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Regular Article
CLINICAL TRIALS AND OBSERVATIONS
Compound mutations in BCR-ABL1 are not major drivers of primary or
secondary resistance to ponatinib in CP-CML patients
Michael W. Deininger,1 J. Graeme Hodgson,2 Neil P. Shah,3 Jorge E. Cortes,4 Dong-Wook Kim,5 Franck E. Nicolini,6
Moshe Talpaz,7 Michele Baccarani,8 Martin C. Müller,9 Jin Li,10 Wendy T. Parker,15 Stephanie Lustgarten,2 Tim Clackson,2
Frank G. Haluska,2 Francois Guilhot,11 Hagop M. Kantarjian,4 Simona Soverini,12 Andreas Hochhaus,13
Timothy P. Hughes,14,15 Victor M. Rivera,2 and Susan Branford15,16
1
Huntsman Cancer Institute University of Utah, Salt Lake City, UT; 2ARIAD Pharmaceuticals, Inc., Cambridge, MA; 3University of California San Francisco,
San Francisco, CA; 4Department of Leukemia, The University of Texas MD Anderson Cancer Center, Houston, TX; 5Seoul St. Mary’s Hospital, The Catholic
University of Korea, Seoul, Korea; 6Centre Hospitalier Lyon Sud, Pierre Benite, & INSERM U1052, Lyon, France; 7Comprehensive Cancer Center,
University of Michigan, Ann Arbor, MI; 8Department of Hematology-Oncology “L. and A. Seragnoli,” S. Orsola-Malpighi University Hospital, Bologna, Italy;
9
III. Med. Klinik, Universitätsmedizin Mannheim, Mannheim, Germany; 10MolecularMD, Portland, OR; 11INSERM Clinical Investigation Center 1402, Centre
Hospitalier et Universitaire de Poitiers, France; 12Department of Experimental, Diagnostic, and Specialty Medicine, University of Bologna, Bologna, Italy;
13
Abteilung Hämatologie/Onkologie, Universitätsklinikum Jena, Jena, Germany; 14SA Pathology and South Australian Health and Medical Research
Institute, Adelaide, Australia; 15Department of Genetics and Molecular Pathology, SA Pathology, Centre for Cancer Biology, School of Medicine and School
of Molecular and Biomedical Science, University of Adelaide, Adelaide, Australia; and 16School of Pharmacy and Medical Science, University of South
Australia, Adelaide, Australia
BCR-ABL1 kinase domain mutations can confer resistance to first- and second-generation
tyrosine kinase inhibitors (TKIs) in chronic myeloid leukemia (CML). In preclinical studies,
clinically achievable concentrations of the third-generation BCR-ABL1 TKI ponatinib inhibit
• Ponatinib induces durable
T315I and all other single BCR-ABL1 mutants except T315M, which generates a single amino
responses regardless of
baseline BCR-ABL1 mutation acid exchange, but requires 2 sequential nucleotide exchanges. In addition, certain compound mutants (containing ‡2 mutations in cis) confer resistance. Initial analyses based
status in CP-CML patients.
largely on conventional Sanger sequencing (SS) have suggested that the preclinical rela• No single or compound
tionship between BCR-ABL1 mutation status and ponatinib efficacy is generally recapitulated
mutant consistently confers
in patients receiving therapy. Thus far, however, such analyses have been limited by the
primary or secondary
inability of SS to definitively identify compound mutations or mutations representing less than
resistance to ponatinib in
∼20% of total alleles (referred to as “low-level mutations”), as well as limited patient follow-up.
CP-CML.
Here we used next-generation sequencing (NGS) to define the baseline BCR-ABL1 mutation
status of 267 heavily pretreated chronic phase (CP)-CML patients from the PACE trial, and
used SS to identify clonally dominant mutants that may have developed on ponatinib therapy (30.1 months median follow-up). Durable
cytogenetic and molecular responses were observed irrespective of baseline mutation status and included patients with compound
mutations. No single or compound mutation was identified that consistently conferred primary and/or secondary resistance to ponatinib in
CP-CML patients. Ponatinib is effective in CP-CML irrespective of baseline mutation status. (Blood. 2016;127(6):703-712)
Key Points
Introduction
Tyrosine kinase inhibitors (TKIs) targeting BCR-ABL1 have substantially improved the prognosis of patients with chronic myeloid leukemia (CML).1 However, resistance to TKI therapy, which manifests
through both BCR-ABL1–dependent and –independent mechanisms,
remains a major challenge.2 The best-characterized mechanism of
resistance involves point mutations in the BCR-ABL1 kinase domain
(KD) that disrupt TKI binding. A range of point mutations can mediate resistance to first- (imatinib) and second- (dasatinib, nilotinib,
and bosutinib) generation TKIs, with the T315I gatekeeper mutation
conferring resistance to all 4 agents.3-5 In addition, compound mutants
(variants containing $2 mutations within the same BCR-ABL1 allele
that presumably arise sequentially) can arise under certain selective
pressures,6 and a subset is highly TKI resistant.5 Although preclinical
data may be used to identify candidate resistance mutations for a particular TKI,7,8 it is the lack of substantial and durable responses in
patients who present with particular BCR-ABL1 mutations (primary
resistance), coupled with the frequent emergence of those mutations at
the time of treatment failure (secondary resistance), that ultimately
define such vulnerabilities.9,10
Historically, Sanger sequencing (SS) has been used clinically to
identify BCR-ABL1 mutations associated with TKI resistance. However, SS is unable to detect mutations present in less than 10% to 20% of
Submitted August 7, 2015; accepted November 10, 2015. Prepublished online
as Blood First Edition paper, November 24, 2015; DOI 10.1182/blood-201508-660977.
The publication costs of this article were defrayed in part by page charge
payment. Therefore, and solely to indicate this fact, this article is hereby
marked “advertisement” in accordance with 18 USC section 1734.
The online version of this article contains a data supplement.
There is an Inside Blood Commentary on this article in this issue.
