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Molecular Biology Methods

Flowcytometric and FISH Markers in


Childhood Acute Lymphoblastic Leukemia
Dr ihan !L Hussieny awish" MSc" #hD $
Acknowledgements
First of all, I cannot give a word to fulfill my deeps love and thanks
to (Allah) for lighting me the way not only throughout this piece of work
but also throughout my whole life.
This work is dedicated to:
My husband; Dr Hussein Al %mer and my Family
I am indebted to &ing Saud 'ni(ersity for support and encouragement to
finish this work.
lso, I wish to e!press my deep thanks to:
Prof. Dr./ Abdelfattah M. Attalah, "rofessor of #enetics $
Immunology, #eorge %ashington &niversity, &' (Former)$ *irector
of +iotechnology ,esearch -enter, .ew *amietta, /gypt, for
continuous advice
Prof. Dr./ Ahmed Abd Al Salam Settin, "rofessor of "ediatrics $
#enetics, Faculty of Medicine, Mansoura &niversity for his
continuous help
Finally, I am indebted to all the team of the honorable #enetics
&nit, Mansoura &niversity -hildren 0ospital, for their continuous
support and encouragement

Gihan El Hussieny Gawish
January 2!
1
Contents
Pa"e )itle
* Introduction
+ ,e(iew o- Literature
+ I. Acute lymphoblastic leukemia
+ 23*efinition
/ 13Incidence of 4eukemia
0 53Types of leukemia
0 63+iological -lassification of 44
1 73-ausing of 4eukemia
2 83The 'igns of 4eukemia
** 93'tages of -hildhood 44
** :3Treatment of -hildhood 44
*+ ;3Four "hases of Treatment
*/ II. Cell cycle and apoptosis
*/ 23-ell cycle
*2 2323-ell cycle and cancer
34 13poptosis and its markers
3* 1323The mechanism of apoptosis
3/ 1313poptosis3targeted therapies for hematologica malignancies
54 1353The apoptosis promoter (p75)
5/ 1363The inhibitor of apoptosis
5/ 2-4-1-Bcl2 proteins
56 2-4-2-C-myc oncogene
+3 III.Flow cytometry
+3 23Introduction
++ 13"rinciples of flow cytometric instrumentation
+0 2-1-Fluidic system
+2 2-2-Illumination system
/5 2-3-Optical and electronics system
/+ 2-4-Data storage and computer control system
/2 53*ata analysis
05
I7. Applications o- -low cytometry
0/ 23-ell cycle analysis
00 1-1-Staining procedure
5
Pa"e
01 1-2-Evaluation o D!" #istogram
1* 13Immunophenotyping pplications
13 2-1-Eryt#rocyte analysis
15 2-2-$I% monitoring
15 2-3-Immunop#enotyping o leu&emias
1/ 2-4'uantiication o stem cells
1/ 2-(-)latelet analysis
10 2-*-+esting or $,"-B2-
10 53Ma<or applications of apoptosis analysis
11 3-1- "poptosis lig#t scatter
16 3-2-"poptosis D!" analysis
64 3-3-"poptosis cell mem.rane analysis
63 3-4-"poptosis en/yme analysis
65 3-(-"poptosis organelle analysis
65 63 *etection of apoptotic markers
60
7. Flourescence in situ hybridi8ation
60 23Introduction
2* 13Three different types of FI'0 probes
2* 2-1-,ocus speciic pro.es
2* 2-2-"lp#oid or centromeric repeat pro.es
23 2-3-0#ole c#romosome pro.es
23 53pplications of FI'0
20 532344 investigation by FI'0
21 3-1-1-)#iladelp#ia
*40 7I. ,e-erences
*++ 7II. Li-e Flowcytometric Figures
*+1 7III.Li-e FISH #ictures
6
List of Figure
Pa"e ,e(iew o- Literature
*6 schematic representation of the mammalian cell cycle Figure 93.*:
33 The intrinsic or mitochondrial pathway Figure 93.3:
3+ The mechanism of apoptosis (poptosis triggered by
e!ternal signals: the e!trinsic or death receptor path way)
Figure 93.5:
30 *iagram of the mitochondrial and death receptor pathways
of cell death
Figure 93.+:
+1 Facscaliblur flow cytometry instrument
Figure 95.*:
+6 Flow cytometer system (Facscalibur)
Figure 95.3:
/4 Flow cytometers use the principle of hydrodynamics
focusing for presenting cells to a laser
Figure 95.5:
/3 simplified illustration of Flow -ytometry
Figure 95.+:
/1 Two parameter histogram and dot plot displaying F423FIT-
on the ! a!is and F413"/ on the y a!is
Figure 95./:
/6 Flow=o program Figure 95.0:
04 nalysis pulse width versus pulse height or area we can
eliminate the ma<ority of #> doublets that appear as #1
Figure 95.1:
0* *. histogram Figure 95.6:
03 *. histogram (aneuphliod population) Figure 95.2:
02 -oeffecient of ?ariation (-.?.) Figure 9+.*:
12 "ropidium iodide and T@3",@35 Figure 9+.3:
6* 'ub #2 peak by propidium iodide staining Figure 9+.5:
66 Fluoresence in situ hypridiAation Figure 9/.*:
7
List o- Abbre(iations
AL cute leukemia
ALL cut lymphoblastic leukemia
AL) lanine amino transaminase
A% cridine orange
AS) spartate amino transaminase
BM +one marrow
CBC -omplete blood picture
CD -luster of differentiation
CML -hronic myeloid leukemia
C7 -oefficient of variation
DA#I 638 diamino 3131phenylindole
DI *. inde!
DMS% *imethylsulfo!ide
D;A *eo!yribonucleic acid
!B /thedium bromide
!D)A /thyline diamine tetraacetic acid
FAB French merican +ritish
FACS Flow activated cell sorter
FISH Fluorescence in situ hybridiAation
FI)C Flourecien isothiocyanate
4<* "hase represents the gap in of *. replication time
between mitosis and the start
3<M "hase represents the gap between the end of *.
replication onest of mitosis
HI7 0uman immunodeficiency virus
HLA 0uman leukocyte antigen
H#7 0uman papilloma virus
LC 4iver cirrhosis
MMC Mithramycin
M,D Minimal residual disease
#BS "hosphate buffer saline
#I "ropidium iodide
#S "hosphatidylserine
S phase *. syntheis
=BC %hite blood cells
Introduction
8
-hildhood acute lymphoblastic leukemia (44) is a disease in
which too many underdeveloped lymphocytes are found in a childBs blood
and bone marrow. 4ymphocytes are infection3fighting white blood cells. 44
is the most common form of leukemia in children, and the most common
kind of childhood cancer #Moorman et al., 2$%.
cute lymphoblastic leukemia (44) represents nearly one third of
all pediatric cancers. nnual incidence of 44 is about 5> cases per million
populations, with a peak incidence in patients aged 137 years. lthough a
small percentage of cases are associated with inherited genetic syndromes,
the cause of 44 remains largely unknown #Jeffrey, 2&%$
Flow cytometry can be applied in basic research and in the clinic to
identify and measure apoptotic cells. The choice of a particular flow method
depends on several variables (cell system, type of flow cytometer, type of
apoptosis inducer, type of information reCuired) #'o"h and Dulin", 2&%.
The cell cycle was subdivided into four consecutive phases; #2 or
pre3synthetic phase, ', #1 or post3synthetic phase, and M phase during which
mitotic division into two daughter cells takes place. The #1 phase represents
the gap in time between the end of *. replication and onset of mitosis. It is
possible to discrimination between #2 vs, ' vs, #1 or M cells because of the
difference in their *. content #(abino)it*h, +!!,%.
The *. content of the cell can provide a great deal of information
about the cell cycle. The measurement of the *. content of cells was one
of the first ma<or applications of flow cytometry #Albro et al., +!!,%.
9
poptosis (programmed cell death) is a physiologic phenomenon
where in the dying cell plays an active part in its own destruction #S*huler
et al., +!!-%. poptosis plays a role in many diseases. There is a great
potential for treatment of these diseases in developing agents that can alter
the apoptotic process and change the natural disease progression. Molecules
whose roles in apoptosis have been investigated include +cl31 and c3myc
proteins, the p75 tumor suppressor gene and various tumor suppressor gene
products #Menende. et al., 2-%.
"75 is a pro3apoptotic genes present in all cells, but has special
significance to cancer cells. It is a tumor repressor gene, meaning that its
presence reduces the occurrence of cancer tumors by promoting apoptosis in
cancer cells #Polya/ et al., +!!0%. +-41 is an important regulator of
apoptosis, The oncogenic activity of the +cl1 gene is carried out via
suppression of lymphocytic apoptosis or programmed cell death #1ory 2
Adams, 22 and (oumier et al., 22%. -3Myc is widely known as a crucial
regulator of cell proliferation in normal and neoplastic cells 93e*hsler et al.,
+!!02 4a**hini and Penn, +!!5%$
The technology of flow cytometry and the discovery of a method to
produce monoclonal antibodies have made possible the clinical use of flow
cytometry for the identification of cell populations. Monoclonal antibodies
(tagged) with the fluorescent dye are commonly used for the identification of
cell surface antigens and fluorescent dyes that directly and specifically bind
to certain components of the cell (i.e. *.) are used for cell cycle analysis
#6han" et al., 2&%.
Fluorescence in situ hybridiAation (FI'0) allows identification of
specific seCuences in a structurally preserved cell, in metaphase or interphase
:
#1hat.imeletiou et al., 2&%. FI'0 is increasingly used for the identification
of 44. FI'0 plays an important role in detecting chromosome changes
#Primo et al., 2,%.
lmost all the chromosome abnormalities in 44 are translocations.
The most common one is "hiladelphia chromosome. It is the main product of
the t(;;11) translocation. This translocation

causes a rearrangement between
the proto3oncogene c3+4 and a gene

called the breakpoint cluster region
(+-,). The +-,D+4 fusion gene resulting from t(;;11) translocation. FI'0
is increasingly used for the identification of +-,D+4 gene rearrangements
#(udol7h et al., 2&%.
>. Acute Lymphoblastic Leukemia
;
cute lymphoblastic leukemia (44) is the most common form of
childhood cancer. It is a type of cancer that starts from white blood cells in
the bone marrow called lymphocytes. In most cases it Cuickly moves into the
blood. It can then spread to other parts of the body including the lymph
nodes, liver, spleen and central nervous system #Moorman et al., 2$%.
4eukemia is a cancer of the blood cells. There are several types of
leukemia and these are classified by how Cuickly they progress and what cell
they affect. cute leukemia is fast3growing and can overrun the body within a
few weeks or months. +y contrast, chronic leukemia is slow3growing and
progressively worsens over years #1arolyn et al., 22%.
.ormal blood cells contain white blood cells, red blood cells,
platelets and fluid called plasma. ll of these products are formed in the bone
marrow, a spongy area located in the center of bones. It contains a small
percentage of cells that are in development and are not yet mature. These
cells are called blasts. @nce the cell has matured, it moves out of the bone
marrow and into the circulating blood. The body has mechanisms to know
when more cells are needed and has the ability to produce them in an orderly
fashion #1arroll et al., 2,%.

2>
*.Incidence o- Leukemia?
cute lymphoblastic leukemia is the most common form of
childhood leukemia where it accounts for about 97E of childhood leukemia
and 17E of all pediatric cancer #8an./ows/y, 2%$ .ational -ancer
Institue, -airo &niversity, 44 represents 15.5E of all pediatric
malignancies and 97E of all pediatric leukemias. In a more recent research in
the "ediatric 0eamatologyD@ncology &nit, in 'hams &niversity 0ospital,
44 constitutes :1E of all leukemic cases #9halifa et al., +!!!%.
The global incidence of leukemias is about : to ; per 2>>,>>> people
each year. ppro!imately 17>,>>> new cases occur annually worldwide.
4eukemia accounts for 1.7E of overall cancer incidence. 0owever, its
incidence among children demonstrates its significance. lthough childhood
cases (through 26 years of age) account for about 21E of all leukemias,
childhood cancer is the second biggest killer of children (after accidents) and
leukemia is the most common form of childhood cancer. The incidence of
childhood 44 in the &nited 'tates has increased appro!imately 1>E over
the past two decades, mostly in the >3 to 63year3old age group. @ver the
course of this century, leukemia rates have also generally increased #Sandler
and (oss, +!!0%.
cute lymphoblastic leukemia affects slightly more boys than girls.
It occurs predominantly in children, peaking at four years of age. It is seen
more freCuently in industrialiAed nations, and it is slightly more common
among white children and boys. 'tudies have suggested that patients who are
younger than thirty five years of age far better than older patients #Jeffrey,
2&%$
22
3.)ypes o- leukemia?
+y considering whether leukemias are acute or chronic, and whether
they are myelogenous or lymphocytic, they can be divided into four main
types. The first one is an acute myeloid leukemia which occurs in both
children and adults. The second one is an acute lymphocytic leukemia which
is the most common type seen in children, but also seen in adultBs over87.The
third one is a chronic myelogenous leukemia which occurs mostly in adults.
-hronic lymphocytic is the fourth type which is the most often seen in people
over age77, can affect younger adults, but almost never seen in children #Pui,
+!!&%.
In acute leukemia, the bone marrow cells are unable to properly
mature. Immature leukemia cells, which are often called blasts, continue to
reproduce and accumulate. In chronic leukemia, the cells can mature but not
completely. They are not really normal. They generally do not fight infection
as well as do normal white blood cells. @f course, they live longer, build up,
and crowd out normal cells. The types of leukemia are also grouped by the
type of white blood cell that is affected, leukemia that affects lymphoid cells
is called lymphocytic leukemia, and leukemia that affects myeloid cells is
called myeloid leukemia or myelogenous leukemia #8i*htman et al., +!!&%.
5 . Biological Classi-ication o- ALL ?
cute lymphoblastic leukemia blasts are derived from either +3cell
or T3cell lineages, as determined by cell surface and other markers. small
percentage of the cells are either so primitive that they do not e!press enough
markers to identify #(oss et al., 2, and Pullen et al., +!!!%.

21
cute lymphoblastic leukemia is categoriAed according to a system
know as the French3merican3+ritish (F+) Morphological -lassification
'cheme for 44. 442 is mature3appearing lymphoblasts (T3cells or pre3+3
cells); these cells are small with uniform genetic material, regular nuclear
shape, nonvisible nucleoli, and little cytoplasm. 441 is immature and
pleomorphic lymphoblasts (T3cells or pre3+3cells), these cells are large,
variable in siAe, varaiable genetic material, irregular nuclear shape, one or
more large nucleoli and variable cytoplasm. 445 is lymphoblast(+3
cells),these are large, genetic material is finely stripped and uniform, nuclear
shape is regular, there are one or more prominent nucleoli, and cytoplasm is
moderately abundant #S*hra77e et al., 2%$

+.Causing o- Leukemia?
The causes of the disease are not known, but e!perts believe that
44 develops from a combination of genetic and environmental factors.
number of genetic mutations associated with 44 have been identified.
Missing or defective genes that suppress tumors are responsible for cases of
44 #Guo et al., 2&%.

