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Blood First Edition Paper, prepublished online December 5, 2012; DOI 10.1182/blood-2012-05-431379
Regular article
BCR-ABL1 compound mutations in tyrosine kinase inhibitor resistant CML: Frequency
and clonal relationships
Jamshid S. Khorashad1, Todd W. Kelley2, Philippe Szankasi3, Clinton C. Mason1, Simona
Soverini 4, Lauren T. Adrian5, Christopher A. Eide5,6, Matthew S. Zabriskie1, Thoralf Lange7,
Johanna C. Estrada1, Anthony D. Pomicter1, Anna M. Eiring1, Ira L. Kraft1, David J. Anderson1,
Zhimin Gu1, Mary Alikian8, Alistair G. Reid8, Letizia Foroni8, David Marin8, Brian J. Druker5,6,
Thomas O’Hare1,9,*, Michael W. Deininger 1,9,*
1
Huntsman Cancer Institute, The University of Utah, Salt Lake City, Utah, USA
Department of Pathology, The University of Utah, Salt Lake City, Utah, USA
3
Research and Development, ARUP Laboratories, Salt Lake City, Utah, USA
4
Department of Hematology/Oncology, “L. e A. Seragnoli”, University of Bologna, Bologna,
Italy
5
Knight Cancer Institute, Oregon Health & Science University, Portland, Oregon, USA
6
Howard Hughes Medical Institute, Portland, Oregon, USA
7
Department of Hematology/Oncology, University of Leipzig, Leipzig, Germany
8
Department of Hematology, Imperial College London, Hammersmith Hospital, London, UK
9
Division of Hematology and Hematological Malignancies, The University of Utah, Salt Lake
City, Utah, USA
* TO and MWD contributed equally.
2
Section: Myeloid Neoplasia
Correspondence:
Michael W. Deininger, M.D., Ph.D.
Address: Huntsman Cancer Institute, 2000 Circle of Hope, Salt Lake City, UT 84112-5550
Email: [email protected]
Phone number: +1 801 581 6363
Fax number: +1 801 585 0900
1
Copyright © 2012 American Society of Hematology
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Abstract
BCR-ABL1 compound mutations can confer high-level resistance to imatinib and other ABL1
tyrosine kinase inhibitors (TKIs). The third generation ABL1 TKI, ponatinib, is effective against
BCR-ABL1 point mutants individually but remains vulnerable to certain BCR-ABL1 compound
mutants. To determine the frequency of compound mutations among CML patients on ABL1
TKI therapy, we examined a collection of patient samples (N=47) with clear evidence of two
BCR-ABL1 kinase domain mutations by direct sequencing. Using a cloning and sequencing
method, we found that 70% (33/47) of double mutations detected by direct sequencing were
compound mutations. Sequential, branching, and parallel routes to compound mutations were
common. Additionally, our approach revealed individual and compound mutations not detectable
by direct sequencing. The frequency of clones harboring compound mutations with more than
two missense mutations was low (10%), while the likelihood of silent mutations increased
disproportionately with the total number of mutations per clone, suggesting a limited tolerance
for BCR-ABL1 kinase domain missense mutations. We report that compound mutations are
common in patients with sequencing evidence for two BCR-ABL1 mutations and frequently
reflect a highly complex clonal network whose evolution may be limited by the negative impact
of missense mutations on kinase function.
2
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Introduction
Tyrosine kinase inhibitors (TKIs) targeting the BCR-ABL1 oncoprotein are the standard therapy
for patients with chronic myeloid leukemia (CML). Imatinib, nilotinib and dasatinib are
approved for the treatment of newly diagnosed CML patients.1-3 However, an estimated 20-40%
of patients receiving first-line imatinib therapy will eventually require an alternative treatment
due to intolerance or resistance to TKIs.3-6 Recent studies in newly diagnosed chronic phase (CP)
patients have reported lower failure rates with dasatinib and nilotinib,1,2 but some patients will
still require salvage treatment. The best-characterized mechanism of resistance is point mutations
within the BCR-ABL1 kinase domain that impair or prevent TKI binding.7-9 Nilotinib and
dasatinib were developed to overcome imatinib resistance, and with exception of the multiresistant T315I mutant, these TKIs exhibit activity against many BCR-ABL1 kinase domain
mutations.10,11 Sanger sequencing, the method most widely used for mutation detection, reveals
only one mutation in the majority of cases of BCR-ABL1 kinase domain mutant-mediated
resistance. However, in a subset of patients, ≥2 mutations are detected by conventional
sequencing, reflecting either multiple BCR-ABL1 mutant clones (polyclonal mutations) or ≥2
mutations in the same BCR-ABL1 molecule (compound mutations; Figure 1). It has been
suggested that sequential therapy with different ABL1 TKIs may inadvertently foster the
development or selection of BCR-ABL1 compound mutations.12 While each of multiple mutant
clones is expected to retain its individual sensitivity to a given TKI, compound mutations can
dramatically affect TKI sensitivity and catalytic fitness of the tyrosine kinase.12-14 Therefore the
distinction between compound versus polyclonal mutations is clinically important as it may
influence the selection of the most suitable TKI to overcome resistance.14 Several compound
mutations have been shown to confer resistance to ponatinib, and this is likely to apply to other
third-line TKIs as well.13 Since the methods currently used for BCR-ABL1 kinase domain
mutation screening cannot definitively distinguish compound from polyclonal mutations, there is
little information available regarding their respective frequencies and clonal relationships.15
Thus, we employed a cloning and sequencing approach to establish the frequency and clonal
relationships of compound mutations in a cohort of CML patients defined by clear evidence of
more than one BCR-ABL1 kinase domain mutation in their conventional Sanger sequencing
trace.
