Analysis of the primary tumor and blood based “Real-Time-Liquid Biopsy”

Trotz Fortschritten in der Behandlung erleiden mehr als 50% der Patientinnen mit Ovarialkarzinom ein Rezidiv, wobei 15 % der Fälle platinresistent sind, was meist jedoch nur retrospektiv erkannt wird. In diesem Zusammenhang war Ziel dieser Dissertation, neue primärtumor- sowie blutbasierte Biomarkerkonzepte im Hinblick auf Platinresistenz und Prognose zu etablieren. Dies wurde zunächst im Rahmen einer primärtumorbasierten LOH-Analyse an vier tumorbiologisch relevanten chromosomalen Regionen realisiert. In diesem Zusammenhang erwies sich LOH proximal zum M6P/IGF2R-Lokus als neuer Biomarkerkandidat für die Tumorzelldisseminierung in das KM. Da Tumorgewebe jedoch nur einmal zum Zeitpunkt der Primärdiagnose zur Verfügung steht und sich nicht für ein Therapiemonitoring eignet, wurden nachfolgend experimentelle Strategien zur Biomarkeridentifikation auf ein blutbasiertes System transferiert. In diesem Sinne wurde die Methodik der primärtumorbasierten LOH-Detektion auf zirDNA im Blutserum der Patientinnen übertragen. Im Rahmen einer zusätzlichen Fraktionierung der zirDNA in HMF bzw. NMF, konnte die Sensitivität der LOH-Detektion deutlich verbessert werden. Des Weiteren zeigte sich nach Chemotherapie eine signifikante Reduktion des zirDNA Gehaltes in der NMF, jedoch nicht in der HMF. Diese Befunde implizierten, dass tumorassoziierte DNA in der Zirkulation vermutlich niedermolekularen Charakter aufweist. Somit könnte die Fraktionierungsmethodik eine selektive Untersuchung tumorassoziierter DNA im Blut erlauben. Ferner wurde LOH am PTEN-Lokus bzw. proximal zum M6P/IGF2R-Lokus als neuer blutbasierte Biomarker für die Tumorzelldisseminierung in das KM sowie für ein verkürztes OS beschrieben. Ergänzend wurde das Profil zirkulierender miRNA im Serum von Ovarialkarzinompatientinnen analysiert. In diesem Zusammenhang konnte miR-1246 im Vergleich zu Normalspenderinnen als signifikant hochreguliert identifiziert werden. Interessanterweise wurde während der experimentellen Phase dieser Dissertation durch eine Kooperationsgruppe gezeigt, dass der miR-1246 Detektionsassay eine starke Kreuzreaktivität mit RNU2-1f im Serum aufweist. Somit war das beobachtete miR-1246 Signal mit großer Wahrscheinlichkeit auf RNU2-1f und nicht auf miR-1246 zurückzuführen. Im Weiteren konnte ein signifikanter Zusammenhang zwischen der RNU2-1f Expression im Serum und dem FIGO-Stadium bzw. dem verbleibenden Tumorrest nach Operation beschrieben werden. Ferner identifizierte eine persistierende bzw. neu erworbene RNU2-1f Positivität nach Chemotherapie eine Patientinnengruppe mit tendenziell verkürztem OS. Dies ist die erste Beschreibung einer nicht kodierenden RNA, die als blutbasierter Biomarker für das Ovarialkarzinom fungieren könnte. Abschließend wurde auf die immunomagnetische Isolation bzw. molekulare Charakterisierung intakter ZTZ aus dem Blut fokussiert. Es konnte bereits gezeigt werden, dass die Präsenz von ZTZ im Blut vor Operation bzw. nach Chemotherapie mit einem verkürzten OS korrelierte. Weiterführend sollte die Hypothese überprüft werden, inwieweit der beobachtete prognostisch-negative Einfluss der ZTZ im Blut auf einen platinresistenzassoziierten Phänotyp dieser Zellen zurückzuführen sein mag. Daher wurde die Expression der DNA-Reparatur Endonuklease ERCC1, die bereits mit Platinsensitivität ovarieller Tumoren in Verbindung gebracht werden konnte, in ZTZ untersucht. ERCC1-Positivität wurde mit einer Inzidenz von 36 % vor Operation und 35% nach Chemotherapie beobachtet, interessanterweise auch in 30% der Patientinnen, die gemäß AdnaTest OvarianCancer, als ZTZ-negativ galten. Ein statistischer Zusammenhang zwischen der ERCC1-Expression und klinisch definierter Platinsensitivität ergab sich nicht, jedoch zeigte sich bei Patientinnen mit persistierend ERCC1-positiven ZTZ ein signifikant verkürztes DFS bzw. OS. Diese Befunde sprechen für eine Implementierung von ERCC1 in die ZTZ-Analyse und beschreiben ERCC1 als möglichen klinisch relevanten Biomarker, der Patientinnen mit besonders schlechter Prognose identifizieren könnte bzw. die Notwendigkeit einer Therapieänderung voraussagen mag. Des Weiteren konnte gezeigt werden, dass ZTZ von Ovarialkarzinompatientinnen zu etwa 14% EMT- bzw. stammzellassoziierte Marker exprimieren. Dies wurde auch bei Patientinnen beobachtet, die gemäß AdnaTest OvarianCancer ZTZ-negativ galten, was für die Heterogenität der ZTZ im Blut spricht. Die Detektion sowie das zielgerichtete Vorgehen gegen stammzellähnliche ZTZ könnte von großer klinischer Relevanz sein, da diese ZTZ-Subgruppe bereits mit Therapieversagen und Rezidiventwicklung in Verbindung gebracht wurde. Zusammenfassend konnte die vorliegende Dissertation einen relevanten Beitrag zur Etablierung neuer primärtumorbasierter Biomarkerkonzepte für das Ovarialkarzinom liefern und die Hypothese unterstützen, dass Patientinnenblut