Master Program in Clinical Laboratory Science Cytogenetic Abnormalities in Palestinian Patients ...

101 Pages·2015·1.61 MB·English

Master Program in Clinical Laboratory Science

Cytogenetic Abnormalities in Palestinian Patients with Hematologic
Malignancies in the Occupied West Bank (Including Jerusalem)
ةلتحملا ةيبرغلا ةفضلا يف نيينيطسلفلا نم مدّلا تاناطرسب نيباصملا يضرملا ىدل ةينيجوتيسلا تلالاتخلاا
)سدقلاو(
By
Ahmed Moh'd Ali Zaid
Advisor
Prof. Mazin Qumsiyeh
"This Thesis was submitted in partial fulfillment of the requirements for the Master’s
Degree in Clinical laboratory sciences, from the Faculty of Graduate Studies at Birzeit
University, Palestine"
June 2014

i
Dedication

I dedicate this work to my dear father Moham'd and my beloved mother Rihab for
inspiring me to continuously seek knowledge and struggle for improvement.

A special word of gratitude to my dear family members; my borhters and my sisters for
their support, endurance, and all kinds of help they offered to me during the time I spent
to complete this work. I am also grateful to my dear friend Dr Mazin for his ongoing
huge support and encouragement.

ii
Acknowledgment

I would like to express my gratitude to my supervisor, Dr. Mazin Qumsiyeh, whose expertise,
understanding, and patience, added considerably to my graduate experience. I appreciate his
vast knowledge, skills, and research experience in various areas related to my research.

I would also like to acknowledge Dr Yacoub Thaher, and Dr Moham'd Farraj, for their valuable
suggestions and assistance in my thesis project.
Special thanks go out to Dr Tamer Eissawi for his revision of my thesis and his important
suggestions. As well as I would like to express my sincere thanks to Dr Lena al-khayri for her kind
help in my thesis.

Distinguished thanks for the laboratory team in Bethlehem university cytogenetics laboratory
Reem Hilal, Ragine Handel, and our volunteering colleague Shaden melhem, as well as Beirzeit
university molecular laboratory team my friends Israr Sabri, and Shadi Rifa'e.
I would like to thank them all for their kind helps and technical assistance.

Finally, I would like to thank Dr Areej Khatib and her laboratory team in the Augusta Victoria
Hospital for their kind assistance and for providing me with valuable information related to my
thesis.

iii
Abbreviations
DNMT DNA methyl transferases
MALT the mucosa-associated lymphoid tissue
ABL Abelson murine leukemia
ALL Acute lymphoblastic leukemia
AML Acute myelogenous leukemia
APL Acute promyelocytic leukemia
ASXL1 Additional sex combs-like 1 gene
ATM Ataxia-telangiectasia mutated
ATO Arsenic trioxide
ATRA All-trans retinoic acid
BCR Break point cluster region
BER Base excision repair
BM Bone marrow
CML Chronic myelogenous leukemia
CMML Chronic myelomonocytic leukemia
DCBA Dual-color break-apart
DCDF Dual-color dual-fusion
DI DNA index
DLBCL Aggressive diffuse large B-cell lymphoma
DNMTase's DNA methyl transferases
EORTC European organization for research and treatment of cancer
FAB French American British
FISH Fluorescence in situ hybridization
FL FLT3 ligand
FLT3 Fms-like tyrosine kinase 3
FNA Fine needle aspiration
HCL Hairy cell leukemia
HD Hyperdiploid
HIP1R Huntingtin interacting protein 1 related
iv
HL Hodgkin lymphoma
HM Haematologic malignancies
Hox Homeobox
HRR Homologous recombinational repair
HRS Hodgkin and Reed-Sternberg
IARC International Agency for Research on Cancer
IGH Immunoglobulin heavy locus
IGHV unmutated immunoglobulin heavy chain
ISCN International System for Human Cytogenetic Nomenclature
ITD Internal tandem duplication
IWF International working formulation
JM Juxtamembrane
LMO1 & 2 LIM domain only gene -1 & 2
LPD Lymphoproliferative disorders
LYL1 Lymphoblastic leukemia-derived gene -1
MDR Minimal deleted region
MDS Myelodysplastic syndrome
MDS Myelodysplastic syndromes
MDS-U Myelodysplastic syndrome – unclassified
M-FISH Multiple-FISH
MGUS Monoclonal gammopathy of undetermined significance
MLL Mixed–lineage leukemia
MM Multiple Myeloma
MMR Mismatch repair
MMSET Multiple myeloma SET domain
MNC Modal number of chromosomes
MPD Myelo proliferative disorders
MPN Myelo proliferative neoplasms
MYF6 Myogenic factor 6
NER Nucleotide excision repair
NHEJ Non-homologous end joining
v
NHL Non-Hodgkin's lymphoma
NOS Not otherwise specified
NOTCH Notch homolog 1
PI3K phosphoinositide 3-kinase
PLL Prolymphocytic leukemia
PML Promyelocytic leukemia
PML-NB's PML- nuclear bodies
PT3K Phosphatidylinositol-3 kinase
RAEB-1 Refractory anemia with excess blasts-1
RAEB-2 Refractory anemia with excess blasts-2
RARA Retinoic acid receptor, alpha
RARS Refractory anemia with ring sideroblasts
RB1 Retinoblastoma-1
RCMD Refractory cytopenia with multilineage dysplasia
RCUD Refractory cytopenia with unilineage dysplasia
RTK Receptor tyrosine kinase
SKY Spectral karyotype
TAL1 T-cell lymphocytic leukemia
t-MDS Therapy-related MDS
TNM Tumor, node, metastasis
T-PLL T cell prolymphocytic leukemia
TRIP Tripartite
WCP Whole-chromosome paint
WHO International Agency for Research on Cancer
XPC Xeroderma pigmentosum complementation

