ChronicMyeloid leukaemiaIntroductionChronicmyeloid leukaemia (CML) is a haematopoietic stem cell disorder categorised asan uncontrolled neoplastic growth of myeloid cells in bone marrow withincreased number of these cells present in peripheral blood (Pasic and Lipton,2017). It is classified as myeloproliferative disorder along with polycythemiaVera, essential thrombocythemia and myelofibrosis. CML is considered a rare disorder compared withother leukaemia. In UK 8600 leukaemia cases are diagnosed per year and 750 ofthese cases are CML (Cancer research, 2018).

The worldwide incidences are 1-2cases per 100000 individuals each year. The incidences are high in populationwith high exposure to radiation, such as victims of atomic bomb. Patients also treatedwith radiotherapy for other leukaemia have increased risk of developing CML (Apperley,2018).CMLis well-studied disorder which is caused by a specific genetic mutation, in which part of chromosome 9 attach tochromosomes 22 to form Philadelphia(Ph) chromosomes ((Vaidya et al., 2011). The Ph chromosomes dysregaulatethe activity of tyrosine kinase protein (TKP).

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TKP is an essential proteinwhich regulates metabolic pathways and acts as a mediator for cellproliferation and apoptosis. Dysregulation ofTKP leads to increased granulocytic stimulating factors (G-CSF) which in turnresults in over production of myeloid cells in marrow (McKenzie and Williams, 2010). The disease progress in three phases, whichinclude chronic phase (CP), accelerated phase (AP) and blastic phase (BP) withdifferent clinical and laboratory presentations. Thompsonet al., 2015 states that morethan 85% of cases are diagnosed at chronic phase. In chronic phase patients aremostly asymptomatic and If not treated appropriately the disease can progressto more advanced accelerated or blastic phase. The diagnosis of CML isinitially suspected in full blood counts and peripheral blood smear which islater confirmed by further laboratory tests including bone marrow biopsy,cytogenetic, PCR and FISH (Baccarani et al.

, 2012). Thereare number of treatments modalities available for patients and they respondwell to treatment at chronic phase and maintain normal health for severalyears. However; as disease progress to advance stage the prognosis decreasewith a reduce survival rate of 6 month or less (Thompsonet al.

, 2015).Pathogenesisof disease Ourunderstanding in CML disease and process has dramatically increased withevolving new molecular biology techniques. Discovery of Philadelphiachromosomes in 1960 by Peter Nowell and David Hungerford was first step towardsunderstanding disease and mechanism in CML. The Philadelphia chromosomes abnormality is present inall CML and associated with the malignant disease. It was not until 1973 whenJanet Rowley, using banding technique, described the translocation betweenchromosomes 9 and 22 (figure 1) (Alikian et al., 2017), and further studies in early 80srevealed that the fusion of BCR-ABL genes is the cause of CML. This BCR-ABL form active tyrosinkinase which endorse proliferation and replication (Jabbourand Kantarjian, 2014).

                                                                                                                                             (Deo, 2015)Figure 1.Shows translocation betweenchromosomes 9 and 22 resulting in Philadelphia chromosomes and molecular aspectof CML disease. ABL is a non-receptor tyrosine kinasethat is present in most tissues. It is found in both cytoplasm and nucleus ofcells, and transport between the two compartments. It regulates cytoskeletonstructure by transducing signals from cell-surface for growth and adhesionreceptors.

BCR on other hand have multiple modular domains and also function assignalling protein. The expression of the ABL1 tyrosine kinase istightly regulated (Chen et al., 2010). In CML fusion of BCR to ABL enhance the tyrosin kinaseactivity of ABL and form new motifs and generate different types of BCR-ABLprotein (Figure 2).   Figure2.  Shows locations of variousbreakpoints in the ABL and BCR genes and structure of the BCR/ABL mRNAtranscripts derived from the various break points (Robertand Schiffer, 2018). The ABL gene breakpoint can be upstream exon 1a, among exon 1a and 1b ordownstream exon 1b, however in CML it is almost always upstream exon 2. Apartfrom rare exception most transcripts of BCR-ABL gene have exon 2-11 of the ABLgene (Deo, 2015).

Due to variable nature of BCR breakpoints, they determine thepathogenic 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 threeregions commonly known as micro cluster, minor cluster and major cluster. Threedifferent types of proteins are synthesized, as shown in figure 2, by BCR genesdepending 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 inpatients with uncommon chronic neutrophilic leukaemia (Deo, 2015).

