chronic myeloid leukemia ( cml ) is a myeloproliferative disorder characterized by overproduction of immature and mature myeloid cells in the peripheral blood , bone marrow and spleen . in more than 90% of cases ,
the disease is diagnosed during the initial chronic phase ( cml - cp ) , which is characterized by expansion of functionally normal myeloid cells . if untreated , cml progresses to an initial accelerated phase ( ap ) , and subsequently to a more aggressive blast phase ( bp ) , with loss of terminal differentiation capacity .
a hallmark of cml is the presence of ( 9 ; 22 ) ( q34 ; q11 ) reciprocal translocation , which is cytogenetically visible as philadelphia chromosome ( ph ) and results in the formation of bcr - abl1 fusion protein .
this fusion protein is a constitutively active tyrosine kinase which is necessary and sufficient for malignant transformation ( 1 ) . in vitro studies
have demonstrated that bcr - abl1 is oncogenic , and leads to leukemic cell proliferation and inhibition of apoptosis ( 2 ) .
it is believed that bcr - abl1 gene is initially generated in a single hematopoietic stem cell ( hsc ) which gives it proliferative advantage over its normal counterparts , eventually leading to an expanded myeloid compartment ( 3 ) .
the introduction of imatinib , a bcr - abl1- targeting tyrosine kinase inhibitor ( tki ) has revolutionized cml therapy . following the success of the pivotal iris ( international randomized study of interferon and sti571 ) trial , imatinib mesylate ( gleevec , novartis , basel , switzerland ) - formerly known as
sti571- rapidly became the preferred first line treatment for patients with newly diagnosed cml in chronic phase ( 4 , 5 ) .
subsequently , two other novel tkis with increased activity against bcr - abl1 were developed , dasatinib ( sprycel , bristol - myers squibb , princeton , nj ) and nilotinib ( novartis ) , which were approved for newly diagnosed cml patients and those with previously treated cml ( 6 , 7 ) .
another bcr - abl1 inhibitor is bosutinib ( tasigna , pfizer , new york , ny ) which has been approved for the treatment of chronic , accelerated , or blast phase of cml ( 8) . ponatinib ( iclusig , ariad , cambridge , ma ) is a potent multitargeted kinase inhibitor that has been approved for the treatment of cml - cp , cml - ap , and cml - bp ( 9 ) .
the first is the development of resistance to bcr - abl1 inhibitors in some patients , which can be due to bcr - abl1 overexpression , differences in cellular drug influx and efflux , activation of alternative signaling pathways , or emergence of bcr - abl1 kinase domain mutations during tki treatment ( 10 ) .
the second is the limited efficiency of bcr - abl1-tkis in blast crisis ( bc ) cml ( 11 ) .
this can be due to generation of additional chromosomal and molecular changes during transition from chronic phase to blast phase . therefore
, these cml blast cells may not depend entirely on bcr - abl1 pathway for survival ( 12 , 13 ) .
the third is the insensitivity of cml stem cells to bcr - abl1 inhibitors ( 14 , 15 ) .
cml is sustained by a population of cd/ bcr - abl1 progenitor cells with stem cell properties .
therefore , conventional chemotherapeutics and bcr - abl1 inhibitors which act by inhibiting cell proliferation and inducing apoptosis , are ineffective against these non - proliferating stem cells ( 16 , 17 ) . to reach an ultimate cure , development of new and more effective therapies involving elimination of cml stem cells is required .
the breakpoints within the abl1 gene occurs either upstream of exon 1b , downstream of exon 1a , or more frequently , between exons 1b and 1a ( figure 1 a ) . in most patients with cml ,
the breakpoints within the bcr gene take place in a 5.8-kilobase area spanning exons 12 - 16 , referred to as the major breakpoint cluster region ( m - bcr ) ( figure 1 b ) ( 1 ) .
