FORSCHUNGSLABORATORIEN - Home | MedUni Wien · The results of this study were published in Mol Hum Reprod. 2019 Feb 1;25(2):76-87 Role of estrogen signaling in macrophage plasticity

Universitätsklinik für Frauenheilkunde | 199

FORSCHUNGSLABORATORIEN

200 | Universitätsklinik für Frauenheilkunde

DAS FORSCHUNGSL ABOR DER UNIVERSITÄTSKLINIK FÜR FR AUENHEILKUNDE

Das Forschungslabor der Universitätsklinik für Frauenheil-kunde entwickelte sich aus den Labors der ehemaligen I. und II. Universitäts-Frauenklinik und ist seit Mitte der 1990er Jah-re in einem Cluster auf Ebene 5Q des AKH Wien zusammen-gefügt. Die Labors sind dem Vorstand der Klinik, Univ.-Prof. Dr. Peter Husslein direkt unterstellt, die wissenschaftliche Koordination obliegt ao. Univ.-Prof. Dr. Christian Egarter, die administrative Leitung ao. Univ.-Prof. Dr. Christian Schneeberger.

Wurde ursprünglich die Routine und Forschung gleicherma-ßen abgedeckt, so liegt heute der Schwerpunkt auf Grundla-gen- und angewandter Forschung. Sieben Arbeitsgruppen, die international in vielen Partnerschaften vernetzt sind und im Folgenden alphabetisch gereiht vorgestellt werden, führen eine Vielzahl von Projekten durch, die sich mit speziellen Fragestellungen im Bereich der Geburtshilfe, der Gynäko-logie, der gynäkologischen Onkologie und gynäkologischen Endokrinologie beschäftigen.

Struktur des Forschungslabors der Universitätsklinik für Frauenheilkunde


ENDOMETRIOSIS GROUP

M I T A R B E I T E R :Dr. Iveta YotovaDr. Quanah HudsonChristoph Hauser, MScManuela Gstöttner, MSc

The current scientific interests of the laboratory of Endome-triosis are focused on understanding the molecular mecha-nisms involved in the pathogenesis of endometriosis and the discovery and validation of new non-invasive biomarkers for diagnosis of the disease. These specific points are developed in the following research projects.

Evaluation of the prediction power of the levels of the mature BDNF protein in serum of women with endometriosis as a non-invasive biomarker for the disease.As a growing body of evidence indicates that BDNF may serve as a putative non-invasive marker for the diagnosis of early stage rASF I and II of the disease, in this study we carried out an ELISA based evaluation of the levels of secre-tion of the mature form of the protein in serum of 77 women with endometriosis and 55 disease free controls. We show

that the mature form of the protein is elevated in serum of patients with endometriosis compared to controls, that this alteration is associated with rASF stage I and II endo-metriosis, is independent of the hormonal changes within menstrual cycle and is not associated with the levels of self-reported pelvic pain. By comparing our data to a similar study published by Wessels et al (Fertility and Sterility, 2016) we not only confirmed their observations, but also report an interesting aspect of postoperative alterations in the levels of BDNF secretion affecting both cases and controls. Further, when we analyzed the ability of the mature BDNF to differ-entiate women with endometriosis and controls, we show that the protein is not an indicative marker for the disease. This observation is in contrast to the findings reported by the group of Wessels et al. where the soluble form of the protein is reported as a promising candidate for clinical application. Our results, however, are in accordance with previously published data and add to an evolving picture in which the changes in the levels of expression of BDNF in the circula-tion are associated with minimal and mild endometriosis.

The results of this study were published in Exp Biol Med (Maywood). 2018 Jan; 243(1):50-56. Epub 2017 Nov 15. PMID: 29141456

The endometriosis group (from left to right):

Christoph Hauser, MSc - Dr. Iveta Yotova - Manuela Gstöttner, MSc - Dr. Quanah Hudson.


Evaluation of the prediction power of the levels of differentially expressed miRNAs in plasma of women with endometriosis and controls as non-invasive biomarkers for the disease.This prospective cohort study explored the possibility of using changes in extracellular miRNA spectra in plasma of 51 patients with endometriosis compared with 41 controls combined with clinical data as non-invasive biomarkers for the disease. The project was divided into three different phases for biomarker screening, discovery and validation. The differences in expression levels of plasma miRNA ob-tained from women with and without endometriosis were analyzed with quantitative PCR-based microarrays. The diagnostic performance of the selected individual and/or combined differentially expressed miRNA candidates and clinical parameters was assessed using in silico bioin-formatics modelling and receiver operating characteristic curve analysis. Data showed that a specific plasma miRNA signature is associated with endometriosis (Figure 1) and that hsa-miR-154-5p, which alone or in combination with hsa-miR-196b-5p, hsa-miR-378a-3p, and hsa-miR-33a-5p and the clinical parameters of body mass index and age, are potentially applicable for non-invasive diagnosis of the dis-ease (Figure 2). Changes in the levels of expression of certain circulating plasma miRNA also occurred within the phases of the menstrual cycle. Based on these observations we have concluded that miRNA seem to be promising candidates for the non-invasive diagnosis of endometriosis. Further, other clinical parameters may help in distinguishing women suf-fering from endometriosis from healthy individuals.

Figure 1. Supervised hierarchical clustering of 49 DE (log2 fold change>1) miRNAs between analyzed groups is shown. Biological samples are on the x-axis and DE miRNAs are on the y-axis with relative levels of expression indicated by color sca-le. Red color repre-sents an expression level above the mean across all samples, black represents mean expression and green represents expression lower than the mean.

Figure 2. Diagnostic value of hsa-miRNA-154-5p for endometriosis ex-pressed by ROC-curve analysis (left upper panel) of the log2 transformed expression data (right upper panel) obtained in a cohort of n=64 cases and controls. The AUC and p-values are indicated in the graph. Diagnostic performance of the combined four DE miRNAs in a cohort of n=83 when analyzed together (left lower panel) or in combination with the clinical parameters age and BMI (right lower panel) is graphically presented. The AUC and corresponding p-values for each test are indicated in the graphs.

The results of this study were published in Reproductive Biomedicine Online. 2018 Oct; 37(4):449-466.

Does the enhanced expression level of TACE contri-bute to elevated levels of sVCAM-1 and I-CAM1 in endometriosis?We and others have shown that serum-soluble (s) VCAM-1 levels are significantly higher in women with endometriosis, compared to disease-free controls. Experimental evidence exists suggesting a role of sICAM-1 and sVCAM-1 in the pathogenesis of endometriosis. TACE was identified as the protease responsible for phorbol 12-myristate 13-acetate (PMA)-induced VCAM-1 release in murine endothelial cells. Additionally, it has recently been shown that TACE is upregulated in the endometrial luminal epithelium of the mid-secretory phase in infertile women. In this project we aimed to experimentally give an answer to the question: Are increased sVCAM-1 and sICAM-1 levels associated with tumor necrosis factor-alpha-converting enzyme (TACE) activ-ity in endometriosis? The study was conducted on a cohort of total number of 97 samples collected from women with and without endometriosis. The results of the study dem-onstrated that TACE protein is overexpressed in epithelium of tissue samples of both eutopic endometrium and ectopic lesions of women with endometriosis compared to disease-free controls (P < 0.001 both) (Figure 1) and that the over-expression of the protein in the lesions is due to activation of TACE gene transcription (P < 0.001). Moreover, epithelial TACE protein was significantly higher in ectopic samples than in corresponding eutopic tissue of women with the


disease (P < 0.001). High endometrial tissue TACE protein expression correlated with higher serum sVCAM-1 levels (P < 0.05) but not with sICAM-1 levels. Inhibition of TACE either by TACE inhibitors or by TACE siRNA knockdown resulted in decreased PMA-induced shedding of sVCAM-1 in vitro (P < 0.005 or P < 0.01, respectively), but the TACE inhibitors did not affect transcription of TACE or VCAM-1. Addition-ally, we observed an upregulation of TACE in proliferative endometrial epithelium of infertile (P < 0.005), compared to fertile women. TACE was increased in infertile women with endometriosis (P = 0.051) but not in infertile women without endometriosis. Based on this data we came up with the conclusion that the dysregulation of TACE substrate shed-ding represents a promising yet relatively unexplored area of endometriosis progression and could serve as a basis for the development of new treatments of the disease.

Figure 1. Immunohistochemical analyses of TACE expression levels in ectopic endometrium of women with and without endometriosis are shown. Anti-TACE antibody was applied at a dilution of 1:2000 and yielded weak, moderate or strong staining in ectopic tissue (D, E, F) of women with and without endometriosis, respectively. The intensity (0–3) and the percentage (0–3) of the stained cells were combined to derive a final IHC score (0–9). For statistical evaluation of the cellular TACE intensity, epithelial (down left panel) and stromal (down right panel) were analyzed separately at magnification = 200× in a cohort of n = 39 control patients (EM controls) and n = 42 women with endometriosis.

The results of this study were published in Mol Hum Reprod. 2019 Feb 1;25(2):76-87

Role of estrogen signaling in macrophage plasticity in Endometriosis Endometriosis (EM) is an estrogen driven chronic inflamm-atory disease. Studies have identified macrophages (MF) as important players in the development of EM, since their activation toward a reparative phenotype allows survival, neovascularization, and growth of ectopic lesions. However, the function of estrogen signaling in MF plasticity and MF-mediated T-cell responses are still not understood. We hypo-thesize that estrogen receptor (ER) signaling in MF is involved in the polarization and repolarization of these cells and the subsequent T cell immune responses, which, together with paracrine action of peritoneal fluid (PF) factors, enforce the formation of a tolerogenic microenvironment allowing development of EM lesions. Our aims include the deter-mination of the role of ER signaling and PF on polarization and repolarization of MF in patients and controls (Figure 1) evaluating the effect of MF plasticity on MF-induced T cell immune responses in the context of ER-signaling modulati-on. The main rationale of the proposed project is the basic understanding of impaired immune surveillance in EM.

Figure 1. Generation and re-polarization of M1 and M2 macrophages in vitro using MO obtained from peripheral blood of healthy volunteers, women with and without endometriosis. Representative microscopy data for polarization of monocyte derived macrophages using cytokines. Left: monocyte activated by M-CSF as M0. Middle Upper: M1 cells differen-tiated by TNF-α and INF-γ. Middle Lower: M2 cells differentiated by IL-4 and IL-13. Right Upper: Re-polarization M1 to M2 cells in secondary stimu-lation using reverse cytokines IL-4 and IL-13. Right lower: Re-polarization M2 to M1 cells in secondary stimulation using reverse cytokines TNF-α and INF-γ.


GYNENDO GROUP

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 018Christian Schneeberger, ao. Univ.-Prof. Mag. Dr. Andrea Kolbus, Univ.-Doz. Dr. Detlev Pietrowski, Dipl. Biol. Dr.Aulona Gaba, Dr. Ladislaus Szabo (BMA)Barbara Widmar (BMA) Mary Frank (BMA)Sophie Wanderer (BSc Studentin)

PROJEKTE 2018

Multi-step Streptamer systems for clinical stem cell researchGemeinsam mit den Projektpartnern aus Deutschland wur-de ein neuartiges System zur Anreicherung/Reinigung von hämatopoietischen Stammzellen aus Nabelschnurblut ent-wickelt, welches eine mehrstufige Anreicherung erlaubt. Die neuartigen Affinitätsreagenzien wurden von Firma IBA entwi-ckelt. IBA stellte auch das Know-how im Mikrofluidikbereich zur Verfügung. Die Hauptaufgabe unserer Arbeitsgruppe ist die Evaluierung von neuen Reagenzien und Methoden mit Stammzellen aus Nabelschnurblut sowie der Vergleich zum derzeitigen „state-of-the-art“ im Bereich der Zellselektion.

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 phar-macologically. 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 Testo-

sterone 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 Testo-sterone on endothelial cells from macrovasculature (HUVEC) are different then on microvasculature and whether migration and proliferation are mediated by the Androgen Receptor.

