Molecular Profiling in Prostate Cancer

This study will include formalin-fixed paraffin-embedded tumor tissue (FFPE) from 250 patients with metastatic, recurrent, locally advanced or "intermediate or high risk" (Gleason \>7), operable prostate cancer. FFPE tumor blocks alongside peripheral blood will be retrieved for all patients from Pathology Laboratories. Available FFPE blocks will be subjected to histological review by an experienced pathologist to evaluate H\&E sections for confirmation of diagnosis, histologic type, grade and tumor cell content (TCC%), as well as mark tumor dense areas for manual macro-dissection, prior to DNA extraction, in order to enrich samples for tumor DNA. Clinicopathologic characteristics of patients with prostate cancer will be retrieved from their respective medical records. Diagnosis will be confirmed through pathology reports, which will also provide information about Gleason Score and histological subtype. In all instances, the collection of patient information will be in compliance with the regulations of the Bioethics committees of participating Hospitals. The study will be conducted in accordance with the principles of the Helsinki Declaration of Human Rights. Following manual macro-dissection, FFPE tumor tissue material will be processed for DNA extraction, according to standard procedures with the QIAamp DNA Mini Kit (Qiagen GmbH, Hilden, Germany). Sequencing will be performed at the Laboratory of Molecular Oncology, using an Ion Torrent Proton Sequencer (Life Technologies/Ion Torrent). For the purpose of targeted NGS genotyping of tumor and available matched germline DNA samples, we designed a custom Ampliseq panel to target coding relevant regions of genes involved in homologous recombination (HR), along with several others. Data retrieval and base calling will be performed on the Torrent Server (v5.8.0.8). Consequently, we will then employ appropriate Ion Reporter Workflows (version 5.10) to automatically annotate single nucleotide variants (SNVs), multiple nucleotide variants (MNVs), small insertions / deletions (INDELs) and copy number variations (CNVs).