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Universitätsklinik für Frauenheilkunde | 189

FORSCHUNGSLABORATORIEN

190 | Universitätsklinik für Frauenheilkunde

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 Jahre in einem Cluster auf Ebene 5Q des AKH Wien zusam-mengefügt.

Es ist dem Vorstand der Klinik direkt unterstellt, die wis-senschaftliche Koordination obliegt ao.Univ.-Prof. Dr. Chri-stian 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 Grund-lagen- und angewandter Forschung.

Sieben Arbeitsgruppen, die international in vielen Partner-schaften vernetzt sind und im Folgenden alphabetisch ge-reiht vorgestellt werden, führen eine Vielzahl von Projekten durch, die sich mit speziellen Fragestellungen im Bereich der Geburtshilfe, der Gynäkologie, der gynäkologischen On-kologie und gynäkologischen Endokrinologie beschäftigen.

Struktur des Forschungslabors der Universitätsklinik für Frauenheilkunde


ENDOMETRIOSIS GROUP

G R O U P M E M B E R S ( L E F T T O R I G H T ) :, PhD (Group Leader)

HUDSON, PhD (Senior Scientist) PROESTLING, PhD (Junior Scientist)

WITZMANN-STERN (Biomedical Assistant)

ProjectsThe scientific interests of the Endometriosis Group are fo-cused on understanding the molecular mechanisms involved in the pathogenesis of endometriosis and the discovery and validation of new non-invasive biomarkers for diagnosis of the disease.

The role of Linc01133 in the pathogenesis of endometriosisEndometriosis is a common gynecological disorder with a still unclear etiology. The disease is characterized by the development of endometrial-like lesions at ectopic sites outside the uterus, and affects 10% of women in reproduc-tive age. Although not life threatening, the disease has a series of malignant behaviors, such as over-proliferation, infiltration, and recurrence. Multiple genetic, epigenetic and environmental factors together with a defective im-mune response have been shown to predispose towards development of the lesions. We and others have shown that changes in epigenome (DNA methylation, chromatin modification changes) and epigenetic regulation of gene transcription by miRNAs are important for establishment and progression of the disease. In a current project, we are evaluating the role of another group of differentially ex-pressed epigenetic regulators called long non-coding RNAs (lncRNAs) in endometriosis. LncRNAs can interact with DNA, RNA and proteins, including epigenetic modifiers, and can

regulate gene transcription and RNAs translation via variety of mechanisms. Therefore, investigation of the role of these epigenetic regulators in endometriosis may help understand pathogenesis of the disease. In this project, we identified the lncRNA linc01133 as an endometriosis candidate gene, and investigated its role in the disease.Real time Q-PCR in tissue of women with and without endo-metriosis revealed that linc01133 is upregulated in ectopic lesions of women with the disease compared to eutopic and control endometrium (Fig.1A). To evaluate the role of linc01133 in pathogenesis of endometriosis we investigated the effects of linc01133 siRNA knockdown in the endome-triosis epithelial cell line (12Z) (Fig.1B).

Figure 1.A.: Graphical representation of the relative expression levels of linc01133 in tissue samples of women with and without endometriosis

levels of expression of linc01133 in endometriosis lesions (ectop) and not in endometrium of woman with endometriosis (eutop), compared to controls.

12Z 72 hrs after transfection.

Analysis of RNA seq data identified 1205 differentially expressed (DE) genes between control and knockdown cells. The subsequent GO analysis showed that decreased linc01133 expression can potentially influence 12Z cell proliferation, migration and invasion by affecting signaling pathways associated with cytoskeleton rearrangement, cell cycle progression and cell migration. The 12Z cells with an efficient knockdown of linc01133 showed significant reduc-tion of cell proliferation associated with S-phase cell cycle arrest (Fig.2A). Mechanistic investigations showed that the phenotypic effect of the linc01133 knockdown is associ-ated with upregulation of cell cycle checkpoint regulatory protein p21 (Fig.2B). Knockdown of linc01133 also caused an increase in cell migration associated with changes in cellular phenotype to more flattened morphology. We are currently evaluating the molecular mechanisms underlying the processes of actin and focal adhesion remodeling lead-ing to activation of epithelial cell migration upon linc01133 knockdown.


Figure 2.A.: Effect of linc01133 knockdown on cell proliferation. The plot is showing significant decrease in cell proliferation of 12Z after linc01133 knockdown. The results are based on CyQUANT direct cell proliferation assay data of transfected cells 72 hrs after transfection with control

protein 72 hrs after linc01133 knockdown in 12Z cells.

Confetti-Cre reporter based mouse model for endometriosis Endometriosis lesions are thought to originate from endome-trial cells introduced into the peritoneal cavity by retrograde menstruation flow, a process that occurs in most women. In order for a lesion to develop, endometrial cells must im-plant into the peritoneal cavity where they proliferate and differentiate. It is not known what types of cells establish the lesion and whether the lesion develops clonally or from multiple progenitors, and what the contribution of implanting and resident cells is to the developing lesion. The origin of tissue resident immune cells, the development and growth of the nerve fibers and the origin of cells involved in neo-vascularization is also unclear. Recently, a mouse model of endometriosis has been reported where lesions develop following injection of endometrium from a donor mouse into the peritoneal cavity. The stochastic multicolor Confetti Cre reporter has been successfully used to map clonality in the intestine, indicating that such an approach can also be taken in endometriosis. Therefore, in collaboration with the laboratory of Prof. Josef Penninger at IMBA, we have established the mouse model for endometriosis using a Confetti mouse as a donor, enabling us to identify donor cells in the recipient, and assess clonality of endometriosis lesion development (Fig3).

Figure 3.: Endometriosis lesion.

A.: Brightfield view of endometriosis lesion on the peritoneum.

B.: The endometriosis lesion expresses EGFP demonstrating that it origi-nates from the donor mouse, while the surrounding peritoneum from the recipient mouse shows no expression.

With this model we aim to solve open questions about the development of endometriosis lesions. Specifically, we want to determine if lesions develop clonally, the contribution of implanting and resident cells to the lesion, and gain a detailed picture of the cell types composing the lesions and how this changes during progression of the disease. We also aim to track the effects of the combination of the nonsteroidal anti-inflammatory drug with dietary supple-ment on the disease. For this part of the project we will use bioluminescence marking of donor cell prior injection in recipient mouse to enable development of the lesions to be tracked in the live mouse. This method has been widely used for live imaging in various mouse cancer models, and so should also work with this model.

Endometrial organoids: A 3D model of endometriosis epithelial glandsOrganoids are self-organizing, genetically stable, 3D culture systems containing both progenitor and differentiated cells that resemble the tissue of origin. Human endometrial orga-noids have been established recently from endometrial and endometriosis eutopic tissue by others. We have used the published protocols to establish the system in our laboratory, successfully generating long-term 3D glandular organoid cultures using endometrium from uterine hysterectomies of women with and without the disease (Fig.4). This system provides us with a more realistic human system in vitro than 2D culture to investigate the pathogenesis of endometriosis.

Figure 4.A.: Phase-contrast images established from endometrium of woman without endometriosis (control) and endometrium of woman with endometriosis (eutopic.)

cytokeratin7 (epithelial cell marker), vimentin (stroma cell marker an dDAPI (nuclear staining).

Figure C.: Immunofluorescence staining of organoids with FOXA2 as marker for glandular endometrial epithelial cells.

Research output 2020This year we published two original papers, the first in Repro-ductive BioMedicine Online entitled “Investigating selected adhesion molecules as urinary biomarkers for diagnosing endometriosis” (doi: 10.1016/j.rbmo.2020.01.014), and the second in Reproductive Sciences entitled “Endometriosis Patients Show an Increased M2 Response in the Peritoneal


CD14+low/CD68+low Macrophage Subpopulation Coupled with an Increase in the T-helper 2 and T-regulatory Cells” (doi: 10.1007/s43032-020-00211-9). We also published an invited review in “Experimental biology and medicine (Maywood) entitled “Challenges in uncovering non-invasive biomark-ers of endometriosis” (doi: 10.1177/1535370220903270).

Finally, we completed the Grants4Targets project funded by Bayer entitled “A large-scale validation analysis of a set of differentially expressed miRNAs in plasma of woman with endometriosis for noninvasive disease diagnosis“. We sub-mitted a final report to Bayer and are currently preparing this work for publication.

D A S T E A M D E R A R B E I T S G R U P P E ( V O N L I N K S N A C H R E C H T S , U N D O B E N N A C H U N T E N ) :

HASLINGER (BMA)FRANK (BMA, bis Juli 2020)

WIDMAR (BMA, 20 Std.).SCHNEEBERGER, ao. Univ.-Prof. Mag. Dr.

PIETROWSKI, Dipl.Biol. Dr.

-neller, räumlicher und wissenschaftlicher Hinsicht sehr eng mit der Endometriose-Gruppe zusammen. So teilen sich bei-spielsweise beide Gruppen die Labors, Frau Barbara Widmar ist fast zur Gänze an die Endometriose Gruppe „verborgt“ und Frau Isabelle Haslinger ist unter anderem für die FACS Analytik der Endometriose Gruppe zuständig.

HAUPTPPROJEKTE 2020

„Cryopreservation-Induced Delayed-Onset Cell Death” in Ovarzellen

Die Kryokonservierung von Ovarialgewebe ist eine medizini-sche Methode zur Erhaltung der Fruchtbarkeit bei Patientin-nen, die unter den gonadotoxischen Nebenwirkungen einer chemotherapeutischen Therapie zu leiden haben. Allerdings ist während der Kryokonservierung das Gewebe physikalischen und chemischen Belastungen ausgesetzt, wie z. B. Kältestress, Bildung von intrazellulären und extrazel-lulären Eiskristallen und osmotischem Stress. Diese Belas-tungen können nach dem Auftauen des Gewebes, obwohl zu diesem Zeitpunkt die Zellen noch vital sind, Stunden später zu einem deutlichen Verlust an lebenden Zellen im Ovarialge-webe führen („Cryopreservation-Induced Delayed-Onset Cell Death“ (CIDOCD)). Die Vitrifikation von Zellen und Gewebe ist eine Methode der Kryokonservierung, die sich vor allen Dingen durch ihre enorme Einfriergeschwindigkeit von ca. 20 000°C/min von anderen Methoden der Kryokonser-vierung unterscheidet. In dem hier beschriebenen Projekt wollten wir untersuchen, in welchem Ausmaß CIDOCD auch nach einer Vitrifikation von unterschiedlichen ovariellen Zel-len auftreten kann.

Wir haben daher eine Granulosazelllinie endothelialen Ur-sprungs (KGN) und eine Epithelzelllinie (OvCar-3) verwendet, um den Effekt der Vitrifikation hinsichtlich der Vitalität direkt nach dem Auftauen und nach der Kultivierung für zwei bis drei Tage zu untersuchen. Wie in den Abbildungen 1 und 2 gezeigt, gibt es einen Verlust von ca. 20% an lebenden KGN Zellen und ca. 40 - 50 % an OvCAR-3 Zellen direkt nach der Erwärmung im Vergleich zur Kontrolle. Nach der Kultivierung (Abb. 2) zeigten die KGN Zellen nur noch einen Anteil von ca. 20% und die OvCar-3 Zellen einen Anteil von ca. 40% lebender Zellen. Die Ergebnisse für die KGN Zellen sind insbesondere bemerkenswert, weil die unbehandelten KGN Zellen eine deutliche Vermehrung zeigten. Dies lässt den Schluss zu, dass das Ausmaß des CIDCOD in diesem System zellspezifisch zu sein scheint und erhebliche Aus-wirkungen auf die Überlebensraten von Zellen nach einer Vitrifikation haben kann.