Vitrifikation IIDer Erfolg einer Vitrifikation, dem ultraschnellen Einfrie-ren von Zellen oder Geweben mit Einfrierraten von bis zu 20000°C pro Sekunde in flüssigem Stickstoff- hängt neben anderen Parametern auch von der Geschwindigkeit des Einfrierprozesses ab. Es ist bisher nicht geklärt, in wieweit auch niedriger Einfrierraten die Vitalität der Zellen oder des Gewebes sichern können. Daher wird in vielen Verfahren eine sogenannte „offene“ Vitrifkation eingesetzt. Hierbei kommen die Zellen in direkten Kontakt mit dem Stickstoff, so dass eine optimale Temperaturübertragung an die Zellen gewähr-leistet ist. Diese Verfahren hat allerdings den potentiellen Nachteil, dass über den Stickstoff, der nicht steril ist oder nur mit sehr großem Aufwand sterilisiert werden kann, diese Zellen mit Mikroorganismen kontaminiert werden können. Gerade im klinischen Bereich kann das eine verheerende Problematik sein. In diesem Projekt soll daher untersucht werden, ob und inwieweit es möglich ist, Zellen auch steril in einem Einfriergefäß zu vitrifizieren. Da hierbei für die Tem-peraturübertragung, neben dem Material der Einfriergefäße, auch die Luftvolumina der Einfriergefäße eine Rolle spielen werden unterschiedliche Gefäßgrößen mit dementsprechend auch unterschiedlichen Luftvolumina eingesetzt. Die Überle-bensraten der Zellen nach der Vitrifikation werden dabei mit Hilfe einer FACS-Analyse nach Fluoreszenzfärbung bestimmt. Sollten sich die Überlebensraten dieser „geschlossenen“ Vitrifikation nicht wesentlich von denen der „offenen“ Vitri-fikation unterscheiden, so wäre dies ein erster Schritt hin zu möglichen klinischen Anwendungen.

Das Team der Arbeitsgruppe (von links nach rechts): Barbara Widmar (BMA) - Univ.-Doz. Dr. Andrea Kolbus - ao. Univ.-Prof. Mag. Dr. Christian Schnee-berger - Dipl.Biol. Dr. Detlev Pietrowski - Mary Frank (BMA) -Sophie Wanderer (BSc Studentin)

Nicht auf dem Foto: Dr. Aulona Gaba und Ladislaus Szabo (BMA)


MOLECUL AR ONCOLOGY GROUP

D A S T E A M D E R A R B E I T S G R U P P E M O L E K U L A R E O N K O L O G I E :

Christiane AgreiterChristina BuchingerThomas DillhofSabrina GrundtnerNicole HeinzlBarbara HolzerJana KalinaGabriele KlamingJessica MarksteinerEva ObermayrValentina PaspaljBettina Savarese-BrennerEva SchusterConradin SchweizerPaul SpeiserPeter SteinRobert Zeillinger

PROJEKTE 2018

Analysis of the predictive ability of six proteins for ovarian carcinoma

Collaborators: Obermayr E, Wallisch Ch, Heinze G, Zeillinger R; Collaborators from the Department of Obstetrics and Gy-necology, Innsbruck Medical University (AT), the Department

of Gynecology, European Competence Center for Ovarian Cancer, Campus Virchow Klinikum Charité Universitätsmedizin Berlin (DE), the Division of Gynecological Oncology, Depart-ment of Obstetrics and Gynecology, Leuven Cancer Institute, University Hospitals Leuven, Katholieke Universiteit Leuven (BE), the Department of Obstetrics and Gynecology, Univer-sitätsklinikum Freiburg (DE)

Recently, our group has evaluated the predictive ability of six plasma proteins (CA-125, HE-4, MIF, OPN, prolactin, leptin) for ovarian cancer. The analysis was based on published data from our group, which showed the discriminative potential of a six-protein panel (including CA-125, MIF, OPN, prolactin, leptin, and IGF-II, but not HE-4) for ovarian cancer (n=220) versus healthy donor samples (n=30) (AUC 0.971) and benign tumours (n=35) (AUC 0.939), and for a small subgroup of FIGO I/II ovarian cancer patients (n=19) versus benign tumours (n=35) (AUC 0.853).We validated the published results in a larger cohort of pa-tients with benign conditions. To that purpose we measured the plasma levels of CA-125, HE-4, MIF, OPN, prolactin, and leptin in samples taken from 119 patients with ovarian cancer and from 130 patients with benign gynaecologic tumours us-ing a multi-plex Luminex bead-based immunoassay. From the resulting data we built a logistic regression model in order to estimate the probability for a malignant tumour. The model revealed a c-index of 0.94 (Figure 1), which was higher than the c-index of 0.91 achieved by CA-125 alone. The calculated model was superior to the ROMA index which estimates the risk for ovarian cancer based on CA-125, HE-4 and menopau-sal status (c-index 0.78).

Das Team der Arbeitsgruppe (von links nach rechts):

Jana Kalina, Eva Schuster, Barbara Holzer, Eva Obermayr, Robert Zeillinger, Nicole Heinzl, Conradin Schweizer, Christina Buchinger, Gabriele Klaming, Sabrina Grundtner

Nicht auf dem Foto: Christiane Agreiter, Bettina Savarese-Brenner, Thomas Dillhof, Jessica Marksteiner, Valentina Paspalj, Paul Speiser, Peter Stein


Figure 1: The predictive probability of the model (left panel) and ROC

curve (right panel)

Plasma levels of C-reactive protein and albumin and their correlation with circulating tumour cells in patients with ovarian cancer

Collaborators: Obermayr E, Schuster E, Pecha N, Aust S, Polterauer S, Grimm Ch, Zeillinger R, and various partners from the OVCAD consortium

Low levels of albumin (ALB) and increased concentrations of the C-reactive protein (CRP) in the blood serum reflect inflammatory activity and have been reported as prognostic factors in various cancer types. In this project we investi-gated the impact of ALB and CRP on the prognosis of the FIGO II-IV ovarian cancer patients included in the OVCAD study cohort. High ALB and CRP levels were associated with the presence of peritoneal carcinomatosis and ascites at diagnosis, and high CRP alone was additionally associated with advanced stage of the disease. A high ratio of CRP/ALB had a strong prognostic impact on overall survival independ-ent from patient age, disease stage and the presence of residual tumour mass after debulking surgery. Furthermore, a high CRP/ALB ratio was associated with the presence of gene transcripts in the blood which indicated the presence of circulating tumour cells.

Figure 2: Kaplan-Meier plots showing the overall (top panel) and progres-sion-free survival (bottom panel) of ovarian cancer patient stratified by the median levels of albumin (left), c-reactive protein (middle) and the ratio of these analytes.

Figure 3a: The plasma levels of ALB, CRP and their ratio in ovarian cancer patients with and without gene transcripts indicative for the presence of circulating tumour cells (CTCs). Differences between CTC positive (n=29) and negative (n=141) cases were calculated using the Mann Whitney test.

Figure 3b: The plasma levels of ALB, CRP and their ratio in ovarian cancer patients with and without gene transcripts indicative for the presence of circulating tumour cells (CTCs). Differences between CTC positive (n=29) and negative (n=141) cases were calculated using the Mann Whitney test.

Detection of circulating tumour cells in blood samples taken from patients with breast cancer

Collaborators: Obermayr E, Agreiter Ch, Schuster E, Krainer M, Singer Ch, Steger G, Marhold M, Zeillinger R

Blood samples were taken from patients with breast cancer and processed using the microfluidic Parsortix™ system (Angle plc. UK). We applied several separation protocols in order to compare the efficiency of the respective protocol to capture circulating tumour cells (CTCs), such as separation cassettes with critical step sizes of 10µm and 6.5µm and processing the blood sample with and without prior density gradient centrifugation. The enriched cells were then lysed and further analysed using qPCR. Preliminary evaluation of the results indicates that direct processing of the blood sample using a microfluidic cassette with a critical step size of 6.5µm without further pre-enrichment of the sample may be most efficient for the enrichment of CTCs and subsequent qPCR analysis.

Detection of circulating tumour cells in blood samples taken from patients with prostate cancer

Collaborators: Obermayr E, Agreiter Ch, Krainer M, Angle plc., Zeillinger R

Patients with castration-resistant refractory prostate cancer (CRPC) are treated with drugs targeting the androgen path-way, like abiraterone or enzalutamide. It has been shown that patients harbouring the androgen receptor splice variant v7 (AR-v7) were resistant to these drugs, and that circulating tumour cells (CTCs) may be used as a liquid biopsy in this regard. In a small pilot study, we processed blood samples from seven CRPC patients using the microfluidic Parsortix™ system (Angle plc. UK), and subsequently assessed the presence of CTCs using immune-fluorescent staining and AR-v7 specific PCR employing the AdnaTest ProstateCancer technology. We observed CTCs in all patients by immune-flu-orescent staining, but in none using the AdnaTest approach.

Albumin < medianAlbumin > median

Albumin < medianAlbumin > median

CRP < medianCRP > median

CRP < medianCRP > median

CRP/ALB < medianCRP/ALB > median

CRP/ALB < medianCRP/ALB > median


Figure 4: A representative cell cluster enriched from a CRPC blood sample with the Parsortix™ system. The image on the left-hand side shows the cluster in brightfield, located on the separation steps of the Parsortix™ cassette. The image in the middle shows the nuclei after staining with DAPI, and the image on the right-hand side shows green fluorescence due to the specific staining of EpCAM, Her2, and CK8.

Detection of circulating tumour cells in blood samples taken from patients with small-cell lung cancer

Collaborators: Obermayr E, Agreiter Ch, Schuster E, Sch-weitzer C, Hochmair M, Hamilton G, Zeillinger R and col-laborators from Angle plc.

Lung cancer is the most common cancer worldwide. 15% of the patients are diagnosed with small-cell lung cancer (SCLC), a highly aggressive neuroendocrine tumour of the lung. Most patients present with metastatic disease with a high number of tumour cells circulating in the blood. Periph-eral blood samples from 76 SCLC patients were processed using the microfluidic Parsortix™ (Angle plc., UK) technology to enrich circulating tumour cells (CTCs). Blood samples from 15 healthy donors processed in the same way served as negative controls. The isolated cells were analysed for the presence of RNA transcripts specific for neuroendocrine (CgA, SYP, NCAM, DLL3) and epithelial cells (EpCAM, CK19) using TaqMan qPCR. ENO2 and NCAM1 transcripts were observed both in the patients and control samples at similar levels; thus, these markers were excluded from further analyses. EpCAM, CK19, SYP, CgA and DLL3 transcript levels beyond the detection limit of qPCR were observed in 34 (45.0%) blood samples; thus, these samples were scored as CTC-positive. 5/34 (15%) were assigned as CTC-positive due the expression of the epithelial markers EpCAM and CK19, and 18/34 (53%) by the expression of neuroendocrine markers CgA, SYP, NCAM, or DLL3. In 11/34 cases (32%) the expression of both epithelial and neuroendocrine markers was observed.The patients were stratified by the respective gene expres-sion into two groups (present vs. absent gene expression levels). We observed a negative impact of DLL3 and CGA on overall survival (median OS 4 vs. 9 months, log-rank p=0.035 and p=0.024), but not of EpCAM and CK19. In conclusion, CTCs in SCLC patients can be assessed using endocrine markers and qPCR. Our study may contribute to the implementation of liquid biopsy in SCLC and finally to improve patient management.

Cultivation of tumour cells in pleural fluid samples taken from patients with small-cell lung cancer after Parsortix™ enrichment

Collaborators: Obermayr E, Agreiter Ch, Schuster E, Hoch-mair M, Hamilton G, Zeillinger R and collaborators from Angle plc.

In lung cancer, tumour tissue samples are often difficult to obtain, and thus in vitro testing of the few promising targets is limited to diverse cell lines and patient derived xenografts. The use of primary cell lines and of CTCs is expected to provide advanced models with possible improved predic-tive power. In the present project we performed a proof of principle study of cultivating tumour cells after enrichment with the Parsortix™ microfluidic technology (Angle plc. UK). We found that the enriched tumor cells retain their viability and can be cultivated using standard cell culture conditions.

Figure 5: Brightfield images of tumour cells enriched from the pleural fluid obtained from four lung cancer patients and after short time cultivation in standard RPMI medium.