'everal things have been identified as risk factors3that is, e!posure
to them puts a person at a higher risk of developing leukemia, but it is not a
certainly that this e!posure will lead to leukemia. These factors include
e!posure to high3energy radiation, like that released from a nuclear accident
or bomb. 'ome genetic syndrome put a person at higher risk. "eople who
work with the chemical benAene over a long period of time also have a
greater chance of getting leukemia. 'ome scientist feel that e!posure to
electromagnetic fields, like those that come from power lines, may put a
person to higher risk, but this has not been proven #Pui et al., 2+%.
25
0eredity, radiation, chemical e!posures, and treatment with
chemotherapeutic agents have been implicated in the development of
leukemia. ?iral infection by at least one known virus, human T3cell
leukemiaDlymphotropic virus type I (0T4?32), is a well3understood cause of
adult T3cell leukemia #4ran*hini, +!!& and Grea)es, +!!0%.
nother group of risk factors includes occupational and
environmental e!posure to radiation or chemicals. The best established cause
of leukemia among children is in utero e!posure to diagnostic F3rays.
4eukemia in adults is strongly associated with occupational e!posure to
ioniAing radiation. There is little evidence, however, that nonioniAing
radiation such as electromagnetic fields (/MF) induces leukemia. Indeed,
two recent studies have shown that /MF e!posure is not a ma<or risk factor
for leukemia in children or in adults. 'ome studies have reported an
association between cancer and #ig# levels of electromagnetic radiation
(/M,). %hether lower levels of radiation (eg, living near power lines, video
screen emissions, small appliances, cell phones) play any ma<or role is
uncertain but probably unlikely #8inet et al., +!!0 and :er/asalo, +!!$%.
+ecause most people in the general population are not e!posed to
chemotherapeutic drugs or occupationally e!posed to radiation or chemical
solvents, e!posure to these agents cannot e!plain the causes of the ma<ority
of leukemia cases diagnosed each year. %e conservatively estimate that the
causes of at least 1>,>>> (appro!imately 9>E) of the 1:,>>> new leukemia
cases that develop annually in the &nited 'tates are une!plained. Thus, the
causes of leukemia remain largely unknown. lthough some success has been
achieved in treating leukemias, especially in children, mortality rates have
remained relatively high (appro!imately 97E in the &nited 'tates) #9a.a/ et
al., +!!0%.
26
#enetic predisposition may play a ma<or role in both adult and
childhood leukemia. lthough the 4eukemia 'ociety of merica emphasiAes
the fact that anyone may develop the disease, an increased risk e!ists among
/astern /uropean =ews, and a decreased risk e!ists among sians
(differences in diet and lifestyle may play a role, however). Individuals with a
family history of leukemia or lymphoma have a 7.83fold increased risk for
M4. "arents of children with *own syndrome also have an increased risk
of leukemia #Grea)es, +!!0 and Shannon et al., +!!2%.
&p to 87E of leukemias contain genetic rearrangements, called
translo*ations, in which some of the genetic material (genes) on a
chromosome may be altered, or shuffled, between a pair of chromosomes.
For e!ample the most common genetic in<ury in 44 is t(21;12), which
means a translocation with a genetic shift between chromosome 21 and 12. It
is also referred to as T/43M42 fusion and occurs in appro!imately 1>E of
44 patients. ,esearchers believe that this translocation may occur during
fetal development in some patients. bout 1>E of adults and about 7E of
children with 44 have a genetic abnormality called the "hiladelphia ("h)
chromosome t(;;11). nother important chromosome translocation is t(6;22)
involving the M44 gene on chromosome II. @ften occurring in children
under one year old #9handa/ar et al., 2&%.
/ . )he Signs and diagnosis o- Leukemia ?
The blast cells are unable to perform their normal function of
fighting infection, so patients may develop fevers or infections that wonBt go
away. s the number of immature cells (blasts) increases, the normal cells are
crowded out. This leads to low red blood cell counts and platelets #Smith et
al., +!!$%.
27
cute lymphoblastic leukemia tends to cause symptoms more rapidly
than chronic leukemia. 'ome common symptoms include fever, chills,
weakness and fatigue, swollen or tender lymph nodes, liver or spleen, easy
bleeding or bruising, swollen or bleeding gums, night sweats, and bone pain.
The abnormal cells can accumulate in the brain or spinal cord, causing
headaches, vomiting, confusion, or seiAures #Ada*hi et al., 2&%.

In acute lymphoblastic leukemia, the doctor asks about medical
history and conducts a physical e!am. *uring the e!am, abnormalities such
as enlarged spleen, liver or lymph nodes may be detected, prompting further
investigation. -omplete blood count would find blast cells present in the
blood, thus suggestion a diagnosis of leukemia. This test can reveal that the
patient has leukemia. sample of bone marrow is determined the type of
leukemia #1ham7lin et al. +!5! and 'ur"er et al., 2,%.

complete blood cell count is the first step in diagnosing 44. This
test will often show various findings, including the following: The presence
of circulatory leukemic blast cells, the presence and severity of anemia and
the count of a variety of blood cell types. ( high white blood cell count
indicates a more severe disease.) These tests will not always show the
presence of leukemic cells. +lood tests do not always detect leukemia, and
about 2>E of patients with 44 have a normal blood cell count #Ada*hi et
al., 2&%.
If the results of the blood tests are abnormal or the physician
suspects leukemia despite normal cell counts, a bone marrow aspiration and
biopsy are the ne!t steps #(e.aei et al., 2,%.
28
If bone marrow e!amination confirms 44, a spinal tap may be
performed, which uses a needle inserted into the spinal canal. sample of
cerebrospinal fluid with leukemia cells is a sign that the disease has spread to
the central nervous system. In most cases of childhood 44, leukemic cells
are not found in the cerebrospinal fluid 9:ieira et al., 2&%.
0 . )reatment o- Childhood ALL ?
The treatment depends on age, the results of laboratory tests, and
whether or not the child has been previously treated for leukemia. &ntreated
44 means that no treatment has been given e!cept to reduce symptoms.
There are too many white blood cells in the blood and bone marrow, and
there may be other signs and symptoms of leukemia. ,emission means that
treatment has been given and the number of white blood cells and other blood
cells in the blood and bone marrow is normal that there no signs or symptoms
of leukemia. ,ecurrent disease means that the leukemia has come back after
going into remission. ,efractory disease means that the leukemia failed to go
into remission following treatment #'assan et al., +!!0%.
There are treatments for all patients with childhood acute
lymphoblastic leukemia. The primary treatment for 44 is chemotherapy.
,adiaion therapy may be used in certain cases. +one marrow transplantation
is being studied in clinical trials #;*/un et al., +!!0%.
cute lymphoblastic leukemia patients should receive chemotherapy
drugs as soon as possible after diagnosis. -hemotherapy uses strong drugs to
kill leukemia cells. The goal of chemotherapy is to achieve remission (no
symptoms of 44) and to restore normal blood cell production. -ommon
chemotherapy drugs include do!orubicin, fludarabine and cyclophosphamide.
29
The drugs used depend on factors such as the patientBs age and the number
and type of leukemia cells in the blood. &nfortunately, chemotherapy also
kills normal cells, so 44 patients receiving chemotherapy may have side
effects, including nausea, tiredness and a higher risk of infections #'aldu..i
et al., 2&%.
For most patients, chemotherapy restores normal blood cell
production within a few weeks, and microscopic e!aminations of their blood
and marrow samples will show no signs of leukemia cells. %hen this
happens, the disease is in remission. lthough chemotherapy often brings
long3lasting remissions in children, in adults, 44 freCuently returns. If the
44 returns, patients and their doctors can consider more chemotherapy or a
marrow or blood cell transplant. -hemotherapeutic agents kill cancer cells by
activating apoptosis,

or programmed cell death. Ma<or apoptotic pathways
and the specific role of key proteins

in this response is described. The
e!pression level of some of these proteins,

such as +cl1, +F, and caspase 5,
has been shown to be predictive

of ultimate outcome in hematopoietic
tumors. .ew therapeutic

approaches that modulate the apoptotic pathway are
now available

and may be applicable to the

treatment of childhood 44
#Donadieu 2 Hill, 2+ and <a/ase et al., 2&%.
,adiation therapy uses 13rays or other high3energy rays to kill
cancer cells and shrink tumors. ,adiation for 44 usually comes from a
machine outside the body (e!ternal beam radiation therapy) #Durrant et al.,
+!!0%.
+one marrow transplantation is a newer type of treatment. First, high
doses of chemotherapy with or without radiation therapy are given to destroy
all of the bone marrow in the body. bone marrow transplant using marrow
2:
from a relative or person not related to the patient is called an allogeneic bone
marrow transplant #;lri*h et al., 2+%.
n even newer type of bone marrow transplant, called autologous bone
marrow transplant, is being studied in clinical trials. *uring this procedure,
bone marrow is taken from the patient and may be treated with drugs to kill
any cancer cells. The marrow is froAen to save it. The patient is then given
high3dose chemotherapy with or without radiation therapy to destroy all of
the remaining marrow. The froAen marrow that was saved is thawed and
given through a needle in a vein to replace the marrow that was destroyed
#Sebban et el., +!!-%.

Treatment outcome is dependent not only on the therapy applied,

but
importantly, also on the underlying biology of the tumor

and the host. /ach of
these

variables must be factored into initial treatment decisions,

as well as
later refinements based on initial response, and

several biological features. It
is recogniAed that with improvements in therapy,

certain variables might lose
their prognostic value; therefore,

risk assignment plans should be routinely
reassessed. Finally

an optimal system should allow for comparison of the
outcomes

of similar or identical patients, treated on different protocols #1hoi
et al., 2&%.
There are generally four phases of treatment for 44. The first
phase, remission induction therapy, uses chemotherapy to kill as many of the
leukemia cells as possible to cause the cancer to go into remission. The
second phase, called central nervous system (-.') prophyla!is, is preventive
therapy, it involves using intrathecal andDor high3dose systemic chemotherapy
to the -.' to kill any leukemia cells present there. It is also used to prevent
2;
the spread of cancer cells to the brain and spinal cord even if no cancer has
been detected there. ,adiation therapy to the brain may also be given, in
addition to chemotherapy, for this purpose. -.' prophyla!is is often given in
con<unction with consolidation therapy. @nce a child goes into remission and
there no signs of leukemia, a third phase of treatment called consolidation or
intensification therapy, is given. -onsolidation therapy uses high3dose
chemotherapy to attempt to kill any remaining leukemia cells. The fourth
phase of treatment, called maintenance therapy, uses chemotherapy for
several years to maintain the remission #Attal et al., +!!&%.


1>
II. Cell cycle and Apoptosis
*.Cell cycle?
The concept of the cycle in its current form is introduced by
Howard and Ple*, #+!&,%. They observed that *. synthesis ('3 phase) in
individual cells was discontinuous and occupied a discrete portion of the cell
life and was constant in duration. Mitotic division (M3phase) was seen to
occur after certain period of time following *. replication. distinct phase
between *. replication and mitosis was also apparent (8oo/ et al., +!!$%.
-ell cycle phase of #2 was historically considered to be a time cells
had little observable activity. 'ince this time precedes *. synthesis, the
term #ap 2 (#2) was coined. They have diploid chromosome (1-G68
chromosome). t a certain point in the cellBs life, the *. synthetic
machinery turns on. This phase of the cellBs life is labeled H'H for synthesis.
s the cell proceeds through this phase, its *. content increases from 1-
to 6-. t the end of ', the cell has duplicated its genome and it is in the
tetraploid state. fter the ' phase, the cell again enters a phase that was
historically thought to be Cuiescent. 'ince this phase is the second gap region,
it is referred to as #1. In the #1 phase, the cell is producing the necessary
proteins that will play a ma<or role in cytokinase. fter a highly variable
amount of time, the cell enters mitosis (M). *. content remains constant at
6- until the cell actually divides at the end telophase #8iblit, +!!,%.
The process of replicating *. and dividing a cell can be described
as a series of coordinated events that compose a Hcell

division cycle,H
illustrated for mammalian cells in Fig 93.*:$ In each cell division cycle,
12
chromosomes are replicated once (*.

synthesis or '3phase) and segregated
to create two genetically

identical daughter cells (mitosis or M3phase). These
events are

spaced by intervals of growth and reorganiAation (gap phases #
2

and #
1
). -ells can stop cycling after division, entering a state

of Cuiescence
(#
>
). -ommitment to traverse an entire cycle is

made in late #
2
. t least two
types of cell cycle control

mechanisms are recogniAed: a cascade of protein
phosphorylations

that relay a cell from one stage to the ne!t and a set of
checkpoints

that monitor completion of critical events and delay progression

to the ne!t stage if necessary #<asmyth, +!!$%.
The first type of control involves

a highly regulated kinase family.
Iinase activation generally

reCuires association with a second subunit that is
transiently

e!pressed at the appropriate period of the cell cycle; the periodic

HcyclinH subunit associates with its partner Hcyclin3dependent

kinaseH (-*I)
to create an active comple! with uniCue substrate

specificity. ,egulatory
phosphorylation and dephosphorylation

fine3tune the activity of -*I3cyclin
comple!es, ensuring well3delineated

transitions between cell cycle stages
#Elled"e, +!!$%.
second type of cell cycle regulation, checkpoint control, is more
supervisory. It is not an essential part of the cycle

progression machinery. -ell
cycle checkpoints sense flaws in critical

events such as *. replication and
chromosome segregation. %hen checkpoints are activated, for e!ample by
underreplicated

or damaged *., signals are relayed to the cell cycle3
progression

machinery. These signals cause a delay in cycle progression, until
the danger of mutation has been averted. +ecause checkpoint function

is not
reCuired in every cell cycle, the e!tent of checkpoint

function is not as
obvious as that of components integral to the

process, such as -*Is #Sherr,
+!!$%.
11
Figure 93.*:? A schematic representation o- the mammalian cell cycle
#<asmyth, +!!$%.
15
*.*.Cell cycle and cancer?
'uperficially, the connection between the cell cycle and cancer is
obvious: cell cycle machinery controls cell proliferation,

and cancer is a
disease of inappropriate cell proliferation. Fundamentally,

all cancers permit
the e!istence of too many cells. 0owever, this

cell number e!cess is linked in
a vicious cycle with a reduction

in sensitivity to signals that normally tell a
cell to adhere,

differentiate, or die. This combination of altered properties

increases the difficulty of deciphering which changes are primarily

responsible for causing cancer #Ja*/s and 3einber", +!!$%.

The first genetic alterations shown to contribute to cancer
development were gain3of3function mutations. These mutations

define a set
of HoncogenesH that are mutant versions of normal

cellular Hprotooncogenes.H
The products of protooncogenes function

in signal transduction pathways that
promote cell proliferation.

0owever, transformation by individual oncogenes
can be redundant

(mutation of one of several genes will lead to
transformation)

or can be cell type3specific (mutations will transform some
cells

but have no effect on others). This suggests that multiple, distinct

pathways of genetic alteration lead to cancer, but that not all

pathways have
the same role in each cell type #3hite, +!!$%.

More recently, the significance of loss3of3function mutations in
carcinogenesis has become increasingly apparent. Mutations

in these so3
called Htumor suppressorH genes were initially recogniAed

to have a ma<or
role in inherited cancer susceptibility. +ecause

inactivation of both copies of a
tumor suppressor gene is reCuired

for loss of function, individuals
heteroAygous for mutations at

the locus are phenotypically normal. Thus,
16
unlike gain3of3function

mutations, loss3of3function tumor suppressor
mutations can be

carried in the gene pool with no direct deleterious
conseCuence.

0owever, individuals heteroAygous for tumor suppressor
mutations

are more likely to develop cancer, because only one mutational

event is reCuired to prevent synthesis of any functional gene

product
#Mor"enbesser et al., +!!-%.
It now appears that tumor suppressor gene mutations are highly
likely to promote, and may even be reCuired for, a large number

of
spontaneous as well as hereditary forms of cancer. 4oss

of function of the
tumor suppressor gene product p,b, for e!ample,

would be predicted to
liberate /1F transcriptional activators

without reCuiring phosphorylation and
thus bypass a normal negative

regulation controlling entry into the cycle.
4oss of

the tumor suppressor gene product p28 would have a similar
conseCuence,

liberating /1Fs by increasing p,b phosphorylation . In

addition,
cell cycle progression can be halted at several points

by the tumor suppressor
gene product p75, activated in response

to checkpoints sensing *. and
possibly also chromosome damage;

loss of p75 would remove this brake to
cycling #Symonds et al., +!!-%$
3.Apoptosis and its markers?
poptosis and necrosis are too distinct, mutually e!clusive, modes
of cell death. poptosis, freCuently referred to as programmed cell death is an
active and physiological mode of cell death, in which the cell itself designs
and e!ecutes the program of its own demise and subseCuent body disposal.
*ifferent patterns of apoptosis (early and delayed apoptosis) many cell types,
cells of hematopoietic origin in particular, undergo apoptosis rapidly, to
17
within few hours following e!posure to relatively high concentration of
cytoto!ic agents #Ma=ino and Joris, +!!&%.
poptosis can be defined as Bgene3directed cellular self3destructionBB
although this is really a phenomenon where cells are programmed to die at a
particular point, e.g. during embryonic development, and even here cells may
go through an apoptotic pathway. 0owever, apoptosis is certainly a distinct
process from other forms of oncosis leading to necrosis #Gerbaulet et al.,
2& and 3alla*h et al., +!!!%.
poptosis affects individual cells, physiological induction e.g. lack
of signals, phagocytosis by macrophages or other cells and there is no
inflammatory response. .ecrosis affects group of cells, non physiological
induction e.g. virus and poison, phagocytosis of macrophages and there is
inflammatory response #3irth et al., 2&%.
There are three different mechanisms by which a cell commits
suicide by apoptosis. In the intrinsic or mitochondarial pathway, the outer
membranes of mitochondria in a healthy cell e!press the protein; +cl1 on
their surface. +cl1 is bound to a molecule of the protein paf32. Internal
damage to the cell (e.g., from reactive o!ygen species) causes +cl1 to release
paf32; a related protein, +a!, to penetrate mitochondrial membranes
causing cytochrome c to leak out. The released cytochrome c and paf32 bind
to molecules of caspase ; Fig$ 93.3:$ The resulting comple! of cytochrome c,
paf32, caspase ; and T" is called the apoptosome. The apoptosome
aggregate in the cytosol #<iu et al., 2& and 8am et al., 2& and
9roemer2 (eed 2%.

