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

12 th Congress of the European Hematology Association
tering software to identify specific proteomic signatures. SELDI-TOFassisted
purification followed by in gel trypsin digestion and LC-MS/MS
peptides identification was used to identify proteins overexpressed in
MCL tumors. Results. The combination of all data obtained with the
two different ProteinChip generated 1300 analyzable protein peaks. The
protein patterns were first analyzed using hierarchical clustering in an
unsupervised fashion revealing a very homogenous protein pattern
among all lymphoma samples. The second analysis using hierarchical
clustering in a supervised method (discriminating score) pointed out tissue
specific protein signatures (node and spleen). Those tissue signatures
were subtracted from the data and specific protein signatures for
SLL, MZL and MCL were found based on the expression level of 34
protein peaks. SELDI-TOF-assisted purification was used to optimize
protein purification before in gel digestion and LC-MS/MS identification.
We identified two core histones as overexpressed proteins in MCL
tumor biopsies: histone H2B and histone H4. Conclusions. The proteomic
profiling of MCL tumor biopsies using SELDI-TOF technology leads
to the characterization of a specific MCL protein signature. Among proteins
overexpressed in MCL, we identified histones H2B and H4. The
overexpression of these two core histones in MCL was concordant with
the cell cycle deregulation previously reported in this lymphoma entity.
0167
HIGH FREQUENCY OF MICROSATELLITE INSTABILITY IN LYMPHOID TUMOUR CELL
LINES
A. Siebert, 1 C. Mielke, 2 S. Fähnrich, 1 Y. Merkhoffer, 1 H.G. Drexler, 1
W.G. Dirks1
1 2 DSMZ, BRAUNSCHWEIG; Heinrich-Heine-University, DÜSSELDORF,
Germany
Background. The mismatch repair (MMR) system is specialized in
removing replication errors which are thought to be recognized by the
heterodimers MutSα (MSH2 and MSH6) and MutSβ (MSH2 and MSH3).
Defective MMR can be detected via microsatellite instability (MSI) and
causes diseases like HNPCC. Aims. With our studies we wanted to gain
insights in the frequency of MSI in tumour cell lines from different tissues
and in the contribution of defective expression of MSH2, MSH3 and
MSH6 to the MSI phenomenon. Methods. Fluorescence PCR in combination
with capillary electrophoresis was used to analyse 500 human
tumour cell lines for length shortening of BAT26, a marker for MSI detection.
MSI cell lines were further investigated for presence of transcripts
and proteins of MSH2, MSH3 and MSH6 by RT-PCR and western blot
analysis, respectively. cDNAs of MSH2, MSH3 and MSH6 were inserted
into a bicistronic vector allowing expression of each protein fused to
EGFP for localization studies by fluorescence microscopy. Results. MSI
was proven in 8% (41/500) of tumour cell lines. The relative amount of
MSI cell lines among cell lines of special tumour groups was significantly
highest in T-cell leukaemia (59%) and ovarian adenocarcinoma (50%),
followed by colon adenocarcinoma (25%) and B-cell leukaemia cell lines
(24%). Further investigation revealed presence of full-length transcripts
for MMR genes in 81% of MSI cell lines, while only 51% expressed the
corresponding proteins. In addition to MSH2, MSH3 and MSH6 fulllength
transcripts seven alternatively spliced variants were obtained
from MMR proficient cell lines. Cloning and stable expression of EGFP
fusion proteins with the wildtype transcripts of MSH2, MSH3 and
MSH6 and their variants showed that only MSH2 WT, MSH3-WT,
MSH3-V3, MSH3-V5, MSH6-WT and MSH6 V1 fusion proteins are
restricted to the nucleus, whereas expressed MSH2 V1, MSH6-V2 and
MSH6-V3 are mainly localized in the cytoplasm and MSH3-V4 fusion
protein is distributed all over the cell. For functional characterization
we set a UV-A beam to a defined area of the nucleus and live imaging
revealed an accumulation of MSH2-WT and MSH6-V1 fusion proteins
but not MSH3-WT fusion protein at the site of the UV-A damage. Summary
and Conclusions. The MSI phenomenon could be shown in an unexpected
high number of T- and B-cell leukaemia cell lines. Genomic deletions
or epigenetic silencing of MSH2, MSH3 and MSH6 genes are not
the predominant mechanisms responsible for absence of MMR activity,
indicating that loss of function mutations may play a bigger role. 10
wildtype and variant transcripts for MSH2, MSH3 and MSH6 demonstrated
diverse localization properties upon heterologous expression,
suggesting additional functions or regulatory mechanisms. MutSα but
not MutSβ seems to be involved in DNA repair after UV-A damage.
