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

12 th Congress of the European Hematology Association
bility is a characteristic of tumor cells. Loss of heterozygosity (LOH) is
an indirect method of detecting inactivation of tumor suppressor genes
and, microsatellite instability (MSI) is the other form to show DNA mismatch
repair system which leads to replication rerrors. Aims. In this report
we examined the LOH and microsatellite instability in patients with MM
in order to point to genomic instability in chromosome 14 and we compared
them with clinical stage and Ig type of disease. Materials and Methods.
We selected the 5 different STR loci of cromosome 14 (14q32). 26
patients were included into the study (10 female, 16 male, mean age 63
year). 7 patients diagnosed as stage one, 7 patients stage two, 12 patients
were stage 3. According to Ig heavy chain 14 patients had IgG, 5 patients
IgA, 5 patients had light chain disease and one had non secretory MM.
DNA was extracted from the bone marrow plasma cells after the separation
procedure with magnetic beads from the residuel bone marrow
cells and hair DNA was used as control. After the PCR with fluorescein
congugated primers, the MSI and LOH was detected by capillary electrophoresis
(ABI 310). Results. MSI was detected %35 of patients in
D14S65 locus. LOH was detected %23 of patients in D14S985 loci. The
other MSI and LOH findings were rare. Fifthy per cent of IgG MM had
MSI in D14S65 locus. Four patients with IgG MM (%28) and one patient
with IgA MM had either MSI or LOH in all 5 locus (%20). No significant
association of any molecular defect to Ig type and clinical stages of disease
was found. Conclusions. In present study we showed that MSI and
LOH are comman findings in MM especially chromosome 14q32 region
which we know that Ig Heavy chain is being encoded.
1034
MONITORING LEUKEMIA PATIENTS' RESPONSE TO CHEMOTHERAPY BY A NOVEL IR
LIGHT SPECTROSCOPIC METHOD
U. Zelig, 1 J. Kapelushnik, 1 I. Nathan, 2 S. Mordechai3 1 Soroka University Medical Center, KIBBUTZ NIR ITZHAK, Israel; 2 Ben-
Gurion University, BEER SHEVE, Israel; 3 Bgn-Gurion University, BEER SEE-
VA, Israel
Background. Diagnosis and monitoring leukemia progression have great
importance in determining prognosis and choosing the appropriate protocol
for treatment. Present diagnostic methods such as FACS and cytogenetics
of bone marrow are expensive and involve invasive procedures
and thus cannot be used for daily monitoring of individual patients'
response to chemotherapy. Fourier transform infrared (FTIR) spectroscopy
is an inexpensive, rapid and simple optical method for daily
monitoring biochemical changes of nucleic acids, lipids and proteins in
mononuclear cells. In preliminary studies, we were able to identify
leukemia patients and follow their reaction to chemotherapy treatment
daily by analyzing FTIR spectra of peripheral blood mononuclear cells.
Aims. We aim to use FTIR for diagnosis and follow-up of leukemic children
undergoing chemotherapy treatment. Furthermore, we will search
for parameters which provide information on the biochemical changes
in mononuclear cells during chemotherapy treatment. To investigate the
nature of FTIR spectral changes following drug treatment, we will use
leukemic cell lines as a model system. Methods. Mononuclear cells from
blood samples were obtained from 30 acute lymphoblastic leukaemia
(ALL) patients and 27 healthy controls by the standard Ficoll-Hypaque
technique. FTIR measurements of mononuclear cells were carried out
using a FTIR microscope IRscope II. CCRF-CEM cells were treated with
doxorubicin in different concentrations. Apoptotic and necrotic morphology
was evaluated by staining CCRF-CEM cells with acridine orange
and ethidiume bromide, and examining them using a fluorescent microscope.
