Next‐generation sequencing of residual cytologic fixative preserved DNA from pancreatic lesions: A pilot study

Abstract

Background Endoscopic ultrasound–guided fine needle aspiration (EUS‐FNA) is a sensitive and specific tool in the risk stratification of pancreatic lesions, including cysts. The sensitivity and specificity of EUS‐FNA has been shown to improve when cytology is combined with next‐generation sequencing (NGS). Ideally, fresh cyst fluid is used for NGS. In this pilot study, we explore the possibility of sequencing DNA derived from residual alcohol‐fixed pancreatic aspirates. Methods Residual cytologic fixatives (n = 42) from 39 patients who underwent EUS‐FNA for pancreatic lesions were collected along with demographics, imaging, and laboratory studies. Samples were designated as nonneoplastic/nonmucinous benign (NB), mucinous cyst (MC), pancreatic ductal adenocarcinoma (PDAC), or well‐differentiated neuroendocrine tumor (NET) on the basis of cytopathologic evaluation and sequenced on the Oncomine platform (ThermoFisher Scientific, Waltham, Massachusetts). Results Ten of 14 (71.4%) MCs exhibited clinically significant variants, including KRAS, GNAS, and TP53. Ten of 15 (66.7%) PDACs had KRAS alterations, and 9 of 15 (60%) showed variants in TP53. No variants were detected in any NETs. Only 1 of 9 (11.1%) NB aspirates showed variants in KRAS and MAP2K. Sequencing of formalin‐fixed, paraffin‐embedded tissue revealed variants identical to those detected in fixative‐derived DNA in 4 of 5 cases (80%). Conclusion Residual DNA from alcohol‐fixed aspirates are an underutilized source for NGS. Sequencing residual fixative‐derived DNA has the potential to be integrated into the workup of pancreatic aspirates, possibly impacting management.

Incorporating cytologic adequacy assessment into precision oncology workflow using telepathology: An institutional experience

“…In addition to ensuring sample quality, implementing ROSE assessment maximizes a patient's material available for diagnosis and molecular studies. Because different types of molecular analyses can be performed on a cytology specimen, 23,24 diligent use of the patients' samples and test prioritization are required to prevent material depletion. This should help increase the possibilities for clinical laboratories to offer a wider range of molecular assays through the integration of cytology and tissue-based tests and to reduce failure caused by suboptimal input.…”

Evaluating Pancreatic and Biliary Neoplasms with Small Biopsy-Based Next Generation Sequencing (NGS): Doing More with Less

“…NGS was performed on distinct small biopsy types, including EUS-FNAs or FNBs, brushings, and pancreatic juice, whereas several NGS panels were utilized. The initial material used for nucleic acid extraction was either fresh, directly collected for further NGS testing [ 25 , 55 , 61 ], frozen [ 23 , 57 ], also derived from formalin-fixed, paraffin-embedded tissue or cell blocks [ 70 , 71 , 85 ], residual liquid-based cytology (LBC) samples [ 58 , 62 ], or cytology slide scraping [ 59 , 60 ]. Mutations in the KRAS, TP53, CDKN2A, and SMAD4 genes were the most common ones detected in the PDAC patients tested [ 22 , 60 , 72 , 77 ].…”

“…In addition, they found that alterations in TSC2, KRAS, and TP53 genes were associated with poor prognosis, whereas they also identified potentially actionable alterations in some members of the mTOR pathway (PTEN, TSC2, and PIK3CA) in 10% of primary and 12.5% metastatic NETs tested [ 74 ]. Whereas KRAS mutations were often in PDACs and IPMNs, they were not detected in the PanNET cases tested in two studies [ 58 , 83 ]. VHL mutations indicated a diagnosis of SCA in some studies.…”

Adequacy evaluation and use of pancreatic adenocarcinoma specimens for next‐generation sequencing acquired by endoscopic ultrasound–guided FNA and FNB

“…Another benefit of EUS-FNA sampling of PDAC is the potential use of the supernatant of EUS-FNA needle rinses, which otherwise is discarded; it could be repurposed to provide genomic material, especially in situations where the cellular material in small specimens is insufficient. As reported by Fulmer et al, 14 the supernatant of an EUS-FNA needle rinse after the sampling of pancreatic lesions could be successfully used to provide genomic material for NGS. On the other hand, EUS-FNB specimens are processed for the preparation of tissue blocks.…”

“…[9][10][11][12][13] Moreover, studies evaluating endoscopic ultrasound-guided tissue acquisition (EUS-TA) for biomarker testing in PDAC cases have been very limited. 5,14,15 As described herein, we compared EUS-FNA and EUS-FNB specimens concurrently acquired from patients with PDAC to evaluate their diagnostic yields, adequacy for biomarker testing, and molecular yields for next-generation sequencing (NGS).…”