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

12 th Congress of the European Hematology Association
Myeloproliferative disorders - Biology
0411
ACQUIRED ISODISOMY IS COMMON IN ATYPICAL MYELOPROLIFERATIVE DISORDERS
C. Curtis, 1 F.H. Grand, 1 S. Kreil, 1 D.G. Oscier, 2 A.G Hall, 3 N.C.P Cross 1
1 Wessex Regional Genetics Lab, SALISBURY, United Kingdom; 2 Royal
Bournemouth Hospital, BOURNEMOUTH, United Kingdom; 3 University of
Newcastle Upon Tyne, NEWCASTLE UPON TYNE, United Kingdom
Recent evidence has indicated that acquired isodisomy is a novel
mechanism by which mutations in cancer may be reduced to homozygosity.
Typically, acquired isodisomy is associated with oncogenic
changes rather than tumour suppressor genes, eg. the activating JAK2
V617F mutation in patients with myeloproliferative disorders (MPD) is
often associated with acquired isodisomy for chromosome 9p. We have
undertaken a screen for regions of acquired isodisomy as a means to
identify genomic regions that may harbour novel oncogenes in patients
with atypical MPD (n=30), blast crisis of chronic myeloid leukaemia
(CML-BC; n=20) and chronic lymphocytic leukaemia (CLL; n=20).
Genomewide SNP analysis was performed using Affymetrix 50K XbaI
arrays and analysed for copy number changes and length of homozygous
tracts. Chromosome deletions and gains identified by cytogenetic
analysis were also detected by SNP analysis. Large tracts of homozygosity
(defined as >20Mb running to a telomere), strongly suggesting
acquired isodisomy, were seen 12 (40%) aMPD patients, a single case
(5%) of CML-BC but were not seen in CLL. The homozygous tracts
encompassed diverse genomic regions in aMPD, but two common
regions (3 cases for each region) were identified at 7q and 11q. Because
of the recurrent involvement of tyrosine kinases in MPDs, we focused
our initial screen on this class of gene. The entire coding regions of
EPHB6 and EPHA1 were sequenced in patients with 7q isodisomy but
no sequence changes were identified. Similarly, no changes were found
in selected regions of BRAF and PIK3CG. Ongoing sequence analysis is
focused on other candidate genes within the two regions.
0412
JAK2V617F-NEGATIVE ET PATIENTS DO NOT DISPLAY CONSTITUTIVELY ACTIVE
JAK/STAT SIGNALLING
S. H. Schwemmers, 1 H. Pahl, 1 B. Will1 C. F. Waller, 1 K. Abdulkarim, 2
P. L. Johansson, 2 B. Andreasson, 2 H. L. Pahl1 1 University Hospital Freiburg, FREIBURG, Germany; 2 Sahlgrenska University
Hospital, GÖTEBORG, Sweden
Background. Presence of the Jak2V617F mutation in only 40-60% of
patients with Essential Thrombocythemia (ET) underscores the heterogeneity
of this myeloproliferative disorder (MPD). Several distinct mutations,
either in Jak2 (exon 12) or in c-Mpl (W515L) have been described
in subsets of other Jak2V617F-negative MPDs, Polycythemia vera (PV)
and Idiopathic Myelofibrosis (IMF). Analogous to Jak2V617F, these
mutations cause constitutive Jak2 and STAT activation. It has therefore
been proposed that constitutive activation of the Jak/STAT pathway
underlies the molecular etiology of all MPDs. Aims. We investigated the
alternative hypothesis that distinct alterations, separate from the
Jak/STAT signal transduction pathway, underlie a subset of Jak2V617Fnegative
ET. We therefore compared gene expression profiles of ET
patients with and without the Jak2V617F mutation. Methods. 32 patients
with ET, diagnosed according to the PVSG criteria, were included in the
study after giving informed consent. Jak2V617F allele expression was
quantified in purified granulocytes by real time qRT-PCR. cDNA
microarrays were processed according to the protocol of Eisen and
Brown using the Lowess (Locally weighted scatter plot smoother) subgrid
normalization method for comparison across slides. Statistical significance
of differences was determined after correction for the false
discovery rate by the method of Benjamini and Hochberg. Differences
in gene expression were verified with qRT-PCR and protein phosphorylation
detected by Western Blotting. Results. Unsupervised clustering
of gene expression patterns in ET patients revealed two distinct subclasses
of patients. These subclasses differed in presence or absence of the
Jak2V617F mutation. Patients lacking the Jak2V617F mutation displayed
significantly lower expression of the STAT target genes Pim-1 and
SOCS2. In addition, Jak2V617F-negative patients showed lower levels
of STAT phosphorylation. Conclusions. These data demonstrate that a
large proportion of Jak2V617F-negative ET patients do not display constitutive
Jak/STAT signaling. Hence, we propose that alterations in dif-
152 | haematologica/the hematology journal | 2007; 92(s1)
ferent signal transduction pathways can lead to the clinical phenotype
of ET. Elucidation of novel ET-inducing changes will facilitate both a
molecular classification of ET and the development of rationally
designed therapies.
