chromosomal abnormalities and epstein barr virus in …

CHROMOSOMAL ABNORMALITIES AND

EPSTEIN BARR VIRUS IN ACUTE LYMPHOBLASTIC LEUKEMIA IN CHILDREN

A thesis submitted in partial fulfillment for the requirements for the degree of Doctor of Philosophy in

Haematology

By

Dr. Ghulam Shah Nizamani

Department of Pathology Faculty of Medicine and Allied Medical Sciences

Isra University, Hyderabad, Sindh

2016

CHROMOSOMAL ABNORMALITIES AND

EPSTEIN BARR VIRUS IN ACUTE LYMPHOBLASTIC LEUKEMIA IN CHILDREN

by


Name of Supervisor and co-supervisors

Dr. Zaheer Ahmed Nizamani

PhD (France)

Prof. Dr. Fatah Din Khand PhD

Prof. Dr. Mohammad Ahmed Azami PhD

DEDICATION

THIS THESIS IS DEDICATED TO MY PARENTS & TEACHERS

CERTIFICATE

This is to certify that DR. GHULAM SHAH NIZAMANI S/O ALLAH BAKHSH

NIZAMANI has carried out research work on the topic “CHROMOSOMAL

ABNORMALITIES AND EPSTEIN BARR VIRUS IN ACUTE

LYMPHOBLASTIC LEUKEMIA IN CHILDREN” under my supervision and that

his work is original and his thesis is worthy of presentation to Isra University for

awarding the degree of “Doctor of Philosophy” in the subject of Haematology.

Dr. Zaheer Ahmed Nizamani, Associate Professor, Pathology Sindh Agriculture University, Tando Jam. Supervisor

v

ACKNOWLEDGEMENT

With the deep and profound sense of gratitude and thanks to the almighty

ALLAH for giving me the chance for completing this thesis, I am greatly indebted to

my respected Supervisor, Dr. Zaheer Ahmed Nizamani Associate Professor

Pathology Sindh Agriculture University, Tando Jam and Co-supervisor, Prof. Dr.

Fatah Din Khand, Prof. Dr. Mohammad Ahmed Azmi for the cooperation, guidance

and constructive criticism in the successful completion of this thesis and without their

help, this manuscript was not possible to complete. I am grateful to Prof. Dr. Ghulam

Qadir Kazi, Vice Chancellor Isra University for his valuable co-operation and support

which enabled me to complete this work.

vi

ABSTRACT

Background: Acute lymphoblastic leukemia (ALL) is a disease typically

characterized by the accumulation of immature abnormal lymphoid progenitor cells

(lymphoblasts) in the bone marrow, which have abnormal proliferation and

differentiation. A number of acquired chromosomal abnormalities arising from

translocations, deletions, duplications and inversions have been identified in 80% of

childhood and 79% of adulthood ALL.

1. Objectives of Study: To determine the frequency of chromosomal

abnormalities in children suffering from ALL, To evaluate structural and

numerical chromosomal abnormalities in patients with ALL, To find out the

frequency of Epstein Barr Virus in ALL cases.

Subjects and Methods: An observational study was conducted at the Liaquat

University of Medical and Health Sciences, Jamshoro and Isra University Hospital,

Hyderabad. 100 diagnosed childhood ALL cases were selected through non-

probability purposive sampling according to inclusion and exclusion criteria. The

Blood samples were collected in bottles containing Ethylene diamine tetra acetic

acid (EDTA) as an anticoagulant and were processed on automatic hematoanalyzer,

Sysmex KX 21. Fixed cell suspensions prepared from diagnostic bone marrow. For

routine cytogenetic analysis and FISH, samples were obtained from the diagnosed

cases of acute lymphoblastic leukemia. Methods for detecting EBV infection were

based on RT-PCR. The data was analyzed on SPSS version 21.0 (IBM, Corporation,

USA) and Microsoft excel. The continuous variables were presented as mean ± SD

and analyzed using student’s t-test. Categorical variables were analyzed by Chi-

square test and results were presented as frequencies and percentages. Data was

vii

presented in tables, graphs and charts. P-value of significance was taken at ≤0.05.

Results: Numerical and structural chromosomal abnormalities were noted in 69%

and 60% of cases respectively (p=0.001). Chromosomal ploidy showed Diploidy

and Aneuploidy in 29% and 69% of cases respectively (p=0.0001). Hyperploidy,

hypoploidy and pseudoploidy were noted in 51%, 6% and 12% of cases respectively

(0.001). Chromosomal structural abnormalities noted were; t (12; 21)(p13; q22)

t(9;22)(q34;q11), t(8;14)(q24;q32), t(5;14)(q31;q32), t(17;19)(q22;q13), t (7;11) (q35;

q13), t (1;7) (p32; q35), t (7;19) (q35; p13), t(1;19)(p13;q23), t(8;22)(q24;q11) and

unknown 13%. Ph + chromosome (t (9; 22) (q34; q11) was noted in 6% of cases and

EBV in 19% of total study population

Conclusion: ALL cases are characterized by leukocytosis and anemia. Epstein Barr

Virus was found in 19% childhood ALL in present study. Present study shows good

prognostic cytogenetic abnormalities like hyperdiploidy and t (12; 21)(p13; q22) in

Pakistani children with ALL and frequency of poor prognostic cytogenetic aberrations

like hypoploidy and t (9; 22) (q34; q11.2) is comparable to previous studies.

Keywords: Childhood ALL Chromosomal abnormalities Epstein Barr Virus

viii

LIST OF ABBREVIATION ABBREVIATION

TERM

AIDS ALL AML CBC CFU CLL CML CSC EBV EDTA EGIL ELISA FAB FISH GM-CSF HD ISH MRD NK PCR PHSC PTLD RBC SCF SD SDF-1 SPSS WBC WHO

Acquired immune deficiency syndrome Acute lymphoblastic leukemia Acute myeloblastic leukemia Complete blood counts Colony forming unit Chronic lymphoid leukemia Chronic myeloid leukemia Committed stem cells Epstein Barr virus Ethylene diamine acetic acid European group for classification of leukemia Enzyme linked immunosorbent assay French American British classification Fluorescence in situ hybridization Granulocyte monocyte colony stimulating factor Hodgkin`s disease In situ hybridization Minimal residual disease Natural killer cells Polymerase chain reaction Pluripotent hematopoietic stem cells Post transplantation lymphoproliferative disease Red blood cells Stem cell factor Standard deviation Stromal derived factor-1 Statistical Package for the Social Sciences White blood cells World Health Organization

EMF Electrical Magnetic field CGH Comparative genomic hybridization FITC Fluorescent iso thiocyanate (Anti EBV Antibodies) ISIS Integrated software for imaging spectrometers

ix

TABLE OF CONTENTS

Page # ACKNOWLEDGEMENT--------------------------------------------------------------- V ABSTRACT------------------------------------------------------------------------------- Vi LIST OF ABBREVIATION-------------------------------------------------------------TABLE OF CONTENTS--------------------------------------------------------------

viii ix

LIST OF TABLES----------------------------------------------------------------------- Xii LIST OF FIGURES--------------------------------------------------------------------- Xiii LIST OF GRAPHS---------------------------------------------------------------------- Xv CHAPTER – I ---------------------------------------------------------------------------

01

INTRODUCTION------------------------------------------------------------------------ 01 1. OBJECTIVES ------------------------------------------------------------- 2. RATIONALE OF STUDY----------------------------------------------------------- 3. HYPOTHESIS -----------------------------------------------------------------------

04 05 06

CHAPTER – II ------------------------------------------------------------------------- 07 LITERATURE REVIEW -------------------------------------------------------------- 07 1. Bone marrow, stem cells & hematopoiesis ---------------------------

1.1. Bone marrow ----------------------------------------------------------- 1.1.1. Red Bone marrow --------------------------------------------- 1.1.2. Yellow bone marrow ------------------------------------------

1.2. Bone marrow stroma -------------------------------------------------- 1.3. Sites of hematopoiesis------------------------------------------------- 1.4. Hematopoietic stem cells & progenitor cells ---------------------

1.4.1 Stem Cell Plasticity ---------------------------------------------- 1.4.2 Hematopoietic growth factors ---------------------------------

07 07 07 07 09 10 11 12 12

2. Leukemia overview ----------------------------------------------------------- 2.1. Acute leukemias-------------------------------------------------------

2.1.1 Acute lymphocytic (Lymphoblastic) leukemia (ALL 2.1.2 Acute myeloblastic leukemia (AML) -------------------

2.2. Chronic leukemias ------------------------------------------------------ 2.2.1 Chronic myeloid leukemia (CML) --------------------------- 2.2.2 Chronic lymphocytic leukemia (ALL) -----------------------

15 15 16 16 16 16 16

3. Acute lymphoblastic leukemias -------------------------------------------- 3.1. Epidemiology of ALL --------------------------------------------------- 3.2. Classification ------------------------------------------------------------

3.2.1 Morphological classification (French American British) 3.2.2 European Group for the immunological classification)

3.3. Cytogenetics in ALL ---------------------------------------------------- 3.3.1 Chromosomal translocations ---------------------------------

16 17 18 19 23 24 25

x

3.2.2 Cooperative mutations ----------------------------------------- 3.4. Etiology of leukemia ----------------------------------------------------

3.4.1 Dietary factors ---------------------------------------------------- 3.4.2 Socio-economic status ----------------------------------------- 3.4.3 Environmental factors ------------------------------------------ 3.4.3.1 Ionizing radiations ------------------------------------- 3.4.3.2 Non-ionizing radiations ------------------------------ 3.4.3.3 Chemicals ----------------------------------------------- 3.4.3.4 Pesticides ----------------------------------------------- 3.4.3.5 Cigarette ------------------------------------------------ 3.4.4 Immunological factors ------------------------------------------ 3.4.5 Genetic factors ---------------------------------------------------

28 32 32 33 33 33 34 34 34 35 35 35

4. Epstein-Barr virus EBV ------------------------------------------------------ 4.1. Types of EBV-------------------------------------------------------------- 4.2. Genome of EBV----------------------------------------------------------

37 38 38

5. Natural History of EBV infection ----------------------------------------- 5.1. Primary EBV infection--------------------------------------------------

5.1.1 Infectious mononucleosis -------------------------------------- 5.1.2 Chronic active EBV infection (CAEBV) --------------------

5.2. Cell entry and exit ------------------------------------------------------ 6. Malignancies associated with EBV ---------------------------------------

6.1. Hodgkin’s disease ------------------------------------------------------- 6.2. Burkett’s lymphoma------------------------------------------------------ 6.3. Post – Transplant Lymphoproliferative disorder------------------ 6.4. EBV associated carcinomas ------------------------------------------ 6.4.1 Nasopharyngeal carcinoma (NPC) -------------------------- 6.4.2 Gastric carcinoma ------------------------------------------------ 6.4.3 Other carcinomas ------------------------------------------------

39 39 39 39 40 42 42 44 45 47 47 47 48

CHAPTER – III ------------------------------------------------------------------------- 49 MATERIALS AND METHODS --------------------------------------------------- 49 1. Study Design ------------------------------------------------------------------- 49 2. Study setting -------------------------------------------------------------------- 49 3. Duration of study--------------------------------------------------------------- 49 4. Sample size --------------------------------------------------------------------- 49 4.1 Sample size calculation ------------------------------------------------- 49 4.2 Sampling technique ------------------------------------------------------ 50 4.3 Sample selection --------------------------------------------------------- 5. Inclusion criteria:--------------------------------------------------------------- 5.1 Exclusion criteria ---------------------------------------------------------

50 50 50

6. Data collection procedure --------------------------------------------------- 51 7. Laboratory investigations ---------------------------------------------------

7.1 Complete blood count (CBC)---------------------------------------------- 7.2 Preparation and staining of peripheral blood smear----------------- 7.2.1 Preparation of staining solution----------------------------------- 7.2.2 Preparation of buffered water-------------------------------------- 7.2.3 Staining of peripheral blood smear------------------------------- 7.2.4 Morphology of peripheral smear ----------------------------------

51 51 52 52 52 53 53

xi

7.3 Bone marrow procedure----------------------------------------------------- 7.4 Karyotyping -------------------------------------------------------------------- 7.4.1 Reagents used ----------------------------------------------------- 7.4.2 Instruments & consumables---------------------------------------- 7.4.3 Sample collection & processing------------------------------------- 7.4.4 Method of culture------------------------------------------------------- 7.5 Fish (Fluorescence in Situ Hybridization)------------------------------- 7.6 Epstein Barr Virus detection by PCR-------------------------------------

53 54 54 54 55 55 58 59

8. Data analysis ---------------------------------------------------------------------- 62 CHAPTER – IV ------------------------------------------------------------------------- 63 RESULTS -------------------------------------------------------------------------------- 63 CHAPTER – V -------------------------------------------------------------------------- 97 DISCUSSION --------------------------------------------------------------------------- 97

CHAPTER – VI ------------------------------------------------------------------------- 110 CONCLUSION -------------------------------------------------------------------------- 110 CHAPTER – VII ------------------------------------------------------------------------- 111 RECOMMENDATIONS --------------------------------------------------------------- 111 REFRENCES ---------------------------------------------------------------------------- 112

xii

LIST OF TABLES Chapter Description Page II–1 WHO classification of ALL ------------------------------------ 22

IV –1 Age distribution of study population --------------------------- 65

IV-2 Gender distribution of study population ------------------ 66

IV-3 Hemoglobin findings of study population --------------- 67

IV-4 Red blood cell counts of study population ------------- 68

IV-5 White blood cell counts of study population------------------ 69

IV-6 Chromosomal abnormalities of study population----------- 70

IV-7 Chromosomal ploidy of study population------------------------- 71

IV-8 Chromosomal numerical abnormalities --------------------------- 72

IV-9 Chromosomal structural abnormalities in study ---------------- 73

IV-10 Philadelphia chromosome of study population ------------------ 75

IV-11 Frequency of Epstein Barr Virus of study population ---------- 76

V-1 Studies conducted on EBV in childhood ALL---------------------

107

xiii

LIST OF FIGURES

Figure Description Page

II – 1 Blood cell production in bone marrow -------------------------- 08

II – 2 Bone marrow trephine biopsy--------------------------------------- 09

II – 3 Pluripotent hematopoietic stem cells -------------------------- 13

II – 4 Hematopoietic stem cells differentiation------------------------- 14

II – 5 FAB-L1smear showing small homogenous cells------------- 20

II – 6 FAB-L2 smear showing small homogenous cells------------- 20

II – 7 FAB-L3 smear showing small homogenous cells------------- 21

II – 8 Relative frequency of chromosomal abnormalities in ALL-- 27

II – 9 Retinoblastoma pathway and p53 tumor suppressors---- 29

II – 10 Notch signaling pathway in normal thymocytes --------------- 31

II – 11 EBV Primary infection and cycles of persistence------------- 46

IV –1 Bone Marrow particle Leishman’s stain X 40---- 77

IV-2

Peripheral smear showing Homogenous --------population of Lymphoblast Leishman’s stain X 400-----------

