Prenatal Medicine and Gyn. Oncology
Prof. Sinuhe Hahn
Preeclampsia: a severe auto-inflammatory disorder?
Preeclampsia is a severe pregnancy related disorder, involving aberrant differentiation of trophoblast tissues, leading to the release of inflammatory micro-debris by the placenta. Members of the innate immune system, such as circulatory polymorphonuclear neutrophils (PMNs), mast cells and eosinophils have been shown to be capable of releasing their DNA into the extracellular environment to generate ETS (extracellular traps). The main purpose of these DNA containing structures appears to be to ensnare and kill micro-organisms.
In this context we made the novel observation that syncytiotrophoblast derived micro-debris can trigger isolated PMNs to generate NETs. That this in-vitro finding may have a physiological consequence was underscored by detection of vast numbers of NETs in the inter-villous space of preeclampsia, but not in pregnancies with normal healthy deliveries. Since preeclampsia is associated with hypoxia-reperfusion damage, it could be that the massive presence of NETs could lead to occlusion of the inter-villous space, thereby contribution to this condition.
As damage of the maternal endothelium is a key feature of preeclampsia, we have explored the interaction of NETS with endothelial cells (EC). These ongoing studies have indicated that the interaction of isolated PMN with pre- activated EC, will lead to netosis. The prolonged of exposure of EC to NETs lead to EC apoptosis. Of interest is that this cytolytic process required intact NETs, as it was abolished by concomitant treatment with DNAse.
In a new SNF funded study we are examining the molecular processes involved in aberrant trophoblast differentiation in PE. Here our focus is on possible epigenetic mechanisms. These studies are carried out in collaboration with Prof. M. Bühler, FMI, Basel.
In ongoing studies in collaboration with Prof. P. Hasler (Rheumatology, KSA, Aarau), we have assessed the release of cell-free DNA (cf-DNA) in auto-inflammatory conditions such as rheumatoid arthritis (RA). Here we observed that cf-DNA levels were significantly higher is cases with RA than in healthy controls. A striking feature of these analyses was that cf-DNA levels were much greater in serum of RA patients than in controls. This implies that more cf-DNA is being during the clotting of RA blood samples than in healthy blood samples. We also observed that the vast proportion of cf-DNA in RA was antibody associated. Since antibody-cf-DNA complexes have been shown to be capable of triggering rheumatoid factor production by B-cells, our observation may have significant clinical importance.
Development of new biomarkers for the detection of pregnancies at risk for preeclampsia
PD. Dr. med. Olav Lapaire Prof. Dr. med. Irene Hoesli University Women’s Hospital, Basel
Currently no reliable tests exist to screen for pregnancies at risk for preeclampsia (PE). As a result the disorder only becomes obvious once the symptoms have become manifest, leaving very few options for therapeutic intervention other than frequent delivery of a very premature baby. As it has been suggested that PE involves a lengthy asymptomatic phase prior to clinical manifestation, it would be important to detect such pregnancies early in gestation, in order to develop effective intervention strategies. Since PE is characterised by aberrant trophoblast differentiation, and since the tissue is in direct contact with the maternal circulation, we have sought to determine whether proteomic analysis of maternal plasma samples could not assist with the detection of potential biomarkers.
For this purpose we developed appropriate quantitative strategies using isobaric labelling (iTRAQ) in combination with MALDI-TOF/TOF analysis. These analyses were carried out in collaboration with Prof. P. Jenö, Biozentrum, Basel. Our analysis of maternal plasma samples obtained in the 1st trimester of pregnancy, indicated that significant differences in a number of placentally derived proteins could be discerned between pregnancies which went on to develop PE and those who delivered normally. A similar analysis has been undertaken of pregnancies bearing Down syndrome fetuses, leading to a set of potential biomarkers. The veracity and usefulness of these biomarkers will be further examined in a large number of samples using SRM (selective reaction monitoring).
In collaboration with Prof. P. Hasler, KSA, Aarau, we have observed quantitative alterations in cf-DNA levels in RA patients in response to therapy with biologic agents such as Infliximab. In ongoing studies, we are no examining whether such analyses could assist in discriminating between patients who respond to therapy and those who don’t. The development of such a theranostic tool would greatly assist with treatment strategies for affected patients.