0413
MPL MUTATIONS IN PRIMITIVE MYELOFIBROSIS ARE NOT RESTRICTED TO MPL 515
MUTATIONS BUT ONLY MPL 515 MUTATIONS ARE ONCOGENIC EVENTS PRESENT AT THE
STEM CELL LEVEL
S. Giraudier, 1 R. Chaligné, 2 C. James, 3 C. Tonetti, 4 J.P. Le Couédic, 2
R. Besancenot, 2 I. Godin, 2 L. Lordier, 2 K. Malloum, 5 P. Mossuz, 6
M.C. Le Bousse-Kerdilès, 7 W. Vainchenker, 2 S. Giraudier1 1 INSERM, U790 and Hematology Laboratory H, VILLEJUIF AND CRÉTEIL;
2 INSERM, U790, VILLEJUIF; 3 INSERM, U217, BORDEAUX; 4 Faculté de
Médecine Paris XII, CRÉTEIL; 5 AP-HP, Laboratoire d'hématologie, La pit,
PARIS; 6 Laboratoire d'Hématologie, GRENOBLE; 7 INSERM, U602, VILLE-
JUIF, France
Background. The MPL 515 mutations have recently been described in
10% of primitive myelofibrosis (PMF) as an essential oncogenic event.
Here we report other MPL mutations found in PMF patients. These new
mutations raise the question whether these molecular events occur in a
true stem cell (ie: lymphoid/myeloid progenitor cell), as it has already
been shown for JAK2 V617F occurrence in PMF and PV. Aims. The aim
of this work was to study the presence of MPL exon 10 mutations in a
cohort of 100 PMF, determine the functions of the different mutations,
and if these mutations occurred in both myeloid and lymphoid lineages
in JAK2 V617F negative PMF. Methods and Results. We therefore
sequenced MPL exon 10 in 100 PMF patients. Six different mutations
were found. In order to determine whether these mutations were only
genetic polymorphism or real gain of function mutations, we introduced
all the mutations in Baf3 cells. We found that only the recently described
MPL 515 mutations and a 506+519 mutant induced factor independence
growth, activation of JAK/STAT, RAS/MAPK, and PI3K transduction
pathways as well as P21 overexpression, a spontaneous cell cycling
with an increase in S and G2M phases, and tumorigenesis in NUDE
mice. Then we looked for the MPL 515 mutations in stem cells compartments:
First in mature myeloid and lymphoid cells and second in lymphoid/myeloid
progenitors progeny after CD34 + and CD34 + CD38 – cell
isolation from peripheral blood. Three PMF patients harboring MPL515
mutations were studied. Peripheral blood granulocytes and platelets
were purified by standard methods and B, T, NK cells and monocytes
were isolated by combined immunomagnetic and flow cytometric procedures.
The same techniques were used to sort CD34+ and
CD34 + CD38 – subpopulations from peripheral blood. Clonal
B/NK/Myeloid differentiation from CD34 + CD38 – cells and T cell differentiation
from CD34 + cells were performed respectively onto a MS5
layer in the presence of cytokines and in Fetal Thymic Organ Cultures
(FTOC). Genotyping of mature cell populations, B/NK/Myeloid clones
and CD34 + derived T cells were performed by direct sequencing. Moreover,
CD34 + cells were cultured in a one cell per well experiment to
determine the sensitivilty of these patient’s cells to low dose TPO. The
MPL515 mutations were present in granulocytes and platelets from all
patients, and in monocytes in one. We detected the mutation in NK
cells in two cases. The MPL 515L and MPL 515K mutations could be subsequently
detected in CD34 + cells and in B/NK/Myeloid and/or
NK/myeloid CD34 + CD38 – derived clones from all IMF patients. Interestingly,
MPL 515L homozygous clones were detected in B/NK/Myeloid
and/or NK/Myeloid clones from 1 patient. However, using the FTOC
assay, the mutations were not detected in T cell fractions derived from
CD34 + cells. At least we found that MPL mutations induce a spontaneous
megakaryocytic growth in cell culture but also a hypersensitivity
to low dose TPO. Summary. These results demonstrate that the MPL
515 mutations occur in a lymphoid/myeloid progenitor cell and give
rise to a hypersensitivity to TPO. Thus, these MPD take their origin in
a true lymphoid/myeloid progenitor cell. In accordance with mouse
model, this represent a good argument for a causative role of MPL mutations
in PMF.