Abb 1.: Prozentualer Anteil lebender Zellen direkt nach der Erwärmung. (KGN-O und KGN-CL sind KGN Zellen, die nach unterschiedlichen

Zellen, die nach unterschiedlichen Vitrifikationsprotokollen vitrifiziert wurden. KGN ctrl und Ovcar ctrl sind nicht vitrifizierte Kontrollen

Abb 2.: Prozentualer Anteil lebender Zellen die nach der Erwärmung für 48 Stunden kultiviert wurden. (KGN-O und KGN-CL sind KGN Zellen, die nach unterschiedlichen Vitrifikationsprotokollen vitrifiziert wurden; OV-O

-tionsprotokollen vitrifiziert wurden. KGN ctrl und Ovcar ctrl sind nicht vitrifizierte Kontrollen

GROUP

Das Team der Arbeitsgruppe Molekulare Onkologie

Sabrina Grundtner

Nicht am Bild: Nina Koppensteiner


Detection of circulating tumor cells in blood samples taken from patients with platinum-resistant ovarian cancer enrolled in the GANNET53 study

Collaborators: Obermayr E, Schuster E, Agreiter Ch, Zeillinger R, GANNET53 consortium (http://www.gannet53.eu/consortium)

The aim of the study is to determine whether the presence of circulating tumour cells (CTCs) in whole blood before and during treatment is a suitable marker for monitoring patients and determining their response to therapy. To tackle this question, blood samples of the 129 patients included in phase II of the GANNET53 study were taken at every second cycle of treatment. On average the blood was taken at four (range 1-9) time points before and during treat-ment. All blood samples were processed using the Parsortix™ technology (Angle plc., UK). The total RNA was extracted from the enriched cells and reverse transcribed into cDNA. To increase the sensitivity of the overall approach, a specific pre-amplification was performed.The presence of all 28 target genes was measured using quantitative PCR. Then the patients were stratified into two groups by presence or absence of the respective gene at each cycle of treatment. The association of the two groups (favourable vs. unfavourable) and progression-free (PFS) and overall survival (OS) was assessed at the various cycles using Kaplan-Meier curves and log-rank (Mantel-Cox) tests. The hazard risks for OS and PFS of some representative genes (with 95% confidence intervals) at each cycle of treatment are shown in Figure 1.

Figure 1: Hazard ratios and 95% confidence intervals of OS and PFS of

(C2), cycle 3 (C3), cycle 5 (C5), and cycle 7 (C7) of treatment.

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

Collaborators: Koppensteiner N, Obermayr E, Schuster E, Hochmair M, Hamilton G, Zeillinger R

Lung cancer is the most common cancer worldwide. About 85% of the patients are diagnosed with the epithelial non-small-cell histological subtype (NSCLC). Distant metastases in the brain or bone are frequently observed in this type of disease. Circulating tumor cells (CTCs) may play a central role in the metastatic spread of lung cancer.The aim of the present study was the detection CTCs in the blood of NSCLC patients using quantitative PCR. The blood samples were taken at the Klinik Floridsdorf (Depart-ment of Internal Medicine and Pneumology) and sent to our department. Likewise, blood samples from 30 healthy donors were included as controls. All blood samples were processed using the Parsortix™ technology (Angle plc., UK) and a CTC-enriched cell fraction was retrieved. After ex-traction of the total RNA, reverse transcription, and a gene specific pre-amplification of the respective targets, the gene expression levels of

and CK19 were assessed using qPCR and correlated with the clinical data of the respective patients. BPIFA1, FAM83A and were not detected in the healthy control samples; for all the other markers a threshold was calculated in order to assess CTC-positivity in the patients’ samples. Finally, 75 from 119 NSCLC patients (63%) were assigned as being CTC-positive due to the expression of at least one marker above the threshold value.The hazard risks of the selected genes for OS (with 95% confidence intervals) are shown in Figure 2.

Figure 2: Hazard ratios and 95% confidence intervals of OS of the

selected NSCLC gene markers.


Blood sample processing for CTC analyses

Collaborators: Obermayr E, Schuster E, Koppensteiner N, Holzer B, Hamilton G, Hastermann G, Hochmair M, Za-harie A, Aust S, Singer C, Krainer M, Steger G, Marhold M, Zeillinger R, GANNET53 consortium (http://www.gannet53.eu/consortium)

Over the past year we obtained blood samples taken from about 210 patients, primarily taken at the Medical University of Vienna (Department of Medicine I, Department of Obstet-rics and Gynecology, Department of Radiooncology), Klinik Floridsdorf (Department of Internal Medicine and Pneumol-ogy), and the Klinik Landstraße (Department of Obstetrics and Gynecology). In addition, we obtained blood samples taken from about 50 patients enrolled in the EUDARIO clini-cal trial in the frame of the GANNET53 project. An overview of the obtained samples is given in Figure 3. From these blood samples, a serum/plasma fraction was retrieved, and/or the whole blood was enriched for circulating tumor cells. The serum/plasma samples from patients with gy-necological cancers (N=44) were included into the Biobank collection at the Klinisches Institut für Labormedizin (AKH Wien). 222 whole blood samples (99 lung cancer patients, 67 breast cancer patients, 6 prostate cancer patients, 50 ovarian cancer patients enrolled within the EUDARIO trial) were further processed to enrich circulating tumor cells us-ing density gradient centrifugation and/or the microfluidic Parsortix™ system (Angle plc., UK).

Figure 3: Overview on blood samples obtained from 01-12/2020

Detection of Hsp90-p53 complexes in ascites, pleural effusions and tumour tissue from patients with platinum-resistant ovarian cancer enrolled in the GANNET53 study

Collaborators: Heinzl N, Zeillinger R, Gannet53 project partners (http://www.gannet53.eu/consortium)

The GANNET53 trial is based on the hypothesis that mutant p53 is stabilized through the heat shock chaperone Hsp90 and can therefore neither fulfil its function, nor be degraded. The stabilization of p53 is a prerequisite for gain-of-function capabilities promoting tumour growth. Especially tumours car-rying a missense mutation show protein stabilization. These tumours develop a dependency on the high protein levels and withdrawal should result in cytotoxicity. The Hsp90 inhibitor Ganetespib is used to release stabilized p53 and target it for degradation. The TP53 mutation status was evaluated in 124 patients and in 115 (92.7%) a TP53 mutation was detected. Of 133 patients included in the GANNET53 trial, a low num-ber of patients suffered from symptomatic ascites result-ing in the clinical indication of ascites drainage. From 8 patients 14 ascites samples and from 5 patients 13 pleu-ral effusions were collected. In 4 patients more than one ascites/pleural effusion was available. In two patients both, ascites and pleural effusions were collected. A Hsp90-p53 proximity ligation assay (PLA) was performed on cytological preparations of the ascites and pleural effusions. In 4 out of 14 (29%) ascites samples Hsp90-p53 complexes could be detected. In contrast, in all pleural effusions, Hsp90-p53 PLA signals were detected. In two patients with consecutive pleural effusions, Hsp90-p53 complexes were present at all given timepoints. Patient 1 had a PFS above the 75%ile and is therefore termed a super-responder, whereas the PFS of patient 2 was between the 25- and 75%ile. In the first two out of three consecutive ascites samples of patient 3 no HSP90-p53 complexes could be detected, however the third sample was positive for these complexes. Again, this patient had a PFS between the 25- and 75% percentile (Figure 4). Nonetheless, the number of available consecutive ascites samples/pleural effusions before/during treatment did not allow robust correlations with treatment response.


Figure 4: Three patients where consecutive ascites or pleural effusions were collected. No correlation with treatment response could be found.

In addition to ascites, FFPE tumour tissue was used for the evaluation of Hsp90-p53 complexes. To do so, three tissue micro arrays (TMAs), including 116 patients, were used for this analysis. An immunofluorescence staining (IF) for Hsp90, p53 and nuclear DAPI staining was performed. The statisti-cal analysis revealed a moderate association between the p53 protein expression determined by IF and the p53 gene mutation status (Cramer‘s V=0.633, Fisher‘s p=0.000). In general, 55% of patients had detectable p53 protein ex-pression, whereas all except one tumour sample where Hsp90 positive. Interestingly, not all p53-positive cells were Hsp90-positive, suggesting that not all tumour cells bear Hsp90-p53 complexes. The co-expression of p53 and Hsp90 (p53+/Hsp90+) was neither associated with a significant overall (OS) nor a progression-free survival (PFS) benefit (Figure 5).

Figure 5: Kaplan-Meier curves stratified by the co-expression of p53 and HSP90 for overall survival (top) and progression-free survival (bottom). Log-rank testing was used to compare outcomes.

As mentioned above not all cells were positive for Hsp90 and p53, leading to only 17 out of 116 (14.7%) patients with at least some PLA signals in single tumour cells. The proximity ligation assay (PLA) determines a direct interaction of two proteins (Hsp90 and p53). There was no statistical difference between chemonaïve patients and patients after neoadjuvant therapy regarding the presence of Hsp90-p53 complexes in tumour cells. Furthermore, no association between primary tumours or relapsed tumours and the number of Hsp90-p53 complexes was detected. The association between Hsp90-p53 complexes and progression-free (PFS) as well as overall survival (OS) was assessed using Kaplan-Meier curves and log-rank tests. Our results showed that the presence of Hsp90-p53 complexes had no influence on the patients’ outcome (Figure 6).

Figure 6: Kaplan-Meier curves stratified by presence of HSP90-p53 complexes for overall survival (top) and progression-free survival (bottom). Log-rank testing was used to compare outcomes.


The effect of a p53 aggregation inhibitor in ovarian and breast cancer cell lines

Collaborators: Heinzl N, Buchinger C, Zeillinger R

Aggregated p53 has emerged as a potential drug target in cancer. A research group from the UCLA developed ReACp53, a cell-penetrating peptide designed to inhibit the p53 aggrega-tion. Using ovarian cancer (OC) cell lines and xenograft mice, they were able to prove that the peptide inhibits the forma-tion of amyloids of mutant p53. Functional p53 can enter the nucleus, induce cell death and a proliferation arrest, and reduces in vivo xenograft growth and metastasis. However, they only targeted two of the three most common p53 hotspot mutations in high-grade serous ovarian cancer (HGSOC). With thousands of different TP53 mutations described, they could not anticipate, which other mutations will respond to ReACp53. Therefore, we aimed at investigating the effect of ReACp53 on several other TP53 mutations in OC and breast cancer (BC) cell lines as well as evaluating the influence of the amount of p53 aggregates on the response. To do so, the cells were exposed to different ReACp53 concentrations and the cell viability was determined by using the EZ4U assay. We could see a wide range of responses to ReACp53 (IC50: 4µM to 16.7µM) depending on the amount of p53 aggregates (Table 1). All cell lines, except MCF-7, which had no detectable p53 aggregates did not respond to the inhibitor, as in contrast to cell lines, which showed p53 aggregation.

Cell line

Cancer type

TP53 status

Amount of p53 aggregates [absor- bance/ total protein]

IC50 [µM]

A2780 Ovarian Wild-type 1.7 4.8

59M Ovarian Wild-type 0 14.4

TOV21G Ovarian Wild-type 0 16.7

ES2 Ovarian Missense mutation (S241F)

12.1 4.0

TOV112D Ovarian Missense mutation (175H)

10.7 6.0

PEO23 Ovarian Missense mutation (C277F)

10.9 8.3

OVCAR-3 Ovarian Missense mutation (R248Q)

6.1 9.5

HBL-100 Wild-type (SV40 transformed)

54 10.33

MCF-7 breast Wild-type 0 6.8

SK-BR-3 breast Missense mutation (R175H)

9.27 4.89

CAMA-1 breast Missense mutation (R280T)

5.49 8.83

Table 1: Amount of p53 aggregates measured with the p53-Seprion-ELISA and IC50 values for each cell line are given.

Currently, the effect of ReACp53 is tested in a panel of ovarian and breast cancer cells with various TP53 mutation status and p53 aggregation levels. Moreover, the evaluation of a possible synergistic effect with carboplatin is ongoing.

Determination of the aggregation propensity of the tumour suppressor BRCA1

Collaborators: Grundtner S, Heinzl N, Zeillinger R

By using a computer algorithm for prediction of aggregating regions in protein sequences, it was found that similar to p53, BRCA1 has the potential to form protein aggregates. It is hy-pothesized that the aggregation of BRCA1 is responsible for the complete loss of the tumour suppressor functions. Up to now, poly (ADP-ribose) polymerase (PARP) inhibitors (PARPi) are one of the most promising new classes of targeted agents in BRCA-mutated and/or homologous recombination repair (HRR) deficient cancers. Since there is a certain number of patients, which still benefit from PARP-inhibitor-based therapy, without an obvious HRR deficiency, the BRCA ag-gregation load can add important information as predictive marker for therapy response. To tackle this question, we developed a proximity ligation assay (PLA) to specifically detect BRCA1 aggregates in 23 ovarian cancer, one breast cancer and one cervical carcinoma cell lines. For the PLA-based BRCA1 aggregate detection an anti-BRCA1 antibody and the anti-amyloid fibrils antibody OC were combined. The average number of PLA signals (“dots”) ranged from 0.1 to 32.8 dots per cell and their localization was quite variable.

