and encouragement throughout the course of the research and writing of the thesis. ...... use in any academic unit. ..... effective for any given clinical situation. [Dewald et al ..... 2- The target areas were covered completely with Pepsin solution.
Break point cluster region / Abelson murine leukemia fluorescence in situ hybridization detection in patients with chronic myeloid leukemia pre and post therapy A Thesis Submitted to College of Medicine and Committee of Graduate Studies University of Baghdad In partial Fulfillment of the Requirements For the Degree of Doctor of Philosophy In Pathology (Molecular Hematology) By Jaffar Nouri Jaffar Alalsaidissa MB. Ch.B., M.Sc. (Hematology) Supervised by Professor Dr. Ban A. Abdul Majeed M.Sc.,Ph.D.(Molecular Pathology) Supervisor
Professor Dr. Ali Al-Mothaffar M.B. Ch.B., CABM, FRCP Professor of Hematology Supervisor College of Medicine University of Baghdad
June
2013
Rajab
1434
)و َما أُو ِتيت ُ ْم ِم ْن ا ْل ِع ْل ِم ِإﻻﱠ قَ ِليﻼً( َ سورة اﻻسراء اﻻية 85
Certification We, certify that this thesis was prepared under our supervision at the Department of Pathology of Medicine, University of Baghdad, as a partial fulfillment of the requirement for the degree of Ph.D. in Pathology (Molecular Hematology).
Professor Dr. Ban A. Abdul Majeed M.Sc.,Ph.D.(Molecular Pathology) Supervisor
Professor Dr. Ali Al-Mothaffar M.B. Ch.B., CABM, FRCP Professor of Hematology Supervisor
College of Medicine University of Baghdad
In review of the available recommendations, I forward this thesis for debate by the examining committee.
Professor DR. Salim Hamoudi MB. Ch.B., Ph.D. Head of the Department of Pathology College of Medicine University of Baghdad
We, the examining committee, after reading this thesis and examining the student in its contents, find it adequate as a thesis for the Degree of Doctor of Philosophy in Pathology (Molecular Hematology). Signature ……….…………….. Dr. Ibrahim Khalil Al-Shammari Consultant Hematologist M.B. Ch.B., FICMS (Hematology) Al-Kadhimiya Teaching Hospital Member
Signature ……….…………….. Dr. Aladdin Mudhafar Zubair Assistant Professor M.B. Ch.B., FICMS-Pathology (Hematology) College of Medicine Al- Mustansiriyah University Member
Signature ……….…………….. Dr. Khudayer A. Al-Khalisi Assistant Professor M.B. Ch.B., MRCP. College of Medicine University of Baghdad Member
Signature ……….…………….. Dr. Raad J. Musa Professor M.B. Ch.B., FICMS-Pathology (Hematology) College of Medicine Al-Nahrain University Member
Signature ……….…………….. Dr. Ban A. Abdul Majeed Professor M.Sc., Ph.D. (Molecular Pathology) College of Medicine University of Baghdad Supervisor
Signature ……….…………….. Dr. Ali Al-Mothaffar Professor of Hematology M.B. Ch.B, CABM, FRCP College of Medicine University of Baghdad Supervisor
Signature ……….…………….. Dr. Salim Hamoudi Professor M.B. Ch.B., Ph.D. College of Medicine University of Baghdad Chairman Approved for the collage committee of post graduate studies. Signature ……….…………….. Professor Dr. Muhi Kadhem Wannas Al Janabi Acting Dean College of Medicine University of Baghdad
To My Parents and Family
Acknowledgments I would like to express my sincere gratitude to my parents and family for their support during the whole period of the study. I would like also to express my sincere gratitude to my supervisors Professor Ban Abbas Abdul Majeed and Professor Ali Al- Mothaffar from the university of Baghdad College of Medicine for their guidance and encouragement throughout the course of the research and writing of the thesis. Their kind care, invaluable advices, creative remarks and generous help are highly appreciated. Special thanks should be expressed for the Head, all doctors and all staff of the Department of Pathology College of Medicine University of Baghdad for their great help and support. Also special thanks should be expressed for all doctors, nurses and staff of the Hematology Department at the Medical city teaching hospital for their great help and support during collection of cases and follow up. Also special thanks should also be expressed for all doctors and staff of the Hematology laboratory at the Medical city teaching hospital for their great support and help during this study. Special thanks should also be expressed to all doctors,colleagues, staff, nurses and patients who helped me during this study. Great appreciations are due to Professor Zaid Al-Madfai in the Department of Community Medicine, College of Medicine, University of Baghdad for his kind help and valuable advise in statistics.
Summary Background and objectives: several factors render Chronic myeloid leukemia an interesting subject for study by researchers. They include marked progress in understanding its molecular biology, the discovery of Philadelphia chromosome and the development of new drug generation, the tyrosine kinase inhibitors (TKI). Break point cluster region –Abelsone murine leukemia translocation gene is present in ninety-five percent of chronic myeloid leukemia patients and involving ninety percent of a granulocyte in most cases. Aim of the study: The aim of this study was to apply the technique of interphase fluorescence in situ hybridization (FISH) technique and detect Break point cluster region –Abelsone murine leukemia translocation and P53 alteration, to assess if there is any significant relationship and to detect Ataxia Telangiectasia Mutated gene (ATM), and isochromosome of
the
long
arm
of
chromosome
seventeen(17q)
and
Myeloperoxidase (MPO) gene. Materials and methods: This is a prospective study in which 104 newly diagnosed chronic phase chronic myeloid leukemia having not received a prior tyrosine kinase inhibitors were included together with 30 normal adult subjects as a control group. Two prognostic relative risk scores were used to calculate the predicted response to treatment, including the Sokal score and the European Treatment and Outcome Study score. The patients were divided into two groups, twenty-four patient (fifteen male and nine female) were studied by using ethylene diamine tetra acetic acid anticoagulant and eighty patients were studied by using sodium heparin as anticoagulant.
Results: All patients in both groups had a positive Break point cluster region –Abelsone murine leukemia translocation signal in their peripheral blood specimen but the percentage of positive cells varies between two groups of patients being 86.6% for patients in the heparin group and 11.33% for patients in the ethylene diamine tetra acetic acid group. The majority of patients were in the high risk group in both relative risk scores. Significant statistical association was found between the European Treatment and Outcome Study score and spleen size, lymphocyte%, basophils% and bone marrow Myeloid: Erythroid ratio. In the ethylene diamine tetra acetic acid group of patients there was a significant
statistical
relationship
between
Fluorescence
in
situ
hybridization test with their white blood cells count, platelet count and bone marrow Myeloid: Erythroid ratio. In the heparin group of patients, two patients were excluded because they were Fluorescence in situ hybridization negative, seventy-eight patients included in this study (forty three male and thirty five female). The mean white blood cell count count of these patients was 189.5 x 109/l. The Fluorescence in situ hybridization score of these patients was 7090% (mean 86.6%). Protien fifty three isochromosome(17q) FISH test was also applied to ninteen of our patients (13male and 6 female), ten of them show positive results . No significant statistical relationship was found between P53 i(17q) with the gender of patients.
A statistically significant relationship was found between P53 i(17q) and Sokal relative risk score while no significant statistical correlation was found with the EUTOS relative risk score. No statistically significant difference was observed between the study sample and the control group regarding the P53/ATM gene signals. All patients were started treatment on Tyrosine kinase inhibitor imatinib mesylate in a dose of 400mg/day and were followed for six to nine months of treatment , thirty six patients of them became Fluorescence in situ hybridization negative after treatment while twenty seven of them remain Fluorescence in situ hybridization positive after treatment . Comparison of the clinical characteristics and laboratory parameters before starting treatment between those group of patients who became Fluorescence in situ hybridization negative and those who continue to be Fluorescence in situ hybridization
positive was statistically significant
regarding the spleen size and the percentage of bone marrow granulocytes. Conclusion: Fluorescence in situ hybridization test is a reliable technique in the detection of Break point cluster region –Abelsone murine leukemia translocation in chronic myeloid leukemia patients pre and post therapy to assess the cytogenetic response. It is important both in the diagnosis and follow up. Both Sokal and the European Treatment and Outcome Study scores are useful prognostic parameters in CML patients. Protien fifty three isochromosome (17q), Ataxia Telangiectasia Mutated gene and myloperoxidase gene abnormalities are important genes to be screened in Iraqi CML patients.
List of Contents Page No.
Title Chapter One: Introduction and aim of study
1
1.1. Introduction
1
1.2 Normal hematopoiesis
2
1.3.Historical aspects
5
1.4. Definition
7
1.5. Incidence and prevalence
9
1.6. Risk factors and aetiology
13
1.7. Pathogenesis and cell biology
16
1.8. Molecular anatomy and pathology
20
1.9.
33
Clinical features
1.10. Molecular Diagnosis
48
1.11. Prognostic Scores
65 1.12. Treatment
68
1.13. Monitoring
73
Chapter Two: Patients Materials and Methods
80
2.1. Selection of Patients
80
2.2. Criteria for the diagnosis and selection of patients
81
2.3. Treatment and follow up
83
2.4. Sample collection
83
2.5. Normal control
83
2.6. Materials
84
2.7. Methods
88
2.8. Fluorescence in situ hybridization test (FISH test)
89
2.9.: Fluorescenet microscope examination
94
2.10. Validation and cut off value
96
2.11. P53
97
2.12. Calculation of the relative risk scores
98
2.13. Statistical analysis
99
2.14. Photography
99
Chapter Three: Results
100
3.1 General clinical and laboratory parameters of patients and control. 3.2. Clinical staging of CML patients 3.3. Distribution of all patients according to the relative risk scores before starting treatment 3.4. Results of Fluorescence in situ Hybridization test for BCRABL fusion gene
100 104 104 104
3.5. Results of FISH study for P53
106
3.6. Patients after treatment
107
3.7. Correlation of the relative risk scores of the two groups of patients, those who became FISH2 negative after treatment and
110
those who continued to be FISH2 positive after treatment Chapter Four: Discussion 4.1. Clinical, hematological and laboratory characteristics of CML patients.
160 160
4.2. Clinical parameters.
162
4.3. Laboratory findings.
163
4.4. FISH test for BCR-ABL translocation.
168
4.5. Relative risk scores.
171
4.6. P53, ATM, iso17q, MPO FISH signals.
173
Conclusions.
178
Recommendations.
179
Chapter Five: References
180
List of Tables Table No.
Title
Page No.
1.1
Myeloproliferative neoplasms (WHO classification)
8
1.2
The commonest ten cancers by site/IRAQ
10
1.3
Symptoms and Signs of Chronic-Phase CML at Presentation
35
1.4
Typical features of Chronic myelogenous leukemia in chronic phase.
38
1.5
Conventional and Molecular Cytogenetics Methods Most Commonly Used in Clinical Laboratories.
50
1.6
Advantages and limitations of cytogenetics and Interphase FISH.
55
1.7
Types of probes for FISH.
59
1.8
Sokal index for predicting survival prognostic indices.
66
1.9
ELN and NCCN Monitoring Guidelines for Patients Receiving TKI Therapy.
74
1.10
The current ELN and NCCN response criteria in chronic myeloid leukemia.
78
2.1
Equipments used and their manufacturers
85
2.2
General materials used in our work.
86
2.3
Kit contents and probes used in our work.
88
2.4
Expected signals of FISH technique employing Poseidon repeat-free BCR-ABL probes.
96
2.5
Expected signals of FISH technique employing Poseiden P53 (17P13) and ATM (11q22) Probes.
97
3.1
Gender distribution of CML patients EDTA group.
113
3.2
Clinical and Lab parameters of CML patients EDTA group.
113
3.3
Gender distribution of patients in the heparin group.
114
3.4
Descriptive table for all patients clinical characteristics before starting treatment (patients who had FISH testing by the heparin method).Number =78
114
Descriptive table for all patients lab characteristics before starting treatment (patients who had FISH testing by the heparin method).Number =78
115
Distribution of Sokal and EUTOS scores for all patients before starting treatment who had FISH test done by the heparin method. Number =78
116
3.7
Detection of the BCR-ABL fusion signal in CML patients in the EDTA group and control group.
116
3.8
Correlations of FISH1 score with all clinical and haematological parameters of EDTA group of patients.
117
3.9
Correlation of the FISH1 score results with patients clinical and laboratory characteristics before starting treatment.
119
3.10
Gender distribution and relation to p53 i17q
120
3.11
The relationship between p53 i(17q) and Sokal Score of patients.
120
3.12
The relationship between p53 i17q and EUTOS score of patients.
121
3.13
Frequency distribution of p53/ ATM in 13 CML patients from the EDTA group and 5 control samples FISH study .
121
Clinical parameters of patients in the group who became FISH2 negative after treatment N=36
122
3.5
3.6
3.14
3.15
3.16
3.17 3.18
Main laboratory parameters of the first group of patients those who became FISH2 negative after treatment N=36
123
Paired hematological parameters before and after treatment of patients who became FISH negative after treatment N=36
124
Paired sample t test of hematological parameters in patients who became FISH negative after treatment. N=36 Wilcoxon Signed Ranks Test statistics of hematological parameters in patients who became FISH negative after treatment N=36
125
126
3.19
Clinical parameters of the second group of patients who continued to be FISH2 positive after treatment N=27
127
3.20
Main laboratory parameters of patients in the second group who continued to be FISH2 positive after treatment N=27
128
The paired hematological parameters of patients in group 2 who continued to be FISH2 positive after treatment N=27
129
Paired samples t test of hematological parameters of patients who continued to be FISH2 positive after treatment N=27
130
Wilcoxon Signed Ranks Test statistics of hematological parameters of patients who continued to be FISH positive after treatment N=27
131
3.24
Comparison of clinical characteristics before starting treatment between 2 groups of patients.
132
3.25
Comparison of lab characteristics before starting treatment between 2 groups of patients.
133
3.26
Comparison of the clinical and laboratory parameters of patients in the two groups , those who become FISH2
134
3.21
3.22
3.23
negative and those who continued to be FISH2 positive after treatment. 3.27
Independent sample t test for comparison of clinical and hematological parameters for the two groups of patients before starting treatment, those who became FISH2 negative and those who continued to be FISH2 positive after treatment.
135
Mann-Whitney U test and Wilcoxon W test statistics for the clinical and hematological parameters of the two groups of patients, those who became FISH2 negative and those who continued to be FISH2 positive after treatment.
136
3.29
Comparison of the Sokal and EUTOS scores before starting treatment between 2 groups of patients.
137
3.30
Distribution of patients according to the Sokal relative risk score.
138
3.31
Description of the clinical and some hematological parameters of patients within the Sokal relative risk score.