BLOOD, 11 FEBRUARY 2016 x VOLUME 127, NUMBER 6
© 2016 by The American Society of Hematology
703
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704
BLOOD, 11 FEBRUARY 2016 x VOLUME 127, NUMBER 6
DEININGER et al
cells and does not allow direct detection of compound mutations,
although their presence can be inferred in certain cases (ie, when $2
mutations are detected at a combined frequency .100%). Indeed, the
use of more sensitive techniques such as mass spectrometry11 or
next-generation sequencing (NGS)12 has demonstrated that mutations
present at levels below the detection limit of SS (ie, low-level [LL]
mutations) can also affect clinical outcomes. For example, mutations
that confer resistance to nilotinib or dasatinib have been associated with
poor outcomes in patients even when present at low levels.11 In addition,
the presence of multiple LL mutations, regardless of their association
with TKI resistance, has been shown to identify patients who respond
relatively poorly to treatment with dasatinib or nilotinib.13 Importantly,
NGS also enables direct detection of BCR-ABL1 compound mutations.
However, it has recently been shown that polymerase chain reaction
(PCR)-mediated recombination may cause compound mutation frequencies to be overestimated when PCR amplicons are used for NGS.14
Thus, the ability of NGS to reliably detect compound mutations, and
therefore their prevalence in CML, remains to be established.
Ponatinib is a third-generation BCR-ABL1 TKI shown to potently
inhibit native BCR-ABL1 as well as all single mutants associated with
resistance to imatinib and second-generation TKIs, including T315I,
in preclinical models.8 However, certain compound mutations, in
particular those including T315I, confer resistance to clinically
achievable ponatinib concentrations.5,8 In phase 115 and phase 2
(PACE16) trials, ponatinib had significant antileukemic activity in
heavily pretreated patients with CML (chronic phase [CP], accelerated
phase [AP], or blast phase [BP]) or Philadelphia chromosome–positive
acute lymphoblastic leukemia (Ph1 ALL), .90% of whom had
previously received at least 2 TKIs. Consistent with its preclinical
profile, ponatinib demonstrated activity in patients with or without
BCR-ABL1 mutation (including T315I) present at baseline, as
determined by SS. In addition, analysis of mutations in a subset of
patients in the PACE trial who discontinued therapy (after a 15-month
median follow-up) failed to identify any single mutations consistently
associated with secondary resistance to ponatinib.16 However, in this
and a separate study,5 certain compound mutations were associated
with secondary resistance, though these were seen predominantly in
patients with advanced CML or Ph1 ALL.
The goals of the current study were to characterize the prevalence
and nature of LL and compound BCR-ABL1 mutations in heavily
pretreated CP-CML patients, and to rigorously explore the relationship
between BCR-ABL1 mutation status and primary and secondary
ponatinib resistance. To achieve this, we used an optimized NGS
analysis to determine the baseline mutation status of all 267 CP-CML
patients in the PACE trial (median follow-up, 30.1 months) and
performed postbaseline SS on all patients, except those who remained
on the trial in continuous response.
BCR-ABL1 mutation analysis was conducted on blood samples from all
patients at baseline using SS and NGS (Figure 1). Postbaseline analysis using SS
was attempted on all patients except those who achieved MCyR and remained on
trial in continuous MCyR.
Sequencing: Sanger sequencing and
next-generation sequencing
SS was conducted as previously described.16 In postbaseline samples, the
presence of a compound mutation was inferred by the detection of 2 (or more)
mutations with a combined frequency .100% (eg, 100% T315I and 30%
F317V).
NGS encompassed the following steps: BCR-ABL1 real-time PCR, random
fragmentation of PCR product and size selection of ;400 base-pair (bp)-sized
fragments, sequencing library construction, NGS using IonTorrent PGM (read
lengths up to 400 bp). The average sequence read depth for all detected variants
was 11 831 reads/base (range, 1000-25 028); variants detected at $1% were
analyzed. Bases with Phred Score ,25 were considered quality control failures
and designated as unknown.
A total of 419 sequence variants were detected in ABL1 (amino acids
27-540). All analyses described here focused on the 266 missense mutations
detected in the BCR-ABL1 KD (amino acids 237-507). Missense mutations
outside the KD were excluded (N 5 55) (supplemental Table 1 available on the
Blood Web site), as were all polymorphisms (N 5 3), deletions (N 5 5),
frameshift (N 5 12), nonsense (N 5 18), and synonymous (N 5 60) mutations
because these are not considered relevant for TKI resistance.17-19
In samples in which multiple BCR-ABL1 missense mutations were detected,
the phase (whether present on the same or different alleles) and frequency of all
possible mutation combinations were calculated as described in the supplemental
Methods.
Identification of false-positive compound mutants
Detailed explanation of the methodology used to eliminate putative false-positive
compound mutations is described in the supplemental Methods. In brief, by
mixing 2 RNA samples from patients, each positive for a distinct single mutation,
a “recombination rate” was calculated based on the frequency at which false
compound mutants were generated. A compound mutation was considered truepositive if the observed frequency was above the expected false compound
mutation frequency.
Kaplan-Meier estimation of maintenance of response
Duration of MCyR, CCyR, MMR, progression-free (PFS), and overall survival
(OS) was estimated using the Kaplan-Meier method. Patients without
documented loss of response were censored at their last response assessment.
Loss of MCyR and CCyR was confirmed by a second consecutive assessment.
Results
Baseline BCR-ABL1 mutation status determined by NGS
Methods
PACE trial and sample analysis
The trial design has been previously described.16 In brief, 449 patients with CML
(any phase) or Ph1 ALL with resistance or intolerance to dasatinib or nilotinib, or
with a T315I mutation, were treated with ponatinib (initial dose of 45 mg once
daily). The analysis described here was limited to the CP-CML patients (N 5 267).
The primary end point was major cytogenetic response (MCyR) by 12 months, and
secondary end points included complete cytogenetic response (CCyR) and major
molecular response (MMR) at any time. The data evaluated are as of January 6,
2014; the median follow-up was 30.1 months (range, 0.1-39.3). Patients’ mutation
history and prior TKI exposure were collected at enrollment. Patient-recorded daily
ponatinib dosing information was also collected.
The baseline BCR-ABL1 mutation status of all 267 CP-CML patients
in the PACE trial has previously been assessed by SS, with 161
mutations detected in 131 patients (49%).16 To allow detection of LL
and compound BCR-ABL1 mutations, the baseline mutation status of
all 267 patients was reassessed by NGS (Figure 1). In total, 266
mutations were detected by NGS in 163 patients (61%) (supplemental
Table 2 and Figure 2A). NGS identified all 161 mutations detected by
SS and 105 additional mutations (ie, LL mutations, which were
detected at frequencies ranging from 1%-11%), in 73 patients (27%).
