DNA and RNA extraction for molecular analyses from EUS-acquired pancreatic neoplasia samples employing different needles: a cohort of more than 300 cases

Aims Despite advances with chemotherapy regimens, Pancreatic adenocarcinoma (PDAC) is becoming the 2^nd cause of cancer-related death. Personalized medicine will be the game changer but molecular characterization of pancreatic neoplasms is yet extremely cumbersome and has been performed on surgical samples or few EUS-tissue acquisition(TA) cases, mostly for DNA analysis. Our aim was to evaluate DNA and RNA extracted for research purposes from pancreatic neoplasms employing Endoscopic Ultrasound (EUS) Fine needle aspiration (FNA) and biopsy (FNB) and adequacy to molecular analyses.

Methods From Oct 2022, patients with solid pancreatic neoplasms suggestive of either PDAC or neuroendocrine neoplasm (NEN) undergoing diagnostic EUS-TA were considered for enrollment. Employed needles were either FNA, or Franseen, Fork-tip or Three-prong, either 22G or 25G based on operator’s preference, with Rapid On Site Evaluation and, in case of adequacy within the first 2 passes, 1 additional pass with the same needle was performed for molecular analyses. DNA and RNA were then extracted with a new technique and nucleic acids were quantified and evaluated for adequacy for molecular analysis. Mann-Whitney was employed for comparison of continuous variables, and Kruskal-Wallis test for comparison between multiple groups; Logistic regression was applied to determine factors associated with adequacy to molecular tests.

Results 310 patients underwent sampling for molecular analyses, 268 with a definitive diagnosis of PDAC and 42 with NEN. 93.5% of patients were sampled with 25G FNA needles. Mean quantity of DNA extracted was significantly higher for PDAC 1764 ng (95%CI: 1544-2479) compared to NEN 704 ng (95%CI: 435-981)(p<0.0001). Mean quantity of RNA extracted was similar, 310 ng for PDAC, and 444 ng for NEN (p=0.07), with a higher median RIN of 6,4 (95%CI:6,2-6,5) for PDAC versus 4,7 (95%CI:3,9-5) for NEN (p<0.0001). In terms of needle type, concerning RNA extraction, 25G FNA needles retrieved a median of 311 ng(RIN 6,3), 25G Franseen needles 464 ng(RIN 5,5), 22G Franseen 884 ng (RIN 5,9), 25G Fork-tip or Three-prong 223 ng (RIN 5,1), with no significant difference. 236 (95,9%) of PDAC samples and 34 (80,9%) of NEN samples were considered adequate for sequencing. The only factor associated to adequacy for molecular analysis was tumor type, with PDAC having an OR of 6.34 (95%CI 2.33-17.26)(p=0.0003), while the type of employed needle was not.

Conclusions Molecular analysis on EUS-acquired tissue of pancreatic neoplasms will be the mainstay of personalized therapies but how to best perform tissue acquisition and sample handling for this specific purpose is yet to be defined. This is the largest study ever performed on DNA and RNA extraction from EUS-acquired samples, with PDAC showing better retrieval of DNA and RNA for molecular analysis compared to NEN. All needle types result similar in terms of performance despite the low number of needles other than 25G FNA needles.

Publication History

Article published online:
27 March 2025