Hematopoietic Stem Cell Transplantation in Clinical Practice

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 Seema Singhal MD André Tichelli MD Paul Veys MRCP FRCPath FRCPCH
x Professor of Medicine, Director, Multiple Professor of Hematology, Division of Reader in Stem Cell Transplantation, Great
Myeloma Program, Feinberg School of Hematology, University Hospitals, Basel, Ormond Street Hospital for Sick Children,
Medicine, Northwestern University, Chicago, Switzerland London, UK
Illinois, USA
Jennifer Treleaven MD FRCP FRCPath Phyllis Warkentin MD
Gérard Socié MD PhD Consultant Hematologist, Royal Marsden Professor of Pathology and Pediatrics,
Professor of Hematology and Head, Hospital, Sutton, Surrey, UK University of Nebraska Medical Center, Omaha,
Hematology/Transplantation Center, Hospitalier Nebraska, USA
Universitaire Saint-Louis, Paris, France Jaap van Laar MD PhD
LIST OF CONTRIBUTORS

Professor of Clinical Rheumatology, Institute of Alan S Wayne MD

Shivani Srivastava MD Cellular Medicine, School of Clinical Medical Clinical Director, Pediatric Oncology Branch,
Assistant Professor of Medicine, Bone Marrow Sciences, Newcastle University, Newcastle, UK National Cancer Institute, National Institutes of
and Stem Cell Transplantation, Indiana Health, Bethesda, Maryland, USA
University School of Medicine, Indianapolis, Frits van Rhee MD PhD MRCP(UK)
Indiana, USA FRCPath Daniel Weisdorf MD
Professor of Medicine, University of Arkansas Professor of Medicine and Director of the Adult
John W Sweetenham MD for Medical Sciences, Little Rock, Arkansas, Blood and Marrow Transplant Program,
Professor of Medicine, Cleveland Clinic Taussig USA University of Minnesota, Minneapolis,
Cancer Center, Cleveland, Ohio, USA Minnesota, USA
Sumithira Vasu MD
Lochie Teague DCH FRACP FRCPA Clinical Fellow, Department of Transfusion Robert Wynn MD MRCP FRCPath
Clinical Director, Pediatric Hematology/ Medicine, Warren G Magnuson Clinical Center, Consultant Pediatric Hematologist, and Director,
Oncology, Starship Children’s Hospital, National Institutes of Health, Bethesda, Blood and Marrow Transplant Unit, Royal
Auckland, New Zealand Maryland, USA Manchester Children’s Hospital, Manchester,
UK
John Theus MD
Assistant Professor, Department of Pathology,
University of Arkansas for Medical Sciences,
Little Rock, Arkansas, USA
FOREWORD PREFACE
xi
Hematopoietic stem cell transplantation and cellular therapy are Hematopoietic stem cell transplantation is still a relatively new treat-
rapidly developing, highly effective modalities of treatment for a ment modality, having been used clinically only since the 1970s. As
broad range of hematologic, immunologic, metabolic and malignant time has passed, many changes and refinements have been introduced
diseases. For many disorders, hematopoietic transplantation is poten- into what was originally an extremely hazardous therapy, and prob-
tially curative for what would otherwise be fatal diseases. Hematopoi- lems have been solved which earlier were not even known to exist.
etic transplantation is the most established form of cellular therapy The patient population to which these techniques are applicable has
and is a cornerstone of treatment for a broad range of hematologic also changed and enlarged as treatment has become safer, with the
malignancies. introduction of improved blood product support, antibiotics and better
Hematopoietic transplantation was originally conceived as a means immunosuppressants, not to mention the expanding role of reduced-
of administering high doses of myelosuppressive chemotherapy and intensity conditioned transplantation and an increased repertoire of
total body radiation, followed by autologous or allogeneic hematopoi- stem cell sources now that cord blood is available.
etic stem cell transplantation to restore hematopoiesis. Much of the Although many of the original difficulties continue to pose prob-
benefit, however, is related to an immune graft-versus-malignancy lems, advances in the field have occurred so rapidly that it is difficult
effect mediated by donor immunocompetent cells. Historically, hema- to keep abreast of all the changes. A regular update is therefore neces-
topoietic transplantation has been a high-risk form of treatment; life- sary, and it is the aim of this book to provide it. Advances in all fields
threatening complications may occur related to drug toxicities, graft of transplantation are discussed and an overview of the relevant litera-
rejection, graft-versus-host disease, and infections related to post- ture is provided. An attempt has been made to provide a practical
transplant immune deficiency. approach to problem solving, and where applicable this has been done
There has been enormous progress in every aspect of the field. with tables and lists with the intention of assisting the reader to assess
Supportive care has markedly improved and non-myeloablative the various options rapidly and effectively.
preparative regimens have been developed which have markedly The book will be of use to healthcare workers in the field of hema-
reduced the toxicity associated with hematopoietic transplantation. topoietic stem cell transplantation, and particularly to those who have
The field has been less successful in improving the eradication of day-to-day responsibility for patients, including doctors, nurses, phar-
malignancy, but there is a plethora of novel strategies currently under macy staff and many others. It is hoped that students of medicine will
investigation. also find it readable, and that it will afford them some insight into the
This book effectively summarizes the progress in ongoing clinical options which now face us when dealing with patients undergoing
and translational research involving hematopoietic transplantation as these complicated forms of treatment.
well as the clinical issues faced in practice. The history of hematopoi-
etic transplantation and its underlying biology is reviewed. Current J.T.
considerations for the clinical use of stem cell transplantation are J.B.
presented and its role versus alternative forms of treatment is dis- 2008
cussed. Important practical information is also presented regarding the
organization and operation of clinical stem cell units and ethical con-
siderations faced in practice.

