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

safety of a slow-release liposomal formulation of cytarabine for intrathecal
(IT) meningeal prophylaxis in patients suffering from ALL. Patients and
Results. From 7/2004 to 01/2007 17 patients aged 20 to 65 years (median=
37) were preventively treated with a total of 44 (range:1-6) single
doses containing 50 mg of liposomal cytarabine on a compassionate use
basis. Diagnoses consisted of Pro-B-ALL (n=6), c-ALL (n=2), T-ALL (n=
6), and Pre-B-ALL (n=3). All patients were treated according to the risk
adapted German Multicenter Protocol/ GMALL 07/2003 including 24
Gy irradiation to the neurocranium as constituent component of CNS
directed therapy. 4 patients expressed the bcr-abl fusion oncogen and
were concomitantly treated with imatinib mesylate as outlined in the
study protocol. All patients received dexamethason for 3'5 days in order
to prevent chemical arachnoiditis. Except for headache grade 2 in one
patient no specific toxicity attributable to IT liposomal cytarabine application
was noted. So far, after an observation period of 11 months (1-
27) 6 patients died, 5 of disease recurrence and one of sepsis. 2 patients
show a refractory course of disease. 9 patients are in complete remission,
one after successful salvage therapy of first relapse and 5 after allogeneic
stem cell transplantation. Only one patient experienced a combined
medullary - leptomeningeal disease recurrence 6 month after primary
diagnosis. None of the surviving ALL patients developed neurological
symptoms or unexpected long term neurological side effects. Conclusions.
IT liposomal cytarabine therapy at a dose of 50 mg with concomitant
dexamethasone appears to be feasible and well tolerated. However,
since all patients received concurrent systemic chemotherapy and
CNS directed irradiation the efficacy of liposomal cytarabine cannot be
assessed separately. Further studies are warranted to determine the exact
schedule for IT prophylaxis in adult patients with ALL.
0024
GENE EXPRESSION PROFILING IN E2A-PBX1-SILENCED T(1;19)+ LEUKEMIA CELLS
G. Casagrande, G. Basso
University of Padova, PADOVA, Italy
Background and Aims. The translocation t(1;19)(q23;p13) is one of the
most common ones in childhood pre-B acute lymphoblastic leukemia
and usually results in the chimeric gene E2A-PBX1. Through extensive
studies, E2A-PBX1 has been shown capable of transformation in fibroblast,
myeloid, and T lineage cells, but its role during leukemogenesis is
not yet fully understood (Fu X, Kamps MP. Mol Cell Biol 1997; Kamps
MP, Baltimore D. Mol Cell Biol 1993; Dedera DA, et al. Cell 1993). We
initially silenced E2A-PBX1, achieving an efficient downregulation of
this fusion in several pre-B leukemia cell lines and obtaining as consequence
a strong downregulation of the Wnt16b and EB-1 genes. The
presented project aims to clarify the mechanism of E2A-PBX1 in
leukemia cells also through the elucidation of its interactions with a panel
of gene involved in tumorigenesis and transformation, based on differential
gene expression array in pre-B acute lymphoblastic leukemia cell
lines (in normal conditions as well as during RNAi silencing). Methods.
The transfection of small interfering RNAs (siRNAs) was monitored by
fluorescence microscopy and FACS analysis. The specific mRNA expression
of the fusion gene was measured using TaqMan real-time quantitative
PCR, while the reduction of the correspondent protein levels was
assessed by Western Blot. To better understand the role of E2A-PBX1 in
human pre-B cell leukemogenesis, the study focused also on genes
whose expression invariantly accompanies the t(1;19) translocation, and
their transcripts were detected by SYBR Green PCR. The expression
profiles were obtained through PCR arrays, considering eighty-four
genes representative of different biological pathways. Results and Conclusions.
The downregulation induced by anti-E2A-PBX1 siRNA in the
fusion gene expressing cells reduced the specific transcript expression by
85-90% (ABL-normalized levels were measured 24 hours after transfection).
In particular, the fusion gene depletion downregulated other two
genes: EB-1, which encodes for a protein that could contribute to the
transformed phenotype of pre-B ALL, and Wnt16b (not the Wnt16a isoform
of the Wnt16 gene); their aberrant expression may therefore be a
key-step in leukemogenesis in t(1;19)-positive pre-B leukemia. The roles
of E2A-PBX1 target genes was characterized through differential gene
expression PCR arrays, investigating genes involved in tumorigenesis,
transformation, growth regulation, proliferation, and allowed to focus on
the most significant pathways and on the interactions of E2A-PBX1 with
specific signal trasduction molecules, transcription factors and target
genes.
