Cytogene

Department of Medical Genetics
Haartman Institute
University of Helsinki
Finland
Cytogenetic and Molecular

Genetic Changes in Childhood
Acute Leukaemias
Tarja Niini
Academic dissertation
To be publicly discussed with the permission of the Medical Faculty of

the University of Helsinki, in the lecture hall of the Department of Oncology,
Helsinki University Central Hospital, Haartmaninkatu 4,
on November 22nd 2002, at 12 oclock noon.
Helsinki 2002
SUPERVISED BY
Professor Sakari Knuutila, Ph.D.
Department of Medical Genetics
Haartman Institute
REVIEWED BY
Docent Eija-Riitta Hyytinen, Ph.D.
Department of Clinical Genetics
Tampere University Hospital
University of Tampere
Docent Eeva Juvonen, M.D., Ph.D.
Department of Medicine
Helsinki University Central Hospital
OFFICIAL OPPONENT
Professor Eeva-Riitta Savolainen, M.D., Ph.D.
Department of Clinical Chemistry
University of Oulu
ISBN 952-91-5212-4 (Print)

ISBN 952-10-0760-5 (PDF)
http://ethesis.helsinki.fi
Helsinki 2002
Yliopistopaino
To my son Aaro
Table of contents
LIST OF ORIGINAL PUBLICATIONS........................................................... 7
ABBREVIATIONS ........................................................................................... 8
ABSTRACT....................................................................................................... 9
INTRODUCTION ........................................................................................... 11
REVIEW OF THE LITERATURE.................................................................. 12
1. Normal development of blood cells ..................................................................12
2. Leukaemias........................................................................................................12
3. Childhood leukaemias .......................................................................................13
3.1. Childhood acute lymphoblastic leukaemia (ALL) .......................................... 13
3.2. Childhood acute myeloid leukaemia (AML)................................................... 14
4. Aetiology of childhood leukaemias...................................................................14

4.1. Prenatal origin................................................................................................ 14
4.2. Inherited predisposition and environmental factors ...................................... 15
5. Genetic aberrations in childhood acute leukaemias ..........................................17

5.1. Translocations ................................................................................................ 18
5.1.1. Translocation t(12;21)(p13;q22) and the TEL and AML1 genes........... 21
5.2. Numerical chromosome aberrations .............................................................. 23
5.3. Gene amplifications ........................................................................................ 23
5.4. Tumour suppressor genes and allelic losses .................................................. 24
6. Genetic methods used in the diagnosis and follow-up of leukaemia ................25

6.1. Conventional cytogenetics.............................................................................. 25
6.2. Molecular genetic methods............................................................................. 25
6.3. Molecular cytogenetic methods ...................................................................... 26
6.3.1. Chromosome painting and multicolour-FISH........................................ 27
6.3.2. Interphase-FISH ..................................................................................... 27
6.3.3. Comparative genomic hybridisation ...................................................... 28
7. Gene expression profiling by DNA arrays........................................................31

7.1. Types of array and principles of the method .................................................. 31
7.2. Problems concerning patient sample and reference ...................................... 32
7.3. Data analysis .................................................................................................. 34
7.4. Applications .................................................................................................... 35
AIMS OF THE STUDY .................................................................................. 37

MATERIAL AND METHODS ....................................................................... 38
1. Material .............................................................................................................38
1.1. Patients and samples ...................................................................................... 38
1.2. References....................................................................................................... 39
2. Methods .............................................................................................................39
2.1. Comparative genomic hybridisation (I and II)............................................... 39
2.2. Fluorescence in situ hybridisation (III and IV).............................................. 40
2.3. Calculation and comparison of overall and event-free survivals (IV) ........... 41
2.4. cDNA array method (V).................................................................................. 42
2.4.1. cDNA array hybridisation ...................................................................... 42
2.4.2. Data analysis .......................................................................................... 42
2.5. Quantitative real-time reverse transcriptase polymerase chain reaction (V) 43
RESULTS ........................................................................................................ 45
1. DNA copy number changes in childhood AML (I) ..........................................45
2. DNA copy number changes in childhood ALL (II) ..........................................45
3. Increased copy number of the AML1 gene in childhood ALL (III) ..................47
4. FISH analysis of the TEL and AML1 genes in ALL patients with loss at
12p (IV) .............................................................................................................48
5. Gene expression in childhood ALL (V) ............................................................48
DISCUSSION .................................................................................................. 50
1. Comparative genomic hybridisation and conventional cytogenetics in
childhood AML (I) ............................................................................................50
2. CGH as a support of conventional cytogenetics in childhood ALL (II) ...........51
3. DNA copy number changes in childhood ALL (II) ..........................................52
3.1. Gains............................................................................................................... 52
3.2. Losses.............................................................................................................. 54
4. Amplification of the AML1 gene in childhood ALL (III) .................................55

5. Association of loss at 12p with the TEL-AML1 fusion in childhood ALL (IV) 56
6. Gene expression in childhood ALL (V) ............................................................57
SUMMARY AND CONCLUSIONS .............................................................. 61
ACKNOWLEDGEMENTS ............................................................................. 63
REFERENCES ................................................................................................ 65
ORIGINAL PUBLICATIONS ........................................................................ 84
List of original publications

This thesis is based on the following publications, which are referred to by their
Roman numerals in the text:
I
Tarja Huhta, Kim Vettenranta, Kristiina Heinonen, Jukka Kanerva,

Marcelo L. Larramendy, Eija Mahlamki, Ulla M. Saarinen-Pihkala and
Sakari Knuutila (1999). Comparative genomic hybridization and
conventional cytogenetic analyses in childhood acute myeloid leukemia.
Leukemia and Lymphoma 35:311-315.
II
Marcelo L. Larramendy*, Tarja Huhta*, Kim Vettenranta, Wa'el El-Rifai,

Johan Lundin, Seppo Pakkala, Ulla M. Saarinen-Pihkala and Sakari
Knuutila (1998). Comparative genomic hybridization in childhood acute
lymphoblastic leukemia. Leukemia 12:1638-1644.
III
Tarja Niini, Jukka Kanerva, Kim Vettenranta, Ulla M. Saarinen-Pihkala

and Sakari Knuutila (2000). AML1 gene amplification: a novel finding in
childhood acute lymphoblastic leukemia. Haematologica 85:362-366.
IV
Jukka Kanerva*, Tarja Niini*, Kim Vettenranta, Pekka Riikonen, Anne

Mkipernaa, Ritva Karhu, Sakari Knuutila and Ulla M. Saarinen-Pihkala
(2001). Loss at 12p detected by comparative genomic hybridization
(CGH): Association with TEL-AML1 fusion and favorable prognostic
features in childhood acute lymphoblastic leukemia (ALL). A multiinstitutional study. Medical and Pediatric Oncology 37:419-425.
Tarja Niini, Kim Vettenranta, Jaakko Hollmn, Marcelo L. Larramendy,

Yan Aalto, Harriet Wikman, Blint Nagy, Jouni K. Seppnen, Anna
Ferrer Salvador, Heikki Mannila, Ulla M. Saarinen-Pihkala and Sakari
Knuutila (2002). Expression of myeloid-specific genes in childhood acute
lymphoblastic leukemia a cDNA array study. Leukemia 16:2213-2221.
* these authors contributed equally to the study
Abbreviations
ABL1
ALL
AML
AML1
BCL2
BCR
CBFB
CDKN1B
CDKN2A
CDKN2B
cDNA
CGH
CLC
CLL
CML
E2A
FAB
FISH
FITC
GCSFR
HLH
LOH
MLL
MYC
PBX1
PCA
PCR
PRTN3
RNASE2
ROC
RT-PCR
S100A12
TEL
WBC
v-abl Abelson murine leukaemia viral oncogene homolog 1

acute lymphoblastic leukaemia
acute myeloid leukaemia
acute myeloid leukaemia-1
B-cell CLL/lymphoma 2
breakpoint cluster region
core binding factor, beta subunit
cyclin-dependent kinase inhibitor 1B
cyclin-dependent kinase inhibitor 2A
cyclin-dependent kinase inhibitor 2B
complementary DNA
comparative genomic hybridisation
Charcot-Leyden crystal protein
chronic lymphatic leukaemia
chronic myeloid leukaemia
immunoglobulin enhancer-binding factors E12/E47
French-American-British
Fluorescence in situ hybridisation
fluorescein-isothiocyanate
granulocyte colony stimulating factor receptor
helix-loop-helix
loss of heterozygosity
mixed lineage leukaemia
v-myc avian myelocytomatosis viral oncogene homologue
pre-B-cell leukaemia transcription factor 1
principal component analysis
polymerase chain reaction
proteinase 3
ribonuclease, RNase A family, 2
receiver operating characteristic
reverse transcriptase polymerase chain reaction
S100 calcium-binding protein A12
Translocation-ets-leukaemia
white blood cell count
Abstract
Numerous recurrent cytogenetic aberrations have been found in childhood acute
leukaemias. Many of them have prognostic impact and their contributory role in
the design of treatment has been valuable. Patients have been classified to
different risk groups according to the existing criteria but the groups have,
however, remained heterogeneous. Moreover, little is known about the causes
of leukaemia at the molecular level.
In this thesis, DNA copy number changes were first studied in 19 children
with acute myeloid leukaemia (AML) and 72 children with acute lymphoblastic
leukaemia (ALL) using comparative genomic hybridisation (CGH). In addition,
the suitability of the method for diagnostics of these diseases was investigated.
In AML, the CGH results agreed well with the conventional cytogenetic results,
but did not give any additional information to the karyotypes. In ALL, CGH
supplemented standard cytogenetic findings in about half of the cases.
Therefore, routine use of the method is recommended for children at the
diagnostic stage of ALL.
In childhood ALL, the most common DNA copy number changes were gains
of whole chromosomes, most frequently affecting chromosomes 21, 18, X, 10,
17, 14, 4, 6 and 8 (14-25%). High-level amplifications were detected only in
two patients (3%). Chromosome 21 was involved in both cases with
amplifications, with minimal common region 21q22-qter. The most common
losses were seen at chromosomal arms 9p (13%) and 12p (11%), with minimal
common regions 9p22-pter and 12p13-pter, respectively.
In order to investigate whether the AML1 gene, located at 21q22, is a target
of the CGH amplifications, fluorescence in situ hybridisation (FISH) with
AML1-specific probe was performed for 112 childhood ALL cases. As a novel
finding in ALL, high-level amplification of AML1 was detected in three (2.7%)
of the patients. These three patients also showed high-level amplification at
21q22 by CGH. In addition, 37 patients (33%) had one or two extra copies of
AML1, apparently reflecting the incidence of the gain of whole chromosome 21.
Translocation t(12;21) resulting in the fusion of the TEL and AML1 genes, is
the most common translocation in childhood ALL. Moreover, the 12p13-pter
region, harbouring TEL (12p13), shows frequent loss by CGH. The nontranslocated TEL allele is known to be often deleted in patients with t(12;21).
Gene-specific FISH was carried out in order to investigate whether the loss at
12p is associated with the TEL-AML1 fusion and the TEL deletion. From the
nine patients with 12p loss, the fusion was detected in eight of the cases and one
allele of TEL was deleted in all cases. In addition, all cases showed favourable
prognostic features and a trend to better overall survival compared to 70
patients with no loss at 12p.
Finally, gene expression profiles of the leukaemic blasts of 17 children with
ALL were studied using cDNA array technology. The analysis of 415 genes
9
related to the function of blood cells or their precursors revealed overexpression compared to mature B-cell reference in several myeloid-specific
genes (S100A12, RNASE2, GCSFR, PRTN3 and CLC). The over-expressed
genes included also AML1, LCP2 and FGF6.
Many of the findings of this thesis, predominantly those in childhood ALL,
are likely to have a role in the development or progression of leukaemia. The
information obtained may further be employed in studies of the pathogenesis
and treatment stratification of childhood ALL.
10
Introduction
Leukaemia is a haematological malignancy, in which malignant blood cells or
their precursors proliferate without control and accumulate in bone marrow and
blood. Leukaemias are divided into acute and chronic types, which are further
divided into lymphoblastic and myeloid types depending on the haematopoietic
lineage of the cells involved. Leukaemia is the most common type of cancer in
children. The great majority of childhood leukaemias are of the acute type,
acute lymphoblastic leukaemia (ALL) comprising 80-85% of childhood cases
and acute myeloid leukaemia (AML) 10-15% (http://www.cancerregistry.fi).
The malignant transformation of a cell occurs as the consequence of changes
in its genetic material. We know today a large number of recurrent cytogenetic
aberrations, mainly translocations, specific to a certain type or even a subtype of
leukaemia. Some of them have been shown to be prognostically significant, and
they are utilized in the design of treatment [for review, see (Ma et al., 1999)].
For example, in childhood ALL, translocation t(12;21) has been associated with
favourable outcome, and the patients with the aberration are usually treated with
less intensive chemotherapy to avoid the side effects of the treatment. On the
other hand, patients with translocation t(9;22) or t(4;11) are considered to have
poor prognosis and need high-dose chemotherapy with stem cell transplantation
to be cured. Despite the extensive knowledge of cytogenetic aberrations, little is
known about the molecular changes that eventually lead to malignant
transformation of the cell and to the development of leukaemia.
In the risk classification of childhood ALL, not only cytogenetic alterations,
but also many other factors are taken into account. These include, for example,
white blood cell count (WBC) at diagnosis, age, response to primary therapy
and the phenotype of the blasts (precursor-B cell / immature B cell / T cell)
(Gustafsson et al., 2000). The groups of patients formed according to the
existing criteria remain, however, quite heterogeneous as regards the outcome
of the patients, leading to excessive treatment of some patients and failure of
treatment in others.
Several new methods to study genetic alterations have been developed in
recent years. One of them is comparative genomic hybridisation (CGH), which
enables genome-wide search for gained and lost chromosomal regions. CGH
has been applied both in research and diagnostics. An even more powerful
research tool is the novel DNA array technology, which allows the study of
expression changes in hundreds to thousands of genes in a single experiment.
The new techniques have already helped and will help us to understand the
molecular events causing leukaemia, and to identify new prognostic markers for
more individualised treatment of the patients.
11
Review of the literature

1. Normal development of blood cells
The development of blood cells is called haematopoiesis. Human
haematopoiesis occurs mainly in the bone marrow, where pluripotent
haematopoietic stem cells diverge both into new self-like cells and into
differentiating stem cells. Through numerous divisions and differentiation, the
differentiating stem cells develop into all types of blood cells (Hoffbrand and
Pettit, 1993; Roitt et al., 1996).
Haematopoiesis can be divided into two main lineages: myeloid and
lymphoid. The myeloid lineage produces monocytes, macrophages, neutrophils,
eosinophils, basophils, red blood cells and platelets. The lymphoid lineage gives
rise to B lymphocytes alias B cells, T lymphocytes alias T cells and natural
killer (NK) cells. (Roitt et al., 1996).
The division, survival, life span, lineage commitment and differentiation of
the haematopoietic cells are controlled by various growth factors as well as the
interaction between the cells and their microenvironment. The effects of these
factors are transmitted through cell surface receptor molecules into the cell
where they cause changes in the function of transcription factors and, thus, in
the activity of genes.
2. Leukaemias
A haematological malignancy arises when something goes wrong in the
regulation of the division or the life span of a blood cell or its precursor. The
cell starts to proliferate uncontrollably and forms a large cell population derived
from a single cell. Haematological malignancies include leukaemias,
lymphomas, multiple myeloma and myelodysplastic syndromes. A typical
feature of leukaemias is that the cells accumulate in the bone marrow and blood.
Leukaemias are divided into acute and chronic types, which are further
classified into lymphoid and myeloid types depending on the cell lineage
represented by the leukaemic clone. In acute leukaemias (acute lymphoblastic
leukaemia and acute myeloid leukaemia) the malignant cells are typically
immature blast cells that are unable to differentiate. In the chronic lymphocytic
leukaemia the malignant cells are morphologically mature and in chronic
myeloid leukaemia, though derived from primitive cells, the differentiation of
leukaemic cells is almost normal in the first stage of the disease.
12
3. Childhood leukaemias
In 1998, 55 new cases of leukaemia in 0- to 19-year-olds were diagnosed in
Finland (http://www.cancerregistry.fi). As mentioned, the most common type of
childhood leukaemia is acute lymphoblastic leukaemia (ALL) (80-85%) and the
second most common acute myeloid leukaemia (AML) (10-15%). Chronic
myeloid leukaemia (CML) is very rare in children. Chronic lymphatic
leukaemia (CLL), the most common type of leukaemia in adults, is not found in
children.
3.1. Childhood acute lymphoblastic leukaemia (ALL)

The division of acute leukaemias to subtypes is based on immunophenotyping,
morphology of the leukaemic cells and cytogenetic aberrations. In
immunophenotyping or surface antigen studies, the cell type of the leukaemic
blasts is identified with monoclonal antibodies. In 80-85% of childhood ALL
cases, the abnormal clone represents an early stage of B-cell lineage. Three
different forms of ALL are derived from B-cell precursors: early precursor-B
cell ALL, common ALL and precursor-B cell ALL (Jaffe et al., 2001).
The earliest cell type gives rise to early precursor-B cell ALL, which comprises
most of infant ALL. In the most common type of childhood ALL, accordingly
tagged common ALL, leukaemic blasts express CD10 antigen (CALLA) on the
surface. In precursor-B cell ALL the blasts express immunoglobulins in the
cytoplasm. In rare cases of childhood ALL, the leukaemic clone represents a
more developed B cell with immunoglobulins on cell surface, and the disease is
known as Burkitt leukaemia. In 15% of the patients, the leukaemic blasts are
derived from the precursors of T-cell lineage, forming a subtype called
precursor-T cell ALL (Jaffe et al., 2001).
Childhood ALL is divided into risk groups according to prognostic factors.
The Nordic classification consists of five risk groups: standard risk,
intermediate risk, high risk, very high risk and infants (Gustafsson et al., 2000).
The risks are classified in relation to the probability of relapse, i.e., the
recurrence of the disease. At present, the risk classification is mostly based on
white blood cell count (WBC), age, immunophenotype, genetic aberrations and
response to treatment. The prognostic impacts of some genetic aberrations are
discussed in Chapter 5.
ALL is treated with chemotherapy and the cases with poor prognosis also
with stem cell transplantation. The form and intensity of the treatment are
determined based on the risk group. Patients with good or standard risk may be
given less intensive conventional chemotherapy in order to minimise the side
effects of the treatment, whereas patients with high risk may receive intensive
treatment including stem cell transplantation. Therefore the differences in the
overall outcomes between the different risk groups have reduced in recent years.
The first aim of the treatment is to reach remission, a condition in which the
clinical symptoms have disappeared and no leukaemic cells can be detected by
conventional methods. Short-term side effects of the treatment are increased
13
predisposition to infections, increased danger of haemorrhage, and nausea.

Long-term side effects include disorders in growth and development, and in
boys, in fertility. Secondary cancers caused by the therapy also appear. The
treatment of childhood ALL takes 2-2.5 years. The treatment results have
significantly improved during the past two decades, and at present up to 80% of
the childhood patients recover (Jaffe et al., 2001). The rate is much higher than
in adults with ALL, of whom only 30-40% are cured [reviewed in (Pui and
Evans, 1998)].
3.2. Childhood acute myeloid leukaemia (AML)

The classification of AML consists of four major categories: (1) AML with
recurrent genetic abnormalities, (2) AML with multilineage dysplasia, (3)
therapy-related AML and (4) AML not otherwise categorized (Jaffe et al.,
2001). The most common genetic abnormalities of AML, included in category
1, are t(8;21), inv(16) or t(16;16), t(15;17) and 11q23 abnormalities. Category 4
encompasses cases that do not fulfil the criteria in any of the previous groups.
The subclassification in this group is primarily based on morphologic and
cytochemical features of the leukaemic blasts and the degree of their
maturation.
AML is often preceded by myelodysplastic syndrome. Myelodysplastic
syndromes are a group of blood diseases with sliding boundary to AML, and in
many cases they progress to AML. The progression is caused by clonal
evolution, the appearance of new genetic alterations in the abnormal cell.
In AML the prognosis is worse than in ALL - only about 40% of children
with AML can be cured [(Pui, 1995) and references therein]. Genetic
aberrations are the most important factor determining the prognosis and the
choice of treatment. Chemotherapy is the cornerstone of treatment also in AML.
Conventional chemotherapy is more intensive than in ALL but the total
duration of the treatment is shorter, lasting usually from seven to ten months.
For patients with an HLA-identical sibling available as donor, stem cell
transplantation is usually recommended in the first remission.
4. Aetiology of childhood leukaemias

4.1. Prenatal origin
The first or initiating event in childhood leukaemia often seems to be a
translocation, which occurs already during foetal development (for information
about translocations, see Chapter 5.1). This is suggested by findings from pairs
of identical twins with leukaemia who share the same non-inherited gene fusion
with identical breakpoints (infants with MLL fusions or older children with
TEL-AML1 fusion; about TEL-AML1, see Chapter 5.1.1) (Ford et al., 1993;
14
Wiemels et al., 1999b). In addition, the same fusion gene that is seen in the
patients leukaemic cells is often present in blood already at birth when the
child is still disease-free. These studies suggest leukaemia to be foetal in origin
in all cases of infant leukaemia (with fusions of MLL), in most cases of the
common form of childhood ALL (with TEL-AML fusion), and in about half of
the cases of childhood AML (with AML1-ETO fusion) (Gale et al., 1997;
Wiemels et al., 1999a; Wiemels et al., 2002).
In the case of infant leukaemia, the concordance of both of identical twins
having leukaemia seems to be almost 100%. However, if one of the identical
twins has leukaemia at 2-6 years of age, the risk of the other to get it is only
about 5%. This suggests that the development of leukaemia requires at least one
additional genetic event after birth besides the initial translocation (two hit
model). The TEL-AML1 fusion is the most common gene fusion of childhood
ALL (see Chapter 5.1.1). Screening of newborn has shown the frequency of the
TEL-AML1 fusion to be as high as about 1%. The rate constitutes 100 times the
risk of TEL-AML1-positive leukaemia, which suggests the second hit or the
appearance of the additional genetic aberration to be a rare event [reviewed in
(Greaves, 2002)].
4.2. Inherited predisposition and environmental

factors
The genetic aberrations leading to leukaemia are not likely to be caused by a
single factor but rather by an interaction of exposure to some factor/s with
inherited genetic predisposition (Greaves, 2002). Although the cause in most
cases of childhood leukaemia is not known, certain factors have been suggested
to contribute to susceptibility.
Hereditary factors are likely to have a role in the predisposition to childhood
leukaemia. The family history of cancer may be a risk factor for childhood
ALL, which is supported by a recent study showing association of childhood
ALL both with family history of haematological neoplasm and of solid tumour
(Perrillat et al., 2001). The association was particularly clear when restricted to
family history of AML. In addition, the inherited variation of some specific
genes has been shown to influence the susceptibility of childhood leukaemia.
The risk of infant leukaemias with the MLL (mixed lineage leukaemia) gene
rearrangement has been associated with the polymorphism of NQO1
(NAD(P)H:quinone oxidoreductase), an enzyme that detoxifies quinones
(Wiemels et al., 1999c), and with the polymorphism of MTHFR
(methylenetetrahydrofolate reductase), an important enzyme in folate
metabolism (Wiemels et al., 2001). According to preliminary evidence, HLA
class II alleles, important in immunity, contribute to predisposition to childhood
ALL in boys [(Greaves, 2002) and a reference therein].
Up to 5% of acute leukaemia cases are associated with inherited,
predisposing genetic syndromes [for review, see (Mizutani, 1998)]. Many of
these disorders, like the Li Fraumeni syndrome, Bloom syndrome and ataxia
telangiectasia, are associated with abnormalities in DNA repair or tumour
15
suppressor mechanisms. The incidence of leukaemia is also significantly higher

in individuals with the Down syndrome with an extra chromosome 21. In
addition, some genetic bone marrow failure syndromes, such as Fanconi
anaemia, entail an increased risk to leukaemia. Another example is familial
platelet disorder caused by haploinsufficiency of the AML1 gene (see Chapter
5.1.1), which predisposes to AML (Song et al., 1999).
Viruses have been shown to cause leukaemia in several different animals. In
humans, HTLV-1 virus (human T-cell lymphotropic virus 1) infections have
been connected with the development of adult T-cell ALL [reviewed in
(Greaves, 1997)]. Considerable, although indirect, evidence exists that many
childhood leukaemias, especially those in the childhood peak of common ALL,
are caused by a rare, abnormal response to a common infection. This kind of
response may happen as a consequence of either population mixing or
delayed mixing with infectious carriers [(Greaves, 1997) and references
therein].
Ionising radiation has been found to predispose to acute leukaemia. This is
proved by the high incidence of leukaemia in Japanese survivors from the
explosion area of nuclear bombs and secondary leukaemias in the individuals
treated by radiotherapy [(Greaves, 1997) and references therein]. A transient
increase in the incidence of infant acute leukaemia was suggested in northern
Greece in association with radioactive fallout from Chernobyl (Petridou et al.,
1996). In addition, X-ray examinations of pregnant women may be associated
with increased risk of subsequent childhood ALL [for review, see (Doll and
Wakeford, 1997)].
Certain cytostatic compounds, like alkylating agents and inhibitors of DNA
topoisomerase II enzyme, increase the risk of leukaemia both in children and
adults (Rubin et al., 1991; Felix et al., 1995). These treatment-related diseases
occur mostly as AML, in some cases also as ALL [reviewed in (Greaves,
1997)]. Alkylating agents damage DNA causing point mutations, chromosome
breakages, translocations and loss of chromosomal material, especially losses of
chromosomes 5 and 7 or their q-arms in AML [reviewed in (Pedersen-Bjergaard
and Rowley, 1994)]. The topoisomerase II inhibitors lead to DNA breaks,
deletions and translocations, the most common of which are translocations of
the 11q23 region creating the MLL (mixed lineage leukaemia) gene fusions
[reviewed in (Rowley, 1998)].
According to preliminary studies, topoisomerase II inhibitors in mothers
nourishment or medicine during the pregnancy may cause leukaemia in infants
(Ross, 1998). Infant leukaemia has also been linked to maternal alcohol intake,
marijuana smoking, prior foetal loss, and parental exposure to pesticides and
carcinogens [(Greaves, 1997) and references therein]. Finally, high weight at
birth has also been associated with increased risk of childhood acute leukaemia
(Yeazel et al., 1997).
16
5. Genetic aberrations in childhood acute

