H396R,
F359V and E255K mutations of the Abl
kinase
domain in imatinib-resistant Nigerian patients with
chronic myeloid leukemia
Dr
Anthony Oyekunle FMCPath.
1, Dr Rahman Bolarinwa
FMCPath.
1, Dr Temilola
Owojuyigbe
MBBS
1,
Dr Oche
Ogbe
FWACP
2,
Dr Lateef
Salawu
FMCPath.,
FWACP 1,
Prof
Julius Faluyi
PhD
3,
Prof Muheez
Durosinmi
FMCPath.,
FWACP.
1
1Department
of
Haematology and Blood Transfusion, Obafemi
Awolowo
University Teaching Hospitals, Ile-Ife, Nigeria.
2Department
of
Haematology, National Hospital, Abuja, Nigeria.
3Department
of
Botany, Obafemi
Awolowo
University, Ile-Ife, Nigeria.
Corresponding
Author: Dr.
Anthony A. Oyekunle, Department of Haematology, Obafemi
Awolowo
University, Ile-Ife, Osun
State, Nigeria; E-mail: oyekunleaa@yahoo.co.uk; Tel: +234 803 239 8360.
Afr
J Haematol Oncol
2010;1(3):79-83
ABSTRACT
AIM
We
have observed therapy failure in some Nigerian
patients with chronic myeloid leukemia (CML). We therefore set out to
determine
the causes of imatinib resistance in these patients.
METHODS
From
August 2003 to July 2010, we registered and
commenced 266 consenting CML patients on imatinib (IM) under the Glivec
International Patient Assistance Program (GIPAP). We investigated the
occurrence of Abl
kinase
domain mutations (KDM) among a cohort of these patients, with features
of
imatinib resistance or intolerance. Peripheral blood samples were
collected
from 14 patients, after informed consent was obtained for Bcr-Abl
quantitative PCR assessment and Abl
KDM
screening.
RESULTS
The
chimeric
Bcr-Abl
gene was detectable in all patients, (range of 0.18
– 141.9%), when compared with the Abl
gene.
Sequencing analysis was done in 12 out of the 14 patients and Abl
KDM were
identified in three
of the 12 patients (one quarter). One mutant (H396R) is known to retain
intermediate sensitivity to imatinib, while the other two (E255K and
F359V) are
insensitive; all are sensitive to nilotinib. Four of these patients
have since
been commenced on nilotinib and are responding well to therapy, while
three
have died of disease progression.
CONCLUSION
An increasing number of Nigerian CML patients on imatinib are
developing
resistance or intolerance to the drug, and some are due to kinase
domain
mutations, while others may have other yet unexplained reasons for
suboptimal
response. Continuous monitoring is mandatory in the care of CML
patients, to
aid early detection of suboptimal therapy outcomes, necessitating
further
molecular studies. Additionally, more work needs to be done to fully
understand
the mechanisms for these therapy failures in Nigerian CML patients.
Keywords:
Leukemia, Myelogenous,
Chronic, BCR-ABL Positive ; imatinib; nilotinib;
Polymerase Chain Reaction; mutation; Drug
Resistance.
INTRODUCTION
Chronic
myeloid leukaemia was the first human malignancy to be linked to a
specific
acquired genetic abnormality, the Philadelphia chromosome which is
formed as a
reciprocal translocation between the long arms of chromosomes 9 and 22.1
This
translocation produces the Bcr-Abl
fusion gene on chromosome 22 that encodes an Abl
tyrosine kinase
with increased activity. The
discovery of imatinib, the first molecularly-targeted drug directed at
this
aberrant tyrosine kinase, has revolutionized the management of CML.2
The clinical and cytogenetic response to imatinib was so remarkable
that it soon
became the first-line therapy for all stages of CML, effectively
relegating
allogeneic stem cell transplantation to a second-line option.3-5
While
most patients achieved significant hematologic and cytogenetic
improvement with
imatinib, resistant CML clones soon emerged,6
leading to the
development of other tyrosine kinase inhibitors (TKIs).7
Also, some
patients experience disease progression during imatinib therapy.8
The most common cause of imatinib resistance is the development of
point
mutations in the Abl
kinase
imatinib-binding domain.9
Imatinib (Glivec, Novartis) became
available in Nigeria in 2003 under the patient assistance programme of
Novartis, and the local patient outcomes have since improved
tremendously.10
However, we have also been recording cases of imatinib resistance. We
therefore
examined our imatinib-resistant patients for the presence of kinase
domain
mutations.