3
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Materials and Methods
Patients
We analyzed samples from 47 CML patients treated with various ABL1 TKIs. The unifying
selection criterion was the presence of more than one BCR-ABL1 kinase domain mutation
detected by Sanger sequencing. Archived RNA or cDNA from the University of Utah (18
patients), Oregon Health & Science University (7 patients), University of Leipzig (5 patients),
Hammersmith Hospital, Imperial College London (9 patients) and the University of Bologna (8
patients) was utilized for analysis. The Institutional Review Boards of the participating centers
approved this study and informed consent was obtained according to the Declaration of Helsinki
where applicable. Serial samples were available for 5 patients, facilitating investigation of the
kinetics and evolution of BCR-ABL1 mutations.
BCR-ABL1 kinase domain amplification, cloning and sequencing
For the RNA samples, cDNA was synthesized as previously described.16 The BCR-ABL1 kinase
domain was amplified in a two-step nested PCR reaction that does not amplify non-translocated
ABL1.16 The amplified fragments were cloned using the TOPO TA cloning system (Invitrogen)
and introduced into E. coli.17 In the standard procedure, ten bacterial colonies corresponding to
BCR-ABL1 kinase domain amplicons from each patient were selected at random and grown in
Luria-Bertani medium, and plasmids were extracted using the Qiagen Plasmid Miniprep kit. The
BCR-ABL1 kinase domain was sequenced in both directions using BigDye terminator chemistry
on an ABI3730 instrument (Applied Biosystems, Inc., Foster City, CA) 17 and compared against
the ABL1 sequence (ENST00000318560) from Ensembl Genome Browser using the BLAST
alignment tool (NCBI). If more than one mutation was detected at similar levels among the
initially surveyed 10 colonies, a greater number of bacterial colonies were examined to gain a
higher resolution for representation of the clonal relationship. Specifically, an expanded number
of clones was sequenced in two instances: 1) sample CML#14 (55 in total) for which the
V299L/M351T compound mutation was detected in similar frequency to the predominant
compound mutation, T315I/F359V, among the original 10 clones sequenced, whereas it was not
detected by direct sequencing. 2) CML#46 (15 in total) for which four different mutations were
detected by direct sequencing.
4
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Definition of mutations
Mutations identified by direct sequencing. Direct sequencing of PCR products is the clinical
standard and detects mutant alleles if they represent at least 20-30% of the amplicon pool. We
refer to all mutations detected with this method as predominant mutations. One mutation
detected by direct sequencing is referred to as a single mutation, while two mutations detected by
direct sequencing is referred to as a double mutation (Table 1).
Mutations identified by sequencing of individual cloned PCR products. Each bacterial clone
carries an mRNA transcript from one individual BCR-ABL1 molecule. Mutations detected by this
method but not by direct sequencing are referred to as low-level mutations. Two or more
mutations present in the same molecule are referred to as a compound mutation in contrast to
polyclonal mutations, which refer to those present in different molecules. An individual mutation
that is part of a compound mutation is designated as a component. In cases where a low-level
mutation (detected only by sequencing of individual PCR products) is detected in addition to a
predominant mutation (detected by direct sequencing), it is referred to as an add-on compound
mutation.
Statistical analysis
Frequency differences across multiple groups were assessed with Fisher’s exact test; differences
within single groups were compared with the exact binomial test. For testing the association of
various factors with the number of mutant colonies, it is important to account for the fact that the
probability of observation of a given factor (e.g. type of mutation) will increase with the number
of mutations being observed. This expected probability is equivalent to 1 – (1-x)n , where n is the
number of mutations being assessed and x is the rate of occurrence of the factor when only a
single mutation is being observed. A chi-square test was used to assess the deviation of
observations from the expected value based on this calculation. A two-sided P-value of 0.05 was
used to determine statistical significance unless otherwise noted. Statistical computations were
performed using SAS ver. 9.2 (Carey, NC).
5
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Results
The majority of double mutations are compound mutations
The frequency of occurrence of ≥2 mutations among a total of 1700 samples harboring BCRABL1 kinase domain mutations detectable by direct sequencing at the 5 participating centers was
11.4% (194/1700). The samples with ≥2 mutations pertained to a total of 47 patients, for which
the most recent clinical sample was investigated for this study. We sequenced a total of 583
colonies (Supplementary Information 1) and identified 218 different mutations, 167 (77%) of
which were missense mutations encompassing 157 residues in the kinase domain
(Supplementary Table 1). The remaining 51 nucleotide changes (23%) were silent mutations.