The minorBCR protein (p190) is associated with Ph- positive ALL and some patients ofchronic myeloid leukaemia. The CML with this minor BCR mutation show increasemonocytosis in aggressive disease (Reckel et al., 2017). The p210BCR gene product is associated with chronic myeloid leukaemia as well as somePhiladelphia (Ph) positive acute lymphoblastic leukaemia (ALL). ThisBCR-ABL gene product (p210) is essential for transformation of CML andaccountable for the phenotypic abnormalities of CML (Maru,2012). The p210 BCR-ABL gene products increase thetyrosine kinase activity leading to phosphorylation of various cellularsubstrate and autophosphorylation which induce binding of several proteins andadaptors molecules. This activation of signals pathways by p210 oncoproteininterfere with cellular process including cell differentiation, proliferation,cell adhesion and survival (Ernst and Hochhaus, 2012).

  Studieshave 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). Itis proposed that the RAS, Jun-kinase, and PI-3 kinase pathways are associatedin transformation and proliferation, while inhibition of apoptosis is thoughtto result from activation of the PI-3 kinase and RAS pathways (Maru,2012). Furthermore p210 effects on CRKL, c-CBL as well asproteins associated with the organization of the cytoskeleton and cell membranethat result in cell adhesion defects and structural abnormalities, which are characteristicof CML cells (Apperley, 2018). It is also thought that cell adhesion and migration proteins arephosphorylated by BCR-ABL genes which may lead to premature appearance ofmyeloid cells in blood circulation. Increased reactive oxygen species in CMLpatients leads to DNA damage by breaking DNA double strands. This leads toaddition mutations which are considered to be responsible for accelerated andblast crisis in chronic myeloid patients (Soverini et al., 2015).

CML progressfrom chronic phase to more aggressive accelerated and blastic phase. Studieshave shown that in 75% of CML cases, the disease progression results due toadditional chromosomal abnormalities (Webersinke, 2016).Genetic mutation in p53 gene which is a tumour suppressor gene arefound in patients in blast phase of disease. Figure 3: Shows BCR-ABL downstream pathways and impact on cellularfunction: Activation of  JAK/STATpathways enhance  cell growth, RASpathway activation  increasesproliferation of BCR-ABL-positive leukemic cells, PI3K activates AKT which causeapoptosis by suppressing proteins such as BAD or FOXO and C/EBP? is a regulatorof myeloid differentiation.  (Webersinke,2016)  DiagnosisChronicmyeloid leukemia usually detected during normal routine health check or bloodtest performed for other medical reasons. Full blood count test is firstscreening test performed in haematology laboratory for evaluation of anyhaematological disorder. In majority of cases the CML diagnosis is incidentalon clinical basis, but prior to starting treatment laboratory studies todetermine the presence of the Philadelphia chromosome (Ph) or BCR-ABL fusionare performed.

There are number of laboratory tests used in the diagnosis ofchronic myeloid leukaemia including full blood count, blood smear, bone marrowaspiration and biopsy, cytogenetics analysis, fluorescence in situhybridization and polymerase chain reaction.Themost striking feature of CML in full blood count is increased number ofwhite blood cell with median count of 175×109 /L. Other indices offull blood count may show moderate normocytic normochromic anaemia with reducedhaemoglobin concentration. The platelets count can be normal or high, as wellas slight increase in red cells. The reticulocyte count can be normal ormoderately high. The blood smear shows normocytic normochromic red cellswith some nucleated red cells. White cell shows left shift with stages ofgranulocyte maturation including myelocytes,metamyelocytes, and bands, as well as varying degrees of eosinophils andbasophils (Figure 4).

Figure 4: Characteristic features of CML in blood filmincluding basophilic and granulocytosis with neutrophils and immaturegranulocytes. In CML the predominant cells observed undermicroscope are myelocytes and segmented neutrophil. However, blast cells canalso be seen with some promyelocytes. Even though neutrophils appear normal on bloodfilm but cytochemically score low on test called leucocyte alkaline phosphate(LAP). The significant of this test is that it helps to differentiate between aleukemoid reactions possibly due to infection as well as from polycythaemiaVera in which LAP activity is high.There is also an increase in number of eosinophiland basophil in the CML patients’ blood smears with moderate increase inmonocytes (Egan and Radich,2016). Increase in numbers ofbasophil is a common finding in the blood smears of CML patients and more than90% patients have eosinophilia.

However, absolute monocytosis is not a commonfinding on peripheral blood smears but some patients who have p190 BCR-ABLfusion protein instead of p210 can have increased monocytosis (Etten,2017). On occasion some overlapping features ofchronic myelomonocytic leukaemia and CML such as monocytosis, micromegakaryocytes and myeloid dysplasia are found, which can be differentiated bycarrying out further tests to identify Ph chromosome (McKenzieand Williams, 2010).BM aspirationand 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 blastcells or basophils that will shift the staging from chronic phase toaccelerated or blast phase. Furthermore, we will not be able to know the otherchromosomal abnormalities apart from Ph chromosomes (Thompsonet al., 2015) Bone marrow reveals hypercellularity (Figure 5) with fat as well asgranulocytic hyperplasia with immature granulocytes, a pattern similar observedin the peripheral smear under microscope.  The differential count of leucocytes in marrowis normally within the range.

