Transcript The Introduction of ABL kinase domain mutation testing in

The introduction of ABL kinase
domain mutation testing in CML
patients showing resistance to
Imatinib
Davina Clavering, Elizabeth Perrott, Julian
Borrow, Joanne Mason, Susanna Akiki and
Mike Griffiths
West Midlands Regional Genetics Laboratory
Talk Outline
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Introduction to CML
Methods for AKD analysis
Mutations identified to date
WMRGL database
Follow up
Future work
Chronic Myeloid Leukaemia
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Characterised by the
Philadelphia
chromosome t(9;22)
Results in fusion of
BCR and ABL genes
Imatinib mesylate is
the frontline therapy
Developing resistance
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Glivec has revolutionised treatment for CML but
resistance is a problem in a minority of patients
Point mutations in the ABL kinase domain are the
most frequently reported cause of resistance
Other mechanisms
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BCR-ABL amplification
Over expression of LYN, changes in drug influx and
efflux proteins
To overcome resistance: increase dose / try
different targeted therapy (new generation kinase
inhibitors e.g. dasatinib, nilotinib), bone marrow
transplant
Setting up an AKD mutation service
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Identification of mutations can direct patient
management
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Increase dose of imatinib
Provide evidence that an alternative drug may be
more suitable
Work currently funded by Novartis for 18 months
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Monitoring user satisfaction with a view to obtaining
local NHS funding
Monitoring of CML patients
BCR/ABL:ABL
Level of BCR/ABL normalised to ABL
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Ratio
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scale
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Samples received at
regular intervals
usually every 3-6
months
Quantitative RT-PCR
carried out and
disease levels
compared to ABL
“housekeeping gene”
values
Good response to imatinib
Level of BCR/ABL normalised to ABL
BCR/ABL:ABL
Sensitivity of detection
10
Ratio
on
log
scale
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Sensitivity of detection
Good response post transplant
Activation of samples for ABL
kinase domain mutation testing
BCR/ABL:ABL
Level of BCR/ABL normalised to ABL
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0.000001
BCR/ABL:ABL
Level of BCR/ABL normalised to ABL
Sensitivity of detection
10
Ratio
on
log
scale
1
0.1
0.01
0.001
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0.000001
BCR/ABL:ABL
Level of BCR/ABL normalised to ABL
Sensitivity of detection
1
Ratio
on
log
scale
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0.01
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treatment failure at 6
or more months
sub-optimal
treatment response
loss of response
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Ratio
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Clinical request
Internal activation
based on ELN
guidelines
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Sensitivity of detection
Current Testing Strategy
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RNA clean-up using DNase
28S rRNA
18S rRNA
DNA
Before
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After
Semi-nested RT-PCR followed by direct sequencing
BCR
ABL
BCR F
ABL F
ABL R
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Analysis of sequencing using Mutation Surveyor
Send report and follow-up form to clinician
ABL kinase domain mutations
T315I/A/D
F311L/I/V F317L
C305S
V299L
L248V
L298V
G250E/A/F
Q252H/R
P296H
G321E
M237I
K285N
E281A
E279K
T277A
L384M
A344V
L387F/M
S348L
A350V
Imatinib binding site
M244V
D241G
G383D
M343T K357R
E292V
Y253H/F
E255K/V V289A/I
P-loop
F382L
L324Q
M388L
C
M351T/L
E355G/D
A397P
H396R/P
E453G/K/A/V
E450Q
F486S
Q447R
Activation loop
V379I
L3641
F359V/C/D/I
A412V
S438C
E459K/Q
Adapted from Melo et al., Cancer Lett 2007 249(2):121-32
E276G
E275K
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Over 100 mutations identified in the literature
T315I is most common
16 mutations account for 86% reported cases
Mutations identified to date
3
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2
1
M244V
L248V
G250E
Y253H
E279K
V299L
T315I
F317L
F359I/V
A412V
G450Q
0
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Mutations
No. of Patients
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51 patients reported
so far
13 mutations
identified in 12
patients
Pick up rate of 24%
WMRGL Database
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Compiled a mutation database based on an
extensive literature search consisting of 3 types
of data
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Mutations which emerge in response to treatment and
the ability of each drug to overcome a particular
mutation
IC50 values
In vitro mutagenesis data
Resistance profiles are created for each mutation
and added to the report
Resistance profiles
Mutation
Imatinib
Dasatinib
Nilotinib
M244V
Partially sensitive
Sensitive
Sensitive
L248V
Resistant
?Sensitive
Partially Sensitive
G250E
Resistant/?Partially Sensitive
Sensitive
Partially sensitive/?Sensitive