Prof. Xiao Yan Zhong
Using Omic-Technologies to Develop Theragnostics for Hormone Dependent Malignancies: Example I – Methylation and Breast Cancer
Our aim is to develop theragnostics combining therapeutics with diagnostics for hormone dependent malignancies, such as breast cancer (BC), ovarian cancer (OVC) and endometrial cancer (EC), using omic-technologies, such as, genomics, transcriptomics and proteomics, in personalized medicine. The first successful development of a theragnostics was the HER-2/neu based treatment of using Trastuzumab, especially for BC, however only ben- efiting 20-25% of the patients. Therefore, more cancer specific biomarkers are expected to develop new drugs and strategies for targeted therapy.
We apply high-throughput assays to analyse DNA (nDNA, mtDNA and DNA methylation), RNA (mRNA and microRNA) and protein changes in cancer for discovering new biomarkers. Herewith, we show our partial research data on DNA methylation status in BC as an example.
We developed a MALDI-TOF MS (matrix-assisted laser desorption/ioniza- tion time-of-flight mass spectrometry) -based assay to analyse DNA methyla- tion status of 22 genes (APC, BIN1, BMP6, BRCA1, BRCA2, CADHERIN 1, CST6, DAPK1, EGFR, ESR2, GSTP1, NES1, Nm23-H1, P16, P21, PR, Prosta- sin, RAR-b, RASSF1, SRBC, TIMP3, TP53). They are mostly tumor suppres- sor genes (TSGs) involved in cancerogenesis, metastasis, drug resistance and response to hormone therapy in BC.
In order to understand the diagnostic value of methylation changes of the genes, we quantified the methylation status in paired cancerous and normal breast tissues, as well as in paired / unpaired blood samples. Using the bioinformatic tools, such as two-way hierarchical cluster analysis, one-way ANOVA test, Mann-Whitney test, paired-wise euclidean distances and link- age algorithm analysis, 10 TSGs (APC, BIN1, BMP6, BRCA1, CST6, ESRb, GSTP1, P16, P21 and TIMP3) were identified as hypermethylated in BC, which enabled distinguishing between cancerous and non-cancerous tissues, suggesting a classification value. High levels of hypermethylated circulating cell free DNA (ccf DNA) from most of the selected genes were found in patients plasma/serum samples. Correlation of TSG hypermethylation could be found between cancerous tissues and circulation in overlapping, but not between normal tissues and circulation, suggesting a possible diag- nostic value in developing blood based test useful for facilitating early diagnosis and monitoring of therapies (Fig. 1).
In order to clarify the therapeutic value of using the identified genes as targets, we developed CpG island methylator phenotype (CIMP) criteria for epigenetic therapy. A methylation inhibitor 5-azacitidiene led to increased apoptosis/toxicity and decreased viability of BC cells in-vitro, and reactivated TSG expression based on the CIMP. Effect of the CIMP criteria based- treatment on tumour seeding, growth and metastasis will be confirmed through in-vivo mouse experiments. While demethylating TSG hypermeth- ylation, the drugs may reactivate proto-oncogene, promoting development and progression of tumour metastasis. Therefore, we perform transcriptomic and proteomic studies to analyse gene expression profile and protein profile before and after treatment in cooperation with Novatis and Prof Lefkovits to understand the mechanism. A long-term in-vitro study to investigate the correlation between transient or reversible changes of the methylation signature and relapses/drug resistance has been scheduled.
In order to explain the interactions between gene-gene, mutation-methylation and mtDNA-nDNA, we analysed the network of genes, for example, the relationship between methylation profile of P14ARF/MDM2/TP53/PTEN/
P21/P16Rb pathways and mtDNA alterations, as well as telomere length in BC. Indeed, we could find “cross-talking” between gene changes, which should be taken into consideration during theragnostics (Fig. 2). In order to validate our observations, several international networks, such as “CANgene” for BC, “EUROTROP” for OVC, and one for EC have been built together with several Universities and Companies in USA, Europe and China. The figure 3 shows our research schedule to gaining our final goal of clinical applications.