forschungslaboratorien

Transcription

FORSCHUNGSLABORATORIEN
Universitätsklinik für Frauenheilkunde
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L ABO R - FO RSCH U N GS EINHEITEN DER U NI V ERSITÄTS K LINIK FÜ R FR AU ENHEILKU NDE
Die Labor-Forschungseinheiten der Universitätsklinik für
Frauenheilkunde entwickelten sich aus den Labors der
ehemaligen I. und II. Universitäts-Frauenklinik und sind seit
Mitte der 1990er Jahre in einem Cluster auf Ebene 5Q zu-
174 | Universitätsklinik für Frauenheilkunde
sammengefügt. Sie sind dem Vorstand der Klinik, o.Univ.
Prof. Dr. Peter Husslein direkt unterstellt. Wurde ursprünglich die Routine und Forschung gleichermaßen abgedeckt,
so liegt heute der Schwerpunkt auf Grundlagen- und angewandter Forschung. Sechs Arbeitsgruppen, die international in vielen Partnerschaften vernetzt sind, führen
eine Vielzahl von Projekten durch, die sich mit speziellen
Fragestellungen im Bereich der Geburtshilfe, der Gynäkologie, der gynäkologischen Onkologie und Endokrinologie
beschäftigen.
Molekulare Onkologie
Die Arbeitsgruppe entwickelte sich aus dem Hormonlabor der I. Universitäts-Frauenklinik, wird von ao.Univ.Prof.
Dr. Robert Zeillinger geleitet und ist eng mit dem „Ludwig
Boltzmann Cluster Translational Oncology“ verknüpft. Der
Forschungsschwerpunkt liegt bei der Erforschung des
Ovarialkarzinoms. Weitere Projekte befassen sich mit dem
Nachweis und der molekularen Charakterisierung im Blut
zirkulierender Tumorzellen und der Früherkennung verschiedener Krebsarten.
Prädiktive Onkologie
Die von Univ.Prof. Dr. Christian Singer geleitete Arbeitsgruppe setzt sich schwerpunktmäßig mit dem erblichen
Brust- und Eierstockkrebs und der Rolle von BRCA 1 und 2
bei verschiedenen Aspekten von Brustkrebserkrankungen
auseinander. Weitere Forschungsprojekte beziehen sich
auf die Rolle von Wachstumsfaktoren bei der mitogenen
Stimulation und die Ausbreitungsmechanismen von Tumorzellen. Darüber hinaus wird intensiv die Rolle der Estrogenrezeptoramplifikation (ESR1 Amplifikation) beim endokrinresponsiven Mammakarzinom untersucht.
Reproductive Biology
Die Arbeitsgruppe wird von ao.Univ.Prof. Mag.Dr. Martin
Knöfler geleitet. Sie hat die Differenzierung und Entwicklung der humanen Plazenta als Schwerpunkt und beschäftigt sich mit molekularen Mechanismen der Plazentadifferenzierung unter physiologischen und pathologischen
Bedingungen, mit plazenta-spezifischen Transkriptionsfaktoren und Signalwegen, die die Invasion des Trophoblasten
kontrollieren, sowie der transkriptionellen Kontrolle von
Schwangerschaftshormonen.
Onkogenomics
Der Arbeitsgruppe steht ao.Univ.Prof. Mag.Dr. Martin
Schreiber vor. Das Forschungsthema der Arbeitsgruppe ist
Brustkrebs, wobei der Schwerpunkt auf genomischen und
molekularbiologischen Forschungsansätzen liegt. Eines
der Hauptziele der Forschungsarbeiten ist es, molekulare
Mechanismen der Invasivität und Metastasierung aufzuklären und Proteine sowie funktionelle RNAs zu identifizieren,
die dabei eine Schlüsselrolle spielen.
Gynäkologische Endokrinologie
Geleitet wird die Arbeitsgruppe von ao.Univ.Prof. Mag.Dr.
Christian Schneeberger. Die Schwerpunkte liegen in der
Erforschung von Signaltransduktionswegen, die an der
Entstehung der Endometriose beteiligt sind, der Bedeutung
von SNPs und miRNAs für verschiedene Fragestellungen in
der gynäkologischen Endokrinologie und in der Isolierung,
Kultivierung und Differenzierung von hämatopoietischen
Progenitorzellen aus Nabelschnurblut.
Medical Genetics
2011 wurde Univ.Prof. Dr. Berthold Streubel der Universitätsklinik für Frauenheilkunde zugewiesen und etablierte
eine Forschungsgruppe mit Schwerpunkt in der genetischen Ursachenforschung von pränatalen und postnatalen
Krankheitsbildern sowie in der Tumorentstehung. Hierbei
kommen entsprechend den technologischen Entwicklungen vermehrt Hochdurchsatzgenomanalysen zur Anwendung.
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M O LEKU L A RE ONKO LO G IE
From left to right:
Top: Andrea Wolf, Stephanie Aust, Eva Obermayr, Dietmar
Pils, Eva Schuster, Barbara Holzer, Nina Pecha, Robert Zeillinger;
Centre: Anna Bachmayr-Heyda, Magdalena Gamperl, Eva
Blümel, Caroline Kreuzinger, Peter Borossay, Grazyna Dudek, Martin Stöger, Dan Cacsire Castillo-Tong;
Bottom: Bettina Savarese-Brenner, Simon Deycmar, Nyamdelger Sukhbaatar, Elisabeth Maritschnegg, Katharina
Auer, Markus Niebuhr, Agnes Reiner, Paul Speiser
PROJEK TE 2014
Collaborators: Maritschnegg E, Obermayr E, Zeillinger R,
Holzer B, Pils D, Deycmar S, Cacsire-Castillo Tong D
GANNET53 – Companion diagnostics of a drug study in metastatic, platinum-resistant ovarian cancer
patients
The FP7 EU-funded study GANNET53 targets the predominant, aggressive Type II ovarian tumours, which are characterized by resistance to platinum-based chemotherapy. In
that course, mutant p53 should be targeted via an innovative Hsp90 (heat shock protein 90) inhibition applying the
second-generation inhibitor Ganetespib. After successful
completion of Phase I, Phase II will start in March 2015. Different aspects are covered in the companion diagnostics,
for example a test, for possible prediction of responsiveness to Ganetespib should be established. Therefore ascites
will be collected and cultured in the presence of different
concentrations, or the absence of Ganetespib. The interaction of p53 with Hsp90 will be
determined by the in situ Proximity ligation assay (PLA).
A strongly reduced signal or no
signal after treatment will be interpreted as in vitro response.
Correlations of in vitro response
with clinical outcome measures
will be performed, allowing evaluation of the assay‘s predictive
value. An example of a PLA performed on ascites cells is
shown, where each red dot represents the interaction between a p53 and HSP90 protein. A second part involves
monitoring of the patients via analysis of circulating tumour
cells (CTCs). Tumour cells will be enriched via the Parsortix microfluidic system and analysed concerning their RNA
expression of specific markers, and detected and characterized by immunofluorescence staining.
Recent publications showed that the p53 protein, especially in its mutant form, aggregates into prion-like amyloid
oligomers and fibrils. These structures might play a role in
the development of therapy resistance. Still, these studies
lack patient data and the methods used for analysis are
limited. We established an ELISA based detection method,
where a special coated plate is able to capture protein aggregates. We’ve applied the assay to various ovarian cancer cell lines, with different p53 status. As expected, in p53
mutant cell lines, p53 prions were more prevalent than in
p53 wild type ones. Cell lines carrying p53 null mutations
showed the lowest levels.
Another side project is the evaluation of the extracellular
HSP90 levels in plasma. Normal cells only secrete eHSP90
under stress, whereas tumour cells show constitutive secretion linked to motility, invasion and metastasis. First
tests in a small number of patients showed, that the plasma HSP90 levels were significantly increased not only in
late stage, but also early stage ovarian cancer patients,
compared to healthy controls.
Evaluation of the Parsortix micro-fluidic technology
to enrich CTCs
Collaborators: Obermayr E, Maritschnegg E, Pecha N,
Schuster E, Borrosay P, Holzer B, Wolf A, Speiser P, Zeillinger R
We intended to develop a protocol combining the novel
micro-fluidic enrichment technology (Parsortix, Angle plc.,
UK; see figure 1) and RT-qPCR for the molecular analysis of
CTCs. Recently we identified CTC-specific mRNA markers
allowing CTC detection in 29% of breast cancer patients
and in 24.5% of ovarian cancer patients at diagnosis. However, the detection of cancer cells was hampered by the
large number of contaminating leukocytes. The necessary
Figure 1: The Parsortix device for the enrichment of CTCs from blood samples
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use of cut-off values for positivity reduced the specificity
of the down-stream PCR assay. By improving the purity of
cancer cells and PCR analysis we sought to increase both
sensitivity and specificity of the diagnostic procedure.
Bio Bank of the Division of Gynecology and Gynecological Oncology for Research, Documentation and
Analysis, in cooperation with the Center for Medical
Statistics, Informatics, and Intelligent Systems.
For reducing the blood sample volume (up to 20ml) density gradient centrifugation was used when needed. The
samples were passed through the micro-fluidic disposable
Parsortix cassette (see figure 2), which captures tumour
cells based on their less deformable nature and larger size
compared to blood cells.
Collaborators: Pecha N, Pieber K, Obermayr E, Schuster
E, Borrosay P, Holzer B, Wolf A, Wrba T, Zeillinger R.
Figure 2: The microscope slide sized microfluidic separation cassette
(left) which is inserted into the Parsortix device. When the blood sample is
passed through the cassette, large and less deformable cells are captured
within the 10µm separation step (right).
Lysis of the captured cells was done directly in the cassette and total RNA was extracted. After a cDNA pre-amplification step gene expression levels of leukocyte-specific
and CTC-related markers were measured using RT-qPCR.
The efficiency of the combined protocol was assessed in
blood samples taken from patients with primary and recurrent malignant diseases. As a result, 7 out of 13 pre-selected RNA markers were not detectable in blood samples
from 11 healthy volunteers. In cancer patients we observed
measurable gene expression of at least one out of these 7
RNA markers. 5 out of 5 breast cancer (primary disease:
N=1; metastatic: N=4), 9 out of 10 primary and 4 out of 8
relapsed ovarian cancer patients were classified correctly by the test. The most striking finding is the detection
rate of 90% for primary ovarian cancer at a specificity of
100%. We conclude that the enrichment of rare cells from
blood samples using micro-fluidics results in a highly pure
cell population. This enables the application of extremely sensitive methods, like RT-qPCR to specifically detect
rare events. By combining the novel micro-fluidic Parsortix
technology for cell enrichment with molecular analysis we
have taken a major step forward, which allows the implementation of ‘liquid biopsies’ in cancer detection studies
and as a companion diagnostic in clinical trials.
“Tissue (bio) banking is what will take us to the next level of
cancer treatment and cure.”
- Judy Garber, MD, MPH, Professor of Medicine, Harvard
Medical School
Bio Banks are facilities that are developed to collect, store
and distribute samples for further use in basic and translational cancer research. Collection of high-quality bio samples, such as tumor tissue and peripheral blood, is the basis
for discovering new biomarkers as well as novel therapy
targets. Recent progress in the diagnosis and treatment of
cancer can be attributed mainly on translational oncology
research. Translational oncology focuses on novel research
investigations which bridge the laboratory and clinical
settings, including risk assessment, cellular and molecular characterization, prevention, detection, diagnosis and
treatment of human cancers with the overall goal of improving the clinical care of oncology patients.
Collecting bio samples for research immediately prompts
the need for sound administration of the collected and
conserved samples, enabling the availability of corresponding clinical data and tracking of the samples.
A research database by the name of “Tumorbank Molekulare Onkologie”, located at the laboratory of the Molecular
Oncology Group, Division of Gynecology and Gynecological Oncology at the University Clinic of Obstetrics and
Gynecology, has been already implemented in 2013. Documentation of all data and setting up queries to enable data
management is an ongoing project, with exportable data
being available for statistical analysis now.
Since December 2014, tissue samples as well as liquid
samples (e.g. blood, ascites, saliva) has been collected
and stored partly already in tubes with a barcoding system.
This allows a unique sample assignment and recording in
our Bio Bank database. As mentioned above, identification
of the individual sample is crucial for a well-maintained Bio
Bank. To fasten this process, the use of barcode scanners
is supported by the RDA application. Barcodes can be read
into any text field available in electronic case report forms.
Hand-written tags, as used at the Molecular Oncology laboratory right now, pose a potential risk for mistakes such
as wrong or unreadable labeling. For a fast and definitive
identification of samples, barcoded sample tubes with barcode scanners are set up within reach at the laboratory
workbenches. For an optimal use of barcoded systems,
the import of scanned character strings into the databa-
se has to be set up, too. Due to a planned cooperation of
the Molecular Oncology Group with the Central Laboratory
of the Vienna General Hospital (AKH), the interface between the RDA and the laboratory information system will
be introduced as well. Along with the occurring of “-omics”
platforms, requirement of high quality cancer tissues or
peripheral blood for research of genome, transcriptome,
proteome and metabolome is increasing rapidly. Therefore,
the creation of high quality banks of biological resources
at large medical centers or institutes is a fundamental and
valuable work. The results of research using tissue samples will not affect therapeutic care of the patient right after
donation of the sample, but if the cancer reoccurs in the
future, however, new treatment options may be available.