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List of tables
Table.1……………………………………………………………………………56
Table.2……………………………………………………………………………58
Table.3……………………………………………………………………………59
Table.4……………………………………………………………………………61
Table.5……………………………………………………………………………62
Table.6……………………………………………………………………………65

List of figures
Figure.1……………………………………………………………………………………………………………………35
Figure.2……………………………………………………………………………………………………………………37
Figure.3……………………………………………………………………………………………………………………57
Figure.4……………………………………………………………………………………………………………………58
Figure.5……………………………………………………………………………………………………………………60
Figure.6……………………………………………………………………………………………………………………63
Figure.7……………………………………………………………………………………………………………………72

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I. Contents
Dedication ........................................................................................................................................ ii
Acknowledgment ............................................................................................................................. iii
Abbreviations .................................................................................................................................. iv
List of tables ................................................................................................................................... vii
List of figures ................................................................................................................................. vii
Abstracts .......................................................................................................................................... 1
II. Introduction ........................................................................................................................... 13
A. Hematologic Malignancies ................................................................................................ 14
1. Definition ....................................................................................................................... 14
2. History of discovery of HM ............................................................................................ 14
3. Epidemiology of hematologic malignancies .................................................................. 15
B. Molecular bases of hematologic malignancies ................................................................. 16
1. Mutagenic agents ........................................................................................................... 16
2. Role of DNA repair systems in HM's ............................................................................ 20
3. Role of Protooncogenes and tumor suppressor genes in HM's .................................... 21
4. Role of epigenetics in the development of HM's ........................................................... 25
C. Acute Lymphoblastic Leukemia (ALL) ................................................................................ 28
D. Acute Myeloid leukemia (AML) ......................................................................................... 32
E. Chronic Lymphocytic Leukemia (CLL) ................................................................................ 38
F. Chronic Myeloid Leukemia (CML) ..................................................................................... 41
G. Myelodysplastic Syndrome (MDS)..................................................................................... 42
H. Multiple Myeloma (MM) ................................................................................................... 45
I. Lymphoma ......................................................................................................................... 48
J. Aims and objectives ........................................................................................................... 51
III. Materials and Methods ..................................................................................................... 51
A. Research Design ................................................................................................................ 51
B. Study population ............................................................................................................... 52
C. Cytogenetic methods ........................................................................................................ 52
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1. G-banding (Giemsa Banding) and screening ................................................................. 53
D. Fluorescence in situ hybridization (FISH) .......................................................................... 55
1. Reagents ........................................................................................................................ 58
2. Equipment ..................................................................................................................... 58
3. Procedure ...................................................................................................................... 59
4. Analysis .......................................................................................................................... 61
IV. Results ............................................................................................................................... 63
A. ALL results .......................................................................................................................... 63
B. AML results ........................................................................................................................ 65
C. CML and FISH Results ........................................................................................................ 67
D. CLL results .......................................................................................................................... 69
E. Myelodysplasia .................................................................................................................. 70
F. Multiple Myeloma and Lymphoma ................................................................................... 70
V. Discussion .............................................................................................................................. 71
A. ALL ..................................................................................................................................... 72
1. The hypodiploid (<46) & pseudodiploid categories ...................................................... 74
2. For the ploidy group (47-50) ......................................................................................... 75
3. For hyperdiploidy groups of more than fifty chromosomes (>=51) .............................. 75
B. AML .................................................................................................................................... 76
C. CML with FISH results ........................................................................................................ 77
D. CLL ..................................................................................................................................... 79
E. MDS ................................................................................................................................... 79
F. MM and Lymphoma .......................................................................................................... 80
VI. Conclusions ........................................................................................................................ 81
VII. References ......................................................................................................................... 83

