-
Mechanisms of Resistance to Imatinib and
Second-GenerationTyrosine Inhibitors in Chronic Myeloid
LeukemiaDragana Milojkovic1 and Jane Apperley2
Abstract Targeted therapy in the form of selective tyrosine
kinase inhibitors (TKI) has trans-formed the approach to management
of chronic myeloid leukemia (CML) and dramat-ically improved
patient outcome to the extent that imatinib is currently accepted
as thefirst-line agent for nearly all patients presenting with CML,
regardless of the phase ofthe disease. Impressive clinical
responses are obtained in the majority of patients inchronic phase;
however, not all patients experience an optimal response to
imatinib,and furthermore, the clinical response in a number of
patients will not be sustained.The process by which the leukemic
cells prove resistant to TKIs and the restorationof BCR-ABL1 signal
transduction from previous inhibition has initiated the pursuit
forthe causal mechanisms of resistance and strategies by which to
surmount resistance totherapeutic intervention. ABL kinase domain
mutations have been extensively implicat-ed in the pathogenesis of
TKI resistance, however, it is increasingly evident that
thepresence of mutations does not explain all cases of resistance
and does not accountfor the failure of TKIs to eliminate minimal
residual disease in patients who respondoptimally. The focus of
exploring TKI resistance has expanded to include the mecha-nism by
which the drug is delivered to its target and the impact of drug
influx and effluxproteins on TKI bioavailability. The limitations
of imatinib have inspired the develop-ment of second generation
TKIs in order to overcome the effect of resistance to thisprimary
therapy. (Clin Cancer Res 2009;15(24):7519–27)
Chronic myeloid leukemia (CML) results from the
balancedtranslocation of c-ABL from chromosome 9 and BCR on
chro-mosome 22 leading to the formation of BCR-ABL1
chimericoncoprotein, the product of the BCR-ABL1 hybrid gene,
withconstitutive tyrosine kinase activity (1, 2). Deregulated
BCR-ABL1 activity results in enhanced cellular proliferation, and
re-sistance to apoptosis and oncogenesis (3, 4). CML
naturallyprogresses through distinct phases from early chronic
phaseto an intermediate accelerated phase followed by a
terminalblast phase. Imatinib, the first tyrosine kinase inhibitor
(TKI)approved for the treatment of CML (5), is a
phenylaminopyri-dimine, which principally targets the tyrosine
kinase activity ofBCR-ABL1, exclusively binding to BCR-ABL1 in the
inactiveconformation in addition to inhibitory effects on KIT,
ARG,and PDGFR kinases (6). The recent update of the phase III
ran-domized IRIS study (International Randomized Study of
Inter-feron-α plus Ara-C versus STI571) prospectively
comparingimatinib with interferon-α and cytarabine in previously
untreat-
ed patients in first chronic phase showed the best observed
ratefor a complete cytogenetic response [CCyR; or an
undetectablenumber of Philadelphia chromosome positive (Ph+)
chromo-somes by conventional metaphase analysis] on imatinib of82%
at 6 years (7), with a declining annual rate of progressionas the
molecular response improved with time.
Clinical Resistance to TKIs
In order to best determine an individual's response to
therapy,an operational set of goals, defined within specific time
periodshave been established for all patients (Table 1; ref. 8). An
initialrequirement is the achievement of a complete hematological
re-sponse (CHR), accepted as a normal peripheral blood countwithin
3 months of imatinib. Further response to treatment issubsequently
monitored by sequential cytogenetic assessmentsof the bone marrow
with the aim to achieve a CCyR by 18months. Subsequent evaluation
of the therapeutic response isrecommended by means of molecular
analysis, with reverse-transcriptase polymerase chain reaction
(RT-PCR). Patients thatachieve a major molecular response (MMR)
equivalent to a re-duction in BCR-ABL1 transcripts to less than
0.1% as defined onthe international scale (9), are predicted to
have a remarkablylow risk of disease progression. Within the
framework of recom-mendations, proposals for the definition of
failure and subopti-mal response are now recognized (8). Resistance
to imatinibencompasses failure to reach CHR, CCyR, and MMR within
anallocated duration of time (primary resistance). A number of
Authors' Affiliations: 1Department of Haematology,
HammersmithHospital, 2Department of Haematology, Imperial College
London, London,United KingdomReceived 7/24/09; revised 10/5/09;
accepted 10/13/09; published online12/15/09.Requests for reprints:
Jane F. Apperley, Imperial College London, Hammer-smith Campus, Du
Cane Road, London, W12 0NN United Kingdom. Phone:44-0-20-8383-4017;
Fax: 44-0-20-8742-9335; E-mail: [email protected].
F 2009 American Association for Cancer
Research.doi:10.1158/1078-0432.CCR-09-1068
7519 Clin Cancer Res 2009;15(24) December 15,
2009www.aacrjournals.org
CCR FOCUS
Research. on June 22, 2014. © 2009 American Association for
Cancerclincancerres.aacrjournals.org Downloaded from
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrUnderline
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrHighlight
AdministratorTypewriter1
http://clincancerres.aacrjournals.org/
-
more rapidly yield imatinib-resistant mutant subclones thancells
with low BCR-ABL1 expression levels (75). Similarly, re-sistance to
nilotinib in vitro has also been found consequentto BCR-ABL1
overexpression in vitro (34).
ABL Kinase Domain Mutations
The emergence of mutations within the kinase domain ofBCR-ABL1
is regularly associated with resistance to TKI ther-apy. The most
frequently described mechanism of acquiredresistance to imatinib is
the occurrence of point mutations,representing a single aa
substitution in the kinase domain,which impair drug binding by
affecting essential residuesfor direct contact with the TKI or by
preventing BCR-ABL1from assuming the inactive conformation
appropriate for im-atinib binding. The published incidence of
mutations remainsvariable and in the order of 40 to 90% as a
consequence ofdifferent methods of detection, nature of resistance,
and dis-ease phase examined (76). Mutations were first identified
in2001, in which restoration of BCR-ABL1 signal transductionon
imatinib therapy was associated with a T315I mutation(72). Thr315
forms a fundamental hydrogen bond with im-atinib, disrupted by a
single aa change with a bulkier isoleu-cine, which prevents
imatinib localization within the ATPbinding pocket by consequent
stearic hindrance. The T315Imutation is one of the most frequent
mutations arising inpatients on imatinib therapy, occurring between
4 to 19%of resistant cases (55, 77, 78) and is resistant to all ABL
ki-nase inhibitors. Although the T315I mutation is generally
ac-cepted as conferring a poor outcome (median survival 12.6months;
refs. 79, 80), sustained cytogenetic responses despiteaccelerated
phase and during therapy with a second TKI haverecently been
reported (78).Four categories of mutations have been recognized to
cor-
relate with clinical resistance to imatinib affecting the: (i)
im-atinib binding site, (ii) P-loop (ATP binding site),
(iii)catalytic (C) domain, and (iv) activation (A) loop (2).
Muta-tions in the phosphate (P-loop; residues 244-255 of ABL),which
account for up to 48% of all mutations in imatinibresistant cases
(81), destabilize the conformation requiredfor imatinib binding,
and have been associated with an in-creased transforming potential
(82) and a worse prognosisregardless of their sensitivity to
imatinib (77, 81, 83, 84).P-loop mutations have been reported to be
associated witha worse prognosis in comparison with other
categories ofmutations (81, 83), however, other observers have not
con-firmed these findings (55), perhaps because of the nature ofthe
criteria used to select patients for mutation screening. An-other
potential explanation for this inconsistency may be onaccount of
the M244V mutation, which may not confer apoor outcome and has been
variably included in the P-loopcategories of mutations (84). A
series of mutations are locat-ed in the catalytic domain (residues
350-363 of ABL) andcan also affect imatinib binding. The activation
loop of theABL kinase is the major regulatory component of the
kinasedomain and can adopt an open and/or active or closed and/or
inactive conformation. Mutations in the activation loopinstigate
the open and/or active configuration, and as the in-
active and/or closed configuration is required for imatinib
ac-tivity, resistance occurs. Nevertheless, aa substitutions at
onlyseven residues [M244V, G250E, Y253F/H, E255K/V (P-loop),T315I
(imatinib binding site), M351T, and F359V (catalyticdomain)]
account for 85% of all resistance-associated muta-tions
(80).Although point mutations have been more frequently de-
scribed in TKI resistance and advanced-phase CML (Table3), they
have also been documented prior to TKI therapy(85), inherently
suggesting that pre-existing mutations donot acquire a survival
advantage until subjected to a TKI.In addition, investigators have
found no difference in muta-tional status in those patients who
have relapsed (74). Therelevance of these observations remains
unclear, specificallyabout whether certain mutations are
responsible for diseaseprogression or whether they occur as a
consequence of theunderlying genomic instability linked with
advanced phasedisease (86). It would seem that gain-of-function
mutationsmay independently contribute to disease progression,
whereas
Table 3. Frequency of ABL-kinase domainmutations by disease
phase
KDMutation
No. ofMutations*
No. of CP(%)†
No. of AP(%)*
No. of BP(%)*
P-loop‡
M244 47 33 (70) 1 (2) 13 (28)L248 13 10 (77) 2 (15) 1 (8)G250 63
31 (49) 6 (10) 26 (41)Q252 14 3 (21) 3 (21) 8 (58)Y253 68 23 (34) 9
(13) 36 (53)E255 63 17 (27) 12 (19) 34 (54)
IM binding siteD276 12 6 (50) 2 (17) 4 (33)F311 5 2 (40) 1 (20)
2 (40)T315 56 9 (16) 12 (23) 35 (63)F317 15 10 (67) 2 (13) 3
(20)
Catalytic domainM351 62 33 (53) 12 (19) 17 (28)E355 22 13 (59) 4
(18) 5 (23)F359 35 21 (60) 5 (14) 9 (26)
Activation loopH396 29 21 (72) 2 (7) 6 (21)
C-terminal lobeS417 3 2 (67) 1 (33) 0 (72)E459 6 2 (33) 0 (72) 4
(67)F486 8 0 (0) 1 (13) 7 (88)
Note: Adapted from Apperley (100).Abbreviations: KD, kinase
domain; CP, chronic phase; AP, acceler-ated phase; BP, blast phase;
IM, imatinib.*Number of mutations detected in a pool of patients
reviewed inApperley (100). Infrequently an individual patient
harbored morethan one KD mutation; any detected- mutation is
included in thetable.†Percentage of all KD mutations detected
related to disease phase.‡P-loop mutations have been inconsistently
reported to be associ-ated with a worse prognosis in comparison
with other categories ofmutations (55, 81, 83). Furthermore, the
M244V mutation maynot confer a poor outcome and has been variably
included in theP-loop categories of mutations (84).
