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

12 th Congress of the European Hematology Association
used per patient for these analyses. Results. The molecular marker (TCRD-
LMO2 breakpoint region, n=1, TCR rearrangement, n=2) was detected
in only three of 11 patients 3,3 years old or younger but in none of the
older ones. The positivity at birth did not correlate with other clinical or
biological features as for example leukemia subtype, presence of a thymus
tumor or extent of bone marrow and peripheral blood infiltration.
Summary and Conclusions. This is the first comprehensive study performed
in children with T ALL to assess the in utero origin of their disease. Our
data provide first evidence for a predominant postnatal origin of T ALL
in the majority of cases with a rare prenatal initiation only in young children.
These data suggest, in line with mouse and zebra fish models for
T ALL and the data derived from the T ALL-like disease in two children
with retroviral gene transfer for SCID-X1, that T ALL is not only a rapidly
proliferating disease when fully malignant, but that the latency, the
time from initiation to the clinical manifestation, might also be short. The
results are in sharp contrast to B cell precursor ALL in children for whom
an in utero initiation was demonstrated not only in the majority of cases
with genetically diverse subgroups but also for children, who were older
at the clinical manifestation of the leukemia.
This work was supported in part by a the Oberösterreichischen Kinderkrebsforschung
(KS), the St. Anna Kinderkrebsforschung (SF, RPG) and the Wilhelm
Sander-Foundation (MM).
0875
EML1-ABL1 IS ACTIVATED BY COILED COIL MEDIATED OLIGOMERIZATION AND INDUCES
T-CELL ACUTE LYMPHOBLASTIC OR CHRONIC MYELOID LEUKEMIA IN A MOUSE BONE
MARROW TRANSPLANT MODEL
K. De Keersmaecker, 1 H. Verachtert, 1 W. Landuyt, 1 C. De Wolf-Peeters, 1
P. Vandenberghe, 1 J. Schwaller, 2 P. Marynen, 1 J. Cools1 1 2 University of Leuven, LEUVEN, Belgium; University Hospital Basel, BASEL,
Switzerland
Background.BCR-ABL1 (BCR-ABL) is a potent oncogenic tyrosine
kinase that is activated by coiled coil mediated oligomerization and by
coiled coil independent mechanisms. BCR-ABL1 is common in CML and
B-ALL, but is rarely found in T-ALL. The EML1-ABL1 fusion is a variant
ABL1 fusion gene that we identified in a T-ALL patient. Aims. We aimed
at comparing the oncogenic properties of BCR-ABL1 and EML1-ABL1
using in vitro and in vivo models. Characterization of ABL1 fusions such
as EML1-ABL1 could also provide valuable insights to better understand
the oncogenic properties of BCR-ABL1. Methods. We generated BCR-
ABL1 and EML1-ABL1 constructs as well as deletion constructs and transduced
those to the murine IL-3 dependent Ba/F3 cell line. EML1-ABL1
and EML1-ABL1 deletion variants were assayed for autophosphorylation
and for their ability to transform Ba/F3 cells to IL-3 independent
growth. Using a mouse bone marrow transplant model, we determined
the transforming properties of EML1-ABL1 in vivo. Results. Both BCR-
ABL1 and EML1-ABL1 transformed Ba/F3 cells to IL-3 independent
growth, although EML1-ABL1 showed weaker autophosphorylation
compared to BCR-ABL1. EML1-ABL1 forms homodimers, and deletion
of the coiled coil domain of EML1 abrogated its transformation in Ba/F3
cells. These observations suggest that EML1-ABL1, in contrast to BCR-
ABL1, only depends on oligomerization for its activation, which may
explain the observation of weaker kinase activity of EML1-ABL1. In addition,
deletion of part of EML1, resulting in a direct fusion between the
EML1 coiled coil domain and ABL1 resulted in increased autophosphorylation.
