Evaluating the Effectiveness of a 50-Gene Hotspot Next-Generation Sequencing Panel for Molecular Cytopathology

Summary: Introduction: Cytology specimens often represent the initial and sole source of diagnostic material in cancer patients. Unlike tissue biopsies fixed in formalin, cytology specimens typically retain higher-quality nucleic acids. However, the diversity of cytology specimen types increases the complexity of assay validation. This study aims to validate the use of the Oncomine Precision Assay (OPA), an amplicon-based, 50-gene hotspot next-generation sequencing (NGS) panel, across various cytological samples. Materials and Methods: We extracted DNA, RNA, and their cell-free fractions from cytology specimens (including body fluids, ThinPrep samples, smears, FFPE cell blocks, and direct collection of FNA material into RNAlater) using commercially available kits (Maxwell, Genexus or QIAmp extraction kits). Body fluids and ThinPrep residual material were extracted using 2-4ml of direct fluid with and without prior centrifugation. For centrifuged cases DNA and RNA were extracted from the pellet and cfDNA and cfRNA were extracted from the supernatant. Yields were compared. Cases with known genetic alterations included in the 50-gene panel were sequenced on a Genexus instrument. Per manufacturer instructions, 3 million reads per sample is recommended to run a total of 4 samples per lane. We explored running 1.5 million and 1 million reads per sample to increase the throughput to 8 and 12 samples per lane, respectively. Comparison analyses and concordance determination were performed. Results: A total of 136 extractions (fluids (108), ThinPrep (12), smears (3), RNALater (3), cell blocks (10)) from 75 cases were performed. The highest DNA extraction yield was obtained from centrifuged cell pellets, followed by vortexed fluid aliquot, and then by cell-free DNA and RNA fractions. Sequencing of 56 samples from 26 patients and 3 controls demonstrated 100% concordance with the results of a commercial whole-exome sequencing reference test for the genes included in the panel. For two cases, the 50-gene panel identified additional mutations not detected by the reference test. We observed no significant differences in quality control metrics, coverage, or allelic frequencies across the different sample types. Sequencing up to 12 samples per lane reduced the mean coverage from ~4000X to ~500X but did not compromise sensitivity for the detection of mutations, fusions or copy number alterations (as seen in Figure 1). Conclusions: DNA, RNA, and their cell-free fractions can be successfully extracted from all cytology specimen types in sufficient quantities for targeted next-generation sequencing. Increasing the number of samples per lane on the Genexus instruments reduces coverage but does not compromise assay sensitivity, offering both testing flexibility and modest cost savings. In conclusion, our findings affirm the effectiveness of the OPA panel for NGS across various cytology specimens, demonstrating high concordance with established reference tests and the capability to detect additional mutations. This study underscores the value of cytology specimens in enhancing diagnostic precision in molecular cytopathology, advocating for their broader integration into clinical practice.