Transcript Slide 1

Imatinib Resistance
Geoffrey L. Uy, M.D.
Associate Professor of Medicine
Division of Oncology
Natural History of CML
Time
Chronic Phase
Accelerated Phase
Blast Phase
Duration
3-5 yrs, untreated
Varies
Median of months
Prognosis
Responsive
Less responsive
Resistant
Symptoms
Asymptomatic
Fatigue
Weight loss
Abdominal pain or
discomfort
Night sweats
Progressive
Splenomegaly
Bleeding
Infections
Marrow fibrosis
Disease Monitoring in CML
• Hematologic response
– Complete normalization of peripheral blood
counts
– No immature cells in peripheral blood (ie. Blasts)
– No signs or symptoms of disease such as
splenomegaly
• Sufficient when therapy was relatively
ineffective
Disease monitoring in CML
(Conventional Cytogenetics)
Grow cells in culture and
arrest in metaphase with
colchicine
Chromosomal banding
• Giemsa-banding
• G-banding regions AT-rich and
gene poor.
Analyze 20-30 cells (~5%
sensitivity)
Good for large gains, losses, &
translocations
Not sensitive for small lesions
Disease monitoring in CML
(Fluorescent in-situ Hybridization, FISH)
Dual color, dual fusion probe
BCR
ABL-BCR
•Sensitivity 1:200-1:500
•Can detect very small gains, losses and
translocations
•Need to know what you are looking for
BCR-ABL
Abl
Disease monitoring in CML
(qRT-PCR)
• Make a cDNA library from the mRNA specimen
• Design quantititive real time PCR to amplify across different fusion
breakpoints
• Detect specific transcript based on use of allele specific probe
• Quantify relative expression to control gene, (B2-microglobulin or Abl)
• Detect 1 in 106 Bcr-Abl transcripts
Monitoring CML
Sensitivity
Hematologic
Cytogenetic
Molecular
Karyotype
(Ph chromosome)
FISH
PCR
(BCR-ABL fusion)
H&E stain
Peripheral Blood
Bone Marrow
Chromosomal translocation
Abnormal BCR-ABL
(with myeloid cells)
(myeloid hyperplasia)
t(9;22)(q34;q11)
Red= BCR
Green= ABL
Yellow=Fusion
Abnormal BCR-ABL
Lane 1= BCR-ABL+ sample
Lane 2= BCR-ABL- sample
Imatinib in CML
Cumulative Best Response to Initial Imatinib Therapy.
Druker et al., N Engl J Med. 2006 Dec 7;355(23):2408-17
Outcome of Imatinib by Response
Druker et al., N Engl J Med. 2006 Dec 7;355(23):2408-17
Therapeutic Landmarks
Months of
treatment
Treatment Failure
Suboptimal response
3
No Hematologic Response
Less than Complete Hematologic
Response
6
Less than Complete
Hematologic Response
Ph+ cells >35 percent
12
Ph+ cells >35 percent
Less than complete Cytogenetic
Response
18
Less than complete
Cytogenetic Response
Less than major Molecular Response
Clinical Observations with Imatinib
• Virtually all patients with chronic phase
disease respond to Imatinib (but to different
degrees)
• Patients with advanced disease / blast crisis
have very transient responses
• When imatinib is stopped, disease returns, ie.
imatinib is NOT a cure
Why doesn’t Imatinib work in everyone?
• Drug Intolerance
– Inability of a patient to continue therapy despite
optimal management of side effects
• Primary Resistance (Treatment-refractory Ph+)
– Lack of efficacy after treatment initiation despite use
of therapy at appropriate doses
• Secondary Resistance (Acquired resistance)
– Loss of efficacy despite use of therapy at optimal
doses
Is Imatinib Resistance BCR-ABL Dependent?
• Expose bone marrow
from patient with CML
to increasing amounts
of imatinib
• Western blot for BCRABL, CRKL and P-CRKL
Gorre et al. Science 2001 Aug 3;293(5531):876-80.
BCR-ABL Activity in Relapsed Patients
• Take samples from patients
with relapsed disease and
look for P-CRKL
• Examine samples before,
during treatment and at
relapse
Imatinib resistance associated with reappearance of functional
BCR-ABL ie. BCR-ABL dependent
Gorre et al. Science 2001 Aug 3;293(5531):876-80.
How does this happen???
BCR-ABL FISH in Relapsed Patients
(A) Patient with blast crisis
during imatinib therapy
(B) 2nd patient before, during
and after therapy
(C) FISH from patient showing
duplicated inverted Phchromosome
Gorre et al. Science 2001 Aug 3;293(5531):876-80.
Sequencing of ABL Kinase Domain
Gorre et al. Science 2001 Aug 3;293(5531):876-80.
Model of Imatinib Binding of ABL
Gorre et al. Science 2001 Aug 3;293(5531):876-80.
BCR-ABL Kinase Domain Mutations
Ba/F3 Model of CML growth
+ IL-3
Ba/F3
- IL-3
BCR-ABL
CMV
Ba/F3
- IL-3
+ Imatinib
Ba/F3 Model of CML growth
CMV
BCR-ABL(T315I)
Ba/F3
- IL-3
+ Imatinib
Mechanisms of Imatinib Resistance
• Gene amplification
• ABL kinase domain mutations
• Other mechanisms observed in vitro only
– activation of BCR-ABL–independent pathways (Src
family proteins)
– increased drug efflux through the multidrug
resistance gene
Overcoming Imatinib
resistance?
Overcoming Imatinib Resistance
• Novel BCR-ABL inhibitor, dasatinib (aka. BMS354825) in Ba/F3
Dasatinib against mutant BCR-ABL in
Ba/F3 cell assays
Most BCR-ABL mutations except T315I are sensitive to dasatinib
Dasatinib in mouse model of CML
• BCR-ABL luciferase
transduced Ba/F3
cells
• Injected into SCID
mice
• Treat with Dasatinib
or vehicle
Shah et al., Science 305, 399 (2004);
Dasatinib in Imatinib-Resistant Philadelphia
Chromosome-Positive Leukemias
Talpaz et al., N Engl J Med. 2006 354(24):2531-41.
Sensitivity of Bcr-Abl Mutations
O'Hare T et al. Blood 2007;110:2242-2249
Clinical Responses to Dasatinib According
to Mutation Type
Targeting T315I - Ponatinib
•
Rationally designed inhibitor of BCR-ABL
Ponatinib
•
Active against T315I mutant
– Unique approach to accommodating
gatekeeper residue
– Binds inactive (closed) ABL conformation
Ponatinib cocrystal structure with ABLT315I
Ponatinib in Ba/F3 Assay
A
B
*
*
*
(*P < 0.01)
(*P < 0.05)
O’Hare T, et al. Cancer Cell. 2009;16:401-412.
Conclusions
• Both primary & secondary resistance is rare in
chronic phase CML but extremely common in
advanced stage, blast crisis CML
• Mutations in Abl kinase domain are dominant
mechanism for disease resistance
• Understanding mechanisms of resistance can lead
to rapid development of novel agents to
overcome resistance
Approach to Treatment of CML
• Routine monitoring of patient cytogenetics,
FISH and RT-PCR for BCR-ABL
• If patients fail to meet therapeutic landmarks
• Evaluate for kinase domain mutations
– Increase dose of imatinib
– Change to dasatinib or nilotinib
– Clinical trial (3rd generation inhibitors)
– Allogeneic stem cell transplant