7523 Clin Cancer Res 2009;15(24) December 15,
2009www.aacrjournals.org
Resistance to Imatinib and TKIs in CML
Research. on June 22, 2014. © 2009 American Association for
Cancerclincancerres.aacrjournals.org Downloaded from
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrUnderline
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrUnderline
http://clincancerres.aacrjournals.org/
-
loss-of-function mutations are more often subject to
selectivepressure by imatinib (82, 87). Specific mutations
consider-ably affect the transformation potency of BCR-ABL1, andin
vitro studies have indicated relative transformation poten-cies of
mutations from distinct sections of the kinase domainto be: Y253F
> E255K (P-loop) > unmutated BCR-ABL1 ≥ T315I(imatinib
binding site) > H396P (activation loop) > M351T(catalytic
domain; ref. 82). Particular mutations, as in thecase of E255K, are
noted to have increased oncogenic potencydespite reduced kinase
activity compared with unmutatedBCR-ABL1 (88). The proliferative
advantage of a given mutantseems multifactorial and determined by
intrinsic kinase activity,substrate specificity, and extrinsic
factors including growth fac-tors and cytokines.Although most of
the clinically relevant mutations are in-
hibited by dasatinib and nilotinib, with the exception ofT315I
(Fig. 1; ref. 89), the presence of existing mutations af-ter
imatinib failure, as well as development of new mutationson a
subsequent second TKI is naturally a potential source ofresistance
to successive TKI (90–93). The influence of base-line BCR-ABL1
mutations on response to nilotinib in patientswith
imatinib-resistant CML in chronic phase has shown aninferior
outcome in patients who harbored mutations thatwere less sensitive
to nilotinib in vitro (Y253H, E255V/K,F359V/C; ref. 94). Recently,
the selective pressure of sequentialTKI therapy has been assessed
in the outcome of imatinib-resistant patients already harboring
imatinib-resistant kinasedomain mutations subsequently treated with
an alternativeTKI on a second or even third occasion and showed
that 83%
of cases of relapse after an initial response were associated
withthe emergence of newly acquired mutations (95). The
T315Imutation was most commonly implicated with a frequency of36%
(95). The inability to achieve a sustained cytogenetic re-sponse
could in part be as a consequence of the developmentof new
therapy-resistant kinase domain mutations as patientsare exposed to
sequential TKIs, although some of the arising mu-tations were
reported as having a relatively good in vitro sensitiv-ity to the
concurrent TKI (96).In summary, the consequence of identifying a
mutation re-
mains unclear and seems relevant only according to the dis-ease
phase and response, with a greater impact in advancedphase CML in
which the mutated clone may be responsiblefor disease progression,
but less certain in cases of on-goingresponse to TKI therapy.
Resistance mechanisms may be over-come with imatinib dose
escalation (97), alternative therapywith a 2G-TKI (98) to which the
mutant has documentedsensitivity, withdrawing TKI therapy to allow
the mutantclone to recede (99), as well as
non-BCR-ABL1-dependenttherapies.
Conclusions
Targeted molecular therapy has afforded exceptional clini-cal
responses in the majority of patients with CML to theextent that
therapeutic regimens have centered on theachievement of a MMR,
early within the start of therapy.As most will continue on imatinib
in CCyR, the emphasishas diverted to overcoming imatinib resistance
and the
Fig. 1. Although equivalentexperimental systems have
beenemployed in a variety of assessmentsto determine BCR-ABL1
kinase domainmutation sensitivity on the basis of IC50values (89,
98), different incubationtimes and TKI concentration rangeshave
been used as well as varyingmethods to measure cell viability
andproliferation. Color-coded schemes toindicate TKI sensitivity
based on in vitroanalyses should be interpreted withclinical
caution as in vitro findingscannot be directly extrapolated to
theclinical setting. (Figure adapted fromO'Hare et al. 89, © the
American Societyof Hematology).
7524Clin Cancer Res 2009;15(24) December 15, 2009
www.aacrjournals.org
CCR FOCUS
Research. on June 22, 2014. © 2009 American Association for
Cancerclincancerres.aacrjournals.org Downloaded from
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrUnderline
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrHighlight
http://clincancerres.aacrjournals.org/
-
have excellent responses on either dose: a TOPScorrelative
study. Blood 2008;112:3187a.
46. White DL, Saunders VA, Dang P, et al. OCT-1-mediated influx
is a key determinant of the intra-cellular uptake of imatinib but
not nilotinib(AMN107): reduced OCT-1 activity is the causeof low in
vitro sensitivity to imatinib. Blood2006;108:697–704.
47. Dulucq S, Bouchet S, Turcq B, et al. Multidrugresistance
gene (MDR1) polymorphisms are as-sociated with major molecular
responsesto standard-dose imatinib in chronic myeloidleukemia.
Blood 2008;112:2024–7.
48. Kim DH, Sriharsha L, Xu W, et al. Clinical rel-evance of a
pharmacogenetic approach usingmultiple candidate genes to predict
responseand resistance to imatinib therapy in chronicmyeloid
leukemia. Clin Cancer Res 2009;15:4750–8.
49. Majlis A, Smith TL, Talpaz M, O'Brien S, RiosMB, Kantarjian
HM. Significance of cytogeneticclonal evolution in chronic
myelogenous leuke-mia. J Clin Oncol 1996;14:196–203.
50. Johansson B, Fioretos T, Mitelman F. Cyto-genetic and
molecular genetic evolution ofchronic myeloid leukemia. Acta
Haematol2002;107:76–94.
51. O'Dwyer ME, Mauro MJ, Blasdel C, et al. Clon-al evolution
and lack of cytogenetic response areadverse prognostic factors for
hematologic re-lapse of chronic phase CML patients treated
withimatinib mesylate. Blood 2004;103:451–5.
52. Cortes JE, Talpaz M, Giles F, et al. Prognosticsignificance
of cytogenetic clonal evolution inpatients with chronic myelogenous
leukemia onimatinib mesylate therapy. Blood 2003;101:3794–800.
53. Cortes J, O'Dwyer ME. Clonal evolution inchronic myelogenous
leukemia. Hematol OncolClin North Am 2004;18:671–84 [x.].
54. Lahaye T, Riehm B, Berger U, et al. Responseand resistance
in 300 patients with BCR-ABL-positive leukemias treated with
imatinib in a sin-gle center: a 4.5-year follow-up. Cancer
2005;103:1659–69.
55. Jabbour E, Kantarjian H, Jones D, et al. Fre-quency and
clinical significance of BCR-ABL mu-tations in patients with
chronic myeloid leukemiatreated with imatinib mesylate. Leukemia
2006;20:1767–73.
56. Geahlen RL, Handley MD, Harrison ML. Molec-ular interdiction
of Src-family kinase signaling inhematopoietic cells. Oncogene
2004;23:8024–32.
57. Danhauser-Riedl S, Warmuth M, Druker BJ,Emmerich B, Hallek
M. Activation of Src kinasesp53/56lyn and p59hck by p210bcr/abl in
myeloidcells. Cancer Res 1996;56:3589–96.
58. Donato NJ, Wu JY, Stapley J, et al. BCR-ABLindependence and
LYN kinase overexpression inchronic myelogenous leukemia cells
selected forresistance to STI571. Blood 2003;101:690–8.
59. Hu Y, Swerdlow S, Duffy TM, Weinmann R,Lee FY, Li S.
Targeting multiple kinase pathwaysin leukemic progenitors and stem
cells isessential for improved treatment of Ph+ leuke-mia in mice.
Proc Natl Acad Sci U S A 2006;103:16870–5.
60. Dai Y, Rahmani M, Corey SJ, Dent P, Grant
SA.Bcr/Abl-independent, Lyn-dependent form of im-atinib mesylate
(STI-571) resistance is associat-ed with altered expression of
Bcl-2. J BiolChem 2004;279:34227–39.
61. Schindler T, Bornmann W, Pellicena P, MillerWT, Clarkson B,
Kuriyan J. Structural mecha-nism for STI-571 inhibition of abelson
tyrosinekinase. Science 2000;289:1938–42.
62. O'Hare T, Eide CA, Deininger MW. PersistentLYN signaling in
imatinib-resistant, BCR-ABL-
independent chronic myelogenous leukemia. JNatl Cancer Inst
2008;100:908–9.
63. Copland M, Hamilton A, Elrick LJ, et al. Dasa-tinib
(BMS-354825) targets an earlier progenitorpopulation than imatinib
in primary CML butdoes not eliminate the quiescent fraction.
Blood2006;107:4532–9.
64. Jiang X, Zhao Y, Smith C, et al. Chronic mye-loid leukemia
stem cells possess multiple uniquefeatures of resistance to BCR-ABL
targeted ther-apies. Leukemia 2007;21:926–35.
65. Jin L, Tabe Y, Konoplev S, et al. CXCR4 up-regulation by
imatinib induces chronic mye-logenous leukemia (CML) cell
migrationto bone marrow stroma and promotes surviv-al of quiescent
CML cells. Mol Cancer Ther2008;7:48–58.
66. Konig H, Copland M, Chu S, Jove R, HolyoakeTL, Bhatia R.
Effects of dasatinib on SRC ki-nase activity and downstream
intracellularsignaling in primitive chronic myelogenous leu-kemia
hematopoietic cells. Cancer Res 2008;68:9624–33.