CellProfiler. The technical negative control (no primary antibodies) was used to set the threshold. Cell lines with a lower dots/cell value than the negative control was classified as negative (red). The remaining cell lines were classified as positive (green). − indicates median of dots/cell.


expressed BRCA1 protein aggregates in the cytoplasm, while the cell lines COV504 and ES-2 show a cluster of BRCA1 protein aggregates in the nucleus. In the remaining cell lines the protein aggregates were equally present in the nucleus and cytoplasm (Figure 8). A significantly higher dots/cell value can be found in cells expressing BRCA1 protein in the nucleus (Wilcoxon test, p = 0.015). Interestingly, the ag-gregation propensity was irrespective of the mutation status as only two cell lines had a mutation in the gene, whereas all others carried the wild-type gene.

Further, the response to Olaparib, a PARP inhibitor was tested. A subset of cell lines was treated with Olaparib rang-ing from 0-40 µM. As hypothesized, the wildtype cell lines 59M, COV644, OAW28, OV56, PEA2, COV318, PEO23, SK-OV-3, OVCAR-3 and TOV21G showed resistance to Olapa-

wildtype BRCA1) showed a dose-dependent sensitivity with IC50 values of 11.18, 13.91 and 9.75 (Figure 9). Surprisingly, the cell lines COV362 and Igrov-1, which have a BRCA1 frameshift mutation, were resistant to Olaparib.

-sented by blue DAPI staining. Cytoplasm is visualized through α-tubulin staining (green). The negative control was incubated with antibody diluent only. Images are obtained by a laser-scanning-microscope 780 (LSM780) with a magnification of 63x. Scale bars indicate 20 µm.

Figure 9: Cytotoxicity assay of Olaparib-sensitive cell lines. Cells were treated with Olaparib ranging from 0-40 µM. Data are means ±SD. IC50 values were calculated using GraphPad Prism.


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 :SCHREIBER, ao. Univ.-Prof., M.Sc.

MIEDL (50%), BMA

Assoziation des Indel Polymorphismus rs150550023 im MDM2 Gen mit der Genexpression im p53-MDM2 SignalwegViele natürlich vorkommende Sequenzvarianten im menschli-chen Genom haben einen Einfluss auf das Krebsrisiko, und die meisten von ihnen liegen außerhalb von proteinkodierenden Bereichen. Höchstwahrscheinlich haben sie einen Effekt auf die Regulation von benachbarten Genen und die Menge an mRNA und Protein, die von diesen Genen abgelesen wird. Wir haben eine solche Sequenzvariante im Promoter des MDM2 Gens (human homolog of mouse double minute 2), rs150550023, analysiert. The Hauptfunktion von MDM2 ist die negative Regulation des zentralen Tumor Suppressors p53 (TP53). Wir genotypisierten rs150550023 in 407 Brustkrebs-Patientinnen und 254 weiblichen Kontrollen. Wir fanden keine Evidenz dafür, dass rs150550023 eine wichtige Rolle als Risikofaktor für Brustkrebs, beim Erkrankungsalter, beim Ge-samtüberleben oder beim metastasenfreien Überleben spielt. Hingegen fanden wir Hinweise für Synergie-Effekte mit einer anderen Sequenzvariante im MDM2 Gen, nämlich SNP309. In ca. 100 Tumorgewebeproben von Patientinnen dieser Studie wurde die Expression von MDM2, p53, sowie drei p53 Tar-

bestimmt. Wir konnten zeigen, dass die mRNA Expression

dem homozygoten varianten rs150550023 Genotyp (D/D) um das 1.5–3 fache erhöht war (Abbildung 1). Allerdings

Proteinexpression, und auch nicht der Mutationsrate des TP53 Gens. Insgesamt sprechen diese Ergebnisse dafür, dass die beobachtete Erhöhung der p53 mRNA die Folge von

Feedback Regulationen sind, die den Effekt von rs150550023 auf die MDM2 Expression kompensieren.

Abbildung 1: Assoziation des rs150550023 Polymorphismus mit der

Brustkrebs-Patientinnen. Gezeigt sind die Ergebnisse für unselektierte Pa-tientinnen, Patientinnen mit einem Wildtyp TP53 Gen (auch in der Graphik ganz rechts), und einem mutierten TP53 Gen. Die drei Genotypen von rs150550023 sind durch Farben verdeutlicht: I/I, blau; I/D, grün; D/D, rot. Die längere horizontale Linie in den Grafiken stellt den Median dar, die beiden kürzeren die erste und dritte Quartile. Die gezeigten p-Werte wurden mit ANOVA berechnet. Signifikante Unterschiede zwischen zwei Genotypen sind zusätzlich durch „**“ dargestellt (p<0.01). Die Expres-sionswerte sind auf logarithmischen Achsen abgebildet (log[2]).

Epigenetische Regulation des E-Cadherin Gens (CDH1)E-Cadherin ist ein zentrales Zell-Zell-Adhäsionsmolekül in epithelialen Geweben. Der Verlust seiner Expression geht mit dem Auflösen von geordneten epithelialen Zellverbänden, einer epithelial-mesenchymalen Transition (EMT) und einer erhöhten Invasivität einher, was E-Cadherin zu einem Tu-morsuppressorgen macht. Allerdings ist E-Cadherin nur in lobulären Mammakarzinomen sowie in gastrointestinalen Stromatumoren (GIST) häufig mutiert, in allen anderen Tu-morarten nur sehr selten. Dennoch geht die E-Cadherin Funktion auch in Tumoren ohne Mutation im Zuge der Tumor-progression oft verloren, und in diesen Fällen müssen andere Mechanismen verantwortlich sein, wie z.B. die Abschaltung der Expression durch epigenetische Mechanismen wie die DNA Methylierung des Promoters. Dies untersuchten wir in Tumor-Gewebeproben von Brustkrebs-Patientinnen mit verschiedenen Methoden, wie der methylierungs-spezifis-chen PCR (MSP), der quantitativen MSP (qMSP), COBRA (combined bisulfite restriction analysis), und der Bisulfit Sequenzierung nach Klonierung. Alle Methoden haben spezi-fische Vor- und Nachteile, wir konzentrierten uns auf die Bisulfit-Sequenzierung, welche die aufwändigste, aber auch die detaillierteste und informativste Methode ist (Abbildung 2). Insgesamt zeigten unsere Tumorproben eine unerwartet niedrige Methylierung des CDH1 Promoters, wobei sich je-doch eine inverse Korrelation zwischen Methylierungsgrad und E-Cadherin Expression abzeichnet.

Abbildung 2: Diagramm der DNA Methylierung des E-Cadherin Promot-ers in einer Brustkrebs-Tumorgewebeprobe. Es wurden 10 individuelle Klone nach Bisulfit-Behandlung sequenziert (Zeile I-X). Die 32 Spalten repräsentieren die 32 CpG Dinukleotide im analysierten Abschnitt des E-Cadherin Promoters, die potentiell methyliert sein können. Weiße Kreise stellen unmethyliertes und schwarze Kreise methyliertes Cytosin dar. Die CpGs Nr. 10 und Nr. 19 sind in 50% der Klone methyliert. Insgesamt ist die


L E I T U N G :SINGER, MPH

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 :MUHR (Biomedizinische Analytikerin)TAN, PhD (Epidemiologin)

KASTNER (Biomedizinische Analytikerin)REICHL (Assistent)

PARGER (Dissertantin)

Obere reihe v.l.n.r.: Muhr D, Tan YY, Kastner M-T

WISSENSCHAFTLICHE PROJEKTE

CIMBA (The Consortium of Investigators of Modifiers of BRCA 1/2)

Coordination partners:

Georgia Chenevix-Trench PhD, NHMRC Senior Principal Research Fellow, The Queensland Institute of Medical Re-search, AustraliaAntonis Antoniou, CR-UK Genetic Epidemiology Unit, Strangeways Research Laboratory, University of Cambridge, Cambridge, UKDouglas Easton, CR-UK Genetic Epidemiology Unit, De-partment of Public Health and Primary Care, University of Cambridge, Cambridge, UK

Cooperation partners:Singer CF, Department of Obstetrics and Gynecology, Center for Familial Breast- and Ovarian Cancer, Medical University of Vienna, Austria

Other cooperation partners:http://apps.ccge.medschl.cam.ac.uk/consortia/cimba//groups/groups.html

Collaborators:Tan YY, Muhr D

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:

Polygenic risk scores and breast and epithelial ova-rian cancer risks for carriers of BRCA1 and BRCA2 pathogenic variants Daniel R. Barnes, Matti A. Rookus, […] on behalf of the Con-sortium of Investigators of Modifiers of BRCA and BRCA2. Genet Med 22, 1653–1666 (2020)

AbstractPurposeWe assessed the associations between population-based po-lygenic risk scores (PRS) for breast (BC) or epithelial ovarian cancer (EOC) with cancer risks for and pa-thogenic variant carriers.MethodsRetrospective cohort data on 18,935 and 12,339 female pathogenic variant carriers of Euro-pean ancestry were available. Three versions of a 313 single-nucleotide polymorphism (SNP) BC PRS were evaluated based on whether they predict overall, estrogen receptor (ER)–negative, or ER-positive BC, and two PRS for overall or high-grade serous EOC. Associations were validated in a prospective cohort.ResultsThe ER-negative PRS showed the strongest association with BC risk for carriers (hazard ratio [HR] per standard deviation = 1.29 [95% CI 1.25–1.33], P = 3×10−72). For , the strongest association was with overall BC PRS (HR = 1.31 [95% CI 1.27–1.36], P = 7×10−50). HR estimates decreased significantly with age and there was evidence for differences in associations by predicted variant effects on protein ex-pression. The HR estimates were smaller than general po-pulation estimates. The high-grade serous PRS yielded the strongest associations with EOC risk for (HR = 1.32 [95% CI 1.25–1.40], P = 3×10−22) and (HR = 1.44 [95%


CI 1.30–1.60], P = 4×10−12) carriers. The associations in the prospective cohort were similar.ConclusionPopulation-based PRS are strongly associated with BC and EOC risks for carriers and predict substantial abso-lute risk differences for women at PRS distribution extremes.

Characterization of the Cancer Spectrum in Men With Germline BRCA1 and BRCA2 Pathogenic Variants Results From the Consortium of Investigators of Modifiers of BRCA1/2 (CIMBA) Valentina Silvestri, Goska Leslie, Daniel R. Barnes, […] on behalf of the Consortium of Investigators of Modifiers of BRCA and BRCA2. JAMA Oncol. 2020;6(8):1218-1230

AbstractImportance The limited data on cancer phenotypes in men with ger-mline and pathogenic variants (PVs) have hampered the development of evidence-based recommen-dations for early cancer detection and risk reduction in this population.Objective To compare the cancer spectrum and frequencies between male and PV carriers.Design, Setting, and Participants Retrospective cohort study of 6902 men, including 3651 and 3251 PV carriers, older than 18 years recruited from cancer genetics clinics from 1966 to 2017 by 53 study groups in 33 countries worldwide collabo-rating through the Consortium of Investigators of Modifiers of BRCA1/2 (CIMBA). Clinical data and pathologic characte-ristics were collected.Main Outcomes and Measures

status was the outcome in a logistic regression, and cancer diagnoses were the independent predictors. All odds ratios (ORs) were adjusted for age, country of origin, and calendar year of the first interview.Results Among the 6902 men in the study (median [range] age, 51.6 [18-100] years), 1634 cancers were diagnosed in 1376 men (19.9%), the majority (922 of 1,376 [67%]) being PV carriers. Being affected by any cancer was associated with a higher probability of being a , rather than a , PV carrier (OR, 3.23; 95% CI, 2.81-3.70; P < .001), as well as de-veloping 2 (OR, 7.97; 95% CI, 5.47-11.60; P < .001) and 3 (OR, 19.60; 95% CI, 4.64-82.89; P < .001) primary tumors. A higher frequency of breast (OR, 5.47; 95% CI, 4.06-7.37; P < .001) and prostate (OR, 1.39; 95% CI, 1.09-1.78; P = .008) cancers was associated with a higher probability of being a PV carrier. Among cancers other than breast and prostate, pan-creatic cancer was associated with a higher probability (OR, 3.00; 95% CI, 1.55-5.81; P = .001) and colorectal cancer with a lower probability (OR, 0.47; 95% CI, 0.29-0.78; P = .003) of being a PV carrier.

Conclusions and Relevance Significant differences in the cancer spectrum were obser-ved in male , compared with , PV carriers. These data may inform future recommendations for sur-veillance of -associated cancers and guide futu-re prospective studies for estimating cancer risks in men with PVs.