The residues most frequently involved in missense mutations were F317 (5.4%), T315 (4.9%),
G250 (3.5%), M351 (3.2%), F359 (2.7%) and V299 (2.4%) (Supplementary Table 1). The
predominant mutations detected by direct sequencing have all previously been associated with
resistance to second generation ABL1 TKIs, except M351T, which has been reported in imatinib
failure, but has not been reported to confer significant resistance to either nilotinib or
dasatinib.10,11,18 90% of the mutations were transitions, with the most common nucleotide
changes being A→G and T→C (Supplementary Table 2). We found that the majority of double
mutations detected by direct sequencing (33/47; 70.2%) were in fact compound mutations as
established by sequencing of individually cloned PCR products (Table 2). Thirty different
compound mutations were detected among the 33 patients shown to harbor compound mutations
(Table 2); the T315I/F359V, Y253H/F317L and G250E/V299L mutations were each detected in
more than one patient. The most common components of the compound mutations were (by
frequency): T315I (27%; 8/30), V299L (20%; 6/30), F359V (17%; 5/30) and M351T (17%;
5/30). In total, these four substitutions account for 40% (24/60) of the components in these 30
compound mutations. Only 13/47 patients contained low-level independent mutations that were
detected by cloning and sequencing but not by direct sequencing. While 1/10 clones with a novel
mutation could result from PCR artifact or other technical issues, it is noteworthy that one such
sample (CML#24) contained 2/10 clones with the same mutation (P310S) that was not detected
by direct sequencing. Additionally, all mutations detected by direct sequencing were detected by
the cloning and sequencing method, indicating the high fidelity of this method.
6
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Compound mutations and exposure to multiple TKIs
Detailed clinical annotation was available for 28 patients: 20 with compound mutations and 8
with polyclonal mutations. Among these, 15/28 had received at least two sequential ABL1 TKI
therapies (Table 3): 3 from the polyclonal mutation group and 12 from the compound mutation
group. There is not a pronounced overrepresentation of BCR-ABL1 compound mutations among
advanced phase (AP) or blastic phase (BP) patients (9/13; 69%) compared to CP patients (11/15;
73%). However, the sample number available for this study does not permit a detailed analysis of
the correlation between the phase of disease and the presence of multiple mutations.
In patients treated sequentially with dasatinib, nilotinib or both TKIs following imatinib failure
who had developed resistance to second-line treatment, analysis of the individual components of
the compound mutations revealed that the identities of component mutations reflected the type of
prior drug exposure. Thus, in all patients treated with dasatinib, at least one component of the
compound mutations was V299L, F317L or E255K, all of which have been reported in clinical
or in vitro resistance to dasatinib.10,11,19 Among these, the role of the E255K mutation in
conferring clinical resistance to dasatinib is the least certain.20,21 In patients treated with nilotinib,
at least one component was associated with clinical or in vitro resistance (E255K, Y253H,
F359V) (Table 3).10,11 Analogous observations were made in the patients with polyclonal
mutations (Supplementary Table 3).
Low-level mutations
Cloning and sequencing of individual clones revealed many additional mutations that were not
detectable by direct sequencing. The categories of mutation patterns that we observed among our
samples are shown as a model in Figure 2. The majority of additional mutations are components
of compound mutations and presumably originate from a BCR-ABL1 clone that carried a
predominant mutation. We refer to this group of mutations as add-on compound mutations
(Figure 2, mutation C, D, and E) since they are acquired in addition to a pre-existing
predominant mutation. A second group of mutations present in clones separate from the
predominant mutations are referred to as independent low-level mutations (Figure 2, mutation F,
G and H). Based on the substantial underlying mutational complexity observed in our study, we
reasoned that patients with multiple mutations may have a heightened risk of acquiring further
mutations. Thus, for comparison, we applied the cloning and sequencing approach to 12
7
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additional patients with single mutations as detected by direct sequencing in order to screen for
the presence of low-level mutations. However, we found no statistically significant difference in
the prevalence of low-level single or compound mutations in patients with a single predominant
mutation (3/12; 25%) compared to the main study group of patients with two or more
predominant mutations detectable by direct sequencing (15/47; 32%; p=0.74). The clinical
significance of these low-level mutations is unknown. A recent study focusing on a selected set
of 31 common BCR-ABL1 kinase domain mutations demonstrated a clear association between
the presence of multiple low-level mutations and poor response to subsequent ABL1 TKI
therapy.22-24 Parker et al.22,
24
specifically assessed for the presence of known TKI-resistant
mutants. The current manuscript does not examine the impact of low-level mutants upon TKI
sensitivity.
Clonal relationships
It is commonly thought that BCR-ABL1 kinase domain mutant clones evolve in a linear fashion
through successive acquisition of different point mutations that incrementally increase their
fitness to withstand selective pressure, for example by conferring resistance to TKI therapy.
Figure 3 gives an example of this linear evolution (shown for patient CML#19), and the clonal
relationships for 61% (20/33) of compound mutant samples were compatible with this model.