78

IV-3 Peripheral smear showing Homogenous population of Lymphoblast Leishman’s stain X 400-----------------------------

79

IV-4 Peripheral smear showing Homogenous population of Lymphoblast Leishman’s stain X 400-----------------------------

80

IV-5 Peripheral smear showing Homogenous population of Lymphoblast Leishman’s stain X 400-----------

81

IV-6 Bone Marrow showing Lymphoblast Leishman’s stain X 1000 - 82

IV-7 Bone Marrow showing Lymphoblast Leishman’s stain X 1000 83






xiv


IV-14 Bone Marrow showing Lymphoblast Leishman’s stainX 1000 90


IV-16 Peripheral Blood Smear. Leishman’s Stain shows leukemic blast of ALL X1000-------------------------------------------------------

92


93

IV-18 Peripheral Blood Smear. Leishman’s Stain shows leukemic blast of ALL X1000 --------------------------------------------------------

94

IV-19 Peripheral Blood Smear. Leishman’s Stain shows leukemic blast of ALLX400 ---------------------------------------------------------

95


96

xv

LIST OF GRAPHS

IV –1 Age distribution of study population --------------------------------

65

IV-2 Gender distribution of study population ---------------------------- 66

IV-3 Hemoglobin findings of study population -------------------------- 67

IV-4 Red blood cell counts of study population ------------------------ 68

IV-5 White blood cell counts of study population------------------------ 69

IV-6 Chromosomal abnormalities of study population----------------- 70

IV-7 Chromosomal ploidy of study population--------------------------- 71

IV-8 Chromosomal numerical abnormalities ---------------------------- 72

IV-9 Chromosomal structural abnormalities in study ----------------- 74

IV-10 Philadelphia chromosome of study population ------------------- 75

IV-11 Frequency of Epstein Barr Virus of study population ----------- 76

1

CHAPTER I

INTRODUCTION

Acute lymphoblastic leukemia (ALL) is a disease typically characterized

by the accumulation of immature abnormal lymphoid progenitor cells

(lymphoblasts) in the bone marrow, which have abnormal proliferation and

differentiation. It is a heterogeneous disease which can be divided into a number

of distinct biological and prognostic subtypes. ALL can develop from any

lymphoid cell, blocked at a particular stage of development, including both

primitive cells with a multilineage potential, as well as more mature cells(1).

The national data on ALL in children is lacking in Pakistan. ALL is common

in children of less than 15 years of age. In a retrospective study at Oncology unit

of National Institute of Child Health and Children Cancer Hospital, Karachi.

Yasmeen et al (2) reported a frequency of 32% of ALL in children.

A number of acquired chromosomal abnormalities arising from

translocations, deletions, duplications and inversions have been identified which

are often associated with deregulated gene expression. The abnormal karyotype

have been detected in more than 80% of children (3) and 79% of adults suffering

from ALL(1, 4).

Aneuploidy, defined as having more or less than the normal diploid

number of chromosomes, is a significant feature of ALL. A high hyperdiploid

karyotype, with 51-65 chromosomes, is found in approximately 30% of childhood

cases and 5% of adult patients(5, 6).The chromosomal gains in the form of

trisomies are restricted to certain chromosomes. In Chromosomes X, 4, 6, 10, 14,

2

17, 18 and 21 (frequently the gain of chromosome 21 is tetrasomic) abnormalities

are frequently found (7).

A second significant chromosomal abnormality in childhood ALL is

hypodiploidy (Ho), where chromosomes are ≤ 45. It is rare, with a reported

incidence of approximately 6%(8, 9). In the majority of reported cases, patients

have 45 chromosomes(8, 9). Overall, hypodiploidy has been linked to a poor

prognosis (8-10). Karyotypic analysis of the group shows chromosomal gains

onto the haploid chromosome set is common with high hyperdiploidy (X, Y, 14,

18 and 21). They show rare structural abnormalities and a co-incident doubled

hypodiploid clone. Conventional chromosomal analysis remains the method of

choice for the initial detection of cytogenetic abnormalities in leukaemic

samples(1, 10).

Epstein Barr Virus (EBV) is known to infect about 90% of the adult

population worldwide and its infection is generally restricted to humans(11, 12).

The virus is shed into the saliva of persistently infected individuals who spread

the virus to uninfected individuals (13, 14).

EBV is a virus of the genus Lymphocryptovirus within the subfamily of

gamma-herpes viruses, which is an enveloped virus. The envelope consists of a

toroid shaped protein core wrapped with DNA, a nucleocapsid, a tegument

protein, and a linear double stranded DNA molecule of 172 kb(14-16).EBV is

linked to a variety of neoplasms,(17, 18) including lymphoid tumors like Burkitt`s

Lymphoma, Hodgkin’s disease (HD), lymphoproliferations in solid organ

transplant, natural killer (NK) T-cell lymphoma or bone marrow recipients (post-

transplantation lymphoproliferative disease, PTLD), AIDS-associated lymphomas,

3

Nasopharyngeal carcinoma, gastric carcinoma, salivary gland tumors, thymic

carcinoma, mesothelial tumors and leiomyosarcoma(18).

Currently national data is seriously lacking on prevalence of chromosomal

abnormalities and possible role of EBV in ALL in children of Sindh, Pakistan. The

present research was designed to study patterns of chromosomal abnormalities

and prevalence of EBV in the children suffering from ALL reporting at the

Oncology Unit/NIMRA, Liaquat University of Medical and Health Sciences,

Jamshoro and Isra University Hospital, Hyderabad.

4

OBJECTIVES OF STUDY

The objectives of this study are

1. To determine the frequency of chromosomal abnormalities in children

suffering from ALL

2. To evaluate structural and numerical chromosomal abnormalities in

patients with ALL

3. To find out the frequency of Epstein Barr Virus in ALL cases.

5

RATIONALE OF STUDY

The acute lymphoblastic leukemia is common in children of less than 15

years of age. The disease has been reported to be increasing throughout the

globe and the country. There is paucity of data pertaining to chromosomal

abnormalities and Epstein Barr virus in acute lymphoblastic leukemia. Epstein

Barr virus has unique association with human malignancies in general and

lympho proliferative disorders in particular. Currently, there is paucity of national

data on acute lymphoblastic leukemia and studies on chromosomal abnormalities

and Epstein Barr virus. The chromosomal abnormalities have proven importance

in the prognosis of diseases. The research designed for studying various

chromosomal abnormalities and role of Epstein Barr virus in acute lymphoblastic

leukemia. The study will help oncologist/physicians in treating patients and

estimating prognosis of disease.

6

HYPOTHESIS

The association between the acute lymphoblastic leukemia and

chromosomal abnormalities and EBV will be determined.

Null Hypothesis (Ho): says that there is no association of dependent variable

(chromosomal abnormalities and EBV) with independent variable (Acute

lymphoblastic leukemia), if any association found is by chance until proved

otherwise.

Alternative Hypothesis (H1): says that there is association of dependent

variable (chromosomal abnormalities and EBV) with independent variable (Acute

lymphoblastic leukemia).

In present study H1 hypothesis will be accepted as true if the researcher

fails to prove the Ho hypothesis.

7

CHAPTER II

LITERATURE REVIEW

1. BONE MARROW, STEM CELLS AND HEMATOPOIESIS

1.1. Bone Marrow

Bone marrow is defined as cellulo-vascular hematopoietic tissue which

occupies medullary cavities and cancellous/trabecular spaces of bones and

produces blood cells. Hematopoisis is defined as the formation of blood cells

which primarily occurs in bone marrow. The bone marrow cavities of all bones

produce blood cells in children, however by the age 20; the marrow cavities of all

long bones, except for the upper humerus and femur, become inactive.

(Figure.II-1).(19, 20) Bone marrow may be classified according to functional

status as;

1.1.1. Red Bone Marrow: An active cellular bone marrow which is

performing the function of producing blood cells.

1.1.2. Yellow Bone Marrow: An inactive bone marrow that is loaded

with/adipose tissue.

The bone marrow is one of the largest organs of body (19, 20), nearly

approximating size and weight of the liver. It is also one of the most active

proliferating tissues. Normally, bone marrow contains 75% cells of myeloid series

which produce white blood cells and only 25% cells of erythroid series which

produce red blood cells. (19, 20)

8

The bone marrow contains multipotent hematopoietic stem cells called

pluripotent hematopoietic stem cells (PHSC), from which all blood cells in

circulation are derived. The PHSC differentiate into one or other type of

progenitor cells known as the committed stem cells (CSC). The committed stem

cells in turn differentiate into colony forming unit for erythrocytes, granulocytes,

monocytes, etc. (Figure.II-6)(19, 20)

Figure.II-1. Figure shows blood cell production in bone marrow at different ages (indifferent bones). (Adapted from: Guyton and Hall 2012) (20)

9

1.2 Bone Marrow Stroma

The stroma of bone marrow is naturally designed to provide a normal

homeostasis necessary for the stem cell survival, growth and proliferation. The

stroma of bone marrow is composed of stromal cells, interwoven fibers and

microvascular network. The Mesenchymal stem cells are thought to be critical in

stroma and stromal cell formation. The stromal cells include vascular endothelial

cells, adipocytes, fibroblasts and macrophages. The fibroblasts produce and

secrete extracelluar fibers such as glycoproteins (fibronectin, thrombospondin),

collagens and glycosaminoglycans (hyaluronic acid, chondroitin sulphate). The

stromal cells produce and secrete a number of growth factor necessary for stem

cell survival.(21)

Figure.II-2. A normal Bone marrow trephine biopsy. H & E staining shows 50% hematopoietic tissue and 50% adipose tissue. (Adapted from: HOFFBRAND A.V. 2006)(21)

10

1.3 Sites of Hematopoiesis

The hematopoiesis takes place in different organs of body but changes

anatomical position from embryonic life to adulthood. The various sites involved

in hematopoiesis and erythropoiesis are described as under:

The yolk sac is the first site of hematopoiesis of most primitive red blood

cells during early weeks of gestation. Yolk sac produces blood cells including

nucleated RBC. However, definitive hematopoiesis is observed in stem cells

located near the dorsal aorta. This site of hematopoiesis is known as AGM

(aorta-gonads-mesonephros) region because of close proximity to the dorsal

aorta, primitive gonads and kidneys. The precursor cells known as

hemangioblasts migrate to seed the liver, spleen, lymph nodes and the bone

marrow cavities. The liver and spleen produce blood cells from 6th week of

gestation till 2 weeks after child is born. The bone marrow begins hematopoiesis

form 6-7 months of fetal life and continues throughout life. The bone marrow is

the only source of hematopoiesis during childhood and adult life.(19, 20)

The bone marrow is red during whole infancy but from childhood onward it

is progressively replaced by fatty tissue. During adolescence and adult life, the

functioning bone marrow is confined to axial skeleton like vertebrae, and most

proximal ends of long bones like humeri and femori. The bone marrow is

approximately 50% loaded with fatty tissue even in these sites. The fatty yellow

marrow is reversible to hematopoiesis and so is the liver and spleen. The liver

and spleen can resume hematopoiesis even in adulthood but during certain

11

disease states, and this is known as extramedullary hematopoiesis. The bone

marrow of different bones in relation to age is shown in figure.II-4. (19-21)

1.4 Hematopoietic stem cells and progenitor cells

The process of hematopoiesis begins with proliferation and differentiation

of Pluripotent Hematopoietic Stem Cell (PHSC) in bone marrow (Figure II-6.) The

PHSC has potential of differentiating into all cell lineages within bone marrow.

The PHSC is rare cell (21), most probably one in every 20 million

nucleated bone marrow cells. The PHSC show immunological cluster designation

marker (CD), predominantly CD34+ and CD38+. The PHSC appear similar to be

a small to medium sized lymphocyte. The differentiation of PHSC occurs through

committed hematopoietic progenitor cells, which are comparative to PHSC

restricted in their developmental potential. The in-vitro culture techniques have

been used to demonstrate the existence of separate progenitor cells. An example

of earliest progenitor cell is the mixed myeloid precursor which gives rise to

granulocytes, erythrocytes, monocytes-macrophage and megakaryocytes cell,

known as colony forming unit (CFU)-GEMM.

The bone marrow is also the primary site of lymphocyte production, which

are derived from a common lymphoid progenitor precursor within bone marrow.

The PHSC are capable of self-replication and self-renewal so that cellularity of

bone marrow remains at a constant steady state in a normal healthy person. It is

said that one stem cell (PHSC) can produce about 106 mature blood cells with 20

cell divisions. (21)

12

The stem cells are capable of moving around whole body in peripheral blood

vessels. The stem cells cross through capillaries of bone marrow and exit into

systemic circulation, a process known as mobilization of stem cells. The

mobilization of stem cells is dependent upon growth factors like granulocyte-

colony stimulating factor (G-CSF) or granulocyte-monocyte colony stimulating

factor (GM-CSF). The reverse process of stems cells of colonizing bone marrow

cavities is known as homing. The homing of stem cells is dependent on the

chemotactic factors like SDF-1 (stromal derived factor 1). The stem cell viability,

proliferation, mobilization and homing are dependent upon interactions of stroma,

and stromal cells with the stem cells itself. The SCF (stem cell factor) and jagged

proteins are expressed on membranes of stromal cells while their receptors c-Kit

receptors and Notch receptors are expressed on stem cells. (21)

1.4.1 Stem Cell Plasticity

It is evident from various studies that adult stem cells in different organs

are pluripotent i.e.; they can differentiate into various types of cells and tissues.

