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

12 th Congress of the European Hematology Association
and the role of hematopoietic stem cell transplantation is still not fully
elucidated. We performed an observational analysis on 26 patients (pts)
with tAML (n=15, FAB M0, M1, M2, M4) and tMDS (n=7 RAEB-2. n=4
RAEB-1) developed after chemo/radiotherapy for lymphoma or breast
cancer. Conventional karyotype analysis was available in 14 pts (n=8
complex, n=1 normal, n=2 isolated del(7q) or monosomy 7, n=1 isolated
del(5q), n=1 trisomy 21, n=1 trisomy 8). All patients were considered
as candidates to allogeneic stem cell transplantation (SCT), and 21 of
them already received the planned SCT. The purpose of this study was
to determine the long-term outcome. Median age was 53 years (range:23
- 68); all pts, because of comorbidities, received a reduced-intensity conditioning
(RIC) followed by allogeneic peripheral blood SCT. One pt
died for disease progression before transplant and 4 pts are still completing
consolidation therapy before allo-SCT. Disease status at transplant
was categorized as low risk (n=7 CR1 or CR2), high risk (n=3 PR, n=5
PD, n=2 refractory) and 4 pts were treated up-front. The median time
from diagnosis to allografting was 6 months (range: 1-80 months). Pts
received allogeneic stem cells from HLA-matched/1ag mismatched siblings
(n=15), or HLA-matched unrelated doors (n=4), or haploidentical
related donors (n=2). All pts engrafted. Acute GVHD grade II-IV occurred
in 6 pts (n=1 post-DLIs), chronic GVHD developed in 6 pts (n=1 post-
DLIs). OS at 5.5 years was 5.3%, the 4-year EFS was 0%, TRM at 100
days and at 1 year were 29.8%. No statistical differences were observed
in TRM, EFS, and OS according to disease status at transplant and diagnosis
of tMDS or tAML. In conclusion, our data show that the outcome
of patients with therapy-related leukemias was very poor even after
allogeneic SCT
1040
EFFECT OF BUSULFAN AND CYCLOPHOSPHAMIDE ON ENDOTHELIAL AND HEPATIC
CELLS IN CULTURE: TOWARDS UNDERSTANDING PATHOGENESIS OF HEPATIC
VENO-OCCLUSIVE DISEASE
C. Vassord, C. Lapoumeroulie, R. Krishnamoorthy
INSERM UMR 763, PARIS, France
Background. Hepatic veno-occlusive disease (HVOD) is the major complication
of Busulfan (Bu)/Cyclophosphamide (CPA)-based conditioning
regimen prior to hematopoietic stem cell transplantation (HSCT).
Although clinical efficacy of Bu/CPA combination is well established,
interindividual differences in susceptibility to drug-induced toxicities
are significant. The pathogenesis of HVOD is complex. Even though
age, sex, preexisting liver damage and type of tranplant have been associated
with this complication, high bioexposure to these drugs is considered
to be the major determinant. Analysis of biological markers suggests
the potential involvement of cytokines and haemostasis in mediating
the drug-induced damage of sinusoidal endothelial cells (SEC) and
adjacent centrilobular hepatocytes in HVOD. In vivo, Bu is metabolised
by conjugation with glutathion (GSH), catalysed by a family of Glutathion
S-transferase (GST) enzymes. Of the 4 main subfamilies of GST
(GST A1, GST M1, GST P1 and GST T1), GST M1 and GST T1 are
highly polymorphic, with homozygous deletion of either one or both
genes found in significant frequencies in different ethnic groups.
Although these isoforms are considered to be less efficient than GST A1
in the metabolism of Bu, we reported earlier that both Bu clearance and
GST M1-null genotype were significantly associated with the incidence
of HVOD. Aims. We hypothetize that Bu and CPA could modulate GST
expression and so act on their own metabolism and that these drugs
and/or their metabolites affect molecules implies in hemostasis (TF),
vasomotricity (ET-1), endothelial adhesion (ICAM-1) and GvHD
(PECAM-1) leading to HVOD. Methods. We have studied the effect of
Bu and CPA on an endothelial cell line (TrHBMEC), as well on primary
endothelial cells (HUVEC) from several donors and an hepatic cell line
(HepG2). These cells had previously been genotyped for GST A1, GST
M1 and GST T1. We analysed the mRNA expression of GSTs, TF, ET-
1, PECAM-1 and ICAM-1 by real-time quantitative PCR (Q-PCR) and
the protein expression by ELISA for ET-1 and ICAM-1. Results. Effect of
CPA on mRNA expression of these genes in these cell types was not significant
but we show that Bu: i) moderately up-regulates GST A1, a
major Bu metabolising enzyme, in the hepatic cell line, ii) does not alter
GST M1 and ICAM-1 expression but down-regulates GST T1 (2 fold),
iii) down-regulates the expression of ET-1 (2 fold), PECAM-1 (2 fold) and
TF (2 fold) Summary/Conclusions. These data are not in agreement with
the previously proposed involvement of TF and ET-1 up-regulation in
the pathogenesis of HVOD. Our study also demonstrated that the
expression status of ET-1, ICAM-1 and TF is not related to the GST
genotype in endothelial cells. Ongoing studies of Bu/CPA-induced functionnal
interactions between EC and hepatic cells must provide further
384 | haematologica/the hematology journal | 2007; 92(s1)
insights into the pathogenesis of HVOD. The striking observation is the
absence of expression of GST A1 in all endothelial cell types, regardless
of GST genotypes and may provide an explanation for the Bu-induced
endothelial desquamation that ensues conditioning in HSCT. A better
understanding of molecular mechanisms of HVOD pathogenesis must
allow designing of novel therapeutic strategy for this lethal complication
in HSCT.
