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Chronic Myeloid leukaemia

Introduction

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Chronic
myeloid leukaemia (CML) is a haematopoietic stem cell disorder categorised as
an uncontrolled neoplastic growth of myeloid cells in bone marrow with
increased number of these cells present in peripheral blood (Pasic and Lipton, 2017). It is classified as
myeloproliferative disorder along with polycythemia Vera, essential
thrombocythemia and myelofibrosis. CML is considered a rare disorder compared with other leukaemia. In UK
8600 leukaemia cases are diagnosed per year and 750 of these cases are CML (Cancer research, 2017). The worldwide incidences
are 1-2 cases per 100000 individuals, each year. The incidences are high in
population with high exposure to radiation, such as victims of atomic bomb.
Patients also treated with radiotherapy for other leukaemia have increased risk
of developing CML (Lavallade, 2017).

CML is well-studied disorder which is caused by a specific genetic mutation, in
which part of chromosome 9 attach to chromosomes 22 to form Philadelphia (Ph) chromosomes (Ref). Ph chromosomes,
dysregaulate the activity of tyrosine kinase protein (TKP). TKP is an essential
protein which regulates metabolic pathways and acts as a mediator for cell
proliferation and apoptosis.
Dysregulation of TKP leads to increased granulocytic stimulating factors
(G-CSF) which in turn results in over production of myeloid cells in marrow (McKenzie and Williams, 2010). The disease progress in three
phases, which include chronic phase (CP), accelerated phase (AP) and blastic phase
(BP) with different clinical and laboratory presentations. Studies (Ref) have shown that more than 85% of cases are
diagnosed at chronic phase. In chronic phase patients are mostly asymptomatic
and If not treated appropriately the disease can progress to more advanced
accelerated or blastic phase (Ref). The
diagnosis of CML is initially suspected in full blood counts and peripheral
blood smear which is later confirmed by further laboratory tests including bone
marrow biopsy, cytogenetic, PCR and FISH (Ref).

There
are number of treatments modalities available for patients and they respond
well to treatment at chronic phase and maintain normal health for several
years. However; as disease progress to advance stage the prognosis decrease
with a reduce survival rate of 6 month or less (Ref).

Pathogenesis
of disease

 

Our
understanding in CML disease and process has dramatically increased with
evolving new molecular biology techniques. Discovery of Philadelphia
chromosomes in 1960 by Peter Nowell and David Hungerford was first step towards
understanding disease and mechanism in CML. The Philadelphia chromosomes abnormality is present in
all CML and associated with the malignant disease. It was not until 1973 when
Janet Rowley, using banding technique, described the translocation between
chromosomes 9 and 22 (figure 1) (Alikian et al 2017)
and further studies in early 80s revealed that the fusion of BCR-ABL genes is
the cause of CML. This BCR-ABL form active tyrosin kinase which endorse
proliferation and replication.(Jabour and Kantrajian, 2014).

                                                                                                    
(Deo, 2015)

Figure 1.Shows
translocation between chromosomes 9 and 22 resulting in Philadelphia
chromosomes and molecular aspect of CML disease.

 

ABL is a non-receptor tyrosine kinase
that is present in most tissues. It is found in both cytoplasm and nucleus of
cells, and transport between the two compartments. It regulates cytoskeleton
structure by transducing signals from cell-surface for growth and adhesion
receptors. BCR on other hand have multiple modular domains and also function as
signalling protein.
The expression of the ABL1 tyrosine kinase is
tightly regulated (Chen et al., 2009). In CML
fusion of BCR to ABL enhance the tyrosin kinase activity of ABL and form new
motifs and generate different types of BCR-ABL protein (Figure 2).

 

 

Figure
2.  Shows locations of various
breakpoints in the ABL and BCR genes and structure of the BCR/ABL mRNA
transcripts derived from the various break points (uptodate).

 

The ABL
gene breakpoint can be upstream exon 1a, among exon 1a and 1b or downstream
exon 1b, however in CML it is almost always upstream exon 2. Apart from rare
exception most transcripts of BCR-ABL gene have exon 2-11 of the ABL gene (Deo,
2015). Due to variable nature of BCR breakpoints, they determine the
pathogenic properties of the BCR-ABL gene as well as the size of the gene
(Table 1). The breakpoints on the BCR gene are present very closely in three
regions commonly known as micro cluster, minor cluster and major cluster. Three
different types of proteins are synthesized, as shown in figure 2, by BCR genes
depending on location of break point on the gene (Kang
et al., 2016).

