The biological progression of pancreatic neuroendocrine neoplasms (PanNENs) is significantly influenced by their grading. Endoscopic ultrasound (EUS) is the gold standard for PanNENs’ grading evaluation but inconsistent EUS/surgery Ki67 agreement rates  have been found. This inconsistency underscores the need for improved diagnostic techniques in EUS. The aim of the project is to evaluate the feasibility of extracting RNA in sufficient quantity and quality to perform genomic analyses from specimens obtained through EUS-fine needle aspiration (EUS-FNA) of PanNENs, comparing three methods of RNA preservation and extraction.

This study prospectively evaluated patients undergoing EUS for suspected PanNENs. A uniform biopsy procedure using a 25-gauge Menghini needle with a slow-pull technique was applied. Sample adequacy was assessed on-site by a cytopathologist. For cases deemed adequate and suspicious of malignancy, an additional biopsy was performed, and RNA preservation and extraction were conducted using three distinct methods: Snap frozen plus Trizol (Method 1), Fresh tissue plus 1-Thioglycerol buffer solution (Method 2), and Snap frozen followed by 1-Thioglycerol buffer solution (Method 3). RNA integrity and concentration were measured using the 2100 Bioanalyzer

The study included 37 PanNEN patients, predominantly male (62.2%), with a median age of 59 years. The median Ki67 proliferation index was 2%, with most tumors graded as G2 (62.2%). RNA extraction yielded a median global RNA concentration of 11,000 pg/ul, and the median RNA Integrity Number (RIN) was 3.7. Method 1 resulted in the highest median RNA concentration, while Method 3 produced the highest median RIN value. Significant differences were observed between the methods in terms of RNA concentration and RIN. Univariate linear regression analysis considering various patient and tumor characteristics found no significant associations with RNA quantity or quality.

Successfully extracting high-quality RNA from EUS-FNA samples of PanNENs represents a significant advancement in the molecular profiling of these tumors. This development is crucial in the context of modern oncology, where molecular characterization increasingly informs treatment and prognostic evaluations.