EACR24 Poster Sessions/European Journal of Cancer 61, Suppl. 1 (2016) S9–S218 S143 636 Performance evaluation of a new integrated blood collection/sample preparation system for the stabilization and extraction of circulating cell-free DNA (ccfDNA) A. Ullius 1 , T. Voss 1 , D. Gr¨ olz 1 . 1 Qiagen GmbH, PreAnalytiX Product Development, Hilden, Germany Background: The application of circulating cell-free DNA (ccfDNA) as biomarker is an emerging field in cancer research, e.g. to monitor and detect tumors. The sensitivity of rare mutant allele fraction analysis and quantification of circulating tumor DNA (ctDNA) is, however, compromised by the release of genomic DNA from lymphocytes due to mechanical lysis or apoptosis during collection, transport and storage of the blood sample. Moreover, the extraction of the low concentrated and highly fragmented ccfDNA is technically challenging. PreAnalytiX has developed the PAXgene ® Blood ccfDNA System, consisting of the PAXgene Blood ccfDNA Tube, a plastic BD Vacutainer ® tube with unique, non-crosslinking chemistry preserving extracellular levels of ccfDNA and preventing the release of intracellular DNA from cells into the plasma, and the QIAsymphony ® PAXgene Blood ccfDNA Kit for automated ccfDNA extraction from up to 5 ml of plasma. The fully automated QIAsymphony PAXgene Blood ccfDNA workflow offers two protocols to choose between purification of predominately small ccfDNA fragments or co-isolation of large ccfDNA fragments to cover the needs for diverse research applications such as organ disease and cancer. Here we present the stabilization and extraction efficiency of the new system compared to blood samples collected in EDTA tubes. Methods: Whole blood from up to 20 donors in three replicates was collected into PAXgene Blood ccfDNA tubes or spray-dried K2EDTA tubes and stored for up to 7 days at 25ºC. For transport studies, blood tubes were rotated for 5 hours, followed by horizontal storage for up to 7 days. ccfDNA was extracted from plasma using the QIAamp Circulating Nucleic Acid Kit or the QIAsymphony PAXgene Blood ccfDNA Kit and the obtained ccfDNA was analyzed by quantitative real-time PCR, using assays with validated performance. Two automated extraction protocols were compared for their efficiency to purify in addition to small ccfDNA also longer ccfDNA fragments. Results: ccfDNA yield from PAXgene Blood ccfDNA tubes was comparable to blood drawn into EDTA tubes, when plasma was processed directly after blood draw. In contrast to blood from EDTA tubes, ccfDNA yield did not increase significantly during simulated transport and storage. A direct comparison between the two protocol lines (STA and LAF) demonstrated efficient co- isolation of large ccfDNA fragments in addition to unbiased isolation of small ccfDNA fragments for the LAF protocol. Conclusions: The PAXgene Blood ccfDNA Tube stabilizes blood cells for transport and short time storage. The fully automated QIAsymphony PAXgene Blood ccfDNA workflow offers two protocols for purification of predominately small ccfDNA fragments or co-isolation of large ccfDNA fragments to provide optimal sample conditions for various research applications. For Research Use Only. Not for use in diagnostic procedures. Conflict of interest: Other Substantive Relationships: QIAGEN employee. 638 Speeding up the S5 XL sequencing system: Sequencing in an hour enables sample to answer in a 8 hr workday R. Qi 1 , C. Parikh 1 , A. Luchetti 2 , D. Mandelman 3 , H. Latif 3 , A. Harris 3 , S. Ghosh 1 . 1 Thermo Fisher Scientific, Ion Torrent, South San Francisco, CA, USA, 2 Thermo Fisher Scientific, Ion Torrent, Carlsbad, CA, USA, 2 Thermo Fisher Scientific, Ion Torrent, San Diego, CA, USA The Ion S5 XL™ Sequencer currently sequences 200 bp in 2.5 hrs with about 1 hr analysis time. Here we focused on optimization of the sequencing and data analysis workflow in an effort to reduce total turn around time (TAT). Since it largely depends on and the number of reagent flows (one flow produces ~0.5 base) and the speed of the flows, we improved the flow speed and utilized the minimum number of flows optimized for the length of the amplicons. We used an existing streamlined library preparation workflow based on AmpliSeq, clonal amplification (templating) with Isothermal Amplification, and the new rapid Ion S5 XL™ Sequencer procedure. The new rapid approach was utilized with two different types of applications that have the potential to benefit from rapid sequencing turn-around times. (1) A rapid infectious disease identification and antibiotic susceptibility panel based on the AmpliSeq highly-multiplexed PCR approach. (2) A whole genome amplification based approach for detection of aneuploidies for pre-implantation Genetic Screening of fresh to allow a same or next day decision for implantation of in vitro fertilized embryos. With Ion S5 XL™ Sequencer and modifications to the on-instrument flow times and total number of flows, we can reduce the time for sequencing and accurate data analysis for applications requiring rapid TAT. For AmpliSeq and ReproSeq assays, sequencing and Torrent Suite analysis could be completed in as short as 55 minutes (200 flows). Paired with fast library and template preparation, the total TAT was at or below a standard workday. We demonstrated that accuracy of detection was not compromised with the reduced TAT. No conflict of interest. 