Notably, approximately one-third of the LL mutations (34/105; 32%)
occurred at amino acids that have not previously been implicated in
resistance to imatinib, dasatinib, or nilotinib (supplemental Table 2).4
Twelve percent (32/267) of patients had LL mutations only
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BLOOD, 11 FEBRUARY 2016 x VOLUME 127, NUMBER 6
COMPOUND MUTANTS AND PONATINIB SENSITIVITY IN CML
705
Figure 1. Schematic representation of the BCR-ABL1
mutation analyses performed in this study. BCRABL1 mutation analysis (Sanger sequencing [SS] and
next-generation sequencing [NGS]) was conducted on
baseline samples from all chronic-phase chronic myeloid
leukemia (CP-CML) patients in the PACE phase 2 study.
Postbaseline analysis (SS) was conducted on any
patient who did not achieve major cytogenetic response
(MCyR) by 1 year, achieved and then lost MCyR, or
achieved MCyR and discontinued while in MCyR.
*Postbaseline (post-BL) analysis was not conducted on
patients who achieved MCyR and remain on study in
continuous MCyR. ^36 patients were not evaluable: 12
did not have a post-BL sample collected, and BCR-ABL1
could not be amplified for sequencing in the remaining
24; 20 of these were associated with low (,1%) BCRABL1 transcript levels. Of the 129 patients with evaluable
post-BL samples, 102 were collected from patients who
did not achieve MCyR by 1 year, 14 from patients
who achieved and lost MCyR, and 13 from patients who
achieved MCyR and discontinued study while in MCyR.
(Figure 2A), whereas 15% (41/267) of patients had $1 high-level
mutations (detected by SS) in addition to LL mutations. Overall, 23%
(62/267) of patients had .1 mutation detected by NGS (Figure 2A).
Thus, consistent with the greater sensitivity of NGS, the percentage of
patients with no detectable BCR-ABL1 mutations by NGS was lower
than that observed by SS (39% vs 51%), and the percentage of patients
with multiple mutations was higher (23% vs 10%) (Figure 2A).
Assessment of compound mutant frequency with an optimized
NGS analysis
In previous studies using NGS and other techniques, as much as 70%
of CML patients with multiple mutations were reported to have
compound mutations.12,20 Somewhat surprisingly, in 60% to 80% of
cases with compound mutations, both individual mutants were also
detected (eg, E255K/T315I detected on the same allele and both E255K
and T315I detected on separate alleles). Noting the difficultly in
explaining such complexity phylogenetically, Parker et al14 reported
the detection of false-positive compound mutations by SS on mixtures
of 2 plasmids or cDNAs that each contained a single mutation. To
confirm these results and explore strategies to overcome limitations of
sequencing protocols when using amplicons, we performed NGS
analysis on mixtures of 2 patient-derived RNA samples positive for
M244V and F359V to recapitulate all steps of the NGS protocol
(supplemental Methods and supplemental Tables 3 and 4). Analyzed
individually, each sample only had 1 mutation detected at high
prevalence (.95%). However, when the 2 samples, whose mutations
were 345 bp apart, were mixed, false-positive compound mutations
Figure 2. Distribution, response rates, and duration
of response according to BCR-ABL1 mutation status
at baseline as determined by SS or NGS. (A)
Percentage of patients with no mutation (0), one
mutation (1), or 2 or more mutations ($2) at baseline,
as determined by SS or NGS. Also shown are subsets
of these groups (separated by a dotted line) that had LL
mutations only, or compound mutations, as determined
by NGS. (B) MCyR rates, (C) MMR rates, and (D)
Kaplan-Meier curves indicating the duration of continuous MCyR, according to baseline mutation status. The
median follow-up after achievement of MCyR was 27.4
months, with the majority of patients (.80%) remaining
in follow-up. BL, baseline; C, compound; LL, low-level;
MCyR, major cytogenetic response; MMR, major molecular response.
N
N
N
N
N
N§
N{
N
Y
Y
Y
Y
191
264
49
263
149
249
242
35
125
262
247
115
83
848
594
298
224
10141
10071
9241
8401
245
226
27
11
8
IDN
I
I (D N) B
IND
IDN
IDN
IDN
IDN
ID
IDN
IND
ID
IDN
Prior TKI*
%†
40
T315I
100
100
E459K
100
F317L
T315I
20
100
F359V
G250E
100
V299L
100
100
10
E279K
V299L
80
H396R
100
F317L
40
H396R
50
T315I
F317L
100
80
F317L
Y253H
100
F359C
90
90
T315I
F359V
100
100
100
F359C
H396R
V379I
Mutation
Baseline SS
1
F359V/E255K
1
T315I/R386M
F317L
2
95
2
F317L/E459K
95
T315I
1
T315I/I432M
27
G250E
97
97
G250E/E279V
F359V/V299L
H396R/V299L
2
1
F317L
20
75
1
F317L/E281K
F317L/E279K
F317L/H396R
T315I
34
1
G250E/F317L
T315I/F317L
94
3
21
Y253H
F359C
F359C/T315I
73
3
F359C/F317L
82
T315I
9
F359V
12
F359C
76
F359C/V299L
F359C/T315I
5
H396R/V299L
3
V379I/E459K
88
3
V379I/H396P
H396R
90
%†
V379I
Mutation
Baseline NGS
MMR
MMR
CCyR
CCyR
None
MMR
CCyR‖
None
None
None
None
None
None
Best
response‡
583
167
175
83
N/A
259
506
N/A
N/A
N/A
N/A
N/A
N/A
Time to
response (d)
82
433
78
1#
N/A
6641
4171
N/A
N/A
N/A
N/A
N/A
N/A
Response
duration (d)
32
45
43
26
27
26
43
13
31
35
45
45
45
PADD (mg)
Withdrawal
Adverse event
Death
Progressive disease
Ongoing
Ongoing
Ongoing
Ongoing
Adverse event
Progressive disease
Progressive disease
Progressive disease
Adverse event
Reason discontinued
%†
100
EOT
Time
10
100
100
100
100
EOT
1y
1y
1y
1y
EOT
EOT
Not evaluable (no amplification)††
Not evaluable (no amplification)††
Not evaluable (no amplification)††
F359V
Y253H
No mutation detected
(no amplification)††
Not evaluable
(no amplification)††
Not evaluable
Y253H
F359C
T315I
sample)
Not evaluable (no postbaseline
sample)
Not evaluable (o postbaseline
V379I
Mutation
Postbaseline SS
DEININGER et al
B, bosutinib; CCyR, complete cytogenetic response; D, dasatinib; EOT, end of treatment; I, imatinib; MMR, major molecular response; N, nilotinib; N/A, not applicable; PADD, ponatinib average daily dose; PCyR, partial cytogenetic
response; Pt, patient; TKI, tyrosine kinase inhibitor.
