1

General introduction and outline of this thesis

 General introduction and outline of this thesis

General introduction 1
1. Acute leukaemia in children and assessment of prognosis following chemotherapy
1.1. General prognostic factors
The vast majority of childhood leukaemia cases are acute and are characterized by rapid
tumour cell proliferation and a predominance of blast cells. The most common form, acute
lymphoblastic leukaemia (ALL) accounts for 75%-80% of all cases of acute leukaemia in
children and adolescents, while acute myeloid leukaemia (AML) accounts for 15-20%. With
modern intensive chemotherapy and supportive care measures the prognosis of ALL, and to
a lesser extent of AML, in children has improved significantly over the past decades.

Figure 1. Disease free survival of children with ALL in the Netherlands (1972-1998); adapted from
the revised SNWLK ALL-9 protocol with permission from the SKION.

To minimize the frequency of treatment-related deaths and sometimes severe late effects of
chemotherapy, risk-group stratification is important. At the same time suboptimal therapy
should be avoided. Therefore, paediatric collaborative study groups try to assess the relapse
risk in individual patients, so that only high-risk cases are treated aggressively, while less
aggressive therapy is reserved for patients at lower risk of relapse(1). To develop adequate
risk stratification many prognostic parameters have been investigated. However, due to
effective contemporary therapy improvements, several of the biological and clinical features,

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 General introduction and outline of this thesis

1 which once were associated with poor prognosis, do not confer a poor outcome anymore.
It is, therefore, necessary to evaluate risk factors within the context of contemporary treatment
strategies and continue the development of new prognostic parameters.
The prognostic determinants for a patient with a malignant disease are heterogeneous and
often tumor-type specific. In general, prognostic factors are related to the characteristics of
the patient, the histopathology and biology of the tumour, and of the efficacy of therapy.
However, the same variable may play a different prognostic role and may have diverse
correlations with the other prognostic indicators in different malignancies(2).
In childhood leukaemia, patient’s age and leukocyte count at diagnosis have consistently
shown prognostic strength, regardless of the treatment regimen used. Although this impact
can in part be explained by the association with specific tumour cytogenetic abnormalities,
these genetic features do not entirely account for treatment outcome. One of the possible
reasons for the unpredictable relationship between the biological characteristics of the disease
and the response to treatment is the influence that pharmacodynamic and pharmacogenetic
factors exert on the effectiveness of treatment. Many studies have indicated that the patient’s
early response to therapy (i.e. initial decrease in leukaemic blasts), reflecting both leukaemic
cell characteristics and patient pharmacogenetics, is an important independent predictor of
long-term outcome(3;4). The sequential monitoring of minimal residual disease (MRD), by
polymerase chain reaction (PCR) analysis of clonal antigen-receptor gene rearrangements,
provides an objective and more sensitive assay than formerly used bone marrow cytology
to monitor the response to treatment and can improve the precision of risk assessment still
further. Several clinical trials currently incorporate MRD assessment as part of risk group
classification, adjusting the intensity of therapy based on MRD levels obtained at various
time points early in treatment. We will need a longer follow-up to determine the prognostic
impact of the therapeutic changes(5).

1.2. Specific prognostic factors

In addition to the patient’s response to treatment as an important prognostic factor, many
other strategies have been used to optimize risk stratification and subsequent therapy. This

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 General introduction and outline of this thesis

chapter will highlight some of these prognostic factors.

