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

0164
EVALUATION OF MULTIPLEX LIGATION-DEPENDENT PROBE AMPLIFICATION (MLPA) TO
DETECT GENOMIC DELETIONS IN CHRONIC LYMPHATIC LEUKEMIA A COMPARISON
WITH CYTOGENETICS, FISH AND PCR
P. Irnstorfer, 1 R. Marschon, 1 S. Heuritsch, 1 , K. Enzenhofer, 2
S. Hochreiter, 2 G. Webersinke 1
1 Krankenhaus der Barmh. Schwestern, LINZ, Austria, 2 Institut für Bioinformatik
JKU Linz, LINZ, Austria
Background. MLPA is a new method to detect losses or gains of genes
or gene fragments. CLL ist the most common leukemia in the western
world. Genetic variances, like deletions, are important prognostic factors
in this form of leukemia. Aims.Was to test the clinical applicability of
MLPA in CLL-cases in comparison with established standards for risk
evaluation like cytogenetics and FISH. Methods. Peripheral blood and
bone marrow samples were collected from 70 CLL patients at various
stages of disease, 22 healthy persons and a human B cell lymphoma cell
line (DOHH-2) showing characteristic chromosomal aberrations as a
control. If possible, karyotyping by GTG banding was performed. The
samples were checked for CLL typical chromosomal aberrations with a
FISH panel containing probes for 11q, 13q, 17p and CEP12. For MLPA,
20-500 ng of genomic DNA were used. After hybridisation of the probes
to their target sequences, a ligation reaction was carried out. The ligation
products were amplified by PCR. The CLL optimised MLPA-Kit
contains probes for the detection of del(11q) (ATM), del(17p) (TP53),
Trisomy 12, Trisomy 19, del(13q), amplification of 2p24 (MYCN), and
6q rearrangements. 1 µL of the PCR product was analysed on an ABI3130
Genetic Analyzer and evaluated with the SeqPilot v1.3.1 program. In
cooperation with the Institute for Bioinformatik of the Johannes Kepler
University of Linz a Feature Selection Analyse with the P-SVM (Potential
Support Vector Machine) program was performed to detect markers
predicting CLL. For this, the raw data from the ABI 3130 was converted
into Excel files and analysed with this program. Results. The relative
peak area (RPA) represents difference of the peak area of the patient file
and the median of the peak area of 19 control data. All RPA data below
75% were scored as deleted, whereas all data over 125% were scored
as amplified. The present MLPA data generated by the SeqPilot Program
show significant correlation to FISH results and karyotyping. Furthermore
we have tested the sensitivity for detecting aberrations in cell
mosaics because this is a common observation in CLL. At least 30 percent
mosaicism is well detectable in cell dilution experiments with the
DOHH-2 cellline; the limited sensitivity has to be considered in the clinical
setting. The (P-SVM) feature selection analyses showed that MYC
(localised on chromosome 8) was the best marker to predict CLL in samples
of unknown origin, 49 of the 70 analysed CLL patient samples are
predicted right (70%). 20 of the 22 control samples were predicted as
negative (91%). Summary and Conclusions. This study shows that MLPA
may be a helpful tool for the assessment of losses or gains of gene fragments
in CLL, although a solitary application of this method in routine
diagnostic is not practical at the moment. The result of the P-SVM shows
that MYC may be an interesting marker in the diagnosis of CLL.
0165
HEMOCHROMATOSIS GENOTYPES AND RISK OF 31 DISEASE ENDPOINTS -
META-ANALYSES INCLUDING 66,000 CASES AND 226,000 CONTROLS
C. Ellervik, 1 H. Birgens, 1 A. Tybjærg-Hansen, 2 B.G. Nordestgaard1 1 Herlev Hospital,University of Copenhagen, HERLEV; 2 Rigshospitalet, University
of Copenhagen, COPENHAGEN, Denmark
Background. Hemochromatosis genotypes have been associated with
liver disease, diabetes mellitus, heart disease, arthritis, porphyria cutanea
tarda, stroke, neurodegenerative disorders, cancer, and venous disease,
but studies have reported conflicting results. Aims. We examined associations
between hemochromatosis genotypes C282Y/C282Y,
C282Y/H63D, C282Y/wild type, H63D/H63D, H63D/wild type vs. wild
type/wild type and 31 disease endpoints. Methods. Meta-analyses including
202 studies with 66,263 cases and 226,515 controls were conducted.