However, cloning and sequencing of individual PCR amplicons revealed the presence of each
individual component as a single mutation in some patients. For example L387M, T315I and the
compound mutation L387M/T315I were all detected in separate clones originating from sample
CML#44, suggesting that either L387M or T315I must have been acquired via two independent
events. This unexpected pattern was observed in 39% (13/33) of compound mutation samples
(Table 4) and may indicate that certain patients are predisposed toward a highly restricted range
of mutations.
In one sample (CML#14), cloning and sequencing of ten colonies revealed a compound mutation
with a frequency similar to that of the predominant mutation but was not detected by direct
sequencing. To better understand the complexity of clonal relationships in this sample, we
sequenced the BCR-ABL1 kinase domain in an additional 45 bacterial colonies. We identified
three independent major clones, each of which could be implicated in further mutational changes
(Figure 4). In total, we found 26 different mutant clones, consistent with a high level of
8
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mutational complexity. None of the clones harbored more than four missense mutations and the
number of silent mutations increased disproportionately with the total number of mutations in
each clone. Based on this observation, we investigated whether there may be a limit to the
number of missense mutations tolerated by BCR-ABL1.
Limited mutation tolerance of the BCR-ABL1 kinase domain
Add-on compound mutations were found in polyclonal and compound mutant samples, with
some samples harboring up to six different components within the context of a compound
mutation. The majority of missense add-on mutations (Supplementary Table 4) were at residues
that have not previously been associated with resistance and most of these mutations were nonrecurrent, suggesting that they were passenger mutations that did not confer a competitive
advantage or increased resistance to TKIs.9,18,25-29 We hypothesized that successively acquired
missense mutations will eventually impair or abrogate kinase function. Given that such reducedfitness clones will be eliminated in vivo, analyzing the frequency of silent mutations, which are
neutral with respect to kinase function, can test this hypothesis only indirectly. If additional
missense mutations are detrimental, then the likelihood of an additional mutation being a silent
mutation should increase disproportionately relative to the total number of mutations in a given
clone. We found that the proportion of silent mutations increased with the total number of
mutations significantly beyond the calculated expectation, suggesting that additional missense
mutations are poorly tolerated by the kinase (Figure 5 and Supplementary Information 2).
9
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Discussion
BCR-ABL1 kinase domain mutations are the most common and best-characterized mechanism of
TKI resistance in BCR-ABL1-positive leukemia. Depending on the clinical setting (primary or
secondary resistance, chronic phase or advanced phase), kinase domain mutations have been
detected in 30-90% of patients with imatinib resistance and 20-80% of patients who failed
nilotinib or dasatinib.12,30-34 While the latter two drugs overcome the majority of mutants
associated with imatinib resistance, they are not universally active and exhibit some
vulnerabilities. It has been suggested that sequential therapy with different ABL1 TKIs may
predispose patients to the acquisition or selection of clones harboring multiple mutations,
including mutations in the same BCR-ABL1 molecule (compound mutations).12 Certain
compound mutations may pose a significant clinical problem due to their ability to confer crossresistance to multiple TKIs, in contrast to mutations in different clones that are individually
susceptible to one or more TKIs. If sequential TKI therapy (imatinib followed by dasatinib
and/or nilotinib) predisposed a patient to multi-resistant compound mutations, this would be a
strong argument for the use of dasatinib or nilotinib as first-line therapy. While double or
multiple BCR-ABL1 mutations have been reported in many studies, it is unknown which
proportion of these represent compound mutations.
In this study we investigated the frequency of compound mutations among 47 CML patients
harboring two or more BCR-ABL1 kinase domain mutations as detected by direct Sanger
sequencing. We confirmed through cloning and sequencing that 70% of the double mutations
were, in fact, compound mutations. However, for polyclonal mutations, both mutations should be
present at higher than 20% to be detectable by direct sequencing. When a stark unequal
representation occurs (for example 15% and 85%), then one of the mutations in a polyclonal
mutation will become undetectable by direct sequencing. From a clinical point of view direct
sequencing may be biased toward detection of compound mutations compared to polyclonal
mutations.
We found that the composition of the compound mutations clearly reflected the TKI exposure
history, with at least one component of each compound mutant in patients exposed to more than
one TKI being associated with clinical or in vitro resistance to nilotinib or dasatinib. Examining
complexity of the clonality of BCR-ABL1 compound mutations according to disease phase is an
10
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interesting next step, but requires profiling within a larger focused cohort of samples not
available from the current study.
We observed different patterns of mutations as shown in Figure 2. One concern regarding lowlevel mutations is the genuineness of these mutations. Since they are detected at a very low-level
one must consider that they could be artifacts associated with cDNA synthesis or PCR. However,
we have evidence that supports the genuineness of these mutations. First, we did not see any
low-level mutations in the sequenced colonies for three samples. Specifically, these three
samples contained exactly and only the mutations detected by direct sequencing, arguing against
frequent introduction of PCR artifact with our methods. Secondly, these low-level mutations are
not random: 39 low-level silent or missense mutations were detected in more than one patient
(Supplementary Table 5). Finally, the rate of silent mutations was lower than expected
(Supplementary Information 2). However, technical artifact can never be ruled out with PCRbased analysis since a replication error early in the reaction will be perpetuated as the population
of product is expanded.
