 131
6 2 12/13/00 4.7 3.1 170
7 2 06/11/99 2.1 1.2 198
8 2 07/26/99 4.3 ND 20
9 2 04/22/99 3.5 2.3 178
10 2 02/15/00 4.3 3.08 185
U 2 12/14/00 4 2.5 181
WBC=white blood count; ANC=absolute neutrophil count; PLT=platelet count
ND indicates no data available
Counts reported X1000
Table 10.2 MEAN, MEDIAN, AND RANGE
FOR PERIPHERAL BLOOD COUNTS AT LAST FOLLOW-UP
Cohort 1 WBC (uL) ANC (uL) PLT mmA3
Mean 4.8 3 147
Median 4.5 3.2 142
Range 1.6 to 8.4 0.6 to 5.5 22 to 211
Cohort 2 WBC (uL) ANC (uL) PLT mmA3
Mean 3.8 2.4 163
Median 3.8 2.4 178
Range 2.1 to 4.7 1.2 to 3.1 102 to 198
Counts reported X 1000
73


Table 10.3 DISEASE-FREE/OVERALL SURVIVAL FOR COHORTS
ID COHORT DFS MONTHS OS MONTHS
1 1 0 28 0 28
2 1 0 28 0 28
3 1 1 27 0 27
4 1 1 19 0 26
5 1 0 12 0 12
6 1 0 10 0 17
7 1 0 25 0 25
8 1 0 26 0 26
9 1 0 24 0 24
10 1 0 24 0 24
1 2 0 23 0 23
2 2 0 18 0 18
3 2 ND ND 0 15
4 2 1 2 1 22
5 2 0 15 0 15
6 2 0 23 0 23
7 2 0 5 0 5
8 2 1 6 1 6
9 2 0 3 0 3
10 2 0 18 0 18
11 2 1 19 0 23
OFS=Disease Freel Survival; 0= No Evidence of Disease, 1=Evidence of
Disease
OS=Overall Survival; 0=Alive, 1=Death
ND indicates no data available
74


Figure 10.1.1-10.1.5 Cohort 1: Absolute Neutrophil Count vs Date
Date of transplant is the first date on the x-axis. Patients' absolute neutrophil
counts (ANC) are near zero at the time of peripheral blood progenitor/stem cell
(PBPC) transplant. Patients are given granulocyte-colony stimulating factor (GCSF)
posttransplant, which causes a rise in neutrophils shortly after transplant. Engraft-
ment occurs when the ANC is > 500/uL for three consecutive days. Long-term
engraftment occurs when blood counts are maintained overtime as shown on the
y-axis.
Figure 10.1.1 Patient 1
Figure 10.1.2 Patient 3
75


'-J
Os
ANC (X10A3)/UL
D
0)
p-*
to
10/12/98
1/12/99
4/12/99
7/12/99
10/12/99
1/12/00
4/12/00
7/12/00
10/12/00
>
z
o
CO
c
<3
o
o
0)
5
3
>
Z
o
Figure 10.1.3 Patient 5


77


Figure 10.2.1-10.2.6 Cohort 2: Absolute Neutrophil Count vs Date
Date of transplant is the first date on the x-axis. Patients' absolute neutrophil
counts (ANC) are near zero at the time of peripheral blood progenitor/stem cell
(PBPC) transplant. Patients are given granulocyte-colony stimulating factor (GCSF)
posttransplant, which causes a rise in neutrophils shortly after transplant. Engraft-
ment occurs when the ANC is > 500/uL for three consecutive days. Long-term
engraftment occurs when blood counts are maintained overtime as shown on the
y-axis.
78


Figure 10.2.4 Patient 7
S?
O)0)O)OO)O>O>O)O)O>
CO
in
CM CM CO CO
in oo
CM CD
O
in
co
CD
-ANC
Date
79


Figure 10.2.5 Patient 9
o
z
<
Date
---ANC
80


Figure 10.3.1-10.3.2 Cohort 1: Platelet Count vs Date
Date of transplant is the first date on the x-axis. Platelet
counts (PLT) are low on the date of transplant. Engraftment
occurs when PLT is > 20,000/mmA3 for three consecutive days
Long-term engraftment is indicated when blood counts are
maintained overtime as shown by values along the y-axis.
Figure 10.3.1 Patient 2
oOT-tsiioco^-CMirico
Date
Figure 10.3.2 Patient 6
-PLT
Date
81


Figure 10.4.1-10.4.2 Cohort 2: Platelet Count vs Date
Date of transplant is the first date on the x-axis. Platelet
counts (PLT) are low on the date of transplant. Engraftment
occurs when PLT is > 20,000/mmA3 for three consecutive days
Long-term engraftment is indicated when blood counts are
maintained overtime as shown by values along the y-axis.
Figure 10.4.1 Patient 2
NNNJjNNNNNN
Date
---PLT
Figure 10.4.2 Patient 6
Date
82


Figure 10.5.1-10.5.2 Graphic overlay of white blood cell
count (WBC) vs Days post-Peripheral Blood Progenitor
Cell Transplant for Cohort 1 and Cohort 2.
Figure 10.5.1. Cohort 1
WBC (xlOA3)/uL
Figure 10.5.2. Cohort 2
WBC (xlOA3)/uL
83


Figure 10.6.1-10.6.2 Graphic overlay of absolute neutrophil
count (ANC) vs Days post-Peripheral Blood Progenitor
Cell Transplant for Cohort 1 and Cohort 2.
Figure 10.6.1. Cohort 1
ANC (x 10A3)/ul
Figure 10.6.2. Cohort 2
ANC (xlOA3)/uL
84


Figure 10.7.1-10.7.2 Graphic overlay of platelet count
(PLT) vs Days post-Peripheral Blood Progenitor
Cell Transplant for Cohort 1 and Cohort 2.
Figure 10.7.2. Cohort 2
PLT (xlOA3)/mmA3
Figure 10.7.1. Cohort 1
PLT (xlOA3)/mmA3
85


CHAPTER 11
DISCUSSION
Results on Early Neutrophil Engraftment
Studies at the University of Colorado Health Sciences Center with the same
cohort of patients have shown that ex vivo expanded peripheral blood progenitor
cells provide rapid neutrophil engraftment (McNiece et al., 2000). The importance
of this finding cannot be understated because patients become immunocompromised
in the period following myeloablative high-dose chemotherapy and are susceptible to
a variety of opportunistic infections. The sooner the patients neutrophil count
reaches acceptable peripheral blood levels, the better they can fight infections.
Acceptable levels of neutrophils were measured by monitoring the absolute
neutrophil count (ANC). Engraftment was indicated when the patient maintained an
ANC of > or = 500/mL The ANC is calculated by multiplying the percentage of
neutrophils times the total white count. The researchers (McNiece et al., 2000) at
the UCHSC found that patients in Cohort 1 engrafted neutrophils in a median of 6
days and Cohort 2 engrafted neutrophils in a median of 8 days. The range for Cohort
1 was 5-14 days, and the range for Cohort 2 was 4-16 days. Historical controls
receiving unmanipulated cells engrafted neutrophils in a median of 9 days with a
86


Full Text

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LONG-TERM FOLLOW-UP ON BREAST CANCER PATIENTS TRANSPLANTED WITH EX VNO EXPAND ED P E RIPHERAL BLOOD CD34 + CELLS b y Julie Ruth Candia B S ., Colorado State University 1988 A thesis submitted to the University of Colorado at Denver in partial fulfillment of the requirements for the degree of Master of Basic Science 2001

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2001 by Julie Ruth Candia All rights reserved

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This thesis for the Master of Basic Science degree by Julie Ruth Candia has been approved by i/3oj() I Date

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Candia Julie Ruth (M B.S., Applied Science) Long-term Follow-up on Breast Cancer Patients Transplanted with Ex Vivo Expanded Peripheral Blood CD34 + Cells Thesis directed by Ian K McNiece PhD ABSTRACT Patients with v arious stages of breast cancer were administered growth factor(s) and underwent leukapheresis to collect peripheral blood progenitor / stem cells for transfusion following high-dose chemotherapy Transplantation is required to establish hematopoiesis The patients in this study were consented and enrolled consecutivel y into two cohorts Cohort 1 consisted of 10 patients who were transfused with both CD34 antigen (CD34 +) selected cells which were expanded ex vivo and with unexpanded CD34 + selected cells The first cohort of patients established the safety of administerin g expanded CD34 + cells Cohort 2 consisted of 11 patients who were transfused only with the CD34 selected cells that were expanded Short-term results indicate that all patients in both cohorts demon s trated rapid engraftment of neutrophils posttransplant and that neutrophil engraftment occurs faster with expanded CD34 + cells than unexpanded cells IV

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The long-term engraftment kinetics were investigated in the present study b y measuring peripheral blood counts The median absolute neutrophil count for Cohort 2 at long-term follow-up was 3.8 X 101\3/ uL and the median platelet count was 178 X 1 0/\3 / mm/\ 3. These results indicate that CD34 + expanded cells can provide durable long-term engraftment. This abstract accurately represents the content of the candidate s thesis I recommend its publication Signed v

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DEDICATION I dedicate this thesis to : Candace B Malet RN, BSN (University of Colorado Health Sciences Center) for her unfaltering understanding patience support and friendship My family (Vina Candia, Robert Gonzalez ; Julian, Jackie, and Sarah Candia) for their love and support, and for tolerating hearing the word "thesis for two years

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r ACKNOWLEDGEMENTS Dr Ian K. McNiece (University of Colorado Health Sciences Center) my advisor for his patience understanding guidance and knowledge. Dr. Elizabeth J. Shpall and the Bone Marrow Transplant Program Research Team for giving me the opportunity to participate in research at the University of Colorado Health Sciences Center. Dr. Zenas Hartvigson for his genuine guidance, support and enthusiasm in the Master ofBasic Science Program at the University of Colorado at Denver. Dr. William Briggs for his patience and participation as a committee member at the University of Colorado at Denver. Dr Anna Baron and Chan Zeng at the University of Colorado Health Sciences Center for their assistance with statistical analysis Tom Porterfield for his friendship, support and patience. Glenda Robles for her lasting friendship understanding and support Raymond Kingery for his computer program assistance Julian Candia for his computer program assistance ; and for having a cute little daughter Sarah to distract me from my studies and to make me laugh and smile

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Figures Tables CHAPTER CONTENTS XIII XIV 1 INTRODUCTION Problem Statement . . . . . . . . . 1 Purpose of the Stud y . . . . . . . . . 2 2 HEMATOPOIESIS ....................................... 4 Hematopoietic Progenitor and Stem Cells .................. 4 Characteristics ofMature Peripheral Blood Cells ............. 8 Bone Marrow Structure and Function . . . . . 10 Granulocytopoiesis Maturation Sequence . . . . 16 Myeloblast . . . . . . . . . . 1 7 Promyelocyte . . . . . . . . . 17 Myelocyte ......................................... I 8 Metamyelocyte . . . . . . . . . 18 Neutrophilic Band . . . . . . . . 18 Segmented Neutrophil . . . . . . . 19 VIII

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3 HISTORY OF PERIPHERAL BLOOD PROGENITOR/STEM CELL TRANSPLANTATION . . . . . . . 21 Cellular Transplants . . . . . . . . 21 E arly Animal Models . . . . . . . . 22 Hematopoietic Stem Cells in Human Peripheral Blood ....... 24 Attempts to Quantitate and Identify the Progenitor / Stem Cell . . . . . . . . 26 CD34 + Antigen . ..... ........................ .... 27 Optimization ofPeriphera l Blood Progenitor / Stem Cell Collection ........................ 27 4 CD34 + CELLS .... ... .. .. ... ........................... 30 Discovery of the CD34 Antigen . . . . . . 30 5 GRANULOCYTE-COLONY STIMULATING FACTOR ....... 33 Function ...,..., . . . . . . . . . . _,_, Structure ........................................... 34 Mobilization ofProgenitor / Stem Cells .................... 34 6 E X VIVO EXPANSION AND SELECTION OF HEMATOPOIETIC PROGENITOR/STEM CELLS . . . . . . . 37 E x Vivo E xpansion and Cytokines ..................... 37 Reduced Tumor Cell Content ........................... 39 Decreased Neutropenia .......................... ..... 41 7 HIGH-DOSE CHEMOTHERAPY .......................... 43 IX

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Purpose ............................................ 43 Characteristics of Oncology Drugs .................. ..... 44 Cyclophosphamide (Cytoxan) .......... .. ............ 44 Cisplatin (Platinol) ................................. 45 Carmustine (BCNU) ............. .................. 45 Docetaxel (Taxotere) ...................... ......... 46 Carboplatin (Parap latin ) . . . . . . . .46 Melphalan ............... ........................ 47 Paclitaxel (Taxol) .................................. 47 8 BREAST CANCER .................................... 49 Types of Breast Cancer .................... ........ 49 Breast Cancer Risk . . . . . . . . 51 9 METHODS ........................................... 53 Patients . . . . . . . . . . . 53 Leukapheresis ....... ..... ........................... 55 Materials and Supplies ............................. 55 Procedure . . . . . . . . . 57 CD34 + Selection . . . . . . . . . 59 Materials and Supplies ........ .................. .. 59 Procedure . . . . . . . . . 59 X

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Ex Vivo Expansion .................................. 60 Materials and Supplies . . . . . . . 60 Procedure . . . . . . . . . 60 High-Dose Chemotherap y ............................. 61 Materials and Supplies . . . . . . . 62 Procedure . . . . . . . . . 62 Peripheral Progenitor / Stem C ell Reinfusion .............. 63 Materials and Procedure . . . . . . . 64 Complications . . . . . . . . . 64 10. RESULTS ............................................ 66 Lon g -term Follow-up on E ngraftment .................... 66 Disease-Free Survival ............................... 69 O v erall Survival .................................... 70 Patient Health Status . . . . . . . . 71 11. DISCUSSION . . . . . . . . . . 86 Results on E arly Neutrophil Engraftment . . . . 86 Platelet Recovery Time . . . . . . . 87 CD34 + Counts . . . . . . . . . 88 Tumor Content . . . . . . . . . 89 Long-term Engraftment . . . . . . . 90 X I

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12. CONCLUSION ...................................... 93 APPENDIX ....................................... ............ 95 A. Appendix A: Follow-up Peripheral Blood Counts for Cohorts 1 and 2 . . . . . . . . . 95 B Appendix B : Thesis Defense Slide Presentation . . . 126 REFERENCES . . . . . . . . . . . . 133 XII

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FIGURES Figure 2.1 Schematic View ofHematopoiesis .......................... .... 5 2.2 Scheme ofHematopoietic Cell Lines ............................ 7 2 3 Fractal Pattern ofBone Marrow ............................... 1 2 2.4 Graphic Presentation of Bone Marrow Tissue . . . . . 14 2 5 Granulocytopoiesis ......... .. ............................. 20 4 1 Schematic of Cell Surface Antigens ...................... ..... 32 9.1 Methods Schema for Cohort Patients . . . . . . 54 10. 1.1-10 .1.5 Cohort 1 : Absolute Neutrophil Count vs Date 75-77 10. 2 .1-1 0.2.6 Cohort 2 : Absolute Neutrophil Count vs Date 78-80 10.3 1-10 3.2 Cohortl:PlateletCountvsDate ................. .... 81 10.4 .1-1 0 4 2 Cohort 2 : Platelet Count vs Date ............ .......... 8 2 10.5. 1-10.5.2 Graphic Overlay of White Blood Cell Count (WBC) vs Days Post-Peripheral Blood Progenitor Cell Transplant for Cohort 1 and 2 . . . . . . . . 83 10. 6 1-10.6.2 Graphic Overla y of Absolute Neutrophil Count (ANC ) vs Days Po s t-Peripheral Blood Progenitor Cell Transplant for Cohort 1 and Cohort 2 . . . . . . 84 10.7 1-10 7 2 Graphic Overlay ofPiatelet Count (PLT) vs Days Post-Peripheral Blood Progenitor Cell Transplant for Cohort 1 and Cohort 2 . . . . . . . . . 85 X Ill

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TABLES Table 10. 1 Peripheral Blood Counts at Last Follow-up .................... 73 10. 2 Mean Median and Range for Peripheral Blood Counts at Last Follow-up . . . . . . . . 73 10.3 Disease-Free/ Overall Survival for Cohorts ..... ............... 74 XIV

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CHAPTER 1 INTRODUCTION Problem Statement Ex vivo expanded progenitor cells used for hematopoietic support provide rapid engraftment of neutrophils in breast cancer patients receiving high-dose chemotherapy as demonstrated by researchers at the University of Colorado Health Sciences Center (UCHSC) (McNiece et al., 2000). In particular specific progenitor / stem cells called CD34 + cells are specifically and magnetically selected from other hematopoietic cells and subsequently expanded in culture ( McNiece et al. 1999). Riche! et al. (2000) have shown that CD34 + cells isolated usi ng magnetically activated cell sorting techniques provide safe and rapid engraftment in breast cancer patients In addition recent studies suggest that expansion and/or selection of CD34 + progenitor cells significantly decreases and ma y even eliminate tumor cells in the final product to be infused for transplantation (Voso et al. 1999 ; Lundell Vredenburgh Tyer, DeSombre & Smith 1998) Studies have shown that there is consistentl y a significant loss in the number of CD34 + progenitor / stem cells to be infused using onl y CD34 + selection techniques (Riche! et al. 2000 ; Martin-Henao et al. 2000) However ex vivo expansion following CD34 + selection

PAGE 16

ameliorates the loss of CD34 + selected cells Thus ex vivo expansion accomplishes several important functions including the possibility of increasing the number of engrafting cells to be infused and decreasing or possibly eliminating, contamination of the transplant product with tumor cells Expanded cells have the potential of reducing the period that patients are thrombocytopenic (experience a low platelet count) which can lead to bleeding complications. In addition the use of CD34 + expanded cells can shorten the time of neutropenia and decrease the amount of time that the patient is immunocompromised following high-dose transplant therapy Thus the possibility of using expanded progenitor / stem cells for transplantation offers substantial benefits for the patient. Purpose of the Study The purpose of the study is to evaluate the long-term engraftment kinetics of growth factor-mobilized autologous peripheral blood prog e nitor stem cells (PBPC) which have been immunomagnetically CD34 antigen selected and expanded ex vivo and are subsequently infused into breast cancer patients following high-dose chemotherapy Patients were followed for up to two years after receiving their PBPC transplant. The rate and duration of sustained hematopoietic reconstitution produced is measured by peripheral blood counts performed on the patients posttransplantation. The data that is of particular interest are white blood counts absolute neutrophil counts and platelet counts in the follow-up of hematopoietic 2

PAGE 17

engraftment. Long-tenn follow-up is important in evaluating sustained engraftment of expanded progenitor / stem cells and may indicate whether or not the most primitive stem cells are maintained in expansion culture Without durable engraftment patients may become aplastic and are unable to maintain repopulation of the blood cells which are essential for life In addition the patient's disease and health status are important to the study 3

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CHAPTER2 HEMATOPOIESIS The dynamic process of blood cell production and development is called hematopoiesis Mature blood cells, such as leukocytes (white cells), erythrocytes ( red cells) and thrombocytes (platelets) must be maintained at adequate levels in the body and are very important to an individual s health Leukocytes are comprised of granulocytes lymphocytes and monocytes. Granulocytes are important in fighting infections lymphocytes play an important role in the immune response and monocytes engulf debris and pathogens Erythrocytes provide a variety of important functions in the body such as oxygen transport and excretion of carbon dioxide and delivery of nutrients to other cells Thrombocytes are important in the mechanism of blood clotting termed hemostasis (Bell & Hughes 1997) Hematopoietic Progenitor and Stem Cells All ofthe above cells are derived from undifferentiated somatic cells called hematopoietic stem cells which are morphologically indistinct. Plurip o t e nt s t e m cells are not only able to self-replicate but they can also differentiate into either the multipol e ntial (my e l oid) s t e m ce ll or th e l y mph oid s t e m c ell. The lymphoid stem cell line only produces the T and B lymphocytes The multipotential (myeloid ) stem cell 4

PAGE 19

is capable of giving rise to several different cell lines Multipotential cells which become committed to a particular cell line are called proge nitor cells These various blood cells lines are granulocytes erythrocytes monocytes and megakaryocytes. Each cell line has morphologically distinct characteristics and functions (Bell & Hughes 1997 ; Hoffman 1995). Stem cells and progenitor cells express an identifying antigen on the cell surface known as the CD34 antigen These cells are termed CD34 + cells ( see Figure 2 1 ). Figure 2.1 Schematic view of hematopoiesis Hematopoiesis Long-term Cells Short-term --------: I Precursor Cells I '-....--5