Potential sources of heterogeneity were explored. Results. For liver disease,
the odds ratio for C282Y/C282Y vs. wild type/wild type was
3.9(99% CI: 1.9-8.1) overall, 11(3.7-34) for hepatocellular carcinoma,
4.1(1.2-14) for hepatitis C, and 10(2.1-53) for nonalcoholic steatohepatitis.
For porphyria cutanea tarda, the odd ratios were 48(24-95) for
C282Y/C282Y, 8.1(3.9-17) for C282Y/H63D, 3.6(1.8-7.3) for C282Y/wild
type, 3.0(1.6-5.6) for H63D/H63D, and 1.7(1.0-3.1) for H63D/wild type
vs. wild type/wild type. Finally, for amyotrophic lateral sclerosis the
odds ratio was 3.9(1.2-13) for H63D/H63D vs. wild type/wild type.
These findings were consistent across individual studies. Conclusions. In
aggregate, C282Y/C282Y associated with liver disease, all 5 hemochromatosis
genotypes associated with porphyria cutanea tarda, while
H63D/H63D associated with amyotrophic lateral sclerosis.
Figure 1.
12 th Congress of the European Hematology Association
0166
IDENTIFICATION OF HISTONES H2B AND H4 OVEREXPRESSION IN MANTLE CELL
LYMPHOMA BY MASS SPECTROMETRY-BASED PROTEOMIC STUDY
D. Rolland, 1 A. Bouamrani, 1 A. Barbarat, 2 M. Arlotto, 1 F. Berger, 3
P. Felman, 2 S. Brugière, 4 J. Garin, 4 R. Houlgatte, 5 F. Berger, 1
C. Thieblemont6 1 INSERM U836 (équipe 7), GRENOBLE, France; 2 Laboratoire d'hématologie,
CHLS, LYON; 3 Laboratoire anatomo-pathologie, CHLS, LYON; 4 EMR-0201
INSERM/CEA, GRENOBLE; 5 INSERM U533, NANTES; 6 Hématologie,
hôpital Saint Louis, PARIS, France
Background. Mantle cell lymphoma (MCL) is a non germinal centre
small B-cell lymphoma that represents about 6-10% of all non-Hodgkin
lymphomas (LNH). The MCL genetic hallmark is the chromosomal
translocation t(11;14)(q13;q32) leading to the overexpression of cyclin D1
an important regulator of the G1 phase of the cell cycle. However, the
cyclin D1 overexpression is not sufficient to induce MCL. Additional
genetic alterations may occur in subsets of MCL. Most of these alterations
appear to disturb the cell cycle machinery / interfere with the cellular
response to DNA damage, thus making MCL a paradigm for cell
cycle and DNA damage response deregulation in cancer in general. Aims.
Our goal was to expand the current understanding of the molecular
pathogenesis of MCL using proteomic strategy and to provide the basis
for identification of biomarkers specific to MCL compared to other small
B-cell lymphomas. Methods. Using Surface Enhanced Laser
Desorption/Ionization - Time of Flight (SELDI-TOF) technology the
MCL proteome was compared to the proteome of 2 other non germinal
centre small B-cell lymphomas, the small lymphocytic lymphoma (SLL)
and the marginal zone lymphoma (MZL). Whole cell lysates obtained
from 18 MCL (17 nodes and 1 spleen), 20 SLL (20 nodes), 20 MZL (1 node
and 19 spleens) fresh frozen biopsies and 7 traumatic normal spleens
were applied on two different ProteinChip array surfaces (CM10 and
Q10) that were bombarded by 4 different laser intensities. All spectra
were aligned and peak intensities were normalized to the total ion current
(M/Z