leukaemias
Present diagnostic methods reveal genetic aberrations in about 90% of ALL and
AML patients. In most cases an aberration, which has been found at the time of
diagnosis, is tightly connected with a specific leukaemia type and often also
with some of its immunological or morphological subtypes (Heim and
Mitelman, 1995). Especially in ALL, the distribution of the abnormalities is
clearly different in children and adults. In addition, the distributions in infants
and older children differ remarkably from each other (Figure 1) [reviewed in
(Ma et al., 1999)]. The differences in frequencies offer a partial explanation to
the different outcomes in different age groups.
Infant
Childhood
Random
25 %
Random
30 %
Hyperdiploidy
>50
25 %
MLL rearrangements
75%
Miscellaneous
10 %
MLL
rearrangements
6%
E2A-PBX1
5%
BCR-ABL
4%
Adult
MLL
rearrangements
7%
TEL-AML1
20 %
Miscellaneous
17 %
Random
40 %
BCR-ABL
25 %
TEL-AML1
2%
Hyperdiploidy>50
6%
E2A-PBX1
3%
Figure 1. Prevalence of genetic changes in ALL with respect to different age

groups (Ma et al., 1999).
17
5.1. Translocations
The most common genetic alterations in leukaemias are translocations. These
translocations create a rearrangement of genes, which leads a so-called protooncogene to transform into an oncogene. The oncogene causes leukaemia either
by stimulating cell division or by inhibiting the programmed cell death called
apoptosis. Most of the proto-oncogenes involved in leukaemia encode
transcription factors, many of which have revealed to be important regulators of
the proliferation, differentiation and survival of blood cell precursors [reviewed
in (Look, 1997; Biondi and Masera, 1998)]. The most frequent translocations of
childhood ALL and AML are presented in Table 1.
A translocation can activate a proto-oncogene by two different mechanisms.
A more frequent event is a merger of two genes to form a fusion gene that
produces abnormal chimaeric protein inducing leukaemia. As an example,
translocation t(1;19) in ALL creates the fusion of E2A (immunoglobulin
enhancer binding factors E12/E47) and PBX1 (pre-B-cell leukaemia
transcription factor 1) genes. In the E2A-PBX1 fusion protein transactivating
domains of E2A are joined to the DNA-binding domain of PBX1, which alters
the transcriptional properties of the PBX1 transcription factor (Kamps et al.,
1990; Nourse et al., 1990; Van Dijk et al., 1993; LeBrun and Cleary, 1994; Lu
et al., 1994). Another example is t(12;21), the most common translocation in
ALL, which is discussed in more detail in Chapter 5.1.1.
Another mechanism by which a translocation causes leukaemia is transfer of
a normally quiet transcription factor gene to the neighbourhood of active
promoter or enhancer elements, which accelerate the function of the gene. For
example, in translocations t(8;14), t(2;8) and t(8;22) in Burkitt leukaemia, the
gene encoding the MYC transcription factor is exposed to the enhancer
elements of an immunoglobulin gene. These enhancer elements cause overexpression of the MYC gene, which is important in the regulation of cell
division and cell death [reviewed in (Knudson, 2000)]. Immunoglobulin and Tcell receptor genes are normally rearranged during B-cell and T-cell
development to generate the enormous variety of immunoglobulins and
receptors necessary for immunity. This explains the high frequency of these
genes in the translocations of lymphoid malignancies.
Besides transcription factors, also tyrosine kinases can be activated in the
translocations of leukaemias. Tyrosine kinases are growth factor receptors or
transmitters of signals inside the cell. For example, translocation t(9;22) that
produces the well-known Philadelphia-chromosome, joins sequences of the
BCR gene encoding a phosphoprotein with sequences of the ABL1 gene (v-abl
Abelson murine leukaemia viral oncogene homolog 1) encoding a tyrosine
kinase (Clark et al., 1987; Fainstein et al., 1987; Hermans et al., 1987). The
BCR-ABL1 protein produced by the fusion gene has higher tyrosine kinase
activity than normal ABL1 protein, and drives cell proliferation independently
of growth factors required normally (Lugo et al., 1990). Translocation t(9;22) is
found in 4% of childhood ALL, 25% of adult ALL and 95% of CML [reviewed
in (Friedmann and Weinstein, 2000)]. In ALL, the breakpoint of the BCR gene
18
Table 1. The most frequent translocations in childhood ALL and AML (modified
from Ma et al., 1999).
ALL
AML
Abnormality
Activated gene Mechanism of

activation
Type of
protein
Frequency Cell type /

(%)
Phenotype
t(12;21)(p13;q22)
TEL-AML1
Gene fusion
TF
25
Precursor B
t(9;22)(q34;q11)
BCR-ABL1
Gene fusion
TK
Precursor B
t(1;19)(q23;p13)
E2A-PBX1
Gene fusion
TF
Precursor B
t(17;19)(q22;p13)
E2A-HLF
Gene fusion
TF
<1
Precursor B
t(4;11)(q21;q23)
MLL-MLLT2
Gene fusion
TF
Precursor B
t(11;19)(q23;p13.3)
MLL-MLLT1
Gene fusion
TF
<1
Precursor B
t(8;14)(q24;q32)
MYC
Relocation to IgH
TF
2-5
Immature B
t(8;22)(q24;q11)
MYC
Relocation to IgL
TF
<1
Immature B
t(2;8)(p12;q24)
MYC
Relocation to IgK
TF
<1
Immature B
t(1;14)(p32;q11)
TAL1
Relocation to TCR
TF
<1
t(1;7)(p32;q34)
TAL1
Relocation to TCR
TF
<1
t(1;7)(p34;q34)
LCK
Relocation to TCR
TK
<1
t(7;9)(q34;q32)
TAL2
Relocation to TCR
TF
<1
t(7;9)(q34;q34)
NOTCH1
Relocation to TCR
TF
<1
t(8;14)(q24;q11)
MYC
Relocation to TCR/
TF
<1
t(11;14)(p15;q11)
LMO1
Relocation to TCR
TF
<1
t(11;14)(p13;q11)
LMO2
Relocation to TCR
TF
<1
t(7;10)(q34;q24)
HOX11
Relocation to TCR
TF
<1
t(7;11)(q34;p13)
LMO2
Relocation to TCR
TF
<1
t(7;19)(q34;p13)
LYL1
Relocation to TCR
TF
<1
t(10;14)(q24;q11)
HOX11
Relocation to TCR
TF
<1
t(8;21)(q22;q22)
AML1-ETO
Gene fusion
TF
12
AML-M2
inv(16)(p13;q22)
CBFB-MYH11
Gene fusion
TF
12
AML-M4Eo
t(3;21)(q26;q22)
AML1-EV11
Gene fusion
TF
1-2
AML
t(3;5)(q25;q35)
NPM-MLF1
Gene fusion
AML
t(6;9)(p23;q34)
DEK-CAN
Gene fusion
TF
AML-M2Baso
t(9;11)(p21;q23)
MLL-MLLT3
Gene fusion
TF
AML-M5
t(8;16)(p11;q13)
MOZ-CBP
Gene fusion
TF
<1
AML-M4/5
Ig, immunoglobulin gene locus; TCR, T cell receptor gene locus; TF, transcription factor; TK, tyrosine kinase
19
differs from that in CML (Clark et al., 1987; Fainstein et al., 1987; Hermans et
al., 1987).
In addition to transcription factors and tyrosine kinases, other possible
oncogenic proteins are, for example, growth factors and anti-apoptotic BCL2family proteins. They, however, seem to have a minor role in acute leukaemias.
Many cytogenetic aberrations are prognostically significant and they are
important factors when planning the treatment of a patient [for review, see (Ma
et al., 1999; Friedmann and Weinstein, 2000; Pui, 2000)]. In childhood ALL,
translocation t(9;22) is associated with poor prognosis (Uckun et al., 1998a;
Forestier et al., 2000). Also t(4;11) and other 11q23 translocations are adverse
risk factors, and the unfavourable outcome seen in infant leukaemias is mostly
attributable to high frequency of these translocations (Chen et al., 1993;
Heerema et al., 1994; Pui et al., 1994; Rubnitz et al., 1994; Forestier et al.,
2000). Translocation t(1;19) has lost part of its prognostic significance along
with intensification of the treatment (Raimondi et al., 1990), but it is still
regarded as a marker of poor prognosis in some treatment protocols (Pui et al.,
2000). The patients with unbalanced der(19)t(1;19) seem to have better outcome
than the patients with balanced t(1;19) (Forestier et al., 2000), and according to
one report only the balanced form indicates inferior prognosis (Uckun et al.,
1998b). Translocation t(12;21) in ALL is generally regarded as a favourable
prognostic sign (Liang et al., 1996; McLean et al., 1996; Borkhardt et al., 1997;
Rubnitz et al., 1997b; Rubnitz et al., 1997c; Loh et al., 1998; Maloney et al.,
1999a; Rubnitz et al., 1999a). Translocations t(8;14), t(2;8) and t(8;22) are
connected to Burkitt leukaemia, in which the prognosis is poor (Jaffe et al.,
2001). The three most common aberrations of AML, t(8;21), inversion(16) and
t(15;17) are all signs of low risk (Grimwade et al., 1998).
Translocations are also utilised in the search for minimal residual disease by
fluorescence in situ hybridisation (FISH) and polymerase chain reaction (PCR)
(see Chapters 6.2 and 6.3) (Campana and Pui, 1995; El-Rifai et al., 1997c).
Minimal residual disease means the existence of a number of malignant cells
while the patient is in remission according to conventional criteria. In many
ALL and AML cases, residual disease is known to presage relapse, both during
treatment and after it. Although further studies are needed to understand the
significance of residual disease and the methods used in its monitoring are still
being developed, it is believed to become one of the most important elements in
the design of individual treatment in acute leukaemias.
Finally, the understanding of the molecular mechanism of the translocation
may in some cases lead into development of a specific form of therapy. This is
shown by the rearrangement of the retinoic acid receptor alpha gene (RAR) in
translocation t(15;17) typical for acute promyelocytic leukaemia (APL), and the
successful treatment of APL with all-trans retinoic acid [(Melnick and Licht,
1999) and references therein]. Furthermore, the treatment of the t(9;22)-positive
patients with a specific inhibitor of the BCR-ABL1 tyrosine kinase has yielded
promising results in CML and adult ALL (Druker et al., 2001a,b). The aim in
the future is to clarify the complicated network effects of the proteins
20
transformed by translocations, which would further provide basis for the

discovery of new effective drugs.
5.1.1. Translocation t(12;21)(p13;q22) and the TEL and AML1
genes
The t(12;21)(p13;q22), the most common translocation of childhood ALL, is
present in about 25% of the patients (Golub et al., 1995; Romana et al., 1995a;
Romana et al., 1995b; Shurtleff et al., 1995; Liang et al., 1996; McLean et al.,
1996; Borkhardt et al., 1997). In adults, it is seen only in 2-3% of the cases
(Aguiar et al., 1996; Kwong and Wong, 1997). Translocation t(12;21) occurs
only in the B-precursor type of ALL (Romana et al., 1995b; Shurtleff et al.,
1995; Liang et al., 1996; McLean et al., 1996; Borkhardt et al., 1997; Raimondi
et al., 1997; Rubnitz et al., 1997a). The translocation fuses the TEL gene
located in 12p13 and the AML1 gene located in 21q22 (Golub et al., 1995;
Romana et al., 1995a). For review about the TEL-AML1 fusion, see (Friedman,
1999; Rubnitz et al., 1999b).
TEL (translocation-ets-leukaemia; ETV6) encodes a sequence-specific
transcriptional repressor, which belongs to ETS family of transcription factors
(Golub et al., 1994; Fears et al., 1997; Fenrick et al., 1999; Lopez et al., 1999).
The TEL protein contains an aminoterminal helix-loop-helix (HLH) domain
and a carboxyterminal DNA-binding domain (Jousset et al., 1997; Poirel et al.,
1997). The HLH domain is responsible for forming protein homodimers with
itself as well as heterodimers with FLI1 (friend leukaemia virus integration 1),
another ETS factor (McLean et al., 1996; Jousset et al., 1997; Kwiatkowski et
al., 1998). Studies of chimaeric mice with TEL-deficient cells have shown that
TEL is critical for the normal transition of haematopoiesis from foetal liver to
bone marrow (Wang et al., 1998). TEL is also involved in several other
leukaemic translocations that generate fusion proteins [for review, see (Rubnitz
et al., 1999b)]. The gene was first identified in t(5;12), present in CML, fusing
TEL with the PDGFRB gene (platelet derived growth factor receptor, beta)
(Golub et al., 1994). In the fusion protein the HLH domain of TEL joins to the
PDGFRB transmembrane and tyrosine kinase domains resulting in constitutive
activation of the PDGFRB kinase (Golub et al., 1994; Jousset et al., 1997).
The AML1 gene (acute myeloid leukaemia-1; RUNX1) encodes a variety of
transcripts giving rise to different proteins, which function as transcription
factors [(Miyoshi et al., 1995; Levanon et al., 2001), for review about AML1,
see (Lo Coco et al., 1997; Speck et al., 1999; Lutterbach and Hiebert, 2000;
Downing, 2001)]. The major transcriptionally active protein isoform, AML1B,
consists of an aminoterminal DNA-binding domain, also called the Runt
domain, and a carboxyterminal transactivation domain (Meyers et al., 1995).
Besides DNA binding, the Runt domain is responsible for heterodimerization of
the protein with CBFB (core binding factor, beta subunit), which enhances the
DNA binding and transactivation of AML1 (Meyers et al., 1993; Ogawa et al.,
1993; Wang et al., 1993). AML1 regulates transcription in a sequence-specific
manner, acting mostly as a transcriptional activator but in some cases also as a
21
repressor (Meyers et al., 1993; Lutterbach and Hiebert, 2000 and references
therein). It is known to activate a number of genes important in haematopoietic
cell development, function and differentiation [(Lutterbach and Hiebert, 2000)
and references therein]. Some of the lineage-restricted genes encode interleukin3, granulocyte-macrophage colony-stimulating factor, receptor for CSF1
(colony stimulating factor 1), myeloperoxidase and subunits of the T-cell and
B-cell antigen receptors. However, AML1 is apparently not capable of
activating transcription alone, but it has been suggested to function as an
organising factor that recruits lineage-specific elements to form transcriptional
activation complexes that stimulate lineage-restricted transcription.
AML1 is one of the most frequently rearranged genes in leukaemias. The
translocations involving AML1 create gene fusions retaining the region
encoding its DNA-binding domain [reviewed in (Speck et al., 1999)]. In
addition to t(12;21), AML1 participates in t(8;21), the most common
translocation of AML, which fuses AML1 to the ETO gene (eight-twenty-one)
(Miyoshi et al., 1991). The function of AML1 is indirectly disrupted by
inversion inv(16), another common rearrangement in AML. The inversion leads
into the fusion of CBFB, the gene encoding the cofactor of AML1, with MYH11
(myosin, heavy polypeptide 11, smooth muscle) (Liu et al., 1993).
In the TEL-AML1 fusion protein formed by t(12;21), the HLH domain of
TEL joins to the full-length AML1 protein (Golub et al., 1995; Romana et al.,
1995a). The transcription of the fusion gene is regulated by the promoter of
TEL. The TEL-AML1 protein is a transcriptional repressor, which dominantly
blocks the AML1-dependent transactivation. Repression by TEL-AML1 is an
active process, in which the HLH domain of the fusion protein associates with
co-repressors and a histone deacetylase protein (Hiebert et al., 1996; Fenrick et
al., 1999; Hiebert et al., 2001). The histone deacetylase activity is thought to
mediate the function of co-repressor complexes [(Lutterbach and Hiebert, 2000)
and references therein]. The fusion of the HLH domain of TEL to AML1 thus
transforms AML1 from a regulated to an unregulated transcriptional repressor
(Hiebert et al., 2001).
The TEL-AML protein is able to form heterodimers with the normal TEL
through the HLH domains, which makes it possible that the fusion also alters
the normal function of TEL (McLean et al., 1996). Interestingly, the nontranslocated allele of TEL is deleted in about 75% of the patients with t(12;21)
(Cav et al., 1997). The deletion seems to be a secondary event in the TELAML1-positive ALL (Raynaud et al., 1996; Romana et al., 1996; Cav et al.,
1997). One hypothesis for the role of this deletion is that the normal TEL
interferes with the function of TEL-AML1 by heterodimerization (McLean et
al., 1996). Another possibility is that the loss of TEL itself contributes to the
development or progression of leukaemia (Friedman, 1999).
22
5.2. Numerical chromosome aberrations

In addition to translocations, altered chromosome numbers are frequently found
in leukaemias. In AML, losses or gains of single chromosomes (monosomies
and trisomies) are common, whereas more radical changes are frequent in ALL
(Heim and Mitelman, 1995). Especially hyperdiploidy (chromosome number
>46) is common in childhood ALL. The number of chromosomes varies
between 47 and 50 in about 15% and is more than 50 in about 27% of the cases.
Hypodiploidy (chromosome number <46) is found in about 7% of the patients
[reviewed in (Ma et al., 1999)].
The numerical chromosome changes are frequent secondary aberrations,
emerged during the progression of leukaemia. Therefore, the change in the
chromosome number is often thought to represent a secondary aberration, even
when it is the only detectable alteration. Were the changes of chromosome
number primary or secondary, their significance for the development or
progression of leukaemia is supported by the non-randomness of the
chromosomes involved. In AML, for example, the monosomy of chromosome 7
and trisomies of chromosomes 8 and 21 are frequent, and the gains of
chromosomes 4, 6, 10, 21 and X appear often in hyperdiploid ALL (Heim and
Mitelman, 1995).
In ALL, hyperdiploidy in excess of 50 chromosomes indicates favourable
prognosis, whereas hypodiploidy is a sign of poor prognosis (Chessels et al.,
1997; Forestier et al., 2000). Hyperdiploidy is also associated with low WBC
[(Pui and Evans, 1998) and references therein]. The better outcome of patients
with hyperdiploidy can result from higher accumulation of active metabolised
form of methotrexate, which is one of the most widely used drugs in childhood
ALL (Whitehead et al., 1990; Synold et al., 1994).
5.3. Gene amplifications

In addition to chromosomal translocations, an amplification can active a protooncogene to produce an excess number of proteins encoded by the gene. Gene
amplification is a frequent event in some solid tumours and lymphomas (Monni
et al., 1997; Schwab, 1999 and references therein).
In conventional cytogenetics (see Chapter 6.1) amplified chromosomal
regions can be seen as intrachromosomal homogenously staining regions (HSR)
or small extrachromosomal double minute chromosomes. There is only one
report of double minute chromosomes in childhood ALL (Murray et al., 1998).
In adult AML, double minute chromosomes are found in about 1% of the cases
(Tanaka et al., 1993), usually involving the amplification of the MYC or the
MLL gene (Alitalo et al., 1985; Asker et al., 1988; Tanaka et al., 1993; Park et
al., 2000; Streubel et al., 2000; Andersen et al., 2001).
23
5.4. Tumour suppressor genes and allelic losses

Another event that may initiate leukaemia is inactivation of a tumour suppressor
gene. Tumour suppressor genes are essential for normal cell development and
they prevent carcinogenesis. Usually the prerequisite of malignant
transformation is inactivation of both alleles. Non-functioning first allele may
be inherited. The activity may be neutralised by deletion, by hypermethylation
of the promoter region or by point mutation. Very few tumour suppressor genes
have been reported in acute leukaemias.
Screening for chromosomal regions with loss of heterozygosity (LOH) is one
way to track novel tumour suppressor genes. In childhood ALL, the regions that
most frequently show LOH are the short arms of chromosomes 9 and 12 (in
about 30-40% and 25-30% of the patients, respectively) (Takeuchi et al., 1995;
Baccichet et al., 1997; Chambon-Pautas et al., 1998).
The short arm of chromosome 9 contains two adjacently located and highly
homologous tumour suppressor genes, CDKN2A (p16) and CDKN2B (p15)
(9p21). These genes encode cyclin-dependent kinase inhibitors, proteins that
negatively regulate cell cycle progression. CDKN2A and CDKN2B are
frequently inactivated in many tumour types. Both of the genes are deleted,
often concordantly, in almost 40% of childhood ALL [reviewed in (Drexler,
1998)]. Furthermore, CDKN2B is inactivated by hypermethylation in about
40% of childhood ALL and 50% of childhood AML, and CDKN2A in 40% of
childhood AML [(Drexler, 1998) and references therein; (Guo et al., 2000)].
According to two studies, deletions of CDKN2A and CDKN2B, and
hypermethylation of CDKN2B are frequently acquired during the time between
diagnosis and relapse in childhood ALL, which suggests that the inactivation of
these genes may be associated with the progression of the disease (Maloney et
al., 1999b; Carter et al., 2001). The prognostic value of CDKN2A and CDKN2B
inactivation is controversial [(Graf Einsiedel et al., 2001; Drexler, 1998) and
references therein]. Recently, CDKN2A protein expression was reported to be
an independent prognostic factor in childhood ALL (Dalle et al., 2002).
However, as pointed by the authors, the CDKN2A expression can be altered not
only by gene deletion and methylation, but also by other, unknown mechanisms.
The region with frequent deletions in the short arm of chromosome 12
(12p12-p13) contains the TEL gene, the counterpart of translocation t(12;21),
and the CDKN1B gene. As mentioned previously, the non-translocated allele of
TEL is deleted in most of the patients with t(12;21) (Raynaud et al., 1996; Cav
et al., 1997). However, homozygous deletions of TEL or mutations in the other
TEL allele in the presence of hemizygous deletion are very rare, which suggests
that it is not a classical tumour suppressor gene (Sato et al., 1995; Stegmaier et
al., 1996). Hemizygous deletion of CDKN1B (cyclin-dependent kinase inhibitor
1B; KIP1; p27) (12p12) has also been found to be a common feature in ALL by
LOH and FISH studies (Pietenpol et al., 1995; Takeuchi et al., 1995; Komuro et
al., 1999). The expression of CDKN1B is frequently reduced in several tumour
types, and its under-expression in most of them is associated with poor
prognosis. In contrast to most tumour suppressor genes, CDKN1B is
24
haploinsufficient for tumour suppression, which means that the absolute level of
CDKN1B expression is critical for the tumour development [reviewed in
(Philipp-Staheli et al., 2001)]. Deletion of only one allele of CDKN1B may thus
be sufficient to cause a malignant effect.
6. Genetic methods used in the diagnosis and

follow-up of leukaemia
6.1. Conventional cytogenetics
Conventional cytogenetics has been used in the diagnosis of leukaemia since
1970s. It is based on the recognition of metaphasic chromosomes according to
their specific banding patterns. The most widely used cytogenetic staining is
Giemsa- or G-banding. Standard cytogenetics is still an important routine
method at the diagnostic stage of leukaemia, giving an overview of genetic
alterations in malignant cells. The method has, however, several weaknesses.
The lack of metaphases may be a problem because the leukaemic cells,
especially the blasts of childhood ALL, often fail to proliferate in the cell
culture. It is also possible that the normal cells from the patient sample divide
as well or even more actively than the malignant cells, which may lead into the
analysis of irrelevant cells. Other drawbacks of cytogenetics are that the quality
is often insufficient for detailed analysis or that the aberration is too complex to
be interpreted. Furthermore, many important chromosomal alterations are
missed. For example the translocation t(12;21) of ALL cannot be detected by
the method because the aberration does not change the banding pattern or the
size of the chromosomes involved. Finally, conventional cytogenetics is
laborious and time-consuming, and only about twenty cells can be analysed per
patient. This is also the reason why the method cannot be used for the detection
of minimal residual disease in patient follow-up. In recent years, several other
techniques have been developed to compensate for the shortcomings of the
banding technique.
6.2. Molecular genetic methods

A specific gene fusion caused by a translocation can be detected by Southern
blotting and polymerase chain reaction (PCR). In Southern blotting, DNA is cut
by sequence-specific restriction enzymes and the resulting fragments are
separated in gel electrophoresis. The size of the fragment recognized by a genespecific probe is different in the fusion gene as compared to the fragment in the
normal gene. The weaknesses of the method are that it is slow and that it
requires large amount of cells.
In most translocations the variance in cleavage sites is so great that PCR
cannot be performed on DNA. RNA is therefore the usual source of PCR to
25
detect gene arrangements. First RNA has to be transformed into more stable
complementary DNA (cDNA) by reverse-transcriptase (RT) enzyme and,
accordingly, this type of PCR is called RT-PCR. Two specific primers, one for
each side of the fusion point are needed for the reaction. No PCR product is
detected in samples without a gene fusion, whereas cDNA is amplified
exponentially in samples that contain the fusion.
Southern blotting and RT-PCR are used as a support for diagnostics based on
the result of G-banding analysis. Because specific probes or primers are
required, the methods can be routinely used only for the most well-defined
leukaemia-specific translocations, such as t(12;21), t(9;22), t(1;19) and t(4;11)
in ALL.
RT-PCR is also widely used to monitor minimal residual disease. PCR is
highly sensitive: the method reveals one leukaemic cell among 10 000-100 000
normal cells. Except for leukaemia-specific gene fusions, the normal
rearrangements of immunoglobulin and T-cell receptor genes can be utilised in
the disease follow-up, when the PCR targets can be identified in 95% of the
patients (Campana and Pui, 1995; van Dongen et al., 1998). The rearrangement
is unique in each cell and it can thus be used to find a monoclonal cell
population. When PCR is performed on DNA, several primer pairs are needed
for the detection of the immunoglobulin or T-cell receptor gene rearrangement.
The detection of the rearrangement from RNA requires that specific primers are
designed for each patient. The task is laborious and interpretation of the results
is often difficult.
In conventional PCR, contamination can be a recurrent problem. In addition,
the method is not quantitative, which makes it deficient especially in residual
disease monitoring. These drawbacks are overcome in quantitative real-time
PCR, performed in a closed system, in which a fluorescent signal is generated
and detected continuously during the amplification. The extent of fluorescence
is directly proportional to the extent of PCR product. Real-time PCR has
already been reported to be applicable to the quantification of residual disease
in childhood ALL (Donovan et al., 2000; Eckert et al., 2000; Drunat et al.,
2001). Primer sets for childhood ALL are now commercially available and
several laboratories are currently using the method in routine clinical studies of
the disease.
6.3. Molecular cytogenetic methods