Stored tissue material of the cancerous disease at the time
of first occurrence will be available. To compare the original tissue with the recurrent disease could help to not
only distinguish between true recurrences versus second
primary tumor, but help to improve the clinical care of the
cancerous disease for two reasons:
In comparison to a pathological institute, that often only
stores paraffin embedded tissue samples, the Bio Bank
of the Molecular Oncology group has been storing fresh
frozen tissue samples and “liquid biopsies” such as blood,
saliva and lavage samples, as well.
The stored samples have already been tested on. New
treatment options as well as novel biomarkers may be
available by the time the patient is diagnosed with metastasis. The primary tissue samples stored already at the Bio
Bank can then be tested with newly discovered diagnostic
and prognostic biomarkers and compared to the recurrent
disease. The patient will therefore profit from the scientific advances made by applied research done on already
stored sample in the past donated by other individuals and
hopefully profit from new advances in translational oncology like new drug targets.
Lavage of the uterine cavity as potential tool for the
diagnosis of ovarian cancer, its precursor lesions
and endometrial cancer.
Figure 1: Laboratory entry form
Figure 1: MPS mutation analysis of lavage specimens depicted as percent
mutant alleles present. Shown are the results obtained from analysis of
patients included in final analysis. Disease type, as well as stage of disease
of EOC patients is listed on the y axis. The fraction of mutant alleles varied
between 0.95% and 50.72%.
Collaborators: Speiser P, Pecha N, Maritschnegg E, Horvat R, Zeillinger R
Epithelial ovarian cancer (EOC) is generally diagnosed at an
advanced stage, translating into a poor survival rate. There is increasing evidence that EOC often originates in the
fallopian tubes and furthermore, tumours in the ovary may
exfoliate cells that are transported into the uterine cavity
via the fallopian tube. We have established an approach
for lavage of the uterine cavity to detect shed cancer cells.
This technique was further optimized, to be performed in
an outpatient setting and in a non-invasive manner. Genetic changes were analysed in the lavage specimens to
confirm the presence of cancer cells. This principle is applied in different studies. The LUDOC study will give information on the specificity and sensitivity of our approach in
detecting EOC and its application in differential diagnosis
of suspicious pelvic masses. Six European centres are involved so far. In the corresponding proof of concept study
we obtained a lavage of the uterine cavity from 42 patients.
Twenty-seven of these had EOCs, six had other malignancies with involvement of gynaecological organs, while nine
had benign tumours. These samples, as well as corresponding tumour tissue if available, were examined for the
presence of somatic mutations in a panel of genes using
massively parallel sequencing. Lavage technique could
successfully be applied and sufficient amounts of DNA
obtained in all patients. Mutations in the genes PPP2R1A,
TP53, CDKN2A, FBXW7, KRAS, PTEN, AKT1, EGFR, FGFR2
were identified in 16/26 (62%) lavage samples of patients
with malignant tumors and only in 1/8 (12.5%) of patients
with benign tumours (P=0.02, Fisher Exact Probability Test,
one-tailed) (manuscript submitted).
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By adding yet three more studies, using the same molecular detection principle, we further extend the potential application spectrum of the Lavage technique: We hypothesize that even premalignant changes from high risk patients,
could be detected applying this procedure, since already
the precursor lesions, so called STICs, show a high exfoliation tendency. In the course of a project funded by the
“Anniversary Fund of the Austrian National Bank” (LUSTIC)
which started in 2014, we will be able to address this question. Six different European centres have already entered
patients in this study.
One pilot study is aiming at detecting exfoliated cells from
endometrial cancer, particularly of type II EC, in the lavage
fluid. The third study tested the feasibility of the procedure,
measuring objective parameters like the amount of liquid
that can be obtained as well as the condition of the lavage
and complications occurring. 87 lavages have been protocoled in Vienna and in four additional cooperating centres
in Europe. No adverse event occurred so far (manuscript
under preparation).
HPV-Papillocheck: Image and PCR based detection
of cervical cancer derived CTC’s
Collaborators: Niebuhr M, Maritschnegg E, Holzer B, Zeillinger R
Human papillomavirus (HPV) is involved in about 99% of
all cervical carcinoma cases. A possible relapse detection
method for HPV caused cervix carcinoma is the analysis of
off-scaled circulating tumour cells in peripheral blood. For
this purpose CTC detection via immunofluorescence and
PCR should be established.
For secondary immunofluorescence based image detection a panel of known CTC marker (EpCAM and Cytokeratin) is extended by the HPV oncogene E7 and upregulated
cell cycle promotor (CCNE2 and p16).
An example of cervical derived CTCs immunofluorescence
staining is shown. Here the overlying signals of HPV E7
(yellow), EpCAM (green) and Cytokeratin (blue) verifying
the cervical cancer offspring. In order to increase the general specificity and throughput a PCR based CTC detection
should be established. Due to its constitutive transcription,
the E7 mRNA represents a reliable PCR marker. Therefore
genotype specific probes targeting HPV E7 should be designed and evaluated. Currently
58 patients are included.
Figure1(HPV-Papillocheck): Immunofluorescence staining of patients enriched
mononuclear blood cell fraction. Clear
distinction between cells of hematopoietic offspring and cervical cancer CTCs.
Grey DAPI, green EpCAM, red CD45,
yellow HPV E7, blue Cytokeratin.
Since the clinical relevance and abundance of HPV is mainly based on the genotypes HPV 16 and HPV 18, a reliable
genotyping for the evaluation of both methods is needed.
Therefore a sub-project started to evaluate the retrospective HPV genotyping method using FFPE starting material.
It is planned to include 30 CIN I to CIN III patients in the
genotyping evaluation.
Correlation of circular RNA abundance with proliferation - exemplified with colorectal and ovarian
cancer, idiopathic lung fibrosis, and normal human
tissues.
Collaborators: Bachmayr-Heyda A, Reiner AT, Auer K,
Sukhbaatar N, Aust S, Bachleitner-Hofmann T, Mesteri I,
Grunt TW, Zeillinger R, Pils D
Circular RNAs are a recently (re-)discovered abundant
RNA species with presumed function as miRNA sponges,
thus part of the competing endogenous RNA network. We
analysed the expression of circular and linear RNAs and
proliferation in matched normal colon mucosa and tumour
tissues. We predicted >1,800 circular RNAs and proved
the existence of five randomly chosen examples using
RT-qPCR. Interestingly, the ratio of circular to linear RNA
isoforms was always lower in tumour compared to normal
colon samples and even lower in colorectal cancer cell lines. Furthermore, this ratio correlated negatively with the
proliferation index. The correlation of global circular RNA
abundance (the circRNA index) and proliferation was validated in a non-cancerous proliferative disease, idiopathic pulmonary fibrosis, ovarian cancer cells compared
to cultured normal ovarian epithelial cells, and 13 normal
human tissues. We are the first to report a global reduction
of circular RNA abundance in colorectal cancer cell lines
and cancer compared to normal tissues and discovered
a negative correlation of global circular RNA abundance
and proliferation. This negative correlation seems to be a
general principle in human tissues as validated with three
different settings. Finally, we present a simple model how
circular RNAs could accumulate in non-proliferating cells
(Figure 1).
Sci Rep. 2015 Jan 27;5:8057. doi: 10.1038/srep08057
Figure 1. Schematic model
of circular and linear RNAs
in proliferating and nonproliferating cells.
Peritoneal Tumor Spread in Serous Ovarian Cancer Epithelial Mesenchymal Status and Outcome
Collaborators: Auer K, Bachmayr-Heyda A, Aust S,
Sukhbaatar N, Reiner AT, Grimm Ch, Horvat R, Zeillinger
R, Pils D
In this study we aimed to analyse the biological mechanisms underlying apparently different modes of peritoneal
tumour spread in serous ovarian cancer: miliary (widespread, millet-like lesions) versus non-miliary (bigger, exophytically growing implants). Tumour tissues and ascites
from 23 chemotherapy naive patients were analysed by
RNA-sequencing and flow cytometry. On the basis of differential gene expression between miliary and non-miliary, gene signatures were developed. A calculated tumour
spread factor revealed a significant independent negative impact of miliary spread on overall survival (HR 3.77;
CI95 1.14-12.39; p=0.029) in an independent cohort of 165
serous ovarian cancer patients.
Comparing previously published epithelial-mesenchymal
transition (EMT) gene signatures, non-miliary spread correlated significantly with a reduced epithelial status.
We conclude that serous ovarian cancer is a heterogeneous disease with distinct modes of peritoneal tumour
spread, differing not only in clinical appearance, but also
in molecular characteristics and outcome. EMT, peritoneal
inflammation status, and therapeutic options are discussed
and findings summarized in Figure 2.
Significance. More than half of serous epithelial ovarian
cancer patients present with a newly described type of intraperitoneal tumour spread, associated with differences
in the inflammation status, activated oncogenic pathways,
Figure 2. Summary of results (flow cytometry and transcriptomics) comparing cancer cells of patients with miliary and non-miliary peritoneal tumour
spread. Non-miliary ascites samples showed more CD44+ cells, lower keratin expression, and higher MMP9/12 expression compared to miliary ascites samples. Ascites tumour cells of non-miliary patients showed a substantial reduced epithelial character compared to tumour cells from all other
origins and tumour spread types, whereas ascites and solid tumour cells
of non-miliary patients a globally increased mesenchymal characteristic
revealed compared to ascites and solid tumour cells from miliary patients.
lack of EMT, and thus reduced overall survival. Both, the
diminished immune reaction and the enhanced epithelial
and malignant characteristics of the tumour cells open new
avenues for therapeutic options and strategies, like Catumaxomab, already in clinical use.
Surface plasmon fluorescence spectroscopy for EV
analysis
Collaborators: Agnes T. Reiner1,2, Wolfgang Knoll1,2, Jakub Dostalek2, Alain Brisson3, Sai-Kiang Lim4, Robert Zeillinger, Dietmar Pils
1Centre for Biomimetic Sensor Science, School of Materials Science and Engineering, Nanyang Technological University, Singapore, Singapore
2BioSensor Technologies, AIT-Austrian Institute of Technology, Vienna, Austria
3Molecular Imaging and NanoBioTechnology, University of
Bordeaux CNRS-UMR-CBMN, Pessac, France
4Institute of Medical Biology, A*STAR, Singapore, Singapore
Extracellular vesicles (EVs) are gaining interest in many
fields of research, but still methods for their analysis are
mostly complex and lack sensitivity or specificity. Recently
plasmonic biosensors appeared as EV analysis tools making use of surface plasmon resonance for detection of
EVs captured by specific antibodies to the sensor surface.
Additionally surface plasmons have the capability of coupling with fluorophores in their proximity and thus enhancing fluorescence emission. This attribute is employed in
surface plasmon enhanced fluorescence spectroscopy
(SPFS), on basis of which we want to develop a method for
EV analysis (setup shown in Figure 3).
EVs were purified by filtration and ultracentrifugation from
cell culture supernatant of different cell lines and from
biological fluids (i.e. ascites and plasma of ovarian cancer
patients and plasma of healthy individuals). The vesicles
were analysed with flow cytometry in fluorescence mode
examining general EV makers and markers for ovarian cancer cell derived EVs. A model system using SPFS for EV
analysis was built, where Annexin-V is used as capturing
molecule on the sensor surface. With the means of flow
cytometry in fluorescence mode EVs purified by ultracentrifugation could be detected and analysed. The general EV
markers were, as expected, present on EVs in all samples.
The ovarian cancer specific markers were only present on
EVs from CaOV3 cell culture supernatant and ascites, but
not from plasma of ovarian cancer patients, indicating that
the concentration of cancer derived EVs in plasma might
be below the detection limit. The SPFS model system could
detect standard vesicles down to fM concentrations.
Combining the specificity of molecular binding, the sensitivity of SPFS and the simplicity of plasmonic sensors, SPFS
can provide a cheap and simple platform for EV analysis for
scientific and clinical application.
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pulation of resistant cells that are ultimately responsible
for relapse and that by targeting this population front-line,
we may prolong disease-free survival or even achieve cure.
Figure 3. Setup of the model system for EV detection and analysis
with SPFS.
OCTIPS: Ovarian Cancer Therapy –
Innovative Models Prolong Survival
FP7 EU Project (01.01.2012 – 31.12.2015)
HEALTH.2011.2.4.1-2: Translational research on cancers
with poor prognosis
Coordinator: Cacsire Castillo-Tong D
Collaborators: Wolf A, Kreuzinger C, Gamperl M,
Reinthaller A and the Onco-Team
Website: www.octips.eu
Concept:
About 75% of advanced epithelial ovarian cancer patients
respond to first-line surgery and chemotherapy but most
relapse and ultimately acquire platinum resistance which
soon leads to death. Relapsed high grade serous ovarian
cancer is the single main cause of epithelial ovarian cancer.