Fine-mapping of 150 breast cancer risk regions iden-tifies 191 likely target genesLaura Fachal, Hugues Aschard, Jonathan Beesley, […] on behalf of the Consortium of Investigators of Modifiers of BRCA and BRCA2.Nat Genet 52, 56–73 (2020)

AbstractGenome-wide association studies have identified breast cancer risk variants in over 150 genomic regions, but the mechanisms underlying risk remain largely unknown. These regions were explored by combining association analysis with in silico genomic feature annotations. We defined 205 independent risk-associated signals with the set of credible causal variants in each one. In parallel, we used a Bayesian approach (PAINTOR) that combines genetic association, linkage disequilibrium and enriched genomic features to determine variants with high posterior probabilities of being causal. Potentially causal variants were significantly over-represented in active gene regulatory regions and transcrip-tion factor binding sites. We applied our INQUSIT pipeline for prioritizing genes as targets of those potentially causal variants, using gene expression (expression quantitative trait loci), chromatin interaction and functional annotations. Known cancer drivers, transcription factors and genes in the developmental, apoptosis, immune system and DNA integrity checkpoint gene ontology pathways were over-represented among the highest-confidence target genes.

Familial Breast Cancer Research Unit Risk Factor Analysis of Hereditary Breast and Ovarian Cancer

Coordinator:Dr. Steven Narod, Toronto, Canada

Cooperation partners:Singer CF, Department of Obstetrics and Gynecology, Center for Familial Breast- and Ovarian Cancer, Medical University of Vienna, Austria


Collaborators: Tan YY, Muhr D

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:

Breastfeeding and the risk of epithelial ovarian can-cer among women with a BRCA1 or BRCA2 mutationKotsopoulos, Joanne; Gronwald, Jacek; McCuaig, Jeanna M; Karlan, Beth Y; Eisen, Andrea; Tung, Nadine; Bordeleau, Louise; Senter, Leigha; Eng, Charis; Couch, Fergus; Fruscio,

Christian F; Pal, Tuya; Foulkes, William D; Neuhausen, Susan L; Sun, Ping; Lubinski, Jan; Narod, Steven A. Gynecol Oncol; 159(3): 820-826, 2020 Dec

AbstractObjective

mutation carriers face a high lifetime risk of developing ovarian cancer. The strong inverse association between breastfeeding and the risk of ovarian cancer is established in the general population but is less well studied among women with a germline or mutation.MethodThus, we conducted a matched case-control analysis to evaluate the association between breastfeeding history and the risk of developing ovarian cancer. After matching for year of birth, country of residence, gene and personal history of breast cancer, a total of 1650 cases and 2702 controls were included in the analysis. Conditional logistic regression was used to estimate the odds ratio (OR) and 95% confidence intervals (CI) associated with various bre-astfeeding exposures.ResultsA history of ever-breastfeeding was associated with a 23% reduction in risk (OR = 0.77; 95%CI 0.66–0.90; P = 0.001). The protective effect increased with breastfeeding from one month to seven months after which the association was relatively stable. Compared to women who never bre-astfed, breastfeeding for seven or more months was as-sociated with a 32% reduction in risk (OR = 0.68; 95%CI 0.57–0.81; P < 0.0001) and did not vary by gene or age at diagnosis. The combination of breastfeeding and oral contraceptive use was strongly protective (0.47; 95%CI 0.37–0.58; P < 0.0001).

ConclusionsThese findings support a protective effect of breast-feeding for at least seven months among women with a or mutation, that is independent of oral contraceptive use.

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 CF, Department of Obstetrics and Gynecology, Center for Familial Breast- and Ovarian Cancer, Medical University of Vienna, Austria and others


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).

Recent studies:


ERA-NET on Translational Cancer Research (TRAN-SCAN)“Translational research on primary and secondary prevention of cancer” – Development of a Compre-hensive 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 CF, Austria, Medical University of Vienna, Wien Van Gils Carla, The Netherlands, Universitity Medical Cen-ter Utrecht, Utrecht


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 is1. 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.

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

Recent results:

Alcohol consumption, cigarette smoking, and risk of breast cancer for BRCA1 and BRCA2 mutation carriers: results from The BRCA1 and BRCA2 Cohort ConsortiumHongyan Li, et alCancer Epidemiol Biomarkers Prev February 1 2020 (29) (2) 368-378

AbstractBackgroundTobacco smoking and alcohol consumption have been in-tensively studied in the general population to assess their effects on the risk of breast cancer (BC), but very few studies have examined these effects in BRCA1 and BRCA2 mutation carriers. Given the high BC risk for mutation carriers and the importance of BRCA1 and BRCA2 in DNA repair, better evidence on the associations of these lifestyle factors with BC risk is essential. MethodsUsing a large international pooled cohort of BRCA1 and BRCA2 mutation carriers, we conducted retrospective (5,707 BRCA1 mutation carriers; 3,525 BRCA2 mutation carriers) and prospective (2,276 BRCA1 mutation carriers; 1,610 BRCA2 mutation carriers) analyses of alcohol and tobacco consumption using Cox proportional hazards models. ResultsFor both BRCA1 and BRCA2 mutation carriers, none of the smoking-related variables was associated with BC risk, except smoking for more than five years before a first full-term pregnancy (FFTP) when compared to parous women who never smoked. For BRCA1 mutation carriers, the HR from retrospective analysis (HRR) was 1.19 (95%CI:1.02,1.39) and the HR from prospective analysis (HRP) was 1.36 (95%CI:0.99,1.87). For BRCA2 mutation carriers, smoking for more than five years before a FFTP showed an association of a similar magnitude, but the confidence limits were wider (HRR=1.25,95%CI:1.01,1.55 and HRP=1.30,95%CI:0.83,2.01). For both carrier groups, alcohol consumption was not asso-ciated with BC risk. ConclusionsThe finding that smoking during the pre-reproductive years increases BC risk for mutation carriers warrants further investigation.

Association of Genomic Domains in BRCA1 and BRCA2 with Prostate Cancer Risk and AggressivenessVivek L.et al.Cancer Res November 13 2019. DOI: 10.1158/0008-5472.CAN-19-1840

Abstract Pathogenic sequence variants (PSV) in BRCA1 or BRCA2 (BRCA1/2) are associated with increased risk and severity of prostate cancer (PCa). We evaluated whether PSVs in


BRCA1/2 were associated with risk of overall PCa or high grade (Gleason 8+) PCa using an international sample of 65 BRCA1 and 171 BRCA2 male PSV carriers with PCa, and 3,388 BRCA1 and 2,880 BRCA2 male PSV carriers without PCa. PSVs in the 3‘ region of BRCA2 (c.7914+) were significantly associated with elevated risk of PCa compared with reference bin c.1001-c.7913 (HR=1.78, 95%CI: 1.25-2.52, p=0.001), as well as elevated risk of Gleason 8+ PCa (HR=3.11, 95%CI: 1.63-5.95, p=0.001). c.756-c.1000 was also associated with elevated PCa risk (HR=2.83, 95%CI: 1.71-4.68, p=0.00004) and elevated risk of Gleason 8+ PCa (HR=4.95, 95%CI: 2.12-11.54, p=0.0002). No genotype-phenotype associations were detected for PSVs in BRCA1. These results demonstrate that specific BRCA2 PSVs may be associated with elevated risk of developing aggressive PCa

Polygenic Risk Modelling for Prediction of Epithelial Ovarian Cancer RiskEileen O Dareng, et al.

doi: https://doi.org/10.1101/2020.11.30.20219220

AbstractPolygenic risk scores (PRS) for epithelial ovarian cancer (EOC) have the potential to improve risk stratification. Joint estimation of Single Nucleotide Polymorphism (SNP) ef-fects in models could improve predictive performance over standard approaches of PRS construction. Here, we imple-mented computationally-efficient, penalized, logistic regres-sion models (lasso, elastic net, stepwise) to individual level genotype data and a Bayesian framework with continuous shrinkage, “select and shrink for summary statistics” (S4), to summary level data for epithelial non-mucinous ovar-ian cancer risk prediction. We developed the models in a dataset consisting of 23,564 non-mucinous EOC cases and 40,138 controls participating in the Ovarian Cancer Asso-ciation Consortium (OCAC) and validated the best models in three populations of different ancestries: prospective data from 198,101 women of European ancestry; 7,669 women of East Asian ancestry; 1,072 women of African ancestry, and in 18,915 and 12,337 patho-genic variant carriers of European ancestry. In the external validation data, the model with the strongest association for non-mucinous EOC risk derived from the OCAC model development data was the S4 model (27,240 SNPs) with odds ratios (OR) of 1.38(95%CI:1.28–1.48,AUC:0.588) per unit standard deviation, in women of European ancestry; 1.14(95%CI:1.08–1.19,AUC:0.538) in women of East Asian ancestry; 1.38(95%CI:1.21-1.58,AUC:0.593) in women of African ancestry; hazard ratios of 1.37(95%CI:1.30–1.44,AUC:0.592) in pathogenic variant carriers and 1.51(95%CI:1.36-1.67,AUC:0.624) in pathogenic vari-ant carriers. Incorporation of the S4 PRS in risk prediction models for ovarian cancer may have clinical utility in ovarian cancer prevention programs.

Analysing outcomes after prostate cancer diagnosis and treatment in carriers of rare germline mutations in cancer predisposition genes

Coordinator:Ros Eeles, The Institute of Cancer Research and the Royal Marsden Hospital NHS Foundation Trust, UK

Partners:Singer CF, Medical University of Vienna, Wien Susan Domchek, Basser Research Center, Philadelphia, USAElena Castro, Spanish National Cancer Research Center, Madrid, SpainAntonis Antoniou, Cambridge University, UKCIMBA Consortium


GENPROS aims to evaluate the outcomes of patients with rare germline genetic variants including

genes & other PCa predisposition gene mutations following prostate cancer (PCa) diagnosis and treatment.

Main aims:

germline mutation have shorter CSS compared to non-carriers

mutations have a shorter biochemical progression free survival (bPFS) and metastasis free survival (MFS) after radical treatment for PCa than non-carriers. The study will also analyse progression free survival (PFS) and CSS from metastasis (CSS_M1).

-mon allele profiles or specific common alleles also have an association with prognosis and treatment outcome.

BRCA-P: A Randomized, Double-Blind, Placebo-Con-trolled, Multi-Center, International Phase 3 Study to determine the Preventive Effect of Denosumab on Breast Cancer in Women carrying a BRCA1 Germline Mutation


Coordinator:Singer CF, Medical University of Vienna, Austria Garber Judy, Dana Farber Cancer Institute, USA

Partners:Rita Schmutzler, University Hospital Cologne, GermanyJosef Penninger, University of British Columbia, CanadaShani Paluch-Shimon, Shaare Zedek Medical Center, IsraelEphrat Levy-Lahad, Shaare Zedek Medical Center, IsraelMona Elzayet, Europa Donna, ItalyD. Gareth Evans, Manchester University Hospital, United KingdomJoan Vidal, Institute of Oncology, Spainand ABCSG

Collaborators: Tan YY, Brandl I

Abstract:Background: Women who carry a germline mutation in the BRCA1 gene (gBRCA1m) have a lifetime breast cancer (BC) risk in the range of 40-80 percent. Most tumors (~70%) are clinically aggressive triple negative BC. Denosumab (DNSB), an anti-RANKL (RANK ligand) monoclonal antibody, is approved to treat osteoporosis and to manage bone metastases. Ex-perimental evidence has shown that progesterone-mediated upregulation of RANKL/RANK signaling plays a critical role in mammary gland mammary stem cell activation and epithelial cell proliferation. We and others have shown that the RANK pathway is a critical regulator of BRCA1-mutationdriven breast cancer. Genetic inactivation of RANK in mouse mam-mary epithelium markedly delayed onset, reduced incidence, and attenuated progression of Brca1/p53 mutation-driven mammary cancer. Moreover, pharmacological inhibition of RANKL reduced Brca1 mutation-driven neoplastic lesions. In humans, a hyperactive RANK-positive progenitor population has been identified in ostensibly normal human breast tissue from gBRCA1 mutation carriers. Taken together, these data suggest that inhibition of RANKL represents an attractive BC prevention strategy for women with gBRCA1m. Overarching challenge: To Prevent Breast Cancer: Women with gBRCA1m have a remarkably high risk of BC, especially triple negative disease. Currently, bilateral prophylactic mastectomy is the only effective riskreduction strategy. In the BRCA-P trial, we will definitively assess the efficacy of DNSB for breast cancer risk reduction. Safety and acceptability will be explored as these are pre-requisite for uptake in the clinic.

