Optimierung der Präanalytik für die NGS-basierte (Next Generati-on Sequenzierung) Nukleinsäurediagnostik an Tumorgewebe

Summary: NGS as a fundament of personalized cancer therapy The investigation of the NGS offers a comprehensive method for the identification of therapeutically attackable target structures in cancer therapy. Currently, fresh tissue is used as starting material, but its extraction is usually associated with invasiveness for the pa-tient. Therefore, the question arises as to whether the NGS examination, with optimal pre-analytics, could also be carried out with another source tissue, e.g. FFPE tissue already generated in the course of routine diagnostics. Which material shows the highest quality results? Furthermore, the results of a local panel sequencing and the much more complex and expensive Whole Exome sequencing were compared. For the establishment of an optimal preanalytics three tissue forms were compared under qualitative, financial and practical aspects (fresh tissue, FFPE and cryo). Fresh tissue was stored with a RNAlater Stabilization Solution and cryopreserved by shock freezing in liquid nitrogen. The FFPE samples have already been established in clinical practice. After the respective nucleic acid isolation, the quality and quantity of the samples were evaluated by spectrometry, tape-gel electrophoresis and an internal sequencing QC assay by Archer. Furthermore, an exempla-ry comparison of the sequencing results of four patients who received a panel sequencing (63 genes) at the University Hospital Marburg from FFPE samples and a WES from fresh tissue at the University Hospital Heidelberg was performed. It was shown that in principle all three methods of tissue preservation can be used for the investigation of NGS. The comparatively lowest amounts and the lowest quality scores were found in the FFPE samples. The highest quantity and quality in combination with low costs and a good feasibility showed the fresh tissue preserved in RNAlater. The comparison of the results of the 4 exemplary examined patients with Panel + WES revealed newly discov-ered mutations in both sequencing approaches, but none of the four patients showed a therapeutic success. In general, the approach of personalized oncology provides the basis for current controversial discussions about advantages and disadvantages (MOSCATO study vs. DRUP trials). In order to integrate these inconsistent findings into a clinically oriented estimate, it would be a comprehensible approach to focus on gene panel diagnos-tics, through which most therapy-relevant mutations can be found. The optimization and continuous updating of panel sequencing with current drug targets and new effective ther-apies should be in the focus. This could be performed on mostly existing FFPE samples without further invasiveness for the patient. Furthermore, in case of therapy failure a more detailed sequencing could be considered individually. Thus, a targeted panel analysis could be equivalent to the more time and cost intensive overall genome sequencing.