18
Figure 93.3:? )he intrinsic or mitochondrial pathway #8am et al., 2&%.
19
-aspase ; is one of a family of over a doAen caspases. They are all
proteases. They get their name because they cleave proteins3mostly each
other at aspiratic acid residues. -aspase ; cleaves and, in so doing, activates
other caspases. The seCuential activation of one caspase by another creates an
e!panding cascade of proteolytic activity (rather like that in blood clotting
and complement activation) which leads to digestion of structural proteins in
the cytoplasm, degradation of chromosomal *. and phagocytosis of the
cell #3ada et al., 2&%.
In the e!trinsic or death receptor pathway, Fas and the T.F receptor
are integral membrane proteins with their receptor domains e!posed at the
surface of the cell. +inding of the complementary death activator (Fas4 and
T.F respectively) transmits a signal to the cytoplasm that leads to activation
of caspase :. -aspase : (like caspase ;) initiates a cascade of caspase
activation leading to phagocytosis of the cell Fig$ 93.5:. For e!ample,
cytoto!ic T cells recogniAe (bind to) their target, they produce more Fas4 at
their surface, this binds with the Fas on the surface of the target cell leading
to its death by apoptosis. In some cases, final destruction of the cell is
guaranted only withits engulfment by a phagocyte #'i=an"i et al., 2& and
:e"a et al., 2&%.
In the third way, neurons, and perhaps other cells, have another way
to self3destruct that unlike the two paths described above, doesnBt use
caspase. poptosis3 inducing factor (IF) is a protein that is normally located
in the inter membrane space of mitochondaria. %hen the cell receives a
signal telling it that it is time to die, IF is released from the mitochondrial, it
is migrates into the nucleus and binds to *., %hich triggers the destruction
of the *. and cell death #;rbano et al., 2&%.
1:

Figure 93.5:? )he mechanism o- apoptosis 9Apoptosis triggered by
e@ternal signals? the e@trinsic or death receptor path way: #'i=an"i et al.,
2&%$

1;
*efects in programmed cell death (apoptosis) mechanisms play

important roles in the pathogenesis and progression of hematological

malignancies, allowing neoplastic cells to survive beyond their

normally
intended life3spans and subverting the need for e!ogenous

survival factors.
poptosis defects also serve as an important

complement to proto3oncogene
activation, as many deregulated

oncoproteins that drive cell division also
trigger apoptosis #E)an and 8ittlewood, +!!5%.
'imilarly, errors in *. repair

and chromosome segregation
normally trigger cell suicide as

a defense mechanism for eradicating
genetically unstable cells,

and thus apoptosis defects permit survival of the
genetically

unstable cells, providing opportunities for selection of
progressively

aggressive clones #>ono) et al., 2%.
-hemotherapy and irradiation trigger apoptosis in tumor cells

and an
understanding of the biochemical pathways involved in

apoptosis provides an
opportunity to classify tumors based on

their response to common induction
regimens. Multiple distinct

signaling pathways regulate apoptosis, but two
ma<or cell death

pathways have been implicated in hematological
malignancies:

the mitochondrial pathway and the death receptor pathway Fig$
93.+: #E)ans et al., 22%.
5>
Figure 93.+:? Diagram o- the mitochondrial and death receptor pathways
o- cell death 9!(ans et al$" 3443:$
52
+oth of these pathways ultimately activate members of

the caspase
family of proteins that are responsible for e!ecuting

the terminal phases of
apoptosis. p75 protein levels rise in

response to various cellular stresses
including chemotherapy.

p75 induces the loss of mitochondrial membrane
potential with

subseCuent release of cytochrome c, which forms a comple!,
the

Hapoptosome,H with the adapter molecule paf32, T", and caspase3;.

This comple!, in turn, activates caspase35 #E)ans et al., 22%.
nother pro!imal pathway of cell death involves death receptor

signaling at the cell surface. +inding of -*;734 and other tumor

necrosis
factor (T.F) family ligands to their death inducing

receptors, -*;7D"@3
2DF' or T.F3 and T,I4 respectively, leads

to receptor trimeriAation and
the recruitment of adapter molecules.

These molecules include
F**DM@,T32 that in turn lead to recruitment

and activation of caspase3:.
This initiator caspase also cleaves

and activates downstream caspases,
including caspase35. lthough

generally described as being distinct, these two
pro!imal pathways

are interconnected. For e!ample, caspase3: cleaves the
pro3apoptotic

protein +I*, which results in translocation to the mitochondria

and release of cytochrome c #9ishi et al., 2,, 'lom, 2, de 4ran*his et
al., 2 and Goto et al., 2+%.
'everal studies have e!amined the prognostic significance of

apoptotic protein e!pression in leukemia. *efects in the p75

pathway are
distinctly rare in childhood malignancies including

44, where mutations are
detected in J 7E of cases at the

time of initial diagnosis. 0owever, relapsed
blasts may harbor

mutations of p75 gene much more commonly. Further,
44 blasts

at relapse have been noted to e!press high levels of the Mdm31

protein, which abrogates p75 signaling #Dir)en et al., +!!& and Pemble et
al., +!!-%.
51
-ancer3associated defects in apoptosis play a role

in chemoresistance
and radioresistance, increasing the threshold

for cell death, and thereby
reCuiring higher doses for tumor

killing #?s*ho77 et al., +!!! and Ma/in et
al., 2%.
Melanoma (skin cancer) cells avoid apoptosis by inhibiting the
e!pression of the gene encoding paf32. 'ome cancer cells, especially lung
and colon cancer cells, secrete elevated levels of a soluble (decoy) molecule
that binds to Fas4, plugging it up so it cannot bind Fas. Thus cytoto!ic T
cells (-T4) cannot kill the cancer cells by the mechanism of death receptor
pathway. @ther cancer cells e!press high levels of Fas4, and can kill any
cytoto!ic T cells (-T4) that try to kill them because -T4 also e!press Fas
(but are protected from their own Fas4) #Mei=er et al., 2&%.
poptosis plays a role in many diseases, such as cancer, viral
infections, and autoimmune and neurodegenerative disorders. There is a great
potential for treatment of these diseases in developing agents that can alter
the apoptotic process and change the natural disease progression. Molecules
whose roles in apoptosis have been investigated include +cl31 and c3myc
proteins, the p75 tumor suppressor gene and various tumor suppressor gene
products, M" kinases, and proteases #Menende. et al., 2-%.
3.*.)he apoptosis promoter 9p/5:?
p75 stimulates

a wide network of signals that act through two ma<or
apoptotic

pathways. The e!trinsic, death receptor pathway triggers the

activation of a caspase cascade, and the intrinsic, mitochondrial

pathway
shifts the balance in the +cl31 family towards the pro3apoptotic

members,
promoting the formation of the apoptosome, and conseCuently

caspase3
55
mediated apoptosis. The impact of these two apoptotic

pathways may be
enhanced when they converge through +id, which

is a p75 target. The
ma<ority of these apoptotic effects are

mediated through the induction of
specific apoptotic target

genes. 0owever, p75 can also promote apoptosis by
a transcription3independent

mechanism under certain conditions. Thus, a
multitude of mechanisms

are employed by p75 to ensure efficient induction
of apoptosis

in a stage3, tissue3 and stress3signal3specific manner #8inda 2
1arol, +!!$ and Susan et al., 2,%2
'ome cancer causing viruses use tricks to prevent apoptosis of the
cells they have transformed. 'everal human papilloma viruses (0"?) have
been implicated in causing cervical cancer. @ne of them produces a protein
(/8) that binds and inactivates the apoptosis promoter p75. +inding of Fas
ligand or agonistic anti3Fas antibody to the death receptor Fas can activate a
caspase3cascade resulting in apoptosis. Fas cell surface e!pression was
determined by flow cytometry #Hou"ardy et al., 2&%.
#enes involved in apoptosis are either pro3apoptotic (promote
apoptosis) or anti3apoptotic (inhibit apoptosis). "75 is a pro3apoptotic genes
present in all cells, but has special significance to cancer cells. It is a tumor
repressor gene, meaning that its presence reduces the occurrence of cancer
tumors by promoting apoptosis in cancer cells. .ormally it induces apoptosis
by activating caspases ;, :, 9, and 5. The loss of p75 decreases caspase
activation and therefore the cell will not undergo apoptosis. Mutation in the
p75 gene is the most common mutation in cancer; it is present in about half
of all cancer tumors, :>E in all colon cancer tumors, 7>E of lung cancer
tumors, and 6>E of breast cancer tumors #Polya/ et al., +!!0%.

56
&nder normal conditions p75 is a short3lived protein. The p75

inhibitor Mdm1 (0dm1 in humans) is largely responsible for keeping

p75 in
this state. Mdm1 inhibits the transcriptional activity

of p75 and, more
importantly, promotes its degradation by the

proteasome #8e)ine, +!!0%.
p75 mutants in tumours have a reduced affinity for *. and a
reduced ability to induce apoptosis. %e describe a mutant with

the opposite
phenotype, an increased affinity for some p753binding

sites and an increased
ability to induce apoptosis. The apoptotic

function reCuires transcription
activation by p75 #Elisabeth et al., +!!!%.
/arly observations suggested that p75 may function as an oncogene,
because overe!pression of p75 appeared to cause oncogenic transformation
of cells. In the late 2;:>s, however, several critical discoveries defined the
normal function of p75 to be anti3oncogenic. %ild3type p75 genes, when
introduced into cells, were found to be growth suppressive #>sabela et al.,
2%.
p75 plays multiple roles in cells. /!pression of high levels of wild3
type (but not mutant) p75 has two outcomes: cell cycle arrest or apoptosis. In
response to genoto!ic stress, p75 acts as an Hemergency brakeH inducing
either arrest or apoptosis, protecting the genome from accumulating e!cess
mutations. -onsistent with this notion, cells lacking p75 were shown to be
genetically unstable and thus more prone to tumors #>sabela et al., 2%.
p75 promotes cytochrome

c release through the induction of target
genes encoding +053only

proteins. Importantly, p75 also induces ")"F-1
e!pression through

a response element within the ")"F-1 promoter #9annan
et al.,

2+%
57
The tumor suppressor gene product p75 is clearly a central player in

many biochemical pathways that are pivotal to human carcinogenesis. The

seCuence3specific *. binding properties of this nuclear phosphoprotein

regulate the transcription of a continually e!panding number

of genes, the
protein products of which regulate cell cycle progression

and apoptosis
#>sabela et al., 2%.
4oss of p75 function by mutation is common in cancer. 0owever,
most natural p75 mutations occur at a late stage in tumor development, and
many clinically detectable cancers have reduced p75 e!pression but no p75
mutations. It remains to be fully determined what mechanisms disable p75
during malignant initiation and in cancers without mutations that directly
affect p75. p75 mutants in tumours have a reduced affinity for *. and a
reduced ability to induce apoptosis #<iu et al., 2&%.
p75 e!pression has important

clinical implications as an indicator of
prognosis and response

to chemotherapy or radiotherapy in different human
tumor types.

The common effect of p75 mutations found in tumours is to
inactivate p75 as a transcription factor. -onseCuently, a great deal of effort
has been e!pended in trying to identify transcriptional targets of p75.
"articular attention has been paid to target genes which may mediate cell3
cycle arrest and apoptosis #9o and Pri)es, +!!$ %.
p75 dependent #
2
and #
1
arrest reCuires induction of the p12 cyclin3
dependent kinase inhibitor. In contrast, no single gene can e!plain p753
induced apoptosis. Many p75 target genes have been identified which
function in known apoptotic pathways, regulate survival factor signalling,
induce apoptosis when over e!pressed or are involved in biochemical events
linked to apoptosis #'u*/binder et al., +!!0 , Miyashita and (eed, +!!& ,
58
@wenAS*haub et al., +!!&, Polya/ et al., +!!0, :armehA6iaie et al., +!!0,
M*1urra*h et al., +!!0 and (am7ino et al., +!!0%.
p75 can activate target genes through a non3canonical seCuence.

The
first such e!ample is in the p753induced gene 5 ()I33),

which has been
implicated in the accumulation of reactive o!ygen

species and apoptosis
induction #Polya/ et al., +!!0%. nother recently

described e!ample is the
gene encoding the pro3apoptotic phosphatase

"-2, which is induced
through binding of p75 to a novel palindromic

binding site #Bin et al., 2,%.
novel insight into the interplay between p75 and its family

members, p85 and p95, in the induction of apoptosis has been

recently
revealed #4lores et al., 22%. The effect of p85 and p95 on p75
transcriptional activity,

using a selection of knockout mouse embryo
fibroblasts (M/Fs),

defined two distinct classes of target gene. %hereas p75
alone

is sufficient for the induction of p21 and 4dm2, the induction

of the
apoptotic genes )E5)6 Ba7 and !o7a reCuires p75 together

with p85 and p95.
This finding demonstrates an essential role

for both p85 and p95 in the
efficient induction of apoptotic

target genes by p75. The mechanism of this
cooperation is currently

unknown, but it may involve an enhanced binding to
andDor stabiliAation

of the transcription comple! on the promoters of p75
apoptotic

target genes by the cooperative action of all three members

#;rist
and Pri)es, 22%.
In addition to the contribution of

p85 and p95 to the apoptotic
function of p75, they play an important

role in the precise control of cell
death during normal mouse

development. p95 also plays a role in the
induction of cell

death in response to *. damage, a process involving
cooperation

between the bl tyrosine kinase and p95 #Shaul,

2%.
59
Immunohistochemical (I0-) detection of p75 e!pression has been

established as a relatively easy and straightforward method

for fresh and
archival tissues. vailable monoclonal antibodies

recogniAe both wild3type
and mutant forms, but there may be

a selective detection of the latter owing to
the very short

half3life of the former #Porter et al., +!!2 and Soussi et al.,
+!!-%.
p75 is a tumor suppressor that is rarely mutated in 44 patients
but whose function is freCuently altered by mutations to genes that code for
proteins that regulate p75 function. ctivation of p75 occurs in response to
cells that have acCuired *. damage that may be engaged in aberrant cell
proliferation. Mutations to proteins that regulate p75 function, like 0*M1,
p26, and p12, are freCuent findings in 44 #(oman et al., 22%.
+ovine papillomavirus type 2 (+"?32)3transformed mouse fibroblast
cell lines were analyAed via flow cytometry (F-M) for e!pression of p75
protein along with their *. content. 'ignificantly elevated levels of the p75
protein was present in some but not all of the transformed cell lines.
Kuantitation of p75 protein in cell lines containing +"?32 *. revealed that
the tumorigenic cell lines e!pressed higher levels of the p75 protein
#A"rawal et al., +!!-%.
The correlation between p75 abnormalities and *. aneuploidy and
that analysis of p75 protein is useful for prediction of clinical course in
esophageal sCuamous cell carcinoma #Gou/on et al., +!!-%.
8iu et al., (2-) evaluated changes in apoptotic proteins e!pression