Molecular cytogenetics refers to the study of DNA or genes at chromosome- or
cell-level. It is based on in situ hybridisation (ISH), in which a DNA probe
binds with a specific region in the genome of the cell. The detection is based
either on a colour produced by enzymatic reaction or on fluorescence, the latter
being more widely used. In fluorescence in situ hybridisation (FISH), the probe
is labelled with fluorochrome and the detection is performed using fluorescence
microscope with specific filters [for detailed information about in situ
hybridisation, see (Andreeff and Pinkel, 1999)].
26
6.3.1. Chromosome painting and multicolour-FISH

In chromosome painting, the whole chromosome or a chromosome arm is
stained. The analysis is made from metaphase cells. The whole chromosomepainting probe is a mixture of fragments from the entire length of a specific
chromosome. Painting probes can be derived from chromosome-specific
libraries, by PCR amplification from chromosome fractions or from
microdissected DNA specific for each chromosome. Chromosome painting is
used to verify and define translocations or numerical aberrations seen in Gbanding. However, the ability of chromosome painting to detect translocations
like t(12;21) that are located near the telomeric regions of the chromosomes is
limited [reviewed in (Kearney, 1999)]. Chromosome painting suits well for
monitoring of minimal residual disease, although the sensitivity of the method
is rather low (1:1000) compared to PCR (1:10 000-100 000) (El-Rifai et al.,
1997c).
In multicolour-FISH alias 24-colour FISH the whole set of chromosomes is
painted in a single hybridisation. Each chromosome is stained with a different
combination of five fluorochromes. Using appropriate filters and software, all
the chromosomes can be visualized in different artificial colours, each
chromosome showing a specific colour. Multicolour-FISH is called multiplex
FISH (M-FISH) (Speicher et al., 1996) or spectral karyotyping (SKY) (Schrck
et al., 1996) depending on the technical details applied to fluorescence
detection. Like the G-banding method, multicolour-FISH gives an overview of
chromosomal changes in the whole of the genome. It is especially useful in
defining complex translocations and so-called marker chromosomes with
unknown origin, and it is used to support standard cytogenetics in diagnostics
[reviewed in (Raap, 1998; Kearney, 1999; Patel et al., 2000)].
6.3.2. Interphase-FISH
Centromere-specific and locus-specific probes provide fluorescent signals that
can be analysed in interphase cells. Centromere-specific probes are targeted for
highly-repetitive DNA present in the centromere of a chromosome. They are
used for the detection of gains and losses of a specific whole chromosome.
Specific gene fusions, gene deletions and gene amplifications can be studied
with locus-specific probes. These probes are produced by cloning them in
different kinds of vectors, such as cosmids, plasmids, phages, yeast artificial
chromosomes (YACs) and bacterial artificial chromosomes (BACs). Locusspecific probes are commercially available for several specific aberrations, e.g.,
the TEL-AML1 fusion of t(12;21). The locus-specific probe is usually a cocktail
of two probes targetted for separate loci and labelled with different
fluorochromes [reviewed in (Werner et al., 1997; Kearney, 1999)]. For
example, in a cocktail that detects the TEL-AML1 fusion, the probe for the TEL
gene is labelled with green fluorochrome and the probe for the AML1 gene with
red fluorochrome. Thus the normal TEL allele produces a green signal and the
normal AML1 allele a red signal. In the TEL-AML1 fusion green and red signals
are fused producing yellow colouring.
27
The study of interphase cells has major advantages over the study of mitotic
cells. As mentioned, especially the leukaemic blasts of ALL proliferate poorly
in cell culture, and mitotic cells may not represent the neoplastic clone
[reviewed in (Kearney, 1999)]. Another advantage of interphase FISH is that it
can be combined with the study of the immunophenotype and morphology of
the cell by immunocytochemical staining [reviewed in (Werner et al., 1997)].
This makes it possible to define the lineage and maturation stage of the cells
with the aberration. Finally, interphase FISH can be used for stored material
including smear preparations, which is beneficial especially in retrospective
studies [reviewed in (Cuneo et al., 1997; Wolfe and Herrington, 1997)].
Interphase-FISH can be used both in diagnostics and follow-up. However,
the method shows a high number of false positive cells, which seriously limits
its sensitivity for monitoring residual disease. For centromeric probes, the falsepositive rate varies from 0.5% (false trisomy) to 2% (false monosomy), and for
locus-specific probes the rate ranges from 2% to 5% [reviewed in (Ma et al.,
1999)]. When searching for fusion genes, the problem can be reduced by using
a series of probes spanning both translocation breakpoints, resulting in two
fusion signals in the presence of the translocation. Another strategy is to use
three or even four different colours. However, the more complex is the colour
scheme, the more difficult is the analysis [reviewed in (Kearney, 1999)].
6.3.3. Comparative genomic hybridisation
Comparative genomic hybridisation (CGH) (Kallioniemi et al., 1992) is a
modification of FISH that allows screening of the whole genome for DNA
sequence gains and losses in a single experiment [for review, see (Forozan et
al., 1997; Raap, 1998; Tachdjian et al., 2000)]. The principle of the method is
illustrated in Figure 2. DNA extracted from a patient sample is cut into small
fragments and labelled with green fluorochrome using nick translation.
Similarly, DNA extracted from normal tissue, usually blood, is cut and labelled,
but red fluorochrome is used for labelling. Equal amounts of differently labelled
patient DNA and normal DNA are mixed and hybridised into normal metaphase
spreads (Figure 2). The patient DNA and normal DNA compete for the same
target sequences, and the hybridisation takes place proportionally to the
amounts of patient DNA and normal DNA present. Consequently, the overrepresented chromosomal regions in the patient are seen as green, and underrepresented as red. For a detailed and reliable analysis, images of the hybridised
metaphases have to be captured by a CCD (charge coupled device) camera
connected to a fluorescence microscope, and the intensities of red and green
fluorescence are calculated using image analysis software. The software
produces profiles of green-to-red fluorescence ratios along the entire length of
each chromosome. The gains and amplifications of patient DNA can be
detected as chromosomal regions with increased ratio, and the losses as regions
with reduced ratio.
28
Green-labelled
patient DNA
Red-labelled
normal DNA
Slide with normal

metaphase spreads
In situ hybridisation
Red
Loss
Green
Gain
Figure 2. The principle of comparative genomic hybridisation (CGH). DNA

extracted from patient sample is labelled with green fluorochrome and DNA
from normal blood with red fluorochrome. Equal amounts of differently
labelled patient and normal DNAs are mixed and hybridised into normal
metaphase chromosomes. Images of the hybridised metaphases are captured
using fluorescence microscope and CCD camera. The profiles of greed-to-red
ratio are calculated along each chromosome using image analysis software.
When the ratio exceeds or falls below certain limits, the region is considered
gained or lost in the patient sample, respectively.
29
CGH is used in clinical diagnostics to supplement conventional cytogenetics. In

addition, the method has many applications in cancer research and it has
provided an enormous amount of data since it was first introduced in 1992
[(Kallioniemi et al., 1992); for review, see (Knuutila et al., 1998, 1999)]. By
revealing chromosomal regions that are recurrently gained or lost, CGH is
valuable for example in pinpointing regions with putative oncogenes or tumour
suppressor genes.
A remarkable advantage of CGH is that no intact cells or metaphases from
the patient are required. Moreover, only a small amount of sample (less than
1 g DNA) is needed. DNA from even a few cells is enough for the method, if
it is first amplified by PCR [reviewed in (Forozan et al., 1997)]. This procedure
is applicable in research, but it is too labour-intensive in routine diagnostics.
Patient DNA can be obtained from fresh, frozen or paraffin-embedded tissue.
The disadvantage of CGH is its inability to reveal balanced translocations
and inversions because they do not cause alterations in DNA copy number.
Another limitation is that an abnormality can be detected only when it is present
in at least 50% of the cells (Kallioniemi et al., 1994). Thus, subclones with
aberrations developed by clonal evolution may be missed by the method.
The reliance on metaphase chromosomes as hybridisation targets reduces the
value of CGH findings due to resolution limitations. The minimum size of gains
and deletions that can be detected is considered to be about 10 Mb
(megabasepairs). For a region with high-level amplification, the sensitivity is
higher: for example, a 5-10 fold amplification of 1 Mb is detectable [reviewed
in (Forozan et al., 1997; Tachdjian et al., 2000)]. It has also been stated that a
deletion of 3 Mb can be easily detected (Kirchhoff et al., 1999).
The resolution of CGH technique can be substantially increased by replacing
the condensed metaphase chromosomes with cloned genomic DNA, arrayed in
small spots on a glass slide. The technique is called matrix-CGH or array-based
CGH (Solinas-Toldo et al., 1997). So far, CGH arrays of specific chromosomal
regions have been used mostly for research purposes, but also a study with a
genome-wide CGH array has been reported (Pollack et al., 1999). Perhaps
focussed disease-specific CGH arrays will be available for clinical diagnostics
in the future.
30
7. Gene expression pro filing by DNA arrays

The sequencing of human genome has identified approximately 19 000 genes
and tens of thousands partial fragments of genes termed expressed sequence
tags (ESTs) (Alizadeh et al., 2001). At present, the number of human genes is
estimated to be about 30 00035 000 (Pennisi, 2000; Claverie, 2001). The
availability of the sequences has made it possible to use novel techniques to
study the function of genes as well as their role in diseases. The most powerful
of these techniques is DNA array technology, first introduced in 1995, which
has made it possible to study the expression of thousands genes in a single
experiment [(Schena et al., 1995; Zhao et al., 1995; Schena et al., 1996), for
review, see (Duggan et al., 1999; Marx, 2000; van Hal et al., 2000; Golub,
2001; Maughan et al., 2001; Rozovskaia et al., 2001; Gershon, 2002;
Ramaswamy and Golub, 2002)]. As vigorous development within the field has
translated into decreasing performance costs, DNA array technology has
become a widely used method to study gene expression in genomic scale. The
method has already provided important insights into human neoplasms by
identifying specific cancer subtypes as well as genes that are important for the
development of various cancers.
7.1. Types of array and principles of the method

A DNA array contains PCR-amplified cDNAs or oligonucleotides spotted
densely and systematically on solid surface, usually filter or glass. Each spot
represents a single gene. The variety of commercially available arrays includes
genes associated with cancer or with functional traits of a specific tissue.
Another alternative is to use a custom-made array with a set of genes
determined by the researcher. Today, also arrays covering genes of nearly the
entire genome are available.
Arrays can be either glass- or filter-based according to surface material.
Moreover, two types of array exist according to the kinds of probe used in the
gene spots. cDNA arrays, alias spotted arrays, are made by mechanical
deposition of solutions containing PCR-amplified cDNA fragments in small
spots on filter or glass. The oligonucleotide arrays are made by deposition or
more often by direct synthesis of oligonucleotides on a glass slide.
In the nylon and plastic filter-based cDNA array method, the cDNA derived
from mRNA is radioactively labelled and the labelled patient and reference
cDNAs are hybridised to separate filters. The principle of the filter-based
system is illustrated in Figure 3.
Filter arrays were appreciated as a relatively economical alternative that can
be assembled easily, often on an already existing basis. However, glass-based
arrays are rapidly replacing filter arrays in gene expression studies. When using
glass-based cDNA arrays, cDNA derived from mRNA is labelled fluorescently.
Fluorochromes of different colours are used for cDNAs from test and reference
samples, usually red for the test cDNA and green for the reference cDNA. The
differently labelled test and reference cDNAs are simultaneously hybridised to
31
the same array slide in a competitive manner. The expression differences

between the test and reference samples are then measured as the ratio of red-togreen fluorescence in each spot [reviewed in (Duggan et al., 1999; Alizadeh et
al., 2001)].
The glass-based cDNA arrays have several advantages over the membranebased arrays. Glass allows higher density of spots, which enables spotting of
more than 50 000 genes on a single microscope slide [reviewed in (Alizadeh et
al., 2001)]. The high-density arrays are called microarrays. Furthermore, the
fluorescence-based procedure is faster, because of the long exposure time
required for detection of radioactivity [reviewed in (Cox, 2001)]. A great
advantage is also the avoidance of problems that arise from handling and
storage of radioactive material. Finally, simultaneous hybridisation of distinctly
labelled test and reference cDNAs intrinsically normalizes for noise and
background in their comparison [reviewed in (Harrington et al., 2000)].
The spots in glass-based arrays can also be oligonucleotides instead of
cDNAs (Fodor et al., 1993; Lipshutz et al., 1999). Oligonucleotides are short
fragments that allow even higher density of spots than cDNAs. Because these
short oligomers frequently cross-hybridise with several genes, the arrays
contain several oligomers for the measurement of each gene [(reviewed in (Wu,
2001)]. The most commonly used oligonucleotide arrays are GeneChips,
manufactured by Affymetrix (Santa Clara, CA, USA). In the Affymetrix
system, the experimental and reference mRNAs are enzymatically amplified,
hybridised to separate arrays, and the detection is based on single colour
fluorescent label. Oligoarrays offer higher specificity than cDNA arrays because
the chances of cross-hybridisation can be minimised [reviewed in (Ramaswamy
and Golub, 2002)]. In addition, smaller amount of total RNA (around 10 g per
sample per hybridisation) is needed than when using glass-based cDNA arrays
(around 50 g) (2 g in filter-based system). Oligonucleotide arrays can also be
used to monitor sequence polymorphisms and splice variants (DeRisi and Iyer,
1999; Lipshutz et al., 1999). A particular advantage of Affymetrix GeneChips is
the ability to recover samples from a chip after hybridisation (Ramaswamy and
Golub, 2002). The disadvantages of oligoarrays compared to glass-based cDNA
arrays are higher price and minor flexibility, and the requirement of a priori
knowledge of the gene sequences.
7.2. Problems concerning patient sample and

reference
The limitation of the glass-based DNA array technique, especially when using
the cDNA system, is the large amount of RNA required. The number of cells in
the bone marrow samples of leukaemia patients, especially children, is limited.
Therefore the amount of RNA obtained from the samples is usually too small for
the glass-based cDNA system. Techniques for amplifying mRNA prior to array
hybridisation are, however, being improved to overcome this problem [reviewed in
(Lockhart and Winzeler, 2000; Hughes and Shoemaker, 2001)]. High levels of
roughly linear amplification have already been achieved (Luo et al., 1999).
32
Patient RNA
Reference RNA
RT-PCR with 33Plabelled nucleotides

33P-labelled
33P-labelled
patient cDNA
reference cDNA
Hybridisation
Exposure
Scanning
Image of reference
hybridisation
Image of patient
hybridisation
Comparison
by software
Intensity differences
between patient and
reference
Figure 3. Principle of gene expression profiling using filter-based cDNA

arrays. mRNA is extracted from the patient sample and the normal reference.
The mRNAs are converted into radioactively labelled cDNA by RT-PCR. The
radioactively labelled patient cDNA and reference cDNA are hybridised to
separate array filters. The hybridised filters are exposured to an imaging plate,
which is scanned using phosphorimager. The intensities of corresponding gene
spots in patient and reference images are compared using software to obtain
the intensity differences between the patient and the reference (reviewed in
Duggan et al., 1999; Cox, 2001). The intensity difference in each gene spot
reflects the difference in the expression of the gene between the patient and the
reference.
33
Most cancer specimens are a mixture of normal and malignant cells, which is a
problem that must be taken into account in gene expression analyses. One
approach to study only a specific cell type of bone marrow is the purification of
cells of specific lineage and differentiation-stage with the help of monoclonal
antibodies (Chen et al., 2001). This is, however, a laborious procedure whereby
a large proportion of cells, and thus RNA, is often lost. Another possibility is
virtual purification that has already been used in solid tumours (Ross et al.,
2000). This can be performed in a computer by clustering and identifying
groups of genes whose expression can be attributed to the normal cells on the
basis of cell-specificity [reviewed in (Alizadeh et al., 2001)].
Another problematic issue in gene expression studies is the choice of correct
reference. When identifying genes with abnormal expression in a specific
cancer type, the ideal reference sample would consist of the same types of
normal cells in the same proportions as in cancer specimen. When using
purified cancer cells, the normal cells of same cell type should be used as
reference. In cancer classification, the same type of reference should be used for
all the samples. The requirements for the reference are not as strict as filtering
of abnormally regulated genes calls for, but an increase in the level of similarity
between the reference and test samples leads to improved accuracy.
7.3. Data analysis

The study of hundreds to thousands of genes in a large series of patients
produces a huge amount of data, which can be analysed only by using
sophisticated software and statistical methods [for detailed information about of
data analysis, see (Hand et al., 2001)]. Before statistical analysis, the data may
need pre-processing. It is often necessary to correct or normalise the differences
in the overall intensities between the arrays. The normalisation can be
performed using housekeeping genes, which are genes that are expressed at the
same level in all the samples studied, including the reference. Another
possibility is to subtract the average of the intensities in one array from each
intensity value, which standardises the average in all arrays to zero.
So-called parametric tests, which usually offer the highest statistical power,
require that data are normally distributed. Generally, gene expression data are
approximated to normal distribution after logarithmic transformation. A
logarithmic transformation is particularly useful when dealing with ratios of
expression between test and reference samples. The problem with ratios is that
over- and under-expression give values of different magnitude. For example, a
two-fold over-expression will have more weight in any comparison than a one
half under-expression. After logarithmic transformation the values will be of
identical magnitude [reviewed in (Dopazo et al., 2001)].
The choice of a correct statistical method is important for the reliability of
the results. Array data analysis is continuously developing. Several different
statistical methods have been used to answer similar kinds of questions.
The search of differentially expressed genes has mainly relied on hypothesis
testing, which employs thresholding rules [reviewed in (Dopazo et al., 2001;
34
Wu, 2001)]. The disadvantage of these methods, however, is loss of

information. Another alternative is to use principal component analysis (PCA),
a technique that reduces the dimensionality of data. The genes are considered to
be points in a multidimensional space with as many dimensions as samples
measured. The aim of PCA is to get a two-dimensional projection of the points
that conserve most of the information of the original data [reviewed in (Dopazo
et al., 2001)]. These points, or scores, of each gene represent its expression
difference between the experimental samples and the reference samples. When
the genes are ranked according to their scores, the most over-expressed genes
appear in one end of the list and the most under-expressed in the other.
In cancer classification and gene-grouping studies, cluster analysis is usually
employed [(reviewed in (Dopazo et al., 2001)]. Two types of cluster analysis
exist: hierarchical and non-hierarchical clustering. Hierarchical clustering
allows the detection of higher order relationships between clusters, whereas
non-hierarchical clustering groups the expression profiles to a predefined
number of clusters. The hierarchical clustering has been preferred in array
studies. The most frequently used method is aggregative hierarchical clustering,
which represents the data in the shape of binary tree, in which the most similar
profiles are clustered in a hierarchy of nested subsets.
7.4. Applications
DNA array technology has several applications in cancer research [for review,
see (Marx, 2000)]. First, it can be applied to searching for genes that are upregulated or down-regulated in a given cancer [for review, see (Wu, 2001)]. In
this way, possible genes and proteins for drug targeting may be identified.
Furthermore, the effect of drugs on gene expression can be studied, which in
turn facilitates finding of markers for drug sensitivity and genes associated with
drug resistance (Zembutsu et al., 2002). The results may eventually lead to
individualised treatment with more effective and less harmful drugs, as well as
to development of new drugs.
In addition to identifying single genes, novel biological pathways can be
found by using DNA arrays. This helps us to understand the complex molecular
processes in a given tissue and the biological roles of the genes with unknown
functions [reviewed in (Lockhart and Winzeler, 2000; Maughan et al., 2001)].
Cancer expression profiling can lead to discovery of novel cancer subtypes
as well as to identification of genes whose differential expression can be used in
classification of cancer or as predictive markers of cancer behaviour [(Golub et
al., 1999): for review, see (Alizadeh et al., 2001)]. The applicability of the DNA
array method to cancer classification was first demonstrated by Golub et al.
(1999) in ALL and AML patients. In the study, gene expression profiling was
first performed on 38 patients (27 ALL, 11 AML) using Affymetrix
oligonucleotide arrays with more than 6800 genes. The authors used an
algorithm to select 50 genes whose expression level differed most between ALL
and AML. The expression patterns of these genes reliably distinguished patients
35
with ALL from those with AML, both in the original group of 38 and also in
another group of 36 patients not previously studied.
Perhaps the most notable example of classification studies is the discovery of
two distinct subtypes of diffuse large B-cell lymphoma (Alizadeh et al., 2000).
When the expression profiles were compared, the subtypes were seen to express
two distinct groups of genes, reflecting different stages of B-cell development.
Moreover, these two groups appeared to have significantly different treatment
responses. The microarray study provided a large set of candidate markers to
distinguish the two subtypes, which will probably help to optimise the treatment
of the patients in the future.
DNA arrays can also be used to study the correlation of gene expression with
known subgroups of cancer. Armstrong et al. used Affymetrix GeneChips with
12 600 genes, and showed 17 patients with MLL rearrangements to have a
characteristic, highly distinct gene expression profile when compared to 20
patients with common childhood ALL or 20 patients with AML (Armstrong et
al., 2002). For example, the homeobox (HOX) genes, important in
haematopoiesis and regulated by MLL, were expressed at higher levels in the
ALL patients with MLL fusions than in the patients with common ALL. Using
cDNA arrays with about 9 700 genes, Rozovskaia et al. also found overexpression of HOXA9 in 14 ALL patients with t(4;11) compared to 10 ALL
patients with similar phenotype but without the translocation (Rozovskaia et al.,
2001). Recently, a comprehensive study was performed by Yeoh et al. on 360
childhood ALL patients using Affymetrix chips with 12 600 genes (Yeoh et al.,
2002). The authors demonstrated distinct expression profiles in prognostically
important ALL subtypes, including T-ALL, t(1;19), t(9;22), t(12;21), MLL
rearrangement and hyperdiploidy >50 chromosomes. In addition, they identified
another ALL subgroup based on its unique expression profile. Furthermore,
they showed expression profiles to differ between the diagnostic samples of
patients who relapse and those who remain in continuous complete remission.
In the future, new molecular prognostic markers provided by arrays should
be useful in diagnosis and in monitoring response to therapy. Less costly and
simpler methods that are already in routine clinical use, such as real-time PCR,
could be applied. Another conceivable possibility is to apply the DNA array
method itself to clinical diagnostics, after commercially available, low-cost,
technically simple arrays and easy-to-use software have become available
(Ramaswamy et al 2002). Furthermore, identification of novel molecular targets
associated with leukemogenesis is likely to lead into development of new
efficient drugs with minimal side effects.
36
Aims of the study

1.
To screen DNA copy number changes in childhood acute leukaemias using

comparative genomic hybridisation (CGH).
2.
To investigate the applicability of the CGH method to the diagnostics of

childhood acute leukaemias.
3.
To expand the characterization of certain genomic regions with recurrent

amplification or frequent loss detected by CGH in childhood acute
lymphoblastic leukaemia (ALL).
4.
To perform expression profiling of genes related to the function of blood

cells or their precursors in childhood ALL using cDNA arrays.
37
Material and methods

1. Material
1.1. Patients and samples
All cytogenetic and molecular genetic analyses were performed from bone
marrow specimens of children with AML (Study I) and with ALL (studies IIV). The number of patients in each study is shown in Table 2. Patients that
were included in two or more studies are shown separately.
Table 2. Number of patients in the studies, the methods used, and the hospitals
where the patients were treated.
Type of
leukemia
Number of
patients
Studies
Methods
Hospital
AML
19
CGH, CC#
HUCH, KUH
ALL
67
II
CGH, CC#
HUCH
99*
III
FISH
HUCH, KUH, OUCH,