Objective/Hypothesis: As described in the Background, we have experimental evi-dence that RANKL/RANK are critical regulators of BRCA1 mutation-driven BC, and that inactivation of RANK signal-ing in mouse mammary epithelium delayed tumor onset in Brca1/p53 mutation-driven mammary cancer. Brief exposure to DNSB in gBRCA1m women was also found to reduce

breast epithelial proliferation. Therefore, we hypothesize that RANKL inhibition with DNSB will reduce the risk of BC among women with gBRCA1m. Specific Aims: The primary objec-tive of the BRCA-P phase III randomized placebo-controlled chemoprevention trial is to evaluate the reduction in risk of any BC (invasive or DCIS) in women with a gBRCA1m treated with DNSB compared to placebo. About 2,900 women from the US and other countries will be enrolled during the fund-ing period, and create a specimen/image/data repository. Although the primary endpoint is unlikely to be achieved during the funding period, key secondary and exploratory aims will be met: 2. To investigate whether serum OPG lev-els in DNSB or placebo-treated pre- and postmenopausal BRCA1 carriers are inversely associated with breast cancer risk. 3. To evaluate the change in mammographic breast density (MBD) in premenopausal gBRCA1m after 12 months of therapy with DNSB versus placebo. 4. To investigate the impact of 24 months DNSB use in women with a BRCA1 germline mutation on healthrelated quality of life (HRQoL), controlling for potential confounders such as menopausal status and age. 5. To evaluate the effect of DNSB versus placebo on peripheral volumetric BMD, cortical and tra-becular microarchitecture, and estimated bone strength as measured by HR-pQCT scanner. 6. In exploratory studies, we will examine the impact of DNSB on tumor phenotype and molecular profile (including tumor infiltrating lymphocytes). Its effect on peripheral blood immune cell subsets will also be studied. Breast tissue will also be studied in women who withdraw from study and undergo mastectomy. ctDNA will be collected and evaluated as a potential screening strategy for gBRCA1m women.

Study Design: Randomized trial of DNSB versus placebo in premenopausal gBRCA1m with healthy breast tissue conducted 7 countries. Sub-studies in this proposal will use specimens and data from specific subsets of trial participants, comparing DNSB to placebo: 1) 1000 women for sRANKL/OPG/Estradiol/Progesterone blood levels; 2) Mammographic density from US women; 3) QOL data from US/UK/Australia (English-speaking) women; 4) Bone data from women on drug/pla-cebo with access to HR-pQCT scanners; 5) tumor and breast tissue (from prophylactic mastectomies from women who withdraw), FACS analysis on peripheral blood mononuclear cells, ctDNA studies in women who develop cancer versus controls.

Impact: At completion of the project, we should have important information about the effects of Denosumab vs placebo on circulating markers, quality of life, bone and breast tissue of women with gBRCA1m, and will have generated a unique and important repository of blood (including germline DNA for future SNP studies), digital images (MRI and MMG), archival tumor and normal breast tissue for further studies.


CONFLUENCE: genetic architecture of breast cancer

Coordinator:Katherine Nathanson, University of Pennsylvania, USAGeorgia Chevenix-Trench, QIMR Berghofer Medical Re-search Institute, Australia

Partners:Singer CF, Austria, Medical University of Vienna, Wien and CIMBA-BCAC Consortium

Collaborators: Tan YY, Muhr D, Reichl F

Abstract Genome wide association studies (GWAS) have been suc-cessful in identifying over 180 common susceptibility loci for breast cancer. However, heritability analyses indicate that breast cancer is a highly polygenic disease with thou-sands of common genetic variants of small effects, and that increasing sample sizes will generate new discoveries. The Confluence project aims to build a large research resource of over 300,000 cases and 300,000 controls of different an-cestries—doubling current sample sizes to study the genetic architecture of breast cancer. This will be accomplished by the confluence of existing and new genome-wide genotyp-ing data to be generated through this project. The specific aims of this project are: (1) to discover susceptibility loci and advance knowledge of etiology of breast cancer overall and by subtypes, (2) to develop polygenic risk scores and integrate them with known risk factors for personalized risk assessment for breast cancer overall and by subtypes, and (3) to discover loci for breast cancer prognosis, long-term survival, response to treatment, and second breast cancer. To be eligible to participate, studies with cases of in situ or invasive breast cancer (females or males) must have genome-wide genotyping data and/or germline DNA for genotyping, core phenotype data, and appropriate eth-ics approval for genetic studies and data sharing. During September-December 2018, we reached out to potential studies through existing GWAS consortia and other means to request interest in participating in this project. We re-ceived an excellent response demonstrating the feasibility of reaching the target number of cases and controls. This large increase in sample size and diversity of populations will enable discoveries that will lead to a better understanding of the etiology of distinct breast cancer subtypes and the role of genetic variation in prognosis and treatment response, thus improving risk stratification, prevention, and clinical care of breast cancer across ancestry groups.

Gray’s Foundation: Determinants of immune activity and molecular features in BRCA1/2 mutation carriers

Coordinator:Katherine Nathanson, University of Pennsylvania, USAGeorgia Chevenix-Trench, QIMR Berghofer Medical Re-search Institute, Australia

Partners:Singer CF, Austria, Medical University of Vienna, Wien and CIMBA-BCAC Consortium

Collaborators: Tan YY

Abstract:This project will utilize several complementary approaches to understanding immune function in BRCA1/2 mutation carri-ers and their tumors. Understanding the interplay between the molecular features of BRCA1/2 mutation associated breast cancers and the immune features of the cancer will lead to essential insights important not only for understand-ing the role of checkpoint blockade for mutation carriers with cancer, but in general as BRCA1/2 mutated tumors display both elevated tumor mutation burden (TMB) and aneuploidy. We will be able to better understand the interplay between these features in determining immunogenicity, with BRCA1/2 mutation-associated tumors serving as a paradigm for all cancer types. The goal of the project is to evaluate the relationship between the molecular and immune features of BRCA1/2 related tumors. Further, we will correlate the data with polygenic risk scores, including those generated from specific sub-groups of breast cancer risk SNPs predicted to target genes in immune cells.

PARP-register Coordinator:Singer CF, Medical University of Vienna, Austria Tan YY, Medical University of Vienna, Austria

Partners:Petra Kuehhaas, Pfizer Inc.

Abstract:Breast cancer (BC) associated with germline mutations of

represent 3-5% of all cases. Evidence regarding the use of status to guide treatment selection (e.g. platinum-based chemotherapy or PARP inhibitors) in BC is particularly strongest in the metastatic disease setting. To date, data for -associated metastatic BC (mBC) has not been systematically collected and assessed in Austria. This registry will therefore be the first Austrian-wide standard-ized documentation of it. Thus, we plan to set up a registry to collect information as precisely as possible concern-ing initial disease progression, initial tumor characteristics of patients, treatment responses, and health outcomes of


-associated mBC patients. We will collect clinicohis-topathological data, mutation status, treatment responses, and health outcomes (e.g. mortality and quality of life) from all mBC patients in Austria. The data collected will play a key role in identifying subtypes of -associated mBC and to provide a more accurate assessment for treatment response and health outcomes for advanced staged cancer patients. Descriptive and inferential statistics will be used to describe and summarize data collected.

Vocabulary Translation Project Coordinator:Tan YY, Medical University of Vienna, AustriaSinger CF, Medical University of Vienna, Austria

Partners:Arcangela De Nicolo, MD PhD, University of Padua, ItalyPaolo Radice, Fondazione IRCCS Istituto Nazionale dei Tu-mori: Milano, Italyand ENIGMA

Abstract:The vocabulary currently used to describe genetic variants and their consequences reflects many years of studying and discovering monogenic disease with high penetrance. With the recent rapid expansion of genetic testing brought about by wide availability of high-throughput massively parallel sequencing platforms, accurate variant interpretation has become a major issue. The vocabulary used to describe single genetic variants in silico, in vitro, in vivo and as a contributor to human disease uses terms in common, but the meaning is not necessarily shared across all these contexts. In the setting of cancer genetic tests, the added dimension of using data from genetic sequencing of tumour DNA to direct treatment is an additional source of confusion to those who are not experienced in cancer genetics. The language used to describe variants identified in cancer susceptibility genetic testing typically still reflects an outdated paradigm of Mendelian inheritance with dichotomous outcomes. Cancer is a common disease with complex genetic architecture; an improved lexicon is required to better communicate among scientists, clinicians and patients, the risks and implica-tions of genetic variants detected. This review arises from a recognition of, and discussion about, inconsistencies in vocabulary usage by members of the ENIGMA international multidisciplinary consortium focused on variant classification in breast-ovarian cancer susceptibility genes. It sets out the vocabulary commonly used in genetic variant interpretation and reporting, and suggests a framework for a common vocabulary that may facilitate understanding and clarity in clinical reporting of germline genetic tests for cancer susceptibility.

Using ovarian cancer tumour characteristics to refine likelihood ratio models for the evaluation of unclassified variants of BRCA1 and BRCA2

Coodinator:Tan YY, Medical University of Vienna, Austria

Partners:Kyriaki Michailidou, PhD, Cyprus School of Molecular Medi-cine, Cyprusand ENIGMA

Abstract:It has been shown that ovarian tumors in individuals that carry a pathogenic variant in or or both exhibit a distinct distribution of pathological features (Lakhani et al. 2004), with a higher percentage being invasive serous adenocarcinoma and very unlikely to be borderline or muci-nous tumors (Lakhani et al 2004, Zhang et al 2011, Song et al 2014, Hirasawa et al 2017). Furthermore, both - and

-related tumors were of higher grade, had a higher percentage of solid component and were more likely to stain strongly for p53 (Lakhani et al. 2004, Soegaard et al. 2008, Alsop et al. 2012). Histopathological and other tumour characteristic features of ovarian cancers have the potential to be used together with other information for statistical modeling to assess pathogenicity of and VUS.

The Multifactorial Likelihood Model developed for the evalu-ation of VUS of and (Goldgar et al 2004) was extended to include likelihood ratio estimates of pathogenic-ity based on tumour histopathological features of breast cancers (Spurdle et al 2008). Large-scale analysis of data from the CIMBA and BCAC consortia was subsequently used to re-derive estimates for breast tumour pathology likelihood ratios to assess pathogenicity of and variants (Spurdle et al 2014). Currently the model does not include any information on any histopathology features of ovarian cancer tumors.

Previous studies exploring the histopathologic and other tumour characteristics of ovarian cancer in mutation carriers compared to non-carriers were from single and relatively small datasets. We aim to conduct analyses of the com-bined pathology datasets accrued by the Consortium of Investigators of Modifiers of BRCA1/2 (CIMBA), OTTA and other ENIGMA collaborators to determine independent pre-dictors of mutation status. We will compare histopathology and tumour characteristics between carriers and non-carriers, in order to estimate likelihood ratios in favour or against pathogenicity for specific ovarian tumour features.

We aim to investigate the distribution of ovarian tumour characteristics in and carriers compared to non-carriers, and calculate likelihood ratios (LRs) to be used in the multifactorial probability model already used for the


classification of Variants of Uncertain clinical Significance (VUS) in BRCA1/2 genes. Conventionally BRCA1 and BRCA2 clinical genetic testing in ovarian cancer patients was mainly performed in high grade serous patients. However, for this investigation we need to use data from sites that performed

testing irrespective of tumour characteristics, or at least in a defined subset that encompassed more than just high grade serous ovarian tumours, in order to derive meaningful LRs for use in the clinical setting.