Stem cell transplants in animals and humans can differentiate into neurons, liver

and muscle. (21)

1.4.2. Hematopoietic growth factors

The hematopoietic growth factors are glycoprotein cytokine hormones

which control and regulate the differentiation and proliferation of progenitor cells

and functions of mature blood cells. The growth factors include GM-CSF, G-CSF,

M-CSF, thrombopoeitin, erythropoietin and interleukins. The major source of

growth factors is the stromal cells except thrombopoeitin and erythropoietin. The

13

erythropoietin is secreted mostly by renal tissue and partly by liver. However

thrombopoeitin is secreted mostly by liver. (21)

Figure.II-3. Figure Shows Pluripotent Hematopoietic Stem Cells, committed

stems cells and colony forming units in bone marrow (Adapted from: Guyton and Hall 2012)(20)

14

Figure II. 4. Schematic illustration of hematopoietic stem cells

differentiating into lymphoid and myeloid series.(21)

15

2. LEUKEMIA- AN OVERVIEW

The term leukemia was first introduced by the German pathologist

,Rudolph Virchow (1856), who described a disease characterized by excess

counts of white blood cell under microscope. Leukemia is not a single entity, but

rather a disorder overlapping multiple types. Leukemia is a cancerous

proliferation of hematopoietic lymphoid cells within the bone marrow. It is

characterized by uncontrolled proliferation of hematopoietic lymphoid cells which

accumulate in bone marrow before spilling into peripheral blood circulation.

Most of cases appear without an evident cause, however, radiation and

toxins have been shown to be leukemogenic. Chromosome and gene

disturbances are related to leukomogenesis.(22) The chromosome and gene

alteration disrupts the proliferation of lymphoid series at some point of maturation.

The most common cancer in childhood is the leukemia accounting for one

out of three cancers. (23)Under 15 years of age, the leukemia is leadingcause of

cancer death and 7th mostcommon form of cancer death.Although the cause of

Leukemia remains usually unknown and uncertain, however thesymptoms are

produced because of pooling of immature lymphocyte cells in the bone marrow

and peripheral circulation. In the bone marrow, the leukemic cells disturb the

normal production of erythrocytes, leukocytes and thrombocytes(24).

From clinical course of leukemia, it is classified as being acutely fast

growing or chronic slowly growing. Almost all of the childhood leukemias run an

acute course.

16

2.1. Acute Leukemias: There are two main types of acute leukemia,

depending upon whether the lymphoid and/or myeloid series is involved.

2.1.1 Acute Lymphocytic (lymphoblastic) Leukemia (ALL): About

60% cases of acute leukemia are ALL type. The leukomogenesis begins

from the lymphoid series hematopoietic tissue.

2.1.2 Acute Myeloblastic Leukemia (AML): Acute myeloblastic

leukemia originates from the myeloid series of hematopoietic tissue.

2.2 Chronic Leukemias:

Chronic leukemias are more common in adults than in children.

Thechronic leukemia is characterized by slow proliferation of hematopoietic

tissue involving erythrocyte, leukocyte and/or megakaryocytic series. The chronic

leukemias show mature cells but in countless numbers.

Chronic leukemias may be further sub typed into:

2.2.1 Chronic Myeloid Leukemia (CML): The CML is most common

leukemia of adulthood and rarely seen in children.

2.2.2 Chronic Lymphocytic Leukemia (CLL): The CLL is

common in adults but is extremely rare in children.(21-25)

3. ACUTE LYMPHOBLASTIC LEUKEMIA (ALL)

Acute lymphoblastic leukemia (ALL) is a neoplastic disorder of lymphoid

series of hematopoietic tissue. It is defined as a malignant proliferation of

lymphoid cells which are blocked at an early stage of differentiation because of

unknown cause. The lymphoblast cells proliferate in uncontrolled fashion and

eventually replace bone marrow cavities. ALL is basically a heterogeneous

disorder with differing characteristics of lymphoblasts. The changes do occur at

17

the level of cell morphology, biochemical characteristics, and cytogenetic

organization, immunological and molecular characteristics of lymphoblastic cells.

Characteristics of leukemic lymphoblasts are essential in establishing diagnosis

of ALL, excluding other causes of bone marrow failure, and finally to divide the

ALL into its respective subtypes. The morphological heterogeneity reveals the

fact that the leukemic changes may occur at any point during the lymphoid cell

differentiation.

3.1. Epidemiology of ALL

ALL is the most common malignancy of childhood. The incidence of ALL

below 14 years of age is 3 to 4/100,000 and approximately 1/100,000 in older

than 15 years, in the United States(22). The peak incidence of ALL is observed at

age of 2-5 years.The ALL is reportedly the single most common cancer in

Pediatric oncology, accounting for nearly 1/3 of total cancers. ALL represents

75% of all acute leukemias in children, which accounts for 34% of cancer in

childhood.(26).

The incidence of ALL is much lower in adult patients, in whom AML and

CLL are reported to be more common (8, 23, 26).ALL predominates male

population in all age groupsand incidence is more among white children

compared to others (22).

It is reported by various studies that the incidence of T-cell ALL is

somewhat higher in boys compared to girls. (27-30). However, incidence of ALL

is slightly higher in girls during first year of life(26, 29). ALL almost always

appears as de-novo disease rather rarely occurring secondary to a primary

leukemogenic process (31). A variety of environmental and genetic causes have

been implicated in the pathogenesis of ALL. ALL is associated congenital genetic

18

syndromes like Neurofibromatosis type, ataxia telangiectasia, 1, Bloom`s

syndrome and Down`s syndrome (32).

Exposure to pesticides, solvents and ionizing radiations during intra-

uterine life has been linked to increased frequency of childhood

leukemia(32).Fusion of leukemic specific genes, the Immunoglobulin (Ig) and

clonal Ig genes have been identified as predisposing factor of developing ALL

(33, 34). The incidence of childhood leukemia varies according to geographical

distribution, age, gender, race and ethnicity in different parts of the World(35-37).

The Childhood leukemia incidence is highest in United States, Germany,

Australia and Costa Rica.While intermediate incidence is reported from the Indian

subcontinent, Europe and among blacks of United States of America. (26, 38)

3.2 Classification

The ALL umbrella encompasses a variety lymphoid precursor cells which

are morphologically and immunologically related to the B-cell and T-cell lineages.

The ALL usually presents with extensive involvement of bone marrow and

peripheral circulation but rarely limited to tissues, with no or limited involvement

of marrow cavities. However, the later cases are classified as lymphoblastic

lymphomas (LBLs).

The current WHO classification of leukemia of hematopoietic tissue is

designated as B-cell or T-cell lymphoblastic leukemia and or lymphomas (39).

Acute leukemia may classify in different way as;

(1) French-American-British (FAB) classification is based upon morphology,

(2) Proposed WorldHealth Organization Classification of Acute Leukemia

19

(3) Byimmunophenotyping alone, as proposed by the European Group for the

immunological classification of leukemias.(41, 42)

3.2.1 Morphological classification (French-American-British

Classification)

A group of French- American and British (FAB) leukemia experts(1970)

divided ALL into three subtypes as; L1, L2 and L. The L1, L2 and L3 were based

on the microscopic appearance of leukemic cells after routing staining properties.

The ALL is subdivided into FAB-L1 occurring in children, FAB-L2 in older

children and adults and FAB-L3 occurring inleukemia secondary to Burkitt's

lymphoma.

The FAB subtypes are classified according to 2 criteria;

i. Individual features of leukemic cells

ii. Degree of leukemic cell heterogeneity

The features considered in ALL include leukemic cell size, content of

chromatin material, shape of nuclei, nucleoli, degree of basophilia and

cytoplasmic vacuolations (40).

FAB-L1: (Leukemic Small Cell): FAB-L1 is the acute leukemia of childhood

which accounts for 70% of all, with 74% of cases occurring under 15 years.

Homogenous leukemic cell population is observed on smear .Cells are

predominantly small, with moderately basophilic cytoplasm, regular nuclear

shape with occasional cleft and rarely visible are the nuclear contents.(43).

20

Figure II-5. FAB- L1 Smear showing small homogenous cells.

(Adapted from: Guenova M, 2013) (43).

FAB-L2: The leukemic cells are large. Nucleus is irregular and nuclear cleft is

common, may be one or more nucleoli visible. Cytoplasm shows variable

staining properties. FAB-L2 accounts for 27% of ALL patients and 66% of

cases occur in children of older than 15 years of age.The FAB-L2 leukemic

blasts may be confused with the blast cells ofacute myeloid leukemia

(AML).(43).



21

FAB-L3: (Burkitt's lymphoma type): The leukemic cells are homogenous,

large size with round to oval nucleus. Prominent nucleoli usually one to three

but sometime up to 5 may be visible. Deeply staining basophilic cytoplasm

with prominent vacuoles is visible. Prominent cytoplasmic vacuolations with

intense basophilia is characteristic feature of leukemic cells. Similarly a high

mitotic index with varying degree of phagocytosis is observed. Cell markers of

mature B-cells are detectable on cell surface.(43).



The FAB classification has been abandoned nowadays, and replaced by

new WHOclassification. The new WHO classification reflects better

understanding of biology and molecular characteristics of leukemic cells. The

WHO utilizes immunophenotyping and divides ALL into three basic types

designated as;

Precursor B cell

Precursor T cell, and

Mature B cell leukemia/lymphoma (44).

22

Table. II-1. WHO CLASSIFICATION(45)

Precursor B-cell ALL/LBL

Cytogenetic subgroups

t(9;22)(q34,q11),BCR/ABL

t(v;11q23);MLL rearranged

t(1;19)(q23;p13),PBX1/E2A

t(12;21)(p13;q22);TEL/AML1

Hypodiploid

Hyperdiploid, >50

Precursor T-cell ALL/LBL

Mature B-cell leukemia/lymphoma

ALL= acute lymphoblastic leukemia; LBL= lymphoblastic lymphoma; MLL= mixed lineage leukemia

23

3.2.2 European Group for the Immunological classification of

Leukemias (EGIL)

The ALL can easily be sub-divided into different types according to

cytoplasmic markers and immunologic surface ligands. The EGIL proposed to

classify ALL by immunophenotyping. (41, 46)

Initially, it was observed that normal lymphoid precursor cells express

common cell surface antigens. Based on expression of cell surface antigens, the

EGIL defined a threshold of at least 20% of positive blast cells to a given

monoclonal antibody.

Pro-B ALL:The B-cell precursor blast cells which are positive for membrane

and cytoplasmic markers CD19, CD22 andCD79a (47) are classified as pro-B

ALL type. By definition, if any two of three above markers are positive then

ALL cells must be titled as pro-B cells ALL.

Common ALL: The presence of CD10 antigen (CALLA) defines the

"common" ALL subgroup of acute lymphoblastic leukemia.

Pre-B group: leukemic cells which show cytoplasmic IgM are labeled as the

pre-B ALL.

Mature B-ALL: The mature B-ALL is labeled if immunoglobulin light chains of

cell membrane are positive.

T-cell ALL: The most immature T lymphoid cells are positive for CD markers

viz; CD1a, CD2, CD3, CD4, CD5, CD7, CD8, CD2, CD5and CD7. However,

none of these are absolutely T cell line specific, hence unequivocal diagnosis

is made of T-cell ALL if surface/cytoplasmic CD3 are positives. The T-cell ALL

comprises 25% of all adult cases of leukemia of ALL type.

24

CD34 marker: The CD 34 marker is expressed on the stem cells of both B

and T lineage. The CD34 has clinical prognostic value but has no diagnostic

value. (48)

The EGIL scoring system addressed characterization of ALL as of B- or T- cell

type based on expression of specific membrane or cytoplasmic markers. The

markers are also helpful in cell differentiation of all stages of maturation of B and

T cell lineages (49).It is reported that these markers can be used for making

diagnosis and sub classification of ALL (50).

3.3 Cytogenetics in ALL


number of chromosomes, is a significant feature of ALL (21).

Hyperdiploid Karyotype with > 46 number of chromosomes (mostly they

are from 51-65 chromosomes). (21)

Hypoploidy: A second significant chromosomal abnormality in childhood

ALL is hypodiploidy (Ho), where chromosomes are ≤ 45. (21)

Structural abnormalities like deletions (5 – syndrome) and translocations

(e.g. Philadelphia chromosome) are also reported genetic abnormalities in

ALL. (21)

Translocation: is defined as transfer one part of a chromosome to

another chromosome. (21)

Deletions: is defined as a loss of a part of chromosome or sequence of

DNA. (21)

Point mutation: is defined as a change in one or a few gene sequence

(21)

Duplication: is defined as a part of chromosome is duplicated. (21)

25

The cytogenetic grouping of ALL improves the understanding of both

etiology and epidemiology of ALL subtypes. The ALL shows distinct cytogenetic

characteristics related to clinical and hematological understanding.

The main reason for getting cytogenetic information in ALL is to obtain

understanding of prognosis and monitoring of disease, and MRD status beyond

the cytomorphologic classification. Genetic abnormalities are hall mark in ALL

and may be recurrent. The genetic abnormalities provide probing into the

molecular mechanisms of leukomogenesis (51).

The genes controlling transcription and tyrosine kinases activity are most

frequent targets of genetic alterations in ALL.(52).The genetic alterations may be

pointmutations and/or deletions. But the main genetic alterations of ALL

aretranslocations and chromosome imbalances which result in hypo- or

hyperploidy. (53).

3.3.1 Chromosomal translocations

Childhood ALL must be analyzed for cytogenetic alteration because these

are of clinical importance. Banded karyotyping is nowadays a routine testing of

ALL. The chromosomal aberrations in ALL can be divided into two groups;

Abnormalities in the number of chromosomes (ploidy) and

Chromosomal structural changes, such as deletions, partial deletions,

partial duplications, translocation, inversions and presence of dendritic

chromosomes.

Numerous primary and secondary cytogenetic abnormalities have been

described in ALL. The cytogenetic abnormalities include both structural changes

and numerical counting of chromosomes. The cytogenetic abnormalities show

correlation with clinical parameters and prognosis.(54, 55).