139
3.32
Statistical significance of the different clinical and some hematological parameters of patients within the Sokal relative risk score.
141
3.33
Multiple comparison of the spleen size and blast1% within groups of the Sokal relative risk score.
143
3.34
Distribution of patients according to the EUTOS relative risk score.
144
3.35
The clinical and some hematological parameters of patients in each risk level group within the EUTOS relative risk score.
145
T - test statistics of the different clinical and hematological parameters of patients in the EUTOS relative risk group.
147
3.28
3.36
3.37
Mann-Whitney U, Wilcoxon W, and Z test statistical correlations of patients in the EUTOS relative risk score.
148
List of Figures Figure No.
Title
Page No.
1.1
The process of hematopoiesis.
5
1.2
The commonest ten cancers in IRAQ 2008
11
1.3
BCR-ABL translocation and the Philadelphia chromosome.
18
1.4
The structure of the normal BCR and ABL1 genes and the fusion transcripts found in CML
23
1.5
suggested model for the involvement of wild type p53
30
1.6
Peripheral blood film in a patient with CML CP
41
1.7
Diagrammatic representation of the typical differential count in untreated cases of CML.
42
1.8
Bone marrow aspirate of patient with CML CP.
45
1.9
Bone Marrow Biopsy of Patient with CML showing hyper cellular marrow.
49
1.10
BCR-ABL- Negative FISH.
54
1.11
Timeline for development of therapies for CML. HSCT, hematopoietic stem cell transplantation.
69
1.12
Disease burden and tests.
77
3.1
Flowchart of CML patients samples in the experimental work.
149
3.2
periphral blood of patient with CML showing the whole spectrum of granulocytic cells.
150
3.3
Diagrammatic representation of the mean differential WBC count in our patients.
151
3.4
Periphral blood of a patient with CML showing granulocytes at different maturation stages including a
152
basophil. 3.5
Bone marrow aspirate showing a hypercellular marrow.
153
3.6
Hypercellular bone marrow aspirate showing granulocytic hyperplasia.
153
3.7
Normal cell with two copies of BCR and ABL genes (2 green and 2 red signals)
154
3.8
The BCR-ABL translocation is detected in interphase nuclei by the prescence of a fusion signal (yellow), along with the BCR gene (green) and the ABL gene (red).
155
3.9
P53 i(17q) FISH test showing one green signal for p53 and three red signals for MPO
156
3.10
P53 ( 17 p13) and ATM (11q22) probe FISH signal
157 & 158
3.11
Distribution of patients according to the FISH2 results.
159
List of Abbreviations ALL
Acute lymphoblastic leukemia
AML
Acute myeloid leukemia
BM
Bone marrow
BCM
Below costal margin
BCR-ABL C CCD Camera
Break point cluster region –Abelsone murine leukemia
CGH
Comparative Genomic Hybridization
CLL
Chronic lymphoid leukemia
cm CML CMPDs CP CP CML DAPI DDW
Centimeter
Celsius Charge coupled device Camera
Chronic myeloid leukemia Chronic myeloproliferative disorders Chronic phase Chronic phase chronic myeloid leukemia 4',6-diamidino-2-phenylindole Deionised distilled water
EDTA
ethylene diamine tetra acetic acid
ELN
European LeukemiaNet
EUTOS score
European Treatment and Outcome Study score
FISH
Fluorescence in situ hybridization
FISH1
Fluorescence in situ hybridization test before treatment
FISH2 FITC IM
Fluorescence in situ hybridization test after treatment Fluorescein-5-thiocyanate Imatinib mesylate (Glivec®)
kcl l
International System for Human Cytogenetic Nomenclature (1995) Potassium chloride Liter
M : E ratio
Myeloid: Erythroid ratio
Min
Minute or Minutes
ISCN 1995
Milliliter
ml NCCN
National Comprehensive Cancer Network
ON probes
Oncology probes
P53
Protein 53
PBS
Phosphate buffered saline
PCV
packed cells volume (Hematocrit)
rpm
Round per minute
RT
Room temperature
RT-PCR
Real time polymerase chain reaction
RtU
Ready to use
SD
Standard deviation
Sec
Seconds United States Surveillance Epidemiology and End
SEER
Results saline sodium citrate
SSC TKI TRITC
Tyrosine kinase inhibitor or inhibitors (Tetramethylrhodamine isothiocyanate)
WBC
White blood cells
Chapter One
Introduction & Review of literature
Introduction and aim of study 1.1. Introduction: Chronic myeloid leukemia (CML) was probably the first form of leukemia to be recognized as a distinct entity, it was first described in the early 19th century [Goldman JM 2003]. According to the WHO classification of tumours of haemopoietic and lymphoid tissues, it is included in the myeloproliferative disorders [Vardiman J 2008.]. In Iraq leukemia ranked third among the commonest ten cancers by site according to the Iraqi cancer registry 2008. [Iraqi cancer registry 2008 ]. In Iraq , Al-Rawi 2001 studied the clinical findings and bone marrow features in chronic myeloid leukemia. Al-Anizi 2003 found low nitroblue tetrazolium reduction
by neutrophils of patients with
chronic myeloproliferative disorders , Kadhom 2010 studied the expression of BCL-2 and P53 in 60 CML patients and concluded that there was relatively high statistically significant relationship between Bcl-2 oncoprotein and mutant P53 protein expression with high bone marrow blast count and advanced stages of the disease , Al-Khafagi 2010
studied
Bone marrow fibrosis in CML by using special
histochemical stains for collagen, Abdullah 2011 studied the Impact of chemotherapy upon quality of life for patients with chronic myeloid leukemia and found a statistically significant association between quality of life and sex, marital status, occupation, crowding index, duration of disease, phase of disease and performance status, while there was no statistically significant association between quality of life and age, socioeconomic status and educational level.
Aim of study
1- To study the response to treatment of patients with chronic myeloid leukemia both clinical and hematological response by application of fluorescence in situ hybridization (FISH) technique to detect BCR-ABL fusion gene in patients cells before and after therapy. 2- To study certain molecular abnormalities in chronic myeloid leukemia patients affecting the prognosis including p53/i(17q) , ATM and MPO.
Review of literature 1.2 Normal hematopoiesis
Hematopoiesis is the formation of blood cells where the bone marrow is the main organ. All blood cells derive from pluripotent hematopoietic stem cells that have the capacity to self-renew and also give rise to myeloid and lymphoid cell lineages (Figure1.1).[ Ioana B 2011].
Although an estimated 1 x 1010 red blood cells and 1 x 109 white blood cells are produced per hour, mature white blood cells have a limited lifespan of only a few hours to a few days and, therefore, require continuous production. Hematopoiesis is the highly regulated process of blood cell production from hematopoietic stem cells (HSCs) within the bone marrow [Bewry NN. 2009].
HSCs are small, non-adherent, rounded cells that possess a rounded nucleus and can be identified by their low cytoplasm-to-nucleus ratio. Characterized by their multipotency and high replicative and differentiation capacity, HSCs are capable of giving rise to all types of blood cells, which are divided into three distinct lineages: lymphoid, myeloid and erythroid. [Bewry NN. 2009]. The proliferation, differentiation and maturation of hematopoietic progenitor cells are regulated by various hematopoietic growth factors, in addition to microenvironment and the interactions between the cells. Growth factors influence transcription factors and implicit associated genes that determine the differentiation and function of mature blood cells. [Ioana B. 2011]. The hematopoietic growth factors include a group of cytokines, which comprise colony stimulating factors (CSFs), hormones, interleukins (IL) and glycoproteins [Wadhwa & Thorpe 2008].
Erythropoietin (EPO) is a hormone-like glycoprotein with the most lineage- specific activity that promotes the maturation and proliferation of erythroid cell lineages (by interacting with the receptors on erythroid burst-forming units and erythroid colony- forming units) [Bociek & Armitage 1996].
Thrombopoietin development
(TPO),
known
as
megakaryocyte
growth
and
factor, specifically regulates the production and
differentiation of megakaryocytes, which also produce platelets. Beside the primary action in megakaryocytopoiesis, TPO also plays
an
important role in the growth and survival of hematopoietic stem cells . [Bociek & Armitage 1996].
The CSFs include granulocyte-macrophage colony-stimulating factor (GM- CSF), granulocyte colony-stimulating factor (G-CSF) and macrophage colony- stimulating factor (M-CSF). GM-CSF stimulates the production of neutrophils, eosinophils, basophils, monocytes and their progenitor cells. G-CSF stimulates the production of granulocytes, and M-CSF stimulates the production of macrophages [Wadhwa & Thorpe 2008]. Interleukins (IL) are secreted by different types of body cells, regulate the immune system and participate in the development and differentiation of hematopoietic cells. [Ioana B 2011].
Fig 1.1 The process of hematopoiesis. [Bewry NN. 2009].
1.3. Historical aspects: In 1845, Bennett in Scotland and Virchow in Germany described patients with splenic enlargement, severe anemia, and enormous concentrations of leukocytes in their blood at autopsy. Bennett initially favored an extreme pyemia as the explanation, but Virchow argued against suppuration as a cause. Additional cases were reported by Craige and others, and in 1847 Virchow introduced the designation weisses Blut and leukämie (leukemia), Greek for "white blood" [Deininger M. 2007]. In 1878, Neumann proposed that the marrow not only was the site of normal blood cell production, but also was the site from which leukemia originated and used the term myelogene (myelogenous) leukemia [Kaushansky K. et al 2010 ]. Subsequent observations amplified the clinical and laboratory features of the disease, but few fundamental insights were gained until the discovery by Nowell and Hungerford [ Nowell & Hungerford 1960] who reported
in 1960 that two patients with the disease had an apparent loss of the long arm of chromosome 21 or 22, an abnormality that was quickly confirmed and designated the Philadelphia chromosome
in honor of the city in
which it was identified [ Nowell & Hungerford 1960]. In 1973, Janet Rowley discovered that the abnormal “Philadelphia” chromosome identified in patients with CML by Nowell and Hungerford was actually a reciprocal exchange of material between two chromosomes (9 and 22), a translocation (Rowley JD 1973). This discovery was enhanced by technical advances in the staining of chromosomes resulting in a unique pattern of alternating dark and light bands for each of the chromosomes [Reichard KK. et al 2009].
This observation led to a new approach to diagnosis, a marker to study the pathogenesis of the disease, and a focus for future studies of the molecular pathology of the disease. The availability of banding techniques to define the fine structure of chromosomes led to the discovery by Rowley [Rowley JD 1973] that the apparent lost chromosomal material on chromosome 22 was part of a reciprocal translocation between chromosomes 9 and 22. The discovery that the cellular oncogene ABL on chromosome number 9 and a segment of chromosome 22, the breakpoint cluster region (BCR), fuse as a result of the translocation provided a basis for the study of the molecular cause of the disease.[ Bartram et al 1983 ]. The appreciation that the fusion gene encoded a constitutively active tyrosine kinase (BCR-ABL) that was capable of inducing the disease in mice established the fusion gene product as the proximate cause of the malignant transformation. The search for, identification of, and clinical
development of a small molecule inhibitor of the mutant tyrosine kinase has provided a specific agent, imatinib mesylate, with which to inhibit the molecule that incites the disease.[ Drucker et al 1996]. Several more potent congeners have also been synthesized. The important landmarks in the study of CML could be summarized in the following points: 1- The discovery of the Philadelphia (Ph) chromosome in 1960. 2- The characterization in 1973 of the t(9;22)(q34;q11) translocation. 3- The identification in the 1980s of the BCR – ABL (now renamed BCR – ABL1 ) chimeric gene and associated oncoprotein. 4- The demonstration that introducing the BCR – ABL gene into murine stem cells in experimental animals caused a disease that simulated human CML [Goldman & Mughal 2011].
1.4. Definition: The term ‘ myeloproliferative neoplasm ’ has recently been introduced by the panel of experts convened by the World Health Organization (WHO) to classify tumours of the haemopoietic and lymphoid systems and chronic myeloid leukaemia (CML) is the most common subtype (Table 1.1)[ Vardiman et al 2008 , Bain BJ. 2010]. Chronic myeloid leukaemia [Faderl et al 1999, Sawyers CL 1999, Alvarez et al2007](also known as chronic myelogenous leukaemia and chronic granulocytic leukaemia) is a clonal disease that results from an acquired genetic change in a pluripotential haemopoietic stem cell. This altered stem cell proliferates and generates a population of differentiated cells that gradually displaces normal haemopoiesis and leads to a greatly
expanded total myeloid mass [Vardiman et al 2008 , Vardiman JW 2009, Goldman & Mughal 2011 ]. It is characterized by anemia, extreme blood granulocytosis and granulocytic
immaturity,
basophilia,
often
thrombocytosis,
and
splenomegaly [Kaushansky et al 2010]. CML is often referred to as the disease of “firsts” .It was the first disease: (a) in which the term leukemia was utilized. (b) to be associated with a consistently recurring chromosomal abnormality. (c) to be recognized as the result of material reciprocally translocated from one chromosome to another. (d) to be the direct result of a specific gene fusion (as a result of the translocation). (e) to have a therapy particularly targeted against the fusion protein. [Nowell PC 1962 , Fialkow et al 1967, Rowley JD 1973 , de Klein et al 1982, Groffen J et al 1984, Daley GQ et al 1990, Druker et al 1996, Mughal & Goldman 2007, Buyukasik et al 2010].
Table 1.1
Myeloproliferative neoplasms (WHO classification).
Chronic myelogenous leukaemia, BCR – ABL1 positive Chronic neutrophilic leukaemia Polycythaemia vera Primary myelofibrosis Essential thrombocythaemia Chronic eosinophilic leukaemia Mastocytosis
Myeloproliferative neoplasm unclassifiable
Source : Vardiman et al (2008).
1.5. Incidence and prevalence: In Iraq the registered cases of all types of leukemia (including ALL, CLL, AML, CML& Non-specified leukemias ) during 2008 ranked third after breast, bronchus and lung cancers and formed 6.77% of the commonest ten cancers by site according to the Iraqi cancer registry, there were 960 registered cases of all types of leukemia (555 males and 405 females with a male to female ratio of 1.37 :1 ,and an incidence of
3.01/100 000 of the population. The age specific
incidence rate for all types of leukemia in Iraq was higher during the 7th and 8th decade of life.[ Table 1.2 ,Fig 1.2]. [ Iraqi cancer registry 2008].
Table 1.2: The commonest ten cancers by site/IRAQ
It also ranked third after bronchus and lung cancers and urinary bladder cancers in Iraqi male
population and second after breast
cancer in Iraqi female population registered in Iraq [Iraqi cancer registry 2008].