PATIENTS
AND METHODS
From
August 2003 to July 2010, 266 consenting CML patients have been
registered with
the Glivec International Patient Assistance Program (GIPAP), and
commenced on
Imatinib (IM). We investigated the occurrence of Abl
kinase
domain mutations (KDM) among a cohort of these
patients, with features of imatinib resistance or intolerance.
Peripheral blood
samples were collected from 14 patients, after informed consent was
obtained
for Bcr-Abl
quantitative PCR (qPCR)
assessment as well as Abl
KDM screening which was
done in 12 of these 14 patients.
This
prospective study included all confirmed cases of Philadelphia
chromosome-positive (Ph+) and/or Bcr-Abl-positive
CML patients in all disease phases managed in
our centre over a seven-year period. At every clinic visit, patients
are
examined to identify those with features suggestive of suboptimal
response to
IM, and thus candidates for possible resistance. Local institutional
approval
was obtained for this study. Written informed consent was obtained from
all
patients according to the Helsinki declaration.
Total
RNA was extracted from mononuclear cells from the peripheral blood
samples, and
subjected to reverse-transcription and quantitative PCR. The resulting amplicon
was sequenced and the Abl
kinase
domain examined for mutations. All qPCR
and KDM screening were carried out at the Hammersmith
Hospital, London.
RESULTS
Fourteen
imatinib-resistant or intolerant patients were screened (male/female
ratio was
6/8),
mean age was 38.1 ± 22.9. The chimeric
Bcr-Abl
gene was
detectable in all patients, (range of 0.18 – 141.9%) when
compared with the Abl
gene. Of the 12 patients in whom KDM screening was
done, three (one quarter) had mutations. Two mutations (H396R and
F359V) are
known to retain intermediate sensitivity to imatinib, while the third
(E255K)
is insensitive; though all are sensitive to nilotinib.11
All
14 patients were recruited to the follow-up program of compassionate
use of
nilotinib (Novartis), and four of them have since been commenced on
nilotinib.
Two of the four commenced have been on nilotinib
for
>16 months
and are in major molecular
remission. Four of the 14 patients have since died, from disease
progression.
There
was an average of 313 ± 984 days from diagnosis to
commencement of imatinib in
all 14 patients, though this was heavily skewed by five patients with a
range
of 375 – 1597 days, while the remaining ranged from 0
– 71 days. They had been
on imatinib for an average of 43.5 ± 46.2 months before
showing features of
therapy failure, necessitating molecular testing (Table
1).
|
Table
1. Detailed summary of Abl-Bcr kinase
domain mutations in all patient |
Of
the five patients starting imatinib more than one year after diagnosis,
three
of them have died, representing three fifths of all deaths from this
cohort.
Using the Kaplan-Meier survival model, commencement of imatinib more
than one
year after diagnosis (p = 0.028) and advanced Hasford risk group (i.e.
intermediate and high grade disease) at diagnosis (p = 0.027) were
found to be
statistically significant in predicting for death events. The presence
of Abl
kinase
domain mutations and Sokal
risk groups were statistically insignificant in
predicting death events in this study (p > 0.05).
DISCUSSION
Imatinib
has been shown to act by binding to the ATP-binding site of the Abl
catalytic domain, resulting in the inhibition of phosphorylation
of Bcr-Abl,
thereby preventing its kinase activity and making the cell susceptible
to
apoptosis. Imatinib resistance is due to the interactions between
several
factors, some Abl-dependent
and others Abl-independent:
treatment compliance, bioavailability, pharmacodynamics,
genetic changes and Bcr-Abl
kinase
domain mutations.11
Plasma levels
of imatinib are reduced in individuals with increased activity of
cytochrome
p450 isoenzymes p3A4 and p3A5 or use of enzyme inducing drugs leading
to
reduced efficacy of imatinib.12,13
Resistance to imatinib is
sometimes primary i.e. no response at the beginning of therapy, or
secondary
i.e. loss of initial response. There are also variations in the
manifestation
of resistance: some patients present with hematologic - lack or loss of
complete haematological remission (CHR), cytogenetic - persistence or
recurrence of Philadelphia chromosome, or molecular - persistence or
recurrence
of Bcr-Abl
transcripts on qPCR.