We hypothesize that the primary tumor includes a small po-
Objectives:
t4VSLJ\SHYS` JOHYHJ[LYPaPUN OPNO NYHKL ZLYV\Z V]HYPHU
cancer, identifying molecules or pathways responsible
for resistance and relapse, and defining new therapeutic
targets and strategies.
t,Z[HISPZOPUN PUUV]H[P]L TVKLSZ JLSS SPULZ _LUVNYHM[Z
which represent resistant high grade serous ovarian cancer.
t,]HS\H[PUN[OLUL^Z[YH[LNPLZPUPUUV]H[P]LTVKLSZ
Results in 2014:
t4HUHNLTLU[ HUK JVVYKPUH[PVU! ( JVUZVY[P\T TLL[PUN
was organized together with a project partner Dr. Charles
Theillet from INSERM, Institut National de la Santé et de
la Recherche Médicale, Montpellier, France. The second
periodic reports were submitted to the European Commission. The project entered its third and the last period.
t,Z[HISPZOTLU[HUKTVSLJ\SHYJOHYHJ[LYPaH[PVUVMV]HYPHU
cancer cell lines:
Definition of the establishment of cell lines
In ascites, tumor cells often appeared in form of clusters
which were easily separated and purified by filtration. In
some primary cultures, tumor cells formed an island-like
structure surrounded by fibroblasts (Figure 1I) which mimicked the tumor structure in vivo. The fibroblasts were
reduced and finally eliminated by repeating selective trypsinisation, until pure tumor cell culture had been established (Figure 1H). All cell lines have been passaged more
than 40 times.
After TP53 mutations were determined by the functional
yeast assay in the corresponding tumor tissues, the purity
of the cell culture was determined using ddPCR. A cell line
was defined when 100% cells in the culture were confirmed to harbor the specific TP53 mutation. To ensure the
authenticity of the cells, short tandem repeat analysis
of 7 markers (TPOX, vWA, CSF180, D16S539, D7S820,
D13S317, D5S818, Applied Biosystems Life Technologies)
was performed regularly.
Morphology of the cells
7 cell lines have been established (examples in figure 1).
Two of them were derived from the same patient, one at
primary diagnosis and the other at the recurrence. The cell
lines presented with irregular sizes and shapes and had
a high ratio of nucleus to cytoplasm (Figure 1A). Most of
the cell lines grew as monolayers. One cell line grew as a
mixture of clusters and adherent “islands”. These clusters
could contain a couple of cells up to several hundred cells
(Figure 1B).
Figure 2: Scratch Assays from all cell lines at 0h and after 48h (brightfield
microscopy images). 0 hour: the scratched area was defined as 100%; 48
hours: proportion of the remaining cell free area was indicated. A: 58%*
remaining cell free area was manually calculated because of cell clustering.
response to the first line chemotherapy (Table 1). Two patients (12370 and 13363) could not be optimally debulked,
having a tumor mass of <5mm and >5mm after surgery,
respectively. Although they showed partial response to the
first line chemotherapy, they died of disease progression at
3 and 9 months after the last dose, respectively.
Mutations in the TP53, KRAS, BRAF and
BRCA1/2 genes
All patients had a somatic TP53 gene mutation (Table 1).
The percentage of the mutant cells was examined to deFigure 1. Morphology of established cell lines (brightfield microscopy images). A: HE staining of 13914_1; B: 12370; C: 13363; D: 15233; E: 13699;
F: 13914_1; G: 14433_1; H: 8587 in pure culture; I: 8587 tumor cell islands
surrounded by fibroblasts.
Growth and mobility of the cells
Split ratios of the cell lines differed from 1:2 to 1:3. Some
cell lines had a shorter doubling time of two to three days
whereas some needed six to nine days.
Scratch assay showed that some cell lines (for example
13699) did not have any mobility (Figure 2D) while other
lines had a similar migration rate, filling around 1/3 to 4/5
of the scratched areas (Figure 2A-2C, 2E-2G). One pair
of the matched cell lines derived from the same patient
(13363 and 15233) had similar high migration ability (Figure
2B and 2C).
Patients and tumor characteristics
Among the seven established cell lines, the 13363 and
15233. One cell line 8587 was derived from tumor tissue
and all others were established from ascites. The age of the
patients at diagnosis ranged from 33 to 67 with a median
age of 55. All primary samples set for cell culture were taken
before any chemotherapy. All patients received standard
treatments and presented with partial or complete clinical
Table 1. Clinical data of patients and clinicopathological characteristics of
tumors
1
cPR=clinical partial response; 2cCR= clinical complete remission; 3Point
Mutation at intron 5 (bp 13239) leading to a splice variant (g.13193_13238del,
Val172Val), which causes a frameshift and a truncated protein (fsX60); 4DBD:
central DNA-binding core domain; 5OD: homo-oligomerization domain
| 183
Table 2. Immunohistochemistry staining in all established cell lines and
their corresponding tumor tissues
Cytokeratins 8, 18 and 19 and EpCAM
were expressed in all tumor cells in
tissues as well as in the cell lines (Figure 3A, 3B and 3G, 3H).
TT = tumor tissue; 0: no expression; 1: weak expression in the minority of the cells; 2: weak expression
in the majority of the cells; 3: strong expression in the minority of the cells, 4: strong expression in
the majority of the cells.
termine the purity of the cell lines. It was confirmed that
the TP53 mutations were stable through all passages. Sequencing of the corresponding blood DNA confirmed that
none of the cell lines were from patients with a germline
TP53 mutation.
A non-sense mutation (c.8557a>t, p.Lys2853X) in BRCA2
and a 11bp deletion (c.3481_3491del, p.Glu1161fsPheX3)
in BRCA1 were found in the cell lines 14433_1 and 13914_1,
respectively, both being germline mutations. The latter was
detected from a patient, who had breast cancer previously.
No mutations in the KRAS and BRAF genes were detected
in the cell lines.
CA125 was expressed in the majority
of the cell lines as well as in tumor tissues (Figure 3M, 3N and 3O, 3P).
Most of the patients had very few CD44 and vimentin positive tumor cells in tissues (Figure 3J, 3L and 3D, 3F). In the
cell lines, the expression of these two proteins was quite
heterogeneous (Figure 3C, 3E and 3I, 3K).
In vitro chemosensitivity
Five cell lines did not show any remarkable difference in
the responsiveness to carboplatin, all being highly sensitive to the drug. One cell line (13914_1) as well as the cell
line established from ascites under treatment (15233) were
highly resistant (Figure 4).
Antigen expression of cell lines and the corresponding tumors
Antigen expression of the established cell lines was compared with that in the corresponding tumor tissues (Table 2).
Figure 4: IC50 +/- SD [µM] values of all cell lines treated with carboplatin.
Prospects in 2015:
t.LULL_WYLZZPVUWYVÊSPUNVMWHPYLKWYPTHY`HUKYLJ\YYLU[
HGSOC will be validated
t*LSSSPULZ^PSSILHUHS`ZLKYLNHYKPUNKPMMLYLU[PHSNLULL_pression in primary and recurrent lines
t;OLÊUHSWYVQLJ[TLL[PUN^PSSILVYNHUPaLK
Figure 3: Immunohistochemistry staining examples from cell lines and corresponding tumor tissue (brightfield microscopy images). A,B: Cytokeratin
8,18,19 staining of 13914_1 with corresponding tumor tissue; C,D: Vimentin
staining of 13699 with corresponding tumor tissue, E,F: Vimentin staining
of 8587 with corresponding tumor tissue; G,H: EpCAM staining of 14433_1
with corresponding tumor tissue; I,J: CD44 staining of 13363 with corresponding tumor tissue; K,L: CD44 staining of 13914_1 with corresponding
tumor tissue; M,N: CA125 staining of 12370 with corresponding tumor tissue; O,P: CA125 staining of 13914_1 with corresponding tumor tissue.
Post OVCAD: Ovarian Cancer –
Diagnosis of a silent killer
FP6 EU Project (01.01.2006 – 30.06.2010);
post period: 01.07.2010 LifeSciHealth: LSH-2004-2.2.0-7
Coordinator: Robert Zeillinger
Collaborators: Dan Cacsire Castillo-Tong, Andrea Wolf,
Eva Schuster
Website: www.ovcad.eu
Background:
After closing the project successfully in 2010, the consortium decided to continue the cooperation on ovarian cancer
research with the materials collected during the project,
including samples from blood, ascites, and tumor tissues
from 275 ovarian cancer patients with advanced epithelial
ovarian cancer.
Objectives:
t;VPKLU[PM`HUK]HSPKH[LIPVTHYRLYZMVYKPHNUVZPZHUKWYVgnosis.
t;VL]HS\H[LJSPUPJHSWHYHTL[LYZYLNHYKPUN[OLV\[JVTLVM
the patients.
t;V Z[\K` [OL TLJOHUPZTZ \UKLYS`PUN [OL [\TVYPNLULZPZ
and progression of ovarian cancer.
Results in 2014:
t7\ISPJH[PVUZ! 6=*(+ JVUZVY[P\T WHY[ULYZ JVU[PU\L [V
do research work with OVCAD resources and publish the
data. In 2014, 5 publications had appeared in scientific
journals.
t<WKH[PUNJSPUPJHSKH[H!+\YPUN[OLWYVQLJ[WH[PLU[Z
have been included into the study. At the end of 2013,
all patients have had a clinical follow-up of at least five
years. The data were thoroughly updated.
Prospects in 2015:
t*VU[PU\PUN[OLJVVWLYH[PVUHUKWYVQLJ[Z
t*VTWVZPUNHU6=*(+W\ISPJH[PVUZ\TTHYPaPUN[OLKH[H
produced from the projects, after the updating of the clinical data of patients.
| 185
REPRODU CT I V E B IO LO GY U NIT
P E R S O N A L S TA N D
W I S S E N S C H A F T L I C H E M I TA R B E I T E R / I N N E N
Mag. Dr. Martin Knöfler
Ao. Univ. Prof.
Mag. Dr. Jürgen Pollheimer
Ass.Prof.
DIin (FH) Valerie Fock
Dissertantin
Mag. Philipp Velicky
Dissertant
Mag.a Gerlinde Otti
Dissertantin
MSc. Kerstin Plessl
Dissertantin
MSc. Leila Saleh
BMA (Klinik)
Peter Haslinger
BMA (MUW, 100%)
BMA (MUW, 75%), Dissertantin
Mag.a Sandra Haider
Mag.a Gudrun Meinhardt BMA (MUW (100%), Dissertantin
DIPLOMSTUDIERENDE
Sophie Herzer
Humanmedizin
Stephanie K. Milla
Humanmedizin
Stefanie Kaltenberger
Biomedizinische Wissenschaften
Ä R Z T L I C H E M I TA R B E I T E R / I N N E N
Dr. Harald Zeisler
Dr. Katharina Worda
Dr. Christof Worda
sen Trophoblasten (EVT). Dieser trophoblastäre Subtyp invadiert die Dezidua sowie Spiralarteriolen, um eine adäquate Nährstoffversorgung und die immunologische Akzeptanz
in einem allogenen Umfeld zu gewährleisten. Die EVT Differenzierung ähnelt teilweise der Krebszellentstehung, unterscheidet sich aber durch strikte Kontrollmechanismen,
deren Ursprung bisher noch nicht vollständig geklärt ist. In
diesem Zusammenhang konnten wir bereits zeigen, dass
invasive Trophoblasten ein bemerkenswertes Repertoire an
ERBB Rezeptoren exprimieren (Figur 1). Interessanterweise,
finden wir ausschließlich ERBB2 und ERBB3 an der Oberfläche dieses trophoblastären Zelltypus. Frau Mag.a Valerie
Fock konnte in einer aktuellen Studie zeigen, dass beide
Rezeptoren vom Wachstumsfaktor Neuregulin 1 (NRG1) stimuliert werden können und dadurch die Apoptose in invasiven Trophoblasten unterdrücken. Darüber hinaus konnten
wir erstmals zeigen, dass NRG1 von dezidualen Stromazellen gebildet und sezerniert wird. In einer weiteren, bereits in
Begutachtung befindlichen Studie assoziierten Frau Mag.a
Fock und Plessl die Expression von EGFR und ERBB4 mit
der proliferativen Kapazität von Trophoblasten. Beide ERBB
Rezeptoren regen, nach Stimulierung mit heparin-binding
epidermal growth factor (HB-EGF) und epidermal growth
factor (EGF), die Zellteilung in primären Trophoblasten an.
In Zuge einer Zusammenarbeit mit Frau Dr. Sabine Dekan
(Institut für Pathologie, MUW) konnten wir des Weiteren
zeigen, dass das hyperproliferative Krankheitsbild der kompletten Blasenmole mit einem starken Anstieg der EGFRpositiven Trophoblastsubtypen einhergeht.
Ao. Univ. Prof.
Priv. Doz
Priv. Doz
Z U S ÄT Z L I C H E M I TA R B E I T E R / I N N E N
Jakob Knöfler
Laborassistenz (Drittmittel, 25%)
WISSENSCHAFTLICHE PROJEKTE
LIGAND-INDEPENDENT CONTROL OF EXTRAVILLOUS TROPHOBLAST DIFFERENTIATION
Im Rahmen des vom FWF geförderten Projektes (P25187)
studieren wir die Rolle der ERBB (EGFR) Rezeptoren in der
invasiven Differenzierung des humanen Trophoblasten.
M I TA R B E I T E R I N N E N :
Fock V, Plessl K, Haider S, Knöfler M und Pollheimer J.
Kurzbeschreibung
Während der Schwangerschaft adhäriert die Plazenta an
der mütterlichen Dezidua und bildet somit den extravillö186 | Universitätsklinik für Frauenheilkunde
Abb. 2. Reziproke Expression von verschiedenen Rezeptor-Tyrosinkinasen
(RTK) in primären nicht invasiven (CTB) und invasiven (EVT) Trophoblasten.
Die „heat map“ zeigt die RTK mRNA expression von verschiedenen CTB und
EVT Isolaten. Jede Reihe repräsentiert ein Probenset. Der Farbcode zeigt
den n-fachen Unterschied im Vergleich zum Median. (rot, überexprimiert;
blau, reprimiert; grau, unverändert).