Fig 1.2 : The commonest ten cancers in IRAQ 2008 Fig 1.2 : The commonest ten cancers in IRAQ 2008
CML is the most common of the CMPDs. It accounts for about 15– 20% of all cases of adult leukemias, but less than 5% of all childhood leukemias. According to the United States Surveillance Epidemiology and End Results data (http://www.seer.cancer.gov) incidence of CML was 1.1 and 2 per 100.000 in men and women, respectively between 2003 and 2007.[ Buyukasik et al 2010]. Chronic myeloid leukemia accounts for approximately 5000 new cases per year in the United States. [ Deininger M. 2007]. Andolina et al reported that CML is composed of 3% of newly diagnosed pediatric leukemias, with an annual incidence of approximately 1 per million children and adolescents younger than 20 years, and CML is even rarer in children younger than age 4, with only 2 published case reports of children in this age group and there appears to be no ethnic or genetic predisposition[ Andolina et al 2012]. It is slightly more common in males than in females and occurs exceedingly rarely during the first decade of life. [Mughal and Goldman 2007]. The incidence of CML appears to be constant worldwide [Muir CS et al 1987]. It occurs in about 1.0 – 1.5 per 100 000 of the population per annum [Morrison VA 1994, Deininger M 2007, Horner MJ et al 2008] in all countries where statistics are adequate accounting for some 5000 new patients per year in the United States [Deininger M.2007].
CML is rare below the age of 20 years but occurs with increasing frequency with each decade of life. Currently, the median age of onset is 50 – 60 years. The incidence is slightly higher in males than in females [Reichard et al. 2009,Goldman & Mughal 2011], the male to- female ratio is 1.4–2.2:1. The clinical course is similar in males and females, [ Cortes JE et al 1996] and CML is slightly more common in Whites and Blacks than in other races. [Guilhot F. 2005].
Median age differs between cancer registries and clinical trials by 10–20 years. Reports of clinical studies underestimate the true age of the CML population [Rohrbacher & Hasford 2009]. Prevalence
rate
has
increased
by
use
of
tyrosine
kinase
inhibitors[Rohrbacher & Hasford 2009]. Some reports suggest a slightly higher prevalence in some parts of India and China, in particular Hong Kong, but these remain unconfirmed. [Mughal and Goldman 2007]. 1.6. Risk factors and aetiology: The mechanism by which the Ph chromosome is first formed and the time required for progression to overt disease are unknown.[ Goldman & Melo 2003]. In the majority of patients with CML a clear etiology is absent and therefore the disease is neither preventable nor inherited. [Quint á s – Cardama et al. 2010]. The following risk factors could be implicated: 1.6.1: Radiation : The risk of developing CML is significantly increased by exposure to high doses of irradiation, exposure to very high doses of ionizing radiation can increase the occurrence of CML above the expected frequency in comparable populations. [ Preston et al 1994,Guilhot& Roy 2005]
1.6.1.1. Atomic bomb radiation: It is well documented that ionizing radiation is leukemogenic and CML has been observed in individuals exposed to the radiation emitted by the atomic bomb explosions in Japan in 1945. In these patients, the incidence of CML was 50 – fold higher than that of non - exposed subjects and it peaked approximately 10 years after the explosion, although patients younger than 15 years of age developed CML earlier than those 30 years of age or older. Nonetheless, in most cases of CML no antecedent radiation exposure is discernible. [Ichimaru M et al 1978, Preston et al.1994,Quint á s – Cardama et al 2010 ]. Approximately 30 percent of the leukemia patients had CML. with a median latent period of 11 years among survivors . [Maloney WC. 1987]. 1.6.1.2.:Diagnostic X-ray exposure has been associated with an increased occurrence of CML. [Preston-Martin et al.1989 ], A similar observation was reported for patients receiving intravenous thorium dioxide for diagnostic X-ray contrast studies. [National Research Council 1990] . 1.6.1.3. Therapy-related exposure to radiation: CML has been described in: 1-British patients with ankylosing spondylitis treated with spine irradiation,[ Smith and Doll 1982 ]. among whom approximately 20 percent of the leukemia cases were CML with a median latent period of approximately 4 years [Maloney WC. 1987].
2- Women with uterine cervical carcinoma who received radiation therapy [ Boice et al 1985] of whom approximately 30 percent had CML with a latent period of approximately 9 years [Maloney WC. 1987]. 3- Also CML has been reported after treatment for Hodgkin disease [Jackson et al 1994, Varady et al 2001]. 4- In vitro, high-dose ionizing irradiation of hematopoietic cell lines can induce the formation of the BCR-ABL fusion gene with corresponding mRNA transcripts. [Aguiar RCT 1998, Numata
et al.2002,]. Although
ionizing radiation is a physical mutagen known to induce DNA doublestrand breaks , little is known of the molecular mechanisms by which it generates leukemia specific fusion genes.[ Deininger MW et al 1998,Guilhot & Roy 2005]. 1.6.2. Chemical agents: In general, no chemical or petrochemical products have been recognized as factors that could increase the occurrence of CML, and in contrast to the high incidence of therapyrelated myelodysplastic syndrome and acute myeloid leukemia, therapyrelated CML is less frequent. 1.6.2.1. Chemical leukemogens: such as benzene and alkylating agents, are not causative agents of CML, although they are well established to produce a dose-dependent increase in acute myelogenous leukemia [Lichtman MA 2008]. In several large studies, no relationship was found between benzene exposure and CML. [Dean MR 1996 ,Raabe & Wong 1996,].
1.6.2.2.
chemotherapy:
CML
was
reported
after
high-dose
chemotherapy with subsequent autologous stem cell transplantation. [Aguiar RCT 1998,Numata et al 2002]. 1.6.3.Genetic factors: There is no evidence of a genetic predisposition although multiple occurrence of CML in a family has been reported. [ Tokuhuta et al 1968].
1.7. Pathogenesis and cell biology: Chronic myeloid leukemia (CML) is probably the most extensively studied human malignancy, [ Deininger et al 2000], and has served as a pacemaker for the development of new concepts and strategies in oncology.[ Deininger M. 2007]. Its biology has been a big challenge for investigators and researchers for many years, specialy during the last few decades. It is generally believed that CML develops when a single, pluripotential, hematopoietic stem cell acquires a Ph chromosome carrying the BCRABL fusion gene, which confers on its progeny a proliferative advantage over normal hematopoietic elements and thus allows the Ph-positive clone gradually to displace residual normal hematopoiesis.[ Idem.2001]. The evidence for this hypothesis derives in part from the consistency of the molecular abnormality in any given patient, but the mechanism by which the molecular and cytogenetic changes occur remains enigmatic. Similarly, the molecular basis of the apparent proliferative advantage is not well defined, but it may relate in part to constitutive expression by
leukemic progenitors of growth-stimulating factors, notably interleukin-3 and granulocyte colony-stimulating factor. Moreover, CML cells seem to survive longer than their normal counterparts, as a result of a defective apoptotic response to stimuli that would otherwise lead to physiologic cell death. [Goldman & Melo 2003]. Hematopoiesis in CML is clonal, derives from a self-renewing, hematopoietic pluripotent stem cell, and is fairly normal with respect to cellular maturation and function [Daley et al.1990]. However, there is a relentless rise in the white blood cell count. The disease is acquired (somatic mutation), given that the identical twin of patients with CML and the offspring of mothers with the disease neither carry the Ph chromosome nor develop the disease. [Whang-Peng & Knutsen 1982 ]. Acquisition of the BCR-ABL fusion gene as a result of the t(9;22) (q34;q11.2) in a single multipotential hematopoietic cell results in the CML stem cell, necessary for the initiation and maintenance of the chronic phase of CML. [Kavalerchik et al. 2008 ,Savona & Talpaz 2008]. This fusion gene product is the genetic hallmark of CML, Its acquisition, presumably during a division of a pluripotent stem cell, is part of the molecular and cellular defects that ultimately produce CML.[ Reichard et al 2009]. The unequivocal demonstration of Ph chromosomes in all blood cell types, including B and T cells, is widely accepted as evidence that the neoplastic clone originates in a very primitive pluripotent stem cell. Most often, this fusion protein is the result of a reciprocal translocation between the Abelson (ABL) oncogene on chromosome 9 and the breakpoint cluster region (BCR) on chromosome 22 (Fig 1.3).
Fig 1.3 BCR-ABL translocation and the Philadelphia chromosome.
Occasionally, there is a complex (e.g., three-way) translocation involving a third (or more) chromosome; however, the BCR/ABL fusion product is always present.[ Reichard et al 2009]. It is presumed that the leukaemic stem cell replicates and that its progeny give rise to increased numbers of myeloid progenitor cells and also of differentiated progeny. Thus, the normal marrow is gradually replaced by a leukaemic myeloid mass that expands to fill normal fat spaces and encroaches on areas of long bones that are normally devoid of haemopoiesis in the adult. The increased myelopoiesis involves primarily the granulocyte series, but megakaryocyte and platelet numbers are also usually increased. Obvious erythroid hyperplasia and polycythaemia occur only rarely .
The numbers of committed progenitors (i.e. CFU - GEMM, BFU - E and CFU - GM) are greatly increased compared with normal, and this increase is proportionately much larger in the blood than in the marrow of untreated CML patients. BFU - E and CFU - GM numbers in the blood are significantly correlated with the leucocyte count; their numbers are restored to normal or subnormal levels by appropriate treatment. [Goldman & Mughal 2011]. Cells that form colonies of neutrophils and macrophages or eosinophils (CFUs) are increased in the marrow and blood. The increase in CFUs in marrow is approximately 20-fold normal and in blood approximately 500-fold normal. [Kaushansky et al 2010]. Theoretically, an apparently autonomous proliferation of myeloid progenitors could be due to increased responsiveness to one or more physiological stimulators of haemopoiesis or to loss of sensitivity to a normal inhibitor. As a consequence of this , many efforts have been directed to assessing the response of CML progenitors to haemopoietic growth factors, notably granulocyte
colony - stimulating factor (G -
CSF), granulocyte/ macrophage colony - stimulating factor (GM - CSF), interleukin (IL) - 3, stem cell factor and erythropoietin. CML progenitors in in vitro culture systems adhere less well to preformed marrow stromal layers than their normal counterparts. This may be due to an abnormality of an integrin or absence of a glycosylphosphatidylinositol - anchored protein that has not yet been defined. Thus, it is possible that the excessive proliferation of CML progenitors is due partly to their premature escape from physiological inhibitory influences in the stem cell niche. [Goldman & Mughal 2011] .
In normal individuals, BCR and ABL protein is expressed in virtually all cells. . [ Melo JV 1996]. Most, if not all, patients with CML have hematopoietic stem cells that, after treatment or culture in vitro, use of special cell isolation techniques, do not have the Ph chromosome or the BCR-ABL fusion gene. [Kaushansky et al 2010]. In CML, a t(9;22) results in a hybrid BCR/ABL gene in which exon 1 of ABL is replaced by 5′ exons of BCR . The breakpoints in the BCR gene region on chromosome 22 are found within three defined regions. Depending on the position of the BCR breakpoint, fusion genes are generated that encode 190-, 210-, or 230-kDa forms of the BCR/ABL tyrosine kinase .Because the ABL component of the fusion gene is largely invariant, it follows that variability in disease phenotype may be due to protein sequences encoded by the translocation partner, BCR.[ Melo JV 1996].
1.8. Molecular anatomy and pathology: The cytogenetic hallmark of CML is the Philadelphia Chromosome (Ph chromosome), which is demonstrable in approximately 90% of patients with a diagnosis of CML based on clinical and morphologic criteria. .[ Mughal and Goldman 2007]. The Ph chromosome arises post-zygotically, as it is found only in hematopoietic tissue. [ Najfeld V. 2008]. The unequivocal demonstration of Ph chromosomes in all blood cell types, including B and T cells, is widely accepted as evidence that the neoplastic clone originates in a very primitive pluripotent stem cell [Caspersson et al 1970].
The classic BCR-ABL gene of CML results from the fusion of parts of two normal genes: the ABL gene on chromosome 9 and the BCR gene on chromosome 22. Both genes are ubiquitously expressed in normal tissues, but their precise functions are not well defined. [ Goldman & Melo 2003]. The Ph chromosome, originally thought to be a shortened chromosome 22 and thus referred to as 22_3 is the result of a reciprocal translocation between the long arms of chromosomes 9 and 22 [t(9;22)(q34;q11)].[ Mughal and Goldman 2007]. The material missing from chromosome 22 was not lost (deleted) from the cell, but was translocated to the distal portion of the long arm of chromosome 9. The amount of material translocated to chromosome 9 was approximately equivalent to that lost from 22, and the translocation was predicted to be balanced. [Rowley 1973] Mutations of the ABL gene on chromosome 9 and of the BCR gene on chromosome 22 are central to the development of CML. [Kaushansky et al 2010] In the translocation that forms the fusion gene, a break occurs in ABL somewhere upstream of exon a2, and simultaneously a break occurs in the major breakpoint cluster region of the BCR gene. As a result, a 5' portion of BCR and a 3' portion Of ABL are juxtaposed on a shortened chromosome 22 (the derivative 22q-,or Ph, chromosome) .[ Goldman & Melo 2003].
The breaks were localized to band 34 on the long arm of 9 and band 11 on the long arm of 22. Therefore, the classic Ph chromosome is t(9;22)(q34;q11), abbreviated t(Ph) (Fig.1.3). The Ph chromosome can develop on either the maternal or the paternal member of the pair. [melo 1996]. This reciprocal chromosomal translocation gives origin to a hybrid BCRABL gene, encoding a p2lO (BCR-ABL) fusion protein with elevated tyrosine kinase activity and transforming abilities [Melo JV 1996]. In 1982, the human cellular homologue ABL of the transforming sequence of the Abelson murine leukemia virus was localized to human chromosome 9. In 1983, ABL was shown to be on the segment of chromosome 9 that is translocated to chromosome 22 by demonstrating reaction to hybridization probes for ABL only in somatic cell hybrids of human CML cells containing 22q– but not those containing 9q+. v-abl is the viral oncogenic homologue of the normal cellular ABL gene. This gene (v-abl) can induce malignant transformation of cells in culture and can induce leukemia in susceptible mice. [Kaushansky et al 2010] In 1984 the precise positions of the genomic breakpoint on chromosome 22 in different CML patients were found to be clustered in a relatively small 5.8 - kb region to which the name ‘ breakpoint cluster region ’ (BCR) was given. Later, it became clear that this region formed the central part of a relatively large gene now known as the BCR gene, whose normal function is not well defined, and the breakpoint region was renamed ‘ major breakpoint cluster region ’ (M- BCR).[ Goldman & Mughal 2011].
