Comparison of cytocentrifugation supernatant fluid and formalin‐fixed paraffin‐embedded tissue for targeted next‐generation sequencing

Abstract

BACKGROUND:The emergence of less invasive procedures coupled with the growth of molecular testing have created a need for clinical laboratories to optimize workflows to enable tissue preservation and ancillary testing. In the preparation of formalin-fixed paraffin-embedded cell blocks (FFPE CBs), there is a cytocentrifugation step for cell pellet extraction that results in postcentrifugation supernatant fluid (SN). This SN, which in most routine workflows is discarded, has been suggested to contain adequate cellular material for molecular testing. In the current study, the authors describe the use of DNA and RNA extracted from SN for the detection of clinically relevant biomarkers by next-generation sequencing (NGS). METHODS: After cell pellet removal, cytocentrifugation SN from 30 endobronchial fine-needle aspiration rinses that were positive for malignancy on FFPE CB were collected. DNA and RNA were extracted from the SN and tested using an in-house NGS Solid Tumor Focus Assay. The NGS results were compared with findings from corresponding FFPE samples. RESULTS: Testing was successful in all 30 samples. There was 100% concordance between variants observed in the SN and corresponding FFPE specimens, which included 50 single-nucleotide variants, 9 copy number amplifications, 3 structural variants, and 2 indels. Furthermore, there was excellent correlation (correlation coefficient, 0.93) between the variant allele frequency of mutations observed in SN compared with that noted in corresponding FFPE CBs. CONCLUSIONS:Cytocentrifugation SN is a valuable source for NGS, is comparable to FFPE that preserves tissue for other ancillary testing, and can reduce the failure rate of testing that may result from insufficient material being available in the CB.We thank team members from the cytology and molecular diagnostic laboratories at University Hospitals of Cleveland.

Use of cytology centrifuged supernatants improves cost and turnaround time for targeted next generation sequencing

“…No further quality measures were used. 9 The CB FFPE sample and direct smears are used for diagnosis. The final report included a statement indicating F I G U R E 1 A, Flowchart of the old workflow using formalin fixed paraffin embedded cell blocks (FFPE CB).…”

“…Then NGS is ordered through EMR, and subsequent nucleic acid extraction and NGS analysis are performed [Color figure can be viewed at wileyonlinelibrary.com] on both workflows were performed as previously described. 9 Information for TAT and sample quality metrics were retrieved from pathology LIMS systems. The cost savings associated with the new workflow were calculated based on hourly wages of staff involved in the by-passed steps with new workflow.…”

“…Although the cytology formalin fixed paraffin embedded (FFPE) cell block (CB) is commonly used for molecular testing, post-centrifugation cytology supernatant (CCS) has been shown to serve as a viable alternative. [5][6][7][8][9][10][11][12][13] Previous reports have suggested that CCS for NGS analysis may be associated with improved turnaround time (TAT) and cost savings. [6][7][8][9] In this study, we reviewed our implementation process to evaluate its performance and share our experience with the improved molecular cytopathology workflow using previously discarded CCS.…”

Current and future trends in non–small cell lung cancer biomarker testing: The American experience

“…Recent studies have reported on the promising role of NSCLC FNA supernatant fluid samples for genomic profiling. [53][54][55] When compared with the mutation profile obtained from plasma ctDNA, the FNA supernatant fluid specimens were able to identify additional genomic alterations with therapeutic targets and detect mutations with higher allele fractions. 54 This increases the potential for the better use of limited-volume FNA specimens, especially in situations in which the diagnostic yield is adequate for a diagnosis of NSCLC but the cellular component is borderline or inadequate for lung cancer biomarker testing.…”

“…The advantage of using FNA supernatant fluid for mutational profiling in conjunction with immunohistochemistry and fluorescence in situ hybridization-based testing using conventional substrates such as cell blocks and/or liquid-based cytology is a more efficient and faster turnaround time for obtaining biomarker test results in these patients with NSCLC. 55 The ability to incorporate FNA supernatant fluid specimens into the clinical molecular workflow provides an opportunity to provide clinically relevant genomic information without the need for a repeat biopsy in patients for whom the tissue biopsy was insufficient.…”