Patients who did not achieve the primary end point are listed first, with each subgroup then arranged by time on study. Bold font denotes exact or alternate (also underlined) substitutions at amino acids previously associated with TKI
resistance4; nonbolded font denotes substitutions at amino acids not previously associated with TKI resistance.
*The order of TKIs from left to right indicates the sequence of TKI treatment in the patient’s history (first to last); parentheses indicate that the treatment order is unknown.
†The percentage indicates the mutation allele frequency in the patient sample.
‡Best response does not include hematologic response.
§Cytogenetic response for this patient was only assessed at 9 months (no MCyR) and 18 months (CCyR).
{In this patient, MMR was achieved at 9 months; however, cytogenetic response was not assessed between months 3 and 12; SS was conducted at 1 year and no amplification of the transcript was achieved.
‖Patients with atypical transcripts in whom molecular response could not be assessed.
#1-day duration indicates patient had one assessment at which criteria for response were met and no further evaluable cytogenetic assessments.
**Postbaseline mutation status was not evaluated for patients who achieved and remain on study in continuous MCyR.
††Patients are in CCyR and/or ,1% BCR-ABL1 transcript levels at the time of sample analysis.
N
Pt ID
Time on
study (d)
706
Achieved
primary
end point
Table 1. Clinical and molecular characteristics of the 25 PACE CP-CML patients harboring compound mutations at baseline
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BLOOD, 11 FEBRUARY 2016 x VOLUME 127, NUMBER 6
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Pt ID
101
7
185
228
76
234
113
112
89
36
159
10
11191
10551
10121
9921
9671
9531
9511
9441
9371
9281
903
875
Time on
study (d)
IN
(D N) I
IN
INB
I
IDN
IDN
I (D N) B
ID
IDN
IND
ID
Prior TKI*
%†
Y253H
100
100
100
E459K
T315I
100
20
T315I
F317L
90
F359V
100
100
T315I
100
F317L
100
100
E459K
F359V
M244V
100
10
G250E
V299L
90
90
E450G
F359I
100
100
F317L
T315I
Mutation
Baseline SS
3
3
2
F359V
M244V/
1
T315I/K262N
96
1
1
1
Y253H
Y253H/E282D
Y456C
3
Y253H/A287V
65
T315I
T315I/M237V
98
2
E459K/F317L
2
F359V/R362T
29
F359V/M237R
T315I
60
1
F359V
92
T315I
C475W
99
1
F317L/E459K
91
F359V
F359V/M244V
G250W
89
M244V
2
V299L/P310L
1
95
V299L
1
E507G
2
84
6
G250E
F359I/T277I
F359I
F359V
92
T315I/E275D
F317L/E450G
93
%†
T315I
Mutation
Baseline NGS
MMR
MMR
MMR
MMR
MMR
MMR
MMR
MMR
MMR
MMR
PCyR
CCyR‖
Best
response‡
83
249
168
84
81
87
336
85
55
524
252
168
Time to
response (d)
10161
7591
7621
8321
8421
8451
5761
8411
7851
3221
170
587
Response
duration (d)
18
16
42
31
44
45
28
16
44
43
5
29
PADD (mg)
Ongoing
Ongoing
Ongoing
Ongoing
Ongoing
Ongoing
Ongoing
Ongoing
Ongoing
Ongoing
Withdrawal
Adverse event
Reason discontinued
%†
Time
N/A**
N/A**
N/A**
N/A**
N/A**
N/A**
N/A**
N/A**
N/A**
N/A**
F317L/ E450G
10
EOT
Not evaluable (no amplification)††
Mutation
Postbaseline SS
BLOOD, 11 FEBRUARY 2016 x VOLUME 127, NUMBER 6
B, bosutinib; CCyR, complete cytogenetic response; D, dasatinib; EOT, end of treatment; I, imatinib; MMR, major molecular response; N, nilotinib; N/A, not applicable; PADD, ponatinib average daily dose; PCyR, partial cytogenetic
response; Pt, patient; TKI, tyrosine kinase inhibitor.
Patients who did not achieve the primary end point are listed first, with each subgroup then arranged by time on study. Bold font denotes exact or alternate (also underlined) substitutions at amino acids previously associated with TKI
resistance4; nonbolded font denotes substitutions at amino acids not previously associated with TKI resistance.
*The order of TKIs from left to right indicates the sequence of TKI treatment in the patient’s history (first to last); parentheses indicate that the treatment order is unknown.
†The percentage indicates the mutation allele frequency in the patient sample.
‡Best response does not include hematologic response.
§Cytogenetic response for this patient was only assessed at 9 months (no MCyR) and 18 months (CCyR).
{In this patient, MMR was achieved at 9 months; however, cytogenetic response was not assessed between months 3 and 12; SS was conducted at 1 year and no amplification of the transcript was achieved.
‖Patients with atypical transcripts in whom molecular response could not be assessed.
#1-day duration indicates patient had one assessment at which criteria for response were met and no further evaluable cytogenetic assessments.
**Postbaseline mutation status was not evaluated for patients who achieved and remain on study in continuous MCyR.
††Patients are in CCyR and/or ,1% BCR-ABL1 transcript levels at the time of sample analysis.
Achieved
primary
end point
Table 1. (continued)
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COMPOUND MUTANTS AND PONATINIB SENSITIVITY IN CML
707
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708
DEININGER et al
were detected in ;11% of the reads. Although several potential
mechanisms may contribute (supplemental Figure 1), we were able to
estimate a “recombination rate” of ;15% per 100 bp, which explains
the observed 11% frequency of false-positive compound mutations
(supplemental Methods and supplemental Table 3).