1
One of the oldest classifications is the French-American-British (FAB) classification(6)
system, that divided AML into 8 subtypes, M0 through to M7, based on the type of cell
from which the leukemia developed and its degree of maturity. This is done by examining
the appearance of the malignant cells under light microscopy and/or by using cytogenetics
to characterize any underlying chromosomal abnormalities. The subtypes have varying
prognoses and responses to therapy. The World Health Organization (WHO) classification of
acute myeloid leukemia attempts to be more clinically useful and to produce more meaningful
prognostic information than the FAB criteria. Although the WHO classification may be more
useful, the FAB system is still widely used for AML. In ALL the FAB classification system(6)
originally defined three subtypes (L1, L2, L3), based solely on morphologic features. Because
of the importance of immunophenotypic, cytogenetic and molecular features of ALL in risk
stratification and the lack of correlation between these features and the FAB subtypes, the
FAB classification of ALL has been largely abandoned in practice(1).
Several cytogenetic abnormalities can be found in many patients with AML and ALL;
the types of chromosomal abnormalities often have prognostic significance. Many of the
rearranged genes encode tyrosine kinases, transcription factors or other proteins that have
relevant activities during the cell cycle and apoptosis. The ultimate consequence of many
of these rearrangements and mutations is the impairment of hematopoietic development
and a “differentiation arrest.” For example, in acute promyelocytic leukemia, the t(15;17)
translocation produces a PML-RARα fusion protein which binds to the retinoic acid
receptor element in the promoters of several myeloid-specific genes and inhibits myeloid
differentiation. In ALL, the abnormal karyotypes are subgrouped according to chromosome
number (ploidy) and structural chromosomal changes. Current treatment protocols use the
presence of specific cytogenetic abnormalities for risk stratification.
Drug cytotoxicity assays are used to test the responsiveness of leukaemic cells to different
classes of both novel and already established cytotoxic drugs in vitro and can be used to
stratify ALL patients in risk groups(7). Children with in vitro-resistant ALL cells at initial
diagnosis have poorer long-term clinical outcome than do in vitro-sensitive patients(8).

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 General introduction and outline of this thesis

1 In contrast, cellular drug resistance at initial diagnosis of paediatric AML has not been
shown to be discriminative for long-term clinical outcome(9). Although the use of in vitro
cellular resistance testing in the contemporary risk stratification has been limited so far, the
generation of cellular models of drug resistance has been fundamental in unravelling the
main effectors of resistance to traditional chemotherapy at the molecular level. Furthermore,
recently a relatively small number of genes was found to be associated with both in vitro
drug resistance and treatment outcome in childhood ALL by gene expression analysis(10),
implicating that genetic profiling may identify patients with poor initial responses.
In acute leukaemia the immature malignant cells accumulate in the bone marrow. Soon after,
they leave the bone marrow, populate the blood, and in some cases localize in extramedullary
sites. In ALL, leukaemic cells have a tendency to migrate to the central nervous system
(CNS), liver, spleen and lymph nodes. In AML, leukaemic spread occurs most commonly
in the skin, gingiva, soft tissue, bone and the CNS. The soft tissue tumours are also referred
to as chloromas, granulocytic sarcomas or myeloblastomas. Especially in childhood AML,
extramedullary disease is frequently reported at diagnosis (in 23-40% of the cases) (11-13).
There is conflicting evidence on the prognostic significance of extramedullary leukemia in
patients with acute myeloid leukemia. In general, they are felt to confer a poorer prognosis,
with a poorer response to treatment and lower survival rate; however, others have reported
that extramedullary disease in childhood AML does not have independent prognostic
significance(11).
Very rarely, chloroma or solid extramedullary tumor can occur without a known pre-
existing or concomitant diagnosis; this is known as primary chloroma. In almost all reported
cases of primary chloroma, acute leukemia has developed shortly afterward (median time
to development of acute leukemia 7 months, range 1-25 months)(14). Therefore, primary
chloroma should be considered an initial manifestation of acute leukemia, rather than a
localized process, and should be treated as such.
More research on the mechanisms of cancer migration and the role of chemokine/ chemokine
receptor interaction in acute leukaemia may offer insights into the development of metastasis
and identification of potential therapeutic targets.

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 General introduction and outline of this thesis

2. Chemokines and their receptors

1
2.1. Chemokines and chemokine receptors in cancer metastasis
Chemokines are a superfamily of small cytokine-like proteins that work by binding to
specific G-protein-coupled receptors (chemokine receptors) on plasma membranes of
target cells. Binding triggers intracellular G proteins to activate a signalling cascade that
results in adherence of leukocytes to the endothelium before they extravasate into the tissue.
Chemokines, along with adhesion molecules, are crucial during inflammatory responses for a
timely recruitment of specific leukocyte subpopulations to sites of tissue damage. Chemokines
and their receptors are also important in dendritic cell maturation, B and T cell development,
Th1 and Th2 responses, and angiogenesis(15). In addition, they also seem to be involved in
the process of tumour cell growth, migration, invasion, and metastasis(16).