Richard Champlin MD 2008

FOREWORD PREFACE
xi
Hematopoietic stem cell transplantation and cellular therapy are Hematopoietic stem cell transplantation is still a relatively new treat-
rapidly developing, highly effective modalities of treatment for a ment modality, having been used clinically only since the 1970s. As
broad range of hematologic, immunologic, metabolic and malignant time has passed, many changes and refinements have been introduced
diseases. For many disorders, hematopoietic transplantation is poten- into what was originally an extremely hazardous therapy, and prob-
tially curative for what would otherwise be fatal diseases. Hematopoi- lems have been solved which earlier were not even known to exist.
etic transplantation is the most established form of cellular therapy The patient population to which these techniques are applicable has
and is a cornerstone of treatment for a broad range of hematologic also changed and enlarged as treatment has become safer, with the
malignancies. introduction of improved blood product support, antibiotics and better
Hematopoietic transplantation was originally conceived as a means immunosuppressants, not to mention the expanding role of reduced-
of administering high doses of myelosuppressive chemotherapy and intensity conditioned transplantation and an increased repertoire of
total body radiation, followed by autologous or allogeneic hematopoi- stem cell sources now that cord blood is available.
etic stem cell transplantation to restore hematopoiesis. Much of the Although many of the original difficulties continue to pose prob-
benefit, however, is related to an immune graft-versus-malignancy lems, advances in the field have occurred so rapidly that it is difficult
effect mediated by donor immunocompetent cells. Historically, hema- to keep abreast of all the changes. A regular update is therefore neces-
topoietic transplantation has been a high-risk form of treatment; life- sary, and it is the aim of this book to provide it. Advances in all fields
threatening complications may occur related to drug toxicities, graft of transplantation are discussed and an overview of the relevant litera-
rejection, graft-versus-host disease, and infections related to post- ture is provided. An attempt has been made to provide a practical
transplant immune deficiency. approach to problem solving, and where applicable this has been done
There has been enormous progress in every aspect of the field. with tables and lists with the intention of assisting the reader to assess
Supportive care has markedly improved and non-myeloablative the various options rapidly and effectively.
preparative regimens have been developed which have markedly The book will be of use to healthcare workers in the field of hema-
reduced the toxicity associated with hematopoietic transplantation. topoietic stem cell transplantation, and particularly to those who have
The field has been less successful in improving the eradication of day-to-day responsibility for patients, including doctors, nurses, phar-
malignancy, but there is a plethora of novel strategies currently under macy staff and many others. It is hoped that students of medicine will
investigation. also find it readable, and that it will afford them some insight into the
This book effectively summarizes the progress in ongoing clinical options which now face us when dealing with patients undergoing
and translational research involving hematopoietic transplantation as these complicated forms of treatment.
well as the clinical issues faced in practice. The history of hematopoi-
etic transplantation and its underlying biology is reviewed. Current J.T.
considerations for the clinical use of stem cell transplantation are J.B.
presented and its role versus alternative forms of treatment is dis- 2008
cussed. Important practical information is also presented regarding the
organization and operation of clinical stem cell units and ethical con-
siderations faced in practice.

Richard Champlin MD 2008

 Introduction CHAPTER
1
Jennifer Treleaven

PART
Stem cell transplantation is now used worldwide in the treatment of of Cooley, the Táin Bó Cúailnge.The charioteer Cethern, an Ulster
many malignant and non-malignant hematologic conditions and in the warrior, was severely wounded in battle and treated by the healer
treatment of various solid tumors. Every year, many hundreds of Fingin. The treatment involved him sleeping in a bath of bone marrow,
patients receive an autologous or allogeneic transplant procedure, and although its effects were going to be short-lived. Some of his ribs were
the numbers have increased vastly since the pool of allogeneic donors
available worldwide widened, enabling a larger number of patients
replaced with chariot parts, and the frame of the chariot was tied to
his belly to keep his insides in. According to the legend, this renewed 1

SETTING THE SCENE

with no sibling donor to undergo an allogeneic transplant prodedure. Cethern’s strength sufficiently to allow him to resume fighting although
Figure 1.1 shows the annual number of transplants reported in the he was killed in battle shortly afterwards.1,2
International Bone Marrow Transplant Registry and how this increased In the 19th century it was understood that bone marrow was
as stem cell transplantation became a realistic treatment possibility. involved in the formation of blood and the idea arose that bone marrow
However, stem cell transplants have only become a therapeutic pos- might have healing properties and could possibly be of use in treating
sibility since the late 1960s. Prior to this, understanding of such topics anemia. In 1896, Quine reported that Brown Sequard & D’Arsenoval
as human leukocyte antigen matching was rudimentary. The concepts had administered bone marrow orally in 1891 to treat defective blood
of immunosuppression and graft-versus-host disease were entirely formation.3 Attempts were also made to treat pernicious anemia using
unexplored and little was known about preparative therapies. Early a glycerol extract of animal bone marrow administered orally, so there
transplants thus invariably met with a woeful lack of success due to would have been no chance of living cells being transferred and any
problems from regimen-related toxicity, graft-versus-host disease and benefit from the treatment would have derived from the nutritional,
lack of availability of support measures, including antibiotics and rather than cellular, aspect of the mixture.4,5 Subsequetly, intramedul-
blood products. lary injections of marrow were used to treat aplastic anemia, which
As knowledge about these fundamental topics was acquired and may have resulted in the transfer of some living cells although they
methods of identifying a suitable donor improved along with support would have remained viable for only a short period of time. Billings
measures and knowledge about immunosuppression, so did the results in 1894 and Hamilton in 1895 probably correctly attributed any posi-
of cell transplantation. Since the 1970s, steady progress has been made tive effects of treatment to the mineral content of the elixir.6,7
and stem cell transplantation is now regarded as a routine, rather than
an experimental, approach in the treatment of a number of conditions Early 20th century: 1920s–1940s
which would have proven fatal earlier on. It is now possible to identify
the risk factors which will predict a good or poor outcome in a par- In 1923, Leake & Leake observed some responses in rabbits and dogs
ticular clinical setting, thereby facilitating the decision of whether or with anemia to saline extracts of bone marrow and spleen administered
not to proceed with the transplant. However, the problems which beset intravenously, particularly when the two were administered as a com-
the early transplanters, in particular disease relapse, graft-versus-host bination.8 Daily oral administration of 1% filtered solution of desic-
disease and overwhelming infection, are still the major causes of treat- cated spleen and powdered red bone marrow in rabbits gave parallel
ment failure in spite of the improvements which have been made to results,9 depending on whether the extracts were given singly or in
support therapies and the immense amount of information now avail- combination. Oral administration of a filtered solution of combined
able regarding the cellular and humoral aspects of transplantation and powdered red bone marrow and desiccated spleen in dogs caused a
our consequent ability to manipulate and control the microenviron- marked rise in the number of circulating erythrocytes. The authors
ment in the transplant setting. Figure 1.2 depicts some of the mile- concluded that splenic and red bone marrow extracts were more
stones in the evolution of stem cell transplantation and therapeutic powerful stimulants of erythropoiesis in combination than separately,
interventions which have become available in the context of the dis- attributing their action firstly to increasing the rate of production or
eases for which transplantation was attempted early on. delivery in existing sites of erythropoiesis, and secondly to causing an
increase in the amount of functioning red marrow.
In 1937 Schretzenmayr administered intramuscular injections of
A brief history of bone marrow transplantation freshly aspirated autologous or allogeneic bone marrow to patients
suffering from parasitic infections, with limited success. This was the
first use of bone marrow administered by a technique likely to result
Before the 20th century in the transfer of living cells.10
One of the earliest references to the therapeutic properties of bone A couple of years later, an attempt was made to treat a patient suf-
marrow is found in the 8th-century Irish epic tale of the Cattle Raid fering from aplastic anemia. A small quantity of bone marrow from
 his brother was infused.11 Prior to this, in 1930, Gloor described the exposure to irradiation (TBI) if they were given an infusion of bone
4 cure of a patient with acute myeloid leukemia by stem cell transplanta- marrow cells removed and stored prior to the TBI.13
tion.12 However, stem cell transplantation as an approach for treating In 1948, Jacobson showed that mice could be protected from bone
leukemia and aplastic anemia was not investigated seriously until the marrow failure by shielding either a portion of marrow in the hindlimb
PART

1960s when it was found that dogs could survive 2–4 times the lethal or the spleen,14 and that provided this were done, a mouse which had
received a lethal dose of irradiation would regenerate its blood count.
He subsequently showed that bone marrow failure following other-
wise lethal doses of irradiation could also be prevented by infusing

1
40000
either spleen cells or bone marrow cells from a litter mate into the
35000 animal.15 At the time, the mechanism involved in this effect was not
SETTING THE SCENE

understood and was thought by some to be due to the transfer of a

30000 humoral factor which stimulated or protected the marrow against the
Autologous effects of irradiation. Others, including Lorenz,16 believed that transfer
Number of transplants

25000 of living cells was responsible for the recovery of the marrow.