We observed compound mutations with up to six components in individual clones. However, if
more than two missense mutations were detected in a single clone, this particular combination
was always limited to one of the ten sequenced clones from each patient, suggesting that the addon mutations failed to promote the clone’s expansion. Moreover, the prevalence of silent
mutations increased disproportionately with the total number of accumulated mutations. Since
silent mutations are not subject to selection pressure, they are a convenient measure of mutation
rate35; their disproportionately increased frequency in clones with multiple mutations suggests
that clones that acquire additional missense mutations are not competitive, possibly due to
impaired kinase function. Altogether, these data suggest that the BCR-ABL1 kinase domain may
have a limited tolerance for missense mutations. The evolution of mutant clones will be
influenced both by the selection pressure imposed by TKIs and the need to maintain kinase
activity. We and others have previously reported that kinase domain mutations can alter kinase
activity and downstream signaling.36,37 While it is conceivable that in some cases fortuitous
combinations of mutations may not attenuate kinase function,12 our data suggest that a lack of
deleterious effects on kinase activity as the number of mutations increases must be the exception.
On the other hand, a surprisingly high proportion of double mutations represent compound
11
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mutations. Given that certain compound mutations such as G250E/T315I and E255V/T315I
confer high level resistance to ponatinib or DCC-2036 in vitro, our data suggest that these
mutations may emerge as a significant clinical challenge in patients on third-line TKIs.13,14
Comprehensive in vitro TKI resistance profiles of BCR-ABL1 compound mutants have not been
established. Among the 30 different BCR-ABL1 compound mutations detected in this study
(Table 2), 8 unique BCR-ABL1T315I-inclusive compound mutations were identified among 9
patients. There were two occurrences of BCR-ABL1T315I/F359V (CML#7 and CML#14). We
recently identified and reported BCR-ABL1T315I-inclusive compound mutants recovered in cellbased screens for resistance to ponatinib13 or DCC-2036.38 Consultation of this dataset revealed
that 6/8 (75%) and 3/8 (38%) of the BCR-ABL1T315I-inclusive mutants from the current study
were recovered in the ponatinib or DCC-2036 resistance screen, respectively, and 3/8 (38%)
were recovered in both resistance screens (Supplementary Table 6). The standard mutation
detection technologies in current use cannot distinguish between polyclonal and compound
mutations, and this limitation hampers clinicians’ ability to choose between TKI options.
In the clonal dominance model, selective pressure exerted by TKIs drives the linear evolution of
clones that acquire ever greater fitness to withstand TKIs and expand at the expense of less
competitive clones. However, our data reveal a high level of clonal diversity. For example, in
sample CML#46 from the polyclonal mutation group, we detected three clones, each with a
different mutation at the same residue (F317C, F317L, and F317V). This indicates independent
acquisition of mutations at the same residue by at least three different clones. Unexpectedly this
does not seem to be a rare event. In 39% of the patients with compound mutations, transcripts
with each of the individual component mutations co-existed, indicating at least two independent
events. This is similar to the pattern observed for some patients with myeloproliferative disorders
in which JAK2 and TET2 mutations were observed in the same clone and also in different
clones.39 One practical implication of this is that one cannot assume identical clones when a
patient relapses with the same mutation after a transient response to TKI therapy.
It has been suggested that cancer development depends on the continuous acquisition of genetic
variation by random mutations, with environmental selection acting on the resultant phenotype
diversity.40 The evolution of BCR-ABL1 kinase domain mutant clones in the TKI environment is
an example of the complex interplay between random mutation and selection in CML.
12
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Fortunately, there appears to be strong constraints on the evolution of the causal oncoprotein
BCR-ABL1, which may contribute to the remarkable success of TKI therapy in CML.
Acknowledgments
We thank Suzanne Wickens for her clerical assistance. This work was supported by HL08297801 (M.W.D.) and CA04963920A2 (M.W.D.), Leukemia and Lymphoma Society grant 7036-01
(M.W.D.) and Howard Hughes Medical Institute. A.M.E. is a Fellow and M.W.D. is a Scholar in
Clinical Research of the Leukemia and Lymphoma Society.
Authorship
Contribution: TO and MWD designed the experiments. JSK, TO and MWD wrote the paper.
JSK, PS, SS, LTA, CAE, MSZ, JE and MA performed the experiments. TWK, SS, CAE, TL,
BJD and DM provided the clinical information for the samples. TWK, BJD, SS, AGR and LF
provided materials. JSK and CCM analyzed the data. JSK, ADP and ILK made the figures.
AME, ADP, DJA, AGR, CAE, ZG and DM commented on the manuscript.