67. Jorgensen HG, Allan EK, Jordanides NE,Mountford JC, Holyoake
TL. Nilotinib exertsequipotent antiproliferative effects to
imatiniband does not induce apoptosis in CD34+ CMLcells. Blood
2007;109:4016–9.
68. Copland M, Pellicano F, Richmond L, et al.BMS-214662
potently induces apoptosis ofchronic myeloid leukemia stem and
progenitorcells and synergizes with tyrosine kinase inhibi-tors.
Blood 2008;111:2843–53.
69. Holtz M, Forman SJ, Bhatia R. Growth factorstimulation
reduces residual quiescent chronicmyelogenous leukemia progenitors
remainingafter imatinib treatment. Cancer Res 2007;67:1113–20.
70. Copland M, Fraser AR, Harrison SJ, HolyoakeTL. Targeting the
silent minority: emergingimmunotherapeutic strategies for
eradicationof malignant stem cells in chronic myeloid leu-kaemia.
Cancer Immunol Immunother 2005;54:297–306.
71. Jamieson CH, Ailles LE, Dylla SJ, et
al.Granulocyte-macrophage progenitors as candi-date leukemic stem
cells in blast-crisis CML. NEngl J Med 2004;351:657–67.
72. GorreME,MohammedM, Ellwood K, et al. Clin-ical resistance to
STI-571 cancer therapy causedby BCR-ABL gene mutation or
amplification.Science 2001;293:876–80.
73. Modi H, McDonald T, Chu S, Yee JK, FormanSJ, Bhatia R. Role
of BCR/ABL gene-expressionlevels in determining the phenotype and
imati-nib sensitivity of transformed human hemato-poietic cells.
Blood 2007;109:5411–21.
74. Hochhaus A, Kreil S, Corbin AS, et al. Molecu-lar and
chromosomal mechanisms of resistanceto imatinib (STI571) therapy.
Leukemia 2002;16:2190–6.
75. Barnes DJ, Palaiologou D, Panousopoulou E,et al. Bcr-Abl
expression levels determine the rateof development of resistance to
imatinibmesylatein chronic myeloid leukemia. Cancer Res
2005;65:8912–9.
76. Quintas-Cardama A, Kantarjian HM, Cortes JE.Mechanisms of
primary and secondary resistanceto imatinib in chronic myeloid
leukemia. CancerControl 2009;16:122–31.
77. Nicolini FE, Corm S, Le QH, et al. Mutation sta-tus and
clinical outcome of 89 imatinib mesylate-resistant
chronicmyelogenous leukemia patients:a retrospective analysis from
the French inter-group of CML (Fi(phi)-LMC GROUP).
Leukemia2006;20:1061–6.
78. Jabbour E, Kantarjian H, Jones D, et al. Char-acteristics
and outcomes of patients with chronicmyeloid leukemia and T315I
mutation following
failure of imatinib mesylate therapy. Blood 2008;112:53–5.
79. Nicolini FE, Hayette S, Corm S, et al. Clinicaloutcome of 27
imatinib mesylate-resistantchronic myelogenous leukemia patients
harbor-ing a T315I BCR-ABL mutation.
Haematologica2007;92:1238–41.
80. Soverini S, Colarossi S, Gnani A, et al. Contribu-tion of
ABL kinase domain mutations to imatinibresistance in different
subsets of Philadelphia-positive patients: by the GIMEMA Working
Partyon Chronic Myeloid Leukemia. Clin Cancer
Res2006;12:7374–9.
81. Branford S, Rudzki Z, Walsh S, et al. Detec-tion of BCR-ABL
mutations in patients withCML treated with imatinib is virtually
alwaysaccompanied by clinical resistance, and muta-tions in the ATP
phosphate-binding loop (P-loop) are associated with a poor
prognosis.Blood 2003;102:276–83.
82. Griswold IJ, MacPartlin M, Bumm T, et al.Kinase domain
mutants of Bcr-Abl exhibit al-tered transformation potency, kinase
activity,and substrate utilization, irrespective of sensitiv-ity to
imatinib. Mol Cell Biol 2006;26:6082–93.
83. Soverini S, Martinelli G, Rosti G, et al. ABL mu-tations in
late chronic phase chronic myeloidleukemia patients with up-front
cytogenetic re-sistance to imatinib are associated with a
greaterlikelihood of progression to blast crisis andshorter
survival: a study by the GIMEMA Work-ing Party on Chronic Myeloid
Leukemia. J ClinOncol 2005;23:4100–9.
84. Khorashad JS, de Lavallade H, Apperley JF,et al. Finding of
kinase domain mutations inpatients with chronic phase chronic
myeloidleukemia responding to imatinib may identifythose at high
risk of disease progression. J ClinOncol 2008;26:4806–13.
85. Roche-Lestienne C, Soenen-Cornu V, Grardel-Duflos N, et al.
Several types of mutations of theAbl gene can be found in chronic
myeloid leuke-mia patients resistant to STI571, and they
canpre-exist to the onset of treatment. Blood 2002;100:1014–8.
86. Khorashad JS, Anand M, Marin D, et al. Thepresence of a
BCR-ABL mutant allele in CMLdoes not always explain clinical
resistance to im-atinib. Leukemia 2006;20:658–63.
87. Willis SG, Lange T, Demehri S, et al. High-sensitivity
detection of BCR-ABL kinase domainmutations in imatinib-naive
patients: correlationwith clonal cytogenetic evolution but not
re-sponse to therapy. Blood 2005;106:2128–37.
88. Skaggs BJ, Gorre ME, Ryvkin A, et al. Phos-phorylation of
the ATP-binding loop directs on-cogenicity of drug-resistant
BCR-ABL mutants.Proc Natl Acad Sci U S A 2006;103:19466–71.
89. O'Hare T, Eide CA, Deininger MW. Bcr-Abl ki-nase domain
mutations, drug resistance, and theroad to a cure for chronic
myeloid leukemia.Blood 2007;110:2242–9.
90. Shah NP, Skaggs BJ, Branford S, et al. Se-quential ABL
kinase inhibitor therapy selectsfor compound drug-resistant BCR-ABL
muta-tions with altered oncogenic potency. J Clin In-vest
2007;117:2562–9.
91. Cortes J, Jabbour E, Kantarjian H, et al. Dynam-ics of
BCR-ABL kinase domainmutations in chron-ic myeloid leukemia after
sequential treatmentwith multiple tyrosine kinase inhibitors.
Blood2007;110:4005–11.
92. Khorashad JS, Milojkovic D, Mehta P, et al.In vivo kinetics
of kinase domain mutations inCML patients treated with dasatinib
after failingimatinib. Blood 2008;111:2378–81.
93. Stagno F, Stella S, Berretta S, et al. Sequentialmutations
causing resistance to both Imatinib
7526Clin Cancer Res 2009;15(24) December 15, 2009
www.aacrjournals.org
CCR FOCUS
Research. on June 22, 2014. © 2009 American Association for
Cancerclincancerres.aacrjournals.org Downloaded from
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrHighlight
AdministratorTypewriterfailure of imatinib mesylate therapy.
Blood 2008; 112:53–5.
userrHighlight
AdministratorRectangle
AdministratorCalloutthis row was moved from right column
http://clincancerres.aacrjournals.org/
-
have excellent responses on either dose: a TOPScorrelative
study. Blood 2008;112:3187a.
46. White DL, Saunders VA, Dang P, et al. OCT-1-mediated influx
is a key determinant of the intra-cellular uptake of imatinib but
not nilotinib(AMN107): reduced OCT-1 activity is the causeof low in
vitro sensitivity to imatinib. Blood2006;108:697–704.
47. Dulucq S, Bouchet S, Turcq B, et al. Multidrugresistance
gene (MDR1) polymorphisms are as-sociated with major molecular
responsesto standard-dose imatinib in chronic myeloidleukemia.
Blood 2008;112:2024–7.
48. Kim DH, Sriharsha L, Xu W, et al. Clinical rel-evance of a
pharmacogenetic approach usingmultiple candidate genes to predict
responseand resistance to imatinib therapy in chronicmyeloid
leukemia. Clin Cancer Res 2009;15:4750–8.
49. Majlis A, Smith TL, Talpaz M, O'Brien S, RiosMB, Kantarjian
HM. Significance of cytogeneticclonal evolution in chronic
myelogenous leuke-mia. J Clin Oncol 1996;14:196–203.
50. Johansson B, Fioretos T, Mitelman F. Cyto-genetic and
molecular genetic evolution ofchronic myeloid leukemia. Acta
Haematol2002;107:76–94.
51. O'Dwyer ME, Mauro MJ, Blasdel C, et al. Clon-al evolution
and lack of cytogenetic response areadverse prognostic factors for
hematologic re-lapse of chronic phase CML patients treated
withimatinib mesylate. Blood 2004;103:451–5.
52. Cortes JE, Talpaz M, Giles F, et al. Prognosticsignificance
of cytogenetic clonal evolution inpatients with chronic myelogenous
leukemia onimatinib mesylate therapy. Blood 2003;101:3794–800.
53. Cortes J, O'Dwyer ME. Clonal evolution inchronic myelogenous
leukemia. Hematol OncolClin North Am 2004;18:671–84 [x.].
54. Lahaye T, Riehm B, Berger U, et al. Responseand resistance
in 300 patients with BCR-ABL-positive leukemias treated with
imatinib in a sin-gle center: a 4.5-year follow-up. Cancer
2005;103:1659–69.
55. Jabbour E, Kantarjian H, Jones D, et al. Fre-quency and
clinical significance of BCR-ABL mu-tations in patients with
chronic myeloid leukemiatreated with imatinib mesylate. Leukemia
2006;20:1767–73.
56. Geahlen RL, Handley MD, Harrison ML. Molec-ular interdiction
of Src-family kinase signaling inhematopoietic cells. Oncogene
2004;23:8024–32.
57. Danhauser-Riedl S, Warmuth M, Druker BJ,Emmerich B, Hallek
M. Activation of Src kinasesp53/56lyn and p59hck by p210bcr/abl in
myeloidcells. Cancer Res 1996;56:3589–96.