26

Two of most important findings are presence of Philadelphia (Ph)

chromosome and hypoploidy (chromosomes <46 per cell). Both of these are

chromosomal abnormalities are risk factors for failure of chemotherapy for ALL.

Massive hyperdiploidy in ALL (chromosomes >50 per cell) is common finding of

childhood ALL under 10 years of age. Massive hyperploidy in ALL estimates the

impact on the prognosis and makes differences in treatment intensity. However,

clinically hyperploidy is an indicator of lower risk factor for chemotherapy.

Chromosome translocations either commonly result in formation of a

chimeric fusion of gene with novel properties or by formation of an oncogene by

changing genes.An exaggerated number of promiscuous genes like NUP 98,

ETB6 or MLL have been reported in ALL. These genes recombine with different

other genes and result in fusion and formation of chimeric genes. Thus, the

number of fusion genes overextends the number of affected genes.

In childhood B-ALL, the commonest genetic alteration is the translocation,

t(12;21)(p13;q22), which results in fusion of ETV6 toRUNX. The t (12; 21)(p13;

q22) is reported to be present in 25% of B-ALLcases.(51). Other cytogenetic

alterations found include the;t (1; 19) (q23; p13)/E2A-PBX1 (TCF3-PBX1), the

t(9;22)(q34;q11)/BCR-ABL1, and hyperdiploidy (Chromosome > 46). The

hyperploidy is often associated with a FLT3 mutation (51). Infant ALL is

associated with 80% MLL gene rearrangements and phenotypically presents as

pro-B ALL. (56-58).

27

Figure II-8: Pie diagram showing relative frequencies of chromosomal

aberrationsfound in childhood B-ALL (Armstrong et al., 2005) (51)

Recently, childhood ALL has been characterized by mutations, deletions

or structural rearrangements in 40% of BCP-ALL genes, which have been

implicated in B-cell development and differentiation (59). The known genes are

the IKZF3(Aiolos),IKZF1 (Ikaros),EBF1, TCF3 (E2A),LEF1, andPAX5 (59-

60).Recurrent deletions of BTG1 are a negative effector of B-cell proliferation.

Genes controlling cell cycle progression (e.g. CDKN2A, CDKN2B, and RB1) are

also frequently affected by losses of chromosomal number. Such deletions are

detected in 86% of T-ALL and 54% of B-ALL (60).Most of T-ALL cases are

initiated postnatal as revealed by analysis of neonatal blood for chromosomal

rearrangements.(61).TheETV6-RUNX1 and the RUNX1-RUNX1T1 (AML1-

ETO)fusion genes can be detected 100 times more often from normal healthy

neonate’s blood that later on may be on risk of developing leukemia. (62). The B-

ALL diagnosed twins showing positive leukemic blasts for ETV6-RUNX1 reveal a

deletion of the ETV6 allele, whereas the ETV6-RUNX1 positive cellsof the

healthy twin carry one intact copy of ETV6. The above data supports the fact that

28

inactivation of the second unrearranged ETV6 allele represents acrucial

cooperative mutation (63).

3.3.2 Cooperative mutations

Formation of fusion genes produced by chromosomal translocations is

mainstay in the pathogenesis of ALL. However, it seems that other genetic

lesions are equally essential in inducing overt leukemia.(64).An example of such

genetic lesion is deletion of a cyclic dependent kinase inhibitor 2 A

genes(CDKN2A) which is located on 9p21.3.This CDKN2Agene encodes the

tumor suppressors like p14ARF and p16INK4A(65, 66).Deletions of CDKN2A are

present in 30% of B-cell precursor ALL and 70% of T-cell precursor ALL. The

alteration of CDKN2A gene makes both the TP53 and retinoblastoma pathways

inactive. The TP53 and retinoblastoma pathways control cell cycle transition from

G1 to S phase. Hence, inactivations of tumor suppressor proteins of these two

genes fail to prevent leukomogenesis.(67).

29

Figure II-9: The Retinoblastoma pathway and p53 Tumor suppressorcross talk

(Pui et al., 2004) (68).

30

The NOTCH1 gene alteration has been reported in <1% of T-cell ALL.

NOTCH1 gene is associated with chromosomal translocation of t (7; 9). (69). The

NOTCH1 gene encodes a membrane receptor which regulates normal T-cell

maturation.(70). Although the association of NOTCH1 in translocations is rare,

but the recent studies have reported its role in T-ALLthrough mutations. The

NOTCH1 gene mutations have been reported to be present in > 50% of T-ALL

patients (71-73). However, the underlying NOTCH1 gene mechanisms which

cause abnormal signaling and T cell proliferation remain unclear.

It is postulated that the expression of MYC oncogene may be playing role

in induction of NOTCH1gene associated T cell leukomogenesis.One study

reports that the MYC oncogene product is apro-growth mediator of NOTCH1

signaling in the developing thymocytes (71). It is reported from experimental

models that the NOTCH1 gene can induce T cell ALL and may be an initiating

gene in human T cell leukemias. (72).

31

Figure II-10: Notch signaling pathway in normal thymocytes

(Pui et al., 2008) (74)

32

3.4 Etiology of leukemia

ALL is a heterogeneous group of leukemias and many previousstudies

lacked sufficient number of possible potentialrisk factors because of study

designs, sample size and statistical power. Thus, there is emerging need of

information to gather about the etiology of childhood ALL. Many external and

internal risk factors for childhood ALL have been reported by various

epidemiological studies. The possible risk factors may be dietary agents, social

andenvironmental factors, genetic and/or immunological alterations. Knowledge

of possible hazards may help to reduce the exposure and control the deadly

dangerous disease.

3.4.1. Dietary factors

Little is known about diet of mothers of children developing childhood ALL.

Many studies have focused on association of ALL with cured meats, (75),

supplementation with folate (76), vitamins like cholecalciferol and retinol(77),

and/or foods containing topoisomerase II inhibitors (78).

The cured meat contains N-nitrosamine precursors which can be

converted into carcinogenic metabolites. The N-nitrosamine compounds are

hypothesized to induce leukomogenesis either through mother diet or food

consumption during early childhood.(75).

A previous study reported that foods such as black tea, cocoa, coffee, soy,

fruits, fresh vegetables, canned vegetables, beans and wine contain DNA

topoisomerase II inhibitors and are potential risk factors in childhood ALL. (78).

33

3.4.2.Socio-economic status

The role of socioeconomic status (SES) has been controversial as

reported by various studies. Previous studies from United States reported that

higher SES was a possible risk factor, while studies from United Kingdom

reported mixed results, some claimed SES as possible risk factor (79). Other

study from UK reported non significant differences between higher and lower

social classes. It was reported that 75% belonged to lower social class. (79)

3.4.3. Environmental factors

3.4.3.1. Ionizing radiations

Various epidemiological studies had reported that ionizing radiation are

carcinogenic and leukemogenic. (80, 81).Causal relationship of ionizing

radiations has been established for the childhood leukemia, particularly AML.(82-

85). Currently, the relationship of intrauterine radiation exposure as risk factor for

childhood cancers is already established, and it is reported that fetal exposure to

ionizing radiations is more leukemogenic compared to childhood exposure. The

magnitude of ionizing radiation as risk for occurrence of leukemia depends upon

duration of exposure, age of child at time of exposure and more over the dose of

ionizing radiations. The relationship of ionizing radiation and leukomogenesis is

now already established.(85, 86). The ionizing radiations may prove potentially

hazardous at the time of conception, or even before, during pregnancy and after

childbirth birth.

34

3.4.3.2 Non- Ionizing Radiations

Various studies had reported causal relationship between electric or

magnetic fields (EMF) and childhood leukemia. However, the basis of such

association remains unclear. (87-91) Other studies had reported no such causal

relationship.(92-94). The controversial results of such association of various

studies might have been introduced due to different methods of assessing

EMF.(95-97). Animal studies conducted with very high levels of exposure to EMF

have not shown any causal association of EMF with bone marrow neoplasia.(97).

3.4.3.3 Chemicals

Occupational exposure of parents to plastics, thinners, paints and

chlorinated solvents may cause leukemia in children. It is reported by previous

studies that benzene can trigger induction of lymphoid cells into leukemia (53).

An occupational study has reported association of benzene with occurrence of

leukemia. Benzene exposure as low as (98) <60 ppm-years could result in

leukomogenesis than previously reported as high as 220 ppm-

years.(99).Occupational and home exposure has been evaluated as possible risk

factors for childhood leukemia.(100, 101). The father’s occupational exposure to

chlorinated solvents, methyl ethyl ketone, spray paints, dyes and cutting oils have

all been considered potential risk factors.

3.4.3.4 Pesticides

Growing evidences are accumulating for possible association of pesticide

exposure and childhood leukomogenesis. Both intrauterine and postnatal

pesticide exposure are suggested as risk factors for childhood leukemia. (102).

Hence neonates and children are at risk of carcinogenic effects of pesticides as

35

their use is now overwhelming. (103). Childhood exposure is usually from home,

lawn and garden pesticides. (104).

Agriculture, seeds, vegetables, occupational exposure, and pet products

are possible source of pesticide poisoning. (100, 105, 106).

3.4.3.5 Cigarette smoking

Maternal or paternal cigarette smoking before or during pregnancy as risk

factor is yet unclear. (107, 108). Some studies have reported association of

smoking with childhood leukemia (109) while other reported no

association(110).Studies had reported that paternal and maternal smoking before

conception is related to elevated risk of childhood leukemia.(108, 109, 111)

3.4.4 Immunological Factors

Previous studies have suggested role of immunological factors in the

leukomogenesis. (112). Others have reported viral agents associated neoplastic

growths in human beings. Epstein Barr virus may change immunological

mechanisms and may induce neoplasm.(112, 113).

3.4.5. Genetic factors

Genetic factors like gene mutations significantly influence the inter

individual variation in tumor incidence (114). Various factors are acting

simultaneously some activating oncogene and other inactivating regulatory genes

resulting in an imbalance and new growth does occur eventually(115).Gene

polymorphisms have been implicated in altering the risk of leukomogenesis. The

gene polymorphisms interact with environmental factors, immune mechanism

and dietary factors for the leukomogenesis.

However,clonal evolution and the modest concordance rate for ALL in

identical twins strongly suggest that additional genetic mutations, occurring in

36

after child birth time period, are required for progression to full blown neoplasm

growth.(112). Consistent with this paradigm, several genes changes because of

incorrect DNA synthesis or abnormal methylation of oncogeneand/or tumor

suppressor genes, had been identified in the pathogenesis of lymphoid cell

cancers.(116-118).

37

4. EPSTEIN-BARR VIRUS (EBV)

EBV is classified as a human gamma herpes virus with a tropism for

epithelium and B lymphocyte. EBV is also known as human herpes virus 4 (HHV-

4), and is the type specimen of Lymphocryptovirus. Similar to other herpes

viruses, it comprises of a nucleoprotein core which is surrounded by a capsid.

The capsid, in turn, is enveloped by tegument and lipid layer containing at least

ten types of viral glycoproteins and host acquired cell proteins.

EBV is first DNA virus whose genome was sequenced and it was known to

be related to the Lymphocrypto virus genus of gamma herpes viruses.EBV

was the first large DNA virus to be sequenced, and was determined to be part of

the Lymphocrypto virus genus of gamma herpes virus. (119)

The diameter of viral particles is approximately 200 nm. It contains a singly

linear genome comprising of 185 kb, and is designated a type C genome among

herpes family(119).EBV is unique viral infection, which can induce transformation

and proliferation of B lymphocytes in humans as well as other primates, in the

absence of other stimuli.EBV is restricted to human beings under natural

conditions. EBV almost always causes symptomatic infection once in the life of

host similar to other herpes viruses; this is known as primary infection. However,

in later part of life it remains latent in host cells may be for decades.

The EBV was first identified in African region in patients of Burkitt’s

lymphoma (BL). The BL is endemic in Africa and EBV was previously unidentified

herpes virus.(120, 121)The EBV is the first herpes virus knows to immortalize

human cells. The EBV is thus believed to be oncogenic virus, though it is not first

oncogneic virus. (122)

38

4.1. Types of EBV

There are two subtypes of EBV; EBV-1 and EBV-2. Both are closely

related to each other and differ in the structure and sequence of EBNA2,

EBNA3A, EBNA3B& EBNA3C.The occurrence of EBV2 is reported from Papua

New Guinea and Africa. However, EBV 1 is dominant subtype in remaining world.

EBV 2 is reported most in homosexual, HIV +ve males of Western world. The

infectivity and disease association are thought to be similar for both EBV.

Although, it is fact that EBV-2 gene EBNA 2 is less pathogenic in causing cell

transformation and proliferation of host. (123, 124)

4.2. Genome of EBV

In latent form, the genome is a circular episome, while in infectious viral

particles it appears in linear form. When the EBV viral particles enter cells,

circularization occurs at the Terminal repeats (TR) and form linear genome.

Super coiled negative DNA is contained in circularized episome, associated with

histones and localizes to chromosomes.EBV genome replicates once per

episome per cell division. Each episome is attached to chromosome with the help

of EBNA1 protein of EBV.(125, 126) Circularization and linearization of EBV

genome during latent and Lytic infectious phases lead to variability of genome

size through a change in terminal repeats. The terminal repeats contain one

important promoter for the EBV latent membrane protein 1 (LMP1). The LMP 1 is

inversely correlated with upstream of the terminal repeat number. (127) Most of

EBV associated neoplasm arise from latent infected cells whose circular episome

replicate by host cells and contain a fixed TR number.Another contributor in the

variability of EBV strains is the major internal repeats in the W region, which vary

in number of copies. (128)

39

5. NATURAL HISTORY OF EBV INFECTION

5.1. Primary EBV infection

The EBV enters body through a breach in oral mucosa. Once inside

epithelia cells, the virus replicates and passes through mucosa into lymphoid

reservoirs of Waldeyer`s ring (adenoids, palatine tonsils, etc). The primary EBV

infection in adults clinically presents as infectious mononucleosis (IM) while in

children it is usually mild or asymptomatic.