To correct for this artifact, the percentage of false-positive compound
mutations (the product of individual mutation frequencies and the
recombination rate) was estimated for 23 patients with a total of 26
“suspect” compound mutations (ie, a compound mutation and both single
component mutations were detected) (supplemental Methods). Using this
approach, 88% (23/26) of suspect compound mutants were identified as
probable false-positives and were excluded, leaving a total of 33 “true”
compound mutants (59% [33/56] of those originally called) in 25 patients
(supplemental Table 4). Thus, 9% (25/267) of patients overall (Figure 2A
and Table 1), and 42% (25/62) of patients with multiple mutations, were
found to have true compound mutations at baseline.
Compound mutations were highly heterogeneous. Only 3 compound
mutations were observed in multiple patients: F317L/E459K in 3
patients, V299L/H396R in 2 patients, and T315I/F359C in 2 patients
(Table 1). Compound mutations invariably involved at least 1 mutation
previously implicated in TKI resistance (Table 1). Notably, T315Iinclusive compound mutations were observed in only 3% of all patients
(7/267) despite enrichment for T315I-positive patients in the PACE trial.
In summary, whereas a majority (61%) of PACE CP-CML patients had at
least 1 BCR-ABL1 mutation detected by NGS at baseline, and 23% had
.1 mutation, only 9% had compound mutations.
Robust and durable major cytogenetic responses to ponatinib
irrespective of baseline mutation status
Previous analyses based on SS revealed high response rates in PACE
CP-CML patients, irrespective of the number of BCR-ABL1 mutations
detected at baseline (ie, 0, 1, or $2), with MCyR rates by 1 year ranging
from 49% to 64% and MMR rates at any time ranging from 29% to 48%
(Figure 2B-C).16 Response rates in patients with 0, 1, or $2
BCR-ABL1 mutations at baseline based on NGS were similar to those
based on SS (50%-61% MCyR by 1 year and 29%-45% MMR at any
time; Figure 2B-C). Moreover, response rates in patients with LL
mutants only (43% MCyR and 31% MMR) were similar to those with
no mutations, and response rates in patients with compound mutations
(64% MCyR and 52% MMR) (Figure 2B-C) were similar to those in
patients with $1 mutations. Importantly, responses were durable
regardless of baseline mutation status (Figure 2D and supplemental
Figure 2). Among patients who achieved MCyR or MMR, the
estimated rates of a sustained response of at least 2 years were 87% and
65%, respectively, for the total population, and 90% and 92% for
patients with compound mutations. Baseline mutation status also had
no significant impact on rates of CCyR, PFS, or OS, estimated to be
79%, 68%, and 86%, respectively, for the total population at 2 years
(supplemental Figure 2). In summary, robust and durable responses
were observed irrespective of baseline NGS mutation status, including
in patients with compound mutants.
No single or compound mutant as a major driver of primary
resistance to ponatinib
To further investigate the relationship between BCR-ABL1 mutation
status and sensitivity to ponatinib, we evaluated responses according to
the specific single or compound mutation present at baseline as
determined by NGS. Evidence of efficacy was observed against all 20
single mutants present in at least 2 patients (Table 2). Ten mutations
were present in at least 5 patients at baseline. Response rates were high
(20%-68% MCyR and 25%-54% MMR) in patients with H396R,
BLOOD, 11 FEBRUARY 2016 x VOLUME 127, NUMBER 6
V299L, E459K, F317L, F359V, M244V, E255K, G250E, and T315I.
Response rates were lower in patients with F359C (14% MCyR and 0%
MMR). Of the 7 patients with F359C present at baseline, 6 had a
postbaseline sample available for analysis. In 2 patients, F359C was still
detectable by SS, whereas in 4 it was not (data not shown), indicating
that the mutant clone had not expanded during therapy. Although
patient numbers were small, response rates were relatively low in
patients with E255V, the mutation with the lowest in vitro sensitivity to
ponatinib of those tested (IC50 5 16 nM; 1/4 and 0/4 achieved MCyR
and MMR, respectively). Overall, no association between response
rates and IC50 was observed (Table 2).
Of the 25 patients found to harbor compound mutations at baseline,
16 achieved MCyR by 1 year (Table 1). Of the 9 patients who did not, 1
with 3 compound mutations (patient 35; F317L/H396R, F317L/E279K,
and F317L/E281K) achieved MMR at 9 months but did not have a
cytogenetic assessment between months 3 and 12, and 1 with a T315I/
F317L compound mutation (patient 242) subsequently achieved CCyR
and remains on study. Of the remaining 7 patients, 5 did not have
compound mutations detected by SS in postbaseline samples, indicating
that these compound mutants did not expand during therapy (Table 1,
patients 191, 263, 149, 249, and 125); the remaining 2 could not be
evaluated. In summary, with the possible exception of F359C, no recurrent
single or compound mutation conferring primary resistance to ponatinib
was detected at baseline in the 267 CP-CML patients from the PACE trial.
No single or compound mutant as a major driver of secondary
resistance to ponatinib
To evaluate whether single or compound mutants undetectable by NGS
at baseline emerged and expanded during ponatinib therapy, postbaseline
samples were analyzed by SS. Postbaseline sequencing was attempted
on all treated patients, except those who achieved MCyR by 1 year and
remain on the trial in continuous MCyR (Figure 1). In total, postbaseline
samples from 129 patients were evaluable. Of these patients, 24 had
ceased ponatinib for at least 1 month before the postbaseline mutation
analysis (34-239 days). Previous studies have demonstrated that some
mutants can be rapidly deselected in the absence of kinase inhibition.21-23
Therefore, some mutations may have become undetectable by SS at the
time of the postbaseline analysis. Of the 129 patients evaluated, 8
harbored mutations that were not detected at baseline by NGS (Table 3).
E255V was detected in 1 patient at the time of MCyR loss (patient 248)
and was associated with a low average daily dose of ponatinib (13 mg).