20000
1950s–1960s
15000 As early as 1956, the idea that allogeneic bone marrow transplants
(BMT) might exert a therapeutic immunologic effect against malig-
10000 Allogeneic
nancies was proposed by Barnes & Loutit,17 who observed an antileu-
5000
kemia effect of transplanted spleen cells in experimental murine
models.18 They also observed that animals who had been given allo-
0 geneic rather than syngeneic marrow cells died of a ‘wasting disease’19
1970 1975 1980 1985 1990 1995 2000 which would now be recognized as being graft-versus-host disease
Year (GvHD).
Figure 1.1 In 1954 major advances were made. Ford, using the T6 chromo-
Annual numbers of blood and marrow transplants worldwide, 1970–2000, from the somal marker which identified the transfused cells, demonstrated that
CIBMTR. the same marker recurred in all the cells derived from the recovering

Years
1950 1960 1970 1980 1990 2000

Auto-BMT BMT for Inborn errors 1981 Unrelated Donor’s lymphocyte SCT for solid
1958 immune Chronic donors 1986 transfusions for tumors 2000
deficiencies Aplastic anemia 1972 leukemias, lymphoma, relapse 1991
1968 myeloma 1984–6
Animal Acute leukemia 1971 Thalassemia Cord blood 1989 Non-myeloablative SCT
experiments in 1985 PBSCT 1989 for hematological
radiation protection malignancies 1997
20 000
Number of transplants per annum
15 000
10 000
5 000

0
1970 1975 1980 1985 1990 1995 2000

Therapeutic agents
Fludarabine
Busulfan
Cyclophosphamide
Whole body irradation
Sirolimus
Tacrolimus
Ciclosporin
Antilymphocyte globin
Campath
OKT3
Methotrexate
Imidazole antifungals
Aciclovir
Ganciclovir / foscarnet

Figure 1.2
Some developmental steps in blood and marrow stem cell transplantation and the introduction of significant therapeutic agents, 1950–2000.
marrow of the lethally irradiated recipient mouse,20 thereby proving patient was big S positive and (Le a-b+). The erythrocyte phenotype
that such cells were donor derived. This proved not only that cellular remained that of the brother 8 months after transplantation, and the 5
engraftment had occurred, but also that the transfer of a few cells patient was also tolerant of a skin graft from this donor whereas he
resulted in complete and stable hematopoietic reconstitution of the rejected skin grafts from his other donors at varying lengths of time

Chapter 1 Introduction
recipient. A few years later, in 1961, Till & McCulloch showed that after transplantation. This paper is probably the first documented case
in irradiated mice, marrow repopulation originated from multipoten- of full donor chimerism to be described after stem cell transfer and it
tial ‘colony-forming units’ which could be detected in the spleen.21 also states that the patient experienced ‘secondary disease’ with weight
Dosing experiments demonstrated that such colonies must have arisen loss, digestive disturbances and desquamative erythroderma, now
from the seeding of a single cell – a stem cell. Single colonies isolated known as graft-versus-host disease.26
from the spleen could reconstitute hematopoiesis in other irradiated Mathé continued working on the comparisons in antileukemia
recipients, thus demonstating that one stem cell could reconstitute the effects of allogeneic adult or embryonic hematopoietic grafts condi-
entire myeloid compartment of a recipient mouse. tioned by irradiation and on attempting to control graft-versus-host
However, in spite of various successes with animal models, up to disease. At this point he was led to observe and describe the graft-
this time almost all attempts to achieve allogeneic grafts in humans versus-leukemia (GvL) effect associated with GvHD and he also
had been unsuccessful although it was recognized that marrow trans- observed a specific antileukemia effect of adoptive immunotherapy,
plantation would be of potential use in the treatment of various bone resulting in a reduced plasma concentration of the Friend leukemo-
marrow failure syndromes and hematologic disorders. Thomas & genic virus.27 This indicated the possibility that lymphoid elements in
Ferrebee had started studies on patients suffering from terminal leu- the engrafted marrow might react against the malignant cells in the
kemia, and in 1957 they reported on six such patients who had been patient to aid in eradicating any remaining malignant cells.28
treated with irradiation and marrow infusion from a single, normal Around the same time, E Donnell Thomas, working on dogs in the
donor, although only one showed transient marrow engraftment.22 USA, defined radiation doses, designed marrow harvesting techniques
Billingham, Brent and Medawar23 provided much of the early infor- and ascertained the support required to carry out marrow transplants
mation concerning graft tolerance, using newborn mice which were in man. Dogs can be physically large and have some immunologic
given allogeneic marrow cells.24 They went on to make the following similarities to man in terms of the fact that they possess a complex
observations with regard to allogeneic marrow transfusion. HLA system. The fact that they come in large families enabled experi-
• Autologous cells did not result in runt disease. ments to be conducted which relied on the infusion of litter mate
• Allogeneic cells had to persist in the recipient in order for runt marrow to a lethally irradiated recipient, and allowed such issues to
disease to develop. be resolved as what constituted a permanently marrow-ablative dose
• The severity of runt disease was determined by antigenic differ- of radiotherapy and the number of cells that it was necessary to infuse
ences between the recipient and donor. to effect marrow reconstitution. The use of autologous marrow as
In addition to the lack of knowledge at the time concerning tissue ‘rescue’ after otherwise lethal irradiation was also documented around
typing and HLA compatibility, treatment success was also limited by this time; marrow from dogs to be irradiated was set aside before the
lack of knowledge of how to administer high-dose therapy and inabil- procedure and reinfused after irradiation had taken place. Control dogs
ity to provide adequate supportive care for marrow failure because which were irradiated and not reinfused with marrow died, whereas
few antibiotics and antifungal agents were available, and the avail- those who had received marrow after irradiation experienced auto-
ability of blood products was very limited. logous reconstitution and lived.29
Georges Mathé was a pioneer in the early development of clinical Thomas and colleagues then went on to develop a regimen of post-
BMT. In 1958, six physicists were accidentally exposed to large doses transplant immunosuppression using methotrexate as GvHD prophy-
of mixed gamma and neutron irradiation at Vinca in Yugoslavia,25 and laxis,30 and they established various facts concerning transplantation
were estimated to have received radiation doses of between 600 and which are still valid and which won Thomas the Nobel Prize for
1000 rads. One, who was judged to have received about 700 rads, Physiology or Medicine in 1990.
died. The sixth one survived with no treatment apart from being placed Elucidation of the human histocompatibility antigen (HLA) system
in a ‘clean’ environment, and he was thought to have received only allowed major steps to be made in the field of transplantation biology,
400 rads. Mathé gave them multiple allogeneic bone marrow infusions much of the early work having been conducted by Dausset & van
from family members. The men all survived, with eventual autologous Rood who recognized that the HLA system was inherited.31 They went
marrow recovery, but the allogeneic bone marrow served to protect on to define the nature of various leukoagglutinins found in certain
the patients until this had taken place. Red cell antigen studies of the human sera,32 eventually constructing the major histocompatibility
transfused allogeneic marrow demonstrated that successful but tem- gene map.33 Again, much of the early work was conducted in dogs.
porary engraftment had taken place, with eventual autologous recon- Antisera were subsequently raised in dogs to various HLA antigens
stitution which was evident from the changes seen in the red cell so that characterization of the HLA system could be undertaken.34
groups in subjects who were not ABO and Rhesus compatible with The miniaturization of the matching system by Terasaki and col-
their donors. Of note was the fact that the red cell output of the donated leagues made HLA typing much more practical,35 since, with the
cells paralleled the amount of marrow initially infused, perhaps indi- ability to tissue match donors and recipients, allogeneic transplants
cating that the subjects who received the larger numbers of stem cells could be carried out in humans between HLA identical siblings. One
experienced enhanced hemopoietic recovery. of the first cases to be reported was of an infant with severe combined
In 1963 Mathé published a case report concerning a patient with immune deficiency disease in Leiden, Netherlands.36 By the end of the
leukemia who was transfused with a mixture of bone marrow and 1960s, all the components were in place for developing and expanding
blood from six family members – mother, father, three brothers and a a new era of clinical bone marrow transplantation.
sister – after preparation with 800 rads of total body irradiation with
cobalt-60 preceded by 4 days of mercaptopurine, 300 mg per day. All 1970s–1980s
the donors and the recipient were red cell ABO group O Rh D positive,
but because of differences in other red cell antigens between the During the 1970s and 1980s, allogeneic stem cell transfer (SCT) was
patient and his donors, it was possible to document engraftment from used to treat many different diseases including congenital immune
one of the brothers who had small s and was (Le a+b−), whereas the deficiency syndromes, severe aplastic anemia, and acute and chronic
 leukemias. Much progress was made in understanding the immunol- more patients with a suitable matched unrelated donor,50 particularly
6 ogy of transplantation, in controlling the problems associated with in view of the establishment of large, unrelated volunteer donor panels
transplantation which arose as a consequence of using high-dose by the Anthony Nolan Research group in London, Europdonor based
chemoradiotherapy, and improved conditioning regimens were worked in Leiden, Holland, and the North American Marrow Donor Pool
PART