Whole blood stabilization for the analysis of circulating tumour cells

Collaborators: Obermayr E, Agreiter Ch, Schuster E, Paspalj V, Zeillinger R and collaborators from Angle plc (London)

The analysis of liquid biopsies based on circulating tumour cells requires the blood to be processed immediately or stabilized with fixatives. Especially in multi-centered studies, when the blood samples are shipped to a central laboratory site for CTC analysis, the selection of the most appropri-ate blood collection tube is crucial for a reliable analysis. Therefore, we recently tested the stability of blood samples taken in EDTA collection tubes and in three commercially available blood collection tubes containing fixatives (Cell-free DNA BCT, Streck, Blood Exo DNA ProTeck® and Blood Exo CTC ProTeck® tubes; CFGenome). We observed that follow-ing enrichment of the blood samples using the microfluidic Parsortix™ system (Angle plc., UK) and qPCR-based analysis of tumour cell specific gene transcripts the EDTA collection tubes and Cell-free DNA BCT tubes are more appropriate than the Blood Exo DNA and Blood Exo CTC ProTeck® tubes.Currently, we evaluated the approach using a smaller amount of tumour cells spiked into the blood samples, and addition-ally we tested a further type of blood storage using ACD tubes at 4°C.The capture rate of spiked tumour cells after storage in Cell-free DNA BCT tubes was higher than after storage in EDTA and ACD tubes (Becton Dickinson). This may largely be due to the effect of the preservative which may lead to an


increased stiffness of the cells and, thus, a higher chance for being trapped within the microfluidic cassette. qPCR analysis of CTC-specific transcripts, among them CK19 and PPIC, revealed that the gene expression levels decrease over time. However, the smallest decrease is observed after storage in EDTA tubes at room temperature. Thus, the recommended procedure in future studies is taking the blood in EDTA tubes and keeping them at room temperature up to three days until further processing.

Figure 6: CK19 and PPIC gene expression levels normalized to the levels at day 0 (no storage) after storage in EDTA (red), Cell-free DNA BCT (green), and ACD (blue) tubes. The capture rate of the spiked tumour cells after storage in the respective blood tubes is shown in the right graph.

Error bars depict standard deviations from triplicate experiments.

Blood sample processing for CTC analyses

Collaborators: Agreiter Ch, Obermayr E, Schuster E, Holzer B, Hamilton G, Stein P, Hastermann G, Hochmair M, Aust S, Singer C, Krainer M, Steger G, Marhold M, Zeillinger R

Over the past year we obtained blood samples taken from about 330 patients, primarily taken at the General Hospital of Vienna (Department of Medicine I, Department of Obstetrics and Gynecology), Sozialmedizinisches Zentrum Baumgartner Höhe - Otto Wagner Spital (Department of Respiratory and Critical Care), and the Krankenanstalt Rudolfsstiftung (De-partment of Obstetrics and Gynecology). From these blood samples, a serum/plasma fraction was retrieved, and/or the whole blood was enriched for circulating tumour cells. The serum/plasma samples from patients with gynaecologi-cal cancers (N=87) were included into the Biobank collec-tion at the Klinisches Institut für Labormedizin (AKH Wien).

Figure 7: Overview on blood samples obtained from 01-12/2018

219 whole blood samples (171 lung cancer patients, 73 breast cancer patients, 12 prostate cancer patients, and 11 patients with gynaecological malignancies) were further processed to enrich circulating tumour cells using density gradient centrifugation and/or the microfluidic Parsortix™ system (Angle plc., UK). In addition, the lung cancer samples were also used for CTC cultivation experiments.

Detection and characterization of p53 aggregates in high-grade-serous ovarian cancer

Collaborators: Heinzl N, Maritschnegg E, Zeillinger R, Ober-mayr E, Schuster E, Holzer B, Reinthaller A, Horvat R, Heinze G, Wallisch C and collaborators from the OVCAD study and from microsens Biotechnologies (London)

Prions are known to play a key role in the development and progression of different serious diseases as, for example, Alzheimer’s disease or Mad Cow disease. Over the last years, evidence has been generated that cancer belongs to this group of diseases as well. Only recently it was described that the tumour-suppressor protein p53 also associates into prion-like aggregates. Our research group has developed a simple, robust method for a high-throughput, quantitative detection of p53 aggregates. In a preliminary study, we were able to show that p53 prions are an independent prognostic marker for survival of high-grade serous ovarian cancer pa-tients. To assess the clinical relevance of prion-like p53 in HGSOC we divided our patient cohort in three groups: nega-tive samples, samples with a moderate p53 aggregation level and patients with extensive p53 aggregation. A multivariable Cox regression analysis was performed, considering other prognostic factors which are significantly associated with overall survival in patients with HGSOC (age, FIGO stage and presence of a residual tumour mass). This analysis showed a superiority of the group with extensive p53 aggregation in overall survival and in progression-free survival as compared to the patients with negative to moderate p53-aggregation levels (P values 0.025 and 0.011). A similar correlation with progression-free survival was observed in the three groups (P values 0.030 and 0.008). Interestingly, a trend towards higher level of homologous recombination deficiency was observed in the group with extensive aggregation. Further-more, this group had a significantly higher Ki67 index com-pared to patients with moderate p53 aggregation (P value 0.033), which could be a gain-of-function effect of mutant aggregated p53. The higher Ki67 proliferation index and the trend towards higher genomic instability in patients with extensive p53 aggregation indicate that these tumours have an increased likelihood to responds to platinum-containing therapies. In conclusion, we have demonstrated the high potential of p53 aggregation as a biomarker for patients’ sur-vival, suggesting that classification of patients based on the quantity of aggregated p53 could guide therapy decisions.


Figure 8: Left: Progression-free survival based on amount of p53 aggre-gates. Extensive aggregation resulted in a prolonged progression-free survival (HR 4.12, 95% CI 1.15-14.777, p=0.030 and HR 5.46, 95% CI 1.57-18.96 p=0.008). Right: Overall survival based on amount of p53 aggregates. Again, extensive aggregation resulted in a significant longer survival (HR 4.37, 95% CI 1.20-15.95, p=0.025 and HR 4.85, 95% CI 1.44-16.39, p=0.011)

Analysis of p53 aggregates in breast cancer

Collaborators: Kalina J, Heinzl N, Zeillinger R, Budczies J*, Villegas S*, Koch I*, Denkert C* (* Charité Berlin)

Worldwide, breast cancer (BC) is the leading type of cancer in women. One in 8 women will develop BC over the course of her lifetime. Although the disease is curable at an early stage, about 50% of the patients are diagnosed with a stage II or III tumour and are potential candidates for a systemic therapy. When it comes to selecting patients, who are likely to respond to a systemic therapy, biomarkers play an essential role. We evaluated the presence of p53 prions in 20 BC cell lines using our newly established p53-Seprion-ELISA. As can be seen in Figure 8a, cell lines carrying a TP53 missense mutation gave a p53 prion specific signal in the ELISA. In contrast, no signal was detected in cell lines carrying a TP53 nonsense mutation, a deletion or an insertion. Further, no signal was detected in wild-type, except the HBL-100 cell line. This cell line was thought to originate from a casein-producing breast cell line. However, its female origin has been disproved due to the presence of a Y chromosome. HBL-100 cells contain two copies of the SV40 genome. The SV40 large T anti-gen inactivates p53 by binding it, which could explain why these cells gave a strong signal in the ELISA measurement.Furthermore, we investigated the aggregation propensity in fresh-frozen tumour tissue collected in the METAcan-cer study (Charité Berlin). Interestingly, also wild-type samples formed p53 aggregates. In total, 71 samples were analysed, out of which 9 of 18 mutated samples, and 10 of 53 wild-type samples tested positive in the p53-Seprion-ELISA (Figure 8b). In conclusion, we showed that with our established ELISA we can quantitatively measure the prevalence of p53 prions not only, as previously demonstrat-ed, in ovarian cancer, but also in BC. The potential of p53 ag-gregation as a biomarker in BC is currently under evaluation.

Figure 8a: p53−Seprion-ELISA analysis of 19 breast cancer cell lines and the HBL-100 cell line. A missense mutation resulted in a p53-aggregate specific signal, whereas wild-type and nonsense-mutated cell lines showed no aggregate formation, with the exception of the cell line HBL-100. Data represent

Figure 8b: p53-Seprion-ELISA analysis of 71 fresh-frozen tumour tissues of breast cancer patients. Data represent

absorbance____________ x 1000. total protein

absorbance____________ x 1000. total protein


ONCOGENOMICS GROUP

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

Mag. Dr. Martin Schreiber, ao. Univ.-Prof., M.Sc.Heidi Miedl (50%), BMABianca Dietrich, B.Sc. (März-November), Master’s StudentinJürgen Lebhard, B.Sc. (Jänner-Oktober), Master’s StudentDr. Aneesa Sultan (Juli-September), Research Fellow (ÖAD)

PROJEKTE 2018

Assoziation der Polymorphismen SNP285 und SNP309 im MDM2 Gen mit dem Brustkrebsrisiko und dem ErkrankungsalterEs wird angenommen, dass die varianten Allele von SNP309 (G) und SNP285 (C) gegensätzliche Effekte auf die Bind-ung des Transkriptionsfaktors SP1 an den Promoter von MDM2, auf die Expression von MDM2 und den Spiegel von p53, sowie auf das Krebsrisiko, das Erkrankungsalter und die Prognose haben. Wir haben SNP309 und SNP285 in 406 Brustkrebspatientinnen und 254 Frauen ohne maligne Erkrankungen analysiert. Der variante Genotyp GG von

SNP309 war mit einem erhöhten Brustkrebsrisiko in p53-negativen (OR, 1,82; 95% CI, 1,09-3,03; p<0.05), jedoch nicht in p53-positiven oder unselektierten Patientinnen assoziiert. Hingegen erkrankten Patientinnen mit dem Genotyp TT von SNP309 im Durchschnitt früher an Brustkrebs (TT, 57.0 ±12.9; TG, 58.6±13.9; GG, 59.7±15.0 Jahre; p<0,05). TP53 war in 31% der Patientinnen mit dem SNP309TT Genotyp, 26% jener mit dem TG Genotyp, aber nur 13% jener mit dem GG Genotyp mutiert (p<0,05). Dies deutet auf einen höheren Selektionsdruck in Tumoren mit dem SNP309TT Genotyp hin, TP53 durch Mutation zu inaktivieren. Darüber hinaus wiesen Patientinnen mit dem Genotyp TT ein verkürztes Metastasen-freies Überleben in der multivariablen Analyse auf. Die Stärke der beobachteten Assoziationen von SNP309 veränderte sich kaum, wenn die (seltenen) Träger des C-Al-lels von SNP285 bei den Analysen weggelassen wurden. Dies spricht gegen die Hypothese, dass SNP285 antagonistische Effekte gegenüber SNP309 hat. Das variante C-Allel von SNP285 selbst war – nicht signifikant – mit einem erhöhten Brustkrebsrisiko und einem verkürzten Krankheits-freien und Metastasen-freien Überleben assoziiert. Da dies mit der ver-muteten molekularen Funktion der Allele von SNP285 nicht im Einklang steht, vermuten wir dass es sich um sekundäre


„bystander“-Effekte der vollständigen genetischen Kopplung von SNP285 mit SNP309 handelt (die beiden SNPs sind nur 24 Basenpaare voneinander entfernt). Aus unseren Ergebnis-sen sowie publizierten Vorstudien leiten wir die Hypothese ab, dass das SNP309 G-Allel die p53-Stressantwort dämpft und dadurch zu einem erhöhten (Brust-)Krebsrisiko, aber auch zu einer niedrigeren Mutationsrate von TP53 und in Folge dessen zu einem höheren Erkrankungsalter und einer guten Prognose führt (Abbildung 1).

Abbildung 1: Modell der Auswirkungen von SNP309 und SNP285. SNP309T führt zu einer schwächeren Expression von MDM2 und damit zu höheren p53-Spiegeln. Diese reduzieren einerseits das Brustkrebsrisiko, erhöhen aber andererseits den Selektionsdruck, TP53 zu mutieren. Diese erhöhte TP53 Mutationsrate führt dann zu einem jüngeren Erkrankung-salter und einer schlechten Prognose. Bei SNP309G ist es genau umgekehrt. SNP285C tritt nie gemeinsam mit SNP309T auf. Es wurde ver-mutet, dass SNP285C die Effekte von SNP309G konterkariert, allerdings ergab unsere Studie keine Hinweise auf diesen antagonistischen Effekt.

