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

12 th Congress of the European Hematology Association
0630
AUGMENTED IN VITRO SENSITIVITY OF MDS PROGENITORS TO THE FARNESYLTRANS-
FERASE INHIBITOR TIPIFARNIB
I. Kotsianidis, I. Basdiara, A. Anastasiades, D. Pantelidou, D. Margaritis,
A. Goutzouvelidis, G. Bouricas, C. Tsatalas
Democritus University of Thrace, ALEXANDROUPOLIS, Greece
Background. Farnesyltransferase inhibitors (FTIs) target, amongst other
proteins needing prenylation for functioning, activating ras mutations
which occur in up to 20% of myelodysplastic syndrome (MDS) patients.
Tipifarnib (Zarnestra ® ), a potent and specific inhibitor of farnesyltransferase
R115777 showed activity in two phase II studies in MDS, but the
optimal dose to avoid significant myelossupression remains to be determined.
Moreover, the exact mechanism of FTI action is not fully clarified,
whereas a direct effect on human MDS progenitors in vitro has not
yet been shown, neither is known if FTI exhibit selective toxicity against
clonal MDS hematopoiesis. Patients and methods. Bone marrow aspirates
of 16 MDS patients, 9 men and 7 women (5 with refractory anemia, 9
refractory anemia with multilineage dysplasia, 2 refractory anemia with
excess blasts I, according to WHO classification), were taken prior to
either cytotoxic or growth factor treatment. The median age was 68
years (range 62-77). MDS was diagnosed on the basis of morphologic
and cytogenetic criteria. The control group consisted of 6 age matched
individuals all of which had anemia of chronic disease (ACD). Mononuclear
BM cells, were enriched for CD34 + cells with positive selection by
using immunomagnetic beads and plated for short term cultures in semisolid
media, or liquid cultures for assessment of apoptosis, in the presence
of either DMSO or 2.5, 10, 25 and 50 nM of FTI. The percentage
of apoptotic, annexin V positive, mature (CD34 + CD38 + ) and immature
(CD34 + CD38 – ) progenitor cells was assessed by flow cytometry after 48
and 72 hours. The significance of the differences was assessed by paired
or unpaired Student t test as appropriate. IC50 values were determined
by linear interpolation. Results. FTI inhibited the colony growth of total
committed MDS progenitors at concentrations of 2.5nM (p=0.017) and
higher, in contrast to normal, non clonal, CD34 + progenitors which were
sensitive only in concentrations of 25 nM and above. Interestingly, the
effect was more prominent in the MDS CFU-GM which significantly
reduced their plating efficiency at 2.5 nM (p=0.018), whereas erythroid
and CFU-GEMM progenitors exhibited significant inhibition at 10nM
(p=0.01) and above. Indicative of the augmented susceptibility of the
MDS CD34 + cells was also the lower IC50 value for MDS (14 nM) compared
to the normals (33 nM). No differences were observed in either
early CD34 + CD38 – or mature CD34 + CD38 + MDS progenitors when cultured
in DMSO or various concentrations of tipifarnib for 48 and 72
hours. Likewise, the same subsets in normal controls displayed no evidence
for increased apoptosis in the presence of tipifarnib. Conclusions.
Commited MDS progenitors are more sensitive to tipifarnib in vitro than
their normal counterparts, the effect being more evident in white cell
progenitors even at low tipifarnib concentrations, whereas this action is
not due to apoptosis induction. Since myelosuppression represents the
main obstacle in the clinical use, in MDS careful determination of the
appropriate tipifarnib dose will help in dissecting the desired control of
the more susceptible leukemic clone from the unwanted inhibition of
normal hematopoiesis.
0631
PREVALENCE OF ERYTHROCYTE HEMOGLOBIN H INCLUSIONS IN UNSELECTED
PATIENTS WITH MYELODYSPLASTIC SYNDROMES OR MYELOPROLIFERATIVE
DISORDERS
P. Steensma, 1 J.C. Porcher, 1 C.A. Hanson, 1 C.L. Lathrop, 1 T.A. Lasho, 1
A. Tefferi, 1 D.R. Higgs2 1 2 Mayo Clinic, ROCHESTER MINNESOTA, USA; University of Oxford,
OXFORD, United Kingdom
Background. Patients with clonal chronic myeloid disorders (CMD),
especially myelodysplastic syndrome (MDS), may develop an acquired
form of α thalassemia - a phenomenon recently linked to somatic mutations
in the chromatin remodeling factor ATRX. The prevalence of erythrocyte
hemoglobin H (Hb H) inclusions, a sensitive marker for α thalassemia,
in a general population of patients with CMD is unknown.
Most reported patients with acquired thalassemia arising in the context
of myeloid neoplasia have exhibited microcytic, hypochromic red cell
indices. However, in the era when reticulocytes were routinely quantified
by supravital staining of peripheral blood - the most sensitive assay
for Hb H inclusions - rather than by modern flow cytometric methods,
patients with CMD and normal erythrocyte indices were sometimes
236 | haematologica/the hematology journal | 2007; 92(s1)
incidentally discovered to have Hb H inclusions. Aims. We sought to
estimate the prevalence of Hb H inclusions in a general population of
patients with CMD, regardless of red cell indices, in order to better
understand the clonal evolution of thalassemic erythoid cells in this setting.
Methods. We prepared fresh brilliant cresyl blue-stained peripheral
blood smears from 355 individuals: 130 patients with CMD (95 with
MDS and 35 with myeloproliferative disorders (MPD) - including idiopathic
myelofibrosis, where unexplained microcytosis is common); 200
patients with an abnormal blood count not associated with a clonal
myeloid disorder or an inherited form of thalassemia; and 25 healthy
persons. Patients were not selected on the basis of red cell indices.
Smears were examined at 4 and 24 hrs for Hb H inclusions. Results.
Among the 95 MDS patients (all WHO subtypes represented), 7 had
small numbers of Hb H inclusions (0.05; Mann-Whitney
test). When MNCs were co-cultured or not with stroma cells and treated
with DAC we observed a downregulation of ARHGAP21 expres-