Fock V, Plessl K, Fuchs R, Dekan S, Milla S, Haider S, Fiala
C, Knöfler M, and Pollheimer J. Trophoblast subtype-specific EGFR/ERBB4 expression correlates with cell cycle progression and hyperplasia in complete hydatidiform moles.
2014, Revision underway for Human Reproduction
Kaltenberger S, Meinhardt G, Fiala C and Pollheimer J.
ERBB2 gene amplification is associated with the invasive
differentiation program in human trophoblasts. 2014, manuscript in preparation
Fock V, Plessl K, Otti G, Fiala C, Knöfler M and Pollheimer J.
Neuregulin-1 suppresses apoptosis in trophoblasts via the
activation of the oncogenic unit ERBB2/ERBB3. 2014, manuscript in preparation
THE ROLE OF ADAM12 IN NORMAL AND
PATHOLOGICAL PREGNANCIES
In diesem von der Nationalbank (P17147) geförderten Projekt untersuchen wir die Funktion der Protease ADAM-12
im humanen extravillösen Trophoblasten.
Fock V, Biadasiewicz K, Knöfler M und Pollheimer J
Kurzbeschreibung:
ADAM-12 wird in zwei verschiedenen Varianten, durch alternatives Splicing, gebildet. ADAM-12L, die längere Form
wird an der Zellmembran exprimiert und die kürzere Variante, ADAM-12S wird als solubles Protein sekretiert. Obwohl sehr wenig über die biologische Bedeutung dieser
Protease bekannt ist gibt es Hinweise, dass ADAM-12L in
der Krebsentstehung und Metastasierung eine Rolle spielt.
In einer erst kürzlich publizierten Arbeit konnten wir zeigen, dass uteroplazentares ADAM-12 ausschließlich vom
Trophoblasten gebildet wird. Des Weiteren konnten wir
ADAM-12 als einen positiven Regulator der Trophoblasteninvasion identifizieren. Hierbei ist es uns gelungen die
ADAM-12 mediierte Aktivierung des pro-migratorischen
Moleküls Integrin beta 1 nachzuweisen. Darüber hinaus
postulieren wir, dass ADAM-12 eine wichtige Rolle in der
Degradation von IGFBP3 einnimmt und somit eine tragende Rolle in der Entstehung von Schwangerschaftserkrankungen spielen könnte.
Biadasiewicz K, Fock V, Dekan S, Proestling K, Velicky P,
Haider S, Knöfler M., Fröhlich C, Pollheimer J. Extravillous
trophoblast-associated ADAM12 exerts pro-invasive properties. Biol Reprod. 2014
Pollheimer J, Fock V, Knöfler M. Review: The ADAM metalloproteinases - Novel regulators of trophoblast invasion?
Placenta. 2013
IDENTIFICATION OF FACTORS CONTROLLING DIFFERENTIATION IN HUMAN DECIDUAL STROMAL CELLS
OF EARLY PREGNANCY
Otti G, Saleh L, Pollheimer J und Knöfler M.
Kurzbeschreibung
Dezidualisierung, der Umbau des uterinen Endometriums
in das Endometrium der Schwangerschaft, ist ein kontrollierter zellulärer Prozess, der im Wesentlichen durch cAMP
und Progesteron gesteuert wird. Da die Versorgung mit
gesunden endometrialen Zellen extrem limitiert ist, haben
wir ein Modellsystem mit dezidualen Stromazellen, die von
Plazenten isoliert werden, aufgebaut. Bislang konnten wir
zeigen, dass diese Zellen analog zu jenen, die aus dem normalen Endometrium isoliert wurden, reagieren (Saleh et
al., 2011). Im Rahmen ihrer Dissertation konnte nun G. Otti
zeigen, dass Notch signalling eine wichtige Rolle bei der
Funktion und Differenzierung dieser Zellen spielt, beispielsweise die Expression von dezidua-spezifischen Hormonen
wie Prolaktin steuert. Diese Daten wurden heuer im internationalen Top-Journal Plos One publiziert (Otti et al., 2014)
Des Weiteren wurde eine entsprechende Arbeit zur Thematik kürzlich publiziert (Saleh et al. 2011). Diese Studie
beschreibt die Etablierung eines primären Zellsystems zur
Simulation der Dezidualisierung. In diesen Modellsystemen
untersuchte G. Otti die Funktion des kanonischen Notch
signalling. Sie konnte zeigen, dass der Rezeptor Notch2
sowie die Liganden Jag1, Dll1 und Dll4 in dezidualen Stromazellen vorhanden sind und deren Expression von cAMP/
Porgesteron abhängt. Ein kanonischer Notch Reporter wird
während der in vitro Differenzierung angeschalten, sodass
die Vermutung nahe liegt, dass der Notch Signalweg eine
kritische Rolle in der humanen Dezidualisierung spielt. Weitere funktionelle Studien zu diesem Thema sind zurzeit in
Arbeit.
Saleh L, Otti G, Fiala C, Pollheimer J, and Knöfler M
Evaluation of human first trimester decidual and telomerase-transformed endometrial stromal cells as model systems of in vitro decidualization.
Reprod Biol Endocrinol. 2011; Dec 7;9:155
Otti G, Saleh L, Fiala C, Pollheimer J, and Knöfler M
Notch signalling controls differentiation of human decidual
stromal cells.
PLoS One. 2014; Nov 14;9(11)
CRITICAL SIGNALLING PATHWAYS REGULATING TROPHOBLAST INVASION OF THE HUMAN PLACENTA
Im Rahmen des vom FWF geförderten Projektes (P22587)
studieren wir die Rolle des konservierten Signalweges
Notch sowie anderer Signalketten in der invasiven Differenzierung des humanen Trophoblasten.
| 187
Haider S, Velicky P, Otti GR, Pollheimer J, Knöfler M
Notch signalling ist generell für Aufrechterhaltung von
Stammzellen, aber auch für Homöostase und Differenzierung von Geweben verantwortlich. Um Notch Signalling
auszulösen bedarf es eines direkten Zell-Zell Kontakts
zwischen Notch Rezeptoren und den ebenfalls Membrangebundenen Notch Liganden. Jüngere Befunde aus der
Literatur weisen weiters darauf hin, dass die Expression
von Notch Rezeptoren und deren Liganden in präeklamptischen Plazenten verändert sein könnten. Die Funktion
des Pathways bei humaner Plazentaentwicklung und Differenzierung ist jedoch gänzlich unbekannt. Im Rahmen
ihrer Dissertation untersucht S. Haider die Produktion von
Notch Signalkomponenten und die Funktion des Notch Signalweges in verschiedenen Trophoblastmodellsystemen.
Bislang konnte sie zeigen, dass die Inhibition des Notch Signallings zu vermehrter Invasion und Proliferation des Trophoblasten führt (Haider et al., 2014). Weiters lassen sich
Trophoblast Subtyp-spezifische Expressionen bestimmter
Notch Rezeptoren feststellen. Neben der Funktion in der
Trophoblastenproliferation dürften die Notch Liganden
auch parakrine Effekte auf deziduale Zellen ausüben. Im
Rahmen seiner Dissertationen untersucht weiters Philipp
Velicky die Funktion des Notch-abhängigen Transkriptionsfaktors RBPJkappa (Velicky et al. 2014), während eine
andere Dissertantin des Labors, Gerlinde Otti, Notch Signaltransduktion in der Dezidua studiert (siehe oben).
Haider S, Meinhardt G, Velicky P, Otti GR, Whitley G, Fiala
C, Pollheimer J, Knöfler M. Notch signaling plays a critical
role in motility and differentiation of human first-trimester
cytotrophoblasts. Endocrinology. 2014 Jan;155(1):263-74
Velicky P, Haider S, Otti GR, Fiala C, Pollheimer J, Knöfler M
Notch-dependent RBPJț inhibits proliferation of human
cytotrophoblasts and their differentiation into extravillous
trophoblasts.
Mol Hum Reprod. 2014 Aug;20(8):756-666
Function of the Wingless ligand Wnt5A in placental
differentiation and senescence
In diesem von der Herzfelderschen Familienstiftung unterstützen Projekt (AP00574OFF) untersuchen wir die Funktion des Wnt-Liganden Wnt5A während der Differenzierung
und Seneszenz der Plazenta
MitarbeiterInnen: Meinhardt G, Knöfler M
Die Wingless (Wnt)- Signalkaskade ist ein hochkonservierter Signalweg, der in Embryogenese und Onkogenese eine
wichtige Rolle spielt und Zellfunktionen wie Wachstum,
Proliferation, Überleben und Zelldifferenzierung steuert.
Der Wnt Ligand Wnt5A wurde kürzlich als Regulator des
Wachstums und der Alterung von Ovarialkarzinoma-Zellen
identifiziert und könnte diese Prozesse auch in anderen reproduktiven Zelltypen steuern. Tatsächlich konnten wir und
andere Wnt5A sowohl in der Plazenta als auch im schwangeren Endometrium, der Dezidua, nachweisen. Die Funktion des Liganden in diesem biologischen System ist jedoch
noch unbekannt. Wir studieren daher mittels verschiedener
molekularbiologischer Techniken und Trophoblastzell-Modellsystemen die Rolle von Wnt5A in der Plazentafunktion
und-differenzierung. Unsere Experimente zeigten, dass der
Ligand in zahlreichen Zelltypen (mesenchymalen, leukozytären und trophoblastären Zellen) an der feto-maternalen
Grenzfläche nachweisbar ist und hier eine wichtige Rolle
zu spielen scheint. In plazentären villösen Gewebskulturen
konnten wir zeigen, dass Wnt5A einerseits die Zellproliferation der epithelialen villösen als auch der Zellsäulentrophoblasten in vitro fördert, andererseits apoptotische Prozesse unterdrücken kann und somit eine wichtige Rolle in
der Gewebshomeostase spielt. Des Weiteren konnten wir
eine Aktivierung der MAPK(ERK) Signalkaskade durch den
Liganden nachweisen.
GY N ÄKOLO G ISCHE
END OK RINOLO G IE
expressed in endometrium of patients with versus without
endometriosis. With this study we aim to search for differences in the plasma of secreted miRNAs of patients
with versus without endometriosis. Additionally, we plan
to evaluate the correlation of these differentially secreted
miRNAs with the disease. This shall allow assessing the potential use of miRNAs as monitoring or prognostic markers
for endometriosis.
Leiter/Verantwortliche des Projekts: I. Yotova, P. Pateisky,
Kooperation mit Gynäkologie
W I S S E N S C H A F T L I C H E M I T A R B E I T E R I N N E N 2 0 14
Christian Schneeberger
ao. Univ.-Prof. Mag. Dr.
Andrea Kolbus
Univ.-Doz. Dipl. Biol. Dr.
Mario Mairhofer
Mag. Dr.
Detlev Pietrowski
Dipl. Biol. Dr.
Iveta Yotova
Dr.
Marco Medjimorec
BSc
Marion Martins
BSc
Gerald Hofstätter
BSc
Daniel Faust
BSc
Barbara Widmar
Ladislaus Szabo
AR
Ä R T Z L I C H E M I T A R B E I T E R I N N E N 2 0 14
Walter Tschugguel
ao. Univ.-Prof. Dr.
Petra Pateisky
Dr.
Aulona Gaba
Dr.
PROJEKTE 2014
Circulating miRNAs as potential biomarkers
for Endometriosis
Endometriosis is a disease which is affecting up to 10% of
women in their reproductive age. The symptoms are very
heterogeneous and may range from dysmenorrhea and
dyspareunia to infertility. Up to now the gold standard
of diagnosis is still laparoscopic evaluation of the pelvis.
Therefore, the search for new and less invasive diagnostic
and/or monitoring markers is an interesting research
field. MicroRNAs are a class of short, non-coding RNAs
known to regulate gene expression post- transcriptional.
There exists evidence that those factors are differentially
EU-Projekt HYPERLAB
Das von der EU im 7. Rahmenprogramm geförderte Forschungsprojekt HYPERLAB (High Yield and Performance
Stem Cell Lab) wurde im September 2009 gestartet. Der
Schwerpunkt dieses Projektes liegt auf der Optimierung
von gegenwärtigen Stammzell-Kultivierungsmethoden.
Innovative neue Techniken (Pipet Robots, PipeBased Bioreactors) werden an die besonderen Anforderungen der
Stammzell-Kultivierung adaptiert und sollen so den Arbeitsaufwand für die Kultivierung reduzieren, sowie ein
High-Throughput-Screening von unterschiedlichen Medien
und Wachstumsfaktoren ermöglichen. An der Universitätsklinik für Frauenheilkunde werden in Zusammenarbeit mit
den Projektpartnern Protokolle zur Expansion und Differenzierung von CD34-positiven hämatopoetischen Stammzellen aus Nabelschnurblut etabliert und optimiert.