Die IL-34 Expression ist mit der Prognose des Mammakarzinoms assoziiert und unterscheidet sich zwischen den molekularen Subtypen

Interleukin-34 (IL-34) ist neben CSF-1 ein Ligand des CSF-1 Rezeptors, und hat auch noch zwei weitere Rezeptoren, nämlich PTPRZ1 und Syndecan-1. IL-34 spielt bei der ange-borenen Immunantwort, Entzündungsprozessen und Krebs eine wichtige Rolle. Wir haben in Kooperation mit Prof. Diet-mar Abraham et al. die bislang noch kaum erforschte Rolle des erst 2008 entdeckten IL-34 beim Mammakarzinom untersucht. Unser Beitrag zu diesem Kooperationsprojekt war die Analyse der Expression von IL-34 in Brustkrebs-Zelllinien und Brusttumoren mittels quantitativer real-time RT-PCR, und die Korrelation dieser Expression mit wichtigen klinischen und histopathologischen Charakteristika, den molekularen Subtypen des Mammakarzinoms, und mit dem Gesamtüberleben, krankheitsfreien Überleben, und Metasta-sen-freien Überleben. Eine hohe IL-34 Expression war über-raschenderweise mit einer guten Prognose assoziiert, und zwar im Gesamtkollektiv sowie in den molekularen Subtypen „Luminal A/B“ und „HER2 type“ (Abbildung 2). Bei Patien-tinnen mit dem basalen Subtyp war es jedoch umgekehrt, hier war eine hohe IL-34 Expression mit einer schlechten

Prognose assoziiert. Weiters wurde eine signifikant erhöhte Expression von IL-34 in Patientinnen mit einem Alter von 55 Jahren (p = 0,002) und in post-menopausalen Patientinnen (p = 0,007) ermittelt. Hingegen fanden wir keine Assoziation mit der Tumorgröße, dem Stadium, dem Tumorgrad, dem Lymphknotenstatus sowie dem Status von p53, HER2, dem Östrogenrezeptor und dem Progesteronrezeptor. Bei der Analyse der molekularen Subtypen zeigte sich, dass sowohl Tumore als auch Zelllinien mit dem Subtyp „Luminal B“ eine deutlich niedrigere Expression aufwiesen als die anderen 4 Subtypen (p = 0.005 bei Tumoren, ANOVA; Abbildung 2). Die Expression von IL-34 war in Brustkrebs-Zelllinien deutlich niedriger als in Tumorgewebe und normalem Brustgewebe, basierend auf den ermittelten Ct (cycle threshold) Werten in der real-time qRT-PCR. Dieser durchschnittliche Ct-Wert betrug bei Zellinien 30,94, bei Tumorgewebe 26,35 und bei Normalgewebe 26,26 (je niedriger der Ct-Wert, desto höher die Expression). Dies deutet darauf hin dass im Gewe-beverband die Mammmaepithel-Zellen nur einen Teil der Expression ausmachen, der Rest kommt vermutlich von den infiltrierenden Immunzellen. Weiters fanden wir heraus, dass die Expression von IL-34 sich signifikant zwischen den molekularen Subtypen des Mammakarzinoms unterscheidet, und dass dies auch einen Einfluss auf die Prognose hat (Abbildung 2).

Abbildung 2: Analyse von IL-34 beim Mammakarzinom. IL-34 ist in Brust-tumoren (a) und Brustkrebs-Zelllinien (b) vom Molekularen Subtyp Lumi-nal B (LumB) deutlich niedriger exprimiert als in den anderen Subtypen. Patientinnen mit niedriger IL-34 Expression haben ein signifikant kürzeres Gesamtüberleben (c) und krankheitsfreies Überleben (d). Molekulare Subtypen: Basal, HER2-Typ (HER2), Luminal A (LumA), Luminal B (LumB), Normal-like (a) bzw. untransformierte Zelllinien (b; Normal).



PREDICTIVE ONCOLOGY GROUP

L E I T U N G :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 :

Frederik Holst, PhD (Molekularbiologe, Karenzvertretung)Marie-Theres Kastner (Biomedizinische Analytikerin)Daniela Muhr (Biomedizinische Analytikerin)MMag. Christine Rappaport-Fürhauser (Dissertantin, derzeit karenziert)Yen Yen Tan, PhD (Epidemiologin)Sigrid Weingartshofer, MSc (Biomedizinische Analytikerin, Dissertantin)

Obere Reihe v.l.n.r.: YY. Tan, F. Holst

Untere Reihe v.l.n.r.: MT. Kastner, C. Rappaport-Fürhauser, D. Muhr, S. Weingartshofer

PROJEKTE 2018

Endometrial cancer risk and survival by tumor MMR statusChristina M Nagle, Tracy A O’Mara, Yen Tan, Daniel D Bu-chanan, Andreas Obermair, Penny Blomfield, Michael A Quinn, Penelope M Webb, Amanda B Spurdle, Australian Endometrial Cancer Study GroupJ Gynecol Oncol. 2018 May;29(3):e39. doi: 10.3802/jgo.2018.29.e39.

The risk of developing endometrial cancer (EC) and/or sur-vival following a diagnosis of EC might differ by tumor DNA mismatch repair (MMR) status. We assessed the association between tumor MMR status (classified as MMR-proficient, somatic MMR-deficient, germline MMR-deficient) and the risk of developing EC and survival following a diagnosis of EC. We analyzed data from women who participated in the Australian National Endometrial Cancer Study (ANECS) conducted between 2005 and 2007. Risk analyses (698 cases/691 population controls) utilized sociodemographic

and lifestyle information obtained from telephone interviews at recruitment. For survival analyses (728 cases), patients’ clinical data was abstracted from medical records, and survival data were obtained via linkage with the Australian National Death Index. We used logistic regression analysis to evaluate the associations between tumor MMR status and EC risk, and proportional hazards models to perform survival analyses with adjustment of known prognostic fac-tors. Established risk factors for EC did not differ significantly by tumor MMR status. In analyses including all EC subtypes, overall and EC-specific survival did not differ by tumor MMR status. Among women with the most common endome-trioid subtype, EC-specific survival was worse for women with somatic MMR-deficient EC compared to women with MMR-proficient EC (hazard ratio [HR]=2.18; 95% confidence interval [CI]=1.19–4.01). The risk of EC is not associated with MMR status. Accurate separation of germline from somatic causes of MMR deficiency suggests that patients with endo-metrioid subtype somatic MMR-deficient tumors have poorer EC-specific survival than those with MMR-proficient tumors, after accounting for other prognostic factors.

Differential Claudin 3 and EGFR expression predicts BRCA1 mutation in triple-negative breast cancerDanzinger S, Tan YY, Rudas M, Kastner M, Weingartshofer S, Muhr D, kConFab, Singer CF Cancer Invest. 2018;36(7):378-388. doi: 10.1080/07357907.2018.1499934.

BRCA-1 mutation-associated triple-negative breast cancer (TNBC) has been hypothesized to exhibit a phenotype that is distinct from non-mutation carriers, since it usually arises at a more differentiated developmental stage. Sporadic TNBC, on the other hand, are believed to be more hetergenous in their developmental origin, and can comprise stem cell-like properties such as a “claudin-low” phenotypes, as well as “basal-like” features. We have analyzed the expression of differentiation markers in BRCA-mutated and in wild type (WT) TNBC in order to find out whether BRCA-1-associated TNBC can be identified using these markers.We have analyzed immunohistochemically detected cy-tokeratins 5 and 14, epidermal growth factor receptor (EGFR), claudin (CLDN) 3, 4, and 7, and E-cadherin in 57 TNBC in order to characterize the developmental stage of these tumors. Of these, 32 tumor samples were obtained from BRCA1 mutation carriers, 8 from BRCA2 mutation car-riers, while 17 tumor samples came from women with BRCA WT genotype. For all tumors, histopathological data of the different subgroups were evaluated, and logistic regression was conducted to investigate tumorbiological parameters associated with BRCA status. EGFR expression was signifi-cantly more common in WT tumors compared to BRCA1-related breast cancer (BC) (53% versus 16%, p=0.004), but was not significantly different from BRCA2 mutation carriers (13%, p=0.089). Conversely, CLDN3-positivity was more common in BRCA1 carriers than in WT controls (66% versus


6%, p<0.001), while no difference was seen in comparison to BRCA2 (25%, p=0.053)). These findings were held in a multivariate regression model, where BRCA1-associated BC expressed CLDN3 22 times more often than tumors of non BRCA1 mutation carriers (BRCA2 and WT tumors) (p=0.006). Interestingly, CLDN3 was positively correlated with E-cadherin (r=0.332, p=0.013), and CK14 was positively correlated with CK5 (r=0.436, p=0.001). The distribution of the phenotypic subtypes is demonstrated in Figure 1. Tumors were characterized by basal-like and claudin-low immunohis-tochemistry BRCA1-associated TNBC had poorer claudin-low features (3%) compared to non BRCA1 carriers (BRCA2 and WT tumors, 12%) (p=0.23). Immunohistochemical staining of CLDN3, CLDN4, and E-cadherin in triple-negative tumors is shown in Figure 2. Finally, EGFR and CLDN3 expression was able to predict the presence of BRCA1 mutation with a farely high sensitivity and specificity (area under curve (AUC) 0.802, p<0.001) (Figure 3).

Figure 1. Distribution of the phenotypic subtypes.

A

C

E

B

D

Figure 2. Expression of Claudin (CLDN) 3 (A) and CLDN 4 (B) in BRCA1-

mutated breast cancer (BC). Positive CLDN 4 in the BRCA2 (C) and the

wild type group (D, E).

Figure 3: ROC analysis of EGFR and CLDN3 expression.


Prognostic and predictive value of ESR1 amplification in postmenopausal receptor-positive women with early breast cancer treated with TAM vs sequential TAM and AIHolst F, Kastner MT, Weingartshofer S, Rudas M, Filipits M, Jahn S, Singer CF

Expression of estrogen receptor alpha (ERα) is predictive for endocrine therapy response and an important prognos-tic factor in breast cancer. Overexpression of ERα can be caused by estrogen receptor 1 (ESR1) gene amplification and was originally reported to be a frequent event associated with a significantly longer survival for ER-positive women treated with adjuvant tamoxifen monotherapy, which was however questioned by subsequent studies. Recently, ESR1 amplification has also been suggested to predict responsive-ness 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. 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 this end, we will utilize an archived subset of breast cancer samples from more than 3200 postmenopausal women who have been randomized 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 pre-menopausal 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 premenopau-sal women. There is growing evidence that ESR1 gain or amplification is a fairly frequent event in breast cancer, although it is difficult to detect with current analysis and threshold techniques, which were initially established for ERBB2 measurement.

Examples of ESR1 status in breast carcinomas as determined by FISH. FISH signals of ESR1 (green) and centromere 6 (red) are shown within tumor cell nuclei (blue): A) ESR1 amplification with approximately 4-10 and 1-2 visible copies of ESR1 and centromere 6 per nucleus respectively. B) Low level ESR1 amplification (gain) with approximately 1-6 and 1-3 visible copies of ESR1 (and centromere 6 per nucleus respectively. C) Normal copy number with 1-2 visible copies of both ESR1 and centro-mere 6.

A

B

C


Interplay between the antitumor effects of nitrogen-containing bisphosphonates (N-BPs) and the endocrine environment in breast cancerWeingartshofer S, Bilban M, Kastner MT, Grunt TW, Tan YY, Singer CF

The nitrogen-containing bisphosphonate (N-BP) zoledronic acid (ZO) has been shown in several studies to influence tumor metastasis by inhibiting angiogenesis, in the field of immunology by targeting directly B cells inhibiting of cell invasion and also inhibiting tumor cell adhesion to extra-cellular bone matrix. Clinical data from the Austrian Breast and Colorectal Cancer Study Group Trial 12 (ABCSG-12) and Zometa-Femara Adjuvant Synergy Trial (ZO-FAST) tri-als suggest an improved disease-free survival (DFS) after ZO treatment in adjuvant breast cancer. Results of subset analyses of Adjuvant Zoledronic Acid to Reduce Recurrence (AZURE) showed a significantly improved overall survival in women after ZOL treatment that were more than five years postmenopausal or older than 60 years at baseline. Similarly, subset analyses of the ABCSG-12 trial data dem-onstrate greater benefits from ZO treatment in patients who theoretically would have achieved more complete ovarian suppression. These observations, together with the AZURE postmenopausal data, suggest that the endocrine environ-ment may affect the potential anticancer activity of ZO.

We analyzed:The influence of E2 and several other hormones on the anti-tumor activity of ZOL on several (hormone sensitive and insensitive) breast cancer cells using an array of in vitro cell viability and toxicity assays.Using whole-genome transcriptome assays combined with bioinformatics analysis to identify the molecular mechanism (genomic, non-genomic, mitochondrial) and signaling pathways that protect hormone-sensitive breast cancer cells from ZOL induced cell death.Find out if ZOL reduces protein farnesylation in the breast cancer cells and if so, determine the affected (signaling) proteins.Finally, based on the genes identified through my transcriptome screen I will study the top-regulated genes using gain- as well as loss of function cell models in combination with pharmacological interventions to verify their involvement in the molecular mechansims that protect hormone-sensitive breast cancer cells from ZOL induced cell death.

Figure 1. Venn diagram showing the distribution of genes regulated by ZO and E2 in MCF-7 cells. Comparison of differential gene expression between untreated (purple), treated with E2 (red) and ZO and E2 (green) MCF-7 cells.

