Supplementary Material for: Pearls and Pitfalls of Real-Life Molecular Testing on FNA and Core Biopsy in Pancreatic Adenocarcinoma Practice

Background/Objectives:Pancreatic ductal adenocarcinoma (PDAC) frequently requires
neo-adjuvant therapy, leaving cytologic preparations—especially endoscopic
ultrasound-guided fine needle aspiration (EUS-FNA) 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 (ROSE)
guided triage in all FNA cases. DNA and RNA isolation 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.