1a , the abl1 gene is located on chromosome 9q34 and spans more than 230 kb .
it contains two alternative first exons , exon 1b and 1a , followed by exons 2 to 11 .
the breakpoints that create the philadelphia chromosome , are scattered over a large area ( more than 300 kb ) at the 5 end of the gene , either upstream of alternative exon 1b , between alternative exons 1b and 1a , or between exons 1a and 2 .
the breakpoint is in the major breakpoint cluster region that spans exons 12 - 16 .
the n - terminal coiled - coil domain of bcr - abl1 has dimerization and trans - autophosphorylation activity ( 18 ) .
autophosphorylation of tyrosine-177 of bcr - abl1 results in formation of a complex between grb2 , gab2 , and sos , leading to activation of ras and phosphatidylinositol 3-kinase ( pi3k ) pathways ( 19 ) . signaling from ras - raf - mek - mapk pathway
pi3k activates the serine - threonine kinase akt , which results in : 1 , promoting survival by inhibiting foxo transcription factors ( 20 ) ; 2 , activation of mtor which increases protein translation ( 21 ) ; and 3 , increasing the expression of skp2 , the recognition protein of scf e3 ubiquitin ligase , which degrades p27 and results in enhanced proliferation ( 22 ) .
another crucial consequence of bcr - abl activation is stat5 activation , either through direct phosphorylation or indirectly through phosphorylation by jak2 or hck ( 23 ) .
lack of stat5 inhibits both myeloid and lymphoid leukemogenesis ( 24 ) ( figure 2 ) .
dimerization of bcr - abl1 induces autophosphorylation and activation of the kinase , generating docking sites for adaptor proteins like grb2 .
this signaling pathway results in activation of multiple downstream targets , leading to increased proliferation , enhanced survival , and perturbation of cell adhesion and migration .
imatinib , dasatinib , and nilotinib bind to the atp - binding pocket of the abl kinase domain and inhibit bcr - abl activity ( 25 - 27 ) .
imatinib binds to the inactive conformation of bcr - abl , and prevents conformational changes needed for bcr - abl activation ( 25 ) .
nilotinib was developed by changing the structure of imatinib and binds to bcr - abl with a higher affinity .
nilotinib is 10 to 50 times more potent than imatinib for inhibiting bcr - abl ( 26 ) .
dasatinib has a different chemical structure and binds to a slightly different region of the atp- binding pocket .
dasatinib binds to the active conformation of bcr - abl and is 325 fold more potent than imatinib and 16 fold more potent than nilotinib ( 27 ) .
conventional cytogenetic analysis of bone marrow aspirates is performed by assessing ph positivity in at least 20 giemsa- stained metaphase cells .
complete cytogenetic response ( ccyr ) is defined as the absence of detectable ph+ metaphase cells , which is a clinically important prognostic marker in cml patients . in patients without complete cytogenetic response ,
the degree of cytogenetic response is classified as partial ( 1% - 35% ph metaphase cells ) , major ( 0% - 35% ph metaphase cells ) , or minor ( > 35% ph metaphase cells ) .
a limitation of cytogenetic testing is the need for an invasive bone marrow biopsy to culture cells and obtain metaphase spreads .
furthermore , the limit of detection of this method is relatively low , because a minimum of 20 metaphase cells are analyzed ( 1:20 or 5% ) .
however , cytogenetic testing has the advantage of detecting , in addition to philadelphia chromosome , other chromosomal abnormalities which may have prognostic value ( 28 ) .
a more sensitive detection method is to assess peripheral blood interphase cells with fluorescence in situ hybridization ( fish ) , thereby analyzing a higher number of cells in comparison with conventional cytogenetic testing .
approximately , 100 to 500 interphase cells are usually assessed with fish , so this method is more sensitive ( 1:500 to 1:100 , or 0.2% to 1% ) .
however depending on the probes used , fish can have a false - positive rate of 1% to 10% .
