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

12 th Congress of the European Hematology Association
uated by flow-cytometry, expressed CD2 and/or CD25 (median 0.041%
of CD45 + BM cells, range 0.004-0.28%). By contrast, MCs from normal
BM (n=2), other hematological malignancies (n=4), and cutaneous mastocytosis
(n=4) did not express CD2 and/or CD25. Cytogenetic analysis
was normal in all cases. One patient revealed typical cutaneous
lesions of CM (confirmed by histology). In summary, the final diagnosis
was indolent SM (ISM) according to WHO criteria in 8/10 patients
(80%). Five patients had both major and minor criteria of SM, whereas
only three minor criteria were found in 3 patients. In one patient only
two minor criteria were found (expression of CD25/CD2 on MC and
tryptase serum levels >20 ng/mL), and one patient showed only the
aberrant expression CD25/CD2 on MC: these patients were considered
affected by Monoclonal Mast Cell activation syndrome (MMCAS), as
proposed by Valent et al. (2007). Summary / Conclusions. Our results show
strong association among anaphylaxis due to hymenoptera bites, abnormal
basal serum tryptase levels and SM. In addition, the multiparametric
flow cytometry analysis seems to be the most sensitive and reliable
method to identify occult BM involvement, especially in patients without
skin lesions and indolent disease.
Table 1.
1061
HIGH EXPRESSION OF APAF-1XL IN BONE MARROW CELLS OF LOW RISK
MYELODYSPLASTIC SYNDROME
B.D. Benites, F. Traina, A.S.S. Duarte, F.F. Costa, S.T.O. Saad
State University of Campinas, CAMPINAS, Brazil
Background. Apoptosis has a crucial role in myelodysplastic syndromes
(MDS). Early disease is associated with excessive apoptosis and the
apoptotic rate diminishes during disease progression. Cytochrome c/
APAF-1/ CASP-9 pathway is the main pathway involved in apoptosis
initiation by several stimuli. Original APAF-1 comprises three functional
domains; APAF-1XL and APAF-1LN isoforms have an insertion
between the CARD and ATPase domains and APAF-1XL also has an
additional WDR. It has been reported that only the isoforms with the
extra WDR activate pro-caspase 9. We hypothesized that APAF-1XL
expression could be related to the higher rates of apoptosis found in
early-stage disease. Aims. To analyse the expression of APAF-1 transcripts
in bone marrow cells from MDS patients and correlate these
findings with IPSS. We also attempt to verify the modulation of APAF-
1 mRNA expression during erythroid differentiation of CD34 + normal
and MDS cells. Methods. Marrow aspirates were obtained from 7 normal
donors, 14 patients with low risk MDS (IPSS: 9 low and 5 intermediate-1),
and 8 patients with high risk disease (IPSS: 5 intermediate-2 and
3 high), out of treatment (11 males, 11 females; 69 [40-91] yo). The
National Ethical Committee Board approved the study; informed-written
consent was obtained from all patients and donors. Total cells were
submitted to RNA extraction and the expression level of mRNA was
detected through real time RT-PCR. The relative quantification value of
gene expression was calculated using 2-DDct. For erythroid differentiation,
bone marrow samples from one MDS patient with refractory anemia
(IPSS low) and one normal donor were collected and CD34 + cells
were separated from mononuclear cells using the MIDI-MACS
392 | haematologica/the hematology journal | 2007; 92(s1)
immunoaffinity columns. CD34 + cells were plated on plastic culture dishes
in methylcellulose medium with appropriate growth factors for 6
days. BFU-E, CFU-E and proerythroblasts were then cultured in α MEM
for an additional 8 days. At days 6 and 14, cells were collected and submitted
to real time RT-PCR and apoptosis analysis. Apoptosis was quantified
with anexin V and propidium iodide; erythroblast differentiation
was observed with transferrin receptor and glycophorin A by flow
cytometry. Results. APAF-1XL expression was significantly higher in low
risk (15.327[3.095-121.1]) when compared to high risk MDS
(4.379[2.412-44.632]; p=0.0103, Wilcoxon rank-sum test). APAF-
1XL/APAF-1LN ratio was also significantly different comparing low and
high risk groups (1.614[0.6598-6.964] versus 0.8904[0.155-2.99],
p=0.0197). Erythroid differentiation of CD34 + cells from low risk MDS
was characterized by increased apoptosis (20% at day 14) and increased
expression of APAF-1XL mRNA (six fold at day 14 compared to day 6).
