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ObjectivesPoor survival of high-grade serous pelvic cancer is caused by a lack of effective screening measures. The detection of exfoliated cells from high-grade serous pelvic cancer, or precursor lesions, is a promising concept for earlier diagnosis. However, collecting those cells in the most efficient way while fulfilling all requirements for a screening approach is a challenge. We introduce a new catheter for uterine and tubal lavage (UtL) and the clinical evaluation of its performance.Methods/MaterialsIn study I, the clinical feasibility of the UtL using the new catheter was examined in 93 patients admitted for gynecologic surgery under general anesthesia. In study II, the safety of the UtL procedure was assessed. The pain during and after the UtL performed under local anesthesia was rated on a visual analog scale by 22 healthy women.ResultsIn study I, the UtL was carried out successfully in 92 (98.9%) of 93 cases by 16 different gynecologists. It was rated as easy to perform in 84.8% of patients but as rather difficult in cancer patients (odds ratio, 5.559; 95% confidence interval, 1.434–21.546; P = 0.007). For benign conditions, dilatation before UtL was associated with menopause status (odds ratio, 4.929; 95% confidence interval, 1.439–16.884; P = 0.016). In study II, the pain during UtL was rated with a median visual analog scale score of 1.6. During a period of 4 weeks after UtL, none of the participants had to use medication or developed symptoms requiring medical attention. The UtL took 6.5 minutes on average. The amount of extracted DNA was above the lower limit for a sensitive, deep-sequencing mutation analysis in all cases.ConclusionsOur studies demonstrate that the UtL, using the new catheter, is a safe, reliable, and well-tolerated procedure, which does not require elaborate training. Therefore, UtL fulfils all prerequisites to be used in a potential screening setting.

RT-qPCR is a highly sensitive approach to detect rare transcripts, as derived from circulating tumor cells (CTCs) in the blood of cancer patients. However, the presence of unwanted leukocytes often leads to false positive results. Here, we evaluated whether the micro-fluidic Parsortix™ technology is appropriate to remove these leukocytes and thereby finally to improve the overall approach. In this study, we established a workflow including the micro-fluidic Parsortix™ technology for the molecular detection of CTC related transcripts. Background levels of EpCAM, PPIC, TUSC3, and MAL2 were efficiently removed due to an up to 106-fold depletion of leukocytes. The presence of these gene markers was observed in Parsortix™-enriched blood samples from patients with primary and recurrent gynecological cancer (32% and 14%), as well as in 86% of the metastatic breast cancer samples, at a very high specificity. In the ovarian cancer samples, PPIC was the most prominent gene marker, contributing to all positive cases and at least to 70% of the positive cases after pre-amplification of the respective target genes. Expanding the analytical panel up to 29 gene markers further increased the positivity rate (primary gynecological cancer: 95%, recurrent gynecological cancer: 100%, metastatic breast cancer: 92%). The established workflow strongly improved the overall molecular analysis of the target cells by the efficient removal of contaminating cells, and, thereby offers great promise for the molecular characterization of CTCs.