PAGE 20

Committed progenitors can only proliferate and mature along a single pathway (Naeim 1998) A committed progenitor can be grown in culture to form colonies of cells called colony-forming ce ll (CFC) or colony-forming units (CF U) In v itro growth of committed progenitors requires specific colony-stimulating factors. CFU-GM i s the progenitor cell for neutrophilic granulocytes and monocytes The CFU-GM becomes even more specific and becomes either a CFU-G, which differentiates into a neutrophil or a CFU-M which differentiates into a monocyte CFU-Eo originate from the multipotential myeloid cell line and different i ate along the eosinophilic pathwa y CFU-Bas originate from the m ye loid cell line to become basophils E rythrocytes ( red blood cells) will be produced from burst-forming unit-erythroid (BFU-E). Megakaryocytes will grow from cell s which ha ve committed to the CFU-Meg line and will eventually become mature thrombocytes ( platelets ) (N aeim 1998 ) (se e Figure 2.2). 6

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Fia=ure 2.2 Schema of hematopoietic ce11 lines: showing development of different cell lines into mature cells. Schema does not detail complete maturation sequence ;--, :=I I =-= ,---..., I r-=--l -l : !--1 ----l' l '-=r--c:l I ::::!E g ic..r> l]fl _jH I 1 r:;-l I L.L.J I I ----' !._ _____ _____ --c:::;) Cltl ____ [_ ______ LY c::: I I l __ _j L _____ l r--u-1 I :2 I co 8-' 1 i?. I I 1 l <.....> L ___ .. ... --! r 1 I <.....> : co L _________ 7

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Studies propose a stochastic model for self-replication and differentiation of hematopoietic cells This model is based on differentiation as a random event and the ability of the stem cell to choose from several options at the time of commitment. A slight change in initial conditions may result in thousands of mature cells formed from one stem cell. Stem cells are influenced towards self-replication or differentiation based on complex interactions with extrinsic and intrinsic factors ( Naeim 1998) Characteristics of Mature Peripheral Blood Cells Granulocytes consist of cells called neutrophils eosinophils and basophils These cells are named for their characteristic granules found in the cytoplasm These granules and other morphologic characteristics become evident when the cell is stained with Wright's stain The granulocytes are very similar in size approximately 14 micrometers (urn) (Bell & Hughes 1997) However each type of granulocyte is unique and can easily be distinguished under the microscope after staining. Neutrophils are the most numerous cell in the peripheral blood comprising 50-70% of the total white cells They have a very important function in fighting bacterial infections The cytoplasm of these cells stains light pink with many tiny, pink granules The nucleus is usually segmented and stains dark purple with Wright's stain. Eosinophils function in allergic reactions The granules ofthis cell 8

PAGE 23

are large and round, and they stain a very distinct bright orange The nucleus is bilobed and stains dark purple Basophils fight parasitic infections They have very large, coarse granules which stain very dark purple to black. Basophils and eosinophils comprise the smallest percentage of cells in normal adult peripheral blood (0-6% total) (Bell & Hughes 1997). Normal adult blood contains 20% to 40% lymphocytes and is the next most numerous cell. Lymphocytes consists ofT and B immune cells ; both are derived from a common lymphoid progenitor cell. There are two different types ofT cells Helper T cells and Cytotoxic T cells When activated, Helper T cells produce polypeptides which help to activate B cells and Cytotoxic T cells B cells are responsible for producing the five different antibodies (IgM IgG lgA IgD lgE) T and B cells are difficult to distinguish morphologically but are easily distinguished by cell surface markers Lymphocytes are smaller than the granulocytes usually 7 to 10 urn The dense round nucleus is large compared to the cytoplasm and stains dark purple The cytoplasm usually has round margins and stains a fairly dark blue However lymphocytes that are reacting to appropriate antigenic stimuli will cause the cell to enlarge and the nucleus to become irregularly shaped and less dense (Bell & Hughes 1997). Monocytes comprise about 2% to 9% of the leukocytes in normal adult blood Monocytes are known for their ability to phagocytize various debris ", such as cell 9

PAGE 24

fragments bacteria or fungi The monocyte is larger than a neutrophil ranging in size from 15 to 18 urn The abundant cytoplasm stains grayish-blue and may be irregular-shaped Also the cytoplasm contains numerous small reddish granules The nucleus is fairly large and is usually kidney-shaped and deeply folded The distinctive monocyte nucleus resembles a brain because of the indentations or convolutions (Bell & Hughes 1997) Monocytes also migrate from the bloodstream to the tissues and are then called macrophages Macrophages perform the same functions in the tissues as in the bloodstream Thrombocytes ( platelets ) are the smallest cells in the peripheral blood varying in size from 1 to 4 urn However this is the mature form of the cell ; the thrombocyte descends from the largest hematopoietic cell in the bone marrow called the megakaryocyte When a megakaryocyte has completed maturation., the cytoplasm fragments to form several thousand individual thrombocytes (or platelets). The platelet has a very important role in the blood clotting cascade for preventing bleeding with injury to vessels Platelets do not have a nucleus since the y are cytoplasmic fragments Platelets stain purple-blue and have small reddish granules ( Bell & Hughes 1997 ) They resemble tiny little cotton balls Bone Marrow Structure and Function All of the various cell lines follow a sequence of maturation Maturation and production of hematopoietic cells in the adult begins in the bone marrow called 10

PAGE 25

medullary h e matopoiesis. Medullary hematopoiesis is usuall y a steady state process. In the adult hematopoietic cells are produced in the sternum ribs, pelvis vertebrae, and skull (Bell & Hughes 1997). Extra m edullary hematopoi es is occurs in the spleen, liver thymus and lymph nodes usually in response to a demand for increased blood cells Hematopoietic stem cells in the bone marrow respond to a variety of regulatory cytokines that cause them to become committed progenitor cells The progenitor cell will become committed to one particular cell line which has morphologically distinct maturation characteristics The granulocytic cell line will be used later to illustrate the maturation process (Bell & Hughes 1997). Bone marrow (BM) is derived from mesenchymal tissue (Naeim, 1998 ) which is a diffuse network of tissue derived from the embryonic mesoderm (Anderson K., & Andeson L., 1998) The spatial organization ofthe bone marrow is a fractal ge ometry which consists of man y spaces and surfaces that wrinkle, branch and fold (see Figure 2 3). 11

PAGE 26

Fi2ure 2.3 Fractal pattern of bone marrow : shows pattern of cell distribution in the bone marrow. 12

PAGE 27

The hematopoietic component supported b y the microenvironment of the bone marrow constantly cycles through proliferation and differentiation The microenvironment is composed of stromal cells which include endothelial cells macrophages fibroblasts adipocytes osteoblasts and osteoclasts (Naeim 1998 ; Link 2000) The stromal cells are distributed randomly and unevenly to form the extracellular matrix (ECM) of the bone marrow Hematopoietic cells are supported by a fine reticulin mesh which is made up of subtances produced by the stromal cells such as collagen fibronectin laminin thrombospondin hemonectin proteoglycans glycoproteins and glycosaminoglycans ( Naeim 1998 ; Link 2 000) (see Figure 2.4) 13

PAGE 28

2.4 Graphic presentation of bone marrow tissue: (A) arteriole (B) endothelial cells (C) central sinus (E) areas of erythropoiesis (F) fat tissue (G) areas of granulocytopoiesis (H) nutrient histiocyte (I) lymphocyte (J) plasma cell (K) megakaryocyte (L) supportive adventitial-reticul urn cells 14

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The stromal substances which comprise the EC M are believed to regulate hematopoietic activity via specific cell surface receptors called adhesion molecules (Link 2000) Thus the EC M is responsible for facilitating cell-cell interactions and binding growth factors to interact with hematopoietic cells In addition the ECM is probabl y inv olved in the migration ofhematopoietic cells (N aeim 1998 ; L ink 2000) Adhesion molecules on the surface of cell membranes of stromal cells also have a role in proliferation activation and homing of hematopoietic cells to the bone marrow microenvironment after intravenous infusion Link (2000) refers to studies that indicate that lectin s on the membrane surface of hematopoietic pro ge nitor cell s may recognize galactose and mannose in the bone marrow microenvironment and this ma y be the molecular basi s for recognition The very late antigen-4 (VL A-4 ) is an integrin thought to be important in homing and is expressed on hematopoietic pro g enitor / s tem cells Link (2000) also refers to s tudies in which antibodi es to VLA-4 inhibit adhesion of CD34 + cells to bone marrow stromal cells Inhibition of a dhesion in the bone marrow leads to mobilization ofCD34+ cells in the peripheral circulation Hematopoiesis is restricted to the extravascular spaces of the bone marrow However hematopoietic cells are interspersed among the thin-walled venous sinuses within the bone marrow E ndothelial cells line the venous sinuses (Naeim 1998 ; Link, 2000). Migration of hematopoietic cells into the intravascular space probably 15

PAGE 30

" occurs near interendothelial cell junctions and regions where the endothelial-cell basement membrane ECM is thinned or absent Link 2000 p 25)." Granulocytopoiesis Maturation Sequence Production of the neutrophil starts in the bone marrow from precursors committed to the m y eloid cell line The neutrophilic cell s tarts as a very immature cell called a myelobla s t and progresses in a continuum of cell maturation to the most mature cell called a seg m e nted n e utr o phil. Several notable cytologic characteristic s signifY the progressive stages of the maturation sequence (Bell & Hughes 1997) (see F igure 2 5 ) : Generall y the diameter of the immature cell is greater than the mature cell. T he overall size of the cell becomes smaller as the cell matures The immature cell will has a high nucleus to cytoplasm (N : C ) ratio ; thus the nucleus is much more prominent than the cytoplasm. The nucleus becomes s maller a s the cell matures and the shape of the nucleus changes The nucleus also shows other obvious changes as the cell matures The immature nuclear chromatin i s v ery fine but as the nucleus matur es it becomes more dense N ucl eo li (contains deoxyribonucleic acid [DNA] templates for ribosomal ribonucleic acid [RNA] ; enlar ged during protein s y nthesis ) are visible in immature cells but not i n more mature cells 16

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The previously mentioned cell characteristics are visible with staining as are other characteristics such as changes in granulation and the intensity and color produced with Wright's stain Myeloblast The myeloblast has a round nucleus which stains predominately red and contains one or more nucleoli The chromatin is very delicate and fine resembling a lacy pattern The slight to moderate cytoplasm is nongranular and stains blue but is darker at the periphery than next to the nucleus The lighter area adjacent to the nucleus is due to the Golgi body ( Bell & Hughes 1997 ) Promyelocyte Development of large primary granules identifies the cell as a promyelocyte. These early granules may cover the entire cell and stain dark blue or reddish blue The granules are usuall y round but can vary in shape. Like the myeloblast the nucleus is usually round and still occupies a large volume of the cell However the chromatin pattern is slightly more coarse and nucleoli ma y be visible but may not be as distinct as in the myeloblast. The nucleus has less red and stains more bluish The cytoplasm is still blue with a lighter area adjacent to the nucleus ( Bell & Hughes 1997) 17

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Myelocyte The initial presence of secondary lysosomal granules is referred to as the dawn to neutrophilia and the cell is classified as a my elocy te. The smaller secondary granu les stain reddish or pinkish ; thus as the cell ages the cytoplasm becomes less bluish and more pinkish. The nucleus may be round or oval or flattened on one side. The chromatin continues to thicken and nucleoli are either absent or indistinct. Also the cytoplasm becomes more abundant than the promyelocyte (Bell & Hughes 1997) Metamyelocyte When the nucleus becomes indented or bean-shaped the cell is classified as a m e tamy e lo cyte The nuclear chromatin is noticeably clumped and the purple nucleus becomes smaller. The cytoplasm stains more pinkish because of the predominance of small pinkish secondary granules (Bell & Hughes 1997 ) Neutrophilic Band The nuclear indentation becomes more marked as the cell continues to mature. Often the now coarser dark purple nucleus is shaped like a horseshoe and shows s igns of degeneration The cytoplasm is filled with small, pink secondary granules 18

PAGE 33

and maybe an occasional dark primary granule ( Bell & Hughes 1997) These defining characteristics now classify the cell as a band Segmented Neutrophil The mature n e utrophil has a nucleus which is separated into lobes which are connected by a narrow filament. The nuclear chromatin is very clumped and appears dark purple The cytoplasm stains a light pink and contains many small pink secondary granules Few primary granules may be present (Bell & Hughes 1997) Neutrophjls are stored in the bone marrow and are released into the peripheral blood for approxjmately 7 to 10 hours At this time the cells exit the blood stream and enter tissues and are subsequently replaced b y other cells from the marrow 19

PAGE 34

2.5 Granulocytopoiesis Sta ges of neutrophilic granulocytopoiesis illustrate sequence starting from an immature precursor the myeloblast to the mature segmented neutrophil. 2 0

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CHAPTER 3 HISTORY OF PERIPHERAL BLOOD PROGENITOR/STEM CELL TRANSPLANTATION Cellular Transplants Exciting research in several different clinical disciplines over the last 50 years has led to the development of more than 20,000 human cellular transplantations being performed annually Cellular transplants include hematopoietic cells from the bone marrow peripheral blood or cord blood The focus of this paper is on hematopoietic pro ge nit or / ste m cells mobilized into the bloodstream which are called p e riph e ral blood progenitor/stem ce ll s (PBPC) PBPC is generall y used as an abbreviation for p e riph era l blood progenitor cells, and encompasses a transplant product that contains both progenitor and stem cells. Initially cellular transplants were onl y carried out on patients with terminal diseases However toda y transplantation may be used preferentially in treating patients in the early stages of disease Cellular transplants have been used to treat patients with a variety of hematologic and oncologic malignancies such as acute and chronic leukemias non-Hodgkin's and Hodgkin s l y mphomas Multiple Myeloma and a variety of solid tumors (Thomas, 1999 ; Mangan 1995). 21

PAGE 36

In 1909 Alexander Maximow a Russian from St. Petersburg, gave a lecture on the possible existence of small, easily transportable cells, which were part of the white blood cell population in the bloodstream and were capable of regaining their full pluripotentiality (able to differentiate into all hematopoietic lineages) His was the first publication to suggest the existence of hematopoietic stem cells in the bloodstream. Forty years later, research begin in several areas which contributed to our knowledge of the physiology of the stem cell (Korbling & Fliedner 1996) Early Animal Models Studies after World War II began to unravel the mysterious nature of the stem cell In 1949 pivotal experiments showed that by shielding the spleen of mice during lethal total body irradiation permitted their survival. Thus, the theory of stem cell migration was demonstrated by showing that hematopoietic cells in the spleen were capable of circulating and repopulating or engrafting the ablated bone marrow (Jacobson Marks, Robson et al., 1949) In addition continuing research supported the existence of stem cells in the circulating blood, and their ability to restore hematopoiesis in several animal models. Radiolabeling cell techniques provided additional evidence that stem cells were permanent residents of the circulating blood (Bond, Fliedner, & Archambeau, 1965) Previously it was believed that these immature cells were only found in the peripheral blood under pathological conditions. In the 1960's, studies in canines 22

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provided even more evidence for the peripheral circulating stem cell. Canines were also used in clinical PBPC transplantation studies Successful autologous transplantation was performed after lethally irradiating dogs and subsequently giving them their own PBPC back. The canine autologous stem cells were collected, and either set aside or cryopreserved before the stored cells were infused intravenously (Cavins Scheer, Thomas, & Ferrebee 1962) Of note autologous human bon e marrow transplants were already being performed in the 1950 s (Kumick et al. 1958) A patient with malignant lymphoma received an autologous bone marrow transplant in 1959 following treatment with high-dose nitrogen mustard and remained in remission for 21 years (Haurani 1997) The first peripheral blood transplant was not performed until the 1970 s (Thomas 1999 ; Mangan 1995 ; Korbling & Fliedner 1996). The canine model contributed to other important observations in hematopoietic stem cell studies which are applicable to human treatment today Dogs that were given supralethal irradiation and all oge n e i c (cells transplanted into a recipient from a donor) transplants experienced various problems in transplantation such as failure to engraft and engraftment with Graft Versus Host Disease ( GVHD) (immune reaction of the engrafted lymphoid cells against the tissues of the host) (Thomas et al. 1962 ) Also studies showed that dogs could be successfully engrafted using chemotherapy only with no prior irradiation (Storb et al. 1969 ) 23

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Hematopoietic Stem Cells in Human Peripheral Blood E. J. Friereich was involved in experiments in the early 60 s which showed that bone marrow engraftment occurred in patients with myelosuppression after being transfused with peripheral blood leukocytes from patients with chronic myelogenous leukemia McCredie Hersh & Freireich (1971) were also involved in previous experiments which showed that engraftment occurred when peripheral blood leukocytes were collected with a cell separator from donors and transfused into patients who were myelosuppressed with chemotherapy McCredie et al. (19 71) took note of these experiments and the earlier experiments with the lethall y irradiated and normal dogs In light of these observations they proposed that stem cells exist in the peripheral blood are capable of repopulating the bone marrow McCredie et al. (1971) collected white blood cells (leukocytes) from normal donors b y three different techniques They collected bone marrow leukocytes (BML), peripheral blood leukocytes (PBL) and peripheral blood leukocytes collected by a blood-cell separator or leukapheresis (LL). The three different leukocyte collections were cultured and observed for colony formation Colony-forming cells (CFC) were grown from all three types of collections CFC grew more regularly in the BML than the PBL or LL. CFC were higher in the LL culture than the PBL culture. This observation indicated that CFC are more 24

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concentrated with the leukapheresis procedure than a peripheral blood draw Furthermore, the majority of circulating leukocytes collected by leukapheresis are large mononuclear cells. Thus the investigators suggested that the CFC (i.e. progenitor cells) must be present in this cell fraction. Furthermore they proposed that peripheral blood collections ma y be as efficient as bone marrow in reconstitution of granulocytic cells in patients that are myelosuppressed (McCredie et al., 1971) Barr Whang-Peng & Perry (1975 ) may have been the first to clearly demonstrate the presence of hemopoietic stem cells in human peripheral blood They harvested mononuclear cells from the peripheral blood of normal donors. Since hemopoietic cells closely resemble lymphocytes the collection of mononuclear cells were se parated from thymus-dependent lymphocytes ( T cells) by rosette formation with sheep erythrocytes The mononuclear cell fraction which failed to exhibit rosetting was further separated based on size A monocyte concentrate was obtained which consisted mainly ofmonocytes and a smaller portion ofbasophils and lymphoid cells. Monocytes bursa-equivalent l y mphocytes ( B cells), and basophils were removed from this concentrate using immunoglobulin G and complement complexes T his caused rosetting formation of these cells Cells that exhibited rosetting characteristics failed to grow in culture and only contained the original cells (i.e l y mphocytes monocytes and basophils ) Residual 25

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cells which failed to rosette were cultured in vivo However these cells proliferated and differentiated into erythrocytic granulocytic, and megakaryocytic progeny thus demonstrating true pluripotentiality of a small fraction of cells in human peripheral blood (Barr Whang-Peng & Perry 1975) Later studies performed with long-term cultures of normal human peripheral blood showed that primitive hematopoietic cells, which are closely related to stem cells are present in the circulation although at very low levels Furthermore these cells are indistinguishable in their properties of differentiation and proliferation from primitive cells in normal bone marrow (Udomsakdi et al. 1992). Attempts to Quantify and Identify the Progenitor / Stem Cell The case for the circulating stem cell, capable of protecting lethally irradiated animals and differentiating into all hematopoietic cell lines, was definitely strong after monumental research in the 1950's and 1960 s The next step was to be able to identify and quantify the progenitor / stem cell in the blood. This would not be an easy feat because the progenitor/stem cell is a morphologically indistinct cell appearing as a medium-sized mononuclear lymphoid-looking cell (Mangan, 1995) The first quantitative experiments used colony forming units (CFUs) for enumeration Today CFUs are known to be characteristic of the progenitor cell not the stem cell. Scientists discovered that colonies formed on the spleen of mice which had been irradiated and subsequently injected with bone marrow. Eventually 26

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an estimate of human progenitor/stem cells was obtained by culturing blood or marrow white blood cells specifically mononuclear cells (Till & McCulloch, 1963). In vitro assays improved by using LTC (long-term cultures) which are indicative of stem cells (Udomaski et al. 1992). LTC further improved the approximation of progenitor/stem cells in the peripheral blood However these culturing techniques were time consuming and thus were impractical for routine identification and quantitation of peripheral blood progenitor / stem cells (Mangan 1995) C34 + Antigen In 1984 an antigen expressed on all types of progenitor and stem cells was discovered and termed the C D34 antigen Cells expressing the CD34 antigen are referred to as C D34 positive cells (CD34 + ). This discovery enabled more rapid identification of the circulating progenitor / stem cells Monoclonal antibodies and flow cytometry were used to identifY subsets of the CD34 + cells, either the committed progenitor cell group (CD38 + ) or the pluripotential cell group (CD38-) Again, these cells commonly express the CD34 antigen, but also express different antigens that further differentiate these cells (Civin et al., 1984). Optimization of Peripheral Blood Progenitor / Stem Cell Collection The discovery ofthe CD34 + antigen helped in the enumeration ofPBPC under steady state conditions ; however researchers discovered that less than 1% of all the 27