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صخّ لملا

ّ،سلاؼناّذٚذححّٔ،ةسٕطخناّىٛٛمحّٔ،ضٛخشخهنّتً ٓيّوذناّثاَاؽشسنّتببسًناّتُٛٛصٕخٛسنأّتُٛٛضناّثلالاخخلااٌّّ إ
ّتناسيٌّإفّتًًٓناسأدلأاِّزّْببسبّ.وذ ناّثاَاؽشسنّتٛػشًناّاٛصٕنٕٛسٛفناّساشسأّفشكنّٔ،ٙػشًناّمبمخسًنأ
ّوذ ناّثاَاؽشسّلٕحّتٛفاكناّثايٕهؼًناّشفٕخحّلاٌّٜاّٗنإّّ َأّشٛغّ،ٍٛطسهفّٙفّٔاًٛناػّآخًْٛأّثدادصاّوذ ناّثاَاؽشس
.ٍٛطسهفّٙفّاَذُػ
ّداحناّ٘ٔافًٛهناّوذناٌّاؽشسّلٕحّتٛناحناّفساؼًناّتًٛهؼناّثاٛبدلأابّضظخًناّءضضناّٙفّساظخخابّاُؼصاسّذمن
ٍّيضًنإّ٘مُناّوذناّعاؼٛبأّّ CLLٍّيضًناّ٘ٔافًٛهناّوذناٌّاؽشسّّٔAMLّداحنإّ٘مُناّوذناّعاؼٛبأّّALL
ّذلّٔ. MDS ّٙػاخُناّشسُخناّمهخّٔMMّةدذؼخًناّايٕهٚاًنأّ LymphomaّتٚٔافًٛهناّدذغناٌّاؽشسّٔCML
ِّزّْٙفّّ.ٍٛطسهفّٙفّةسٕكزًناّوذ ناّثاَاؽشسٍّػّآخٚافكّوذػّٔ تفشؼًناّّح شّعٕػًٕناّثاٛبدلأّاُخؼصاشيّثشٓظا
ّتساسذبّاًُلّٔ)طذمنأّتٛبشغناّتفؼناّتمطُيّاذٚذحح(ٍّٛطسهفّٙفّوذ ناّثاَاؽشسبّتباظًناّثلااحناّغًضبّاَأذبّتناسشنا
ّقلاؽاّمصأٍّئّلاضًناّازّْٙفّٙهبمخسًناّذحبهنّقلاطَاّتطمَّاُخساسدٌّٕكخنّآٛفّتفشخكًناّتُٛٛصٕخٛسناّثلالاخخلاا
.ٍٛطسهفّٙفّثلااحناِّزٓنّذحبنأّكٛرٕخنأّذطش هنّت ٚذصّتٛهًػ
ّFISHّتُٛمخبّثاطٕحفّاُفػأّاًكّتٛيٕسٕئشكناّثلالاخخلااّغؼبّاُفشكّٔثلااحهنّاُٛٛصٕخٛسّلاٛهححّاُيذلّذمن
ّشرأّآنٌّٕكٌّٚاّتساسذناِّزٍّْيٍّٛؼلٕخيّّق دأّثلالاخخاٍّػّفشكناّفذٓبّCMLّٔAMLٍّيّثلااحناّغؼبن
.آنٕحّتٛنٔ أّةشظبحّوذ محٌّأٍّٔٛطسهفّٙفّوذناّثاَاؽشسّغلأّٗهػّٙباضٚا
ّ،ALLّصٕظخبّ.ٖشخلأاّىناؼناّلٔدّغيٍّٛطسهفّٙفّوذ ناّثاَاؽشسّغلأّٙفّاي اػّآباشحّتساسذناّشئاخَّثشٓظأّذمن