58. Donato NJ, Wu JY, Stapley J, et al. BCR-ABLindependence and
LYN kinase overexpression inchronic myelogenous leukemia cells
selected forresistance to STI571. Blood 2003;101:690–8.
59. Hu Y, Swerdlow S, Duffy TM, Weinmann R,Lee FY, Li S.
Targeting multiple kinase pathwaysin leukemic progenitors and stem
cells isessential for improved treatment of Ph+ leuke-mia in mice.
Proc Natl Acad Sci U S A 2006;103:16870–5.
60. Dai Y, Rahmani M, Corey SJ, Dent P, Grant
SA.Bcr/Abl-independent, Lyn-dependent form of im-atinib mesylate
(STI-571) resistance is associat-ed with altered expression of
Bcl-2. J BiolChem 2004;279:34227–39.
61. Schindler T, Bornmann W, Pellicena P, MillerWT, Clarkson B,
Kuriyan J. Structural mecha-nism for STI-571 inhibition of abelson
tyrosinekinase. Science 2000;289:1938–42.
62. O'Hare T, Eide CA, Deininger MW. PersistentLYN signaling in
imatinib-resistant, BCR-ABL-
independent chronic myelogenous leukemia. JNatl Cancer Inst
2008;100:908–9.
63. Copland M, Hamilton A, Elrick LJ, et al. Dasa-tinib
(BMS-354825) targets an earlier progenitorpopulation than imatinib
in primary CML butdoes not eliminate the quiescent fraction.
Blood2006;107:4532–9.
64. Jiang X, Zhao Y, Smith C, et al. Chronic mye-loid leukemia
stem cells possess multiple uniquefeatures of resistance to BCR-ABL
targeted ther-apies. Leukemia 2007;21:926–35.
65. Jin L, Tabe Y, Konoplev S, et al. CXCR4 up-regulation by
imatinib induces chronic mye-logenous leukemia (CML) cell
migrationto bone marrow stroma and promotes surviv-al of quiescent
CML cells. Mol Cancer Ther2008;7:48–58.
66. Konig H, Copland M, Chu S, Jove R, HolyoakeTL, Bhatia R.
Effects of dasatinib on SRC ki-nase activity and downstream
intracellularsignaling in primitive chronic myelogenous leu-kemia
hematopoietic cells. Cancer Res 2008;68:9624–33.
67. Jorgensen HG, Allan EK, Jordanides NE,Mountford JC, Holyoake
TL. Nilotinib exertsequipotent antiproliferative effects to
imatiniband does not induce apoptosis in CD34+ CMLcells. Blood
2007;109:4016–9.
68. Copland M, Pellicano F, Richmond L, et al.BMS-214662
potently induces apoptosis ofchronic myeloid leukemia stem and
progenitorcells and synergizes with tyrosine kinase inhibi-tors.
Blood 2008;111:2843–53.
69. Holtz M, Forman SJ, Bhatia R. Growth factorstimulation
reduces residual quiescent chronicmyelogenous leukemia progenitors
remainingafter imatinib treatment. Cancer Res 2007;67:1113–20.
70. Copland M, Fraser AR, Harrison SJ, HolyoakeTL. Targeting the
silent minority: emergingimmunotherapeutic strategies for
eradicationof malignant stem cells in chronic myeloid leu-kaemia.
Cancer Immunol Immunother 2005;54:297–306.
71. Jamieson CH, Ailles LE, Dylla SJ, et
al.Granulocyte-macrophage progenitors as candi-date leukemic stem
cells in blast-crisis CML. NEngl J Med 2004;351:657–67.
72. GorreME,MohammedM, Ellwood K, et al. Clin-ical resistance to
STI-571 cancer therapy causedby BCR-ABL gene mutation or
amplification.Science 2001;293:876–80.
73. Modi H, McDonald T, Chu S, Yee JK, FormanSJ, Bhatia R. Role
of BCR/ABL gene-expressionlevels in determining the phenotype and
imati-nib sensitivity of transformed human hemato-poietic cells.
Blood 2007;109:5411–21.
74. Hochhaus A, Kreil S, Corbin AS, et al. Molecu-lar and
chromosomal mechanisms of resistanceto imatinib (STI571) therapy.
Leukemia 2002;16:2190–6.
75. Barnes DJ, Palaiologou D, Panousopoulou E,et al. Bcr-Abl
expression levels determine the rateof development of resistance to
imatinibmesylatein chronic myeloid leukemia. Cancer Res
2005;65:8912–9.
76. Quintas-Cardama A, Kantarjian HM, Cortes JE.Mechanisms of
primary and secondary resistanceto imatinib in chronic myeloid
leukemia. CancerControl 2009;16:122–31.
77. Nicolini FE, Corm S, Le QH, et al. Mutation sta-tus and
clinical outcome of 89 imatinib mesylate-resistant
chronicmyelogenous leukemia patients:a retrospective analysis from
the French inter-group of CML (Fi(phi)-LMC GROUP).
Leukemia2006;20:1061–6.
78. Jabbour E, Kantarjian H, Jones D, et al. Char-acteristics
and outcomes of patients with chronicmyeloid leukemia and T315I
mutation following
failure of imatinib mesylate therapy. Blood 2008;112:53–5.
79. Nicolini FE, Hayette S, Corm S, et al. Clinicaloutcome of 27
imatinib mesylate-resistantchronic myelogenous leukemia patients
harbor-ing a T315I BCR-ABL mutation.
Haematologica2007;92:1238–41.
80. Soverini S, Colarossi S, Gnani A, et al. Contribu-tion of
ABL kinase domain mutations to imatinibresistance in different
subsets of Philadelphia-positive patients: by the GIMEMA Working
Partyon Chronic Myeloid Leukemia. Clin Cancer
Res2006;12:7374–9.
81. Branford S, Rudzki Z, Walsh S, et al. Detec-tion of BCR-ABL
mutations in patients withCML treated with imatinib is virtually
alwaysaccompanied by clinical resistance, and muta-tions in the ATP
phosphate-binding loop (P-loop) are associated with a poor
prognosis.Blood 2003;102:276–83.
82. Griswold IJ, MacPartlin M, Bumm T, et al.Kinase domain
mutants of Bcr-Abl exhibit al-tered transformation potency, kinase
activity,and substrate utilization, irrespective of sensitiv-ity to
imatinib. Mol Cell Biol 2006;26:6082–93.
83. Soverini S, Martinelli G, Rosti G, et al. ABL mu-tations in
late chronic phase chronic myeloidleukemia patients with up-front
cytogenetic re-sistance to imatinib are associated with a
greaterlikelihood of progression to blast crisis andshorter
survival: a study by the GIMEMA Work-ing Party on Chronic Myeloid
Leukemia. J ClinOncol 2005;23:4100–9.
84. Khorashad JS, de Lavallade H, Apperley JF,et al. Finding of
kinase domain mutations inpatients with chronic phase chronic
myeloidleukemia responding to imatinib may identifythose at high
risk of disease progression. J ClinOncol 2008;26:4806–13.
85. Roche-Lestienne C, Soenen-Cornu V, Grardel-Duflos N, et al.
Several types of mutations of theAbl gene can be found in chronic
myeloid leuke-mia patients resistant to STI571, and they
canpre-exist to the onset of treatment. Blood 2002;100:1014–8.
86. Khorashad JS, Anand M, Marin D, et al. Thepresence of a
BCR-ABL mutant allele in CMLdoes not always explain clinical
resistance to im-atinib. Leukemia 2006;20:658–63.
87. Willis SG, Lange T, Demehri S, et al. High-sensitivity
detection of BCR-ABL kinase domainmutations in imatinib-naive
patients: correlationwith clonal cytogenetic evolution but not
re-sponse to therapy. Blood 2005;106:2128–37.
88. Skaggs BJ, Gorre ME, Ryvkin A, et al. Phos-phorylation of
the ATP-binding loop directs on-cogenicity of drug-resistant
BCR-ABL mutants.Proc Natl Acad Sci U S A 2006;103:19466–71.
89. O'Hare T, Eide CA, Deininger MW. Bcr-Abl ki-nase domain
mutations, drug resistance, and theroad to a cure for chronic
myeloid leukemia.Blood 2007;110:2242–9.
90. Shah NP, Skaggs BJ, Branford S, et al. Se-quential ABL
kinase inhibitor therapy selectsfor compound drug-resistant BCR-ABL
muta-tions with altered oncogenic potency. J Clin In-vest
2007;117:2562–9.
91. Cortes J, Jabbour E, Kantarjian H, et al. Dynam-ics of
BCR-ABL kinase domainmutations in chron-ic myeloid leukemia after
sequential treatmentwith multiple tyrosine kinase inhibitors.
Blood2007;110:4005–11.
92. Khorashad JS, Milojkovic D, Mehta P, et al.In vivo kinetics
of kinase domain mutations inCML patients treated with dasatinib
after failingimatinib. Blood 2008;111:2378–81.
93. Stagno F, Stella S, Berretta S, et al. Sequentialmutations
causing resistance to both Imatinib
7526Clin Cancer Res 2009;15(24) December 15, 2009
www.aacrjournals.org
CCR FOCUS
Research. on June 22, 2014. © 2009 American Association for
Cancerclincancerres.aacrjournals.org Downloaded from
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrHighlight
http://clincancerres.aacrjournals.org/
-
Perspectives
Selecting optimal second-line tyrosine kinase inhibitor therapy
for chronicmyeloid leukemia patients after imatinib failure: does
the BCR-ABL mutationstatus really matter?