fish method can be used for monitoring response to therapy until fish levels are less than 5% to 10% .
this method is not useful for analyzing further reduction in ph metaphases ( 28 ) .
real - time quantitative pcr is an effective and clinically validated method to quantify bcr - abl1 transcript levels and assess molecular responses .
guidelines from national comprehensive cancer network ( nccn ) and european leukemianet ( eln ) recommend serial bcr - abl1 rt - qpcr assays at regular 3 to 6-month intervals for routine minimal residual disease ( mrd ) monitoring of cml treatment ( 28 , 29 ) .
molecular monitoring includes rna extraction from a bone marrow or peripheral blood sample and subsequent rt - qpcr to measure the level of bcr - abl1 transcripts relative to a reference gene . in comparison with cytogenetic testing , pcr - based molecular
monitoring offers exquisite sensitivity , almost 100 to 1000 times greater than fish or conventional cytogenetic analysis
. it can assess bone marrow or peripheral blood samples , and provides quantitative results with validated clinical response thresholds ( 30 , 31 ) .
a significant proportion of cml patients do not achieve a satisfactory response to first - line tki therapy , which is mostly due to drug resistance .
tki resistance is classified as primary ( lack of initial response to tki ) or acquired ( loss of response ) .
primary imatinib resistance is defined as failure to achieve any of the following criteria : a complete hematologic response ( chr ) within 3 to 6 months , any cytogenetic response ( cyr ) by 6 months , a major cytogenetic response ( mcyr ) by 12 months or a complete cytogenetic response ( ccyr ) by 18 months on imatinib treatment .
primary hematologic resistance is very rare in newly diagnosed cml - cp patients , whereas primary cytogenetic resistance is seen in 15% to 25% of patients .
suboptimal response is defined as : no cyr at 3 months , less than mcyr at 6 months , less than ccyr at 12 months , or less than major molecular response at 18 months .
acquired resistance is defined as loss of chr or ccyr or progression of cml at any time on treatment ( 28 , 29 , 32 ) .
it has been shown that the risk of imatinib resistance varies depending on cml phase : the risk is lowest in cml - cp and highest in cml - bp ( 4 , 33 ) .
mutations in the abl1 tyrosine kinase domain of bcr - abl1 are responsible for a substantial proportion of imatinib resistance .
bcr - abl1 mutations have been detected in 35% - 45% of cml patients with imatinib resistance ( 4 , 34 , 35 ) .
these mutations are more frequently found in patients with acquired resistance rather than primary resistance ( 36 ) .
furthermore , bcr - abl1 mutations occur more often in patients with advanced cml compared to cml - cp ( 37 ) .
less frequently , amplification of bcr - abl1 gene or increased bcr - abl1 expression can also lead to tki resistance ( 34 , 38 ) .
bcr - abl1 independent mechanisms include activation of the src family of kinases , cytogenetic clonal evolutions with emergence of additional chromosomal abnormalities in ph cml cells , or the presence of quiescent stem cells may also contribute to tki resistance ( 34 , 39 ) .
point mutations in the abl1 kinase domain are the most frequent mechanism of tki resistance .
these mutations impair drug binding by changing essential amino acids for direct contact with the tki or by preventing bcr - abl1 from adopting the inactive conformation appropriate for tki binding .
the t315i mutation is one of the most frequent mutations detected in 4% to 19% of resistant cases ( 37 , 40 ) .
some investigators have suggested that t315i is associated with disease progression and poor survival ( 40 , 41 ) different mutations in the abl1 kinase domain have been classified into four categories : a , in the imatinib binding site ; b , in the p - loop ( atp binding site ) ; c , in the catalytic domain ; d , in the activation ( a ) loop .
mutations in the p - loop ( residues 244 - 255 of abl1 ) accounts for about 48% of all mutations in imatinib resistant cases , a worse prognosis regardless of sensitivity to imatinib ( 42 - 44 ) .