During the last 10 years many new chemokines and chemokine receptors have been identified
(Table 1). The small (8-10 kDa) chemokine proteins are classified into four highly conserved
groups (CXC, CC, C and CX3C), based on the position of the first two cysteines that are
adjacent to the amino terminus. More than 50 chemokines have been discovered so far and
there are at least 19 seven-transmembrane-domain chemokine receptors(16).
Although metastases to organ sites such as lymph nodes, lung and liver are to a certain extent
due to specific anatomic pathways such as lymphatic or vascular channels that mechanically
direct tumour cell migration, it has been known since the early 1900s that some tumour
cells have a propensity to metastasize to specific organs(17) and that they do not migrate
randomly. In the last few years, the involvement of chemokines and their receptors in cancer,
particularly metastasis, has been firmly established(18). The role of chemokines and their
receptors in cancer include (i) providing directional clues for migration, (ii) shaping the
tumour environment and (iii) providing survival and/or growth signals(16).

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 General introduction and outline of this thesis

Table 1. Summary of chemokine receptors and some of their known ligands

1 Chemokine receptors*
CXCR1
Ligands*
CXCL6, CXCL8
Old nomenclature(15)
GCP-2, IL-8
CXCR2 CXCL1-3; CXCL5-8 Groα/Groβ/Groγ; ENA-78/GCP-2/NAP-2/
IL-8
CXCR3 CXCL9-11 Mig/IP-10/I-TAC
CXCR4 CXCL12 SDF-1
CXCR5 CXCL13 BLC
CXCR6 CXCL16 HCC-4
CXCR7 CXCL12 SDF-1
CCR1 CCL3-5; CCL7, CCL8, CCL13-16; Groγ/PF4/ENA-78; NAP-2, IL-8, BLC/
CCL23 Leukotactin/HCC-4; MPIF-1
CCR2 CCL2, CCL7, CCL8, CCL13 MCP-1, MCP-3, MCP-2, MCP-4
CCR3 CCL5, CCL7, CCL8, CCL11, CCL13, RANTES, MCP-3, MCP-2, Eotaxin, MCP-
CCL15, CCL24, CCL26 4, Leukotactin, MPIF-2, SCYA26
CCR4 CCL17, CCL22 TARC, MDC
CCR5 CCL3, CCL4, CCL5. CCL8, CCL14 MIP-1α, MIP-1β, RANTES, MCP-2,
HCC-1
CCR6 CCL20 MIP3α
CCR7 CCL19, CCL21 MIP3β
CCR8 CCL1, CCL4, CCL17 I-309, MIP-1β, TARC
CCR9 CCL25 TECK
CCR10 CCL26-28 SCYA26/CTACK/MEC
XCR1 XCL1-2 Lymphotactin/SCM-1β
CXCR3 CX3CL1 Fractalkine

*CXC, CC, C and CX3C reflect the four families of chemokine receptors based on the pattern of cysteine residues
in the ligands. R (receptor) or L (ligand) is added to these letters accordingly.

When Müller et al. highlighted the role for chemokines in directing organ-specific
metastasis, it became clear that chemokine receptor expression patterns on cancer cells
and the localization of the corresponding ligands could provide clues for understanding
directional metastasis(19). In many types of cancer the malignant cells were shown to exhibit
an increased or aberrant expression of particular chemokine receptors relative to their normal
counterparts(20), resulting in migration of the tumour cells by mechanisms similar to normal
lymphocyte trafficking (Table 2).
Various reasons for altered chemokine receptor expression have been identified. The tumour
microenvironment, mutant proteins or altered signalling in the cancer cell itself can affect

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 General introduction and outline of this thesis

Table 2. Some of the chemokine receptors that are expressed on cancer cells(16;22)
Chemokine
receptor
Cancer cell expression Normal-cell expression 1
CXCR4 23 different haematopoietic Haematopoietic stem cells, thymocytes, T cells, B
and solid cancers cells, immature and mature dendritic cells, some
endothelial cells, macrophages and neutrophils
CCR5 Breast cancer cell lines Thymocytes, B lymphocytes, immature and
mature dendritic cells, and macrophages
CCR7 Breast cancer, CLL, gastric B cells, T cells and mature dendritic cells
cancer, non-small-cell lung and
oesophageal cancer
CCR10 Melanoma Plasma cells and skin-homing T cells
CXCR2 Melanoma Macrophages, eosinophils and neutrophils

chemokine receptor levels(16). For example, hypoxia induces up-regulation of CXCR4

transcription(21). Secondly, chemokines and their receptors play roles in shaping the tumour
microenvironment by their ability to attract leukocytes, which promote angiogenesis and
activate other enzymes and cytokines. Thirdly, expression of chemokine receptors on cancer
cells can provide the cells with more than a mechanism for migration. Receptor signalling
may also provide a survival advantage. It has been demonstrated that CXCL12 [formerly
known as SDF-1α (stromal-cell-derived factor-1α)]
activates the specific signalling pathways that promote survival effects(23). These pro-
tumourigenic pathways are likely to be particularly important for the ability of metastatic
tumour cells to thrive in a foreign environment. Furthermore, chemokines that serve as
migratory signals to control homing of lymphocytes to protective niches in the bone marrow
and lymph nodes may also promote survival of leukaemic cells.

