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

12 th Congress of the European Hematology Association
ues whereas 18 (52,9%) revealed a positive value for CRAC. This distribution
is comparable to the pattern found in the analysis of acute
lymphoblastic leukemia in our previous study (N=78, CRAC positive
n=35, 44,9%; CRAC negative n=43, 55,1%). Conclusions. The analysis of
cytochrome c related caspase activation (CRAC) identifies leukemia
samples with deficient apoptosis signaling in a group of pediatric AML
patients. The detection of such patients by assessment of the parameter
CRAC may be used for additional treatment stratification in childhood
acute myeloid leukemia.
This work is supported by an EHA research fellowship grant to L.H. Meyer.
0473
FLT-1 ACTIVATION BY VEGF/PLGF INDUCES LEUKEMIA CELL MIGRATION VIA
P38/ERK1/2 KINASE PATHWAY, RESULTING IN RHO GTPASES ACTIVATION AND
CAVEOLAE FORMATION
C. Casalou, R. Fragoso, J.F. Nunes, S. Dias
Portuguese Institute of Oncology, LISBON, Portugal
Vascular endothelial growth factor (VEGF) receptors -1 (FLT-1) and -
2 (KDR) are expressed by subsets of malignant hematopoietic cells,
where they signal in a paracrine and/or autocrine manner to induce cell
survival, proliferation and migration. We recently demonstrated that
acute lymphoblastic leukemia (ALL) migration in response to VEGF via
FLT-1 modulates the onset of extramedulary disease, and thus has clinically
predictive value. However the molecular mechanisms involved in
leukaemia cell migration were not yet characterized and are the subject
of this study. We demonstrate that FLT-1 activation by VEGF or PLGF
stimulation results in a significant increase of AML migration, having a
minor proliferative effect. This FLT-1 mediated migration of AML cells
resulted in the formation of actin membrane protrusions (as assessed by
phalloidin/FLT-1 immuno-staining and confocal microscopy) with concomitant
increased ERK1/2 and P38 phosphorylation and activation of
Rho-GTPases and PAK/Cofilin. Also, by immunoprecipitation studies,
we have shown that PLGF promoted the recruitment of Hsp90 and its
binding to FLT-1, actin and caveolin-1. While PLGF stimulation of AML
cells induced the formation of caveolae-like structures, cytochalasin D
treatment, used as control to block cell migration, was shown to block
the molecular interactions between FLT-1, actin, Hsp90 and caveolin-
1.Taken together, these data highlight some of the molecular mechanisms
whereby VEGF/PLGF stimulation of FLT-1 on AML cells results
in cell migration. As such, our data reveal important biological and mechanistic
outputs that may be used to monitor leukemia responses to
cytoskeleton-disrupting agents or anti-angiogenic therapies. Our ongoing
studies are know focusing on a screening of kinases/phosphatases
that affect VEGF production in acute leukemia cells, using a system of
retrovirus-based vectors that achieve with high-efficient siRNA-dependent
silencing in leukaemia cells.
0474
A RECURRENT IN-FRAME INSERTION IN CEBPA TAD2 REGION IS A POLYMORPHISM
THAT DOESNT HAVE A PRONOSTIC VALUE IN AML. A STUDY FROM THE FRENCH ALFA
GROUP
V. Biggio, 1 A. Renneville, 2 O. Nibourel, 2 N. Philippe, 2 L. Terriou, 2
C. Roumier, 2 P. Amoyuel, 3 D. Cottel, 3 D. Cottel, 3 S. Castaigne, 4
H. Dombret, 5 X. Thomas, 6 C. Preudhomme2 1 INSERM, LILLE; 2 INSERM U837, Lab of Hemat. CHRU, LILLE; 3 INSERM
U744, INSTITUT PASTEUR, LILLE; 4 Dep of Hem., Hôpital André Mignot,
VERSAILLES; 5 Dep of Hem. Hôpital Saint-Louis, PARIS; 6 Dep of Hem. Hôpital
Edouard Herriot, LYON, France
Background. CCAAT/enhancer-binding protein-α (CEBPa) is a transcription
factor strongly implicated in myelopoiesis through control of
proliferation and differentiation of myeloid progenitors. In patients with
acute myeloid leukemia (AML), this gene is often mutated (about 8%).
Those mutations are in-frame or out of frame types and the consequences
are a loss of function of CEBPa. Among those mutations, an inframe
insertion of 6bp in the TAD2 domain, resulting in a His-Pro duplication
(HP196-197ins), was found in patients at diagnosis and remission
time, letting think of a constitutive abnormality, that may induce
AML susceptibility. Surprisingly this 6bp insertion was observed in
healthy voluntaries too. Recently Delwel et al. have described this insertion
as being a polymorphism. They report that 6bp insertion frequency
in AML patients is between 3.2% (9/282) and 3.9% (12/305), while
in non-leukemic blood samples, the frequency is 8% (22/274). Further,
they didn’t found any correlation with CEBPa gene expression signature
176 | haematologica/the hematology journal | 2007; 92(s1)
and more, patients with insertion didn’t show any particular expression
profile. Aim of study and Methods. In our study we have investigated 325
AML patients enrolled in ALFA 98-02 protocol and 994 healthy voluntaries
for the presence of this insertion by nucleotide sequencing, and we
studied its prognostic value. Results. First, we looked for the frequency
of this insertion between the healthy whole population and the AML
cohort; we didn’t found any difference between AML patients and
healthy voluntaries: 6.5% (21/325) versus 6.6% (66/994) respectively.
