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

SO-005
Prevalence of mutations in signaling pathway components
downstream of the epidermal growth-factor receptor (EGFR) in
colorectal cancer
J . Neumann 1 , L . Wehweck 1 , S . Maatz 1 , F . Mourched 1 , A . Hendrowarsito 1 ,
J . Engel 2 , T . Kirchner 1 , A . Jung 1
1 Ludwig-Maximilians-Universität München, Department of Pathology, München,
2 Ludwig-Maximilians-Universität München, Institut für medizinische
Informationsverarbeitung, Biometrie und Epidemiologie, München
Aims. In metastatic colorectal cancer (mCRC) KRAS-mutations are associated
with clinical resistance to treatment with monoclonal antibodies
targeting the epidermal growth factor receptor (EGFR). However,
up to 50% of these patients do not respond to this therapy. To identify
novel biomarkers, downstream effectors of the EGFR-pathway are currently
validated according their predictive potential. Aim of this study
was to determine the frequency and overlap of key-mutations in the
EGFR-pathway. Furthermore, the concordance between primary tumor
and distant metastasis was analyzed.
Methods. Key mutations of KRAS, BRAF, AKT and PIK3CA were analyzed
by pyrosequencing in a collection of 63 mCRC patients in primary
tumor samples and corresponding metastases, respectively. Furthermore
PTEN- and EGFR-Immunohistochemistry were applied.
Results. Nine of the investigated 63 tumors (14.3%) had mutations in more
than one gene of the EGFR-pathway. KRAS- and BRAF-mutations were
detected in 50.8% and 7.9%, respectively, and were mutually exclusive.
Mutations in the PIK3CA-gene (15.9%) showed huge overlap with KRASmutations
(8 of 10 cases). Only one case with a mutation in the AKT-gene
(1.6%) could be detected showing a simultaneous BRAF-mutation. Mutation
analysis for KRAS, BRAF and AKT revealed a 100% concordance,
whereas PIK3CA exhibited one discordant case showing a mutation in
the primary tumor which could not be detected in the matched distant
metastasis (Κ=0.9). Immunohistochemistry revealed in 49.2% high levels
of EGFR-expression and in 42.9% loss of PTEN-expression, both showing
huge overlap with KRAS-, BRAF-, AKT and exon 9 PIK3CA-mutations
but not with exon 20 PIK3CA-mutations. Furthermore, high rates
of discordant cases were found.
Conclusions. We could demonstrate that KRAS- and PIK3CA-mutations
as well as BRAF- and AKT-mutations can co-occur within a single
tumor. However, the predictive value of these individual tumor profiles
for anti-EGFR targeted therapies has to be validated in further clinical
studies. Additionally, our data indicate that for molecular analysis of
KRAS, BRAF and AKT either the primary tumor or the distant metastasis
is suitable. Due to the lower concordance rates of PIK3CA-mutations
the primary tumor site should be selected. Analysis of PTEN and EGFR
protein expression are afflicted with a huge amount of discordant cases.
Therefore, before these markers can be used in pathological routine diagnostics,
standardized protocols are needed.
SO-006
Synergistic cytotoxicity of recombinant HMGB1 and pro-apoptotic
drugs in colon carcinomas
G . Gdynia1 , C . Zhang1 , M . Keith1 , J . Kopitz2 , P . Schirmacher2 , W . Roth1 1Institute of Pathology, University Hospital Heidelberg, and German Cancer
Research Center, Heidelberg, 2Institute of Pathology, University Hospital
Heidelberg, Heidelberg
Aims. Colon carcinoma cells are highly resistant to chemotherapeutic
drugs. We recently described a new form of necrosis-like cell death in
cancer cells induced by the HMGB1 protein. The aim of this study was to
investigate whether the co-activation of cell death pathways by apoptosis
inducers and HMGB1 can overcome the intrinsic resistance of colon carcinoma
cells to pro-apoptotic therapeutics.
Methods. ATP luciferase assay, LDH-linked lactate accumulation measurement,
OXPHOS flux, FACS, western blot, immunoprecipitation,
electron microscopy, subcellular fractionation, liposome transfection,
generation of stably Flag-/Myc-tagged-HMGB1 and stably Bcl-2 overexpressing
cells, crystal violet cytotoxicity assay, oxygen consumption
measurements.
Results. Colon carcinoma cell lines were differentially sensitive to recombinant
HMGB1. HMGB1 treatment resulted in the formation of giant
mitochondria and a steady decline of ATP. Overexpressed HMGB1
specifically bound to cytochrome c oxidase (COX IV) thus impairing
mitochondrial respiration measured by a marked decrease of mitochondrial
oxygen consumption. Colon carcinoma cells with depleted
mitochondrial DNA (rho zero cells) were less susceptible to the cytotoxic
effects of HMGB1. Combined treatment of colon cancer cells with
subtoxic concentrations of HMGB1 and the death ligand TRAIL or the
Bcl-2 inhibitor ABT-737 resulted in a strongly synergistic induction of
cytotoxicity. The cell death was accompanied by an early activation of
caspase-3 and a continuous decline of ATP. However, no cytochrome c
release was measured in the co-treated cells, and the overexpression of
the mitochondrial outer membrane associated anti-apoptotic Bcl-2 protein
only moderately inhibited the HMGB1-mediated cytotoxicity.
Conclusions. HMGB1 inhibits oxidative respiration of colon carcinoma
cells thus depleting intracellular ATP levels. The administration of
HMGB1 and pro-apoptotic compounds greatly increases their cytotoxic
effects on colon carcinoma cells by activating both the necrotic and
apoptotic cell death machinery.
SO-007
Opposite effects of tissue inhibitor of metalloproteinases- 1
(TIMP-1) overexpression and knockdown on colorectal liver
metastases
O .R . Bandapalli1 , E . Paul1 , P . Schirmacher1 , K . Brand1 1Institute of Pathology, Heidelberg
Aims. Tissue inhibitors of metalloproteinases (TIMPs) and the corresponding
metalloproteinases are integral parts of the protease network
and have been shown to be involved in cancer development and metastasis.
Paradoxically, for TIMP-1, tumor promoting as well as tumor inhibitory
effects have been observed.
Methods. To address this paradox, we utilized the BALB/c/CT26 mouse
model that reliably leads to liver metastasis after splenic tumor cell
injection and variegated the type of target cells for therapeutic intervention
and the modalities of gene transfer. Since we have observed before
that overexpression of TIMP-1 in liver host cells leads to efficient tumor
growth inhibition in this model, we now examined whether targeting
the tumor cells themselves will have a similar effect.
Results. In concordance with the earlier results, TIMP-1 overexpression
in tumor cells led to a dramatic reduction of tumor growth as well. To
evaluate any influence of treatment modality, we further examined
whether TIMP-1 knockdown in the same animal model would have the
opposite effect on tumor growth than TIMP-1 overexpression. Indeed,
TIMP-1 knockdown led to a marked increase in tumor burden.
Conclusions. These data indicate that in the BALB/c/CT26 model, the
modification of TIMP-1 has concordant effects irrespective of the type
of target cell or the technique of modulation of TIMP-1 activity, and that
TIMP-1 is unequivocally tumor inhibitory in this model.
Der Pathologe · Supplement 1 · 2012 |
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