News & Events UPCOMING SCIENTIFIC PRESENTATION Johan Skog, Ph.D., chief scientific officer and founding scientist of Exosome Diagnostics, will present at the upcoming EORTC-NCI-AACR Symposium on Molecular Targets and Cancer Therapeutics in Barcelona, Spain in November. Please see below for details on Dr. Skog’s presentation, as well as information on two poster sessions that will be presented by Exosome Diagnostics at the meeting. In addition, please visit us at booth #B15 to learn more about our company, our technology and our breakthrough biofluid-based diagnostics in development. For more information, please contact [email protected] or +1 617 588 0528. 26TH EORTC-NCI-AACR Symposium on Molecular Targets and Cancer Therapeutics November 18 – 21, 2014 Barcelona, Spain Exosome Diagnostics Presentation Details Date: Wednesday, November 19, 2014 Session: “Liquid Biopsies” in Solid Tumors Time: 10:15 a.m. – 12 p.m. local time (Dr. Skog’s talk is scheduled to begin at 10:35 a.m.) Title: Cancer Mutation and Transcriptome Analysis in Exosomes Exosome Diagnostics Poster Sessions (See abstracts below) Date: Thursday, November 20, 2014 Session: Molecular Targeted Agents I Title: Development of a one-step isolation platform for exosomal RNA and circulating cellfree DNA from cancer plasma samples D. Enderle, K. Brinkmann, T. Koestler, S. Bentink, C. Berking, J. Skog, M. Noerholm ……………………………………………………………………………………………………………………. Exosome Diagnostics TH 26 EORTC-NCI-AACR Symposium on Molecular Targets and Cancer Therapeutics (Nov. 18 – 21, 2014) Date: Friday, November 21, 2014 Session: Molecular Targeted Agents II Title: Monitoring therapy response and resistance mutations in circulating RNA and DNA of plasma from patients with malignant melanoma D. Enderle, K. Brinkmann, T. Koestler, S. Bentink, K.T. Flaherty, J. Skog, M. Noerholm About the Symposium Hosted by the European Organisation for Research and Treatment of Cancer (EORTC), the National Cancer Institute (NCI) and the American Association for Cancer Research (AACR), the 2014 Symposium will bring together around 2000 academics, scientists and pharmaceutical industry representatives from across the globe to discuss innovations in drug development, target selection and the impact of new discoveries in molecular biology. http://www.ecco-org.eu/Events/EORTC_NCI_AACR_2014 Symposium Venue Centre de Convencions Internacional Barcelona (CCIB) Rambla Prim 1-17 ES-08019 Barcelona Spain Tel: +34 (0) 93 230 10 00 POSTER SESSION ABSTRACTS Poster Session 1: Subject: Development of a one-step isolation platform for exosomal RNA and circulating cellfree DNA from cancer plasma samples Abstract: Background: Circulating nucleic acids in the bloodstream of cancer patients are of great interest to medical research because of their potential to yield information on the patient's disease status and treatment options without requiring a tissue biopsy. Any diagnostic test that seeks to utilize body fluids for mutation analysis needs a platform that can maximize the capture of tumor derived mutations in circulation. Blood plasma contains at least two cell-free sources of nucleic acids: circulating cell-free DNA (cfDNA), generated from apoptotic or necrotic cells, and RNA enclosed in extracellular vesicles including exosomes (exoRNA), which are actively secreted by cells in the Exosome Diagnostics TH 26 EORTC-NCI-AACR Symposium on Molecular Targets and Cancer Therapeutics (Nov. 18 – 21, 2014) body. Since the total amount of nucleic acids in biofluids is very limited and tumor mutations are reflected on both RNA and DNA, we devised a method to co-isolate all exoRNA and cfDNA out of blood plasma samples into a volume small enough for effective downstream processing by RTqPCR and targeted re-sequencing by NGS. Materials and Methods: We developed a novel spin column-based method, EXO52, to co-isolate the RNA and DNA from 1-4 mL of blood plasma and serum. The purification is based on membrane affinity-binding of extracellular vesicles, which protect the exoRNA from degradation, and simultaneous capturing of the circulating cfDNA. Purification of high quality nucleic acids and subsequent reverse transcription allows the analysis of mutations present in both fractions of nucleic acids by allele specific RT-qPCR and ultra deep sequencing (UDS) of disease-specific gene panels. The spectrum of mutations covering 15 mutation hotspots from 8 genes was analyzed using a custom library preparation method and bioinformatics pipeline. Results: EXO52 spin columns efficiently capture high quality nucleic acids from biofluids and a single column can be used to deplete cfDNA and exoRNA from plasma volumes