that occur in response to chemotherapy in pediatric cases with acute

leukemia
<ust prior to and 2, 8 and 16 hours following the administration

of multiagent
5:
chemotherapy. They found great heterogeneity

in the patterns of apoptotic
protein e!pression in the initial

response to chemotherapy among individual
patient samples. Importantly,

no increases in p75, p12 or Mdm31 protein
e!pression were seen

in leukemic blasts from the standard risk patients whose
initial

treatment consisted of the non3p753dependent drugs, vincristine

and
prednisone.
In the subgroup of children who received at

least one p75 dependent
drug, patients could be segregated into

two groups, one group that showed
up3regulation of p75 protein

and its target p12, and another group that
showed no increase

following therapy, thus identifying at least two distinct
pathways

leading to apoptosis #1hen et al., +!!$%.
2A2A'*l2 7roteins
Members of the +cl31

protein family play pivotal roles in the
decision and e!ecution

phases of apoptosis in the mitochondrial pathway. To
date,

16 +cl31 family members have been identified as either pro3

(e.g., +a!,
+ak, +cl3F
'
, +id, +ad, and .o!a) or anti3 (e.g.,

+cl31 and +cl3F
4
) apoptotic
proteins. +cl31 proteins form

homo3 and heterodimeric comple!es to regulate
mitochondrial

channel formation and subseCuent release of cytochrome c
from

the mitochondria #9ishi et al., 2,, 'lom, 2, de 4ran*his et al.,
2, Goto et al., 2+ and 1ryns et al., +!!!%.
The +cl1 family proteins are the central regulators of the
mitochondrial pathway. +cl1 is an inhibitor of apoptosis. +cl1 and its human
homolog introduce a new category of oncogenes that act by decreasing cell
death. @ver e!pression of +cl1 promotes oncogensis by repressing cell death
5;
and e!tending cell life. 0owever, overe!pression can also lead to retardation
of cell cycling via prolongation of the #2 phase of the cycle #3ebb et al.,
2& and Green 2 (eed, +!!5%.
The +cl1 family of intracellular proteins is the central regulator of
caspase activation, and its opposing factions of anti3 and pro3apoptotic
members arbitrate the life3or3death decision. The oncogenic activity of the
+cl1 gene is carried out via suppression of lymphocytic apoptosis or
programmed cell death. #1ory 2 Adams, 22 and (oumier et al., 22%.
+-41 is an important regulator of apoptosis, first identified from its
involvement in follicular + cell lymphoma, where the common t(::26)
translocation causes the activation of the +-41 oncogene. +-41 is now
recognised as a survival factor for many types of cell, notably neurons.
/!pression of +-41 is widespread during embryogenesis but is restricted to
long3lived cells in the adult. critical mediator of +-41 apoptosis is
interleukin32 beta3converting enAyme (I-/) a cysteine protease that
processes I432 beta during the inflammatory response #(oumier et al., 22%.
+-41 is a member of a multigene family (highly conserved
evolutionarily with viral homologues). @ther proteins in the family (+-4F,
+*, +F, +* etc) antagonise inhibition of apoptosis by binding to
+-41. 0ence the balance of various members of the +-4 family determines
the e!tent to which cell death is promoted or prevented. This model is
consistent with the findings of high levels of +-41 in a variety of solid
tumours #Jian" and Milner, 2,%.
poptosis can also be induced by a variety of cytokines e.g. T#F
beta family, which inhibit the proliferation of a wide variety of cell types that
6>
may undergo concomitant cell death. T#F beta induced apoptosis is blocked
in myeloblastic leukaemia cells by +-41 e!pressed at a level that does not
block but merely delays p753induced apoptosis. This may reflect the fact that
both T#F beta and p75 suppress +-41 but only p75 has the ability to
activate +F, thus deflecting the e!pression pattern towards apoptosis
#Se*/in et al., 22%.
ctive cell suicide (apoptosis) is induced by events such as growth
factor withdrawal and to!ins. It is controlled by regulators, which have either
an inhibitory effect on programmed cell death (anti3apoptotic) or block the
protective effect of inhibitors (pro3apoptotic). Many viruses have found a
way of countering defensive apoptosis by encoding their own anti3apoptosis
genes preventing their target3cells from dying too soon. ll proteins
belonging to the +cl31 family contain either a +02, +01, +05, or +06
domain. ll anti3apoptotic proteins contain +02 and +01 domains, some of
them contain an additional .3terminal +06 domain (+cl31, +cl3! (4), +cl3
w), which is never seen in pro3apoptotic proteins, e!cept for +cl3!('). @n the
other hand, all pro3apoptotic proteins contain a +05 domain (e!cept for +ad)
necessary for dimeriAation with other proteins of +cl31 family and crucial for
their killing activity, some of them also contain +02 and +01 domains (+a!,
+ak). The +05 domain is also present in some anti3apoptotic protein, such as
+cl31 or +cl3! (4). "roteins that are known to contain these domains include
vertebrate +cl31 (alpha and beta isoforms) and +cl3! (isoforms (+cl3!(4) and
+cl3!(')) #Poliseno et al., 22%.
ntiapoptotic + cell leukemiaDlymphoma (+-41) family
proteins are e!pressed in many cancers, but the circumstances under which
these proteins are necessary for tumor maintenance are poorly understood.
novel functional assay that uses +cl1 homology domain (+05) peptides to
62
predict dependence on antiapoptotic proteins was e!ploiteded , a strategy,
+05 profiling. +05 profiling accurately predicts sensitivity to +cl1
antagonist +T3959 in primary chronic lymphocytic leukemia (-44) cells.
+05 profiling also accurately distinguishes myeloid cell leukemia seCuence
2 (M-42) from +cl1 dependence in myeloma cell lines. It was shown that
the special sensitivity of -44 cells to +cl1 antagonism arises from the
reCuirement that +cl1 tonically seCuester proapoptotic +IM in -44. +T3
959 displaced +IM from +cl1Bs +053binding pocket, allowing +IM to
activate +F, induce mitochondrial permeabiliAation, and rapidly commit
the -44 cell to death. It was demonstrated that +cl1 e!pression alone does
not dictate sensitivity to +T3959. Instead, +cl1 comple!ed to +IM is the
critical target for +T3959 in -44. n important implication is that in
cancer, +cl1 may not effectively buffer chemotherapy death signals if it is
already seCuestering proapoptotic +053only proteins. Indeed, activator
+053only occupation of +cl1 may prime cancer cells for death, offering a
potential e!planation for the marked chemosensitivity of certain cancers that
e!press abundant +cl1, such as -44 and follicular lymphoma #Del Ga.io et
al., 20%.
The relationship between gene e!pression of +cl 1 and +a! and the
therapeutic effect in oral cancer patients had investigated. significant
correlation between +cl31D+a! gene e!pression ratio in the peripheral blood
mononuclear cells ("+M-s) from the patients, and the therapeutic effect of
radiation therapy These findings suggested that +cl31 and +a! gene
e!pression in "+M-s may be useful as a prognostic factor in oral cancer
patients #@shi/awa et al., 2$%.
/pstein3+arr virus (/+?), the cause of mononucleosis and cause of
+urkittBs lymphoma produces a protein similar to +cl1 and produces another
61
protein that causes the cell to increase its own production of +cl1. +oth these
actions make the cell more resistant to apoptosis (thus enabling the cancer
cell to continue to proliferate). /ven cancer cells produced without the
participation of viruses may have tricks to avoid apoptosis #8u et al., 2&%.
'ome +3cell leukemias and lymphomas e!press high levels of +cl1,
thus blocking apoptotic signals they may receive. The high levels result from
a translocation of the +cl1 gene into an inhancer region for antibody
production #Menende. et al., 2-%.
+cl13421 contributes to the classical tumor biological features
of #lioblastoma (#+M) such as intense apoptosis resistance and florid
necrosis, and may provide a target for enhanced therapeutic responsiveness
of this lethal cancer #Ste"h et al., 20%.
2A,1Amy* on*o"eneC
The c3myc gene was discovered as the cellular homolog of the retro
viral v3myc oncogene 1> years ago. The c3myc proto3oncogene was
subseCuently found to be activated in various animal and human tumors. It
belongs to the family of myc genes that includes +3myc, 43myc, .3myc, and
s3myc; however, only c3myc, 43myc, and .3myc have neoplastic potential
93e*hsler et al., +!!0 and 4a**hini 2 Penn, +!!5%$ Targeted homoAygous
deletion of the murine c3myc gene results in embryonic lethality, suggesting
that it is critical for development. 0omoAygous inactivation of c3myc in rat
fibroblasts caused a marked prolongation of cell doubling time, further
suggesting a central role for c3myc in regulating cell proliferation #Mateya/
et al., +!!0%.
65
+ovine papillomavirus type 2 (+"?32)3transformed mouse
fibroblast cell lines were analyAed via flow cytometry (F-M) for e!pression
of c3myc protein along with their *. content. 'ignificantly elevated levels
of the c3myc protein was present in some but not all of the transformed cell
lines. Kuantitation of c3myc protein in cell lines containing +"?32 *.
revealed that the tumorigenic cell lines e!pressed higher levels of the c3myc
protein #A"rawal et al., +!!-%.
The role of c3Myc in the cell cycle has been a confusing area due to
the collection of data from different e!perimental models, although it is well
established that c3myc is an early serum response gene. It should be noted
that models of serum or growth factor stimulation of starved cells primarily
address the #
>
D#
2
and #
2
D' transitions. Therefore, early studies implicated c3
Myc in the #
>
D#
2
transition. In cycling cells, however, the participation of c3
Myc in the cell cycle may be different. Furthermore, in anchorage3dependent
cell growth, c3Myc may affect other components of the cell cycle #Amati et
al., +!!5%.
It is proposed that c3Myc induces apoptosis through separate Bdeath
primingB and Bdeath triggeringB mechanisms in which Bdeath primingB and
mitogenic signals are coordinated. Investigation of the mechanisms that
underlie the triggering steps may offer new therapeutic opportunities
#Prender"ast, +!!!%.
The antiapoptotic effect of /pstein3+arr virus (/+?) was associated
with a higher level of +cl31 e!pression and an /+?3dependent decrease in
steady3state levels of c3ML- protein. lthough the /+? /+.32 protein is
e!pressed in all /+?3associated tumors and is reported to have oncogenic
potential, enforced e!pression of /+.32 alone in /+?3negative kata cells
66
failed to restore tumorigenicity or /+?3dependent down3regulation of c3
ML-. These data provide direct evidence that /+? contributes to the
tumorigenic potential of +urkitt lymphoma and suggest a novel model
whereby a restricted latency program of /+? promotes +3cell survival, and
thus virus persistence within an immune host, by selectively targeting the
e!pression of c3ML- #>n"rid et al., +!!!%.
Much recent research on c3Myc has focused on how it drives
apoptosis. c3Myc is widely known as a crucial regulator of cell proliferation
in normal and neoplastic cells, but until relatively recently its apoptotic
properties, which appear to be intrinsic, were not fully appreciated. Its death3
dealing aspects have gained wide attention in part because of their potential
therapeutic utility in advanced malignancy, where c3Myc is freCuently
deregulated and where novel modalities are badly needed. lthough its e!act
function remains obscure, c3Myc is a transcription factor and advances have
been made in characteriAing target genes which may mediate its apoptotic
properties #Herme/in", 2,%.
/ctopic e!pression of c3Myc (Myc) in most primary cell types

results in programmed cell death, and malignant transformation

cannot occur
without additional mutations that block apoptosis.

The development of Myc3
induced lymphoid tumors was studied. Myc can be upregulated in acute

myeloid leukemia (M4), but its e!act role in myeloid leukemogenesis

is
unclear. To study its role in M4, a murine stem

cell virus (M'-?) retroviral
gene transferDtransplantation system

was used to broadly e!press Myc in the
bone marrow of mice either alone

or in combination with antiapoptotic
mutations. Myc e!pression

in the conte!t either of rfDInk6a loss or +cl31
coe!pression

induced a mi!ture of acute myeloid and acute lymphoid
leukemias

(M4M44). In the absence of antiapoptotic mutations however,

67
all mice transplanted with 4SC%-4yc developed

M4 e!clusively. 4SC%-
4yc3induced M4 was polyclonal, readily

transplantable, possessed an intact
rf3p75 pathway, and did

not display cytogenetic abnormalities by spectral
karyotyping

analysis. 4astly, it was found that Myc preferentially stimulated

the growth of myeloid progenitor cells in methylcellulose. These

data
provided the first direct evidence that Myc is a critical

downstream effector of
myeloid leukemogenesis and suggested that

myeloid progenitors are
intrinsically resistant to Myc3induced

#Hui et al., 2&%.

68
III.Flow cytometry
Flow cytometry is a laser3based technology that is used to measure
characteristics of biological particles. This technology is used to perform
measurements on whole cells as well as prepared cellular constituents such as
nuclei and organelles #Melamed et al., +!!, ?ileney et al., +!!$ and
M*1oy, 22%.

The flow cytometer is an instrument for measuring scattered and
fluorescent light from single particles. The physics of the interaction of light
with single particles provides the scientific foundation for the design and
operation of the flow cytometer and for the critical evaluation of flow
cytometric data #S*orni/ et al., +!!-%.

Flow cytometry uses the principles of light scattering, light e!citation,
and emission of fluorochrome molecules to generate specific multi3parameter
data from particles and cells in the siAe range of >.7um to 6>um diameter.
-ells are hydro3dynamically focused in a sheath of phosphate buffer saline
("+') before intercepting an optimally focused light source. 4asers are most
often used as a light source in flow cytometry #?albot, +!!,%.
The technology of flow cytometry and the discovery of a method to
produce monoclonal antibodies have made possible the clinical use of flow
cytometry for the identification of cell populations. 4ight scatter is utiliAed to
identify the cell populations of interest, while the measurement of
fluorescence intensity provides specific information about individual cells.
Monoclonal antibodies (tagged) with the fluorescent dye are commonly used
for the identification of cell surface antigens and fluorescent dyes that
69
directly and specifically bind to certain components of the cell (i.e. *.) are
used for cell cycle analysis #(e*/enwald, +!!, and Sha7iro, +!!&%.
-ells or particles are prepared as single3cell suspension for flow
cytometric analysis. This allows them to flow single file in a liCuid stream
past a laser beam. s the laser strikes the individual cells. First light
scattering occurs that is directly related to structural and morphological cell
features. 'econd, fluorescence occurs if the cells are attached to a fluorescent
probe. Fluoresent probes are typically monoclonal antibodies that have been
con<ucated to fluorochromes; they can also be fluorescent stains reagents that
are not con<ugated to antibodies #Par/s and Her.enber", +!5! and
(e*henwald et al, +!!,%.
Fluorescent probes are reacted with the cells or particles of interest
before analysis; therefore, the amount of fluorescence emitted as a particle
passes the light source is proportional to the amount of fluorescent probe
bound to the cell or cellular constituent #(ad*liff and Jaros.es/i, +!!5%.
fter acCuisition of light scattering and fluorescence data for each
particle, the resulting information can be analyAed utiliAing a computer and
specific software that are associated with the cytometer #(ose et al,. +!!2
and 8on"obardiAGi)en, +!!2%.
There are two distinct types of flow cytometers that can be used to
acCuire data from particles. @ne type can perform acCuisition of light
scattering and fluorescence only. The other type is capable of acCuiring
scattering and fluorescence data but also has the powerful ability to sort
particles. +oth types function in a similar manner during acCuisition, for
e!ample F-'can (+ecton *ickinson), this eCuipped with an air Ncooled 27
6:
mw argon ion laser emitting at 6:: nm. Three fluorescence channels can be
measured as well as two light scatter parameters. The F-'can is also
eCuipped with a doublet discrimination module allowing the analysis of the
cell cycle. The F-'can is user3operated (after instruction) and is available
for use 16 hours per day #9andathil et al., 2&%.
0owever, sorting instruments have the powerful ability to physically
separate particles based on light scattering andDor fluorescent emission
characteristics. -ytometers were originally designed to sort, for e!ample
F-' caliber 2, 1 (+ecton *ickison), this used for analysis only. &nlike the
F-'can which is a dual laser system. The primary laser is an air3cooled 27
mw argon ion laser emitting at 6:: nm thus allowing two light scatter
parameters and three fluorescence channels to be measured. The second laser
is ared diode laser emitting at 857nm. Thus allowing for the e!citation of
other dyes such as allophycocyanin or to3pro35 with power Macintosh #6
running system ;.> and cell Kuest v 5.5. Thus, cytometers that perform
acCuisition without sorting are the most common of the two types #(ose et
al., +!!2%.
*.#rinciples o- -low cytometric instrumentation?
Flow cytometers are very comple! instruments that are composed of
four closely related systems. The fluidic system transports particles from a
suspension through the cytometer for interrogation by an illumination
system. The resulting light scattering and fluorescence is collected, filtered,
and converted into electrical signals by the optical and electronics system.
The data storage and computer control system saves acCuired data and is also
the user interface for controlling most instrument functions. These four
systems provide a very uniCue and powerful analytical tool for researchers
6;
and clinicians. This is because they analyAe the properties of individual
particles, and thousands of particles can be analyAed in a matter of seconds.
Thus, data for a flow cytometric sample are a collection of many
measurements instead of a single bulk measurement #(ad*liff and
Jarose.es/i, +!!5%..

