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

12 th Congress of the European Hematology Association
compared to known models such as β2 M/chr 13 deletion and ISS. The
-2Log (likelihood) scores calculated on 155 patients were 1107.885,
1113.256 and 1116.829 for Ki-67/β2 M model, ISS model and β2 M/del13
model, respectively. Conclusions. Ki-67 index is easy to perform in routine
practice, and is a good prognostic marker, which provides additional
survival prognostic information to β2 M into the ISS model. (TG and
XL are fisrt author)
0691
COMPARISON OF CLASSIC CYTOGENETICS AND METAPHASE FISH WITH INTERPHASE
FISH ANALYSIS ON UNSELECTED AND SELECTED PLASMA CELLS IN MULTIPLE
MYELOMA. THE NEED FOR A UNIFORMLY ACCEPTED METHOD
A. Lazaridou, 1 S. Mavroudi, 1 A. Nikitidou, 1 P. Salamourtzi, 1 E. Katodritou,
1 C.H. Kartsos, 1 V. Gastari, 1 M. Kotsopoulou, 2 P. Repousis, 3
M.C. Kyrtsonis, 4 E. Terpos, 5 G. Pangalis, 4 Chr. Mitsouli, 2 K. Zervas1 1 Cancer Center of Thessaloniki, THESSALONIKI; 2 Cancer Hospital of Pireaus
Metaxa, ATHENS; 3 Cancer Hospital of Pireaus, ATHENS; 4 Laikon General
Hospital, ATHENS; 5 251, General Airforce Hospital, ATHENS, Greece
Multiple myeloma (MM) is an incurable disease with heterogeneity
in survival and prognosis. The establishment of prognostic factors is
very important in handling the disease and so far cytogenetic status has
been proved as the most important one. However, conventional cytogenetics
has a limited extent because of the low proliferation of plasma
cells (PC), the reduced extent of marrow involvement and the difficulty
to detect cryptic translocations. Recently, classical cytogenetics has
been replaced by the fluorescence in situ hybridization (FISH) technique,
which allows the identification in a short time of specific target regions
at diagnosis and the evolution of the disease. A number of diagnostic
centers have proposed in the literature different FISH procedures in order
to ameliorate FISH results and to overcome the problem of PC low infiltration
of the marrow. In addition, there is an argument on the cut off
levels for the identification of aberrations. We studied 185 patients with
MM using G-banding, M-FISH, metaphase FISH on bone marrow aspirates
and interphase FISH on cytospin centrifuged cells either by unseparated
bone marrow cells or PCs selected by Magnetic cell separation
(MACS) using the CD 138 monoclonal antibody. The aim of the study
was to compare these methods and to find the more accurate, less
expensive and time consuming technique. For FISH we used commercial
probes for the detection of the deletion of 13q14 and 17p13 (p53)
regions, and for the detection of rearrangements of IGH locus with the
FGFR3, the cyclinD1 and the MAF genes. Our results were in accordance
with previously published studies. In most cases, excluding the
detection of aneuploidy, cytogenetics failed to reveal specific chromosome
rearrangements. M-FISH in the abnormal cases helped us to identify
the complex chromosome abnormalities. Metaphase FISH was very
informative in detecting cryptic abnormalities in metaphases. Interphase
FISH in unseparated cells proved to be of equal reliability for the detection
of cryptic abnormalities in comparison with interphase FISH in
selected PCs. However, the cut off levels in unseparated cells was 5-8%
in accordance to the level of bone marrow infiltration, and significantly
lower than that proposed for selected PCs. Also, FISH proved to be a
useful method in detecting aneuploidy for the chomosomes examined.
Conclusions. Convetional cytogenetics is without a doubt the only
method that can give information on the aneyploidy status in MM. We
need to ameliorate our classic cytogenetic methods in order to obtain
better chromosome quality. Metaphase FISH can serve in the detection
of cryptic rearrangements and in this method can be used on already
existed cytogenetics material. Interphase FISH on cytospins using unseparated
bone marrow cells is a reliable, easy and rapid technique, less
expensive and could be performed in cytogenetic labs, where elegant
techniques such as magnetic cell separation are difficult to be established.
From the study arises the need for a uniform procedure for the
detection of chromosome abnormalities in MM taking in consideration
simplicity, time and cost effectiveness.