mutations in the catalytic domain ( residues 350 - 363 of abl1 ) can also affect imatinib binding .
the activation loop is the major regulatory component of abl1 kinase domain and can adopt an open - active or closed - inactive conformation .
mutations in the activation loop impair adopting the inactive conformation which is required for imatinib activity .
amino acid substitutions at seven residues [ m244v , g250e , y253f / h , e255k / v ( p - loop ) , t315i ( imatinib binding site ) , m351 t , and f359v ( catalytic domain ) ] account for 85% of all resistance associated mutations ( 36 , 45 ) .
although point mutations have more frequently been detected in tki resistant and advanced cml , they have also been detected prior to tki treatment .
these findings suggest that prior to tki therapy ; these mutations do not confer a survival advantage ( 46 ) .
it is unclear whether certain mutations are responsible for cml progression or they are a consequence of genomic instability in advanced disease ( 47 ) .
it seems that gain of function mutations may contribute to cml progression , whereas loss of function mutations are frequently subject to selective pressure by imatinib ( 48 , 49 ) .
table 2 summarizes in vitro sensitivity of bcr - abl1 kinase domain mutations to imatinib , nilotinib and dasatinib .
although nilotinib and dasatinib inhibit most of the clinically relevant abl1 mutations , with the exception of t315i mutation , the presence of mutations after imatinib failure as well as development of new mutations on subsequent tki treatment is a potential source of resistance to successive tki ( 50 - 53 ) .
it has been shown that in imatinib - resistant patients , subsequently treated with an alternative tki , 83% of cases after an initial response are associated with emergence of newly acquired mutations .
patients already harboring imatinib - resistant kinase domain mutations , had higher likelihood of relapse associated with development of further mutations compared to patients who did not have mutations ( 54 ) .
the presence of additional chromosomal abnormalities besides ph chromosome is defined as clonal evolution and is considered to be a feature of advanced cml ( 55 ) .
the most frequent chromosomal abnormalities include , in order , an additional ph chromosome ( 38% ) , trisomy 8 ( 34% ) , and isochromosome 17q ( 56 ) ; which have been associated with bcr - abl1 overexpression , myc overexpression , and loss of 17p , respectively ( 57 , 58 ) .
other aberrations , such as trisomy 19 , trisomy 21 , trisomy 17 , and deletion of 7 , have been reported in less than 10% of patients with clonal evolution .
it has been reported that cml - cp patients harbor 0.47 copy number alterations per case ( range 0 - 8) , but cml - bp patients carry 7.8 copy number alterations ( range 0 - 28 ) , supporting the notion that multiple genomic alterations accumulate during cml progression to bp phase ( 59 ) .
clonal evolution is associated with reduced response to imatinib , increase in relapse of disease , and reduction in overall survival ( 60 , 61 ) .
it is proposed that clonal evolution reflects the genomic instability of the highly proliferative cml progenitor cells associated with disease progression .
the frequency of clonal evolution increases with advancing stage , from 30% in accelerated phase to 80% in blast phase ( 55 ) .
cml stem cells originate from hematopoietic stem cells ( hscs ) by acquiring the bcr - abl1 mutation .
this fusion oncoprotein can transform hscs , but is not sufficient to transform committed myeloid progenitors which lack inherent self - renewal capability ( 62 ) .
cml stem cells are cd/ cd cells which have entered the g0 phase of the cell cycle and are therefore quiescent .
these cells account for less than 1% of cd cells present at diagnosis ( 15 ) .
it is postulated that this quiescent fraction sustains the disease with constant potential to aggravation . during transition from chronic phase to blast phase , cml stem cells acquire further genetic and/or epigenetic aberrations that provide survival advantage and resistance to programmed cell death .
the resistance of quiescent stem cells to tki seems to be multifactorial and include altered drug influx and efflux ( decreased expression of oct1 and increased expression of abcb1 and abcg2 ) ( 63 ) , increased bcr - abl1 transcript level and decreased degradation of bcr - abl1 transcript ( 15 ) .