0istograms are the simplest modes of data representation.
0istograms allow visualiAation of a single acCuired parameter. Mean
fluorescence and distributional statistics can be obtained based on markers
that the user can graphically set on the plot. Multiple histograms can be
overlaid on one another to depict Cualitative and Cuantitative differences in
two or more samples. Two3parameter data plots are somewhat more
complicated than histograms; however, they can yield more information.
Two3parameter dot plots of F'- vs ''- allow visualiAation of both light3
scattering parameters that are important for identifying populations of
interest. +ivariate fluorescent plots allow discrimination of dual3labeled
populations that might remain hidden if histograms were used to display
fluorescent data. Two3parameter plots that combine one light3scattering
parameter and a fluorescent parameter are useful for analyAing control
samples to elucidate the origin of nonspecific binding. *ata analysis is very
graphically oriented. /!perience and pattern recognition become important
when using two3parameter data plots for Cualitative as well as Cuantitative
analysis. The techniCue of gating or drawing regions on dual parameter light3
scatter plots allows one to e!clude information and e!amine the population
of interest by disallowing particles that might confound or interfere with
analysis. This is one of the fundamental uses for gating #(ad*liff and
Jarose.es/i, +!!5%.
7>
Flow cytometers can be described as four interrelated systems which
are shown in Fig$ 95.*:$ these four basic systems are common to all
cytometers regardless of the instrument manufacturer and whether or not the
cytometer is designed for analysis or sorting #Melamed et al., +!! and
8on"obardiAGi)en, +!!2 @wens 2 8o/en, +!!&%.
+A+A4luidi* systemC
The fluidic system is the heart of a flow cytometer and is responsible
for transporting cells or particles from a prepared sample through the
instrument for data acCuision Fig$ 95.3:$ The primary component of this
system is a flow chamber. The fluidic design of the instrument and the flow
chamber determine how the light from the illumination source ultimately
meets and interrogates particles. Typically, a diluent, such as phosphate
buffered saline, is directed by air pressure into the flow chamber. This fluid is
referred to as sheath fluid and passes through the flow chamber after which it
is intersected by the illumination source. Then, the sample under analysis, in
the form of a single particle suspension, is directed into the sheath fluid
stream prior to sample interrogations. The sample then travels by laminar
flow through the chamber #@rmerod, +!!-%.
72
Figure 95.*:? Facscaliblur -low cytometry instrument$
71
Figure 95.3:? Flow cytometer system 9Facscalibur: #@rmerod, +!!-%.
75
The pressure of the sheath fluid against the suspended particles
aligns the particles in a single file fashion. This process is called
hydrodynamic focusing and allows each cell to be interrogated by the
illumination source individually while traveling within the sheath fluid
stream #(ad*liff and Jaros.es/i, +!!5%.
The flow cell is the functional core of the fluidic system because it
presents cells in a single file for interrogation by the cytometer illumination
system. typical flow cell Fig$ 95.5: consists of a converging noAAle in
which sample is introduced at low flow rates into a larger laminar flow of
isotonic saline or sheath fluid. The cells in the sample follow the converging
streamlines and are hydrodynamically focused into alignment. The sample is
in<ected into the center of a sheath flow. The combined flow is reduced in
diameter, forcing the cell into the center of the stream. This the laser one cell
at a time. This schematic of the flow chamber in relation to the laser beam in
the sensing area #Phili7, 22%.
+A2A>llumination systemC
Flow cytometers use laser beams that intersect a cell or particle that
has been hydro dynamically focused by the fluidic system. 4ight from the
illumination source passes through a focusing apparatus before it intersects
the sample stream. This apparatus is a lens assembly that focuses the laser
emission into a beam with an elliptical cross3section that ensures a constant
amount of particle illumination despite any minor positional variations of
particles within the sample stream #6immermann and ?russ, +!0!%.
76
Figure 95.5:? Flow cytometers use the principle o- hydrodynamics
-ocusing -or presenting cells to a laser #Phili7, 22%$