258 | haematologica/the hematology journal | 2007; 92(s1)
0692
MOLECULAR TARGETING OF THE PKC-β INHIBITOR ENZASTAURIN (LY317615)
IN MULTIPLE MYELOMA
D. Verdelli, 1 R.L. Nobili, 2 M. Civallero,3 M. Cosenza, 3 K. Todoerti, 1
J. Bertacchini, 3 L. Lombardi, 1 S. Marmiroli, 3 M. Federico, 3 A. De Pol, 3
G. Lambertenghi-Deliliers, 2 P. Tassone, 4 A. Neri, 1 S. Sacchi3 1 Fondazione IRCCS Policlinico, MILAN; 2 Università degli Studi di Milano,
MILAN; 3 Università di Modena e Reggio Emilia; 4 Università di Catanzaro,
CATANZARO, Italy
Background. Over the last few years, great efforts have been made
searching for novel agents that specifically target signalling pathways
which regulate multiple myeloma (MM) cells growth and survival. The
deregulation of phosphatidylinositol 3-kinase (PI 3-K)/Akt pathway, a
prominent regulatory pathway governing the apoptotic response, may
play an oncogenic role in MM. Constitutive activation of the PI 3-K/Akt
pathway has been shown to frequently occur in MM in vivo and has been
also observed in factor-independent MM cell lines. Although the biological
mechanisms that lead to PI 3-K deregulated activation are still
unknown in MM, recently a link between protein kinase C (PKC) activity
and the activity of the PI 3-K/Akt pathway has been reported. Aims.
The purpose of the present study was to test the oral PKCβ inhibitor,
Enzastaurin (LY317615 - Eli Lilly) for its effect on proliferation and survival
of a wide panel of human myeloma cell lines (HMCLs). The possible
mechanisms by which enzastaurin exerts its effects were also
investigated by analysing the transcriptional pattern altered following
enzastaurin treatment. Methods. IC50 values in 20 HMCLs cultured in
10% FCS containing medium, were calculated from curves based on
enzastaurin concentrations ranging from 2.5 to 12.5 µM using both
WST-1 assay and cell viability assessment by Trypan Blue exclusion.
Cell apoptosis, as suggested by an increase in the proportion of cells
with a sub G0/G1 DNA content and by membrane permeability by PI
versus FSC, was assessed by flow cytometry. The effect of enzastaurin
on caspases activation as well as on AKT and GSK3β phosphorylation
was evaluated by Western blotting. The gene expression profiling (GEP)
data generated by means of high-density oligonucleotide arrays
(Affymetrix U133A arrays) were analysed by the Significant Analysis of
Microarrays software for the supervised analyses. Results. Enzastaurin
showed a clear growth inhibition effect in 19 out of 20 HMCLs. Based
on the sensitivity to enzastaurin and PKCβ expression, five cell lines
were then selected for further studies: AMO1, KMS-26 (both expressing
the two PKCβ isoforms) and MM1.S (expressing PKCβ) among the
most sensitive cell lines; the intermediate sensitive KMS-18, expressing
PKβ and the less sensitive RPM showing a weak PKCβ expression Enzastaurin
induced apoptosis in sensitive cell lines was partially caspase
dependent. Phosphorylation status analysis of AKT and of GSK3β, a
downstream AKT substrate, up to 48 h of treatment showed the inhibition
of AKT phosphorylation and a marked decrease of phosphorylated
GSK3β levels in all sensitive cell lines. GEP data analysis of KMS-26
cell line treated or not with enzastaurin for 24 hours, showed that 62
genes were upregulated and 32 were downregulated in the treated samples.
Conclusions. Our data suggest that, in HMCLs, enzastaurin elicits its
antitumor effect through the AKT signaling pathway. A functional analysis
of deregulated genes following enzastaurin treatment revealed a significant
fraction of genes involved in signal transduction, in the immune
and inflammatory responses, in transcription regulation, in cellular adhesion
and apoptosis processes.
0693
CHROMOSOMAL ABNORMALITIES OF PLASMA CELLS AND CORRELATION WITH
IMMUNOPHENOTYPE IN MULTIPLE MYELOMA
P. Omede, 1 M. Ruggeri, 2 M. Brunetti, 2 S. Caltagirone, 2 S. Bringhen, 2
F. Cavallo, 2 M. Gilestro, 1 F. Ferro, 2 M. Spagnolo, 2 C. Di Bello, 2
A. Fantauzzo, 2 B. Bruno, 2 A. Palumbo, 2 M.T. Petrucci, 3 A.M. Leberati, 4
M. Boccadoro2 1 ASO San Giovanni Battista, TORINO; 2 Divisione di Ematologia, TORINO;
3 Dip Biotecn Ematol. La Sapienza Roma, ROMA; 4 Cl. Medica I- Policl. Monteluce-Perugia,
PERUGIA, Italy
Background and Aims. Multiple Myeloma (MM) is characterized by a
marked heterogeneity of genetic lesions, including numerical and structural
chromosomal abnormalities. In the present study we have investigated
the relationship between the immunophenotypic profile of
myelomatous plasma cells and their specific genetic features by FISH.
Materials and Methods. Between August 2002 and January 2007, 840 con-