G250E &
A412V*
Resistant/?Partially Sensitive
Unknown
Sensitive
Unknown
Partially sensitive/?Sensitive
Unknown
Y253H
Resistant
Sensitive
Resistant/?Partially Sensitive
E279K
Resistant
Unknown/?Sensitive
Unknown/?Sensitive
V299L &
K247R
Sensitive
Neutral
Resistant/?Partially Sensitive
Neutral
Unknown
Neutral
F317L
Partially sensitive
Resistant
Sensitive
F359I
Unknown
Unknown
Unknown
F359V
Partially sensitive
Sensitive
Partially sensitive
E450Q
Unknown
?Sensitive
Unknown
IC50 value: the concentration of drug required to inhibit 50% of enzymatic activity
Imatinib:
Nilotinib:
Dasatinib:
Sensitive <4, Partially Sensitive ≥4, <9 Resistant ≥9
Sensitive <4, Partially Sensitive ≥4, <30, Resistant ≥30
Sensitive <4, Partially Sensitive ≥4, <6, Resistant ≥6
Cut-offs: Numbers refer to fold increases in IC50 compared to “wild-type” BCR-ABL
*unique mutation
Case report - Patient VK
Level of BCR/ABL normalised to ABL
Ratio
on
log
scale
BCR/ABL:ABL
Sensitivity of detection
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1
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0.01
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0.0001
0.00001
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0.000001
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r.730A>G
p.Met244Val; M244V
Patient diagnosed June 05,
aged 18
On imatinib for 15 months
Underwent bone marrow
transplant Oct 06
Tested 1 month later; low
disease levels
4 weeks later loss of response
Mutation analysis unavailable
in 2006
Jan 07 – clinical relapse, drug
dose restricted due to
cytopaenias
Mutation analysis requested
Resistance Profile
Imatinib: Partially sensitive
Dasatinib: Sensitive
Nilotinib: Sensitive
Case report – Patient SH
BCR/ABL:ABL
Level of BCR/ABL normalised to ABL
Sensitivity of detection
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Ratio
on
log
scale
10
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0.1
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0.001
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0.0001
0.00001
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/1
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0.000001
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Patient diagnosed with
CML Oct 04
Previous ALL in early
childhood
No response to treatment
after 32 months on
imatinib
Mutation analysis initiated
upon clinician request
r.742C>G
p.Leu248Val
r.742_822del81
Patient SH cont…
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Resistance Profile
Imatinib: Resistant
Dasatinib: ?Sensitive
Nilotinib: Partially sensitive
Reported in the literature
in 2 patients
2 forms
Activates a cryptic 5’
donor splice site within
exon 4
Yields a splice variant
with a deletion
Important to be aware of
this mutation due to the
mixed isoforms present,
producing a messy
sequence trace
Follow up form
This patient has been tested for BCR/ABL kinase domain mutations,
either at your request or because of treatment failure, sub-optimal
response, or loss of response as defined by ELN guidelines.
In order to audit the effectiveness of this service, with implications for
future commissioning (as the service is currently only grant funded for 18
months), we would be grateful if you could provide feedback on the
usefulness of the enclosed result.
Please confirm current treatment (including dose):
…………………………………………………………………….
When was treatment last changed?
…………………………………………………………………….
Has this result been useful?
YES / NO
Comment:
…………………………………………………………………………………………………….
Will this result alter your patient management?
YES / NO
Comment:
……………………………………………………………………………………………………..
If yes, what changes to this patient’s treatment are planned?
…………………………………………………………………………………………………………………
…………………………………………………………………………………………………………………
Follow up form responses
Mutation?
Useful? Alter treatment?
Comments
G250E & A412V
Y
Y
May change to dasatinib if
BCR/ABL levels increase
E279K
Y
Y
Will change to dasatinib if can
get funding
F359V
Y
N
Confirms clinical decision
G250E
Y
N
Patient already on dasatinib
E450Q
Y
N
Thought non-compliance an
issue so changed to dasatinib
N
Y
Potentially
Pt referred for psychiatric
review for non-compliance
N
Y
N
Patient scheduled for BMT
N
Y
N
Would consider
nilotinib/dasatinib
N
Y
N
Possible allograft depending on
response to dasatinib
N
Y
Y
Considering non-compliance
N
N
N
Patient has died
N
-
N
Limitations in quantification of
mutations
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Sequencing cannot easily
be used to quantify the
level of mutation
 Report 100% or less
than 100%
Currently testing other
samples to identify when
mutation emerges
Pyrosequencing to be
used in the future
100%
<100%
Kinetics of AKD mutations
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BCR/ABL wild type clone decreases and another resistant clone
emerges in response to treatment
After time resistant transcripts constitute all mutant transcripts
Pyrosequencing will be able to assess whether this mutant transcript
decreases when alternative drugs are used
Leukemia (2006) 20, 658–663
Acknowledgements
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Mike Griffiths
Joanne Mason
Julian Borrow
Elizabeth Perrott
Elizabeth Ormshaw
Sue Rose
Susanna Akiki
Molecular Oncology team
Molecular Genetics Sequencing
team
 Work funded by
Novartis