ٍّيّجَاكّ%4..1ّثلااحناّبهغأّ.ثلااحناٍّيّ%13.3ّٙفّثلالاخخاٍّػّفسكناٍّيّاُكًحّّٔتناحّ47ّاُٚذنٌّاك
ّّبشَّّٕ٘ٔؾًُبّثلااحّ4ّٔ،ذمؼيَّّٕ٘ٔؾًَّثارّhypodiploidّتَٛافؼؼناّٙخطٕمُيٍّٛخناحّاَذصّّٔ.B-لٕطأ
ّثلااحناّغؼبّt(4:12)ّّٔ(1:19)(q23;p13)ّمًشحّتٛبٛكشحّثلالاخخاّجهزيّآُيّ1ّ؛ّpseudodiploid َّٙافؼػ
ّ%1.-52ّجهزيّ)ّ>=51)ّْٙhyperdiploid ّتَٛافؼؼناّتغٛؼناّغفحشئّّ (47-50),ّ ّّتَٛافؼؼناّتػًٕضيٍّي
ّ.ALL ّثلااحّعًٕضيٍّي
ّمبمخسيّثارّثلااحّاَذصّٔذلّٔ.ّم خخيَّّٕ٘ٔؾًُبّجَاكّثلااحناٍّيّ%5..2ّتناحّ12ّٗهػّاُهظحّ،AMLّايأ
ّٗهػّمظحَّىنّ inv(16)(p13q22)ّّٔ t(15;17)(q22;q12)ٔ t(8;21)(q22;q22)ّآٛفّٔز بحيّٙػشي
ّ.لٕ حًناّٗفشًناّٖذنّٙػاًُناّضٛخشخناّتٚدٔذحيّببسبّثلااحناّىظؼًن FAB ّفُٛظخنا
ّوٕسٕئشكنّتبصًٕناّثلااحناّّْٙٔمخخيَّّٕ٘ٔؾًَّثارّجَاكّآُيّ%73.5ّ.تناحّ17ّٗهػّاُهظحف CML ّايا
ّدلارّٔ.del(11)ّّٔdel(22) ,ّآٛفّاًبّذمؼيَّّٕ٘ٔؾًُبّآًُيٌّاخناحّCLL ٍّيّثلااحّ7ّاُٚذنّجَاكّ.اٛفندلاٛف
ّٙػشيّمبمخسيّثارّآُيّةذحأّتناحّٙفّاذمؼيّإَّٚٔؾًَّذصّٔذٛحّMDSٍّيّّٛهػّاُهظحّايّّْٙجَاكّثلااح
ّمبمخسيّثارّٔذمؼيّٙؼٛبؽّشٛغّٕ٘هخّؾًَّثارّجَاكّةذحأّتناحّ،تناحّ11ّآُيّاُٚذنٌّاكّةدذؼخًناّايٕهٚاًناّ.ءٙ س
ّّ.ُٕ٘ٔناّؾًُناّٙفّثلالاخخاّٗهػّآٛفّشزؼَّىنّ.تناحّ35ّآُيّ اُٚذنٌّاكّايٕفًٛهناّ.ءٙ سّٙػشي
ّٙٛئاظخأّٔ،ٍٛ ُٛٛضناٍّٛٛئاظخلأأّءابؽلأاّدٕٓصّساخححٍّٛطسهفّٙفّوذناّثاَاؽشسٍّػّثاساسذناٌّاّشخُخسَ
ّغيّىٛهسناّمياؼخناّمصأٍّيّتًْٛلأاّتغنابّّْٙثاساسذناِّزّْٔٙحظ ناّلاضًناّٙفٍّٚشخٜاٍّٛهياؼنأّّ،ثاٛئابٕنا
.ىٓنّتٛحطّتٚاػسّمؼفأّىٚذمحّٔٗػشًنا
ّ
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