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

12 th Congress of the European Hematology Association
feasible alternative to bone marrow (BM) transplantation in adults, when
no sibling is available. Aims. We report a case of donor derived acute
myeloid leukaemia following cord blood transplantation for chronic
myeloid leukaemia in a 32 year old female. Methods. BM samples were
analysed by G-banding and FISH. DNA was extracted from BM cells preand
after transplantation and analysed by array comparative genomic
hybridisation (aCGH) (Agilent Technologies). ACGH results were confirmed
by real-time quantitative PCR (qPCR). Results. Cytogenetic and
FISH analysis at diagnosis showed a female karyotype with the t(9;22)
translocation and an inv(7) with unusual breakpoints in both arms. Seventeen
months following a sex mismatch UCB transplantation cytogenetic
analysis revealed an abnormal male karyotype with monosomy 7,
loss of 21 and an aberrant chromosome der(17)t(17;21). Further FISH and
aCGH analysis elucidated the mechanism by which the complex der(17)
occurred and maps the breakpoint at 17p13.3. Moreover, we detected an
amplification of the 21q11.2-q22.1 region with breaks at the flanking
sites. The amplifications and deletions were confirmed by qPCR. Conclusions.
We confirm the occurrence of donor cell leukaemia following UCB
allogeneic transplantation. This study represents an extremely rare event
and to our knowledge this is only the fifth case described for a donor cell
leukaemia after allogeneic cord blood transplantation.
1015
PRAME EXPRESSION IN CHRONIC MYELOID LEUKEMIA
Yu.P. Finashutina, 1 E.V. Aksenova, 1 A.V. Misyurin, 1 A.G. Turkina, 1
N.D. Khoroshko, 1 M.I. Lukashina, 2 A.Yu. Baryshnikov2 1 National Research Center for Hematology, MOSCOW, Russian Federation;
2 Cancer Research center RAMS, MOSCOW, Russian Federation
Background and aims. Tumor antigens recognized by CTLs have been
identified several years ago and are major targets for the creating of anticancer
vaccines. PRAME is an antigen which expresses at high levels in
various malignant tumors including melanomas and hematopoietic
malignancies such as acute and chronic leukemias (AML, CML etc.) The
aim of this study is to determine the frequency and clinical importance
of the expression of this antigen in CML as well as to detect the PRAME
protein in tumor cells. Methods. PRAME mRNA was measured by realtime
RT-PCR using TaqMan technique. Periferal blood mononuclear cell
(PBMC) samples from 245 cases with chronic myeloid leukemia and 25
controls were used as study material. Chi-square test for independent
samples was used for statistical analysis. The monoclonal antibodies
raised against truncated recombinant PRAME were used for PRAME protein
analysis by Western blot and immunocytochemistry on the various
tumor cells. Results. Seventy four of 245 cases with CML (30%) showed
PRAME expression whereas all the healthy donors did not. PRAME
expression was found in six of 20 newly diagnosed CML samples (30%),
55 of 209 CML-chronic phase (26%), and 13 of 16 CML with acceleration
and blastic phase (80%). Highest expression was found in cases with
CML-blastic phase. For some of positive PBMC samples PRAME protein
expression was confirmed by Western blot assay. The control samples
were negative in the same assay. Finally we have revealed the nuclear
localization of PRAME protein in K562 cell line (chronic myeloid
leukemia) and melanoma cell lines by immunocytochemistry and Western
blot. Summary. PRAME is nuclear protein that expressed approximately
in one third of the cases with CML. Low expression levels in
remission and high levels in relapse suggest that PRAME is an important
marker to detect the minimal residual disease and predict relapse.Our
findings support the suggestion that this antigen may be furhter used as
a target for diagnostic and therapeutic approaches.
1016
IMATINIB RESISTANCE POINT MUTATION LEU248VAL IN BCR-ABL GENE GIVES
RISE TO MICRODELETION OF THE SAME REGION WITHOUT FRAMESHIFT
M.V. Suchkova, A.V. Misyurin, E.V. Aksenova, Yu.P. Finashutina,
V.V. Tikhonova, A.A. Krutov, E.Yu. Chelysheva, A.G Turkina,
N.D Khoroshko
National Research Center for Hematology, MOSCOW, Russian Federation
Background. The main disadvantage of mesylate imatinib is the development
of resistance to this drug in patients with chronic myeloid
leukemia (CML) and with Ph-positive acute lymphoblastic leukemia
(ALL). It has been found that point mutations in the ATP binding pocket
of the ABL kinase domain of BCR-ABL gene confer imatinib resistance
in relapsed patients. Aims. To study drug resistance mutations in BCR-
ABL gene in CML patients that turned to be imatinib resistant according
376 | haematologica/the hematology journal | 2007; 92(s1)
to data of quantitative RQ PCR analysis of BCR-ABL gene expression.
