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Genetic and drug sensitivity assays on primary cultures are not only of basic but also of translational interest and could eventually aid oncologists in the selection of treatments. However, cancer cells need to be identified and differentiated from the non-tumor cells always present in primary cultures. Also, successive passages can change the proportions of these two subpopulations. In this study, we propose fluorescence in situ hybridization (FISH) analysis on cell smears to determine the presence of tumor cells in primary cultures obtained from patients carrying translocations or copy number gains. FISH proved to be an easy, fast, economic, and reliable method of characterizing cell populations, which could be used repeatedly at different passages to monitor variations and to confirm the maintenance of translocations and copy number gains throughout the culture process. [ABSTRACT FROM AUTHOR]

Simple Summary: We here performed a comprehensive comparison of commonly used approaches for EV-DNA extraction, by assessing DNA quantity, its quality, and its suitability for downstream analyses. We found that the tested methods resulted in different DNA yields and fragment sizes. As a consequence, we found that different EV-DNA extraction methods had an impact on the detection of specific mutations by qPCR and on the suitability of next-generation sequencing (NGS), which could impact downstream clinical applications. (1) Background: Extracellular vesicles (EVs) have emerged as crucial players in the communication between cells in both physiological and pathological scenarios. The functions of EVs are strongly determined by their molecular content, which includes all bioactive molecules, such as proteins, lipids, RNA, and, as more recently described, double-stranded DNA. It has been shown that in oncological settings DNA associated with EVs (EV-DNA) is representative of the genome of parental cells and that it reflects the mutational status of the tumor, gaining much attention as a promising source of biomarker mutant DNA. However, one of the challenges in studies of EV-DNA is the lack of standardization of protocols for the DNA extraction from EVs, as well as ways to assess quality control, which hinders its future implementation in clinics. (2) Methods: We performed a comprehensive comparison of commonly used approaches for EV-DNA extraction by assessing DNA quantity, quality, and suitability for downstream analyses. (3) Results: We here established strategic points to consider for EV-DNA preparation for mutational analyses, including qPCR and NGS. (4) Conclusions: We put in place a workflow that can be applied for the detection of clinically relevant mutations in the EV-DNA of cancer patients. [ABSTRACT FROM AUTHOR]