However, erythropoiesis is normal with reducednumber of normoblast.  Etten et al, (2017) states that in both peripheral blood smears and bonemarrow biopsy blast cells between 10-19% are considered diagnostic foraccelerated phase of disease whereas over 20% of blast cells are consistentwith blast phase of the disease.Figure5:Shows granulocytic hyperplasia in bone marrowThe cytogenetic of CML provides crucialinformation for its diagnosis as well as prediction of prognosis and treatmentoutcomes. The test provide the information about number and structure of thechromosomes. A bone marrow sample is used for cytogenetic test due to itsrequirement of dividing cells. Majority of BCR-ABL translocations are readily identified byconventional cytogenetics (figure 6). However, in small number of caseswhich 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. Thetranslocation results in a slightly longer chromosome 9 and a shorterchromosome 22 known as Philadelphia (Ph) chromosome. The cytogenetics test have both advantageand disadvantage.

A big advantage of cytogenetic test is its ability to detectother chromosomal structural abnormalities that may indicate advance disease.The down side of this test is it only visualised 20 cells and not suitable fordisease monitoring and progression analysis as compared with FISH and PCR.As compared with cytogenetic testing the FISHuses probes fluorescently labelled for detection BCR-ABL genetic material(figure). FISH has advantage over conventional cytogenetic tests as it quickand can be performed on bone marrow as well as peripheral blood sample.Furthermore, FISH has superior detection capability for BCR-ABL translocationas compared with cytogenetic (Morris, 2011). However, the disadvantage of this technique is that as probesused are specifically designed for BCR-ABL translocation, therefore any otherrearrangement that may be present will not be detected by this method and mayrequire conventional cytogenetic test (Eganand Radich, 2016).  a)       Normal                                                                            b) BCR-ABL                                                                                  Adopted from: Shah and Areci(2014)Figure 7: a) normal cells, two red and two green signals shows normalABL and BCR genes, respectively. b) The BCR-ABL fusionis visualized through the fusion of the red and green signals, which isdetected as a yellow fluorescence.

 PCRdetection of BCR-ABL is most sensitive method for diagnostic purpose of CML. Itdetects 1 CML cell per 105 cells. This high sensitivity of PCR allows use ofblood sample than bone marrow for diagnosis and treatment monitoring. The PCRmethod is considered a backbone in the clinical decision-making (Luu andPress, 2013). Appropriatereverse and forward primers are designed which specifically binds with BCR-ABLtranscript and amplify them. Although there is a significant heterogeneityamong BCR/ABL breakage in CML disease, but majority of patients exhibit cloneswhere exon 1e14 or 1e13 of BCR fuse with ABL exon 2e11 and resulting BCR-ABLtranscript is detected by single test reaction (Thompson et al., 2015). Differential diagnosisThe most common cause of persistently high whitecell count with left shift and mild thrombocytosis seen in neoplasm andinfection diseases.

These conditions are called leukemoid reaction to label anycondition that mimic leukaemia but in reality are benign conditions. Therefore,a collective term leukemoid reaction is used to differentiate CML fromnon-leukemic conditions. LAP test is used to differentiate between theseconditions. The LAP test score is low or absent in CML and high in leukemoidreaction. There are several other disease which have similar presentation asCML as shown in Table 2 below.Table 2.

Laboratoryfeatures 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 characteriseinto 3 phases.-      Chronic phase-      Accelerated phase-      Blast phaseIn CML most of patient diagnosed atchronic phase which lasts three to six years if un- treated. The characteristicfeatures of this stage is a persistently high white cell count and may beplatelets with less than 10% of blast cells in the bone marrow. The next phaseis the accelerated phase in which splenomegaly and leucocytosis are evidentwith low platelets count and blast cells between15%- 20% in bone marrow (Cortesand Kantarjian, 2012). The most fatal and advance stage of CML is blast phase with mediansurvival is between two to four months.

The hallmark of this phase is more than30% of blast cells appearing in both peripheral blood and bone marrow (Zhouand Xu, 2015).Table 3 highlights the characteristiclaboratory features of each stage in CML. Table 3: Shows diagnostic features of each stage inCML.                                                                                                                                           (Gratton,2018)                  TreatmentsChronic myeloid leukaemia has gone throughmany revolutionary phases in last two centuries (figure 8). Thetreatment of chronic leukaemia was first introduced in 19th centuryusing arsenic compounds and splenomegaly was treated using radiation in 19thcentury.

The first evidence based treatment for CML was initiated in 1960 withbusulfan which is an alkylation agent (Apperley, 2018). Introduction ofbusulfan for CML was based on first randomised research for CML treatment.However later studies proved that busalfan was unable to significantly reduceblood counts and considered a possible mutagen which may lead to blast crisis(Goldman, 2009). Buslfan was replaced with hydroxycarbamide.

