Table Of ContentCYTOGENETIC AND MOLECULAR ANALYSIS OF
CHRONIC AND ACUTE LEUKEMIA
CYTOGENETISCH EN MOLECULAIR ONDERZOEK VAN
CHRONISCHE EN ACUTE LEUKEMIE
PROEFSCHRIFT
TER VERKRUGING VAN DE GRAAD VAN DOCTOR
AAN DE ERASMUS UNIVERSITEIT ROTTERDAM
OP GEZAG VAN DE RECTOR MAGNIFICUS
PROF.DR. C.J. RIJNVOS
EN VOLGENS BESLUIT VAN HET COLLEGE VAN DEKANEN.
DE OPENBARE VERDEDIGING ZAL PLAATSVINDEN OP
WOENSDAG 30 OKTOBER 1991 OM 15.45 UUR
DOOR
DOROTHEA CHRISTINA VAN DER PLAS
GEBOREN TE HOORN
PROMOTIECOMMISSIE
promotoren: Prof.Dr.A.Hagemeijer
Prof.Dr.D.Bootsma
Overige !eden: Dr. G. C. Grosveld
Prof.Dr.R.Willemze
Prof.Dr.B.Uiwenberg
y:ted
by: Haveka B.V .• Alblasserdam. The Netherlands.
Dit proefschrift werd bewerkt binnen de vakgroep Celbiologie en Genetica van de
Faculteit der Geneeskunde en Gezondheidswetenschappen van de Erasmus Universiteit
Rotterdam
Aan mijn ouders
Aan Arend
CONTENTS
Abbreviations 7
Chapter 1 General introduction 9
1.1 The Philadelphia chromosome 9
1.2 N annal and abnonnal hematopoiesis 9
L3 Chromosome aberrations in cancer 12
1.4 Genes involved in leukemia 13
1.5 Introduction to the experimental work 15
Chapter 2 Cytogenetics of leukemia 19
2.1 Cytogenetics of chronic leukemia (CLL, CML), and MDS. 19
2.2 Clinical relevance of cytogenetics in acute leukemia 23
2.3 The prognostic significance of karyotype at diagnosis in 33
childhood ALL
Chapter 3 Molecular biology of the Plilladelphia chromosome 53
3.1 The Ph chromosome in CML 53
3.2 Unique fusion of bcr and c-abl genes in Philadelphia
chromosome positive Acute Lymphoblastic Leukemia 59
3.3 Immunological characterization of the tumor specific
bcr-abl junction in Philadelphia chromosome positive
Acute Lymphoblastic Leukemia 69
Chapter 4 Molecular diagnosis of Ph positive CML 77
4.1 -Molecular investigations of Ph positive CML 77
-Breakpoints on chromosome 22 outside the BCR region 81
-Simultaneous expression of two different bcr-abl mRNAs 83
4.2 Bcr-abl mRNA lacking abl exon a2 detected by polymerase
chain reaction in a CML patient 87
Chapter 5 Ph conversion: disappearance of the Ph chromosome 95
Chapter 6 Cytogenetic and molecular diagnosis of Ph negative CML 109
6.1 Cytogenetic and molecular analysis in Ph negative CML 109
6.2 Philadelphia negative Chronic Myeloid Leukemia (CML):
Comparison with Ph positive CML and Chronic
Myelomonocytic Leukemia 119
6.3 Review of clinical, cytogenetic and molecular aspects of
Ph negative CML !29
Chapter 7 Cytogenetic and molecular diagnosis of Ph positive
Acute Leukemia 145
7.1 Bcr-abl rearrangement in acute leukemia 145
7.2 Molecular analysis of a new chromosomal rearrangement
in Acute N onlymphocytic Leukemia 147
Chapter 8 Cytogenetic and molecular diagnosis of Ph positive
Myelodysplastic Syndrome 151
8.1 Cytogenetic and molecnlar studies of the Philadelphia
translocation t(9;22) observed in a patient with
Myelodysplastic Syndrome 151
Chapter 9 General discussion !57
Summary 165
Same nv atting 167
Curriculum Vitae 169
Publications 171
Daukwoord 173
Appendix Clinical evaluation of a DNA probe assay for the Ph
translocation in Chronic Myelogenous Leukemia 175
ABBREVIATIONS
aCML atypical chronic myelogenous leukemia