1. Magic marker

2. MCF-7 0nM E2/0nM ZO

3. MCF-7 0nM E2/6.25µM ZO


5. MCF-7 1nM E2/0nM ZO



8. MCF-7 10nM E2/0µM ZO

9. MCF-7 10nM E2/6.25µM ZO

10. MCF-7 10nM E2/62.5µM ZO

Figure 2. Compensation of the mevalonate pathway. Western blot analysis showing the amount of FDPS in untreated and treated MCF-7 cells.


Figure 3. Expression of E2-responsive Genes in MCF-7 cells. Two-dimen-sional hierarchical clustering of E2 responsive genes in MCF-7 untreated or E2 treated. Each row represents a single gene. Red, genes with high expression levels, blue, genes with low expression levels The similarities in the expression pattern among sublines represented as a “condition tree” on the side of the matrix.


Figure 4. Sensitivity and specificity. Sensitivity and specificity of the combination of five biomarkers (CA125, MIF, Leptin, HE4, OPN) in differentiating bet-ween healthy women and OC patients (A), healthy wildtype women and wildtype OC patients (B), healthy BRCA1 mutation carriers and BRCA1 mutation carriers with OC (C), healthy wildtype women and healthy BRCA1 mutation carriers (D) and wildtype OC patients and BRCA1 mutation carriers with OC (E).


RAD51C in österreichischen HochrisikofamilienRappaport-Fürhauser C, Muhr D, Weingartshofer S, Wein-häusel A, Singer C

Bisherige Studien zeigten, dass Keimbahnmutationen im Reparaturgen RAD51C zu einem erhöhten Risiko für Brust- und Eierstockkrebs führen In dieser Studie soll die Rolle von RAD51C-Mutationen in österreichischen Brust- und Eierstockkrebsfamilien untersucht werden. Dafür wurde RAD51C in 907 Hochrisikopersonen (aus 773 Familien), die keine Mutation in BRCA1 oder BRCA2 tragen, mittels Kapillarsequenzierung und Multiplex ligation-dependent probe amplification (MLPA) untersucht. Zudem wurden 101 Kontrollproben von gesunden Personen sowie von 159 BRCA1- und 76 BRCA2-MutationsträgerInnen analysiert. Die Pathogenität der gefundenen Veränderungen wurde mit in silico- und in vitro-Methoden beurteilt. Insgesamt wurden 17 verschiedene Veränderungen gefunden. Zwei davon, c.224_225insA in einer Familie mit Brust- und Ei-erstockkrebsfällen und c.905-2delA in einer Familie mit Eierstockkrebsfällen, wurden als pathogen eingestuft. Eine dritte Veränderung, c.428A>G in einer Familie mit Brust-krebsfällen, ist wahrscheinlich pathogen. In keiner Probe konnte eine copy number variation nachgewiesen werden. Die Häufigkeit von RAD51C-Mutationen in der österreichi-schen Hochrisikopopulation beträgt somit 0,2 %, was zeigt, dass RAD51C-Mutationen sogar in der Hochrisikopopulation selten sind.

CIMBA (The Consortium of Investigators of Modifiers of BRCA1/2)

C O O R D I N A T I O N P A R T N E R S :Georgia Chenevix-Trench PhD, NHMRC Senior Principal Research Fellow, The Queensland Institute of Medical Research, AustraliaAntonis Antoniou, CR-UK Genetic Epidemiology Unit, Strangeways Research Laboratory, University of Cam-bridge, Cambridge, UKDouglas Easton, CR-UK Genetic Epidemiology Unit, Department of Public Health and Primary Care, Univer-sity of Cambridge, Cambridge, UK

C O O P E R A T I O N P A R T N E R S :Singer Christian, Department of Obstetrics and Gyne-cology, Center for Familial Breast- and Ovarian Cancer, Medical University of Vienna, Austria

O T H E R C O O P E R A T I O N P A R T N E R S :http://apps.ccge.medschl.cam.ac.uk/consortia/cimba//groups/groups.html

Collaborators: Muhr, Weingartshofer, Rappaport, Tan

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 car-riers. 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 cou-pled 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:The prevalence and spectrum of germline mutations in BRCA1 and BRCA2 have been reported in single popu-lations, with the majority of reports focused on White in Europe and North America. The Consortium of Investigators of Modifiers of BRCA1/2 (CIMBA) has assembled data on 18,435 families with BRCA1 mutations and 11,351 families with BRCA2 mutations ascertained from 69 centers in 49 countries on six continents. This study comprehensively describes the characteristics of the 1,650 unique BRCA1 and 1,731 unique BRCA2 deleterious (disease-associated) muta-tions identified in the CIMBA database. We observed sub-stantial variation in mutation type and frequency by geo-


graphical region and race/ethnicity. In addition to known founder mutations, mutations of relatively high frequency were identified in specific racial/ethnic or geographic groups that may reflect founder mutations and which could be used in targeted (panel) first pass genotyping for specific popu-lations. Knowledge of the population-specific mutational spectrum in BRCA1 and BRCA2 could inform efficient strate-gies for genetic testing and may justify a more broad-based oncogenetic testing in some populations.

Hum Mutat. 2018 May;39(5):593-620. doi: 10.1002/humu.23406.

BRCA1/2 mutations confer high lifetime risk of breast can-cer, although other factors may modify this risk. Whether height or body mass index (BMI) modifies breast cancer risk in BRCA1/2 mutation carriers remains unclear. We used Mendelian randomization approaches to evaluate the as-sociation of height and BMI on breast cancer risk, using data from the Consortium of Investigators of Modifiers of BRCA1/2 with 14 676 BRCA1 and 7912 BRCA2 mutation carriers, including 11 451 cases of breast cancer. We created a height genetic score using 586 height-associated variants and a BMI genetic score using 93 BMI-associated variants. We examined both observed and genetically determined height and BMI with breast cancer risk using weighted Cox models. All statistical tests were two-sided. Observed height was positively associated with breast cancer risk (HR = 1.09 per 10 cm increase, 95% confidence interval [CI] = 1.0 to 1.17; P = 1.17). Height genetic score was positively associated with breast cancer, although this was not statistically significant (per 10 cm increase in genetically predicted height, HR = 1.04, 95% CI = 0.93 to 1.17; P = .47). Observed BMI was inversely associated with breast cancer risk (per 5 kg/m2 increase, HR = 0.94, 95% CI = 0.90 to 0.98; P = .007). BMI genetic score was also inversely associated with breast cancer risk (per 5 kg/m2 increase in genetically predicted BMI, HR = 0.87, 95% CI = 0.76 to 0.98; P = .02). BMI was primarily associated with premenopausal breast cancer.

CONCLUSION:Height is associated with overall breast cancer and BMI is associated with premenopausal breast cancer in BRCA1/2 mutation carriers. Incorporating height and BMI, particularly genetic score, into risk assessment may improve cancer management.J Natl Cancer Inst. 2018 Oct 12. doi: 10.1093/jnci/djy132

Large-scale genome-wide association studies (GWAS) have identified approximately 35 loci associated with epithelial ovarian cancer (EOC) risk. The majority of GWAS-identified disease susceptibility variants are located in noncoding re-gions, and causal genes underlying these associations re-main largely unknown. Here, we performed a transcriptome-wide association study to search for novel genetic loci and plausible causal genes at known GWAS loci. We used RNA

sequencing data (68 normal ovarian tissue samples from 68 individuals and 6,124 cross-tissue samples from 369 individuals) and high-density genotyping data from European descendants of the Genotype-Tissue Expression (GTEx V6) project to build ovarian and cross-tissue models of geneti-cally regulated expression using elastic net methods. We evaluated 17,121 genes for their cis-predicted gene expres-sion in relation to EOC risk using summary statistics data from GWAS of 97,898 women, including 29,396 EOC cases. With a Bonferroni-corrected significance level of P < 2.2 × 10−6, we identified 35 genes, including FZD4 at 11q14.2 (Z = 5.08, P = 3.83 × 10−7, the cross-tissue model; 1 Mb away from any GWAS-identified EOC risk variant), a potential novel locus for EOC risk. All other 34 significantly associated genes were located within 1 Mb of known GWAS-identified loci, including 23 genes at 6 loci not previously linked to EOC risk. Upon conditioning on nearby known EOC GWAS-identified variants, the associations for 31 genes disappeared and three genes remained (P < 1.47 × 10−3). These data identify one novel locus (FZD4) and 34 genes at 13 known EOC risk loci associated with EOC risk, providing new insights into EOC carcinogenesis.

Significance: Transcriptomic analysis of a large cohort confirms earlier GWAS loci and reveals FZD4 as a novel locus associated with EOC risk.Cancer Res. 2018 Sep 15;78(18):5419-5430. doi: 10.1158/0008-5472.CAN- 18-0951.

Familial Breast Cancer Research Unit

Risk Factor Analysis of Hereditary Breast and Ovarian Cancer

Coordinator:Dr. Steven Narod, Toronto, Canada

Cooperation partners:Singer Christian, Department of Obstetrics and Gynecolo-gy, Center for Familial Breast- and Ovarian Cancer, Medical University of Vienna, Austria and others

Collaborators: Muhr, Weingartshofer, Tan

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 here-ditary 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 studies: For women at high risk of developing ovarian cancer, it is important to provide an accurate recommendation for the optimal age for preventive surgery in order to maximize the preventative effect while delaying symptoms associated with early surgical menopause. The goal of the current study was to estimate age-specific incidence rates of ovarian cancer among women with a BRCA1 or BRCA2 mutation.From our international registry, we identified 5689 women with no previous diagnosis of ovarian or fallopian tube can-cer or preventive oophorectomy. Women were followed from the date of completion of the baseline questionnaire until either a diagnosis of ovarian or fallopian tube cancer, prophylactic oophorectomy, death or last follow-up. The annual and cumulative incidence rates of ovarian cancer were estimated.Over a mean follow-up period of 4.7 years (ranges 0–22.6), 195 incident ovarian or fallopian tube cancers were diagno-sed (169 [86%] ovarian cancers, 22 [11%] fallopian tube can-cers and four [2%] cancers that involved both the ovaries and fallopian tubes). Of these, 45 (23%) cancers were diagnosed at preventive surgery (occult cancers). The cumulative risk of ovarian cancer to age 80 was 49% for BRCA1 and 21% for BRCA2 mutation carriers. The mean age at diagnosis was 51.3 years (ranges 33–84) among women with a BRCA1 mu-tation and 61.4 years (ranges 44–80) among women with a BRCA2 mutation.

ConclusionBased on a cumulative risk of 0.55% to age 35 for BRCA-1mutation carriers and of 0.56% to age 45 for BRCA2 mu-tation carriers, we recommend bilateral salpingo-oophorec-tomy before age 40, but ideally by age 35, for women with a BRCA1 mutation and by age 45 for those with a BRCA2 mu-tation to maximize prevention and to minimize adverse ef-fects.

Gynecol Oncol. 2018 Jul;150(1):85-91. doi: 10.1016/j.ygy-no.2018.05.011.

ENIGMA – Evidence-based Network for the Interpretation of Germline Mutant Alleles

Coordination partners:David E. Goldgar, University of UtahAmanda Spurdle, Queensland Institute for Medical ResearchFergus J. Couch, Mayo Clinichttp://www.enigmaconsortium.org/steering-committee.html

Cooperation partners:Singer Christian, Department of Obstetrics and Gynecolo-gy, Center for Familial Breast- and Ovarian Cancer, Medical University of Vienna, Austria and others

Collaborators: Muhr, Rappaport, Weingartshofer, Tan

ENIGMA is a consortium of investigators focused on deter-mining 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 faci-litate classification of variants through collaborative large-scale projects by sharing data and improving classification methods. To do this there are different working groups (WG), focusing on the development and maintenance of Databa-ses 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 likeli-hood 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

Coordinator:Rookus Matti, The Netherlands, The Netherlands Cancer Institute, Amsterdam

Partners:Andrieu Nadine, France, INSERM, ParisEaston Douglas, United Kingdom, Cambridge University, CambridgeJakubowska Anna, Poland, Pomeranian Medical University, SzczecinKast Karin, Germany, Universitatsklinikum Carl Gustav Carus, DresdenSinger Christian, Austria, Medical University of Vienna, Wien Van Gils Carla, The Netherlands, Universitity Medical Cen-ter Utrecht, Utrecht

Collaborators: Muhr D, Weingartshofer S, Tan Y


BRCA1/2 mutation carriers have high risks of early onset Breast Cancer (BC) and ovarian cancer (OvC), but age-spe-cific risks vary strongly between and among families. Cur-rently, 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 cur-rent 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 predic-tion 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.