Leiter/Verantwortliche des Projekts: A. Kolbus,
M. Mairhofer
E-rare Projekt EMINA-2
Das E-rare Projekt EMINA-2 (Analysis of VPS13A function
in iPS-derived erythroid cells) wurde im Februar 2013 gestartet und widmet sich der Aufklärung der molekularen
Mechanismen der seltenen Erkrankung Neuroacanthozythose. PatientInnen mit dieser Erkrankung, welche durch
Defekte/ Mutationen im VPS13A Gen ausgelöst wird,
zeigen neben Huntington´s Disease-ähnlichen neuro-degenerativen Symptomen typisch sternförmig deformierte
Erythrozyten. Die Funktion des VPS13A Proteins in humanen Zellen ist noch völlig unklar und soll im Rahmen des
internationalen Kooperationsprojektes mit Partnern aus
Deutschland, Holland und Israel untersucht werden. Als
Modellsystem wird mit patientenspezifischen induzierten
pluripotenten Stammzellen gearbeitet, welche in-vitro in
erythroide Zellen differenziert werden. Als weiteres Modellsystem stehen primäre erythroide Zellen aus Nabelschnurblut zur Verfügung. Weitere Informationen zum Projekt sind
auf der Projekthomepage unter http://www.neuro.med.
tu-dresden.de/emina2/ zu finden.
Leiter/Verantwortlicher des Projekts: M. Mairhofer
| 189
EUROSTARS-Projekt MUSST (Multi-step Streptamer
systems for clinical stem cell research)
Im Projekt MUSST wird zusammen mit Projektpartnern
aus Deutschland ein neuartiges System zur Anreicherung/
Reinigung von hämatopoietischen Stammzellen aus Nabelschnurblut entwickelt, welches eine mehrstufige Anreicherung erlaubt, die in derzeit verwendeten Selektionssystemen nicht möglich ist. Die neuartigen Affinitätsreagenzien
werden von Firma IBA entwickelt, und Projektpartner IBA
stellt das Know-how im Mikrofluidikbereich zur Verfügung.
Die Hauptaufgabe unserer Arbeitsgruppe ist die Evaluierung von neuen Reagenzien und Methoden mit Stammzellen aus der Nabelschnur sowie der Vergleich zum derzeitigen „state-of-the-art“ im Bereich der Zellselektion.
Leiter/Verantwortlicher des Projekts: M. Mairhofer
Mathematische Modellierung des Einflusses von
Genvarianten im VEGF-VEGF Rezeptorsystem bei
OHSS Patienten
In Vorarbeiten haben wir zeigen können, dass das Auftreten
des ovariellen Überstimulations-syndroms (OHSS) mit genetischen Unterschieden im VEGF-VEGF-Rezeptorsystem
zusammenhängt. Diese Unterschiede lassen sich auch auf
Proteinebene im Serum von Patienten nachweisen (siehe
Projekt Nr. 4; Nouri et al (2014), Pietrowski et al (2012) Im
Rahmen dieses Nachfolgeprojekts soll untersucht werden,
ob sich ein mathematisches Modell für einen direkten Genvarianten-Proteinexpressions-Zusammenhang in dieser
Patientengruppe darstellen lässt. Damit würde erstmalig
für OHSS Patienten der Einfluss einzelner Genvarianten auf
die Proteinexpression im VEGF-VEGF-Rezeptorsystem berechenbar sein und könnte damit auch als Prognosefaktor
in der IVF-Therapie eingesetzt werden.
Leiter/Verantwortliche des Projekts: D. Pietrowski, M.
Schreiber (Kooperation mit AG Schreiber)
Effect of testosterone on Human Uterine Vein Endothelial Cells (HUVEC)
Androgens are the most abundant sex steroids in men,
as well as in women after menopause and are also used
pharmacologically. While the effects of estrogens on vasculature are studied extensively, the mechanisms implicated in the androgen regulation of endothelial cell behavior
are far from being thoroughly understood. A growing body
of evidence suggests that androgens affect the behavior
of endothelial cells, modulating proliferation, migration
and angiogenesis. The results of different studies remain
contradictory with regard to the involvement of Androgen
Receptor in Testosterone mediated signaling. The effects
of androgens on vasculature seem to be tissue and vessel
caliber dependent. Our project aims to investigate whether
the effects of Testosterone on endothelial cells from mac190 | Universitätsklinik für Frauenheilkunde
rovasculature (HUVEC) are different then on microvasculature and whether migration and proliferation are mediated
by the Androgen Receptor.
Leiter/Verantwortliche des Projekts: A. Gaba
Analyse des Auftauverfahrens von Ovargewebe im
Rahmen des Ovarian Tissue Banking
Die Durchführung einer Chemotherapie ist in der Regel
gonadotoxisch. Im Rahmen des Ovarian Tissue Bankings
(OTB) wird daher insbesondere jüngeren Patientinnen, angeboten, ein Teil ihres Ovargewebes als Fertilitätsreserve,
einfrieren zu lassen.
Es werden hierbei Kryoprotokolle genutzt, die im wesentlichen DMSO als Kryoprotektant enthalten. Die zurzeit erfolgreich genutzten Auftauprotokolle hingegen enthalten
Sucrose als wesentliches Medium. Schon aus physikalischen Gründen ist davon auszugehen, dass der Wechsel
von DMSO haltigem Einfriermedium hin zu einem Sucrose
haltigem Auftaumedium für die Zellen im Ovargewebe einen
zusätzlichen osmotischen Stress beinhaltet, der zu einem
deutlich erhöhten Anteil an apoptotischen und/oder nekrotischen Zellen führen kann. Im Rahmen dieses Projekts
soll daher untersucht werden, inwieweit es möglich ist optimierte Bedingungen zu schaffen, um den Stress für die Zellen und damit auch die Erfolgsrate einer Retransplantation
zu verbessern. Modellhaft werden die experimentellen Arbeiten an einer Granulosa Zelllinie durchgeführt. Für dieses
Projekt ist bereits ein Ethikvotum vorhanden.
Leiter/Verantwortliche(r) des Projekts: D. Pietrowski,
M. Kokotsaki, Ch. Schneeberger
ONCO G ENOMI C S
W I S S E N S C H A F T L I C H E M I TA R B E I T E R I N N E N
Martin Schreiber, ao.Univ.-Prof. Mag. Dr. M.Sc.
Andrea Friesenhengst, B.Sc.
Tamara Pribitzer-Winner, B.Sc.
Wissenschaftliche Projekte (Auszug)
Assoziation des G473A Polymorphismus und der Expression von Lysyl Oxidase mit dem Brustkrebsrisiko und der Prognose
Die Lysyl Oxidase (LOX) ist ein extrazelluläres Enzym mit
einer essentiellen Rolle in der kovalenten Vernetzung von
extrazellulären Matrixproteinen sowie wahrscheinlich noch
weiteren Funktionen. Das sekretierte LOX-Proenzym wird
proteolytisch zum fertigen, funktionellen Enzym prozessiert. Die LOX-Expression kann im Tumor sowohl erhöht als
auch vermindert sein und ist sowohl mit der Tumorsuppres-
sion als auch mit der verstärkten Ausbildung von Metastasen assoziiert. Der G473A Polymorphismus (rs1800449)
führt zu einer Arg158Gln Aminosäure-Substitution im LOXPropeptid, welche die tumor-suprimierende Wirkung von
LOX beeinträchtigt und in einer chinesischen Studienpopulation mit erhöhtem Brustkrebsrisiko assoziiert ist. In
unserer erstmals mit europäischen Frauen durchgeführten
Studie konnten wir zeigen, dass das A-Allel dieses Polymorphismus mit einem höheren Manifestationsalter, schlechterer Prognose hinsichtlich krankheitsfreier und metastasenfreier Überlebensrate, jedoch nicht mit einem erhöhten
Brustkrebsrisiko assoziiert ist. Die LOX mRNA-Expression
war in Tumoren von Patientinnen von über 55 Jahren, von
postmenopausalen Patientinnen, in Östrogen-Rezeptor
(ER) positiven Tumoren und in p53-negativen Tumoren signifikant erhöht. Der G473A Genotyp hatte jedoch keinen
signifikanten Einfluss auf die LOX Expression, weder in Tumorgewebe noch in Brustkrebszelllinien. Eine hohe LOXExpression war mit einer schlechten Prognose bei Patientinnen mit ER-negativen Tumoren, aber nicht bei solchen
mit ER-positiven Tumoren assoziiert. Bei multivariablen
Analysen zeigte sich die LOX-Expression als unabhängiger
prognostischer Parameter, der G473A Genotyp hingegen
nicht. Eine kleine Subgruppe von Östrogenrezeptor (ER)negativen Patientinnen mit erhöhter LOX-Expression wies
eine sehr ungünstige Prognose hinsichtlich des krankheitsfreien (p=0,001) und Metastasen-freien (p=0,0003) Überlebens auf und stellt somit ein vielversprechendes Kollektiv
für zukünftige, individualisierte Ansätze der Brustkrebsdiagnose und –therapie dar
wir, dass eine zytoplasmatische Überexpression von ILEI
mit der Metastasierung und mit einer schlechteren Prognose bei Brustkrebspatientinnen assoziiert ist. Entscheidend
für den biologischen Effekt auf den Krankheitsverlauf war
dabei die genaue subzelluläre Lokalisation von ILEI. Diese
Entdeckungen konnten wir nun in einem fast doppelt so
großen Patientinnenkollektiv bestätigen. Unsere Kooperationspartner entdeckten zusätzlich, dass die Lokalisation
von ILEI massgeblich durch extrazelluläre Proteasen, insbesondere Plasminogen und den Rezeptor für Plasminogen-Aktivator vom urokinase-Typ (uPAR) beeinflusst wird.
Imunhistochemische Analysen unseres Patientinnenkollektivs bestätigten dass die Lokalisation von ILEI deutlich
mit der Expression von uPAR in Tumorzellen korreliert, und
dass die hohe prognostische Aussagekraft der ILEI-Lokalisation durch gleichzeitige Auswertung der uPAR Expression
und Berücksichtigung beider Marker in Überlebensanalysen weiter verbessert werden kann (Abbildung 2).
Abbildung 1: Kaplan-Meier Analysen des krankheitsfreien und des Metastasen-freien Überlebens von Patientinnen mit hoher vs. niedriger LOXExpression sowie positivem und negativem Östrogenrezeptor (ER) Status.
ILEI (Interleukin-like epithelial-to-mesenchymal
transition inducer) und das plasminogen–uPAR (urokinase plasminogen activator receptor) System als
prognostische Marker des Mammakarzinoms
In einer früheren Studie konnten wir in einer Kooperation
mit dem IMP Wien zeigen, dass das Protein interleukin-like
EMT inducer (ILEI) notwendig und hinreichend für die epithelial-mesenchymale Transition, Tumorbildung und Metastasierung normaler Epithelzellen ist. Außerdem fanden
Abbildung 2: Kaplan-Meier Analysen des Metastasen-freien Überlebens
basierend auf immunhistochemischen Färbungen von ILEI (oben), uPAR
(Mitte), oder einer Kombination aus ILEI und uPAR (unten).
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Assoziation des TT Genotyps des rs10046 Polymorphismus der Aromatase (CYP19) mit einem verstärkt
prämenopausalen Auftreten von Brustkrebs
Das CYP19 Gen kodiert für Aromatase, ein Enzym das die
Produktion von Östrogenen aus Androgenen katalysiert.
Der rs10046 „single nucleotide polymorphism“ (SNP)
befindet sich in der 3´untranslatierten Region des CYP19
Gens. Wir untersuchten in diesem Projekt den Einfluss
dieses SNP auf das Brustkrebsrisiko und das Erkrankungsalter, sowie die Assoziation mit klinischen und histopathologischen Charakteristika des Mammakarzinoms. Wir
fanden heraus dass ein signifikant erhöhter Teil der Patientinnen mit dem TT Genotyp des rs10046 SNPs bereits
in einem jungen Alter von unter 50 Jahren an Brustkrebs
erkrankten (41.8% der Patientinnen mit dem TT Genotyp,
aber nur 26.6% der Trägerinnen eines oder zweier C-Allele;
p = 0.018, Chi-Quadrat Test; Abbildung 3A). Im Gegensatz
dazu war keiner der drei rs10046 Genotypen signifikant mit
einem erhöhten Brustkrebsrisiko, oder mit weiteren klinischen Parametern assoziiert. Beim Vergleich der Kurven
der kumulativen Brustkrebsinzidenz zeigten sich charakteristische Unterschiede zwischen dem TT Genotyp und den
beiden anderen Genotypen. Die Kurve der TT Patientinnen
zeigte zunächst einen deutlich steileren Anstieg bei einem
Alter zwischen 40 und 50 Jahren, wies dann aber einen
auffälligen Knick und eine Plateauphase bei einem Alter
von 50-55 Jahren auf, welches in etwa dem typischen Alter
der Menopause entspricht (Abbildung 3B). In postemenopausalen Patientinnen waren die drei Genotypen wieder im
Gleichklang, sodass es in der Gesamtinzidenz keine signifikanten Unterschiede gab (Abbildung 3B).
Abbildung 3: Einfluss des CYP19 rs10046 SNP auf das Erkrankungsalter
beim Brustkrebs. (A) Anteil der Patientinnen, die bereits in einem Alter unter 50 Jahren erkrankten. (B) Kurven der kumulativen Brustkrebsinzidenz
nach Erkrankungsalter der drei Genotypen.
MEDI CAL G ENE T I C S
LEITER:
Univ.Prof.Dr. Berthold Streubel
Wissenschaftliche Projekte (Auszug)
(16 Publikationen 2014)
Perinatal
Global and single gene DNA methylation in umbilical cord blood cells after elective caesarean: a pilot
study.
OBJECTIVE:
To evaluate global and single gene methylation patterns as
a sign for epigenetic modulation of the immune system in
infants born by elective cesarean section (CS) and vaginal
delivery (VD).
