Pearls and pitfalls of real-life molecular testing on fine-needle aspiration and core biopsy in pancreatic adenocarcinoma practice

Abstract

Pancreatic ductal adenocarcinoma (PDAC) frequently requires neo-adjuvant therapy, leaving cytologic preparations – especially endoscopic ultrasound-guided fine-needle aspiration smears – as the only naïve tissue available for molecular testing. However, their applicability remains underappreciated due to limited data and concerns about specimen adequacy. This study aimed to evaluate the feasibility of performing molecular analysis on cytologic smears to detect targetable alterations in PDAC. Methods: Molecular analysis was conducted on 120 PDAC samples: 41 cytology specimens, 50 core biopsies, and 29 resections. KRAS mutations and homologous recombination repair gene alterations were assessed. Rapid on-site evaluation guided triage in all FNA cases. DNA and RNA isolations were performed, followed by quality control (QC) assessment and sequencing. Results: DNA isolation succeeded in 92/95 cases (97%), with a 100% success rate in cytologic specimens. RNA isolation passed QC in 71/84 samples (83%), with failures more common in smears (n = 8). KRAS mutations were detected in 71/85 patients (82%), with the highest detection in cytologic specimens (92%) compared to biopsies (78%) and resections (80%). Conclusion: Molecular testing is feasible and may even be more successful in cytologic smears than in biopsies or resections. High diagnostic yield and rapid processing favor their integration into routine molecular workflows. The superior performance of smears may relate to reduced stromal content and minimal processing delays. Cytologic specimens showed 100% DNA QC success, even when RNA QC failed, supporting their reliability. Although RNA analysis had a modest failure rate, its overall success suggests it can be incorporated into routine testing, particularly as fusion-driven targets gain clinical relevance.