ALL acute lymphoblastic leukemia
AML acute myeloblastic leukemia
ANLL acute non-lymphoblastic leukemia:
APL acute promyelocytic leukemia
BC blast crisis
BCR 5.8 Kb breakpoint cluster region = M-bcr-1
her her gene
BM bone marrow
BMT bone marrow transplantation
bp base pairs
BV173 cell line derived from a CML patient in blast crisis;
expresses b2a2 mRNA
c-abl cellular abl oncogene
eDNA complementary desoxyribonucleic acid
C terminus carboxy terminus
CLL chronic lymphocytic leukemia
CML chronic myelogenous leukemia
CMML chronic myelomonocytic leukemia
del deletion
DNA deoxyribonucleic acid
el first exon of the her gene
FAB French-American-British Cooperative Group
GM-CSF granulocyte macrophage colony stimulating factor
GVHD graft versus host disease
GVL graft versus leukemia effect
HL60 human acute promyelocytic leukemia cell line
HLA human leucocyte antigen
i iso chromosome
1FN interferon
IgH immunoglobulin heavy chain
IL interleukin
IU international units
J joining region of immunoglobulin gene family
K562 cell line derived from a patient with CML in blast crisis;
Expresses b3a2 mRNA.
Kb kilo bases
kD kilo Dalton
LAP leucocyte alkaline phosphatase
LPS lipopolysaccharide
Mar marker
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m-bcr-1 minor breakpoint cluster region= ALL breakpoint cluster region
M-bcr-1 major breakpoint cluster region=CML breakpoint cluster region=BCR
M-CSF macrophage colony stimulating factor
MDS myelodysplastic syndrome
MPD myeloproliferative disease
mRNA messenger ribonucleic acid
N terminus amino terminus
pl90 190 kD bcr-abl protein
p210 210 kD bcr-abl protein
PB peripheral blood
PCR polymerase chain reaction
PDGF platelet derived growth factor
PHA phytohemagglutinin
PWM pokeweed mitogen
R receptor
RA refractory anemia
RAEB refractory anemia with excess of blasts
RAEBt refractory anemia with excess of blasts in transformation
RAR retinoic acid receptor
RARS refractory anemia with ring sideroblasts
RBC red blood cell count
RNA ribonucleic acid
SH src homology
t translocation
Tom-I cell line derived from Ph positive ALL patient; expresses ela2 mRNA
TPA 12-0-tetradecanoylphorbol-13 acetate
v-abl viral abl oncogene
WBC white blood cell count
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CHAPTER 1
GENERAL INTRODUCTION
1.1 THE PIDLADELPHIA CHROMOSOME
In 1960 Nowell and Hungerford reported the first chromosomal rearrangement
associated with cancer. They discovered that a small chromosome, which they called
Philadelphia (Ph) chromosome, was consistently present in the leukemic cells of
patients with CML. In 1973 Rowley found that this Ph chromosome was chromosome
22q-, that resulted from a reciprocal translocation between chromosome 9 and 22.
This translocation was called the Philadelphia (Ph) translocation or t(9;22)(q34;qll).
It took unti11982 before de Klein et al discovered, that as a result of this translocation
the abl oncogene was translocated from its normal position on chromosome 9 to the
22 q-chromosome. These findings formed the basis for the cytogenetic and molecular
investigations in leukemia, that are described in this thesis.