im Sinne einer „Real-Time-Liquid-Biopsy“ genutzt werden könnte.Despite advances in treatment, more than 50% of patients with epithelial ovarian cancer will experience recurrence. Moreover, resistance to platinum based chemotherapy occurs in about 20% of cases and can only be assessed retrospectively during post-chemotherapy follow-up. In this regard, intention of this PhD thesis was to identify novel biomarkers, predicting therapy response and prognosis. This was initially realized by a primary tumor based LOH-analysis at four ovarian cancer relevant chromosomal regions. In this context, allelic loss proximal to the M6P/IGF2R locus was revealed as a novel biomarker candidate, predicting tumor cell dissemination into the bone marrow (BM). Given that primary tumor tissue is available uniquely by resection and does not allow therapy monitoring, experimental strategies for biomarker identification were subsequently transferred to a blood based context. In this regard, methods for primary tumor based LOH-detection were transferred to circulating DNA (cirDNA) in the blood serum of ovarian cancer patients. Fractionation of cirDNA into high- and low molecular weight fraction (HMWF, LMWF) broadly improved sensitivity of LOH-detection in the blood and cirDNA content in the LMWF but not in the HMWF significantly decreased after chemotherapy. These data implied that tumor-associated cirDNA in the circulation might primarily be of low molecular weight character and that cirDNA fractionation technique could allow selective analysis of tumor related DNA in the blood. Moreover, LOH at the PTEN-locus and proximal to M6P/IGF2R were suggested as novel blood based biomarkers for tumor cell dissemination into the BM and a reduced overall survival (OS). In addition to this approach, the profile of circulating microRNA in the blood of ovarian cancer patients was analyzed. In this context, miR-1246 was found to be significantly overexpressed in the blood of cancer patients compared to healthy donors. Interestingly, during the experimental stage of the experiments, a cooperation group showed that the miR-1246 detection assay was highly cross-reactive with U2 small nuclear RNA fragments (RNU2-1f). Consequently, the observed miR-1246 signal in the circulation was likely derived from RNU2-1f than from miR-1246. Circulating RNU2-1f levels at primary diagnosis paralleled the tumor stage, whereas RNU2-1f expression after chemotherapy correlated with residual tumor burden left after surgery. Moreover, persisting or newly acquired RNU2-1f positivity after chemotherapy trended to identify a patient group with reduced OS. This is the first report, suggesting a small non coding RNA as blood-based biomarker for ovarian cancer patients. Finally, experimental strategies focused on the immunomagnetic isolation and molecular characterisation of circulating tumor cells (CTC) in the blood. In this regard, the presence of CTC before surgery and after chemotherapy was previously ascertained to predict a reduced overall survival. Continuatively, it should be investigated, whether negative prognostic impact of CTC may arise from a cellular phenotype being associated with platinum resistance. In this regard, we assessed expression of Excision repair cross-complementing rodent repair deficiency, complementation group 1 (ERCC1) in CTC, which takes part in the DNA nucleotide excision repair pathway and has already been described as a primary tumor-based biomarker for platinum resistance in ovarian cancer. ERCC1-positivty rate was 36% before surgery and 35% after chemotherapy. Interestingly, 30% of patients with positive CTC-status, according to AdnaTest OvarianCancer, were positive for ERCC1-expression. No association between clinically defined platinum resistance and ERCC1-positivity was observed. However, patients with persistently positive ERCC1-expression in paired blood samples before surgery and after chemotherapy had a significantly decreased OS and DFS. Thus, implementing ERCC1 expression in CTC analysis of patients may identify a subgroup of ovarian cancer patients with worse prognosis. Apart from this, ERCC1 might serve as a valuable biomarker for monitoring disease to possibly change treatment in case of ERCC1-persistent CTC. Moreover, the expression of EMT- and stem cell associated transcripts was demonstrated in 14% of ovarian cancer patients. This was also observed in case of CTC-negative patients, suggesting the heterogeneity of CTC in the blood. Given that stem cell like CTC may be associated with therapy failure and recurrence of ovarian carcinomas, detecting and targeting of these cells could be of prior interest for the clinic. Conclusively, this PhD-thesis contributed to the identification of novel primary tumor based biomarker concepts for ovarian cancer and corroborates the hypothesis that the patient’s blood could be utilized in terms of a “Real-Time-Liquid-Biospy”

