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

tinguish between genotypic B-cell leukemia subgroups with different
responses to chemotherapy. Methods. Methylation Specific PCR, RQ-
PCR, 5’-Aza-2’dC treatment, Western Blot. Results. Analysis of methylation
and gene expression patterns of DLX3 in 64 specimens of B-lineage
ALL revealed that DLX3 presents aberrant methylation in paediatric
B-ALL patients. in vitro experiments with 5’-Aza-2’dC on leukemia cell
lines, confirmed by western blot analysis, indicated that the methylation
of DLX3 CpG islands has a functional role and interferes with DLX3
gene and DLX3 protein expression in B-ALL cells. To validate this data
we studied two groups of patients with the two better known paediatric
chromosomal rearrangements: MLL-AF4 and TEL-AML1 respectively.
Hypermethilation of DLX3 significantly reduces its expression in
MLL/AF4 rearranged leukemias while methylation is almost absent in
TEL-AML1 positive ALL specimens. Conclusions. Our results show for the
first time the aberrant methylation of DLX3 gene and its functional inactivation
role in the specific cohort of paediatric B-ALL with the MLL-AF4
chromosomal aberration. This finding proposes DLX3 as a new epigenetic
candidate marker involved in leukemiogenesis of this high-risk
acute lymphoblastic leukemia.
0153
LBH589, A NOVEL DEACETYLASE INHIBITOR (DACI), IN THE TREATMENT OF CUTANEOUS
T-CELL LYMPHOMA (CTCL): CHANGES IN TUMOR GENE EXPRESSION PROFILES RELATED
TO CLINICAL RESPONSE AFTER THERAPY
Y. Pan, 1 H.M. Prince, 1 J. George, 2 R.W. Johnstone, 1 L. Ellis, 1
C. McCormack, 1 G.K. Smyth, 3 R. Williams-Truax, 2 P. Atadja, 4
D. Butterfoss, 5 M. Dugan, 5 K. Culver5
1Peter MacCallum Cancer Centre, EAST MELBOURNE, Australia; 2Duke
University, DURHAM, USA; 3Walter and Eliza Hall Institute, MELBOURNE,
Australia; 4Novartis Institutes for Biomedical Res, CAMBRIDGE, United States
of America, 5Novartis Pharmaceuticals, EAST HANOVER, USA
Background. In preclinical and Phase I studies, deacetylase inhibitors
(DACIs) have shown promise in the treatment of hematological malignancies,
in particular cutaneous T-cell lymphoma (CTCL); however, the
target genes affected by DACIs remain unknown. LBH589, a novel
DACI, is currently undergoing clinical trials for the treatment of CTCL.
Aims. To determine the maximum tolerated dose (MTD) and dose-limiting
toxicity (DLT) of oral LBH589 in patients with CTCL, and to characterize
the biologic activity of oral LBH589 when administered to this
patient population. Methods. This is an ongoing Phase I study of LBH589
in patients with advanced-stage CTCL. Patients were entered into either
an oral DLT regimen of 30 mg three-times weekly (M, W, F; n=1), or an
MTD regimen of 20 mg three-times weekly (M, W, F; n=9). Treatment
was discontinued in the event of disease progression or unacceptable
toxicity. Gene expression profiling using Affymetrix U133A plus 2.0
GeneChip? with 54,675 probe sets was carried out in 3-mm punch biopsies
of CTCL skin lesions taken from patients 0, 4, 8, and 24 hours after
LBH589 administration. F-statistics were used to adjust for differences
between the subjects, allowing for the prioritization of genes that
responded consistently over time to LBH589. Assessment of hyperacetylation
of histone H3 as a biological marker of LBH589 activity in patient
samples was conducted by immunohistochemistry and assessment of
blood mononuclear cells. Results. All 10 patients were evaluable for
response: 2 achieved a complete response (CR), 4 achieved a partial
response (PR), and 2 had stable disease (SD) (RR = 6/10; 60%). Median
duration of treatment was 4 weeks in the DLT arm and 23 weeks in the
MTD arm. Duration of response was 178.5 days for both arms. One
patient in the DLT arm experienced grade 4 diarrhea (n=1). Patients in
the MTD arm experienced grade 3 diarrhea (n=1), grade 3 neutropenia
(n=3), and grade 3 thrombocytopenia (n=1). Microarray data from 6
patients demonstrated that within individual patient tumors, alterations
in gene expression were observed at all time points in the first 24 hours
post-treatment. Global changes in gene expression patterns were also
observed when all patients and all time points were investigated. The following
were major findings following LBH589 treatment: there were
rapid changes in gene expression; more genes were repressed than were
activated; gene expression changes were observed in both responders
and non-responders. qRT-PCR confirmed array analysis. Hyperacetylation
levels were observed up to 72 hours following LBH589 administration.