In a murine retroviral bone marrow transplant model, 50% of
the mice transplanted with EML1-ABL1 transduced bone marrow cells
developed a transplantable fatal T-ALL, characterized by leukocytosis,
splenomegaly, and massive infiltration of bone marrow and spleen by
CD4 + /CD8 + blast cells. The remaining 50% of recipients of EML1-ABL1
transduced bone marrow cells developed either a non-transplantable
CML-like disease (30% of mice) with leukocytosis, splenomegaly, and
infiltration of bone marrow and spleen by Mac1 + /Gr1 + maturing granulocytes,
or a mixed T-ALL/CML-like disease (20%). All EML1-ABL1 associated
diseases had a latency of 96 -119 days, which is significantly longer
than the 15-21 days latency associated with BCR-ABL1 induced CMLlike
disease. The longer latency for EML1-ABL1 associated disease indicates
that the acquisition of additional mutations is required. Some of the
mice displayed expression of transcription factors LYL1, TAL1 and
LMO2, known to be associated with human T-ALL. Activating NOTCH1
mutations could not be detected. In contrast to EML1-ABL1 induced disease,
mice transplanted with the more active deletion variant with the
EML1 coiled coil domain fused to ABL1 developed a CML-like disease
with much shorter latency (36 days). Conclusions. In contrast to BCR-
ABL1 which is activated by coiled coil dependent and independent mechanisms,
coiled coil mediated oligomerization is necessary and sufficient
326 | haematologica/the hematology journal | 2007; 92(s1)
for EML1-ABL1 kinase activity and transforming properties. Our data
suggest that kinase activity of ABL1 fusions may determine in part the
disease phenotype. The mouse model we describe for EML1-ABL1
induced T-ALL can be further explored to identify co-operating events in
the pathogenesis of T-ALL and for testing novel therapies.
0876
ANALYSIS OF PAIRED PRESENTATION AND RELAPSE SAMPLES IN T-ALL SUGGESTS THAT,
AT LEAST IN SOME PATIENTS, NOTCH-1 MUTATIONS MAY BE ACQUIRED AS A SECONDARY
EVENT
M. Mansour, 1 V. Duke, 2 B. Patel, 2 P. Ancliff, 3 L. Foroni, 2 R.E. Gale, 1
D.C. Linch1 1 University College London, LONDON; 2 Royal Free Hospital, LONDON;
3 Great Ormond Street Hospital, LONDON, United Kingdom
Background.Activating mutations in Notch-1 are present in over 50%
of patients with T-cell acute lymphoblastic leukaemia (T-ALL). It has
been proposed that acquisition of these mutations may be an early event
in leukaemogenesis, committing common lymphoid progenitors towards
a T-cell fate before the acquisition of other secondary mutations. They
may thus represent potential targets for minimal residual disease (MRD)
analysis which can be limited, particularly in adults, by the inability to
identify suitable and stable gene rearrangements in the T-cell receptor
(TCR). Aims. To determine the stability of Notch-1 mutations in paired
presentation-relapse samples. Methods. Presence of Notch-1 mutations in
the HD-N, HD-C, TAD and PEST domains was investigated in 51 presentation
T-ALL samples (34 adults, 17 children) and 14 paired relapse
samples using PCR and denaturing HPLC (Transgenomic WAVE ® ).
Abnormal chromatograms were sequenced to identify the mutation,
with purification of low level mutations where necessary. Relative
mutant level was quantified in products with a size difference (insertions
or deletions) using fragment analysis (Beckman Coulter CEQ8000). T-cell
receptor (TCR) clonality was performed at the TCRα locus using standard
techniques. Same patient identity was confirmed in the paired presentation-relapse
samples using analysis at 4 short tandem repeat loci.
Results. At presentation a total of 49 mutations were detected in 36
patients (70%), 12 of whom had more than one mutation. Seven of the
mutations appeared to be low level from the WAVE pattern. Quantification
was possible in four of them and confirmed that the relative mutant
level was 6-10% of total despite the presence of greater than 85% blasts.
Furthermore, 3 of the patients with low level mutations also had a high
level mutation in another domain, suggesting the former were acquired
as a secondary event in a subclone. Of 14 matched pairs, 7 were wild type
at both presentation and relapse. Four of the 7 mutant-positive patients
at presentation relapsed with the same mutation(s), with no change in
mutant level which was high. Three patients had evidence of a change
in mutant at relapse. One patient lost a low-level HD-C mutation but
gained a high level HD-N mutation which could not be detected at presentation
using mutant-specific qPCR at a sensitivity of 1×10 –4 . The patient
had the same two TCR Vα1 clones at both presentation and relapse.
Another patient lost a high level PEST mutation at relapse whilst a low
level HD-C mutation progressively increased at both first and second
relapse (6%, 21% and 33% respectively). The same TCR Vα11 clone
was detected at each time point. A third patient lost both a high level HD-
N and PEST mutation and gained a different PEST mutation; in this case,
the TCR clone at relapse was different, suggestive of a secondary T-ALL.
Summary. These data suggest, at least in some cases, Notch-1 mutations
post-date the TCR rearrangement and can be acquired as a secondary
event. The loss of mutations at relapse in 3 of 7 mutant-positive patients
indicates that caution must be exercised in using them as single MRD targets.