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

1063
ABNORMAL METHYLATION STATUS OF THE PROMOTER OF APAF-1 IN DE NOVO AML
PATIENTS
A. Valencia, J. Cervera, E. Such, Z. Garcia-Casado, J.C. Pajuelo,
F. Moscardó, M.L. Paciello, M. Scaff, G. Orti, G.F. Sanz-Santillana,
M.A. Sanz
Hospital La Fe, VALENCIA, Spain
Background. Apaf-1, the apoptotic protease activating factor 1, is an
important signaling protein involved in the activation of the Caspase 9.
During DNA damaging stimuli-induced apoptosis, Apaf-1 is released in
the citosol from its interaction with Bcl-2. Apaf-1 forms the so called
apoptosome complex with Pro-Caspase 9 in the presence of cytosolic
cytochrome c and dATP. This association ultimately leads the Caspase
9 activation and subsequent activation of Caspase 3, one of the key executioners
of apoptosis. It has been reported that the inactivated Apaf-1
gene in malignant melanoma could be switch off by DNA methylation
and can be turned back by inhibitors of DNA methylation. Similar findings
found in other malignances point to the possibility that abnormalities
of Apaf-1 play a role in the pathogenesis of a wide variety of cancer.
Besides, recently it has been shown that methylation silencing is a
mechanism of inactivation of Apaf-1 in several human leukemia-lymphoma
cell lines (Furukawa et al. Mol Cancer Res 2005;3(6):325). Objective.
Studying the abnormal methylation status of the promoter of Apaf-
1 in patients with de novo AML by means of the methylation specific PCR
method (MSP) (Herman et al. Proc Natl Acad Sci USA,1996; 93:9821).
Patients and Methods. We have studied bone marrow samples from 88
patients at the time of diagnosis [(48 M / 40 F, median age: 61 yr (range:
18-88), FAB subtype: 0 M0, 18 M1, 23 M2, 10 M4, 15 M5, 14 M6, 1 M7,
2 unknown)]. Genomic DNA was extracted using standard protocols
(Quiamp DNA Mini kit). After bisulfite technic prodedure (Epitect Bisulfide,
Quiagen) the DNA was PCR amplified with primers specific for the
methylated and unmethylated alleles of the gene. The PCR products
were separated on 2% agarosa gel. Bone marrow DNA from healthy
donors was used as negative control. CpGenome Universal Methylated
DNA (Chemicon, Millipore) was used as a positive control. Results.
Aberrant methylation of Apaf-1 promoter region was found in 19 out of
88 samples (22%). Hypermethylation of Apaf-1 did not correlated significantly
with any clinical, biological, morphological, immunophenoypic
or molecular characteristics between the methylated and unmethylated
group. Moreover, no significant differences in overall survival and
relapse risk were observed between both groups. Conclusions. In agreement
with previous studies carried out in AML cell lines our data show
that hypermethylation of the promoter of Apaf-1 is a frequent event in
de novo AML patients. However, no correlation with relevant clinical or
biological data was found. Relevance of this finding for the treatment
with hypomethylating agents should be explored in the clinical setting.
This study was partially supported by the grants AP: 098/06 (Generalitat
Valenciana), BEFI 03/200, FIS PI030400, FIS 060657 (ISCIII).
1064
ABT-737 ACTIVITY ON PRIMARY MULTIPLE MYELOMA SAMPLES
M.R. Ricciardi, 1 C. Gregorj, 1 F. De Cave, 1 E. Calabrese, 1 S. Santinelli, 1
V. Federico, 1 P. Bergamo, 1 S. Decandia, 1 M. Milella, 2 R. Foà, 1 A. Tafuri, 1
M.T. Petrucci1 1 2 University La Sapienza of Rome, ROME, Italy; Regina Elena National Cancer
Institute, ROME, Italy
Background. The bcl-2 family proteins are key regulators of cell survival
and are frequently found aberrantly expressed in lymphoid malignancies
and in multiple myeloma (MM) conferring resistance to chemotherapy.
Aims. In the present study we investigated the cell cycle and apoptotic
effects of ABT-737 (kindly provided by Abbott Laboratories), a Bcl-2 (BH3)
inhibitor, on MM cell lines and on primary CD138+ malignant plasma
cells. Results. The KMS18 MM cell line was exposed to increasing concentrations
of ABT-737 (from 0.1 to 1 µM) up to 72 hours. A dose- and timedependent
cell growth inhibition was documented (IC50s 0.286 microM
at 72 hours) due to a significant increase of apoptotic cells as demonstrated
by the increment of Annexin V+ cells, at 24 hours, from 17.3%±4.4 in
DMSO to 31.7%±12.9, 45.8%±9.1 (p=0.032), 49.8%±7.8 (p=0.017) and
60.8%±5.8 (p=0.0026) in the presence of ABT-737 at 0.1, 0.25, 0.5 and 1
microM, respectively. These data were confirmed by measuring the
subG0/1 peak (Acridine-Orange). Cell cycle analysis demonstrated at 72
hours a significant G1-phase depletion: from 54.6%±4.5 (DMSO) to
26.9%±1.4 (1 µM ABT-737) (p=0.021). The effects of ABT-737 were then
examined on primary cells from untreated patients: 6 MM and 1 plasma
12 th Congress of the European Hematology Association
cell leukemia (PCL). Bone marrow aspirate cells, following CD138 enrichment
(>80% of purity), were cultured in vitro with ABT-737 (at scalar concentrations
from 0.1 to 1 µM) up to 72 hours. Effective apoptosis was
observed in all 6 MM samples. Importantly, low concentrations (0.1
microM) of ABT-737 significantly (p=0.01) increased CD138 + apoptotic
cells from 19.8%±9.0 to 52.9%±17.2 (24 hours) in MM samples. ABT-
737 triggered similar effects in the PCL sample (a 3- and 5-fold increase of
apoptotic cells at 0.1 microM and 0.5 microM). Summary. In conclusion,
ABT-737 shows potent in vitro growth-inhibitory and pro-apoptotic activity
at nanomolar concentrations in MM cells, indicating that it has great
potential for the treatment of this disease. A further pre-clinical/clinical
development of this compound is warranted.