out. At the same time, progress was made in chemotherapy induction (NAMDP) in the USA.51 Blood product support also improved with,
for the acute leukemias, resulting in higher remission rates. Hence, for example, the availability of partially HLA-matched platelet trans-
there was renewed interest in autologous BMT for acute leukaemia. fusions for patients who were failing to increment because of platelet
By the end of the 1970s, numerous centers worldwide had active BMT antibodies. The first attempts at transplant engineering were under-

1 programs and two influential multicenter bodies had become estab-

lished: the International Bone Marrow Transplant Registry and the
taken – the depletion of T-cells from the marrow inoculum to prevent
GvHD – although this maneuver, while reducing GvHD, was soon
SETTING THE SCENE

European Bone Marrow Transplant Group. noted to be associated with an increase of both graft rejection and
The potential of BMT as a means of curing patients with otherwise leukemia relapse.52–54
incurable diseases was clear. However, the shortcomings of the pro- The demonstration that patients with chronic myeloid leukemia
cedure – leukemic relapse, GvHD, graft failure and early toxicity – could be reconstituted with autologous peripheral blood cells55
were still a problem. Improvements were made in conditioning regimes heralded the later widespread use of peripheral blood stem cells for
as radiotherapy and various alkylating agents were combined in an transplantation. Autologous transplantation with marrow rescue was
attempt to overcome the underlying disease, and dose-limiting toxici- used increasingly to treat lymphomas, myeloma and solid tumors56–58
ties became apparent. Methods of immunosuppressing recipients (see Fig. 1.1), and attempts were made to purge the marrow of malig-
advanced beyond the use of steroids and methotrexate. Ciclosporin nant cells by various techniques including the use of immunotoxins,
was introduced into the transplant forum in the early 1970s after work complement-conjugated antibodies and magnetic microspheres.59–61
had been carried out using dog models. It was initially used in solid
organ transplantation,37,38 where the side-effect of nephrotoxicity was
noted, and subsequently in bone marrow transplantation, where it was
The 1990s
observed to improve the situation with regard to GvHD, although The 1990s were a period of rapid development in BMT. Recombinant
many side-effects were apparent which resolved when the drug was growth factors had become available, including granulocyte-
stopped or the dose reduced.39,40 macrophage colony-stimulating factor (GM-CSF) and granulocyte
Improvements were also made in infection prophylaxis with the colony-stimulating factor (G-CSF), which could be used to speed up
introduction of new antibacterial agents and the antiviral drug aciclo- white cell recovery after chemotherapy or after transplantation, thereby
vir which had been shown to be active against the herpes virus in shortening the length of hospital stay.62–64 The use of peripheral blood
animal models.41 Used on a number of patients who had received stem cells (PBSC) mobilized into the blood by G-CSF virtually
treatment for malignant disease or a bone marrow transplant, the drug replaced the use of bone marrow for transplantation, avoiding the need
was noted to arrest the progress of the herpes infections and was found for a general anesthetic and arguably resulting in more rapid myeloid
to be most effective when given early. Although some patients showed reconstitution so that it even became feasible to undertake autologous
transient increases in blood urea, possibly the result of aciclovir, the PBSC transplants on an outpatient basis, provided that medical back-
drug was observed to be remarkably non-toxic at the doses used. up was near at hand.65
The antifungus drug amphotericin was also introduced into the Cord blood transplantation also started to become popular, follow-
transplant setting around this time,42 having first been isolated in 1955 ing the first successful cord blood stem cell transplant in 1988.66
from Streptomyces nodosus, a filamentous bacterium. Since then, Increasing numbers of patients have received cord blood transplants,
numerous antimicrobials designed to treat a vast array of infecting although their use may have to be restricted to lower body weight
organisms have become available. recipients because of problems with the cell dose obtainable from a
Further strides in stem cell transplantation were possible when cord in relation to recipient body weight and the possibility of delayed
understanding of the essentials involved in freezing living cells engraftment. However, cord blood stem cell transplants appear to be
improved.43 Obviously, cooling tissue to below 0°C resulted in freez- associated with a relatively low incidence of acute GvHD and their
ing, which greatly reduced the number of viable cells when these were use has greatly expanded the donor pool available worldwide now that
thawed. As far back as 1949, the discovery had been made that the both public and private cord blood banks have been established in
addition of glycerol to human and bull sperm greatly improved cell many countries.67–69
viability after freezing to −79° and, using infant mouse spleen cells, New understanding of the pathogenesis of GvHD and of the basic
Barnes & Loutit later showed that hematopoietic cells would survive mechanisms involved in alloreactions has dramatically changed the
low-temperature preservation after slow cooling to −79°C in 15% way acute GvHD prevention is being investigated in experimental
glycerolized medium.44 Subsequently, dimethyl sulfoxide was dis- studies. To prevent GvHD, most therapies rely on the elimination of
covered to be a superior cryopreservant to glycerol and it is this agent donor T-cells from the graft or immunosuppression of the host. Un-
which is currently used for freezing stem cells45 in programmed freez- fortunately, these approaches can result in poor engraftment, a loss
ers which use liquid nitrogen. Cells could now be stored in liquid of GvL activity, and a higher risk of leukemic relapse. Instead of the
nitrogen for an indefinite period, if necessary, prior to reinfusion. global immunosuppression conferred by pharmacologic agents and
During the 1980s and early 1990s there was rapid expansion of non-selective T-cell depletion methods, new approaches focus on the
clinical BMT programs worldwide and it became possible to analyze induction of specific tolerance, which prevents the development of
large patient series so as to define specific patient/donor and trans- acute GvHD during the establishment of donor immunity. If GvHD
plant-related risk factors for various diseases. Antibiotic and antiviral does develop, a number of targeted monoclonal antibodies are now
support continued to improve with, for example, the introduction of available which block the host immune response, such as daclizumab
ganciclovir for the treatment of cytomegalovirus,46,47 and liposomal and inolimomab, which are IL-2 receptor blockers,70–72 and infliximab
amphotericin for the treatment of fungal infections without the which is an antitumor necrosis factor antibody (TNF),73,74 thereby
nephrotoxicity associated with regular amphotericin.48,49 There was a hindering tissue damage.
consequent reduction in transplant-related morbidity and, as further Another approach is the use of keratinocyte growth factor (KGF),
understanding of the HLA system developed, it was possible to provide also called fibroblast growth factor 7 (FGF-7).75 This is important in
tissue repair and wound healing and its administration has been shown availability as long-acting preparations, it should be possible not only
to be protective against radiation- and bleomycin-induced lung injury to significantly reduce the need for transfusion support and eliminate 7
in rats,76 possibly by facilitating repair of DNA damage or increasing neutropenic episodes, but also to mobilize larger numbers of stem cells
time of stem cell survival. for autologous or allogeneic donation.

