Supernatant Fluid from Endobronchial Ultrasound-Guided Transbronchial Needle Aspiration for Rapid Next-Generation Sequencing

Abstract

Abstract Introduction/Objective The aims of our study were to optimize the workflow of non-small cell carcinoma (NSCC) endobronchial ultrasound-guided bronchoscopy with transbronchial needle aspiration (EBUS-TBNA) samples to maximize tissue available for next-generation sequencing (NGS), preserve formalin-fixed paraffin-embedded (FFPE) cell blocks (CBs) for future testing, and shorten turnaround time (TAT) of NGS results. We evaluated the performance of supernatant fluid (SNF) processed from a dedicated aspirate for NGS testing. Methods/Case Report 20 EBUS-TBNA samples positive for NSCC on rapid on-site evaluation were collected and processed using a new workflow (Figure 1). Five aspirates were collected in formalin. One additional dedicated pass was collected fresh and centrifuged. The resulting cell pellet was added to the passes in formalin for FFPE CB processing. The SNF was recentrifuged. DNA and RNA were extracted from concentrated SNF for targeted testing using the Oncomine™ Precision Assay (Thermo Scientific™, Waltham, MA). NGS results from the corresponding FFPE CBs were used as “controls” for comparison. Results (if a Case Study enter NA) A total of 31 mutations were detected in SNF (Table 1). The most frequently mutated genes were TP53 (35%), EGFR (23%), KRAS (13%), CTNNB1 (6%), and ERBB2 (6%). EGFR and KRAS amplification, CDKN2A deletion, and SQSTM1-NTRK3 fusion alteration were also detected. There was 100% concordance between the mutations detected in SNF and corresponding FFPE CBs with comparable variant allele frequencies. TAT of NGS results was 1 day for SNF compared to 4 – 10 days for FFPE CB. Conclusion In our study, we were able to demonstrate the usefulness of NGS on SNF to provide reliable, rapid molecular results. This testing strategy was successfully incorporated into the workflow for tissue handling and processing between our clinical, cytopathology, and molecular teams. Molecular results were available at the same time as the cytologic diagnosis, allowing for timely reporting of a comprehensive diagnosis. This approach is particularly useful in patients with advanced disease requiring urgent management.