2.2. Chemokines and their receptors in acute leukaemia

To date most studies, aimed at the involvement of chemokines and their receptors in the
migration and survival of leukaemic cells in humans, have concentrated on the interaction
of CXCL12 and CXCR4(24). CXCL12 is a “homeostatic” chemokine that is constitutively
secreted by marrow stromal cells. It signals through CXCR4, a receptor, which is broadly
expressed by normal and malignant cells of haematopoietic and non-haematopoietic lineages.
The CXCR4-CXCL12 axis is particularly important in the marrow microenvironment. Rapid

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 General introduction and outline of this thesis

1 mobilization of haematopoietic stem cells by CXCR4 antagonists (AMD3100), as shown

in recent clinical trials, supports the hypothesis that regulation of the homing and retention
of haematopoietic progenitor cells in the distinct stromal niches of the marrow is one of its
essential functions(25). It was generally thought that CXCL12 mediates its effects solely
through CXCR4. However, recent studies characterized a novel receptor for CXCL12, termed
CXCR7(26;27). CXCR7 is expressed on malignant cell types and foetal liver cells, but on
few other cell types(27) Furthermore, it is highly expressed on tumour-associated blood
vessels, but not on normal vasculature(28). Unlike many other chemokine receptors, ligand
activation of CXCR7 does not cause Ca2+-mobilization or cell migration(27;29). However,
interaction of CXCL12/SDF-1 with CXCR7 results in a proliferative effect, in contrast to
CXCR4 which mediates chemotaxis. Further, CXCR7 has been attributed a potential role
in tumour development because its expression provides cells with a growth and survival
advantage and increased adhesion properties.
The CXCL12-CXCR4 interaction has been shown to be functional in ALL cells and important
for marrow homing of ALL cells(24). Crazzolara et al reported that a higher expression
level of CXCR4 on ALL cells, as determined by flow cytometry, correlated with more organ
infiltration at diagnosis that was independent from the peripheral lymphoblast count(30).
Schneider et al. detected a high expression of CXCR4 on ALL blasts of children with a
high white blood cell count and organ infiltration at diagnosis. Higher levels of CXCR4
expression on blast cells at diagnosis did not constitute a prognostic factor of extramedullary
relapse, but appeared to confer a poorer outcome in this small study(31).
Also, CCR5 was recently studied in relationship to acute lymphoblastic leukaemia. A
polymorphism in the CCR5 genotype was positively correlated with the clearance of
minimal residual disease, indicating that CCR5 may play a role in the early response to
chemotherapy(32).
Also on myeloid cells, the CXCL12-CXCR4 interaction provides a strong chemotactic signal.
Apart from this chemotactic activity, CXCL12 has also been suggested to suppress apoptosis
and promote G0/G1 transition in myeloid cells. Furthermore, some evidence suggests that
CXCL12 induces an arrest in myeloid differentiation. This mechanism provides a potential

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 General introduction and outline of this thesis

way of leukaemia cell evasion from chemotherapy(33). This hypothesis is supported by

1
the impact of high CXCR4 expression on AML cells, determined by flow cytometry, as an
adverse prognostic indicator in AML, independent from other prognostic factors, particularly
cytogenetics and white blood cell count(33;34). In addition, polymorphisms in the CXCL12
coding gene is associated with higher counts of circulating AML cells and a higher frequency
of extramedullary disease(35).
Given the crucial roles of chemokines and their receptors in a wide array of physiological
functions and their association with many malignant diseases, the receptors have become
important targets for risk stratification and cancer treatment. Confirmation of the prognostic
value of CXCR4 expression and investigation of the expression of other chemokine
receptors in relationship to outcome in childhood leukaemia is needed and may lead to new
risk stratification strategies. Moreover, to successfully develop therapeutic strategies for
modulating chemokine action, we need to gain a detailed understanding of the mechanisms
of chemotaxis mediated by chemokines and their receptors in acute leukaemia.