crkl phosphorylation is a downstream target of bcr - abl1 and dasatinib , contrary to imatinib , can inhibit its phosphorylation in cd cd cells .
dasatinib can also inhibit an earlier progenitor population , but is unable to eradicate the most primitive quiescent stem cells ( 15 , 64 ) .
the breakpoints within the abl1 gene occurs either upstream of exon 1b , downstream of exon 1a , or more frequently , between exons 1b and 1a ( figure 1 a ) . in most patients with cml ,
the breakpoints within the bcr gene take place in a 5.8-kilobase area spanning exons 12 - 16 , referred to as the major breakpoint cluster region ( m - bcr ) ( figure 1 b ) ( 1 ) .
1a , the abl1 gene is located on chromosome 9q34 and spans more than 230 kb .
it contains two alternative first exons , exon 1b and 1a , followed by exons 2 to 11 .
the breakpoints that create the philadelphia chromosome , are scattered over a large area ( more than 300 kb ) at the 5 end of the gene , either upstream of alternative exon 1b , between alternative exons 1b and 1a , or between exons 1a and 2 .
the breakpoint is in the major breakpoint cluster region that spans exons 12 - 16 .
the n - terminal coiled - coil domain of bcr - abl1 has dimerization and trans - autophosphorylation activity ( 18 ) .
autophosphorylation of tyrosine-177 of bcr - abl1 results in formation of a complex between grb2 , gab2 , and sos , leading to activation of ras and phosphatidylinositol 3-kinase ( pi3k ) pathways ( 19 ) . signaling from ras - raf - mek - mapk pathway
pi3k activates the serine - threonine kinase akt , which results in : 1 , promoting survival by inhibiting foxo transcription factors ( 20 ) ; 2 , activation of mtor which increases protein translation ( 21 ) ; and 3 , increasing the expression of skp2 , the recognition protein of scf e3 ubiquitin ligase , which degrades p27 and results in enhanced proliferation ( 22 ) .
another crucial consequence of bcr - abl activation is stat5 activation , either through direct phosphorylation or indirectly through phosphorylation by jak2 or hck ( 23 ) .
lack of stat5 inhibits both myeloid and lymphoid leukemogenesis ( 24 ) ( figure 2 ) .
dimerization of bcr - abl1 induces autophosphorylation and activation of the kinase , generating docking sites for adaptor proteins like grb2 .
this signaling pathway results in activation of multiple downstream targets , leading to increased proliferation , enhanced survival , and perturbation of cell adhesion and migration .
imatinib , dasatinib , and nilotinib bind to the atp - binding pocket of the abl kinase domain and inhibit bcr - abl activity ( 25 - 27 ) .
imatinib binds to the inactive conformation of bcr - abl , and prevents conformational changes needed for bcr - abl activation ( 25 ) .
nilotinib was developed by changing the structure of imatinib and binds to bcr - abl with a higher affinity .
nilotinib is 10 to 50 times more potent than imatinib for inhibiting bcr - abl ( 26 ) .
dasatinib has a different chemical structure and binds to a slightly different region of the atp- binding pocket .
dasatinib binds to the active conformation of bcr - abl and is 325 fold more potent than imatinib and 16 fold more potent than nilotinib ( 27 ) .
complete hematologic response is assessed by analysis of peripheral blood counts . conventional cytogenetic analysis of bone marrow aspirates is performed by assessing ph positivity in at least 20 giemsa- stained metaphase cells .
complete cytogenetic response ( ccyr ) is defined as the absence of detectable ph+ metaphase cells , which is a clinically important prognostic marker in cml patients . in patients without complete cytogenetic response ,
the degree of cytogenetic response is classified as partial ( 1% - 35% ph metaphase cells ) , major ( 0% - 35% ph metaphase cells ) , or minor ( > 35% ph metaphase cells ) .
