Development of a NGS workflow for diagnostic applications in oncology

The Ph.D. research work is an integral part of a Horizon 2020 project, called HERCULES project (CompreHEnsive chaRacterisation and effeCtive combinatorial targeting of high-grade seroUs ovarian cancer via singLE-cell analysiS). The topics of the Horizon 2020 project are to comprehensively characterize high grade serous ovarian cancer (HGS-OvCa) by integrating and modeling clinical and biological data (e.g., genetics, transcriptomics, protein binding, drug screens) from primary, metastatic and relapsed tumors from various anatomical sites of HGS-OvCa patients, and establish combinatorial treatment modalities that effectively kill HGS-OvCa tumor cell subpopulations. The role of the Company in HERCULES project is to develop and validate a marketable prototype biomarker kit for predicting HGS-OvCa patients response to combinatorial therapeutic modalities. In detail, the kit hypothesized for this application is a NGS gene panel, based on an Illumina platform and technology, capable to predict the outcome of a pharmacological therapy using high grade serous ovarian cancer subpopulation genetic biomarkers. A further aim of the Ph.D. project is more generally to study the processes required for developing an in vitro diagnostic (IVD) workflow finalized to a next generation sequencing analysis for oncology applications, not only focused on high grade serous ovarian cancer, having diagnostic, prognostic and predictive purposes. In order to achieve this goal, the following activities were carried out: 1. Evaluation of the “state of art” regarding the presence of patents relevant for the HERCULES project. 2. Definition of a gene panel design based on several genes involved in HGS-OvCa. 3. Identification and selection of FFPE DNA and RNA extraction kits having features, in terms of nucleic acid yields, quality and purity, that are compatible with the NGS downstream analysis and an IVD workflow. 4. Study of the processes necessary to sequence 12 human FFPE samples, having both KRAS wild type and mutated, on the Illumina platform using amplicon technology. As a result of this work three commercial column-based DNA extraction kits have been identified as the most effective when included in a NGS workflow based on Illumina technology. The study and the development of the NGS workflow were done employing available clinical FFPE sections of human colorectal cancer with already determined KRAS mutational status using established IVD assays. The results obtained with the NGS analysis have also demonstrated that the gene panel design for the library preparation kit, which uses both amplicon and UMI (unique molecular identifier) technologies, provides high UMI incorporation, high coverage depth of the regions of interest. This are fundamental aspects required for the identification of false-positive and mutations expressed at very low levels. These results will be used for developing clinical custom genes panel, intended to be used by pathologists and oncologists, capable to provide: -Prediction cancer onset. -Characterization of different tumor types. - Precise information about the exact therapeutic treatment for different cancer types (HGS-OvCa included). - Prognosis.