Unfortunately,
in the clinical setting and for individual patients, only the
assessment of
changes in the Abl
kinase
domain is feasible. In this study, we have found that only about 25% of
our
IM-resistant or -intolerant patients have a resistance-conferring
mutation. The
values reported by different workers have unfortunately varied widely
from 20%
to 90%, due largely to differences in the stringency criteria for
detection. In
fact, it has been claimed that the ability to detect KDMs in these
patients is
highly dependent on patient selection and the assay methods, and
particularly
on the sensitivity of the method used. It has been estimated that
20-30% of CML
patients will eventually develop resistance to imatinib,11
and up to 90% of these are probably due to kinase domain mutations.14
Some
studies on the CML stem cell have shown them to be insensitive to
Imatinib, and
they thus persist in a quiescent stage 15
despite adequate clinical response,
hence they can proliferate when the drug is discontinued and this can
be a
source of resistance.
Additionally,
some novel mutations have been observed in some CML patients, which are
associated with rapid proliferation of cells, blastic transformation
and
resistance to imatinib. Some patients have even been demonstrated by Gorre
et al to have increased expression of the Bcr-Abl
gene; the importance and clinical effects of which
were explored in their report.16
It was shown that the H396R
(detected in patient 2) impairs conformational change of the activation
loop,
while the E255K (detected in patient 3) impairs conformational change
of the P
loop. The F359V (detected in patient 11) inhibits imatinib binding
leading to
resistance. Fortunately, all three mutations are known to retain
sensitivity to
Nilotinib and interestingly responded initially to escalated doses of
Imatinib.
Two of the patients died while awaiting drug supplies, while the third
is doing
well on nilotinib. Only the T315I mutation is known to confer
resistance to
imatinib, dasatinib,
nilotinib
and bosutinib.17
Our
study also shows that there is a much higher risk of death in patients
who were
diagnosed more than one year before they commenced imatinib and
patients with
advanced disease using the Hasford scoring system. However, these
results are
to be interpreted with caution since our numbers are rather small, and
hence
with limited statistical power. In any case, it is prudent to commence
patients
on targeted therapy as soon as a diagnosis is made; and to mandatorily
monitor
for early detection of resistance i.e. complete blood counts every 4-6
weeks
and bone marrow cytogenetic analysis every 6 months.8
It is equally
vital to properly counsel patients to ensure compliance and to withdraw
imatinib in patients who for any reason are intolerant.
Whenever
imatinib failure or resistance is suspected, quantitative molecular
assessment
of the Bcr-Abl
transcript and Abl
kinase domain mutations should be investigated.18
Plans for
second-generation TKI should be commenced at the same time as these
tests are
done. These agents have been proven to be effective in imatinib
resistance.19,20
Considering the favourable responses reported and
also noted in this study, it is advisable to escalate imatinib doses to
600
mg/d and 800 mg/d, while other TKIs are being expected. This is
particularly
so, since most cases are negative for KDMs. The combination of imatinib
with
other drugs like hydroxyurea and cytarabine is also a good strategy.
Patients
in blastic
crises are to be treated with acute
leukaemia induction regimen, in combination with imatinib. Allogeneic
stem cell
transplantation may be considered if T315I mutation is discovered, as
this
mutation does not respond to any of the TKIs. Supportive therapy should
be
given as necessary in form of blood products support, antibiotics and
growth
factors.
ACKNOWLEDGEMENT
We
acknowledge the kind assistance of Novartis Pharma, the
Max Foundation and Axios International for providing
imatinib for these patients. We also appreciate
the medical and nursing staff of the department of Haematology,
OAUTHC
for the meticulous care offered to these patients.
FOOTNOTES
Conflicts of interest: The authors declare no competing conflicts of interest
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