a limitation of cytogenetic testing is the need for an invasive bone marrow biopsy to culture cells and obtain metaphase spreads .
furthermore , the limit of detection of this method is relatively low , because a minimum of 20 metaphase cells are analyzed ( 1:20 or 5% ) .
however , cytogenetic testing has the advantage of detecting , in addition to philadelphia chromosome , other chromosomal abnormalities which may have prognostic value ( 28 ) .
a more sensitive detection method is to assess peripheral blood interphase cells with fluorescence in situ hybridization ( fish ) , thereby analyzing a higher number of cells in comparison with conventional cytogenetic testing .
approximately , 100 to 500 interphase cells are usually assessed with fish , so this method is more sensitive ( 1:500 to 1:100 , or 0.2% to 1% ) .
however depending on the probes used , fish can have a false - positive rate of 1% to 10% .
fish method can be used for monitoring response to therapy until fish levels are less than 5% to 10% .
this method is not useful for analyzing further reduction in ph metaphases ( 28 ) .
real - time quantitative pcr is an effective and clinically validated method to quantify bcr - abl1 transcript levels and assess molecular responses .
guidelines from national comprehensive cancer network ( nccn ) and european leukemianet ( eln ) recommend serial bcr - abl1 rt - qpcr assays at regular 3 to 6-month intervals for routine minimal residual disease ( mrd ) monitoring of cml treatment ( 28 , 29 ) .
molecular monitoring includes rna extraction from a bone marrow or peripheral blood sample and subsequent rt - qpcr to measure the level of bcr - abl1 transcripts relative to a reference gene . in comparison with cytogenetic testing
, pcr - based molecular monitoring offers exquisite sensitivity , almost 100 to 1000 times greater than fish or conventional cytogenetic analysis
. it can assess bone marrow or peripheral blood samples , and provides quantitative results with validated clinical response thresholds ( 30 , 31 ) .
a significant proportion of cml patients do not achieve a satisfactory response to first - line tki therapy , which is mostly due to drug resistance .
tki resistance is classified as primary ( lack of initial response to tki ) or acquired ( loss of response ) .
primary imatinib resistance is defined as failure to achieve any of the following criteria : a complete hematologic response ( chr ) within 3 to 6 months , any cytogenetic response ( cyr ) by 6 months , a major cytogenetic response ( mcyr ) by 12 months or a complete cytogenetic response ( ccyr ) by 18 months on imatinib treatment .
primary hematologic resistance is very rare in newly diagnosed cml - cp patients , whereas primary cytogenetic resistance is seen in 15% to 25% of patients .
suboptimal response is defined as : no cyr at 3 months , less than mcyr at 6 months , less than ccyr at 12 months , or less than major molecular response at 18 months .
acquired resistance is defined as loss of chr or ccyr or progression of cml at any time on treatment ( 28 , 29 , 32 ) .
it has been shown that the risk of imatinib resistance varies depending on cml phase : the risk is lowest in cml - cp and highest in cml - bp ( 4 , 33 ) .
mutations in the abl1 tyrosine kinase domain of bcr - abl1 are responsible for a substantial proportion of imatinib resistance .
bcr - abl1 mutations have been detected in 35% - 45% of cml patients with imatinib resistance ( 4 , 34 , 35 ) .
these mutations are more frequently found in patients with acquired resistance rather than primary resistance ( 36 ) .
furthermore , bcr - abl1 mutations occur more often in patients with advanced cml compared to cml - cp ( 37 ) .
less frequently , amplification of bcr - abl1 gene or increased bcr - abl1 expression can also lead to tki resistance ( 34 , 38 ) .
bcr - abl1 independent mechanisms include activation of the src family of kinases , cytogenetic clonal evolutions with emergence of additional chromosomal abnormalities in ph cml cells , or the presence of quiescent stem cells may also contribute to tki resistance ( 34 , 39 ) .