Table1.Shows
BCR-ABL proteins and associated disease.

 

Synonym

Location on gene (Exons)

Type of protein

Disease

Major-cluster

M-BCR

12-16

p210

– CML
– Ph+ALL
-Thrombocytosis (in e14a2)
 

Minor-cluster

m-BCR

Between (2, e2′ and e2)

p190

-PH+ALL
-CML (monocytosis and aggressive disease)

Micro-cluster

u-BCR

Between (e19 – e20)

p230

-Chronic Neutrophilic leukaemia
 

 

 

The p230 is
one of the largest BCR-ABL1 transcripts and occurs rarely. It is associated
with much slower course of disease and mainly present in patients with uncommon
chronic neutrophilic leukaemia (Ref). The minor BCR protein (p190) is associated with Ph- positive ALL and
some patients of chronic myeloid leukaemia. The CML with this minor BCR
mutation show increase monocytosis in aggressive disease (Ref). The p210
BCR gene product is associated with chronic myeloid leukaemia as well as some
Philadelphia (Ph) positive acute lymphoblastic leukaemia (ALL). This
BCR-ABL  gene  product (p210) is essential for transformation
of CML and accountable for the phenotypic abnormalities of CML (Ref).
The p210 BCR-ABL gene product increase the tyrosine kinase activity leading to
phosphorylation of various cellular substrate and autophosphorylation which
induce binding of several proteins and adaptors molecules. This activation of
signals pathways by p210 oncoprotein interfere with cellular process including
cell differentiation, proliferation, cell adhesion and survival (Ref). 

Studies
(Webersinke,2016) have
shown that p210 activates signal transduction pathways including RAS/MAPK, CRKL
pathways, PI-3 kinase, JAK-STAT and the Src pathway as shown in figure 3. It is
proposed that the RAS, Jun-kinase, and PI-3 kinase pathways are associated in
transformation and proliferation, while inhibition of apoptosis is thought to
result from activation of the PI-3 kinase and RAS pathways (Ref). Furthermore
p210 effects, on CRKL, c-CBL as well as proteins associated with the
organization of the cytoskeleton and cell membrane that result in cell adhesion
defects and structural abnormalities, which are characteristic of CML cells (Ref). It is also
thought that cell adhesion and migration proteins are phosphorylated by BCR-ABL
genes which may lead to premature appearance of myeloid cells in blood circulation.
Increased reactive oxygen species in CML patients leads to DNA damage by breaking
DNA double strands. This leads to addition mutations which are considered to be
responsible for accelerated and blast crisis in chronic myeloid patients (Ref).
CML progress from chronic phase to more aggressive
accelerated and blastic phase. Studies have shown that in 75% of CML cases, the
disease progression results due to additional chromosomal abnormalities (Ref).Genetic
mutation in p53 gene which is a tumour suppressor gene are found in patients in
blast phase of disease.

 

Figure 3: Shows BCR-ABL downstream pathways and
impact on cellular function: Activation of 
JAK/STAT pathways enhance 
cell growth, RAS pathway activation 
increases proliferation of BCR-ABL-positive
leukemic cells, PI3K activates AKT which cause apoptosis by suppressing
proteins such as BAD or FOXO and C/EBP? is a regulator of myeloid
differentiation.  (Webersinke,2016)

 

 

Diagnosis

Chronic
myeloid leukemia usually detected during normal routine health check or blood
test performed for other medical reasons. Full blood count test is first
screening test performed in haematology laboratory for screening of any
haematological disorder. A lot of time,
the CML diagnosis is incidental on clinical basis, but prior to starting
treatment laboratory studies to determine the presence of the Philadelphia
chromosome (Ph) or BCR-ABL fusion are performed. There are number of laboratory
tests used in the diagnosis of chronic myeloid leukaemia including full blood
count, blood smear, bone marrow aspiration and biopsy, cytogenetics analysis,
fluorescence in situ hybridization and polymerase chain reaction.