TAUH, TUCH
5**
II, III, IV
CGH, CC , FISH
HUCH
III, IV
FISH
KUH
13
cDNA array,
real-time RT-PCR
HUCH
III, V
FISH, cDNA array, HUCH

real-time RT-PCR
*, 87 smear preparations; **, smear preparations; CC (conventional cytogenetics) was

performed according to Knuutila et al. (1981). The karyotype data were retrieved from clinical
records.; HUCH, Helsinki University Central Hospital; KUH, Kuopio University Hospital;
OUCH, Oulu University Central Hospital; TAUH, Tampere University Hospital; TUCH, Turku
University Central Hospital
In Study I, frozen bone marrow specimens from 19 children with AML were
investigated by CGH (Table I, Publication I). All the patients were studied at
the diagnostic stage of the disease. The mean age of the patients was 8.5 years
(range 1.0-15.6 years). In Study II, CGH was performed on frozen samples
from 72 patients with ALL at the time of diagnosis (Table I, Publication II). The
mean age of the patients was 6.5 years (range from two months to 15.7 years).
In Study III, FISH was performed for 112 children with ALL, including three
patients with high-level amplification at chromosome 21 shown by CGH.
Ninety-two of the patients were studied retrospectively from smear preparations
and 20 from cultured cells. All patients were studied at diagnosis except three,
38
who were studied at the time of relapse. The mean age was 6.2 years (range 0.2 14.9 years).
Study IV focussed on nine ALL patients who had shown loss at 12p in CGH
studies (Table IV, Publication IV). Cultured cells from four patients and smear
preparations from five patients were analysed by FISH. All samples were
obtained at diagnosis and the mean age of the patients was 6.5 (range 2.2-11.7).
The overall and event-free survivals of the nine patients were compared with
survival of 70 other patients with no loss at 12p in CGH. The mean age of the
70 patients was 4.5 (range 0.6-15.7).
In Study V, frozen bone marrow specimens from 17 ALL patients were
investigated by the cDNA array technique (Table I, Publication V). All patients
showed a common phenotype except one (Patient 8) who had a precursor-B
disease. The samples were diluted 1:10 in RNA/DNA stabilisation reagent for
blood and bone marrow (Boehringer Mannheim GmbH, Mannheim, Germany)
for simultaneous cell lysis and stabilisation of the nucleic acids. Fourteen of the
patients were studied at the time of diagnosis and three at relapse. The mean age
was 5.0 years (range 1.2-12.7 years).
Most of the samples were obtained from the Hospital for Children and
Adolescents, Helsinki University Central Hospital. In studies II, III and IV, part
of the samples was received from four other Finnish university hospitals (Table
2). The patients were diagnosed and treated following the guidelines set by the
Nordic Organization for Paediatric Haematology-Oncology (NOPHO)
(Gustafsson et al., 1998). However, the majority of the patients who were
studied retrospectively from the smear preparations were diagnosed and treated
during 1975 to 1981 according to the conventional protocols of that time.
1.2. References
In CGH studies (I and II), peripheral blood from healthy donors was used as
reference. Because of the very limited number of precursor-B cells in normal
bone marrow, two pools of mature B cells from adenoid samples of healthy
children were used as references in the cDNA array study (V).
2. Methods
2.1. Comparative genomic hybridisation (I and II)
CGH was performed using direct fluorochrome-conjugated probes according to
a protocol described by Kallioniemi et al. (1992, 1994) with minor
modifications. DNA extracted from bone marrow samples of the patients was
labelled green with fluorescein-isothiocyanate (FITC)-conjugated dCTP and
dUTP (DuPont, Boston, MA, USA) by nick translation to obtain fragments
ranging from 600 to 2000 base pairs as described by El-Rifai et al. (1997b).
Similarly, the reference DNA extracted from normal peripheral blood was
39
labelled red with Texas-red-conjugated dCTP and dUTP (Du-Pont). Equal

amounts (1 g) of labelled patient and reference DNAs together with 20 g of
unlabelled Cot-1 DNA to block the binding of repetitive sequences (Gibco
BRL, Life Technologies, Gaithersburg, MD, USA) were precipitated with 1/10
volume of 3 M sodium acetate and 9 volumes of absolute ethanol at -20C
overnight. The DNAs were dissolved in 10 l of hybridisation buffer [50%
formamide/10% dextran sulphate/2xSSC (pH 7)] at 37C for two hours. The
slides with normal metaphase spreads were denatured in 50%
formamide/2xSSC (pH 7) at 65C for 2 minutes, followed by dehydratation in
increasing alcohol series (3 minutes each, on ice). The hybridisation mixture
was denatured at 75C for 5 minutes and applied onto denatured slides.
Hybridisation was performed in a moist chamber at 37C for two days. After
hybridisation the slides were washed three times in 50% formamide/2xSSC
(pH 7), twice in 2xSSC, and once in 0.1xSSC at 45C, followed by 2xSSC, PN
buffer [0.1 M NaH2PO4/0.1 M Na2HPO40.1% NP-40 (pH 8)] and distilled
water at room temperature for 10 minutes each. The slides were mounted with
VectaShield-DAPI (Vectashield, Vector Laboratories, Burlingame, CA, USA),
an antifading medium containing the counterstain (DAPI).
The hybridisations were analysed using an Olympus fluorescence
microscope and the ISIS digital image analysis system (MetaSystems GmbH,
Altlussheim, Germany), which consists of an integrated high-sensitivity
monochrome charge-coupled device (CCD) camera and automated CGH
analysis software. Three-colour images of 8-12 metaphases with good
morphology and strong even hybridisation were acquired from each sample.
The chromosomes were recognized and arranged based on the banding pattern
in the DAPI counterstaining. The green-to-red fluorescence intensity ratios were
calculated along each of the chromosomes and visualised as ratio profiles. In the
chromosomal areas with no copy number change, the ratio was approximately
1. The chromosomal region was considered to be over-represented (gained) if
the ratio exceeded 1.17 and under-represented (lost) if the ratio was below 0.85.
A ratio in excess of 1.5 was interpreted as a high-level amplification. The cut-off
levels were determined by experiments in which two differently labelled normal
DNAs were hybridised to same metaphase spreads. The centromeric regions of
chromosomes 1, 9 and 16, short arms of acrocentric chromosomes and whole
chromosome Y were excluded from the analysis because they contain a large amount
of heterochromatin. The results were confirmed by estimating the error probability and
using 99% confidence intervals for the ratio profile.
2.2. Fluorescence in situ hybridisation (III and IV)

The FISH studies were performed using a commercial dual-colour probe
cocktail with spectrum green-conjugated probe specific to the TEL gene and
spectrum orange-conjugated probe specific to the AML1 gene (Vysis, Downers
Growe, IL, USA). The hybridisations and washings were performed following
the manufacturers instructions. Briefly, the slides were denatured in 70%
formamide/2xSSC at 73C for 5 minutes and the probe at 73C for 5 minutes.
40
The probe was applied to the slide and hybridised at 37C overnight. The slides
were washed in 50% formamide/2xSSC (three times, 10 minutes each), 2xSSC
(10 minutes) and 2xSSC/0.1% NP-40 (5 minutes) at 46C. The slides were
counterstained and mounted with VectaShield-DAPI (Vectashield).
The smear preparations were pre-treated before FISH. First, the coverslips
were removed by xylene and the slides were dehydrated in increasing alcohol
series, followed by fixation in methanol/acetic acid (3:1) at 4C overnight. Then
the slides were treated in 1 M natriumthiocyanate for 10 minutes at 65C and
washed in 2xSSC for 5 minutes at room temperature. Next the slides were
treated in 0.01 N HCl for 10 minutes and in 0.05 N HCl with pepsin
(0.05 mg/ml) for 8 minutes at 37C. Finally, the slides were washed under cold
running tap water for 5 minutes and dehydrated in increasing alcohol series.
The analysis was performed from three-colour images acquired using a
fluorescence microscope and the ISIS digital image analysis system
(MetaSystems) developed for CGH. Filters specific to FITC, Texas-Red and
DAPI were used (Chroma Technology Corp., Brattleboro, VT, USA). In Study
III, at least 50 interphase cells were analysed from the images of each sample.
In addition, 20 metaphases were studied from the three samples with more than
four copies of AML1 and from three of the samples with 3-4 copies. In Study
IV, at least 30 interphases from the TEL-AML1 fusion positive samples and 50
interphases from the fusion negative sample were analysed.
2.3. Calculation and comparison of overall and event free survivals (IV)
The overall and event-free survivals of the nine patients with loss at 12p were
compared to those of 70 patients who showed no change at 12p by CGH. The
Kaplan-Meier method was used to analyse the outcome and standard errors (SE)
were calculated according to Greenwoods formula. The event-free survival was
calculated from the date of diagnosis to the date of an adverse event or time of
last contact. Relapse, death in remission, and second malignancy were
considered as events. The survival of the patients without events was calculated
from the date of diagnosis to the time of last contact or to the date of stem cell
transplantation in the first complete remission. The log-rank test was employed
to compare the outcome between the groups. The data were analysed with SPSS
for Windows software (version 8.0.1.).
41
2.4. cDNA array method (V)

2.4.1. cDNA array hybridisation
Total RNA was extracted from patient and reference samples. First, nucleic
acids were isolated using mRNA isolation kit (Boehringer Mannheim)
according to the first steps in the manufacturers protocol. After this, the DNA
was removed following Clontechs (Palo Alto, CA, USA) instructions for
DNase treatment of total RNA. The quality of the total RNA was checked by
gel electrophoresis.
The study was performed using AtlasTM Human Hematology/Immunology
cDNA expression arrays (Clontech) with duplicate cDNA spots of 415 genes
immobilised on a nylon membrane. The genes are known to be expressed in
normal haematopoietic cells, haematopoietic cell lines or in haematological
disorders.
Total RNA (3-4 g) was reverse-transcribed into 33P-labelled cDNA using
the AtlasTM pure total RNA labelling system (Clontech) with gene-specific
primers following the manufacturers instructions. The labelled cDNA probes
were purified from unincorporated 33P-labelled nucleotides and small cDNA
fragments by column chromatography.
The cDNA probes were hybridised to the array according to the
manufacturers instructions. Briefly, the array was first pre-hybridised with
sheared salmon testes DNA in hybridisation buffer (ExpressHyb) for 30
minutes at 68C. The probe was denatured in denaturing solution at 68C for 20
minutes followed by pre-hybridisation with Cot-1 DNA in neutralisation
solution at 68C for 10 minutes. Next the probe was added to the prehybridisation solution and hybridised with the array overnight at 68C. The
non-specifically bound probe was removed by washing the array three times in
2xSSC/1% SDS and once in 0.1xSSC/0.5% SDS, for 30 minutes each at 68C,
and finally in 2xSSC for 5 minutes at room temperature. The arrays were
exposed to an imaging plate (BAS-MP 2040S; Fuji, Kanagawa, Japan) for 3-7
days, after which images of the hybridised arrays were acquired by scanning the
plate with a phosphorimager (Bio-Imaging Analyser, BAS-2500; Fuji).
2.4.2. Data analysis
In order to find the genes that were differently expressed in the patient samples
as compared to the reference, principal component analysis (PCA) was applied
to score the genes according to the expression differences (Hilsenbeck et al.,
1999). Before PCA, the data were pre-processed. First, the brightness of the
hybridisation images was reduced to get the background intensity
approximately to the same level in all the images. Then the intensities of the
gene spots were measured using the Atlas Image 1.5 software (Clontech). The
intensity of each spot on the patient array was compared to the intensity of the
corresponding spot in the reference array in the following manner. First, the
background intensity of the hybridisation image was subtracted form all intensity
values. Next, the intensity of each gene spot in the reference array was subtracted from
42
the corresponding intensity in the patient array to get the intensity difference,
which represents the expression difference of a given gene between the patient
sample and the reference. The data was globally normalised by subtracting the
average of the intensity differences in one array from each intensity difference
value, standardising the average of intensity differences in all arrays to zero. In
addition, the variance was standardised to one by dividing each of the
normalised intensity differences of an array by the standard deviation.
PCA was applied to normalised intensity differences. By projecting the data
on the first principal component, each gene was given a score to represent its
overall expression difference between the patient samples and the reference.
To describe the differential expression in individual patients, thresholds to
determine under- and over-expression were used. The thresholds were obtained
by calculating the percentiles of the pooled expression arrays. The 10th and 90th
percentiles were used to determine the thresholds, which were normalised
intensity differences of 5555 and 4005 for under- and over-expression,
respectively.
To confirm that over-expression detected in five myeloid-specific genes was
not caused by the presence of myeloid cells in the patient samples, possible nonrandom effect of the proportion of myeloid cells on the expression levels of
these genes was investigated. The patients were divided into two groups
according to the proportion of myeloid cells in their bone marrow samples, six
patients with 10% of myeloid cells into one group and eleven with <10% to
another group. Considering each gene separately, it was checked whether its
expression level could be used to allocate the sample to the correct myeloid cell
group. To test the degree of accuracy with which gene expression could predict
the group assignment, the Receiver Operating Characteristic (ROC) Curve and
the area under the curve were calculated (Hanley and McNeil, 1982; Swets,
1988). To check whether this diagnostic accuracy was statistically significant,
the following randomisation experiment was performed. The group labels of the
patients were randomly permuted and the diagnostic accuracy calculated for the
random problem using the same method as for the original data. This
experiment was repeated 10 000 times.
2.5. Quantitative real-time reverse transcriptase

polymerase chain reaction (V)
The validity of the cDNA array results was confirmed by performing
quantitative real-time RT-PCR on eight of the genes. The real-time RT-PCR
experiment was performed on samples from 15 of the 17 patients because not
enough RNA was available from the other two. RNA (500 ng) was reversetranscribed into cDNA from each patient sample and from one of the reference
pools using the 1st strand cDNA synthesis kit for RT-PCR containing AMV
enzyme (Roche Diagnostic Corp., Indianapolis, IN, USA).
The real-time PCR was carried out in the LightCycler rapid thermal cycler
system (Roche Diagnostics GmbH, Mannheim, Germany). The primers were
designed and prepared by TIB Molbiol (Berlin, Germany). The PCRs were
43
prepared in a 10 l volume with 1 l Hot Start reaction mix from the

LightCycler-FastStart DNA Master SYBR Green I kit (Roche Diagnostics
GmbH), 2.6 mM MgCl2, 0.5 mM each primer (0.25 mM for the MYC gene) and
1 l of diluted cDNA (1:5, 1:10 or 1:50).
The LightCycler run was started with an initial denaturation at 95C for 7
minutes. The cDNA was amplified by performing 45 cycles of denaturation at
95C for 15 seconds, annealing at 58-66C for 5 seconds, and elongation at
72C for 10 seconds. To verify the amplification specificity, melting curve
analyses were performed using an initial denaturation at 95C for 10 seconds,
followed by 20 seconds at 55C, and then slow heating of the samples to 95C
at the rate of 0.1C/s with continuous fluorescence detection.
Each patient sample was run simultaneously with the reference sample and a
negative control without cDNA was included in each run. In addition, to obtain
standard curves for quantification, a dilution series of the globin gene
(LightCycler-Control Kit DNA; Roche Diagnostics GmbH) was run in each
assay according to the manufacturers instructions. The relative concentration
values for each PCR product were calculated using the Second Derivative
Maximum method included in the LightCycler software.
In order to compare the results of cDNA array and real-time RT-PCR, the
logarithmic ratios of patient-to-reference intensities in the array and logarithmic
ratios of patient-to-reference concentrations in the PCR were calculated. The
equivalence between the results was visualised by plotting the logarithmic PCR
ratios against the corresponding logarithmic array ratios.
44
Results
1. DNA copy number changes in childhood AML (I)
The results from CGH and standard cytogenetic analysis of the 19 children with
AML are presented in Table I of Publication I. The karyotypes were retrieved
from clinical records. Taking into account the limitations of CGH, no
discrepancies were found between the two methods.
CGH showed changes in 13 (68%) out of the 19 patients with an average of
1.9 aberrations per patient (range, 1 to 5). Gains were 2.6 times more frequent
than losses. Eighteen (72%) of the changes affected a whole chromosome, five
(20%) a chromosome arm, and only two (8%) of the aberrations were regional.
The most frequent changes were gains of whole chromosomes 6, 8 and 21, all
of which were detected three times. Two of the gains of chromosome 21 were
seen in patients with the Down syndrome. The only losses detected more than
once were those of whole chromosomes 7 and X, both of which appeared in two
patients. No high-level amplifications were observed.
2. DNA copy number changes in childhood ALL (II)

CGH revealed changes in 45 (63%) out of the 72 patients with a mean of 4.6
aberrations per patient (range, 1 to 22). All the findings are presented in Figure I
of Publication II. Gains were about six times more frequent than losses. Table 3
shows the most frequent gains and losses. Most of the gains (87%) comprised
whole chromosomes. Regional gains were most frequently observed at 1q with
minimal common region 1q31-q32 (8.3%). High-level amplifications were
detected only in two patients, four in one patient (chromosomes 8, 10, 18 and
21) and one (21q22-qter) in the other. Thus, chromosome 21 was the only
chromosome with more than one high-level amplification.
Losses were frequently observed at chromosomal arms 9p and 12p. Nine
patients (13%) showed loss at 9p with minimal common region 9p22-pter. In
eight patients (11%), the losses affected 12p with minimal common region
12p13-pter. In three patients, 9p and 12p were affected simultaneously.
Table 4 shows a comparison of results obtained by CGH and conventional
cytogenetic analysis. CGH complemented chromosomal banding in 34 (47%)
out of the 72 patients. In 25 of these, CGH showed additional changes or
defined the changes seen in banding analysis. In addition, CGH revealed
changes in five patients with normal karyotype and in four patients with no
result in G-banding. In nine patients standard cytogenetics showed unbalanced
aberrations that were not detected by CGH. One of the patients showed trisomy
6 in G-banding but no changes in CGH. The result was confirmed by
45
Results
chromosome painting, which showed that the trisomy was present only in 6% of
the cells, which explains why the aberration was not detected by CGH.
Table 3. The most frequent DNA copy number changes detected in 72 children
with ALL (II).
Chromosome or
chromosome arm/
minimal common
region
Number of
patients
Frequency
(%)
21
18
25
18
16
22
14
19
10
14
19
17
14
19
14
13
18
12
17
12
17
10
14
- regional
1q / 1q31-q32
LOSSES
9p / 9p22-pter
13
12p / 12p13-pter
11
GAINS
- whole chromosome
affected
Table 4. Similarities and discrepancies between CGH and chromosome

banding analyses in 72 children with ALL (II).
Findings
No of cases
(Proportion)
Normal in both techniques
23
(32%)
Identical changes in both techniques
(10%)
CGH supplemented G-banding

- G-banding unsuccessful / CGH successful
- G-banding normal / CGH abnormal
- CGH gave additional information to G-banding
4
5
25
(6%)
(7%)
(35%)
G-banding showed changes not seen in CGH

- CGH unsuccessful / G-banding successful
- G-banding showed balanced translocation
- G-banding showed unbalanced changes not seen in CGH
3
9
(4%)
(13%)
46
Results
3. Increased copy number of the AML1 gene in

childhood ALL (III)
In the CGH study of ALL (II), high-level amplification at chromosome 21 with
minimal common region 21(q22-qter) was detected in two of the patients. The
region harbours the AML1 gene that participates in the translocation t(12;21). It
was therefore investigated whether the copy number of AML1 is increased in
childhood ALL.
Forty (36%) of the 112 patients were shown to have at least one extra copy
of the AML1 gene by FISH (Table I, Publication III). Two of the patients (1.8%
of all 112 patients) showed high-level amplification of AML1 in most of the
cells, one with at least 10-15 copies of the gene and the other with six copies.
One patient had four copies of AML1 in most of the cells, but at least five
copies in 6% of the cells. In 20 patients (18%) four copies of the gene were
detected in at least 40% of the cells. Seventeen patients (15%), including two
patients with the Down syndrome, showed three copies of AML1.
The TEL-AML1 fusion was detected in 31 (28%) of the 112 patients. The
fusion was present in seven (18%) of the 40 patients with extra AML1 copies
(Table I, Publication III) and in 24 (33%) of the 72 patients with no extra copies
of the gene. Six of the 31 patients with the fusion showed two copies of the
fusion gene in part of the fusion-positive cells, and in one patient, 2-4 copies of
the fusion gene were seen in all of the cells. The other TEL allele was deleted in
19 (61%) of the 31 fusion-positive patients but only in three (4%) of the 81
fusion-negative patients.
In two patients with high-level amplification of AML1, a derivative of chromosome
21 was seen in conventional cytogenetic analysis. The extra copies of the gene were
located in the derivative chromosome. In the patient with 10-15 AML1 copies, the
extra copies were tandemly located in two sites of the derivative, and in the patient
with six copies, as two separate tandem duplicates. Chromosome painting with a
chromosome 21-specific probe stained the whole der(21) of the patient with 10-15
copies and four distinct regions in der(21) of the patient with six copies. In the patient
with 4-5 copies of the gene, the copies were located in different chromosomes. The
poor quality of the metaphases with five copies made it impossible to distinguish the
type and size of the chromosomes. In addition, the karyotype of the patient was
incomplete. According to chromosome painting analysis the maximum number of
copies of chromosome 21 was four. No material from this chromosome was seen in
any other region.
Metaphases were studied also from three patients with 3-4 copies of AML1.
In all three patients, the gene copies were located in separate chromosomes,
probably in copies of chromosome 21. Karyotypes from conventional
cytogenetic analysis were available from twelve of the patients with 3-4 AML1
copies (Table I, Publication III). Four of them showed an extra chromosome 21
in G-banding. Two of these patients had the Down syndrome.
CGH results were available for 26 of the 112 patients and eleven of them
showed a gain at chromosome 21 (Table I, Publication III). All of these eleven
patients also showed extra copies of the AML1 gene in FISH. In the three
47
Results
patients with more than four copies of AML1 (10-15, 6 or 4-5), a high-level
amplification at chromosome 21 was seen in CGH.
4. FISH analysis of the TEL and AML1 genes in

ALL patients with loss at 12p (IV)
The short arm of chromosome 12 showed a frequent loss in the ALL patients by
CGH (II). The minimal common region of the losses (12p13-pter) contains the
TEL gene (12p13) that is fused to AML1 in t(12;21). Because the nontranslocated TEL allele is deleted in most of the patients with t(12;21), the
association of this translocation and the TEL deletion with the 12p losses was
investigated.
FISH results of nine patients who showed a loss at 12p in CGH studies are
presented in Table IV of Publication IV. Eight of the nine patients showed the TELAML1 fusion and the deletion of the non-translocated allele of the TEL gene. In one
patient, the fusion was not seen, but the other allele of TEL was deleted. In seven of
the eight fusion-positive patients, the fusion was detected in most interphase cells (74100%) and in one patient in 27% of the interphase cells. The non-translocated TEL
allele was deleted in all fusion-positive cells in five patients, and in about 60% of the
fusion-positive cells in three patients. The 12p deletion was detected by conventional
cytogenetics only in one of the cases.
Seven of the nine patients survived in continuous complete remission with a
median follow-up of 74 months (range, 51-121 months). Patients with 12p loss
showed a trend to better overall survival compared to 70 other patients with no
12p loss. In addition, all patients with 12p loss showed good early response to
therapy, precursor-B immunophenotype and L1 morphology, and only one of
them had high (>50x10 9/litre) WBC at diagnosis. None of the nine patients were
hyperdiploid.
5. Gene expression in childhood ALL (V)

The gene expression data was analysed using the PCA method. Each of the 415
genes in the array was given a score, representing the difference in its
expression between the test and reference samples. The genes with the highest
scores were considered to be over-expressed and those with the lowest scores
under-expressed as compared to the reference. Based on the PCA projection, 25
(6%) most over-expressed genes were chosen for closer observation. The genes
are listed in Table III of Publication V with their functions and the types of
haematopoietic cell that normally express the genes. The expression pattern of
these genes in all the patients is shown in Figure II of Publication V. The number of
patients with over-expression of the genes ranged from 7 to 17. The 20 genes that
48
Results
were ranked to be the most under-expressed are presented in Table IV of

Publication V.
The cDNA array results were confirmed by real-time RT-PCR for seven of
the over-expressed genes (ERG, AML1, ENG, TEL, MLLT2, DAPK1 and MYC)
and for one under-expressed gene (BAX; BCL2-associated X protein). Overall,
the PCR results agreed well with the cDNA array results.
Five of the 25 most over-expressed genes (S100A12, RNASE2, GCSFR,
PRTN3, and CLC) are known to be expressed in myeloid cells but not in Blymphoid cells. To ascertain that the over-expression of these myeloid-specific
genes was not caused by myeloid cells present in the bone marrow samples, it
was investigated whether a non-random effect exists between the proportion of
myeloid cells and the expression of these genes. As no such effect could be
demonstrated (ROC = 0.29-066, p = 0.13-0.46), it was concluded that the small
amount of myeloid cells did not have a significant effect on the expression level
of the myeloid-specific genes.
49
Discussion
1. Comparative genomic hybridisation and
conventional cytogenetics in childhood AML (I)
In the present study of 19 children with AML, the concordance between the
results obtained by CGH and conventional cytogenetics was high.
Chromosomal banding analysis was successful, and CGH did not give
additional information to the karyotypes obtained. On the other hand, in 13
patients (68%) the banding technique detected changes not seen in CGH. These
changes included balanced translocations, inversions and marker chromosomes.
Moreover, polyploidy and aberrant subclones were detected by conventional
cytogenetics.
In one previous study of eight adults with AML, CGH complemented
conventional cytogenetics only in one case with no result in banding analysis
(Bentz et al., 1995). However, in one previous and two later studies of adult
AML, CGH was found to be successful in unravelling complex karyotypes,
especially in identifying the composition of marker chromosomes (El-Rifai et
al., 1997a; Kim et al., 2001; Verdorfer et al., 2001). Overall, the karyotypes in
those studies were considerably more complex than in the present study and
contained numerous marker chromosomes. In the present study, marker
chromosomes were only seen in two of the patients. Different degrees of
complexity in karyotypes may explain why CGH failed to complement the
banding analysis in the present study like it did in adult AML studies. In
addition, in all three studies of adult AML, deletions of chromosomal arms 5q
and 7q were among the most frequent changes both by standard cytogenetics
and CGH, whereby CGH yielded more details of the deletions found by the
banding analysis. These aberrations are more rare in childhood AML, and
neither of them was found in the present study (Heim and Mitelman, 1995;
Perkins et al., 1997). On the other hand, the balanced translocation t(8;21), the
most common alteration in childhood AML, is more frequent in children than in
adults (Heim and Mitelman, 1995). Furthermore, high-level amplifications, not
observed in the present study, appear more often in adult AML (Alitalo et al.,
1985; Asker et al., 1988; Tanaka et al., 1993; Park et al., 2000; Streubel et al.,
2000; Andersen et al., 2001). One high-level amplification has been reported in
a later CGH study of adults (Verdorfer et al., 2001) and two high-level
amplifications in another (Kim et al., 2001). Considering these differences
between childhood and adult AML, the CGH method may be more useful in the
diagnostics of adult patients.
In Study II, CGH gave additional information to standard cytogenetics for a
great proportion of the patients with ALL. The main reasons why CGH
supplemented banding analysis in childhood ALL patients but not in the
childhood AML patients are probably the differences between the frequency of
50
Discussion
numerical changes, and the number and quality of the metaphases obtained. In
childhood ALL, the proliferation rate of the blasts is usually weaker and
morphology of the chromosomes poorer. Poor quality of metaphases makes it
often impossible to identify the extra chromosomes frequently occurring in
ALL, resulting in incomplete karyotypes.
On the basis of these results, there seems to be no need to apply CGH to the
routine diagnostics of children with AML. However, CGH can be of great help
in cases with no metaphases available for the banding analysis and in resolving
complex karyotypes in cases with incomplete cytogenetic results.
2. CGH as a support of conventional cytogenetics in

childhood ALL (II)
In about half of the 72 ALL cases studied, CGH supplemented chromosomal
banding analysis. CGH detected copy number changes in five (7%) patients
with a normal karyotype and defined changes or detected additional alterations
in 25 (35%) patients with an abnormal karyotype. Furthermore, changes were
detected by CGH in four (6%) cases, which yielded no metaphases for Gbanding.
In most of the cases, the cytogenetic analysis succeeded in estimating the
number of chromosomes, but the morphology of the chromosomes was often
too poor to enable recognition of the chromosomes that were gained or lost.
CGH was successful in identifying these chromosomes as well as in clarifying
the composition of the marker chromosomes. In addition, CGH revealed small
losses not detected by banding analysis. CGH turned out to be especially
valuable in finding FISH markers for patient follow-up. Thus, CGH lends
significant support to conventional cytogenetic analysis in childhood ALL. This
is in agreement with two previous and several later CGH studies of childhood
ALL (Karhu et al., 1997; Paszek-Vigier et al., 1997; Haas et al., 1998; Wong et
al., 1998; Jarosov et al., 2000; Scholz et al., 2001). The clear supplementary
benefits of CGH to the banding technique are mainly explained by the high
frequency of hyperdiploidy, poor quality of metaphases and low proliferation
rate of leukaemic blasts in childhood ALL. Accordingly, it is highly
recommended to perform CGH routinely for children at the diagnostic stage of
ALL.
In 12 (17%) of the 72 patients, conventional cytogenetic analysis showed
changes that were not detected by CGH. In three (4%) of the cases,
chromosomal banding revealed a balanced translocation, which CGH is unable
to detect because the translocation leaves the DNA copy number unchanged.
The failure of CGH to detect the unbalanced aberrations, found in nine (13%)
cases by G-banding, is probably explained by aberrations that were present only
in subclones that are beyond the detection capacity of CGH (minimum 50% of
the cells), as confirmed by FISH in one case. Because of its limitations, CGH
51
Discussion
cannot replace standard cytogenetics, which continues to be an important

diagnostic tool in ALL.
3. DNA copy number changes in childhood ALL (II)