3. Treatment of acute leukaemia in children

3.1. Treatment of ALL in childhood
Childhood leukaemias are among the most drug-responsive of human malignancies. More
than 70% of children with ALL can now be cured, largely by modern combinations of
chemotherapeutic drugs, administered according to international protocols(36). For all
ALL, except mature B-cell ALL, treatment includes remission induction (RI) followed by
consolidation therapy to eliminate residual leukaemia, eradication or prevention of CNS
leukaemia, and maintenance treatment to ensure continuation of remission. In most studies,
patients are stratified into risk groups, based on cytogenetic abnormalities and response to RI
treatment, although there is no consensus on the most valid criteria. However, measurement
of minimal residual disease (MRD) by leukaemia-specific PCR-based quantitative techniques
has emerged as a very powerful post-RI prognostic factor.
The goal of RI-therapy is to induce a complete remission by eradicating over 99% of the
initial leukaemic cell population, and by restoring normal haematopoiesis. With modern

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 General introduction and outline of this thesis

1 chemotherapy and supportive care, 97% to 99% of children can reach a complete remission
(i.e. ≤5% blasts in the bone marrow on cytology). Patients who fail to achieve a cytological
remission at the end of remission induction have a poor prognosis. These patients, as well
as patients with more than 1% blasts identified by MRD studies as applied in some centres,
should be candidates for allogeneic stem cell transplantation(1).
With restoration of normal hematopoiesis, patients in remission become candidates for
intensification (consolidation) therapy. The most successful post-RI intensification regimens
are generally administered continuously, whereas high-dose pulse therapy with long rest
periods due to myelosuppression appears to be less effective. For reasons that are poorly
understood, children with ALL require long-term continuation treatment. Attempts to
shorten the duration of treatment have resulted in a high risk of relapse after cessation of
therapy(37).
Response to treatment at relapse is much inferior to that in newly diagnosed ALL, because
of drug resistance already present in subclones at diagnosis or acquired after exposure to
antileukaemic drugs. Well-established prognostic factors in relapsed ALL are time from
reaching complete remission to relapse, site of relapse, immunophenotype and specific
cytogenetic translocations. In most children with relapsed ALL, second remissions can be
induced. However, in many patients, chemotherapy is not sufficient to maintain this remission.
Stem cell transplantation has therefore been used as intensive post remission treatment.

3.2. Treatment of AML in childhood

Although the outcome of treatment of children with AML has improved markedly over the
last three decades(38;39), the cure rate continues to lag behind that of children with ALL.

Although for several years uncontrolled proliferation was considered the distinguishing
property of any malignant disease, recent studies have shown that AML is initiated and
sustained by a small, self-renewing population of leukemic stem cells (LSCs), which produce
progeny consisting of a heterogeneous population of progenitor cells; the vast majority of the
LSCs is in a quiescent state and, thus, is insensitive to the effects of most chemotherapeutic

20
 General introduction and outline of this thesis

agents. This latter feature explains, at least in part, the difficulties in eradicating such a cell
1
population by conventional chemotherapy(41;42).

Figure 2. Overall survival of children younger than 15 years of age who had acute myeloid leukemia treated in
MRC trials during the past 3 decades(40); with permission from Nature Publishing Group.

Clinical trials of AML feature intensive chemotherapy with or without subsequent stem cell
transplantation(43). Therapy consists of a limited number of intensive courses of chemotherapy.
Increasing the intensity of the remission induction has not significantly improved remission
rates, but the induction “intensity” may well improve ultimate outcome(44). In contrast to
ALL treatment, most cooperative study groups have abandoned the use of maintenance
therapy in AML(1). Allogeneic stem cell transplantation is a feasible and effective alternative
to chemotherapy as post-remission therapy for AML. Many studies have demonstrated that
relapse-free survival probabilities for patients who have AML and a suitable family donor,
irrespective if they undergo matched sibling donor (MSD-)SCT or not are better than those
of patients who receive only intensive chemotherapy(44). However, others have shown no
such advantage, primarily because of transplantation related mortality (38). Although MSD-
SCT is often recommended, considerable controversy remains as to which patients in first
remission should undergo this procedure. Currently, most European investigators agree that
intermediate-risk and high-risk patients (based on cytogenetic abnormalities and response to

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 General introduction and outline of this thesis

1 treatment) should undergo MSD-SCT during first remission of AML but do not recommend
SCT for low-risk patients(45).