up to 4 mL. The eluate contains cfDNA as well as a non-degraded small and large RNAs including mRNA, miRNA, and rRNA. As a proof of principle, we demonstrate the detection of high and ultra-low frequency mutations in blood samples of three different cancer types including malignant melanoma and colorectal cancer. Somatic mutations can readily be detected in both exoRNA and cfDNA, with the combination of both as the superior choice for mutation signal and yield. Conclusions: In this study we demonstrate the utility of a blood-based liquid biopsy using EXO52 spin columns for detection of somatic mutations in the co-purified exoRNA and cfDNA. Since the total amount of genetic material in plasma is a limiting factor for current development of genetic tests, we show the combination of high quality RNA from vesicles and cfDNA to be of advantage for detection of somatic mutations in various cancers in addition to enabling expression-based analysis and detection of fusion transcripts. Co-authors: D. Enderle1, K. Brinkmann1, T. Koestler1, S. Bentink1, C. Berking2, J. Skog3, M. Noerholm1. 1 Exosome Diagnostics GmbH, Martinsried/Munich, Germany. Ludwig-Maximilian University, Dept. of Dermatology and Allergology, Munich, Germany. 3 Exosome Diagnostics Inc., Cambridge, USA. 2 Poster Session 2: Subject: Monitoring therapy response and resistance mutations in circulating RNA and DNA of plasma from patients with malignant melanoma Exosome Diagnostics TH 26 EORTC-NCI-AACR Symposium on Molecular Targets and Cancer Therapeutics (Nov. 18 – 21, 2014) Abstract: Background: Activating BRAF-mutations (V600E) are frequent events in malignant melanoma, occuring in about 60% of all patients. Mutated BRAF is an important drug target for specific inhibitors, (e.g. Vemurafenib), and monitoring the BRAF mutation status in patients can be useful for informed treatment decisions. Performing repeated tissue biopsies is expensive and an invasive procedure for the patient and does not assess the status of the complete tumor burden including metastases. Isolation of the tumor's genetic material from blood samples in a 'liquid biopsy' is an actively researched option. Blood plasma contains at least two sources of cell-free nucleic acids: circulating cell-free DNA (cfDNA), generated by apoptotic or necrotic cells, and RNA enclosed in extracellular vesicles including exosomes (exoRNA), actively secreted by cells in the body – including the cancerous tissue. Combining both sources of nucleic acids does not only maximize the yield of the limited amount of genetic material circulating in plasma, it also allows to monitor both, biologically different, sources for treatment response and the early occurrence of resistance mutations. Materials and Methods: We applied a novel, spin-column based method (EXO52) to co-isolate all exoRNA and cfDNA out of blood plasma samples from longitudinal clinical samples of patients with malignant melanoma. Purification of high quality nucleic acids and subsequent reverse transcription allows the analysis of mutations present in both fractions of nucleic acids by ultra deep sequencing (UDS) of a gene panel covering 15 mutation hotspots from 8 genes with NGS. The UDS data was generated using a custom library preparation method and analyzed with a novel bioinformatics pipeline to efficiently call the rare mutations. Results: Somatic mutations from melanoma tumors can be readily detected on both exoRNA and cfDNA from plasma samples and was used here to study the mutations over the course of up to 8 months of treatment. Combining both exoRNA and cfDNA is beneficial due to the limited amount of genetic material present in the sample material, especially in patients with lower amounts of circulating mutations. Differential isolation of either RNA or DNA were used to define sets of mutations that are detected earlier in one or the other source since they are generated by different biological mechanisms. Conclusions: The co-isolation of exoRNA and DNA with EXO52 spin-columns is well suited to monitor somatic mutations in melanoma patients over the course of treatment. Combining the information from both mutation sources in the EXO52 eluate and the downstream analysis allows for a more complete picture of the treatment response and recurrence. Co-authors: D. Enderle1, K. Brinkmann1, T. Koestler1, S. Bentink1, K.T. Flaherty2, J. Skog3, M. Noerholm1. 1 Exosome Diagnostics GmbH, Martinsried/Munich, Germany. Massachusetts General Hospital Cancer Center, Boston, USA. 3 Exosome Diagnostics Inc., Cambridge, USA. 2 Exosome Diagnostics TH 26 EORTC-NCI-AACR Symposium on Molecular Targets and Cancer Therapeutics (Nov. 18 – 21, 2014)