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

KNÖFLER, PIPOLLHEIMER, PI

HAIDER, PhD, PostDoc HASLINGER, BMA

KUNIHS, BMAMEINHARDT, PhD, BMA

SALEH, BMADIETRICH, PhD Studentin

HÖBLER, PhD StudentinLACKNER, PhD Student

VONDRA, PhD StudentinWINDSPERGER, PhD Studentin

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

, Universität Wien, Master FRITZ, FH Campus Wien, Bachelor

JUNG, FH Campus Wien, Master

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

EBNER, Veterinärmedizinische Universität Wien, Praktikantin

SCHUSTER, Ferialarbeitnehmer


WISSENSCHAF TLICHE PROJEKTE

PLAZENTATIONS-ABHÄNGIGE EFFEKTE AUF DEZIDUALE MAKROPHAGEN

M I T A R B E I T E R I N N E N :

Saleh L, Bauer R, Husslein H, Knöfler M, Pollheimer J

KurzbeschreibungDie Fortpflanzung bei höheren Säugetieren ist von einer gut koordinierten Wechselwirkung zwischen Plazenta und Uterus abhängig. Während der Frühschwangerschaft lösen sich extravillöse Trophoblasten (EVTs) von der Plazenta ab und dringen in das dezidualisierte Gebärmuttergewebe ein, um die maternal-fetale Schnittstelle herzustellen. EVTs er-weitern uterine Spiralarterien, welche somit den wachsen-den Feten mit größeren Mengen Blut versorgen können. Fehler in der EVT Funktion sind assoziiert mit intrauteriner Wachstumsrestriktion, Präeklampsie und einer erhöhten Rate an Totgeburten. Die Etablierung der maternal-fetalen Schnittstelle ist auf die Decidua basalis (decB) beschränkt, während die Decidua parietalis (decP) auf der gegenüber-liegenden Seite nicht von der Plazentation betroffen ist. Aus diesem Grund stellen wir uns die Frage, ob die Plazen-tation regional spezifische Veränderungen in den Zellen der decB bewirkt. Aus detaillierten Analysen wissen wir bereits, dass Makrophagen (MΦ) der dominante Immunzelltypus an der maternal-fetalen Schnittstelle sind. Darüber hinaus zeigten RNAseq Analysen, dass MΦ, welche aus der decB isoliert wurden, sich substanziell von decPMΦ unterschieden (Abb. 1). Unser Ziel ist es, den Phänotyp von decB- und decPMΦ gründlich zu charakterisieren und durch meta-

bolisches „RNA labeling“ die EVT-induzierte de novo Tran-skription in decMΦ Subtypen zu determinieren. Außerdem planen wir die phagozytische Eigenschaften von decBMΦ und decPMΦ zu beforschen und sie nach ihrer Fähigkeit, Antigene zu präsentieren, regulatorische T-Zellen auszubil-den oder andere Immunzellen anzulocken, zu untersuchen. Die Proliferation von Gewebsmakrophagen wird in situ und in vitro untersucht. Ein breites Spektrum an Patientinnenma-terial, darunter archivierte Fälle von spontanen Aborten und Gebärmutterproben sowie rekrutierte, knochenmarkstrans-plantierte Schwangere, soll es uns ermöglichen, spezifische Eigenschaften von decMΦ und ihren Ursprung weiter zu entschlüsseln.

IMMUNZELLDYNAMIKEN AN DER FETO-MATERNALEN SCHNITTSTELLE

M I T A R B E I T E R I N N E N : Lackner AI., Vondra S., Höbler AL., Kunihs V, Windsperger K, Saleh L, Knöfler M, Bauer R, Pollheimer J

KurzbeschreibungGenerell zeigt die Plazentaentwicklung große Unterschiede zwischen den einzelnen Säugetierspezies, sowohl anato-misch und morphologisch als auch in der Expression von spezifischen Transkriptionsfaktoren. Daher sind humane Modellsysteme für die Erforschung spezifischer Plazenta-funktionen von großem Wert. Andererseits können solche ex-vivo Systeme die Wechselwirkung mit umgebenden phy-siologischen Gewebsdynamiken, einschließlich stromaler Wechselwirkungen, Kommunikation mit ortsansässigen Immunzellen und perfundierten Gefäßsystemen, nicht re-kapitulieren. In diesem Sinne eignen sich humane in-vitro Systeme nicht um etwa Mechanismen der Krebsmetasta-sierung zu erforschen oder, relevant für dieses Projekt, kon-text- und organspezifischen Akkumulation von peripheren

Abbildung 1: A) Darstellung von signifikant unterschiedlich exprimierten Genen in in decPMΦs (rot) und decBMΦs (grün) B) Heat Map von vorselek-tionierten, MΦ-assoziierten, differentiell exprimierten Genen in decPMΦs und decBMΦs.


Immunzellen aus dem Knochenmark zu studieren. Kürzlich hat unsere Gruppe ein sich selbst organisierendes und er-neuerndes humanes Plazenta-Organoidsystem etabliert. Diese Plazentaorganoide spiegeln die zelluläre Heterogenität innerhalb der Plazenta, durch spontane Bildung von synzyti-otrophoblastären Schichten und der kontrollierten Bildung von invasiven, extravillösen Trophoblasten bei Entzug von Wnt-abhängigen Signalen, wider. Im Zuge dieses Projektes, planen wir zum ersten Mal, humane Plazenta-Organoide in NSG (NOD-SCID-Gamma) Mäuse zu transplantieren. Ziel ist es den Effekt der Plazentation auf die Rekrutierung von kno-chenmarkstämmigen myeloiden Zellpopulationen zu unter-suchen. Wir gehen außerdem davon aus, dass menschliche Plazenta-Organoide auch spezifische Veränderungen der lokalen Immunzellpopulationen im umliegenden Geweben bewirken (Abb.2)

Abbildung 2: Versuchsaufbau für das oben beschriebene Projekt. Zu Beginn der Studie, planen wir intrakutane Transplantationen von humanen Plazenta-Organoiden in NSG Mäuse zu etablieren (1). Ein weiteres Ziel ist

-rung von myeloiden Knochenmarksstammzellen mittels transplantierter

transplantierter humaner CD34+ Nabelschnurblutzellen zu untersuchen (2-3). Außerdem interessieren wir uns in diesem Projekt für plazentaspe-zifische Effekte auf lokale gewebsansässige Immunzellpopulationen und infiltrierende myeloide Zellen (4).

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, Dekan S, 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 Gewebe 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. Trimenons (6.-13.SSW) mittels spezifischer Antikörper (MECA-79, SELE, ICAM-I, EPHB4, Willebrand Faktor) zu charakterisieren (Abb. 3). Im Detail, korreliert in beiden Gewebsarten die Anzahl an HEVs mit der Ratio lokaler CD11c+/CD45+-myeloider Immunzellen, 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 3: 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.

Densities of decidual high endothelial venules correlate with T-cell influx in healthy pregnancies and idiopathic recurrent pregnancy lossesWindsperger K, Vondra S, Lackner AI, Kunihs V, Haslinger P, Meinhardt G, Dietrich B, Dekan S, Fiala C, Knöfler M, Saleh L, Pollheimer J. Hum Reprod. 2020 Nov 1;35(11):2467-2477


NEUROSTEROIDE ALS NEUE MARKER IN DER DIAGNOSTIK DER PRÄEKLAMPSIE SOWIE IN DER PROGNOSE DES NEONATALEN OUTCOMES?

M I T A R B E I T E R I N N E N : -

heimer J

KurzbeschreibungMit einer Inzidenzrate von 2-8% zählt die Präeklampsie (PE) in den industrialisierten Ländern zu den führenden Ursachen mütterlicher und neonataler Morbidität sowie Mortalität. Trotz großer Forschungsbemühen zeigt sich die Pathogenese bis dato unvollständig geklärt und so stellt die zeitgerechte Diagnostik eine besondere Herausforderung in der klinischen Praxis dar.

Kennzeichnend für ein frühes Krankheitsstadium (plazen-tares Stadium), in welchem sich die Schwangeren noch asymptomatisch präsentieren, ist die fehlerhafte Plazentati-on, insbesondere die verminderte Trophoblasteninvasion in die mütterliche Dezidua und Spiralarterien. Folglich resultie-ren ein unvollständiger Umbau der uterinen Gefäße, ein hypo-xisches Milieu und letztendlich eine plazentare Dysfunktion.

Abbildung 4: Strukturformel von Allopregnanolone (ALLO)

Von diesen Fehlfunktionen betroffen, zeigt sich u.a. die Se-kretion plazentarer Faktoren in die mütterliche und fetale Zirkulation abnorm verändert. Der 3,5-reduzierte Progesteron-Metabolit Allopregnanolone (ALLO) ist ein Neurosteroid, der durch seine via GABA-A-Rezeptoren mediierte neuroprotektive Wirkung, u.a. für eine adäquate fetale Hirnentwicklung verantwortlich ist. Um eine ausreichende Menge an diesem Neurosteroid in der Schwan-gerschaft sicherzustellen, produziert die Plazenta nicht nur das Neurosteroid selbst, sondern auch dessen Vorstufen und sezerniert diese in die mütterliche und fetale Zirkulation. Die-se können in weiterer Folge im fetalen Gehirn direkt in ALLO metabolisiert werden. Für die Erfüllung dieser Funktion expri-miert die Plazenta die Enzyme 5α-Reduktase I (SRD5A1), II (SRD5A2) und 3α-Hydroxysteroidoxidoreduktase (3α-HSD). Interessanterweise zeigen zahlreiche tierexperimentelle Stu-dien, dass die Plazenta auf akute hypoxische Reize (z.B. zu Beginn einer Präeklampsie) mit einer rapiden Synthesestei-gerung von ALLO reagiert. Chronische hypoxische Stressoren hingegen, bedingen erniedrigte ALLO-Konzentrationen und

sind mit einem schlechten entwicklungsneurologischen, ne-onatalen Outcome assoziiert. Umfassende Analysen dieses vielversprechenden Markers in humanen Schwangerschaften sind bis dato jedoch ausständig.

Daher plant die, von der AGFMM (mit-)finanzierte Studie, die bisher unbekannte Dynamik von ALLO in humanen Schwan-gerschaften mit unkompliziertem Verlauf versus Schwanger-schaften, die mit einer Präeklampsie verkompliziert sind, zu vergleichen sowie die prognostische Aussagekraft von ALLO bezüglich des neonatalen Outcomes zu beleuchten.

ENTWICKLUNG DER PLAZENTA UND EXTRAVILLÖSEN TROPHOBLASTEN WÄHREND DER FRÜHSCHWANGERSCHAFT Im Rahmen des vom FWF geförderten Projektes (APP-31470FW) studieren wir die Entwicklung des Trophoblasten in einem 3-D Modell sowie die Rolle des Notch1-abhängigen Signalweges.


In Kooperation mit Dr. Paulina Latos, Zentrum für Anatomie und Zellbiologie, konnten wir 2018 ein neues 3D „in vitro“ Modell der frühen humanen Plazenta entwickeln (Haider et al. 2018; ). Durch Optimierung der Zell-kulturbedingungen ist es uns gelungen, 3D Organoide des Trophoblasten aus humaner Plazenta zu etablieren. Die Tro-phoblast Organoide enthalten trophoblastäre Stammzellen, die innerhalb von 30 Tagen ein aus mehreren tausend Zellen bestehendes Organoid ausbilden können. 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 Differen-zierung zum invasiven Trophoblasten. B. Dietrich studiert im Rahmen ihrer Dissertation die Funktion von Notch1 in Organoiden. Hierzu werden Notch1 dominant-positive und -negative Konstrukte induzierbar exprimiert und Notch1 Zielgene identifiziert und funktionell untersucht. Das Projekt soll zu einem besseren Verständnis der Plazentaentwicklung und der damit verbundenen Fehlfunktion bei Schwanger-schaftserkrankungen beitragen. Die neuesten Erkenntnisse der Regulation der Plazentaentwicklung wurden auch kürzlich in einem Review zusammengefasst (Knöfler et al. 2019, Cell Mol Life Sci). Das 3D Modell wurde von B. Dietrich und V. Kunihs auch für die Etablierung von JEG-3 Zellorganoiden verwendet. In einer kürzlichen Publikation (Dietrich et al., in press) konnten diese beide Mitarbeiterinnen zeigen, dass JEG-3 Organoide ähnliche den trophoblastären Stammzellen langsamer wachsen und die Differenzierung in extravillöse Trophoblasten (EVT) unterdrücken. Bei Entfernung des Wnt Stimulators werden die EVTs neu gebildet. Das System er-laubt daher die Differenzierung in gerichteter Art und Weise zu untersuchen.


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

Human placenta and trophoblast development: key mole-cular mechanisms and model systemsKnöfler M, Haider S, Saleh L, Pollheimer J, Gamage TKJB, James JCell Mol Life Sci. 2019 Sep;76(18):3479-3496

Dietrich B, Kunihs V, Haider S, Pollheimer J, Knöfler M3-Dimensional JEG-3 choriocarcinoma cell organoids as a model for trophoblast expansion and differentiation. Pla-centa, in press

DIE ROLLE DES KO-AKTIVATORS TAZ IN DER ENTWICKLUNG DER PLAZENTA Im Rahmen des vom FWF geförderten Projektes (APP-31470FW) studieren wir Funktion von TAZ in der extravillösen Trophoblasten Entwicklung.