77
Laser options
4ight and fluorescence are generated when the focused laser beam
strikes a particle within the sample stream. These light signals are then
Cuantitated by the optical and electronics system to yield data that is inter3
prOt able by the user #Sha7iro, +!!&%.
Two systems are used in flow cytometry to focus the illuminating
light to the point at which it intersects the cell stream. @ne type of system
uses a spherical lens to give a focal spot siAe of 5>3 8> Pm. The second
system uses a pair of crossed cylindrical lenses to focus the light to a sheet
about 21> Pm wide and 639 Pm deep #1ledat et al., 2-%.
Most flow cytometers utiliAe a single laser, however, some systems
support the simultaneous use of two or more different lasers. The most
commonly used laser is an argon ion laser that has been configured to emit
light in the visible range of the spectrum. 6::3 nm. 4aser emission is used
for most standard applications. The ma<ority of fluorochrom that are available
on the market today can be e!cited using this wavelength. Thus, laser is
e!cellent e!citation sources because they provide a single wavelength beam
that is also stable, bright, and narrow. 'ome type of lasers present in flow
cytometers can be turned to &.?. or other wavelengths. If the e!iting is not
tunable, then another laser source that emits the desired wavelength is
reCuired. t the measuring point in a typical flow cytometer the stream of
cells intersects a beam of light from a laser or arc lamp Fig$ 95.+:$ %hen light
interacts with biological particles some of the light is scattered out of the
incident beam and this scattered light may be collected over a range of angles
by detectors positioned around the measuring point #Gerstner et al., 2&%.
78
Figure 95.+:? A simpli-ied illustration o- Flow Cytometry #Gerstner et al.,
2&%.
79
+A,A@7ti*al and ele*troni*s systemC
The e!citation optics consists of the light source and the optical
components that serve to interrogate or e!cite the hydrodynamically focused
sample stream in the flow cell. The 6:: nm line of the argon laser is used as a
light source in many commercially available cytometers, but any light source
that provides the reCuisite intensity, e.g. , the mercury vapor or the !enon are
lamp can be used. @ptical components are used to e!pand, shape and focus
the light which then interacts with the sample in the flow cell. The flow cell
is usually made of CuartA and is designed to minimiAe diffraction and to
ma!imiAe the collection of the optical signals. The light source is often a
laser. 4aser is used because they provide a very concentrated and intense
beam of monochromatic character of the light is especially important in
making fluorescence measurements #?elford, 2-%.
The amount of light that is scattered by a cell is a comple! function
of its siAe, shape and refractive inde!. The sensitivity of light scattering to
each of these factors is dependent upon the range of angles over which the
scattered light. The light scattered at small angles (i.e. forward light scatter)
could be successfully used to determine relative volume distributions for
populations of cells #3an" et al., 2-%.
4ight scattered and emitted in all directions (58>Q) after the laser
beam strikes an individual cell or particle that has been hydrodynamically
focused. The optical and electronics system of a typical flow cytometer is
responsible for collecting and Cuantitating at least five types of parameters
from this scatter light and emitted fluorescence. Two of these parameters are
light scattering properties. 4ight that is scattered in the forward direction (in
the same direction as the laser beam) is analyAed as one parameter, and light
scattered at ;>Q relative to the incident beam is collected as a second
7:
parameter. Forward3scattered (F'-) light is a result of diffraction. *iffracted
light provides basic morphological information such as relative cell siAe that
is referred to as forward angle light scatter (F'-). 4ight that is scattered at
;>Q to the incident beam is the result of refracted and reflected light. This
type of light scatters is referred to as side3angle light scatter (''-). This
parameter is an indicator of granularity within the cytoplasm of cell as well
as surface membrane irregularities to topographies #Phili7, 22%.
Most current laboratory bench3top flow cytometers are capable of
detecting fluorescence from three different regions of the visible spectrum.
-utometers are optically conCuered to detect a narrow range of wave lengths
in each region. Fluorescence emission is detected simultaneously along with
F'- and ''- data; therefore, up to five parameters can be simultaneously
measured for each analyAed sample #8on"obardiA Gi)en, +!!2%
Fluorescence is detected using networks of mirrors, optics, and beam
splitters that direct the emitted fluorescent light toward highly specific optical
filters. The filters collect light within the range of wave lengths associated
with each of the three fluorescent channels. Filtered light is directed toward
photo multiplier tubes or "MTs for conversation into electrical signals
#?elford, 2-%.
+A-AData stora"e and *om7uter *ontrol systemC
fter light scattering and fluorescence is converted to electrical
signals by the optical and electronics system, the information is converted
into digital data that the computer can interpret. The signals generated from
cells or particles are referred to as events and are stored by the computer
#(ose et al., +!!2%.
7;
fter the different signals or pulses are amplified they are processed
by an nalog to *igital -onverter (*-) which in turn allows for events to
be plotted on a graphical scale (@ne "arameter, Two parameter 0istograms).
Flow cytometry data outputs are stored in the form of computer files
#(ad*liff and Jaros.es/i, +!!5%.
0istogram files can be in the form of one3parameter or two3
parameter files. 0istogram files consist of a list of the events corresponding
to the graphical display specified in your acCuisition protocol. one3
parameter histogram is a graph of cell count on the y3a!is and the
measurement parameter on !3a!is. ll one3parameter histograms have 2,>16
channels. These channels correspond to the original voltage generated by a
specific HlightH event detected by the "MT detector. In other words, the *-
assigns a channel number based on the pulse height for individual events.
Therefore, brighter specific fluorescence events will yield a higher pulse
height and thus a higher channel number when displayed as a histogram.
graph representing two measurement parameters, on the !3 and y3a!es, and
cell count height on a density gradient. This is similar to a topographical map.
Lou can select 86 or 178 channels on each a!is of two3parameter histograms.
"article counts are shown by dot density or by contour plot. Fig$ 95./:
#(oederer et al., 2-%.
4ist3mode files consist of a complete listing of all events
corresponding to all the parameters collected, as specified by your acCuisition
)rotocol. This file follows a format specified by the F-' 5.> standard. ,aw
list3mode data files can be opened or replayed using any program designed
for analysis of flow cytometry data. Lou should keep in mind that a )rotocol
serves as a template. It allows you to collect specified )arameters (i.e. F4',
F42, F41, etc.), and how these parameters are displayed. )rotocols also serve
8>
to determine how the data is gated, and contains all the 5egions from which
your statistics will be generated. In addition, )rotocols contain other specific
information that serves as direct interface between the computer workstation
and the cytometer. These pertain to high voltage settings for the "MT
detectors, gains for amplification of linear parameters, sample flow rates,
fluorescence compensation, discrimination settings, etc. @nce your data has
been collected and written into a list3mode file you can replay the file either
using the specific )rotocol used for collection or any other program
specifically designed for analysis of flow cytometry data. 0owever, you
should keep in mind that you can only ad<ust 5egions, 3ating, and
)arameters to be displayed. 'ettings such as amplification, fluorescence
compensation, etc., can not be modified. Therefore, when collecting data
make sure that your instrument settings are correct. Finally, if you open your
listmode files using a programs such as Flow=o Fig$ 95.0:, %I.MI*I, andDor
/!"@ you will have to specify parameter displays, and create 5egions and
3ating corresponding to the )rotocol used for collecting the data #M*1oy,
22%.
82
Figure 95./:? )wo parameter histogram and dot plot displaying FL*.
FI)C on the @ a@is and FL3.#! on the y a@is #(oederer et al., 2-%.
81
Figure 95.0:? FlowAo program #M*1oy, 22%.
85
The number of events acCuired for each sample is always determined
before analysis and is usually set using software designed to control
cytometer operation. conventional acCuisition value is 2>>.>>> events per
sample. 0owever this value may vary and range upward of events per sample
depending on the e!perimental ob<ective #Melamed et al., +!!%.
3.Data analysis?
*ata analysis is a very critical part of any e!periment that utiliAes
flow cytometry. *ata is analyAed using a computer and software is usually
specific to flow cytometric data and is often part of the same computer
system that is used to control the instrument during acCuision. The most
common display is a histogram. typical histogram data plot is shown in
Fig$ 95.1" 5.6: #AbuA Absi et al., 2,%.
It is also possible to display two parameters simultaneously such as
F'- vs ''c or F42 vs F41. For two parameter plots, data from a population
of individual particles can be displayed in the form of dots or as contours
shown in Fig$ 95.2: #Par/s and Hen.enber", +!5!%.
-ontour density plots display the data from a population of cells as a
series of concentric lines that correlate to different cell or particle densities
within the a!es. *ot3plots are probably the most common type of two3
parameter plots, and they are also the easiest to understand #(obinson,
+!!,%.
86
Figure 95.1:? Analysis pulse width (ersus pulse height or area we can
eliminate the maBority o- 4 doublets that appear as 3 #AbuA Absi et al.,
2,%.
87
Figure 95.6:? D;A histogram #AbuA Absi et al., 2,%.
88
Figure 95.2:? D;A histogram 9aneuphliod population: #Par/s and
Hen.enber", +!5!%$
89
I7. Applications o- -low cytometry
Flow cytometers are very comple! instruments that are composed of
four closely related systems. They provide a very uniCue and powerful
analytical tool for researchers and clinicians. Therefore, the flow cytometer is
widely used in research as well as in clinical immunology and hematology to
perform rapid immunophenotyping, cell sorting, and *. analysis
#8on"obardiAGi)en, +!!2 and 'o"h 2 Dulin", 2&%.
Flow cytometry is used for immunophenotyping and *. content of
a variety of specimens including whole blood, bone marrow, serous cavity
fluids, cerebrospinal fluid, urine and solid tissues. -haracteristics that can be
measured include cell siAe, cytoplasmic comple!ity, *. or ,. content,
and a wide range of membrane3bound and intracellular portents and sorting
the cells #(e*htnwald, +!!,, @rmerod, +!!- and Assun*ao et al., 2&%.
The use of flow cytometry in the clinical laboratory has grown
substantially in the past decade. This is attributed in part to the development
of smaller user friendly, less e!pensive instruments and a continuous increase
in the number of clinical applications as shown in )able 9+.*: #'rown and
3ittwer, 2%.
Flow cytometry provides rapid analysis of multiple characteristics of
single cells. The information obtained is both Cualitative and Cuantitative.
%here as in the past flow cytometers were found only in larger academic
8:
centers, advances in technology now make it possible for community
hospitals to use this methodology #@rfao et al., +!!& and M*1oy, 22%.
)able 9+.*:? Common clinical uses o- -low cytometry #'rown and 3ittwer,
2%.
Field Clinical application
Common characteristic
measures
Immunology 0istocompitability crossmatching IgM, Ig#
Transplantation re<ection -*5, -I,-&4TI.# @IT5
043+19 detection 043+19
Immunodeficiency studies -*6, -*:
%ncology *. content and ' phase of tumors *.
Measurements of profilation markers Ii389, "-.
Hematology 4eukemia and 4ymphoma phenotyping 4eukocyte surface antigens
Identification of prognostically important
subgroups
Tdt, M"@
0ematopiotic progenit or cell enumeration -*56
*iagnosis of systematic mastocytosis -*17,-*8;
,eticylocyte enumeration ,.
utoimmuneand alloimmune disorders
ntiplatelats disorders Ig#, IgM
nti3neutrophils antibodies Ig#
Immune comple!es -omplement, Ig#
Feto3maternal hemorrge Cuantification 0emoglobin F, rhesus *
8;
Immunohematology /rthrocyte surface antigens
ssessment of leukocyte contamination of blood
products
Forward .* 'ide scatter
enetic
disorders
".0 -*77,-*7;
Functions of cells can be defined through the application of
fluorochrome dyes that have an affinity for cellular components.
Traditionally, common clinical applications are immunophenotyping of cells
of the hematopoietic system with fluorescent3labeled antibodies raised
against specific cell surface proteins #Da)is et al., 22%.
@ther approaches have been used to elucidate changes in cell
function and *. content. /!amples of clinical applications in eCuine
patients include immune3mediated hemolytic anemia, immune3mediated
thrombocytopenia (IMT), chronic inflammatory disease, and neoplasia
#Da)is et al., 22%.
The great advantage of flow cytometry is that its applications are
highly amenable to standardiAation. The efforts that have been made for flow
cytometric applications in four ma<or fields of clinical cell analysis: -*6M T3
cell enumeration, -*56M hematopoietic stem and progenitor cell
9>
enumeration, screening for the 043+19 antigen and leukemiaDlymphoma
immunophenotyping #9eeney et al., 2-%.
The diagnosis of many primary immunodeficiency diseases reCuires
the use of several laboratory tests. Flow cytometry is applicable in the initial
workup and subseCuent management of several primary immunodeficiency
diseases #>lloh, 2-%.
*. Cell cycle analysis?
The measurement of the *. content of cells was one of the first
ma<or applications of flow cytometry and is still one of the biggest
applications in this laboratory today #Albro et al., +!!,%.
Flow cytometry offers the possibility to study several parameters
simultaneously, e.g. cell cycle modulation, mode of cell death, activity of
mitochondria. The phases of the cell cycle were determined and the induction
of apoptosis and necrosis was demonstrated by anne!in binding, analysis of
mitochondrial membrane potential and *. strand breaks #?us*h and
S*hwab, 2&%.
*. ploidy and proliferative activity ('3phase fraction) are the two
biological parameters commonly measured by *. flow cytometric
analysis. The prime purpose of most studies is the investigation of the
prognostic value of *. flow cytometry in addition to the information
provided by conventional clinicopathological factors known to affect disease
prognosis. The general statement, for tumors in the same histopathological
stage of the disease, is that diploid andDor low proliferative tumors have a
more favourable prognosis than aneuploid andDor high proliferative tumors,
92
suggesting an important role of *. flow cytometry in the assessment of
tumor behaviour and in the outcome evaluation of the disease. #Pinto et al.,
22%.
+A+AStainin" 7ro*edureC
The preparation and staining of cell suspension are the ma<or factors
determining the validity and reproducibility of flow cytometric analysis.
There is no flow cytometric staining procedures which is universally
accepted and a number of different protocols have been advocated. ll of the
*. specific stains and the phenanthridinium dyes have been used for total
*. staining of chromosomes. The former group has the potential
disadvantage that &? e!citation is reCuired but this constitutes no problem
for mercury arc lamp based system or those with a laser tunable to &? lines
#Hartwell, +!!5%.
The *. fluorochromes in current use were classified into groups.
'tains that intercalate with double stranded nucleic acid and include ("I),
(/+) and (@);and *. specific stains that have a particular specificity for
moieties in *. and include mithramiycin (MM-), ethedium
bromideDmithramicin (/+DMM-), a bisbenAimidaAole derivative and 638
diamino313 phenylindole (*"I) #?aylor and Mithor7e, +!5%.
"ropidium iodide is bound to polynucleotide both by means of
intercalation. This is only affected to a limited e!tent by high ionic strengths
and electrostatically to secondary binding sites. The binding contents of these
later sites are greatly depending on the ionic strength of the medium and can
be eliminated by using sufficiently high ionic strengths. nd also have
optimal e!citation at a 6:: nm laser and produce good results with ,.se
treatment #Hartwell, +!!5%.
91
+A2AE)aluation of D<A histo"ramC
The *. histogram is a very simple data set which
characteristically contains two peaks separated by a trough Fig.
(632). The first peak, which is usually the larger, corresponds to cells with
#>D#2 *. content and the second, which should be at double the
fluorescence intensity of the first, corresponds to cells with #1MM *.
content. ny cell scored in the trough has a *. content intermediate
between #2 and #1MM and these usually represent cells in '3phase. In a
perfect data set, which doesnBt e!ist, all #2 and #1MM cells would be scored
in single channels and any cells between or immediately ad<acent to these
would be in '3 phase #Henderson, +!!5%.
'ince the cell material always contained normal diploid cells such as
leukocytes or normal kidney cells, these were used as an internal standard
and regarded as diploid (1-) #?ribu/ati, +!5-%.
%hen in doubt, lymphocytes should be added to establish the diploid
*. value. 0uman Ficol3prepared lymphocytes, fi!ed in ethanol, were used
as e!ternal standard. The magnitude of the signal was ad<usted so as to have
the standard diploid peak in a certain channel. In necessary the illumination
was ad<usted so that the coefficient of variation (-?) of the resulting
lymphocyte diploid peak was less than 5E Fig$ 9+.*:$ ll cell population with
#2 ma!imum deviating less than 2>E form the standard value were regarded
as diploid #Gustafson, +!52%.
@nly #>D#2 peak is observed in *. diploid. broad peak
described by a large coefficient of variation may obscure a second peak. The
coefficient of variation of the #>D#2 peak must be less than 7E for single
cell suspensions prepared from freshD froAen tissues, and less than :E for
95
nuclear suspension prepared from fi!ed, paraffin embedded specimens.
%here a diploid peak only is observed, one should ensure that tumor cells are
present in the clinical sample analyAed #3einber", +!!$%.
Full width at half ma!imum(c3a)
-.?. G R SSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSS 61.7 E
96
"eak (or mean) channel(b)
Figure 9+.*:? Coe--ecient o- 7ariation 9C$7$: #Gustafson, +!52%$
n aneuploid cell population was considered to be present when a
distinct peak was found constituting at least 1.7E of the total cell material
and deviating more than 2>E from the diploid standard. *. aneuploid is
reported when at least two separate #>D#2 peaks are demonstrated. For some
samples the diploid normal peak might be almost non e!istent; hence care
should be taken to assign peaks #@rmerod, +!!-%.
The degree of ploidy of this cell line was calculated by relating the
#2 ma!imum of these cells to *. of the diploid #2 cells which are always
present. These diploid cells can be leukocytes, fibroblasts, normal urothelial
cells.
3 channel o- aneuploid peak
Degree o- ploidyC DDDDDDDDDDDD
Channel number o- diploid peak
The degree of aneuploid was determined also by the *. inde!
which represents the ratio of fluorescence intensity of aneuploid cells to the
diploid cells. The *. inde! of a diploid tumor is 2.>, whereas, aneuploid
tumors are designated by progressively higher indices #GDtte, 2+ and
Gorden et al., 2,%.
97
/stimation of the proportion of #2, ' and #1MM cells made by
automatic integration of the cells in corresponding channels in the multi
channel analyAer. The values are corrected for background noise. To estimate
the proportions of cells in the '3phase of aneuploid tumor cell lines where
cells from the cell line coincide, the normal cell lines are subtracted.
-alculation of the phase distribution reCuires a minimum of about 2>>> cells
combined with a low background noise resulting from cell fragments. These
calculations may also be rendered more difficult when the peaks for the
deploid or aneuploid cell population, partially or completely overlap. @n the
other hand, there is no ambiguity in any of these cases to establish an
aneuploid cell line. n additional proof of the e!istence of an aneuploid
hyperdiploid cell line is the e!istence of a #1 peak occurs to the e!treme
right of the histogram without interfering with the normal cell population
#'rown and 3ittwer, 2%.
n aneuploid cell population with a tetraploid amount of *. was
considered to e!ist when a peak e!ceeded the #1M M peak found in normal
cells by three standard deviations or more. To Cuantitate the number of nuclei
normally found in the 6- or #1D M peak, a number of control tissues were
studied. The mean percentage of nuclei in the 6- peak were 1.96M 2.62
(standard deviation) for nuclei e!tracted from fresh normal lymphocyte.
These control data provide a firm basis for using greater than 2>E of nuclei
in the 6- peak as a criterion for *. polyploidy in the specimens
#(abino)it*h, +!!-%.
3. Immunophenotyping Applications?
The most common applications of flow cytometry are measurement
of *. content in tumors and immunophenotyping of haematopoietic
98
malignancies. Flow cytometry has shown to be a suitable method for
immunophenotyping of canine lymphomas #1ulmsee and <alte, 22%.
Immunophenotyping of abnormal cells is now considered a
fundamental tool to establish the cell lineage assignment and to obtain a more
precise identification of the various cell subtypes. *iagnostic
hematopathology depends on the applications of flow cytometric
immunophenotyping and immunohistochemical immunophenotyping
combined with the cytomorphology and histologic features of cases. The
availability of monoclonal antibodies directed against the surface proteins
permits flow cytometric analysis of erythrocytes, leukocytes and platelets
#'rown 2 3ittwer, 2, 1hianese, 22 and Dun7hy, 2-%.
Multiparameter flow cytometry with optimally selected antibody
combinations has e!panded the use of this techniCue beyond traditional
applications in hematopathology. +y analyAing Cualitative patterns of antigen
e!pression on discrete populations or Hclusters,H one can detect
immunophenotypic aberrancy in specific cell populations relative to normal
and reactive populations. /valuation of patterns of antigen e!pression can
also be used to supplement conventional methodologies in the diagnosis and
subclassification of certain types of hematologic neoplasia. Finally, the
diagnosis of some congenital disorders affecting the hematolymphoid system
can be facilitated by the detection of characteristic immunophenotypic
changes #9roft, 2-%.
2A+AErythro*yte analysisC
Tests that appear to have the greatest potential for routine application
of flow cytometry include reticulocyte and reticulated platelet enumeration,
detection of erythrocyte3bound immunoglobulin, immunophenotyping of
99
leukemias and lymphomas, and bone marrow differential cell counting
#'rown and 3ittwer, 2 , 3eiss, 22%.
Flow cytometric methods were first applied to laboratory
hematology with the improvement in reticulocyte counting and the creation
of the immature reticulocyte fraction for better anemia evaluation and
therapeutic monitoring #Da)is, 2+%.
9:
2A2AH>: monitorin"C
More than 57 million people in developing countries are living with
0I? infection. %hile drug prices have dropped considerably, the cost and
technical comple!ity of laboratory tests essential for the management of 0I?
disease, such as -*6 cell counts, remain prohibitive. .ew, simple, and
affordable methods for measuring -*6 cells that can be implemented in
resource3scarce settings are urgently needed #Dieye et al., 2&, 3al/er et
al., 2& and Pattana7anyasat 2 ?ha/ar, 2&%.
2A,A>mmuno7henoty7in" of leu/emiasC
Immunophenotyping has become common in the diagnosis and
classification of acute leukemias and is particularly important in the proper
identification of cases of minimally differentiated acute myeloid leukemia. To
evaluate the immunophenotype of adult M4, cases were studied by
cytochemical analysis and by flow cytometry with a panel of antibodies
#9halidi et al., +!!5%.
-haracteriAation of leukemias by immunotyping is particularly
helpful when the morphology is difficult to interpret. The ma<or advantage of
using immune markers by flow cytometry is the identification of particular
leukemia subtype, not recogniAed by morphologic criteria, which may have
prognostic significance #(e.aei et al., 2,%.
Flow cytometric immunophenotypic analysis allowed to establish
diagnosis in cytomorphologically unclassified cases, identify acute mi!ed3
lineage leukemias (M44) with a freCuency similar to that reported in other
series, and confirm the heterogeneity of acute leukemia (4) #Piedras et al.,
+!!0 and GDtte, 2+%.
9;
Flow cytometry may be used to detect minimal residual disease
(M,*) in acute lymphoblastic leukemia because leukemic cells often display
aberrant phenotypes when compared to normal cells. Flow cytometry is a
sensitive and specific method for detecting M,* of childhood 44, and
could predict the coming relapse #6han" et al., 2&%.
%ith the advent of monoclonal antibodies and a uniform
nomenclature system defining antibody reactivity in terms of clusters of
differentiation (-*), an independent means of characteriAing acute leukemias
using cellular antigen e!pression has evolved. Immunophenotyping is usually
performed using immunofluoresence techniCue and is complementary to the
light microscopic based morphologic classification. This is especially true of
the lymphoid leukemias where morphology and cytochemistry cannot
distinguish among different lineage of lymphoid cells, such as + versus T
cells. %ith Immunophenotyping lineage is assigned using a panel of
monoclonal antibodies that identify the e!pression of cell surface antigens.
The panel of monoclonal reagents must include antibodies reactive with both
myeloid and lymphoid cells to distinguish between the two most important
groups. The reactivity pattern of the leukemia cells for all reagents is then
e!amined for the final assignment of lineage: +3 lymphoid, T3 lymphoid,
myeloid or undifferentiated #Masla/ et al., +!!-%.
-omparative studies of cell surface antigen e!pression between
normal and leukemic cells indicate that most if not all leukemias e!press
phenotypes that are not observed in most normal maturing cells. This
aberrant e!pression of cellular antigens suggests that leukemias are not
proliferations of cells arrested at one state of normal maturation; rather
leukemic cells maintain a genetic program that can produce e!pression of
:>
antigens of any lineage. .early all laboratories performing
immunofluoresence analysis use different reagents #?ersta77en et al., +!!+%.
2A-AEuantifi*ation of stem *ellsC
2>> years ago, hematopoietic stem cells were postulated as blood
lymphocyte3like cells. %ithin the last 1> years, the freCuency of autologous
and allogeneic transplantation of hematopoietic stem cells has increased.
0ematopoietic growth factors allow the stem cellsB mobiliAation from the
bone marrow into the peripheral blood. Kuantification of these hematopoietic
stem cells by means of flow cytometry can be achieved within hours
#Golds*hmidt et al., 2,%.
Flow cytometry has become the ma<or techniCue for the Cuality
control of stem cell3containing products such as apheresis concentrates, bone
marrow or cord blood #Grieson et al., +!!&%. 'tem cells can be easily
identified with flow cytometry due to their uniCue characteristics. They
demonstrate a medium level of -*56 e!pression, a low level of -*67
e!pression and a low forward side scattered #Jennin"s 2 4oon, +!!0 and
Masla/ et al., +!!-%.
2A&APlatelet analysisC
The analysis of platelets by flow cytometry is becoming more
common in both research and clinical laboratories. "latelet3associated
immunoglobulin assays by flow cytometry can be direct or indirect assays,
similar to other platelet3associated immunoglobulin immunoassays. In
autoimmune thrombocytopenic purpura, free serum antibodies are not found
as freCuently as platelet3bound antibodies #Ashman et al., 2,%.
:2
Immunofluorescent flow cytometry was used to measure the
percentage of activated platelet populations (-*81", -*85), the percentage
of plt3monocyte aggregates (pma) (-*62D-*67), and activated monocytes
(-*22b, -*26, -*28) in the blood #Panasui/ et al., 2&%.
2A$A?estin" for H8AA'20C
0uman leukocyte antigen +19 (043+19) is a ma<or
histocompatibility comple! class 2 molecule that is strongly associated with
the disease ankylosing spondylitis. The performance of the two flow
cytometric antigen assays depends on the antibody used and the positive
cutoff values assigned #Sei77 et al., 2&%. flow cytometric 043+19 test
is much faster than the classical microcyto!icity test #Jennin"s and 4oon,
+!!0 and GDtte, 2+%.
biannual e!ternal Cuality assurance scheme for flow cytometric
typing of the 043+19 antigen is operational in The .etherlands and
+elgium since 2;;7. For flow cytometry, the most widely monoclonal
antibody used was F*9>7, followed by #'267.1 and +-3m5. The ma<ority
of laboratories used more than 2 anti3043+19 monoclonal antibody for
typing #Sei77 et al., 2&%.
5. MaBor applications o- apoptosis analysis?
There are many ways of detecting apoptosis by flow cytometry.
poptotic cells can be recogniAed by a characteristic pattern of
morphological (cell shrinkage, cell shape change, condensation of cytoplasm,
nuclear envelope changes, nuclear fragmentation, loss of cell surface
structures, apoptotic bodies, cell detachment and phagoctosis of remains),
:1
biochemical and molecular changes (free calcium ion rise, bcl1D+a!
interaction, cell dehydration, loss of mitochondrial membrane potential,
proteolysis, phosphatidylserine e!ternaliAation, lamin + proteolysis, *.
denaturatuin, 7>35>>kb cleavage, intranucleosomal cleavage and protein
cross3linking #Huban/ et al., 2- and 8iu et al., 2-%.
The methods of detecting apoptosis by flow cytometry are based on
the measurement of light scatter, the detection of changes in the plasma
membrane, the analysis of cell organelles or the sensitivity of *. to
denaturation #Sedla/ et al., +!!!%.
,A+AA7o7tosis li"ht s*atterC
s cells die or become apoptotic the refractive inde! of the internal
cytoplasm becomes more similar to that of the e!tracellular medium this
manifests itself as a reduction in forward scatter signal. t the same time,
intracellular changes and invagination of the cytoplasmic membrane lead to
an increase in side (or orthogonal or ;>Q) scatter. If a dead cell discriminatory
dye is added, cells that have become permeable can be identifying. In this
way low level resolution of dead and apoptotic cells can be get. number of
dead cell dyes are available for use and the one used will depend on any other
fluorochromes that are being measured. 'ome e!amples are; 'yto! #reen
(6::nm e!citation; green fluorescence emission), "ropidium Iodide (6::nm
e!citation; orangeDred fluorescence emission), 93minoactinomycin3* (93
*) (6::nm e!citation; red fluorescence emission) and T@3",@35 (855nm
e!citation; red fluorescence emission) #1ohen and AlA(ubeai, +!!&%.
:5
,A2AA7o7tosis D<A analysisC
*uring apoptosis, calcium and magnesium dependent nucleases are
activated which degrade *.. This means that within the *. there are
nicks and fragmentation. %e can detect these in three ways using *.
analysis to look at the sub #2 peak, using strand break labeling (T&./4) to
detect broken *. or using 0oechst binding to detect *. conformational
changes #Ma=ino and Joris, +!!&%.
The sub3#2 Fig$ 9+.3) method relies on the fact that after *.
fragmentation, there are small fragments of *. that are able to be eluted
following washing in either "+' or a specific phosphate3citrate buffer. This
means that after staining with a Cuantitative *. Nbinding dye, cells that
have lost *. will take up less stain and will appear to the left of the #2
peak. The advantage of this method is that it is very rapid and will detect
cumulative apoptosis and is applicable to all cell types #Dar.yn/iewi*.,
+!!0%.
0owever in order to be seen in the sub #2 area, a cell must have lost
enough *. to appear there, so if cells enter apoptosis from the ' or #1DM
phase of the cell cycle or if there is an aneuploid population undergoing
apoptosis, they may not appear in the sub #2 peak #S*hwart. and @sborne,
+!!,%.
:6
Figure 9+.3:? Sub * peak by propidium iodide staining #Dar.yn/iewi*,
+!!0%$
:7
lso cells that have lost *. for any other reason e.g. death by some
other form of oncosis, will appear in the sub #2 region so we have to be
careful about how we define the sub #2 peak #<i*oletti et al., 2+%.
,A,AA7o7tosis *ell membrane analysisC
In normal cells, phosphatidylserine ("') residues are found in the
inner membrane of the cytoplasmic membrane. *uring apoptosis, the "'
residues are translocated in the membrane and are e!ternaliAed. In general
though not always, this is an early event in apoptosis and is though to be a
signal to neighboring cells that a cell is ready to be phagocytosed #(obinson,
+!!,%.
nne!in3? is a specific "'3binding protein that can be used to detect
apoptotic cells. nne!in ?3 is available con<ugated to a number of different
fluorochromes. /arly apoptotic cells are anne!in positive but "I negative.
+ecause the cells arenBt fi!ed we can e!clude dead cells and it is possible to
add further markers if the cytometer set up are appropriate. s with all live
cell assays, we have to remember that we are only looking at a snapshot of
the cells as they are at time of analysis and generally all apoptotic
e!periments should be performed over a time course; Fig$ 9+.5: #?elford et
al., 2-, Hombur" et al., +!!& and :ermes at al., +!!&%.
:8
Figure 9+.5:? !arly apoptotic cells are anne@in positi(e but 9in this case:
#I 9negati(e) #?elford et al., 2-%.
:9
0oechst 55561 is a *.3binding dye that is able to Cuantitatively
stain the *. of live cells. 0owever it has also been found that if the
concentration of 0oechst is low, the apoptotic cells take up the 0oechst more
rapidly. If we also add "I or T@3",@35 we can specifically identify the dead
cells. This is a rapid and Cuantitative method but reCuires the use of a &?
laser. The advantage of using T@3",@35 is that cell phenotyping using FIT-3
and "/3labelled antibodies is also possible. Thymocytes labelled with -*63
"/ and -*:3FIT- can be assessed for apoptosis using 0oechst and T@3
",@35 #9oo7man et al., +!!-%.
third way of assessing the membrane changes in apoptosis is to use
L@3",@32 (Molecular "robes). s this fluorochrome emits in the green, it
can be combined with propidium iodide to identify dead cells. The rationale
here is that cells in early apoptosis are unable to pump out L@3",@32 but are
still not permeable to other dead cells discriminatory dyes #9oo7man et al.,
+!!-%.
,A-AA7o7tosis en.yme analysisC
Two genes (ced35 and ced36) were crucial to the process of
apoptosis. The ced36 gene product has homologues in mammalian cells,
especially a family of cysteine proteases that are now known as caspases.
There are a number of caspases in mammalian cells that have been shown to
be involved in the early stages of apoptosis e.g. (caspase1, caspase5, caspase
8, caspase ; and caspase 2>). The functions of these enAymes are not yet
entirely clear but it appears that after an initial signal to the cell to undergo
apoptosis, they may be responsible for the activation, amplification and
e!ecution of the apoptotic cascade #1ohen and AlA (ubeai, +!!&%.
::
+ecause of the central importance of the caspases in apoptosis, their
detection by flow cytometry has become widespread. %e can detect the
activity of enAymes implicated in apoptosis in three ways; by detecting the
active form of the enAyme using a specific antibody #Smolews/i et al., 22%,
by using a fluorochrome labelled peptide that binds to the active site of the
enAyme #Po.arows/i et al., 2,% and by using a non3fluorescent substrate
for the enAyme which yields a fluorescent product if the enAyme is active
#?elford et al., 2-%.
,A&AA7o7tosis or"anelle analysisC
*uring apoptosis there is often a collapse of the mitochondrial
membrane potential. This can be detected in a number of ways by flow
cytometry. Two dyes in particular are useful3 -MF,os (also known as Mito
tracker ,ed from Molecular probes) and 4*s3972 (from /!ction). -MF,os
has a chloromethyl group which allows accumulation in active mitochondria.
4ive cells that have active mitchondria are able to take up -MF,os but in
cells that are undergoing apoptosis, the mitochondrial membrane potential
decreases which means less dye accumulates in the mitchondria leading to a
decrease in fluorescenc #1ha7man et al., +!!&%.
+. Detection o- apoptotic markers?
*etermination of p75 e!pression by immunohistochemistry (I0-)