Results. By calculating the ratio of the functional groups CH3/CH2,
which are related to biochemical changes of proteins and lipids respectively,
we were able to distinguish between leukemic and healthy groups
(Figure 1a). This ratio also served as a parameter to monitor the effect of
chemotherapy on mononuclear cells of the leukemic patients. Figure 1b
depicts the response of two leukemic children to chemotherapy monitored
by FTIR. According to the analysis, child #1 responded faster than
child #2 (owing to the development of a blood infection in child #2) to
the treatment as reflected by the return of the CH2/CH3 ratio to normal
values. In order to understand the effects of chemotherapy on an FTIR
spectrum of mononuclear cells, we conducted in vitro experiments on
CCRF-CEM cells treated with doxorubicin. As seen in Figures 1c and 1d,
several spectral changes appeared due to modifications in quantity/conformation
of membrane lipids, nucleic acids and proteins following doxorubicin
treatment. These modifications correlated with the rate of apoptotic
and necrotic cells monitored using a fluorescent microscope. Additional
experiments with other known cell death inducers such as Ara-C,
H2O2 and KCN were carried out using FTIR spectroscopy and were
compared with biochemical standard methods (data not shown). Conclu-
382 | haematologica/the hematology journal | 2007; 92(s1)
sions. We conclude that FTIR is a powerful biochemical tool that can be
used for leukemia diagnosis as well as for monitoring the effects of
chemotherapy on mononuclear cells.
Figure 1.
1035
PRE-TRANSPLANT LOW DOSE ATG AS PROPHLYLAXIS FOR CHRONIC GVHD AFTER
PERIPHERAL BLOOD STEM CELL MYELOABLATIVE SIBLING TRANSPLANTS IN AML:
A SINGLE CENTER EXPERIENCE
G Specchia, 1 D. Pastore, 2 A. Mestice, 2 P. Carluccio, 3 T. Perrone, 2
F. Gaudio, 2 A.M. Mazzone, 2 P. Mongelli, 2 P. Manduzio, 2 M. Leo, 2
V. Liso, 2 G. Specchia2 1 2 3 Hematology, BARI, Italy; Hematology Section, BARI; Hematology Unit, BARI,
Italy
Background. Chronic graft versus host disease (cGvHD) incidence and
severity are generally found to be greater after peripheral blood stem cell
transplantation (PBSCT) than after bone marrow transplantation, and
some studies increased mortality from cGvHD, particularly the extensive
type. In vivo, T-cell depletion with antithymocyte globulin (ATG) is an
effective strategy for decreasing the incidence of cGvHD but may include
side effects such as high risk of infection and relapse. The main utilization
of ATG has been as prophylaxis of GvHD in unrelated bone marrow
transplantation but few data are available on myeloablative HLA-identical
sibling PBSCT. Aims. In our study we evaluated the incidence of infections,
relapse and cGvHD in 30 patients with acute myeloid leukemia
(AML) in first complete remission after a peripheral stem cell myeloablative
HLA-identical sibling transplant. Patients and Methods. Conditioning
regimen was oral busulfan (16 mg/Kg), cyclophosphamide (120 mg/Kg)
and ATG-Thymoglobuline (4 mg/Kg total dose given on days -2 and -1);
the median dose of CD34 + cells and CD3 + cells infused were 4.6×10 6 /Kg
(range 3.1-9) and 250×10 6 /Kg (range 84-442), respectively. GvHD prophylaxis
was Cyclosporin A (2 mg/Kg/die) and short-term Methotrexate (15
mg/m 2 on day +1, 10 mg/m 2 on day +3,+6,+11). Results. The median time
to neutrophil (>0.5×10 9 /L) and platelet (>20×10 9 /L) engraftment was 15
(range 13-17) and 18 (range 15-20) days, respectively. Acute GvHD grade
I-II was observed in 6/30 (20%); no patients experienced aGvHD grade
III-IV. CMV antigenemia was monitored twice a week for the first 100
days; 18/30 (60%) patients developed CMV reactivation but no CMV disease
occurred. As to immunological recovery, the median number of CD3 +
and CD4 + after 1, 3 and 6 months after transplantation was 690, 850, 930
and 125, 214, 256×10 9 /L, respectively; the median number of CD8 and NK
cells after 1, 3 and 6 months was 430, 538, 653 and 216, 156, 230 ×10 9 /L,
respectively. At a median observation time of 34 months (range 8-60) the
overall cGvHD was 8/30 (26%) (limited 5/8 or 17% and extensive 3/8 or
9%); the relapse rate was 8/30 (26%). Summary. Our data suggest that low
dose of ATG is effective in preventing acute and chronic GvHD without
increase in relapse; prospective randomized trials are needed to evaluate
the role of ATG in AML undergoing allogeneic stem cell transplantation
from HLA-identical sibling.