Chapter 1 Introduction
Mesenchymal stem cells have also recently been shown to be of Progress can be expected in the control of T-cell recovery by deple-
use in treatment therapy-refractory GvHD in humans,77,78 and probably tion, and add-back techniques of T-cells selected to avoid GvHD, the
act by inhibiting proliferation and cytokine secretion of primary T- use of vaccines or adoptively transferred T-cells to induce antitumor
cells in response to mitogens and allogeneic T-cells.79,80 and antiviral responses in the donor or the autograft recipient, and the
It first became accepted in the 1980s that GvL was a clinical as well use of gene therapy for selection or elimination of designated lympho-
as an experimental reality, and since that time, much progress has been cyte cell populations. Improvements have already been made in the
made in understanding and characterizing the GvL reaction.81 While field of tissue typing, with molecular typing rather than serologic
the separation of GvL from GvHD is theoretically possible and has, allowing much more precise characterization of the MHC genes in
in limited circumstances, been achieved clinically, lack of ability to donors and recipients. It has thus become possible to match unrelated
identify antigens promoting leukemia-specific alloresponses remains donors to a degree not previously achievable, thereby permitting a
the biggest single obstacle to improving the strength and specificity much wider range of transplants to take place with relative safety.
of GvL in clinical practice. However, reduced-intensity conditioned Continuing improvement in support therapies, including more
allogeneic transplants are now in widespread use, relying for efficacy sophisticated antimicrobial agents and more refined and safer blood
on the graft-versus-tumor effect rather than the conditioning therapy product support, will allow transplants to be undertaken more safely,
to eliminate disease. An additional advantage of this approach is that, and monoclonal antibodies targeting the biologic mechanisms involved
because conditioning is less intensive, older and less fit patients who in the graft-versus-host reaction will hopefully also further reduce
would not previously have been considered suitable for transplantation transplant-related morbidity. Finally, with the development of targeted
have now become eligible. therapies such as tyrosine kinase, FLT-3 inhibitors and so on, it may
Advances in the field of molecular biology have made it possible be possible that fewer patients with hematologic malignancies will
to transfect genes into human cells using an adenovirus vector. With rely upon stem cell transplantation to cure their disease and that this
this technique, a new gene is inserted into an adenovirus vector which will be achievable with drug therapy.
has been genetically altered to carry normal human DNA, and which
is then used to introduce the modified DNA into a human cell. If the
treatment is successful, the new gene will make a functional protein. References
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PART

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 Essential biology of stem cell
transplantation
CHAPTER
A John Barrett 2

PART
Introduction transplant: donor B-cells can reactivate Epstein–Barr virus (EBV) and
cause lymphoproliferative syndromes and, if ABO mismatched with
In addition to an all-round knowledge of internal medicine, the practice the recipient, can generate red cell antibodies against host group A or
B red cells, leading to massive intravascular hemolysis of host red
1
of clinical stem cell transplantation (SCT) demands an understanding
of the hematologic and immunologic principles that form the basis of cells.4,5 Transplanted natural killer (NK) cells may promote engraft-
transplant patient management. Our current knowledge of SCT biology ment, and mesenchymal stromal cells (MSC) in bone marrow trans-

SETTING THE SCENE

derives from a vast body of experimental data extending back more plants have immunomodulatory functions.6,7
than 50 years. Here, we describe only the essentials of transplantation
science as it pertains to the daily clinical practice of SCT and outline
the biologic basis of factors which determine transplant outcome. The
Hematopoietic stem cell sources
reader is referred to the references at the end of this chapter for the more
Three transplant stem cell sources are in current use: bone marrow
experimental aspects of SCT hematology and immunology.
(BM), mobilized peripheral blood stem cells (PBSC) and umbilical
cord blood (UCB).8–10 These sources differ considerably both quanti-
Cellular components of the transplant (Fig. 2.1) tatively and qualitatively, as shown in Table 2.1.