Der Hippo Signalweg ist für die Funktion von Trophoblasten essentiell. In einer kürzlichen Publikation konnten wir zeigen,

von Trophoblaststammzellen und Vorläuferzellen in der sich entwickelnden Plazenta von entscheidender Bedeutung ist (Meinhardt et al., 2020).

-blast stemness of the developing human placenta Meinhardt G, Haider S, Kunihs V, Saleh L, Pollheimer J, Fiala C, Hetey S, Feher Z, Szilagyi A, Than NG, Knöfler M. Proc Natl Acad Sci U S A. 2020 Jun 16;117(24):13562-13570

In diesem Folgeprojekt studieren wird die Rolle des zweiten Hippo Ko-Aktivators TAZ in der humanen Plazenta. Fr. Dr. Meinhardt konnte zeigen, dass TAZ spezifisch in den ex-travillösen Trophoblasten (EVTs) produziert wird und dort mit seinem Bindungspartner TEAD1 interagiert. TAZ dürfte multiple Aufgaben in EVTs erfüllen. Zum einen konnte gezeigt werden, dass TAZ die Produktion des Oberflächenmarkers HLA-G und damit Differenzierung steuert, aber auch für das Überleben der Zellen und die Ausprägung der Polyploidie in EVTs von entscheidender Bedeutung ist. Die wissenschaft-liche Arbeit hierzu befindet sich in Vorbereitung.

Abbildung 5: Extravillöse Trophoblasten (EVT) exprimieren TAZ/TEAD1: Immunfluoreszenzfärbungen (A) zeigen, dass Trophoblasten der Früh-schwangerschaft im Laufe der Differenzierung zum HLA-G positiven (rot) Extravillöse Trophoblasten (EVT) den Co-Aktivator TAZ (weiß) und den Transkriptionsfaktor TEAD1(weiß), hochregulieren. DAPI-gefärbte Zell-kerne sind in blau zu sehen. (B) Immunpräzipitationen beweisen, dass bei-de Proteine miteinander in EVTs, nicht aber in den weniger differenzierten, villösen Cytotrophoblasten (vCTBs) Komplexe bilden und miteinander interagieren können. VS: villöses Stroma, CC Zellsäulentrophoblast, EVT: extravillöser Trophoblast.

Abbildung 6: EVTs, entstanden aus Trophoblastorganoidkulturen (TB-

der EVTs wenn sie unter differenzierenden Bedingungen kultiviert werden. Mittels Immunfluoreszensfärbungen kann die starke Zunahme von HLA-G (weiß) und kernständigem TAZ (rot) in den außenliegenden Zellen nach-gewiesen werden. Zellkerne sind mit DAPI (blau) gefärbt. Die Vermessung der Zellkerngrößen (B) der stark TAZ-positiven EVTs ergab eine deutliche Zunahme der Kerngrößen. Zellzyklusanalysen, durchgeführt mittels DAPI- Flowzytometrie, bekräftigt die Anzeichen einer Polyploidisierung in HLA-G positiven Zellen, wie sie auch in vivo vorkommt. Das Histogramm zeigt DNA-Profile von HLA-G- positiven (violette Linie) und negativen (grün) Trophoblasten isoliert aus TB-Oganoidkulturen unter differenzierenden Bedingungen.


-

Trophoblaststammzellen zeigen in Immunfluorezenfärbungen (A) unter differenzierenden Bedingungen weniger HLA-G (grün) und kernständiges

(blau) gefärbt. (B) Die Vermessung der Kerngrößen ergab eine deutliche

behandelten Zellen eindeutig geringer war. Mittels HLA-G Flowzytometrie -

renden EVT-Zellkulturen (D) weniger HLA-G positive Zellen nachweisbar.

3D OPTICAL COHERENCE TOMOGRAPHY (OCT) TO MEASURE TROPHOBLAST ORGANOIDS


In Kooperation mit Abigail Deloria, MSc und Dr. Richard Haindl vom Zentrum für Medizinische Physik und Biome-dizinische Technik wurde eine 3-dimensionale optische Kohärenz Tomographie (OCT) für die Analyse von in-vitro kultivierten Trophoblast Organoiden (TB-ORG) entwickelt. TB-ORG sind einzigartige Modellsystem der frühen humanen Plazenta, bei welchen außen-liegende undifferenzierte Tro-phoblasten in Richtung Zentrum zu mehrkernigen, hormon-produzierenden Synzytiotrophoblasten hin differenzieren (Haider et al., 2018, Stem Cell Reports). Für diese Studie wurde ein „ultra-high-resolution spectral domain OCT Sys-tem“ mit einer räumlichen und axialen Auflösung von 2.48 nm individuell für TB-ORG angepasst. Der Vorteil in dieser Technik liegt darin, dass die Morphologie der TB-ORG wäh-rend der Kultivierung hochauflösend gemessen werden kann ohne den Versuch dadurch beenden zu müssen (Abb. 8). In der Studie wurde durch Paralleluntersuchungen gezeigt, dass die mittels OCT detektierten inneren Strukturen der TB-ORG mit dem Grad der Synzytien-Differenzierung perfekt korre-lieren. Mit dieser neuen OCT Technik kann man erstmals

den Grad der Differenzierung und die optische Struktur im Inneren der Organoide auf nicht-invasive Weise vermessen und auswerten.

Abbildung 8: Die OCT Technik erlaubt eine detaillierte Analyse der inneren

100 µm. Die Länge des Kubus beträgt 500 µm

Ultra-high-resolution 3D optical coherence tomography re-veals inner structures of human placenta-derived trophoblast organoidsAbigail Joyce Deloria, Sandra Haider, Bianca Dietrich, Victoria Kunihs, Simon Oberhofer, Martin Knofler, Rainer A Leitgeb, Mengyang Liu, Wolfgang Drexler, Richard HaindlIEEE Trans Biomed Eng 2020 Nov 17;PP.doi:10.1109/TBME.2020.3038466

INTERAKTION VON VILLÖSEN ZELLEN WÄHREND DER HUMANEN FRÜHSCHWANGERSCHAFT


Die Interaktion von epithelialen und stromalen villösen Zel-len während der Frühschwangerschaft ist ein essentieller Vorgang während der frühen Plazentaentwicklung. Fehler-hafte Prozesse diesbezüglich werden zu den Risikofaktoren für die Entwicklung von Schwangerschaftskomplikationen wie z.B. Aborte, intra-uterine Wachstums Retardierung und Präeklampsie gezählt. Aus diesem Grund ist eine lücken-lose Erforschung der einzelnen Plazentazellen und deren Interaktionsvorgängen, die letztendlich Entwicklungs- und Differenzierungsprozesse steuern, unabdingbar. In dem ein-gereichten FWF Projekt (P 34588-B) planen wir, anhand neuester Methoden (single-cell und spatial Transcriptomics)


und Zellmodellen (Stammzellen und Organoide; Trophoblast-Organoide, TB-ORG, Haider et al., 2018, Stem Cell Reports) in einer Kooperation mit Dr. Jian Shu, Massachusetts General Hospital, eine detaillierte Zell-Landkarte während der ersten Schwangerschaftsmonate zu konstruieren. Weiters werden wir mittels deskriptiven und funktionellen Methoden die Interaktion einzelner Plazentazellen erforschen (Abb. 9 und 10). Dies ermöglicht uns in weiterer Folge, physiologische und eventuelle pathophysiologische Prozesse während der Frühschwangerschaft besser zu verstehen. Unser langfri-stiges Ziel liegt auch darin, bei jenen Schwangerschaftskom-plikationen, deren Pathologien sich erst später manifestieren (Präeklampsie, IUGR) bessere Früherkennungs- und ggf. Therapieansätze zu studieren.

villösen mesenchymalen Zellen (Stromazellen). (A) Durchlicht-Bilder von Ko-Kulturen über 9 Tage zeigt die rasche Interaktion von villösen Stroma-

Abbildung 10: Immunfluoreszenz Bilder von 3D Ko-Kulturen bestehend

Stromazellen an die Organoide anheften (obere Bildreihe) und diese miteinander vernetzen (untere Bildreihe). Auf der linken Seite sind 3D

gelb (Stromazellen, Vimentin) gefärbt. Auf der rechten Seite sind zusätz-

mittels DAPI blau markiert.

INTERAKTION VON MÜTTERLICHEN UND FETALEN ZELLEN WÄHREND DER HUMANEN PLAZENTAENTWICKLUNG IN DER FRÜHSCHWANGERSCHAFT


Eine unkomplizierte Schwangerschaft hängt von einer ganzen Reihe unterschiedlicher Faktoren während der er-sten Schwangerschaftswochen ab, wie z.B. die korrekte Entwicklung der Föten und der Plazenta sowie eine adäquate Adaption der Mutter an die Schwangerschaft. Maßgeblich trägt hier die Invasion spezialisierter Trophoblasten, der sog. extravillösen Trophoblasten (EVT), in die mütterliche Dezidua bei.

Abbildung 11: Illustration der EVT Entwicklung, Invasion und Interaktion mit maternalen Zellen. Die lokale Proliferation von villösen Zytotropho-blasten (vCTB) führt zur Formierung von Zellsäulen (pCC, proximale Zellsäulen). Distal kommt es zur Differenzierung von pCC in HLA-G-posi-tive Subtypen. Diese wanderen in die mütterliche Dezidua ein und kommu-nizieren mit Immunzellen (Makrophagen, uterinen NK-Zellen), Stromazel-len, Endothelien von Arterien, Venen und Lymphen sowie mit Drüsenzellen.

Durch eine aktive Interaktion der EVTs mit maternalen Zel-len (Abb. 11) werden nicht nur maternalen Spiralarteriolen optimal für die weitere Schwangerschaft umgebaut, auch wird das mütterliche Immunsystem modifiziert, um eine Abstoßung des „genetisch halb-fremden“ Föten zu ver-hindern. Interessanter Weise interagieren EVTs auch mit endometrialen Drüsen, wodurch besonders in den ersten Wochen der Schwangerschaft, wenn die Versorgung über das Gefäßsystem der Mutter noch nicht etabliert ist, eine ausreichende Ernährung von Kind und Plazenta gewährleistet wird. Tatsächlich sind aber besonders diese frühen Wochen in der humanen Schwangerschaft unzureichend erforscht und viele Fragen offen. Deshalb haben wir einen weiteren FWF Antrag eingereicht, um mittels Einsatzes modernster deskriptiver Techniken (single-cell und spatial Transcripto-mics) und Zellmodellen (Stammzellen, Organoide) einerseits die Entwicklung und Differenzierung der EVTs als auch deren Interaktion mit maternalen Stromazellen und endometrialen Drüsen zu erforschen (Abb. 12). Hier kommt uns unsere Ex-pertise bei Organoid-Modellen der Trophoblasten (TB-ORG, Haider et al., 2018, Stem Cell Reports) und endometrialen Drüsen (EG-ORG, Haider et al., 2019, Endocrinology) zugute.


Abbildung 12: EVTs interagieren mit endometrialen Drüsenorganoiden

Drüsenlumen vordringen (invaded iCTBs, rechts).

BIO-ENGINEERING DER HUMANEN PLAZENTA DES ERSTEN SCHWANGERSCHAFTSTRIMESTERS


Die Entwicklung von in-vitro Kulturbedingungen von Tropho-blast Organoiden (TB-ORG, Haider et al., 2018, Stem Cell Reports) und Trophoblast Stammzellen (TSC, Okae et al., 2018, Cell Stem Cell) war ein Meilenstein in der humanen Plazentaforschung. Trotz der Vielzahl an neuen Möglichkeiten (siehe oben), gibt es aber auch hierbei Anwendungsgrenzen, welche die Erforschung wichtiger Fragestellungen derzeit noch erschwert. Tatsächlich kann man durch die Natur von TB-ORG (3-dimensionale, kompakte Trophoblast-Strukturen) und TSC (2-dimensionale, herkömmliche Zellkultur) Trans-portvorgänge und pathogene Durchtritts-Möglichkeiten durch die frühe Plazentaschranke nur sehr unzureichend studieren. Plazenta-Perfusionsmodelle, welche u.a. an der Universität in Graz durchgeführt wird, können aufgrund der unterschiedlichen Struktur zwischen frühen und späten Pla-zenten (Abb. 13) nicht als Transport-Modellsystem für das erste Trimester verwendet werden. Auch repräsentieren Plazenta-on-a-chip Modelle derzeit ausschließlich die Bar-riere der Terminplazenta.