Conflict-of-interest disclosure: S.S. is a consultant for Novartis, BMS and ARIAD. T.L. is a
consultant for Novartis, BMS and Pfizer and receives research funding from Novartis. M.W.D. is
a consultant for BMS, Novartis, ARIAD and Incyte and his laboratory receives research funding
from BMS and Novartis. BJD is principal investigator or co-investigator on Novartis, BristolMyers Squibb, and ARIAD clinical trials. His institution has contracts with these companies to
pay for patient costs, nurse and data manager salaries, and institutional overhead. He does not
derive salary, nor does his lab receive funds from these contracts. OHSU and BJD have a
financial interest in MolecularMD. OHSU has licensed technology used in some of these clinical
trials to MolecularMD. This potential individual and institutional conflict of interest has been
reviewed and managed by OHSU.
13
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Table 1. Definitions of BCR-ABL1 mutations used in this manuscript
BCR-ABL1 Mutation
Classification
Definition
Predominant mutation
Mutation detectable by direct Sanger sequencing of PCR amplicon
pool.
Single mutation
Single missense mutation detectable in a sample subjected to
Sanger sequencing of PCR amplicon pool.
Double mutation
Two missense mutations detectable in a sample subjected to
Sanger sequencing of PCR amplicon pool.
Mutation detected only by sequencing of an individually cloned
PCR amplicon.
Missense mutation emerging in addition to predominant mutation.
Low-level mutation
1) Add-on mutation
2) Independent low-level
mutation
Compound mutation
Polyclonal mutation
Component
Missense mutations present in separate clones relative to clones
harboring the predominant mutation(s).
Two or more missense mutations in the same sample and the same
BCR-ABL1 molecule (Figure 1).
Two or more missense mutations in the same sample, but different
BCR-ABL1 molecules (Figure 1).
A mutation that is part of a compound mutation.
17
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Table 2. BCR-ABL1 compound mutations in patients with ≥2 mutations
Patient IDa
(N= 47)
1. CML#1
2. CML#2
3. CML#3
4. CML#4
5. CML#5
6. CML#6
7. CML#7
8. CML#8
9. CML#9
10. CML#10
11. CML#11
12. CML#12
13. CML#13
14. CML#14
15. CML#15
16. CML#16
17. CML#17
18. CML#18
19. CML#19
20. CML#20a
21. CML#23
22. CML#24a
23. CML#27
24. CML#28
25. CML#29
26. CML#30
27. CML#31
28. CML#32
29. CML#33
30. CML#34
31. CML#35
32. CML#36
33. CML#37
34. CML#38
35. CML#39
36. CML#40
37. CML#41
38. CML#42
39. CML#43
40. CML#44
41. CML#45
42. CML#46
43. CML#47
44. CML#48
45. CML#49
46. CML#50
47. CML#51
a
≥2 mutations detected by
direct sequencing
V299L, M351T
F317I, F359V
Q252H, Y253H, T315I
V299L, L384M
E255V, T315I
F311L, T315I
T315I, F359V
E255K, T315I
V268A, F359V
M244V, T315I
G250E, M351T
T315I, F317L
E255K, F317L
T315I, E355G, F359V
T315I, D325G
L298V, V299L
T315I, F359C
G250E, V299L
E255K, M351T
H396R, F317L
G250E, T315I
G250E, T315A
V338F, L384M
V299L, E459K
G250E, E255V
Y253H, F317L
M244V, F317L
M244V, M351T
M351T, F359V
L248V, G250E
Y253H, F359V
T315I, H396R
V299L, F359V
V299L, F359V
Y253H, T315I
M244V, E459K
E255K, T315I
G250E, Y253H, F317L
F317L, M351T
T315I, L387M
F317L, Y253H
T315A, F317C, F317L, F317V
Y253H, E255V
T315I, F359V
G250E, V299L
G250E, E459K
F311L, H396R
Confirmed BCR-ABL1 compound
mutation(s)b, c
V299L/M351T
F317I/F359V
None
V299L/L384M
None
F311L/T315I
T315I/F359V
None
V268A/F359V
M244V/T315I
G250E/M351T
None
E255K/F317L
T315I/F359Vd
None
L298V/V299L
T315I/F359C
G250E/V299L
E255K/M351T
None
G250E/T315I
G250E/T315A
V338F/L384M
V299L/E459K
None
Y253H/F317L
M244V/F317L
M244V/M351T
None
None
Y253H/F359V
T315I/H396R
V299L/F359V
None
None
M244V/E459K
E255K/T315I
G250E/F317L
F317L/M351T
L387M/T315I
F317L/Y253H
None
None
None
G250E/V299L
G250E/E459K
F311L/H396R
Discontinuous CML#s reflect omission of sequential samples.
18
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b
Confirmed by cloning and sequencing as described in the Materials and Methods and Results.
c
Compound mutations were confirmed among 33 patients. Within the 33 compound mutations,
three pairings occurred twice (G250E/V299L; Y253H/F317L; T315I/F359V). Therefore, a total
of 30 different compound mutations are represented in this data set.
d
K294K/V299L/M351T compound mutation was detected at similar level to T315I/F359V
during
cloning
and
sequencing
of
the
10
colonies
(K294K/V299L/M351T:4/10,
T315I/F359V:4/10) although undetected by direct sequencing so is not considered a predominant
mutation according to our definition (see Table 1).
