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

• Published in: Cancer cytopathology 2021-12, Vol.129 (12), p.956-965
• Main Authors: Perrone, Marie E., Alvarez, Rebeca, Vo, Tawnie T., Chung, Moon‐Wook, Chhieng, David C., Paulson, Vera A., Colbert, Brice G., Q. Konnick, Eric, Huang, Eric C.
• Format: Article
• Language: English
• Subjects: Biomarkers; Cell-Free Nucleic Acids - genetics; Cellular biology; cell‐free DNA; cytology; DNA - genetics; Formaldehyde; High-Throughput Nucleotide Sequencing - methods; Humans; Melanoma - diagnosis; Melanoma - genetics; Mutation; next‐generation sequencing; Paraffin Embedding - methods; Pathology, Molecular; Prospective Studies; supernatant; validation
• ISSN: 1934-662X; 1934-6638
• DOI: 10.1002/cncy.22491

Background 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. Methods cfDNA was extracted prospectively from residual supernatants of 30 cytology samples (29 neoplastic cases and 1 benign ascitic fluid from a patient with a history of melanoma). Samples were tested using clinically validated next‐generation–sequencing platforms and the results were compared with data from paired FFPE cell 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). Results 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. Conclusions This study confirmed that cfDNA from supernatant is a viable alternative to FFPE cell blocks for molecular biomarker testing using both amplicon‐based and capture‐based assays with potential for decreasing additional tissue sampling and faster turnaround time. Cell‐free DNA from supernatant is a viable alternative to cell blocks for molecular biomarker testing using both amplicon‐based and capture‐based assays.