STUDY DESIGN:
For this prospective pilot study a two-step approach was
chosen. Initially 41 newborn infants comprising 23 delivered by VD and 18 delivered by elective CS were included.
Global DNA methylation of umbilical cord blood was determined. In a second step, methylation status of 96 single genes linked to T cell activation, cytokine production,
inflammatory response, and stem cell transcription was
evaluated in 48 newborn infants, 20 delivered by VD and
28 delivered by CS.
RESULTS:
Global methylation did not differ significantly between
CS and VD (p=0.732). The methylation status was low
(threshold: )3%) for the majority of single genes (n=92)
in both groups. FOXP3, CD7, ELA2, and IRF1 showed
hypermethylation in both groups. In the CS group, ELA2
(p<0.001) and IRF1(p =0.017) showed significantly higher
methylation compared to the VD group.
CONCLUSION:
We found no difference in global methylation between
newborn infants in the VD group compared to the elective CS group. Methylation of single genes was significantly
higher in newborn infants delivered by elective CS. Further
research is needed to determine the significance of these
findings.
Tumor
FOLFOX4 Plus Cetuximab for Patients With Previously
Untreated Metastatic
Colorectal Cancer According to Tumor RAS and BRAF
Mutation Status: Updated
Analysis of the CECOG/CORE 1.2.002 Study.
BACKGROUND:
This updated analysis of the CECOG/CORE 1.2.002 study
investigated the association between clinical outcome and
RAS and BRAF mutations in metastatic colorectal cancer
(mCRC) patients treated with FOLFOX4 plus cetuximab.
PATIENTS AND METHODS:
Available DNA samples from CECOG/CORE 1.2.002 study
patients with KRAS exon 2 wild type (wt) (at codons 12 and
13) tumors were screened for mutations at other loci in the
KRAS and NRAS (RAS) coding regions by Sanger sequencing, and for BRAF codon 600 mutations by Sanger sequencing and pyrosequencing. Clinical outcome was compared
among different mutation subgroups.
RESULTS:
Of 152 KRAS wt mCRC patients, 148 were evaluable for
RAS and BRAF mutation status. Eleven RAS mutations
were detected in 10 patients‘ tumors (7%). BRAF mutations
were detected in 14 patients‘ tumors (9%). RAS and BRAF
tumor mutations were mutually exclusive. Compared with
patients with RAS wt/BRAF wt tumors (n = 124; median
overall survival, 28.5 months), those with RAS mutations
(n = 10; median, 16.3 months; hazard ratio, 0.43; 95% confidence interval, 0.20-0.89; P = .020) or BRAF mutations (n
= 14; median, 11.7 months; hazard ratio, 0.23; 95% confidence interval, 0.12-0.41; P < .0001) had worse overall survival, which remained significant (P < .04) when adjusting
for differences in baseline characteristics among the mutation subgroups.
CONCLUSION:
These findings support those from recent studies that RAS
and BRAF mutations are associated with poor outcome in
patients receiving an epidermal growth factor receptortargeted monoclonal antibody in combination with oxaliplatin-based chemotherapy. Furthermore, mutation testing
should not only include RAS codons 12 and 13 but should
also be extended to the entire coding regions.
PR Ä DIK T I V E ONKOLO G IE
LEITUNG:
Univ. Prof. Dr. Christian SINGER, MPH
W I S S E N S C H A F T L I C H E M I TA R B E I T E R I N N E N :
Marie-Theres Kastner (Biomedizinische Analytikerin)
Daniela Muhr (Biomedizinische Analytikerin)
MMag. Christine Rappaport-Fürhauser (Dissertantin)
Sigrid Weingartshofer, MSc (Biomedizinische Analytikerin)
BACHELOR STUDENTINNEN:
Jennifer Jankovich
Veronika Weiler
WISSENSCHAFTLICHE PROJEKTE
Prognostic and predictive value of ESR1 Amplification in postmenopausal receptor-positive women
with early breast cancer treated with TAM vs sequential TAM and AI
Singer, Holst, Rudas, Filipits, Stahl, Weingartshofer
Several independent studies have now also confirmed the
existence of ESR1 amplifications in breast cancer, and the
presence of intratumoral ESR1 gene copy number alterations has been suggested to harbour relevant prognostic
information. Recently, ESR1 amplification has also been
suggested to predict responsiveness to endocrine therapy
although the data are somewhat contradictory. We have
therefore performed an ESR1 gene amplification in breast
cancer retrospective biomarker trial in women with endocrine-responsive early breast cancer who had been randomized into the tamoxifen-only arm of the prospectively designed endocrine ABCSG-06 trial and in whom FFPE tumor
samples were available. When ESR1 gene amplification status was evaluated by FISH analysis, we found copy number
gains and amplifications in a significant fraction of tumor
specimen and detected a strong and independent association with the clinical outcome. At a median follow-up of 10
years, women with intratumoral ESR1 copy number gains
had a significantly longer distant recurrence-free survival
and breast cancer-specific survival when compared to women with normal ESR1 copy numbers. We therefore concluded that the ESR1 gene copy status is an independent
and powerful predictor for long-term distant recurrencefree and breast cancer-specific survival in postmenopausal
women with endocrine-responsive early-stage breast cancer. In the present research initiative we propose to confirm the prognostic role of ESR1 in postmenopausal breast
cancer patients, which we have previously demonstrated in
the preliminary experiments described above, and to evaluate the predictive role of ESR1 in tamoxifen and AI-treated
postmenopausal women. To this end, we will utilize an archived subset of breast cancer samples from more than
3200 postmenopausal women who have been randomized
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into the ABCSG 8 trial and who have either received 5 years of tamoxifen or 2 years tamoxifen followed by 3 years
of the aromatase inhibitor anastrozole. We will then extend
our research objective to premenopausal women and investigate the prognostic and predictive utility of ESR1 in
tumor samples from the ABCSG 12 trial. In this prospectively designed phase III trial, more than 1800 premenopausal women with endocrine-responsive early breast
cancer have received either tamoxifen or the aromatase
inhibitor anastrozole in combination with GnRH for 3 years,
which provides us with a unique possibility to validate our
initial findings in tamoxifen- and AI-treated premenopausal
women. ABCSG 12 is the only prospectively randomized
clinical trial worldwide that has directly compared tamoxifen with an AI in this patient setting. While the previous research strategies comprise retrospective biomarker analyses rom prospective trials, we will also measure ESR1 copy
numbers in a predefined, prospective manner in a neo-adjuvant trial in which postmenopausal women with luminal A
tumors receive 6 months of an AI prior to curative surgery.
The efficacy of the studied AI letrozole will be measured by
the rate of pCR or near pCR and will be correlated with the
ESR1 amplification status in order to investigate whether a
gene dose effect exists also for AI treatment.
We hypothesize that the analysis of ESR1 copy number
alterations allows to predict response to endocrine treatment (tamoxifen and AI) in pre- and in postmenopausal women with endocrine-responsive early breast cancer.
We hypothesize that the analysis of ESR1 copy number alterations has a prognostic role in premenopausal women
with endocrine-responsive early breast cancer We hypothesize that the analysis of ESR1 copy number alterations
has a role in predicting near or complete pathological response in postmenopausal women with endocrine responsive, luminal A tumors that receive preoperative endocrine
therapy with an AI for 6 months.
To evaluate the predictive role of ESR1 copy number alterations in pre- and postmenopausal women with endocrine responsive early breast cancer who receive endocrine
treatment in a prospective-retrospective study design.
To evaluate the prognostic role of ESR1 copy number alterations in premenopausal women with endocrine-responsive early breast cancer who receive either tamoxifen or
AI in combination with GnRH for 3 years in a prospectiveretrospective study design.
To evaluate the utility of ESR1 copy number alterations in
predicting near or complete pathological response in postmenopausal women with endocrine responsive, luminal A
tumors that receive preoperative endocrine therapy with
an AI for 6 months.
To establish a standardized protocol for the treatment and
analysis of ESR1 FISH, and to develop and cross-validate
an analysis algorithm ESR1 gene amplification in breast
cancer.
Figure 1. FISH analysis on FFPE samples of invasive breast carcinomas. (left side) ESR1 gene amplification as indicated by multiple green
ESR1 specific signals in each nucleus. The centromere 6 is red, ESR1 gene
is green labeled, 100x. ER-expression performed by immunohistochemistry. (right side) Transmitted-light microscopic image (4x) of an
immunohistochemical stained cryo section (4µm). ER-protein is colored
brown in the nucleus.
Methyl-binding domain protein-based DNA isolation
from human blood serum combines DNA analyses
and serum-autoantibody testing
Wielscher, Pulverer, Peham, Hofner, Rappaport-Fürhauser,
Singer, Jungbauer, Nähammer, Weinhäusel
Circulating cell free DNA in serum
as well as serum-autoantibodies
and the serum proteome have great
potential to contribute to early cancer diagnostics via noninvasive
blood tests. However, most DNA preparation protocols destroy the protein fraction and therefore do not allow subsequent protein analyses. In this study a novel approach
based on methyl binding domain protein (MBD) is described to overcome the technical difficulties of combining
DNA and protein analysis out of one single serum sample.
Serum or plasma samples from 98 control individuals
and 54 breast cancer patients were evaluated upon silica
membrane- or MBD affinity-based DNA isolation via qPCR
targeting potential DNA methylation markers as well as by
protein-microarrays for tumor-autoantibody testing.
In control individuals, an average DNA level of 22.8 ± 25.7
ng/ml was detected applying the silica membrane based
protocol and 8.5 ± 7.5 ng/ml using the MBD-approach,
both values strongly dependent on the serum sample preparation methods used. In contrast to malignant and benign tumor serum samples, cell free DNA concentrations
were significantly elevated in sera of metastasizing breast
cancer patients. Technical evaluation revealed that serum
upon MBD-based DNA isolation is suitable for protein-array analyses when data are consistent to untreated serum
samples. MBD affinity purification allows DNA isolations
under native conditions retaining the protein function, thus
for example enabling combined analyses of DNA methylation and autoantigene-profiles from the same serum sample
and thereby improving minimal invasive diagnostics.
Figure 1. Extraction of cell free DNA from control individuals. Box
plot of DNA amount isolated from 1 ml serum or plasma from (1) Austrian
Institute of Technology (n = 12), (2) Austrian Red Cross (n = 24) and (3) AKH
(n = 24). Serum DNA levels were dependent on serum source. Using the
silica-based extraction protocol, mean amounts of DNA could be isolated
ranging from (1), 11.9 ± 10.9ng/ml (mean ± SD) and (3), 12.2 ± 9.7 ng/ml to
(2), 39.7 ± 32.8 ng/ml. By contrast using the MBD-based protocol, serum
DNA concentrations of (1) 2.5 ± 1.9 ng/ml, (2) 11.5 ± 7.3 ng/ml and (3) 2.4
± 1 ng/ml were observed.
Figure 2. Increased level of cell free DNA in serum of Breast cancer
patients with metastasizing disease. Amount of cell free DNA isolated
from 1 ml serum of breast cancer patients with metastasizing tumors and
control individuals obtained from AKH (source 3). An increased serum DNA
amount was detected in sera from metastasizing tumors with both isolation
strategies. (A), silica based isolation protocol (P = 0.0043, Wilcoxon test);
(B) MBD loaded bead based purification (P = 0.0021, Wilcoxon test).
Figure 2. Increased level of cell free DNA in serum of Breast cancer
patients with metastasizing disease. Amount of cell free DNA isolated
from 1 ml serum of breast cancer patients with metastasizing tumors and
control individuals obtained from AKH (source 3). An increased serum DNA
amount was detected in sera from metastasizing tumors with both isolation
strategies. (A), silica based isolation protocol (P = 0.0043, Wilcoxon test);
(B) MBD loaded bead based purification (P = 0.0021, Wilcoxon test).
Figure 3. Quality of cell free serum DNA. (A) reflects the amplification
success for each sample using MBD or silica membrane based serum DNA.
A maximum of 12 fragments per sample was possible and in sum 34 samples per isolation approach were analyzed. (B) shows the amplification success of each fragment getting amplified across all analyzed samples. Both
plots (A, B) are based on the analysis of two multiplex PCRs performed on
serum or plasma DNA isolates (source 1, 2).
Figure 4. Autoantibody tests of MBD processed serum. Pearson correlation plots upon X-values of autoantibody protein micro arrays analyzing
serum and plasma samples with and without MBD treatment. All samples
originate from source 1 (AIT). Comparison of plasma and serum samples
were performed on samples obtained from one single blood withdrawal.
The cor-value states the Pearson‘s correlation.
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Antiestrogenic effects of the natural fetal estrogen
estetrol (E4) in women with estrogen-receptor-positive early breast cancer
Singer, Bennink, Natter, Steurer, Moinfar, Rudas, Visser,
Appels, Kubista
Estetrol (E4) is a fetal estrogen which exerts estrogenic effects on vaginal epithelium, hot flushes and bone, but has
estrogen-antagonistic effects on breast cancer cell lines in
vitro and in the rat DMBA model. Therefore E4 may be suitable for Hormone Replacement Therapy (HRT) in women
with breast cancer including women treated with aromatase inhibitors.
We have investigated the effect of 14 days pre-operative
treatment with 20 mg E4 per day on proliferation, apoptosis, ER-receptors and sex steroid levels in a prospective,
randomised, double-blind, placebo-controlled, neo-adjuvant study in 15 pre- and 15 postmenopausal women with
estrogen-receptor positive early breast cancer.