The most
striking feature of CML in full blood count is highly raised white blood
cell count with median count of 175×109 /L. Other indices of full blood count
may show a moderate normocytic normochromic anemia with reduced haemoglobin
concentration. The platelets count can be normal or high, as well as slight
increase in red cells. The reticulocyte count can be normal or moderately high.
The blood smear shows normocytic normochromic red cells with some
nucleated red cells. White cell shows left shift with stages of granulocyte
maturation including myelocytes, metamyelocytes, and bands, as well as varying
degrees of eosinophils and basophils (Figure 4).

Figure 4: Characteristic features of
CML in blood film including basophilic and granulocytosis with neutrophils and
immature granulocytes.

 

In
CML the predominant cells observed under microscope are myelocytes and
segmented neutrophil (Mckenzie).
However, blast cells can also be seen with some promyelocytes. Even though neutrophils appear normal on blood film but cytochemically
score low on test called leucocyte alkaline phosphate (LAP). The significant of
this test is that it helps to differentiate between a leukemoid reactions
possibly due to infection as well as from polycythaemia Vera in which LAP
activity is high.

There
is also an increase in number of eosinophil and basophil in the CML patients’
blood smears with moderate increase in monocytes (Egan
and Radich,2016). Increase in numbers of basophil is a common finding in the blood
smears of CML patients and more than 90% patients have eosinophilia. However,
absolute monocytosis is not a common finding on peripheral blood smears but
some patients who have p190 BCR-ABL fusion protein instead of p210 can have
increased monocytosis (Etten, 2017). On occasion some overlapping features of
chronic myelomonocytic leukaemia and CML such as monocytosis, micro
megakaryocytes and myeloid dysplasia are found, which can be differentiated by
carrying out further tests to identify Ph chromosome(Mckenzie, ).

BM
aspiration
and cytogenetic analysis are essential tests for the diagnosis of CML.
Without these two tests, we are unable to tell if there is an increase in blast
cells or basophils that will shift the staging from chronic phase to
accelerated or blast phase. Furthermore, we will not be able to know the other
chromosomal abnormalities apart from Ph chromosomes (Cortes
and Kantarjian, 2016.) Bone marrow reveals hypercellularity (Figure) with fat as well as granulocytic
hyperplasia with immature granulocytes, a pattern similar observed in the
peripheral smear under microscope.  The
differential count of leucocytes in marrow is normally within the range.
However, erythropoiesis is normal with reduced number of normoblast.  Etten
et al, (2017) states that in both peripheral blood smears and bone marrow
biopsy blast cells between 10-19% are considered diagnostic for accelerated
phase of disease whereas over 20% of blast cells are consistent with blast
phase of the disease.

Figure: Shows granulocytic hyperplasia in bone marrow

The cytogenetic
of CML provides crucial information for its diagnosis as well as prediction of
prognosis and treatment outcomes. The test provide the information about number
and structure of the chromosomes. A bone marrow sample is used for cytogenetic
test due to its requirement of dividing cells. Majority of BCR-ABL translocations are readily identified by conventional
cytogenetics (figure). However, in small number of cases which involve complex changes
that still result in formation of a BCR-ABL transcript but without any detectable Philadelphia chromosome.

Figure: Shows the Chronic myeloid
leukaemia chromosome translocation. The translocation results in a slightly
longer chromosome 9 (first arrow at 9) and a shorter chromosome 22 (second
arrow at 22) known as Philadelphia (Ph) chromosome.

 

The cytogenetics test have both advantage and disadvantage. A big
advantage of cytogenetic test is its ability to detect other chromosomal
structural abnormalities that may indicate advance disease. The down side of
this test is it only visualised 20 cells and not suitable for disease
monitoring and progression analysis as compared with FISH and PCR.

As compared with cytogenetic testing the FISH
uses probes fluorescently labelled for detection BCR-ABL genetic material
(figure). FISH has advantage over conventional cytogenetic tests as it quick
and can be performed on bone marrow as well as peripheral blood sample.
Furthermore, FISH has superior detection capability for BCR-ABL translocation
as compared with cytogenetics (Swansbury, 2013).
However, the disadvantage of this technique is that as probes used are
specifically designed for BCR-ABL translocation, therefore any other
rearrangement that may be present will not be detected by this method and may
require conventional cytogenetic test (Egan and Radich, 2016).