Susan Branford,1 Junia V. Melo,1 and Timothy P. Hughes1
1Centre for Cancer Biology, Departments of Molecular Pathology
and Haematology, SA Pathology, Adelaide, Australia
Preclinical studies of BCR-ABL mutationsensitivity to nilotinib
or dasatinib sug-gested that the majority would be
sensitive.Correspondingly, the initial clinical trialsdemonstrated
similar response rates forCML patients after imatinib failure,
irrespec-tive of the mutation status. However, oncloser
examination, clinical evidence nowindicates that some mutations are
less sen-sitive to nilotinib (Y253H, E255K/V, andF359V/C) or
dasatinib (F317L and V299L).T315I is insensitive to both. Novel
mutations
(F317I/V/C and T315A) are less sensitive/insensitive to
dasatinib. We refer to thesecollectively as second-generation
inhibitor(SGI) clinically relevant mutations. By invitro analysis,
other mutations confer a de-gree of insensitivity; however,
clinical evi-dence is currently insufficient to define themas SGI
clinically relevant. Here we examinethe mutations that are clearly
SGI clinicallyrelevant, those with minimal impact on re-sponse, and
those for which more data areneeded. In our series of patients with
muta-
tions at imatinib cessation and/or at nilotinibor dasatinib
commencement, 43% had SGIclinically relevant mutations, including
14%with T315I. The frequency of SGI clinicallyrelevant mutations
was dependent on thedisease phase at imatinib failure. The
clini-cal data suggest that a mutation will often bedetectable
after imatinib failure for whichthere is compelling clinical
evidence thatone SGI should be preferred. (Blood.
2009;114:5426-5435)
Introduction
The outcome for patients with chronic myeloid leukemia (CML)
whofail imatinib has improved since the availability of
second-generationBCR-ABL kinase inhibitors (SGIs). The most common
mechanisms ofimatinib resistance are mutations within the BCR-ABL
kinase domainand protein overexpression by gene amplification.1-12
Resistance is alsoassociated with other genetic events, as
indicated by the detection ofcytogenetic abnormalities in the
Philadelphia chromosome–positive(Ph�) clone in more than 50% of
imatinib-resistant patients.13 It hasbeen suggested that some
BCR-ABL mutations play no causal role inresistance.14-16 However,
approximately half of the patients who com-mence SGIs after
imatinib therapy have detectable imatinib-resistantBCR-ABL
mutations. Imatinib binds to the inactive conformation ofBCR-ABL,
leading to disruption of the adenosine triphosphate (ATP)binding
site and blockade of the catalytic activity.17,18 BCR-ABLmutations
that impair imatinib binding while still enabling ATP binding,or
that alter the specific protein conformation required for
imatinibbinding, are selected in the presence of imatinib.19-21 In
the absence ofimatinib, these mutations do not confer a growth
advantage.22
For patients commencing nilotinib or dasatinib after imatinib
cessa-tion, clinical trials have demonstrated similar responses for
patients withor without mutations, except for T315I for which
neither drug isactive.23-31 This mutation demonstrates
cross-resistance to imatinib,nilotinib, and dasatinib.32-34
However, a closer examination of responsesto SGI therapy for
individual mutations has identified a limited number,other than
T315I, that are less sensitive to either nilotinib or
dasat-inib.35-37 Furthermore, in vitro studies have identified
mutations thatconfer a degree of insensitivity38 or
resistance.39
How well do the problematic mutations identified by in
vitrostudies correlate with those identified by clinical studies?
More-over, does the in vitro sensitivity of mutations provide a
reliable
indication of the probable response to SGIs? Undoubtedly, in
vitrosensitivity of imatinib-resistant mutations can be a useful
guidewhen considering an increased imatinib dose.40 Here we
assessBCR-ABL mutations in the context of their impact on
responseafter a change to SGI therapy by an examination of the
availableclinical data. The mutation status may contribute to
therapeuticdecisions after imatinib failure or indeed after failure
of an SGI. Weassess the frequency that mutations conferring a
degree of clinicalinsensitivity to SGIs are detectable at the time
of imatinibcessation. These are collectively referred to as SGI
clinicallyrelevant mutations. We also examine whether the disease
phaseinfluences their frequency. Last, we examine the occurrence
ofmultiple mutations in imatinib-treated patients and the extent
towhich disease phase influences their detection.
BCR-ABL mutations in the era of SGIs:type still matters
Mutant sensitivity assessed by in vitro studies
Preclinical studies of nilotinib against 33 BCR-ABL mutants
predictedthat the inhibitor would have clinical activity in
patients harboring thesemutations, except for T315I.33,34,41
Similarly, among 19 imatinib-resistant mutants tested against
dasatinib, T315I was the only clearlyresistant mutation.32,33 The
in vitro results were similar to the earlier invitro studies of
imatinib, in that mutants displayed various degrees
ofsensitivity.15,42 SGI sensitivity was assessed by various
methods, includ-ing the degree of inhibition of BCR-ABL
autophosphorylation or cellproliferation after transfection of
mutants into Ba/F3 cells.32-34Although
Submitted August 10, 2009; accepted October 5, 2009.
Prepublished online asBlood First Edition paper, October 30, 2009;
DOI 10.1182/blood-2009-08-215939.
© 2009 by The American Society of Hematology
5426 BLOOD, 24 DECEMBER 2009 � VOLUME 114, NUMBER 27
userrTextbox2. Branford2009 (truncated)
userrHighlight
userrHighlight
userrUnderline
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrUnderline
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrUnderline
userrHighlight
userrHighlight
userrHighlight
AdministratorTypewriterBlood, 2009, Vol. 114, pp. 5426-5435)
AdministratorTypewriter2
-
Perspectives
Selecting optimal second-line tyrosine kinase inhibitor therapy
for chronicmyeloid leukemia patients after imatinib failure: does
the BCR-ABL mutationstatus really matter?
Susan Branford,1 Junia V. Melo,1 and Timothy P. Hughes1
1Centre for Cancer Biology, Departments of Molecular Pathology
and Haematology, SA Pathology, Adelaide, Australia
Preclinical studies of BCR-ABL mutationsensitivity to nilotinib
or dasatinib sug-gested that the majority would be
sensitive.Correspondingly, the initial clinical trialsdemonstrated
similar response rates forCML patients after imatinib failure,
irrespec-tive of the mutation status. However, oncloser
examination, clinical evidence nowindicates that some mutations are
less sen-sitive to nilotinib (Y253H, E255K/V, andF359V/C) or
dasatinib (F317L and V299L).T315I is insensitive to both. Novel
mutations
(F317I/V/C and T315A) are less sensitive/insensitive to
dasatinib. We refer to thesecollectively as second-generation
inhibitor(SGI) clinically relevant mutations. By invitro analysis,
other mutations confer a de-gree of insensitivity; however,
clinical evi-dence is currently insufficient to define themas SGI
clinically relevant. Here we examinethe mutations that are clearly
SGI clinicallyrelevant, those with minimal impact on re-sponse, and
those for which more data areneeded. In our series of patients with
muta-
tions at imatinib cessation and/or at nilotinibor dasatinib
commencement, 43% had SGIclinically relevant mutations, including
14%with T315I. The frequency of SGI clinicallyrelevant mutations
was dependent on thedisease phase at imatinib failure. The
clini-cal data suggest that a mutation will often bedetectable
after imatinib failure for whichthere is compelling clinical
evidence thatone SGI should be preferred. (Blood.
2009;114:5426-5435)
Introduction
The outcome for patients with chronic myeloid leukemia (CML)
whofail imatinib has improved since the availability of
second-generationBCR-ABL kinase inhibitors (SGIs). The most common
mechanisms ofimatinib resistance are mutations within the BCR-ABL
kinase domainand protein overexpression by gene amplification.1-12
Resistance is alsoassociated with other genetic events, as
indicated by the detection ofcytogenetic abnormalities in the
Philadelphia chromosome–positive(Ph�) clone in more than 50% of
imatinib-resistant patients.13 It hasbeen suggested that some
BCR-ABL mutations play no causal role inresistance.14-16 However,
approximately half of the patients who com-mence SGIs after
imatinib therapy have detectable imatinib-resistantBCR-ABL
mutations. Imatinib binds to the inactive conformation ofBCR-ABL,
leading to disruption of the adenosine triphosphate (ATP)binding
site and blockade of the catalytic activity.17,18 BCR-ABLmutations
that impair imatinib binding while still enabling ATP binding,or
that alter the specific protein conformation required for
imatinibbinding, are selected in the presence of imatinib.19-21 In
the absence ofimatinib, these mutations do not confer a growth
advantage.22
For patients commencing nilotinib or dasatinib after imatinib
cessa-tion, clinical trials have demonstrated similar responses for
patients withor without mutations, except for T315I for which
neither drug isactive.23-31 This mutation demonstrates
cross-resistance to imatinib,nilotinib, and dasatinib.32-34
However, a closer examination of responsesto SGI therapy for
individual mutations has identified a limited number,other than
T315I, that are less sensitive to either nilotinib or
dasat-inib.35-37 Furthermore, in vitro studies have identified
mutations thatconfer a degree of insensitivity38 or
resistance.39
How well do the problematic mutations identified by in
vitrostudies correlate with those identified by clinical studies?
More-over, does the in vitro sensitivity of mutations provide a
reliable
indication of the probable response to SGIs? Undoubtedly, in
vitrosensitivity of imatinib-resistant mutations can be a useful
guidewhen considering an increased imatinib dose.40 Here we
assessBCR-ABL mutations in the context of their impact on
responseafter a change to SGI therapy by an examination of the
availableclinical data. The mutation status may contribute to
therapeuticdecisions after imatinib failure or indeed after failure
of an SGI. Weassess the frequency that mutations conferring a
degree of clinicalinsensitivity to SGIs are detectable at the time
of imatinibcessation. These are collectively referred to as SGI
clinicallyrelevant mutations. We also examine whether the disease
phaseinfluences their frequency. Last, we examine the occurrence
ofmultiple mutations in imatinib-treated patients and the extent
towhich disease phase influences their detection.
BCR-ABL mutations in the era of SGIs:type still matters
Mutant sensitivity assessed by in vitro studies
Preclinical studies of nilotinib against 33 BCR-ABL mutants
predictedthat the inhibitor would have clinical activity in
patients harboring thesemutations, except for T315I.33,34,41
Similarly, among 19 imatinib-resistant mutants tested against
dasatinib, T315I was the only clearlyresistant mutation.32,33 The
in vitro results were similar to the earlier invitro studies of
imatinib, in that mutants displayed various degrees
ofsensitivity.15,42 SGI sensitivity was assessed by various
methods, includ-ing the degree of inhibition of BCR-ABL
autophosphorylation or cellproliferation after transfection of
mutants into Ba/F3 cells.32-34Although
Submitted August 10, 2009; accepted October 5, 2009.
