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

12 th Congress of the European Hematology Association
ing the question weather all or only FDG PET positive patients should
receive post-transplant rituximab consolidation.
1262
ADOPTIVE TRANSFER OF CMV-SPECIFIC T-CELLS SELECTED FOR IFN-GAMMA
SECRETION BY MAGNETIC LABELING: A CLOSER LOOK ON THE SELECTED CELLS
S. Borchers, 1 M. Heuer, 2 B. Hertenstein, 3 J. Alves, 1 A. Ganser, 1
E.M. Weissinger1 1 MHH, HANNOVER, Germany; 2 Universität Würzburg, WÜRZBURG, Germany;
3 Klinikum Bremen, BREMEN, Germany
Background. The reactivation of Cytomegalovirus (CMV) and other
viruses is one of the major complications after (stem cell) transplantation.
In an immuncompromised host recurrent CMV infections occur, due to
the lack of antigen-specific T-cells, if T-cell depleted grafts or immunosuppressive
therapy is administered. Despite the possibility of treating
recurrent CMV infections with antiviral drugs, control of reactivation is
not always successful with medication. Repeated treatment may lead to
resistant virus strains and high doses of e.g. ganciclovir or foscarnet can
lead to severe side effects like graft rejection/loss or renal failure. The
adoptive transfer of antigen-specific T-cells has been investigated over
the past 12 years, initially using T-cell clones, more recently by transfer
of specific T-cell lines. Aims. Here we present preclinical data using magnetic
selection for IFN-γ producing cells after stimulation with antigen.
For enrichment procedures that can be up-scaled for later production of
clinical grade samples different media were tested and the stimulation
of the T-cells was optimised. The enriched cell fraction can contain different
cell types, since enrichment is based solely on the capacity of cells
to secret IFN-γ. Methods. Peripheral blood mononuclear cells (PBMNC)
of healthy, HLA-typed, CMV-seropositive blood donors were stimulated
over night using recombinant viral protein pp65 and/or whole viral
lysate (strain AD169). For assessment of different media (one standard
medium, two GMP-grade media), cell viability, stimulation rates and
expansion of IFN-γ secreting fractions were checked. Furthermore, cryopreserved
material was retested in two different assays (magnetic selection
and intracellular staining). To check the formation of the IFN-γ
secreting population, cells were stained for CD3, CD8, CD4, CD14,
CD16, CD19, CD20, CD25, CD56 and IFN-γ in different steps of the
assay. Results. Both GMP-grade media showed similar performance.
Retesting of cryopreserved cells revealed high variability in percentage
of IFN-γ secreting cells using a secretion assay, while intracellular staining
led to results comparable with the secretion assay when using fresh
cells. Testing of IFN-γ secreting populations is still ongoing. The recombinant
pp65 usually leads to preferential activation of CD8 + cells, while
lysate rather activates CD4 + cells. Usage of pp65 as antigene alone will
not lead to a feasible stimulation rate in all cases, this may be an HLAdependant
effect. Conclusions. Adoptive transfer of CMV-specific T-cells
can be the only cure for patients with recurrent drug-resistant CMVreactivations.
Though the benefits of this technique are obvious, there
are risks like transfer of alloreactive T-cells. To minimize the risk of this
treatment and optimise performance of selected T-cells, knowing the
exact formation of the enriched fraction is crucial. FACS-based assays
like antibody staining or proliferation testing are suitable for quality
testing of cells prior to transfusion. Furthermore stimulation rates and
applicability of the assay could be upgraded by usage of a protein cocktail
instead of single proteins. Therefore cloning, expression and stimulation
testing of several viral proteins has been started in our institution.
1263
BONE MARROW MICROCASCULAR DENSITY AND FIBROSIS ARE RELATED
IN POLYCYTHAEMIA VERA
S. Theodoridou, 1 I. Venizelos, 1 T. Vyzantiadis, 2 S. Vakalopoulou, 1
V. Perifanis, 1 E. Mandala, 1 E. Leukou, 1 I. Klonizakis, 1 D. Markala, 3
V. Garypidou1 1 Hippokration Hospital, THESSALONIKI; 2 Aristotle University1st Microbiology
Dep, THESSALONIKI; 3 Theagenion Hospital, THESSALONIKI, Greece
The bone marrow microvessel density (MVD) has been shown to be
increased in various hematologic malignancies and is correlated with
unfavorable prognosis in multiple myeloma. There are evidences of augmented
angiogenetic process in polycythaemia vera (PV) either by angiogenic
factors measurements or by MVD bone marrow count estimation.