Methods. PCR fragment containing BCR-ABL breakpoints and juxtaposing
ABL region with ATP binding pocket of the ABL kinase domain was
amplified using conventional primers. PCR fragments were purified with
6% PAAG and then directly sequenced using the same forward and
reverse primers. We analyzed 2 CML patients with primary and 25 CML
patients with secondary imatinib resistance. Results. In 2/2 CML patients
with primary imatinib resistance we did not find any BCR-ABL point
mutations (there was only a polymorphism not giving rise to amino acid
substitution). In 19/25 (76%) CML patients with secondary imatinib
resistance we found point mutations that are usual for cases of imatinib
resistance. Among them there were 4 mutations known to be very significant
to render imatinib resistance due to localization within ATP binding
pocket of ABL P-loop (Gly250Glu, Tyr253Phe, Leu248Val,
Gly250Glu). The other 2 point mutations were also common for CML
patients resistant to imatinib but of mere significance as they situated outwards
of P-loop region (Met244Val, Phe359Val). It was of great interest
that in the same patient carrying point mutation Leu248Val we also found
a microdeletion of 739-819 bp. Due to this deletion 82 bp of P-loop was
removed without frameshift. The resultant BCR-ABL protein was predicted
to be 23 aa shorter then usual. Point mutation Leu248Val made
change of AAGC for AAGG. The same AAGG repeat was found on the
other end of deletion. We suppose that point mutation Leu248Val may
facilitate that deletion due to creating two perfect AAGG repeats on both
ends of deletion. Conclusions. It is still unclear if that microdeletion may
have any clinical significance. Nevertheless we consider our finding to be
an interesting example of genetic instability.
1017
CURE OF VERY RARE PATIENTS WITH CHRONIC MYELOID LEUKEMIA (CML) ACHIEVED
BY BUSULFAN ALONE. A HINT FOR A ROLE OF NKT CELLS IN SUCH CASES
J. Tanzer, 1 J.C. Chomel, 2 F. Brizard, 2 A.J. Brizard, 2 A.G. Turhan, 2
J.M. Gombert2 1 University of poitiers medical school, POITIERS, France; 2 UE 38 05 University
Medical School, POITIERS, France
Background. CML is considered to have been consistently fatal prior
to development of bone marrow transplantation. An ineluctable evolution
to a rapidly lethal acute transformation led to death that occurred
even after an unusually prolonged chronic phase in 10 cases of the literature
and in 4 personal cases, 20 to 31 years after diagnosis. Aims. To
show that the term cure or operational cure is perhaps to be used for a
patient whose CML was treated by busulfan 50 years ago and who is
perfectly well in 2007 despite the persistence of very rare circulating
clonogenic BCR-ABL+ cells. Preliminary results indicate that NKT cells
may play a role in this situation as well as in very rare similar cases. Case
records, methods and results. One of us has been following PEL. B since he
was hospitalized in july 1956, at the age of 3 for an adult- type CML.
Busulfan, given for 3 months caused a severe aplasia from which he
recovered in November 1957. From 1967 to 1987, bone marrow studied
5 times showed a total of 167 metaphases, all of them normal. However
in serial examinations since 1996, a small amount of b2-a2 messenger
was detected in the blood leucocytes (Bcr-Abl/Abl 0.01%) and 2-5%
of BCR-ABL cells+ were found by D-FISH. In cultures in presence of 4
growth factors granulocytic and erythroid colonies were obtained; 20%
of which expressed b2-a2, that were suppressed by imatinib, showing
their dependence on an active Bcl tyrosine-kinase. The transcript was
also detected in the blood of 3 out of 6 mouse 3 months after IV inoculation
of blood cells from the patient. Searching for an immune reaction
we were only able to demonstrate, using flow cytometry, an unusually
considerable in vitro expansion (294 fold) stimulated by α-galactosylceramide(α-GC)
of his NKT cells as defined by the CD1d tetramer-invariant
Vα24+staining, that moreover produced high levels of IFNγ, TNFα
and perforine. The value of our findings is confirmed by similar molecular
and immunologic results achieved in 2 other patients who have
been surviving for 3 and 4 decades respectively. We are currently trying
to look whether such α-GC-expanded NKT cells would inhibit the
growth of colonies from our patients’ blood cells. Conclusion. We suggest
that carrying such investigations in 5 patients surviving in good health,
17 to 48 + years after diagnosis, whose previously reported observations
were kindly updated for us by D. Amikam, M. Djaldetti, C. Fegan,
TA. de Witte, RS. Stein and JT. Reilly, would be of great interest. At least
it is thus demonstrated that an obsolete drug may allow rare patients to
be considered as operationally cured, which remains of importance even
in the era of kinase inhibitors. Our observations obviously raise a number
of questions, one being whether NKT cells or α-GC might be
employed as therapeutic agents in the future in some cases of CML.