The position of the genomic breakpoint in the ABL gene (now often referred to as the ABL1 gene to distinguish it from the ABL1- related gene, ARG or ABL2 ) is very variable, but it always occurs upstream of the second (common) exon (a2). Thus, the Ph translocation results in juxtaposition of 5 ′ sequences from the BCR gene with 3 ′ ABL1 sequences derived from chromosome 9 (Figure1.4 ). It produces a chimeric gene, designated BCR – ABL , or better BCR – ABL1 , that is transcribed as an 8.5 - kb mRNA and encodes a protein of 210 kDa. This p210 BCR – ABL1 oncoprotein has far greater tyrosine kinase activity than the normal ABL1 gene product.[ Deininnger et al 2000, Goldman & Mughal 2011].
Fig. 1.4 . The structure of the normal BCR and ABL1 genes and the fusion transcripts found in CML [Goldman & Mughal 2011]
The ABL gene is the human homologue of the v-abl oncogene carried by the Abelson murine leukemia virus (A-MuLV), and it encodes a nonreceptor tyrosine kinase. [Deininger et al 2000].
It was shown in the early 1980s that the ABL1 proto- oncogene, which encodes a non - receptor tyrosine kinase, was normally located on chromosome 9 but was translocated to chromosome 22 in CML patients. .[ Goldman & Mughal 2011].
In normal individuals, BCR and ABL protein is expressed in virtually all cells. In CML, a t(9;22) results in a hybrid BCR/ABL gene in which exon 1 of ABL is replaced by 5′ exons of BCR.[ Reichard et al 2009]
The genetic hallmark of CML is the presence of the BCR-ABL fusion gene product. Its acquisition, presumably during a division of a pluripotent stem cell, is part of the molecular and cellular defects that ultimately produce CML. [Caspersson et al 1970]. Breakpoints within BCR localize to three main breakpoint cluster regions ( bcr ). In most patients with CML and in one - third of those with Ph positive acute lymphoblastic leukemia (ALL), the breakpoint maps to the major breakpoint cluster region (M - bcr ), which spans BCR exons 12– 16 (formerly called b1 – b5), giving rise to a fusion transcript with either b2a2 or b3a2 junctions that translates into a 210 – kDa protein (p210 BCR - ABL1 ). In two - thirds of patients with Ph - positive ALL and rarely in CML, the breakpoints within BCR localize to an area of 54.4 kb between exons e2 ′ and e2, termed the minor breakpoint cluster region (m - bcr ), which translates to a 190 - kDa protein (p190 BCR ABL1 ). A third breakpoint cluster region ( μ - bcr ) has been identified
giving rise to a 230 - kDa fusion protein (p230 BCR - ABL1 ) associated with some cases of chronic neutrophilic leukemia [Melo et al 1994, Mughal and Goldman 2007 , Quint á s –Cardama et al 2010]. Breaks in the major bcr (Mbcr)result in e13a2 or e14a2 fusion mRNAs (previously referred to as b2a2 and b3a2, according to the original numbering of exons within the major breakpoint cluster region) and a 210-kd protein (p210BCR-ABL). p210BCR-ABL is typical of CML but also occurs in one-third of patients with Ph-chromosome-positive ALL. [Deininger et al 2000]. The Ph chromosome is associated with a BCR-ABL fusion gene expressed as an oncoprotein, p210BCR-ABL, which is generally considered as the initiating event for the chronic phase of CML. .[ Mughal and Goldman 2007]. The reciprocal translocation t(9;22)(q34;q11) gives rise to the BCR ABL1 oncogene that encodes for the constitutively active BCR - ABL1 protein kinase. Several experimental models, such as BCR - ABL1 expressing CD34 + cells in culture or retrovirally transduced BCR ABL1 - positive mouse cells, have established a causal relationship between BCR - ABL1 and human leukemia. [Quint á s – Cardama et al 2010]. The fact that the Abl portion in the different fusion proteins is constant while the Bcr portion is variable provides circumstantial evidence that the transforming principle is likely to reside in Abl, while the Bcr part appears to modify the disease phenotype, with retention of a larger BCR portion rendering the disease less aggressive. Although the three major types of BCR-ABL fusion mRNA account for more than 99% of cases of Bcr-Abl-positive leukemia, many more BCR-ABL variants have been seen in anecdotal cases or small series of patients. Most of these variants have atypical breakpoints in BCR, generating fusion mRNAs, such as
e6a2 or e8-insert-a2, where the open reading frame is retained by interposition of intronic sequences. However, fusions between BCR exon 1 and ABL exon 3 have also been described in CML patients. .[ Mughal and Goldman 2007 ]. The clinical significance of different breakpoints in CML is not well defined. However, some intriguing correlations have been observed. Both childhood and adult CML are virtually always of the p210 type. However, in contrast to adults, in whom two thirds of cases have the b3a2 transcript, children with CML have a predominance of b2a2 fusion junctions. [Reichard et al 2009]. Approximately 10% of patients with typical CML are negative for the t(9;22)(q34;q11) by conventional G- or R-banding techniques. In approximately half of these patients, the BCR-ABL translocation is detectable by fluorescence in situ hybridization (FISH) or reverse transcription polymerase chain reaction (RT-PCR), a situation referred to as cryptic or silent Ph translocation. The clinical course of these individuals is not different from classical Ph-chromosome-positive CML, while the remaining 5% of patients have truly BCR-ABL-negative disease. .[ Mughal and Goldman 2007]. According to the World Health Organization, CML is defined by presence of the BCR-ABL translocation, and thus the term Ph-negative or BCR-ABL-negative CML should not be used any longer. Depending on specific features, it may be possible to classify such patients as having chronic myelomonocytic leukemia if there is persistent monocytosis of more
than
109/L,
or
atypical
CML
if
there
is
prominent
dysgranulopoiesis. Otherwise, the disease should be referred to as chronic myeloproliferative disease, unclassifiable. [Mughal and Goldman 2007]. The exclusive definition of CML as the BCR-ABL-positive disease has become even more important with the advent of imatinib
as a specific targeted therapy for this disorder. [ Mughal and Goldman 2007]. The messenger RNA (mRNA) molecules transcribed from this hybrid gene usually contain one of two BCR-ABL junctions, designated e13a2 (formerly b2a2) and e14a2 (or b3a2). There is no evidence that the type of junction has any prognostic significance. Both BCR-ABL mRNA molecules are translated into an 210-kD oncoprotein, usually referred to as p210 BCR-ABL. [ Goldman and Melo 2003]. The structure of the BCR-ABL protein and the biochemical pathways in which it is involved have been extensively studied.[ Goldman and Melo 2003]. One of the most striking differences between the normal ABL protein and BCR-ABL is in their contrasting subcellular locations. The ABL protein is found in both the nucleus and the cytoplasm and can shuttle between these two compartments under the influence of its nuclear-localization signal and nuclear-export signal domains, whereas BCR-ABL is exclusively cytoplasmic. [ Goldman and Melo 2003]. Most often, this fusion protein is the result of a reciprocal translocation between the Abelson (ABL) oncogene on chromosome 9 and the breakpoint cluster region (BCR) on chromosome 22 .Occasionally, there is a complex (e.g., three-way) translocation involving a third (or more) chromosome; however, the BCR/ABL fusion product is always present. [Reichard et al 2009]. Other variant breakpoints and fusions can give rise to full-length, functionally oncogenic BCR-ABL proteins, notably p190 BCR-ABL (associated with an e1a2 mRNA junction) and p230 BCR-ABL
(associated with an e19a2 mRNA junction), but they are rather rare in classic CML. . [ Goldman and Melo 2003]. Notably, BCR - ABL1 can transform hematopoietic stem cells, but not committed progenitors lacking self - renewal capacity. Different breakpoints within ABL1 at 9q34 have been described, located either upstream of exon Ib, downstream of exon Ia, or more frequently between the two (Figure1.4). [Quint á s – Cardama et al 2010]. The leukemogenic potential of p210 BCR-ABL resides in the fact that the normally regulated tyrosine kinase activity of the ABL protein is constitutively activated by the juxtaposition of alien BCR sequences. [ Goldman and Melo 2003]. Bcr-Abl tyrosine kinase activity maintains a complicated and redundant network of signaling pathways, individual components of which are frequently dispensable for malignant transformation. The biological hallmarks of CML cells, increased proliferation, reduced apoptosis, and perturbed adhesion to extracellular matrix, are almost exclusively dependent on Bcr-Abl’s tyrosine kinase activity, indicating that the latter is an ideal therapeutic target. The mechanisms underlying disease progression are not well understood.[ Mughal and Goldman 2007]. Nuclear ABL is an essentially proapoptotic protein, playing a key part in the cellular response to genotoxic stress. BCR-ABL, in contrast, is largely antiapoptotic and, although it retains the ABL nuclear-localization and nuclearexport sequences, seems unable to enter the nucleus. [ Goldman and Melo 2003]. The main reason the BCR-ABL protein is retained in the cytoplasm is its constitutively activated tyrosine kinase. [ Goldman and Melo 2003].
BCR acts by promoting dimerization of the oncoprotein, such that the two adjacent BCR-ABL molecules phosphorylate each other on tyrosine residues in their kinase-activation loops. The uncontrolled kinase activity of BCR-ABL then usurps the physiologic functions of the normal ABL enzyme by interacting with a variety of effector proteins, the net result of which is deregulated cellular proliferation, decreased adherence of leukemia cells to the bone marrow stroma, and a reduced apoptotic response to mutagenic stimuli. Unfortunately, the relative contributions of these effects to the phenotype of chronic-phase CML is still poorly understood. [ Goldman and Melo 2003]. P53: P53 is a tumor-suppressor gene .The human p53 gene is located on chromosome 17 p13, which is one of the more frequent targets for chromosomal alterations found in human tumors. [Prokocimer and Rotter 1994]. It encompasses 20 kb of DNA, consisting of 11 exons, the first noncoding exon separated from the cluster of the 10 exons by a large intron of 10 kb. [Prokocimer and Rotter 1994]. The protein was named p53 because of its size.[ Imamura et al.1994]. The p53 gene codes for a 2.6-kb mRNA molecule that contains a large 3’ noncoding region that is probably involved in the stabilization of the molecule. [Prokocimer and Rotter 1994]. P53 is a cell cycle control protein. [Prokocimer and Rotter 1994]. It is associated with the DNA repair machinery and that it functions as a "guardian" of the genome . It halts the cell cycle upon DNA damage. In addition, it is also a key regulator of apoptosis. [Prokocimer and Rotter 1994,Krug U et al 2002].
The effect of wild-type p53 (wt-P53) on cellular repertoire of response is heterogenous and includes the following: differentiation, apoptosis, and growth arrest, along with DNA repair. Cellular decision between these various pathways is probably cell-type dependent and stimulus-specific. [Prokocimer and Rotter 1994]. Further
functional
studies
determined
that
wt-p53
suppressed
transformation of cells and overexpression of wt-p53 blocked cells in the G, phase of the cell cycle. [ Imamura et al.1994]. In case the DNA damage is very substantiated and unrepairable, p53 induces cells to undergo apoptosis. When DNA damage is rather low and can be repaired, p53 will arrest the cell in the GI phase to permit the activity of the DNA repair machinery (Fig1.5). [Prokocimer and Rotter 1994].
Fig 1.5 A suggested model for the involvement of wild type p53 [Prokocimer and Rotter 1994].
Tumor suppressor genes are altered via different mechanisms, including deletions and point mutations, which may result in an inactive or dominant negative protein. [Krug et al 2002]. If p53 is missing when cell damage occurs , cells do not undergo p53 – mediated arrest or apoptosis. Thus , cells that have sustained mutations in oncogenes or tumor –suppressor genes as a result of the damage obtain a growth advantage and in turn fuel the development of cancer [Dyke 2007]. P53 is involved in differentiation of hematopoietic cells, experiments suggest that endogenous wild-type p53 protein plays a role in hematopoietic cell maturation, possibly by contributing to the inhibition of proliferation that occurs during terminal differentiation. [Prokocimer and Rotter 1994]. It appears that alterations in the p53 gene are involved directly or indirectly in the majority of human malignancies. [Prokocimer
and
Rotter 1994]. Additional careful analysis of the p53 gene has shown that it is frequently mutated in more than 50 varieties of human cancers including lung, breast,
thyroid,
gastrointestinal,
and
ovarian
cancers,
lymphomas/leukemias, and brain tumors. [ Imamura et al 1994]. Mutations in the TP53 gene are a feature of 50% of all reported cancer cases. In the other 50% of cases, the TP53 gene itself is not mutated but the p53 pathway is often partially inactivated. [Cheok et al 2011]. Since the first detection of alterations of p53 in human tumors it has become clear that p53 is the most frequently altered tumor suppressor in human non-hematopoietic malignancies. More than 50% of solid tumors have loss of wild type p53 expression due to deletions or point mutations. In contrast, hematopoietic malignancies are less likely to home a p53 alteration. [Krug et al 2002]. This p53 alterations is reported to occur in
10-20% of hematopoietic malignancies.[preudhomme and Fenaux 1997 , Boyapati et al 2004]. P53 was found to be one of the most common genetic alterations (about 50%)
of
human
cancers,
including
hematologic
malignancies.
[Prokocimer and Rotter 1994]. It was observed that more than 50% of human primary tumors exhibit mutations in the protein and that these mutated products are inactive in rendering cells growth arrest. [Prokocimer and Rotter 1994]. Mutations in the p53 gene may occur at a late or early stage of tumor development. [Prokocimer and Rotter 1994]. These, p53 mutations occur moderately often in hematopoietic malignancies. They are particularly associated with progression of disease in both lymphoid and myeloid leukemias as well as lymphomas. [ Imamura et al 1994]. P53 in CML: The finding of a p53 mutation in myeloid cells during the chronic phase of CML is rare and is probably a grave prognostic sign. [ Imamura et al 1994]. CML frequently shows a loss of the short arm of chromosome 17, often through the formation of an isochromosome 17, i(17q), and acquires a p53 mutation in the progression towards blast crisis .This occurs especially during the progression to myeloid blast crisis . [Krug et al 2002]. Nevertheless, presence of p53 mutation was correlated with a poor clinical outcome. [Krug et al 2002]. i(17q) occurs in
30%
of
patients , causing a deletion of 17p.
Chromosome 17 alterations involving the short arm raised the possibility that p53 is associated with progress of the disease in at least some of the patients. [Prokocimer and Rotter 1994].
Transition from chronic phase to blast crisis is accompanied by a spectrum of alterations in p53 gene
structure and expression in
substantial number of patients. Loss of p53 expression may be responsible for many
of the features of acute-phase CML cells.