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mononuclear cells in the bloodstream were CD34 +, and of the CD34 + cells less than 0 1% were actual stem cells (Sienna Pregni Brando et al. 1991 ) The feasibility of collecting PBPC under steady state conditions was indeed questionable In the 1970 s blood separation devices called apheresis machines were being used in most blood banks for collecting platelets and white blood cells Cells are separated based on cell density and collected by centrifugation The apheresis technique was adapted for the collection ofPBPC. Central venous catheters and the automation of the apheresis instrument allowed convenient access to large volumes of peripheral blood processing Ne v ertheless this meant that large volumes ofblood had to be processed in the laboratory This cumbersome method was not cost-effective and was potentially toxic because of the large volume of cells cryopreserved with chemicals and subsequently infused Therefore, under steady s tate conditions man y apheresis procedures were required for the collection of an adequate number ofPBPC (Mangan 1995). Studies conducted in the 1970 s and early 80 s showed that stem cells were regulated by numerous growth factors The factors are named for the cell line that they effect such as g ranulocyt e co lon y s timulatin g fac tor ( GCSF) which stimulates the production of a particular a group of white cells called granulocytes Clinical use of this growth factor in the early 80 s demonstrated that it was capable of causing the bone marrow to release progenitor / stem cells into the peripheral blood a process termed mobili za tion In 2 8

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addition in 1976 progenitor / stem cells were found to be increased in the peripheral blood after the adminjstration of chemotherapy This rebound recovery of cells in the bone marrow subsequently caused the release of progenitor/stem cells into the circulation (Richman, Weiner & Yankee 1977) Further research showed that chemotherapy followed by administration of growth factors resulted in even greater numbers of progenitor/stem cells in the peripheral blood (Pettingill Testa Swindill et al. 1993). With recent scientific developments in the last 20 years autologous PBPC transplants have practically replaced bone marrow transplants Mobilized peripheral blood transplants offer several advantages over bone marrow transplants including more rapid recovery of circulating granulocytes and platelets Peripheral blood collections via apheresis do not require sedation and the procedure is not as invasive as bone marrow harvest surgery. Furthermore a BM harvest requires general anesthesia with more complications than apheresis inc! uding death 29

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CHAPTER4 CD34 + CELLS Discovery of the CD34 + Antigen Civin et al. (1984) was one of the first to describe the antigenic analysis of hematopoietic progenitor cells Civin used mice to produce a monoclonal antibody (anti-My-10) against the immature human myeloid leukemic cell line (KG-1a). KG-1a are immature undifferentiated white cells were introduced into mice to stimulate the production of antibodies to cell surface antigens Analysis of anti-My-10 showed the antibody to belong to the IgG class (Civin et al. 1984). Indirect immunofluorescence was used to measure binding of the antibody to the KG-1a cell line as well as mature granulocytes from the peripheral blood of normal donors. Among other cells tested against the antibody were lymphocytes monocytes and marrow cells from normal donors Results of the experiment revealed that the KG-1a cells were as fluorescent as the positive control thus the cell line contains large amounts of the M y-1 0 antigen ." The granulocytes, monocytes and lymphocytes from the peripheral blood were not labeled by the antibody Although progenitor and stem cells are known to circulate in the peripheral blood in extremely small numbers they were not detectable as a subpopulation of the 30

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peripheral blood by immunofluorescence in this experiment. However a small percent (1.3%) was detectable as My-10 antigen marrow cells (Civin et al. 1984). Morphologic and cytochemical analysis of the positive normal human marrow cells showed that they were predominately very immature cells, such as myeloblasts and monoblasts Expression of the antigen decreased as the myeloid cell line matured because only rare more mature cells were found to be My-1 0-positive ( Civin et al. 1984) The "My-1 0 antigen was radio labeled and immunoprecipitated from the surface of the KG-1a cells with the anti-My-10 antibody. SDS polyacrylamide gel electrophoresis and autoradiography was performed on the antigen ; a protein with a molecular radius of about 115 kilodaltons (KD) was revealed the CD34 antig e n ( Civin et al. 1984) (see Figure 4.1). The authors definitely saw the potential for further hematopoietic research and the isolation of an immature hematopoietic cell population for human bone marrow transplantation. 31

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Figure 4.1 Schematic of cell surface antigens Presentation includes some of the signjficant cell surface antigens on stem progenjtor, and precursor cells Stem Cell cd34+, cd33, cd38, cd45ro+, cd45raphoid Progenitor CFU-M, cd34+, Monocyte cd45ra+ B-cd34+, cd 1 Progenitors 10+ B-cell Neutrophil 32 T-cell 3-T -cell

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CHAPTERS GRANULOCYTE-COLONY STIMULATING FACTOR Function Granulocyte-colony stimulating factor (GCSF ) is a hematopoietic growth factor ( cytokine ) which regulates the proliferation ( production) and differentiation ( maturation ) of neutrophils Neutrophils comprise approximatel y 70 % of the leukocytes in the circulation They have a v ery important role in providing protection from bacterial pathogens However these cells have a short half-life ; thus neutrophils must be continuously replenished from pluripotent stem cells in the bone marrow (Nagata 1994). Morstyn et al. (1998 ) state that native human GCSF is produced b y stromal c e lls endothelial cell s fibroblasts and monocytes ( p 4 2 4 ) Bagby and Heinrich ( 1999 ) list the following functions of GCSF ( p 3 -4 ) : Stimulates growth of progenitor cells committed to the neutrophil lineage Stimulates neutrophil maturation of certain leukemic cells Activates phagocytic function of mature neutrophils Stimulates quiescent pluripotent hematopoietic progenitor cells to enter G 1-S Stimulates mobilization of stem cells and progenitors from hematopoietic niches into peripheral blood 33

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Maintenance of steady-state neutrophil numbers Structure GCSF protein is encoded on the long arm of chromosome 17 and has core molecular weight of 18. 6 kilodaltons Human GCSF exists in two forms a form with 177 amino acids and another form with 174 amino acids which may be more active Native human GCSF is glycosylated and the sugar chain is attached to the C-D loop at the threonine 134 position Glycosylation does not appear to be important for the biological function of the molecule but may play a role in protecting the GCSF protein from proteolytic degradation Further, filgrastim (rHuG-CSF) is a non-glycosylated commercial hematopoietic growth factor and was the GCSF used in this study (Morstyn et al. 1998 ) As a member of the a superfamily of cytokines GCSF has a protein structure consisting of a four alpha-helical bundle ; the helices are labeled A-D starting at the N-terminus Also a short helix exists in the A-B loop (Layton et al. 1999). Furthermore human GCSF contains four disulfide bonds which are essential for proper folding and biological activity of the molecule (Nagata 1994 ) Mobilization of Progenitor / Stem Cells Hematopoietic cytokines such as GCSF GM-CSF stem cell factor (SCF), Interleukin 1 (IL-1), IL-7 IL-8 IL-11, and IL-12 are capable of mobilizing 34

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hematopoietic cells into the circulation. Chemotherapy also causes rebound mobilization of hematopoietic cells into the circulation (Link 2000) For e x ample GCSF is capable of inducing a 20-fold increase in peripheral blood neutrophils and a 100-fold increase in peripheral blood hematopoietic cells SCF causes a 24-fold increase in peripheral blood neutrophils and a 20-fold increase in peripheral blood hematopoietic cells Another comparison shows that IL-l causes a 5-fold and 30-fold increase respectively Mobilization with chemotherapy alone produces a 8-fold and 80-fold increase respecti v ely (Link 2000) GCSF is most commonly used mobilization agent because this cytokine is potent and lacks serious toxic effects (Link 2000 ) The fact that hematopoietic cytokines with distinct biologic activities can mobilize hematopoietic cells leans towards a common mechanism Link (2000 ) describes the following common characteristics of cytokine mobilization with few exceptions existing : Increased circulating hematopoietic cells Broad spectrum mobilization ofhemapoietic cells including primitive pluripotent to committed progenitors of different cell lines Comobilization of neutrophils with hematopoietic cells Cytokine mobilization of hematopoietic cells into the circulation may occur by phenotypic changes in hematopoietic cells or b y changes in the actual BM microenvironment. Evidence exists for both scenarios Studies have indicated 35

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differences in the phenotypic characteristics of mobilized hematopoietic cells compared to hematopoietic cells found in the steady-state environment of the BM. However experiments with the IL-8 chemokine indicate that changes in the BM microenvironment lead to mobilization (Link, 2000) GCSF-R (GCSF receptor) deficient mice are defective in granulopoiesis leading to neutropenia In addition the GCSF-R has been shown to act synergistically with other agents. For example GCSF-R is necessary for hematopoietic cell mobilization in mice treated with chemotherapy specifically cyclophosphamide ; GCSF-R-deficient mice are deficient in circulating hematopoietic cells The GCSF-R is also required for mobilization of hematopoietic cells from the BM into the circulation by IL-8 Although other cytokines do not require the presence of the GCSF-R for mobilization The author believes that GCSF-R-dependent signals are produced by hematopoietic cells in response to stimuli (such as cytokines), which in turn cause changes in the BM microenvironment, and thus lead to hematopoietic cell mobilization Therefore the model favors BM microenvironment changes in mobilization and places less importance on the cells themselves (Link, 2000) Furthermore evidence suggests that VLA-4 adhesion molecules are down-regulated in the BM and allow release of cells into the peripheral circulation 36

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CHAPTER6 EX VIVO EXPANSION AND SELECTION OF HEMATOPOIETIC PROGENITOR/STEM CELLS Ex vivo expansion/selection of hematopoietic progenitor / stem cells from purified CD34 + cells offer several benefits in transplantation (Brugger Scheding Ziegler Buring & Kanz, 2000 ; Andrews Briddell Hill Gough & McNiece 1999) : Only a small volume of peripheral blood collection is required Reduced tumor cell load in reinfusion product Rapid engraftment leading to decreased duration and severity of neutropenia Ability to perform repeated clinical applications as with multiple high-dose chemotherapy The possibility of increasing stem cells in allogenic cases where human leukocyte antigen is mismatched allowing decreased graft rejection Ex Vivo Expansion and Cytokines Brugger et al. (2000) state that optimal hematopoietic growth factor conditions have not yet been elucidated in progenitor / stem cell ex vivo expansion SCF is thought to be an important growth survival factor for hematopoietic progenitor / stem cells Some cytokines such as IL-l IL-3 and IL-6 act in the early stages of 37

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expansiOn. On the other hand GCSF GM-CSF and erythropoietin (EPO ) are late-acting growth factors The Brugger team (2000) found from previous experiments that a combination of SCF IL-l IL-3, IL-6 and EPO in a stroma-free liquid culture was capable of expanding CD34 + cells collected from the peripheral blood In this system progenitor cells were expanded well but more primitive progenitor and stem cells identified b y LTC-IC ( long-term culture-initiating cells) were not expanded ; nevertheless these cells were maintained in culture However other studies indicate that a cytokine combination of SCF FL, thrombopoietin (TPO) and IL-3 greatly improve generation of more primitive (noncommitted) progenitor / stem cells TPO not only effects primitive progenitor / stem cell generation but also enhances the megakaryocytic cell line responsible for replenishing platelets GCSF GM-CSF and E PO appear to play an important role in expansion of committed progenitor cells (Brugger et al. 2000). Brugger et al. ( 2000 ) deem that controvers y still exists in the ex vivo expansion of hematopoietic progenitor / stem cells mainly the ability of the transplanted cells to home and provide terminally differentiated cells in vivo especially in patients receiving myeloablative therapy Committed progenitors are probably responsible for the rapid kinetics seen in the recovery of peripheral blood cells However Brugger et al. (2000 ) propose that stem cells may be spent from growth factor 38

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addition in culture Albella Segovia Guenechea Pragnell & Bueren (1999) invested combinations of cytokines that prevent differentiation of stem cells and enhance self-renewal in the stem cell compartment. They used mouse BM expanded ex vivo with the SCF and IL-11 (in combination with or without other factors known as Flt3 ligand and macrophage inflammatory protein-1 alpha) cytokines The results showed significant increase in both short-term and mid-term murine hematopoietic repopulating ability 40 days and 180 days posttransplantation respectively The authors conclude that stem cells are not expanded in culture ; however stem cells are maintained in culture and thus not differentiated Though the maintenance of long-term repopulating ability in the mouse model was observed one year following transplantation. Reduced Tumor Cell Content Tumor cell content in ex vivo expanded cells may be reduced because less volume of blood is required In addition culture conditions which favor expansion of normal hematopoietic progenitor / stem cells may inhibit propagation of malignant cells (called pas siv e purging) (Brugger et al. 2000 ; Carlos et al. 1999). Lundell Vredenburgh Tyer DeSombre, & Smith (1998) refer to previous studies which indicate decreased survival in breast cancer patients receiving BM or PBSC with detectable tumor cells Furthermore other studies noted indicate that reduction in the number of tumor cells infused in the transplant product may delay disease 39

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relapse in these patients However, reinfused tumor cells must be distinguished from the possibility of residual tumor cells Lundell et al. ( 1998) expanded tumor cell positive BM from stage IV breast cancer patients and subsequently immunostained the expanded cells for identification of tumor cells ( immunostaining can detect one tumor in 10/\6 normal BM cells) Pre-expansion samples contained 6-2128 tumor cells in 5 x 10/\6 nucleated cells evaluated. Post-expansion samples revealed no tumor cells in four of seven different samples. The other three samples had a decrease of 44 to two, 40 to two, and 2128 to four per 5 x 10/\6 nucleated cells examined This experiment demonstrates passive purging resulting in tumor cell reduction of one to four logs Carlos et al. ( 1999) also demonstrated significant decreased tumor cell content after ex vivo expansion of BM cells from breast cancer patients with detectable tumor cell contamination prior to expansion Additionally studies have addressed whether concomitant clonogenic growth of tumor cells occurs with expansion of CD34 + cells under the influence of cytokines Brugger et al. ( 2000) conclude that tumor cells do not expand significantly in cytokine-induced cultures for CD34 + cells. Besides these researchers found that no tumor cells were detected in previous tumor-positive samples after CD34 + cell selection or after expansion of these CD34 + cells 40

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Decreased Neutropenia Studies on the use ex vivo expanded CD34 + PBPC cells plus posttransplant growth factor have been shown to significantly decrease neutropenia in lethally irradiated primate models (Andrews Briddell Hill Gough & McNiece 1999) Reiffers et al. (1999 ) found similar results in myeloma patients treated with or without irradiation and high-dose chemotherapy and transplanted with CD34 + ex vivo expanded cells McNiece et al. (2000) have also demonstrated decreased time of neutropenia in breast cancer patients transplanted with CD34+ expanded cells Abrogation of neutropenia in these studies is probably due to the high numbers of committed progenitor cells (Andrew et al. 1999 ; Reiffers et al. 1999 ) Thus some studies are aimed at determining how to specifically generate granulopoietic postprogenitor cells (GPPC) that would be additionally transplanted with progenitor / stem cells to help abrogate neutropenia (Stefan et al. 2000). Nevertheless treatment protocols that require high-dose myeloablative chemotherapy would benefit from ex vivo expanded cells because of the morbidity associated with infection during the induced neutropenic phase Andrews et al. ( 1999 ) demonstrated that juvenile baboons transplanted with expanded CD34 + PBPC and subsequently given GCSF and megakaryocyte growth and development factor ( MGDF) significantly shortened the time of neutropenia 41

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This shortened time of neutropenia was much more considerable when compared to baboons who received unmanipulated cells of the same type along with growth factor or in animals that received only expanded cells but no growth factor. However animals receiving either unmanipulated cells or expanded cells demonstrated similar duration of neutropenia without the administration of growth factor. Notably the scientists found that thrombocytopenia seemed to be prolong in animals that received expanded cells compared to animals receiving unmanipulated cells Though, this is somewhat puzzling since megakaryocytes (platelet precursors) are also expanded in culture Furthermore the majority of animals receiving expanded cells maintained sufficient peripheral blood counts for six months posttransplant and these findings may imply that stem cells were maintained in culture (Adrews et al., 1999). 42

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CHAPTER 7 HIGH-DOSE CHEMOTHERAPY Purpose Chemotherapy drugs interfere with cell replication and division, and can act at different phases in the cell cycle. However these drugs are non-discriminating and they also effect normal cells which divide rapidly such as bone marrow gastrointestinal mucosa gonads and hair follicles (Wilkes et al. 1999) High-dose chemotherapy (HDT) is the use of chemotherapeutic agents to cause myeloablation of the bone marrow for the treatment of cancer and other diseases. Thus, the goal of high-dose chemotherapy is to effectively eradicate tumors and/or to allow the immune system to fight residual amounts of tumor (Colvin & Petros, 2000). Since the bone marrow is myeloablated, peripheral blood progenitor / stem cell or bone marrow transplants are required to reestablish hematopoiesis HDT regimens usually include a minimum of two agents which differ in action Use of HDT regimens may serve several important functions : 1) to improve tumor toxicity by synergistic activity 2) to decrease drug toxicity 2) to prevent tumor drug resistance (Colvin & Petros, 2000). 43

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Characteristics of Oncology Drugs Patients in this study received several different high-dose chemotherapy regimens based on the stage of their disease. Several regimens used for high-dose chemotherapy consisted of the following oncology drugs : cyclophosphamide cisplatin BCNU Taxotere carboplatin melphalan and taxol. Three different combinations were used : 1) cyclophosphamide cisplatin and BCNU 2) Taxotere carboplatin and melphalan 3 ) taxol cyclophosphamide and cisplatin The drug class mechanism of action pharmacokinetics dosage and toxicities / side-effects of each drug will be discussed. Cyclophosphamide (Cytoxan) Cyclophosphamide is an alkylating agent that prevents DNA synthesis by causing crosslinkage in DNA strands. The drug is activated in the liver and by serum enzymes The drug and its metabolites are excreted by the kidneys and the half-life of the drug is 6-12 hours The dosage range is from 100-1500 milligrams per meter squared (mg/m/\2) High-dose cyclophosphamide used in this study was 1875 mg/m/\2 /day intravenous (IV) x 3. Possible toxicities/ side-effects include : infection and bleeding caused by bone marrow depression ; accumulation of drug metabolites in the bladder causing 44

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hemorrhagic cystitis ; nausea vomiting and diarrhea leading to malnutrition or anorexia ; alopecia and changes in skin and nails ; amenorrhea and reproductive hazards due to mutagenicity and probable tetragenicity ; secondary malignancy ; alterations in cardiac function ; pulmonary toxicity (Wilkes Ingwersen & Burke 1999) Cisplatin (Platinol) Cisplatin is a heavy metal acting similar to an alkylating agent and also inhibits DNA synthesis by crosslinking and by denaturing the double helix structure The drug is rapidly distributed to the liver and kidneys and the drug is excreted in the urine within 24 hours Dosage ranges from 15-120 mg/m/\2 Dosage in this study was 55 m g/ m/\2 / day IV x3 Possible toxicities / side-effects include : renal damage ; nausea and vomiting and taste alterations ; anaphylaxis (tachycardia wheezing hypotension facial edema rash urticaria erythema pruritus); neuropathy ; anemia ; infection and bleeding ; reproductive hazard ; alterations in cardiac function (Wilkes et al. 1999 ) Carmustine (BCNU) BCNU is classified as a nitrosourea which acts as an alkylating agent and also inhibits DNA repair. This drug is rapidly distributed to tissues and is rapidly metabolized with a half-life of one hour However the drug s lipid solubility allows 45

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the drug to remain in the cerebrospinal fluid nine times longer. Excretion occurs mainly by the kidneys Dosages range from 40-225 mg/m 2 or 450-600 mg/m 2 with transplants. The BCNU dose used in this study was 600 mg/m 2 / day IV x I Possible toxicities / side-effects are: infection and bleeding; pulmonary toxicity ; nausea vomiting, and reversible liver dysfunction ; irritation causing venospasms and skin flushing ; kidney damage ; reproductive hazards ; ocular toxicity causing damage to optic nerve retinal hemorrhage and neuroretinitis (Wilkes et al. 1999). Docetaxel (Taxotere) Docetaxel a mitotic spindle poison acts by arresting cell division in metaphase by enhancing microtubule assembly and inhibiting depolyrnerization. The drug is metabolized by an isoenzyme and is highly protein-bound in the plasma Excretion of the drug occurs mainly by the intestines with waste The dosage range is 60-100 mg/m 2 and the dosage administered in this study was 200-300 mg/m 2 x 1. Possible toxicities / side-effects include : anaphylaxis ; infection and bleeding; fluid retention causing edema pleural effusion and ascites ; alopecia and skin rash ; neuropathy ; nausea vomiting diarrhea and stomatitis (Wilkes et al. 1999) Carboplatin (Paraplatin) This alkylating agent is similar to cisplatin and acts by causing intrastrand and interstrand DNA crosslinking The majority of carboplatin is excreted in the urine 46