The aim of this study was to evaluate the correlation of bone marrow
microvascular density and bone marrow fibrosis in patients with PV.
A total of 24 patients with PV (mean age 55,6±13,4 years) and of 10
458 | haematologica/the hematology journal | 2007; 92(s1)
propositus (control group) with no hematologic malignancy were included
to the study. The bone marrow slides were prepared from paraffinembedded
blocks. The endothelial antigen of choice was CD34 which
has been found superior to other antigens that have been used in literature.
The MVD was estimated by visual microvessel grading at 400x
(HPF) by two of the authors. The MVD in all patients was found 4,2±0,2
vessels per HPF and there was no correlation with age, time from diagnosis,
spleen enlargement and treatment. The MVD in the control group
was found statistically significantly lower 1,6±0,2 vessels per HPF
(p=0,00002). We divided the group of patients in two subgroups according
to the grade of fibrosis (grade 0-1 and grade 1-2) of their bone marrows.
In the first subgroup we found 3,4±0,9 vessels per HPF while in
the second subgroup we found 5,3±2,1 vessels per HPF. The difference
among the two groups is statistically significant (p=0,005) and we found
a positive correlation between MVD and reticulin fibrosis grade in
patients with PV (r=0,59, p=0,0025). We also measured the angiogenetic
factors that are implicated in fibroblast proliferation, bFGF (basic
Fibroblast Growth Factor) and VEGF (Vascular Endothelial Growth Factor),
in the sera of the two groups of PV patents. We found no statistically
significant difference among the two subgroups (p=0,42 and
p=0,84) respectively. Even though we found no difference among serum
angiogenetic growth factors while similar cytokine mediated effect has
been found in myelofibrosis with myeloid metaplasia, the evidences of
augmented MVD count in PV patients with higher grade of fibrosis in
comparison to PV patients with lower grade of fibrosis, could be
explained by the hypothesis that the abnormal angiogenic cytokine
milieu acts stimulating fibroblasts enhancing fibrosis.The possible prognostic
relevance of these findings deserves further research.
1264
MANAGEMENT OF GRAM- POSITIVE INFECTIONS WITH LINEZOLID
IN IMMUNOCOMPROMIZED PATIENTS
C. Lalayanni, 1 N. Neokleous, 1 N. Stavroyianni, 1 A. Sirigou, 2
A. Karpouza, 1 A. Papalexandri, 1 A. Bitsioni, 1 C. Smias, 1 R. Saloum, 1
A. Fassas, 1 A. Anagnostopoulos1 1 2 G. Papanicolaou Hospital, THESSALONIKI; G. Papanicolou, THESSA-
LONIKI, Greece
Infections constitute an important cause of morbidity and mortality in
immunocompromized patients. Antibiotic-resistant strains have become
a common problem in every-day practice. Linezolid, an oxazolidone,
inhibits protein synthesis in bacteria with a unique mechanism of action
and is active against resistant strains. We assessed the efficacy and safety
of linezolid in the management of 106 documented Gram-positive
infections in 87 immunocompromised patients. Forty two women and
45 men were included, aged 8-73 (median 36) years, suffering from: acute
leukemia: 58; lymphoma, 14; Hodgkin’s disease, 6; aplastic anemia, 2;
multiple myeloma, 5; and CML 2 patients. Forty one patients had undergone
stem cell transplantation (12 autologous, 29 allogeneic). Seventy
three 73 infections occurred during neutropenia. Linezolid was administered
at a dose of 600 mg twice daily for 4-26 (median 10) days. Parenteral
linezolid was switched to the oral formulation in 36 patients.
Most patients had already been treated unsuccessfully with an antibiotic
against Gram + bacteria (64 with teicoplanine, 14 with vancomycin),
while in 28 linezolid was administered as first line therapy, based on sample
culture. The most common infection was bacteremia (n=45) caused
by methicilline-resistant staphylococcus and associated with the presence
of a central line catheter in 35 cases. Infections caused by enterococcus
were: urinary, 22; soft tissue, 8; respiratory, 6; bacteremia, 6.
Twenty two of 42 strains (52%) were resistant to vancomycin/ teicoplanine.
The clinical response rate was 78% (83/ 106) and microbiological
response rate was 82% (79/96). The most common toxicity was hepatic
(increased liver function tests: 8 patients), while there was no discontinuation
due to intolerance. On multivariate analysis, the factors predicting
for treatment failure where: refractory disease, prior transplantation
and co-infection by Gram – pathogens. In conclusion, linezolid administration
was safe and effective in this group of immunocompromised
patients. Linezolid played a significant role in sterilizing the central line
catheters and in the management of vancomycin resistant enterococci.