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

0523
DNA REPAIR INHIBITION IN RESTORING CHRONIC LYMPHOCYTIC LEUKEMIA LYMPHO-
CYTES SENSITIVITY TO FLUDARABINE AND RITUXIMAB
T. Sergienko, V. Smolnikova, A. Bakun, I. Taras, A. Svirnovski
Center for Hematology and Transfusiology, MINSK, Rebublic of Belarus
Background. Combined application of cytotoxics and monoclonal antibodies
not always enhances therapeutic activity in leukemia cells. Modified
DNA repair contributes to resistance development in chronic lymphocytic
leukemia (CLL) lymphocytes. Majority of cytotoxic drugs,
including fludarabine (Frn), influence DNA synthesis and repair. Rituximab
(Rtx) has multiple mechanisms of inducing in vivo cytotoxicity and
direct apoptotic signaling is one of them. Lack of appropriate response
to Frn and Rtx can be associated with enhanced DNA repair in peripheral
blood lymphocytes. Usage of DNA-dependent protein kinase (DNA-
PK) inhibitor vanillin (Vnl) may contribute to Frn and Rtx resistance overcoming.
Aims. To investigate CLL cell response in vitro to the action of Frn
and Rtx under the DNA repair inhibition with vanillin. Methods. MTT
assay was used to evaluate cell sensitivity to purine nucleoside analog
and monoclonal antibody (in concentrations similar to therapeutic),
vanillin (in nontoxic concentration to normal lymphocytes) in vitro.
According to lymphocyte viability, cell samples were divided into groups
with different sensitivity to Frn and Rtx: sensitive (cell viability less than
35%), intermediate (35-70% viable cells) and resistant (cell viability more
than 70%). Results. Vnl not significantly enhanced Frn activity in CLL
cells irrespective of lymphocyte response to Frn (shown in table). In cells
with high and intermediate sensitivity to Rtx negligible protective effect
of Vnl was detected. On the contrary, in Rtx resistant CLL lymphocytes
combined action of Vnl and Rtx significantly suppressed cell viability
twice as much as a monoclonal antibodies alone. In Frn resistant group
strong correlations between cell sensitivity to Vnl and Frn were observed
(r=0.83-0.93, p0.05). Correlation analysis confirmed
unidirectionality of CLL cells response to Van and Frn, and lack of interdependence
between Van and Rtx action. Cell sensitivity and clinical
data dependence was studied. No significant correlations were observed
in cell susceptibility to drugs and peripheral blood CD5 + and CD20 + cell
number. Apoptosis marker bcl-2 level was also measured. Intrinsic bcl-
2 level ex vivo didn’t correlate with cell sensitivity to Frn, Rtx, and Vnl.
Most likely Frn and Rtx don’t directly influence bcl-2 pathway. Consequently
endogenous bcl-2 lymphocyte level can not be used as strong
prognostic factor for cell sensitivity. Summary and conclusions. Vanillin is
an effective agent to overcome Rtx resistance in CLL lymphocytes, but
it doesn’t influence Frn activity. DNA repair inhibition is one of the possible
pathways that leads to lymphocyte sensitivity restoration to Rtx,
where as Frn unreceptiveness overcoming is achieved with DNA-PK
inhibitor suggesting another pathways of Frn resistance formation.
Table 1. Viability modification by vanillin in cells with different
sensitivity to Frn and Rtx.
12 th Congress of the European Hematology Association
0524
EFFICACY OF FLUDARABINE IN A NOVEL NOD/SCID XENOTRANSPLANTATION MODEL OF
CHRONIC LYMPHOCYTIC LEUKEMIA
P. Ebeling, F. Grabellus, S. Seeber, U. Duehrsen, T. Moritz, J. Duerig
University of Duisburg-Essen, ESSEN, Germany
Background. Chronic lymphocytic leukemia (CLL) represents the most
frequent leukemia in the western world. Although improvements in
therapeutic strategies has been achieved in recent years, this desaese
still remains incurable. Therefore novel therapeutical stragegies to
improve patient outcome need to be established. However, preclinical
in vivo drug testing, a crucial prerequisite for the develompent of such
novel strategies in CLL, so far still is hampered by the lack of a suitable
animal model. We have previously reported reliable engraftment of
CD19/CD5/CD23/CD45 positive human B-CLL cells in the spleens of
NOD/SCID mice (NSS-CLL cells) following i.v. and/or i.p. transplantation
of 1×108 peripheral blood derived mononuclear cells from CLL
patients (PB-CLL). On histology, in the majority of the samples, NSS-CLL
cells formed focal aggregates which also contained various amounts
(range:0-50%) of human CD45/CD3 + T cells (ASH 2005; #52). Aims. We
now have further characterized these NSS-CLL cells. Additionally, drug
testing experiments were performed to determine if this novel model
may be suitable to preclinically investigate therapeutic effects of established
treatment strategies in CLL. Methods. For further characterization
of NSS-CLL cells, a panel of surface and intracellular marker molecules
like Ki67, Survivin, CD100, CD10, CD11c, CD127 and CD49d was utilized.
Drug testing experiments in this model were performed using fludarabine
(Flud), a chemotherapeutical agent with proven activity against
CLL. For theses latter experiments 1×108 PB-CLL were injected i.v. into
the tail vein of NOD/SCID mice. Starting two weeks after transplantation
the animals were allocated to receive therapy with either Flud or no
therapy, respectively. Flud was given at a dosis of 35mg/kg ip. for 5 consecutive
days. Four weeks after transplantation animals were sacrificed
and BM and spleens were analyzed for the presence of CLL cells using
multicolor flow cytometry. Results. Concerning the further characterization
of NSS-CLL cells, immunohistological analysis showed positive
staining for the proliferation marker Ki67 in 18.5±3.5% of these cells.
Further, markers related to proliferation/activation of B-CLL proliferation
center cells such as survivin (mean MFI: 5.5±0.8 vs. 2.3±0.2; p=0.028;
n=6) and CD100 (semaphorin 4d; mean MFI: 3.3±0.5 vs. 1.1±0.2;
p=0.043; n=5) were significantly up-regulated in NSS-CLL vs. PB-CLL
cells. In contrast, no expression of human CD10, CD11c, CD127 or
CD49d was detected in B-CLL cells derived from either PB- or NSS-CLL
cells, while both sources uniformly stained positive for CD40. With
regard to 8 individual drug testing experiments a significant lower
amount of 2.2±1.2 vs. 11.4±2.4×105 (p=0.003) CLL cells could be recovered
from the spleens of treated animals when compared to the control
group. Thus, treatment with Flud resulted in a relative reduction of total
amount of CLL cells recovered from the murine spleen of 85.5±8.1%
(p=0.003). Summary. NSS-CLL cells display certain similarities of CLL
proliferation centers like expression/upregulation of proliferation/activation
markers and therefore this model could serve as a valuable tool to
investigate CLL tumour biology. In addition the model allowed for reliable
in vivo drug testing of a conventional therapeutic agent and thus
argues for a preclinical evaluation of this new model also to investigate
novel chemotherapeutic strategies.
haematologica/the hematology journal | 2007; 92(s1) | 195