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with a half-life of about 100 minutes The dosage range is 300-360 mg/m/\2 and the dosage used in this study was 1000 mg/m/\2 / day x 3. Possible toxicities / side-effects include : infection and bleeding ; nephrotoxicity ; nausea vomiting, diarrhea stomatitis and reversible liver dysfunction ; neurologic dysfunction; reproductive hazards ; anaphylaxis ( Wilkes et al. 1999) Melphalan This alkylating agent is a derivative of a nitrogen mustard which prevents cell replication via DNA crosslinking and breakage Melphalan has variable bioavailability The drug is excreted by both the intestines and the kidneys with a half-life of about two hours Dosages ranges for bone marrow transplantation is usually 50-60 mg/m/\2 but the dosage administered in this study was 150 mg/m/\2 / day x 3 Possible toxicities / side effects are : infection and bleeding ; nausea and vomiting ; anaphylaxis causing cardiac problems; reproductive hazards ; secondary malignancy ; pulmonary toxicity ; alopecia, skin rash, and urticaria (Wilkes et al. 1999). Paclitaxel (Taxol) Paclitaxel causes cell death by the same mechanism of action as docetaxel. This drug is highly protein-bound in the plasma and is metabolized in the liver. Drug metabolites are excreted mainly in the bile. Dosage varies from 135-250 mg/m/\2 47

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The dosage used in this study was 725 mg/m/\2 / day x 1 Possible toxicities / side-effects are as follows : anaphylaxis ; bleeding and infection ; sensory neuropathy ; alopecia ; nausea vomiting diarrhea stomatitis and liver toxicity (Wilkes et al. 1999). 48

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CHAPTERS BREAST CANCER Types ofBreast Cancer Breast cancer occurs when a malignant tumor develops from previously normal breast tissue The female breast is comprised of four main tissue components : lobul es which are the milk-producing glands; ducts through which milk passes to the nipple ; s tr o ma which consists of fatty tissue and ligaments ; l y mphphatic vessels which lead to the axillary lymph nodes (American Cancer Society [ACS] 1999) There are many different types of breast cancers effecting different cells and tissues of the breast ; thus the types of breast cancers vary in their prognosis and treatment options Ade no c ar cinoma starts in the glandular tissue and includes ductal ca r c inoma s and lobular ca r c inomas. Most breast cancers are adenocarcinomas because they originate in the glandular tissue Ductal carc inoma in s itu (DCIS) is a form of noninvasive breast cancer where cancer cells are present inside the ducts but have not spread to other breast tissue DCIS can be subclassified according to grade and type Grade distinguishes the severity of the cancer cells detected under the microscope. C o m e docar ci noma describes DCIS with areas of dead or degenerating tumor cells In situ breast cancer indicates an early 49

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stage of cancer where the tumor is confined only to the area where the cancer began Infiltratin g ductal c ar c inoma (IDC) began in the duct but has passed through the duct wall and invaded fatty tissue. Metastasis is now possible through the lymphatic and circulatory system. This type of cancer is responsible for approximately 80% of breast cancers Infiltrating lobular c arcinoma (ILC) starts in the milk-producing gland and has the potential to metastasize Lobular carc inoma refers to a special type of invasive ductal or lobular carcinoma with a better prognosis than usual ILC or IDC. Inflammatory br e ast cancer occurs when cancer cells block lymphatic vessels in the skin over the breast. The blockage causes the breast skin to become red and warm with a thick pitted appearance. This type of invasive breast cancer is rare but it is aggressive Lobular carc inoma in si tu ( LCIS ) begins in the breast gland but does not penetrate the lobule wall. This type is a neoplasia rather than cancer and is usually not invasive However, women with this classification may have an increased risk for invasive breast cancer Medullary carcinoma of the breast is invasive but with a clear delineation between tumor tissue and normal tissue This type of invasive breast cancer has a good prognosis but accounts for only 5 % of breast cancers Mucinous carcinoma is a rare type of invasive breast cancer consisting of mucus-producing cancer cells Paget's disease of th e nippl e originates in the duct and metastasizes to the nipple and areola skin. This rare type of breast cancer can cause the skin of the nipple and areola to become crusty scaly, and red 50

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with areas of bleeding. Phyllodes tumor is a very rare type of tumor that forms from connective breast tissue These type of breast tumors are usually benign and rarely become malignant (ACS 1999) Breast Cancer Risk According to the American Cancer Society the following are risk factors for breast cancer that cannot be changed by the individual: female aging, genetics family history of breast cancer, personal history of breast cancer race previous breast biopsy previous breast irradiation, and early menarche or late menopause ( ACS 1999) Breast cancer is about 100 times more common in women than men Seventy-seven percent of women with breast cancer are over 50 years when diagnosed About 50% of women with inherited mutations of the BRCA 1 or BRCA2 will get breast cancer by age 70 Inherited mutations of p53 tumor suppressor gene also increases a women s risk of developing several types of cancers including breast cancer A woman's risk is doubled if she has a first-degree relative (mother daughter sister) with breast cancer Women with cancer in one breast have an increased risk for new cancer developing in the opposite breast. Caucasian women have a slightly greater risk than African-American women but African-Americans are more likely to die Asian and Hispanic women have a lower risk (ACS 1999). 51

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The ACS reports that women with a previous breast biopsy have increased risk for breast cancer but the degree of risk depends on the classification or diagnosis of the breast biopsy Parker et a!. ( 1999 ) found that 82% of women in a study with known breast cancer had undergone a previous breast biopsy or fine-needle aspiration which was initially negative for cancer. Also, women who received chest radiation at a young age as treatment for other cancers have a significant increased risk for breast cancer (ACS, 1999) The ACS also states that early menarche (before age 12) and late menopause ( after age 50) contribute a slightly higher risk for breast cancer. Health Net conducted a recent survey study to determine risk factors for breast cancer in younger women. A Breast Health Assessment survey was sent to women from age 34 to 49 years Risk factors were compared for women with known breast cancer and the general population The study concluded that breast cancer risk was increased by age, first-degree relative with breast cancer and previous breast biopsy for cysts No statistical difference between the two groups was related to menarche nulliparity first pregnancy after age 30 or breast feeding (Parker et al. 1999) 52

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CHAPTER 9 METHODS Patients The study was approved by the Combined Multiple Institutional Review Board at the University of Colorado Health Sciences Center. The study group consisted of21 women with advanced breast cancer ranging from stage II to IV disease The subjects were consecutively assigned to either Cohort 1 (N =lO) or Cohort 2 (N=ll). The subjects in Cohort 1 and Cohort 2 both underwent leukapheresis over a period of five days to collect peripheral blood progenitor / stem cells which had been mobilized from the bone marrow with GCSF (Neupogen ). Cohort 1 patients received both ex vivo expanded CD34 + selected cells and CD34 + selected unexpanded cells Cohort 2 received only ex vivo expanded CD34+ selected cells as the sole hematopoietic support. Both groups received various regimens of high-dose chemotherapy before being infused with the CD34+ selected cells Cohorts 1 and 2 also received recombinant human GCSF posttransplantation (Figure 9 1 ) 53

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Fieure 9.1 Methods schema for cohort patients (A) Cohort 1: CD34+ selected and CD34 + ex vivo expansion infusion (B) Cohort 2 : CD34+ ex vivo expansion infusion (Reprinted with permission ofMcNiece et al., 2000). 54

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Leukapheresis The COBE Spectra Apheresis System version 6 was used to collect mononuclear cell-rich peripheral blood progenitor / stem cells The COBE Spectra collects cells in the same manner as centrifugation where heavier cells such as red cells fall to the bottom and lighter cells settle on the top layer known as the buffy coat. The COBE Spectra only has the capability to collect the buffy coat layer and cannot morphologicall y distinguish cell types Blood is continuously pumped through the machine as the PBPC are removed and the remainder of the blood is returned to the patient. Materials and Supplies Materials and supplies used in the procedure consist of the following : COBE Spectra disposable Auto PBPC blood tubing set # 777-006-100 ; COBE SpectraTHERM disposable blood warmer set # 777-000-200 ; Anticoagulant Citrate Dextrose to prevent clotting of the blood in the apheresis machine ; 0.9% normal saline used to initially prime the PBPC and blood warmer tubing; Baxter Fenwal sampling Site Coupler # 4C2405 to gain sterile access to final product bag ; sterile drapes Betadine alcohol wipes and latex exam gloves to maintain sterility of the catheter site and product throughout the apheresis procedure Blood collection 55

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tubes sterile needles and syringes to collect required peripheral blood specimens and to collect a sample from the final apheresis product. Equipment used in the procedure in addition to the COBE Spectra includes the SpectraTHERM used to warm blood as it is returned to the auto donor and the Sebra Tube Sealer to close off all tubing at the conclusion of the procedure and to seal and detach the product from the rest of the apheresis tubing set. All autologous patients for the autologous apheresis procedure have orders signed by the Bone Marrow Transplant attending physician Subjects also signed an informed consent for the leukapheresis procedure and for high-dose chemotherapy Accordingly all patients in the study signed an informed consent for participation in this particular study and the study was previously approved by the International Review Board at the UCHSC. In addition subjects must complete specific requirements before starting apheresis All subjects were required to complete the same standards as all autologous apheresis patients The following was required for all subjects in the study : health and physical exam ; height and weight ; comprehensive infectious disease panel ; ABO blood group and Rh type ; pregnancy assessment on all females of childbearing potential ; completed HIV questionnaire central line placement ; GCSF mobilization four days before the start of apheresis 56

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Procedure The leukapheresis procedure normally takes four to six hours to complete Cells are usually collected on five consecutive days The COBE Spectra is loaded with the PBPC tubing set and primed in accordance with the COBE Operational Manual-Section 3 Once the machine has been primed with normal saline alarm tests will performed to insure proper loading and functioning of the machine Also the blood warmer tubing is inserted on the SpectraTHERMTM and connected to the PBPC tubing Data must be entered into the COBE Spectra computer to calculate the volume of product that each individual patient will collect in the product bag Data required for the procedure includes : height and weight sex white count the percent of mononuclear cells (the total monocytes lymphocytes myelocytes metamyelocytes promyelocytes and blasts), and hematocrit. The total blood volume (TBV) of each individual patient to be processed is based on sex, height and weight. The patient's TBV is processed a total of four times through the instrument. Vital signs are taken before and after apheresis. The patient is monitored closely during the procedure for any adverse reactions that may occur The most common reaction is citrate sensitivity due to the anticoagulant used during the procedure The anticoagulant works by binding ionized calcium in the bloodstream Calcium is a necessary component in hemostasis thus removal of calcium prevents blood from 57

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clotting in the instrument tubing and prevents clotting of the final product containing the progenitor / stem cells The anticoagulant is eventually metabolized when the processed blood in returned to the patient. However during apheresis the patient may experience a sensation of tingling or numbness in varying degrees and in several areas of the body This occurs as the calcium levels drop in the bloodstream and the body begins to remove blood from the muscles, which require calcium for muscle contraction The tingling sensation is usually located around the mouth hands feet arms and legs Some degree of tingling, or citrate sensitivity is expected and patients are given calcium supplements prophylactically at the beginning of the procedure and as needed thereafter Ionized calcium peripheral blood levels are monitored if symptoms are more severe and intravenous calcium may be required to treat hypocalcemia Patients may experience other adverse reactions but most of these reactions are less common : muscle tetany hypotension, blurred vision nausea, and vasovagal syncope. The patient receives GCSF while undergoing the apheresis procedure to mobilize progenitor / stem cells GCSF also has several side-effects, most commonly bone pain and headache 58

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CD34 + Selection Materials and Supplies Materials and supplies used in CD34 + selection process include the following : Isolex 300! Magnetic Cell Separator ; Isolex Stern Cell Reagent Kit and other necessary laboratory reagents ; Isolex 300! Magnetic Cell Separator Disposable Set ; MPC-1 Dynal Magnetic Particle Concentrator ; Coulter Zl Cell Counter ; Sebra Tube Sealer ; other minor laboratory supplies Procedure The Isolex 3001 Cell Selection System is used to irnmunornagnetically select CD34 + cells from other cell populations contained within the apheresis collection product. First the cells are washed and concentrated. Second the concentrated cells are incubated with murine antihuman CD34 + monoclonal antibody and subsequently washed to remove any excess antibody. Third the sensitized cells are incubated with tiny paramagnetic beads which are coated with another antibody and the beads form rosettes containing the CD34 + cells when exposed to a primary magnet. Finally the bead/cell rosettes are incubated with a stern cell releasing agent and the cells are washed 59

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Ex Vivo Expansion Materials and Supplies Materials and supplies included an ex-vivo cell processing bag, defined medium and several cytokines: Stem Cell Factor (SCF), Granulocyte Stimulating Factor (GCSF), Megakaryocyte Growth and Differentiation Factor (MGDF) (Amgen Inc.). Procedure Cohort 1 received unexpanded CD34 + selected cells that were frozen at -180 degrees Celsius in liquid nitrogen until the product was released for infusion or expansion (1 0% dimethyl sulfoxide [DMSO] was used as a cryopreservant) Cohort 1 patients were also infused with CD34 + cells that were frozen thawed and then expanded in culture. Cohort 2 patients received only CD34 + cells that were frozen thawed, and then ex vivo expanded Unrnanipulated PBPC fractions were frozen as back-up cells for both cohorts Prior to expansion a cell count was performed to determine the number of cells available following CD34 + selection. The cell culture procedure was performed using sterile technique under laminar air flow hood Nutrient media was transferred to the cell processing bag. The cytokines were reconstituted according to 60

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manufacturers instructions. The volume of each cytokine to be added to the culture was calculated using the culture volume and cytokine concentration. Cells were injected into the processing bag and the culture bag was placed in a 37 degrees Celsius humidified incubator with five percent carbon dioxide for ten days The cultures were checked daily for unusual turbidity and media indicator color change Also cultured cells were tested for sterility including mycoplasma detection After ten da y s an aliquot of cultured cells is removed for viability testing and a cell count is performed The cultured cells were processed using the COBE 2991 After washing the cells a cell count viability and sterility testing is performed on the supernatant. The final product is only released for infusion if sterility testing is negative visual inspection is acceptable and the viability is greater than 70 % High-Dose Chemotherapy Several patient criteria are required before the administration of high-dose chemotherapy Chemotherapy dose is based on the patients body surface area ( BSA). Body surface area is calculated using the individual patients height and weight. The patients BSA and the chemotherapy drug dosage / m/\2 is recalculated prior to infusion In addition several tests are required prior to the administration of chemotherapy : serum creatinine ; creatinine clearance ; electrocardiogram (EKG) ; liver function studies 61

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Consent forms are signed by the attending physician the patient and a witness Chemotherapy orders must be signed by the attending physician, and nurse practitioner ( NP ) or fellow Protocol eligibility and cryopreservation forms are signed by the attending Also the patient must be well hydrated the previous six hours to chemotherapy and the bladder must be irrigated for the previous four hours if applicable Urine output should be at least 200 cc!hr for three hours Intravenous ( IV ) access must be available other than the central line catheter that is used for chemotherapy administration usually a peripheral IV or arterial line This access is used to draw blood samples for pharmacokinetics Materials and Supplies Materials and supplies include the following : pumps for bladder irrigation and cc / cc replacement ; pumps for infusion of chemotherapy agents ; cardiac monitor ; chemotherap y gloves and gown ; disposable underpad ; alcohol wipes gauze needles syringes tubing with Luer-lock connectors ; normal saline for flush solution Procedure Permanent venous catheters and central lines are preferred for the administration of chemotherapy If a venous catheter or central line is not available a peripheral IV must be inserted with careful consideration given to the site of insertion ( i e avoiding areas with hematomas inflammation sclerosing phlebitis etc.). 6 2

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Chemotherapy drugs that are v e si c ant s (agents that cause blistering or tissue necrosis) must be infused through a central line. Chemotherapy infusion is most often performed using a central line catheter. The line is permanently placed through the patient's chest and is tunneled into the s uperior vena cava Tubes are held in place by suture on the outside of the patient's chest. The tubes (lines ) on the outside of the chest have caps and clamps for access to v enous circulation Blood return is checked prior to chemotherapy infusion to insure proper line placement. A separate tubing set is filled with normal saline and this tubing is connected to a bag containing the chemotherap y drug which is in fluid form Aseptic technique is used to attach the tubing and bag to the central line or peripheral line if applicable The infusion is usually continuous and a TV pump is used to provide consistent administration of the drug The patient is monitored clos e ly during the infusion for signs of hypersensitivity and anaphylaxis At completion of the infusion the line is flushed with 10 cc of sterile solution. The patient is monitored periodically thereafter for signs of e xtr avas ation (leakage or infiltration of a v esicant chemotherap y agent into local tissue). Peripheral Blood Progenitor / Stem Cell Reinfusion The peripheral stem/progenitor cells are infused through the patients central line into the venous circulation and eventuall y find their way back to the bone marrow 63

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where they engraft. The patients receive their cells back after receiving six to eight days of high-dose chemotherapy Materials and Procedure The patient is hydrated and medicated with Tylenol Benadryl and other medications if necessary A cardiac monitor is used to prior to infusion and for two hours after the infusion is complete Free flowing normal saline ( NS) is attached to the patient's central line with intravenous (IV) tubing. The previously prepared cells are contained in a syringe and are simply pushed into a port in the free flowing NS tubing at a rate of 1 cc every 10 seconds At completion of the infusion the central line is flushed with 10-20 cc NS to completely flush the residual cells into the blood stream Complications The patient is monitored for signs of any adverse reactions during and after the PBPC infusion Symptoms of adverse reactions include the following : hives temperature > 38 3 degrees Celsius chills dyspnea cough bronchospasm hyper/hypotension bradycardia chest pain back or flank pain, hematuria The above signs of adverse reactions can be attributed to several factors directly related to the infusion product. Bacterial contamination can occur via normal skin flora or waterborne flora such as coagulase negative S taphylo cocc us and 64

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P se udomona s pauc imobili s respectively The DMSO used for cryopreservation can cause coughing or a choking sensation, bronchial spasms and dyspnea, and nausea and vomiting. Also the DMSO can cause a garlic-like taste in the mouth However not all patients received DMSO preserved cells Allergic reactions may be attributed to reagents or antibiotics used in the procedure Pulmonary edema may result form circulatory overload of transplanted product and saline infusions Anticoagulants contained in the infusion product can caus e bleeding due to inactivation of coagulation proteins and platelets On the other hand the product may contain microaggregates of fibrin bone and cell clumps which could lead to thrombotic problems and possibly cause pulmonary embolism However these microaggregates w ould mor e likel y be found in bone marrow aspiration product s than peripheral progenitor / stem cell collections Furthermore metabolic possible complications include : hypothermia citrate toxicity ( affects muscle s, including the heart ) acidosis and hyperkalemia ( increased potas s ium ) or hypokalemia ( decreased potassium ) due to h e molysis durin g cryopreservation of the product. Hemolyzed blood may also cause hemoglobinuria chill s fever, disseminated intra v ascular coagulation ( DIC ) and renal failure How e ver perinephria! blood progenitor / stem cell collections typically have a very low percentage of red blood cells in the final transplant product. Finally, there is the critical hematopoietic cell lines that are necessary to sustaining life 65

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CHAPTER 10 RESULTS Long-term Follow-up on Engraftment All patients received peripheral blood progenitor transplants between July 1998 and January 1999 Cohort 1 patients were transplanted with a CD34 + expanded and unexpanded fraction between July 1998 and November 1998 Cohort 2 patients received onl y a CD34 + expanded fraction between November 1998 and January 1999. Thus patients are at various stages posttransplantation Patients were followed for the past two years to obtain information on peripheral blood counts to monitor sustained hematopoietic engraftment All follow-up data obtained for white blood counts (WBC), absolute neutrophil counts (ANC) and platelet counts are presented in Appendix A Mean WBC for Cohort 1 patients at last follow-up was 4 8 (range 1 6 to 8.4 ), and the mean ANC was 3 0 (range 0 6 to 5.5). Median ANC was 3 .2. Mean platelet count for patients in Cohort 1 at last follow-up was 147 000 ( range 22 000 to 211 000). Patient 3 was excluded from mean and median calculation due to recent gastrointestinal bleed However she has still maintained a PLT > 20,000 / mm/\3 Surviving Cohort 2 patients had a mean WBC and ANC of 3 8 (range 2 1 to 4 7) and 2.4 (range 1.2 to 66