19
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Table 3. Mutations, TKI and disease phase
Patient ID
BCR-ABL1 Mutations
d
Im
Im
Im
Im
Im
Im
Das
Pon
Im, Das
Im, Das
Im, Das
Im, Das
Im, Das
Im, Das
Im, Nil, Das
Im, Nil, Das
Im, Nil, Das
Im, Nil, Das
Im, Nil, Das, Bos
Im, Nil, Das, Bos
Im
Im
Im
Das
Das
CP
CP
CP
CP
CP
CP
BP
BP
BP
CP
BP
AP
BP
CP
CP
CP
BP
BP
BP
CP
CP
BP
AP
CP
BP
Type of
BCR-ABL1
Mutations
Compound
Compound
Compound
Compound
Compound
Compound
Compound
Compound
Compound
Compound
Compound
Compound
Compound
Compound
Compound
Compound
Compound
Compound
Compound
Compound
Polyclonal
Polyclonal
Polyclonal
Polyclonal
Polyclonal
Im, Das
Im, Das
Im, Nil, Das
BP
CP
CP
Polyclonal
Polyclonal
Polyclonal
TKI Therapy
Disease
Phase
CML#23
CML#27
CML#32
CML#40
CML#50
CML#51
CML#24
CML#36
CML#28
CML#31
CML#41
CML#43
CML#44
CML#49
CML#19
CML#37
CML#42
CML#45
CML#30
CML#35
CML#33
CML#39
CML#48
CML#20
CML#46
CML#34
CML#47
CML#38
G250Ec/T315Ic
V338F/L384Mb
M244V/M351T
M244V/E459K
G250Ec/E459K
F311L/H396Rb
G250Ec/T315Aa
T315Ic/H396Rb
V299La/E459K
M244V/F317Lc
E255Kc/T315Ic
F317Lc/M351T
T315Ic/L387M
G250Ec/V299La
M351T/E255Kc
V299La/F359Vb
G250Ec/F317Lc
Y253Hb/F317Lc
Y253Hb/F317Lc
Y253Hb/F359Vb
M351T, F359Vb
Y253Hb, T315Ic
T315Ic, F359Vb
H396Rb, F317Lc
T315Aa, F317Ca,
F317Lc, F317Va
L248Vc, G250Ec
Y253Hb, E255Vc
V299La, F359Vb
e
a
In vitro or clinical resistance to dasatinib according to published data as shown in
Supplementary Table 3.
b
In vitro or clinical resistance to nilotinib according to published data as shown in Supplementary
Table 3.
c
In vitro or clinical resistance to nilotinib and dasatinib according to published data as shown in
Supplementary Table 3.
d
Im: imatinib; Das: dasatinib; Nil: nilotinib; Pon: ponatinib; Bos: bosutinib
e
CP: chronic phase; AP: advanced phase; BP: blastic phase
20
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Table 4. Coexistence of BCR-ABL1 mutations as compound and polyclonal mutations in separate
clones originating from the same patient sample
Patient
ID
Clones with compound
mutation
Clones with first component
mutation
Clones with second
component mutation
CML#10
M244V/T315I/I347T/T406I
(1/10)
M244V (4/10)
T315I (2/10)
CML#11
CML#13
G250E/M351T/L429P (1/10)
G250E/M351T (1/10)
E255K/F317L (1/10)
G250E/H396R (1/10)
G250E (1/10)
E255K (2/10)
CML#23
G250E/T315I (1/10)
G250E (8/10)
M351T (4/10)
M351T/L341P (1/10)
F317L (2/10)
F317L/D391G/E334K
(1/10)
F317L/T389A (1/10)
T315I (1/10)
CML#30
Y253H/F317L (1/10)
Y253H (2/10)
F317L (4/10)
CML#31
M244V/F317L (1/8)
F317L (2/8)
CML#35
CML#37
Y253H/F359V/A366V (1/10)
Y253H/F359V (2/10)
V299L/F359V (2/10)
A288T/
V299L/F359V/W423Stop (1/10)
M244V (3/8)
M244V/D391G (1/8)
Y253H (3/10)
CML#40
F359V (3/10)
V299L (2/10)
V299L/V377A (1/10)
F359V (3/10)
M244T/F359V/ T389A
(1/10)
M244V/E459K (1/10)
M244V (1/10)
CML#41
CML#44
CML#50
E255K/T315I (8/10)
L387M/T315I (6/10)
G250E/E459K (1/9)
CML#51
F311L/L341P/H396R (1/10)
E255K (1/10)
L387M (2/10)
G250E (1/9)
G250E/F317L (1/9)
E279G/F311L/E355G/W405R
(1/10)
E459K (2/10)
W423R/E459K (1/10)
K247R/E459K (1/10)
E238G/L273S/E459K
(1/10)
T315I (1/10)
T315I (2/10)
E459K (3/9)
L266P/E459K (1/9)
H396R (1/10)
L266P/K356R/H396R
(1/10)
H396R/N414S (1/10)
The predominant compound mutations and their components are shown in bold and color. Silent
mutation components are not shown in this table. The frequency of each clone is shown in
proportion to the number of sequenced clones per sample.