1.2 NORMAL AND ABNORMAL HEMATOPOIESIS
Blood cell formation
Blood cell formation or hematopoiesis takes place primarily in the bone marrow. All
elements of blood and lymph are derived from the pluripotent stem cell. Pluripotent
stem cells are present in low numbers in the bone marrow. Each of them has the
potential to proliferate and differentiate, giving rise to all lymphoid and myeloid blood
white blood cells as well as to erythrocytes and platelets. However, under normal
conditions the majority of the pluripotent stem cells are in a quiescent state, and only
a few are active in blood cell formation. The processes of selfrenewal, differentiation
to a more restricted phenotype, and cell death are regulated very strictly. These
regulation processes concern also the more mature offspring of the stem cell e.g. the
committed progenitor cell, which is a cell type whose developmental lineage is already
restricted to one lineage, but is still capable of self renewal.
The regulation mechanisms are not yet fully known, but are thought to be essential for
understanding normal hemopoiesis as well as the etiology of leukemia (reviewed by
Sawyers, 1991). The last few years several soluble factors have been identified, that
are involved in this regulation process e.g. growth factors, and small peptides that are
produced by blood cells or by bone marrow stromal cells. GM-, G-, M-CSF and
several interleukins such as ll-1, 3 and 6 have been identified as positive regulators
(i.e. stimulators) of bone marrow stem cells and committed precursor cells. Examples
of negative regulators (inhibitors) of these cells are transforming growth factor Jl (TGF
Jl), which is produced by marrow stromal cells, and small peptides such as stem cell
inhibitor (SCI), which is produced by macrophages (Graham et a!, 1990, Zebo et
al,1990, Williams et al, 1990, Huang et al !990, Dexter et al, 1977, Clark et al,
1987).
9
In addition to these factors cell-cell contact and specific effects of the extracellular
matrix play a role in regulation of stem cell function, the latter possibly by binding
growth factors (Roberts eta!, 1988). Disturbances in these regulation mechanisms can
result in uncontrolled cell proliferation and/o r failure of the progenitor cells to
differentiate to mature cells.
Blood cancer
Blood cancer or leukemia is a heterogeneous group of diseases resulting from
neoplastic transformation of hematopoietic cells. The main characteristics are
uncontrolled proliferation of hematopoietic cells, that do (in most cases) not retain the
capacity to normally differentiate to mature blood cells. This differentiation arrest can
occur in every maturation stage and in every cell lineage of blood cell differentiation,
resulting in distinct forms of leukemia.
Throughout the years several attempts have been made to devise a classification
system of the different types of leukemia.
Based on length of survival of the patients and degree of maturation of the cells the
leukemias are classified as acute or chronic. A further subclassification is made
according to the predominant cell lineage involved, e.g. lymphoid, myelogenous, or
monocytic and to morphological and immunological characteristics of the leukemic
cells.
- Acute leukemia
The origin of acute leukemia is a single transformed progenitor cell. This might be a
pluripotent stem cell or a committed precursor.
In acute leukemia the number of circulating blood cells is increased and the maturation
is arrested at early stage of differentiation. The disease is characterized by the
presence of large numbers of immature lymphoid or myeloid precursors in the bone
marrow and the peripheral blood. These precursors subsequently replace normal bone
marrow. Thereafter they often invade other tissues such as central nervous system, the
eye, the skin and the testis. Displacement of normal bone marrow by large numbers
of undifferentiated or immature leukemic cells results in insufficient production of
mature blood cells such as granulocytes, neutrophils, eosinophils, basophils,
erythrocytes, and thrombocytes, causing anemia, and susceptibility to infections and
hemorrhage. Without treatment acute leukemia patients die within weeks to months
after diagnosis. Acute leukemia occurs at all ages.
As a consequence of the fact that the primary cause of acute leukemia might be in the
stem cell or a committed precursor and that differentiation arrest can occur in every
stage of blood cell differentiation, a great variability is found with regard to the
clinical, morphologic, cytochemical, cytogenetical, and immunological features.
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Description:Cytogenetic and molecular analysis in Ph negative CML. 109. 6.2 Philadelphia negative Chronic Myeloid Leukemia (CML):. Comparison with Ph