 

a)     
Normal                                                                            
b) BCR-ABL

                                                                                   
Adopted from: Shah and Areci (2014)

Figure: a) normal cells, two red and two green
signals shows normal ABL and BCR genes, respectively. b) The BCR-ABL fusion is
visualized through the fusion of the red and green signals, which is detected
as a yellow fluorescence.

 

PCR detection of BCR-ABL is most
sensitive method for diagnostic purpose of CML. It detects 1 CML cells per 105
cells. This high sensitivity of PCR allows use of blood sample than bone marrow
for diagnosis and treatment monitoring. The PCR method is considered a backbone
in the clinical decision-making. Appropriate reverse and forward primers are
designed which specifically binds with BCR-ABL transcript and amplify them.
Although there is a significant heterogeneity among BCR/ABL breakage in CML disease,
but majority of patients exhibit clones where exon 1e14 or 1e13 of BCR fuse
with ABL exon 2e11 and resulting BCR-ABL transcript is detected by single test
reaction (EGAN and RADICH,2016).

 

Differential
diagnosis

The most common cause of persistently high white cell count with left
shift and mild thrombocytosis seen in neoplasm and infection diseases. These
conditions are called leukemoid reaction to label any condition that mimic
leukaemia but in reality are benign conditions. Therefore, a collective term
leukemoid reaction is used to differentiate CML from non-leukemic conditions.
LAP test is used to differentiate between these conditions. The LAP test score
is low or absent in CML and high in leukemoid reaction. There are several other
disease which have similar presentation as CML as shown in Table below.

Table Laboratory features of CML and
other conditions in differential diagnosis

Conditions

Ph chromosomes

LAP score

Basophilia

Myelocyte bulge

CML

Positive

Low/absent

1-3+

+

Leukemoid reaction

Negative

High

0

Chronic
neutrophilic leukemia 

Negative

High

0

Atypical CML

Negative

 

0

+

 

Stage of disease

 

Chronic myeloid leukaemia is characterise into 3
phases.

–      
Chronic phase

–      
Accelerated phase

–      
Blast phase

In CML most of patient diagnosed at chronic phase
which lasts three to six years if un- treated. The characteristic features of
this stage is a persistently high white cell count and may be platelets with
less than 10% of blast cells in the bone marrow. The next phase is the
accelerated phase in which splenomegaly and leucocytosis are evident with low
platelets count and blast cells between15%- 20% in bone marrow (Kantrajian, 2006). The most fatal and advance
stage of CML is blast phase with median survival is between two to four months.
The hallmark of this phase is more than 30% of blast cells appearing in both
peripheral blood and bone marrow (Zhou,2011).Table highlights the characteristic laboratory
features of each stage in CML.

 

Table: Shows diagnostic features of each stage in CML.

   Adopted from: 
(Gratton, 2018)

                  Treatments

Chronic
myeloid leukaemia has gone through many revolutionary phases in last two
centuries (figure).
The treatment of chronic leukaemia was first introduced in 19th
century using arsenic compounds and splenomegaly was treated using radiation in
19th century. The first evidence based treatment for CML was
initiated in 1960 with busulfan which is an alkylation agent (Apperley, 2018).
Introduction of busulfan for CML was based on first randomised research for CML
treatment. However later studies proved that busalfan was unable to
significantly reduce blood counts and considered a possible mutagen which may
lead to blast crisis (Goldman, 2009). Buslfan was replaced with
hydroxycarbamide. Combination of these two drugs relived the symptoms and
normalised the full blood count, but did not succeed in slowing down disease
progression to achieve cytogenetic remission (Jabbour
and Kantarjian, 2012). It was not until 1970s and later in 1980
when stem cell transplant and interferon alpha respectively, were introduced in
treatment modality for CML and not only shown a complete cytogenetic response
but also prolonged the life expectancy to six to seven years (Bonifazi et al, 2001 & Allan et al 1995).
A comparison study by Guilhot et al
(1997) shown a good cytogenetic response for CML patients treated with
interferon alpha and cytarabine than interferon alpha as a sole treatment.
These findings were further supported by chen
et al, (2011) by demonstrating a complete cytogenetic and haematological
response with prolonged survival of four to five years, when CML patients
treated with combination of interferon alpha and cytarbine as opposed to
interferon alpha only. However study reported some severe side effects of
combined therapy which included weight loss, nausea vomiting and diarrhea.