The primary infection is characterized by proliferation of infected B

lymphocyte of Waldeyer’s ring lymphoid tissue. The B cells express immunogenic

EBV antigens and present to T cells and Natural Killer (NK) cells, which in turn

start killing EBV infected B cells.(129)

5.1.1. Infectious mononucleosis:

The symptoms of acute Infectious mononucleosis (IM) last for 1-3 weeks

from onset.The symptoms include pharyngitis, high grade fever, cervical

lymphadenopathy, splenomegaly, hepatomegaly and jaundice. Malaise and sever

fatigue last for months after resolution of acute symptoms. The antibody

response against EBV persists for rest of life(129).

5.1.2. Chronic active EBV infection (CAEBV)

CAEBV severe EBV illness is defined as lasting longer than six months.

The blood from CAEBV shows low titers of antibodies against EBNAs or high titer

against lytic antigens. CAEBV is characterized by lymphadenopathy, chronic

hepatitis, splenomegaly, etc. High EBV antibody titers with impaired T or NK cell

responses are often evident (130). CAEBV shows abnormal proliferation of EBV

infected mature T and NK cells. The underlying mechanisms of non-B

lymphocytes infection by EBV remain unclear. (131)

40

Once infected in life, the person becomes carrier throughout life. The

latency occurs by EBV residing within B lymphocyte. EBV infected B

lymphocytes can be divided into two;

Memory B lymphocytes circulating in vessel

CD10+CD77+ B lymphocytes in the germinal center of lymph nodes

(132)

The number of EBV+ B lymphocytes decreases exponentially throughout

the life of person. Shedding of EBV occurs intermittently in saliva of carriers. This

occurs when infected B cells differentiate into antibody producing plasma cells in

Waldeyer’s ring. The proliferating B cells release lytic EBV particles into saliva.

The infectious EBV secreted in saliva is made by epithelial cells rather than by

plasma cells. (132)

5.2. Cell entry and exit

The EBV binds to complement receptor 2 (CR2), also known as CD2,

which promotes stimulation, proliferation and survival of B lymphocytes by

stimulation of cell surface immunoglobulin. The complement receptor 2 (CR2) is

present on some neutrophils; hence neutrophils can be infected with EBV.

However, neutrophils can be diseased as they express high levels of death

receptors FAs and LMP 1 which cause apoptosis.The EBV glycoprotein

gp350/220 mediates B cell attachment through interaction with CR32. This leads

to cell activation, helps in cell survival through CD 19and PI3K/Akt pathway.

TheCD19 and PI3K/Akt pathwaystimulates endocytosis via non-clathrin-coated

membrane vesicles. (119, 132)

The EVB proteins gp42, gL, and gHmediate fusion of endocytosed vesicle

through interaction with class II MHC. The viral capsid carried to nucleus and

linear genome is transferred. New circular episomes are detected after 16 hours.

41

The new circular episomes function as templates for latent gene expression.The

intracellular survival of EBV inside B cells is dependent upon inactivation of

apoptotic pathways. The B cell apoptotic pathways are inactivated through viral

Bcl-2 expression.(119, 132)

Within 24 hrs of EBV infection, resting B cells show expression of the

BZLF1 protein. The BZLF1stands for Z-encoded broadly reactive activator

(ZEBRA). The BZLF1 serves as the primary lytic switch gene.Upon EBV

infection, latent viral genes expression occurs. Latent viral gene expression is

activated B cells CD23, CD44 and CD10 markers. The expression of these

markers indicates that B cells are about entering cell cycle phase of

differentiation.

The infectious virus production does not occur soon after infection of B

lymphocytes with wild-type EBV. Virus production may take 3 days and enough

virus production is evident after 5 days and 9 days before any virus is

secreted.The long life cycle and multiple steps of lytic viral cycle are peculiar to

herpes viruses.

EBV release proceeds in similar way to other herpes viruses.The viral

genome enters capsid via a dodecameric complex of Portal (BBRF1) protein.

The capsid complex fuses with inner nuclear membrane, de-enveloped by

fusion with outer nuclear membrane. Nuclear egression depends on disassembly

of nuclear lamina by different protein kinases (PK). These kinases include protein

kinase C (PKC) and the Cdk1 homologue BGLF4, which is the only EBV-

encoded PK.(119, 132)

Once in the cytoplasm, the capsid becomes complexed with an

amorphous network of tegument proteins. The complex cellular proteins include

tubulin, cofilin, actin, Hsp90, and Hsp70. Finally, the tegument-coated capsid

42

buds into regions of the trans-Golgi network, acquiring a lipid envelope containing

numerous viral glycoproteins. Secretory vesicles traffick mature virions to the cell

membrane, where the infectious particles are released. (119, 132)

6. MALIGNANCIES ASSOCIATED WITH EBV

EBV possesses potential of transforming B lymphocytes into proliferating

immortalized cell lines.LMP1 and BARF1 are the two latent proteins related to

EBV which are known oncogenic. When both are expressed the neoplastic

growth of B cells lineage begins. The LMP1 is CD40 ligand and is found in

latency II and III while the BARF1 is a CSF-1 receptor found in lytic programming

of EBV (133, 134).

The EBV is suggested as an oncogenic viral agent for cancers of

nasopharyngeal epithelium and lymphoid collections. EBV is strongly associated

with carcinomas and lymphomas (133, 134), however it is also one of the

suggested etiological agents for rare cancers like NK cells, T cells and

leiomyosarcoma associated with AIDS. (133, 134)

The plasmablastic lymphoma and lymphoma presenting as primary

effusion are tumors of large B cell lineage, which contain EBV genome and show

irregular production of LMP2A and or LMP1. (135-138)

EBV is suggested etiological agent in causing some of tumors like;

Hodgkin`s disease, Burkitt`s lymphoma, nasopharyngeal carcinoma, gastric

carcinoma and post transplant lymphoproliferative disorders (135-138).

6.1. Hodgkin’s disease (HD)

Classical HD is a lymphoma localized to lymph nodes or spleen and is

usually treatable. HD is suggested to be derived from germinal center B cells

43

which have been arrested at some stage of maturation. HD is more in male than

female. Male cases are more likely to be EBV+.

Older patients, a history of infectious mononucleosis and HD in developing

countries show high positivity for EBV genome. The classical histological picture

of HD is a giant cell known as the Reed-Sternberg (RS) cells (139). The RS cells

were once considered as granulocytes or macrophages based on shape, size

and cell markers. Later on, the surface immunoglobulin showed a B cell

decendency. The RS cells CD30 and CD15 positive contrary to most B cells.

The RS cells lack normal B cell markers like CD19 and CD20. The RS cells also

lack CD40 and CD80 markers which are necessary for T cell interactions.(139)

The RS cells comprise of 0.1- 10% of total cell population in the lesion. The RS

cells are usually surrounded by normal lymphocytes, and this makes RS genetic

abnormalities very difficult.

The primary EBV infection with symptoms of infectious mononucleosis is

at risk of developing EBV+ HD but not for EBV- HD. The EBV- HD are

characterized by excess of tyrosinase activity and blockage of A 20, both of

which activate STAT and NFκB pathways. In EBV- HD, the genetic alterations

are frequent and often associated with cytokine gene polymorphism and

autoimmune disorders. In EBV+ HD, the EBV is suggested to contribute to

surrounding milieu and probably inhibits normal immune functions. In EBV+

cases the virus is likely contributing to the milieu that surrounds the HRS cells

and prevents resolution by the immune system. (135-138)

The EBV surrounded RS-milieu contains IL-21 and IL-21R, both of which

cause activation of STATs pathway, and increase proliferation factors such as IL-

10, BAFF, APRIL and cytokines which attract T helper and T regulatory cells.

LMP1 induces IL-10 production and EBV-EBNA1 up regulates regulatory T cell

44

chemokine CCL20. EBV infection up regulates autotoxins and LPA, both are HD

growth factors. The LMP1 and LMP2A activate various signal transduction

pathways, which stimulate cell proliferation. Activated T cells, T helper cells and

antiviral immune factors are observed more in EBV+ cases compared to EBV-

HD. The B cell aberration producing RS cell phenotype is produced by down

regulation of B cell factors. EBV LMP2A activates the NOTCH pathway in mouse

models, which is normally associated with T cells rather than with B cells.(135-

138).

6.2. Burkitt’s lymphoma (BL)

Histologically, the BL shows cells similar to germinal center B

cellsi.e.;gene rearrangement for somatic Ig, BCL6 transcriptional repressor gene

levels are elevated and CD10 and CD77 positive phenotype.

BL is found in non nodal areas more often than most lymphomas. The BL

is characterized by translocation of c-myc oncogene on chromosome 8. The

translocation is often between c-myc and Ig gene and usual translocation is

t(8:14), however, t(2:8), and t(8:22) are also noted.

The BL exists in sporadic and endemic types. Theendemic BLis common

in boys of Papua New Guinea and central Africa. The EBV+ is noted in 95% of

cases of endemic BL. The sporadic BLoccurs during childhood commonly and

EBV+ is noted in only about 30%. The somatic mutations and hypermutation of

GC-B cells are associated with occasional translocations which cause

mitogenesis.

The fact that EBV is associated specifically with BL implicates EBV latent

proteins in tumorigenesis and maintenance of the tumor, and there have been

many hypotheses about how this can happen. The EBV helps to sustain BL after

oncogenesis. When EBNA1 is expressed without LMP1 as in BL, it reducesMHC

45

I loading and presentation of viral antigens on the cell surface. EBNA1 also

contributes to chromosomal instability by up regulating enzymes which generate

ROS (reactive oxygen species). The above changes cause translocations which

augment c-myc activity, such as inhibiting the p53-suppressor pathway.(135-138)

6.3. Post-transplant lymphoproliferative disorder (PTLD)

In immunocompromised persons, the cells showing the Latency III

program are killed easily. Primary immunodeficiency disorders, acquired

immunodeficiency syndrome and immunosuppressive drugs like methotrexate

often show lymphoma disease. The PTLD in transplant recipients is a frequent

adverse effect of immunosuppressant (119, 140).

In solid organ transplants, the PTLD is treatable and reducing

immunosuppressant is adding factor. While in Bone marrow transplants, the

PTLD is donor derived, with very good prognostic and treated with donor derived

T cell or EBV specific CTLs cultured ex-vivo.

In bone marrow recipients the PTLD is usually donor-derived and the

prognosis is usually very poor and treatable only by donor T lymphocyte infusion

or ex vivo-cultured EBV-specific CTLs. (119, 140)

PTLD is a diagnosis comprises various types of lesions.

Monomorphic PTLD includes malignancies which are often reported in

immunocompetent people like BL, T cell lymphoma; B-ALL and B-CML.

Monomorphic PTLD are characterized by chromosomal abnormalities.

Polymorphic PTLD is commonest type and EBV + associated with

Latency II program. It is either monoclonal or polyclonal. The B cells appear in

different stage of development.

46

HD-like PTLDs are also reported. They are not always malignant, some

are characterized by plasma blast overproduction and some resemble to EBV

infected proliferating cells as in infectious mononucleosis.

Figure II-11. The EBV primary infection and cycles of persistence(119)

They resemble to hyperplasia’s rather than tumors, but are often

associated with Latency III. The HD-like PTLDs appear soon after

transplantation. They can be controlled by reducing immunosuppressants. But

47

may appear months or years later characterized by monoclonality and or

chromosomal abnormalities. (141)

6.4. EBV associated carcinomas

6.4.1. Nasopharyngeal carcinoma (NPC):

The NPC is a malignancy of the nasal mucosal epithelium. It is prevalent

in south East Asia, and particularly among Chinese. Undifferentiated and Non-

keratinizing NPCs are always EBV+, however, some squamous type NPC are

EBV –ve. The NPC is associated with immunosuppressive microenvironment.

The lesions are characterized by presence of large number of T-regulatory cells

and surprisingly lack T-cytotoxic cells against latent EBV. Anti-EBV serology is of

diagnostic importance in NPC, specifically of the Ig A type. (142)The lesions of

NPC harbor cytokines which function as growth factors for tumor, and are

indicators of lytic EBV reactivation. The cytokines include IL-1α, IL-6, and IL-8; in

addition to the anti-inflammatory IL-10 induced by LMP1, and a lytic viral IL-10

homologue. (143-145)

6.4.2. Gastric carcinoma

The gastric carcinomas show EBV+ in 10% cases. The EBV+ gastric

carcinomas are far higher than EBV+ colon or esophageal carcinoma. The

endemicity of EBV+ gastric carcinoma is not reported. However, EBV+ gastric

carcinomas are common in Europe and Hispanic population.

One review suggests that the EBV infects basal cell layer of disrupted gastric

mucosa and EBV genome becomes methylated. If host genes are also hyper

methylated, it induces carcinogenesis.(143-146)

48

6.4.3. Other carcinomas

The EBV+ve esophageal and colon carcinomas have been reported, but

are much less compared to gastric carcinoma. (119, 140, 147)

49

CHAPTER III

MATERIALS AND METHODS

1. Study Design:

Observational study

2. Study Settings:

Liaquat University of Medical and Health Sciences, Jamshoro and Isra

University Hospital, Hyderabad.

3. Duration of Study:

Three years from January 2013 to December 2015.

4. Sample Size:

One hundred diagnosed cases of acute lymphocytic leukemia (N=100)

4.1 Sample size calculation:

The sample size for the study was calculated by the formula for sampling

for proportions. Following formula was used:

n = (z1-a/2)2 x p (1- p) d2.

n= Number of sample size.

(Z1 –a/2)= is the probability level of confidence level is taken for 95% (1.96).

p= is the probability of an event that is occurring.

1-p= is the probability of an event that is not occurring i.e. (1-p = 1-0.17 = 0.83).

50

d is the margin of sampling error (taken 5%)

n = (1.962)2 x 0.10 (1- 0.10) 0.0035

n = 3.84 x 0.10 x0.9 0.0035

n = 99

Approximated sample

Sample size (N) = 100

4.2 Sampling technique:

Non probability- purposive sampling.

4.3 Sample Selection:

Patient were selected in a systemic manner for which inclusion and

exclusion criteria were delineated.

5. Inclusion criteria

Diagnosed cases of Acute lymphoblastic leukemia

Children of <15 years of age of both genders.

