96. Jahrestagung der Deutschen Gesellschaft für Pathologie e. V ...

or without different concentrations of BI2536 (10 nM, 50 nM, 100 nM,
200 nM and 500 nM). Cell cycle analysis was performed after 24 and
48 hours of culture using FACS (PI-staining). Western blot analysis
was conducted after 24 (H3Ser10ph) and 48 hours (cleaved caspase-3) of
BI2536 treatment. Immunofluorescence staining of cells was done using
antibodies detecting Plk1 (24 h, 100 nM BI2536), cleaved caspase-3 (48 h,
200 nM BI2536) and alpha-tubulin (24 and 48 h).
Results. Cell cycle analyses of AML cells after 24 h of treatment revealed
an accumulation of mitotic cells (4N) and a decrease of cells in G0/
G1 phase (2N) of the cell cycle. Furthermore, elevated protein levels of
the mitosis specific phosphorylated histone 3 (Ser10) were detected by
western blot after 24 h. Immunofluorescence staining of PLK1 and alpha-tubulin
showed an increase of mitotic figures exhibiting monopolar
spindles without Plk1 localization at centrosomes or kinetochores
of prometaphase chromosomes. A strong increase of cleaved caspase-3,
which was used for the detection of apoptotic cells, was visible after 48 h
by western blot and immunofluorescence analyses in inhibitor treated
cells. Furthermore, the less proliferative LCLs arrested in mitosis to a
lower extent and showed fewer apoptotic cells after 48 h compared to the
analyzed AML cells.
Conclusions. Our data confirm the sensitivity of leukemic cells to Plk1
inhibition by BI2536. Treatment with variable doses of BI2536 results in
prometaphase arrest and a pronounced cell death after 48 h in AML cell
lines. In addition, our experiments with lymphoblastoid cell lines indicate
that less proliferative hematopoietic non-leukemic cells show a weaker
response to therapeutic Plk1 inhibition by BI2536.
FR-P-156
Analysis of BCL6 and MYC coexpression in diffuse large B-cell
lymphoma
L . Culemeyer1 , C . Stuhlmann-Laeisz1 , W . Klapper1 1UKSH Campus Kiel, Institute of Pathology, Kiel
Aims. The transcription factor BCL6 is the master regulator of the germinal
centre reaction. cmYC is a transcription factor, which is often overexpressed
in diffuse large B-cell lymphomas (DLBCL), but is not expressed
in the germinal centre under physiological conditions. Herein we aimed
to quantify the unphysiological co-expression of BCL6 and cmYC in
DLBCL. Furthermore, functional effects of this unphysiological coexpression
of BCL6 and cmYC will be estimated by evaluating BCL6 target
gene expression.
Methods. Fluorescence multi-stainings using the combination of BCL6
and cmYC were quantified by digital image analysis (Tissuequest©).
Results. Unphysiological co-expression of BCL6 and cmYC in lymphoma
cells was detected in DLBCL. This co-expression occurred in DLBCL
with and without BCL6 or cmYC translocations and influences BCL6
target gene expression.
Conclusions. The unphysiological co-expression of transcription factors
of the B-cell differentiation can be detected in DLBCL without genetic
alterations affecting these genes. We suggest that this co-expression interferes
with the activation or inhibition of the respective target genes.
We consider this mechanism to be a reason for the oncogenic potential
of transcription factors, which are also expressed under physiological
conditions.
FR-P-157
Conditional PHD2 deficiency leads to non-lethal erythrocytosis
and alters the hematopoietic stem cell fate
B . Wielockx1 1TU Dresden – Pathology, Dresden
Aims. Hypoxia is a prominent feature during development and physiological
as well as pathological conditions in adults. An oxygen-sensing
machinery is therefore very important to help the cells adapt instantaneously
to any inacceptable O2 level. Such a system relies on the oxygen
dependent HIF-prolyl hydroxylases (PHD1-3), enzymes that can inactivate
the alpha subunit of the hypoxia inducible transcription factor
(HIF). In case of low oxygen availability, PHDs lose their functionality
and allow the HIF complex to promote biochemical and physiological
changes including anaerobic glycolysis, angiogenesis and hematopoiesis.
Results. Our research unit produced a mouse line that lacks PHD2 in
a broad spectrum of cell types (e.g. hematopoietic cells, epithelial cells)
which resulted in an unexpected hematologic phenotype. The mice display
strongly elevated hematocrit levels together with high EPO concentrations
in the blood although mice don’t show any premature lethality.
Moreover, we found that the hematopoietic stem cell (HSC) compartment
in the bone marrow was significantly altered compared to WT
mice.
Conclusions. Indeed, detailed FACS analyses demonstrate that cKO mice
contain much more multipotent progenitors (MPPs). Moreover, under
stress conditions in vivo, cKO HSCs are pushed towards self-renewal.
Double deficient cKO mice with one of the two HIFα revealed that the
erythrocytosis phenotype is exclusively driven by HIF2α, whilst HIF1–α
is responsible for the HSC/MPP phenotype.
FR-P-158
Tumor infiltrating T-cells in high risk chronic lymphocytic leukemia
(B-CLL): a clinicopathological study of CD3, CD8 and FOXP3
expression
C . Schrader1 , C . Pflüger1 , S . Stilgenbauer2 , H . Döhner2 , M . Ritgen1 , J . Claasen1 ,
P . Dreger3 , W . Klapper4 1 2 2 University Hospital of Kiel, nd Department of Medicine, Kiel, University of
Ulm, Department of Hematology, 3University of Heidelberg, Department of
Hematology, 4UKSH, Campus Kiel, Department of Pathology
Aims. The prognostic value of the mutation status of the immunoglobulin
heavy chain variable region (IgVH) and cytogenetic abnormalities in
chronic lymphatic leukemia is well known. We investigated the tumour
associated reactive T-cell infiltrates in bone marrow and lymph nodes
biopsies of patient with high risk B-CLL in correlation to other clinical,
biological and genetic markers including age, sex, binet stage, bone marrow
involvement (infiltration pattern and grade), β2 microglobulin level,
leucocytes account, lymphocytes double time, thymidinkinase level, cytogenetic
aberrations (e.g. 11q deletion), IgVH mutations status and ZAP
70 expression
Methods. Bone marrow (n=51) and lymph node biopsies (n=8) from 59
untreated patients (43 men and 16 women) were investigated immunohistochemically
with monoclonal antibodies against CD20, CD5, CD23,
CD3, CD8, FOXP3 and ZAP70. Cells with clear positive staining were
counted and the percentage was calculated. Molecular analysis of IgVH
mutation and FISH analysis was done from fresh peripheral blood tumor
cells.
Results. In 58 biopsies the CD 3 staining, 57 cases of the CD 8 and in
all 59 cases the FOXP staining was evaluable. The CD3 staining had a
range of 0.4% to 35.2% with a median of 9.3% and a mean of 11.2%. In
lymph node tissue a significant higher number of CD3 cells than in bone
marrow biopsies (p=0.0054) was found. Similar results were found in the
CD8 (p=0.0052) and FOXP3 (p=0.0087) stainings with higher T-cells account.
In the analysis of the bone marrow biopsies the nodular pattern/
involvement showed a significant higher number of CD3 cells (p=0.013)
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