                                                                                    
Hamad et al (2013)

Figure: Shows historic
moments in the evolution of CML treatment

 

Thus until
1990 CML patients were treated with combination of interferon alpha and
cytarabine or interferon alpha alone. However young and healthy CML patients
were treated with allogenic stem cell transplant despite its toxicity and risk
of host versus graft disease (Goldman,
2009).  The treatment of CML
revolutionised following breakthrough in discovery of BCR-ABL oncoprotein which
lead to development of drugs known as imatinib which inhibit activities of
these oncoprotein (Bollmann and Giglio,
2011).
Druker et al (1996) published a
report on the first data on tyrosine kinase inhibitor 2-phenylaminopyrimidine
Abl1 named as signal transduction inhibitor 571 or STI571, now known as
Imatinib, for its effectiveness in inhibition of tyrosin kinase in BCR-ABL
oncoprotein. Imatinib works as a competitive inhibitor as shown in figure1 (B)
on BCR-ABL oncoprotein at adenosine triphospate binding (ATP) site for its
substrate which inhibits phosphorylation of protein engaged in signal transduction.
This action of imatinib hinders the oncoprotein function and signals for stem
cell growth factors and platelets derived growth factors (Fentie et al, 2017) and
restores normal cellular function by inhibiting cell proliferation and inducing
cell apoptosis in CML patients. Several studies have demonstrated the effectiveness of imatinib as compared with
other treatments choices measured by haematological, cytogenetic and molecular
response to disease (Table).

Table: Defines treatment response in CML patients

                                                               
Adapted from: Boliman and Giglio (2011)

 

 

In 2002 Kantarjian et al., studied imatinib outcomes in 454 patients who failed
to respond to interferon alpha in chronic phase of CML. The study shown a
complete haematological response in 95%  
of patients (430/454 patients) with no disease progression, to
accelerated phase or blast crisis, in 89% of patients over a period of 18
months. These findings were consistent with recent studies.  Houshhaus et al., (2017) conducted a randomised trial
on newly diagnosed CML patients treated with imatinib and interferon alpha
combined with cytarabine for efficacy and safety to include treatment response,
survival and serious complication.  The
results shown a complete cytogenetic response in 82.8% of patients and 83%
estimated to have overall survival of 10 years.

 

                                                                                   Tamascar and Ramanarayanan (2009)

Figure: Shows imatinib action mechanism: A) In CML the phosphorylation and activation of tyrosine residue
following binding of adenosine triphosphate (ATP) in the kinase domain on the
BCR-ABL oncoprotein. B) Imatinib
occupies the ATP binding sites on BCR-ABL oncoprotein and prevents substrate
phosphorylation and signal transduction pathways which inhibit proliferation
and survival which is basis of CML pathogenesis.

These findings are considered highly significant to support
imatinib as first line of treatment in chronic phase CML.

Despite excellent
treatment results are achieved with imatinib, however findings are not
consistent among some patients who developed imatinib resistance (Ref). The drug resistance in CML is
BCR-ABL dependant or independent (Figure)
and occurs through various mechanisms including BCR-ABL over expression and
genetic mutation. The resistance can be overcome by giving high dose imatinib
or treating with second generation tyrosin kinase drugs (Ref).The second generation drugs include bosutinib, dasatinib and
nilotinib which are considered more effective tyrosin kinase inhibitor. These
drugs are highly active against all most all CML mutations. Patients newly
diagnosed with CML, and in chronic phase of disease with Ph positive and
resistant to imatinib, are treat with dasatinib and nilotinib whereas Bosutinib
used for patients who are resistant to imatinib and are in accelerated or blast phase of
disease(Krishnan,2018).

 

                                                                                               
Huang et al 2016

Figure:
CML resistance mechanisms

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