5.1 Exclusion criteria:

Children with mixed lymphoproliferative disorders

Children with concomitant systemic disease.

Children with an acute infectious disease

Adult cases of acute lymphocytic leukemia

51

6.DATA COLLECTION

Liaquat University of Medical and Health Sciences, Jamshoro and Isra

University Hospital, Hyderabad. Informed consent was taken from parents of

study population.

A detailed patient history regarding duration, drugs, and symptoms related

to the acute lymphoblastic leukemia. Complete bio-data, residential address,

family history, exposure to chemicals and history of previous feverish illness was

taken and recorded in proforma.

The weight and height measurements for the calculation of body mass

index (BMI=weight/height) were performed.

7. LABORATORY INVESTIGATIONS

The study was conducted at Isra University Hospital Hyderabad and Liaquat

University of Medical and Health sciences, Jamshoro.

Detailed patients history regarding duration symptoms and drugs related with

acute lymphoblastic leukemia were noted on proforma. Complete biodata family

history, exposure to chemicals and history of previous illness was taken.

LABORATORY INVESTIGATIONS

7.1 Complete blood counts (CBC)

CBC of all samples were determined for basic hematological

parameters; this include Hb estimation, red cell count, white cell count, platelet

count, packed cell volume(PCV), mean cell volume (MCV), mean cell hemoglobin

52

(MCH), mean cell hemoglobin concentration (MCHC) and red cell distribution

width (RDW) using automated cell analyzer (Sysmex XN 1000i Tokyo, Japan).

7.2 Preparation and staining of peripheral blood smear

Peripheral smears were made, air dried and stained with Leishman’s stain.

7.2.1 Preparation of staining solution

Briefly, 0.2 g of Lieshman’s powder was placed in a volumetric flask of 100

ml. A small amount of acetone free methanol was added to dissolve the powder;

final volume was then adjusted to 100 ml by adding more methanol. The staining

solution was filtered before use.

7.2.2 Preparation of buffered water

Disodium hydrogen phosphate : 3.76 g

Potassium dihydrogen phosphate : 2.1 g

Distilled water : 1000 ml

Disodium hydrogen phosphate and potassium dihydrogen phosphate were

placed in a volumetric flask. Small amount of distilled water was added to

dissolve the salts, final volume was then adjusted to 1000 ml by adding more

distilled water.

53

7.2.3 Staining of peripheral blood smear

Leishman’s stain was poured on dried blood smears and was

allowed to stain for 2 minutes. Next, buffered water was added to the slides

having stain. The diluted stain was allowed to stay for 5-10 minutes. Slides were

then washed in running tap water, air dried and mounted.

7.2.4 Morphology of peripheral smear

Morphology of the stained blood smears were observed under the

microscope.

7.3 Bone marrow procedure

1. After taking informed consent from parents of patients, the procedure was

performed from lower end of tibia bone.

2. A small incision was made over the intended biopsy site.

4. A bone marrow aspiration needle was inserted at incision site and fixed in the

bone. Bone marrow sample was drawn using 50ml disposable syringe.

5. Using a specialized hollow needle, a bone marrow core biopsy was obtained in

cases where needed. This sample was sent to the pathology lab for examination.

6. Once the core bone marrow sample was obtained, pressure was applied for 5

minutes to the biopsy site with gauze to stop bleeding.

7. A sterile dressing or bandage was applied to the biopsy incision site.

8. Biopsy site was kept dry for 48 hours. Patients were discharged after the

procedure.

54

7.4 Karyotyping

Karyotyping is the arrangement of chromosomes according to their size,

banding pattern and centomeric position.

7.4.1 Reagents used:

• RPMI 1640 Basal Medium

• Fetal Bovine Serum

• Pencillin/Streptomycin

• Phytoheamaglutinin

• Colcemid

• Pottasiun Chloride 0.075 M Solution

• Fixative: 3 parts Methanol & 01 part Galcial Acetic Acid ( 3:1 Ratio )

Coronoy’s Fixative Freshly Prepared

• Ethanol 70%

7.4.2. Instruments & consumables

• Laminar Flow Hood ( Safety Cabinet Class II )

• Culture flasks T-25

• Co2 incubator

• Co2cylinder

• Centifuge tubes

• Centrifuge machine

• Water bath

• Micro pipettes &adjusters 5-20l, 10-100ul & 100-1000ul

• Syringes 01 c.c, 05 c.c & 10 c.c

• Serological pipettes 02 ml, 05 ml & 10 ml.

• Rack

• Bottle top dispensers

• Refrigerator 2-8oc

• Freezer -20oc

55 • Pippette aid

• Graduated cylinders 100ml,500ml & 1000 ml

• Suction machine

• Plastic Pasteur pipettes 03ml

• Coplinjars

• Frosted slide box

• Coverslips 24x50mm

• Oil immersion

• DPX mounting medium

• Serological glass Pasteur pipettes

• Permanent marker

7.4.3. Sample collection & processing:

Bone Marrow samples were aspirated and then delivered into bottle containing

appropriate amount of ethylene diaminetetra - acetic acid (EDTA) for

cytogenetics.

7.4.4. Method of culture:

The bone marrow was added to the culture tubes having 5ml cell culture medium.

Correct amount of cell suspension was added to each tube. All tubes were

labeled with the case numbers and incubated in a 370C incubator with 5% CO2

for 48 hours. Next, 50ul of 5ug/ml colcemid was added and mixed well in each

tube and reincubated at 370C.

Harvesting: The tubes were centrifuged for 10 minutes at 900 rpm. The

supernatant was discarded with a Pasteur pipette and then pellet was

resuspended . Then, 8 ml of pre-warmed 0.075M KCl was added to tubes which

were left at room temperature for 15 minutes. The tubes were then centrifuged for

10 minutes at 900 rpm. The supernatant was removed and suspension was

vortexed to keep the cell pellet moving. Freshly prepared cold fixative (3 volume

56

ethanol:1 volume acetic acid) was added drop by drop to avoid cell clumping. The

fixative to make up a final volume of 10 ml.

Preparation of slides: The pellet was gently diluted and resuspended with

additional fixative to give a slightly cloudy suspension.

The cell suspension was assessed using a microscope. If the preparation was

too dense, remake slide using a diluted cell suspension. If suspension was too

dilute, it was spinned down again and resuspended in less fixative. If the spreads

were clumped, these were washed with fixative one more time and the slides

were remade.

Two to three drops of cell suspension transferred on to a clean wet slide, spread

out and then air dried. About 2-4 slides were made per case.

Excess cell suspension was stored in fixatives at- 200C for remaking of slides if

needed. For that purposed the suspension was spinned down and fixative was

changed once before making slides.

Banding and Staining: Trypsin solution 0.125% with pH7.3 phosphate buffered

saline (PBS) was freshly prepared.

For staining, Leishman’s stain diluted with `1:7 phosphate buffer pH 6.8 was

prepared.

Coplin jars each containing the following solutions were prepared :

Phosphate Buffer Saline 1

Trypsin solution

PBSII

PBS III

Slides were dipped in PBS 1 for 1 minute. Next slides were dipped in trypsin for

5-10 seconds. Slides were then rinsed in the two Coplin jars containing PBS II

and PBS III. The slides were then stained immediately with fresh Leishman’s

57

stain for 4 minutes. Then slidess were rinsed in running water and air dried.

Cover slips were mounted on slides. The slides were examined under

microscope for chromosome bands. When slides were found under banded, time

of trypsin treatment was increased. If it is overbanded, the duration of trypsin

treatment or the PBS timing were increased, whichever was appropriate. Slides

were screened for good quality metaphase spreads.

Screening of metaphase spreads

Each banded slide was screened for well banded metaphase spreads using a

bright field microscope. The position of the metaphase spreads recorded. During

screening the position for at least 20 good metaphase spreads were recorded.

Capturing of metaphase spreads:

At least 10 metaphase spreads were captured using a satellite capture station.

Image was transferred the to an image analyzer. Karyotyping of chromosome

spreads was done. The patients’ particulars were entered in the image analyzer.

Karyotyping was performed using an image analyzer. Human cytogenetic

nomenclature used in reporting was according to ISCN (1995).

CytoVision Image Analysis and Capture System Version 7.4

CytoVision is a network based Imaging system which used for imaging

during karyotyping. It comprised of application software and hardware modules

for human metaphase finding, image capture, computer aided chromosome

presentation, data management and information output.

Sample treatment and staining

Chromosome visualization requires an appropriate banding and staining

technique being applied to the sample once on the microscope slide. In the

58

present study G-Banding with Giemsa stain and imaging using Brightfield

microscopy were used.

7.5 Fluorescence in Situ Hybridization (FISH)

FISH (Fluorescence in situ hybridization) is a molecular pathology

technique that allows detection of a specific DNA sequence in situ. It relies on the

principle that a fluorescently labeled single stranded DNA probe will anneal

(hybridize) to a complementary single stranded target DNA. The target DNA from

either metaphase chromosomes or non-dividing interphase nuclei can be

visualized using a fluorescent microscope.20

The FISH studies were performed using a commercial dual color probe

cocktail with spectrum green conjugated probe specific. The hybridization and

washings were performed following the manufacturer’s instructions. Briefly, the

slides were denatured in 70% form amide/2xSCC at 730C for 5 minutes and the

probe also in the same way. The slides were washed in 50% formamide (3 times,

10 minutes each) /2xSCC (10 minutes) and /2xSCC/0.1% NP-40 (5 minutes) at

460C. The slides were counterstained and mounted with VectaShiled DAPI. The

smears preparations were pre-treated before FISH.

1st step, the covers slips were removed by Xylene and the slides were

dehydrated in increasing alcohol series, followed by fixation in

methanol/acetic acid (3:1) at 40C overnight. Then, the slides were treated in

1M natrium thiocyanate for 10 minutes at 650C and washed in 2xSCC for 5

minutes at room temperature. Next the slides were treated in 0.01N HCl for 10

minutes and in 0.05 N HCl with pepsin (0.05 mg/ml) for 8 minutes at 37 0C

59

Final step, the slides were washed under cold running tap water for 5

minutes and dehydrated in increasing alcohol series. The analysis was

performed from three color images acquired using a fluorescence microscope

and the ISIS digital image analysis system (Meta System) developed for

CGH. Filters specific to FITC, Texas-Red and DAPI were used (Chroma

Technology Corp., Brattleboro, VT, USA).

7.6 Epstein Barr virus detection by PCR

Epstein - Barr virus (EBV) specific DNA.

Quantitative EBV PCR Kit 1.0 consisted of:

Two Master reagents (Master A and Master B)

Template Internal Control (IC)

Four Quantification Standards (QS1 – QS4)

PCR grade water

Master A and Master B reagents contained all components (buffer,

enzymes, primers and probes) to allow PCR mediated amplification and

target detection of EBV specific DNA and Internal Control in one reaction

setup.

The Quantification Standards contained standardized concentrations of

EBV specific DNA. These Quantification Standards were calibrated

against the 1st WHO International Standard for Epstein - Barr virus for

Nucleic Acid Amplification Techniques (NIBSC code: 09/260). The

Quantification Standards were used individually as positive controls, or

together to generate a standard curve, which was used to determine the

concentration of EBV in the sample.

60 Master Mix Setup

All reagents and samples were thawed completely, mixed (by pipetting or

gentle vortexing) and centrifuged briefly before use.

The Real Star® EBV PCR Kit 1.0 contained a heterologous Internal

Control (IC), which was either used as a PCR inhibition control or as a

control of the sample preparation procedure (nucleic acid extraction) and

as a PCR inhibition control.

When the IC was used as a PCR inhibition control, but not as a control for

the sample preparation procedure, the Master Mix was set up according to

the following pipetting scheme:

Number of

Reactions (rxns)

1 12

Master A 5 μl 60 μl

Master B 15 μl 180 μl

Internal Control 1 μl 12 μl

Volume Master

Mix

21 μl 252 μl

When the IC was used as a control for the sample preparation procedure

and as a PCR inhibition control, the IC was added during the nucleic acid

extraction procedure.

The IC was added to the specimen/lysis buffer mixture. The volume of the

IC which was added depended on the elution volume. It represented 10%

of the elution volume. For instance, if the nucleic acid was going to be

61

eluted in 60 μl of elution buffer or water, 6 μl of IC per sample, it was

2added into the specimen/lysis buffer mixture.

Reaction Setup

Pipette 20 μl of the Master Mix into each required well of an appropriate

optical 96-well reaction plate or an appropriate optical reaction tube.

Add 10 μl of the sample (eluate from the nucleic acid extraction) or 10 μl of

the controls (Quantification Standard, Positive or Negative Control).

At least one Positive and one Negative Control were used per run.

For quantification purposes all Quantification Standards (QS1 to QS4)

were used.

Thoroughly mixed the samples and controls with the Master Mix by up

and down pipetting.

Closed the 96-well reaction plate with an appropriate optical adhesive film

and the reaction tubes with appropriate lids.

Centrifuged the 96-well reaction plate in a centrifuge with a micro titer

plate rotor for 30 seconds at approximately 1000 x g (~ 3000 rpm).

62

8. DATA ANALYSIS

The data was analyzed on SPSS version 21.0 (IBM, Corporation, USA)

and Microsoft excel. The continuous variables is presented as mean ± SD and

has been analyzed using student’s t-test. Categorical variables were analyzed by

Chi-square test and results are presented as frequencies and percentages. Data

is presented in tables, graphs and charts. P-value of significance has been taken

at ≤0.05.

SPSS is a software for statistical analysis of research data.

63

CHAPTER IV

RESULTS

The present study observed the Mean ± SD of age as 7.5±3.2 years. Out

of 100 cases, most frequent age groups belonged to 5-10 years noted in 59

(59%) of total cases where as rest of 35 (35%) of cases belonged to < 5years of

age and only 6 (6%) cases to ≥10 years (p=0.0001).Table IV-1 and Graph IV-1

shows the age distribution of study population.

The gender distribution of study population (n= 100 cases) showed that of

of males were 57 (57%) and 43 (43%) were females (p=0.001). Male to female

ratio was 1.32:1. Gender distribution is shown in table IV-2 and graph IV-2.