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3 1 ), respectively at last follow-up. Median ANC was 2.4 Cohort 2 had a mean platelet count of 163 000 (102 000 to 198,000). Blood count results at last follow-up are presented in Table 1 0 1 Mean, median and range for peripheral blood counts are shown in Table 10. 2 The normal reference range for WBC is 4 8 to 10.8 (X 101\JiuL), and the normal range for ANC is 2 0 to 8 1 (X 10/\3 uL). The normal reference range for platelet count is 150 000 to 450 000 ( /mm/\3) (Harmening 1997) Normal ranges for peripheral blood counts will vary depending on methodology used at different institutions. Patients were considered neutropenic when the ANC was < 500 per microliter (uL) of peripheral blood Thrombocytopenia occurred when patients had a platelet count < 20 000 per millimeter cubed (/ mm/\3) of peripheral blood Patients are already compromised with their disease and prior chemotherapy Furthermore about 60% of patients undergo chest wall radiation therapy subsequent to their PBPC transplant. Accordingly patient counts cannot be evaluated solely on the basis of what is considered normal for the rest ofthe population Therefore both cohorts have mean counts that are acceptable and thus capable of providing protection against infection and bleeding The data shows that the majority of patients in both cohorts have maintained peripheral blood counts at adequate levels over long-term follow-up However Patient 4 in Cohort 1 had problems with pancyt ope nia (decreased counts in all cell lines) She had disease recurrence and there is a possibility that bone marrow 67

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involvement was preventing normal cells from growing properly Despite disease last follow-up counts were adequate. Further, Patient 8 in Cohort 2 had problems with slow platelet engraftment and failed to obtain adequate platelet counts She was reinfused with back-up cells approximately 5 months after the first transplant. Figure 1 0 .1.1 to 10. 1.5 and Figure 10.2.1 to 1 0.2 6 represent WBC and ANC for both cohorts (odd numbered patients chosen for examples) from date of transplant to date oflast known follow-up Figure 10 3 1 to 10.3.2 and Figure 10.4.1 to 10.4 2 represent examples of platelet counts for both cohorts, monitored over time The first date shown on the graphs is the date of the peripheral blood transplant commonly referred to as day 0 ." Initially patients peripheral blood counts are dramatically decreased due to high-dose chemotherapy just prior to infusion of cells All patients were given GCSF after infusion of cells The spike early in the graphs for both cohorts represents response to growth factor and initial engraftrnent of cells Long-term durable engraftrnent is indicated by a stable leveling off in the graphs and also demonstrates maintenance ofthe hematopoietic cell line ; thus the results indicate that the bone marrow can maintain function after myeloablation followed by progenitor / stem cell rescue Surviving patients in both cohorts have maintained peripheral blood counts over time which indicates sustained engraftment of the hematopoietic cell lines 68

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Figures 10. 5 1-10.7 2 represent overlay graphs of all data (WBC, ANC PLT) for all patients in Cohort 1 and Cohort 2. All patients are graphed from the same starting point (day 0) instead of individual transplant date Patient 3 (Cohort 1) was excluded from the graphs in Figures 10.5.1 and 10.6.1 for statistical representation of the majority of the data Likewise patients 3 and 4 (Cohort 2) were excluded from the graphs in Figures 10.5.2 and 10.6 2 Disease-Free Survival Medical records were researched to monitor patients' disease and survival status Patients were monitored for recurring disease by physical examination tumor markers and C omput e d Tomography (CT scan) of the brain chest, abdomen, pelvis and bone Data was collected for all 10 patients in Cohort 1 8 out of 10 patients remain with no evidence of disease at last follow-up Thus the proportion of Cohort 1 patients remaining in remission was 0.80 Patient 3 had metastatic disease to the liver and brain Patient 4 had metastasis to the lungs. Disease free survival (DFS) in months is recorded in Table 10. 3 At last known follow-up Cohort 1 patients with no evidence of disease have remained in remission from 10 to 28 months The proportion ofDFS for Cohort 2 was 0 70 Patients 3 did not have recent documentation on disease status at last follow-up Seven out of the 10 remaining patients have no evidence of disease at their individual follow-up and remain in remission from 3 to 23 months Patient 4 had additional chemotherapy for 69

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secondary disease to the lungs two months following transplant. She received a stem cell reinfusion one year later and was well engrafted but brain metastasis was disco v ered five months later. A BM biopsy one month after this revealed myelodyspla s ti c sy ndr o m e (MDS) or preleukemic syndrome Patient 8 had metastatic disease to the liver approximately 6 months posttransplant. Patient 11 had slow very slow disease progression in the bone only, at last follow-up DFS recorded is based on la s t written documentation of disease status from follow-up clinics ; some patients may actually be in remission longer than indicated. Overall Survival Overall posttransplantation survival (OS) recorded in months was based on last known follow-up of peripheral blood counts obtained from patients (Table 10. 3). All Cohort 1 patients were alive at last known follow-up (proportion of overall survival = 1 0). Based on the last known documentation of peripheral blood counts patients range of overall survival (OS) in Cohort 1 is 12 to 28 months. Proportion of OS for patients in Cohort 2 is 0 .82; nine out of the 11 patients were alive at last follow-up. Patient 4 developed MDS and Patient 8 had liver metastasis Cohort 2 patients recei v ed their transplants up to 6 months later than Cohort 1 ; range of OS is 3 -23 months at last individual patient documentation. OS is based on last documentation of blood count data from follow-up clinics ; thus patients may have a longer OS than presently indicated 70

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Patient Health Status Patients experienced some significant changes in health status following treatment during peripheral blood cell transplant. At last follow-up the most common complaints in both cohorts were pulmonary toxicity, peripheral neuropathy and post herpetic neuralgia Pulmonary injury and neuropathies were caused by drugs used during the high-dose therapy regimen of treatment. Pulmonary injury manifests in complaints of shortness of breath and oxygen desaturation with exertion Pulmonary toxicity was treated with steroids, such as prednisone Neuropathjes are most commonly manifested as numbness and tingling in hands and feet. Neuropathies were treated with a drug called Neurotonin Pulmonary toxicity and neuropathies may resolve with treatment ; however the degree of recovery may not be complete. Further some patients complained of fatigue and musculoskeletal pain A few patients also experienced a decrease in short-term memory Memory loss is occasionally seen shortly after transplant ; however it is unusual for this to persist in the long-term Some patients required treatment for depression Further a few patients experienced vision problems including blurred vision and ischemic optic neuritis resulting from chemotherapy drugs One patient tried to get pregnant after transplant but was diagnosed with primary infertility. 71

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Furthermore, several patients complained of hot flashes which were secondary to Tamoxifen therapy in the posttransplant period Tamoxifen is an antiestrogen usually used in the palliative treatment of patients with advanced breast cancer who have estrogen-dependent tumors On the other hand, a number of patients were reported as doing very well at last follow-up One patient had a one year remission of asthmatic symptoms following transplant. Some patients had no major complaints at last follow-up and several patients returned to work part-time and full-time 72

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i i : I I Table 10.1 PERIPHERAL BLOOD COUNTS AT LAST FOLLOW-UP ID I COHOR T I DATE i WBC (uL) 1 ANC (uL) I PL T mm"3 I 1 1 11/27/00 8 3.41 I 211 i 2 I 1 12/15/00 I 4 i 2 9 i 156 3 i 1 l 12/26/00 l 8.4 i 5.5 22 ; 4 I 1 11/21/00 I 4.5 3 69 l 103 5 1 I 10/06/00 I 5.4 i 4 6 i 140 I 6 I 1 03/27/00 I 4 4 i 2.93 142 7 i 1 11/09/00 i 3 6 1.4 174 8 I 1 12/21/00 l 2 1 .21 i 121 9 I 1 10/31/00 I 6 5 I 4 23 145 10 : 1 11/07/00 1 6 l 0.6 I 132 1 2 11/13/00 37 I 1 9 194 2 2 06/01/00 4.2 2.7 102 3 i 2 03/13/00 3 5 I 2 1 131 I 4 2 10/05/00 6.2 5 160 5 i 2 04/20/00 3 8 2.4 131 6 2 12/13/00 4 7 3.1 170 7 I 2 06111/99 2 1 I 1 2 198 8 2 07/26/99 4.3 NO 20 9 2 04/22/99 3.5 2.3 178 10 2 02/15/00 4.3 3 .08 185 11 i 2 12/14/00 4 2 5 181 WBC=white blood count ; ANC=absolute neutrophil count ; PL T=platelet count NO indicates no data available Counts reported X 1 000 Table 10.2 MEAN, MEDIAN, AND RANGE FOR PERIPHERAL BLOOD COUNTS AT LAST FOLLOW-UP Cohort 1 WBC (uL) ANC (uL) PLT mm"3 Mean 4.8 3 147 Median 4.5 3.2 142 Range 1.6 to 8.4 0 6 to 5 5 22to211 Cohort 2 WBC (uL) ANC (uL) PLT mm"3 Mean 3.8 2.4 163 Median 3 8 2.4 178 Range 2.1 to4.7 1.2 to 3.1 102 to 198 Counts reported X 1 000 73 I I I

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Table 10.3 DISEASE-FREE/OVERALL SURVIVAL FOR COHORTS 10 COHORT DFS MONTHS OS MONTHS 1 1 0 28 0 28 2 1 0 28 0 28 3 1 1 27 0 27 4 1 1 19 0 26 5 1 0 12 0 12 6 1 0 10 0 17 7 1 0 25 0 25 8 1 0 26 0 26 9 1 0 24 0 24 10 1 0 24 0 24 1 2 0 23 0 23 2 2 0 18 0 18 3 2 NO NO 0 15 4 2 1 2 1 22 5 2 0 15 0 15 6 2 0 23 0 23 7 2 0 5 0 5 8 2 1 6 1 6 9 2 0 3 0 3 10 2 0 18 0 18 11 2 1 19 0 23 OFS=Disease Freel Survival ; 0= No Evidence of Disease 1 =Evidence of Disease OS=Overall Survival ; O=Aiive 1 =Death NO indicates no data available 74 -

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Figure 10.1.1-10.1.5 Cohort 1: Absolute Neutrophil Countvs Date Date of transplant is the first date on the x-axis Patients' absolute neutrophil counts (ANC ) are near zero at the time of peripheral blood progenitor / stem cell (PBPC) transplan t Patients are given granulocyte-colony stimulating factor (GCSF) posttransplant which causes a rise in neutroph i ls shortly after transplant Engraft ment occurs when the ANC is> 500/ul for three consecutive days. Long term engraftment occurs when blood counts are maintained over t i me as shown on the y axis ...J 2 ;;;< 0 u 2 Figure 10 .1.1 Patient 1 15 ,.----14 13 12 1 1 10 9 8 7 1\ 6 5 4 3 2 1 ,. \ r----... / ,..___ 0 co co (Jl (Jl (Jl (Jl 0 0 0 0 !;2 !;2 !;2 !;2 0 0 0 0 0 0 0 0 0 0 1'-0 ...,. 1'-0 ...,. 1'-0 Date Figure 10.1.2 Patient3 15 ,.,....,....._..__-.,.--.-------------14*----------------------------...J 11 :::.. 10 r 1 -ANC I u 6 -tl----------1-+-------2 5 4 :f'\M 1/ 1 co co (Jl (Jl (Jl (Jl 0 0 0 0 !;2 !;2 (") (") (") (") 0l "' (0 Oi 0l "' (0 Oi 0l Date 7 5

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15 14 13 ..J 12 11 10 ;;;9 < 0 8 .... 7 6 (.) 5 z 4
PAGE 91

Figure 10.1.5 Patient 9 15 14 13 ...J 12 ::J 11 ::::.. 10 M 9 < 0 8 ..... 7 X -6 (.) 5 z 4
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Figure 10.2.1-1 0.2.6 Cohort 2: Absolute Neutrophil Count vs Date Date of transplant is the first date on the x-axis Patients' absolute neutrophil counts (ANC) are near zero at the time of peripheral blood progenitor/stem cell (PBPC) transplant Patients are given granulocyte-colony stimulating factor (GCSF) posttransplant which causes a rise in neutrophils shortly after transplant Engraft ment occurs when the ANC is> 500/ul for three consecutive days Long-term engraftment occurs when blood counts are maintained over time as shown on the y-axis Figure 10.2.1 Patient 1 Figure 1 0.2.2 Patient 3 15 14 11 :::::.. 10 +\-.,.-------------------------9 +1-1----------------------------0 8 +1-1-------------------------x 7 +1-!--------------------------(,) 5 +t-111------------------------...___ -.._, 1 +H--------------------------0> 0> 0> 0> 0> 0> 0 !2 c;; ;;;: co a; 0 c;; c;; Date 78 \-ANC\ \-ANc\1

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Figure 1 0.2.3 Patient 5 15 1 4 ...J 12 +1-------------------,-."------1 11 ;;; 10 +1--------------,-",------1 < 9 -++-----------'--------; 0 8-++----------------i -+11-----._,-------------i I -.......... 0 5 z 4 ct 3 2 1 0 15 14 13 (J) ,.....J 12 11
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..J ::I ;;< Cl ..... (.) z <( ...J ;;;< Cl ..... (.) z <( 15 14 13 12 11 10 9 8 7 6 5 4 3 2 1 0 m 15 14 13 12 11 10 9 8 7 6 5 4 3 2 1 0 en ,..._a \1 m C:! v Figure 1 0.2.5 Patient 9 i I l-ANe I \. \. \. ."\. ...... -J I en en en en en en en en ::!: co co 10 ?i N i'i ?i Date Figure 10.2.6 Patient 11 '" .. n I I ...... ...... ---m m 0 0 0 0 e e e 10 10 10 10 C:! C:! {:::! {:::! C:! {:::! 1'-0 v 1'-0 Date 80

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Figure 10.3.1-1 0.3.2 Cohort 1: Platelet Count vs Date Date of transplant is the first date on the x axis Platelet counts (PL T) are low on the date of transplant Engraftment occurs when PL Tis> 20 000/mm113 for three consecutive days Long -t erm engraftment is indicated when blood counts are maintained over time as shown by values along they-axis. Figure 10.3.1 Patient 2 250 M 1 :!: 150 < 50 1 a. a) en en en en 0 0 0 0 en en en en en 0 0 0 0 ;::: ;::: ;::: ;::: ;::: ;::: ;::: ;::: ;::: -i\i ;o Cia -i\i ;o Cia Date Figure 1 0.3.2 Patient 6 250 ..................... M j 200 + -----------------------------i1 ;:;< 0 ..... 1--1 a. 100 50 0 a) en i\i 0 a) en en en m i\i i\i i\i i\i en m en en 0 en m en m 0 i\i i\i i\i i\i i\i to Cia 0 i\i i\i Date 81 J-PLTJ J -PLTJ

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Figure 10.4.1-10.4.2 Cohort 2: Platelet Count vs Date Date of transplant is the first date on the x-axis Platelet counts (Pl T) are low on the date of transplant. Engraftment occurs when Pl T is > 20 000/mrrr'3 for three consecutive days Long-term engraftment is indicated when blood counts are maintained over time as shown by values along the y-axis Figure 1 0.4.1 Patient 2 f 150 0 -100 .... ..J 50 Q. 0 Ol c;; c;; c;; c;; ;::: ;::: Date Figure 10.4.2 Patient 6 250 .., 200 +---1 150 +-f!, A-:--f----------< 100 -+-1-'-t\rl-------------; .... 50 ..J Q. 0 Ol 0 Date 8 2 1-PLTI 1-PLT I

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10.5.1-10.5.2 Graphic overla y of white blood cell count ( WBC ) vs Days post-Peripheral Blood Progenitor Cell Transplant for Cohort 1 and Cohort 2 60 50 40 0 20 10 0 -100 Figure 10.5.1. Cohort 1 WBC (x10"3)/uL 300 500 700 Days Figure 10.5.2. Cohort 2 WBC (x10"3)/uL 0 100 200 300 400 500 600 700 Days 83 900

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Fi2ure 10.6.1-10.6.2 Graphic overlay of absolute neutrophil count ( ANC) vs Days post-Peripheral Blood Progenitor Cell Transplant for Cohort I and Cohort 2. 20 15 < 10 5 0 50 40 30 0 z < 20 10 0 0 100 Figure 10.6.1. Cohort 1 ANC (x 10AJ)/ul 300 500 700 Days Figure 10.6.2. Cohort 2 ANC (x10AJ)/uL 100 200 300 400 500 600 Days 84 900 700

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Figure 10.7.1-10.7.2 Graphic overlay of platelet co unt (PLT) vs Days post-Peripheral Blood Progenitor Cell Trans plant for Cohort 1 and Cohort 2 400 300 t1[200 100 0 Figure 10.7.2. Cohort 2 PLT (xlO"J)/mm"J 0 100 200 300 400 500 600 700 Days Figure 10.7.1. Cohort 1 PLT (x10 3)/mm"3 1 00 300 500 700 Days 85 900

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CHAPTER 11 DISCUSSION Results on Early Neutrophil Engraftment Studies at the University of Colorado Health Sciences Center with the same cohort of patients have shown that ex vivo expanded peripheral blood progenitor cells provide rapid neutrophil engraftment (McNiece et al., 2000) The importance of this finding cannot be understated because patients become immunocompromised in the period following myeloablative high-dose chemotherapy and are susceptible to a variety of opportunistic infections The sooner the patient's neutrophil count reaches acceptable peripheral blood levels, the better they can fight infections Acceptable levels of neutrophils were measured by monitoring the absolute neutrophil count (ANC) Engraftment was indicated when the patient maintained an ANC of > or = 500 /uL The ANC is calculated by multiplying the percentage of neutrophils times the total white count The researchers (McNiece et al., 2000) at the UCHSC found that patients in Cohort 1 engrafted neutrophils in a median of 6 days and Cohort 2 engrafted neutrophils in a median of 8 days The range for Cohort I was 5 -14 days and the range for Cohort 2 was 4 -16 days Historical controls receiving unmanipulated cells engrafted neutrophils in a median of9 days with a 86

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range of 7 30 days This study also demonstrates that ex vivo expanded cells can be safely infused without toxicity to the patient. Platelet Recovery Time No effect of expanded CD34 + cells was seen on the platelet recovery time The median time to platelet engraftment was 18.5 days and 15 days for Cohort 1 and Cohort 2 respectively (McNiece et al., 2000) The historical group had a median platelet engraftment of 15 days Platelets are essential to hemostasis and a faster platelet engraftment time would be desirable The reason for this outcome has not yet been elucidated since immunohistochemical analysis indicated that the expanded CD34 + cells did contain large numbers of mature megakaryocytes the precursors to functioning mature platelets McNiece et al. (2000) postulate that the expanded cells may require more specific growth factors to induce platelet development after the transplantation process. Paquette et al. (2000) demonstrated that patients receiving 4 5 X 10/\7 expanded unselected cells per kilogram had a median time of thrombocytopenia of only 8 8 days. They used megakaryocyte growth and development factor (MGDF) along with GCSF and stem cell factor (SCF) in their culture media The cohorts in the present study received CD34 + selected cells which were expanded using the same growth factors Rutella et al. (2000) found that platelet reconstitution occurred faster in patients receiving unselected cells than those 87

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I I ; ... ii receiving selected Thus expansion may not adversely effect the development of platelets but CD34 + selection may have some yet unknown effect on megakaryocytes CD34 + Counts Scheid et al. ( 1999) have investigated the minimum number of CD34 + cells to be infused to decrease duration of neutropenia and thus decrease the incidence of infection They demonstrated that patients receiving at least 5 x 10/\6 CD34 + cells per kilogram (kg ) had a decreased incidence of fever along with shorter duration of antibiotic use when compared with patients receiving 2 x 10/\6 CD34 + cells/kg but less than 5 x 10/\6 CD34 + cells/kg Patients in the Scheid study had various malignant diseases including solid tumors The patients in the present study received a median of3.8 X lQ/\6 CD34 + cells/kg and 6 6 X 10/\6 CD34 + cells/kg respectively for Cohorts 1 and 2. However statistical analysis did not find a correlation between neutrophil engraftment time and the number of CD34 + cells infused for either cohort (McNiece et al. 2000) Perez-Simon et al. (1999) demonstrated that the CD34 + dose also effects long-term engraftment. Patients in the Perez-Simon study who received more than 0 75 X 10 /\6 CD34 + cells/kg sustained permanent engraftrnent after one year follow-up However transitory loss of engraftrnent was observed in some patients who received less than 1 1 X 10 1\6 CD34 + cells/kg Therefore these researchers suggest that 1.1 X 1 0/\6 cells/kg is the 88