[Prokocimer and Rotter 1994]. Circumstantial evidence strongly suggests that a p53 mutation in the CML clone can result in disease transformation to myeloid blast crisis. [ Imamura et al 1994]. The structure and expression of the p53 gene is altered in about 20% to 30% of samples from patients in myeloid blast crisis of CML, whereas chronic-phase CML cells only rarely have detectable p53 alterations. [ Imamura et al 1994]. 1.9.
Clinical features
1.9.1 Stages of Disease CML evolves typically in three phases: 1- Chronic phase (CP). 2- Accelerated phase (AP). 3- Blast phase (BP). However, with current medical practice it is becoming more common for patients to be diagnosed before the development of any symptoms (preclinical stage) because of periodic routine medical evaluation with physical examination and laboratory testing. By following multiple clinical parameters in untreated patients, previous investigators have constructed regression curves describing the chronology of clinical events in CML. These observations indicate that ∼6 years elapse from the time of the initial chromosomal translocation until patients become
symptomatic. Once a leukocytosis is present, it takes ∼19 months (range, 7 to 24 months) for the WBC to reach 100,000/μl. [Reichard et al 2009]. The first clinical stage is a chronic phase that has historically lasted for an average of 4 years before transforming into an aggressive (accelerated)/blast phase with an overall grim prognosis. [Reichard et al. 2009]. Approximately 90% Of patients with CML are diagnosed in chronic phase (CP),characterized by overproduction of immature myeloid cells and mature granulocytes in the bone marrow and peripheral blood. At this stage, the leukemic burden is approximately1012 cells, which replace the normal hematopoietic tissue in the bone marrow. [Quint á s – Cardama et al 2010]. 1.9.2 Symptoms The symptoms are vague, nonspecific, and gradual in onset (weeks to months). [Kaushansky et al 2010]. In the past, the majority of patients presented with symptoms, usually attributable to splenomegaly, haemorrhage or anaemia. In recent years, CML has been diagnosed in almost 50% of patients before the onset of symptoms as a result of routine blood tests performed as part of medical examinations in healthy persons, for pregnancy, before blood donation or in the course of investigation for unrelated disorders. [Quint á s – Cardama et al 2010 ,Goldman and Mughal 2011 ,]. Patients in CP are typically asymptomatic, but if symptoms are present these usually relate to the presence of splenomegaly (e.g., abdominal fullness, early satiety, pain) [Quint á s – Cardama et al 2010]. These findings are the direct result of the expansion of granulocytes and precursors in the blood, bone marrow, and extramedullary sites (e.g., spleen). [Reichard et al 2009].
The most frequent complaints include easy fatigability, loss of sense of well-being, decreased tolerance to exertion, anorexia, abdominal discomfort, early satiety (related to splenic enlargement), weight loss, and excessive sweating [Thompson and Stainsby 1982, Cortes et al 1996, Goldman JM 1997]. (Table 1.3).
Table 1.3 Symptoms and Signs of Chronic-Phase CML at Presentation
Percent of Patients Symptoms Fatigue Weight loss Abdominal fullness and anorexia Easy bruising or bleeding Abdominal pain Fever Signs Splenomegaly Sternal tenderness Lymphadenopathy Hepatomegaly Purpura Retinal hemorrhage [Reichard K. 2009].
83 61 38 35 33 11 95 78 64 48 27 21
Other signs and symptoms that may occur are anorexia, weight loss, fever, fatigue, or anemia, which is usually normochromic and normocytic [Quint á s – Cardama et al 2010].
When present, symptoms may include lethargy, loss of energy, shortness of breath on exertion or weight loss or haemorrhage from various sites. Increased sweating is characteristic. Spontaneous bruising or unexplained bleeding from gums, intestine or urinary tract are relatively common. Visual disturbances may occur. Fever and lymphadenopathy are rare in chronic phase. The patient may have severe pain or discomfort in the splenic area, often associated with splenic infarction, or may have noticed a lump or mass in the right upper abdomen. Visual disturbances may be due to retinal haemorrhages. Sudden hearing loss occurs very rarely. Patients may present with features of gout or priapism in males, both of which are also rare. [Goldman and Mughal 2011]. Uncommon
presenting
symptoms
include
those
of
dramatic
hypermetabolism (night sweats, heat intolerance, weight loss) simulating thyrotoxicosis; acute gouty arthritis, presumably related in part to hyperuricemia; priapism, tinnitus, or stupor from the leukostasis associated with greatly exaggerated blood leukocyte count elevations; left upper quadrant and left shoulder pain as a consequence of splenic infarction and perisplenitis; vasopressin-responsive diabetes insipidus; and acne urticata associated with hyperhistaminemia. Acute febrile neutrophilic dermatosis (Sweet syndrome), a perivascular infiltrate of neutrophils in the dermis, can occur. In the latter situation, fever accompanied by painful maculonodular violaceous lesions on the trunk, arms, legs, and face are characteristic. Spontaneous rupture of the spleen is a rare event. Digital necrosis has been reported as a rare paraneoplastic event. [Kaushansky et al 2010]. 1.9.3 Signs A physical examination may detect pallor and splenomegaly. The latter was present in approximately 90 percent of patients at diagnosis, but with
medical care being sought earlier, the presence of splenomegaly at the time of diagnosis is decreasing in frequency. [Cortes et al 1996], reported to be present in 50-70% of patients [ Goldman and Mughal 2011]. The spleen varies from just palpable to being so large that it occupies all the left side of the abdomen and is palpable also in the right iliac fossa. [Goldman and Mughal 2011]. Approximately 50% of patients have splenomegaly extending more than 10 cm below the costal margin at the time of diagnosis. Spleen size correlates reasonably well with the magnitude of the leukocyte count. The spleen is quite firm and usually nontender (unless splenic infarction is present), and the notch may be palpable. [Reichard et al 2009]. Splenic infarct is common and can herald the presentation of the illness. [Reichard et al 2009]. The liver is frequently also enlarged but with a soft edge that is difficult to define. [Goldman and Mughal 2011]. But it is less common. [Reichard et al 2009]. Sternal
tenderness,
especially the lower
portion,
is
common;
occasionally, patients notice it themselves. [Kaushansky et al 2010]. It is a reliable sign of disease and usually is limited to a small area of the sternum. If true sternal tenderness is present, the patient withdraws or complains spontaneously when firm pressure is applied to the tender area., and signs of infection could be present. [Reichard et al 2009]. Lymph nodes are palpable in most patients but rarely are >1 cm in largest diameter. [Reichard et al 2009].
There may be no other abnormal findings. Ecchymoses of varying sizes and ages may be present and may form discoloured subcutaneous lumps. Some patients have asymptomatic retinal haemorrhages. Patients with very high leucocyte counts may have features of leucostasis, with retinal vein engorgement and respiratory insufficiency. [Goldman and Mughal 2011]. 1.9.4 Laboratory findings: The diagnosis of CML rests on the examination of a peripheral blood smear and marrow biopsy. The documentation of either a Ph+ chromosome by karyotypic analysis or the presence of the BCR-ABL translocation by Southern blot or polymerase chain reaction (PCR) assays confirms the diagnosis. [Reichard et al 2009]. The great majority of patients with CML in the CP who have a p210 BCR-ABL protein will show the following haematoloical features. (Table 1.4) [Melo JV 1996]. Table 1.4 Typical features of Chronic myelogenous leukemia in chronic phase.[Hasserjian RP 2010].
1.9.4.1. Peripheral blood: The presumptive diagnosis of CML can be made from the results of the blood cell counts and examination of the blood film . [Thompson and Stainsby 1982, Cortes et al 1996]. 1.9.4.1.1 The red cells The blood hemoglobin concentration is decreased in most patients at the time of diagnosis. Red cells usually are only slightly altered, with an increase in variation from small to large size and only occasional misshapen (elliptical or irregular) erythrocytes. Small numbers of nucleated red cells are commonly present. The reticulocyte count is normal or slightly elevated, but clinically significant hemolysis is rare. Rare cases of mild erythrocytosis or erythroid aplasia have been documented. [Guilhot and Roy 2005,Kaushansky et al 2010]. The hematocrit is decreased in most patients [Guilhot and Roy 2005], Rarely the Hb is elevated. [ Bain BJ 2010]. 1.9.4.1.2 The white blood cells Leukocytosis is a common finding in patients with CML in the CP, The leucocyte count at diagnosis is usually in the range 20 – 200*109/ L, but the diagnosis of CML can be established by appropriate investigations in patients with persistent leucocytosis in the range 10 – 20*10 9/L; at the other extreme, occasional patients may present with leucocyte numbers in the range 200 – 800*109/L. [ Goldman and Mughal 2011]. The total leukocyte count is always elevated at the time of diagnosis and is nearly always greater than 25,000/
L (25 x 109/L); at least half the
patients have total white counts greater than 100,000/
L (100 x 109/L)
[Thompson and Stainsby 1982, Cortes et al 1996], but this high count only occasionally leads to signs and symptoms of hyperviscosity (priapism, cerebrovascular accidents, dizziness, confusion) or retinal hemorrhage. [Quint á s – Cardama et al 2010]. The total leukocyte count rises progressively in untreated patients[Bain BJ 2010]. Rare patients show cyclical changes in the count, e.g. from 30 to 500*109/l with a periodicity of about 50 days, suggesting a partially intact negative feedback mechanism ;in this patient there was a similar cycle in the platelet count but 1 week in advance of the changes in the WBC . In some patients with cyclical changes, haematological remissions are sometimes prolonged although haemopoietic cells remain Ph positive . [Bain BJ 2010]. Granulocytes at all stages of development are present in the blood and are generally normal in appearance (Fig. 1.6).
Nucleated red cell
Myelocyte
Band form Neutrophil
Fig 1.6 Peripheral blood film in a patient with CML CP
The mean blast cell prevalence is approximately 3 percent but can range from 0 to 10 percent; progranulocyte prevalence is approximately 4 percent;
myelocytes,
metamyelocytes,
and
bands
account
for
approximately 40 percent; and segmented neutrophils account for approximately 35 percent of total leukocytes. [Kaushansky et al 2010]. The two predominant cell types are the myelocyte and the mature neutrophil (Fig.1.7).
Fig 1.7 Diagrammatic representation of the typical differential count in untreated cases of CML.[Bain BJ 2010].
Earlier granulocyte precursors are also present but promyelocytes are fewer than myelocytes and blasts are fewer than promyelocytes. [Bain BJ 2010]. Often, there is a "myelocyte bulge" in which the differential count shows an exaggerated proportion of myelocytes compared to the proportion observed in normal persons. Hypersegmented neutrophils are commonly present. [Kaushansky et al 2010]
Neutrophil alkaline phosphatase activity is low or absent in more than 90 percent of patients with CML. [DePalma et al 1996 , Bain BJ 2010,Kaushansky et al 2010], The NAP score may cycle inversely to the white cell count.[ Bain BJ 2010]. The proportion of eosinophils usually is not increased, but the absolute eosinophil count nearly always is increased. [Kaushansky et al 2010]. An absolute increase in the basophil concentration is present in almost all patients, and this finding can be useful in preliminary consideration of the differential diagnosis. Basophilic progenitor cells are increased in the blood. The proportion of basophils usually is not greater than 10 to 15 percent during the chronic phase .[ Kaushansky et al 2010 ]. Dysplastic features are lacking during the chronic phase of the disease. [Bain BJ 2010] Functional
abnormalities
of
neutrophils
(adhesion,
emigration,
phagocytosis) are mild; are compensated for by high neutrophil concentrations; and do not predispose patients in chronic phase to infections by either the usual or opportunistic organisms. [Kaushansky et al 2010], and In CP, CML cells retain their ability to differentiate and produce morphologically normal blood elements, capable of carrying out the physiological functions of normal counterparts. [Quintá s – Cardama et al 2010]. Absolute numbers of lymphocytes and monocytes are slightly increased, but both are reduced as percentages in the differential count. [Goldman and Mughal 2011]. Occasional nucleated red cells are present in the circulation in some patients. [Bain BJ 2010 , Goldman and Mughal 2011]. Platelets tend to be normal or increased in number and occasionally may exceed 1 million/μl. [Reichard et al 2009]. The platelet count is elevated in approximately 50 percent of patients at the time of diagnosis and is
normal in most of the rest. [Kaushansky et al 2010], their number are usually in the range 300 – 600 *109/L[Goldman and Mughal 2011], The median value in patients at diagnosis is approximately 400,000 cells/ L (400 x 109 cells/L). but may be normal or even reduced. [Goldman and Mughal 2011].A reduced platelets count at the time of diagnosis usually signals an impending progression to the accelerated phase of the disease. [Kaushansky et al 2010]. Thrombohemorrhagic complications of thrombocytosis are infrequent, and Platelet dysfunction can occur but is not associated with spontaneous or exaggerated bleeding [Kaushansky et al 2010]. 1.9.4.2. Bone marrow: Examination of the bone marrow by aspiration or trephine biopsy is not necessary to confirm the diagnosis of CML, but is usually carried out to assess the degree of marrow fibrosis, perform cytogenetic analysis on marrow cells and exclude incipient transformation. [Goldman and Mugahl 2011]. 1.9.4.2.1 Bone marrow aspirate: The bone marrow aspirate and biopsy are intensely hypercellular and demonstrate granulocytic and megakaryocytic hyperplasia, basophilia, and a blast percentage of 5% or less. [Quint á s – Cardama et al 2010] ,their number is 2-10%.[ Goldman and Mughal 2011]. (Fig. 1.8).
Myelocyte Neutrophil Fig. 1.8: Bone marrow aspirate of patient with CML CP
When small hypercellular fragments were spread on a glass slide, the trails show a cellular composition resembling that of CML blood. [Goldman and Mughal 2011]. Granulopoiesis is dominant, with a granulocytic-to-erythroid ratio between 10:1 and 30:1, rather than the normal 2:1 to 4:1. Erythropoiesis usually is decreased, and megakaryocytes are normal or increased in number. [Kaushansky et al 2010]. Their average size and nuclear lobe count is reduced in comparison with normal megakaryocytes. [Bain BJ 2010]. Eosinophils and basophils may be increased, usually in proportion to their increase in the blood. Mitotic figures are increased in number. [Kaushansky et al 2010]. Pseudo-Gaucher cells and sea blue histiocytes may be increased. [Bain BJ 2010].