Prepublished online asBlood First Edition paper, October 30, 2009;
DOI 10.1182/blood-2009-08-215939.
© 2009 by The American Society of Hematology
5426 BLOOD, 24 DECEMBER 2009 � VOLUME 114, NUMBER 27
userrTextbox2. Branford2009 (truncated)
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrHighlight
AdministratorTypewriterBlood, 2009, Vol. 114, pp. 5426-5435)
AdministratorSquiggly
AdministratorSquiggly
AdministratorSquiggly
-
bosutinib, which is under clinical trial as SGI therapy.39
However,G250E is described as sensitive to nilotinib and dasatinib
in anotherassessment.38 F317L is classified as moderately resistant
or resis-tant to the 4 inhibitors in one study39 and sensitive to
nilotinib andinsensitive to dasatinib in another.38 These
discrepancies may bethe result of methodologic differences or
differences in the cutoffvalues for the classification of mutant
sensitivity and may causedifficulty for interpretation.
Mutations identified from resistance screens
Resistance screens have identified a limited number of
mutationsthat emerge in the presence of increasing doses of
nilotinib anddasatinib,43-46 and these correspond, to a degree, to
the mutantsensitivity determined in cell proliferation assays.38,39
Mutations atdasatinib contact residues appeared to be particularly
relevant,including V299L. In 2 resistance screens, mutations at
T315 andF317 accounted for 95% of all mutants recovered, including
novelmutations F317V/I/S/C and T315A, which had not been reported
inimatinib-treated patients.44,45 In one study, F317V and T315A
werethe most frequent to emerge (41% and 30%, respectively) and
had40- to 90-fold reduced dasatinib sensitivity compared with
unmu-tated BCR-ABL.45 In accord with these results, T315A and
F317Vhad the highest IC50 values, except for T315I, in the in
vitroassessment of dasatinib by O’Hare et al.33
In 3 in vitro resistance screens of increasing doses of
nilotinib,T315I emerged most frequently and represented 49% of
allmutations recovered.43,44,46 However, the common
imatinib-resistant mutation, Y253H, was also among the most
frequent toemerge and had the highest nilotinib IC50 value in each
of thesestudies, apart from T315I. E255K, Y253H, and T315I were
theonly mutations to emerge in all 3 screens, and E255V and
Q252Hemerged in 2 of 3 studies. All other mutations were confined
to oneof the screens. With the exception of T315I, all mutations
wereeffectively suppressed by nilotinib concentrations of
2000nM,which falls within the peak-trough plasma levels
(3600-1700nM)measured in patients treated with 400 mg nilotinib
twice daily.24
Over the past few years, clinical studies have identified
alimited number of mutations that may be relevant for response
tonilotinib and dasatinib as second- or third-line
inhibitortherapy35,36,47,48 and are implicated in resistance.47-51
In one report,a new mutation was acquired in 83% of patients who
relapsed aftera response.48 The available clinical data present an
opportunity toassess how effectively in vitro studies predict the
SGI clinicallyrelevant mutations and their validity for determining
appropriatetherapy after imatinib failure.
Mutation sensitivity assessed by clinical studies of
dasatinib
The majority of imatinib-resistant mutations remain sensitive
todasatinib.23,26-29,31 However, consistent with the dasatinib
resis-tance screens and cell proliferation assays, clinical reports
con-firmed T315I/A, F317L/I/V/C, and V299L as relevant for
de-creased clinical efficacy, either as preexisting or as
emergingmutations.37,48-51
One of the most frequent mutations to emerge with
clinicaldasatinib resistance was V299L.48,50 This mutation was
reportedvery rarely in imatinib-treated patients.8,52 V299L and
F317L werealso preferentially associated with dasatinib failure in
a study ofmutation dynamics after sequential inhibitor therapy.51
Interest-ingly, V299L was only detected at a frequency of 1% in
resistancescreens44,45 and only at lower dasatinib
concentrations.44
The largest analysis of clinical response to dasatinib
afterimatinib failure to date involved 1043 CML patients treated
inchronic phase (CP).37 The presence of T315I or F317L at the
timeof commencing dasatinib was associated with the least
favorableresponses. Furthermore, the most frequently detected new
muta-tions were T315I, F317L, and V299L. A conclusion of the
studywas that alternative treatment options should be considered
forpatients with these mutations.37 Consistent with these
findings,other studies reported low response rates for patients
with F317L.47,48
In one study, 8 of 16 dasatinib-treated patients after imatinib
failureacquired F317L, and this mutation was deemed
dasatinib-resistantbut sensitive to other inhibitors.47
Mutant sensitivity assessed by clinical studies of nilotinib
Clinical studies have demonstrated that the majority of
imatinib-resistant mutations remain sensitive to nilotinib.24,25,30
Neverthe-less, several mutations are less sensitive, which
influences theresponse.36 The mutations that emerged in the
nilotinib resistancescreens have corresponded, to a degree, with
the clinical findings.In an evaluation of 281 CP patients in the
nilotinib phase 2registration study, those with T315I, Y253H,
E255K/V, andF359V/C (n � 31) at nilotinib commencement had the
leastfavorable responses. These mutations had the highest IC50
values incell proliferation assays as assessed by Weisberg et al( �
150nM).34,41 No patient with these mutations achieved acomplete
cytogenetic response (CCyR) by 12 months, 6 (19%)achieved a major
cytogenetic response, and 10 (32%) a completehematologic response
(CHR).36 In contrast, 32 of 74 patients (43%)with any other
mutation and 35 of 87 (40%) with imatinibresistance but no mutation
achieved CCyR. These mutations werealso among the most common new
mutations during nilotinibtherapy and were associated with a higher
risk of progression. Inanother study, 13 of 14 patients who
relapsed with new mutationson nilotinib as second- or third-line
inhibitor therapy had one ofthese mutations.48
The poor response associated with Y253H, E255K/V, andF359V/C
when present at the time of commencing nilotinib wasconfirmed in
patients treated in accelerated phase (AP) CML.53
Of 17 of 87 AP patients with these mutations, only 24%achieved a
CHR. In contrast, 55% without mutations and 58%with other mutations
(excluding T315I) achieved a CHR. Fromthe nilotinib clinical
response data, the suggestion was thattherapies other than
nilotinib should be considered for patientswith these
mutations.36,53 Consistent with these findings, themost frequent
mutations detected in patients with nilotinibfailure in the study
of Cortes et al51 were at residues 253, 255,359, and 311. A
mutation at residue 311 was also detected in anilotinib resistance
screen.43
Which mutations are relevant for response and resistance
tonilotinib and dasatinib from clinical studies?
The current clinical data suggest the SGI clinically
relevantmutations are T315I for both inhibitors: F317L/I/C/V,
V299L, andT315A for dasatinib and Y253H, E255K/V, and F359V/C
fornilotinib. Sensitive mutation detection of these specific
mutationsto aid therapeutic choices may be beneficial. Several
techniquesmoderately improve the sensitivity to 1.5% to 10%,
includingpyrosequencing,16,51 ligation-dependent competitive
polymerasechain reaction (PCR),54 and SEQUENOM MassARRAY.55
Highlysensitive techniques have a detection limit from 0.0003% to
0.1%,including mutation-specific PCR based on the Taqman
platform,56
5428 BRANFORD et al BLOOD, 24 DECEMBER 2009 � VOLUME 114, NUMBER
27
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrUnderline
userrUnderline
userrHighlight
userrHighlight
userrHighlight
-
PCR–restriction fragment length polymorphism,57 polymerasecolony
assay,58 allele-specific PCR,59 and a nanofluidic platform.60
Whether highly sensitive detection of SGI clinically
relevantmutations before SGI therapy will always correlate with
theirclonal expansion and resistance is unknown. This was not
alwaysthe case using highly sensitive mutation detection before
imatinibtherapy.59
Do the SGI clinically relevant mutations correspond to thein
vitro data?
Based on current clinical information, the answer to this
question isyes, to a degree. Why are some mutations clinically
relevant forSGIs and not others that either emerged more frequently
in in vitroresistance screens and/or those with greater in vitro
insensitivity?In the case of dasatinib, identification of
clinically relevantmutations at residues T315 and F317 is
consistent with theiremergence in resistance screens. However,
despite the high fre-quency of F317V and T315A in a dasatinib
resistance screen andtheir significantly reduced sensitivity to
dasatinib compared withF317L,45 F317V was not detected in any
patient and T315A in only2 patients in the initial reports.49,50
Could this be related to reducedoncogenicity of these mutations?
Severely attenuated transformingactivity of T315A was demonstrated
relative to wild-type BCR-ABL.61 However, F317L was only marginally
more transformingthan T315A. Furthermore, reduced transforming
activity does notappear to be related to frequency of detection of
imatinib-resistantmutations: M351T displays reduced transforming
activity61,62 yet isamong the most commonly detected
imatinib-resistant mutations.63
The sensitivity rankings by cell proliferation assays
consistentlysuggest that the P-loop mutations Q252H and E255K/V may
berelevant for dasatinib (Table 1).38,39 In dasatinib-treated CP
pa-tients, the CCyR rates for patients with these mutations
rangedfrom 17% to 38%.37 These mutations have rarely been
associatedwith clinical dasatinib resistance or as new mutations
duringdasatinib therapy.37,48-51 However, E255K and Q252H were
amongthe mutations recovered in in vitro dasatinib resistance
screens butwere the only noncontact residues.44,45 The BCR-ABL
crystalstructure in complex with dasatinib suggested that
interactionsbetween the P-loop and dasatinib were less critical for
binding.64
Manley et al65 proposed that it is doubtful mutations of Q252
andE255 could cause a change in the structure of the P-loop to
interferewith dasatinib binding, without also disturbing binding of
ATP,which is critical for BCR-ABL reactivation. Clearly,
additionalclinical information is required before the significance
for dasatinibresponse of E255K/V and Q252H is elucidated.
