Validating cell-free DNA from supernatant for molecular diagnostics on cytology specimens.

Abstract

Cytology specimens are often used for biomarker testing in the setting of neoplasia. On occasion, formalin-fixed paraffin-embedded (FFPE) cell blocks unfortunately may not yield sufficient material for testing. Recent studies have suggested that residual supernatant fluid from cell block preparation is a valuable source of DNA: both cellular and cell-free DNA (cfDNA). In the present study, the use of cfDNA from supernatant is compared against DNA from FFPE materials. cfDNA was extracted prospectively fromresidual supernatants of 30 cytologysamples (29 neoplastic cases and 1 benign ascitic fluid from a patient with a history of melanoma).Samplesweretested using clinically validated next-generation-sequencing platforms and the results werecompared with data from paired FFPEcell blocks in a real-time prospective clinical setting. Thirteen samples were tested on an amplicon-based assay (Solid Tumor Hotspot), and 17 samples were tested using a comprehensive capture-based assay (UW-Oncoplex). Neoplastic content was estimated by mutational variant allele fraction, with a mean content of 24.0% and 25.8% in supernatant and FFPE, respectively. The variant concordance between paired samples was 90%, and identical results were detected in both supernatant and FFPE samples in 74% of cases. This study confirmed that cfDNA from supernatant is a viable alternativeto FFPE cell blocksfor molecular biomarker testing using both amplicon-based and capture-based assays with potential for decreasing additional tissue sampling and faster turnaround time.