Evaluation and diagnostic value of next‐generation sequencing analysis of residual liquid‐based cytology specimens of pancreatic masses

Abstract

Background Liquid‐based cytology (LBC) is a widely used method for processing specimens obtained by endoscopic biopsy. This study evaluated next‐generation sequencing (NGS) analysis of LBC specimens to improve the diagnostic accuracy of pancreatic lesions. Methods Upon the diagnosis of a suspected pancreatic mass, LBC residues were used retrospectively. The quantity and quality of DNA extracted from residual LBC samples were evaluated, and an NGS analysis targeting 6 genes (KRAS, GNAS, TP53, CDKN2A, SMAD4, and PIK3CA) was performed. Results The library was prepared from LBC specimens taken from 52 cases: 44 were successful, and 8 preparations failed. An analysis of DNA quantity and quality suggested that the success or failure of NGS implementation depended on both properties. The final diagnosis was achieved by a combination of the pathological analysis of the surgical excision or biopsy material with clinical information. Among the 33 cases of pancreatic ductal adenocarcinoma (PDAC), KRAS, TP53, CDKN2A, and SMAD4 mutations were identified in 31 (94%), 16 (48%), 3 (9%), and 2 (6%), respectively. Among the 11 benign cases, only a KRAS mutation was identified in 1 case. On the basis of NGS results, 18 of 33 PDACs (55%) were classified as highly dysplastic or more, and 10 of 11 benign lesions were evaluated as nonmalignant, which was consistent with the final diagnosis. Conclusions NGS analysis using LBC specimens from which DNA of appropriate quantity and quality has been extracted could contribute to improving the assessment of pancreatic tumor malignancies and the application of molecular‐targeted drugs.

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

“…According to the PDAC progression model, KRAS mutations are found early, whereas TP53 and DPC4 mutations occur later during the PDAC carcinogenesis [ 18 , 19 ]. Thus, although KRAS mutations could be detected in PanINs and IPMNs/MCNs of any grade or even in non-neoplastic cases, TP53 and SMAD4 alterations indicate the presence of HGD or cancer, triaging eligible patients for surgery [ 21 , 22 , 23 , 24 , 25 , 26 , 27 ]. Notably, Hosoda et al selected 23 isolated HG-PanIN cases characterized by the absence of concurrent PDAC to perform molecular analysis.…”

“…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 ]. Although KRAS mutations, as an early carcinogenic step, were also found in non-malignant cases, TP53 and SMAD4 alterations indicated HGD or carcinoma, triaging surgically fit patients for surgery [ 22 , 23 , 24 , 25 , 26 , 27 ].…”

“…Mutations in the KRAS, TP53, CDKN2A, and SMAD4 genes were the most common ones detected in the PDAC patients tested [ 22 , 60 , 72 , 77 ]. Although KRAS mutations, as an early carcinogenic step, were also found in non-malignant cases, TP53 and SMAD4 alterations indicated HGD or carcinoma, triaging surgically fit patients for surgery [ 22 , 23 , 24 , 25 , 26 , 27 ].…”

“…Of interest, although KRAS mutations could be detected in low- or high-grade PanINs and IPMNs/MCNs, they could also be found in non-neoplastic disorders such as in chronic pancreatitis. In contrast, TP53 and SMAD4 alterations generally indicate the presence of HGD or cancer [ 21 , 22 , 23 , 24 , 25 , 26 , 27 ]. While evaluating a pancreatic cyst, finding a KRAS mutation favors a mucinous (e.g., IPMN or MCN) vs. a non-mucinous cyst (e.g., SCA or pseudocyst); moreover, an additional GNAS mutation indicates the presence of IPMN rather than MCN [ 28 , 29 , 30 ].…”

Impact of LBC Fixative Type and Fixation Time on Molecular Analysis of Pancreatic Cancer Cells

“…[ 23 24 ] For pancreatic cancer, NGS analysis using LBC specimens was reported to contribute to a possible improvement in the evaluation of pancreatic tumor malignancies and the application of molecular-targeted therapeutic agents. [ 25 ] Preservation of samples is a key step to successful NGS analyses. In this study, the nuclear morphology was not significantly different between CR and CB.…”