T315I was detected in 3 patients: in 2 at the time of MCyR loss (patients
29 and 155) and in 1 after 1 year of treatment in a patient (patient 121)
with a low average daily dose of ponatinib (6 mg) and a history of T315I
before commencing ponatinib. Finally, compound mutations were
detected at the end of treatment in 4 patients—Y253H/F359V in 2
patients, and T315I/M351T and T315I/F359V each in 1 patient (in each
case 1 mutation was detected at a frequency of 100% and the second was
detected at 40% to 100%, using SS). Notably, there was no evidence of
F359C being newly acquired in any patient. Thus, at a median follow-up
of 30.1 months, emergence of previously undetected single and compound mutants during ponatinib therapy is a rare event.
Discussion
BCR-ABL1 KD mutations are a major mechanism of CML resistance
to TKIs, and the BCR-ABL1 genotype is used to rationalize selection of
salvage therapy after first-line TKI failure.4 KD mutants present at low
levels in specimens obtained before switching (“switch samples”) can
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BLOOD, 11 FEBRUARY 2016 x VOLUME 127, NUMBER 6
COMPOUND MUTANTS AND PONATINIB SENSITIVITY IN CML
709
Table 2. Response rates according to baseline BCR-ABL1 mutation for the 20 mutants detected by NGS in at least 2 patients
Unique NGS
baseline
mutations
Patients (NGS)
Patients (SS)
Patients (LL)
IC50 (nM)*
T315I
74
64
10
6
MCyR rate (%)†
50/74 (68)
MMR rate (%)†
40/74 (54)
F317L
29
22
7
4
13/29 (45)
12/29 (41)
11/20 (55)
F359V
20
13
7
4
E255K
13
8
5
6
8/13 (61.5)
6/13 (46)
8/20 (40)
G250E
12
8
4
5
8/12 (67)
4/12 (33)
M244V
9
5
4
3
4/9 (56)
3/9 (33)
V299L
8
5
3
4
3/8 (37.5)
2/8 (25)
F359C
7
4
3
6
1/7 (14)
0/7 (0)
E459K
7
3
4
5
3/7 (43)
3/7 (43)
H396R
5
5
0
4
1/5 (20)‡
2/5 (40)
E255V
4
2
2
16
1/4
F359I
4
4
0
11
3/4
1/4
L248V
2
2
0
4
1/2
1/2
Q252H
2
1
1
ND
2/2
2/2
Y253H
2
2
0
5
1/2
1/2
E279K
2
1
1
ND
0/2§
1/2
M351T
2
1
1
4
1/2
1/2
E355A
2
2
0
7
1/2
1/2
H396P
2
1
1
ND
1/2
0/2
M437I
2
0
2
ND
1/2
1/2
0/4
ND, not determined.
The mutations are sorted by number of patients with the corresponding mutant as detected by NGS, and then by ascending amino acid sequence.
*IC50 values are based on ponatinib potency in Ba/F3 cells harboring the corresponding BCR-ABL1 mutation.29
†MCyR rate by 12 months or MMR rate at any time is reported based on patients with mutations detected by NGS in this study; percent of MCyR or MMR is shown when
the number of evaluable patients was $5. Bold font denotes exact substitutions at amino acids previously associated with TKI resistance4; nonbolded font denotes a
substitution at an amino acid not previously associated with TKI resistance.
‡One patient achieved MMR at 12 months but did not have documented evidence of MCyR by 12 months because CyR was not assessed between 3 and 12 months;
therefore, this patient was not included as achieving MCyR.
§In one patient, MMR was achieved at 9 months; however, cytogenetic response was not assessed between months 3 and 12.
be selected on therapy if not covered by the second-generation TKI
chosen for salvage therapy.11 In contrast, we show that the genotype of
the switch sample, whether assessed by SS or NGS, has no impact on
cytogenetic or molecular responses of CP-CML patients treated with
ponatinib. These data are consistent with the in vitro profile of ponatinib
that predicts activity against all single mutants previously associated
with resistance to imatinib, including the T315I, F317L, Y253H,
F359V/C, E255K/V, and V299L mutants associated with resistance to
second-generation TKIs.2,5 Response rates were notably lower in the
7 patients with the F359C mutation. Based on its in vitro ponatinib IC50
of 6 nM, F359C should be responsive to the ;64 nM trough plasma
concentrations achieved with the 45-mg daily initial ponatinib dose
used in the PACE study.15 The fact that F359C was undetectable in 4 of
6 patients with evaluable follow-up samples argues against a causative
role of F359C for resistance, although deselection remains possible in
1 patient whose last dose of ponatinib predated the mutation analysis
sample by 85 days. Responses were generally durable regardless of
baseline mutation status: overall 87%, 79%, and 66% of patients were
estimated to remain in MCyR, CCyR, and MMR, respectively, for
2 years, with similar durations observed across the various subgroups
(compound, LL only, 0, 1, $2 mutants). Consistent with this, baseline
mutation status had no impact on PFS and OS.
BCR-ABL1 compound mutations, particularly those including
T315I, have recently been shown to confer high-level resistance against
all approved TKIs, including ponatinib, raising the question of whether
preexisting compound mutants may be selected on therapy.5 Although
SS cannot distinguish between polyclonal and compound mutations,
unless the combined mutant allele burden clearly exceeds 100%, NGS
identifies compound mutations as long as the average read length
exceeds the distance between the 2 single nucleotide variations. With an
average read length of 400 bp, our assay is expected to detect the vast
majority of compound mutations, although some long-distance combinations may be missed. Recent reports have drawn attention to
“recombination” events that can cause false-positive compound
mutation calls.14 In our analysis, a substantial percentage of compound
mutation calls appear to be false-positives (41%; 23/56), which
increased to 88% (23/26) when a compound mutation and both single
mutations were detected in the same sample. The precise mechanisms
responsible for these recombination events remain to be defined
(supplemental Figure 1). Based on a direct measurement of a falsepositive “recombination rate” between 2 mutations separated by a
known distance, we developed a simple algorithm that predicts the
frequency with which false-positive compound mutants are observed
with remarkable precision (supplemental Methods). Still, after
eliminating all suspect compound mutation calls, we observed no
correlation between compound mutation detection at baseline and
cytogenetic and molecular response to ponatinib. This surprising result
may be explained by the very low frequency of compound mutations
containing T315I, and compound mutants without T315I remaining
sensitive to ponatinib.5 Conversely, in the 4 patients with compound
mutations at end of treatment, NGS failed to detect the respective
compound mutations at baseline, suggesting they were below the
detection limit or were acquired on therapy.