Conclusions. LBH589 induces CR in patients with CTCL. Ongoing
disease regression is apparent weeks after discontinuation of therapy.
CTCL provides a unique scenario in which the effect of a drug on genes
can be observed over time using microarray analysis. Treatment with
LBH589 results in the differential expression of numerous genes, which
may help elucidate the biologic activity of LBH589 in the CTCL patient
population.
12 th Congress of the European Hematology Association
0154
WNT PATHWAY MUTATIONS IN ACUTE LEUKEMIA PATIENTS
M. Sayitoglu, O. Hatirnaz, F. Atalar, Y. Erbilgin, U. Ozbek
Istanbul University, ISTANBUL, Turkey
Aims. WNT signaling pathway proteins function as haematopoietic
growth factors and regulate proliferation in normal T-cell and B-cell
development. Not much is known about activation of this pathway, its
ligands and receptors in leukaemia pathogenesis. Genetic alterations in
WNT pathway associate with different types of cancers as well as acute
leukemias. Here we determined β-catenin expression levels and mutational
alterations in the β-catenin, AXIN1 and APC genes in acute
leukemia patients. Patients and Method; Peripheral blood and/or bone
marrow samples were taken at diagnosis. β-catenin mRNA levels were
determined by quantitative real time PCR (QRT-PCR). Acute lymphoblastic
(n=126) and myeloblastic leukemia patients (n=30) showed
~4 fold increased β-catenin mRNA levels. Using denaturing high-performance
liquid chromatography (DHPLC) analysis and DNA sequencing,
acute lymphoblastic leukemia and acute myeloblastic leukemia
patients were screened for inactivating WNT pathway mutations.
Sequence variations of AXIN1 (exons 1-5, APC, MKK, GSK3 β and βcatenin
binding domains) and APC (exon 15, AXIN and β-catenin binding
domains) were determined. Results and Conclusion; These variations
were found to be 35% for AXIN1 (exons 1-5) and 50% for APC
(exon 15) in ALL patients and 19% for AXIN1 and 54% for APC genes.
There was no β-catenin (exon 3) mutation in AML patients, and one
ALL patient have this mutation. Cytoplasmic B-catenin accumulation
frequently occurs in leukemia. According to our findings β-catenin mutations
are rare in leukemia samples. Our preliminary results also support
that, abnormal β-catenin accumulation via ligand-independent manner
in acute leukemias seem to closely associated with AXIN1 and APC
mutations.
0155
WNT5A GENE EXPRESSION AND PROMOTER METHYLATION IN ACUTE LEUKEMIA
PATIENTS
O. Hatirnaz, M. Sayitoglu, F. Atalar, Y. Erbilgin, U. Ozbek
Istanbul University, ISTANBUL, Turkey
WNT5A which is a member of WNT family, functions as a tumor
suppressor gene in haematopoietic tissue. It promotes intracellular Ca ++
release and activation of protein kinase C. WNT5A was found to be
over-expressed in various cancer types, where as acute lymphoblastic
(ALL) and acute myleoblastic (AML) leukemia patients showed loss of
WNT5A gene expression. Previous results indicated that WNT5A/Ca ++
dependent pathway suppresses cyclin D1 expression and WNT5A null
mice develop B-cell lymphoma and chronic myeloid leukemia. We determined
WNT5A and its downstream targets c-MYC and cyclin D1
expression levels in acute leukemia patient. Bone marrow and/or peripheral
blood samples were obtained from ALL (n=126) and AML (n=34),
at the time of diagnosis. Also normal bone marrow (n=6), normal peripheral
blood (n=10) and CD19 + cells samples were used as controls. The
relative expression levels of WNT5A, c-MYC and cyclin D1 genes were
detected by quantitative-real time PCR (QRT-PCR). Methylation status
of WNT5A promoter is determined by methylation specific PCR (MSP).
Our results indicated that the mRNA levels of WNT5A and cyclin-D1
were decreased, but level of c-MYC was increased. This confirms the
finding that c-MYC expression is independent of WNT5A presence, but
cyclin-D1 is WNT5A dependent. To investigate the decrease in WNT5A
mRNA expression levels, we performed MS-PCR and found hypermethylation
of WNT5A promoter in 86% of ALL’s and 85,7% of AML’s.
Loss of WNT5A gene dosage was observed in several primary tumors
depending on the homozygous deletions of WNT5A, RNA or promoter
silencing. Here we determined decreased WNT5A expression depending
on the WNT5A promoter hypermethylation in ALL and AML
patients.
haematologica/the hematology journal | 2007; 92(s1) | 55