1041
ASSESSMENT OF THE RISK FOR THROMBO-EMBOLIC COMPLICATIONS
BY QUANTITATIVE ALLELE-SPECIFIC PCR FOR THE JAK2 V617F MUTATION
IN PATIENTS WITH ESSENTIAL THROMBOCYTHEMIA
H. Hauser, 1 O. Zach, 1 M. Fridrik, 2 D. Lutz1 1 2 Krankenhaus der Elisabethinen, LINZ; Allgemeines Krankenhaus, LINZ,
Austria
Thrombo-embolic events are considered the most relevant complications
in patients with essential thrombocythemia (ET). Thus, cytoreductive
therapy is widely recommended for patients with the major
riskfactors for thrombosis, i.e. age over 60, platelet counts over 1,5 M/l
or a history of prior thrombo-embolic events. Recently, the JAK2 V617F
mutation has been associated with an enhanced risk for thrombosis in
patients with ET. The objective of this study was to test for a correlation
of peripheral blood JAK2 burden with thrombo-embolic events.
Nucleated peripheral blood cells from 21 patients (7 male, 14 female;
median age 69 yrs, range 27-87) with newly diagnosed or hitherto
untreated ET were tested for occurrence of the JAK2 V617F mutation by
quantitative allele-specific PCR. The proportion of JAK2 V617F was calculated
by correlation with GAPDH. The sensitivity of this assay was
below 1%. To establish the risk of thrombo-embolic events in untreated
patients, JAK2 mutational status and JAK2 V617F burden in peripheral
blood was correlated to the patients history of thrombo-embolic
complications. With this highly sensitive test the V617F mutation was
found in 17 out of 21 patients (81%) with a quantitative range from 1,1
to 52,7% (median 8,0%). 10 of these 21 patients had a history of thrombo-embolic
complications. 3 patients had suffered apoplectic strokes, 4
patients had a history of acute coronary syndrome, including 2 myocardial
infarctions, 2 patients had peripheral arterial occlusive symptoms
and one patient suffered from sinus vein thromboses. The mutated allele
was found in 9 of 10 patients with trombo-embolic events. Statistical
analysis with the Mann-Whitney U-Test showed a significant association
of thrombo-embolic events with with age (p 0,015) as well as with
the burden of mutated JAK2 in peripheral blood (p 0,036). Due to the
low number of patients with extremely high platelet counts, the role of
platelet numbers could not be addressed. The JAK2 V617F mutation is
a risk factor for thrombo-embolic complications in patients with ET.
The question, whether occurrence of this mutation is an indication for
cytoreductive therapy, should be addressed in larger studies. However,
if mutated JAK2 is considered a clinically relevant prognostic factor, the
detection methods should be highly sensitive, since thrombo-embolic
events are also prevalent in patients with lower copy numbers of mutated
JAK2.
1042
USE OF CD34 + CELL COLLECTION EFFICIENCY CALCULATIONS FOR DOSE PREDICTION,
PROCESS QUALIFICATION AND PRODUCT SPECIFICATION DURING PBSC COLLECTION
USING THE MNC PROGRAMME ON THE COBE SPECTRA CELL SEPARATOR
W. Douglas, 1 M. McGarvey, 1 E. Sinclair, 1 W. Drummond2 1 2 Glasgow Royal Infirmary, GLASGOW; Western Infirmary Glasgow, GLAS-
GOW, United Kingdom
Background. The clinical heterogeneity of PBSC collection, which is
carried out in widely different patient groups, using widely different
mobilising regimes and with a very wide range of peripheral CD34 cell
counts, makes validation, process qualification and definition of a product
specification potentially difficult. We describe the use of calculated
CD34 cell collection efficiency values to address these issues. Methods.
330 consecutive peripheral blood stem cell collections were audited, 32
allogeneic collections (donors mobilised with G-CSF only) and 298 autologous
collections (mobilised using a wide variety of chemotherapy
regimes plus G-CSF). The MNC collection programme on the Cobe
Spectra cell separator was used for all procedures. Absolute number of
CD34 + cells processed by the machine was calculated as the product of
peripheral CD34 + count and the blood volume processed by the machine
(final inlet flow minus final AC volume from the machine’s end-run data).
Absolute CD34 + cell count in the PBSC collection bag is measured rou-