4. This project is based on the International BRCA1/2 mutation Carrier Cohort Study, the largest available prospective BRCA1/2 cohort study (IBCCS).

Recent results:For BRCA1 and BRCA2 mutation carriers, the association between oral contraceptive preparation (OCP) use and bre-ast cancer (BC) risk is still unclear.Breast camcer risk associations were estimated from OCP data on 6030 BRCA1 and 3809 BRCA2 mutation carriers using age-dependent Cox regression, stratified by study and birth cohort. Prospective, left-truncated retrospective and full-cohort retrospective analyses were performed.For BRCA1 mutation carriers, OCP use was not associated with BC risk in prospective analyses (hazard ratio [HR] = 1.08, 95% confidence interval [CI] = 0.75 to 1.56), but in the left-truncated and full-cohort retrospective analyses, risks were increased by 26% (95% CI = 6% to 51%) and 39% (95% CI = 23% to 58%), respectively. For BRCA2 mutation carriers, OCP use was associated with BC risk in prospective analyses (HR = 1.75, 95% CI = 1.03 to 2.97), but retrospective analy-ses were inconsistent (left-truncated: HR = 1.06, 95% CI = 0.85 to 1.33; full cohort: HR = 1.52, 95% CI = 1.28 to 1.81). There was evidence of increasing risk with duration of use, especially before the first full-term pregnancy (BRCA1: both retrospective analyses, P <.001 and P =.001, respectively; BRCA2: full retrospective analysis, P =.002).

Prospective analyses did not show that past use of OCP is associated with an increased BC risk for BRCA1 mutation carriers in young middle-aged women (40–50 years). For BRCA2 mutation carriers, a causal association is also not

likely at those ages. Findings between retrospective and prospective analyses were inconsistent and could be due to survival bias or a true association for younger women who were underrepresented in the prospective cohort. Given the uncertain safety of long-term OCP use for BRCA1/2 mutati-on carriers, indications other than contraception should be avoided and nonhormonal contraceptive methods should be discussed.

JNCI Cancer Spectrum. 2018; Apr; Vol.2(2)1

Genomic profiles of matched primary breast tumors and metastases using next-generation sequencing Deutschmann C, Holst F, Bago-Horvath Z, Singer CF

As breast cancer mortality is caused by metastatic spread of the disease - analysis of the genetic landscape of metastatic lesions is of high scientific interest. However, comparative studies of primary breast tumors and corresponding meta-stases - applying next generation sequencing and additional advanced -omic technologies -transcriptomics, proteomics, epigenomics, etc. - are scarce.

Methods: In this study formalin-fixed paraffin embedded (FFPE) samples of matched pairs of primary breast tumors and metastases using next-generation sequencing will be analysed.

Objectives: The objective of this study is to define the con-cordance of the mutational profiles of primary breast can-cers and paired distant metastases and among metastases themselves, respectively. The studies´ results will give further insight in to which extend the metastatic tumor genome is derived from the primary tumor cells as opposed to being an independent tumor. In addition, consistently altered genes are sought in order to define metastasis-site specific alte-rations. Furthermore, the optimal tissue source (primary tumor versus metastatic lesion) for collection of biopsies will be defined.

Association of Cytokeratin 5 and Claudin 3 expression with BRCA1 and BRCA2 germline mutations in women with early breast cancer (submitted)Danzinger S, Tan YY, Rudas M, Kastner M, Weingartshofer S, Muhr D, kConFab, Singer CF

BackgroundIt is important to identify biomarkers to pre-screen women with early breast cancer (BC) for BRCA testing to improve early identification of mutation carriers. Thus, in this study, we investigated the protein expression of claudin (CLDN) 3, CLDN4, CLDN7, and E-cadherin. Moreover, we analyzed additional histopathological variables and their associations in familial BC.


MethodsImmunohistochemical analysis for CLDNs and E-cadherin was performed on 237 BC cases of three different subsets of BC tumors: 62 BRCA1-associated BC, 59 BRCA2associated BC, and 116 tumors from patients with BRCA wild type (WT) as controls. Histopathological data were also analyzed in the different subgroups. Logistic regression and receiver operation characteristic (ROC) curve were conducted to investigate factors associated with BRCA tumors.

ResultsExpression of CLDN3 positively correlated with BRCA-mu-tated BC. CLDN3 was expressed in 58% of BRCA1-mutated tumors compared to only 7% in BRCA2-mutated tumors (p<0.001) and 1% in WT tumors (p<0.001). CK5 and CK14 expression were also more likely to arise in BRCA1 tumors (44% and 16%, respectively) than in the control group (8%

and 4%) (p<0.001, p=0.012, respectively). We also found a significantly higher proportion of CK5+ among BRCA1 tumors (44%) in comparison with BRCA2-related BC (8%) (p<0.001). There was also a significant difference between both groups regarding CK14: positive expression in 16% and 5%, respectively (p=0.030). CK5 and CK14 did not differ between the BRCA2 group and the WT tumors significantly. In a multivariate regression model, expression of CK5 (Odds ratio (OR): 6.46; 95% confidence interval (CI): 1.52-27.43; p=0.011), and CLDN3 (OR: 200.48; 95% CI: 21.52-1867.61; p<0.001) were associated with BRCA1 mutation status.

ConclusionsOur data suggests that CLDN3, CK5, and CK14 in combi-nation with ER, PR and HER2 are associated with BRCA1 mutation status.

REPRODUCTIVE BIOLOGY GROUP

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

Ao. Univ. Prof. Mag. Dr. Martin Knöfler, PIAssoc. Prof. Mag. Dr. Jürgen Pollheimer, PIMag. Sandra Haider, PhD, BMA B.Sc. Peter Haslinger, BMAM.Sc. Viktoria Kunihs, BMAMag. Gudrun Meinhardt, BMA, PhD StudentinM.Sc. Leila Saleh, BMAM.Sc. MM. Sc. Sigrid Vondra, PhD StudentinDr. Mag. Karin Windsperger, PhD Studentin

D I P L O M S T U D I E R E N D E

Viktoria Greifeneder, Biomedizinische Analytik, MasterJasmin Rief, Biomedizinische Analytik, Master

Ä R Z T L I C H E M I T A R B E I T E R / I N N E N

Dr. Dana Muin, MSc

Z U S Ä T Z L I C H E M I T A R B E I T E R / I N N E N

Andreas Lackner, Studentischer Mitarbeiter


PROJEKTE 2018

Critical signalling pathways regulating trophoblast invasion of the human placenta

M I T A R B E I T E R I N N E N : Haider S, Velicky P, Saleh L, Pollheimer J, Knöfler M

KurzbeschreibungNotch signalling ist für die Aufrechterhaltung von Stamm-zellen, aber auch für Homöostase und Differenzierung von Geweben verantwortlich. Um Notch Signalling zu initiieren bedarf es eines direkten Zell-Zell Kontakts zwischen Notch Rezeptoren und den ebenfalls Membran-gebundenen Notch Liganden. In den letzten Jahren lag unser Fokus auf der Charakterisie-rung des Notch Signalweges in Zellen der Dezidua und Pla-zenta (FWF geförderten Projektes (P22587, abgeschlossen 2013; Otti et.al.2014; Velicky et al. 2014; Plessl et.al. 2015)

Weiterführende Studien konnten zeigen, dass in vivo Notch Rezeptor 1 (Notch1) ausschließlich in Vorläuferzellen der extravillösen Trophoblasten (EVT) und in spezifischen Re-gionen villöser Trophoblasten, die in weiterer Folge zu EVT differenzieren werden, zu finden ist. Unter Zuhilfenahme verschiedener Trophoblastmodellsysteme wie Gewebe Ex-plantate und primäre Zellkulturen, konnten in vitro Expe-rimente demonstrierten, dass Notch1 Gene unterdrückt, die für die Selbsterneuerung fusionierender Vorläuferzellen verantwortlich ist, während er in EVT-Vorläuferzellen Prolife-ration, Überleben und Differenzierung förderte (Abb. 1). Die

Ergebnisse identifizierten Notch1 als essentiellen Regulator in der Determination der EVT Differenzierung (Haider et. al., Proc Natl Acad Sci U S A. 2016).

Otti G, Saleh L, Fiala C, Pollheimer J, and Knöfler MNotch signalling controls differentiation of human decidual stromal cellsPLoS One. 2014 Nov 14;9(11)

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 differen-tiation 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 RBPJκ inhibits proliferation of human cytotrophoblasts and their differentiation into extravillous trophoblastsMolecular Human Reproduction. 2014, Aug;20(8):756-66.

Plessl K, Haider S, Fiala C, Pollheimer J, Knöfler MExpression pattern and function of Notch2 in different sub-types of first trimester cytotrophoblastPlacenta. 2015 Apr;36(4):365-71

Haider S, Meinhardt G, Saleh L, Fiala C, Pollheimer J, Knöfler M

Notch1 controls development of the extravillous trophoblast lineage in the human placentaProc Natl Acad Sci U S A. 2016 Nov 29;113(48):E7710-E7719

Abbildung 1: Funktion von Notch 1 während der Trophoblastendifferenzierung. Notch intrazelluläre Domäne1 (N1ICD) programmiert villöse Trophoblasten (vCTB) in Zellsäulentrophoblasten (CCT). Dabei werden die Stammzellmarker myc und VE-cadherin exprimiert. Zusätzlich verhindert N1ICD die Differen-zierung der CCT in extravillöse Trophoblasten (EVT) durch Aufrechterhaltung von Proliferation und Überleben dieser Zellen. N1ICD unterdrückt p63 (durch Erhöhung von IRF6, ein p63 Repressor) und TEAD4 wodurch sowohl Selbsterneuerung als auch Zellfusion der vCTBs inhibiert werden.


Transcriptional co-activators at the intersection of signalling pathways controlling extravillous trophoblast differentiation

Im Rahmen des vom FWF geförderten Projektes (APP-28417FW) studieren wir die Funktion der transkriptionellen Koaktivatoren Yap und Taz während der invasiven Differen-zierung des humanen Trophoblasten

M I T A R B E I T E R I N N E N : Meinhardt G, Kunihs V, Haider S, Saleh L, Knöfler M

KurzbeschreibungDie Bildung der humanen Plazenta und insbesondere der verschiedenen Subtypen des Trophoblasten ist für die em-bryonale Entwicklung und eine erfolgreiche Schwangerschaft unerlässlich. Invasive extravillöse Trophoblasten (EVTs) stam-men von verankerten Zotten der Plazenta ab und migrieren in das Gewebe und die Gefäße des mütterlichen Uterus. Die Zellen spielen eine zentrale Rolle bei der immunologischen Akzeptanz des Föten und beim Umbau der Spiralarteriolen der Mutter. Letztere werden in der Frühschwangerschaft durch EVTs verschlossen um einen vorzeitigen Blutfluss zur wachsenden Plazenta zu verhindern. Ab der zehnten Schwangerschaftswoche ist der EVT am Gefäßumbau be-teiligt, der durch Weitstellung der Arteriolen für eine adä-quate Versorgung des Föten mit Nährstoffen und Sauer-stoff sorgt. Fehler in diesem Prozess treten bei schweren Schwangerschaftskomplikationen, wie der Präeklampsie und der intrauterinen Wachstumsretardierung auf, die in der westlichen Welt die Hauptursachen der fötalen und mater-nalen Morbidität und Mortalität darstellen. Veränderungen im Wachstum und der Differenzierung des EVTs während der Frühschwangerschaft könnten hierfür ein Grund sein. Die molekularen Mechanismen, die die Entwicklung des invasiven Trophoblasten steuern sind jedoch größtenteils unbekannt.In diesem Projekt wollen wir die Rolle zweier transkripti-oneller Ko-aktivatoren, „Yes-associated protein“ (YAP) und „transcriptional co-activator with PDZ-binding motif“ (TAZ) studieren, die eine Schlüsselrolle in den Signalwegen „transforming growth factor-beta (TGF-beta)“, „Wingless (Wnt)“ und „Hippo“ spielen, die embryonale Entwicklung steuern. Es wurde gezeigt, dass YAP/TAZ die Translokation und Aktivität bestimmter Transkriptionsfaktoren wie den TGF-beta-abhängigen SMADs, den Wnt/beta-catenin-ab-hängigen T-Zell Faktoren (TCFs) sowie den Hippo-abhängigen TEAD Proteinen steuern. Betreffend Plazentaentwicklung ist bekannt, dass TEAD-YAP Komplexe für die Entwicklung des Trophektoderms, dem Vorläufer der Plazenta, essentiell sind. Unsere Daten zeigen, dass YAP/TEAD4 hauptsächlich in Kernen humaner villöser Trophoblasten (vCTB), nukle-äres TAZ jedoch vorwiegend in extravillösen Trophoblasten (EVT) zu finden ist. Wir schließen daraus, dass die Koakti-vatoren eine zentrale Rolle in Funktion und Differenzierung der Trophoblasten Subtypen spielen könnten. Um diese

