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

dasatinib. Methods. We studied Ikaros gene expression in bone marrow
and peripheral blood samples from 29 patients with Ph + ALL: 5 adult de
novo patients, 16 patients resistant to imatinib and 8 resistant to dasatinib
after imatinib failure. Reverse transcription-polymerase chain reaction
(RT-PCR) using primers specific for exon 1 and exon 7 of Ikaros and
nucleotide sequencing were performed to identify the specific isoforms.
Genomic sequencing of the regions surrounding the predominant splice
donor and acceptor sites at the exon-intron splice junctions was performed
in search for mutations. BCR-ABL transcript levels were monitored
in each patient by real-time quantitative PCR (RQ-PCR). Results.
We detected expression of the full-length Ik1, Ik2 DNA binding isoforms
in cells from healthy volunteers and in 3 (10%) Ph + ALL patients (2 resistant
to dasatinib and 1 to imatinib). In the 26/29 (90%) remaining patients
the Ik6 isoform lacking all DNA-binding domains was detected and in
13/26 (50%) it was the predominant isoform, demonstrating its dominant
activity. In 5/26 patients we detected also the Ik4 isoforms. Genomic
sequencing of splice junction regions demonstrated no mutations. We
confirmed the identification of a SNP that affects the third base of the
triplet codon for a proline (CCC or CCA) in the highly conserved bipartite
activation region of the exon 7. Bi-allelic expression pattern of the
various Ikaros isoforms suggested that trans-acting factors were involved
in the generation of the non-DNA binding isoforms. Molecular monitoring
showed that the dominant negative Ik6 expression correlated with
the BCR-ABL transcript levels suggesting that this alteration could
depend on the Bcr-Abl activity. The majority of patients with Ik6 expression
had also point mutations in the ABL kinase domain. Conclusions. The
Bcr-Abl oncoprotein may induce the expression of aberrantly spliced
oncogenic Ikaros isoforms which arrest the leukemic cells at the pre Bcell
stage and contribute to the tyrosine kinase inhibitor resistance interfering
with proteins in pathways that are normally regulated by the fulllength
Ikaros protein.
Supported by: European LeukemiaNet, COFIN 2003 (M. Baccarani), AIL,
AIRC, Fondazione Del Monte di Bologna e Ravenna.
0007
MLL TRANSLOCATION IN A MULTIPOTENT PROGENITOR CAUSING ACUTE
LYMPHOBLASTIC LEUKAEMIA - TWO-STEP MODEL OF THE DISEASE
J. Zuna, 1 T. Burjanivova, 1 Z. Zemanova, 2 S. Horsley, 3 L. Kearney, 3
K. Muzikova, 1 C. Meyer, 4 E. Mejstrikova, 1 A. Houdkova, 5 S. Colman, 3
H. Ptoszkova, 6 R. Marschalek, 4 J. Stary, 1 M. Greaves, 3 J. Trka1 1 CLIP, Charles Univ. Prague, 2nd Med. Sch, PRAGUE, Czech Republic;
2 Charles Univ. Prague, 1st Med. School, PRAGUE, Czech Republic; 3 Institute
of Cancer Research, SUTTON, United Kingdom; 4 Univ. of Frankfurt, FRANK-
FURT/MAIN, Germany; 5 University Hospital Motol, PRAGUE, Czech Republic;
6 University Hospital Ostrava, OSTRAVA, Czech Republic
Background. Leukaemias with MLL gene rearrangement are usually
considered prognostically unfavourable and the clinical symptoms typically
follow the translocation formation rapidly. MLL rearrangement is
thus thought to be a major hit in leukaemogenesis that is either sufficient
to cause the disease or it is a very strong and rapid inducer of the subsequent
hit(s) required for the malignant transformation. Methods and
Results. We report an unusual presentation of secondary acute lymphoblastic
leukaemia (sALL) with MLL rearrangement. Our patient was
diagnosed originally with acute myeloid leukaemia (AML-M3) characterised
by PML/RARα fusion and an internal tandem duplication of FLT3
(FLT3/ITD). After 30 months of complete remission of AML, she developed
sALL with MLL/FOXO3A fusion gene. Bone marrow (BM) samples
taken during AML therapy were analysed for the presence of these
aberrations. Both the PML/RARα fusion and FLT3/ITD disappeared
shortly after AML onset and did not reappear. However, FISH and quantitative
RT-PCR showed the presence of the MLL/FOXO3A fusion 20
months before the diagnosis of sALL, present in 10-90% of BM cells.