Precipitation with polyethylene glycol followed by washing and pelleting by ultracentrifugation enriches extracellular vesicles from tissue culture supernatants in small and large scales

Extracellular vesicles (EVs) provide a complex means of intercellular signalling between cells at local and distant sites, both within and between different organs. According to their cell-type specific signatures, EVs can function as a novel class of biomarkers for a variety of diseases, and can be used as drug-delivery vehicles. Furthermore, EVs from certain cell types exert beneficial effects in regenerative medicine and for immune modulation. Several techniques are available to harvest EVs from various body fluids or cell culture supernatants. Classically, differential centrifugation, density gradient centrifugation, size-exclusion chromatography and immunocapturing-based methods are used to harvest EVs from EV-containing liquids. Owing to limitations in the scalability of any of these methods, we designed and optimised a polyethylene glycol (PEG)based precipitation method to enrich EVs from cell culture supernatants. We demonstrate the reproducibility and scalability of this method and compared its efficacy with more classical EV-harvesting methods. We show that washing of the PEG pellet and the re-precipitation by ultracentrifugation remove a huge proportion of PEG co-precipitated molecules such as bovine serum albumine (BSA). However, supported by the results of the size exclusion chromatography, which revealed a higher purity in terms of particles per milligram protein of the obtained EV samples, PEG-prepared EV samples most likely still contain a certain percentage of other non-EV associated molecules. Since PEG-enriched EVs revealed the same therapeutic activity in an ischemic stroke model than corresponding cells, it is unlikely that such co-purified molecules negatively affect the functional properties of obtained EV samples. In summary, maybe not being the purification method of choice if molecular profiling of pure EV samples is intended, the optimised PEG protocol is a scalable and reproducible method, which can easily be adopted by laboratories equipped with an ultracentrifuge to enrich for functional active EVs

Research and Design of a Routing Protocol in Large-Scale Wireless Sensor Networks

无线传感器网络，作为全球未来十大技术之一，集成了传感器技术、嵌入式计算技术、分布式信息处理和自组织网技术，可实时感知、采集、处理、传输网络分布区域内的各种信息数据，在军事国防、生物医疗、环境监测、抢险救灾、防恐反恐、危险区域远程控制等领域具有十分广阔的应用前景。 本文研究分析了无线传感器网络的已有路由协议，并针对大规模的无线传感器网络设计了一种树状路由协议，它根据节点地址信息来形成路由，从而简化了复杂繁冗的路由表查找和维护，节省了不必要的开销，提高了路由效率，实现了快速有效的数据传输。 为支持此路由协议本文提出了一种自适应动态地址分配算——ADAR（AdaptiveDynamicAddre...As one of the ten high technologies in the future, wireless sensor network, which is the integration of micro-sensors, embedded computing, modern network and Ad Hoc technologies, can apperceive, collect, process and transmit various information data within the region. It can be used in military defense, biomedical, environmental monitoring, disaster relief, counter-terrorism, remote control of haz...学位：工学硕士院系专业：信息科学与技术学院通信工程系_通信与信息系统学号：2332007115216