PAGE 103

minimum number of CD34 + cells needed for permanent engraftment. Additionally they found that patients receiving more than 0 .75 X lQ/\6 CD34 + cells/kg required Jess blood product transfusions such as red blood cells and platelets than patients receiving more than this number. A higher CD34 + dose also correlated with decreased incidence of fever and antibiotic treatment during the late posttransplant period Tumor Content Cited studies suggest that relapse may be due to reinfusion of tumor cells in peripheral / stem cell collections thus CD34 + selection may aid in sustaining remission (Gandhi et al. 1 999) In the present study tumor cells were found in unmanipulated cell collections but not in the final expanded fraction However the tumor content of the CD34 + expanded cells specifically could not be evaluated ( McNiece et al., 2 000). Thus tumor depletion may be due to culture expansion resulting in a purging effect or to CD34 + selection or both Voso et al. (1 999) analyzed the efficacy of the Isolex 300 device (the same system used in the present study) for CD34 + selection in peripheral progenitor / stem cell collections from patients with various malignancies including breast cancer. They demonstrated that I immunomagnetic selection of the collections leads to transplant products with a reduced tumor content. Martin-Henao et al. (2000) discuss specific techniques in = .. 89 =

PAGE 104

using the Isolex 300 system, to achieve highly isolated CD34 + cells rapidly and effectively. Long-term Engraftment The present study at the UCHSC demonstrates that long-term engraftment is capable with expanded CD34 + selected cells Recall that patients in Cohort 2 received only expanded CD34 + cells. Long-term hematopoietic engrafting cells may be established from one of three different sources First long-term engraftment may be established from the transplanted graft Second, long-term engraftment may originate from endogenous quiescent cells that are not affected by high-dose chemotherapy. Third long-term engraftment may arise from both the transplant graft and residual primitive hematopoietic cells One patient in Cohort 2 failed to permanently engraft her platelets and the reason for this is unknown. Anyhow the patient did have a lower CD34 + count transplanted than most patients in the study A second patient in Cohort 2 had recurring disease after a few months, but was well engrafted following two different transplants One patient in Cohort 2 had problems with pancytopenia and this may have been due to progressive disease involving the BM. Albella, Segvia Guenechea, Pragnell & Bueren (1999) transplanted irradiated nonobese diabetic / severe-combined immunodeficient (NOD / SCID) mice with ex vivo expanded human CD34 + cord blood cells and found no adverse effect on 90

PAGE 105

long-term repopulation and differentiation However Szilvassy Bass VanZant, & Grimes (1999) used cell staining techniques and found that homing of progenitor cells was adversely effected by ex vivo expansion Transplanted cells must efficiently find their way back to the bone marrow and spleen in order to regenerate and proliferate On the other hand another study demonstrated that ex vivo expanded peripheral and cord blood CD34 + cells can produce large numbers of committed cells without significantly affecting immature progenitor cells ( David et al. 1997) which are necessary for long-term engraftment. Lanzkron Collector & Sharkis ( 1999) compared short-term and long-term hematopoietic progenitor cells by staining and tracking transplanted cells in lethally irradiated mice This group previously separated long-term and short-term repopulating cells on the basis of size. Large cells are responsible for short-term repopulation and smaller cells are responsible for long-term reconstitution of the hematopoietic cell line The researchers found that more labeled long-term cells were found in the bone marrow 48 hours after transplant and that these cells were quiescent in their cell cycle activity. Small-sized cells given alone were not able to provide repopulating protection of the mice from previous lethal irradiation. Furthermore large-sized cells given alone failed to provide long-term repopulation and all mice died b y 12 weeks Onl y the small-sized cells were capable of providing long-term engraftment and only small numbers of cells were required 91

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Thus the Lanzkron study emphasizes the importance of transplanting both early and late hematopoietic precursors Late progenitor cells are more mature and more active in their cell cycle activity and they provide necessary rapid recovery of cells after myeloablative therapy ; white cells red cells and platelets are more quickly replenished to protect the host. In addition early progenitor and stem cells are necessary for long-term hematopoiesis or engraftment. 92

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CHAPTER 12 CONCLUSION The study at the UCHSC demonstrated that patients rapidly engraft clinically important neutrophils with infusion of ex vivo expanded CD34 + cells Patients with a shorter time of neutropenia are at decreased risk for opportunistic infections following myeloablative therapy Rapid platelet engraftrnent was not demonstrated in this study ; however most patients recovered adequate platelet counts at the same rate as unrnanipulated cell transplants Furthermore, infusion of ex vivo expanded cells was shown to be safe. Most of the side-effects experienced by patients were caused by the high-dose chemotherapy drugs used prior to PBPC transplantation. In addition ex vivo expansion of cells may be an effective method of purging tumor cells from the PBPC product ; ex vivo culture may not provide ideal conditions for clonogenic growth of breast cancer cells In this study tumor cells were detected in unmanipulated PBPC products, but not in expanded PBPC fractions Thus far all surviving patients have demonstrated long-term engraftment posttransplant with ex vivo expanded CD34 + cells Future tracking studies are necessary to determine whether long-term engraftrnent originates from residual endogenous cells or from 93

PAGE 108

PBPC transplantation or both However the importance of durable long-term engraftment, in either case supersedes knowledge of the exact source of sustained hematopoietic reconstitution 94

PAGE 109

-ID 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 2 2 2 2 2 2 2 APPENDIX A: FOLLOW-UP PERIPHERAL BLOOD COUNTS FOR COHORTS 1 AND 2 COHORT DATE WBC (ul) ANC (ul) PLT mmA3 1 07/30/98 1 1 1 02 1 07/31/98 1 7 1 66 1 08/01/98 0 3 1 08/02/98 0 3 1 08/03/98 0.3 1 08/04/98 1 2 0 79 1 08/05/98 2.4 1 8 1 08/06/98 4 3 3 35 1 08/06/98 NO NO 1 08/07/98 9 5 7 98 1 08/08/98 21 17 22 1 08/09/98 27 21. 87 1 08/10/98 15 7 10 67 1 08/12/98 6.5 3 83 1 08/15/98 3 7 1 7 1 08/18/98 3.5 2 2 1 09/15/98 10.8 8 3 1 11/30/98 4.4 2 8 1 12/29/98 3 9 2 1 1 02/06/99 4 6 2 .71 1 04/23/99 3 6 1.48 1 08/19/99 4.2 1 85 1 10/21/99 6 8 3 9 1 04/15/00 5.9 2.3 1 07/17/00 5 9 2 4 1 11/27/00 8 3.41 1 08/17/98 0 1 1 08/18/98 0 1 1 08/18/98 0 1 1 08/19/98 0 1 1 08/19/98 0 1 1 08/19/98 NO NO 1 08/20/98 0 1 ANC not calculated for WBC <500/uL NO i ndicates no data provided or assay not performed Counts reported X 1 000 95 150 92 54 31 9 32 13 8 62 38 19 11 12 24 85 132 160 138 107 152 156 219 254 189 200 211 70 35 17 13 8 50 26

PAGE 110

ID 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 APPENDIX A: FOLLOW-UP PERIPHERAL BLOOD COUNTS FOR COHORTS 1 AND 2 COHORT DATE WBC (ul) ANC (ul) PLT mmA3 1 08/20/98 0.3 1 08/21/98 0 1 1 08/21/98 0 1 1 08/21/98 NO NO 1 08/22/98 0 1 1 08/22/98 0 2 1 08/23/98 2.4 1 08/23/98 0 1 1 08/23/98 0 1 1 08/23/98 NO NO 1 08/24/98 0 1 1 08/24/98 0 1 1 08/25/98 0 1 1 08/25/98 0 1 1 08/25/98 NO NO 1 08/26/98 0 1 1 08/26/98 0.1 1 08/27/98 0.2 1 08/27/98 NO NO 1 08/27/98 0 3 1 08/28/98 0 3 1 08/28/98 0 4 1 08/28/98 0.4 1 08/29/98 0 5 0.45 1 08/29/98 0 6 0 52 1 08/29/98 NO NO 1 08/30/98 0 5 0.42 1 08/30/98 NO NO 1 08/30/98 0 8 0 66 1 08/30/98 0 8 0.65 1 08/31/98 NO 0 6 1 08/31/98 NO NO 1 08/31/98 1 2 0 9 ANC not calculated for WBC <500/uL NO indicates no data provided or assay not performed Counts reported X 1 000 96 18 17 9 34 29 18 11 6 46 29 19 15 9 32 28 14 8 35 32 21 16 8 15 8 16 9 20 16 10 9 6 15

PAGE 111

APPENDIX A: FOLLOW UP PERIPHERAL BLOOD COUNTS FOR COHORTS 1 AND 2 10 COHORT DATE WBC {ul) ANC {ul) PLT mm"3 2 2 2 2 2 2 2 2 2 --2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 1 09/01/98 1.6 1 04 1 09/01/98 2.4 1 82 1 09/02/98 3 2 .31 1 09/02/98 NO NO 1 09/03/98 5 3 4 39 1 09/05/98 10 8 3 1 09/06/98 10 9 9 15 1 09/09/98 3 1 1.8 1 09/16/98 2 3 1 29 -1 09/24/98 1 8 0 9 1 10/01/98 4 2 .28 1 10/30/98 3 2 2 37 1 11/09/98 4.2 3 23 1 11/20/98 2 5 1 73 1 12/10/98 3.9 2.81 1 01/07/99 5 1 4 03 1 02/04/99 3.8 2 62 1 03/04/99 4 3 04 1 04/01/99 3 9 3 1 04/29/99 4 2 2 77 1 05/27/99 3 8 2 85 1 08/26/99 3 9 2 7 1 09/23/99 3 2 2 3 1 10/21/99 3 6 2 23 1 12/20/99 3 2 2 2 1 01/17/00 3 3 2 2 1 02/17/00 3 6 2 45 1 03/16/00 5 3 75 1 04/27/00 5 1 3 7 1 05/26/00 5 3 4 1 06/23/00 4.4 3 2 1 08/18/00 4 1 3 1 1 09/22/00 4 7 3 5 ANC not calculated for WBC <500/uL NO indicates no data provided or assay not performed Counts reported X 1 000 97 14 13 10 12 13 15 18 37 110 125 216 142 157 122 133 162 152 135 127 144 144 146 156 134 124 125 134 136 156 116 125 134 150

PAGE 112

ID 2 2 2 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 APPENDIX A: FOLLOW UP PERIPHERAL BLOOD COUNTS FOR COHORTS 1 AND 2 COHORT DATE WBC {ul) ANC {ul) PLT MM"3 1 10/20/00 4.4 3 2 1 11/17/00 4 6 3 2 1 12/15/00 4 2 9 1 09/13/98 9 7 56 1 09/13/98 ND ND 1 09/14/98 17 15 .81 1 09/15/98 36 5 29.56 1 09/16/98 46.4 35 .72 1 09/17/98 46 7 35 95 1 09/21/98 9 3 5 02 1 09/22/98 5 8 2 .61 -1 09/29/98 5.4 3 3 1 11/05/98 2 9 1 94 1 11/12/98 3 2 2 53 1 11/17/98 2 9 2 03 1 11/25/98 3 3 2 .71 1 12/02/98 3 5 2 84 1 03/09/99 3 9 2 7 1 06/15/99 4 2 5 1 09/13/99 5 7 4 1 1 11/01/99 5 7 3 8 1 11/29/99 4 2 2 9 1 12/17/99 5 2 3 3 1 12/30/99 2 6 1.4 1 01/24/00 5 1 3 2 1 02/07/00 6.9 3 17 1 02/21/00 9 8 7 7 1 03/17/00 6.7 4 5 1 04/17/00 5.3 3 4 1 05/15/00 4 9 3 2 1 12/08/00 6 1 ND 1 12/18/00 6 7 3 9 1 12/26/00 8.4 5 5 ANC not calculated for WBC <500/uL ND indicates no data provided or assay not performed Counts reported X 1 000 98 147 132 156 5 53 42 48 67 110 253 298 411 175 232 152 142 143 200 189 189 203 151 178 168 145 156 100 130 148 125 23 39 22 -

PAGE 113

ID 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 APPENDIX A: FOLLOW-UP PERIPHERAL BLOOD COUNTS FOR COHORTS 1 AND 2 COHORT DATE WBC (ul) ANC (ul) PLT mm"3 1 09/15/98 0 2 1 09/15/98 0 1 1 09/16/98 0 1 1 09/16/98 0 2 1 09/17/98 0 1 1 09/17/98 0 2 1 09/17/98 NO NO 1 09/18/98 0.1 1 09/18/98 0 1 1 09/19/98 0 2 1 09/19/98 0.4 1 09/19/98 0 5 0 37 1 09/20/98 0 8 0.52 1 09/20/98 0 7 0.4 1 09/21198 0 7 0.51 1 09/21/98 0 7 0 5 1 09/21/98 1 0.7 1 09/22/98 NO NO 1 09/22/98 1 4 1 09 1 09/23/98 2 9 2.4 1 09/23/98 4.8 3 69 1 09/24/98 5 8 4 87 1 09/24/98 5 7 5 3 1 09/24/98 10 1 8.68 1 09/24/98 NO NO 1 09/25/98 10 6 9.32 1 09/25/98 18 1 16.29 1 09/26/98 20 2 17 97 1 09/27/98 17 7 15.22 1 09/28/98 12. 9 10 83 1 09/29/98 9 7 8.43 1 10/01/98 7 3 6.57 1 10/03/98 4 7 NO ANC not calculated for WBC < 500/uL NO indicates no data provided or assay not performed Counts reported X 1 000 99 42 31 27 17 13 9 53 40 23 15 10 51 38 3 1 22 14 10 81 63 34 24 14 11 9 67 53 35 28 19 16 16 18 26

PAGE 114

ID 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 APPENDIX A: FOLLOW-UP PERIPHERAL BLOOD COUNTS FOR COHORTS 1 AND 2 COHORT DATE WBC (ul) ANC (ul) PLT mmA3 --1 10/05/98 5 1 4 5 1 10/07/98 4 5 3.6 1 10/09/98 3 3 2 2 1 10/10/98 2.9 1 8 1 10/20/98 3.6 2 98 1 11/03/98 3 1 1 52 1 11/10/98 3 6 2 02 1 11/18/98 3.4 1 63 1 11/24/98 3 8 2 .01 1 12/03/98 3 1 92 1 12/15/98 3.4 NO 1 12/22/98 4.4 3 57 1 01/13/99 4 3.18 1 01/25/99 3.4 2.55 1 02/02/99 3 2.37 1 02/23/99 2 7 2.13 1 04/13/99 2 1 1.39 1 06/21/99 3 3 2 44 1 08/09/99 3 6 2.77 1 09/13/99 3 9 2 82 1 11/16/99 4 2 2.81 1 12/07/99 4 5 3.33 1 02107100 6 1 4 94 1 03/07/00 4.4 2 89 1 03/22/00 4 3 3 .31 1 04/04/00 3 7 3 68 1 04/11/00 2 6 1 14 1 04/18/00 3 5 2.03 1 04/25/00 3 1 95 1 05/02/00 3 2 1 05/09/00 3 2 08 1 05/16/00 2 5 1.67 1 06/02/00 4 3 3 3 ANC not calculated for WBC <500/uL NO indicates no data provided or assay not performed Counts reported X 1 000 100 25 28 36 34 52 7 12 28 42 -55 61 54 100 105 44 25 31 47 66 62 105 92 88 74 93 93 38 16 51 38 26 34 92

PAGE 115

10 4 4 4 4 4 5 5 5 5 r-5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 6 6 APPENDIX A: FOLLOW UP PERIPHERAL BLOOD COUNTS FOR COHORTS 1 AND 2 COHORT DATE WBC (ul) ANC (ul) PLT mml\3 1 06/26/00 4 9 3.68 1 08/18/00 4 5 NO 1 09/28/00 4 3 3 14 1 10/26/00 4.6 3 45 1 11/21/00 4 5 3 69 1 09/28/98 0.4 1 09/28/98 0 3 1 09/29/98 0 1 1 09/30/98 0 1 1 09/30/98 NO NO 1 10/01/98 0 2 1 10/02/98 0 4 1 10/02/98 0.4 1 10/03/98 0 9 0 78 1 10/04/98 1 4 1 2 1 10/04/98 NO NO 1 10/05/98 2 1 1.91 1 10/06/98 4 3 3.95 1 10/07/98 6 2 5 89 1 10/07/98 NO NO 1 10/08/98 9.4 8 55 1 10/09/98 14 9 12. 96 1 10/10/98 12 10 08 1 10/11/98 9 2 8 37 1 10/13/98 4 2 2 9 1 10/14/98 3 1 2 1 1 11/17/98 5 5 4 2 1 02/09/99 7 2 6 6 1 04/28/99 6 3 5 4 1 09/20/99 5 4 2 1 10/06/99 5.4 4 6 1 10/12/98 1 1 1 .01 1 10/12/98 0 7 0 65 ANC not calculated for WBC <500/uL NO indicates no data provided or assay not performed Counts reported X 1 000 101 137 111 96 119 103 41 29 10 6 40 16 6 44 21 6 44 22 14 6 48 33 20 13 15 18 18 150 68 135 145 140 68 63

PAGE 116

ID 6 6 6 6 6 6 6 6 6 r-6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 7 7 7 7 7 7 7 7 APPENDIX A: FOLLOW UP PERIPHERAL BLOOD COUNTS FOR COHORTS 1 AND 2 COHORT DATE WBC (ul) ANC (ul) PLT mml\3 1 10/13/98 0 7 0 65 1 10/13/98 0.4 1 10/14/98 0 3 1 10/14/98 0 2 1 10/15/98 0.2 1 10/15/98 ND ND 1 10/16/98 0 3 1 10/17/98 0 6 0 5 1 1 10/18/98 1 3 1 15 1 10/18/98 ND ND 1 10/19/98 1 6 1 34 1 10/20/98 1 6 1 39 1 10/21/98 2 1 66 1 10/21/98 ND ND 1 10/22/98 3 8 3 34 1 10/23/98 4 9 4 26 1 10/24/98 7 4 6.36 1 10/25/98 10 6 9 75 1 10/25/98 ND ND 1 10/26/98 12.8 9.85 1 10/28/98 4 8 3 36 1 10/30/98 3 1 9 1 08/20/99 6 6 4 92 1 10/28/99 4 2 2.82 1 03/27/00 4.4 2 93 1 10/12/98 0 5 0.27 1 10/12/98 1 0 38 1 10/13/98 0 3 1 10/13/98 0.5 0 07 1 10/14/98 0 3 1 10/14/98 0 2 1 10/14/98 ND ND 1 10/15/98 0 1 ANC not calculated for WBC < 500/uL ND i ndicates no data provided or assay not performed Counts reported X 1 000 102 50 37 30 18 9 60 32 15 8 49 33 17 10 56 35 16 12 10 73 44 25 19 101 140 142 61 50 38 24 15 8 50 38

PAGE 117

ID 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 f---7 7 APPENDIX A: FOLLOW UP PERIPHERAL BLOOD COUNTS FOR COHORTS 1 AND 2 COHORT DATE WBC (ul) ANC (ul) PLT mm"3 1 10/15/98 0 2 1 10/16/98 0 1 1 10/16/98 0 2 1 10/16/98 NO ND 1 10/17/98 0 2 1 10/17/98 0 3 1 10/17/98 ND ND 1 10/17/98 0 3 1 10/18/98 0.4 1 10/18/98 NO NO 1 10/18/98 0.4 1 10/19/98 0 6 ND 1 10/19/98 ND ND 1 10/19/98 0 6 0.45 1 10/20/98 0 9 0 72 1 10/20/98 1 0 .78 1 10/21/98 ND ND 1 10/21/98 1 6 1 26 1 10/22/98 1 7 1 34 1 10/22/98 2 9 2 .11 1 10/23/98 3.8 2 73 1 10/23/98 5 6 4 .81 1 10/24/98 8.4 6.8 1 10/25/98 15 9 12.72 1 10/26/98 25 20 1 10/28/98 4 8 2 73 1 11/02/98 1 6 0 56 1 11/04/98 11.4 9 7 1 11/09/98 2 5 1 42 1 11/13/98 3 8 1 9 1 01/21/99 2 3 1 2 1 04/29/99 2.6 1 2 1 08/12/99 3.4 1 7 ANC not calculated for WBC <500/uL ND i ndicates no data prov i ded or assay not performed Counts reported X 1 000 103 29 16 13 55 26 14 44 29 14 -55 33 1 9 54 40 28 17 45 38 26 19 15 11 12 17 28 69 271 338 357 240 215 191 200