12 th Congress of the European Hematology Association
0025
TOLERANCE TO METHOTREXATE THERAPY IN CHILDHOOD ACUTE LYMPHOBLASTIC
LEUKAEMIA: IMPORTANCE OF GENETIC POLYMORPHISMS IN FOLATE METABOLIC
PATHWAY
E. Oliveira, 1 S. Quental, 2 F. Ferreira, 3 S. Alves, 4 V. Gomes, 1 A. Amorim, 2
M.J Prata 2
1 IPATIMUP, PORTO, Portugal; 2 IPATIMUP/FCUP, PORTO, Portugal; 3 Hospital
S. João, Hematologia Clinica, PORTO, Portugal; 4 Instituto de Genética
Médica, PORTO, Portugal
Background. Present days, approximately 80% of children diagnosed
with acute lymphoblastic leukemia (ALL) are cured. Almost all therapeutic
protocols use methotrexate (MTX) as antifolate agent and this drug
is administred in high doses (5 g/m 2 ) during interval therapy. It has been
observed that only some patients experience therapy-related toxicity,
developing intolerance to MTX such as hepatic toxicity and/or mucositis.
MTX targets several critical aspects of folate metabolism inhibiting
enzymes in the folate cycle and leading to the decrease of folate pool.
The folate metabolic pathway is crucial in purine and pyrimidine synthesis,
as well as in the provision of methyl groups for DNA, RNA and
protein methylation. SNPs in genes encoding enzymes involved in
methotrexate metabolism have been implicated in relapse or toxicity in
ALL patients. Aims. We hypothesized that toxicity to MTX could be
influenced by genetic polymorphisms in the folate-metabolizing pathway
because MTX induces a low folate state. In this work we investigated
how seven genetic polymorphisms in five genes encoding
enzymes involved in folate metabolism (Methylenetetrahydrofolate
Reductase - MTHFR, Thymidylate Synthase - TYMS, Methionine Synthase
- MS, Methionine Synthase Reductase - MTRR and Serine Hydroxymethyltransferase
- SHMT), influence the response to MTX in childhood
ALL therapy. Methods. Using PCR/RFLP and direct sequencing
based methods, we characterized DNA sample from 34 children diagnosed
with ALL, and retrospectively analyzed their clinical histories. All
children were following the same protocol for ALL therapy. Intolerance
to MTX was defined as the occurrence of hepato-toxicity (increase in liver
transaminases - TGO/TGP) and mucositis. Results. The MTRR C524T
variant was found to be significantly associated with a decrease of
mucositis during MTX administration and an over-representation of the
MTHFR C677T allele in the group of patients who developed mucositis
was also observed although not reaching statistical significance. Conclusions.
While these results seem to indicate that pretherapeutic testing
of MTRR and MTHFR may predict toxicity reactions to MTX treatment
in children undergoing ALL therapy, enlargement of sample sizes (which
is currently ongoing) is needed to validate these preliminary observations.
0026
DASATINIB INDUCES RAPID AND DURABLE RESPONSES IN PATIENTS WITH PH+ ALL
RESISTANT OR INTOLERANT TO IMATINIB: UPDATED RESULTS FROM CA180015
(START-L) TRIAL
O. Ottmann, 1 A. Hochhaus, 2 G. Saglio, 3 R. Paquette, 4 B. Simonsson, 5
K. Porkka, 6 J.M.A. Van Tornout, 7 A.M. Apanovitch, 7 P. Rousselot8 1 Johann Wolfgang Goethe Universitaet, FRANKFURT MAIN, Germany; 2 University
Heidelberg, MANNHEIM, Germany; 3 Universita di Torino, ORBAS-
SANO-TORINO, Italy; 4 Universite de Montreal, MONTREAL, Canada;
5 Uppsala University Hospital, UPPSALA, Sweden; 6 University of Helsinki,
HELSINKI, Finland; 7 Bristol-Myers Squibb Co., WALLINGFORD, USA;
8 Hôpital Saint-Louis, PARIS, France
Background. Relapsing patients with Ph + ALL who have previously
received treatment with imatinib and/or chemotherapy have a particularly
poor prognosis. Dasatinib is a novel multi-targeted kinase inhibitor
of BCR-ABL and SRC family kinases, with in vitro potency some 325-fold
greater than imatinib against BCR-ABL and proven efficacy in this
patient population. Aims. This study was designed to assess the efficacy
and safety of dasatinib. Methods. Phase-II, open-label, international,
multicenter study where patients with Ph + ALL with resistance or intolerance
to imatinib were treated with dasatinib 70 mg BID, administered
orally. Dose escalation to 100 mg BID was allowed for inadequate
response, and dose reduction to 50 mg or 40 mg BID for adverse events.
All patients provided written informed consent. Results. From January
through July 2005, 46 patients (median age 48 years; 59% male) enrolled
and received treatment, 96% of whom were imatinib-resistant; 46%
had received doses of >600 mg/d per day, and 52% had received imatinib
therapy for ≥12 months. Median time from initial diagnosis of Ph +
ALL was 18 months and 37% of patients had undergone prior stem-cell
haematologica/the hematology journal | 2007; 92(s1) | 9