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

12 th Congress of the European Hematology Association
in the disease pathogenesis, in particular the role of a cytoplasmic tyrosine
kinase JAK2. It has been shown that >80% of Polycythaemia Vera
(PV) patients and 30% of Essential Thrombocythaemia (ET) patients
demonstrate a clonal and recurrent mutation V617F in the JH2 pseudokinase
domain of JAK2 located on 9p24.1. Array CGH investigations of
cell lines, established from lymphoid and myeloid blast phase samples
revealed a complex pattern of imbalances affecting the short arm of
chromosome 9: on the background of 2-3-fold amplification, a loss of the
9p24.1 region, which harbours the JAK 2 along with a number of other
kinases, was repeatedly observed. The aim of this investigation was to
clarify the aCGH observations using BAC DNA probes from the 9p24.2
and 9p23 region. FISH mapping was carried out to map the JAK2 gene
and the flanking sequences. Results so far indicate the presence of both
imbalances (amplifications & deletions) and rearrangements involving
the JAK2 region in 5 cell lines. This work is now being extended to
include BAC clones telomeric and centromeric to JAK2. An on going
FISH study of CMPD patients (CML, PV, ET and MF) with normal and
abnormal karyotypes has revealed 9p24 imbalances in 18% (5 out of 28).
The pathegenomic significance of aberrations found in the region surrounding
JAK2 has yet to be defined and our study demonstrates the
importance of molecular cytogenetics in exploring the importance of 9p
in the CMPD disease model.
0573
ANALYSIS OF 482 PATIENTS WITH ACUTE MYELOID LEUKEMIA (AML) FOR THE IDENTIFI-
CATION OF MUTATIONS IN THE PROMOTER REGION AND THE CODING SEQUENCE OF
THE CEBPA GENE BY CAPILLARY ELECTROPHORESIS
C. Juhl-Christensen, 1 M. Bomberg, 1 D. Melsvik, 1 M. Oestergaard, 1
H. Hasle, 2 P. Hokland, 1 C. Nyvold1 1 2 Laboratory of Immunohematology, AARHUS; Department of Pediatrics,
AARHUS, Denmark
Background. The CEBPA gene encodes a transcription factor,
CCAAT/enhancer binding protein (C/EBP)α. The wildtype (WT) protein
is essential for the lineage specific differentiation of myelocytic
haematopoietic precursors into mature neutrophils. Approximately 9%
of all AML patients harbour one or more mutations in this gene, while
the frequency in the cytogenetically negative group of patients is reported
to comprise up to 15%. Until recently, the mutational status of the
gene has been determined using cloning and sequencing. Aims. We
designed a fragment analysis using capillary electrophoresis that enabled
us to discriminate as little as a single base pair insertion or deletion
accounting for about 90% of all CEBPA mutations. Methods. Diagnostic
bone marrow (BM), or when not available peripheral blood (PB), from
444 adults and 38 children diagnosed with AML at a single centre from
1980 to date and from whom bio banked material was available were
assessed for mutations employing fragment analysis. Mononuclear cells
were isolated, cryopreserved, and stored until use at -80°. Genomic DNA
was extracted from approximately 1 million mononuclear cells and eluted
in a volume of 100 µL using MagNa-Pure LC Robot (Roche Diagnostics,
Basel CH). Four overlapping and tailed primer pairs were designed
to cover the entire coding sequence and the region upstream the intronless
gene using Oligo (Primer Analysis Software, version 6.83, Molecular
Biology Insights, CO, USA) and purchased from Applied Biosystems
(Foster City, CA, USA). PCR was performed for each primer pair and
subsequently the PCR products for each patient were pooled and transferred
to capillary electrophoresis on a 3130 Genetic Analyzer (Applied
Biosystems). The T-Cell line 8402 was included as a wildtype control.
The results were analysed using GeneMapper software version 3.7
(Applied Biosystems). Samples harbouring insertions or deletions were
identified and sequenced directly on both forward and reverse strand.
Results. We analyzed 482 patients hereof 38 children. In total, we identified
45 abnormal PCR fragments in the CEBPA gene in 37/444 (8,3%)
and 1/38 (2,6%) adult and childhood AML patients respectively. Using
the method described above we were able to distinguish between Nand
C- terminal mutations and reached the same mutational frequency
as described in the literature. Conclusions. We conclude that fragment
analysis by capillary electrophoresis can be used as an easy and high
throughput diagnostic procedure for the mutational status in the CEB-
PA gene.