Abbildung 13: Unterschiede der frühen (1. Trimester) und späten (3.

Trimester) Plazenta. Während das Trophoblast (TB) Epithel (grün) in der Frühschwangerschaft 2-reihig ist und aus villösen Zytotrophoblasten (vCT-Bs, rote Kerne) und Synzytiotrophoblasten (STBs, blaue Kerne) besteht, findet man in der Terminplazenta nur mehr vereinzelt vCTBs. Dadurch ändert sich die Dicke der Plazentabarriere, welche 20 – 30 µm im ersten und 2 – 4 µm im dritten Trimester beträgt.

Deshalb sind wir derzeit dabei, in Kooperation mit Experten der Materialwissenschaft der TU Wien (Prof. Ovsianikov und Dr. Markovic) ein Modellsystem zu etablieren, dass den Aufbau der frühen Plazentaschranke repräsentiert. Hierbei

verwenden wir eine spezielle, methacrylierte Gelatine (gel-MA), welche sich in Vorversuchen als ideale Matrix für die Ansiedlung von TSCs herausgestellt hat (Abb. 14).

Abbildung 14: Illustration des in-vitro Modells für das 2-reihige Tropho-blastepithel der humanen, frühen Plazenta. Villöse Stromazellen (VCC) werden mit methacrylierter Gelatine (gel-MA) vermischt und die Struktur wird mittels UV gefestigt (I-II). TSCs werden angesiedelt (III) und nach einigen Tagen bildet sich ein multinukleäres Synzytiotrophoblastepithel (STB) über der TSC Schichte (IV).

In Vorversuchen konnten wir bisher sehen, dass die angesie-delten TSCs tatsächlich ein 2-reihiges Epithel bilden, welches der frühen Plazenta entspricht (Abb. 15).

Abbildung 15: In-vitro generiertes 2-reihiges Trophoblast (TB) Epithel. (A) Ähnlichkeiten zwischen in-vitro Modell (oben) und in-situ Plazenta des ersten Trimesters. (B) Das 2-reihige, in-vitro TB Epithel besteht aus villösen Zytotrophoblasten (vCTBs) (positiv für p63, TEAD4, E-cadherin) und eine darüber liegende Synzytiotrophoblastschichte (STB, positiv für CG', ENDOU).

Zu diesem Thema werden wir in Kooperation mit der TU Wien einen FWF Antrag einbringen um, unter anderem Trans-portvorgänge über die frühe Plazentaschranke sowie deren Suszeptibilität für Pathogene studieren zu können (Placenta-on-a-chip, Abb. 16).


Abbildung 16: Plazenta-on-a-chip des frühen Trophoblast Epithels. TSCs und Endothelzellen (EC) werden auf der jeweils gegenüberliegenden Seite einer semipermeablen Membran angesiedelt. TSCs bilden nach einigen Tagen ein Epithel bestehend aus Membran-kontaktierenden villösen Zytotrophoblasten (vCTBs) und darüberliegenden Synzytiotrophoblasten (STBs). Durch die Kontrolle des Kulturmediums im upper channel und lower channel kann man den Transport von gewünschten Substanzen detektieren.

IN VITRO ZELLKULTURMODELL ZUR ANALYSE DER GLANDULÄRE DEZIDUALISIERUNG

M I T A R B E I T E I N N E N :

Eine Grundvoraussetzung für eine erfolgreiche Schwanger-schaft ist ein auf die Implantation korrekt adaptiertes En-dometrium. Dieser Differenzierungsprozess, die sogenannte Dezidualisierung, führt zu morphologischen und bioche-mischen Veränderungen aller Zellpopulationen des Endome-triums und erlaubt die Aufnahme des semi-allogenen Fötus. Während die Dezidualisierung der Stromazellen von uns und anderen über die letzten Jahre intensiv studiert wurde (Saleh et al. 2011, Otti et al. 2014) ist der Differenzierungsprozess in den endometrialen Drüsen noch weitgehend unerforscht. Letztere spielen aber eine essentielle Rolle, da in den ersten Wochen der Implantation, erodierte Drüsen die primäre Nah-rungsquelle für die Blastozyste darstellen. Zudem kommt es in weiterer Folge zu einer intensiven Interaktion zwischen

dezidualen Drüsen und einer bestimmten Plazentazellpopu-lation, den extravillösen Trophoblasten (EVTs). Wie auch in deziduale Gefäße, invadieren die EVTs in die Drüse wobei der Grund für die glanduläre Invasion noch unbekannt ist.

Abbildung 17: Immunfluoreszenzfärbung des Endometriums während des Zyklus (proliferative Phase, sekretorische Phase), sowie während der Schwangerschaft (Dezidua, 8 SSW). Markerproteine sind in grün dargestellt, die korrespondierenden nukleären Färbungen mit DAPI (blau) darunter. Fox A2

zeigt eine positive Kernfärbung in jeder Zyklusphase und der Dezidua während das Stroma negativ ist. Progesteronrezeptor □□□/□ (dritte

□□/□), ein Markerprotein für Drüsen der proliferativen Phase, verschwindet in Drüsen der sekretorischen Phase und Dezidua während das Stroma positiv bleibt. Glykodelin hingegen färbt spezifisch Drüsen


P E R S O N A L S T A N D 2 0 2 0WOLF (BMA)KENDLER (Master Student)BARTL (PhD Student)

ALBERTS (Master Studentin)CASTILLO-TONG (ao.Univ.-Prof.Dr.)

Project 1: Molecular characterization and identification of tumor specific markers of mucinous ovarian cancer

Background and aims of the project:

Ovarian cancer is the fifth most prevalent cause of cancer-related death in women and the gynecological malignant disease with the highest mortality rate and poorest prognosis in the western world. Whereas the predominant subtype of high-grade serous epithelial ovarian cancer (HGSOC) is well characterized, studies on tumorigenesis, disease progression and treatment strategies for the subtype mucinous ovarian cancer (MOC) are rare. MOC contributes to approximately 3% of all ovarian malignancies with a distinct molecular back-ground, spread pattern and chemosensitivity. Biologically, it is considered closer to gastrointestinal mucinous carcinoma

Despite both its distinct mutational background and clini-cal behavior, patients with MOC are treated with standard adjuvant platinum-based chemotherapy due to a lack of potential alternatives. Obviously, MOC is less sensitive to cytotoxic chemotherapy regimens than HGSOC, resulting in an impaired prognosis with median overall survival rates of only 12-14 months for patients with advanced stages. A major reason for the shortcomings in research and treatment is the low incidence of this histotype of cancer.

Our project on MOC has the following scientific and tech-nologic aims:

FFPE blocks);

sequencing of enriched tumor cells from primary tumor materials and RNA from FFPE;

disease progression of MOC by molecular analysis of the RNA sequencing data;

cell lines;

Methods and results:

Collection of biological materialsAbout 40 FFPE blocks of MOC tumor tissues, the correspon-ding clinical information and follow-up data of the patients have been collected. Currently, two cell lines have been established from MOC. Tumor cells form ascites of the pa-tients will be enriched and deposited at -80°C for further processing (on going).

DNA and RNA extractionDNA and RNA can be isolated from FFPE sections. DNA can be used to sequence the hot-spot mutation regions in the

and TP53 genes. RNA could be obtained in sufficient quantity to perform gene expression analyses. The quality of the RNA is being examined.

Determination of biomarkers on the FFPE sectionsIn order to examine the potential therapeutic targets, we carried out IHC staining to determine the protein expression of ER, Mek1/2, and HER2. Preliminary results showed that ER was not expressed in the MOC in contrast to HGSOC, which has higher expression in nucleus of almost all tumor cells (Figure 1). Nevertheless, the MOC seemed to have higher number of ER+ stromal cells. HER2 however, was expressed in MOC, but not as strong as in HGSOC and it seemed that it was not expressed in all tumor cells (Figure 2). Mek1/2 had a strong expression in most of the MOC tumor cells, but was not expressed in all tumors (Figure 3).


nucleus in most of the tumor cells can be observed. C.-F.: MOC, Tumor Figure 3. IHC staining of Mek1/2. A. B: HGSOC; C.-F.: MOC.

Project 2. Combating ovarian cancer by hampering tumor cell proliferation

Background and objectives of the projects:

High grade serous ovarian cancer (HGSOC) accounts for about 75% of all epithelial ovarian cancer. Standard first line treatment includes cytoreductive surgery and platinum-based chemotherapy. Although most patients show primary response, the disease frequently recurs. The patients will not be operated by recurrent disease and are treated with carboplatin in most of cases. Many of the patients show clinical resistance, presenting increasing concentration of tumor marker CA125, and die of disease progression. The five year survival rate of HGSOC is between 35-40%. Cancer recurrence and therapy resistance are considered the major causes of death.Evidence is accumulated that disease-free interval (DFI) of the patients is directly correlated or determined by the cell proliferation rate. As shown in Figure 4, Patient1 had an over 5 years of DFI, while P7 had 3 times of recurrence with every DFI of about one year. The Ki67, which is an indicator of cell proliferation, was shown to have significant higher expression in tumors of Patient 7 than in that of Patient 1.


Figure 4. Clinical follow up and Ki67 IHC staining of tumors by two pati-ents with HGSOC.

In the current study, we propose a new therapeutic concept based on an inhibition of the proliferation of tumor cell. We hypothesize if we could reduce or even stop the tumor cell proliferation, DFI can be prolonged, and thus the quality of life of the patients will be dramatically increased. Further-more, we hypothesize that therapeutics aiming at inhibiting cell proliferation instead of killing them might be less toxic as platinum-based drugs.

We have the following scientific and technological objectives.

patient-derived HGSOC cell lines;

by gene set enrichment analyses (GSEA) of the RNA sequencing data;

Methods and results:

The proliferation rate of 34 established cell lines were determinedUsing fluorescent labelling methods (CyQuant cell prolife-ration assay, ThermoFisher Scientific; CytoTox Cytotoxicity Assay, Promega), we measured the numbers of total cells and dead cells separately in HGSOC cells and calculated the doubling time (Figure 5).

Figure 5. Doubling time of the established patient-derived HGSOC cell lines.

GSEA analyses revealed predominant pathways for rapid proliferation of tumor cellsBoth continues and categorical methods were used to per-form the GSEA analyzes. In the first case the doubling time in hours were used without taking the multiple presentations of cell lines from the same patient into consideration. In the second case, gene expression profiles were compared bet-ween 8 fast growing versus 9 slowly growing cell lines, taken the mean of multiple presented cell lines from one patient. Both analytical results showed that DNA replication and homologous recombination pathways were significantly over-presented in fast growing tumor cells than the slowly growing ones (Table 1, Table 2, Figure 6).

Gene set SIZE NESNOM p-val

FDR q-val

1HOMOLOGOUS RECOMBINATION

27 -1.9 0.004 0.097

2METABOLISM

94 -1.83 0 0.108

3 DNA REPLICATION 35 -1.82 0.009 0.084

4 121 -1.76 0.011 0.128

5NUCLEOTIDE

42 -1.74 0.023 0.133

6 PROTEASOME 40 -1.72 0.023 0.135

7REPAIR

34 -1.67 0.009 0.171

8DRUG METABOLISM

30 -1.65 0.027 0.179

9 MISMATCH REPAIR 22 -1.61 0.054 0.231

Table 1. Differentially presented pathways in regard of cell doubling time (as continues parameter)


Gene set SIZE NESNOM p-val

FDR q-val

1HOMOLOGOUS RECOMBINATION

27 2.13 0 0.002

2 121 1.9 0.019 0.114

3 DNA REPLICATION 35 1.86 0.014 0.12

Table 2. Differentially presented pathways in regard of cell doubling time (as categorical parameter)

Figure 6. Enrichment plot of homologous recombination pathway and DNA replication pathway by GSEA analyses.

Determinant genes for proliferation were validated in the DNA replication pathwayAs the first step, we selected seven genes, which play key roles in the DNA replication pathways and validated their expression using RT-qPCR. As shown in Figure 7, there was indeed a correlation of the expression of these genes with the quick proliferation of tumor cells.

gene expression and cell proliferation (presented in doubling time).

Future aspects:siRNAs will be selected to interfere with the targeted mR-NAs. The change in cell proliferation rate will be measured.

AKH Jahresbericht 2021 WEB...z. B. Kältestress, Bildung von intrazellulären und extrazel-lulären...

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