4. Haematopoietic stem cell transplantation

Over the last three decades, haematopoietic stem cell transplantation (HSCT) has become
an important treatment modality for a wide range of life-threatening haematological and
immunological disorders in both children and adults, with over 40,000 transplants worldwide
each year. With increased understanding of histocompatibility and development of novel
immunosuppressive drugs, HSCT has become a therapeutic option for an increasing number
of patients with otherwise incurable leukaemias. Other indications include certain immune
deficiencies, hemoglobinopathies and inborn errors of metabolism. On the following pages
some background information will be given on a few basic aspects of allogeneic stem cell
transplantations, which are relevant to this thesis.

4.1. Conditioning regimen and eradication of leukaemia

The chemotherapy or irradiation given immediately prior to a transplant is called the
conditioning or preparative regimen, the purpose of which is to eradicate the residual
leukaemic cells and provide adequate immunosuppression needed for successful engraftment
of allogeneic haematopoietic stem cells. The most commonly used preparative regimens
prior to allogeneic HSCT for leukaemia include various doses of total-body irradiation
(TBI) and cyclophosphamide, without or with additional chemotherapeutical agents(46).
Non-TBI-based regimens usually include busulphan and cyclophosphamide. Sometimes
other chemotherapeutic agents are added for extra anti-leukaemic effect such as cytosine-
arabinoside or etoposide.
TBI is also one of the most important causes of late effects after HSCT in children, such
as interstitial pneumonitis and pulmonary fibrosis, cataract, stunted growth, endocrine
dysfunction, and secondary tumours(47-49). In the paediatric population, besides the risk of
diminished neuropsychological functioning, the damage of irradiation to growth plates and
gonads has important effects on growth and gonadal development. For that reason an age-

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 General introduction and outline of this thesis

dependent total dose is applied in the paediatric SCT centre of the LUMC(50).
1
In an attempt to reduce the effects of radiation-induced toxicity, most centres have replaced
single fraction TBI by fractionated TBI. Fractionation of the total radiation dose allows
higher total doses without increasing the damage to normal tissue. Some haematological
malignancies, however, are not very radiosensitive, as was demonstrated by Cosset et al.
(51), resulting in reduced tumour kill if fractionation is applied.

4.2. Graft-versus-Host Disease and Graft-versus-Leukaemia effect

Since the start of allogeneic HSCT prevention and management of acute Graft-versus-Host
Disease (GVHD) has been an important challenge. GVHD is a complex immunological
reaction of immunocompetent cells in the graft against tissues of the host. The incidence of
this complication increases with increasing HLA mismatches between donor and recipient.
Acute GVHD, per definition starting within 50 days after HSCT, is mostly first visible in the
skin. Other major target organs are the liver and gastrointestinal tract, but other tissues can also
be involved. A grading system based on signs and symptoms for acute GVHD was originally
proposed by Glucksberg et al(52). To prevent acute GVHD, originally methotrexate (MTX)
was used, later on cyclosporine A (CsA), a calcineurin inhibitor with an immune-modulating
effect on donor T cells, was introduced(53), and the combination was clearly superior than
single drugs in the prevention of GVHD after SCT (54). Nowadays, in case the graft is not
depleted of T cells, a short course of MTX in combination with daily administration of CsA
for several months is usual.
At the same time, HSCT provides an anti-leukaemic effect caused by a reaction of donor
immune cells against residual leukaemic cells in the recipient. This effect is called the Graft-
versus-Leukaemia (GVL) effect(55). Its effect differs for different types of leukaemia, and
is most pronounced in chronic leukaemia. Unfortunately, GVL is mostly associated with
GVHD. Measures to reduce or prevent GVHD, e.g. T-cell depletion of the graft, were
associated with a higher relapse rate(56).
A lot of research is directed towards the development of strategies to exploit the GVL effect
without increasing Graft-versus-Host Disease-related mortality. Optimizing the GVL effect

23
 General introduction and outline of this thesis

1 has been demonstrated by using a reduced dose of cyclosporine A, which was associated
with a significant reduction in the leukaemia relapse rate in an prospective randomized trial
in children with acute leukaemia (57). The intensity of post-transplant immunosuppression
may well affect the GVL effect and, therefore, appears to be an important determinant for
a potential relapse of leukaemia after HSCT. An alternative strategy to augment an anti-
leukaemia immune response is the infusion of lymphocytes from the original stem cell donor
(DLI) after the transplant.