In contrast, erythroid differentiation of CD34 + normal hematopoietic
cells was characterized by a low rate of apoptosis (8% at day 14) and
low expression of APAF-1XL mRNA (half fold on day 14 compared to
day 6). Conclusions. High levels of APAF-1XL mRNA expression in low
risk disease and its positive correlation with the apoptotic rate, observed
during erythroblast differentiation of low risk MDS cells, suggest that
APAF-1 participates in the augmented susceptibility of myelodysplastic
cells to intramedullary death, since APAF-1XL, instead of APAF-1LN, is
the isoform constitutionally capable of activating pro-caspase 9.
1062
MYELOID SARCOMA: A CLINICO-PATHOLOGICAL STUDY INCLUDING IMAGING
OF STROMAL COMPONENTS
H. Schmitt-Graeff, 1 M. Metzger, 1 F. Feuerhake, 2 E.J. Juttner, 1 S. Haxelmans,
3 B. Strahm, 4 H. Bertz, 5 M. Lübbert, 5 J. Burger6 1 University of Freiburg, FREIBURG, Germany; 2 Institute of Neuropathology,
Universityh, FREIBURG, Germany; 3 Department of Biology I, University,
FREIBURG, Germany; 4 Department of Pediatrics and Adolescent, FREIBURG,
Germany; 5 Dept. of Medicine, Div. Hematology/Oncol, FREIBURG, Germany;
6 Department of Leukemia, Unit 428 The Uni, HOUSTON, USA
Background.The term myeloid sarcoma (MS) is currently used to designate
extramedullary tumors composed of immature myeloid cells. Aims.
Our purpose was to analyse clinico-pathological aspects of a large series
of MS. Since the homing to extramedullary tissues resulting in MS may
be promoted by the local microenvironment, we further characterized
stromal cell populations of MS occurring in different anatomic sites. Methods.
Paraffin-embedded specimens containing representative formalinfixed
MS biopsy material were retrieved from our files. Tissue sections
stained for a panel of antigens including myeloperoxidase, lysozyme,
CD3, CD4, CD20, CD34, CD41, CD61,CD68, CD117, CD99, CD 56,
TdT , hemoglobin were reviewed. Clinical and laboratory informations
including CXCR4 expression and survival data were obtained. Additional
imaging studies focused on the spatial localization of microvessels and
of stromal cells expressing stromal cell-derived factor-1 (SDF-1), cellular
retinol-binding protein-1 (CRBP-1) and α-smooth muscle actin (SMA).
Results. We obtained representative samples and clinical informations from
61 patients. MS developed simultaneously with bone marrow infiltration
as initial manifestation of acute myeloid leukaemia (AML, n=11), as
relapse after chemotherapy or allogeneic bone marrow transplantation of
a previously diagnosed AML (n=12), as extramedullary transformation of
a myelodysplastic syndrome (n=11) or as blast phase disease in typical or
atypical myeloproliferative disorders (n=16). Moreover, 11 patients presented
with an isolated MS without any bone marrow involvement. The
myeloid infiltrates showed phenotypic features of an acute myelomonocytic
or monoblastic leukaemia in 48% while the remaining cases corresponded
to various AML categories including megakaryoblastic leukaemia
or were classified as blastic type, not further specified. In contrast to the
large variety of phenotypic profiles and clinical settings, a common feature
of all samples was an increased density of SDF-1, CRBP-1 and SMAexpressing
stromal cells.The spatial organization of the stroma was highlighted
by confocal imaging. Summary/Conclusions. According to our observations,
the functional interaction of local stromal cells with myeloid population
via chemokines and chemokine-receptors may be crucial for blast
cell infiltration of extramedullary sites in MS.