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

12 th Congress of the European Hematology Association
Bone marrow failure
0436
IMPACT OF SOMATIC CELL MOSAICISM IN FANCONI ANEMIA ON DEVELOPMENT OF
BONE MARROW FAILURE
J. Tolar, 1 M.L. MacMillan, 1 S.D. Batish, 2 C. Eide, 1 Y. Flit, 2 S.M. Davies, 3
B.R. Blazar, 1 A. Auerbach, 2 J.E. Wagner1 1 University of Minnesota, MINNEAPOLIS; 2 The Rockefeller University, NEW
YORK; 3 Cincinnati Children's Hospital Med Ctr, CINCINNATI, USA
Background. Determination of the degree of somatic mosaicism providing
functional correction of Fanconi anemia (FA) hematopoiesis has
direct implications for gene therapy for FA: it may help assess the percentage
of FA hematopoietic cells corrected by gene therapy approaches
that are needed to achieve clinically meaningful effects. Aims. Hypersensitivity
to DNA inter strand cross-linking agents, such as diepoxybutane
(DEB) and mitomycin C (MMC), is a cellular marker for diagnosis
of FA. However, in some FA patients a population of DEB-resistant PHAstimulated
lymphoblasts (PHA-L) was observed, and this population
sometimes varied over time. Our aim was to assess the significance of
this finding on hematopoietic function. Methods. To that end, we evaluated
the MMC sensitivity of bone marrow mononuclear cells (BMMC)
and DEB sensitivity of PHA-L and cultured lymphoblastoid cell lines
(LCL) in 42 consecutive FA patients referred to the University of Minnesota.
In cases where LCL were DEB-resistant, cultured fibroblasts
were also studied. BMMC were cultured in the presence of increasing
concentrations of MMC. PHA-L and LCL were cultured in DEB at 0.1
µ/ml. Results. Wild type BM progenitors (N=17 subjects) proliferated
regardless of increasing MMC concentrations (albeit at decreased efficiency
at the highest concentrations) as follows: 0 MMC (normalized to
100%), 5 nM MMC (99% [standard deviation, SD, 16%]), 10 nM MMC
(90% [SD 22%]), 25 nM MMC (77% [SD21%]), and 50 nM MMC (44%
[SD 30%]). Of the 42 FA patients, BMMC failed to proliferate at 0 nM
MMC in 10 patients and at 5 nM MMC in 20 patients. Twelve FA
patients had MMC resistant BMMC: cells cultured in 5, 10, 25 and 50
nM MMC grew 44% (SD 28%), 35% (SD 24%), 24% (SD 30%) and
17% (SD 32%) of colony numbers in MMC free culture, respectively.
Six of these 12 subjects were PHA-L mosaics as determined by DEB
sensitivity testing. Four patients with no growth of BMMC at 0 or 5 nM
MMC were also somatic mosaics in their PHA-L and LCL. Thus there
was no clear correlation between somatic mosaicism as demonstrated
by DEB testing in peripheral blood and sensitivity of BMMC to growth
in MMC. Clinically, two patients with hematopoietic somatic
mosaicism (out of six subjects with simultaneous sensitivity of BMMC
to MMC and PHA-L to DEB) developed severe marrow aplasia, one of
which received hematopoietic stem cell transplantation. The remaining
four hematopoietic mosaic patients had normal or near normal peripheral
blood counts. While patients with hematopoietic somatic mosaicism
had mixed populations of DEB sensitive cells in their peripheral blood,
all their fibroblast cultures were DEB sensitive. Summary. These data
show that the presence of somatic mosaicism per se does not necessarily
prevent bone marrow failure. Moreover, the data suggest that
patients with stigmata of FA may have chromosomal breakage studies
showing few cells (or no cells) with the characteristic changes of FA; in
these cases, skin fibroblasts should be tested as well.
162 | haematologica/the hematology journal | 2007; 92(s1)
0437
INCREASED PRODUCTION OF TGF-Β1 BY BONE MARROW STROMAL CELLS IS
ASSOCIATED WITH DOWNREGULATION OF IL-10 AND OVEREXPRESSION OF SOLUBLE
FLT-3 LIGAND IN PATIENTS WITH CHRONIC IDIOPATHIC NEUTROPENIA
H. Papadaki, K. Pyrovolaki, I. Mavroudi, M. Spanoudakis, C.
Pontikoglou, M. Ximeri, C. Kalpadaki, G. Gvazava, M. Velegraki,
G.D. Eliopoulos
University of Crete School of Medicine, VOUTES, HERAKLION, Greece
Background. Chronic idiopathic neutropenia (CIN) is a bone marrow
(BM) failure syndrome characterized by low number of circulating neutrophils
for prolonged period of time. The disease has been attributed
to low number of BM myeloid progenitor cells due to an inflammatory
BM microenvironment. Among multiple cytokines normally involved in
regulation of myelopoiesis, transforming growth factor-β1 (TGF-β1),
interleukin-10 (IL-10), and soluble flt-3 ligand (sFL) play a significant
role. The involvement of these cytokines in the pathogenesis of CIN has
not been extensively studied. Aims. The aim of the study was to evaluate
the production of TGF-β1, IL-10 and sFL in the BM microenvironment
of CIN patients and investigate the possible involvement of these
cytokines in the pathogenesis of neutropenia in the affected subjects.
Materials and Methods. BM was obtained from 70 patients with CIN and
35 healthy controls. All patients fulfilled the diagnostic criteria for CIN
as we have previously published (Blood 2003; 101: 2591-2600). Aliquots
of BM mononuclear cells (BMMCs) were evaluated by flow cytometry
for CD34 + /CD33 + expression. Long-term BM cultures (LTBMCs) were
initiated from BMMCs according to a standard technique and TGF-β1,
IL-10 and sFL were measured in the supernatants by means of ELISA.
LTBMC supernatant IL-10 and sFL were also measured in patient cultures
after the addition of anti-human TGF-β1 mouse monoclonal neutralizing
antibody. Results. The levels of TGF-β1 in LTBMC supernatants
were significantly increased in CIN patients compared to controls
(p