3.1. Gains
The most common aberrations shown by CGH were gains of whole
chromosomes. This outcome could be expected in view of the high frequency of
hyperdiploidy in childhood ALL. Chromosome 21 (25%) was gained most
frequently. Other chromosomes with frequent whole chromosome gains were
18, X, 10 and 17 (19-22%) and, at lower frequencies, chromosomes 14, 4, 6 and
8 (14-18%). The results are in agreement with previous findings by
conventional cytogenetics, as well as with two previous and several later CGH
studies (Raimondi et al., 1992; Raimondi et al., 1996; Karhu et al., 1997;
Paszek-Vigier et al., 1997; Haas et al., 1998; Larramendy et al., 1998; Jarosov
et al., 2000; Scholz et al., 2001). Table 5 summarises the most frequent findings
in all the CGH studies.
Regional gains were rare. The most frequent were gains at 1q with minimal
common region 1q31-q32, observed in six (8%) of the patients. The gain of
1q24-qter in three patients was probably a result of the unbalanced translocation
t(1;19), a well-known translocation in ALL (Heim and Mitelman, 1995).
Unbalanced translocations can easily be missed in standard cytogenetics due to
poor quality of metaphases. In these cases, CGH would help to reveal them.
High-level amplifications were found only in two patients, which supports the
concept of high-level amplifications being rare in childhood ALL. In the other
CGH studies, only four patients with altogether six high-level amplifications were
reported (Karhu et al., 1997; Larramendy et al., 1998; Wong et al., 1998). In the
present study, the only chromosome with more than one high-level amplification
was chromosome 21. In one patient the whole chromosome was amplified and in
the other the affected region was 21q22-qter. Interestingly, the chromosomal band
21q22 contains the AML1 gene, which is fused to the TEL gene in the t(12;21)
translocation. Later, additional high-level amplifications affecting the whole
chromosome 21 have been found in one of 36 patients and two of 14 patients
(Larramendy et al., 1998; Wong et al., 1998).
52
Discussion
Table 5. Summary of the frequencies (%) of most frequent CGH findings in eight publications of childhood
ALL.
Reference
Most frequent gains
Most frequent losses
(No. of patients)
1q
10
14
17
18
21
6q
9p
12p
13q
A (72)
B (36)
C (72)
D (13)
E (65)
F (71)
19
42
28
38
31
15
8
11
3
9
8
17
31
17
15
23
11
17
31
18
8
25
15
14
8
11
23
8
6
19
36
15
23
26
20
18
28
19
23
22
15
19
28
7
15
25
14
22
33
22
23
28
15
25
44
24
31
35
20
3
1
8
9
4
13
6
18
14
11
14
11
8
4
9
1
Total A-F
(329)
26
18
19
10
22
20
17
23
28
10
G (14)*
H (14)**
36
93
7
-
29
71
43
86
29
57
36
79
36
86
21
71
43
11
36
100
14
-
A, Study II; B, Larramendy et al. 1998; C, Paszek-Vigier et al. 1997; D, Karhu et al. 1997; E, Jaroov et al. 2000;
F, Scholz et al. 2001; G, Wong et al. 1998; H, Haas et al. 1998; *selected group of patients showing unbalanced
changes by standard cytogenetics; **selected group of patients showing hyperdiploidy by standard cytogenetics.
53
Discussion
3.2. Losses
The two most frequent losses affected 9p in nine patients (13%) and 12p in eight
patients (11%) with minimal common regions 9p21-pter and 12p13-pter, respectively.
Surprisingly, no losses at 9p or 12p were detected in three CGH studies (Karhu et al.,
1997; Paszek-Vigier et al., 1997; Haas et al., 1998). More recently, however, losses at
9p and 12p have been reported in three and two studies, respectively (Table 5) (Wong
et al., 1998; Jarosov et al., 2000; Scholz et al., 2001). The aberrations of 9p have
been shown to be associated with poor overall survival (Heerema et al., 1999). The
frequencies of 9p loss (18% and 14%) and 12p loss (11% and 8%) in the studies by
Jarosov et al. (2000) and Scholz et al. (2001) showing both aberrations corresponded
well with the findings of the present study. Jarosov et al. (2000) also showed
relatively frequent losses at 13q (9%), 6q (9%) and 7p (8%), findings that were less
common in the other studies, including the present study (Table 5). The losses at 9p
and 12p, including deletions and unbalanced translocations, had both previously been
seen in about 8% of the patients using conventional cytogenetics (Raimondi, 1993;
Raimondi et al., 1997). Furthermore, 9p and 12p are the regions that most frequently
show LOH in childhood ALL (in about 30-40% and 25-30% of the patients,
respectively) (Takeuchi et al., 1995; Baccichet et al., 1997; Chambon-Pautas et al.,
1998). The absence of these losses in some of the CGH studies is probably due to a
methodological failure. This emphasises how important it is to control the technical
aspects when using the method.
Despite the rather high frequency of losses at 9p and 12p in standard
cytogenetic studies by Raimondis group (Raimondi, 1993; Raimondi et al.,
1997), only one of the nine 9p losses and two of the eight 12p losses were
revealed by banding analysis in the present study. Also, in a later study by
Jarosov et al. (2000), only two 9p losses out of 12 and one 12p loss out of
seven detected in CGH were seen by chromosome banding. In addition, CGH
helped to define the region of the losses seen in cytogenetic analysis.
The minimal common region of the 9p losses contains the chromosomal
band 9p21 with two tightly linked tumour suppressor genes, CDKN2A and
CDKN2B. Both genes are deleted, usually simultaneously, in about 40% of
children with ALL [(Drexler, 1998) and references therein]. Other candidate
tumour suppressor genes in the region, often co-deleted with CDKN2A and
CDKN2B, are MTAP (methylthioadenosine phosphorylase) and IFN (interferon)
gene cluster (Dreyling et al., 1995).
The minimal common region of the 12p losses involves the chromosomal
band 12p13 with the TEL gene. One TEL allele is deleted in about 75% of the
patients with the TEL-AML1 fusion (Cav et al., 1997), which is present in
about 25% of the patients (Romana et al., 1995b; Shurtleff et al., 1995; Liang et
al., 1996; McLean et al., 1996; Borkhardt et al., 1997). The hemizygous
deletion of CDKN1B (KIP1; p27), mapped to 12p12, is also common in
childhood ALL (Pietenpol et al., 1995; Takeuchi et al., 1995; Komuro et al.,
1999). Furthermore, hypermethylation of LRP6 (low-density lipoprotein-related
protein 6) in 12p12.3 has been reported in two patients with hemizygous 12p
deletion (Baens et al., 1999).
54
Discussion
The much lower frequency of the 9p- and 12p losses seen in CGH, as
compared to the frequencies of LOH and gene deletions in the region, is
explained by the inability of CGH to detect deletions smaller than 3-10 Mb
(Kallioniemi et al., 1994; Kirchhoff et al., 1999). However, a great proportion
of the deletions seem to be large enough for the resolution of the method.
Because these deletions seem to be significant prognostic markers, it is crucial
to detect them.
4. Amplif ication of the AML1 gene in childhood

ALL (III)
CGH revealed high-level amplifications at chromosome 21 in two children with
ALL (Study II). Chromosome 21 contains the AML1 gene involved in t(12;21).
The aim of the present study was to investigate whether AML1 is a target of
amplification in childhood ALL.
Three (2.7%) of the 112 patients studied showed high-level amplifications of
AML1 with 10-15, six, and five copies of the gene. In all of these patients, highlevel amplification was also detected at chromosome 21 by CGH, the minimal
common region being 21q22-qter. In two patients with 10-15 and six copies of
AML1, the extra copies were found to be located in tandem in two distinct sites
of a derivative chromosome 21. Thus, the increase in the copy number seems to
have occurred through intrachromosomal amplification, followed by duplication
of the region with tandem repeats of the gene.
The present study was the first publication to describe high-level
amplification of AML1 in ALL. In addition, gene amplification had not been
reported in childhood ALL before. Later the finding has been confirmed by
several other studies, which demonstrated childhood ALL cases with
amplification of AML1 (Busson-Le Coniat et al., 2001; Dal Cin et al., 2001; Ma
et al., 2001; Mathew et al., 2001; Nordgren et al., 2002). In the reports the copy
number of the gene varied from five to 15. The frequency seen in the present
study agrees with the findings of Nordgren et al. (2002) who detected AML1
amplification (7-15 copies) in two out of 70 patients studied. In addition to
childhood ALL, the amplification of AML1 has been reported in an adult with
myelodysplastic syndrome (Kakazu et al., 1999), and recently also in an adult
with AML (Streubel et al., 2001).
Thirty-seven (33%) out of the 112 patients showed three or four copies of
AML1. Probably all or almost all of these patients had one or two extra copies
of whole chromosome 21. This is supported by the high frequency of gains of
chromosome 21 by CGH (Table 5) and conventional cytogenetics (Raimondi et
al., 1992, 1996; Berger, 1997; Karhu et al., 1997; Paszek-Vigier et al., 1997;
Haas et al., 1998; Larramendy et al., 1998; Jarosov et al., 2000; Scholz et al.,
2001; Study II).
In the present study, none of the patients with high-level amplification of
AML1 showed the TEL-AML1 fusion. The amplification was, however, reported
55
Discussion
in a fusion-positive patient by Ma et al. (2001). The amplification was present

both in cells with and without the fusion, suggesting that it had occurred before
the translocation. The frequency of the TEL-AML1 fusion in the patients with 34 copies of AML1 was clearly lower (19%) than in those with two copies (33%).
This agrees with previous studies, which showed that the patients with the
fusion have much lower frequency of hyperdiploidy (51+) (Shurtleff et al.,
1995; Raimondi et al., 1997). The result is further supported by a report of the
absence of TEL-AML1 fusion in eleven patients with the Down syndrome
(Lanza et al., 1997).
The amplification of AML1 is likely to increase the expression of the gene.
Busson-Le Coniat et al. (2001) studied three patients with five copies of the
AML1 gene and searched for point mutations in its exons 3-5, where all the
published point mutations are located (Osato et al., 1999; Song et al., 1999).
They detected no point mutations in these exons, which suggests that the
amplified AML1 was of wild-type. Over-expression of the AML1B isoform has
been shown to cause neoplastic transformation in fibroblasts (Kurokawa et al.,
1996) and to shorten the cell cycle in myeloid progenitor cells (Strom et al.,
2000) suggesting that the amplification of wild-type AML1 has a pathogenic
effect. Further studies are, however, needed to investigate whether AML1 is
mutated when amplified, and also whether the 21q22-qter amplicon harbours
other leukaemia-specific genes.
5. Association of loss at 12p with the TEL-AML1

fusion in childhood ALL (IV)
In Study II, loss at 12p was frequently observed in ALL patients by CGH. In
order to investigate whether this loss is associated with the TEL-AML1 fusion
and the deletion of the other TEL allele, FISH was performed for nine patients
with the 12p loss. Eight of the nine patients showed the TEL-AML1 fusion with
the deletion of the non-translocated TEL allele. This proportion is slightly
higher than in two previous studies using standard cytogenetics, in which the
TEL-AML1 fusion was detected in 17 (68%) of 25 (Raimondi et al., 1997) and
six (67%) of nine (O'Connor et al., 1998) patients with loss of 12p material.
Furthermore, 15 (94%) of 16 (Raynaud et al., 1996) and 34 (81%) of 42 (Cav
et al., 1997) patients with LOH at the TEL locus were reported to show the
TEL-AML1 fusion.
In three patients of the present study, the TEL deletion was present only in
part of the cells with the fusion. This supports previous studies that show the
deletion to be a secondary event after the translocation (Raynaud et al., 1996;
Romana et al., 1996; Cav et al., 1997).
In the present study, one of the nine patients did not show the TEL-AML1
fusion but had lost one TEL allele. Because point mutations of TEL in cases
with LOH at 12p as well as homozygous deletions of TEL seem to be rare (Sato
et al., 1995; Stegmaier et al., 1996), CDKN1B and/or another putative tumour
56
Discussion
suppressor genes in this region were probably co-deleted with TEL in this
patient (Pietenpol et al., 1995; Baens et al., 1999).
In the present study, the 12p loss seemed to be associated with favourable
prognostic features. All the patients had good early response to therapy, L1
morphology and precursor-B immunophenotype. In addition, the patients with
12p loss showed a trend toward a better overall survival. This trend is likely to
be related with the TEL-AML1 fusion, which has been associated with
favourable outcome in most studies (Liang et al., 1996; McLean et al., 1996;
Borkhardt et al., 1997; Rubnitz et al., 1997b, c; Loh et al., 1998; Maloney et
al., 1999a; Rubnitz et al., 1999a). Some studies have, however, questioned the
prognostic significance of the fusion (Harbott et al., 1997; Seeger et al., 1998,
2001). The discrepancies between the findings are probably due to differences
in treatment (Loh et al., 1998).
6. Gene expression in childhood ALL (V)

Gene expressions of a total of 415 genes related to normal or abnormal function
of blood cells and their precursors were analysed in the leukaemic blasts of 17
children with precursor-B ALL using cDNA arrays. Because the number of Blineage cells in normal bone marrow is very limited, normal mature B cells
from palatine tonsils were used as the reference.
At least 16 out of the 20 most under-expressed genes (Table IV, Publication
V), including LCP1 (lymphocyte cytosolic protein 1), CD83, LYN (v-yes-1
Yamaguchi sarcoma viral related oncogene homolog), SPIB (Spi-B
transcription factor) and CD48 are known to be expressed in mature B cells
(Hamaguchi et al., 1982; Yokoyama et al., 1991; Zhou et al., 1992; Su et al.,
1996; Hibbs and Dunn, 1997). Although some of the under-expressed genes are
also known to be expressed in the precursors of B cells, the under-expression of
most of them was probably related to the use of mature B cells as reference for
the precursor-B cell type of leukaemia. Consequently, closer examination of the
results was focussed on over-expressed genes.
The 25 most over-expressed genes (Table III, Publication V) included four
genes that are expressed only in the cells of the myeloid lineage: GCSFR,
PRTN3, RNASE2 and CLC (Weller et al., 1982; Bories et al., 1989; Nagata and
Fukunaga, 1991; Rosenberg, 1998). In addition, over-expression was detected
in S100A12, which encodes a peptide released from sensory nerve endings to
bone marrow (Bjurholm et al., 1988), but it is also expressed in myeloid cells
(Guignard et al., 1995; Robinson and Hogg, 2000). Using statistical tests it was
shown that the small amount of myeloid cells in the bone marrow samples of
the patients did not significantly affect the expression level detected in these
myeloid-specific genes. It was therefore concluded that the over-expression of
myeloid-specific genes was related to their up-regulation in the malignant cells.
The results are in agreement with flow-cytometric studies that show variable
degrees of phenotypic expression of myeloid markers in a large proportion of the
57
Discussion
patients with B-lineage ALL (Pui et al., 1991). Out of the 17 patients of the
present study, immunophenotyping showed expression of CD34 and CD13
antigens in 16 and 10 patients, respectively. The array method detected overexpression of CD34 and CD13 in 15 and six patients, respectively.
Out of the five over-expressed myeloid-specific genes, GCSFR and PRTN3 encode
proteins that are known to regulate cell proliferation. Thus, these genes are good
candidates to have a role in the pathogenesis of ALL. These two genes function in a
same pathway. GCSFR encodes a receptor for G-CSF, a myeloid growth factor, and
PRTN3 is known to be up-regulated by G-CSF (Suda et al., 1987; Lutz et al., 2000).
GCSFR has been reported to be up-regulated in precursor-B ALL by the t(1;19)specific oncoprotein E2A-PBX1, providing evidence that over-expression of GCSFR
may be related to leukemogenesis (de Lau et al., 1998). Constitutive over-expression
of PRTN3 causes G-CSF independent growth of myeloid progenitors, the G-CSF
receptor being essential for this process (Lutz et al., 2000). PRTN3 has not previously
been reported to be associated with ALL.
One of the numerous functions of S100A12 is to inhibit B-cell development
(Fernandez et al., 2000). Over-expression of S100A12 was previously detected
in CLL using cDNA arrays (Aalto et al., 2001). No involvement of S100A12 in
ALL has been reported before.
Because mature B cells were used as reference for leukaemic blasts derived
from precursor-B cells, over-expression was expected in the genes that are more
highly expressed in the early stages of B-cell development. Accordingly, overexpression was detected in genes that function in the rearrangement of
immunoglobulin genes (TDT and RAG1) and genes encoding cell surface
antigens of lymphoid precursors (CD34, SPN, CD9 and CALLA). In addition,
the expression of murine homologues of ERG, TEL and MLLT2 has been found
to be higher in B-cell precursors as compared to mature B cells (Baskaran et al.,
1997; Anderson et al., 1999). All three are genes that encode transcription
factors and are rearranged in recurrent translocations of leukaemias (Golub et
al., 1995; Heim and Mitelman, 1995). Furthermore, the expression of MYC has
been reported to correlate with the stage of differentiation and the proliferation
activity of B cells (Larsson et al., 1991). On the other hand, MYC has been
shown to be up-regulated as a result of translocations in Burkitt lymphoma and
leukaemia, and T-cell ALL, and by amplification or other mechanisms in AML
and in various solid tumours (Alitalo et al., 1985; Erikson et al., 1986; Preisler
et al., 1987; Baer et al., 1992; Heim and Mitelman, 1995).
Interestingly, AML1 was one of the most over-expressed genes. The previous
finding of high-level amplification of AML1 by FISH (Study III) suggests that
over-expression of the gene may be related to leukemogenesis. Furthermore, as
mentioned in Chapter 4, over-expression of AML1 shortens the cell cycle in
myeloid cells and causes transformation in fibroblasts (Kurokawa et al., 1996;
Strom et al., 2000). However, haploinsufficiency of AML1 has been reported to
cause autosomal dominant platelet disorder with predisposition to AML (Song
et al., 1999). What has to be taken into account is that AML1 encodes a number
of alternative transcripts with different and even opposite functions, and that
besides the absolute amount of AML1 mRNA, the relative amounts of distinct
58
Discussion
mRNA forms affect the function of the gene (Miyoshi et al., 1995; Levanon et
al., 2001). The primers that were used recognise several AML1 mRNA
isoforms, thus their individual expression could not be determined.
Over-expression was also detected in LCP2, which encodes an adapter
protein in precursor-T and T-cell receptor signalling [(Rudd, 1998) and
references therein]. The gene has been found to be up-regulated in a Burkitt
lymphoma B-cell line after B-cell receptor cross-linking, and the protein has
been suggested to function in B-cell receptor signalling as well (Mizuno et al.,
1996; Nakayama et al., 2000). LCP2 has been found to be over-expressed in
CLL using the cDNA array method (Aalto et al., 2001). The gene has not been
reported to be associated with ALL.
The present study was the first publication about the expression of FGF6 in
leukaemic blasts of ALL. Because FGF6 does normally not seem to be
expressed in bone marrow at all, it is highly probable that the over-expression
detected in the gene is related to its up-regulation in leukaemic blasts.
Expression of FGFR1 and FGFR4, which encode the receptors of FGF6, has
been reported in precursor-B cell lines, and expression of FGFR1 also in mature
B cells (Bikfalvi et al., 1992; Allouche et al., 1995; Ornitz et al., 1996). FGF6
in turn has been seen to up-regulate the expression of FGFR1 in myoblasts
(Pizette et al., 1996). Interestingly, over-expression of FGFR1 was detected in
five out of the 12 patients showing over-expression of FGF6, but in none of the
patients without FGF6 over-expression. FGFR1 is rearranged in several 8p12
translocations leading to fusion proteins with constitutive tyrosine kinase
activity of FGFR1 in a myeloproliferative disorder generally progressing to
AML (Popovici et al., 1998, 1999; Guasch et al., 2000). In addition, overexpression of FGFR1 was found in the absence of these translocations in AML
patients using cDNA arrays (Larramendy et al., 2002). Neither FGF6 nor
FGFR1 have been connected to ALL before.
The number of genes in the present study was very limited and restricted to genes
known to have some role in haematopoiesis, in the function of blood cells or in blood
disorders. The expression of other types of gene, such as those functioning in cell
cycle or apoptosis, is also likely to be altered in childhood ALL. Probably because of
the difficulties in obtaining a relevant reference, the DNA array studies of childhood
ALL have so far been focussed mostly on classification and not on identifying genes
abnormally expressed in the leukaemic blasts (Rozovskaia et al., 2001; Armstrong et
al., 2002; Yeoh et al., 2002). Yeoh et al. (2002) performed a comprehensive study of
360 patients using arrays with 12 600 genes but without using any reference. Chen et
al. (2001), on the other hand, purified precursor-B cells and used them as reference,
but only four patients were studied. A study with large number of genes and patients
using precursor-B cells as reference would greatly enhance our knowledge about the
changes in gene expression in precursor-B cell ALL.
The number of patients in the present study was too small to perform
classification studies. In addition, a larger number of patients will be required to
study whether the expression levels of the genes correlate with some clinical
features. The study of the possible pathogenic and prognostic roles of the genes
could be focussed to the most interesting ones and be performed using a less
59
Discussion
complicated method with lower expenses, such as real-time RT-PCR. The

requirement of smaller amount of RNA in real-time RT-PCR could also
enhance the possibility to use precursor-B cells as a reference.
60
Summary and conclusions