PAGE 118

ID 7 7 7 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 APPENDIX A: FOLLOW UP PERIPHERAL BLOOD COUNTS FOR COHORTS 1 AND 2 COHORT DATE WBC (ul) ANC (ul) PLT mmAJ 1 11112/99 5 1 4 3 1 05/11/00 2.9 1 2 1 11/09/00 3 6 1.4 1 10/19/98 0 5 0.48 1 10/19/98 0.4 1 10/20/98 0 6 0 5 7 1 10/20/98 0 2 1 10/21/98 0 1 1 10/21/98 0 1 ---1 10/22/98 0 1 1 10/23/98 0 1 1 10/23/98 NO NO 1 10/24/98 0 2 1 10/25/98 0 1 1 10/25/98 NO NO 1 10/26/98 0 1 1 10/27/98 0.1 1 10/28/98 0 1 1 10/29/98 0.2 1 10/30/98 0.3 1 10/31/98 0.4 1 11/01/98 0 5 0.4 1 11/01198 NO NO 1 11/02/98 0 9 0 65 1 11/03/98 1.2 1 02 1 11/04/98 1 8 1 38 1 11/05/98 2 6 1 97 1 11/06/98 2 9 2.29 1 11/06/98 NO NO 1 11107/98 3 6 2 .91 1 11/07/98 NO NO 1 11/08/98 4 1 3 19 1 11/09/98 6.4 5.18 ANC not calculated for WBC < 500/uL NO indicates no data provided or assay not performed Counts reported X 1 000 1 04 194 194 174 61 47 46 30 28 27 12 5 14 13 7 19 6 17 14 8 28 15 7 32 8 22 13 9 5 14 5 33 19 12

PAGE 119

APPENDIX A: FOLLOW-UP PERIPHERAL BLOOD COUNTS FOR COHORTS 1 AND 2 ID COHORT DATE WBC {ul) ANC {ul) PLT mmAJ 8 8 8 8 8 8 8 8 8 8 8 -8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 9 1 11/10/98 6.8 5 3 1 11/11/98 9.7 8 14 1 11/12/98 5 2 3 9 1 11/13/98 3.2 1 9 1 11/14/98 2.6 1 3 1 11/16/98 2 5 1.37 1 11/19/98 2 7 1 62 1 11/25/98 3 8 1 76 1 11/30/98 3 6 2 54 --1 12/07/98 8 2 5 9 1 12/14/98 8 4 7.48 1 12/21/98 4 7 4.46 1 12/28/98 4 7 4 22 1 01/04/99 4 6 NO 1 01/06/99 2 9 2 19 1 01/11/99 3 2.42 1 01/15/99 3 1 2.44 1 01/18/99 2 5 1.96 1 01/20/99 2 1 1 .61 1 01/22/99 1.8 1.24 1 01/25/99 1 9 1.42 1 01/27/99 3 5 3 15 1 01/29/99 2.4 1 73 1 02/01/99 3 1 2 68 1 02/11/99 4 9 4 42 1 03/25/99 6 5 5 3 1 09/30/99 1 7 0 93 1 10/07/99 2.3 1 56 1 01/13/00 2 7 1 78 1 05/25/00 3 2 2.28 1 11/15/00 4 5 3 .21 1 12/21/00 2 1 .21 1 11/02/98 NO NO ANC not calculated for WBC <500/uL NO indicates no data provided or assay not performed Counts reported X 1 000 105 10 11 13 19 25 32 56 96 166 136 117 106 72 45 31 34 32 42 34 43 42 50 60 59 84 83 72 90 114 116 118 121 118

PAGE 120

ID 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 10 10 10 APPENDIX A: FOLLOW-UP PERIPHERAL BLOOD COUNTS FOR COHORTS 1 AND 2 COHORT DATE WBC (uL) ANC (uL) PLT mmAJ 1 11/02/98 1 3 ND 1 11/03/98 3.4 3 29 1 11/04/98 0 6 0 39 1 11/05/98 0 3 1 11/06/98 0.4 1 11/07/98 0 6 0 34 1 11/07/98 ND ND 1 11/08/98 1 0 79 1 11/09/98 1.7 1 25 1 11/10/98 3.4 2 58 1 11/11/98 6 8 5 .78 1 11/12/98 9 3 7 .71 1 11/13/98 17 6 13 02 1 11/16/98 4 6 2.99 1 11/17/98 4 8 3 1 11/25/98 3 9 2 3 1 12/06/98 17 6 16 1 01/12/99 7 6 6.4 1 02/11/99 6.9 5 6 1 03/01/99 5 5 4 1 03/15/99 3 3 2 3 1 03/30/99 3 7 3 03 1 05/07/99 4.4 3 3 1 07/21/99 4.4 3 1 1 09/22/99 4 9 3 3 1 11/23/99 4.8 3 2 1 02/16/00 5.8 4 1 04/27/00 5.6 3 6 1 07/11/00 5 7 3 6 1 10/31/00 6 5 4 23 1 11/16/98 0 2 1 11/17/98 0 2 1 11/18/98 0 2 ANC not calculated for WBC <500/uL ND indicates no data provided or assay not performed Counts reported X 1 000 106 93 89 62 37 16 8 34 28 14 11 33 25 24 30 56 143 244 161 153 168 132 145 165 144 153 148 163 158 164 145 84 55 34

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ID 10 10 10 10 10 10 10 10 10 r-----1 0 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 APPENDIX A : FOLLOW-UP PERIPHERAL BLOOD COUNTS FOR COHORTS 1 AND 2 COHORT DATE WBC (uL) ANC (uL) 1 11/19/98 0 1 1 11/20/98 0 1 1 11/21/98 0 1 1 11/21/98 0.2 1 11/21/98 0.1 1 11/22/98 0.1 1 11/22/98 0 1 1 11/22/98 ND ND 1 11/23/98 0 1 11/24/98 1 0 1 1 11/25/98 0 1 1 11/25/98 ND ND 1 11/26/98 0.2 1 11/27/98 0.4 1 11/27/98 ND ND 1 11/28/98 0.5 0 43 1 11/28/98 ND ND 1 11/29/98 0 8 0 59 1 11/30/98 1 2 1.03 1 12/01/98 2.2 1 82 1 12/02/98 2.8 2.4 1 12/02/98 ND ND 1 12/03/98 3.4 2 82 1 12/03/98 ND ND 1 12/04/98 4.4 3.34 1 12/05/98 5 9 4.89 1 12/06/98 5 8 4 75 1 12/07/98 7 1 6.31 1 12/08/98 9.5 7 98 1 12/09/98 14.4 12 67 1 12/10/98 10.6 7 2 1 12/11/98 7 3 ND 1 12/14/98 5 2 35 ANC not calculated for WBC <500/uL ND indicates no data provided or assay not performed Counts reported X 1 000 107 PLT mmAJ 19 11 45 42 40 18 11 40 31 -11 3 34 17 9 18 4 33 6 24 16 10 33 10 42 12 7 18 12 15 14 19 37 76

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10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 APPENDIX A : FOLLOW-UP PERIPHERAL BLOOD COUNTS FOR COHORTS 1 AND 2 COHORT DATE WBC (ul) ANC (ul) PLT mm"3 1 12/21/98 5.5 3 7 1 02/04/99 2 2 1 6 1 02/10/99 2 1 1.7 1 02/18/99 1 6 1 1 02/19/99 1.8 1 1 1 02/22/99 2.8 2 3 1 02/25/99 1.8 1 3 1 03/01/99 2 2 1.5 1 04/06/99 1 6 0 7 1 04/23/99 1 2 0.8 1 05/06/99 2 1 4 1 06/16/99 1.7 1 3 1 09/08/99 1 8 1 3 1 12/16/99 2 1 1 3 1 02/07/00 2 5 1.5 1 04/17/00 3.5 1 9 1 05/26/00 2 3 1.4 1 11/07/00 1 6 0.6 2 11/25/98 0 2 2 11/26/98 0 2 2 11/27/98 0 1 2 11/28/98 0 3 2 11/29/98 0.4 2 11/29/98 NO NO 2 11/30/98 0 5 0 43 2 12/01/98 0 4 2 12/02/98 0 5 NO 2 12/03/98 0 6 0 54 2 12/04/98 1 0 .91 2 12/04/98 NO NO 2 12/05/98 1 3 1 22 2 12/06/98 2 1 .78 2 12/07/98 2 3 2 04 ANC not calculated for WBC <500/uL NO indicates no data provided or assay not performed Counts reported X 1 000 108 178 111 89 115 122 124 126 127 120 121 125 1 46 173 130 142 137 154 132 83 49 27 13 6 50 46 24 13 13 9 56 59 40 24

PAGE 123

ID 1 1 1 1 1 1 1 1 1 t--1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 APPENDIX A: FOLLOW UP PERIPHERAL BLOOD COUNTS FOR COHORTS 1 AND 2 COHORT DATE WBC (ul) ANC (ul) PLTmm"3 -2 12/08/98 2.2 2 2 12/09/98 2.4 2 2 2 12/09/98 ND ND 2 12/10/98 2.7 2 48 2 12/11/98 2 7 2 59 2 12/12/98 3.8 3.45 2 12/13/98 3 3 2.77 2 12/14/98 3 1 2 44 2 12/15/98 3.3 2 54 2 12/16/98 4 2 76 -1--2 12/17/98 5 7 4 .78 1--2 12/18/98 6 4 4 54 2 12/19/98 7 6 5.85 2 12/20/98 7 9 6 39 2 12/21/98 10 8 9 2 12/21/98 ND ND 2 12/23/98 2.7 1 7 2 12/26/98 2.4 1 3 2 12/29/98 2 4 1 5 2 12/31/98 4.1 3 1 2 01/04/99 3 1 1 92 2 01/04/99 4.1 3.24 2 01107/99 3 7 3 1 2 01/10/99 5 ND 2 01/18/99 6 7 5 63 2 01/24/99 6.4 ND 2 02/21/99 5 4 ND 2 03/15/99 4.2 3 2 2 03/19/99 4.6 3 27 2 03/20/99 4 3.2 2 03/23/99 3 8 3 1 2 04/12/99 5 8 5 05 2 05/15/99 3 5 2 7 ANC not calculated for WBC <500/uL ND indicates no data provided or assay not performed Counts reported X 1 000 109 14 7 44 38 25 12 8 59 43 -28 20 13 12 12 10 77 61 57 42 58 69 64 79 90 119 151 135 116 115 107 100 105 115

PAGE 124

ID 1 1 1 1 1 1 1 1 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 APPENDIX A: FOLLOW UP PERIPHERAL BLOOD COUNTS FOR COHORTS 1 AND 2 COHORT DATE wee (uL) ANC (ul) PLT mmAJ 2 06/09/99 3 6 2 62 2 07/09/99 2 5 1 6 2 08/13/99 3 2 1 .92 2 11/05/99 4 3.1 2 02/21/00 3 7 2.1 2 05/12/00 3 3 1.4 2 07/31/00 3.3 1 8 2 11/13/00 3.7 1 9 2 11/26/98 2 1 .98 --2 11/26/98 1 1 NO 2 11/27/98 1.2 NO 2 11/27/98 0 6 0 58 2 11/28/98 0.2 2 11/29/98 0.3 2 11/29/98 NO NO 2 11/30/98 0 4 2 12/01/98 0 8 0.63 2 12/02/98 2 1 7 2 12/02/98 NO NO 2 12/03/98 3 3 2.93 2 12/04/98 5.4 4 .91 2 12/04/98 NO NO 2 12/05/98 7 6 7.29 2 12/06/98 11. 2 8 96 2 12/06/98 NO NO 2 12/07/98 12 2 10 12 2 12/08/98 8 2 6 .31 2 12/09/98 4 1 2.91 2 12/10/98 5 3 4.13 2 12/10/98 NO NO 2 12/11/98 5 9 4.13 2 12/13/98 5.1 3.67 2 12/14/98 5 4 3.51 ANC not calculated for WBC <500/uL NO indicates no data provided or assay not performed Counts reported X 1 000 110 148 137 151 161 140 155 210 194 75 63 42 35 23 10 50 17 7 10 46 11 12 55 14 7 37 12 8 13 10 57 29 15 16

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ID 2 2 2 2 2 2 2 2 2 t--2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 APPENDIX A: FOLLOW-UP PERIPHERAL BLOOD COUNTS FOR COHORTS 1 AND 2 COHORT DATE WBC (ul) ANC (ul) PLT mm"3 2 12/15/98 4 5 3 28 2 12/17/98 4.3 2 62 2 12/19/98 4 9 2 64 2 12/21/98 4 3 2.45 2 12/23/98 5 2 7 2 12/29/98 6.3 3 96 2 12/31/98 6 1 NO 2 01/03/99 4 6 2 8 2 01/06/99 5 8 NO ---2 01/11/99 4 7 3 52 2 01/18/99 5 3 3.9 2 01/21/98 5 1 2.9 2 01/25/99 5 7 3 1 2 01128/99 6.9 4 3 2 02/01/99 6 8 4 7 2 02/08/99 7 7 5 2 2 02/16/99 5 9 2 7 2 02/22/99 4.7 NO 2 03/01/99 4 1 2 5 2 03/15/99 4.7 2.4 2 03/22/99 4 1 2 5 2 03/29/99 4 1 2 3 2 04/19/99 4 2 6 2 04/22/99 3 9 2 2 05/05/99 5 7 NO 2 05/13/99 6.3 3 6 2 05/25/99 6 2 4 2 06/08/99 4 5 2 5 2 07/08/99 5.4 3 7 2 08/10/99 3.8 2.4 2 10/07/99 3.9 3 3 2 11/03/99 3.7 2.3 2 12/08/99 3.5 2 6 ANC not calculated for WBC <500/uL NO indicates no data provided or assay not performed Counts reported X 1 000 111 18 16 16 20 24 20 28 23 34 33 48 68 79 94 89 102 157 136 157 216 181 188 123 133 164 187 161 126 181 150 94 79 78

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ID 2 2 2 2 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 APPENDIX A: FOLLOW-UP PERIPHERAL BLOOD COUNTS FOR COHORTS 1 AND 2 COHORT DATE WBC (ul) ANC (ul) PLT mml\3 2 01/10/00 2 6 1 9 2 02/03/00 3 8 2 5 2 04/03/00 4 2 6 2 06/01/00 4.2 2 7 2 12/11/98 49 6 44 64 2 12/11/98 51. 7 49 .11 2 12/14/98 8 8 NO 2 12/15/98 7 5 NO 2 12/15/98 7.4 NO 2 12/16/98 7 3 NO 2 12/16/98 6 1 NO 2 12/17/98 5 7 NO 2 12/17/98 5 1 NO 2 12/18/98 5 2 NO 2 12/18/98 4 2 NO 2 12/19/98 4 8 NO 2 12/19/98 7 3 7 22 2 12/20/98 4 5 4 36 2 12/20/98 3 2 85 2 12/21/98 2.2 2 .11 2 12/21/98 0 9 NO 2 12/22/98 1 1 1 05 2 12/23/98 0 2 2 12/24/98 0 2 2 12/25/98 0 3 2 12/26/98 0 5 0.41 2 12/27/98 0 7 0 65 2 12/27/98 NO NO 2 12/28/98 0 8 0 65 2 12/29/98 1 0 9 2 12/30/98 1 6 1.34 2 12/30/98 NO NO 2 12/31/98 2.3 2 13 ANC not calculated for WBC <500/uL NO indicates no data provided or assay not performed Counts reported X 1 000 1 12 48 95 110 102 104 79 142 115 131 150 158 158 144 148 155 161 167 171 163 160 139 101 64 38 17 26 12 41 24 12 5 51 37

PAGE 127

APPENDIX A: FOLLOW UP PERIPHERAL BLOOD COUNTS FOR COHORTS 1 AND 2 10 COHORT DATE WBC (ul) ANC (ul) PLT mmAJ 3 2 01/01/99 5 3 4 5 3 2 01/02/99 7 5 95 3 2 01/04/99 14 8 9.76 3 2 01/04/99 ND ND 3 2 01/06/99 6.6 4 2 3 2 01/08/99 4.5 1.6 3 2 01/11/99 5 3 2.4 3 2 01/11/99 2 9 1 6 3 2 01/18/99 3 7 2 I----13 2 01/25/99 4 3 1 9 3 3 3 3 3 3 3 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 2 02/01/99 6 3.6 2 08/11/99 4.4 3.3 2 09/15/99 4.1 3 1 2 11/16/99 3 7 2 3 2 01/14/00 2.6 1 5 2 01/27/00 3.8 2.4 2 03/13/00 3 5 2 1 2 12/25/98 2 3 ND 2 12/25/98 1.5 1.48 2 12/26/98 1.8 ND 2 12/26/98 1 6 1.55 2 12/27/98 0 5 0 38 2 12/27/98 0 1 2 12/28/98 0 1 2 12/29/98 0 2 2 12/30/98 0 3 2 12/31/98 0 3 2 01/01/99 0 5 0.4 2 01/01/99 ND ND 2 01/02/99 0 9 0 79 2 01/03/09 1 8 1 54 2 01/04/99 3 ND 2 01/05/99 3 5 3 32 ANC not calculated for WBC < 500/uL ND indicates no data provided or assay not performed Counts reported X 1 000 113 23 17 12 52 31 35 61 42 127 203 196 152 149 138 185 131 131 75 64 53 42 30 25 18 7 27 17 7 44 29 16 9 24

PAGE 128

APPENDIX A: FOLLOW UP PERIPHERAL BLOOD COUNTS FOR COHORTS 1 AND 2 10 COHORT DATE WBC (ul) ANC (ul) PLT mmAJ 4 4 4 4 4 4 4 4 4 ----4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 2 01/06/99 5 9 5 13 2 01/07/99 8.4 8 06 2 01/08/99 9 9 9 2 01/09/99 4.1 NO 2 01/10/99 3 1 NO 2 01/11199 2.2 NO 2 01/13/99 2 9 NO 2 01/15/99 3 1 NO 2 01/25/99 3 7 2.41 2 02/08/99 8 9 7 39 2 02/15/99 7 3 5 69 2 02/22/99 4 5 3.47 2 03/01/99 4.4 3 26 2 03/08/99 3 7 2 37 2 04/05/99 6 6 NO 2 04/06/99 9 NO 2 04/07/99 7.7 NO 2 04/08/99 6 5 NO 2 04/09/99 5.5 NO 2 04/10/99 4 8 NO 2 04/11199 4 9 NO 2 04/12/99 4 9 4 8 2 04/13/99 4.6 NO 2 04/13/99 5.6 NO 2 04/14/99 5 2 NO 2 04/15/99 11. 9 NO 2 04/16/99 7 1 NO 2 04/17/99 9 3 NO 2 04/21/99 11. 2 10.86 2 04/23/99 8 7 NO 2 04/30/99 6 6 NO 2 05/05/99 5 4 NO 2 05/10/99 7 1 6 2 ANC not calculated for WBC <500/uL NO indicates no data provided or assay not performed Counts reported X 1 000 114 18 12 21 22 20 22 43 67 148 130 161 153 129 103 94 102 84 80 81 80 81 80 84 86 89 94 87 97 94 91 59 66 77

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ID 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 5 5 5 APPENDIX A: FOLLOW-UP PERIPHERAL BLOOD COUNTS FOR COHORTS 1 AND 2 COHORT DATE WBC (ul) ANC (ul) PLT mmA3 2 05/17/99 7 6 2 05/21/99 9 8 2 2 06/10/99 9 9 9 2 08/18/99 9 1 8 3 2 09/25/99 6 5 5 1 2 11/22/99 35 8 28.8 2 12/01/99 5 3 9 2 01/17/00 4 1 2 9 2 01/21/00 5 1 3.8 2 02/09/00 7 3 NO 2 02/10/00 5 NO 2 02/11/00 5 3 NO 2 02/12/00 4 7 NO 2 02120100 0 6 0 55 2 02122100 NO NO 2 02/23/00 0 6 0 54 2 02/26/00 0 1 2 02127100 0 1 2 03/04/00 0 5 0 19 2 03/05/00 0 9 0 55 2 03/09/00 3 1 2 2 2 03/11/00 5.4 3 94 2 03/12/00 7 5 6 52 2 05/26/00 6 9 5 8 2 07107100 5 2 4 1 2 07/18/00 10 8 9.4 2 09/06/00 3 3 2 3 2 09/14/00 4 1 3 2 2 09/25/00 7 2 5 6 2 10/05/00 6 2 5 2 01/04/99 1 6 NO 2 01/04/99 0 9 NO 2 01/05/99 0 8 0 72 ANC not calculated fo r WBC < 500/uL ND indicates no data provided or assay not performed Counts reported X 1 000 115 66 79 129 152 125 132 159 160 179 149 147 131 160 27 71 80 21 10 23 17 9 19 17 109 124 205 28 72 135 160 149 152 116