Hemoglobin and hematocrit values are shown in table IV-3 and graph IV-3

respectively. Hemoglobin values of <8, 8-10 and >10 g/dl were noted in 9 (9%),

49 (49%) and 42 (42%) of cases respectively (p=0.0001). Anemia was noted in

90% and hematocrit (<20%) in 91% of cases.

Red blood cell (RBC) counts are shown in table IV-4 and graph IV-4. RBC

counts of > 5 million/µL were noted in 33% of cases. RBC counts of 2-5 and <2

million/µL were noted in 61% and 6% of cases respectively (p=0.001).

White blood cell counts (WBC) are shown in table IV-5 and graph IV-5.

WBC counts of < 10, 000, 10, 000-50,000, > 50, 000 < 100,000 and >100,000/µL

were observed in 9%, 17%, 15% and 59% of cases of respectively (p=0.0001).

Chromosomal abnormalities

Chromosomal abnormalities found in present study are summarized in

tables and graphs IV-6 to IV-10. Numerical and structural chromosomal

64

abnormalities were noted in 69% and 60% of cases respectively (p=0.001) (Table

IV-6 and Graph IV-6). Chromosomal ploidy included Diploidy, Aneuploidy and

unknown in 29%, 69% and 2% of cases respectively (p=0.0001) (table IV-7 and

graph IV-7).

Hyperploidy, hypoploidy, pseudoploidy and unknown were noted in 51%,

6%, 12% and 2% of cases respectively (0.001) (table IV-8 and graph IV-8).

Philadelphia chromosome

Ph + chromosome (t (9; 22) (q34; q11) was noted in 6% of cases of

childhood acute childhood lymphoblastic leukemia as shown in table and graph

IV-10.

Epstein Barr Virus

EBV was detected in 19% of total study population in present study as

shown in table and graph IV-11.

Bone marrow and Peripheral Blood Smear

Findings of microscopic examination of peripheral blood smears are

shown in figure IV-1 to IV-6. Immature blast cells of acute lymphoblast leukemia

are shown with hyperchromasia in L1, L2, and L3. Blast cells of variable size,

prominent nuclei, nucleoli and reticular chromatin are shown in figure IV-3. Figure

IV-5 shown immature, small blast cells with high N/C ratio, scanty cytoplasm and

open chromatin material.

65

Table IV-1. Age distribution of study population (n=100)

Age (years) No. of Pt. % p-value

< 5years 35 35

0.0001

5-10 years 59 59

>10 years 6 6

Mean ± SD 7.5±3.4

Graph. IV-1. Age distribution of study population

35

59

6

0

10

20

30

40

50

60

70

< 5years 5-10 years >10 years

Age distribution (n=100)

66

Table IV-2. Gender distribution of study population (n=100)

Gender No. of Pt. %

Male 57 57

Female 43 43

Graph. IV-2. Gender distribution of study population

57

43

0

10

20

30

40

50

60

Male Female

Gender distribution (n=100)

No. of Pt.

67

Table IV-3. Hemoglobin findings of study population (n=100)

Hemoglobin No. of Pt. % p-value

< 8 g/dl 9 9

0.0001 8-10 g/dl 49 49

>10 g/dl 42 42

Graph. IV-3. Hemoglobin distribution of study population

9

49

42

0

10

20

30

40

50

60

< 8 g/dl 8-10 g/dl >10 g/dl

Hemoglobin (n=100)

No. of Pt.

68

Table IV-4. Red blood cell counts of study population

RBC No. of Pt. % p-value

<2 million/µL 6 6

0.001

2-5 million/µL 61 61

>5 million/µL 33 33

Graph. IV-4. Red blood cell counts of study population

6

61

33

0

10

20

30

40

50

60

70

<2 million/µL 2-5 million/µL >5 million/µL

Red blood cells (n=100)

No. of Pt.

69

Table IV-5. White blood cell counts of study population

WBC No. of Pt. % p-value

< 10,000/µL 9 9

0.0001

10,000- 50,000/µL 17 17

> 50,000/µL to < 100,000 15 15

> 100,000/µL 59 59

Graph. IV-5. White blood cell counts of study population

9

17 15

59

0

10

20

30

40

50

60

70

< 10,000/µL 10,000-50,000/µL

> 50,000- <100,000/µL

> 100,000/µL

White blood cells (n=100)

No. of Pt.

70

Table IV-6. Chromosomal abnormalities in study population

Yes No Unknown p-value

Numerical chromosomal abnormality 69 29 2

0.001 Structural chromosomal abnormality 60 27 13

Graph. IV-6. Chromosomal abnormalities in study population

69

29

2

60

27

13

0

10

20

30

40

50

60

70

80

Yes No Unknown

No

. of

Pat

ien

ts

Chromosomal abnormalities (n=100)

Numerical chromosomalabnormality

Structural chromosomalabnormality

71

Table IV-7. Chromosomal ploidy of study population

No. of Pt. % p-value

Diploidy 29 29

0.0001 Aneuploidy 69 69

Unknown 2 2

Graph. IV-7. Chromosomal ploidy of study population

29

69

2

0

10

20

30

40

50

60

70

80

Diploidy Aneuploidy Unknown

No. of Pt.

72

Table IV-8. Chromosomal Numerical abnormalities

No. of Pt. % p-value

Hyperploidy 51 51

0.001

Hypoploidy 6 6

Pseudoploidy 12 12

Unknown 2 2

Graph. IV-8. Chromosomal numerical abnormalities of study population

51

6

12

2

0

10

20

30

40

50

60

Hyperploidy Hypoploidy Pseudoploidy Unknown

No

. of

Pat

ien

ts

Chromosomal numerical abnormalities (n=100)

73

Table IV-9. Chromosomal structural abnormalities in study population

(n=100)

Chromosomal abnormality

No. of Pt.

%

t(12;21)(p13;q22) 18 18

t(9;22)(q34;q11) 6 6

t(8;14)(q24;q32) 5 5

t (1;19) (p13; q23) 2 2

t(5;14)(q31;q32) 3 3

t(17;19)(q22;q13) 3 3

t(8;22)(q24;q11) 6 6

t (7;11) (q35; q13) 5 5

t (1;7) (p32; q35) 7 7

t (7;19) (q35; p13) 5 5

Unknown 13 13

74

Graph. IV-09. Chromosomal structural abnormalities of study population

18

65

23 3

65

7

5

13

0

2

4

6

8

10

12

14

16

18

20

No

. of

Pat

ein

ts

Chromosoal structural abnormalities (n=100)

75

Table IV.10. Philadelphia chromosome in study population (n=100)

Philadelphia chromosome t(9;22)(q34;q11)

No. of Pt. %

6 6

Graph. IV-10. Philadelphia chromosome in study population

6

94

0

10

20

30

40

50

60

70

80

90

100

Positive Negative

No

. of

Pa

tie

nts

Philadelphia chromosome (n=100)

t(9;22)(q34;q11)

76

Table IV-11. Frequency of Epstein Barr virus in study population (n=100)

EBV No. of Pt. % p-value

No 81 81

0.0001 Yes 19 19

Graph. IV-11. Epstein Barr virus in study population

0

10

20

30

40

50

60

70

80

90

yes No

Epstein Barr virus

No. of Pt.

77

Photomicrograph IV-1. Bone Marrow X 40

78

Photomicrograph IV-2. Peripheral smear showing Homogenous population

of Lymphoblast. X 100

79



80



81


of Lymphoblast . X 400

82

Photomicrograph IV-6. Bone Marrow showing Lymphoblast X 1000

83


84


85


86


87


88

Photomicrograph IV-12. Bone Marrow showing Lymphoblast s X 1000

89

Photomicrograph IV-13. Bone Marrow showing Leukemic blast cells X 1000

90

Photomicrograph IV-14. Bone Marrow showing leukemic blast cells X 1000

91


92

Photomicrograph IV-16. Peripheral Blood Smear. shows leukemic blast of

ALL X1000

93

Photomicrograph IV-17. Peripheral Blood Smear. Shows leukemic blast of

ALL X1000

94


ALL X1000

95


ALLX400

96

Photomicrograph IV-20. Peripheral Blood Smear. shows leukemic blast of

ALL X1000

97

CHAPTER V

DISCUSSION

The present research is conducted to analyze chromosomal abnormalities

and Epstein Barr Virus (EBV) in Childhood ALL at Isra University Hyderabad,

Sindh, Pakistan. Therefore it will help for future studies and for better

management of childhood ALL as the cytogentic characteristics are vital in

patient management.

Acute lymphoblastic leukemia (ALL) is a malignant disease characterized

by accumulation of lymphoblasts. ALL is common in children of less than 15

years of age. It accounts for 75-80% of childhood leukemias and various

subtypes of the disease can be defined based on cell morphology,

immunophenotype, and karyotype and gene expression characteristics. Over the

past several years, diagnosis and treatment of ALL in children has improved

significantly and approximately 80% of children with ALL now survive into

adulthood (149-152).

The national data on ALL in children is lacking in Pakistan.(2) Of a few

studies available, the Yasmeen et al (2) reported a frequency of 32% of ALL in

children in a retrospective study at Oncology unit of National Institute of Child

Health and Children Cancer Hospital, Karachi.(2)

Mean± SD of age was noted as 7.5±3.4 years. Of 100 cases, most

frequent age groups belonged to 2-10 years noted in 94% of total. 35 (35%)

belonged to < 5years of age and only 6 (6%) to ≥10 years (p=0.0001).Table IV-1

and Graph IV-1 shows the age distribution of study population. The findings are

consistent with Shaikh et al (149) and Yasmeen et al (2). Shaikh et al (149) had

reported mean± SD of age 7±4.4 which is comparable to present and previous

98

studies. (2, 153-155)

Of 100 cases, 57 (57%) were male and 43 (43%) were female (p=0.001).

Male to female ratio was 1.32:1. Gender distribution is shown in table IV-2 and

graph IV-2. The findings are consistent to previous studies from Pakistan (2)

(149). Shaikh et al (149) has reported male to female children ratio of 1.8:1 while

Yasmeen et al a ratio of 1.7:1. The boys predominance is a universal fact, the

findings of present study are comparable to male gender predisposition as

mentioned above. (153-155)

Hemoglobin and hematocrit values are shown in table IV-3 and graph IV-3

respectively. Hemoglobin <5, 5-10 and >10 g/dl were noted in 9 (9%), 49 (49%)

and 42 (42%) of cases respectively (p=0.0001). Anemia was noted in 90% and

hematocrit (<20%) in 91% of cases.

Red blood cell counts (RBC) of > 5 million/µL were noted in 33% of cases.

RBC counts 2-5 and <2 million/µL were noted in 61% and 6% of cases

respectively (p=0.001).

White blood cell counts (WBC) are shown in table IV-5 and graph IV-5.

WBC counts < 10, 000, 10, 000-50,000, > 50, 000 and >100,000/µL were

observed in 9%, 17%, 15% and 59% of cases of respectively (p=0.0001).

CHROMOSOMAL ABNORMALITIES

Cytogenetic analysis in hematological malignancies like many other

diseases plays a significant role in understanding the pathophysiology as well as

clinical behavior of the condition (156, 157)In fact, for ALL, like other malignant

conditions, karyotype is one of the prognostic indicators(158, 159). Other

important prognostic indicators in ALL include age (good prognosis in 1-9 years)

99

(160, 161), gender (better prognosis in girls) (162), white blood cell count

(163)(good prognosis if <50x 109/L at presentation), immune-phenotype and

minimal residual disease (MRD) detection (164) (high relapse risk with MRD of

1% or more at the end of remission induction therapy and those with MRD of

0.1% or more during continuation therapy). Numerous cytogenetic abnormalities

have been found associated with distinct immunologic phenotypes of ALL and

characteristic outcomes (150, 165-166).

Both structural and numerical chromosomal abnormalities are detected

recurrently in approximately 80 percent of ALL (167, 168).There are considerable

differences in types of cytogenetic abnormalities detected in different age groups.

For instance, t(9;22) is detected more commonly in adults (167, 168) as

compared to children. Whereas, t(4; 11), t (12; 21) and hyperdiploidies are more

common in children(167-169).

These cytogenetic abnormalities also differ in overall prognosis of the

disease including response to chemotherapy and subsequent chances of

relapse. For example, certain translocations, such as t (4;11) and t(9;22), are

associated with resistant disease and may require intensive chemotherapy

(170).In comparison, the t (12; 21), t (1; 19), and hyperdiploidy (47 to 57

chromosomes) are associated with encouraging outcomes (162, 167, 171).

The clinical and cytogenetic analyses have a crucial role in diagnosis, risk

stratification, treatment and prognosis of ALL (153, 173).

Cytogenetic data of Pakistani children with ALL at national level is

unavailable. Therefore, this study aimed in determining the cytogenetic profile of

Pakistani children with ALL in order to provide an insight into the prognosis and

furthermore proper management of the patients.

100

This study underscored several important facts regarding ALL in Pakistani

children. Literature search revealed that, it is the largest study detailing

cytogenetic profile of Pakistani children with ALL in association with search of

EBV genome in ALL.

A number of acquired chromosomal abnormalities arising from

translocations, deletions, duplications and inversions have been identified which

are often associated with deregulated gene expression. Currently an abnormal

karyotype has been detected in more than 80% of childhood (3) and 79% of

adults suffering from ALL (1, 4). 45% of chromosomal abnormalities of present

study as shown in table IV-6 are comparable to above mentioned studies.

Chromosomal abnormalities noted in present study are summarized in

table and graphs IV-6 to IV-10. Numerical and structural chromosomal

abnormalities were noted in 69% and 60% of cases respectively (p=0.001) (Table

IV-6 and Graph IV-6).

Chromosomal study showed Diploidy, Aneuploidy and unknown in 29%,

69% and 2% of cases respectively (p=0.0001) (table IV-7 and graph IV-7).

Hyperploidy, hypoploidy, pseudoploidy and unknown were noted in 51%,

6%, 12% and 2% of cases respectively (0.001) (table IV-8 and graph IV-8).

Overall, both numerical and structural cytogenetic abnormalities were

detected in 65% of patients. Findings of hyperploidy of present study are highly

comparable to studies (149, 165).