In childhood AML, the CGH results were well in concordance with the
karyotypes obtained by chromosomal banding analysis. However, CGH did not
complement the results of conventional cytogenetics. Although CGH is
certainly helpful in the cases with complex karyotypes, the results do not
advocate performing CGH routinely for all children with AML.
In childhood ALL, by contrast, CGH was shown to supplement standard
cytogenetics in about half of the patients studied, especially by identifying the
chromosomes gained in hyperdiploid cases. The method clearly facilitated the
finding of markers for disease follow-up by FISH. The use of CGH is thus
highly recommended to support conventional cytogenetics when studying
children at the diagnostic stage of ALL. The difference in the utility of the
method between the two forms of acute leukaemia is mostly explained by the
higher frequency of numerical changes in ALL and the better success rate of
standard cytogenetics in AML.
The most common DNA copy number changes in childhood ALL were gains
affecting whole chromosomes 21, 18, X, 10, 17, 14, 4, 6 and 8 (14%-25%).
Regional gains were rare, the most frequent of them being gains at 1q (8%).
High-level amplifications were detected only in two patients (3%).
Chromosome 21, with minimal common region 21q22-qter, was the only one
involved in both of these cases. Two frequent losses affected chromosomal
arms 9p (13%) and 12p (11%) with minimal common regions 9p22-pter and
12p13-pter, respectively. Further studies with larger patient material are needed
for the evaluation of prognostic significance of the individual CGH findings.
The AML1 gene was shown to be highly amplified in three (2.7%) of 112
children with ALL. In all of the three patients, high-level amplification at 21q
was detected by CGH. In addition, one or two extra copies of AML1 were
detected in 33% of the patients studied, probably in consequence of the gain of
whole chromosome 21, which was found to be the most frequent gain by CGH.
The frequency of the TEL-AML1 fusion was lower in the patients with extra
AML1 copies (18%) than in other patients (33%). Study III was the first
publication of high-level amplification of AML1 in ALL, and this was also the
first gene amplification reported in childhood ALL. In order to understand the
possible role of the amplification in the pathogenesis of ALL, it should be
investigated whether AML1 is mutated in the cases with amplification and
whether the amplified region harbours some other gene(s) with possible
leukaemic effect.
The loss at 12p detected by CGH was found to be strongly associated with
the TEL-AML1 fusion and the deletion of another TEL allele in childhood ALL.
Eight of nine patients with 12p loss showed the TEL-AML1 fusion and the
deletion of the non-translocated TEL allele. One TEL allele was lost also in one
patient without the fusion. In addition, the results support the association of 12p
61
Summary and conclusions
aberrations and the TEL-AML1 fusion with favourable prognostic features and
good overall survival in children with ALL.
The cDNA array study provided novel information about gene expression in
the malignant cells of childhood ALL. Several myeloid-specific genes
(S100A12, RNASE2, GCSFR, PRTN3 and CLC) were shown to be up-regulated
in leukaemic blasts. Interestingly, AML1, whose high-level amplification was
demonstrated by FISH, was over-expressed in comparison to the B-cell
reference. The most over-expressed genes included also LCP2 and FGF6. The
data obtained can be utilised in further studies with a larger number of patients
and using other techniques, to investigate which of the findings are related to
the pathogenesis and/or prognosis of childhood ALL.
62
Acknowledgements
This work was carried out at the Department of Medical Genetics in Haartman
Institute, University of Helsinki, during the years 1997-2002. I wish to express
my sincere gratitude to all who made this study possible, especially to:
Sakari Knuutila, my supervisor for initiating me with the mysteries of cancer
genetics. I am grateful for his inspiring guidance and encouragement spiced
with humour.
Albert de la Chapelle, Juha Kere, Pertti Aula, Anna-Elina Lehesjoki and
Leena Palotie, the former and present heads of the Department of Medical
Genetics, for providing me with excellent working facilities.
Eija-Riitta Hyytinen and Eeva Juvonen, the official pre-examiners of this
thesis for both fast and thorough review and valuable comments.
Pirjo Pennanen, for language revision of this book and the articles, as well as
for her help in whatever the practical matter. By her great sense of humour she
cheered me up in the middle of the often tedious polishing of the manuscript.
My co-authors, for fruitful collaboration. Ulla Saarinen-Pihkala, Kim
Vettenranta and Jukka Kanerva, for offering most of the patient material for
these studies and for sharing their expertise in clinical aspects of childhood
leukaemias. Marcelo Larramendy, for his indispensable contribution especially
on Study II and for teaching me the method of CGH. Jaakko Hollmn, for
performing the tricky statistics of DNA arrays and for patiently trying to make
me understand what he had done. Wael El-Rifai, for not only his impact on
Study II but also for always finding time to help in various matters. Yan Aalto,
Kristiina Heinonen, Ritva Karhu, Johan Lundin, Eija Mahlamki, Heikki
Mannila, Anne Mkipernaa, Blint Nagy, Seppo Pakkala, Pekka Riikonen,
Anna Ferrer Salvador, Jouni Seppnen and Harriet Wikman, for their valuable
contribution.
Everybody who has belonged to the CMG group in these years, for the
friendly atmosphere and the share of your knowledge. I especially want to thank
Yan Aalto, Henrik Edgrn, Kowan Ja Jee, Eeva Kettunen, Tuija Lundn, AnnaMaria Nissn, Asta Varis, Anna Ferrer Salvador, Harriet Wikman, Maija Wolf
and Ying Zhu, for their help, support and friendship. It certainly was my lovely
P-floor family, who prevented me from going crazy during the last year of
intensive work under big pressure, complaints of pregnancy and scarcity of
social contacts.
The people of the chromosome laboratory, for their kindness and helpfulness. I will
never forget the pleasant years when I worked with them in the routine.
Aki Vyrynen, for his help in the FISH analyses, not forgetting his friendship in
these years.
Anita Ikonen, Eeva Kettunen, Paula Kvick and Asta Varis, for many great
laughters both at work and on the unforgettable cruises, as well as for listening
to me when I had hard days in my life. Special thank to Paula for excellent
63
Acknowledgements
CGH slides. Probably the greatest thank I owe to Asta who started with me a
project, the finding of which is my husband today.
All my friends outside the laboratory, for sharing the ups and downs with
me. I greatly appreciate that they remained my friends despite the fact that I did
not have so much time and energy to keep in touch in recent years.
My wonderful parents Marja-Liisa and Veikko, for their love, care and
encouragement. I would never have finished this book by the fixed date without
my mother, who was nursing our baby while I was working. I also wish to
thank my brother Tommi for love and happy moments.
My warmest thoughts go to my dear husband Ilkka, whom I thank for his
love and understanding, and for making me believe that I will get through this.
The way you make me to feel beloved every day carries me over the bad times
and completes the good times of my life.
I dedicate this work to my dear son, Aaro, who was growing in my belly
while I was writing this book. At the writing of these final lines, Aaro is a sweet
little baby of three months. I am happy that he is healthy, as I hope he always
will be. I have worked in the laboratory without ever seeing the children whom
I studied, and it was difficult to comprehend the real meaning of what I was
doing. Motherhood opened me a whole new perspective to these studies.
This study was financially supported by the Finnish Cancer Institution
Foundation, the Helsinki University Science Fund, the Nona and Kullervo Vre
Foundation, the Biomedicum Helsinki Foundation, the Sigrid Juslius
Foundation and Helsinki University Central Hospital.
Helsinki, October 2002
64
References
Aalto, Y., El-Rifai, W., Vilpo, L., Ollila, J., Nagy, B., Vihinen, M., Vilpo, J. and Knuutila, S. Distinct
gene expression profiling in chronic lymphocytic leukemia with 11q23 deletion. Leukemia, 15: 1721-8.
(2001).
Aguiar, R.C., Sohal, J., van Rhee, F., Carapeti, M., Franklin, I.M., Goldstone, A.H., Goldman, J.M. and
Cross, N.C. TEL-AML1 fusion in acute lymphoblastic leukaemia of adults. M.R.C. Adult Leukaemia
Working Party. Br J Haematol, 95: 673-7. (1996).
Alitalo, K., Saksela, K., Winqvist, R., Alitalo, R., Keski-Oja, J., Laiho, M., Ilvonen, M., Knuutila, S. and
de la Chapelle, A. Acute myelogenous leukaemia with c-myc amplification and double minute
chromosomes. Lancet, 2: 1035-9. (1985).
Alizadeh, A.A., Eisen, M.B., Davis, R.E., Ma, C., Lossos, I.S., Rosenwald, A., Boldrick, J.C., Sabet, H., Tran, T., Yu,
X., Powell, J.I., Yang, L., Marti, G.E., Moore, T., Hudson, J., Jr., Lu, L., Lewis, D.B., Tibshirani, R., Sherlock, G.,
Chan, W.C., Greiner, T.C., Weisenburger, D.D., Armitage, J.O., Warnke, R., Levy, R., Wilson, W., Grever, M.R.,
Byrd, J.C., Botstein, D., Brown, P.O. and Staudt, L.M. Distinct types of diffuse large B-cell lymphoma identified
by gene expression profiling. Nature, 403: 503-11. (2000).
Alizadeh, A.A., Ross, D.T., Perou, C.M. and van de Rijn, M. Towards a novel classification of human
malignancies based on gene expression patterns. J Pathol, 195: 41-52. (2001).
Allouche, M., Bayard, F., Clamens, S., Fillola, G., Si, P. and Amalric, F. Expression of basic fibroblast
growth factor (bFGF) and FGF-receptors in human leukemic cells. Leukemia, 9: 77-86. (1995).
Andersen, M.K., Christiansen, D.H., Kirchhoff, M. and Pedersen-Bjergaard, J. Duplication or
amplification of chromosome band 11q23, including the unrearranged MLL gene, is a recurrent
abnormality in therapy-related MDS and AML, and is closely related to mutation of the TP53 gene and
to previous therapy with alkylating agents. Genes Chromosomes Cancer, 31: 33-41. (2001).
Anderson, M.K., Hernandez-Hoyos, G., Diamond, R.A. and Rothenberg, E.V. Precise developmental
regulation of Ets family transcription factors during specification and commitment to the T cell lineage.
Development, 126: 3131-48. (1999).
Andreeff, M. and Pinkel, D., Introduction to fluorescence in situ hybridization - principles and clinical
applications, 1st ed., Wiley-Liss, New York (1999).
Armstrong, S.A., Staunton, J.E., Silverman, L.B., Pieters, R., den Boer, M.L., Minden, M.D., Salla n, S.E.,
Lander, E.S., Golub, T.R. and Korsmeyer, S.J. MLL translocations specify a distinct gene expression
profile that distinguishes a unique leukemia. Nat Genet, 30: 41-7. (2002).
Asker, C., Mareni, C., Coviello, D., Ingvarsson, S., Sessarego, M., Origone, P., Klein, G. and Sumeigi, J.
Amplification of c-myc and pvt-1 homologous sequences in acute nonlymphatic leukemia. Leuk Res,
12: 523-7 (1988).
Baccichet, A., Qualman, S.K. and Sinnett, D. Allelic loss in childhood acute lymphoblastic leukemia.
Leuk Res, 21: 817-23. (1997).
65
References
Baens, M., Wlodarska, I., Corveleyn, A., Hoornaert, I., Hagemeijer, A. and Marynen, P. A physical,
transcript, and deletion map of chromosome region 12p12.3 flanked by ETV6 and CDKN1B:
hypermethylation of the LRP6 CpG island in two leukemia patients with hemizygous del(12p).
Genomics, 56: 40-50 (1999).
Baer, M.R., Augustinos, P. and Kinniburgh, A.J. Defective c-myc and c-myb RNA turnover in acute
myeloid leukemia cells. Blood, 79: 1319-26. (1992).
Baskaran, K., Erfurth, F., Taborn, G., Copeland, N.G., Gilbert, D.J., Jenkins, N.A., Iannaccone, P.M. and
Domer, P.H. Cloning and developmental expression of the murine homolog of the acute leukemia
proto-oncogene AF4. Oncogene, 15: 1967-78. (1997).
Bentz, M., Dhner, H., Huck, K., Schtz, B., Ganser, A., Joos, S., du Manoir, S. and Lichter, P.
Comparative genomic hybridization in the investigation of myeloid leukemias. Genes Chromosomes
Cancer, 12: 193-200. (1995).
Berger, R. Acute lymphoblastic leukemia and chromosome 21. Cancer Genet Cytogenet, 94: 8-12.
(1997).
Bikfalvi, A., Han, Z.C. and Fuhrmann, G. Interaction of fibroblast growth factor (FGF) with
megakaryocytopoiesis and demonstration of FGF receptor expression in megakaryocytes and
megakaryocytic-like cells. Blood, 80: 1905-13. (1992).
Biondi, A. and Masera, G. Molecular pathogenesis of childhood acute lymphoblastic leukemia.
Haematologica, 83: 651-9. (1998).
Bjurholm, A., Kreicbergs, A., Brodin, E. and Schultzberg, M. Substance P- and CGRP-immunoreactive
nerves in bone. Peptides, 9: 165-71. (1988).
Bories, D., Raynal, M.C., Solomon, D.H., Darzynkiewicz, Z. and Cayre, Y.E. Down-regulation of a serine
protease, myeloblastin, causes growth arrest and differentiation of promyelocytic leukemia cells. Cell,
59: 959-68. (1989).
Borkhardt, A., Cazzaniga, G., Viehmann, S., Valsecchi, M.G., Ludwig, W.D., Burci, L., Mangioni, S.,
Schrappe, M., Riehm, H., Lampert, F., Basso, G., Masera, G., Harbott, J. and Biondi, A. Incidence and
clinical relevance of TEL/AML1 fusion genes in children with acute lymphoblastic leukemia enrolled
in the German and Italian multicenter therapy trials. Associazione Italiana Ematologia Oncologia
Pediatrica and the Berlin-Frankfurt-Munster Study Group. Blood, 90: 571-7. (1997).
Busson-Le Coniat, M., Nguyen Khac, F., Daniel, M.T., Bernard, O.A. and Berger, R. Chromosome 21
abnormalities with AML1 amplification in acute lymphoblastic leukemia. Genes Chromosomes Cancer,
32: 244-9. (2001).
Campana, D. and Pui, C.H. Detection of minimal residual disease in acute leukemia: methodologic
advances and clinical significance. Blood, 85: 1416-34. (1995).
Carter, T.L., Reaman, G.H. and Kees, U.R. INK4A/ARF deletions are acquired at relapse in childhood
acute lymphoblastic leukaemia: a paired study on 25 patients using real-time polymerase chain reaction.
Br J Haematol, 113: 323-8. (2001).
66
References
Cav, H., Cacheux, V., Raynaud, S., Brunie, G., Bakkus, M., Cochaux, P., Preudhomme, C., La, J.L.,
Vilmer, E. and Grandchamp, B. ETV6 is the target of chromosome 12p deletions in t(12;21) childhood
acute lymphocytic leukemia. Leukemia, 11: 1459-64. (1997).
Chambon-Pautas, C., Cav, H., Grard, B., Guidal-Giroux, C., Duval, M., Vilmer, E. and Grandchamp, B.
High-resolution allelotype analysis of childhood B-lineage acute lymphoblastic leukemia. Leukemia,
12: 1107-13. (1998).
Chen, C.S., Sorensen, P.H., Domer, P.H., Reaman, G.H., Korsmeyer, S.J., Heerema, N.A., Hammond,
G.D. and Kersey, J.H. Molecular rearrangements on chromosome 11q23 predominate in infant acute
lymphoblastic leukemia and are associated with specific biologic variables and poor outcome. Blood,
81: 2386-93. (1993).
Chen, J.S., Coustan-Smith, E., Suzuki, T., Neale, G.A., Mihara, K., Pui, C.H. and Campana, D.
Identification of novel markers for monitoring minimal residual disease in acute lymphoblastic
leukemia. Blood, 97: 2115-20. (2001).
Chessels, J.M., Swansbury, G.J., Reeves, B., Bailey, C.C. and Richards, S.M. Cytogenetics and prognosis
in childhood lymphoblastic leukaemia: results of MRC UKALL X. Medical Research Council Working
Party in Childhood Leukaemia. Br J Haematol, 99: 93-100. (1997).
Clark, S.S., McLaughlin, J., Crist, W.M., Champlin, R. and Witte, O.N. Unique forms of the abl tyrosine
kinase distinguish Ph1-positive CML from Ph1-positive ALL. Science, 235: 85-8. (1987).
Claverie, J.M. Gene number. What if there are only 30,000 human genes? Science, 291: 1255-7. (2001).
Cox, J.M. Applications of nylon membrane arrays to gene expression analysis. J Immunol Methods, 250:
3-13. (2001).
Cuneo, A., Bigoni, R., Roberti, M.G., Bardi, A., Balsamo, R., Piva, N. and Castoldi, G. Detection of
numerical aberrations in hematologic neoplasias by fluorescence in situ hybridization. Haematologica,
82: 85-90. (1997).
Dal Cin, P., Atkins, L., Ford, C., Ariyanayagam, S., Armstrong, S.A., George, R., Cleary, A. and Morton,
C.C. Amplification of AML1 in childhood acute lymphoblastic leukemias. Genes Chromosomes
Cancer, 30: 407-9. (2001).
Dalle, J.H., Fournier, M., Nelken, B., Mazingue, F., La, J.L., Bauters, F., Fenaux, P. and Quesnel, B.
p16(INK4a) immunocytochemical analysis is an independent prognostic factor in childhood acute
lymphoblastic leukemia. Blood, 99: 2620-3. (2002).
de Lau, W.B., Hurenkamp, J., Berendes, P., Touw, I.P., Clevers, H.C. and van Dijk, M.A. The gene
encoding the granulocyte colony-stimulating factor receptor is a target for deregulation in pre-B ALL
by the t(1;19)-specific oncoprotein E2A-Pbx1. Oncogene, 17: 503-10. (1998).
DeRisi, J.L. and Iyer, V.R. Genomics and array technology. Curr Opin Oncol, 11: 76-9. (1999).
Doll, R. and Wakeford, R. Risk of childhood cancer from fetal irradiation. Br J Radiol, 70: 130-9. (1997).
Donovan, J.W., Ladetto, M., Zou, G., Neuberg, D., Poor, C., Bowers, D. and Gribben, J.G.
Immunoglobulin heavy-chain consensus probes for real-time PCR quantification of residual disease in
acute lymphoblastic leukemia. Blood, 95: 2651-8. (2000).
67
References
Dopazo, J., Zanders, E., Dragoni, I., Amphlett, G. and Falciani, F. Methods and approaches in the analysis
of gene expression data. J Immunol Methods, 250: 93-112. (2001).
Downing, J.R. AML1/CBFbeta transcription complex: its role in normal hematopoiesis and leukemia.
Leukemia, 15: 664-5. (2001).
Drexler, H.G. Review of alterations of the cyclin-dependent kinase inhibitor INK4 family genes p15, p16,
p18 and p19 in human leukemia-lymphoma cells. Leukemia, 12: 845-59. (1998).
Dreyling, M.H., Bohlander, S.K., Le Beau, M.M. and Olopade, O.I. Refined mapping of genomic
rearrangements involving the short arm of chromosome 9 in acute lymphoblastic leukemias and other
hematologic malignancies. Blood, 86: 1931-8. (1995).
Druker, B.J., Sawyers, C.L., Kantarjian, H., Resta, D.J., Reese, S.F., Ford, J.M., Capdeville, R. and
Talpaz, M. Activity of a specific inhibitor of the BCR-ABL tyrosine kinase in the blast crisis of chronic
myeloid leukemia and acute lymphoblastic leukemia with the Philadelphia chromosome. N Engl J Med,
344: 1038-42. (2001a).
Druker, B.J., Talpaz, M., Resta, D.J., Peng, B., Buchdunger, E., Ford, J.M., Lydon, N.B., Kantarjian, H.,
Capdeville, R., Ohno-Jones, S. and Sawyers, C.L. Efficacy and safety of a specific inhibitor of the
BCR-ABL tyrosine kinase in chronic myeloid leukemia. N Engl J Med, 344: 1031-7. (2001b).
Drunat, S., Olivi, M., Brunie, G., Grandchamp, B., Vilmer, E., Biche, I. and Cav, H. Quantification of
TEL-AML1 transcript for minimal residual disease assessment in childhood acute lymphoblastic
leukaemia. Br J Haematol, 114: 281-9. (2001).
Duggan, D.J., Bittner, M., Chen, Y., Meltzer, P. and Trent, J.M. Expression profiling using cDNA
microarrays. Nat Genet, 21: 10-4. (1999).
Eckert, C., Landt, O., Taube, T., Seeger, K., Beyermann, B., Proba, J. and Henze, G. Potential of
LightCycler technology for quantification of minimal residual disease in childhood acute lymphoblastic
leukemia. Leukemia, 14: 316-23. (2000).
El-Rifai, W., Elonen, E., Larramendy, M., Ruutu, T. and Knuutila, S. Chromosomal breakpoints and
changes in DNA copy number in refractory acute myeloid leukemia. Leukemia, 11: 958-63. (1997a).
El-Rifai, W., Larramendy, M.L., Bjorkqvist, A.M., Hemmer, S. and Knuutila, S. Optimization of
comparative genomic hybridization using fluorochrome conjugated to dCTP and dUTP nucleotides.
Lab Invest, 77: 699-700. (1997b).
El-Rifai, W., Ruutu, T., Vettenranta, K., Elonen, E., Saarinen, U.M., Volin, L. and Knuut ila, S. Follow-up
of residual disease using metaphase-FISH in patients with acute lymphoblastic leukemia in remission.
Leukemia, 11: 633-8. (1997c).
Erikson, J., Finger, L., Sun, L., ar-Rushdi, A., Nishikura, K., Minowada, J., Finan, J., Emanuel, B.S.,
Nowell, P.C. and Croce, C.M. Deregulation of c-myc by translocation of the alpha-locus of the T-cell
receptor in T-cell leukemias. Science, 232: 884-6. (1986).
Fainstein, E., Marcelle, C., Rosner, A., Canaani, E., Gale, R.P., Dreazen, O., Smith, S.D. and Croc e, C.M.
A new fused transcript in Philadelphia chromosome positive acute lymphocytic leukaemia. Nature, 330:
386-8. (1987).
68
References
Fears, S., Gavin, M., Zhang, D.E., Hetherington, C., Ben-David, Y., Rowley, J.D. and Nucifora, G.
Functional characterization of ETV6 and ETV6/CBFA2 in the regulation of the MCSFR proximal
promoter. Proc Natl Acad Sci U S A, 94: 1949-54. (1997).
Felix, C.A., Hosler, M.R., Winick, N.J., Masterson, M., Wilson, A.E. and Lange, B.J. ALL-1 gene
rearrangements in DNA topoisomerase II inhibitor-related leukemia in children. Blood, 85: 3250-6.
(1995).
Fenrick, R., Amann, J.M., Lutterbach, B., Wang, L., Westendorf, J.J., Downing, J.R. and Hiebert, S.W.
Both TEL and AML-1 contribute repression domains to the t(12;21) fusion protein. Mol Cell Biol, 19:
6566-74. (1999).
Fernandez, S., Knopf, M.A. and McGillis, J.P. Calcitonin-gene related peptide (CGRP) inhibits
interleukin-7-induced pre-B cell colony formation. J Leukoc Biol, 67: 669-76. (2000).
Fodor, S.P., Rava, R.P., Huang, X.C., Pease, A.C., Holmes, C.P. and Adams, C.L. Multiplexed
biochemical assays with biological chips. Nature, 364: 555-6. (1993).
Ford, A.M., Ridge, S.A., Cabrera, M.E., Mahmoud, H., Steel, C.M., Chan, L.C. and Greaves, M. In utero
rearrangements in the trithorax-related oncogene in infant leukaemias. Nature, 363: 358-60. (1993).
Forestier, E., Johansson, B., Gustafsson, G., Borgstrm, G., Kerndrup, G., Johannsson, J. and Heim, S.
Prognostic impact of karyotypic findings in childhood acute lymphoblastic leukaemia: a Nordic series
comparing two treatment periods. For the Nordic Society of Paediatric Haematology and Oncology
(NOPHO) Leukaemia Cytogenetic Study Group. Br J Haematol, 110: 147-53. (2000).
Forozan, F., Karhu, R., Kononen, J., Kallioniemi, A. and Kallioniemi, O.P. Genome screening by
comparative genomic hybridization. Trends Genet, 13: 405-9. (1997).
Friedman, A.D. Leukemogenesis by CBF oncoproteins. Leukemia, 13: 1932-42. (1999).
Friedmann, A.M. and Weinstein, H.J. The role of prognostic features in the treatment of childhood acute
lymphoblastic leukemia. Oncologist, 5: 321-8 (2000).
Gale, K.B., Ford, A.M., Repp, R., Borkhardt, A., Keller, C., Eden, O.B. and Greaves, M.F. Backtracking
leukemia to birth: identification of clonotypic gene fusion sequences in neonatal blood spots. Proc Natl
Acad Sci U S A, 94: 13950-4. (1997).
Gershon, D. Microarray technology: an array of opportunities. Nature, 416: 885-91. (2002).
Golub, T.R. Genomic approaches to the pathogenesis of hematologic malignancy. Curr Opin Hematol, 8:
252-61. (2001).
Golub, T.R., Barker, G.F., Bohlander, S.K., Hiebert, S.W., Ward, D.C., Bray-Ward, P., Morgan, E.,
Raimondi, S.C., Rowley, J.D. and Gilliland, D.G. Fusion of the TEL gene on 12p13 to the AML1 gene
on 21q22 in acute lymphoblastic leukemia. Proc Natl Acad Sci U S A, 92: 4917-21. (1995).
Golub, T.R., Barker, G.F., Lovett, M. and Gilliland, D.G. Fusion of PDGF receptor beta to a novel etslike gene, tel, in chronic myelomonocytic leukemia with t(5;12) chromosomal translocation. Cell, 77:
307-16. (1994).
69
References
Golub, T.R., Slonim, D.K., Tamayo, P., Huard, C., Gaasenbeek, M., Mesirov, J.P., Coller, H., Loh, M.L.,
Downing, J.R., Caligiuri, M.A., Bloomfield, C.D. and Lander, E.S. Molecular classification of cancer:
class discovery and class prediction by gene expression monitoring. Science, 286: 531-7. (1999).
Graf Einsiedel, H., Taube, T., Hartmann, R., Eckert, C., Seifert, G., Wellmann, S., Henze, G. and Seeger,
K. Prognostic value of p16(INK4a) gene deletions in pediatric acute lymphoblastic leukemia. Blood,
97: 4002-4. (2001).
Greaves, M. Childhood leukaemia. Bmj, 324: 283-7. (2002).
Greaves, M.F. Aetiology of acute leukaemia. Lancet, 349: 344-9. (1997).
Grimwade, D., Walker, H., Oliver, F., Wheatley, K., Harrison, C., Harrison, G., Rees, J., Hann, I.,
Stevens, R., Burnett, A. and Goldstone, A. The importance of diagnostic cytogenetics on outcome in
AML: analysis of 1,612 patients entered into the MRC AML 10 trial. The Medical Research Council
Adult and Children's Leukaemia Working Parties. Blood, 92: 2322-33. (1998).
Guasch, G., Mack, G.J., Popovici, C., Dastugue, N., Birnbaum, D., Rattner, J.B. and Pbusque, M.J.
FGFR1 is fused to the centrosome-associated protein CEP110 in the 8p12 stem cell myeloproliferative
disorder with t(8;9)(p12;q33). Blood, 95: 1788-96. (2000).
Guignard, F., Mauel, J. and Markert, M. Identification and characterization of a novel human neutrophil
protein related to the S100 family. Biochem J, 309: 395-401. (1995).
Guo, S.X., Taki, T., Ohnishi, H., Piao, H.Y., Tabuchi, K., Bessho, F., Hanada, R., Yanagisawa, M. and
Hayashi, Y. Hypermethylation of p16 and p15 genes and RB protein expression in acute leukemia.
Leuk Res, 24: 39-46. (2000).
Gustafsson, G., Kreuger, A., Clausen, N., Garwicz, S., Kristinsson, J., Lie, S.O., Moe, P.J., Perkki, M.,
Yssing, M. and Saarinen-Pihkala, U.M. Intensified treatment of acute childhood lymphoblastic
leukaemia has improved prognosis, especially in non-high-risk patients: the Nordic experience of 2648
patients diagnosed between 1981 and 1996. Nordic Society of Paediatric Haematology and Oncology
(NOPHO). Acta Paediatr, 87: 1151-61. (1998).
Gustafsson, G., Schmiegelow, K., Forestier, E., Clausen, N., Glomstein, A., Jonmundsson, G., Mellander,
L., Mkipernaa, A., Nygaard, R. and Saarinen-Pihkala, U.M. Improving outcome through two decades
in childhood ALL in the Nordic countries: the impact of high-dose methotrexate in the reduction of
CNS irradiation. Nordic Society of Pediatric Haematology and Oncology (NOPHO). Leukemia, 14:
2267-75. (2000).
Haas, O., Henn, T., Romanakis, K., du Manoir, S. and Lengauer, C. Comparative genomic hybridization
as part of a new diagnostic strategy in childhood hyperdiploid acute lymphoblastic leukemia. Leukemia,
12: 474-81. (1998).
Hamaguchi, H., Yamada, M., Noguchi, A., Fujii, K., Shibasaki, M., Mukai, R., Yabe, T. and Kondo, I.
Genetic analysis of human lymphocyte proteins by two-dimensional gel electrophoresis: 2. Genetic
polymorphism of lymphocyte cytosol 64K polypeptide. Hum Genet, 60: 176-80 (1982).
Hand, D., Mannila, H. and Smyth, P., Principals of Data Mining, MIT Press, Cambridge, MA, USA
(2001).
70
References
Hanley, J.A. and McNeil, B.J. The meaning and use of the area under a receiver operating characteristic
(ROC) curve. Radiology, 143: 29-36. (1982).
Harbott, J., Viehmann, S., Borkhardt, A., Henze, G. and Lampert, F. Incidence of TEL/AML1 fusion gene
analyzed consecutively in children with acute lymphoblastic leukemia in relapse. Blood, 90: 4933-7.
(1997).
Harrington, C.A., Rosenow, C. and Retief, J. Monitoring gene expression using DNA microarrays. Curr
Opin Microbiol, 3: 285-91. (2000).
Heerema, N.A., Arthur, D.C., Sather, H., Albo, V., Feusner, J., Lange, B.J., Steinherz, P.G., Zeltzer, P.,
Hammond, D. and Reaman, G.H. Cytogenetic features of infants less than 12 months of age at
diagnosis of acute lymphoblastic leukemia: impact of the 11q23 breakpoint on outcome: a report of the
Childrens Cancer Group. Blood, 83: 2274-84. (1994).
Heerema, N.A., Sather, H.N., Sensel, M.G., Liu-Mares, W., Lange, B.J., Bostrom, B.C., Nachman, J.B.,
Steinherz, P.G., Hutchinson, R., Gaynon, P.S., Arthur, D.C. and Uckun, F.M. Association of
chromosome arm 9p abnormalities with adverse risk in childhood acute lymphoblastic leukemia: A
report from the Children's Cancer Group. Blood, 94: 1537-44. (1999).
Heim, S. and Mitelman, F., Cancer Cytogenetics, 2nd ed., Wiley-Liss, New York (1995).
Hermans, A., Heisterkamp, N., von Linden, M., van Baal, S., Meijer, D., van der Plas, D., Wiedemann,
L.M., Groffen, J., Bootsma, D. and Grosveld, G. Unique fusion of bcr and c-abl genes in Philadelphia
chromosome positive acute lymphoblastic leukemia. Cell, 51: 33-40. (1987).
Hibbs, M.L. and Dunn, A.R. Lyn, a src-like tyrosine kinase. Int J Biochem Cell Biol, 29: 397-400. (1997).
Hiebert, S.W., Lutterbach, B., Durst, K., Wang, L., Linggi, B., Wu, S., Wood, L., Amann, J., King, D.
and Hou, Y. Mechanisms of transcriptional repression by the t(8;21)-, t(12;21)-, and inv(16)-encoded
fusion proteins. Cancer Chemother Pharmacol, 48 Suppl 1: S31-4. (2001).
Hiebert, S.W., Sun, W., Davis, J.N., Golub, T., Shurtleff, S., Buijs, A., Downing, J.R., Grosveld, G.,
Roussell, M.F., Gilliland, D.G., Lenny, N. and Meyers, S. The t(12;21) translocation converts AML -1B
from an activator to a repressor of transcription. Mol Cell Biol, 16: 1349-55. (1996).
Hilsenbeck, S.G., Friedrichs, W.E., Schiff, R., O'Connell, P., Hansen, R.K., Osborne, C.K. and Fuqua,
S.A. Statistical analysis of array expression data as applied to the problem of tamoxifen resistance. J
Natl Cancer Inst, 91: 453-9. (1999).
Hoffbrand, A.V. and Pettit, J.E., Essential Haematology, 3rd ed., Blackwell Scientific Publications,
Oxford (1993).
Hughes, T.R. and Shoemaker, D.D. DNA microarrays for expression profiling. Curr Opin Chem Biol, 5:
21-5. (2001).
Jaffe, E.S., Harris, N.L., Stein, H. and Vardiman, J.W., Pathology and genetics of tumours of
haematopoietic and lymphoid tissues, IARC Press, Lyon (2001).
Jarosov, M., Holzerov, M., Jedlickov, K., Mihl, V., Zuna, J., Star, J., Pospsilov, D., Zemanov, Z.,
Trka, J., Blazek, J., Pikalov, Z. and Indrk, K. Importance of using comparative genomic hybridization
71
References
to improve detection of chromosomal changes in childhood acute lymphoblastic leukemia. Cancer