Thus, our data show that the increased sensitivity of mutation
detection afforded by NGS is of no clinical utility in terms of predicting
response to ponatinib in patients with CP-CML. However, because
current guidelines3 suggest using ponatinib in patients with the T315I
mutation, and low-level detection of T315I is associated with failure of
dasatinib and nilotinib, it is conceivable that NGS, similarly to targeted
approaches such as mass spectrometry,11,13 may identify patients likely
to benefit from ponatinib. Importantly, relapse rates were much higher
in PACE patients with AP-CML or BP-CML compared with CP-CML,
From www.bloodjournal.org by guest on June 16, 2017. For personal use only.
BLOOD, 11 FEBRUARY 2016 x VOLUME 127, NUMBER 6
B, bosutinib; D, dasatinib; EOT, end of treatment; I, imatinib; N, nilotinib; N/A, not applicable.
Mutations detected postbaseline that were not detected at baseline are bolded; if such mutations had been detected before baseline (in the patient’s history), they are also underlined.
*The order of TKIs from left to right indicates the sequence of TKI treatment in the patient’s history (first to last).
†IC50 values are based on in vitro ponatinib activity against cells harboring bolded BCR-ABL1 mutations.29
‡Patient achieved primary end point; however, loss of response before discontinuation was not documented.
Compound mutant at EOT
Compound mutant at EOT
45
30
5
27
EOT
EOT
Adverse event
Other
N/A
N‡
Y
N
T315I/M351T (100%/40%)
T315I/F359V (100%/90%)
None
ID
167
T315I (41%)
B
142
M244V (4%)
Compound mutant at EOT
Compound mutant at EOT
34
26
5
5
EOT
Other
N/A
N
Y253H/F359V (100%/100%)
IDN
158
F359V (93%)
7
EOT
Progressive disease
N‡
Y
E355A (10%)
Y253H/F359V (100%/100%)
IDN
262
V299L/F359V (97%)
Possible BCR-ABL–independent resistance
Low drug exposure and re-emergence of T315I
6
6
1y
On study
N/A
N
T315I (10%)
IN
Comment
Possible BCR-ABL–independent resistance
121
E355A (81%)
44
43
6
6
EOT
EOT
Progressive disease
Withdrawal by subject
Y
Y
Y
Y
T315I (100%)
T315I (100%)
ID
155
None
IN
29
None
13
16
EOT
Adverse event
Y
Y
E255V (100%)
G250E (1%)
IDN
Postbaseline SS (frequency)
Prior TKI*
Patient ID
248
Average daily
dose (mg)
IC50 (nM)†
Sample
analysis at
Reason for discontinuation
Lost
primary
end point
Achieved
primary
end point
Baseline NGS
(frequency)
Low drug exposure
DEININGER et al
Table 3. Clinical and molecular characteristics of patients with postbaseline mutations that were not identified by NGS at baseline
710
and compound mutations were detected by SS in many AP- and BPCML patients at the time of discontinuation,5 suggesting that NGS may
have predictive utility in these more advanced cases.
Although 161 mutations detected by SS occurred at amino acids
previously implicated in TKI resistance, 34 of the 105 LL mutations
(31%) occurred at amino acids that were not.4 The fact that none of
these “novel” mutants was found to expand during ponatinib therapy to
become detectable by SS strongly suggests they are not relevant to
resistance. Similarly, responses to ponatinib were independent of the
number of BCR-ABL1 mutations detected at baseline by NGS (0, 1, or
$2; Figure 2B-C) and substantially exceeded the 26% MCyR and 3%
MMR rates observed in these patients on their prior line of therapy.16
Interestingly, response rates in patients with LL mutants only (43% and
31% MCyR by 1 year and MMR at any time, respectively) were more
similar to those with no mutations (50% and 29%) than to patients with
compound mutations (64% and 52%) or with $1 mutations (57%-64%
and 44%-48%). We speculate that the relatively low response rates in
patients with no BCR-ABL1 mutations may reflect the presence of
BCR-ABL1–independent mechanisms of resistance, and that this may
also be the case in patients with exclusively LL mutations. BCRABL1–independent resistance may also account for the fact that ;30%
of T315I patients did not respond to ponatinib, although this mutant
should be sensitive to ponatinib plasma concentrations expected with
45-mg daily dosing. Low drug exposure as a result of pharmacokinetic
factors and/or dose reductions caused by adverse events may be contributing factors. Inducing rapid and deep reductions in disease burden
may ultimately reduce the probability of randomly acquiring additional
resistance mutations and achieve better outcomes.24-26
Acquired resistance to imatinib and second-generation TKIs is
frequently associated with the emergence of new BCR-ABL1 mutations. We analyzed postbaseline samples from 129 patients (all except
those who remain on trial in continuous MCyR at a median follow-up of
30.1 months). In total, 8 of 129 patients were found to have mutations
that were not detectable at baseline: 3 of 102 who did not achieve
MCyR by 1 year, 3 of 14 who achieved and lost MCyR, and 2 of 13 who
achieved MCyR and discontinued study while in MCyR. In 4 of the
patients, a single mutation emerged (T315I in 3 patients and E255V in 1
patient with relatively low exposure to ponatinib [average dose 5 13
mg]). High response rates were seen in patients with T315I (MCyR
68%), suggesting that BCR-ABL1 kinase–independent mechanisms
are responsible or at least contributed to resistance in the T315I cases.
Although the number of patients with E255V at baseline was small, the
response rate was low (MCyR 25%), which suggests that E255V may
contribute more directly to ponatinib resistance. However, 2 of 2
patients with AP-CML in the PACE study who had an E255V mutation
at baseline achieved MCyR,16 demonstrating that E255V does not
confer uniform resistance to ponatinib. Compound mutants emerged in
the remaining 4 patients. Y253H/F359V was observed in 2 patients
previously successively treated with imatinib, dasatinib, and nilotinib,
and T315I/M351T and T315I/F359V were observed in 1 patient each.