Long-lived cell lineages BM harvesting

In infants and children, red marrow is distributed throughout the
Transplants contain a variety of cell types but the hematopoietic stem skeleton, including the long bones. Later, the red marrow recedes to
cells (HSC), conveniently identified by the CD34 surface antigen, and the axial skeleton. The anterior and posterior spines of the pelvis, the
CD3+ T-lymphocytes, responsible for immunologic memory, are the upper sternum in adults, and the head of the tibia in infants are the
most important because they can self-replicate and survive a lifetime sites most frequently used for marrow harvesting. The process of
in the recipient. Cells within the CD34 compartment establish lifelong multiple aspirations from these sites ruptures marrow sinusoids allow-
hematopoiesis and regenerate an entire immune system comprising ing marrow cells to be aspirated along with some bone spicules. BM
dendritic cells (including the specialized Langerhans cells), tissue collections are a mixture of venous blood enriched with marrow cells.
macrophages, B-cells, T-cells and natural killer (NK) cells. In the first The maximum yield is achieved by limiting the volume of each aspira-
few months after transplant, T-cells are derived from transfused mature tion and performing multiple punctures, a process that lasts 1–2
(post-thymic) T-cells from the donor. Later, CD34-derived prethymic hours.11
T-lymphocyte precursors requiring maturation in the host thymus
repopulate the peripheral T-cell compartment.1 Mobilization of PBSC
In recent years, there has been much interest in the possibility
Circulating HSC are normally present at frequencies 1/00 to1/1000
that SCT contain precursors of cells able to develop into non-hemato-
times lower than they are in the marrow. HSC are retained adherent
poietic cells such as angiocytes, endothelial cells, fibroblasts, neurone
to the marrow stroma by the binding of CXCR4 on their surface with
and muscle cells. The surface marker characteristics of the precursors
VCAM-1 on stromal cells. HSC will pass from the stromal compart-
of non-hematopoietic tissues and their relationship to HSC are not fully
ment into the sinusoids and thence into the circulation if the VCAM-
established. Some data suggest that HSC have plasticity to redifferenti-
1/CXCR4 adhesion is interrupted. In PBSC collection the hematopoietic
ate along alternative developmental pathways; other data suggest that
growth factor granulocyte colony-stimulating factor (G-CSF) mobi-
small populations of non-hematopoietic stem cells in the transplant
lizes HSC by reducing CXCR4 expression. Large numbers of circulat-
inoculm are responsible for repopulating non-hematopoietic tissues.
ing HSC can be collected by apheresis after six daily injections of
Whatever mechanism is invoked, it appears that the efficiency with
G-CSF. PBSC collections are heavily mixed with peripheral blood
which non-hematopoietic tissues are replaced by donor-derived cells is
cells and contain about 10-fold more lymphocytes than do BM
very low and of little significance in routine SCT.2,3
collections.12

Short-lived cell lineages UCB collection

Other cells with limited capacity to establish long-term engraft- At birth, the umbilical vein is rich in HSC but the total volume avail-
ment accompanying the stem cells can have immediate effects after able for collection is usually less than 100 ml. This is because most
 Cellular content
of transplant
Early regeneration
(first 100 days)
Late regeneration
(3–12 months)
Hematopoietic stem cell homing, engraftment
10
mo
DC
and reconstitution of hematopoiesis13
gran RBC
PART

Following intravenous infusion, HSC circulate and accumulate in the

CD34 c lungs before homing within 24 h to hematopoietic sites. Stem cell
MK New T-cell
CD34 c NK repertoire homing involves endothelial rolling within marrow sinusoids, passage
c
through the endothelium of the sinusoid and lodging in a stem cell

1 c niche. Within the niche, the HSC can proliferate and establish foci of
hematopoiesis as well as self-replicate (Fig. 2.2).
SETTING THE SCENE

CD3 Thymus The localization of HSCT in the marrow is directed by specific adhe-
CD3 Post-thymic sion molecule interactions. Endothelial rolling, which is the first step
New B-cell
T-cell repertoire repertoire to immobilization of the HSC and its passage through the endothelial
wall, is controlled by interaction between selectins such as vascular cell
NK adhesion molecule-1 (VCAM-1) on the endothelial cell, and integrins,
sialomucins and CD44 on the HSC. Stromal-derived factor 1 (SDF1)
Cells with no sustained is a chemokine produced by stromal cells which enhances adhesion and
capacity to proliferate
transendothelial migration of the HSC through expression of its recep-
Figure 2.1 tor on HSC CXCR4. Despite the well-established practice of giving the
Cellular content of the stem cell transplant and fate of long- and short-lived cell SCT intravenously, animal experiments suggest that the process is rela-
lineages in the recipient. CD34, hematopoietic stem cells; CD3, post-thymic T-cells;
NK, natural killer cell; B, B-cell; mo, monocyte; gran, granulocytes; MK, tively inefficient, with only a proportion of transfused HSC reaching
megakaryocytes; DC, dendritic cells; c, committed progenitor cells; RBC, red blood their niches.13 Hematologic recovery (>500 neutrophils/μl) takes about
cells. 10 days following SCT from BM and PBSC, while UCB transplants
require a median of 21 days.
To engraft in the recipient, HSCT must overcome both immuno-
logic and non-immunologic barriers. The most important factor deter-
mining engraftment of allotransplanted stem cells is a favorable
immune environment: predominance of donor T-cells promotes even-
tual engraftment of the donor’s stem cells. HSC engraftment can be
Table 2.1 Bone marrow, peripheral blood and umbilical cord blood stem cell blocked by circulating host antibodies, alloreacting NK cells and
sources compared donor-specific cytotoxic T-cells. In autologous SCT, engraftment
Property BM PBSCT UCB depends on the vigor of the autologous stem cells, which may have
been compromised by previous chemotherapy and radiotherapy,
Collection Multiple G-CSF mobilization Placental blood
leading to incomplete hematopoietic reconstitution. HSC may fail to
aspirates
engraft in a marrow full of malignant cells or damaged by fibrosis or
HSC minimum for graft chemotherapy. A large spleen can trap circulating HSC, leading to
×106/kg recipient wt 1.0 1.0 0.1
delayed and incomplete engraftment. Once engrafted, human HSC
Neutrophils > 500/μl appear to maintain hematopoiesis over many years and with follow-up
Median days post SCT 14 12 21
of some patients transplanted more than 30 years ago, there are no
Platelets > 20,000/μl reports of late graft failure from stem cell exhaustion.
Median days post SCT 21 18 28

Immunologic characteristics
GvHD risk
(for equivalent match) ++ +++ +
Reconstitution of immunity
Recovery of immunity involves the reconstitution of a diverse family
of cells and molecules: the innate immune system of the NK cell, the
adaptive immunity of the T- and B-cells, the regeneration of antigen-
presenting cells and the production of antibodies. We now understand
much more about the process of immune reconstruction in both autolo-
of the placental blood must be allowed to drain into the newborn infant gous and allogeneic SCT, which show some similarities. However,
before cutting the cord, to avoid anemia. Cord blood HSC have strong engraftment of an allogeneic immune system into the recipient intro-
proliferative potential which partly compensates for the very much duces further complexities.14,15
lower cell numbers collected. UCB lymphocytes are largely naíve
(non-antigen experienced) but have strong proliferative potential.
UCB collections also contain endothelial cells.10 Recovery of innate immunity
In both autologous and allogeneic SCT, NK cells are the first immune
Cryopreservation cells to reach normal blood levels, often overshooting in the first
HSC and lymphocytes can be readily stored frozen in liquid nitrogen month after SCT. Increased production of lymphocyte growth factors,
– for years if necessary – and retain their viability on thawing. Cryo- especially IL-12 and IL-15, stimulates rapid neogenesis of NK cells
preservation requires slow, controlled-rate freezing and the addition from CD34 cells.15,16 Antigen-presenting cells (APC) derived from
of an agent (usually dimethyl sulfoxide – DMSO) which prevents CD34 cells include monocyte-macrophages, dendritic cells (myeloid
intracellular ice crystal cell damage. When the cells are required for and plasmacytoid), Langerhans cells and B-cells. Recovery of trans-
transplantation they must be rapidly thawed, and rapidly transfused to plant-origin APC begins within weeks of transplantation and is com-
minimize toxicity from DMSO.11 plete within about 6 months of transplant.16
 Sequestration marrow and processed by the recipient thymus to form a new immune
repertoire. The recovery of CD8+ T-cells outpaces the CD4+ T-cells, 11
SCT with an associated deficiency of cell-mediated immunity. After trans-
plant, there is rapid proliferation of grafted lymphocytes, driven by