Morphological examination showed no blast infiltration of the BM at
this time. Experiments combining FISH and morphology confirmed the
presence of an MLL rearrangement in myeloid as well as lymphoid cells,
indicating that the fusion arose in a multipotent progenitor. In order to
identify potential secondary genetic events precipitating sALL in this
patient, we used Affymetrix 50K single nucleotide polymorphism (SNP)
array analysis on DNA from the diagnostic sALL sample versus the
preleukaemic (remission AML) sample taken 16 months before. This
analysis revealed a 10 Mb amplification on 19q13.32 in the sALL sample,
not present in the preleukaemic sample: this was confirmed by FISH
with a BAC from the amplified region. A difference between the preleukaemic
and leukaemic cells is also demonstrated by the incomplete
rearrangement of IgH gene (DH1/JH) present only at the diagnosis of
12 th Congress of the European Hematology Association
sALL. There are about 450 genes in the amplified region on 19q and several
of them might be involved in deregulation of the preleukaemic cell
if overrepresented (e.g. FLT3 ligand, interleukin 11, Ras interacting protein
1, Stem cell growth factor, Aurora C). Summary and conclusions. The
long latency period prior to the onset of the secondary leukaemia in our
case resembles the mouse model of MLL/FOXO3A. However, in contrast
to the animal model and also to the previous reports of
MLL/FOXO3A patients (2 cases described so far, both secondary AMLs
after Hodgkin's disease), our child developed leukaemia from the lymphoid
lineage. Taken together, these results indicate that the
MLL/FOXO3A fusion alone is not sufficient to cause leukaemia and that
second hit is required to the onset of the disease. A responsible gene is
possibly located on the telomeric part of the 19q.
Grant support: MSMT 21620813.
0008
V(D)J-MEDIATED TRANSLOCATIONS MORE EXCEPTION THAN THE RULE?
K. Vanura, 1 T. Le, 1 M. Marusic Vrsalovic, 2 R. Marculescu, 3 U. Jäger, 1
B. Nadel4 1 Medical University of Vienna, VIENNA, Austria; 2 Dubrava University Hospital,
ZAGREB, Croatia; 3 Rudolfstiftung Hospital, VIENNA, Austria; 4 CNRS-
INSERM-Université de la Méditerrané, MARSEILLE, France
Background. Translocations of proto-oncogenes to the B-cell or T-cell
antigen receptor loci (BCR and TCR) usually are accompanied by an
overexpression of the proto-oncogene involved which leads to the development
of lymphoid neoplasms. The mechanisms held accountable for
these translocations are either V(D)J- or class switch recombination,
depending on the receptor region the oncogene is translocated to. Arguments
for one or the other mechanism range from a simple statement
with no additional comments to very detailed discussions why the
authors think that a particular mechanism is responsible for the translocation
or not. In most cases, however, arguments are rather indiscriminating
and do not take into account mechanistic definitions characteristic
for the recombination mechanism. Aims. To assess the potential of
cryptic sites in the proto-oncogenes LMO2/RBTN2, LCK/p56,
HOX11/TLX1, and E2A/TCF3 to undergo illegitimate V(D)J recombination.
LMO2, LCK, and HOX11 translocations to the TCR occur in 7, 4
and 1% of T-ALL, respectively. The E2A-PBX or E2A-HFL fusions are
found in 25% of pre-B cell leukemias and in 5% of childhood and adult
ALL. While not rearranged to one of the antigen receptor loci, cryptic
sites have been found close to the breakpoints at E2A. Methods. An ex
vivo recombination substrate assay was used to assess potential and efficiency
of the cryptic sites to undergo V(D)J-recombination with a genuine
TCR element. Results. LMO2 cryptic site, a site 1,3 kb downstream
of a cryptic site that had been proven to function as a 12-RSS, appeared
to be a 23-RSS target for V(D)J-recombination. Interestingly, this region
contained additional cryptic sites which also engaged in V(D)J-recombination.
While not as attractive a target as the 12-RSS site, these sites
showed high recombinogenic efficiency in our assay. For LCK, two sites
have been reported to be involved in translocations to the TCRβ locus.
LCK site #1 functioned as a 12-RSS with high efficiency. Site #2, situated
30 kb upstream of site #1, only showed unspecific breaks scattered
over the region used in our assay, indicating that it was not a direct target
for V(D)J-recombination. The breakpoints of more than 6 patients
were found close to a putative 23-RSS cyptic site 300 bp upstream of
HOX11 exon 1. Recombination in our assay, however, resulted in unspecific
breaks scattered over the region tested indicating that, as for LCK
site #2, it was not a direct target for V(D)J-recombination. Finally, E2A
contains two putative heptamers close to the breakpoint region found
in translocations to PBX and HLF. These heptamers did not function as
direct targets and recombination was unspecific in our experiments. Conclusions.
While being potentially dangerous due to the initiation of DNA
breaks, V(D)J-recombination is a tightly controlled mechanism that
keeps the percentage of directly V(D)J-mediated chromosomal aberrations
as low as possible. Other factors are involved causing doublestrand
breaks which result in DNA ends that subsequently are incorrectly ligated
to one of the immune receptor loci thus leading to overexpression
of a proto-oncogene and the development of leukemia.
haematologica/the hematology journal | 2006; 91(s1) | 3