214 | haematologica/the hematology journal | 2007; 92(s1)
0574
GENE POLYMORPHISMS OF METHYLENTETRAHYDROFOLATREDUCTASE IN MALIGNANT
DISEASE
R. Simanek, R. Vormittag, P. Bencur, C. Ay, G. Alguel, C. Zielinski,
U. Jaeger, I. Pabinger, C. Mannhalter
Medical University of Vienna, VIENNA, Austria
Background. Methylentetrahydrofolatreductase (MTHFR) plays a role
in the human folate metabolic pathway. The MTHFR 677 C>T polymorphism
is associated with a reduced enzyme activity, which leads to a
reduced probability of DNA double-strand breakage. A number of studies
focused on the polymorphism’s influence as a risk factor for different
malignancies, however, data are conflicting. Aims. It was the aim of
our study to analyse the prevalence of mutations in patients with malignant
disease and healthy controls. Methods. Between October 2003 and
July 2006, 427 patients with solid malignant tumours (225 female/202
male; mean age=62y) of the breast, lung, stomach, colon and prostate
and 80 patients with haematological malignancy (32 female/48 male;
mean age=56y) including Hodgkin’s lymphoma, non-Hodgkin’s lymphoma
and multiple myeloma were enrolled. Seventy-four healthy controls
were matched for age and sex (36 female/38 male, mean age=61y).
MTHFR genotype was analysed with MS-PCR. Results. Genotype distribution
was in Hardy-Weinberg equilibrium and 41% CC, 45% CT
and 13% TT in patients with solid tumours, 38% CC, 54% CT and 9%
TT in those with haematological malignancies and 31% CC, 51% CT
and 18% TT in healthy controls (p-values for pair-wise comparisons
not significant). TT genotype was not associated with a significantly
reduced risk for malignancy in univariate analyses (solid tumours:
OR=0.6 [95% CI=0.3-1.2], p=0.14, haematological malignancies:
OR=0.4 [0.1-1.2], p=0.10, respectively). In a subgroup analysis for colorectal
carcinoma (n=97, 46% CC, 44% CT and 9% TT) MTHFR 677
TT was associated with a significantly lower risk in univariate (OR= 0.35
[0.13-0.95], p=0.039) and bivariate analyses corrected for age (OR=0.37
[0.137-0.997], p=0.049). Summary/Conclusions. MTHFR 677 C>T polymorphism
seems to modulate the risk for selected malignancies, but it
does not seem to influence the risk for developing cancer in general.
0575
A HIGHLY SENSITIVE METHOD FOR DETECTION OF HAEMOPOIETIC CHIMERISM IN
ALLOGENEIC HAEMOPOIETIC STEM CELL TRANSPLANT RECIPIENTS
A. Abdallah, 1 L. Pearce, 2 A. Mijovic, 3 G. Mufti, 2 K. Tobal2 1 National Blood Bank Service, King’s College Hospital LONDON; 2 King's
College London, Rayne Institute, LONDON; 3 National Blood Bank Service,
LONDON, United Kingdom
Background. The ability to detect donor cell engraftment in reduced
intensity conditioning allogeneic hematopoietic stem cell transplant
recipients with high sensitivity would enable early detection of patients
at high risk of relapse. Methods. Sixteen specific biallelic polymorphic
markers were selected to identify informative markers for analysis of
chimerism in transplant donor/recipient pairs. Four informative markers
were then used in SYBR green Q-PCR to quantify post-transplantation
chimerism in serial samples from these patients. The percentage of
donor cells was calculated using a standard curve, constructed using artificially
mixed donor/recipient chimeric DNA in 8 serial dilutions (0.01-
100%). A linear correlation with r higher than 0.98 and a sensitivity of
0.1% proved reproducible. DNA from 9 donor/recipient pairs was retrospectively
screened for informative markers and 38 post-transplant
samples were monitored for chimerism using the SYBR green Q-PCR
method. Results. We were able to find at least 4 different informative
markers for 7 donor/recipient pairs, 2 markers for 1 donor/recipient pair
and 1 marker for 1 donor/recipient pair. These results were compared
to the existing method using PowerePlex 16 microsatellite STR analysis
kit (Promega). The detection limit of the Q-PCR method was 0.1%,
which is significantly higher than that achieved with STR methodology
(5%). This high sensitivity enabled us to predict relapses up to 4
months earlier than the STR method. Conclusions. this assay is highly
sensitive and provides an accurate quantitative assessment of mixed
chimerism that can be useful in evaluating patients’ response to transplantation
and distinguishing those at high risk of relapse at an early
stage to enable implementation of additional treatments. Detailed analysis
of data from large cohort of transplant patients will be presented.