Genet Cytogenet, 123: 114-22. (2000).
Jousset, C., Carron, C., Boureux, A., Quang, C.T., Oury, C., Dusanter-Fourt, I., Charon, M., Levin, J.,
Bernard, O. and Ghysdael, J. A domain of TEL conserved in a subset of ETS proteins defines a specific
oligomerization interface essential to the mitogenic properties of the TEL-PDGFR beta oncoprotein.
Embo J, 16: 69-82. (1997).
Kakazu, N., Taniwaki, M., Horiike, S., Nishida, K., Tatekawa, T., Nagai, M., Takahashi, T., Akaogi, T.,
Inazawa, J., Ohki, M. and Abe, T. Combined spectral karyotyping and DAPI banding analysis of
chromosome abnormalities in myelodysplastic syndrome. Genes Chromosomes Cancer, 26: 336-45.
(1999).
Kallioniemi, A., Kallioniemi, O.P., Sudar, D., Rutovitz, D., Gray, J.W., Waldman, F. and Pinkel, D.
Comparative genomic hybridization for molecular cytogenetic analysis of solid tumors. Science, 258:
818-21. (1992).
Kallioniemi, O.P., Kallioniemi, A., Piper, J., Isola, J., Waldman, F.M., Gray, J.W. and Pinkel, D.
Optimizing comparative genomic hybridization for analysis of DNA sequence copy number changes in
solid tumors. Genes Chromosomes Cancer, 10: 231-43. (1994).
Kamps, M.P., Murre, C., Sun, X.H. and Baltimore, D. A new homeobox gene contributes the DNA
binding domain of the t(1;19) translocation protein in pre-B ALL. Cell, 60: 547-55. (1990).
Karhu, R., Siitonen, S., Tanner, M., Keinnen, M., Mkipernaa, A., Lehtinen, M., Vilpo, J.A. and Isola, J.
Genetic aberrations in pediatric acute lymphoblastic leukemia by comparative genomic hybridization.
Cancer Genet Cytogenet, 95: 123-9. (1997).
Kearney, L. The impact of the new fish technologies on the cytogenetics of haematological malignancies.
Br J Haematol, 104: 648-58. (1999).
Kim, M.H., Stewart, J., Devlin, C., Kim, Y.T., Boyd, E. and Connor, M. The application of comparative
genomic hybridization as an additional tool in the chromosome analysis of acute myeloid leukemia and
myelodysplastic syndromes. Cancer Genet Cytogenet, 126: 26-33. (2001).
Kirchhoff, M., Gerdes, T., Maahr, J., Rose, H., Bentz, M., Dhner, H. and Lundsteen, C. Deletions below
10 megabasepairs are detected in comparative genomic hybridization by standard reference intervals.
Genes Chromosomes Cancer, 25: 410-3. (1999).
Knudson, A.G. Chasing the cancer demon. Annu Rev Genet, 34: 1-19 (2000).
Knuutila, S., Aalto, Y., Autio, K., Bjrkqvist, A.M., El-Rifai, W., Hemmer, S., Huhta, T., Kettunen, E.,
Kiuru-Kuhlefelt, S., Larramendy, M.L., Lushnikova, T., Monni, O., Pere, H., Tapper, J., Tarkkanen,
M., Varis, A., Wasenius, V.M., Wolf, M. and Zhu, Y. DNA copy number losses in human neoplasms.
Am J Pathol, 155: 683-94. (1999).
Knuutila, S., Bjrkqvist, A.M., Autio, K., Tarkkanen, M., Wolf, M., Monni, O., Szymanska, J.,
Larramendy, M.L., Tapper, J., Pere, H., El-Rifai, W., Hemmer, S., Wasenius, V.M., Vidgren, V. and
Zhu, Y. DNA copy number amplifications in human neoplasms: review of comparative genomic
hybridization studies. Am J Pathol, 152: 1107-23. (1998).
72
References
Knuutila, S., Vuopio, P., Elonen, E., Siimes, M., Kovanen, R., Borgstrom, G.H. and de la Chapelle, A.
Culture of bone marrow reveals more cells with chromosomal abnormalities than the direct method in
patients with hematologic disorders. Blood, 58: 369-75. (1981).
Komuro, H., Valentine, M.B., Rubnitz, J.E., Saito, M., Raimondi, S.C., Carroll, A.J., Yi, T., Sherr, C.J.
and Look, A.T. p27KIP1 deletions in childhood acute lymphoblastic leukemia. Neoplasia, 1: 253-61.
(1999).
Kurokawa, M., Tanaka, T., Tanaka, K., Ogawa, S., Mitani, K., Yazaki, Y. and Hirai, H. Overexpression
of the AML1 proto-oncoprotein in NIH3T3 cells leads to neoplastic transformation depending on the
DNA-binding and transactivational potencies. Oncogene, 12: 883-92. (1996).
Kwiatkowski, B.A., Bastian, L.S., Bauer, T.R., Jr., Tsai, S., Zielinska-Kwiatkowska, A.G. and Hickstein,
D.D. The ets family member Tel binds to the Fli-1 oncoprotein and inhibits its transcriptional activity. J
Biol Chem, 273: 17525-30. (1998).
Kwong, Y.L. and Wong, K.F. Low frequency of TEL/AML1 in adult acute lymphoblastic leukemia.
Lanza, C., Volpe, G., Basso, G., Gottardi, E., Perfetto, F., Cilli, V., Spinelli, M., Ricotti, E., Guerrasio, A.,
Madon, E. and Saglio, G. The common TEL/AML1 rearrangement does not represent a frequent event
in acute lymphoblastic leukaemia occuring in children with Down syndrome. Leukemia, 11: 820-1.
(1997).
Larramendy, M.L., Huhta, T., Heinonen, K., Vettenranta, K., Mahlamaki, E., Riikonen, P., SaarinenPihkala, U.M. and Knuutila, S. DNA copy number changes in childhood acute lymphoblastic leukemia.
Haematologica, 83: 890-5. (1998).
Larramendy, M.L., Niini, T., Elonen, E., Nagy, B., Ollila, J., Vihinen, M. and Knuutila, S.
Overexpression of translocation-associated fusion genes of FGFR1, MYC, NPMI, and DEK, but
absense of translocations in acute myeloid leukemia. A microarray analysis. Haematologica, 87: 569577 (2002).
Larsson, L.G., Schena, M., Carlsson, M., Sllstrm, J. and Nilsson, K. Expression of the c-myc protein is
down-regulated at the terminal stages during in vitro differentiation of B-type chronic lymphocytic
leukemia cells. Blood, 77: 1025-32. (1991).
LeBrun, D.P. and Cleary, M.L. Fusion with E2A alters the transcriptional properties of the homeodomain
protein PBX1 in t(1;19) leukemias. Oncogene, 9: 1641-7. (1994).
Levanon, D., Glusman, G., Bangsow, T., Ben-Asher, E., Male, D.A., Avidan, N., Bangsow, C., Hattori,
M., Taylor, T.D., Taudien, S., Blechschmidt, K., Shimizu, N., Rosenthal, A., Sakaki, Y., Lancet, D. and
Groner, Y. Architecture and anatomy of the genomic locus encoding the human leukemia-associated
transcription factor RUNX1/AML1. Gene, 262: 23-33. (2001).
Liang, D.C., Chou, T.B., Chen, J.S., Shurtleff, S.A., Rubnitz, J.E., Downing, J.R., Pui, C.H. and Shih,
L.Y. High incidence of TEL/AML1 fusion resulting from a cryptic t(12;21) in childhood B-lineage
acute lymphoblastic leukemia in Taiwan. Leukemia, 10: 991-3. (1996).
Lipshutz, R.J., Fodor, S.P., Gingeras, T.R. and Lockhart, D.J. High density synthetic oligonucleotide
arrays. Nat Genet, 21: 20-4. (1999).
73
References
Liu, P., Tarl, S.A., Hajra, A., Claxton, D.F., Marlton, P., Freedman, M., Siciliano, M.J. and Collins, F.S.
Fusion between transcription factor CBF beta/PEBP2 beta and a myosin heavy chain in acute myeloid
leukemia. Science, 261: 1041-4. (1993).
Lo Coco, F., Pisegna, S. and Diverio, D. The AML1 gene: a transcription factor involved in the
pathogenesis of myeloid and lymphoid leukemias. Haematologica, 82: 364-70. (1997).
Lockhart, D.J. and Winzeler, E.A. Genomics, gene expression and DNA arrays. Nature, 405: 827-36.
(2000).
Loh, M.L., Silverman, L.B., Young, M.L., Neuberg, D., Golub, T.R., Sallan, S.E. and Gilliland, D.G.
Incidence of TEL/AML1 fusion in children with relapsed acute lymphoblastic leukemia. Blood, 92:
4792-7. (1998).
Look, A.T. Oncogenic transcription factors in the human acute leukemias. Science, 278: 1059-64. (1997).
Lopez, R.G., Carron, C., Oury, C., Gardellin, P., Bernard, O. and Ghysdael, J. TEL is a sequence-specific
transcriptional repressor. J Biol Chem, 274: 30132-8. (1999).
Lu, Q., Wright, D.D. and Kamps, M.P. Fusion with E2A converts the Pbx1 homeodomain protein into a
constitutive transcriptional activator in human leukemias carrying the t(1;19) translocation. Mol Cell
Biol, 14: 3938-48. (1994).
Lugo, T.G., Pendergast, A.M., Muller, A.J. and Witte, O.N. Tyrosine kinase activity and transformation
potency of bcr-abl oncogene products. Science, 247: 1079-82. (1990).
Luo, L., Salunga, R.C., Guo, H., Bittner, A., Joy, K.C., Galindo, J.E., Xiao, H., Rogers, K.E., Wan, J.S.,
Jackson, M.R. and Erlander, M.G. Gene expression profiles of laser-captured adjacent neuronal
subtypes. Nat Med, 5: 117-22. (1999).
Lutterbach, B. and Hiebert, S.W. Role of the transcription factor AML-1 in acute leukemia and
hematopoietic differentiation. Gene, 245: 223-35. (2000).
Lutz, P.G., Moog-Lutz, C., Coumau-Gatbois, E., Kobari, L., Di Gioia, Y. and Cayre, Y.E. Myeloblastin is
a granulocyte colony-stimulating factor-responsive gene conferring factor-independent growth to
hematopoietic cells. Proc Natl Acad Sci U S A, 97: 1601-6. (2000).
Ma, S.K., Wan, T.S. and Chan, L.C. Cytogenetics and molecular genetics of childhood leukemia. Hematol
Oncol, 17: 91-105. (1999).
Ma, S.K., Wan, T.S., Cheuk, A.T., Fung, L.F., Chan, G.C., Chan, S.Y., Ha, S.Y. and Chan, L.C.
Characterization of additional genetic events in childhood acute lymphoblastic leukemia with
TEL/AML1 gene fusion: a molecular cytogenetics study. Leukemia, 15: 1442-7. (2001).
Maloney, K., McGavran, L., Murphy, J., Odom, L., Stork, L., Wei, Q. and Hunger, S. TEL-AML1 fusion
identifies a subset of children with standard risk acute lymphoblastic leukemia who have an excellent
prognosis when treated with therapy that includes a single delayed intensification. Leukemia, 13: 170812. (1999a).
Maloney, K.W., McGavran, L., Odom, L.F. and Hunger, S.P. Acquisition of p16(INK4A) and
p15(INK4B) gene abnormalities between initial diagnosis and relapse in children with acute
lymphoblastic leukemia. Blood, 93: 2380-5. (1999b).
74
References
Marx, J. Medicine. DNA arrays reveal cancer in its many forms. Science, 289: 1670-2. (2000).
Mathew, S., Rao, P.H., Dalton, J., Downing, J.R. and Raimondi, S.C. Multicolor spectral karyotyping
identifies novel translocations in childhood acute lymphoblastic leukemia. Leukemia, 15: 468-72.
(2001).
Maughan, N.J., Lewis, F.A. and Smith, V. An introduction to arrays. J Pathol, 195: 3-6. (2001).
McLean, T.W., Ringold, S., Neuberg, D., Stegmaier, K., Tantravahi, R., Ritz, J., Koeffler, H.P., Takeuchi,
S., Janssen, J.W., Seriu, T., Bartram, C.R., Sallan, S.E., Gilliland, D.G. and Golub, T.R. TEL/AML-1
dimerizes and is associated with a favorable outcome in childhood acute lymphoblastic leukemia.
Blood, 88: 4252-8. (1996).
Melnick, A. and Licht, J.D. Deconstructing a disease: RARalpha, its fusion partners, and their roles in the
pathogenesis of acute promyelocytic leukemia. Blood, 93: 3167-215. (1999).
Meyers, S., Downing, J.R. and Hiebert, S.W. Identification of AML -1 and the (8;21) translocation protein
(AML- 1/ETO) as sequence-specific DNA-binding proteins: the runt homology domain is required for
DNA binding and protein-protein interactions. Mol Cell Biol, 13: 6336-45. (1993).
Meyers, S., Lenny, N. and Hiebert, S.W. The t(8;21) fusion protein interferes with AML-1B-dependent
transcriptional activation. Mol Cell Biol, 15: 1974-82. (1995).
Miyoshi, H., Ohira, M., Shimizu, K., Mitani, K., Hirai, H., Imai, T., Yokoyama, K., Soeda, E. and Ohki,
M. Alternative splicing and genomic structure of the AML1 gene involved in acute myeloid leukemia.
Nucleic Acids Res, 23: 2762-9. (1995).
Miyoshi, H., Shimizu, K., Kozu, T., Maseki, N., Kaneko, Y. and Ohki, M. t(8;21) breakpoints on
chromosome 21 in acute myeloid leukemia are clustered within a limited region of a single gene,
AML1. Proc Natl Acad Sci U S A, 88: 10431-4. (1991).
Mizuno, K., Katagiri, T., Hasegawa, K., Ogimoto, M. and Yakura, H. Hematopoietic cell phosphatase,
SHP-1, is constitutively associated with the SH2 domain-containing leukocyte protein, SLP-76, in B
cells. J Exp Med, 184: 457-63. (1996).
Mizutani, S. Recent advances in the study of the hereditary and environmental basis of childhood
leukemia. Int J Hematol, 68: 131-43. (1998).
Monni, O., Joensuu, H., Franssila, K., Klefstrom, J., Alitalo, K. and Knuutila, S. BCL2 overexpression
associated with chromosomal amplification in diffuse large B-cell lymphoma. Blood, 90: 1168-74.
(1997).
Murray, J.C., Poplack, D.G., Mahoney, D.H., Jr., Gresik, M.V., Harrison, W.R. and Cooley, L.D.
Detection of double minute chromosomes in a child with acute lymphoblastic leukemia. Cancer Genet
Cytogenet, 101: 138-42. (1998).
Nagata, S. and Fukunaga, R. Granulocyte colony-stimulating factor and its receptor. Prog Growth Factor
Res, 3: 131-41 (1991).
Nakayama, Y., Iwamoto, Y., Maher, S.E., Tanaka, Y. and Bothwell, A.L. Altered gene expression upon
BCR cross-linking in Burkitt's lymphoma B cell line. Biochem Biophys Res Commun, 277: 124-7.
(2000).
75
References
Nordgren, A., Heyman, M., Sahln, S., Schoumans, J., Sderhll, S., Nordenskjld, M. and Blennow, E.
Spectral karyotyping and interphase FISH reveal abnormalities not detected by conventional Gbanding. Implications for treatment stratification of childhood acute lymphoblastic leukaemia: detailed
analysis of 70 cases. Eur J Haematol, 68: 31-41. (2002).
Nourse, J., Mellentin, J.D., Galili, N., Wilkinson, J., Stanbridge, E., Smith, S.D. and Cleary, M.L.
Chromosomal translocation t(1;19) results in synthesis of a homeobox fusion mRNA that codes for a
potential chimeric transcription factor. Cell, 60: 535-45. (1990).
O'Connor, H.E., Butler, T.A., Clark, R., Swanton, S., Harrison, C.J., Secker-Walker, L.M. and Foroni, L.
Abnormalities of the ETV6 gene occur in the majority of patients with aberrations of the short arm of
chromosome 12: a combined PCR and Southern blotting analysis. Leukemia, 12: 1099-106. (1998).
Ogawa, E., Inuzuka, M., Maruyama, M., Satake, M., Naito-Fujimoto, M., Ito, Y. and Shigesada, K.
Molecular cloning and characterization of PEBP2 beta, the heterodimeric partner of a novel Drosophila
runt-related DNA binding protein PEBP2 alpha. Virology, 194: 314-31. (1993).
Ornitz, D.M., Xu, J., Colvin, J.S., McEwen, D.G., MacArthur, C.A., Coulier, F., Gao, G. and Goldfarb,
M. Receptor specificity of the fibroblast growth factor family. J Biol Chem, 271: 15292-7. (1996).
Osato, M., Asou, N., Abdalla, E., Hoshino, K., Yamasaki, H., Okubo, T., Suzushima, H., Takatsuki, K.,
Kanno, T., Shigesada, K. and Ito, Y. Biallelic and heterozygous point mutations in the runt domain of
the AML1/PEBP2alphaB gene associated with myeloblastic leukemias. Blood, 93: 1817-24. (1999).
Park, J.P., Ladd, S.L., Ely, P., Weiner, N.J., Wojiski, S.A., Hawk, A.B., Noll, W.W. and Mohandas, T.K.
Amplification of the MLL region in acute myeloid leukemia. Cancer Genet Cytogenet, 121: 198-205.
(2000).
Paszek-Vigier, M., Talmant, P., Mchinaud, F., Garand, R., Harousseau, J.L., Bataille, R. and AvetLoiseau, H. Comparative genomic hybridization is a powerful tool, complementary to cytogenetics, to
identify chromosomal abnormalities in childhood acute lymphoblastic leukaemia. Br J Haematol, 99:
589-96. (1997).
Patel, A.S., Hawkins, A.L. and Griffin, C.A. Cytogenetics and cancer. Curr Opin Oncol, 12: 62-7.
(2000).
Pedersen-Bjergaard, J. and Rowley, J.D. The balanced and the unbalanced chromosome aberrations of
acute myeloid leukemia may develop in different ways and may contribute differently to malignant
transformation. Blood, 83: 2780-6. (1994).
Pennisi, E. Human Genome Project. And the gene number is...? Science, 288: 1146-7. (2000).
Perkins, D., Brennan, S., Carstairs, K., Bailey, D., Pantalony, D., Poon, A., Fernandes, B. and Dub, I.
Regional cancer cytogenetics: a report on 1,143 diagnostic cases. Cancer Genet Cytogenet, 96: 64-80.
(1997).
Perrillat, F., Clavel, J., Jaussent, I., Baruchel, A., Leverger, G., Nelken, B., Philippe, N., Schaison, G.,
Sommelet, D., Vilmer, E., Bonaiti-Pellie, C. and Hemon, D. Family cancer history and risk of
childhood acute leukemia (France). Cancer Causes Control, 12: 935-41. (2001).
76
References
Petridou, E., Trichopoulos, D., Dessypris, N., Flytzani, V., Haidas, S., Kalmanti, M., Koliouskas, D.,
Kosmidis, H., Piperopoulou, F. and Tzortzatou, F. Infant leukaemia after in utero exposure to radiation
from Chernobyl. Nature, 382: 352-3. (1996).
Philipp-Staheli, J., Payne, S.R. and Kemp, C.J. p27(Kip1): regulation and function of a haploinsufficient
tumor suppressor and its misregulation in cancer. Exp Cell Res, 264: 148-68. (2001).
Pietenpol, J.A., Bohlander, S.K., Sato, Y., Papadopoulos, N., Liu, B., Friedman, C., Trask, B.J., Roberts,
J.M., Kinzler, K.W., Rowley, J.D. and Vogelstein, B. Assignment of the human p27Kip1 gene to 12p13
and its analysis in leukemias. Cancer Res, 55: 1206-10. (1995).
Pizette, S., Coulier, F., Birnbaum, D. and DeLapeyrire, O. FGF6 modulates the expression of fibroblast
growth factor receptors and myogenic genes in muscle cells. Exp Cell Res, 224: 143-51. (1996).
Poirel, H., Oury, C., Carron, C., Duprez, E., Laabi, Y., Tsapis, A., Romana, S.P., Mauchauffe, M., Le
Coniat, M., Berger, R., Ghysdael, J. and Bernard, O.A. The TEL gene products: nuclear
phosphoproteins with DNA binding properties. Oncogene, 14: 349-57. (1997).
Pollack, J.R., Perou, C.M., Alizadeh, A.A., Eisen, M.B., Pergamenschikov, A., Williams, C.F., Jeffrey,
S.S., Botstein, D. and Brown, P.O. Genome-wide analysis of DNA copy-number changes using cDNA
microarrays. Nat Genet, 23: 41-6. (1999).
Popovici, C., Adlade, J., Ollendorff, V., Chaffanet, M., Guasch, G., Jacrot, M., Leroux, D., Birnbaum,
D. and Pbusque, M.J. Fibroblast growth factor receptor 1 is fused to FIM in stem-cell
myeloproliferative disorder with t(8;13). Proc Natl Acad Sci U S A, 95: 5712-7. (1998).
Popovici, C., Zhang, B., Grgoire, M.J., Jonveaux, P., Lafage-Pochitaloff, M., Birnbaum, D. and
Pbusque, M.J. The t(6;8)(q27;p11) translocation in a stem cell myeloproliferative disorder fuses a
novel gene, FOP, to fibroblast growth factor receptor 1. Blood, 93: 1381-9. (1999).
Preisler, H.D., Kinniburgh, A.J., Wei-Dong, G. and Khan, S. Expression of the protooncogenes c-myc, c-fos, and cfms in acute myelocytic leukemia at diagnosis and in remission. Cancer Res, 47: 874-80. (1987).
Pui, C.H. Childhood leukemias. N Engl J Med, 332: 1618-30. (1995).
Pui, C.H. Acute lymphoblastic leukemia in children. Curr Opin Oncol, 12: 3-12. (2000).
Pui, C.H., Behm, F.G., Downing, J.R., Hancock, M.L., Shurtleff, S.A., Ribeiro, R.C., Head, D.R.,
Mahmoud, H.H., Sandlund, J.T., Furman, W.L., Roberts, W.M., Crist, W.M. and Raimondi, S.C.
11q23/MLL rearrangement confers a poor prognosis in infants with acute lymphoblastic leukemia. J
Clin Oncol, 12: 909-15. (1994).
Pui, C.H., Boyett, J.M., Rivera, G.K., Hancock, M.L., Sandlund, J.T., Ribeiro, R.C., Rubnitz, J.E., Behm,
F.G., Raimondi, S.C., Gajjar, A., Razzouk, B., Campana, D., Kun, L.E., Relling, M.V. and Evans, W.E.
Long-term results of Total Therapy studies 11, 12 and 13A for childhood acute lymphoblastic leukemia
at St Jude Children's Research Hospital. Leukemia, 14: 2286-94. (2000).
Pui, C.H. and Evans, W.E. Acute lymphoblastic leukemia. N Engl J Med, 339: 605-15. (1998).
Pui, C.H., Raimondi, S.C., Head, D.R., Schell, M.J., Rivera, G.K., Mirro, J., Jr., Crist, W.M. and Behm,
F.G. Characterization of childhood acute leukemia with multiple myeloid and lymphoid markers at
diagnosis and at relapse. Blood, 78: 1327-37. (1991).
77
References
Raap, A.K. Advances in fluorescence in situ hybridization. Mutat Res, 400: 287-98. (1998).
Raimondi, S.C. Current status of cytogenetic research in childhood acute lymphoblastic leukemia. Blood,
81: 2237-51. (1993).
Raimondi, S.C., Behm, F.G., Roberson, P.K., Williams, D.L., Pui, C.H., Crist, W.M., Look, A.T. and
Rivera, G.K. Cytogenetics of pre-B-cell acute lymphoblastic leukemia with emphasis on prognostic
implications of the t(1;19). J Clin Oncol, 8: 1380-8. (1990).
Raimondi, S.C., Pui, C.H., Hancock, M.L., Behm, F.G., Filatov, L. and Rivera, G.K. Heterogeneity of
hyperdiploid (51-67) childhood acute lymphoblastic leukemia. Leukemia, 10: 213-24. (1996).
Raimondi, S.C., Roberson, P.K., Pui, C.H., Behm, F.G. and Rivera, G.K. Hyperdiploid (47-50) acute
lymphoblastic leukemia in children. Blood, 79: 3245-52. (1992).
Raimondi, S.C., Shurtleff, S.A., Downing, J.R., Rubnitz, J., Mathew, S., Hancock, M., Pui, C.H., Rivera,
G.K., Grosveld, G.C. and Behm, F.G. 12p abnormalities and the TEL gene (ETV6) in childhood acute
lymphoblastic leukemia. Blood, 90: 4559-66. (1997).
Ramaswamy, S. and Golub, T.R. DNA microarrays in clinical oncology. J Clin Oncol, 20: 1932-41.
(2002).
Raynaud, S., Cav, H., Baens, M., Bastard, C., Cacheux, V., Grosgeorge, J., Guidal-Giroux, C., Guo, C.,
Vilmer, E., Marynen, P. and Grandchamp, B. The 12;21 translocation involving TEL and deletion of
the other TEL allele: two frequently associated alterations found in childhood acute lymphoblastic
leukemia. Blood, 87: 2891-9. (1996).
Robinson, M.J. and Hogg, N. A comparison of human S100A12 with MRP-14 (S100A9). Biochem
Biophys Res Commun, 275: 865-70. (2000).
Roitt, I., Brostoff, J. and Male, D., Immunology, Mosby (1996).
Romana, S.P., Le Coniat, M., Poirel, H., Marynen, P., Bernard, O. and Berger, R. Deletion of the short
arm of chromosome 12 is a secondary event in acute lymphoblastic leukemia with t(12;21). Leukemia,
10: 167-70. (1996).
Romana, S.P., Mauchauff, M., Le Coniat, M., Chumakov, I., Le Paslier, D., Berger, R. and Bernard,
O.A. The t(12;21) of acute lymphoblastic leukemia results in a tel-AML1 gene fusion. Blood, 85: 366270. (1995a).
Romana, S.P., Poirel, H., Leconiat, M., Flexor, M.A., Mauchauffe, M., Jonveaux, P., Macintyre, E.A.,
Berger, R. and Bernard, O.A. High frequency of t(12;21) in childhood B-lineage acute lymphoblastic
leukemia. Blood, 86: 4263-9. (1995b).
Rosenberg, H.F. The eosinophil ribonucleases. Cell Mol Life Sci, 54: 795-803. (1998).
Ross, D.T., Scherf, U., Eisen, M.B., Perou, C.M., Rees, C., Spellman, P., Iyer, V., Jeffrey, S.S., Van de
Rijn, M., Waltham, M., Pergamenschikov, A., Lee, J.C., Lashkari, D., Shalon, D., Myers, T.G.,
Weinstein, J.N., Botstein, D. and Brown, P.O. Systematic variation in gene expression patterns in
human cancer cell lines. Nat Genet, 24: 227-35. (2000).
Ross, J.A. Maternal diet and infant leukemia: a role for DNA topoisomerase II inhibitors? Int J Cancer
Suppl, 11: 26-8 (1998).
78
References
Rowley, J.D. The critical role of chromosome translocations in human leukemias. Annu Rev Genet, 32:
495-519 (1998).
Rozovskaia, T., Feinstein, E., Mor, O., Foa, R., Blechman, J., Nakamura, T., Croce, C.M., Cimino, G. and
Canaani, E. Upregulation of Meis1 and HoxA9 in acute lymphocytic leukemias with the t(4;11)
abnormality. Oncogene, 20: 874-8. (2001).
Rubin, C.M., Arthur, D.C., Woods, W.G., Lange, B.J., Nowell, P.C., Rowley, J.D., Nachman, J.,
Bostrom, B., Baum, E.S., Suarez, C.R., Shah, N.R., Morgan, E., Mauer, H.S., McKenzie, S.E., Larson,
R.A. and Le Beau, M.M. Therapy-related myelodysplastic syndrome and acute myeloid leukemia in
children: correlation between chromosomal abnormalities and prior therapy. Blood, 78: 2982-8. (1991).
Rubnitz, J.E., Behm, F.G., Pui, C.H., Evans, W.E., Relling, M.V., Raimondi, S.C., Harrison, P.L.,
Sandlund, J.T., Ribeiro, R.C., Grosveld, G. and Downing, J.R. Genetic studies of childhood acute
lymphoblastic leukemia with emphasis on p16, MLL, and ETV6 gene abnormalities: results of St Jude
Total Therapy Study XII. Leukemia, 11: 1201-6. (1997a).
Rubnitz, J.E., Behm, F.G., Wichlan, D., Ryan, C., Sandlund, J.T., Ribeiro, R.C., Rivera, G.K., Hancock,
M.L., Relling, M.V., Evans, W.E., Pui, C.H. and Downing, J.R. Low frequency of TEL-AML1 in
relapsed acute lymphoblastic leukemia supports a favorable prognosis for this genetic subgroup.
Leukemia, 13: 19-21. (1999a).
Rubnitz, J.E., Downing, J.R., Pui, C.H., Shurtleff, S.A., Raimondi, S.C., Evans, W.E., Head, D.R., Crist,
W.M., Rivera, G.K., Hancock, M.L., Boyett, J.M., Buijs, A., Grosveld, G. and Behm, F.G. TEL gene
rearrangement in acute lymphoblastic leukemia: a new genetic marker with prognostic significance. J
Clin Oncol, 15: 1150-7. (1997b).
Rubnitz, J.E., Link, M.P., Shuster, J.J., Carroll, A.J., Hakami, N., Frankel, L.S., Pullen, D.J. and Cleary,
M.L. Frequency and prognostic significance of HRX rearrangements in infant acute lymphoblastic
leukemia: a Pediatric Oncology Group study. Blood, 84: 570-3. (1994).
Rubnitz, J.E., Pui, C.H. and Downing, J.R. The role of TEL fusion genes in pediatric leukemias.
Leukemia, 13: 6-13. (1999b).
Rubnitz, J.E., Shuster, J.J., Land, V.J., Link, M.P., Pullen, D.J., Camitta, B.M., Pui, C.H., Downing, J.R.
and Behm, F.G. Case-control study suggests a favorable impact of TEL rearrangement in patients with
B-lineage acute lymphoblastic leukemia treated with antimetabolite-based therapy: a Pediatric
Oncology Group study. Blood, 89: 1143-6. (1997c).
Rudd, C.E. Lymphocyte signaling: adapting new adaptors. Curr Biol, 8: R805-8. (1998).
Sato, Y., Suto, Y., Pietenpol, J., Golub, T.R., Gilliland, D.G., Davis, E.M., Le Beau, M.M., Roberts, J.M.,
Vogelstein, B., Rowley, J.D. and Bohlander, S.K. TEL and KIP1 define the smallest regi on of deletions
on 12p13 in hematopoietic malignancies. Blood, 86: 1525-1533 (1995).
Schena, M., Shalon, D., Davis, R.W. and Brown, P.O. Quantitative monitoring of gene expression
patterns with a complementary DNA microarray. Science, 270: 467-70. (1995).
Schena, M., Shalon, D., Heller, R., Chai, A., Brown, P.O. and Davis, R.W. Parallel human genome
analysis: microarray-based expression monitoring of 1000 genes. Proc Natl Acad Sci U S A, 93: 106149. (1996).
79
References
Scholz, I., Popp, S., Granzow, M., Schoell, B., Holtgreve-Grez, H., Takeuchi, S., Schrappe, M., Harbott,
J., Teigler-Schlegel, A., Zimmermann, M., Fischer, C., Koeffler, H.P., Bartram, C.R. and Jauch, A.
Comparative genomic hybridization in childhood acute lymphoblastic leukemia: correlation with
interphase cytogenetics and loss of heterozygosity analysis. Cancer Genet Cytogenet, 124: 89-97.
(2001).
Schrck, E., du Manoir, S., Veldman, T., Schoell, B., Wienberg, J., Ferguson-Smith, M.A., Ning, Y.,
Ledbetter, D.H., Bar-Am, I., Soenksen, D., Garini, Y. and Ried, T. Multicolor spectral karyotyping of
human chromosomes. Science, 273: 494-7. (1996).
Schwab, M. Oncogene amplification in solid tumors. Semin Cancer Biol, 9: 319-25. (1999).
Seeger, K., Adams, H.P., Buchwald, D., Beyermann, B., Kremens, B., Niemeyer, C., Ritter, J., Schwabe,
D., Harms, D., Schrappe, M. and Henze, G. TEL-AML1 fusion transcript in relapsed childhood acute
lymphoblastic leukemia. The Berlin-Frankfurt-Munster Study Group. Blood, 91: 1716-22. (1998).
Seeger, K., Stackelberg, A.V., Taube, T., Buchwald, D., Krner, G., Suttorp, M., Drffel, W., Tausch, W.
and Henze, G. Relapse of TEL-AML1-positive acute lymphoblastic leukemia in childhood: a matchedpair analysis. J Clin Oncol, 19: 3188-93. (2001).
Shurtleff, S.A., Buijs, A., Behm, F.G., Rubnitz, J.E., Raimondi, S.C., Hancock, M.L., Chan, G.C., Pui,
C.H., Grosveld, G. and Downing, J.R. TEL/AML1 fusion resulting from a cryptic t(12;21) is the most
common genetic lesion in pediatric ALL and defines a subgroup of patients with an excellent progn osis.
Leukemia, 9: 1985-9. (1995).
Solinas-Toldo, S., Lampel, S., Stilgenbauer, S., Nickolenko, J., Benner, A., Dhner, H., Cremer, T. and
Lichter, P. Matrix-based comparative genomic hybridization: biochips to screen for genomic
imbalances. Genes Chromosomes Cancer, 20: 399-407. (1997).
Song, W.J., Sullivan, M.G., Legare, R.D., Hutchings, S., Tan, X., Kufrin, D., Ratajczak, J., Resende, I.C.,
Haworth, C., Hock, R., Loh, M., Felix, C., Roy, D.C., Busque, L., Kurnit, D., Willman, C., Gewirtz,
A.M., Speck, N.A., Bushweller, J.H., Li, F.P., Gardiner, K., Poncz, M., Maris, J.M. and Gilliland, D.G.
Haploinsufficiency of CBFA2 causes familial thrombocytopenia with propensity to develop acute
myelogenous leukaemia. Nat Genet, 23: 166-75. (1999).
Speck, N.A., Stacy, T., Wang, Q., North, T., Gu, T.L., Miller, J., Binder, M. and Marn-Padilla, M. Corebinding factor: a central player in hematopoiesis and leukemia. Cancer Res, 59: 1789s-93s. (1999).
Speicher, M.R., Gwyn Ballard, S. and Ward, D.C. Karyotyping human chromosomes by combinatorial
multi-fluor FISH. Nat Genet, 12: 368-75. (1996).
Stegmaier, K., Takeuchi, S., Golub, T.R., Bohlander, S.K., Bartram, C.R., Koeffler, H.P. and Gilliland,
D.G. Mutational analysis of the candidate tumor suppressor genes TEL and KIP1 in childhood acute
lymphoblastic leukemia. Cancer Res, 56: 1413-7. (1996).
Streubel, B., Valent, P., Jger, U., Edelhuser, M., Wandt, H., Wagner, T., Bchner, T., Lechner, K. and
Fonatsch, C. Amplification of the MLL gene on double minutes, a homogeneously staining region, and
ring chromosomes in five patients with acute myeloid leukemia or myelodysplastic syndrome. Genes
Chromosomes Cancer, 27: 380-6. (2000).
80
References
Streubel, B., Valent, P., Lechner, K. and Fonatsch, C. Amplification of the AML1(CBFA2) gene on ring
chromosomes in a patient with acute myeloid leukemia and a constitutional ring chromosome 21.
Strom, D.K., Nip, J., Westendorf, J.J., Linggi, B., Lutterbach, B., Downing, J.R., Lenny, N. and Hiebert,
S.W. Expression of the AML-1 oncogene shortens the G(1) phase of the cell cycle. J Biol Chem, 275:
3438-45. (2000).
Su, G.H., Ip, H.S., Cobb, B.S., Lu, M.M., Chen, H.M. and Simon, M.C. The Ets protein Spi-B is
expressed exclusively in B cells and T cells during development. J Exp Med, 184: 203-14 (1996).
Suda, T., Suda, J., Kajigaya, S., Nagata, S., Asano, S., Saito, M. and Miura, Y. Effects of recombinant
murine granulocyte colony-stimulating factor on granulocyte-macrophage and blast colony formation.
Exp Hematol, 15: 958-65. (1987).
Swets, J.A. Measuring the accuracy of diagnostic systems. Science, 240: 1285-93. (1988).
Synold, T.W., Relling, M.V., Boyett, J.M., Rivera, G.K., Sandlund, J.T., Mahmoud, H., Crist, W.M., Pui,
C.H. and Evans, W.E. Blast cell methotrexate-polyglutamate accumulation in vivo differs by lineage,
ploidy, and methotrexate dose in acute lymphoblastic leukemia. J Clin Invest, 94: 1996-2001. (1994).
Tachdjian, G., Aboura, A., Lapierre, J.M. and Vigui, F. Cytogenetic analysis from DNA by comparative
genomic hybridization. Ann Genet, 43: 147-54. (2000).
Takeuchi, S., Bartram, C.R., Wada, M., Reiter, A., Hatta, Y., Seriu, T., Lee, E., Miller, C.W., Miyoshi, I.
and Koeffler, H.P. Allelotype analysis of childhood acute lymphoblastic leukemia. Cancer Res, 55:
5377-82. (1995).
Tanaka, K., Takechi, M., Nishimura, S., Oguma, N. and Kamada, N. Amplification of c-MYC oncogene
and point mutation of N-RAS oncogene point mutation in acute myelocytic leukemias with double
minute chromosomes. Leukemia, 7: 469-71. (1993).
Uckun, F.M., Nachman, J.B., Sather, H.N., Sensel, M.G., Kraft, P., Steinherz, P.G., Lange, B.,
Hutchinson, R., Reaman, G.H., Gaynon, P.S. and Heerema, N.A. Clinical significance of Philadelphia
chromosome positive pediatric acute lymphoblastic leukemia in the context of contemporary intensive
therapies: a report from the Children's Cancer Group. Cancer, 83: 2030-9 (1998a).
Uckun, F.M., Sensel, M.G., Sather, H.N., Gaynon, P.S., Arthur, D.C., Lange, B.J., Steinherz, P.G., Kraft,
P., Hutchinson, R., Nachman, J.B., Reaman, G.H. and Heerema, N.A. Clinical significance of
translocation t(1;19) in childhood acute lymphoblastic leukemia in the context of contemporary
therapies: a report from the Children's Cancer Group. J Clin Oncol, 16: 527-35. (1998b).
Van Dijk, M.A., Voorhoeve, P.M. and Murre, C. Pbx1 is converted into a transcriptional activator upon
acquiring the N- terminal region of E2A in pre-B-cell acute lymphoblastoid leukemia. Proc Natl Acad
Sci U S A, 90: 6061-5. (1993).
van Dongen, J.J., Seriu, T., Panzer-Grmayer, E.R., Biondi, A., Pongers-Willemse, M.J., Corral, L., Stolz,
F., Schrappe, M., Masera, G., Kamps, W.A., Gadner, H., van Wering, E.R., Ludwig, W.D., Basso, G.,
de Bruijn, M.A., Cazzaniga, G., Hettinger, K., van der Does-van den Berg, A., Hop, W.C., Riehm, H.
and Bartram, C.R. Prognostic value of minimal residual disease in acute lymphoblastic leukaemia in
childhood. Lancet, 352: 1731-8. (1998).
81
References
van Hal, N.L., Vorst, O., van Houwelingen, A.M., Kok, E.J., Peijnenburg, A., Aharoni, A., van Tunen,
A.J. and Keijer, J. The application of DNA microarrays in gene expression analysis. J Biotechnol, 78:
271-80. (2000).
Wang, L.C., Swat, W., Fujiwara, Y., Davidson, L., Visvader, J., Kuo, F., Alt, F.W., Gilliland, D.G.,
Golub, T.R. and Orkin, S.H. The TEL/ETV6 gene is required specifically for hematopoiesis in the bone
marrow. Genes Dev, 12: 2392-402. (1998).
Wang, S., Wang, Q., Crute, B.E., Melnikova, I.N., Keller, S.R. and Speck, N.A. Cloning and
characterization of subunits of the T-cell receptor and murine leukemia virus enhancer core-binding
factor. Mol Cell Biol, 13: 3324-39. (1993).
Weller, P.F., Bach, D. and Austen, K.F. Human eosinophil lysophospholipase: the sole protein component
of Charcot-Leyden crystals. J Immunol, 128: 1346-9. (1982).
Verdorfer, I., Brecevic, L., Saul, W., Schenker, B., Kirsch, M., Trautmann, U., Helm, G., Gramatzki, M.
and Gebhart, E. Comparative genomic hybridization-aided unraveling of complex karyotypes in human
hematopoietic neoplasias. Cancer Genet Cytogenet, 124: 1-6. (2001).
Werner, M., Wilkens, L., Aubele, M., Nolte, M., Zitzelsberger, H. and Komminoth, P. Interphase
cytogenetics in pathology: principles, methods, and applications of fluorescence in situ hybridization
(FISH). Histochem Cell Biol, 108: 381-90. (1997).
Whitehead, V.M., Rosenblatt, D.S., Vuchich, M.J., Shuster, J.J., Witte, A. and Beaulieu, D. Accumulation
of methotrexate and methotrexate polyglutamates in lymphoblasts at diagnosis of childhood acute
lymphoblastic leukemia: a pilot prognostic factor analysis. Blood, 76: 44-9. (1990).
Wiemels, J.L., Cazzaniga, G., Daniotti, M., Eden, O.B., Addison, G.M., Masera, G., Saha, V., Biondi, A.
and Greaves, M.F. Prenatal origin of acute lymphoblastic leukaemia in children. Lancet, 354: 1499503. (1999a).
Wiemels, J.L., Ford, A.M., Van Wering, E.R., Postma, A. and Greaves, M. Protracted and variable
latency of acute lymphoblastic leukemia after TEL-AML1 gene fusion in utero. Blood, 94: 1057-62.
(1999b).
Wiemels, J.L., Pagnamenta, A., Taylor, G.M., Eden, O.B., Alexander, F.E. and Greaves, M.F. A lack of a
functional NAD(P)H:quinone oxidoreductase allele is selectively associated with pediatric leukemias
that have MLL fusions. United Kingdom Childhood Cancer Study Investigators. Cancer Res, 59: 40959. (1999c).
Wiemels, J.L., Smith, R.N., Taylor, G.M., Eden, O.B., Alexander, F.E. and Greaves, M.F.
Methylenetetrahydrofolate reductase (MTHFR) polymorphisms and risk of molecularly defined
subtypes of childhood acute leukemia. Proc Natl Acad Sci U S A, 98: 4004-9. (2001).
Wiemels, J.L., Xiao, Z., Buffler, P.A., Maia, A.T., Ma, X., Dicks, B.M., Smith, M.T., Zhang, L., Feusner,
J., Wiencke, J., Pritchard-Jones, K., Kempski, H. and Greaves, M. In utero origin of t(8;21) AML1ETO translocations in childhood acute myeloid leukemia. Blood, 99: 3801-5. (2002).
Wolfe, K.Q. and Herrington, C.S. Interphase cytogenetics and pathology: a tool for diagnosis and
research. J Pathol, 181: 359-61. (1997).
82
References
Wong, N., Chen, S.J., Cao, Q., Su, X.Y., Niu, C., Wu, Q.W., Leung, T.W., Wickham, N., Johnson, P.J.
and Chen, Z. Detection of chromosome over- and underrepresentations in hyperdiploid acute
lymphoblastic leukemia by comparative genomic hybridization. Cancer Genet Cytogenet, 103: 20-4.
(1998).
Wu, T.D. Analysing gene expression data from DNA microarrays to identify candidate genes. J Pathol,
195: 53-65. (2001).
Yeazel, M.W., Ross, J.A., Buckley, J.D., Woods, W.G., Ruccione, K. and Robison, L.L. High birth
weight and risk of specific childhood cancers: a report from the Children's Cancer Group. J Pediatr,
131: 671-7. (1997).
Yeoh, E.-J., Ross, M.E., Shurtleff, S.A., Williams, W.K., Patel, D., Mahfouz, R., Behm, F.G., Raimondi,
S.C., Relling, M.V., Patel, A., Cheng, C., Campana, D., Wilkins, D., Zhou, X., Li, J., Liu, H., Pui, C.H., Evans, W.E., Naeve, C., Wong, L. and Downing, J.R. Classification, subtype discovery, and
prediction of outcome in pediatric acute lymphoblastic leukemia by gene expression profiling. Cancer
Cell, 1: 133-43 (2002).
Yokoyama, S., Staunton, D., Fisher, R., Amiot, M., Fortin, J.J. and Thorley-Lawson, D.A. Expression of
the Blast-1 activation/adhesion molecule and its identification as CD48. J Immunol, 146: 2192-200.
(1991).
Zembutsu, H., Ohnishi, Y., Tsunoda, T., Furukawa, Y., Katagiri, T., Ueyama, Y., Tamaoki, N., Nomura,
T., Kitahara, O., Yanagawa, R., Hirata, K. and Nakamura, Y. Genome-wide cDNA microarray
screening to correlate gene expression profiles with sensitivity of 85 human cancer xenografts to
anticancer drugs. Cancer Res, 62: 518-27. (2002).
Zhao, N., Hashida, H., Takahashi, N., Misumi, Y. and Sakaki, Y. High-density cDNA filter analysis: a
novel approach for large-scale, quantitative analysis of gene expression. Gene, 156: 207-13. (1995).
Zhou, L.J., Schwarting, R., Smith, H.M. and Tedder, T.F. A novel cell-surface molecule expressed by
human interdigitating reticulum cells, Langerhans cells, and activated lymphocytes is a new member of
the Ig superfamily. J Immunol, 149: 735-42. (1992).
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Department of Medical Genetics