21
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Figure Legends
Figure 1: Polyclonal versus compound mutations. In a subset of patients who develop clinical
resistance to ABL1 TKIs, more than one point mutation in the kinase domain of BCR-ABL1 is
detectable by direct sequencing. In the case of polyclonal mutations, these BCR-ABL1 mutations
(green and red stars; upper panel) exist separately in different clones. By contrast, BCR-ABL1
compound mutants exhibit two mutations within the same BCR-ABL1 molecule (green and red
stars; lower panel).
Figure 2: Mutational patterns revealed by cloning and sequencing of the BCR-ABL1 kinase
domain region. Hypothetical mutation identities are designated A through H for
discussion/explanation purposes. The range of mutation patterns revealed by cloning and
sequencing when two BCR-ABL1 kinase domain mutations, A and B (green and red stars in the
trace) are detected by direct sequencing are represented. This model encompasses all observed
mutational patterns among the patients with two mutations evident by direct sequencing in this
study. Detection of mutations A and B by direct sequencing can reflect their presence in the
same BCR-ABL1 molecule (detected in one clone) or in different BCR-ABL1 molecules (detected
in separate clones). The presence of both A and B in the same or different clones is signified as
A/B or as A and B, respectively. In some compound mutant patients, clone A and clone B were
observed separately in addition to clone A/B. Mutation A or B was sometimes co-existent with
add-on mutations, for example A/D and B/E. Predominant compound mutants could also acquire
further add-on mutations as exemplified by A/B/C. Some mutations such as F (single
independent mutants) or G/H (compound independent low-level mutants) were observed in
clones that did not carry either predominant mutation, A or B.
Figure 3: Successive acquisition of mutations. Five serial samples were available for patient
CML#19. The clones with thick circles represent the mutations detected by direct sequencing.
The number of sequenced clones for each sample is shown on the left. The number of each
mutated clone is shown inside each clone unless it was detected only once. The unmutated or
independent low-level mutant clones are not shown here. M351T and Y253F were detected by
direct sequencing in the first sample under imatinib (IM) therapy (41 months). Cloning and
sequencing confirmed M351T/Y253F as a compound mutation but also revealed other low-level
clones presumably derived from M351T that were undetectable by direct sequencing. The red
22
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line indicates a 30-month gap between stoppage of imatinib therapy and start of nilotinib (NI)
therapy during which the patient was treated with homoharringtonine. Direct sequencing in the
next three samples taken after start of nilotinib therapy detected only M351T. Similar to the first
sample, cloning and sequencing revealed add-on compound mutants derived from M351T not
detected by direct sequencing. An E255K/M351T compound mutation was first observed under
dasatinib (DA) therapy. E255K/M351T was the only mutation detected in the last sample, and
was evident by direct sequencing as well as the more sensitive cloning and sequencing method.
M351T/E255K developed along with resistance to dasatinib in this patient. The patient’s disease
progressed to blast crisis a few months after detection of M351T/E255K and loss of response to
dasatinib.
Figure 4: Simultaneous evolution of compound mutant clones at comparable levels. Cloning
and sequencing of 55 colonies for CML#14 revealed a complex pattern of compound mutations.
There were at least three major classes of clones (F359V/T315I, K294K/M351T/V299L and
E355G) and their derivatives, which formed the majority of the BCR-ABL1 clones in this sample.
The thick red, green and blue circles represent 11, 6 and 14 clones respectively. The dotted circle
represents a clone that was not detected but expected to be the parent of the two observed clones.
The remaining circles represent clones detected a single time. These single clone results should
be interpreted with caution since PCR error can never be rigorously excluded. Silent mutations
are labeled in red. There is a marked rise in the proportion of silent mutations as clones acquire
additional mutations. One clone presumably destined for immediate elimination has a stop
mutation (E355G/Q300Stop) expected to abolish kinase activity in the middle of the kinase
domain.
Figure 5: The percentage of silent mutations increases disproportionately with the total
number of mutations per clone. The X-axis represents the number of mutations per clone and
the Y-axis represents the percentage of clones with at least one silent mutation (gray bars)
compared to the expected percentage (white bars).
23
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Prepublished online December 5, 2012;
doi:10.1182/blood-2012-05-431379
BCR-ABL1 compound mutations in tyrosine kinase inhibitor resistant CML:
frequency and clonal relationships
Jamshid S. Khorashad, Todd W. Kelley, Philippe Szankasi, Clinton C. Mason, Simona Soverini, Lauren T.
Adrian, Christopher A. Eide, Matthew S. Zabriskie, Thoralf Lange, Johanna C. Estrada, Anthony D.
Pomicter, Anna M. Eiring, Ira L. Kraft, David J. Anderson, Zhimin Gu, Mary Alikian, Alistair G. Reid,
Letizia Foroni, David Marin, Brian J. Druker, Thomas O'Hare and Michael W. Deininger
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