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ID 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 6 6 6 APPENDIX A: FOLLOW UP PERIPHERAL BLOOD COUNTS FOR COHORTS 1 AND 2 COHORT DATE WBC (ul) ANC (ul) PLT mml\3 2 01/05/99 0.4 2 01/06/99 0 3 2 01/06/99 0 2 2 01/06/99 0 1 2 01/07/99 0 1 2 01/07/99 0 3 2 01/08/99 0 2 2 01/08/99 0 2 2 01/09/99 0 2 2 01/09/99 0 2 2 01/10/99 0 3 2 01/10/99 NO NO 2 01/10/99 0 3 2 01/11/99 0 6 0 46 2 01/11/99 NO NO 2 01/11/99 0 9 0 63 2 01/12/99 1 5 1 29 2 01/12/99 NO NO 2 01/12/99 2 3 2 04 2 01/13/99 3 9 3 35 2 01/13/99 4.8 4 12 2 01/14/99 9 3 7.81 2 01/15/99 12 2 9 27 2 01/16/99 18 5 14.61 2 01118/99 6 2 4 09 2 04/10/99 2.9 1 9 2 04/27/99 6 8 6 1 2 11/03/99 4.8 3 1 2 02/09/00 4.6 3 .61 2 04/20/00 3 8 2.4 2 01/11/99 2 7 2 .61 2 01/11199 1.9 NO 2 01/12/99 2 1 94 ANC not calculated for WBC <500/uL NO indicates no data provided or assay not performed Counts reported X 1 000 116 104 81 67 65 49 43 33 25 17 12 10 20 14 4 33 24 9 27 24 21 17 13 15 20 29 156 214 151 221 131 85 74 57

PAGE 131

ID 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 7 APPENDIX A: FOLLOW-UP PERIPHERAL BLOOD COUNTS FOR COHORTS 1 AND 2 COHORT DATE WBC {ul) ANC {ul) PL T mm-1\3 2 01112/99 1.1 1 06 2 01/13/99 0 4 2 01/14/99 0 3 2 01/15/99 0 7 0 5 2 01/16/99 2 3 1 72 2 01/17/99 4 9 4.06 2 01/18/99 7 9 7 03 2 01/19/99 11. 7 9 94 2 01/21/99 13 3 11. 17 2 01/23/99 5 4 1 2 01/25/99 5 3 3.5 2 02/08/99 5.4 2 9 2 02/10/99 8 8 7 5 2 02/18/99 9 9 7 3 2 02/22/99 9 8 7 7 2 03/01/99 7.1 5 5 2 03/11/99 5.3 4 2 2 03/23/99 4 8 4 1 2 03/26/99 4 7 3 8 2 03/30/99 10.6 10 2 04/09/99 9 3 8 9 2 04/13/99 8.5 8 2 04/20/99 7 1 6 7 2 04/27/99 7 9 7 2 2 05/04/99 8 9 8 2 05/12/99 6 7 5 9 2 05/18/99 11. 3 10 2 2 06/09/99 6 9 ND 2 06/16/99 8 1 6 8 2 09/02/99 5 5 4.4 2 06/08/00 5 7 3 9 2 12/13/00 4 7 3 1 2 01/14/99 1 0 84 ANC not calculated for WBC <500/uL ND indicates no data provided or assay not performed Counts reported X 1 000 117 52 30 24 13 8 72 54 41 31 30 48 117 159 156 102 151 174 114 92 140 135 144 101 93 85 106 120 185 223 181 187 170 154

PAGE 132

ID 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 APPENDIX A: FOLLOW UP PERIPHERAL BLOOD COUNTS FOR COHORTS 1 AND 2 COHORT DATE WBC (uL) ANC (uL) PLT mmJ\3 2 01/14/99 1 0 .81 2 01/15/99 0.4 2 01/15/99 0 3 2 01/16/99 0 3 2 01/16/99 0 2 2 01/17/99 0 1 2 01/17/99 0 1 2 01/18/99 0 1 2 01/18/99 0 2 2 01/19/99 0 1 2 01/19/99 0 1 2 01/20/99 0 1 2 01/20/99 NO NO 2 01/20/99 0 2 2 01/21/99 0.2 2 01/21/99 NO NO 2 01/21/99 0.5 NO 2 01/22/99 0 6 0 53 2 01/22/99 NO NO 2 01/22/99 0 6 0 46 2 01/22/99 0 9 0.81 2 01/23/99 1 1 NO 2 01/23/99 NO NO 2 01/23/99 1 5 1 33 2 01/24/99 2 1 1 84 2 01/25/99 3 6 3 2 2 01/25/99 NO NO 2 01/26/99 4 7 3 85 2 01/26/99 NO NO 2 01/27/99 6 8 6.05 2 01/29/99 11.4 8 55 2 01/30/99 16 9 13 35 2 02/01/99 6.9 4 69 ANC not calculated for WBC <500/uL NO indicates no data provided or assay not performed Counts reported X 1 000 118 139 130 117 97 83 65 71 51 42 30 24 17 32 11 9 5 11 4 9 6 23 2 46 34 20 5 21 8 49 21 13 17 29

PAGE 133

10 7 7 7 7 7 7 7 7 7 7 7 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 APPENDIX A : FOLLOW UP PERIPHERAL BLOOD COUNTS FOR COHORTS 1 AND 2 COHORT DATE WBC (ul) ANC (ul) PLT mmA3 2 02/03/99 4.3 1 8 2 02/05/99 4 1.2 2 02/06/99 4 2 1 09 2 02/09/99 4.4 0.48 2 04/15/99 5 5 2 2 05/07/99 3 5 1 7 2 05/14/99 2 5 1 3 2 05/17/99 2 8 1 6 2 05/20/99 3 1 8 2 05/27/99 2 7 1 6 2 06/11/99 2 1 1.2 2 01/17/99 0 1 2 01/18/99 0 2 2 01/19/99 0 2 2 01/19/99 NO NO 2 01/20/99 0.4 2 01/21/99 0.5 0.42 2 01/22/99 0 7 0 52 2 01/23/99 0 6 0 46 2 01/24/99 1 1 0 93 2 01/25/99 1 1 0 95 2 01/25/99 NO NO 2 01/26/99 1 2 1 06 2 01/27/99 1 6 1 .31 2 01/28/99 1 6 1 4 2 01/29/99 1 7 1 39 2 01/30/99 2 4 2 .01 2 01/31/99 2 9 2.29 2 02/01/99 2.6 2.41 2 02/05/99 4 3 3 .31 2 02/06/99 4 4 3 .38 2 02/07/99 4 2 88 2 02/08/99 4 3 3 48 ANC not calculated for WBC < 500/uL NO indicates no data provided or assay not performed Counts reported X 1 000 1 19 44 63 79 107 308 23 1 196 182 197 172 198 39 18 6 70 46 29 12 8 25 8 69 44 30 86 43 22 10 56 18 7 14 8

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10 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 APPENDIX A: FOLLOW-UP PERIPHERAL BLOOD COUNTS FOR COHORTS 1 AND 2 COHORT DATE WBC (ul) ANC (ul) PLT mmA3 2 02/09/99 5 9 4.42 2 02/10/99 4.8 3.07 2 02/11/99 6 3 4.41 2 02/12/99 8.4 6.13 2 02/13/99 8 9 NO 2 02/14/99 8.9 7.12 2 02/14/99 NO NO 2 02/16/99 3.2 NO 2 02/18/99 4 3 NO 2 02/20/99 2.2 NO 2 02/22/99 2 6 NO 2 02/24/99 1 7 0.66 2 02/26/99 7.7 NO 2 02/26/99 NO NO 2 03/01/99 1 7 0.44 2 03/04/99 5.7 NO 2 03/06/99 3 1 NO 2 03/08/99 3 NO 2 03/11/99 2 4 NO 2 03/15/99 2.2 1.18 2 03/18/99 4 1 NO 2 03/25/99 3.7 NO 2 03/26/99 4 5 NO 2 03/30/99 3 9 NO 2 04/07/99 7 4 NO 2 04/14/99 5 NO 2 04/21/99 3 6 NO 2 05/06/99 3 1 2.26 2 05/10/99 2 1 1 44 2 05/10/99 NO NO 2 05/13/99 2 6 NO 2 05/17/99 2.4 NO 2 05/17/99 NO NO ANC not calculated for WBC <500/uL NO indicates no data provided or assay not performed Counts reported X 1 000 120 20 6 53 32 18 9 84 48 22 12 60 31 11 101 38 16 23 24 27 23 22 27 34 27 32 27 25 21 22 112 52 23 34

PAGE 135

10 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 9 9 9 9 9 9 9 9 9 9 9 APPENDIX A: FOLLOW-UP PERIPHERAL BLOOD COUNTS FOR COHORTS 1 AND 2 COHORT DATE WBC {uL) ANC {uL) PLT mm"3 2 05/18/99 NO NO 2 05/21/99 2 1 NO 2 05/24/99 2 5 1 97 2 05/24/99 NO NO 2 05/27/99 2 1.48 2 05/29/99 1 7 1 2 2 05/31/99 1 6 1.05 2 05/31/99 NO NO 2 06/04/99 1 7 NO 2 06/07/99 2 9 NO 2 06/10/99 2.5 NO 2 06/14/99 1 8 NO 2 06/17/99 2 1 3 2 06/19/99 3 2 2 06/24/99 1.6 1 16 2 06/27/99 3.2 2 56 2 06/30/99 1 9 NO 2 07/08/99 2 NO 2 07/14/99 2 1 1 2 07/21/99 4 6 3 6 2 07/23/99 3 4 2 6 2 07/26/99 4.3 NO 2 01/21/99 0 3 2 01/21/99 0.2 2 01/22/99 0 1 2 01/22/99 0 1 2 01/22/99 0 2 2 01/23/99 0 1 2 01/23/99 0 2 2 01/24/99 0.2 2 01/24/99 0.1 2 01/25/99 0 1 2 01/25/99 NO NO ANC not calculated for WBC <500/uL NO indicates no data provided, or assay not performed Counts reported X 1 000 121 101 64 31 82 54 71 41 88 38 23 23 23 24 25 24 30 27 25 25 26 21 20 78 58 30 38 19 13 32 14 32 14 45

PAGE 136

ID 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 10 10 10 10 APPENDIX A: FOLLOW UP PERIPHERAL BLOOD COUNTS FOR COHORTS 1 AND 2 COHORT DATE WBC {ul) ANC {ul) PLT mmAJ 2 01/25/99 0 1 2 01/26/99 0 2 2 01/26/99 0 1 2 01/26/99 NO NO 2 01/27/99 0 2 2 01/27/99 0.3 2 01/27/99 NO NO 2 01/28/99 0 4 2 01/28/99 0 3 2 01129/99 0 5 0 44 2 01/29/99 0 5 NO i-----2 01/29/99 NO NO 2 01/30/99 0.4 2 01/30/99 0 5 0.23 2 01/31/99 1 1 0 79 2 02/01/99 2 1 1 .51 2 02/02/99 4 7 3 57 2 02/03/99 11. 5 8 .51 2 02/04/99 31. 3 27 54 2 02/05/99 58.7 48 .72 2 02/09/99 12 9 7 74 2 02/11199 9 1 5 8 2 02/22/99 3 7 1 8 2 03/01/99 3 9 2 3 2 03/08/99 3 1 1 6 I2 03/15/99 2.4 1.4 2 03/22/99 3 1 9 2 03/29/99 3 3 2 4 2 04/22/99 3 5 2 3 2 01/22/99 1 2 NO 2 01/23/99 1.4 1 35 2 01124/99 0 2 2 01/25/99 0 1 ANC not calculated for WBC <500/uL NO indicates no data provided or assay not performed Counts reported X 1 000 122 24 14 8 28 24 13 40 38 26 -14 9 35 NO 11 25 14 17 29 63 95 203 296 370 194 188 170 138 148 178 106 71 46 ___ 25

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ID 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 11 11 11 APPENDIX A : FOLLOW-UP PERIPHERAL BLOOD COUNTS FOR COHORTS 1 AND 2 COHORT DATE WBC (ul) ANC (ul) PLT mmAJ 2 01/26/99 0 1 2 01/27/99 0 1 2 01/27/99 NO NO 2 01/28/99 0 3 2 01/29/99 0.3 2 01/30/99 0 1 2 01/30/99 NO NO 2 01/31/99 0 1 2 02/01/99 0 2 2 02/01/99 NO NO 2 02/07/99 NO 0 76 2 02/08/99 1 9 1 67 2 02/09/99 4 9 4 .31 2 02/10/99 7 6 6 15 2 02/11/99 17 13.6 2 02/12/99 32 23.68 2 02/16/99 2 7 1 56 2 04/07/99 4 3 1 2 04/13/99 5 3 4.97 2 04/16/99 5 3 4.94 2 04/20/99 7 6.53 2 04/23/99 5 3 4 96 2 04/27/99 5 3 5 .01 2 05/11/99 6.8 NO 2 06/24/99 3 7 NO 2 07/26/99 3 3 2.48 2 10/07/99 3.8 2.69 2 10/29/99 4.3 2.98 2 01/11100 5 1 3.67 2 02/15/00 4 3 3.08 2 01/25/99 3 1 2 .91 2 01/25/99 1.9 1 84 2 01/26/99 1 2 1 .11 ANC not calculated for WBC <500/uL NO indicates no data provided or assay not performed Counts reported X 1 000 123 11 6 37 20 9 7 40 18 6 28 NO 28 54 49 59 66 143 136 136 154 147 124 138 137 167 176 167 176 145 185 127 109 88

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ID 11 11 11 11 11 11 11 11 11 11 11 11 11 11 11 11 11 11 11 11 11 11 11 11 11 11 11 11 11 11 11 11 11 APPENDIX A: FOLLOW-UP PERIPHERAL BLOOD COUNTS FOR COHORTS 1 AND 2 COHORT DATE WBC (ul) ANC (ul) PLT mmJ\3 2 01/26/99 0 6 0 .51 2 01/27/99 0 2 2 01/28/99 0.3 2 01/29/99 0.4 2 01/30/99 0.6 0 28 2 01/31/99 0 5 0 29 2 02/01/99 0 7 0 53 2 02/02/99 2 1 1 76 2 02/03/99 4 5 3.78 2 02/04/99 4 6 4.14 2 02/05/99 6.4 5.5 2 02/06/99 7 8 ND 2 02/07/99 9 2 7 63 2 02/08/99 5 6 4 03 2 02/10/99 2 8 1.4 2 02/12/99 3.4 1 8 2 02/17/99 3 3 1 6 2 02/18/99 2.5 0.9 2 02/26/99 2.4 1 15 2 03/12/99 3 9 2 96 2 03/29/99 4 9 3.48 2 04/20/99 4 2 95 2 05/11199 4 3 3 05 2 05/14/99 4.4 3 74 2 05/19/99 3 2 2 34 2 05/21/99 2 2 1 .72 2 05/25/99 2 5 2 03 2 05/28/99 2 7 2 24 2 06/01/99 2 6 2 02 2 06/04/99 2 5 2 08 2 08/17/99 4 2 7 2 09/14/99 11. 7 9.4 2 10/12/99 5 8 4.4 ANC not calculated for WBC <500/uL ND indicates no data provided or assay not performed Counts reported X 1 000 124 82 63 35 32 9 40 38 26 23 17 13 11 51 43 40 53 168 177 175 138 151 178 160 180 126 122 96 96 93 103 118 193 138

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ID 11 11 11 11 11 11 11 11 11 APPENDIX A: FOLLOW-UP PERIPHERAL BLOOD COUNTS FOR COHORTS 1 AND 2 COHORT DATE WBC (ul) ANC (ul) PLT mm.l\3 2 11/30/99 4 2 3 3 2 02/02/00 3 7 2 5 2 03/03/00 4 2 7 2 05/03/00 4 7 3 2 2 07/19/00 4 7 3 2 2 08/25/00 5 2 3 7 2 09/29/00 5.4 4 2 11/09/00 4 6 3 1 2 12/14/00 4 2 5 ANC not calculated for WBC <500/uL ND indicates no data provided or assay not performed Counts reported X 1 000 1 25 176 132 142 146 144 149 186 171 181

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APPPENDTX B : SLIDE PRESENTATION : Thesis Defense nresi s oetense for M.B.S. in Applted Sctence Julie Candia Long-term Follow-up on Breast Cancer Patients Transplanted with Ex Vivo Expanded Peripheral Blood CD34+ Cells / ervtew d ; ........... .. ,i ........ ..... .. 1 Patient:S vario L s stages (stage II to stage N disease) 1 1 I I I I f trreaSt:jtancer were givE'!n t1ei11atopdietic growth factor I to mobili i e blood ; (PBPC) cells into t H e circulation Leukapheresis was performetl to coliectt the PBFt. PatientS the PBPC tiahsfusion hlgh-dose themotherapy. i Periphe,rcJI counts to i engraftinbnt kinetk:s. res Jtts i that a1n 1 1 i patientS 6emonstrclted rapid neutrophil engraftrhent. : llong"tenhengraftrnent kinetics were in this stydy. 1 1 i 126

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APPPENDIX 8 (cont.) : SLID E PRESENTATION : Thesis Defense COHORTS .,, Consecutively Enrolled From J uly 1998 to January 1999 COHORT 1 Transfused with CD34+ selected cells which were expanded and Transfused with CD34+ selected unexpanded cells COHORT 2 Transfused o nly with CD34+ selected cells whiCh were expanded ;;!;,,., 1;11:1! lll ''r ;1;.,
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APPPENDIX B ( cont. ) : SLIDE PRESENTATION : Thesis Defense .Important ,.,,. ........ ,. NE\JTROPHil. j S (50 -lO % o f WBC) protectj againsUnfection from 11 of Blood :cells 1 1 -RBC (red biO
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APPPENDIX B ( cont.) : SLlDE PRESENTATION : Thesis Defense Hematopoiesis _. +Stem Cells + Progenitor Cells + Mature Cells Granulocytopoiesis : Pluripotential stem cell All cell lines : Multipotentia l myeloid stem i> RBc,i wsc, PL T ,.;. Committed p r ogenitor cell (WBC) Granulocytes: heutrophils, I eosir\ophils basophiiS l Neutrophil Maturation o Myeloblast o Promyelocyte o Myelocyte o Metamyelocyte o Band o Segmented Neutrophil 129 w 11

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APPPENDIX B (cont.) : SLIDE PRES ENTATION: Thesis Defense Methods to collect PBPC r;nobilized from the bone marrow by recombinant GCSF1 (growth rcolony facto r J> Immunomagnetic CD34+ selection of the PBPC product of cells for J>High-dose chemotherapy to eradicate tumor cells Reinfusion o(Progenitor/Stem Cells PBPC are infused through a central venous line into the circulation Cells "home" t o the bone marrow where they engraft ll i4 Results l>t_ll t /!I!IJI/!1 '1" 111'''''1' 11'111!)'1" J!IIP!IPJI/1'."!!1'.1!!.' '! Peripheral Blood Counts iat Last fiollow-up WBC ANC Cohort 1 PLT ,.. (UL) (UL) MM"3 r--------'-----I Mean 4.8 3 147 -I Median: 14.5 3.2 142 ,----I Range I 11.6-8.4 0.655 22-211 I I I I Cohort 2 1 WBC ANC PLT Mean ,3.8 2.4 I 163 I I Median .3.8 2.4 I 178 ----Range 2 .1-4,7 1.2-3.1 102198 130

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APPPENDIX B (c ont. ) : SLIDE PRESENTATION: Thesis Defense Long-term Engraftment OL.ongterm engraftment was demonstrated fo r .. bo th cohorts. Blood counts w ere mainta ined over tim e with few exceptions. Patients have maintained adequa te rounts ranging from 12 up to 2 8 months atlast follow-up for Cohort 1 and 3 to 23 month s at last follow up for Cohort 2. .. ;;,,.,,.,.,.,,;,;,.,.) ,;),.;;;,1! "''"" ,,, .. ,,,,,,; .. ,,., .... i Disease Free Survivai/Overallt !Survival : .. 1 COhlORT 2 ;"8 out!of 10 wi t h 'ilO evidente of disease at last I follow up (Pn;JPprt;i()n 0.8.0) ..-All patients ali v e at last follow-up ( proportion Lo) 7 out of 10 survi viljg : patients in remission 1at last fo liow up : t (prop ortion 0 70) : out of 11 al ive a t last fo ll ow !UP (proPQrtion 17 Patient Health Status c;o Pulmonary toxicity (shortness of brea th) Neuropathies (tingling/numbness in hands and feet) Other: fatigue short-term memory loss, depression, and vision problems. Most sideeffects caused by high -dose chemotherapy drugs Number of patients reported as "doing very well" 131 lB

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APPPENDIX B (c ont.): SLIDE PRESENTATION: Thesis Defense Sustained Long-te rm Engrafting Cells May .. be established From Three Possible Sources: I I Transp!anted graft i 'Endogenous quiescent cells that are resistant to high ; dose chemotherapy Knowledge of the tra n splant graft ahd resid uql primitive cells ct source of sustaiijed hematoppietic recqnstitution is superseded by the value of murable long-term engraftmeflt Conclusion (cont.) ,/Researchers at the University of Colora do Health Sciences Center (M cNiece et. al. 2000) that patients rapidly engraft neutrophils with infusion of ex vivo expanded CD34+ cells. ,/ Infusion of ex vivo expanded cells has been shown to be safe deplet ion possible with CD34+ selection and expansion. ,I Surviving patients in both cohorts den10nstrated durable engraftm ent, thus far. 132 li

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