Combination ofthese two drugs relived the symptoms and normalised the full blood count, butdid not succeed in slowing down disease progression to achieve cytogeneticremission (Jabbourand Kantarjian, 2012). Itwas not until 1970s and later in 1980 when stem cell transplant and interferonalpha respectively, were introduced in treatment modality for CML and not onlyshown a complete cytogenetic response but also prolonged the life expectancy tosix to seven years (Bonifazi et al., 2001).A comparison study by Guilhot et al.,(1997) shown a good cytogenetic response for CML patients treated withinterferon alpha and cytarabine than interferon alpha as a sole treatment.These findings were further supported by chen et al, (2011) bydemonstrating a complete cytogenetic and haematological response with prolongedsurvival of four to five years, when CML patients treated with combination ofinterferon alpha and cytarbine as opposed to interferon alpha only.

Howeverstudy reported some severe side effects of combined therapy which includedweight loss, nausea vomiting and diarrhoea.                                                                                     Hamad et al (2013)Figure 8: Shows historic moments in the evolution ofCML treatment Thusuntil 1990 CML patients were treated with combination of interferon alpha andcytarabine or interferon alpha alone. However young and healthy CML patientswere treated with allogenic stem cell transplant despite its toxicity and riskof host versus graft disease (Goldman, 2009). The treatment of CML revolutionised following breakthrough in discoveryof BCR-ABL oncoprotein which lead to development of drugs known as imatinibwhich inhibit activities of these oncoprotein (Bollmann and Giglio, 2011).

Druker et al., (1996 & 2001)published a report on the first data on tyrosine kinase inhibitor2-phenylaminopyrimidine Abl1 named as signal transduction inhibitor 571 orSTI571, now known as Imatinib, for its effectiveness in inhibition of tyrosinkinase in BCR-ABL oncoprotein. Imatinib works as a competitive inhibitor asshown in figure 9  on BCR-ABL oncoproteinat adenosine triphospate binding (ATP) site for its substrate which inhibitsphosphorylation of protein engaged in signal transduction. This action ofimatinib hinders the oncoprotein function and signals for stem cell growthfactors and platelets derived growth factors ((Mulu Fentie et al., 2017)and restores normal cellular function by inhibiting cell proliferation andinducing cell apoptosis in CML patients. Several studies have demonstrated the effectiveness of imatinib as compared withother treatments choices measured by haematological, cytogenetic and molecularresponse to disease (Table 4). Table 4 : Defines treatment response in CMLpatients                                                                                                                Bolimanand Giglio (2011)  In2002 (Kantarjian et al.

,) studiedimatinib outcomes in 454 patients who failed to respond to interferon alpha inchronic phase of CML. The study shown a complete haematological response in95%   of patients (430/454 patients) withno disease progression, to accelerated phase or blast crisis, in 89% ofpatients over a period of 18 months. These findings were consistent with recentstudies.  Houshhaus et al., (2017) conducted a randomised trial on newly diagnosed CMLpatients treated with imatinib and interferon alpha combined with cytarabinefor efficacy and safety to include treatment response, survival and seriouscomplication.  The results shown acomplete cytogenetic response in 82.

8% of patients and 83% estimated to haveoverall survival of 10 years.                                                                                     Tamascar and Ramanarayanan (2009)Figure 9: Shows imatinib action mechanism: A) In CML the phosphorylation and activation of tyrosine residuefollowing binding of adenosine triphosphate (ATP) in the kinase domain on theBCR-ABL oncoprotein. B) Imatiniboccupies the ATP binding sites on BCR-ABL oncoprotein and prevents substratephosphorylation and signal transduction pathways which inhibit proliferationand survival which is basis of CML pathogenesis.These findings are considered highlysignificant to support imatinib as first line of treatment in chronic phaseCML. Despite excellent treatment results are achieved with imatinib, howeverfindings are not consistent among some patients who developed imatinibresistance (Ref).

The drugresistance in CML is BCR-ABL dependant or independent (Figure) and occurs through various mechanisms including BCR-ABLover expression and genetic mutation. The resistance can be overcome by givinghigh dose imatinib or treating with second generation tyrosin kinase drugs (Vaidya et al., 2011) ).The second generation drugsinclude bosutinib, dasatinib and nilotinib which are considered more effectivetyrosin kinase inhibitor. These drugs are highly active against all most allCML mutations. Patients newly diagnosed with CML, and in chronic phase ofdisease with Ph positive and resistant to imatinib, are treat with dasatiniband nilotinib whereas Bosutinib used for patients who are resistant to imatiniband are in accelerated or blast phase of disease(Krishnan,2018).

                                                                                                Huang et al., 2016Figure: CMLresistance mechanisms