Myeloid leukaemia


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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, 2018). 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 (Apperley,

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 ((Vaidya et al., 2011). The 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. Thompson
et al., 2015 states 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. 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 (Baccarani et al., 2012).

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 (Thompson
et al., 2015).

of disease


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 (Jabbour
and Kantarjian, 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., 2010). 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).



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 (Robert
and Schiffer, 2018).


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.



Location on gene (Exons)

Type of protein






– Ph+ALL
-Thrombocytosis (in e14a2)



Between (2, e2′ and e2)


-CML (monocytosis and aggressive disease)



Between (e19 – e20)


-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 (Deo, 2015). 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 (Reckel et al., 2017). 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 (Maru,
2012). The p210 BCR-ABL gene products 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 (Ernst and Hochhaus, 2012). 

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 (Webersinke, 2016). 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 (Maru,
2012). 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 (Apperley, 2018). 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 (Soverini et al., 2015). 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 (Webersinke, 2016).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)




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 evaluation of any
haematological disorder. In majority of cases 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.

most striking feature of CML in full blood count is increased number of
white blood cell with median count of 175×109 /L. Other indices of
full blood count may show moderate normocytic normochromic anaemia 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


In CML the predominant cells observed under
microscope are myelocytes and segmented neutrophil. 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
and Williams, 2010).

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 (Thompson
et al., 2015) Bone marrow reveals hypercellularity (Figure 5) 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.

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 6). 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 6: Shows the Chronic myeloid leukaemia chromosome translocation. The
translocation results in a slightly longer chromosome 9 and a shorter
chromosome 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 cytogenetic (Morris, 2011). 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                                                                            

Adopted from: Shah and Areci

Figure 7: 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.


detection of BCR-ABL is most sensitive method for diagnostic purpose of CML. It
detects 1 CML cell 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 (Luu and
Press, 2013). 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 (Thompson et al., 2015).


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 2 below.

Table 2. Laboratory
features of CML and other conditions in differential diagnosis


Ph chromosomes

LAP score


Myelocyte bulge










Chronic neutrophilic leukemia 




Atypical CML






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 (Cortes
and Kantarjian, 2012). 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
and Xu, 2015).Table 3 highlights the characteristic
laboratory features of each stage in CML.


Table 3: Shows diagnostic features of each stage in



Chronic myeloid leukaemia has gone through
many revolutionary phases in last two centuries (figure 8). 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).
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 diarrhoea.

Hamad et al (2013)

Figure 8: Shows historic moments in the evolution of
CML treatment


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 & 2001)
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 figure 9  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 ((Mulu 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 4).

Table 4 : Defines treatment response in CML

and Giglio (2011)



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 9: 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

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 (Vaidya et al., 2011) ).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