has been incorporated into routine practice and its reliability

has been
consolidated. 0owever, flow cytometric (F-M) analysis

might represent an
important ob<ective and rapid approach. F-M may provide important
information about p75 protein e!pression

in the different subpopulations and
cell cycle phases. In most breast, lung,

and colon aneuploid tumors (99E),
:;
p753positive cells were detected

only in the subpopulations with abnormal
*. content #El)ira et al., +!!5%.
+ovine papillomavirus type 2 (+"?32)3transformed mouse fibroblast
cell lines were analyAed via flow cytometry (F-M) for e!pression of p75 and
c3myc proteins along with their *. content. t least ;,>>>32>,>>> p75 or c3
myc protein molecules per cell were detected in the transformed tumorigenic
cell lines. These results show that Cuantitative F-M can be reliably used to
detect very low levels (5,>>> molecules per cell) of specific protein, and
F-M is a useful tool to study the virus3induced changes in the levels of
nuclear proteins within a cell population and in tumorigenesis #A"rawal et
al., +!!-%.
In human follicular lymphoma, nalysis of transgenic +cl1
e!pression used biotinylated

+cl132>> monoclonal antibody for the surface
phenotyping of hematopoietic

cells by flow cytometry.

-ells (2>
8
per

analysis)
were stained with relevant antibodies labeled with

fluorochromes (fluorescein
isothiocyanate TFIT-U, phycoerythrin

T"/U, or cyanin 7 T-y7U) or biotin using
2E normal rat serum

to block Fc receptors. 'treptavidin con<ugated to FIT-

or "/ was used as a secondary

reagent for biotinylated antibodies. nalyses
were performed

on a 4ife 'ciences ,esearch (4',) or a F-'tar II flow
cytometer

(+ecton *ickinson, 'an =ose, -) #AleFander et al., 2-%.
The e!pression of bcl31 was e!amined by multicolor flow
cytometry in samples including lymph node or other tissue biopsy specimens
containing follicular lymphoma (F4), reactive hyperplasia (,0), or other
malignant lymphomas, as well as bone marrow aspirates. For all of the
aforementioned reasons, a reliable flow cytometric assay for e!pression of
;>
bcl31 would be a useful additional techniCue for establishing a diagnosis of
F4. 0owever, the measurement of bcl31 by flow cytometric techniCues has
received only scant attention. It was described a 13color flow cytometric
assay using antibodies against bcl31 that demonstrated promise in the
recognition of F4. #James et al., 2,%.
;2
7.Flourescence in situ hybridi8ation
*.Introduction?
FI'0 provides researchers with a way to visualiAe and map the
genetic material in an individualBs cells, including specific genes or portions
of genes. This is important for understanding a variety of chromosomal
abnormalities and other genetic mutations. &nlike most other techniCues used
to study chromosomes, FI'0 does not have to be performed on cells that are
actively dividing. This makes it a very versatile procedure. The first step is to
prepare short seCuences of single3stranded *. that match a portion of the
gene the researcher is looking for. These are called probes. The ne!t step is to
label these probes by attaching one of a number of colors of fluorescent dye
#S*hrD*/ et al., +!!$ and 4oF et al., +!!$%.
*. is composed of two strands of complementary molecules that
bind to each other like chemical magnets. 'ince the researchersB probes are
single3stranded, they are able to bind to the complementary strand of *.,
wherever it may reside on a personBs chromosomes. %hen a probe binds to a
chromosome, its fluorescent tag provides a way for researchers to see its
location #3hite et al., +!!& and 'loom, 2&%.
Fluorescent in situ hybridiAation (FI'0) represents a modem
molecular pathology techniCue, alternative to conventional cytogenetics
(karyotyping). Fluorescence in situ hybridiAation (FI'0) allows identification
of specific seCuences in a structurally preserved cell, in metaphase or
interphase #9onto"eor"os, 2- and 1eledaet al., +!!-%.
;1
The probe, bound to the target, will be developed into a fluorescent
signal. The fact that the signal can be detected clearly, even when fi!ed in
interphase, improves the accuracy of the results, since in some cases it is
e!tremely difficult to obtain mitotic samples Fig$ 9/.*: #Muhlmann, 22%.
The power of in situ hybridiAation can be greatly e!tended by the
simultaneous use of multiple fluorescent colors. Multicolor fluorescence in
situ hybridiAation (FI'0), in its simplest form, can be used to identify as
many labeled features as there are different fluorophores used in the
hybridiAation. +y using not only single colors, but also combinations of
colors, many more labeled features can be simultaneously detected in
individual cells using digital imaging microscopy #(aa7 et al., +!!&%.
Fluorescence, a phenomenon whereby a chemical e!cited at one
light wavelength emits light at a different and usually longer wavelength, is
used throughout the life sciences to study a wide variety of structures and
intracellular activities. dvances in probe and microscope technology have
led to the rapid development of techniCues for fluorescence over the past
decade #?ras/, +!!+%.
The accuracy of cytogenetic diagnosis in the management of
hematological malignancies has improved significantly over the past 2>
years. Fluorescence in situ hybridiAation (FI'0), a techniCue of molecular
cytogenetics, has played a pivotal role in the detection of uniCue sub3
microscopic chromosomal rearrangements that helped in the identification of
chromosomal loci, which contain genes involved in leukemogenesis #Amare
et al., 2+%.