Hypothese zu verifizieren, wurde YAP/TAZ mittels „small interfering RNAs“ sowie „CRISPR-Cas9 genomic editing“ in verschiedenen humanen Trophoblasten-Modellsystemen ausgeschaltet und mögliche Veränderungen in Differenzie-rung, Proliferation und Invasivität anhand von spezifischen Trophoblastenmarkern analysiert. Die Ergebnisse zeigten, dass nukleäres YAP die Zellproliferation von vCTBs fördert, ihre Differenzierung jedoch inhibiert. Im Gegensatz dazu war die EVT Differenzierung mit einer Induktion von nukleären TAZ, TEAD1 und 2 assoziiert.Außerdem wollen wir in diesen Zellen die YAP/TAZ-abhän-gige transkriptionelle Aktivität von SMADs, TCFs und TEAD Proteinen bestimmen, sowie mögliche Zielgene ermitteln. Erste Resultate bezüglich Expressionsmuster und Phos-phorylierungsstatus von SMAD2/3 in den verschiedenen Trophoblast-Subtypen konnten kürzlich veröffentlicht werden (Haider et al., 2017). Weiters ist geplant die Lokalisation und Expression von YAP/TAZ in verschiedenen Schwangerschaftserkrankungen wie Plazenta accreta/percreta, kompletten Molen sowie in Pla-zenten mit abnormaler Vaskularisierung zu messen. Mit diesem Projekt wollen wir neue Erkenntnisse in der EVT Differenzierung gewinnen, um das Verständnis der Entste-hung von Schwangerschaftserkrankungen mit defekter Pla-zentation zu verbessern.

Haider S, Kunihs V, Fiala C, Pollheimer J, Knöfler MExpression pattern and phosphorylation status of Smad2/3 in different subtypes of human first trimester trophoblastPlacenta. 2017 Sep;57:17-25

Cellular senescence in human placental trophoblastsIn diesem von der Österreichischen Nationalbank (AP-165170NB) und der Herzfelder´schen Stiftung geförderten Projekt untersuchen wir die Seneszenz im humanen Tro-phoblasten

M I T A R B E I T E R I N N E N : Velicky P, Plessl K, Vondra S, Knöfler M, Pollheimer J

KurzbeschreibungIm Verlauf dieser bereits abgelaufenen Studie konnten wir gemeinsam mit WissenschafterInnen der Universität Stan-ford und mit der St. Anna Kinderkrebsforschung erstmals wesentliche Zusammenhänge des Zell-Zyklus und der Al-terung in der menschlichen Plazenta entschlüsseln. Das zentrale Ergebnis der Studie: Der Zellteilungs-Zyklus der Trophoblasten in der Plazenta hört in der Schwangerschaft nach einer Verdoppelung des gesamten Genoms auf, um unkontrolliertes Wachstum zu vermeiden. Gleichzeitig las-sen unsere Studienergebnisse den Schluss zu, dass die zelluläre Seneszenz einen wichtigen Mechanismus darstellt, um eine unkontrollierte Zellteilung und –Wucherung des Trophoblasten zu unterdrücken. Interessanterweise konnten wir ein Ausbleiben dieses wichtigen Kontrollmechanismus bei


der so genannten Molenschwangerschaft feststellen. Diese gestationsbedingte Trophoblasterkrankung betrifft weltweit etwa eine Frau von 1.000. Dabei wird ein leeres Ei in der weiblichen Gebärmutter befruchtet, wodurch zwar eine Pla-zenta entsteht, sich aber kein Embryo bildet. Die Folge: Die Plazenta wuchert wild, die Trophoblasten altern nicht. Dafür werden sie explosionsartig polyploid, tragen hunderte Kopien ihres Genoms in sich und teilen sich völlig unkontrolliert. Die Ergebnisse dieser Studie tragen also einerseits zu einem ausführlicheren Verständnis der humanen Plazentation bei und könnten möglicherweise zu einer verbesserten, thera-peutischen Behandlung von Molschwangerschaften führen. Philipp Velicky, der Erstautor dieser Studie, ist mittlerweile am IST Austria (Institute of Science and Technology – Aus-tria) beschäftigt. Die Daten dieser Studie konnten heuer im Top Journal PlosGenetics publiziert werden (Velicky et. al., PLoS Genet. 2018 Oct).

Abbildung 2: Postuliertes Modell der Tetraploidisierung und Induktion von zellulärer Seneszenz im gesunden (A) und pathologischen (B) (CHM, complete hydatidiform mole) Verlauf der humanen EVT Differenzierung.

Velicky P, Meinhardt G, Plessl K, Vondra S, Weiss T, Haslinger P, Lendl T, Aumayr K, Mairhofer M, Zhu X, Schütz B, Hannibal RL, Lindau R, Weil B, Ernerudh J, Neesen J, Egger G, Mikula M, Röhrl C, Urban AE, Baker J, Knöfler M, Pollheimer J. Ge-nome amplification and cellular senescence are hallmarks of human placenta development. PLoS Genet. 2018 Oct 12;14(10):e1007698. doi: 10.1371/journal.pgen.1007698. eCollection 2018 Oct.

Humane Organoidsysteme aus dezidualen Geweben des ersten Schwangerschaftstrimesters

M I T A R B E I T E R I N N E N : Haider S, Knöfler M

KurzbeschreibungOrganoidsysteme entstehen aus isolierten Stammzellen, die sich in einer 3-dimensionalen Matrix zu einer Gewebestruktur

formieren. Der Vorteil gegenüber herkömmlichen 2D Zell-kultursystemen liegt in der Bewahrung der Strukturintegrität und somit der räumlichen Orientierung der Zellen zueinander. Bis vor Kurzem war die Kultivierung des endometrialen Drü-senepithels nicht möglich, wodurch die Erforschung patho-physiologischer Prozesse ausschließlich auf den stromalen Anteil dieses Gewebes begrenzt war. In unserem Projekt liegt der Fokus auf der Etablierung und Manipulation von Organoiden aus Deziduagewebe des ersten Trimesters (Abb. 3). Zunächst wollen wir die physiologischen Prozesse des humanen Menstruationszyklus durch Zugabe der weiblichen Schlüsselhormone Östrogen und Progesteron imitieren. Da-durch sollen die Phasen der Menstruation, die Proliferations- und Sekretionsphase, simuliert und deren Auswirkungen auf Wachstum und Differenzierung der endometrialen Drüsen genau studiert werden. Durch dieses Zellkultursystem kön-nen wir nun erstmals die Rolle der Endometriumsdrüsen bei Pathologien wie Infertilität, Endometriose und Endome-triumskarzinomen besser charakterisieren, um in weiterer Folge mögliche Behandlungen zielgerichtet zu etablieren.

Abbildung 3: Organoide aus dezidualen uterinen Drüsen. Das linke Bild zeigt eine Phasenkontrastaufnahme von 3-D Organoidkulturen endometri-aler Drüsen (20-fache Vergrößerung). Das rechte Bild zeigt eine Immunflu-oreszenzfärbung fixierter und in Paraffin eingebetteter Drüsenorganoide. Detektiert wurden Epithelzellen (Zytokeratin 7, grün) und nukleäre DNA

(DAPI, blau).

Etablierung von Trophoblast-Organoiden aus Plazentagewebe des ersten Schwangerschaftstrimesters

Im Rahmen des vom FWF geförderten Projektes (APP-31470FW) studieren wir die Differenzierung des Tropho-blasten in einem 3-D Modell.

M I T A R B E I T E R I N N E N : Haider S, Knöfler M

In Kooperation mit Dr. Paulina Latos, Zentrum für Anatomie und Zellbiologie, haben wir ein neues 3D „in vitro“ Modell der frühen humanen Plazenta entwickelt (Haider et al. 2018; Stem Cell Reports). Durch Optimierung der Zellkulturbedin-gungen ist es uns gelungen den Trophoblast-spezifischen Aufbau der Plazenta in der Petrischale zu rekapitulieren (Abb. 4)


Abbildung 4: Organoide aus Plazentagewebe. Das linke Bild zeigt eine Phasenkontrastaufnahme von humanen Organoiden aus der Plazenta des ersten Schwangerschaftstrimesters. Im rechten Bild sieht man eine Häma-toxylin-Eosinfärbung eines Gewebeschnittes von fixierten und in Paraffin eingebetteten Trophoblastorganoiden.

Anhand dieses Modellsystems konnten wir zeigen, dass der WNT-Signalweg eine duale Rolle in der Plazentaentwicklung spielt, nämlich sowohl in der Selbsterneuerung als auch in der Differenzierung der Trophoblasten (Abb. 5). Dieses Kultursystem ist erstmals pharmakologisch und genetisch manipulierbar und eröffnet völlig neue Möglichkeiten, phy-siologische und pathologische Vorgänge in der humanen Plazenta zu untersuchen. Dadurch rücken medizinische Behandlungsmöglichkeiten bei gefährlichen Schwanger-schaftserkrankungen in greifbare Nähe.

Abbildung 5: Organoide aus Plazentagewebe. Das linke Bild zeigt eine Phasenkontrastaufnahme von humanen Organoiden aus der Plazenta des ersten Schwangerschaftstrimesters unter Standardkonditionen (indu-ziertes Wnt Signalling, Erhaltung des Stammzellcharakters). Im rechten Bild sieht man Organoide ohne Wnt-Induktion, deren Trophoblasten zu extravillösen Trophoblasten differenzieren.

Haider S, Meinhardt G, Saleh L, Kunihs V, Gamperl M, Kaindl U, Ellinger A, Burkard TR, Fiala C, Pollheimer J, Mendjan S, Latos PA, Knöfler MSelf-Renewing Trophoblast Organoids Recapitulate the De-velopmental Program of the Early Human Placenta. Stem Cell Reports. 2018 Aug 14;11(2):537-551

DAO - EIN NEUER MARKER ZUR FRÜHERKENNUNG FÜR PRÄEKLAMPSIE

M I T A R B E I T E R I N N E N : Velicky P, Pollheimer J

KurzbeschreibungDas vom AOC1 Gen kodierte Enzym Diaminooxidase (DAO) katabolisiert Histamin und Polyamine wie Putreszin oder Spermidin. Im Humansystem, bei nicht-schwangeren Frauen und ebenso bei Männern, wird DAO gewebsspezi-fisch im Magen und Darm exprimiert, wo es zum Beispiel durch die Nahrung aufgenommenes Histamin abbaut. Die

höchste DAO Expression findet sich allerdings während der Schwangerschaft in der Plazenta und im Serum der Mutter. Es besteht die Annahme, dass das Enzym den Embryo vor einem Übermaß an Histamin, das zu Uteruskontraktionen und im schlimmsten Fall zum Tod des Foeten führen kann, schützt. Tatsächlich wurden erniedrigte Plasma DAO Spiegel mit Schwangerschaftserkrankungen wie Präeklampsie und Spontanaborten assoziiert (Maintz et al., 2008). In diesem Projekt studieren wir die DAO Expression und Regulation während der Schwangerschaft. Jahrzehntelang wurden de-ziduale Fibroblasten als DAO Quelle propagiert. Wir haben allerdings sogenannte extravillösen Trophoblasten (EVT), jene Plazentazellen, die in die maternale Dezidua invadieren, als alleinige DAO Produzenten identifiziert. Immunfluores-zenzfärbungen zeigten, dass speziell perivaskuläre EVT hohe DAO Expressionen aufweisen. Mittels eines, an der MUW entwickelten (Prof. Bernd Jilma und Dr. Thomas Böhm, Uni-versitätsklinik für Klinische Pharmakologie), ELISA konnten wir zeigen, dass die DAO Plasma Werte im ersten Trimester von gesunden Schwangeren signifikant ansteigen. In der Folge untersuchten wir Blutplasma-Proben von gesunden und an Präeklampsie erkrankten schwangeren Frauen (in Kollaboration mit Prof. Thomas F McElrath, Harvard Medical School). Interessanterweise stellte sich heraus, dass jene Patientinnen, die später an Preäklampsie („early-onset pree-clampsia“) erkrankt waren, schon um die zehnte Schwan-gerschaftswoche signifikant weniger DAO im Blut hatten. Es konnte also gezeigt werden, dass sich die Krankheit schon in der Frühschwangerschaft manifestiert, noch be-vor Symptome auftreten. Zusammenfassend zeigen unsere Resultate, dass das histaminabbauende Enzym DAO ein EVT-spezifischer Faktor ist, der sowohl an der feto-maternalen Schnittstelle, als auch in der maternalen Zirkulation agiert und möglicherweise ein interessanter Kandidat zur prädik-tiven Diagnose besonders schwerer Fälle der Preäklampsie („early-onset preeclampsia“) sein könnte.