Estetrol induced a significant increase of SHBG, a significant decrease of FSH in postmenopausal women and no
increase of gonadotrophins in premenopausal women. Estetrol had no effect on Ki67 expression and on apoptosisrelated Bax and Bcl-2, but the apoptosis index in tumor
tissue increased significantly. Systemic IGF-1 levels decreased significantly. Surprisingly the intratumoral epithelial
ER-alpha expression decreased significantly, whereas the
ER-beta expression showed a trend to increase.
Fig. 2. Representative immunohistochemistry photomicrographs
depicting ERĮ staining before (a)
and after (b) 2 weeks of E4 treatment, and ERȕ staining before
(c) and after (d) 2 weeks of E4
treatment.
This data show that E4 has estrogenic endocrine effects.
The data support the hypothesis that E4, may be suitable
and safe for HRT in women with spontaneous or induced
menopausal symptoms, since apoptosis increases, IGF-1
decreases and no unfavourable effects are observed on
Ki67, Bax and Bcl-2. The decrease of ER-alpha and the
increase of ER-beta suggest a mechanism of action, explaining why the natural fetal estrogen E4 has estrogenantagonistic effects on breast cancer tissue.
Carcinogenesis vol.35 no.11 pp.2447–2451, 2014
doi:10.1093/carcin/bgu144
Advance Access publication July 5, 2014
Fig. 1. Ki67 expression (%) in breast tumor tissue before (day 0) and after
14 days of oral treatment with 20 mg E4 or placebo per day.
Fig. 3. Relative changes (%) in SHBG (a),
FSH (b), bioavailable
testosterone (c) and
IGF-1 (d) serum concentrations after 14
days of treatment with
E4 or placebo in preand post-menopausal
BC patients.
Serum-Autoantibody testing for early diagnosis of
Breast Cancer
Singer, Weinhäusel (AIT), Zeillinger, Pecha, Koch
Early detection of breast cancer will
be the main focus of this projectproposal, although prognosis and
prediction of clinical outcomes
might be a potential task using our
immunological approach. Early detection reduces the suffering and cost to society associated with the disease. A
sensitive assay to identify biomarkers that can accurately
diagnose the onset of breast cancer using non-invasively
collected clinical specimens is ideal for early detection.
The earlier and more accurate the diagnostic biomarker
can predict disease onset, the more valuable it becomes.
Autoantibodies and abnormal tumor-specific DNA methylation found in cell-free serum DNA may provide the best
opportunity for constructing multiplexed tests, which will
be sufficiently specific and sensitive for early detection of
breast cancer. Although we have identified DNA-methylation based classifiers in primary tumors and tested those
along with published breast cancer methylation markers
for detection of abnormal tumor-specific DNA methylation
in serum, we could not distinguish females with benign and
malignant breast tumors (unpublished results). Although
PCR testing is relatively simple and robust, we have found
that a simpler assay might be advantageous. This is especially true for presymptomatic screening or improving diagnostic testing of females with nodular (including benign
and malignant) breasts. In these settings, a minimal invasive serum test would be of great value, especially when
a breast tissue biopsy for histopathological assessment of
nodules could be avoided. Therefore we aim to develop a
technically simple and cheap immunologic serum test for
minimal invasive diagnostics, and expect that technologies
developed for breast cancer detection will be useful for
other types of cancer.
5) a peptide-based proto-type assay will be developed,
transferred to the clinical research laboratory and validated on a retrospective study-cohort (n=1200). All these
techniques and methods are already established and will
be combined for the most comprehensive approach for
autoantibody-based serum diagnostics.
In this cooperational project AIT has successfully conducted immune-profiling of cases and controls using AITs 16k
protein microarray for distinguishing malignant, benign and
healthy controls from each other. The significant proteins were then used for a tiling-design of a peptide array.
Together with peptides deduced from antigenic protein
classifiers for colon-, lung, and prostate cancer, together
with peptides presenting the top 500 known mutations in
Our innovative approach will
1) provide a protein-microarray which enables highly reproducible identification of marker-candidates,
2) integrate already defined candidate markers derived
from macro-array- and SEREX- pre-screenings, and
3) evaluate peptides deduced form marker-candidates. In
addition we will
4) apply phage-display-technology for selection of seroreactive peptides from controls and clinico-pathologically
different patient-groups defined during clinical examinations. Phage-display will elucidate antigenic peptides in a
very efficient manner without the need of tumor derived
cDNA libraries. Both the microarray-derived and the phage display derived antigenic-peptides panels of candidate
markers will be chemically synthesized and used for confirmation and elucidation of a classifier using state-of the art
statistical bioinformatics. Thus
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cancer (derived from the COSMIC database), a high density array was presenting 179000 different peptides was
designed. This array was then probed with breast-cancer,
benign breast-tumor and healthy control serum-samples
(n=72). The 179k peptide array data enabled correct classification of all the three different patient groups with high
accuracy (see ROC curves depicted below). Then serum
from initial diagnostics of patients representing cases and
hospital control were collected and provided to partners
at AIT for testing the candidate peptides to generate a targeted assay using Luminex bead array technology. These
assays, when providing high accuracy will be provided to
MUW for cross-validation early in 2015.
Evaluation and fine mapping of FISH-detected lowlevel ESR1 amplification using SNP-GeneChip analysis to reveal amplicon breakpoints in fresh frozen
breast cancer tissue specimens
Weingartshofer, Holst, Ambros (CCRI), Ambros (CCRI),
Rudas, Kriegner (AIT), Weinhäusel (AIT), Singer
Breast cancer is still the most common cancer in women worldwide.
The rapid development in the field
of diagnostic and therapeutic possibilities regarding to the underlying
causes and mechanisms of the disease there are many
questions still unresolved. It is known that estrogen positive breast cancer is associated with estrogen receptor
overexpression, which is linked to estrogen receptor 1
(ESR1) amplification. In a previous study, it was particularly
striking that women who received tamoxifen monotherapy,
in terms of breast cancer recurrence and 5-year survival
had a better prognosis than women who had no ESR1 amplification. Therefore, it was of great interest in this work,
to elucidate the underlying mechanism of amplification,
which is responsible for the copy number increase. In addition, we wanted to ascertain if the start and end points
(break points) of the additional copy of the gene happen
randomly or are localized to specific sites on the gene.
For the study we used 25 fresh frozen breast carcinoma
specimens of women with early breast cancer who had
been naïve to any therapies, neither chemotherapy nor endocrine therapy to exclude any negative influences or other
potential effects regarding the sample quality as a result
of treatment. Additionally, a neuroblastoma control cell line
(SHEP cells) was cultured and used for further specific investigations. For the detection of ESR1 amplifications a fluorescence in situ hybridization (FISH) protocol was developed with and without RNase pretreatment and the amplified
regions on correlating frozen sections then were marked.
FISH analysis samples with cell nuclei showing tight as well
as confluent signal clusters and distinguishable signals,
have been included into the study. An average ratio * 2 was
rated as amplification, and * 1.3 as gain. Cases showing
defragmented DNA or normal signals were deleted. Six tumors, which were gained and one amplified sample, could
be selected for the following comparative genomic hybridization (CGH) array (Figure 1). To avoid dilution effects
performing SNP-GeneChip analysis, marked areas were
lasered out by laser microdissection (Figure 2), DNA then
was isolated therefrom and subsequently a SNP GeneChip
analysis was performed to locate the exact breakpoints of
an additional copy within and around the ESR1 gene.
To investigate the potential involvement of intrachromosomal low copy repeats (LCRs) in recurrent chromosomal
translocation formation, we performed computational Basic Local Alignment Search Tool (BLAST) analysis that can
potentially mediate chromosomal translocations (Figure
3). BLAST analysis was accomplished within ESR1 gene regions using the long and a short isoform of ESR1. For this,
we downloaded the genomic sequence of the ESR1 ±10 kb
from UCSC and generated a BLAST database with 17,311
fragments. These fragments then were blasted against
whole ESR1 gene sequence including 10 kb upstream and
downstream sequences with selected criteria including Evalue 1E-10, homology * 95 % and hit length of 100 % and
resulted in 508 hits. We also wanted to know how much
of these 508 hits are within a repeat region identified by
RepeatMasker and found 10 (0.058 %) hits. It was also of
great interest to identify potential in amplification involved
repetitive sequences in ESR1 flanking regions. Here, genomic sequence of ESR1 ± 200 kb was downloaded from
UCSC followed by BLAST analysis and homology mapping.
Several candidates such as AluYa5 and L1MB8 that were
adjacent and showed homologous sequences could be
identified in ESR1 flanking regions. But very exceptional
was the highest level of sequence homology of all ESR1
flanking RepeatMasker sequences we could observe in
AluYb8 with only two bases mismatches over 312 bp. This
was unique in this area.
To confirm the exact location of the suspected break points
in this range, the results were compared with the data of
SNP-GeneChip analysis and further a PCR analysis was
carried out. In laser micro dissected samples we could not
identify break points within or in expected flanking regions
of ESR1. However, in the neuroblastoma cell line we found
a small and partial gain (Figure 4). Based on the data of the
amplified region identified with array CGH it was of great
interest to know if starting and endpoint of the gained region is correlating to AluYb8. There were identified homologies but none, which really correlated to the gained region.
Results showed that there are missing fragments in a size
of 300 kb on the left and right side from ESR1. Nevertheless, we could identify the MLT1C, a retrotransposon, within
the gained region, but none homologous element confining
this area.
Beside FISH analysis, microdissection and further SNP array we wanted to investigate, if the recombination of amplified samples is localized to the assumed homologous
sequences beside the ESR1 gene. For this, the Alu element
AluYb8, where the highest level of sequence homology of
all ESR1 flanking RepeatMasker sequences was shown,
was used for primer design. The primers were designed
rather PCR analysis was so performed that if a recombination would take place in AuYb8 area, a corresponding
amplification product would be produced. Primer C was
corresponding to the 3’ ESR1 sequences directed to the
‘right side’ and primer B was corresponding to 5’ ESR1 sequence with the direction to the ‘left side’. So if here would
be held recombination from our DNA samples (non-laser
and laser microdissected), we would have an amplification
product. However, no PCR amplification products could be
detected using this kind of analysis.
Although within the cell line a „gain“ could be found by
CGH-array, we could not visualize amplification by RNAse
FISH analysis. With this result the limitation of FISH analysis
could be demonstrated. Sequences may were directly adjacent and consequently no additional FISH signal could be
identified because the hybridized sequences by FISH would
appear together as one signal. That means that the FISH
analysis could not detect additional gene signals, suggesting that the additional ESR1 sequences were not translocated, but probably lie directly behind in the original locus.
Summing up, ESR1 amplification, its underlying mechanisms and its role in therapy and prognosis as potential
biomarker is still an exciting topic and has to be resolved.
Figure 2. Selected cryo sample before and after the laser microdissection. Light microscopy image (4x) of a histological cryo section (7µm) of
a breast tumor before and after microdissection. The red outlined tumor
regions in this picture (left) are isolated after microdissection (right).
Figure 4. ESR1 homology regions overview. H = homology (black)
e.g. H1 upstream + H1 downstream or H8 upstream + H8 downstream
are homologous. These data were performed by using BLAST analysis; U =
upstream (blue), e.g. U4 upstream against downstream analysis (second
attempt) for long isoform (above ESR1 isoforms). U = upstream (blue)
e.g. U4 = d2, d4, d13. There is a minimum homology from fragment U4
with fragment d2, d4 and d13 downstream for short isoform (below ESR1
isoforms). ESR1 (blue lines): Long ESR1 isoform including the five short
isoforms is shown.
Figure 4: Neuroblastoma cell line with partial gain in ESR1 gene.
Figure 1. Selected tumor samples with ESR1 copy number increase.
RNase FISH was performed for laser microdissection. Images A to F show
RNase FISH with low-level copy number increase (gain). (ESR1 signal is
green, Centromere 6 is red, 100x)
| 199
The role of G-CSF and GM-CSF as biomarker
in breast cancer
Singer, Salama, Jankovich, Weiler, Weingartshofer
Among women in the western industrial countries, breast
cancer is the most common type of cancer. Biomarkers
are used to obtain more detailed information concerning
diagnosis and prognosis or in order to improve treatment.
In this respect, G-CSF becomes more and more interesting, as it was found that there are differences regarding
the serum levels of breast cancer patients compared to
healthy women. In several studies, positive effects of GCSF in terms of stimulating growth and development of the
tumor have been demonstrated. To prevent severe forms
of neutropenia caused by chemotherapy, prophylactic GCSF is used. Therefore it is vital to evaluate the influence of
this growth factor on the progression of breast cancer. As
higher concentrations of G-CSF are expected in advanced
stages, the serum levels of breast cancer patients with and
without lymph node involvement were compared to a control group of women without breast cancer.
The granulocyte macrophage colony-stimulating factor
(GM-CSF) and its function as biomarker in breast cancer
was also determined. Until today there are only two biomarkers which are used for the interpretation of the status
of breast cancer uPA and PAI-1. For this reason it is important to find other biomarkers which support cancer diagnosis. GM-CSF was found to change its concentration in
patients with colorectal cancer. Because of this fact, GMCSF serum concentrations maybe also changes in patients
with breast cancer. This growth factor is used combined
to chemotherapies because of its ability to raise the cell
population of granulocytes and macrophages. Due to the
fact that GM-CSF has a very low serum concentration it
is necessary to compare the characteristics between the
multiplex systems and the common ELISA if detecting cytokines. Also a trouble shooting is a central theme in this
study because it helps to prevent problems when using this
kind of assay.