Introduction/Background Poor survival of high-grade serous pelvic cancer (HGSC) is caused by a lack of effective screening measures. Uterine and tubal lavage (UtL) is the sampling technique for HGSC detection with the highest sensitivity ever reported. We combined UtL with the highest accuracy sequencing technology for TP53 mutations, Duplex Sequencing (DS) in order to develop a test potentially feasible for HGSC screening. Methodology In study I, the clinical feasibility of UtL using a new catheter was examined in 93 patients undergoing surgery for suspected HGSC. In study II, pain at UtL under local anesthesia was evaluated in 22 healthy women. In Study III, uterine lavages from 10 HGSC patients and 11 controls were subjected to DS to identify TP53 mutations. Results In study I, UtL was performed successfully in 92 (98.9%) of 93 cases by 16 gynecologists. In 84.8% of patients no cervical dilatation was required. For benign conditions, the need for dilatation was associated with menopause status (OR, 4.929; 95% CI, 1.439–16.884; P=0.016). In study II, pain during UtL was rated with a median VAS score of 1.6. During 4 weeks after UtL, no serious complications occurred. UtL took 6.5 minutes on average. The amount of extracted DNA was above the lower limit for sensitive, deep-sequencing mutation analysis in all cases. In study III, in 80% of all HGSC tumor-derived TP53 mutations in UtL samples were detected by DS. However, we detected low frequency TP53 mutations in nearly all samples tested. Mutations increased with age and shared the selection traits of clonal TP53 mutations commonly found in human tumors. However, tumor-derived TP53 mutations were present at frequencies at least 10-fold higher than background mutations. Conclusion The UtL concept fulfils all prerequisites to be used in a potential screening setting. Interpretation of DS results needs to consider TP53 biologic background mutations. Disclosure Philipp Harter COI: Honoraria: Astra Zeneca, Roche, Sotio, Tesaro, Stryker, ASCO, Zai Lab, MSD; Advisory Board: Astra Zeneca, Roche, Tesaro, Lilly, Clovis, Immunogen, MSD/Merck; Research funding (Inst): Astra Zeneca, Roche, GSK, Boehringer Ingelheim, Medac, DFG, European Union, DKH, Tesaro, Genmab Rosana Risques COI: RAR shares equity in NanoString Technologies Inc. and is the principal investigator on an NIH SBIR subcontract research agreement with TwinStrand Biosciences Inc. Florian Heitz COI: Honoraria: AstraZeneca, Roche, Tesaro, Clovis; Advisory Board: Astra Zeneca, Roche, Tesaro, Clovis Christoph Grimm COI: Consultant: AstraZeneca, Celgene, MSD, PharmaMar, Roche, Tesaro, Vifor Pharma Speaker: Amgen, AstraZeneca, MSD, PharmaMar, Roche, Tesaro Direct research funding: Meda Pharma, Roche Diagnostics Ignace Vergote COI: CONSULTING = ADVISORY BOARD: Advaxis, Inc., Eisai Inc., MSD Belgium, Roche NV, Genmab, F. Hoffmann-La Roche Ltd, Pharmamar, Millennium Pharmaceuticals, Clovis Oncology Inc., AstraZeneca NV, Tesaro, Oncoinvent AS, Immunogen Inc, Sotio CONTRACTED RESEARCH (via KULeuven): Oncoinvent AS, Genmab GRANT = CORPORATE SPONSORED RESEARCH: Amgen, Roche, Stichting tegen Kanker Accomodations, travel Fabian Trillsch COI:Consultant: AstraZeneca, Roche, Tesaro Speaker/Travel Expenses: AstraZeneca, Medac, PharmaMar, Roche, Tesaro Paul Speisr: Share Holder OVARTEC GmbH.

Purpose Type II ovarian cancer (OC) and endometrial cancer (EC) are generally diagnosed at an advanced stage, translating into a poor survival rate. There is increasing evidence that Müllerian duct cancers may exfoliate cells. We have established an approach for lavage of the uterine cavity to detect shed cancer cells. Patients and Methods Lavage of the uterine cavity was used to obtain samples from 65 patients, including 30 with OC, five with EC, three with other malignancies, and 27 with benign lesions involving gynecologic organs. These samples, as well as corresponding tumor tissue, were examined for the presence of somatic mutations using massively parallel sequencing (next-generation sequencing) and, in a subset, singleplex analysis. Results The lavage technique could be applied successfully, and sufficient amounts of DNA were obtained in all patients. Mutations, mainly in TP53, were identified in 18 (60%) of 30 lavage samples of patients with OC using next-generation sequencing. Singleplex analysis of mutations previously determined in corresponding tumor tissue led to further identification of six patients. Taken together, in 24 (80%) of 30 patients with OC, specific mutations could be identified. This also included one patient with occult OC. All five analyzed lavage specimens from patients with EC harbored mutations. Eight (29.6%) of 27 patients with benign lesions tested positive for mutations, six (75%) as a result of mutations in the KRAS gene. Conclusion This study proved that tumor cells from ovarian neoplasms are shed and can be collected via lavage of the uterine cavity. Detection of OC and EC and even clinically occult OC was achieved, making it a potential tool of significant promise for early diagnosis.

High-grade serous ovarian cancer (HGSOC) is the most aggressive type of ovarian cancer and is responsible for most deaths caused by gynecological cancers. Numerous candidate biomarkers were identified for this disease in the last decades, but most were not sensitive or specific enough for clinical applications. Hence, new biomarkers for HGSOC are urgently required. This study aimed to identify new markers by isolating different extracellular vesicle (EV) types from the ascites of ovarian cancer patients according to their affinities for lipid-binding proteins and analyzing their protein cargo. This approach circumvents the low signal-to-noise ratio when using biological fluids for biomarker discovery and the issue of contamination by large non-EV complexes. We isolated and analyzed three distinct EV populations from the ascites of patients with ovarian cancer or cirrhosis and observed that Annexin V-binding EVs have higher levels of matrix metalloproteinase 9 in malignant compared to portal-hypertensive ascites. As this protein was not detected in other EV populations, this study validates our approach of using different EV types for optimal biomarker discovery. Furthermore, MMP9 in Annexin V-binding EVs could be a HGSOC biomarker with enhanced specificity, because its identification requires detection of two distinct components, that is, lipid and protein.