Nilotinib in vitro sensitivity classifications38,39 correlate
veryclosely with clinical data (Table 1). T315I, Y253H, E255K/V,
andF359V have the highest IC50 values (F359C was not tested).
Themajor inconsistency is for the classification of G250E
(sensitive38/resistant39). The IC50 reported by Weisberg et al41
for G250E was145nM, which is close to the cutoff value of 150nM
used to definemutations less sensitive to nilotinib in the clinical
evaluation of CPpatients.36 There were only 5 CP patients with this
mutation atnilotinib start, and 3 (60%) achieved a CCyR.36 G250E
was amongthe most common mutations to emerge with nilotinib.36
However, itwas not among the most common mutations associated
withprogression. Mutations that are less sensitive, but still
responsive,to nilotinib or dasatinib may mistakenly appear to be
newlyacquired as more sensitive alleles disappear more rapidly.
G250Edid not emerge in another study in which 13 patients with
nilotinibresistance acquired new mutations.48 Inconsistency is also
apparentfor in vitro sensitivity classification of G250E for
dasatinib
(sensitive38/resistant39). This mutation was the most
commonlydetected in CP patients at the start of dasatinib, and 20
of 60 (33%)achieved a CCyR.37 G250E did not emerge in the
resistance screensof dasatinib.44,45 There is currently no strong
clinical evidence tosuggest that the presence of G250E would
influence the response tonilotinib or dasatinib. Q252H and Y253F
are consistently classifiedas moderately insensitive38 or
resistant39 to nilotinib by in vitroassessment, and Q252H emerged
in the nilotinib resistancescreens.43,46 Further clinical data are
required for adequate assess-ment of their response to
nilotinib.
Validation of in vitro sensitivity of different mutations
wasrecently demonstrated.35 In vitro sensitivity was predictive
ofresponse and long-term outcome for patients treated with
nilotinibor dasatinib. Mutations for which there was a discrepancy
inreported sensitivity among in vitro studies were classified
accord-ing to the highest IC50 to the corresponding inhibitor.
However,G250E was classified as a sensitive mutation to both
inhibitorsdespite the discrepancy in the in vitro sensitivity
classifications.38,39
In Figure 1, CCyR rates of CP patients with various mutations
atthe start of dasatinib therapy in the large clinical study of
Müller etal37 are plotted according to in vitro sensitivity
classifications.CCyR rates were only partially predicted by in
vitro sensitivity.
There may be rare imatinib-resistant mutations not included inin
vitro studies that could also be less sensitive to SGIs, such
asF359I. This mutation emerged in one nilotinib resistance
screen.43
Furthermore, rapid progression was observed in a
nilotinib-treatedpatient harboring F359I at commencement of
nilotinib.66 The IC50value of this mutation is unknown.
From the available studies, we now have a clearer understand-ing
of the BCR-ABL mutations for which there is compellingclinical
evidence that response could be compromised by treatmentwith one
and/or another of the SGIs if present after imatinib failure.These
are T315I, F317L, V299L, Y253H, E255K/V, and F359V/C,the finding of
which would influence the therapeutic decision.These mutations are
classified in Table 2 as either class D (no rolefor SGI therapy) or
class C (compelling clinical evidence torecommend an alternative
inhibitor). At this stage, the presence ofother mutations should
have no impact on clinical decisions.Nevertheless, there are
mutations where further clinical evidencemay reveal relevance for
an inhibitor (class B, Table 2). However,additional clinical
assessment is required before an alternativeinhibitor would be
recommended for class B mutations. Table 2lists the frequency of
mutations at our institution, whereas theclassifications are based
on published literature. Of course, muta-tion status is only one
factor that needs to be considered whenselecting SGIs, which
include issues of tolerance and the diseasephase.
How frequently will the mutation status be anissue when
considering therapeutic optionsafter imatinib failure?
We have performed BCR-ABL mutation analysis at our
institutionfor imatinib-treated patients since 2001, which allows
an assess-ment of the frequency of SGI clinically relevant
mutations at asingle institution. Mutations were detected in 386
patients usingdirect sequencing5,67,68 at the time of imatinib
cessation (n � 159)and/or at commencement of nilotinib or dasatinib
after imatinibfailure (n � 227). The assay has a mutation
sensitivity of 10% to20%, and the approximate percentage of mutant
nucleotide relativeto unmutated nucleotide is calculated either by
the mutation
BCR-ABL MUTATION STATUS AND THERAPEUTIC DECISIONS 5429BLOOD, 24
DECEMBER 2009 � VOLUME 114, NUMBER 27
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrUnderline
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrHighlight
-
PCR–restriction fragment length polymorphism,57 polymerasecolony
assay,58 allele-specific PCR,59 and a nanofluidic platform.60
Whether highly sensitive detection of SGI clinically
relevantmutations before SGI therapy will always correlate with
theirclonal expansion and resistance is unknown. This was not
alwaysthe case using highly sensitive mutation detection before
imatinibtherapy.59
Do the SGI clinically relevant mutations correspond to thein
vitro data?
Based on current clinical information, the answer to this
question isyes, to a degree. Why are some mutations clinically
relevant forSGIs and not others that either emerged more frequently
in in vitroresistance screens and/or those with greater in vitro
insensitivity?In the case of dasatinib, identification of
clinically relevantmutations at residues T315 and F317 is
consistent with theiremergence in resistance screens. However,
despite the high fre-quency of F317V and T315A in a dasatinib
resistance screen andtheir significantly reduced sensitivity to
dasatinib compared withF317L,45 F317V was not detected in any
patient and T315A in only2 patients in the initial reports.49,50
Could this be related to reducedoncogenicity of these mutations?
Severely attenuated transformingactivity of T315A was demonstrated
relative to wild-type BCR-ABL.61 However, F317L was only marginally
more transformingthan T315A. Furthermore, reduced transforming
activity does notappear to be related to frequency of detection of
imatinib-resistantmutations: M351T displays reduced transforming
activity61,62 yet isamong the most commonly detected
imatinib-resistant mutations.63
The sensitivity rankings by cell proliferation assays
consistentlysuggest that the P-loop mutations Q252H and E255K/V may
berelevant for dasatinib (Table 1).38,39 In dasatinib-treated CP
pa-tients, the CCyR rates for patients with these mutations
rangedfrom 17% to 38%.37 These mutations have rarely been
associatedwith clinical dasatinib resistance or as new mutations
duringdasatinib therapy.37,48-51 However, E255K and Q252H were
amongthe mutations recovered in in vitro dasatinib resistance
screens butwere the only noncontact residues.44,45 The BCR-ABL
crystalstructure in complex with dasatinib suggested that
interactionsbetween the P-loop and dasatinib were less critical for
binding.64
Manley et al65 proposed that it is doubtful mutations of Q252
andE255 could cause a change in the structure of the P-loop to
interferewith dasatinib binding, without also disturbing binding of
ATP,which is critical for BCR-ABL reactivation. Clearly,
additionalclinical information is required before the significance
for dasatinibresponse of E255K/V and Q252H is elucidated.
Nilotinib in vitro sensitivity classifications38,39 correlate
veryclosely with clinical data (Table 1). T315I, Y253H, E255K/V,
andF359V have the highest IC50 values (F359C was not tested).
Themajor inconsistency is for the classification of G250E
(sensitive38/resistant39). The IC50 reported by Weisberg et al41
for G250E was145nM, which is close to the cutoff value of 150nM
used to definemutations less sensitive to nilotinib in the clinical
evaluation of CPpatients.36 There were only 5 CP patients with this
mutation atnilotinib start, and 3 (60%) achieved a CCyR.36 G250E
was amongthe most common mutations to emerge with nilotinib.36
However, itwas not among the most common mutations associated
withprogression. Mutations that are less sensitive, but still
responsive,to nilotinib or dasatinib may mistakenly appear to be
newlyacquired as more sensitive alleles disappear more rapidly.
G250Edid not emerge in another study in which 13 patients with
nilotinibresistance acquired new mutations.48 Inconsistency is also
apparentfor in vitro sensitivity classification of G250E for
dasatinib
(sensitive38/resistant39). This mutation was the most
commonlydetected in CP patients at the start of dasatinib, and 20
of 60 (33%)achieved a CCyR.37 G250E did not emerge in the
resistance screensof dasatinib.44,45 There is currently no strong
clinical evidence tosuggest that the presence of G250E would
influence the response tonilotinib or dasatinib. Q252H and Y253F
are consistently classifiedas moderately insensitive38 or
resistant39 to nilotinib by in vitroassessment, and Q252H emerged
in the nilotinib resistancescreens.43,46 Further clinical data are
required for adequate assess-ment of their response to
nilotinib.
Validation of in vitro sensitivity of different mutations
wasrecently demonstrated.35 In vitro sensitivity was predictive
ofresponse and long-term outcome for patients treated with
nilotinibor dasatinib. Mutations for which there was a discrepancy
inreported sensitivity among in vitro studies were classified
accord-ing to the highest IC50 to the corresponding inhibitor.
However,G250E was classified as a sensitive mutation to both
inhibitorsdespite the discrepancy in the in vitro sensitivity
classifications.38,39
In Figure 1, CCyR rates of CP patients with various mutations
atthe start of dasatinib therapy in the large clinical study of
Müller etal37 are plotted according to in vitro sensitivity
classifications.CCyR rates were only partially predicted by in
vitro sensitivity.
There may be rare imatinib-resistant mutations not included inin
vitro studies that could also be less sensitive to SGIs, such
asF359I. This mutation emerged in one nilotinib resistance
screen.43
Furthermore, rapid progression was observed in a
nilotinib-treatedpatient harboring F359I at commencement of
nilotinib.66 The IC50value of this mutation is unknown.