Velicky P, Windsperger K, Petroczi K, Pils S, Reiter B, Weiss T, Vondra S, Ristl R, Dekan S, Fiala C, Cantonwine DE, McElrath TF, Jilma B, Knöfler M, Boehm T, Pollheimer JPregnancy-associated diamine oxidase originates from extravillous trophoblasts and is decreased in early-onset preeclampsiaSci Rep. 2018 Apr 20;8(1):6342

THE ROLE OF TISSUE-RESIDENT UTERINE MACROPHAGES IN HEALTHY AND PATHOLOGICAL PREGNANCIES

M I T A R B E I T E R I N N E N : Vondra S, Kunihs V, Knöfler M, Pollheimer J

KurzbeschreibungGewebsansässige Makrophagen (GaMφ) siedeln sich wäh-rend der Embryogenese an und erneuern sich unabhängig vom Knochenmark. Obwohl die Funktionalität von GaMφ


mit mehreren Erkrankungen assoziiert ist, weiß man sehr wenig über ihre physiologische Funktion. Tatsächlich weisen unsere Daten auf die Präsenz von uterinen GaMφ, sowohl vor als auch während der Schwangerschaft hin, da ein we-sentlicher Teil dieser Mφ proliferative Aktivität zeigt. Die Schwangerschaft ist durch eine leukozytäre Expansion im Uterus gekennzeichnet, der von Plazenta-stämmigen extra-villösen Trophoblasten (EVT) invadiert wird. Dementspre-chend postulieren wir eine funktionelle Verbindung zwischen EVTs und lokaler Mφ Expansion, da Orte mit erhöhter Mφ Proliferation mit der Präsenz von invasiven Trophoblasten koinzidieren. Zusammenfassend planen wir proliferative, uterine Mφs zu charakterisieren, EVT-spezifische Effekte auf deren Teilungsrate zu studieren und Veränderungen von Mφ Populationen idiopathischer Aborte zu analysieren.

Presence of High Endothelial Venules in Endometrium and Decidua: Association with Reduced Numbers of Myeloid Cells and Infiltrating T Cells in Recurrent Spontaneous Abortions

M I T A R B E I T E R I N N E N : Windsperger K, Vondra S, Sabine Dekan, Knöfler M, Pollheimer J

KurzbeschreibungDie Etablierung der mütterlichen Toleranz gegenüber dem semiallogenen Feten in der Frühschwangerschaft stellt ein immunologisches Enigma dar. Ein Charakteristikum dieses Phänomens ist der kontrollierte Influx verschiedener Im-munzelltypen in die Dezidua, in der mütterliche und fetale Zellen miteinander in Kontakt treten. Es ist bekannt, dass

hochendotheliale Venolen (HEVs), welche physiologischer Weise in lymphatischen Geweben präsent sind, über die konstitutive Expression bestimmter Adhäsionsmoleküle die Fähigkeit haben, die Rekrutierung von Leukozyten, v.a. T-Lymphozyten, in das umliegende Gewebe zu steuern. Ob HEVs auch im endometrialen und dezidualen Gewebe eine signifikante Rolle spielen, versucht das folgende Projekt zu beantworten.

Mittels Immunfloureszenzanalysen ist es bereits gelungen, erstmalig sowohl die Lokalisation, als auch die Häufigkeit von HEVs im endometrialen und dezidualen Gewebe von gesunden Frauen bzw. Schwangerschaften des 1. Trime-nons (6.-13.SSW) mittels spezifischer Antikörper (MECA-79, SELE, ICAM-I, EPHB4, Willebrand Faktor) zu charakterisieren. Im Detail korreliert in beiden Gewebsarten die Anzahl an HEVs mit der Ratio lokaler CD11c+/CD45+-myeloider Im-munzellen, welche über die Expression von Lymphotoxin-ß eine signifikante Rolle bei der Neubildung von HEVs spielen dürften. Interessanterweise finden sich im dezidualen, aber auch bereits im endometrialen Gewebe von Frauen, die unter der Diagnose eines Abortus habitualis leiden, die Anzahl an myeloiden Immunzellen, und konsekutiv, die der HEVs signi-fikant verringert. Folglich weist das pathologische Gewebe, im Vergleich zu den gesunden Kontrollen, eine signifikant verringerte T-Zell-Migration auf. Die derzeit laufenden wei-terführenden Analysen dieser identifizierten immun-regulato-rischen Achse beschreiben nicht nur eine neue Pathogenese des Abortus habitualis, sondern könnten zukunftsblickend auch vielversprechende Möglichkeiten hinsichtlich neuer Therapieoptionen, z.B. im Sinne einer immun-gesteuerten Induktion von HEVs, aufdecken.

Abbildung 6: Immunfluoreszenz-basierende Doppel-Färbungen von dezidualen Gewebsschnitten mit spezifischen Antikörpern gegen (A) vWF (magenta), MECA-79 (grün) und (B) ICAM-I (magenta) und MECA-79 (grün). DAPI Färbung zeigt Zellkerne in blau.


TR ANSL ATIONAL GYNECOLOGY GROUP

P E R S O N A L S T A N D 2 018Andrea Wolf (BMA)Stephanie Pfaffinger (Master Studentin)Isabel von der Decken (Master Studentin)Sofia Papadopoulos (Master Studentin)Thomas Bartl (PhD Student)Dan Cacsire Castillo-Tong (ao.Univ.-Prof.Dr.)

Projects 2018

Establishment of experimental models to study the molecular mechanisms of mucinous ovarian cancer

Background and aims of the projectMucinous epithelial ovarian carcinoma (MOC) is a rare ova-rian malignancy with both distinct biological characteristics and clinical behavior than other types of epithelial ovarian cancer, like high grade serous ovarian cancer (HGSOC). Due to a lack of solid evidence on possible treatment alternatives, MOC patients receive standard care as the HGSOC patients, including radical cytoreductive surgery followed by adjuvant platinum-based chemotherapy. MOC patients have a rather poor prognosis compared to HGSOC, which is probably due to a lower chemosensitivity of MOC to platinum-based drugs. There is an urgent medical need to define MOC specific targets so that targeted therapies might be developed and the efficiency of treatment might be improved. So far, the molecular mechanism of the disease progression of MOC is poorly understood, partly due to the lack of well characte-rized experimental models, which represent the tumors. This project aims at establishing cell lines from tumor materials and analyzing them at molecular levels.

Methods and resultsEstablishing cell lines: For comparing the molecular fea-tures of MOC with HGSOC, we established 3 cell lines from 2 patients with MOC and 8 cell lines from 7 patients with HGSOC (Table 1, Figure 1). Tumor materials (ascites or tumor tissues) were obtained from the Department of Obstetrics and Gynecology, Medical University of Vienna. In addition, we also used two colorectal cancer cell lines in the study.

Table 1. Established cell lines

Histological typePatient’s code

Cell line name

Tumor material

MOC1

M1a Tissue

M1b Tissue

2 M2 Tissue

HGSOC

3HGS3a Tissue

HGS3b Ascites

4 HGS4 Tissue

5 HGS5 Tissue

6 HGS6 Tissue

7 HGS7 Ascites

8 HGS8 Ascites

9 HGS9 Ascites

Figure 1. Examples of morphology of the cell lines. MOC: mucinous ovarian cancer; HGSOC: high grade serous ovarian cancer; CC: colorectal cancer. The name of the cell lines is indicated above each figure.


Clinical outcome of the corresponding patients: We examined the plasma courses of two biomarkers cancer antigen 125 (CA125) and carbohydrate antigen 19-9 (CA 19-9) in all patients, from whom the cell lines were established. CA125 is the most commonly used biomarker in HGSOC and elevated plasma concentration could be observed in all patients by primary diagnosis and by recurrence (Figure 2). However, it did not represent the disease progression in MOC. In case of CA 19-9, elevated plasma concentrati-

on could be observed in the two MOC patients by primary diagnosis and recurrence of the disease. In contrast, all HGSOC patients showed normal CA 19-9 plasma values with the only exception of Patient 6 (Figure 2). A drop of the CA125 or CA19-9 could be observed following treatment with carboplatin in all patients. However, by P1, P2, P4 and P6, an increase of tumor marker concentration could be overserved by drugs other than carboplatin.

Figure 2. Clinical information of the patients. The green and gray lines indicate the cut-off concentration of the CA125 (35U/ml) and of the CA 19-9 (37U/ml), respectively. “Others” are none platinum-based drugs including paclitaxel, docetaxel, caelyx and yondelis.


Mutations in the TP53 and KRAS genes: With Sanger Sequencing, we examined the coding region of the TP53 and KRAS genes. All HGSOC harbored a specific TP53 mutation and had wild type KRAS. All MOC had a specific mutation of KRAS at the codon 12. One of the MOCs had an additional mutation in the TP53 gene. Two TP53 mutations were found in the colorectal cell lines SW480 in addition to a KRAS mutation at codon 12 and the cell line LS174T presented a KRAS mutation (Table 2).

Table 2. Mutations in the TP53 and KRAS genes

A*: c.672+1_672+2insATCTG; B*: p.Ser261_Gly262insArg; C*: c.782_783insTCG

Mucinous cell lines presented lower sensitivity to carboplatin: We measured the IC50 value of the cell lines with established methods. It showed that in comparison with HGSOC, MOC cell lines are less sensitive to carboplatin. But they were more sensitive to carboplatin than the two colorectal cancer cell lines (Figure 3).

Figure 3. Sensitivity of the cell lines to carboplatin. The tests were repea-ted for at least 3 times. The average IC50 ± SD is given.

mRNA expression of relevant genes: We examined the mRNA expression of several genes with RT-qPCR. As shown in Figure 4, MOC had a comparable expression level of MUC16 and EGFR with HGSOC, but had no expression of ESR1. They seemed to have higher expression of CK7 and CK20 than HGSOC. We also determined the expression of EpCAM and PAX8 to assure the epithelial origin of the tumors and to exclude that the histological type of the MOCs were correctly diagnosed.

Patient Cell line

TP53 U94788.1 KRAS NG007524.1

Mutation at RNA Protein changesMutation at mRNA

Protein changes

MOC1

M1ac.841G>A p.Asp281Asn c.34G>C p.Gly12Arg

M1b

2 M2 WT WT c.35G>C p.Gly12Val

HGSOC

3HGS3a

c.488A>G p.Tyr163Cys WT WTHGS3b

4 HGS4 c.722C>A p.Ser241Tyr WT WT

5 HGS5 A* B* WT WT

6 HGS6C* p.Val225Ilefs*24

WT WTc.817C>T p.Arg273Cys

7 HGS7 c.711G>T p.Met237Ile WT WT

8 HGS8 c.743G>A p.Arg248Gln WT WT

9 HGS9 c.97_375del pSer33_Thr125del WT WT

CC

LS174T WT WT c.35G>A p.Gly>Asp

SW480c.818G>A p.Arg273His

c.35G>T p.Gly>Valc.925C>T p.Pro309Ser


ConclusionWe established two cell lines from mucinous ovarian cancer, analyzed mutations in the TP53 and KRAS genes, studied their sensitivity to carboplatin and measured the mRNA expression of several relevant genes. Mucinous ovarian cancer indeed does not only present clinical behavior other than HGSOC, it also show lower sensitivity to carboplatin, and have distinct molecular characteristics in comparison to HGSOC and colorectal cancer. The cell line models pro-vide a useful tool to further investigate molecular pathways that determine the tumor progress and eventually to define specific therapeutic targets.

Figure 4. mRNA expression of relevant genes in MOC, HGSOC and CC cell lines. The grey columns indicate two MOC cell lines, the blank ones HGSOC cell lines and the black ones two CC cell lines.

Documents

FORSCHUNGSLABORATORIEN - Home | MedUni Wien · The results of this study were published in Mol Hum Reprod. 2019 Feb 1;25(2):76-87 Role of estrogen signaling in macrophage plasticity