Background: As outlined by the Kyoto guidelines, targeted DNA-based NGS of pancreatic cyst fluid (PCF) is an important adjunct to the evaluation of pancreatic cyst patients. For instance, DNA mutations in KRAS are specific for mucinous cysts, while DNA alterations in TP53 are associated with advanced neoplasia (high-grade dysplasia and adenocarcinoma). Recently, retrospective studies have shown RNA-based NGS can further improve preoperative pancreatic cyst classification, but this technology has not been applied in clinical practice. We therefore performed a prospective, multi-institutional study to integrate a DNA/RNA NGS panel into the routine clinical assessment of PCF.

Methods: A 74-gene DNA/RNA-targeted NGS panel (PancreaSeq GC) was utilized to evaluate molecular alterations to include gene fusions and gene expression (e.g., CHGA [for cPNETs] and CEACAM5 [for CEA]). Between July 2022 to February 2024, 4312 patients were tested from >40 institutions and diagnostic performance was compared to clinical, imaging, cytologic, and surgical follow-up.

Results: Within 19 months, 4235 (98%) PCFs were satisfactory for DNA/RNA-targeted NGS, and a molecular alteration was identified in 3339 (79%) cysts. Mutations in KRAS, GNAS, BRAF, and/or RNF43 were identified in most cases (n=2645, 62%) and correlated with elevated CEA (>192 ng/ml) and mucin by cytology (p<0.001). However, RNA testing found fusion genes and elevated CEACAM5 in an additional 48 (1%) and 383 (9%) cysts. The most common fusion genes were PRKACA/B, BRAF, and ALK. Both fusion genes and elevated CEACAM5 correlated with elevated CEA and mucin by cytology (p<0.001). Further, 338 (8%) PCFs had alterations in TP53, CDKN2A, SMAD4, CTNNB1, and/or the mTOR genes that were correlative with worrisome features (p=0.033), high-risk stigmata (p=0.026), and malignant cytology (p=0.025). Low-level mutations in these high-risk genes were observed in 93 (28%) of 338 cysts, but while correlative of high-risk stigmata (p=0.031), they were not associated with malignant cytology (p=0.102). Based on follow-up, the sensitivity and specificity of DNA alterations associated with a mucinous cyst was 89% and 100%, respectively. In conjunction with RNA testing, the sensitivity improved to 95%, while the specificity remained high at 98%. Alterations in high-risk genes were associated with malignancy but improved with the inclusion of fusion genes in PRKACA/B, which achieved a sensitivity of 95% and a specificity of 97%. Interestingly, mutations in MEN1 had 36% sensitivity and 100% specificity for cPNETs, but elevated CHGA by RNA testing had a sensitivity and specificity of 100%.

Conclusions: Through a prospective, multi-institutional study, the addition of RNA testing improved the molecular assessment of patients with pancreatic cysts, especially the identification of mucinous cysts, advanced neoplasia, and cPNETs.

Anne Marie O'Broin-Lennon

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