PURPOSE OF THE STUDY: The detection of circulating tumor cells (CTCs) in the blood of cancer patients has raised considerable interest over the last decade as a promising biomarker of minimal residual disease in various solid tumors. Although hematogenous spread has not been deemed a major issue for ovarian cancer, there are several studies demonstrating the presence and prognostic impact of CTCs in ovarian cancer patients. Recently, we developed a protocol for the enrichment of CTCs from ovarian cancer blood samples, achieving a high purity of the target cells and enabling high specificity. EXPERIMENTAL PROCEDURES: Blood samples were enriched using a density gradient centrifugation (OncoQuick, Greiner Bio–One) followed by a micro–fluidic based isolation of the target cells using the Parsortix™ system (Angle plc.). The captured target cells were lysed, RNA was extracted (RNeasy Micro Kit, Qiagen) and transcribed into cDNA (ssVILO, Life Technologies). After a pre–amplification step (PreAmp Master Mix, Life Technologies), the transcript levels of selected genes (EpCAM, PPIC, TUSC3, EMP2, LAMB1, MAL2, FN1) were measured using RT–qPCR. Additionally, density gradient enriched cells were immuno–fluorescently stained targeting CK8/18/19, PPIC, TP53 and CD45. SUMMARY: By combining a density gradient based pre–enrichment and the micro–fluidic Parsortix™ technology we achieved an up to 106–fold depletion of blood cell contamination. The analysis of CTC–related transcripts in the enriched samples indicated the presence of CTCs in 78% of the ovarian cancer blood samples taken at primary diagnosis (N=15) and in 80% of the samples taken at relapse (N=9). Concordant results of immuno–fluorescent staining and RT–qPCR were obtained in just 30% of ovarian cancer samples. By adding further 22 RT–qPCR markers, 92% of all cancer patients (n=13) and 100% of the ovarian cancer patients (n=7) were classified as being CTC–positive by RT–qPCR. CONCLUSIONS: In conclusion, we developed a protocol which enabled the detection of CTC–related transcripts in the majority of ovarian cancer blood samples, which is a clear improvement in sensitivity as compared to earlier studies employing density gradient centrifugation as the only enrichment step. Our results are currently validated in the large cohort of platinum resistant ovarian cancer patients provided by a multi–center Gannet53 study. Citation Format: Eva Obermayr, Elisabeth Maritschnegg, Paul Speiser, Eva Schuster, Barbara Holzer, Nina Pecha, Robert Zeillinger. THE MOLECULAR DETECTION OF CIRCULATING TUMOR CELLS IN THE BLOOD OF OVARIAN CANCER PATIENTS [abstract]. In: Proceedings of the 11th Biennial Ovarian Cancer Research Symposium; Sep 12-13, 2016; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(11 Suppl):Abstract nr AP04.

Specimen collection method and quality insurance are pivotal in biomarker discovery. Pre-analytical variables concerning blood collection and sample handling might affect analytical results and should be standardised prior application. In this study, we examine pre-analytical characteristics of blood samples using protein microarray. The influences of 1) standby times until centrifugation (1 h, 4 h, 24 h and 48 h), 2) four blood collection methods, and 3) IgG purified from those samples on differentially reactive antigens between samples (“DIRAGs”) were investigated. Spearman correlation analyses of intra-individual arrays demonstrated remarkable differences (0.75–0.98 vs. 0.5–0.75) of antibody reactivities within and between serum and plasma samples. Class comparison showed that reactive antigen profiles were best preserved using IgG purified samples of serum tubes without separation gel as after 24 h 83% of the 1 h baseline DIRAGs were re-found. Testing dilution series with protein microarrays and Luminex xMap® Technology, we found linear correlations (R2 = 0.94–0.99) between IgG concentration and read-out when using purified IgG instead of serum. Therefore, we highly recommend standardising pre-analytics and proposing the use of purified IgG for autoantibody immune-profiling with protein microarrays to reduce potential unspecific binding of matrix proteins abundant in serum and plasma samples. Significance Although purified IgG cannot completely compensate the influence of pre-analytics, in highly parallel immune-profiling IgG enables reduction of unspecific effects, which occur when using serum or plasma for analysis on protein microarrays. Reproducibility problems due to pre-analytical processing of blood samples might explain discrepant results reported in various biomarker studies.