Cytogenetic and Molecular

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4. Aetiology of childhood leukaemias...................................................................14

5. Genetic aberrations in childhood acute leukaemias ..........................................17

6. Genetic methods used in the diagnosis and follow-up of leukaemia ................25

7. Gene expression profiling by DNA arrays........................................................31

AIMS OF THE STUDY .................................................................................. 37

4. Amplification of the AML1 gene in childhood ALL (III) .................................55

List of original publications

Tarja Huhta, Kim Vettenranta, Kristiina Heinonen, Jukka Kanerva,

Marcelo L. Larramendy*, Tarja Huhta*, Kim Vettenranta, Wa'el El-Rifai,

Tarja Niini, Jukka Kanerva, Kim Vettenranta, Ulla M. Saarinen-Pihkala

Jukka Kanerva*, Tarja Niini*, Kim Vettenranta, Pekka Riikonen, Anne

Tarja Niini, Kim Vettenranta, Jaakko Hollmn, Marcelo L. Larramendy,

* these authors contributed equally to the study

v-abl Abelson murine leukaemia viral oncogene homolog 1

Review of the literature

Review of the literature

3.1. Childhood acute lymphoblastic leukaemia (ALL)

Review of the literature

predisposition to infections, increased danger of haemorrhage, and nausea.

3.2. Childhood acute myeloid leukaemia (AML)

4. Aetiology of childhood leukaemias

Review of the literature

4.2. Inherited predisposition and environmental

Review of the literature

suppressor mechanisms. The incidence of leukaemia is also significantly higher

Review of the literature

5. Genetic aberrations in childhood acute

Figure 1. Prevalence of genetic changes in ALL with respect to different age

Review of the literature

Review of the literature

Activated gene Mechanism of

Frequency Cell type /

Review of the literature

Review of the literature

transformed by translocations, which would further provide basis for the

Review of the literature

Review of the literature

5.2. Numerical chromosome aberrations

5.3. Gene amplifications

Review of the literature

5.4. Tumour suppressor genes and allelic losses

Review of the literature

6. Genetic methods used in the diagnosis and

6.2. Molecular genetic methods

Review of the literature

6.3. Molecular cytogenetic methods

Review of the literature

6.3.1. Chromosome painting and multicolour-FISH

Review of the literature

Review of the literature

Slide with normal

Figure 2. The principle of comparative genomic hybridisation (CGH). DNA

Review of the literature

CGH is used in clinical diagnostics to supplement conventional cytogenetics. In

Review of the literature

7. Gene expression pro filing by DNA arrays

7.1. Types of array and principles of the method

Review of the literature

the same array slide in a competitive manner. The expression differences

Marcelo L. Larramendy, Tarja Huhta, Kim Vettenranta, Wa'el El-Rifai,

Jukka Kanerva, Tarja Niini, Kim Vettenranta, Pekka Riikonen, Anne