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

formation (in total 28 samples). Time to AML progression was 2-40
months (mean 13 months). The methodology utilized for CGH was a
variation of the basic protocol described by Kallioniemi et al (Genes
Chrom C 1994;10:231-43). DNA copy number changes were described
according to the ISCN (2005) guidelines. Results. Average number of
chromosome imbalances per MDS sample was 6 (range 0-17). Average
number of chromosome imbalances per AML sample was 12 (range 3-
21). X and Y chromosomes were excluded from analysis. Unexpectently,
a common pattern of chromosomal loci gains was observed in different
MDS patients during AML transformation (Table 1) namely 20p11.2,
21q11.2, 21q21, 4q12, 16p11.2, 7q11.2, 18p11.2, 5q11.2, 20q11.2,
18q11.2, 14q13, 16q13. Chromosomal losses were less frequently detected
in this series. Confirmation of Ch-CGH Results. In 2 patients with
abnormal karyotypes (1 patient with trisomy 8 and 1 with monosomy
7) Ch-CGH showed amplification/loss of the relevant chromosomal loci.
In 2 patients with 5q31 deletion in Ch-CGH was verified by FISH analysis
using a 5q31 probe hybridised to bone marrow smears. In a patient
with amplification of 22q11.2 in Ch-CGH 3 signals were obsereved in
44% of cell nuclei when hybridised with 22q11.2 probe. Ch-CGH results
were also verified by array-CGH in a sample (Gene Chip Affymetrix).
Discussions. The finding of the existence of common chromosomal loci
gains who appear only during the transition of MDS to AML is extremely
important, given the extreme clinical and genetic heterogeneity of
MDS patients. Identification of oncogene(s) located in the amplified
chromosomal areas that may contribute to the transformation of MDS
to AML will give new therapeutic targets.
Table 1.
0629
INCREASED BONE MARROW ENDOTHELIAL PROGENITOR CONTENT IN MDS PATIENTS
CORRELATES WITH DISEASE STAGE, AND MAY BE INVOLVED IN THE ANGIOGENESIS
RESPONSE LEADING TO ACUTE LEUKEMIA
C. Osório, 1 A.P. Elias, 1 M. Gomes da Silva, 2 S. Dias1 1 IPO/IGC, LISBON; 2 Haematology Department, IPO, LISBON, Portugal
Myelodysplastic syndromes (MDS) are bone marrow (BM) disorders
considered as pre-leukemic stages; an increase in BM angiogenesis has
been suggested to contribute towards leukaemia progression. However,
the mechanisms that regulate the vascular increase in MDS are poorly
understood. In the present study we hypothesized changes within the
MDS BM microenvironment might contribute to increased BM angiogenesis,
and eventually result in AML onset and progression. For this purpose,
we determined the cellular content and cell apoptosis of MDS BM
samples (at diagnosis), and compared these to BM samples from patients
with other BM diseases (lymphomas, CLL, others). In parallel, we
analysed the BM expression of factors that might contribute towards
increased vascularisation and/or altered cell turnover, such as VEGF, PlGF,
TNF-α and TGF-β, respectively. Our criteria for BM collection and analysis,
restricted to MDS BM samples collected at diagnosis (before any
therapeutic intervention), allowed us to study these parameters in 12
MDS patients and in 10 samples from patients with other BM diseases.
Flow cytometry analysis of BM biopsies revealed a significant increase
in the percentage of CD34 + , CD117 + and AC133 + progenitor cells which
were related with MDS stages. Although MDS BM had globally a low-
12 th Congress of the European Hematology Association
er BM cellular content, the proportion of other cell lineages remained
largely unchanged between MDS BM (at diagnosis) and other diseases.
In detail,AC133 + and KDR + cells percentage was significantly higher in
MDS patients (5,6 and 1,7-fold respectively), suggesting an increase in
endothelial cells or progenitors (EPC) BM content. Accordingly, the apoptotic
index of AC133 + cells was lower in MDS marrows (8% apoptotic
AC133 + cells in MDS versus 30% in other bone marrow malignancies).
