The development of breast and ovarian cancer is strongly connected to the inactivation of the BRCA1 and BRCA2 genes by different germline and somatic alterations, and their diagnosis has great significance in targeted tumor therapy, since recently approved PARP inhibitors show high efficiency in the treatment of BRCA-deficient tumors. This raises the need for new diagnostic methods that are capable of performing an integrative mutation analysis of the BRCA genes not only from germline DNA but also from formalin-fixed and paraffin-embedded (FFPE) tumor samples. Here we describe the development of such a methodology based on next-generation sequencing and a new bioinformatics software for data analysis. The diagnostic method was initially developed on an Illumina MiSeq NGS platform using germline-mutated stem cell lines and then adapted for the Ion Torrent PGM NGS platform as well. We also investigated the usability of NGS coverage data for the detection of copy number variations and exon deletions as a replacement of the conventional MLPA technique. Finally, we tested the developed workflow on FFPE samples from breast and ovarian cancer patients. Our method meets the sensitivity and specificity requirements for the genetic diagnosis of breast and ovarian cancers both from germline and FFPE samples.

Circulating tumor DNA (ctDNA) is increasingly employed in the screening, follow-up, and monitoring of the continuously evolving tumor; however, most ctDNA assays validated for clinical use cannot maintain the right balance between sensitivity, coverage, sample requirements, time, and cost. Here, we report our BC-monitor, a simple, well-balanced ctDNA diagnostic approach using a gene panel significant in breast cancer and an optimized multiplex PCR-based NGS protocol capable of identifying allele variant frequencies below 1% in cell-free plasma DNA. We monitored a cohort of 45 breast cancer patients prospectively enrolled into our study receiving neoadjuvant chemotherapy or endocrine therapy or palliative therapy for metastatic diseases. Their tumor mutation status was examined in the archived tumor samples and plasma samples collected before and continuously during therapy. Traceable mutations of the used 38-plex NGS assay were found in approximately two-thirds of the patients. Importantly, we detected new pathogenic variants in follow-up plasma samples that were not detected in the primary tumor and baseline plasma samples. We proved that the BC-monitor can pre-indicate disease progression four–six months earlier than conventional methods. Our study highlights the need for well-designed ctDNA monitoring during treatment and follow-up, integrated into a real-time treatment assessment, which could provide information on the active tumor DNA released into the blood.