5. Pharmacokinetics of HSCT-related drugs in children

5.1. Paediatric drug research
Most drugs that are currently prescribed for children have not been tested in children.
Various approaches to determine paediatric drug dosing are being used. Many of these
apply allometric principles (i.e., based on relative body size), that generally assume that
there are predictable, linear relations between body weight and body-surface area among
infants, children, adolescents and adults(58). However, since growth is not a linear process
and the body composition changes through childhood, these simplified dosing strategies are
not adequate to reach optimal treatment results or to achieve appropriate systemic exposure
to drugs in case of a narrow therapeutic window and/or high interpatient variability. The
testing of medications in children, however, presents a dilemma. Nonetheless, children may
be harmed if they are given medications that have been inadequately studied. Therefore,
future paediatric patients’ clinical studies will be needed for a better understanding of the
pharmacokinetics of drugs used in infants and children in order to optimize dosing and
increase safety for children taking these drugs. There are several HSCT-related drugs, that
need pharmacologic research when applied in children, e.g. busulfan, already investigated
by us and others in the past(59-61) and CsA, only scarcely studied in the paediatric graft
recipients(62).

5.2. Pharmacokinetic analysis

Pharmacokinetics is the study of the absorption, distribution, metabolism, and excretion of

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 General introduction and outline of this thesis

drugs. Interpatient variability characterizes the disposition of many drugs. In drugs with a
1
narrow therapeutic window, such variability is likely to affect either clinical efficacy and/
or toxicity. Clinical pharmacology studies often need a series of measurements (e.g. serum
concentration) in subjects at consecutive time points. Subsequently, pharmacokinetic
(PK) analysis is performed without specific models (e.g. non-compartment models) or by
estimating the parameters of mathematical models that describe that particular profile (e.g.
compartmental methods)(63). Non-compartmental methods estimate the exposure to a drug
by estimating the area under the curve (AUC) of a concentration-time graph (Figure 2).

Figure 2. Example of a concentration-time graph.

The total drug exposure can be estimated with the trapezoid rule. This area estimation is
highly dependent on the sampling schedule; i.e. the closer the sampling points are, the closer
the trapezoids are to the actual shape of the concentration-time curve. This approach may
be satisfactory if data are extensive for each subject and if there is only minor between-
subject variability. In contrast, compartmental PK analysis uses kinetic models, similar to
models used in other scientific disciplines such as chemical kinetics and thermodynamics,
to describe and predict the concentration-time curve. Especially in the pediatric setting,
compartmental population PK modeling offers several advantages. Rather than obtaining
rich data from a selection of individuals, sparse data from many individuals can be analyzed.
This approach generally can use data from the children being given the drug therapeutically.
Furthermore, the model is flexible, does not depend on exact blood sampling time-points

25
 General introduction and outline of this thesis

1 and enables interpretation of sets with missing data. It is therefore useful in clinical practice.
Consequently, a Bayesian maximum a posteriori fitting uses individual patient covariables
and the information obtained from a prior population analysis, to estimate individual PK
parameters(64).
Thus, population PK studies can describe variabilities in response and/or concentration,
determine the dose that achieves a target concentration, derive maximum a posteriori Bayesian
individual predictions during therapeutic drug monitoring and predict the best sampling
protocols for future studies(63). Also in the setting of HSCT, children have benefitted and
will continue to benefit from this approach.

5.3. Pharmacokinetics of cyclosporine A

CsA is a highly lipophilic drug whose pharmacokinetics are characterized by slow, incomplete
and variable absorption, large volume of distribution, liver biotransformation via the 3A4
isoenzyme of cytochrome P450 (CYP) and low clearance (Cl)(65). Despite the extensive
use of CsA for HSCT in adults and children, only limited information is available about its
pharmacokinetics. The narrow therapeutic index of CsA and its high intra- and interindividual
pharmacokinetic variability have made therapeutic drug monitoring essential(66). To prevent
drug toxicity, trough level monitoring usually is applied. Systemic exposure, however, is
poorly reflected by this measurement alone(67). Furthermore, in solid organ transplantation,
systemic exposure of CsA, as measured by the area under the curve (AUC), has been shown
to correlate well with clinical outcome(68;69). It has not been established yet how systemic
exposure, as reflected by the AUC, correlates with the clinical effects of CsA following
allogeneic SCT.