FNTB Promoter Polymorphisms Are Independent Predictors of Survival in Patients with Triple Negative Breast Cancer

In breast cancer, the promising efficacy of farnesyltransferase inhibitors (FTIs) in preclinical studies is in contrast to only limited effects in clinical Phase II–III trials. The objective of this study was to explore the clinical relevance of farnesyltransferase β-subunit (FNTB) single nucleotide promoter polymorphisms (FNTB-173 6G > 5G (rs3215788), -609 G > C (rs11623866) and -179 T > A (rs192403314)) in early breast cancer. FNTB genotyping was performed by pyrosequencing in 797 patients from a prospective multicentre observational PiA trial (NCT 01592825). In the total cohort, the FNTB-173 6G > 5G polymorphism was an independent predictor of RFI (HR = 0.568; 95% CI = 0.339–0.949, p = 0.031), OS (HR = 0.629; 95% CI = 0.403–0.980, p = 0.040) and BCSS (HR = 0.433; 95% CI = 0.213–0.882; p = 0.021), whereas the FNTB-609 G > C polymorphism was an independent predictor of RFI (HR = 0.453; 95% CI = 0.226–0.910, p = 0.026) and BCSS (HR = 0.227; 95% CI = 0.075–0.687, p = 0.009). Subtype analysis revealed the independent prognostic relevance of FNTB promoter polymorphisms, particularly in TNBC but not in luminal or HER2-positive intrinsic subtypes. Finally, we used electrophoretic mobility shift assays (EMSAs) to confirm in vitro that the polymorphism FNTB-173 6G > 5G resulted in the differential binding of nuclear proteins from five different breast cancer cell lines. This is the first study on breast cancer suggesting that FNTB promoter polymorphisms (i) are independent prognostic biomarkers, particularly in patients with early TNBC, and (ii) could modulate FNTB’s transcriptional activity

The conjugated antimetabolite 5-FdU-ECyd and its cellular and molecular effects on platinum-sensitive vs. -resistant ovarian cancer cells in vitro

Background: Resistance to platinum-based chemotherapy is a clinical challenge in the treatment of ovarian cancer (OC) and limits survival. Therefore, innovative drugs against platinum-resistance are urgently needed. Our therapeutic concept is based on the conjugation of two chemotherapeutic compounds to a monotherapeutic pro-drug, which is taken up by cancer cells and cleaved into active cytostatic metabolites. We explore the activity of the duplex-prodrug 5-FdU-ECyd, covalently linking 2'-deoxy-5fluorouridine (5-FdU) and 3'-C-ethynylcytidine (ECyd), on platinum-resistant OC cells. Methods: In vitro assays and RNA-Sequencing were applied for characterization of 5-FdU-ECyd treated platinum-sensitive A2780 and isogenic platinum-resistant A2780cis and independent platinum-resistant Skov-3-IP OC cells. Results: Nano molar 5-FdU-ECyd concentrations induced a rapid dose-dependent decline of cell viability in platinum-sensitive and -resistant OC cells. The effect of 5-FdU-ECyd was accompanied by the formation of DNA double strand breaks and apoptosis induction, indicated by a strong increase of pro-apoptotic molecular markers. Moreover, 5-FdU-ECyd efficiently decreased migration of platinum-resistant OC cells and inhibited clonogenic or spheroidal growth. Transcriptome analysis showed early up-regulation of CDKN1A and c-Fos in both, platinum-resistant and -sensitive cells after 5-FdU-ECyd treatment and de-regulation of distinct cellular pathways involved in cell cycle regulation, apoptosis, DNA-damage response and RNA-metabolism. Combined treatment of 5-FdU-ECyd and cisplatin did not show a synergistic cellular response, suggesting the potential use of 5-FdU-ECyd as a monotherapeutic agent. Conclusion: Our data provide novel mechanistic insight into the anti-tumor effect of 5-FdU-ECyd and we hypothesize that this duplex-prodrug could be a promising therapeutic option for OC patients with resistance to platinum-based chemotherapy