1065
JAK2 V617F MUTATION SCREENING IN PATIENTS WITH CHRONIC MYELOPROLIFERATIVE
DISORDERS AND CORE BINDING FACTOR ACUTE MYELOID LEUKEMIAS (CBF-AML)
J. Marková, 1 P. Michková, 1 J. Star˘, 2 J. Schwarz1 1 Institute of Hematology & Blood Transf., PRAGUE 2, Czech Republic; 2 Dept.
of Pediatric Hematology & Oncology, PRAGUE 5, Czech Republic
Background. An acquired V617F mutation of the JAK2 gene is of diagnostic
and prognostic value in myeloproliferative disorders (MPD). Low
incidence of JAK2 mutation has been also described in patients with
CBF-AML, negatively affecting their prognosis. Aims. We have updated
our results of JAK2 V617F mutation screening in a cohort of 604 patients
with suspected Ph- MPD and 50 patients with CBF-AML. Methods. For
detection of the JAK2 V617F mutation, allelic discrimination real-time
RT-PCR assay with specific locked nucleic acid (LNA)-modified Taq-
Man probes was used. The expression of AML1/ETO and CBFβ/MYH11
in CBF-AML was assessed by quantitative real-time PCR. C-KIT mutations
in AML patients were determined by gel electrophoresis and direct
sequencing. Results. 604 samples of patients with suspected Ph- MPD
were analyzed. In 267 patients (44.2%), JAK2 mutation was detected. 29
of 267 JAK2 mutations (10.9%) were homozygous. Out of 113 patients
suffering from polycythemia vera (PV), 112 had mutations (99.1%),
which were homozygous in 21 cases (18.8%). In WHO criteria-defined
essential thrombocythemia (ET), JAK2 mutations were demonstrated in
33/63 (52.4%) patients, none of them was homozygous. 34 of 65
(52.3%) individuals with idiopathic myelofibrosis (IMF) had JAK2 mutations
(5 were homozygous). Of another 96 MPD patients with thrombocythemia
not discriminated according to WHO criteria, 77 (80.2%)
had detectable JAK2 mutations. Within patients with thrombocythemia
of unknown reason (i.e., patients that might have MPD or secondary
thrombocythemia), 5/90 (5.6%) had mutated JAK2 genes, whereas none
of 14 patients with well defined secondary thrombocythemia had their
JAK2 genes mutated. Of 73 patients with polyglobulia of unknown reason
(i.e. patients that might have PV, secondary polyglobulia, or idiopathic
erythrocytosis), none had mutated JAK2 genes. Likewise, 11 patients
with well defined secondary polyglobulia had their JAK2 genes unmutated.
Among the remaining 79 patients with miscellaneous diagnoses,
6 cases with JAK2 mutation were found. In the cohort of 50 CBF-AML
patients, having either AML1/ETO (n=29) or CBFβ/MYH11 (n=21)
fusion genes, 2 (4.0%) had JAK2 mutation. The first of them was 12-yearold
female with AML1/ETO positivity, diagnosed in July 2004. She carried
a heterozygous JAK2 mutation. After one month of induction
chemotherapy, she became JAK2 negative but remained AML1/ETO
positive, with a decreasing trend until December 2004 when she reached
molecular remission. In May 2005, she relapsed clinically with comparatively
high AML1/ETO expression as at diagnosis, but with undetectable
JAK2 mutation. She underwent allogeneic stem cell transplantation
in September 2005 and since November 2005 she remains in
molecular remission. The second patient was a 22-year-old male presenting
with extramedullar disease, AML1/ETO positivity, heterozygous
JAK2 mutation and D820G exon 17 C-KIT gene mutation. Following
induction chemotherapy, he became AML1/ETO and JAK2 negative.
Conclusions. Our results confirm that incidence of JAK2 mutation is
extremely high in PV (99%) and moderate (about 50%) in either ET or
IMF, in case allele-specific PCR is employed. The rather low percentage
of CBF-AML patients with detectable JAK2 mutation may share a course
with clinical problems (extramedullar disease, relapses).
Supported by the Czech Ministry of Health Research project 00237360001.
haematologica/the hematology journal | 2007; 92(s1) | 393