1.9.4.2.2 Bone marrow biopsy: The marrow biopsy shows complete loss of fat spaces with dense hypercellularity. [Goldman and Mughal 2011] , The hematopoietic tissue takes up 75 to 90 percent of the markedly hypercellular marrow volume,. [Lorand-Metze et al 1987]. there is an increase in cells of all granulocyte lineages and, in some patients, increased megakaryocytes. [Bain BJ 2010]. (Fig.1.9) The marrows of CML patients have a mean doubling of microvessel density compared to healthy controls and have more angiogenesis in marrow than other forms of leukemia. This increased marrow vascularity decreases to normal after treatment. [Kaushansky et al 2010] Nearly half of the patients, show increase Collagen type III (reticulin fibrosis) at the time of diagnosis, this fibrosis takes the silver impregnation
stain
and
is
correlated
with
the
proportion
of
megakaryocytes in the marrow. Increased fibrosis also is correlated with larger spleen size, more severe anemia, and a higher proportion of marrow and blood blast cells. [Dezmezian et al 1987]. 1.9.4.3 Chemical Abnormalities: Uric Acid: Usually there is an increased production of uric acid with hyperuricemia and hyperuricosuria in untreated CML. [Kaushansky et al 2010]. If aggressive therapy leads to rapid cell lysis, excretion of the additional purine load may produce urinary tract blockage from uric acid precipitates. Formation of urinary urate stones is common in patients with CML. Serum Vitamin B12-Binding Proteins and Vitamin B12: Patients with myeloproliferative diseases have an increased serum level of B12-binding
capacity, and the source of the protein is principally mature neutrophilic granulocytes. The increase in transcobalamin level and the resultant increase in vitamin B12 concentration are particularly notable in CML, although any increase in the number of neutrophilic granulocytes, as in leukemoid reactions, can be accompanied by an increase in serum B12binding protein levels and vitamin B12 concentration. [Kaushansky et al 2010].]. Whole Blood Histamine Mean histamine levels are markedly increased in patients in chronic phase (median: ~5000 ng/mL) compared to healthy individuals (median: ~50 ng/mL); and, this elevation is correlated with the blood basophil count. [Kaushansky et al 2010]. Serum Lactic Dehydrogenase, Potassium, Calcium, and Cholesterol: The level of serum lactic acid dehydrogenase (LDH) is elevated in CML. Pseudohyperkalemia and hypercalcemia could occur during the chronic phase of cml but are rare,and Serum cholesterol is decreased in patients with CML. [Kaushansky et al 2010]. Serum Angiogenic Factors Angiogenin, endoglin (CD105), vascular endothelial growth factor (VEGF), fibroblast growth factor, and hepatocyte growth factor are increased strikingly in the serum of CML patients. [Kaushansky et al 2010].
1.9.4.4. Flow Cytometric Analysis: Flow cytometric analysis does not play a significant role in the chronic phase of CML. Aberrant expression of CD56 on the granulocytes and precursors may be seen; however, the blast count is not elevated in chronic phase. As the disease enters a more aggressive phase or blast crisis, flow cytometry is very useful in determining the lineage of the increased blasts that may be present. [Reichard et al 2009]. 1.10. Molecular Diagnosis: Molecular biology has found major applications in pathology, notably as applied to oncology. This has been a field of enormous expansion, where pure science has found a place in clinical practice and is now of everyday use in any academic unit. Presumably, we are looking at a series of methods which are novel in the diagnostic laboratory. [ Crocker J. 2002]. Over the past 35 years, cytogenetic analysis of malignant hematological disorders has been one of the most rapidly growing areas in cancer. More than 45,000 cytogenetically abnormal neoplastic disorders have been reported and the evidence accumulated clearly demonstrates that karyotype information provide both biological and significant clinical value .[ Najfeld V. 2008]. Molecular pathology is the study of disease at the fundamental level in relation to nucleic acid abnormalities or by means of techniques involving DNA or RNA analysis. [ Crocker J. 2002]. Molecular cytogenetics has now made it possible to identify genes involved at translocation breakpoints in specific chromosomal rearrangements. And the hypothesis put forward by Boveri at the turn of the century, namely, that an abnormal chromosome pattern is intimately associated with the malignant phenotype of the tumor cell has proved correct for many malignant disorders. . [ Najfeld V. 2008].
The important molecular cytogenetic methods most commonly used in clinical laboratories include: 1- Conventional cytogenetics G- Banding 2-Fluorescence in situ hybridization (FISH). 3-Hypermetaphase FISH 4-Fiber FISH 5- Comparative Genomic Hybridization (CGH). 6-Array CGH 7-Multicolor karyotyping( M-FISH,SKY). 8-PCR
Hypercellular marrow
Fig. 1.9 Bone Marrow Biopsy of Patient with CML showing hyper cellular marrow.
Table 1.5 Conventional and Molecular Cytogenetics Methods Most Commonly Used in Clinical Laboratories.
Characteristics Protein indigestion &/or special dye generate specific banding pattern for each chromosome
Applications Identification of both numerical and structural rearrangement of the entire genome
Advantages 1-Screening of the entire genome for chromosome level abnormalities 2-Low cost of reagents 3-Simple and robust procedure
Limitations Low resolution. Dependant on mitotic index
FISH
A small DNA fragment is used as a probe to search for homologus target sequence in chromosomal DNA
Detection of the number copies per cell and localization of probe DNA
Tested target must be known High cost in instrumentation
Hypermetaphase FISH
FISH application on accumulated large number of metaphase cells A small DNA fragment is used as a probe to search for homologous target sequence on chromatin fiber Comparative hybridization of differentially labeled total genomic DNA and normal reference DNA to normal human metaphase cell used as template
Detection of probe on large number of chromosomes
Cell culture not required Fast analysis and scoring Quantitative method Simple and robust Applicable to paraffinembeded tissue Large number of metaphase cells available (50D-2000)
Fine physical mapping DNA-protein interactions
High resolution mapping
Mainly used in research due to the complexity of the procedures involved
Detection of DNA copy number at the chromome bandlevel
Cell culture not required Genome-wide screening for genetic inbalances
Array CGH
The test and normal DNA are differentially labelled and cohybridized to a microarray
Disease-specialized arrays Chromosome arm-specific array Detection of DNA copy number
Genome-wide scanning;
Multicolor karyotyping (M-FISH, SKY)
Hybridization with 24 differentially labeled chromosome-specific probes for paining of every human chromosome in a distinct color
Detection of one or multiple rearrangements within a single metaphase cell
Identification of marker chromosomey Accurate identification of all segments in complex rearrangements
High cost in dedicated instrument Low resolution, dependent on chromosomal condensation of normal template Balanced rearrangements and low level of inbalances cannot be detected Not suitable for balanced translocations, inversions Automated high-throughput instrumentation Not practical for clinical use Vast amounts of data Requires mitotic cells and well spread chromosomes Limited accuracy in determination of breakpoints; Intrachromosomal changes remain undetected; High cost in instrumentation High cost in probes
Conventional Cytogenetics G Banding
FiberFISH
CGH
[ Najfeld V. 2008].
Not suitable for conventional cytogenetics
1-Conventional Cytogenetics Cytogenetic analysis in malignant disease is based on studies of the tumor cells themselves. Obtaining metaphase cells of tumor specimen is sine qua non of cancer cytogenetics. In hematologic malignancies usually a bone marrow aspirate is a required tissue while in lymphoma, it is usually a lymph node biopsy, and in solid tumors it is a tumor biopsy, either processed immediately (direct preparation) or cultured for 24-96 hours. Peripheral blood is not useful for cytogenetic analyses of the majority of patients with lymphoma and solid tumors. Chromosomes obtained from leukemic bone marrow cells from lymph nodes or solid tumors may be fuzzy with indistinct bands. [ Najfeld V. 2008]. 2-Fluorescence in situ hybridization(FISH) Flouresence In Situ Hybridization (FISH) provides an important adjunct to conventional cytogenetics and molecular studies in the evaluation of chromosome abnormalities associated with hematologic malignancies. In addition, FISH analysis offers one of the most sensitive, specific, and reliable strategies for identifying acquired genetic abnormalities such as characteristic gene fusions, aneuploidy, loss of a chromosomal region associated with hematologic disorders, and serving as a technique that can help in both the diagnosis of a genetic disease or suggesting prognostic outcomes. . [Tastemir et al 2011]. It is especially important for cells of patients with leukemia, where the quality of metaphases is often not so good, and is frequently used to monitor the response to therapy in various hematological malignancies. Thus, FISH is widely used today in clinical practice to help in diagnosis and selects appropriate treatments for patients with hematological malignancies . [Tastemir et al 2011].
In research, FISH studies are used to investigate the origin and progression of hematological malignancies, and to establish which hema topoetic compartments are involved in neoplastic processes. [Tastemir et al 2011]. The acronym FISH has been coined to describe the extremely powerful technique of fluorescence in situ hybridisation that has an ever increasing range of applications in medicine and biology. The technique allows the visualisation of quantitative genetic alterations on a cell by cell basis.[ Waters et al 1998]. • FISH is a molecular method that allows detection of the number, size, and location of DNA and RNA segments within individual cells in a tissue sample. It is based on the ability of single-stranded DNA to anneal to complementary DNA. [ Najfeld V. 2008]. • In malignant disorders, the target DNA is the tumor nuclear DNA of interphase cells or the DNA of metaphase chromosomes that are fixed on a microscope slide[ Najfeld V. 2008]. • In FISH technology, a specific DNA segment is converted into a probe through the attachment of a fluorescent tag or a reporter molecule that later in the procedure will be conjugated with the fluorescent tag [ Najfeld V. 2008]. • The probe (and the target DNA) is denatured and under proper hybridization conditions recognizes and binds to the homologous sequences in the target DNA [ Najfeld V. 2008]. • After hybridization of the copy number and the localization of the fluore scent tags, and consequently of the target homologous regions, are recognized under fluorescent microscopy both in chromosme spreads and interphase nuclei. FISH technology is simple, with high sensitivity,
and it phenomenal contribution to the cancer field largely relies on its applicability to interphase cells [ Najfeld V. 2008]. Fluorescence in situ hybridization (FISH) has become an important tool both for defining initial chromosomal abnormalities within a disease process, and for monitoring response to therapy as well as minimal residual disease.
[Tastemir et al 2011].
The FISH method involves hybridization of fluorescently labeled DNA probes onto interphase (nondividing) or metaphase cells that are adhered to a microscopic glass slide. [ Reichard et al 2009]. FISH uses fluorescently labelled probes specific for genes or whole chromosomes. It is based on the ability of a single-stranded DNA probe to anneal to its complementary sequence in a target genome. Many probes are now commercially available, offering reliable detection of the range of significant chromosomal abnormalities. It is possible to access detailed genomic information and develop FISH probes for virtually any known DNA sequence. [ Konn ZJ. 2009]. Fluorescence in situ hybridisation (FISH) is a specialised form of ISH and has become almost the province of the cytogeneticist. By using one or more probes, labelled with differently coloured fluorochromes, it is possible to demonstrate whole chromosomes or chromosomal loci in chromosome spreads or even in paraffin wax embedded archival tissue sections. This has revolutionised the analysis of tumour chromosomal abnormalities in tissue specimens at the molecular genetic level. [ Crocker J 2002]. These probes can be targeted to specific genetic loci (e.g., the ABL oncogene on the long arm of chromosome 9) or to repeat sequences that characterize centromeres. [Reichard et al 2009]. Interphase FISH
One of the main advantages of FISH is its ability to use non-dividing interphase cells as DNA targets, enabling the screening of large numbers of cells and providing access to a variety of cells with different hematopoetic activity .[ Kolialexi et al 2005]. • Interphase cytogenetics is the term used to describe detection and visualization of chromosomal abnormalities in non-dividing, interphase nucleus that allows the analysis of genome of individual cells. Chromatin in interphase cells is almost a tenth as condensed as in metaphase chromosomes, allowing for ordering of probes over shorter distances (50 kb-2 Mb) [ Najfeld V. 2008]. (Fig. 1.10 ) (Table 1.6).
Fig 1.10 BCR-ABL- Negative FISH.
Table 1.6 Advantages and limitations of cytogenetics and Interphase FISH [Najfeld V. 2008].
• Six aspects of interphase FISH are particularly important . - Interphase cytogenetics allows screening of a large number of cells. This permits investigation of hematological malignancies with a low mitotic yield, such as chronic lymphocytic leukemia (CLL) or multiple myeloma (MM). It also allows investigation of paraffin embedded tissues without any mitotic figures. Most of formalin-fixed, paraffin-embedded solid tissue that has not been de-calcified may be used for I-FISH evaluation . [ Najfeld V. 2008]. - Interphase cytogenetics permits detection of chromosomal rearrangements in hematologic malignancies in peripheral blood samples, thus obviating the need for bone marrow aspiration. For instance, in chronic myelogenous leukemia (CML), which rarely yields a large number of dividing cells in peripheral blood, conventional cytogenetics is usually uninformative. However, detection of BCR-ABL, a molecular equivalent of the Ph chromosome, in peripheral blood using interphase FISH provides reliable, fast, and quantitative results . [ Najfeld V. 2008]. - Interphase FISH offers a quantitative assay for monitoring disease progression or detection of minimal residual disease after ablative chemotherapy or stem cell transplantation, thus allowing detection of rare
and small abnormal cell population. This aspect is particularly useful for detection of micrometastasis and detection of disseminated cells in patients with early stage cancers. Studies of disseminated cell by interphase cytogenetics revealed a high degree of cellular heterogeneity even in early stages of tumor progression. [ Najfeld V. 2008]. - The use of specific probe sets allows detection of specific disease associated abnormalities such as t(8;21), which denotes the M2 sub-type of acute myelogenous leukemia (AML) or t(l5;17) associated with acute promyelocytic leukemia .[ Najfeld V. 2008]. - Abnormalities can be detected accurately in archival specimens kept up to 15 years at -20 to -70°C. - Lastly, simultaneous use of interphaseFISH and immunophenotyping is a powerful tool for the investigation of lineage involvement in diseases, such as myelodysplasia and to determine which cell population carries the specific chromosome abnormality. [ Najfeld V. 2008]. Fish analysis in the peripheral blood is a simple and reliably sensitive test for the detection and quantitative monitoring of the M-bcr/abl fusion gene in CML in routine clinical practice,although this can not entirely replace karyotype analysis of bone marrow cells.[Yanagi et al 1999]. Fiber FISH • A higher resolution of chromosomal abnormalities can be achieved when fluorescently labeled probes are hybridized to extended DNA or free chromatin (chromatin strands released from their chromosomal scaffold) or free DNA fibers. DNA fibers are chromatins from which proteins such as histones are removed allowing it to unfold and extend[ Najfeld V. 2008].