Epithelial ovarian cancer (EOC) is diagnosed in more than 70% of the cases in advanced FIGO stages which renders EOC the most deadly gynecologic cancer. Up to date there are no specific symptoms and no screening tests for general population or for patients at risk. The development of early diagnostic tools that will improve clinical outcome and differentiate between benign gynecological diseases and EOC are still an unmet need. In a multicenter study financed by the Seventh Framework Program of the European Commission (TRANSCAN) called ImPECT, three European EOC centers (Medical University of Vienna, Charité in Berlin, and the European Institute of Oncology in Milan) validated a “combined blood based gene expression and plasma protein abundance signature for diagnosis of epithelial ovarian cancer” (Pils et al. BMC Cancer, 13:178). In total, blood of 576 women were enrolled in this validation study, including healthy controls, patients with EOC, with breast cancer (to assess tumor specificity), with benign gynecologic diseases (to assess the potential for differential diagnosis), and with inflammatory diseases and diabetes (to assess non-malignant immune changes). A specific blood leukocyte fraction and plasma were isolated and RNA prepared. The expression of 23 genes (13 from the diagnostic signature, seven from an unpublished prognostic signature and three house-keeping) were measured by a multiplexed hybridization based method (Nanostring's nCounter Elements™) and the abundance of six proteins from plasma by a multiplexed bead-based technology (Luminex). Due to evidence for substantial fluctuations of gene expression values by RT-qPCR, especially of the reverse transcription (RT) reaction, we replaced the RT-qPCR method by a hybridization based method without RT. Also, one protein of the protein panel (IGF2) was replaced by another protein (HE4) for two reasons: measurement of IGF2 cannot be multiplexed with the other proteins and HE4 was shown to have a higher discriminative power compared to IGF2. In a first step the published discriminative models were validated with 96 healthy controls and 96 EOC patients. All models showed significant area under the receiver operating characteristic (ROC) curve (AUC) values (p To assess the technical reproducibility of the Nanostring method, each 84 samples were measured a second time and correlated to the first measurement: Interestingly, the median Pearson's r correlations over all 20 genes was only 0.689 (0.337-0.799) but the SVM model between controls and EOC patients was more robust: AUC of 0.811 compared to 0.848 (p=0.394). A clinical validation of these models with independent patient and control cohorts (healthy controls and patients with benign diseases) and the specificity compared to breast cancer and diseases with immune system involvement will be presented. Citation Format: Anna Bachmayr-Heyda, Stefanie Aust, Katharina Auer, Peter Borossay, Nina Pecha, Ugo Cavallaro, Ioana Braicu, Sarah Igidbashian, Costanza Savino, Franchi Dorella, Jalid Sehouli, Sonia Zaafrani, Florian Fitzal, Christian Singer, Michael Gnant, Robert Zeillinger, Dietmar Pils. Validation of a combined 13 gene six protein blood signature for earlier detection of ovarian cancer. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research: Exploiting Vulnerabilities; Oct 17-20, 2015; Orlando, FL. Philadelphia (PA): AACR; Clin Cancer Res 2016;22(2 Suppl):Abstract nr B28.

Objectives of the study We intended to develop a protocol combining a novel micro-fluidic enrichment technology and RT-qPCR for the molecular analysis of CTCs. Recently we identified CTC-specific mRNA markers allowing CTC detection in 29% of breast cancer patients and in 24.5% of ovarian cancer patients at diagnosis. However, the detection of cancer cells was hampered by the large number of contaminating leukocytes. The necessary use of cut-off values for positivity reduced the specificity of the down-stream PCR assay. By improving the purity of cancer cells and PCR analysis we sought to increase both sensitivity and specificity of the diagnostic procedure. Methodology For reducing the blood sample volume (up to 20ml) density gradient centrifugation was used when needed. The samples were passed through a micro-fluidic disposable cassette, which captures tumor cells based on their less deformable nature and larger size compared to blood cells. Lysis of the captured cells was done directly in the cassette and total RNA was extracted. After a cDNA pre-amplification step gene expression levels of leukocyte-specific and CTC-related markers were measured using RT-qPCR. The efficiency of the combined protocol was assessed in blood samples taken from patients with primary and recurrent malignant diseases. Results 7 out of 13 pre-selected RNA markers were not detectable in blood samples from 11 healthy volunteers. In cancer patients we observed measurable gene expression of at least one out of these 7 RNA markers. 5 out of 5 breast cancer (primary disease: N = 1; metastatic: N = 4), 9 out of 10 primary and 4 out of 8 relapsed ovarian cancer patients were classified correctly by the test. The most striking finding is the detection rate of 90% for primary ovarian cancer at a specificity of 100%. Conclusions The enrichment of rare cells from blood samples using micro-fluidics results in a highly pure cell population. This enables the application of extremely sensitive methods, like RT-qPCR to specifically detect rare events. By combining a novel micro-fluidic cell enrichment and molecular analysis we have taken a major step forward, which allows the implementation of ′liquid biopsies′ in cancer detection studies and as a companion diagnostic in clinical trials. Citation Format: Eva Obermayr, Elisabeth Maritschnegg, Paul Speiser, Christian Singer, Eva M. Schuster, Sabine Danzinger, Nina Pecha, Robert Zeillinger. Circulating rare cells enable highly efficient cancer detection. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr LB-197. doi:10.1158/1538-7445.AM2015-LB-197