Cytogenetic analysis demonstrated in 69.5% ALL patients had a

hyperdiploid condition. Structural abnormalities observed included: t (9; 22) (q34;

q11) and t(1; 19)(q23; p13.3) (153). In present study, hyperploidy was observed

101

in 51% of cases, findings and these findings are comparable to the results of

Raimondi et al (173) which is a Western population based study.

A similar proportion of hyperdiploid karyotype has been reported in Iranian

children by Farkhondeh et al (174). This and the present study therefore by

obvious reasons show the important finding of a greater prevalence of

hyperdiploidy in Asian region of the world. However, hyperdiploidy is generally

associated with favorable prognosis. It is known that trisomies 4, 10, and 17 are

usually associated with a potentially favorable prognosis (153, 175, 176).

A study from China (177) reported frequency of hyperdiploidy of 10.61%

vs. 20-38%) pediatric ALL and 2.36% vs. 6.77-12% in adult ALL. The findings of

hyperploidy are a controversial finding and do not match with present and

previous studies (153, 173) which are also reported from Asia.

Another study from China by Chai et al (178) has reported cytogenetic

analysis in 124 cases of pediatric ALL and found 60% had clonal abnormalities,

32% had hyperploidy, 12.5% had hypoploidy and 16% pseudoploidy.

Chromosomal translocations found in 13 patients were: 4; 11, 9; 22 and 1; 19

(178). The findings of above study are in contrast to present study in terms of

chromosomal abnormalities and hyperploidy (51%) as shown in table IV-8 and

hypoploidy was found in 6% of total cases which is less compared to above

study.

A study from Denmark by Forestier et al (179) examined 1425 pediatric

ALL cases aged 2-7 years, reported high hyperploidy (51-61 chromosomes) and

a translocation t(12; 21) (p13; q22). The study concluded a high frequency of

102

cytogenetic abnormalities and hyperploidy. The findings of present study are in

full agreement with above study.

In a study in Taiwanese children with ALL, in 78 patients of under 18 years

of age, 20.5% had normal diploidy; 35.9% had pseudodiploidy; 7.7% with

hyperdiploidy (47-50 chromosomes); 24.4% with hyperdiploidy (>50

chromosomes) and 99.4% had hypodiploidy. Most frequent structural abnormality

detected was t (9; 22) (180).


number of chromosomes, is a significant feature of ALL. In present study diploidy

and aneuploidy were found in 29% and 69 % of cases respectively (p=0.0001)

(table IV-7 and graph IV-7).

A high hyperdiploidy karyotype, with 51-65 chromosomes, is found in

approximately 30% of childhood cases and 5% of adult patients (5, 6).

Hyperploidy (table IV-8) shows a frequency of 51% in present study is a

comparable finding to above mentioned studies as above.

A significant chromosomal abnormality in childhood ALL is hypodiploidy

(Ho), where chromosomes are ≤ 45. It is rare, with a reported incidence of

approximately 6%.8 In the majority of reported cases, patients have 45

chromosomes. (8, 9) Overall, hypodiploidy has been linked to a poor prognosis

(8-10). Finding of 6% of hypoploidy is comparable to previous studies (8, 9).

Padhi et al (155) reported a high proportion of patients (51.2%) had hypodiploidy

karyotype (modal number of chromosomes<46). Above findings are contrary to

present study.

103

The chromosomal gains in the form of trisomies are restricted to certain

chromosomes. Chromosomes X, 4, 6, 10, 14, 17, 18 and 21 (frequently the gain

of chromosome 21 is tetrasomic) are frequently found abnormalities (7). t (12; 21)

(p13; q22) which is the commonest translocation in children with ALL and carries

good prognosis was found in 18% of cases present study which is contrary to a

previous study by Shaikh et al (149).

Ph + chromosome (t (9; 22) (q34; q11) was noted in 6% of cases of

childhood ALL in present study as shown in table and graph IV-10.The findings is

comparable to reported medical literature on the topic. A study by Schultz et al

(181) has reported a prevalence of 3-5% in Pediatric ALL, while a recent study

(149) from Pakistan has reported a prevalence of 7.08% which is much higher.

The controversies might have been introduced by bias at some step in patient

selection, data collection, analysis and presentation. Philadelphia positive ALL

was seen in one of the two adults with ALL in a previous study by Arico et al

(182). It is known to be found in 15 to 30 % of adults with ALL.

EPSTEIN BARR VIRUS

Epstein Barr Virus (EBV) is known to infect about 90% of the adult

population worldwide and its infection is generally restricted to humans (11, 12).

The virus is shed into the saliva of persistently infected individuals who spread

the virus to uninfected individuals.(13)

EBV is linked to a variety of neoplasms (17, 18)including lymphoid tumors

like Burkitt`s Lymphoma, Hodgkin’s disease (HD), lymphoproliferations in solid

organ transplant, natural killer (NK) T-cell lymphoma or bone marrow recipients

(posttransplantation lymphoproliferative disease, PTLD), AIDS-associated

lymphomas, Nasopharyngeal carcinoma, gastric carcinoma, salivary gland

104

tumors, thymic carcinoma, mesothelial tumors and leiomyosarcoma (18).

Currently national data is seriously lacking on possible role of EBV in ALL in

children.

In recent years some convincing leads have been obtained on a causal

relationship between EBV and a variety of lympho reticular malignancies.

Patients with acute infectious mononucleosis document an increased

susceptibility to Hodgkin's disease (184). Patients with AIDS are known to have

an increased incidence of EBV infection as well as lymphomas (185-187).

In present study, the EBV was detected in 19% of total study population of

Childhood ALL in present study as shown in table and graph IV-10.

In a previous study from India by Sehgal et al (183), the PCR for EBV was

positive in 32% of ALL cases (183). Western blot test using anti ZEBRA

antibodies were positive in 20% cases of ALL. Considering PCR as the gold

standard, 32% of the children with ALL had evidence of active EBV replication.

In India, although nasopharyngeal carcinoma and Burkitt type of

lymphoma are uncommon, EBV infection is common as indicated by the

ubiquitous presence of IgG antibodies to VCA in control subjects (183). One

striking observation in all these malignancies is the long latent period between

the primary infection and development of malignancy. Two exceptions, however,

are the immunoblastic lymphomas occurring after acute EBV infection in

immunocompromised hosts like transplant recipients and those with XLP

syndrome (183).

PCR followed by hybridization was considered the gold standard for

compiling data in this study. The study showed evidence of active EBV replication

105

in some patients with ALL and Hodgkin's as EBV PCR specific for replicating EB

virus was positive in 38% of the children. In this study six /11 children even

without therapy or with a short course of therapy showed evidence of EBV

infection, There was no correlation between the duration of therapy and EBV

infection in ALL. The number is, however, small to conclusively prove that

therapy was not directly or indirectly related to positivity in these children. Ideally,

age matched patients with solid tumors should also have been included but it was

not done in this preliminary study. This study was initiated because a wide variety

of hematological malignancies have been linked to EBV. Such molecular studies

pertaining to EBV in healthy children are wanting in India (183).

Venkitaraman et al (188) reported age specific prevalence of IgG

antibodies to VCA in 80% by the age of 5 years. No data on molecular studies on

EBV in cancers is available in India barring stray case reports (188).

Roy et al (189) also reported a significant number of adult controls and

cancer patients positive for EBV by ELISA assays. However, the point of active

replication was not addressed and no molecular studies were done. There are

controversial reports in the world literature regarding EBV coinfection in childhood

ALL (189).

Schlehoefer et al (190) reported increased incidence of anti VCA

antibodies and anti EBNA antibodies in children with ALL in Germany but it is

known that serological tests may not be very specific and antibodies do not

indicate active viral replication.

Wolf et al(191) documented EBV mRNA IN 4/6 cases of hairy cell

leukemia, another indication of oncogenic potential of EBV. In the single largest

106

epidemiological study conducted on 550000 mothers and 7 million years follow-

up in Finland and Iceland.

Lehtinen et al (192) concluded that activation of maternal Herpes virus

infection increased the risk of ALL in the offspring. Only EBV immunoglobulin M

positivity in EBV-immunoglobulin-G-positive mothers was associated with a

highly significant increased risk of ALL in the offspring (adjusted odds ratio=2.9,

95% confidence interval: 1.5, 5.8). These observations were supported by EBV

DNA studies. No other study has, however, substantiated these observations.

Loufty et al. from Egypt documented that HSV 1 and HSV 2 but not EBV was

linked to ALL(192).

In a more recent study from Sweden, Altieri et al (194) reported that ALL

was positively correlated with the number of siblings; the younger sibs were

strongly protected from the risk of malignancies suggesting an infectious etiology.

Sakajiri reported increased EBV infection in a patient with T ALL employing

Southern blotting and in situ hybridization (ISH) (195).

Table V-I summarizes the relevant information available from the world

literature. This study, using ELISA techniques, indicates that EBV infection is

present in 19% of childhood ALL.

Miyagi (196) from Japan documented EBV in 11/12 cases of Hodgkin's

disease using in situ hybridization (ISH). It appears that EBV may be an

important coinfection in some patients of ALL. Our findings corroborate

observations of Lehtinen et al (192).

EBV is an important opportunistic infection and the role of chemotherapy

in these patients could not be ignored. However, since some untreated patients

http://www.ijpmonline.org/viewimage.asp?img=IndianJPatholMicrobiol_2010_53_1_63_59186_b2.jpg

107

or those in the induction phase also revealed PCR positivity, it points against

EBV being an opportunistic infection due to therapy in this group of children. It is

quite possible that EBV by itself may not cause ALL but may be an important

cofactor at least in some patients. Similarly, 12 patients with AML, seven of them

on chemotherapy, were also negative. In the available literature, there is no

convincing evidence of EBV infection causing AML in children. It would be

relevant to conduct a large multicenter study on drug naïve children with ALL,

which may give key additional information on the role of EBV in childhood

leukemia in India. With tremendous advances in vaccine research there could be

a drastic change in the management of these patients in the foreseeable future.

The disease is a formidable economic burden on society. If an infectious agent is

involved in ALL, there is hope that in future these would be preventable just as

the incidence of hepatocellular carcinoma due to HbsAg can be drastically

reduced by HBV immunization (183).

Table V-I. Studies conducted on EBV in childhood ALL

Country Author EBV - ALL

India Sehgal et al 2010 (183) EBV +ve in ALL

Finland Lehtinen et al 2003 (192) EBV +ve in ALL

Egypt Loufty et al 2006 (193) EBV +ve in ALL

Japan Sakajiri et al 2002 (195) EBV +ve in ALL

Okinawa Miyagi et al 2002 (196) EBV +ve in ALL

Germany Schlehofer et al 1996 (198) EBV +ve in ALL

Qi X (199) studied Effects of Epstein-Barr virus and cytomegalovirus

infection on childhood acute lymphoblastsic leukemia gene methylation.

108

Compared with those in non-infected group and EBV- or HCMV-infected group.

In children with acute lymphoblastic leukemia, EBV and HCMV co-infection cause

changes in the methylation levels of PTEN and hTERT. These results may be

associated with epigenetic changes caused by viral infections, and further studies

are needed to further verify this hypothesis (199).

Central nervous system (CNS) involvement of Epstein-Barr virus (EBV)-

associated lymphoproliferative disease is a rare and serious complication in

children with leukemia. Although rituximab therapy seems to be promising in

these cases,persistent hypogammaglobulinemia may appear after treatment due

to complete depletion of normal B lymphocytes inthe peripheral blood. Here we

report isolated CNS involvement of EBV-associated lymphoproliferative disorder

in a 4-year-old boy with acute leukemia. The patient was treated with rituximab

and interferon alpha; however, persistenthypogammaglobulinemia developed as

a complication. Given the rarity of the complication in children receiving these

agents, our experience with such a case may be helpful to others (200).

Besides being the largest cytogenetic study in Pakistani children with ALL,

strength of our study is the combined use of polymerase chain reaction (PCR) –

for EBV detection along with cytogenetic method for karyotype determination. We

could not compare our findings with immunophenotype (B or T lineage) of ALL

however, specific cytogenetic abnormality when present, independently provides

strong predictions as far as the prognosis of the disease is concerned. The

relevance of gene set analysis also remains unclear (201)

As the national data on childhood on ALL is lacking and studies on role of

EBV in childhood ALL is absolutely lacking, hence present study will help in

109

making local data and may help for proper management of childhood acute

lymphoblastic leukemia.

110

CHAPTER VI

CONCLUSION

Most of ALL cases were characterized by leukocytosis and anemia.

Chromosomal aberrations made up 64.5% of ALL cases among which

numerical abnormalities were found in 69 % and structural abnormalities in

60% of the karyotypes of ALL cases.

Prevalence Epstein Barr Virus was 19% amongst ALL cases.

This study showed good prognostic cytogenetic abnormalities like

hyperdiploidy and t (12; 21) (p13; 22) in Pakistani children with ALL.

Prevalence of poor prognostic cytogenetic aberrations like t (9; 22) (q34;

q11.2), hypoploidy was comparable to available international literature.

The present study will help for better management of childhood ALL as the

cytogenetic abnormalities are of proven prognostic value in patient

management.

111

CHAPTER VII

RECOMMENDATION

Further studies are recommended to be conducted to evaluate frequency

of cytogenetic abnormalities and role of EBV in childhood ALL in our

population

Prognostic value of each type of chromosomal abnormality should be

determined by follow-up of such cases.

112

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Proforma

CHROMOSOMAL ABNORMALITIES AND EPSTEIN BARR VIRUS

IN ACUTE LYMPHOBLASTIC LEUKEMIA IN CHILDREN

Department of Pathology/Hematology, Isra University Hospital

MR# Date:

Name: Age :

Sex: Religion:

Ethnicity:

Socioeconomic status:

Address:

Phone No:

General health status:

General Physical Examination:

Pulse:

B.P:

Anemia:

Clubbing:

Koilonychias:

Dehydration:

Pedal edema

129

Laboratory Investigations:

1. Complete Blood Counts

Hemoglobin

Hematocrit

ESR

RBC:

WBC:

Platelet count

2. Karyotyping (Chromosomal studies)

3. Epstein Barr Virus


chromosomal abnormalities and epstein barr virus in …

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