60 | haematologica/the hematology journal | 2007; 92(s1)
0168
METALLOPROTEINASE POLYMORPHISMS IN PH-NEGATIVE CHRONIC
MIELOPROLIFERATIVE DISORDERS: NEW GENETIC FACTORS?
M.L. Biondi, 1 G.V. Melzi d'Eril, 2 F. Pateri, 1 L. Ferri, 1 M. Erario, 1
G. Fontana, 2 N.N. Fantini, 2 G.C. Gerli2 1 2 Laboratorio Chimica-Clinica e Microbiolo, MILAN; Università degli Studi,
MILANO, Italy
Background. Philadelphia-negative chronic myeloproliferative disorders
(MPDs) are characterized by clonal proliferation of haematopoietic
progenitor cells in the bone marrow and by different degrees of
extramedullary haematopoiesis, which is associated with increased
CD34 + circulating cells. The transient mobilization of haematopoietic
stem cells and progenitor cells has been attributed to the induction of a
highly proteolytic environment within the bone marrow. Matrix metalloproteinases
(MMPs) are central factors in the control of extracellular
matrix turnover and have been implicated in connective tissue destruction
and remodelling associated with cancer invasion and metastasis. A
common insertion polymorphism (an additional guanidine) in the
nucleotide sequence of the MMP-1 gene promoter has been reported.
The 2G homozygotes show an increased transcription activity compared
to 1G homozygotes ad controls. On the promoter of the MMP-3
gene has been described a polymorphism that in vitro assays demonstrate
that the promoter activity of the 5A allele had 2-fold higher promoter
activity than the 6A allele. Aims. We investigated whether the
MMP-1 and/or the MMP-3 promoters polymorphisms are associated
with susceptibility and/or progression of MPDs. Methods. 46 patients: 21
with Polycythemia Vera (PV); 18 with Essential Thrombocythemia (ET)
and 7 with Idiopathic Myelofibrosis (cIMF) and 133 healthy controls
were genotyped for the two polymorphisms. Genotypes were determined
with PCR-RFLP methods. Results MMP-1 genotype was statistically
different between patients and controls (patients vs controls ;1G/1G
vs 1G/2G+2G/2G 6% vs 26%; OR 4.78; CI% 1.35-25.22; p=0.007); for
MMP3 the 5A/5A genotype was less represented (13% vs 27%; OR
2.59: CI% 0.97-8.07; p=0.04)).No correlation was observed with spleen
size, CD34 + percentage, and JAK2-V617F mutation. Conclusions. Our
data suggest a role of MMP1 and MMP3 in the matrix remodeling associated
with CMD. However, no firm conclusion regarding the clinical
significance of these Metalloproteinase promoter polymorphisms can be
drawn until much larger cohorts have been analysed.
0169
NEW DATA ON ROBUSTNESS OF GENE EXPRESSION SIGNATURES IN LEUKEMIA:
COMPARISON OF THREE DISTINCT TOTAL RNA PREPARATION PROCEDURES
M. Campo Dell'Orto, 1 A. Zangrando, 1 L. Trentin, 1 R. Li, 2 W. Liu, 2
G. Te Kronnie, 1 G. Basso, 1 A. Kohlmann2 1 2 University of Padua, PADUA, Italy, Roche Molecular Systems, Inc.,
PLEASANTON, USA
Background. Gene expression microarrays had been used to classify
known tumor types and various hematological malignancies enforcing
the objective that microarray analysis could be introduced soon in the
routine classification of cancer. However, there are still doubts about
gene expression experiments performance in clinical laboratory diagnosis.
For instance, the quality of starting material is a major concern in
microarray technology and there are no data on the variation in gene
expression profiles ensuing from different RNA extraction procedures.
Aims. Here, as part of the internal multicenter MILE Study program, we
want to assess the impact of different RNA preparation procedures on
gene expression data, analyzing 27 patients representative of nine different
subtypes of pediatric acute leukemias. We compared the three currently
most used protocols to isolate RNA for routine diagnosis and
microarray experiments. Methods. The methods are named as method
A: lysis of mononuclear leukemia cells, followed by lysate homogeniziation,
followed by total RNA isolation; method B: TRIzol RNA isolation,
and method C: TRIzol RNA isolation followed by total RNA purification
step. The methods were analyzed in triplicates for each sample (24)
and additional three samples were performed in technical replicates of
three data sets for each preparation (HG-U133 Plus 2.0). Results. Method
A results in better total RNA quality as demonstrated by 3’/5’ GAPD
ratios and by RNA degradation plots. High comparability of gene expression
data is found between samples in the same leukemia subclasses and
collected with different RNA preparation methods thus demonstrating
that sample preparation procedures do not impair the overall signal distribution.
Unsupervised analyses showed clustering of samples first by