To understand how the MDS BM microenvironment might contribute
to towards an increase in BM vasculature, the total level of BM VEGF
was determined (by ELISA), and the proportion of the different VEGF
isoforms was assessed (by RQ-PCR). Although the bone marrow
mononuclear cell number was lower in MDS BM, VEGF levels per cell
were 2-fold higher in MDS marrows than in the other disases group.
Interestingly, besides a clear increase in total VEGF, it was also observed
that the proportion between the VEGF isoforms changed: isoforms
VEGF145 and VEGF189 increased significantly in MDS BM, and with
MDS progression. The quantification of factors that might be involved
in the changes in cell turnover within the MDS BM samples, favouring
endothelial cell and EPC expansion in relation to other lineages, revealed
that TNF-α expression was lower in MDS patients while TGF-β levels
were similar in both groups, and PlGF was marginally expressed.
Although obtained from a restricted group of patients, these findings
suggest that an increase in the EPC pool is directly related with MDS
stages, may contribute to the angiogenic profile in MDS, which in turn
may lead to disrupted BM homeostasis, eventually resulting in leukemia
onset and disease progression. Myelodysplastic syndromes (MDS) are
bone marrow (BM) disorders considered as pre-leukemic stages; an
increase in BM angiogenesis has been suggested to contribute towards
leukaemia progression. However, the mechanisms that regulate the vascular
increase in MDS are poorly understood. In the present study we
hypothesized changes within the MDS BM microenvironment might
contribute to increased BM angiogenesis, and eventually result in AML
onset and progression. For this purpose, we determined the cellular content
and cell apoptosis of MDS BM samples (at diagnosis), and compared
these to BM samples from patients with other BM diseases (lymphomas,
CLL, others). In parallel, we analysed the BM expression of factors
that might contribute towards increased vascularisation and/or
altered cell turnover, such as VEGF, PlGF, TNF-α and TGF-β, respectively.
Our criteria for BM collection and analysis, restricted to MDS BM
samples collected at diagnosis (before any therapeutic intervention),
allowed us to study these parameters in 12 MDS patients and in 10 samples
from patients with other BM diseases. Flow cytometry analysis of
BM biopsies revealed a significant increase in the percentage of CD34 + ,
CD117 + and AC133 + progenitor cells which were related with MDS
stages. Although MDS BM had globally a lower BM cellular content,
the proportion of other cell lineages remained largely unchanged
between MDS BM (at diagnosis) and other diseases. In detail, AC133 +
and KDR + cells percentage was significantly higher in MDS patients (5,6
and 1,7-fold respectively), suggesting an increase in endothelial cells or
progenitors (EPC) BM content. Accordingly, the apoptotic index of
AC133 + cells was lower in MDS marrows (8% apoptotic AC133 + cells
in MDS versus 30% in other bone marrow malignancies). To understand
how the MDS BM microenvironment might contribute to towards
an increase in BM vasculature, the total level of BM VEGF was determined
(by ELISA), and the proportion of the different VEGF isoforms
was assessed (by RQ-PCR). Although the bone marrow mononuclear cell
number was lower in MDS BM, VEGF levels per cell were 2-fold higher
in MDS marrows than in the other disases group. Interestingly, besides
a clear increase in total VEGF, it was also observed that the proportion
between the VEGF isoforms changed: isoforms VEGF145 and VEGF189
increased significantly in MDS BM, and with MDS progression. The
quantification of factors that might be involved in the changes in cell
turnover within the MDS BM samples, favouring endothelial cell and
EPC expansion in relation to other lineages, revealed that TNF-α expression
was lower in MDS patients while TGF-β levels were similar in both
groups, and PlGF was marginally expressed. Although obtained from a
restricted group of patients, these findings suggest that an increase in the
EPC pool is directly related with MDS stages, may contribute to the
angiogenic profile in MDS, which in turn may lead to disrupted BM
homeostasis, eventually resulting in leukemia onset and disease progression.
haematologica/the hematology journal | 2007; 92(s1) | 235