T315I/M351T and T315I/F359V confer significant resistance to
ponatinib (IC50 ;100 nM), consistent with a causal role for clinical
resistance in these patients, whereas Y253H/F359V has thus far not
been characterized.5 Overall, however, mutational escape is rare in CPCML patients treated with ponatinib, despite the fact that many of these
patients were pretreated with multiple TKIs. In contrast, certain
compound mutants have been found to emerge frequently in BP-CML
and Ph1 ALL patients treated with ponatinib or other TKIs.5,16
Compound mutants that arise through sequential mutation of a single
amino acid residue have also been observed (eg, T315M, which can
arise from a T315-I315, followed by an I315-M315 mutation event).5
Higher levels of genomic instability and greater clonal diversity are
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BLOOD, 11 FEBRUARY 2016 x VOLUME 127, NUMBER 6
likely to contribute to the higher frequency of compound mutations in
advanced BCR-ABL1–positive leukemias. In our study, NGS was
limited to the baseline samples, and it is unknown whether ponatinib
exposure changes the frequency and spectrum of LL mutations—for
example, by improving genetic stability through profound inhibition of
BCR-ABL1 kinase activity.
Ponatinib is the most potent BCR-ABL1 TKI, inducing rapid, deep,
and durable responses in CP-CML patients; however, ponatinib
treatment is associated with considerable cardiovascular toxicity that
may be dose-dependent—as described in a post hoc multivariate analysis
of the PACE trial.27 The results discussed in this study are based on the
analysis of patients treated with a starting dose of 45 mg ponatinib, though
the average daily dose (over the median follow-up of 30.1 months) was
only ;30 mg owing to dose reductions. As strategies to lower average
dose intensity are explored, it will be important to determine whether this
results in an increase in mutation-based resistance. Although preliminary
analyses of PACE patients suggest that responses are maintained in most
patients after dose reductions to 15 or 30 mg, more follow-up is required
to maximize the benefit/risk ratio in a rational manner.28
In summary, we show that pretherapeutic BCR-ABL1 mutation
profiles, whether obtained by SS or by NGS, have little impact on
ponatinib response, and no single or compound mutant has been
identified as a major driver of primary and secondary resistance to
ponatinib in CP-CML patients. The role of NGS in this setting may be to
identify patients with LL T315I who are unlikely to derive lasting benefit
from second-generation TKIs, but have a high likelihood of achieving
durable cytogenetic and molecular responses to ponatinib, an important
factor for balancing risks and benefits of salvage therapy selection.
Acknowledgments
The authors thank all the patients, their families and caregivers, the
site investigators, and research personnel for their participation in the
PACE trial. They acknowledge their colleagues at ARIAD Pharmaceuticals, Inc. and in the CML community for their contributions.
They also thank Thihan Padukkavidana (ARIAD Pharmaceuticals,
Inc.) for scientific writing and editorial assistance.
This study was funded by ARIAD Pharmaceuticals, Inc.
COMPOUND MUTANTS AND PONATINIB SENSITIVITY IN CML
711
Authorship
Contribution: M.W.D., S.B., V.M.R., and J.G.H. designed experiments, performed research, analyzed data, and wrote the manuscript;
S.L. performed statistical analyses and contributed to writing the
manuscript; N.P.S., J.E.C., D.-W.K., F.E.N., M.T., M.B., M.C.M.,
J.L., W.T.P., F.G., H.M.K., S.S., A.H., and T.P.H. analyzed data and
contributed to writing the manuscript; and T.C. and F.G.H. reviewed
the manuscript, provided critical feedback, and contributed to
writing the manuscript.
Conflict-of-interest disclosure: M.W.D. researched research
funding from BMS, Novartis, Celgene, Genzyme, and Gilead, and
is on the advisory board and consultant for BMS, ARIAD, Novartis,
Incyte, and Pfizer. J.G.H., S.L., T.C., F.G.H., and V.M.R. are
employees and hold equity ownership at ARIAD Pharmaceuticals
Inc. N.P.S. received research funding from ARIAD and BMS. J.E.C.
is a consultant for ARIAD, BMS, Novartis, and Pfizer, and received
research funding from ARIAD, BMS, Novartis, Pfizer, and Teva.
D.-W.K. is a consultant for BMS and Novartis, and received
honoraria and research funding from BMS, Novartis, and Wyeth.
F.E.N. is a consultant for Novartis, BMS, and ARIAD; received
research funding from Novartis; and received honoraria from
Novartis, BMS, and ARIAD. M.T. received research funding from
ARIAD, BMS, Sanofi, Incyte, and Pfizer. M.B. is a consultant for
ARIAD, Novartis, and BMS, and received honoraria and is on the
speakers bureau for ARIAD, BMS, Novartis, Pfizer, and Teva.
M.C.M. is a consultant to and received research funding and
honoraria from ARIAD, BMS, and Novartis. J.L. is an employee of
MolecularMD, Inc. F.G. received honoraria from ARIAD. H.M.K.
received research funding from ARIAD. S.S. is a consultant for
ARIAD, Novartis, and BMS. A.H. received honoraria and research
funding from ARIAD, Novartis, BMS, and Pfizer. T.P.H. and S.B.
received honoraria and research funding from ARIAD, Novartis,
BMS. W.T.P. declares no competing financial interests.
Correspondence: Michael Deininger, Division of Hematology
and Hematologic Malignancies, Huntsman Cancer Institute, The
University of Utah, Salt Lake City, UT 84112; e-mail: michael.
[email protected].
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From www.bloodjournal.org by guest on June 16, 2017. For personal use only.
2016 127: 703-712
doi:10.1182/blood-2015-08-660977 originally published
online November 24, 2015
Compound mutations in BCR-ABL1 are not major drivers of primary or
secondary resistance to ponatinib in CP-CML patients
Michael W. Deininger, J. Graeme Hodgson, Neil P. Shah, Jorge E. Cortes, Dong-Wook Kim, Franck
E. Nicolini, Moshe Talpaz, Michele Baccarani, Martin C. Müller, Jin Li, Wendy T. Parker, Stephanie
Lustgarten, Tim Clackson, Frank G. Haluska, Francois Guilhot, Hagop M. Kantarjian, Simona
Soverini, Andreas Hochhaus, Timothy P. Hughes, Victor M. Rivera and Susan Branford
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