point mutations in the abl1 kinase domain are the most frequent mechanism of tki resistance .
these mutations impair drug binding by changing essential amino acids for direct contact with the tki or by preventing bcr - abl1 from adopting the inactive conformation appropriate for tki binding .
the t315i mutation is one of the most frequent mutations detected in 4% to 19% of resistant cases ( 37 , 40 ) .
some investigators have suggested that t315i is associated with disease progression and poor survival ( 40 , 41 ) different mutations in the abl1 kinase domain have been classified into four categories : a , in the imatinib binding site ; b , in the p - loop ( atp binding site ) ; c , in the catalytic domain ; d , in the activation ( a ) loop .
mutations in the p - loop ( residues 244 - 255 of abl1 ) accounts for about 48% of all mutations in imatinib resistant cases , a worse prognosis regardless of sensitivity to imatinib ( 42 - 44 ) .
mutations in the catalytic domain ( residues 350 - 363 of abl1 ) can also affect imatinib binding .
the activation loop is the major regulatory component of abl1 kinase domain and can adopt an open - active or closed - inactive conformation .
mutations in the activation loop impair adopting the inactive conformation which is required for imatinib activity .
amino acid substitutions at seven residues [ m244v , g250e , y253f / h , e255k / v ( p - loop ) , t315i ( imatinib binding site ) , m351 t , and f359v ( catalytic domain ) ] account for 85% of all resistance associated mutations ( 36 , 45 ) .
although point mutations have more frequently been detected in tki resistant and advanced cml , they have also been detected prior to tki treatment .
these findings suggest that prior to tki therapy ; these mutations do not confer a survival advantage ( 46 ) .
it is unclear whether certain mutations are responsible for cml progression or they are a consequence of genomic instability in advanced disease ( 47 ) .
it seems that gain of function mutations may contribute to cml progression , whereas loss of function mutations are frequently subject to selective pressure by imatinib ( 48 , 49 ) .
table 2 summarizes in vitro sensitivity of bcr - abl1 kinase domain mutations to imatinib , nilotinib and dasatinib .
although nilotinib and dasatinib inhibit most of the clinically relevant abl1 mutations , with the exception of t315i mutation , the presence of mutations after imatinib failure as well as development of new mutations on subsequent tki treatment is a potential source of resistance to successive tki ( 50 - 53 ) .
it has been shown that in imatinib - resistant patients , subsequently treated with an alternative tki , 83% of cases after an initial response are associated with emergence of newly acquired mutations .
patients already harboring imatinib - resistant kinase domain mutations , had higher likelihood of relapse associated with development of further mutations compared to patients who did not have mutations ( 54 ) .
the presence of additional chromosomal abnormalities besides ph chromosome is defined as clonal evolution and is considered to be a feature of advanced cml ( 55 ) .
the most frequent chromosomal abnormalities include , in order , an additional ph chromosome ( 38% ) , trisomy 8 ( 34% ) , and isochromosome 17q ( 56 ) ; which have been associated with bcr - abl1 overexpression , myc overexpression , and loss of 17p , respectively ( 57 , 58 ) .
other aberrations , such as trisomy 19 , trisomy 21 , trisomy 17 , and deletion of 7 , have been reported in less than 10% of patients with clonal evolution .
it has been reported that cml - cp patients harbor 0.47 copy number alterations per case ( range 0 - 8) , but cml - bp patients carry 7.8 copy number alterations ( range 0 - 28 ) , supporting the notion that multiple genomic alterations accumulate during cml progression to bp phase ( 59 ) .
clonal evolution is associated with reduced response to imatinib , increase in relapse of disease , and reduction in overall survival ( 60 , 61 ) .
it is proposed that clonal evolution reflects the genomic instability of the highly proliferative cml progenitor cells associated with disease progression .