6. Chimerism after stem cell transplantation

In zoology, a chimera is an animal that has two or more different populations of genetically
distinct cells that originated in different zygotes. In medicine, this condition is either natural
(inherited or acquired through pregnancy), or artificial, resulting from a medical intervention
such as an allogeneic HSCT(70).

26
 General introduction and outline of this thesis

Natural chimerism includes chimerism of blood cells caused by an exchange of hematopoietic

1
stem cells via communications between the two fetal blood systems or resulting from passage
of cells between maternal and fetal circulation. Fetal progenitor cells can survive for many
years in maternal blood and vice versa. Based on certain similarities between GVHD and
auto-immune diseases, microchimerism is considered to be a potential trigger for certain
types of auto-immune diseases, however, the mechanisms responsible for this relationship
remain unclear(71).
Allogeneic HSCT leads to complete, and sometimes incomplete replacement of the
haematopoietic cell lineages(72). Chimerism of distinct haematopoietic cell lineages after
HSCT is a critical observation because it provides information about the status of the graft.
Accumulating evidence suggests that bone marrow-derived progenitor cells are capable of
differentiating into other cells than strictly haematopoietic cells, e.g. endothelial cells(73-75).
In recipients of allogeneic hematopoietic stem cells, donor-derived endothelial cells were
detected in the skin and gut after SCT(76). Bone marrow-derived cells can also take on the
phenotype of epithelial cells in multiple organs(77). However, these findings have generated
controversy. One of the main concerns is the technical challenge to prove a cell is (i) donor-
derived, (ii) expresses epithelial markers and (iii) to rule out overlay of cells. Furthermore,
it is not clear which subpopulation of cells present in the graft is capable of engrafting
as epithelial cells. Also little is known regarding the mechanism, kinetics, and potential
clinical utility or pathology associated with this phenomenon(78). However, to successfully
develop strategies for using of bone marrow-derived stem cells for therapeutic purposes e.g.
regenerative medicine and drug delivery, we need to gain a detailed understanding of the
exact subpopulation of the bone marrow precursor cells involved, the cell type into which
they have ability to evolve and by which mechanism this occurs first.

27
 General introduction and outline of this thesis

1 Outline of this thesis

To gain a better understanding of the role that chemokine receptors play in the migration
and survival of leukaemic blasts in paediatric ALL and AML, we investigated the pattern
of chemokine receptor expression on these cells. In chapter 2, we describe the chemokine
receptor expression on the leukaemic blast cells of 11 paediatric T-ALL patients at diagnosis
in relationship to homing to the gut. Chapter 3 describes the chemokine receptor expression
on AML blasts in children with and without spread of the leukaemic blasts to the skin, as well
as the expression of the specific ligands at the site of migration.
To analyze prognostic factors for relapse after SCT, we evaluated, retrospectively, the
outcome of 132 children with acute leukaemia that were treated with HLA-identical HSCT
in the paediatric SCT centre of the Leiden University Medical Centre (LUMC) in chapter 4.
With the aim of improving therapeutic drug monitoring of Cyclosporine A (CsA) in children
after SCT, we described in chapter 5 a pharmacokinetic model and developed a limited
sampling strategy in order to determine the AUC of CsA. With this pharmacokinetic model,
we retrospectively estimated the area-under-the-curve (AUC) of CsA in children transplanted
for a haematological malignancy. Chapter 6 evaluates CsA systemic exposure (~AUC) during
the early post HSCT period on clinical outcome i.e. occurrence of acute GVHD and relapse
of the haematological malignancy after HSCT.
Although, the objective of allogeneic stem cell transplantation is to graft hematopoietic
precursor cells in patients, recent evidence indicates that bone marrow-derived cells may have
potential to fuse with or differentiate into endothelial and epithelial cells. The potential future
therapeutic applications of these findings call for a better understanding of the mechanisms
of chimerism induction and its context in the HSCT-setting. Therefore, in chapter 7, we
investigated the appearance of donor-derived endothelial and epithelial cells after HSCT in
relation to conditioning regimen, time course after SCT and occurrence of GVHD.
Finally, the results and implications of the different studies are discussed and summarized in
chapter 8, followed by a summary in Dutch in chapter 9.

28
 General introduction and outline of this thesis

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 General introduction and outline of this thesis

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35
 General introduction and outline of this thesis

36