639 Cold atmoshperic plasma as an approach to retinoblastoma R. Silva-Teixeira 1 , M. Laranjo 2 , G. Brites 1 , A.M. Abrantes 2 , T. Rodrigues 3 , A.C. Gon¸ calves 4 , S. Raquel 3 , A.B. Sarmento-Ribeiro 4 , P. Matafome 5 , F. Caramelo 1 , M.F. Botelho 2 . 1 Faculty of Medicine of University of Coimbra, Institute of Biophysics/Biomathematics, Coimbra, Portugal, 2 Faculty of Medicine of University of Coimbra, Institute of Biophysics/Biomathematics- CIMAGO- CNC.IBILI, Coimbra, Portugal, 3 Faculty of Medicine of University of Coimbra, Laboratory of Physiology- CNC.IBILI, Coimbra, Portugal, 4 Faculty of Medicine of University of Coimbra, Applied Molecular Biology Unit, Coimbra, Portugal, 5 Instituto Polit´ ecnico de Coimbra- Coimbra Health School ESTeSC, Department of Complementary Sciences, Coimbra, Portugal Background: Retinoblastoma is the most common primary intraocular tumor in children. Histologically, necrosis found in relatively avascular areas demonstrate the dependence of retinoblastoma on its blood supply. Unfortunatelly, available therapies lack selectivity and are associated to serious side effects such as loss of vision and secondary neoplasias. In order to circumvent these effects our group is studying plasma, an ionized gas, as an option. Therefore, the aim of this work was to evaluate the effect and selectivity of cold atmospheric plasma (CAP) in human retinoblastoma. Materials and Methods: An electronic device capable of generating high output voltage (HV; ~4kV) was designed to produce an electrical discharge between the HV electrode and multiwell plates. Human retinoblastoma Y79 cells were seeded in multiwell plates. CAP was generated in open air, 2 mm above the surface of the cell cultures medium, during several short periods of time, ranging from 15 s to 180 s. In order to evaluate the cytotoxicity of the plasma flair, Alamar Blue and MTT assays were performed. In addition, cell viability was assessed by SRB assay. Moreover, annexin V/ propidium iodide (PI) apoptosis detection kit and PI/RNAse staining solution for Flow Citometry were used to study type of cell death and cell cycle, respectively. Concerning selectivity, human fibroblasts HFF1 were treated similarly and Alamar Blue was performed. Furthermore, to evaluate the potential anti-angiogenic effects of CAP, an aortic ring assay was carried out in excised mouse thoracic aorta segments. All the tests were performed 24 hours after CAP exposure. Results and Discussion: The metabolic activity of retinoblastoma cells significantly decreased accordingly to the CAP exposure time. After 60 s of CAP exposure, the metabolic activity was (37.5±19.7)% (p < 0.001) and (57.2±21.2)% (p = 0.047) as evaluated by Alamar Blue and MTT assay, respectively. Furthermore, the time to reduce this activity in 50% (IT50) was 68.1 s and 79.2 s, accordingly to the previous techniques. Moreover, after 60 s of CAP exposure a viability of (56.3±19.6)% (p = 0.048) was obtained SRB assay, which reveals cell death as the main cause of obtained results. Preliminary results show both the contribution of apoptosis and necrosis to cell death and a shift of cells populations in each phase of the cell cycle. However, regarding the fibroblasts, after 60 s of CAP exposure the metabolic activity was still (74.5±13.1)% (p = 0.058) with an IT50 of 109.6 s. Furthermore, our preliminary results on aortic ring assay revealed that CAP might have anti- angiogenic effects. Conclusions: These results suggest that CAP is cytotoxic and might be selective to tumor cells (p < 0.001). Furthermore it might have a deleterious effect on tumor neoangiogenesis. Acknowledgment: Funding: FCT, Portugal (UID/NEU/04539/2013), COMPETE- FEDER. No conflict of interest. 640 Selective inhibition of HDAC1 and HDAC2 counteracts medulloblastoma cell growth in mouse models through Gli acetylation S. Coni 1 , A.B. Mancuso 1 , L. Di Magno 2 , G. Sdruscia 2 , D. Rotili 3 , A. Mai 3 , G. Canettieri 1 . 1 Sapienza University, Molecular Medicine, Rome, Italy, 2 Italian Institute of Technology, Center for Life Nanoscience@Sapienza, Rome, Italy, 3 Sapienza University, Department of Drug Chemistry and Technologies, Rome, Italy Introduction: Medulloblastoma (MB) is one of the most aggressive pediatric brain tumor and, despite a combination of surgery and chemotherapy, the prognosis is still poor for a significant fraction of patients. Hedgehog pathway is a crucial regulator of cerebellar development and its aberrant activation is a leading cause of MB. Pharmacological inhibition of Hedgehog pathway using Smo inhibitors has been proven effective in clinical trials, although resistance eventually occurs. Thus, targeting downstream Hh effectors appears to be a preferable option. In this view, we previously identified Gli acetylation as a critical druggable inhibitory modification for Hedgehog signaling, selectively regulated by HDAC1 and HDAC2. Aim of this work was to test the efficacy of HDAC1/2 inhibitors in MB growth in vitro and in preclinical mouse models. Material and Methods: Allografts, RNAi, proliferation curves, were performed as described in D’Amico et al. 2015. RNA studies, BrDu proliferation assays, immunohistochemistry and western blots were performed as described in Canettieri et al. 2010. Results: We first tested whether inhibition of HDAC1 and 2 is sufficient to suppress Hh signaling. Knockdown of HDAC1 or HDAC2 caused a strong