Assessing and Evaluating the Scope and Constraints of Idylla Molecular Assays by Using Different Source Materials in Routine Diagnostic Settings.

For cancer treatment, diagnostics concerning tumor type and determination of molecular markers in short TAT is critical. The fully automated, real-time PCR-based molecular diagnostic Idylla assays are well established in many laboratories for qualitative detection, short TAT and routine screening of clinically relevant oncogenic mutations. According to the manufacturer, all IVD assays are recommended for use only with FFPE tissue samples of 5-10 µM dissections with at least 10% tumor content. In this study, we tested the performance and accuracy of the IVD assays along with the gene fusion assay (RUO) with different tissue/source materials like isolated DNA/RNA, cryomaterial, etc. The study also included testing archival FFPE tissue sections dating back from 20 years and a performance check for different pan-cancer samples individually. All the assays tested with FFPE sections and gDNA/RNA input showed above 96% accuracy and sensitivity, individually with 100% specificity. The Idylla assays also performed exceptionally well on the archival FFPE tissues, and the use of assays for other solid tumors was also remarkable. The performance test and accuracy of Idylla assays showed high efficiency with certain limitations. For the use of Idylla assays, both qualitative and quantitative applicability of different tumor source materials could produce efficient results in different diagnostic settings within a short TAT.

Investigation of genomic DNA methylation by ultraviolet resonant Raman spectroscopy.

Cytosine plays a preeminent role in DNA methylation, an epigenetic mechanism that regulates gene expression, the misregulation of which can lead to severe diseases. Several methods are nowadays employed for assessing the global DNA methylation levels, but none of them combines simplicity, high sensitivity, and low operating costs to be translated into clinical applications. Ultraviolet (UV) resonant Raman measurements at excitation wavelengths of 272 nm, 260 nm, 250 nm, and 228 nm have been carried out on isolated deoxynucleoside triphosphates (dNTPs), on a dNTP mixture as well as on genomic DNA (gDNA) samples, commercial from salmon sperm and non-commercial from B16 murine melanoma cell line. The 228 nm excitation wavelength was identified as the most suitable energy for enhancing cytosine signals over the other DNA bases. The UV Raman measurements performed at this excitation wavelength on hyper-methylated and hypo-methylated DNA from Jurkat leukemic T-cell line have revealed significant spectral differences with respect to gDNA isolated from salmon sperm and mouse melanoma B16 cells. This demonstrates how the proper choice of the excitation wavelength, combined with optimized extraction protocols, makes UV Raman spectroscopy a suitable technique for highlighting the chemical modifications undergone by cytosine nucleotides in gDNA upon hyper- and hypo-methylation events.