• The hybridized signals have the appearance of a "string of pearls" along the fiber rather than tight fluorescing spots observed in interphase cells. [ Najfeld V. 2008]. Multi-Color Karyotyping Multi-color karyotyping permits examination of the entire genome in a single analysis. In 1996, it became possible to identify 24 different human chromosomes (22 autosomes and the X and the Y sex chromosomes) each with a unique color with the help of fluorochrome-specific optical filters . This method is called M-FISH. [ Najfeld V. 2008]. When interferometer-based spectral imaging is used, the method is called SKY-FISH or spectral karyotyping. [ Najfeld V. 2008]. Comparative Genomic Hybridization (CGH) • Another powerful method used to identify locations of chromosomal gains, losses, deletions or amplifications, without prior knowledge of the chromosomal target that may be altered is CGH • Briefly, isolated DNA from leukemic bone marrow or tumor tissue is labeled with a one color fluorochrome (e.g., red) while DNA isolated from normal control tissue is labeled with a different color (e.g., green) . These differently labeled DNAs are hybridized against each other in a competitive hybridization reaction onto normal metaphase spreads. [ Najfeld V. 2008]. Array CGH (aCGH) • A newly emerging investigational method is aCGH. In aCGH metaphase chromosomes are replaced as the target by large number of mapped cloned DNA fragments and sequence-verified genomic clones (±100-200 kb) arrayed on the standard glass slide as hybridization target.
Thus, the main limitation of low resolution of CGH is substantially increased and radically transformed the CGH technique • In aCGH both the normal and the reference DNA are differentially labeled and co-hybridized to microarray, most often genomic DNA or eDNA, which are spotted on a glass slide (the array). The DNA copy number aberrations are subsequently measured by detecting intensity differences in the hybridization patterns of both DNAs. The resolution of the analysis is restricted only by clone size and by the density of the clones on the array. [ Najfeld V. 2008]. Types of FISH Probes There are three groups and five types of probes (as shown in Table1.7), which are usually used alone or combined to determine both numerical and structural rearrangements. [ Najfeld V. 2008].
Table 1.7 Types of probes for FISH [Najfeld V. 2008].
I-Repetitive sequence: (1): Centromeric enumeration probes (CEP), as the name implies, are used most frequently in interphase nuclei for detection of numerical chromosome anomalies II- Telomeric probes: (2): Pantelomeric (p-Tel). (3): Subtelomeric ( sub-T). Subtelomeric probes and unique gene loci probes may be applied both to interphase and metaphase cells in single, dual, triple or multi-colors to determine specific chromosomal rearrangements, deletions, or amplifications.
III-Locus-specific probes: (4): Unique sequence ,locus-specific identifier (LSI). (5): Whole chromosome painting probes (WCP) are used only on the metaphase cells and are never used in interphase cells. They are very useful in delineating complex rearrangements or the origin of marker chromosome [ Najfeld V. 2008]. Three basic types of DNA probes are generally employed: centromeric probes, whole chromosome probes, and locus-specific probes. (i)Centromeric probes, also known as chromosome enumeration probes (CEPs), hybridize to highly repetitive 171-base-pair sequences of alpha satellite DNA present within the centromeres of each chromosome. The probes are carefully selected to target sequences unique to specific chromosomes, allowing the detection and enumeration of specific chromosomes in interphase nuclei or metaphase cytogenetic preparations. Because of the presence of highly homologous satellite sequences, not all chromosomes can be specifically identified in this manner. In particular, the similarity between chromosomes 13 and 21 and between chromosomes 14 and 22 precludes the use of specific centromeric probes for these chromosomes. [ Cook JR. 2009].
(ii)Whole chromosome probes (sometimes called whole chromosome paints) are a complex mixture of probes, all labeled with one fluorochrome, that hybridize to sequences distributed along the length of a given chromosome. Analysis of metaphase chromosomes with a whole chromosome paint therefore identifies the chromosome of interest, allows enumeration of the chromosome, and can identify regions of the
chromosome of interest that may be located elsewhere because of structural karyotypic abnormalities (i.e., translocations). In an interphase nucleus, the DNA material from any given chromosome is widely spread throughout the nucleus. Whole chromosome paints therefore yield only diffuse staining in interphase cells, and this type of probe is suitable only for analysis of metaphase chromosome spreads. [ Cook JR. 2009]. (iii) locus-specific probe (also known as the locus-specific identifier, or LSI probe). It is the type of FISH probe most oftenly employed. This probe hybridizes to specific genes of interest, allowing the assessment of copy number and location of particular chromosomal regions. By using the LSI probe in two color or multicolor assays with other reference probes, one can assess for amplification of a target gene or design probes to detect translocations involving specific loci. Locusspecific probes are generally in the range of 100 to 300 kilobases to produce an easily visible signal. Locus-specific probes may be prepared from a variety of sources, including bacterial artificial chromosomes (BACs), P1 artificial chromosomes, and yeast artificial chromosomes. Particularly useful are the recently available FISH-mapped BAC libraries, which allow rapid identification of BACs hybridizing to particular areas of interest that may be subsequently grown, purified, and labeled for “home-brew” applications. In addition, the number of commercially available locus-specific probes relevant to common inherited conditions and neoplasms continues to grow rapidly.[ Cook JR. 2009].
FISH Probe Strategies • Four strategies are used in probe design to detect chromosomal translocations in hematologic malignancies and soft-tumor tissues: They include : - Conventional strategy - Extra sensitive strategy (ES) - Dual fusion strategy - Breakapart strategy • Conventional strategy: the first application of FISH technology for detection of chromosomal translocation in hematologic malignancy was in 1990 when BCR-ABL hybrid gene was identified using two-color FISH in interphase cells as well as in metaphase bone marrow derived CML cells . In the standard strategy for interphase evaluation of chromosomal translocation, a DNA probe, comprising sequences mapped proximally to the breakpoint in one of the chromosomes involved in reciprocal translocations, is combined with differentially labeled DNA probe that includes sequences mapped distantly to the breakpoint in the other chromosome. Positive nuclei for the translocation display one dual color fusion signal, representing one of the derivative chromosomes generated by the translocation, and two single color signals, one for each of the normal alleles. This standard FISH strategy has been used in hematological malignancies and lymphoma translocations at diagnosis. • ES: for detection of residual disease standard approach lacks specificity because cells with random spatial colocalization of normal signals with different colors, usually found in frequency from I to 10% of scored nuclei, are seen as false-positive. To minimize this problem, a strategy for ES method was developed in which a probe for one abnormal chromosome is designed in such way to generate an extra, smaller signal
in positive nuclei. Hybridization with this strategy results in abnormal cell showing colocalization of two signals in dual color, additional two signals in one single color and one signal in another single color • A dual fusion strategy was developed not only to minimize false positive results but also to detect additional deletions at the translocation breakpoints. Dual color dual fusion strategy includes the probe set with DNA sequences that encompass proximally and distally the translocation breakpoints on both chromosomes involved in translocation. The sequences for each chromosome are labeled with specific color, and the translocation generates fused signals in both derivative chromosomes. Positive nuclei are showing two copies of fusion signals and one copy of each of the single signals representing the normal alleles . • Multiple translocation partners are well known for leukemic genes such as myeloid lymphoid lineage (MLL) , RARA, or lymphoma gene ALK, and thus the fourth FISH strategy, "breakapart" probe, was developed to address this issue . In this approach, the breakapart probe includes DNA sequences mapped proximally and distally to the breakpoint within a critical gene (the 3' end and the 5' end) labeled with two different fluorochrome. In this approach, the fused fluorescent signals represent a normal gene, whereas nuclei with rearrangements within the target gene show one single color signal, one for each derivative chromosomes, regardless of which chromosome is the partner in translocation[ Najfeld V. 2008]. Conventional Cytogenetics and Varying FISH Methodologies These technologies are complimentary; each has its own advantages and limitations in investigating genome rearrangements of malignant cells . While conventional cytogenetics is the comprehensive study of all chromosomes, it requires a large number of proliferating and dividing
cells, which in some diseases is difficult to obtain particularly in solid tumors . Furthermore, many small deletions or structural rearrangements are either beyond the microscopic level of detection or chromosomal banding is not distinct enough for accurate detection due to the fuzzy appearance of chromosomes obtained from leukemic or tumor tissue. Moreover, complex rearrangements involving two or more chromosomes are often difficult to decipher accurately, which leads to underestimation of chromosomal abnormalities. FISH can be used in conjunction with conventional cytogenetics both in interphase and metaphase cells. It is a more sensitive method and can detect rearrangements as small as I kb. The main disadvantage of interphase FISH is that it cannot be used unless a known abnormality is suspected: "FISHing in the dark" is not a recommended clinical test. When the abnormality is known , interphase FISH can pinpoint the clonal aberration at the single cell level in a very short period of time. Other investigative FISH methods provide further refinement. Undoubtedly, in the future, automation will help to more accurately classify and diagnose hematological malignancies as well as solid tumors [ Najfeld V. 2008]. Genetic Testing Strategies for CML Cytogenetic, molecular genetic, and FISH are all useful techniques to study specimens from patients with CML. Each of these methods has strengths and weaknesses. However, no single testing method fulfills all the needs of clinicians who treat patients with CML. It is important to develop combinations of testing strategies that are accurate and cost effective for any given clinical situation. [Dewald et al 1998] If the patient has CML, it is important to perform FISH studies to establish a pretreatment base line for the percentage of interphase nuclei
with BCR/ABL fusion. It is useful to perform FISH studies on bone marrow and blood to permit appropriate comparisons with post-treatment specimens. Pretreatment specimens provide a good opportunity to examine a few Ph-positive metaphases to establish the type of signal pattern and thus to assure appropriate scoring criteria for subsequent specimens. For patients on therapy, D-FISH can be performed on peripheral blood at periodic intervals to assess the effectiveness of therapy. Consequently, bone marrow may not need to be collected to monitor therapy as frequently as in current practice. [Dewald et al 1998]
1.11. Prognostic Scores: The recognition of prognostic scores at diagnosis is essential. Most important is accurate identification of the disease stage (or phase), but in early chronic phase important prognostic information is derived from clinical and laboratory features. [Hehlmann et al 2007]. Many attempts have been made to subclassify or ‘ stage ’ chronic phase CML at diagnosis, in a manner that would permit some prediction of the duration of chronic phase in individual patients. [Goldman and Mughal 2011]. Several prognostic scales have been proposed in CML, These scores differentiate between patients with low, intermediate, and high risk of short survival. [Hehlmann et al 2007]. There are three prognostic scores: 1-Sokal score. 2-Hasford or Euro score 3-EUTOS score.
At diagnosis, it is important to collect all data relevant to calculate the CML risk scores (Sokal, Euro or Hassford , and EUTOS scores) as they have an important impact on the outcome of CML patients. [ Guilhot et al 2012]. For patients in CP, it is common to use one of the available risk stratification scores, such as Sokal, Hasford, or the more recently developed EUTOS score. [Cortes and Kantarjian 2012]. 1-Sokal score The most commonly used
classification, devised by
Sokal and
colleagues in 1984 , is based on a formula that takes account of the patient’ s age, blast cell percentage, spleen size and platelet count at diagnosis (Table 1.8 ). [ Sokal et al 1984, Reichard et al 2009, Bain BJ 2010, Goldman and Mughal 2011].
Table 1.8 Sokal index for predicting survival prognostic indices [ Goldman and Mughal 2011].
It was developed much earlier during the busulfan era of treatment and was less accurate in patients treated with IFN. [Kaushansky et al 2010].
Although the Sokal score was validated for use with patients treated with HU and busulphan, it remains the most common method of assigning prognostic factors worldwide [Au WY et al 2009]. Developed almost 30 years ago, the Sokal score has been calculated to prognosticate by defining patients as low, intermediate, and high risk. [Andolina et al 2012 ]. It is preferred for use by many workers as it has been more consistently predictive of outcome. [Cortes and Kantarjian 2012], and it maintains prognostic value in patients treated with imatinib. [Reichard et al 2009]. There is evidence that a patient with chronic phase CML and a favorable Sokal Score at the time of diagnosis has a higher proportion of hematologic and cytogenetic responses than other patients. [Kaushansky et al 2010]. The Sokal score correlated well with the likelihood of achieving a complete cytogenetic response (CCR): 91% for low-, 84% for intermediate-, and 69% for high-risk patients. In a subsequent report, with a median follow-up of 54 months, 72% of the 553 randomized patients remain on initial imatinib therapy. [Reichard et al 2009]. 2-Hasford Score: A similar classification, which may or may not prove more useful than that of Sokal, was introduced by Hasford in 1998 and called the Hasford or Euro score; it makes use of eosinophil and basophil numbers in
addition to the values included in the Sokal system. [Goldman and Mughal 2011]. The system of Hasford et al. may be more appropriate for interferontreated patients. [Bain BJ 2010].
3-EUTOS Score: The EUTOS score has the beauty of simplicity, including only 2 factors: spleen size and basophils. Unfortunately, 2 independent series have not confirmed its value, albeit with some methodologic differences in the analysis. [Cortes and Kantarjian 2012]. The EUTOS score was the only one developed in a sample of patients treated with TKIs, and it is suggested to put it into focus when CCgR at 18 months and PFS are analyzed. [ Guilhot J et al 2012].
1.12. Treatment A depiction of the timeline of the development of therapies in CML is presented in Fig. 1.11. The initial therapy for patients presenting in chronic phase requires controlling the elevated white blood cell count, reducing the symptoms of concomitant splenomegaly, and treating any metabolic
complications
caused
by
the
profound
marrow
proliferation.[Reichard et al 2009]. Historically, therapy for CML was empirically based. During the late 1800s, the mainstay of therapy for CML was Fowler solution, which was developed by Dr Thomas Fowler in the mid1700s. The active ingredient in Fowler solution was probably potassium arsenite and there has been a resurgence of interest in the use of arsenic preparations for CML. During
the 1900s, radiation, busulfan, hydroxyurea, interferon-_ (IFN-_), and stem cell transplantation were developed for other indications, tried broadly, and found to have activity in CML. Allogeneic stem cell transplantation is the only proven curative therapy, but is associated with significant morbidity and mortality. [Druker BJ. 2008, Goldman JM 2009].
Figure 1.11: Timeline for development of therapies for CML. HSCT, hematopoietic stem cell transplantation. [Reichard et al 2009].