In this study we compared patient groups of a total of 149.
The first group consisted of healthy women with changes in
their breastparenchym. The remaining groups were sera of
women with breast cancer (with and without involvement
of lymph nodes). G-CSF and GM-CSF serum concentrations were detected by using the Bio-Plex multiplex system (Bio Rad/Luminex). This assay is based on the binding
between the analyte and the specific coated bead, which
is detected by flow cytometry. There was no significant elevation in the serum concentrations of G-CSF of breast carcinoma patients compared to women without breast cancer (p=0.74). Moreover, the serum levels in patients with
lymph node involvement in comparison to women without
lymph node metastasis showed no significant difference
(p=0.421). The same applies to subgroups of other clinical
relevant parameters like the ER- or PR-status (p=0.494;
p=0.428), the HER2-status (p=0.755), the tumor grading
(p= 0.282) and the menopausal status (p=0.89). However,
controversial results have been shown by other studies.
The statistical analysis in the serum concentrations of GMCSF has represented that there is a significant difference
(p=0.002) between healthy women and breast cancer
patients (Figure 1). All other clinical relevant parameters
(age, menopause status, hormones) had no significant difference (Figure 2).
This study was able to show that the Bio-Plex multiplex System (Luminex) is an appropriate method to detect the GMCSF serum concentrations. Furthermore it was possible to
confirm the literarily known significant difference between
healthy persons and breast cancer patients.
Further research is needed to evaluate the true value of
G-CSF as prognostic biomarker and its influence on tumor
development.
Figure 2. Significant difference (p=0.002) in GM-CSF serum concentrations (Obs Conc) between healthy women and women with Mamma carcinomas using the Mann-Whitney-U-Test.
Figure 3. No correlation between age and GM-CSF serum concentration.
Sponsion:
Foto: Sponsion am FH Campus Wien.
Links: Maga Martina Fondi; Mitte: Sigrid Weingartshofer,
MSc; Rechts: Ao. Univ.-Prof. Mag. Dr. Arthur Mettinger
Im Rahmen eines berufsbegleitenden Studiums hat die
Biomedizinische Analytikerin, Sigrid Weingartshofer, den
akademischen Grad „Master of Science“ mit der Arbeit
“Evaluation and fine mapping of FISH-detected low-level
ESR1 amplification using SNP-GeneChip analysis to reveal
amplicon breakpoints in fresh frozen breast cancer tissue
specimens” erreicht.
Diese Arbeit beschäftigte sich mit der Detektion von
„low-level“ ESR1-Amplifikationen in Gefrierschnittpräparaten von Mammakarzinomen. Für die Detektion der
ESR1-Amplifikationen wurden verschiedene Methoden wie
die FISH-Analyse, Lasermikrodissektion, PCR und SNPGeneChip-Analyse durchgeführt. Mittels bioinformatischer
BLAST-Analyse konnten flankierende Regionen am ESR1Gen mit repetitiven homologen Sequenzen festgestellt
werden. Um die vermutete exakte Position der Bruchpunkte in diesem Bereich zu bestätigen, wurden die Ergebnisse mit den Daten der SNP GeneChipanalyse verglichen.
Verwertbare FISH-Analysen aus den Tumoren zeigten eine
„low-level“ Amplifikation, jedoch keine keinen Genkopiezugewinn in der SNP-GeneChip-Analyse. In einer Zelllinie
konnte zwar ein „gain“ mittels CGH-Array festgestellt werden, jedoch konnte dieser wiederum nicht mittels RNAseFISH visualisiert werden. Weiters zeigte der Vergleich mit
der BLAST-Analyse, dass die gefundenen Bruchpunkte
nicht mit den identifizierten homologen Sequenzen übereinstimmten. Zusammenfassend kann man sagen, dass
diese Ergebnisse letztendlich darauf schließen lassen, dass
diese ESR1-Amplifikationssignale einem anderen, als dem
angenommenen Mechanismus unterliegen.
CIMBA (The Consortium
of Investigators
of Modifiers of BRCA1/2)
Coordination partners:
Georgia Chenevix-Trench PhD, NHMRC Senior Principal
Research Fellow, The Queensland Institute of Medical Research, Australia
Antonis Antoniou, CR-UK Genetic Epidemiology Unit,
Strangeways Research Laboratory,
University of Cambridge, Cambridge, UK
Douglas Easton, CR-UK Genetic Epidemiology Unit, Department of Public Health and Primary Care, University of
Cambridge, Cambridge, UK
Cooperation partners:
Singer Christian, Department of Obstetrics and Gynecology, Center for Familial Breast- and Ovarian Cancer, Medical University of Vienna, Austria
Other cooperation partners:
http://apps.ccge.medschl.cam.ac.uk/consortia/cimba//groups/groups.html
Collaborators:
Muhr, Weingartshofer, Rappaport
The Consortium of Investigators of Modifiers of BRCA1/2
is a collaborative group of researchers working on genetic modifiers of cancer risk in BRCA1 and BRCA2 mutation
carriers. The aim of CIMBA is to provide sufficient sample
sizes to allow large scale studies in order to evaluate reliably the effects of genetic modifiers. BRCA1 and BRCA2
mutation carriers are at substantially increased risk for
developing breast and ovarian cancer. The incomplete
penetrance coupled with the variable age at diagnosis in
carriers of the same mutation suggests the existence of
genetic and nongenetic modifying factors. In this study, the
putative role of variants in many candidate modifier genes
will be evaluated.
Recent results showed that DNA glycosylases involved in
the first steps of the BER pathway may be associated with
cancer risk in BRCA1/2 mutation carriers and should be
more comprehensively studied.
A retrospective cohort approach of genotyping from 15,252
BRCA1 and 8,211 BRCA2 mutation carriers, for known variants (n = 3,248) located within or around 445 candidate
genes showed that none of the variants was significantly associated with breast or ovarian cancer risk in either
BRCA1 or BRCA2 mutation carriers, also after multiple testing adjustments.
| 201
Familial Breast Cancer
Research Unit
ENIGMA – Evidence-based Network for the
Interpretation of Germline Mutant Alleles
Risk Factor Analysis of Hereditary Breast
and Ovarian Cancer
Coordinator:
Dr. Steven Narod, Toronto, Canada
Singer Christian, Department of Obstetrics and Gynecology, Center for Familial Breast- and Ovarian Cancer, Medical
University of Vienna, Austria
and others
Collaborators: Muhr, Weingartshofer
This is the largest long-term study of women who carry a
mutation in one of the two breast cancer genes (BRCA1/
BRCA2). This study was started in 1995 by Dr. Steven Narod and now has upwards of 9,000 participants from across Canada, the United States, Europe, and Asia. Its purpose is to better understand the prevention and treatment
of hereditary breast and ovarian cancers. We hope to gain
a better understanding of the interaction between various
hormonal, reproductive, and lifestyle factors that may be
associated with the development of breast and ovarian
cancer in high-risk families.
Recent result showed the following:
t:OVY[[LYT \ZL VM [HTV_PMLU MVY JOLTVWYL]LU[PVU PU
BRCA1 and BRCA2 mutation carriers may be as effective
as a conventional 5-year course of treatment to reduce
the risk of contralateral Breast Cancer.
t7YL]LU[P]LVVWOVYLJ[VT`^HZHZZVJPH[LK^P[OHUYLduction in the risk of ovarian, fallopian tube, or peritoneal
cancer in BRCA1 or BRCA2 carriers and a 77% reduction
in all-cause mortality.
t6YHS JVU[YHJLW[P]L \ZL ILMVYL HNL PUJYLHZLZ [OL
risk of early-onset breast cancer among women with a
BRCA1 mutation and the risk increases with duration of
use. Therefore caution should be taken when advising
women with a BRCA1 mutation to take an oral contraceptive prior to age 25.
t;OLYPZRVMJVSVYLJ[HSJHUJLYPZPUJYLHZLKPUMLTHSLJHYriers of BRCA1 mutations below the age of 50 years but
not in women with BRCA2 mutations or in older women.
t;OL L_WLYPLUJL VM NLUL[PJ [LZ[PUN PU (\Z[YPHU ^VTLU
with a BRCA1 or BRCA2 mutation in terms of preventive
measures taken and incident cancers diagnosed showed that the majority of healthy women with a BRCA1 or
BRCA2 mutation opt for preventive oophorectomy and
MRI screening to manage their breast cancer risk; few
have preventive mastectomy or take tamoxifen.
Coordination partners:
David E. Goldgar, University of Utah
Amanda Spurdle, Queensland Institute for
Medical Research
Fergus J. Couch, Mayo Clinic
http://www.enigmaconsortium.org/steering-committee.
html
Singer Christian, Department of Obstetrics and Gynecology, Center for Familial Breast- and Ovarian Cancer, Medical
University of Vienna, Austria
and others
Collaborators: Muhr, Rappaport, Weingartshofer
ENIGMA is a consortium of investigators focused on determining the involvement of all unclassified variants (UV),
also called variants of uncertain significance (VUS), in the
BRCA1 and BRCA2 tumor suppressor genes, in predisposition to breast and ovarian cancer.
The purpose of this research-based Consortium is to facilitate classification of variants through collaborative largescale projects by sharing data and improving classification
methods. To do this there are different working groups
(WG), focusing on the development and maintenance of
Databases and applying statistical analysis (Analysis/Database WG), the integration of the clinical aspects (Clinical
WG), developing functional analysis (Functional WG), identifying tumor markers to be integrated into the multifactorial likelihood model (Pathology WG) and studying splicing
Variants (Splicing WG).
ERA-NET on Translational
Cancer Research (TRANSCAN)
“Translational research on
primary and secondary
prevention of cancer” - Development of a Comprehensive Risk Prediction Model for BRCA1 and BRCA2
mutation carriers
UPA und PAI – Mammakarzinomexpression
Das Ziel der Studie ist es prognostische Zusammenhänge
beim Nachweis von Urokinase-Typ Plasminogen-Aktivator
(uPA) und seinem Inhibitor PAI-1 nachzuweisen. Um sich
nach der Operation für oder gegen eine ergänzende Chemotherapie zu entscheiden, soll die Färbung der Biomarker
uPA und PAI-1 im Tumorgewebe helfen.
Prädiktive Marker sollen Therapieentscheidung verbessern, hohe Konzentration ist mit schlechter Prognose verbunden
Coordinator:
Rookus Matti, The Netherlands, The Netherlands Cancer
Institute, Amsterdam
Partners:
Andrieu Nadine, France, INSERM, Paris
Easton Douglas, United Kingdom, Cambridge University,
Cambridge
Jakubowska Anna, Poland, Pomeranian Medical University, Szczecin
Kast Karin, Germany, Universitatsklinikum Carl Gustav
Carus, Dresden
Singer Christian, Austria, Medical University of Vienna,
Wien
Van Gils Carla, The Netherlands, Universitity Medical
Center Utrecht, Utrecht
Collaborators: Muhr, Weingartshofer
BRCA1/2 mutation carriers have high risks of early onset
Breast Cancer (BC) and ovarian cancer (OvC), but age-specific risks vary strongly between and among families. Currently, we are rapidly generating knowledge on genetic and
hormonal modifiers of BC and OvC risks among BRCA1/2
carriers. However, the new risk modifiers cannot yet be
used in the counselling of BRCA1/2 mutation carriers as
the current risk prediction models do not take them into
account. In this project the aim is
1. to assess the independent and combined associations of
common genetic variants, reproductive/hormonal risk factors, breast density and risk reducing surgeries and risks of
breast and ovarian cancer in BRCA1 and BRCA2 mutation
carriers,
2. to examine if a (lack of) decrease in breast density after
a risk reducing oophorectomy may help to define a hormone-(in)sensitive group, and
3. to develop a novel online comprehensive risk prediction
tool that provides valid individualized age specific cancer
risk estimates and uses for the first time the combined information of common genetic variants, reproductive/hormonal factors, breast density and risk reducing surgeries.
This project is based on the International BRCA1/2 mutation Carrier Cohort Study, the largest available prospective
BRCA1/2 cohort study (IBCCS).
Während man bei einem niedrigen Rezidivrisiko in der Regel keine Chemotherapie empfiehlt, raten Ärztinnen und
Ärzte bei einem hohen Rezidivrisiko fast immer zu einer ergänzenden Therapie. Bei Patientinnen mit einem mittleren
Risiko reichen die etablierten Faktoren (Grad der Entartung
der Tumorzellen u. a.) jedoch für eine solche Therapieempfehlung nicht aus. Deshalb ist in den Leitlinien auch keine
enthalten. Um bei Patientinnen mit mittlerem Rezidivrisiko bessere Therapieentscheidungen zu treffen, ihnen gegebenenfalls eine belastende Chemotherapie ersparen zu
können, untersuchen wir die Zusammenhänge von uPA und
PAI-1. Diese sollen helfen, diejenigen Patientinnen zu identifizieren, die mit hoher Wahrscheinlichkeit einen Nutzen
von der Chemotherapie haben.
Umfang der Studie:
Es wurden gut 1000 Patientinnen mit 3 verschiedenen AK
(1x uPA und 2x PAI-1) gefärbt.
Kooperationspartner:
Dr Stefan Jahn, MUW Graz
Prof.Dr. Manfred Schmitt – TU München
| 203

forschungslaboratorien

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