Chapter 2 Essential biology of stem cell transplantation

the release of lymphocyte growth factors IL-2, IL-12, IL15, and IL-18
in response to lymphopenia.15,16 Studies of immune responses to cyto-
megalovirus after SCT show that in transplants from donors previ-
ously exposed to CMV, memory cells expand rapidly and can control
reactivating CMV within a few weeks after SCT. Conversely, the
acquisition of immunity against reactivating CMV following trans-
plantation from a donor not previously exposed to CMV is much
slower, sometimes taking months to generate immune competence.15
Engrafting T-cells have special functions after allo SCT – they interact
SCT with recipient APC and cells of skin, gut and liver as well as residual
Homing host T-cells and marrow cells, resulting in GvHD, and the establish-
Rolling ment of space for the graft through a graft-versus-marrow effect.
Sticking
Importantly, engrafting T-cells also recognize and kill malignant cells
through the so-called graft-versus-leukemia or graft-versus-tumor
effect (Fig. 2.3).13–17
Passage through
sinusoid endothelium
Humoral immunity
Full reconstitution of antibody production follows the generation of
Self-replication/ new B-cell precursors from engrafted stem cells. Humoral immunity
differentiation after transplant is slow to recover, with immunoglobulin levels reach-
ing the normal range at between 6 and 12 months, often longer for
IgA and for patients who develop chronic GvHD.18

The preparative regimen

Niche
The preparative or conditioning regimen has three functions.
Figure 2.2 1 It is used to treat the patient’s malignant disease intensively and
Steps in stem cell engraftment.
reduce disease burden to minimal levels.
2 It immunosuppresses the recipient to allow engraftment of the
Immune intolerance Thymic function donor hematopoietic and immune system.
Cytokine
and tolerance 3 It makes space for incoming stem cells in hematopoietic niches
storm in the marrow.
Donor APC The requirement for one or all of these three functions according to
Host APC
the specific patient–donor situation determines choice of regimen.19
The intensity of the regimen can also be selected according to the
NK cells
ability of the patient to tolerate the regimen.20 Thus, older or debili-
tated patients may be given reduced-intensity regimens to prevent
CD3 cells mortality from high-dose treatment but which are nevertheless suffi-
Clonal
expansion ciently immunosuppressive to ensure engraftment. While the three
CD8
functions are distinct, the agents used in the regimens (for example,
cyclophosphamide) often have overlapping properties of myelo-
CD4 suppression and immunosuppression (see Fig. 2.4).
Recent thymic
emigrants Treatment to control malignant disease
Weeks 1 2 3 4 5 6 Months
Some regimens are specific to the particular malignant disease but
Figure 2.3
Immune recovery after stem cell transplantation. The first few weeks are characterized many regimens use total body irradiation to a dose of 12–15 Gy or
by a period of lymphopenia followed by rapid recovery of NK cells and a slower busulfan up to 12 mg/kg iv over 2–4 days in association with immu-
recovery of T-cells which are mainly CD8+. In the first few months the T-cell recovery nosuppressive agents such as cyclophosphamide, fludarabine or anti-
is irregular with numerous clonal expansions as well as defects in the repertoire. A CD52 (Campath) monoclonal antibody, or antilymphocyte globulin
year or more after transplant thymic function restores CD4 leves and broadens the
(ATG).
T-cell repertoire with new thymic-derived T-cells. B-cell recovery and restoration of
immunoglobulin levels begin in the first year.
Immunosuppression
Immunosuppression is required to achieve engraftment in all alloge-
Recovery of adaptive immunity neic SCT patients, with the exception of recipients with severe com-
Immediate T-cell immune reconstitution following SCT derives from bined immunodeficiency disease who cannot reject an allograft, or
transfused post-thymic T-cells (largely a memory T-cell compartment) recipients of stem cells from an identical twin. Patients who have been
and later from T-cell precursors generated from the CD34 cells in the sensitized by prior transfusions and children who have greater ability
 Table 2.2 Comparison of T-cells and NK cells
Myeloablation Immunosuppression
12 T-cells NK cells
Melphalan
Origin CD34 cells – prethymic CD34 cells
PART

precursors
Busulfan
Mature in thymus Mature in marrow

Development Naive Recognition/activation

Total body irradiation

1
Central memory Limited expansion

Cyclophosphamide Effector memory Death

SETTING THE SCENE

End effector

Fludarabine Stimulator cells APC (DC, B-cells, monocytes) Hematopoietic cells

Target cells Any MHC class I/II expressing Hematopoietic cells

Total lymph node irradiation cell

Recognition structures MHC class I and II MHC class I

T-cell antibodies +9–15 mer peptides Non-classic MHC (MicA/B)
Campath
OKT3 Receptors T-cell receptor complex KIR molecules (inhibitory)
ATG
CD4 + CD8+ NKG2D and others
costimulatory molecules (activating)

Figure 2.4 Receptor diversity Very high (1011–12 TCR Low (18 KIR types)
Agents used in preparative regimens sorted by their relative ability to immunosuppress sequences)
or myelosuppress.
Clinical impact:
GvHD + None
GvL + + (myeloid leukemias)
Engraftment + +
to reject transplants may need more intensive immunosuppression to
ensure engraftment.