;5

Figure 9/.*:?
Fluoresence in situ hypridi8ation #Muhlmann, 22%$

;6
The use of FI'0 is growing rapidly in genomics, cytogenetics,
prenatal research, tumor biology, radiation labels, gene mapping, gene
amplification, and basic biomedical research. In principle, the techniCue is
Cuite straightforward #Attarbas*hi et al., 2-%.
The hybridiAation reaction identifies, or labels, target genomic
seCuences so their location and siAe can be studied. *. or ,. seCuences
from appropriate, chromosome3specific probes are first labeled with reporter
molecules, which are later identified through fluorescence microscopy. The
labeled *. or ,. probe is then hybridiAed to the metaphase
chromosomes or interphase nuclei on a slide. fter washing and signal
amplification, the specimen is screened for the reporter molecules by
fluorescence microscopy #Hohman and Gundla*h, +!!-%.
FI'0 probes are commonly used to detect the presence of specific
*. seCuences either when *. is condensed into metaphase
chromosomes or dispersed in non dividing interphase cells. The fact that
hybridiAation of probes to metaphase chromosomes is visualiAed in two
dimensions while interphase targets are three dimensional has implications
for both validations of assays and the development of baseline reference
ranges #Pauletti et al., +!!$%.
Metaphase applications generally yield clear yesDno answers while
interphase applications commonly reCuire reportable reference ranges before
interpreting of results. In addition to determining the presence or absence of
particular seCuences in the genome, FI'0 is useful in assessing gene copy
number in some disorders #Massod et al., +!!5%.
;7
nalytical uncertainty over *. probe assays also may stem from
issues related to inherent population variation. The use of some repeat
seCuence probes has been discontinued because of inability to detect targeted
seCuences in individuals who possess very few repeats, leading to insufficient
probe label in the targeted region which precludes visualiAed of the signal.
'uch probes have been eliminated #Myrata et al., +!!0 and 'ossuyt et al.,
+!!&%.
FI'0 allows very precise spatial resolution of morphological and
genomic structures. The techniCue is rapid, simple to implement, and offers
great probe stability. The genome of a particular species, entire
chromosomes, chromosomal3specific regions, or single3copy uniCue
seCuences can be identified, depending on the probes used #Attarbas*hi et
al., 2-%.
&ntil recently, FI'0 was limited by the hardware, software,
reagents, probe technology, and cost involved in implementing the techniCue.
-ommercially available microscope hardware optimiAed for multicolor FI'0
was not available until the mid32;;>s. "rior to that, microscopes had to be
customiAed for FI'0 applications. Most microscope optics was not designed
to detect the low light levels inherent in FI'0 signals. s the genomic
resolution of the techniCue has increased dramatically, the reCuirements on
microscope optics have further increased. -hromatic aberrations among
multiple wavelengths have been a problem. For multicolor analysis in
particular, all lenses, including the collector lens, had to be chromatically
corrected. In addition, epi3fluorescence light sources were difficult to align
for uniform illumination #Amare et al., 2+%.
;8
nalysis of multicolor FI'0 images reCuires isolation of the various
signals either with individual filter cubes; or utiliAation of an e!citation filter
wheel with multipass dichroic and barrier filters. ,ecent developments in
filter technology corrected some of the previous problems encountered
through optical misalignments caused by mechanical switching of individual
filter cubes. /!citation filter wheels used with multi3pass dichroic and barrier
filters can be used effectively for up to three colors by employing separate
e!citation filters for each color with no registration shift. +ut, for more than
three colors, single3pass filters still had to be used #(a*e)s/is, 2&,
>aro)aia et al., 2& and >ouro) et al., 2&%.
3.)hree di--erent types o- FISH probes?
2A+A8o*us s7e*ifi* 7robes
They bind to a particular region of a chromosome. This type of
probe is useful when scientists have isolated a small portion of a gene and
want to determine on which chromosome the gene is located #H=almar, 2&
and 3an" et al., 2&%.
2A2AAl7hoid or *entromeri* re7eat 7robes
They are generated from repetitive seCuences found in the middle of
each chromosome. ,esearchers use these probes to determine whether an
individual has the correct number of chromosomes. These probes can also be
used in combination with Hlocus specific probesH to determine whether an
individual is missing genetic material from a particular chromosome
#Edward et al., 2&%.
;9
2A,A3hole *hromosome 7robes
They are actually collections of smaller probes, each of which binds
to a different seCuence along the length of a given chromosome. &sing
multiple probes labeled with a mi!ture of different fluorescent dyes, scientists
are able to label each chromosome in its own uniCue color. The resulting full3
color map of the chromosome is known as a spectral karyotype. %hole
chromosome probes are particularly useful for e!amining chromosomal
abnormalities, for e!ample, when a piece of one chromosome is attached to
the end of another chromosome #Du"an et al., 2&%.
5.Applications o- FISH?
The clinical uses of FI'0 were considered in three main areas;
diagnosis of individuals with birth defects and mental retardation, prenatal
diagnosis and screening, and identification and monitoring of acCuired
chromosome abnormalities in leukemiaD cancer. In each area the critical
consideration remains a clear understanding of the capabilities and
limitations of a test to provide useful information #'ossuyt et al., +!!& and
Pauletti et al., +!!$%.
Traditional cytogenetic analysis, detecting deletions, duplications,
rearrangement and the identifications of unknown material of marker or
derivative chromosomes, in individuals with birth defects andDor mental
retardation has led to an understanding of the etiology of a number of
syndromes. The clinical utility and limitations of these tests are both general
and disease specific #8edbeteer et al., +!50, 1allen et al., +!!2, (ibeiro et
al., +!!0 and 1assidy et al., +!!5%.
;:
"renatal applications of FI'0 testing include both screening tests
and diagnostic tests. Technical issues are few, and clinical utility raises
Cuestions as to the intended use of FI'0 in testing. The application of FI'0
to prenatal screening for common autosomal trisomies and se! chromosome
anomalies is becoming increasingly common. The primary considerations
involve differing clinical sensitivity between the abnormalities detected by
classical cytogenetic versus these detected by FI'0 based assays #E)ans et
al., +!!+ and 9lin"er et al., +!!2%.
mong cases ascertained via ultrasonographically identified fetal
anomalies, some may be conclusive for a syndromes diagnosis and may be
approached by a (diagnostic) FI'0 test. Families in which subtle or
submicroscopic chromosomal abnormalities, detectable by FI'0, are known
to segregate will benefit greatly from prenatal FI'0 studies #9onto"eor"os et
al., 2 and 8ewin et al., 2%.
Fluorescence in situ hybridiAation (FI'0) has become one of the
ma<or techniCues in environmental microbiology. The original version of this
techniCue often suffered from limited sensitivity due to low target copy
number or target inaccessibility #6wir"lmaier, 2&%.
The reagents and probes themselves were not sufficient for all
applications. For instance, the efficiency of hybridiAation site detection
decreased with decreasing probe siAe, creating significant limits to what
could be observed via fluorescence microscopy. The number of differently
colored fluorescent dyes was limited, and the photostability of the dyes was
poor. +ut new developments in fluorescent dye technology and spin3off
technology from the federally funded 0uman #enome "ro<ect are now
having an impact. There are probes for all the human chromosomes and a
;;
growing number of new gene3specific probes are available. >n situ
hybridiAation kits and fluorescently labeled probes are commercially
available from several companies #Sarrate et al., 2&%.
The ability of FI'0 to rapidly test interphase and metaphase
chromosome defects makes it especially useful in the study of cancer. In solid
tumors, conventional cytogenetics is rarely used because obtaining
metaphases is difficult and those cells that do proceed to mitosis may not be
representative of the tumor. @ther molecular techniCues, such as "-, and
'outhern, .orthern, and %estern analysis, reCuire e!traction of the tissue.
/!traction procedures net both normal and abnormal cells, so sensitivity is
lower and Cuantitation less reliable than with FI'0 probes #'os*h et al.,
2&%.
FI'0 allows cell3by3cell analysis and thus provides for a more
sensitive and reliable assessment of chromosomal aneuploidy, gene
amplifications and deletions, and chromosome translocations. reliable
determination of whether a gene is amplified in a specimen is often possible
with evaluation of only 1> to 7> cells #@"il)ie et al., 2&%.
The accuracy of cytogenetic diagnosis in the management of
hematological malignancies has improved significantly over the past 2>
years. FI'0 has played a pivotal role in the detection of uniCue sub3
microscopic chromosomal rearrangements that helped in the identification of
chromosomal loci, which contain genes involved in leukemogenesis #Amare
et al., 2+%.
FI'0 was performed with specific probes to make the rapid prenatal
diagnosis of *own syndrome. FI'0 was performed respectively with locus3
2>>
specific probe (4'I) and centromeric probe (-/") FDL on the uncultured
amniotic fluid. FI'0 is a rapid and reliable method to detect *own syndrome
in uncultured amniotic fluid #3an" et al., 2&%
Fluorescence in situ hybridiAation assay and to correlate the genetic
findings with the pathologic grade and stage were used to investigate the
chromosomal abnormalities present in bladder carcinoma #Pla*er et al.,
2&%.
novel application of FI'0 to isolated nuclei is described. The
method detects gene amplification and chromosome aneuploidy in e!tracted
nuclei from paraffin3embedded tissue of human cancer with greater
sensitivity and specificity than e!isting FI'0 methods. The method is applied
to signal detection of the 0/,31Dneu (c3erb+31) gene, whose amplification is
one of the most common genetic alterations associated with human breast
cancer #(ossi et al., 2&%.
Tumor3specific chromosomal abnormalities are attracting a large
interest owing to the diagnostic, prognostic, and therapeutic importance. The
development of FI'0 has improved the detection of specific chromosomal
abnormalities in chronic lymphocytic leukemic (-44). +y using FI'0, the
problem with tumor cells with low mitotic rate is avoided since this method
readily detects clonal aberrations also in nondividing, interphase cells. Three
different types of probes are used centromeric probes for numerical
chromosome abnormalities, whole chromosome paints, and locus3specific
probes for numerical chromosome abnormalities, whole chromosome paints,
and locus3specific probes. #H=almar, 2&%
2>2
FI'0 of *.3*. or *.3,. using post3mortem brain samples
is one approach to study low3level chromosomal aneuploidy and selective
e!pression of specific genes in the brain of patients with neuropsychiatric
diseases. FI'0 could be applied to e!tended studies of chromosomal
aneuploidy, abnormal patterns of chromosomal organiAation and functional
gene e!pression in situ in the neurons of the brain in different psychiatric and
neurodevelopmental diseases #Buro) et al, 2+%.
5.*.ALL in(estigation by FISH?
To investigate patients with acute lymphoblastic leukemia (44) for
T/4DM42 fusion, +-,D+4 fusion, M44 gene rearrangements, and
numerical changes of chromosomes 6, 2>, 29 and 12 by fluorescence in situ
hybridiAation (FI'0) and to determine the relationship and the significance of
those findings #6han" et al., 2,%.
Interphase fluorescence in situ hybridiAation (iFI'0) is increasingly
used for the identification of +-,D+4 gene rearrangements in chronic
myeloid leukemia (-M4) and acute lymphoblastic leukemia (44). FI'0
plays an important role in detecting chromosome changes, especially in some
cryptic chromosome translocations and patients with culture failures #Primo
et al., 2, and 6han" et al., 2,%.
44 blasts routinely contain somatically acCuired genetic
abnormalities

that provide insight into pathogenesis and strongly influence

prognosis. ppro!imately one third of cases of 44 show an increase

in the
modal chromosome number (e.g., hyperdiploid, V 69

chromosomes, and
HhighH hyperdiploid, V 7> chromosomes) blasts

make up a uniCue biologic
subset associated with increased in

vitro apoptosis and sensitivity to a variety
2>1
of chemotherapeutic

agents #Heerema et al., 2 and ?rueworthy et al.,
+!!2%.
lmost one third of 44 blasts show chromosomal translocations

in
the absence of changes in chromosome number. Four ma<or translocations

have been observed, and each defines a uniCue biological subset

of patients.
The t(2;2;)(C15;C25) is a hallmark of some pre3+

(cytoplasmic PM) 44, and
is characteriAed by fusion of

the E2" and )B1 genes #;*/un et al., +!!5%.
*espite the adverse prognostic impact

of this translocation in older
studies, recent intensification

of therapy has resulted in an improved survival
for these children.

Translocations between the mi!ed lineage leukemia (4,,)
gene

at 22C15 and over 5> different partner chromosomes characteriAe

8E of
44 cases. 4,, translocations, most commonly t(6;22)(C12;C15),

are seen in
the vast ma<ority of infant patients with 44.

recent, large series
demonstrates that any rearrangement of

22C15 is associated with a worse
prognosis (e.g., 1>E to 17E) #Pui et al., 22%.
,A+A+APhiladel7hia
The presence

of the t(;;11)(C56;C22) translocation, commonly
known as "hiladelphia

chromosome ("h), in about 5E to 7E of all children
with 44

is considered as one of the molecular markers associated with

a
particularly high risk for treatment failure #(ibeiro et al., +!50, 1rist et
al.,+!!, Pui et al., +!!, 4let*her et al., +!!+, (eiter et al., +!!-, and
1hessells et al., +!!&% .
This translocation

causes a rearrangement between the
protooncogene c3+4 and a gene

called the breakpoint cluster region (+-,).
%hereas the breaks

in c3+4 occur mainly in the same region (between the
2>5
e!ons a2

and a1) on chromosome ;, two different ones affect the breakpoint

cluster region on chromosome 11: the more freCuent one (appro!imately

in
1 of 5 of all cases) shows a break in the minor breakpoint

cluster region (m3
+-,) between the e!ons e2 and e1. This is predominant

in 44. In 2 of 5 of
all "h
M
44 cases, the ma<or (M3) +-, found between e!ons b1 and b5 or

e!ons b5 and b6 is affected. M3+-, is also found in nearly all

patients with
chronic myelogenous leukemia (-M4). -himeric proteins

of 12> k* (p12>)
and 2;> k* (p2;>) result from the M3+-,D+4 and

m3+-,D+4
rearrangements, respectively #9antar=ian et al., +!!+%.

These fusion proteins

cause a deregulation of protein tyrosine kinase
activity. +oth

forms of the chimeric gene (+-,D+4) can be detected by
polymerase

chain reaction ("-,) and fluorescent in situ hybridiAation.

#Maurer et al., +!!+, Dewald et al., +!!,, S*hlieben et al., +!!$%.
Most patients with "hiladelphia ("h)3positive acute lymphoblastic
leukemia (44) show evidence of secondary chromosome aberrations that
may influence the course of disease and response to treatment. To better
understand how these secondary chromosomal aberrations occur and to
investigate whether the p2:7Dp2;> +-,3+4 fusion protein may directly
induce an increased chromosomal instability and subseCuently the
appearance of clonal chromosome aberrations, three +,-3+4 (p2:7D
p2;>)3transduced mouse pre3+ cell lines were analyAed by spectral
karyotyping and fluorescence in situ hybridiAation. The human wild3type
+-,3+4 gene was e!pressed at a level comparable with that in human "h3
positive leukemias at diagnosis. ll +-,3+43transduced cell lines acCuired
similar clonal chromosomal aberrations. Trisomy 7 was always present,
followed by loss of the L chromosome, trisomy of chromosomes 21 and 2:,
and an unbalanced translocation between chromosomes F and 21. Thus,
2>6
ectopic p2:7Dp2;> +-,3+4 e!pression, such as p12> +-,3+4, "M43
,,, or -3ML- transduction, may induce an increased chromosomal
instability leading to clonal karyotypic evolution, which may mimic
secondary chromosome aberrations in human "h3positive 44 #(udol7h et
al., 2&%.
The "hiladelphia ("h) chromosome, the main product of the (;;11)
(C56;C22) translocation, is the cytogenetic hallmark of chronic myeloid
leukemia (-M4), a clonal myeloproliferative disorder of the hematopoietic
stem cell; the "h chromosome is also found in a siAeable portion of acute
lymphoblastic leukemia (44) patients and in a small number of acute
myeloid leukemia (M4) cases. Three different breakpoint cluster regions
are discerned within the +-, gene on chromosome 11: M3bcr, m3bcr, and
mu3bcr #DreFler et al., +!!!%.
.early all "h M 44 cell lines have the m3bcr e23a1 fusion gene
(only two 44 cell lines have a b53a1 fusion) whereas all -M4 cell lines, but
one carry the M3bcr b13a1, b53a1 or both hybrids. The mu3bcr e2;3a1 has
been detected in one -M4 cell line. Four cell lines display a three3way
translocation involving chromosomes ;, 11 and a third chromosome.
dditional "h chromosomes (up to five) have been found in four "h M 44
cell lines and in 2: -M4 cell lines; though in some cell lines the e!tra "h
chromosome(s) might be caused by the polyploidy (tri3 and tetraploidy) of
the cells. nother modus to acCuire additional copies of the +-,3+4 fusion
gene is the formation of tandem repeats of the +-,3+4 hybrid as seen in
-M4 cell line I3781. +oth mechanisms, selective multiplication of the
der(11) chromosome and tandem replication of the fusion gene +-,3+4,
presumably lead to enhanced levels of the fusion protein and its tyrosine
kinase activity (genetic dosage effect). The availability of a panel of "h M cell
2>7
lines as highly informative leukemia models offers the uniCue opportunity to
analyAe the pathobiology of these malignancies and the role of the "h
chromosome in leukemogenesis #DreFler et al., +!!!%.
Treated children with acute lymphoblastic leukemia were analysed
for chromosomal abnormalities with conventional #3banding, spectral
karyotyping ('IL) and interphase fluorescent in situ hybridisation (FI'0)
using probes to detect M44, +-,D+4, T/4DM42 rearrangements and
I.I6 locus deletions. Three novel T/4 partner breakpoints on 2C62, :C16
and 12p21 were identified, and a recurrent translocation t(2;21)(p51;p25) was
found. In addition, two cases displayed amplification (9327 copies) of M42.
,esults were demonstrated the usefulness of 'IL and interphase FI'0 for
the identification of novel chromosome aberrations and cytogenetic
abnormalities that provide prognostically important information in childhood
44 #<ord"ren et al., 22%.
The +-,D+4 and M44DF6 fusion genes33resulting from t(;;11)
(C56;C22) and t(6;22)(C12;C15) translocations, respectively33are considered as
a high risk prognostic factors in children with acute lymphoblastic leukaemia
(44). Their presence in malignant cells indicates patient for the most
intensive antileukaemic therapy regardless of the other criteria. In contrast,
the most common non3random chromosomal aberration in paediatric 4433
translocation t(21;12)(C21;C11)33is associated with a favourable prognosis.
The e!amination of these rearrangements is important for the stratification of
patients to the risk groups and also provides the most sensitive and specific
tool for minimal residual disease (M,*) follow3up #?r/a et al., +!!! and
Po7la*/, +!!,%.
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7II. LIF! FL%=CE)%M!),IC FI',!S
() (+)
Figure 9*:? Flow cytometric analysis o- c.myc e@pression on mononuclear
cells showing diagram 9A: and dot plot 9B: o- positi(ely stained cells in
relation to negati(e ones$
() (+)
Figure 93:? Flowcytometric analysis o- p/5 e@pression on mononuclear
cells showing histogram 9A: and dot plot 9B: o- positi(ely stained cells in
relation to negati(e ones$
267
M1
M2
R1
R2
M1
M2
Diploid? *44$44F
*ip #>3#2: ;5.;5 E at 55.6;
*ip #13M: 6.79 E at 82.8;
*ip ': 2.7> E #1D#2: 2.:6
*ip E-?: 1.:6
Diploid? *44$44F
*ip #>3#2: ;>.65 E at 57.52
*ip #13M: 9.:6 E at 87.5>
*ip ': 2.95 E #1D#2: 2.:7
*ip E-?: 5.29
Figure 95:? Histogram showing cell cycle parameters 9diploid: using -low
cytometer FACS caliber program mod-it $
268
Diploid? 03$46 F
*ip #>3#2: 2>>.>> at55.2>
*ip #13M: >.>> E at 88.12
*ip ': >.>> E #1D#2:1.>>
*ip E-?: 5.28
Aneuploid *? 51$23 F
neup #>3#2: ;7.59 E at67.2;
neup #13M: 2.98 E at 95.;7
neup ': 1.:: E #1D#2: 2.86
neup E-?: 5.65
neup *I: 2.59
Diploid? 6/$3/ F
*ip #>3#2: 2>>.>> E at 5:.71
*ip #13M: >.>> E at 99.>7
*ip ': >.>> E #1D#2: 1.>>
*ip E-?: 5.61
Aneuploid *? *+$1/ F
neup #>3#2: 87.26 E at 8;.97
neup #13M: 1:.91 E at 2>6.95
neup ': 8.25 E #1D#2: 2.7>
neup E-?: 5.52
neup *I: 2.:2
Figure 9+:? Histogram showing cell cycle parameters diploid and
aneuploid using -low cytometer FACS caliber program mod-it$
269
7III. LIF! FISH #IC)',!S
Figure A
Figure B
Figure C Figure D
Figure 9A"B"C and D:? !ach childhood acute lymphoblastic leukemia
case shows red signal which is ABL on chromosome 2 and green signal
which is the breakpoint cluster region 9BC,: on chromosome 33 -or
children with #hiladelphia negati(e acute lymphoblastic leukemia 9#h\
ALL:$
26:
Figure ! Figure F
Figure 9! and F:? !ach childhood acute lymphoblastic leukemia case
shows red signal which is ABL on chromosome 2" green signal which is
the breakpoint cluster region 9BC,: on chromosome 33 and pale orange
signal which is the -usion 9BC,<ABL: -or children with #hiladelphia
positi(e acute lymphoblastic leukemia 9#h
G
ALL:$
26;