the frequency of clonal evolution increases with advancing stage , from 30% in accelerated phase to 80% in blast phase ( 55 ) .
cml stem cells originate from hematopoietic stem cells ( hscs ) by acquiring the bcr - abl1 mutation .
this fusion oncoprotein can transform hscs , but is not sufficient to transform committed myeloid progenitors which lack inherent self - renewal capability ( 62 ) .
cml stem cells are cd/ cd cells which have entered the g0 phase of the cell cycle and are therefore quiescent .
these cells account for less than 1% of cd cells present at diagnosis ( 15 ) .
it is postulated that this quiescent fraction sustains the disease with constant potential to aggravation . during transition from chronic phase to blast phase ,
cml stem cells acquire further genetic and/or epigenetic aberrations that provide survival advantage and resistance to programmed cell death .
the resistance of quiescent stem cells to tki seems to be multifactorial and include altered drug influx and efflux ( decreased expression of oct1 and increased expression of abcb1 and abcg2 ) ( 63 ) , increased bcr - abl1 transcript level and decreased degradation of bcr - abl1 transcript ( 15 ) .
crkl phosphorylation is a downstream target of bcr - abl1 and dasatinib , contrary to imatinib , can inhibit its phosphorylation in cd cd cells .
dasatinib can also inhibit an earlier progenitor population , but is unable to eradicate the most primitive quiescent stem cells ( 15 , 64 ) .
the emphasis is currently on overcoming imatinib resistance and the development of alternative tki engineered to surmount this problem .
combination of tkis and different targeting agents , including those targeting self - renewal and those inducing apoptosis increases the efficacy of tkis on cml cells
. a better understanding of the mechanisms that underlie tki resistance , progression to bp , genomic instability and stem cell quiescence is essential to develop curative strategies for patients with cml .
 context : chronic myeloid leukemia ( cml ) is a myeloproliferative disorder characterized by overproduction of immature and matured myeloid cells in the peripheral blood , bone marrow and spleen.evidence acquisition : a hallmark of cml is the presence of ( 9 ; 22 ) ( q34 ; q11 ) reciprocal translocation , which is cytogenetically visible as philadelphia chromosome ( ph ) and results in the formation of bcr - abl1 fusion protein . 
 this fusion protein is a constitutively active tyrosine kinase which is necessary and sufficient for malignant transformation . 
 the introduction of imatinib , a bcr - abl1- targeting tyrosine kinase inhibitor ( tki ) has revolutionized cml therapy . 
 subsequently , two other tkis with increased activity against bcr - abl1 , dasatinib and nilotinib , were developed and approved for cml patients . 
 nevertheless , cml therapy faces major challenges.results:the first is the development of resistance to bcr - abl1 inhibitors in some patients , which can be due to bcr - abl1 overexpression , differences in cellular drug influx and efflux , activation of alternative signaling pathways , or emergence of bcr - abl1 kinase domain mutations during tki treatment . 
 the second is the limited efficiency of bcr - abl1-tkis in blast crisis ( bc ) cml . 
 the third is the insensitivity of cml stem cells to bcr - abl1 inhibitors . 
 conventional chemotherapeutics and bcr - abl1 inhibitors which act by inhibiting cell proliferation and inducing apoptosis , are ineffective against quiescent cml stem cells.conclusions:a better understanding of the mechanisms that underlie tki resistance , progression to bc , genomic instability and stem cell quiescence is essential to develop curative strategies for patients with cml . 
1. Context
2. Evidence Acquisition
3. Results
3.1. BCR-ABL1 Signaling Pathway
3.2. Mechanism of Action of BCR-ABL Inhibitors
3.3. Cytogenetic and Molecular Response During CML Treatment
3.4. Mechanisms of Resistance to TKI Inhibitors
3.5. ABL1 Kinase Mutations
3.6. Clonal Evolution
3.7. CML Stem Cells
4. Conclusions