Myelosuppression and C), but only some cells (including professional antigen-presenting
Complete myeloablation is not required to achieve full engraftment cells) express MHC class II molecules (HLA-DR, DP, DQ). Antigenic
but it is usually a consequence of high-dose treatment used to treat peptides presented by class I MHC molecules represent the ‘self’ of
malignant disease prior to transplant. Myeloablation is often avoided the cell. They are derived from cellular proteins which are degraded
in patients receiving reduced-intensity transplants. These patients into short peptide sequences during the course of protein turnover by
show rapid initial recovery of autologous hematopoiesis, followed the proteasome. Antigens presented by MHC class II molecules are
several months later by a T-cell mediated elimination of host hemato- derived from exogenous proteins as well as some self proteins. They
poiesis and a switch to donor hematopoiesis.21 Thus, in an allogeneic are degraded into peptides in lysosomal vacuoles. Endogenous and
SCT there is no prerequisite for the conditioning regimen to ‘make exogenous peptides complex with MHC class I molecules in the endo-
space’ for engrafting donor stem cells as long as cellular immunity plasmic reticulum and with MHC class II molecules in lysosomal
has switched to that of the donor. vacuoles. The T-cell receptors of CD8+ T-cells engage mainly with
MHC class I molecules, while those of CD4+ T-cells interact with
MHC class II molecules. The peptide in the MHC molecule is called
The basis of alloimmune reactions a minor histocompatibility antigen (mHag) while the MHC is the
major antigen. Minor histocompatibility antigens are important
Alloimmunity describes the immune interaction between genetically because in HLA identical sibling transplants, disparity between host
distinct individuals. Because of genetic diversity, all allografts have and donor mHag alone can cause lethal GvHD, graft rejection or
the potential for donor-versus-host or host-versus-donor immune powerful GvL effects. The molecular basis of antigen presentation is
responses which can cause graft-versus-host disease (GvHD), graft illustrated in Figure 2.5.23
rejection or more favorable graft-versus-leukemia (GvL) responses.
Allorecognition involves an adaptive immune response by CD3+ T- The innate immune response
cells and an innate immune response mediated by CD16+ CD56+ NK Although the NK repertoire is clonal, its diversity is much less than
cells. The two immune systems are contrasted in Table 2.2. that of the T-cell. Unlike T-cells, which can only be effective if there
is clonal expansion of the relevant antigen-specific T-cell clones, NK
The adaptive immune response immune effects on their target cells occur without prior clonal expan-
The adaptive immune response involves the interaction of T-lympho- sion. NK cells interact with other cells through inhibitory and activa-
cytes with antigens from the other individual.22 Upon antigen recogni- tion pathways. The activating interactions are mediated through a
tion, T-cells become activated and proliferate to generate expanded number of molecules, notably NKG2D encountering the non-classic
clones of effector and helper cells reacting to the cells bearing the MHC class I molecules MIC A/B on the target.24 An activated NK cell
antigen. Once established, these immune responses remain in the T- kills its target cell by lysis through perforin-granzyme release. To
cell memory, and further contact with the antigen initiates a new wave prevent autoimmune attack by self NK cells against self tissues, NK
of effector and helper cell generation. T-cell recognition of alloanti- cells also have a predominating inhibitory interaction with other
gens involves engagement of the T-cell receptor molecule with an cells.
HLA-peptide molecular complex on the surface of cells from another Inhibition of NK activation occurs when one of a family of killer
individual. All cells express MHC class I HLA molecules (HLA-A, B immunoglobulin-like receptors (KIR) on the NK cell engages with a
 A B NK KIR MHC I Target
Cytosolic SHP-1 KIR MHC class 1 13
protein
T-cell 2DL3
Ubiquination Group 1 Cw1,3,7,8
2DL3

Chapter 2 Essential biology of stem cell transplantation

Adhesion
molecules Perforin NKG2D MIC A/B 3DL1 Group 2 Cw2,4,6,15
CD3 No Lysis 3DL2 BW4, HLA-A
Proteasome TCR KIR MHC I
KIR mismatch
Peptides Class I
vacuole KIR2DL1
CD8
TAP KIR2DL2
Lysis
MHC
class 1
β2 TCR-peptide Missing MHC
recognition
Peptide Golgi
binding
Antigen-presenting
cell Stable HLA Lysis

Figure 2.5
Molecular basis of alloreactivity. (a) Adaptive immunity: generation of self peptides from intracytoplasmic degradation of self protein by ubiquination and digestion in the
proteasome. Short peptides are actively pumped into the endoplasmic reticulum, where they become incorporated into HLA class I molecules, undergo glycosylation in the golgi
and reach the cell surface in a class I vacuole to be scrutinized by passing CD8 T-cells which bind to the MHC molecule through a weak interaction via CD3 and CD8, and a
strong interaction between the peptide and T-cell receptor (TCR) hypervariable region. (b) Innate immunity: positive and negative signals control NK reactivity. When KIR groups
are matched with an appropriate MHC ligand, SHP-1 is upregulated and blocks the process of perforin-granzyme release, otherwise induced by the NKG2D/MICA/B activating
interaction. When there is a KIR mismatch or the target T-cell lacks MHC class I molecules, there is no negative signal to control NK-mediated lysis.

relevant class I MHC molecule on the target, delivering an inhibitory molecular or serologic typing of blood leukocytes to determine HLA
signal to the cell and blocking activation of its lytic machinery. NK- A, B, C (MHC class I) and DR, DP, DQ types (MHC class II). Clinical
target compatibility involves many KIR types interacting with many transplant results clearly show the advantage, in both related and
HLA class I molecules, but the most important functionally appears unrelated donor searches, of finding the closest HLA identity possible.
to be the inhibitory interaction between KIR group 1 and HLA-C1,-3, In HLA identical siblings, identity at HLA- A, B and DR is sufficient
-7, -8 and KIR group 2 and HLA-C2,-4,-6,-15. The behavior of an to determine HLA compatibility between donor and recipient, because
individual NK cell with its target depends on the balance between the MHC complex is inherited from parent to child in a single raft of
surface-expressed effector and inhibitory molecules characterizing genes constituting the parental haplotype. So closely are these genes
that particular NK clone. The conditions required for NK alloreactivity arrayed that only rarely (<2% of cases) do crossovers occur during
are fulfilled when the responder NK cell fails to engage with an inhibi- meiosis, creating mismatches, usually at the A locus. In unrelated
tory MHC class I molecule – either because the molecule is not donors the situation is different because, while haplotypes tend to be
expressed or because it belongs to a non-compatible group. NK allo- generally conserved, genetic recombination over many generations
reactivity is well characterized for HLA mismatched transplants, but leads to a legacy of rearrangements through crossovers not shared
may also occur between HLA identical donor–recipient pairs because between patient and donor. To fully define HLA compatibility in
KIR groups are inherited differently from the MHC complex. Thus, unrelated donors, further typing for HLA-C, DQ, and DP is also per-
the donor may lack the KIR group corresponding to that of the recipi- formed (Fig. 2.6).
ent, or the recipient may not express MHC molecules compatible with HLA typing was originally performed on lymphocytes using a
the donor’s KIR groups.25 panel of antibodies with defined reactivity to certain MHC molecules
NK cells differ from T-cells in their alloimmune responses in (serotyping). HLA typing is now increasingly performed by molecular
important ways. Critically, NK cells predominantly recognize cells of typing, which provides high definition characterization of individual
hematopoietic lineage. This explains the importance of NK cells in MHC molecules.27–29
engraftment: host NK cells can destroy an incoming graft, while donor
NK cells facilitate engraftment by eliminating residual host hemato-
poietic and lymphoid cells.26 A second consequence of the hematopoi- Functional tests of compatibility
etic specificity of NK cells is that they do not directly cause GvHD.
In fact, their ability to deplete host antigen-presenting cells reduces While selecting the best matched (or fully matched) donor is the first
the ability of the host to stimulate alloresponses in donor T-cells.25 step in limiting potential alloreactivity between donor and recipient,
it is clear from the preceding description of the alloresponse that HLA
typing does not exclude alloreactions occurring from mHag disparities
Tissue typing and KIR-MHC incompatibility. Functional compatibility tests can
The MHC locus is highly polymorphic, representing over 800 alleles complement HLA typing by identifying alloreactions between donor
described to date, and new molecules continue to be described. The and recipient without the need to characterize the genetic disparity.
reason for this diversity is believed to be the evolutionary pressure on The classic test is the mixed lymphocyte reaction (MLR) which mea-
genetic change required for adapting molecules to emerging variations sures proliferative response of the donor lymphocytes to the recipient
in micro-organisms encountered during the human diaspora into new in a 4–6 day assay. It is relatively easy to perform, but suffers from
environmental niches over the last 100,000 years. HLA typing involves wide variability and requires careful controls. Furthermore, the test