In due course, hydroxycarbamide replaced busulfan, but interferon-alfa, the first agent to induce any degree of Ph-chromosome negativity in the bone marrow, was introduced in the early 1980s and became the
treatment of choice for patients not eligible for allogeneic stem cell transplantation. Between 1980 and 2000, allografting, despite the risks of morbidity and mortality, was the recommended initial treatment for younger patients with HLA-matched donors. [Goldman JM 2009]. Conventional
chemotherapeutic
agents
such
as
busulfan
or
hydroxycarbamide (hydroxyurea) are no longer used, except as a means to achieve initial hematological control.[ Quintas –Cardama et al 2010]. The management of the newly diagnosed CML patient has changed very greatly in the last decade.[ Quintas –Cardama et al 2010,Goldman and Mughal 2011]. Therapy has now been “revolutionized” by the introduction of imatinib (imatinib mesylate, IM), the original Abl tyrosine kinase inhibitor (TKI), which was used first in the clinic in 1998. [Goldman JM 2007]. The emergence of imatinib mesylate has brought about a change in the therapeutic algorithm for CML, to the point that IFN - α has been superseded by imatinib as frontline therapy, and imatinib has spurred the development of an array Of tyrosine kinase inhibitors (TKIs) with higher potency against BCR - ABL1 kinase. Allogeneic stem cell transplantation (SCT) remains a curative modality and a valid option for patients who fail TKI therapy.[ Quintas –Cardama et al 2010]. Imatinib mesylate Imatinib mesylate (Glivec or Gleevec; previously known as STI571), a 2 - phenylaminopyrimidine compound [Goldman and Mughal 2011]. It was developed for clinical use as a result of a collaboration between Brian Druker and investigators at Ciba-Geigy (subsequently merged with Sandoz to form Novartis Pharma) in Basel that started in the early 1990s. [Goldman JM 2007]. Imatinib, the first tyrosine kinase inhibitor, was developed for chronic myeloid leukemia (CML) and has also been found to be active in
gastrointestinal stromal tumors and some rare myeloproliferative neoplasms. [Druker BJ. 2008]. From 2000 onward imatinib at 400 mg daily became the preferred initial treatment [Glivec information sheet 2002,Goldman JM 2009 ]. It is an ABL1 tyrosine kinase inhibitor that entered clinical trials in 1998. It was thought originally to act by occupying the ATP – binding pocket of the ABL1 kinase component of the BCR – ABL1 oncoprotein, thereby blocking the capacity of the enzyme to phosphorylate downstream effector molecules; it is now thought to act also by binding to an adjacent domain in a manner that holds the ABL1 component of the BCR – ABL1 oncoprotein molecule in an inactive or
‘ closed ’
conformation [Goldman and Mughal 2011]. Imatinib mesylate is an orally bioavailable 2 – phenylaminopyrimidine relatively selective for and moderately potent against the constitutively Active tyrosine kinase of the BCR ABL1 fusion protein .In addition to BCR - ABL1, imatinib also inhibits other kinases such as KIT, platelet derived growth factor receptor (PDGFR) α and PDGFR β , and ABL – related gene (ARG). [Quntas –Cardama et al 2010 ]. Imatinib mesylate (IM) is a specifically targeted drug that inhibits the tyrosine kinase activity of Bcr-Abl protein. Imatinib has impressive results in newly diagnosed patients with CML and also in previously treated CML patients in late chronic phase (CP) [Holzerova et al 2009]. Drug treatment should ideally be started soon after the diagnosis is confi rmed. The best single agent for patients in chronic phase is imatinib. Hydroxycarbamide is a reasonable alternative in the very short term if imatinib is not immediately available. [Goldman and Mughal 2011].
Imatinib is also recommended as the first line of treatment in patients with chronic myeloid leukemia by the National Comprehensive Cancer Network in a daily single oral dose of 400mg.[ NCCN guidelines 2013]. Side -effects include nausea, headache, rashes, infraorbital oedema, bone
pains
and,
sometimes,
more
generalized
fluid
retention.
Hepatotoxicity is also reported and very rarely fatal cerebral oedema . Patients with black skin may sustain areas of depigmentation, and repigmentation of grey hair, has been reported in a small group of responders.
[Goldman
and
Mughal
2011].
Neutropenia
,thrombocytopenia and anemia are very common adverse effects.[ Glivec information sheet 2002]. Treatment should continue as long as the patient continues to benefit.[ Glivec information sheet 2002]. Second generation TKIs: Second generation TKIs are also recommended as the first line of treatment for patients with CP CML by the NCCN [NCCN 2013] , and for those patients resistant or intolerent to Imatinib [Quntas –Cardama et al 2010 ]. The common denominator of most of these agents is their superior potency relative to imatinib and their activity against a wide spectrum of imatinib – resistant BCR - ABL1 kinase mutations, which represent the most common mechanism of resistance encountered in patients with CML undergoing TKI therapy. [Quntas –Cardama et al 2010 ]. These drugs are: 1- Nilotinib (Tasigna, formerly AMN107) is a phenylaminopyrimidine derived from the crystal structure of imatinib in complex with ABL1 kinase . Nilotinib has 20 –t o 30 – fold improved affinity and inhibitory activity against unmutated BCR - ABL1 and inhibits the tyrosine kinase
activity of 32 of 33 BCR - ABL1 mutants tested, the exception being T315I. [Quintas –Cardama et al 2010 ]. A dose of 300mg BID is recommended . [NCCN 2013]. 2- Dasatinib (Sprycel, formerly BMS - 354825) is a sub nanomolar inhibitor of BCR - ABL1 and SFKs, with potent activity against KIT (IC50 13 nmol/L), PDGFR β (IC50 28 nmol/L), and ephrin receptor EPHA2 (IC50 17
nmol/L) tyrosine kinases . Like nilotinib, dasatinib
inhibits multiple imatinib – resistant BC- ABL1 mutant isoforms,except for T315I. [Quintas –Cardama et al 2010 ]. The recommended dose is 100 mg daily. [NCCN 2013]. Third generation TKIs: e.g. Ponatinib is a potent, oral pan-BCRABL TKI which is under active investigation. Importantly, it is active against T315I and other imatinib-resistant mutations.[ Bhamidipati et al 2013]. 1.13. Monitoring Monitoring the response to CML therapy is a continuum that begins at diagnosis and carries on serially throughout the entire course of treatment, as detailed in recently published expert consensus guidelines. [Baccarani et al 2009 , Vigil et al 2011]. Molecular monitoring in chronic myeloid leukemia is a powerful tool to document treatment responses and predict relapse. [Radich JP 2009]. The ELN and the NCCN monitoring guidelines for patients receiving TKI therapy are shown in table 1.9.
Table 1.9 ELN and NCCN Monitoring Guidelines for Patients Receiving TKI Therapy [Akard LP.2011]. Indication NCCN Guidelines Diagnosis
Method
qRT –PCR ; bone marrow cytogenetics During TKI therapy qRT- PCR Bone marrow cytogenetics
After CCyR achieved
qRT-PCR Bone marrow cytogenetics FISh not recommended ≥1-Log increase in Evaluate compliance; BCR-ABL transcripts repeated qRT-PCR if MMR; bone marrow Cytogenetics if no MMR; Consider mutation testing ELN Guidelines Diagnosis Hematologic ;bone Marrow cytogenetics During TKI therapy Hematologic Bone marrow cytogenetics
qRT-PCR
After CCyR achieved Bone marrow cytogenetics
qRT-PCR Suboptimal Response or faliure Change in therapy
Mutation analysis Mutation analysis
Every 3 months Every 6,12 and 18 month Until CCyR is Achieved Every 3 to 6 months As clinically indicated
Every 15 day un l CHR then at least every 3 Months or as required At 3 and 6 months , and Every 6 months Thereafter, until CCyR Is confirmed Every 3 months un l MMR is confirmed Then at least every 6 Months thereafter Every 12 month if regular molecular monitoring cannot be assured;for treatment failure and unexplained anemia, leucopenia,or thrombocytopenia Every 3 months un l MMR,then at least every 6 months therea er
The unique t(9;22) reciprocal translocation forming the Ph chromosome forms the basis of monitoring in CML. Because all CML cells will harbor the Ph, but normal hematopoietic cells should not, the Ph is a unique genetic fingerprint for CML detection. [Radich JP 2009]. Molecular monitoring is used in the TKI trials as a measure of disease response, and such monitoring is now advocated for the routine clinical care of CML. [Radich JP 2009]. However, there is some controversy and confusion about the merits and use of molecular monitoring: what test, how often, and what to do with the results? [Radich JP 2009]. Multiple genetic testing methods are used in clinical practice to assess response to therapy in chronic myeloid leukemia , but no one technique accurately detects and quantifies disease at diagnosis and at all times during treatment. [Dewald et al 1998 ]. There are several tests to measure disease state and burden, and, as one might expect, greater complexity is required to gain sensitivity. [Radich JP 2011] The following monitoring methods are used routienly for monitoring patients with CML 1-Conventional metaphase cytogenetics. 2-Fluorescence in situ hybridization. 3-RT-PCR. 4- Abl mutation. Conventional metaphase cytogenetics looks for the Ph and can detect other chromosomal changes associated with advanced-phase disease.
Molecular cytogenetics using fluorescence in situ hybridization (FISH) is a more sensitive method to detect the fusion BCR-ABL gene and has the advantage of routinely interrogating 50 to 200 metaphase or interphase cells, although additional chromosomal changes cannot be detected unless specific probes for those abnormalities are added to the FISHing trip. [Radich JP 2009] The method of fluorescence in situ hybridization , now appears to meet this need in clinical practice. This method uses FISH and commercially available differently colored BCR and ABL probes that span the common breakpoints of t(9;22)(q34;q11.2) and show double BCR/ABL fusion (DFISH) in cells with this translocation, one on the abnormal chromosome 9 and one on the Ph chromosome. With D-FISH, the number of falsepositive and false-negative cells approaches zero.[Dewald et al 1998 ]. The most sensitive approach to detect CML is the RT-PCR of the chimeric BCR-ABL mRNA, which can detect one CML cell in approximately 100 000 to 1 million cells. The assay has well-documented pitfalls, mostly revolving around the complexity and the fact that there is little standardization across laboratories. On an extremely positive note, peripheral blood can be used instead of bone marrow for monitoring. [Radich J P 2009]. Although most CML patients treated with imatinib have an excellent response, monitoring through hematological, cytogenetic and molecular testing is recommended by the ELN and NCCN to promptly identify and optimize treatment for the minority of patients who respond slowly. [Mu¨ller et al 2009]. In a newly diagnosed CML patient, TKI therapy progressively reduces the disease burden. Therefore, as the number of leukemia cells decrease,
the sensitivity of the techniques used to effectively monitor the disease must increase accordingly (Figure 1.12). [Radich JP 2009].
Figure 1.12 Disease burden and tests. [Radich 2011]
Response to treatment: There are three levels of responses to TKI treatment in patients with CP CML. (Table 1.10) Table 1.10 :The current ELN and NCCN response criteria in chronic myeloid leukemia.[Tibes R. and Mesa RA. 2012]. 2009 ELN guidelines Hematologic response Complete (CHR)
WBC count 0.05 G represents green FISH signal for P53 ; R represents red FISH signal for ATM 1 [2G,1G,Absent G, more than2 copies of G ;2R,1R,absent R ,more than 2 copies R] 2 [2G,1G,Absent G;2R,1R,Absent R] 3 [2G,1G,Absent G, more than 2copies of G; 2R,1R,Absent R] 4 [2G,1G,Absent G;2R,1R,Absent R ,more than 2 copies of R]
Table 3.14 Clinical parameters of patients in the group who became FISH2 negative after treatment Parameter
Group 1
Age in years Mean + SD
42.5±14.8
Gender Male : n (%)
20 (55.6)
Female: n (%)
16 (44.4)
Spleen size in cm Mean +SD
14.7±4.8
Liver size in cm Mean +SD
1.8±2.1
N=36
Table 3.15 Main laboratory parameters of the first group of patients those who became FISH2 negative after treatment Parameter
Group 1
PCV Mean+SD
31.8±7.0
WBC count x109/l
202.5±106.3
Mean+SD Basophils%
3.7±2.7
Mean+SD Blast%
2.5±2.4
Mean+SD Myelocyte%
24.6±9.2
Mean+SD BM Granulocytes %
89.5±3.5
Mean+SD Score of initial FISH Before treatment
87.4±7.1
N=36
Table 3.16: Paired hematological parameters before and after treatment of patients who became FISH negative after treatment
N=36
Paired Samples Statistics Mean Pair 1
N
Std. Deviation
Std. Error Mean
PCV1
31.8444
36
7.00532
1.16755
PCV2
36.3278
36
3.11793
.51966
WBC1
202.494444
36
106.3168712
17.7194785
WBC2
5.8556
36
1.52061
.25343
Neutrophil1 %
56.3429
35
12.34776
2.08715
Neutrophil2 %
53.4286
35
9.96844
1.68497
Lymphocyte 1%
5.4286
35
4.49369
.75957
Lymphocyte2 %
39.9429
35
9.42445
1.59302
Monocyte1 %
1.1143
35
1.49059
.25196
Monocyte2 %
4.2857
35
1.88760
.31906
Eiosinophil1 %
2.2000
35
1.79542
.30348
Eiosinophil2 %
2.0000
35
3.00979
.50875
Basophil1 %
3.6000
35
2.74612
.46418
Basophil2 %
.3429
35
.80231
.13561
Promyelocyte1 %
2.8000
35
1.98227
.33507
Promyelocyte2 %
.0000
35
.00000
.00000
Myelocyte1 %
24.1143
35
8.78090
1.48424
Myelocyte2 %
.0000
35
.00000
.00000
Pair 10 Metamyelocyte1 %
1.8857
35
2.70915
.45793
Metamyelocyte2 %
.0000
35
.00000
.00000
Pair 11 Blast1 %
2.5143
35
2.46590
.41681
Blast2 %
.0000
35
.00000
.00000
Pair 12 Platelets1
404.4857
35
231.01520
39.04870
Platelets2
226.4857
35
41.21491
6.96659
Pair 2 Pair 3 Pair 4 Pair 5 Pair 6 Pair 7 Pair 8 Pair 9
Table 3.17: Paired sample t test of hematological parameters in patients who became FISH negative after treatment. N=36 Paired Samples Test t
df
Sig. (2-tailed)
Pair 1
PCV1 - PCV2
-3.748-
35
.001
Pair 2
WBC1 - WBC2
11.148
35
.000
Pair 3
Neutrophil1 % - Neutrophil2 %
1.227
34
.228
Pair 4
Lymphocyte 1% - Lymphocyte2 %
-20.280-
34
.000
Pair 5
Monocyte1 % - Monocyte2 %
-8.732-
34
.000
Pair 6
Eiosinophil1 % - Eiosinophil2 %
.333
34
.741
Pair 7
Basophil1 % - Basophil2 %
7.633
34
.000
Pair 8
Promyelocyte1 % - Promyelocyte2 %
8.357
34
.000
Pair 9
Myelocyte1 % - Myelocyte2 %
16.247
34
.000
Pair 10 Metamyelocyte1 % Metamyelocyte2 %
4.118
34
.000
Pair 11 Blast1 % - Blast2 %
6.032
34
.000
Pair 12 Platelets1 - Platelets2 P