From the available studies, we now have a clearer understand-ing
of the BCR-ABL mutations for which there is compellingclinical
evidence that response could be compromised by treatmentwith one
and/or another of the SGIs if present after imatinib failure.These
are T315I, F317L, V299L, Y253H, E255K/V, and F359V/C,the finding of
which would influence the therapeutic decision.These mutations are
classified in Table 2 as either class D (no rolefor SGI therapy) or
class C (compelling clinical evidence torecommend an alternative
inhibitor). At this stage, the presence ofother mutations should
have no impact on clinical decisions.Nevertheless, there are
mutations where further clinical evidencemay reveal relevance for
an inhibitor (class B, Table 2). However,additional clinical
assessment is required before an alternativeinhibitor would be
recommended for class B mutations. Table 2lists the frequency of
mutations at our institution, whereas theclassifications are based
on published literature. Of course, muta-tion status is only one
factor that needs to be considered whenselecting SGIs, which
include issues of tolerance and the diseasephase.
How frequently will the mutation status be anissue when
considering therapeutic optionsafter imatinib failure?
We have performed BCR-ABL mutation analysis at our
institutionfor imatinib-treated patients since 2001, which allows
an assess-ment of the frequency of SGI clinically relevant
mutations at asingle institution. Mutations were detected in 386
patients usingdirect sequencing5,67,68 at the time of imatinib
cessation (n � 159)and/or at commencement of nilotinib or dasatinib
after imatinibfailure (n � 227). The assay has a mutation
sensitivity of 10% to20%, and the approximate percentage of mutant
nucleotide relativeto unmutated nucleotide is calculated either by
the mutation
BCR-ABL MUTATION STATUS AND THERAPEUTIC DECISIONS 5429BLOOD, 24
DECEMBER 2009 � VOLUME 114, NUMBER 27
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrUnderline
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrHighlight
-
irrespective of the disease phase at imatinib start.5-7,9,12
Therefore,the higher rate of detection of these mutations in
patients whoprogressed to advanced phases in our study is highly
consistent.However, there are some inconsistencies in our mutation
fre-quency. In AP, some mutation frequencies were
intermediatebetween CP and BC (Figure 2), which could be associated
withtransition between the phases. However, F359V was the
mostfrequent mutation detected in AP but detected at a relatively
lowfrequency in BC. The frequencies of individual mutations
inspecific disease phases should be considered with caution,
andvalidation is required.
The differences evident in BC phenotype, if validated,
raiseseveral questions. Why were these particular mutations related
toeither a lymphoid or myeloid phenotype? Could the BCR-ABLmutant
genotype drive the lineage phenotype in some cases or viceversa? A
known genetic determinant of LBC is homozygousdeletion of the
p16INK4A/p19ARF gene locus that occurs in asubstantial proportion
of LBC patients, but not MBC.70-74 It waspostulated that p16INK4A
and/or p19ARF mutations could induce theselective expansion of
B-cell progenitors by favoring their cellcycle entry, rather than
myeloid progenitors.75
In the case of BCR-ABL mutations, in vitro transformationassays
have demonstrated a gain or loss of function relative tounmutated
BCR-ABL.61,62 Mutations can alter the substrates thatbind to
BCR-ABL and activate alternate signal transductionpathways that
influence disease progression. Interestingly, E255Kand Y253F
mutations showed a pronounced increase of transforma-tion potency
in primary B-lymphoid progenitor cells.62 However,the
transformation potency of these mutations was not
significantlyincreased over unmutated BCR-ABL in the myeloid
lineage. In ourcohort, the frequency of E255K was markedly
increased inLBC/Ph� ALL (19.2%) compared with MBC (2.0%).
However,Y253F was detected at a low frequency in all disease
phases. TheSRC kinases, LYN, HCK, and FGR, have also been
implicated inthe molecular pathogenesis of Ph� ALL76 and are
critical for
transition of CML to LBC.77 Whether BCR-ABL mutations that
arecharacteristic of BCR-ABL� lymphoid leukemia in our
cohortenhance the activation of SRC kinases and hence the
transition to alymphoid phenotype is unknown.
Frequency of mutations that would influence the
therapeuticdecision
In our cohort of patients with mutations, 166 of 386 (43%) had
oneor more SGI clinically relevant mutations. T315I was detected
in53 of 386 patients (14%). In 110 of 386 patients (28%), one or
moreof their mutations were clinically relevant for either
nilotinib ordasatinib, but not to both. For these patients, there
may be anadvantage for one or other inhibitor. The remaining 3 of
386patients (0.8%) had 2 mutations, one of which was
clinicallyrelevant for dasatinib and the other for nilotinib
(neither wasT315I).
Among the disease phases, there were significant differences
infrequency of SGI clinically relevant mutations: 63% LBC/Ph�
ALL, 32% MBC, 49% AP, and 35% CP (P � .001, �2; Figure 3).When
patients with LBC/Ph� ALL were subdivided, the frequencywas 59% for
LBC and 67% for Ph� ALL.
Our analysis was performed in patients with detectable
muta-tions; and from this, we can estimate the percentage of
patients withan SGI clinically relevant mutation among all
imatinib-resistantpatients. For imatinib-resistant CP patients
commencing nilotinibor dasatinib, 48% to 55% had a mutation.36,37
Similarly, forimatinib-resistant patients in AP who commenced
nilotinib ordasatinib, the frequency of mutations was 62% to
64%.27,53
Mutations in patients with LBC/Ph� ALL were detected in 62%
to83%9,28,78 and up to 75% of patients with MBC.9 From
thesemutation frequencies, the estimated percentage of all
imatinib-resistant patients with an SGI clinically relevant
mutation is 18%for CP, 31% for AP, up to 29% for MBC, and 39% to
52% forLBC/Ph� ALL. For imatinib-intolerant CP patients, the
frequency
Table 2. Most frequent mutations detected at a single
institution, which accounted for 88% of all mutations
Mutation No. detected Percentage of patients with mutations (n �
386) Percentage of all mutations (n � 503)
Mutation class for therapeutic decision*
Nilotinib Dasatinib
T315I 53 13.7 10.6 D D
M351T 47 12.2 9.4 A A
G250E 46 11.9 9.2 A A
F359V 35 9.1 7.0 C A
M244V 33 8.5 6.6 A A
Y253H 32 8.3 6.4 C A
E255K 27 7.0 5.4 C B
H396R 26 6.7 5.2 A A
F317L 22 5.7 4.4 A C
E355G 16 4.1 3.2 A A
Q252H 15 3.9 3.0 B B
E255V 14 3.6 2.8 C B
E459K 14 3.6 2.8 A A
F486S 13 3.4 2.6 A A
L248V 10 2.6 2.0 A A
D276G 10 2.6 2.0 A A
E279K 10 2.6 2.0 A A
Y253F 6 1.6 1.2 B A
F359C 6 1.6 1.2 C A
F359I 6 1.6 1.2 B A
*Class A indicates currently no compelling clinical evidence to
suggest that the mutation would not respond to the inhibitor. Class
B, In vitro assessment consistentlyindicates that the mutation may
confer intermediate insensitivity38/resistance39 to the inhibitor,
or clinical evidence may be suggestive of reduced sensitivity. At
this stage, thepresence of these mutations should have no impact on
clinical decisions and additional clinical assessment is required
before an alternative inhibitor would be recommended.Class C,
Compelling clinical evidence to recommend an alternative inhibitor;
V299L, which is very rarely detected in imatinib-treated patients,
is a dasatinib class C mutation.Class D, No role for SGI
therapy.
BCR-ABL MUTATION STATUS AND THERAPEUTIC DECISIONS 5431BLOOD, 24
DECEMBER 2009 � VOLUME 114, NUMBER 27
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrHighlight
userrUnderline
userrHighlight
userrHighlight
userrHighlight
-
irrespective of the disease phase at imatinib start.5-7,9,12
Therefore,the higher rate of detection of these mutations in
patients whoprogressed to advanced phases in our study is highly
consistent.However, there are some inconsistencies in our mutation
fre-quency. In AP, some mutation frequencies were
intermediatebetween CP and BC (Figure 2), which could be associated
withtransition between the phases. However, F359V was the
mostfrequent mutation detected in AP but detected at a relatively
lowfrequency in BC. The frequencies of individual mutations
inspecific disease phases should be considered with caution,
andvalidation is required.
The differences evident in BC phenotype, if validated,
raiseseveral questions. Why were these particular mutations related
toeither a lymphoid or myeloid phenotype? Could the BCR-ABLmutant
genotype drive the lineage phenotype in some cases or viceversa? A
known genetic determinant of LBC is homozygousdeletion of the
p16INK4A/p19ARF gene locus that occurs in asubstantial proportion
of LBC patients, but not MBC.70-74 It waspostulated that p16INK4A
and/or p19ARF mutations could induce theselective expansion of
B-cell progenitors by favoring their cellcycle entry, rather than
myeloid progenitors.75
In the case of BCR-ABL mutations, in vitro transformationassays
have demonstrated a gain or loss of function relative tounmutated
BCR-ABL.61,62 Mutations can alter the substrates thatbind to
BCR-ABL and activate alternate signal transductionpathways that
influence disease progression. Interestingly, E255Kand Y253F
mutations showed a pronounced increase of transforma-tion potency
in primary B-lymphoid progenitor cells.62 However,the
transformation potency of these mutations was not
significantlyincreased over unmutated BCR-ABL in the myeloid
lineage. In ourcohort, the frequency of E255K was markedly
increased inLBC/Ph� ALL (19.2%) compared with MBC (2.0%).
However,Y253F was detected at a low frequency in all disease
phases. TheSRC kinases, LYN, HCK, and FGR, have also been
implicated inthe molecular pathogenesis of Ph� ALL76 and are
critical for
transition of CML to LBC.77 Whether BCR-ABL mutations that
arecharacteristic of BCR-ABL� lymphoid leukemia in our
cohortenhance the activation of SRC kinases and hence the
transition to alymphoid phenotype is unknown.
Frequency of mutations that would influence the
therapeuticdecision
In our cohort of patients with mutations, 166 of 386 (43%) had
oneor more SGI clinically relevant mutations. T315I was detected
in53 of 386 patients (14%). In 110 of 386 patients (28%), one or
moreof their mutations were clinically relevant for either
nilotinib ordasatinib, but not to both. For these patients, there
may be anadvantage for one or other inhibitor. The remaining 3 of
386patients (0.8%) had 2 mutations, one of which was
clinicallyrelevant for dasatinib and the other for nilotinib
(neither