This data strongly suggest that this insertion is a polymorphism, but
familiar molecular study should be performed to confirm this hypothesis.
Second, we made a comparison between true mutation of CEBPa
and this 6bp polymorphism. Patients who harbored only the polymorphism
have heterogeneous cytogenetic finding (8 normal, 5 complex
and 1 intermediate karyotypes, 4 CBF-AML and 4 unknown), while
patients with a true abnormality of CEBPa gene (n=23) show significant
association to normal karyotypes (p=0.03). Mutations of CEBPa are often
associated to M1 and M2 FAB subtypes, but we didn’t find an association
to FAB subtype for the group of patients with 6bp polymorphism.
Regarding other molecular prognostic factors, CEBPa is rarely associated
to NPM mutations (p=0.03) while polymorphism doesn’t show this
particularity (p>0.2). Regarding the EFS, we didn’t find any correlation
between this polymorphism, true CEBPa mutations and others patients.
However regarding the OS only the true CEBPa mutation patients presented
a trend for longer survival. Conclusions. In conclusion, we confirm
that HP196-197ins is a common polymorphism spread in normal population
and AML patients, without any prognostic value in our cohort
of AML patients.
0475
CAN HYPERACTIVE SIGNALLING THROUGH THE RAS PROTEINS INDUCE AML IN MICE?
A. Cutts, A.M. Wahlstrom, C. Karlsson, B. Swolin, M.O. Bergo
Sahlgrenska University Hospital, GÖTEBORG, Sweden
Background. Mutationally activated forms of the RAS proteins, especially
the N and K isoforms, are implicated in the pathogenesis of several
haematological malignancies, including acute myeloid leukaemia
(AML). However, in AML, the mutations in RAS genes are always
accompanied by other genetic abnormalities (e.g. MDR1). In mice,
hyperactive signalling through the RAS proteins, e.g., via expression of
endogenous oncogenic K-RAS or inactivation of the tumor suppressor
NF1, results in myeloproliferative disorders (MPD). At this point, it is
unclear if hyperactive RAS signalling can induce AML in the absence of
other genetic abnormalities. Aims. To test the hypothesis that hyperactive
signalling through the RAS proteins can induce AML in mice, without
other genetic abnormalities. To accomplish this, we will determine
if inactivation of Nf1 - which increases signalling through the N- and K-
RAS proteins - will transform a K-RAS-induced MPD into AML. Methods.
For these studies, we use Cre-loxP techniques in mice. Cre-induced
activation of endogenous oncogenic K-RAS in bone marrow cells results
in a rapidly progressing MPD with leukocytosis, splenomegaly, and tissue
infiltration; without an increase in blasts in peripheral blood. Similarly,
Cre-induced inactivation of the RAS-GAP NF1 results in increased
levels of RAS-GTP, hyperactive RAS signalling, and a slowly progressing
MPD; without an increase in blasts. In this study, we simultaneously
activate the expression of oncogenic K-RAS and inactivate the tumour
suppressor NF1 in myeloid cells. This approach allows us to determine
if higher levels of RAS-GTP would result in transformation of MPD into
AML with > 20% blasts in peripheral blood. Results and Conclusions. This
is an ongoing project. By June, 2007, we will have all the necessary data
that will allow us to accept or reject our hypothesis.
0476
INCREASED RISK AND EARLY ONSET OF ACUTE MYELOID LEUKAEMIA IN BRAZILIAN
INDIVIDUALS WITH NAD(P)H:QUINONE OXIDOREDUCTASE 1 (NQO1) AND CYTOCHROME
P450 A1 (CYP1A1) GENE DEFECTS
G.G. Yamaguti
State University of Campinas (UNICAMP), CAMPINAS, Brazil
Background. Acute myeloid leukaemia (AML) has been linked to
chronic exposure to benzene and tobacco’s polycyclic aromatic hydrocarbons
(PAH). NAD(P)H:quinone oxidoreductase 1 (NQO1) is an
enzyme that detoxifies benzene-derived quinones and reduces oxidative
stress on hematopoietic cells. A C-->T substitution polymorphism at
nucleotide 609 of the NQO1 gene has been linked to a decreased activity
of the coded enzyme. On the other hand, PAH are bioactivated by
the cytochrome P450 A1 (CYP1A1) enzyme. An A-->G substitution