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

12 th Congress of the European Hematology Association
in CD3 + T-cells obtained from imatinib treated CML patients and
healthy individuals. These results are given in the attached Figure 1.
Conclusions. Most likely the observed difference in gene expression of
apoptosis-related genes can be linked to the imatinib treatment, since:
(i) all patients were in CCgR and (ii) all had a negative interphase FISH
for the bcr-abl fusion gene. Fifteen of 94 studied apoptosis related genes
were downregulated in T-cells from imatinib treated patients and only
one gene was upregulated. Functional assays are needed to explain how
these alterations affect the T-cell function, e.g. proliferation and activation
induced cell death (AICD) - an important regulator of the immune
response.
Figure 1.
0543
BCR-ABL KINASE MUTATIONS IN RESPONSE TO DASATINIB
J. Sorouri Khorashad, P. Mehta, D. Milojkovic, D.C. Marin, J.S. Kaeda,
B. Swale, D. Stevens, J.M. Goldman, J.F. Apperley
Imperial College of London, LONDON, United Kingdom
Background. Dasatinib is a novel Src-Abl kinase inhibitor with efficacy
in patients with CML resistant to imatinib. In vitro studies show that
dasatinib is active against nearly all the known BCR-ABL kinase mutations
except T315I. Aims. Analysis of the kinetics of BCR-ABL kinase
mutations in patients treated with dasatinib. Methods. Karyotyping for
evaluation of cytogenetic response, RT-PCR for measurement of BCR-
ABL transcript levels, direct sequencing and pyrosequencing for detection
and quantification of the mutations in serial samples of patients
with a suboptimal response to dasatinib. Results. 47 imatinib-resistant
CML patients received dasatinib. Patients were followed for 3-23
months. 12 patients had mutations in the BCR-ABL kinase domain, prior
to treatment with dasatinib. Of these 12, 10 (82%) achieved MCyR
with dasatinib usually within three months of starting therapy. 22%
later lost their MCyR. Kinetic analysis of the mutant clones revealed 3
patterns of response. Group 1 (n=6): patients initially responded to dasatinib
with a 1-3 log reduction in BCR-ABL/ABL ratio, but this was not
maintained. A new mutation was noted either in isolation or in addition
to pre-existing mutations. Group 2 (n=4): patients achieved a MCyR
with dasatinib, which was accompanied by disappearance of the mutant
clone (E453V, H396R, F359V). Group 3 (n=2): patients achieved a MCyR
with dasatinib. However, the mutant clone persisted with fluctuating
levels after more than 18 months follow up. Nearly all the patients with
mutations known to be sensitive to dasatinib in cellular studies achieved
MCyR with dasatinib. This response was accompanied with decline or
disappearance of the mutant clone (group 2), or a fluctuating level of the
mutant clone (group3). The only exception was one patient with L248V
(group1), in whom L248V remained dominant during the follow-up
with a transient MCyR. Group 1 included patients who had an initial
response, but subsequently lost this as demonstrated by an increase in
the mutant BCR-ABL positive clone. In two patients in this group, a
pre-existing mutant clone disappeared along with > 2 log reduction in
BCR-ABL/ABL ratio. This response was later lost following the reappearance
of the previously detected mutation along with T315I,
Quantitative analysis showed that these mutations co-existed in the
same clone. In a further 3 patients in Group 1 there was an initial reduction
in the BCR-ABL/ABL ratio but this was lost with the emergence of
a new mutation, F317L. The appearance of F317L coincided with disap-
202 | haematologica/the hematology journal | 2007; 92(s1)
pearance of the pre-existing mutant clones, M244V, M351T and H396R.
The F317L induces a 9 to 13.5-fold increase of dasatinib IC50 in comparison
to wild-type BCR-ABL in cellular assays. These results show the
emergence of new mutations under dasatinib and document their different
kinetics of mutation.
0544
WHEN IS A DOUBLE PHILADELPHIA NOT A DOUBLE PHILADELPHIA
D. Brazma, 1 C. Grace, 1 A. Virgili, 1 J. Howard, 1 A. Chanalaris, 1
J.V. Melo, 2 J.F. Apperley, 2 E.P. Nacheva1 1 Royal Free and UCL Medical School, LONDON; 2 Imperial College&Hammersmith
Hospital, LONDON, United Kingdom
Background. Chronic myeloid leukaemia (CML) is a pluripotent
haematopoietic stem cell disorder defined by expression of the BCR-ABL
fusion gene, a constitutively activated tyrosine kinase. The fusion gene
commonly results from formation of the Philadelphia chromosome (Ph)
after a t(9;22)(q34;q11) or related variant rearrangement. We studied 48
samples and 12 cell lines from CML patients at various stages of the disease
by arrayCGH using a 1 mbase BAC array (Spectral Genomics,
Houston, USA), 44k 60 mer oligo arrays (Agilent) and by molecular cytogenetics
techniques. Gains in 9q34.1-qter distal to the ABL breakpoint
along with gains of 22pter-c-22q11.2 were detected in 1/22 CP, in 3/26
BC samples and in 6 cell lines. This is in keeping with the presence of a
second Ph chromosome as confirmed by both G banding, chromosome
painting and D-FISH. Contrary to expectations, 5 of these samples (50%)
did not display the aCGH profile corresponding to the presence of extra
Ph markers instead, various gains were identified from the 9q34.1-9qter
segment that (a) failed to cover the whole 9q material of the Ph marker
and (b) disagreed with the G banding data. Aim. In the absence of BAC
array coverage of the ABL breakpoint region, a more precise assessment
of the 9q34 imbalances was sought using a high resolution oligo based
aCGH analysis of three BC patients and two cell line samples. Results.
The oligo’s aCGH profile confirmed the presence of gains from the
9q34.12 sub-band and delineated a common amplicon with an estimated
size of 330 Kbp. Three genes - ABL, LAMC3, NUP214 are located in
this amplicon. We constructed a dual colour FISH probe from clones
RP11-83J21 (containing ABL from the 5’ end to exon 1), RP11-143H20
(covering LAMC3) and RP11-544A12 (partially LAMC3 & NUP214
genes), and applied it to the mitotic figures of bone marrow from 15
CML patient’s samples in accelerated phase/blast crisis and with 10
CML cell lines. Extra copies of the region covered by the probe were
detected as follows: a) High copy number gains were seen as ‘tandem
duplications’ within similar acrocentric marker chromosomes in 2 cell
lines, K562 and KU812; b) Duplication of the ABL-NUP14 region within
the Ph chromosome with an apparently normal G-banding morphology
in (3/15) patient’s samples; similar changes were found in the cell
lines MC3, MEG-01, EM-2 and KYO while affecting one of the two
copies of the Ph chromosome that were indistinguishable by G-banding;
c) The ABL-NUP214 region was found inserted within one of the
apparently morphologically normal chromosome 22 homologues at the
22q11.2 sub-band in two instances. Conclusions. These findings clearly
demonstrate that the Philadelphia chromosome is an unstable structure
and prone to rearrangements that commonly involve sequences downstream
of the ABL breakpoint, specifically the ABL-NUP214 region.