Sequential evaluation of MUC promoter methylation using next‐generation sequencing‐based custom‐made panels in liquid‐based cytology specimens of pancreatic cancer

• Published in: Diagnostic cytopathology Vol. 50; no. 11; pp. 499 - 507
• Main Authors: Yokoyama, Sieya, Iwaya, Hiromichi, Akahane, Toshiaki, Hamada, Taiji, Higashi, Michiyo, Hashimoto, Shinichi, Tanoue, Shiroh, Ohtsuka, Takao, Ido, Akio, Tanimoto, Akihide
• Format: Journal Article
• Language: English
• Published: Hoboken, USA John Wiley & Sons, Inc 01.11.2022; Wiley Subscription Services, Inc
• Subjects: Cellular biology; DNA; Endoscopic Ultrasound-Guided Fine Needle Aspiration - methods; Formaldehyde; Genes; High-Throughput Nucleotide Sequencing - methods; Humans; liquid‐based cytology; Methylation; methylation panel; MUC; Mutation; next‐generation sequencing; Pancreatic cancer; Pancreatic Neoplasms; Pancreatic Neoplasms - diagnosis; Pancreatic Neoplasms - genetics; Pancreatic Neoplasms - pathology; Promoter Regions, Genetic - genetics; liquid-based cytology; pancreatic cancer; MUC; methylation panel; next-generation sequencing
• ISSN: 8755-1039; 1097-0339
• DOI: 10.1002/dc.25022

Background As liquid‐based cytology (LBC) specimens preserve high‐quality DNA, clinical sequencing of LBC specimens using next‐generation sequencing (NGS) is becoming a common strategy. This study aimed to evaluate the feasibility of NGS‐based custom‐made panels for evaluating MUC promoter methylation in LBC specimens. Methods Thirty‐one patients with pancreatic cancer were enrolled in the study. Cancer tissue samples were obtained using endoscopic ultrasound‐guided fine‐needle aspiration/biopsy (EUS‐FNA/FNB). LBC, formalin‐fixed paraffin‐embedded (FFPE), and fresh frozen specimens were prepared for DNA extraction after pathological diagnosis. These specimens were then subjected to NGS analysis using custom‐made cancer gene screening and methylation panels comprising 28 cancer‐related genes and 13 gene promoter regions, including MUC1, MUC2, and MUC4. Results The success rate of NGS using the cancer gene panel was comparable among the LBC, FFPE, and frozen specimens, and the presence of cancer cell‐derived somatic mutations in each specimen was confirmed. The specimens were then tested using a methylation panel that revealed the sequential methylation status of CpG islands located in the promoter regions of MUC genes. The methylation status results obtained from LBC specimens were almost comparable with those from FFPE and frozen specimens. Conclusions MUC and other gene methylation analyses using an NGS‐based panel were successfully performed in residual LBC specimens obtained by EUS‐FNA/FNB. Therefore, this approach provides an alternative source of molecular tests for gene mutations and methylation, especially in the pancreatic cancers, which are often unresectable and unsuitable for obtaining FFPE specimens.