12th Congress of the European Hematology ... - Haematologica

Ph + cells in the bone marrow). We also evaluated the possible influence
of SVV levels on the clinical outcome (time to progression, response to
therapy and overall survival) of the pts. All CML pts underwent IM therapy
at 400 mg/die and their SVV levels were assessed by quantitative RT-
PCR prior to the first IM treatment. Complete hematologic response
(CHR), cytogenetic response (Cy-R) and molecular response (MolR) were
scored as previously described (Baccarani et al. Blood 2006;108:1809).
Results and Conclusions. All CML pts expressed the SVV transcript (median
SVV=727 units). Nineteen out of 20 CML pts (95%) reached a CHR
at 3 months; 10/20 pts (50%) obtained a Cy-R (complete + partial) at 12
months; 7/20 pts (35%) achieved a MolR (complete + major) at 12
months. Four pts (20%) displayed a failure to treatment within 12
months. In our analysis, SVV transcripts produced a median time to progression
and survival of 31 (p=0.04) and 53 months (p=0.6), respectively.
These findings indicate that increased SVV expression reduces IM
therapeutic efficacy, in agreement with our previous in vitro observation
that high SVV levels significantly reduce cell death in CML cells. Thus,
our data suggest that strategies aimed at lowering SVV expression may
improve the efficacy of IM, therefore representing a possible therapeutic
option for both IM-sensitive and IM-resistant CML patients.
0534
DETECTION OF SINGLE NUCLEOTIDE POLYMORPHISMS IN THE ABL KINASE DOMAIN OF
CML PATIENTS
T. Ernst, M.C. Müller, P. Erben, N. Härtel, C. Walz, R. Hehlmann,
A. Hochhaus
Universitätsklinikum Mannheim, MANNHEIM, Germany
Background. BCR-ABL kinase domain mutations constitute the leading
cause of resistance in CML patients treated with imatinib. Detection of
BCR-ABL mutations prior to and during the course of imatinib therapy
may aid in risk stratification as well as in determining optimal individual
therapeutic strategies. More than 50 different point mutations have
been described so far. The K247R change has been identified as a rare
gene polymorphism occurring also in normal control patients and other
cell types. Despite its position near to the P-loop, biochemical and cellular
assays of imatinib and dasatinib sensitivity showed no alteration
compared to wild-type ABL. Aims. Since gene polymorphisms do not
automatically necessitate a change in the therapeutic strategy we sought
to investigate if other BCR-ABL mutations are rather polymorphisms
than mutations associated with resistance to tyrosine kinase inhibitors.
Methods. In comparison to real BCR-ABL kinase domain mutations, single
nucleotide polymorphisms (SNPs) also have to occur on the normal,
untranslocated ABL allele. Therefore, we designed an allele-specific PCR
for amplification of the normal ABL allele following mutation analysis
by denaturing high-performance liquid chromatography (D-HPLC) and
subsequent sequencing. A multiplex PCR was performed to ensure that
both Ib and Ia ABL splice variants were amplified. Ninety-one imatinibresistant
CML patients (chronic phase, n=88; accelerated phase, n=1;
blast crisis, n=2) with 65 different BCR-ABL kinase domain mutations
leading to 50 different amino acid changes were investigated in our
study. 39 of 65 different mutations were evaluable in two different
patients and 26 different mutations in one patient, respectively. Results.
Amplification of the normal ABL allele succeeded in all cases. The D-
HPLC analysis indicated a mutation in the normal ABL allele in eight cases.
The nucleotide exchanges were confirmed by direct sequencing in
both directions. In all cases the same mutation of the BCR-ABL allele was
confirmed in the normal ABL allele (mutated ABL (%)/ mutated BCR-
ABL (%)): T240T (50/100), F311V (50/100), T315T (40/100), Y320C
(90/30), K247R (100/20; 40/100) and E499E (100/100; 40/100). In three
cases the SNP did not lead to an amino acid change (silent mutation). Five
patients had a wild-type as well as a mutated ABL allele leading to 50%
mutated ABL allele. In these patients the SNP including ABL allele seems
to be translocated to BCR-ABL thus containing 100% mutated BCR-
ABL. Another patient harbouring the F311V mutation at the BCR-ABL
allele did not show the mutation at the normal ABL allele. Conclusions.
In addition to the previously described polymorphism K247R we uncovered
five new polymorphisms, two of these led to amino acid changes.
SNPs should be taken into consideration for design of mutation-specific
PCRs, particularly the T315T polymorphism for T315X amplifications.
Newly identified mutations should be confirmed by amplifying
the normal ABL allele to exclude polymorphisms. Clinicians should be
aware that polymorphisms do not reflect mutations that automatically
demand a change in therapeutic strategy, unless there are other signs of
inadequate response to treatment.
12 th Congress of the European Hematology Association
0535
MAPK-ACTIVATION IN RESPONSE TO BCR-ABL INHIBITION IS CYTOKINE-DEPENDENT
AND CAN BE OVERCOME BY DASATINIB
P. La Rosée, N. Härtel, T. Klag, R. Hehlmann, A. Hochhaus
University of Heidelberg, MANNHEIM, Germany
Background. Inhibition of BCR-ABL by imatinib mesylate is current
standard treatment for patients with chronic myelogenous leukaemia
(CML). Despite favourable response rates, imatinib fails to eradicate
minimal residual disease in the majority of patients, which is believed
to be caused by stem-cell resistance. A proposed mechanism of human
CML stem-cell survival is growth-factor- (GF) dependent activation
(phosphorylation) of the MAPK Erk1/2 in response to imatinib (Chu et
al., Blood 2004). Aims. We used IL3-dependent murine haematopoietic
cell lines and their IL3-independent BCR-ABL-expressing progenies to
study MAPK-signalling in response to imatinib and the high affinity
BCR-ABL inhibitor dasatinib. Primary CD34 + enriched human CML cells
were used, to validate cell-line results in a clinically relevant cellular context.
Methods. Murine Baf3 and 32D cells transformed by p210Bcr-Abl
were grown in the presence of 5 µM imatinib with and without WEHIconditioned
media as source of IL3. Cytotoxicity was determined by
trypan blue viability count. Cells were harvested after 3h, 6h, and 24h
and lysed for Western blot analysis. Human CML mononuclear cells
derived from 5 patients with first chronic-phase CML prior to treatment
were enriched by magnetic separation for CD34 + . MAPK-signalling after
treatment with BCR-ABL-inhibitors for 16h was studied in serum-free
media in the presence of standard GF-mix. Results. Baf3p210 and
32Dp210 cells treated with 5 µM imatinib for 24h were rescued by IL3
as determined by trypan blue viability count. Western blot analysis after
3h, 6h, and 24h of treatment demonstrated time-dependent significant
activation of Erk1/Erk2 with peak phosphorylation after 6h in the presence
of IL3. In the absence of IL3, Erk1/Erk2 phosphorylation was abolished
at any time. Simultaneous detection of phosphotyrosine (pTyr)
and BCR-ABL demonstrated complete inhibition of BCR-ABL with and
without IL3. Similar results were obtained in both cell lines. Exposure of
Baf3p210 cells with 60nM dasatinib induced marked MAPK-inhibition
independent of IL3 as early as 3h after start of treatment. Treatment of
CD34+ enriched CML cells confirmed imatinib-induced MAPK-activation.
Exposure of patient (n=5) cells with dasatinib (12 nM, 62 nM) in
contrast showed significant (p