胰腺囊液的超灵敏CRISPR基NGS（MUTE-Seq）：KRAS变异等位基因分数作为恶性潜能的早期标志物

Background: Conventional cytology of pancreatic cystic fluid from endoscopic ultrasound-guided fine-needle aspiration offers limited sensitivity for malignant potential of pancreatic cystic neoplasm. This proof-of-concept study aims to explore the clinical utility of cyst fluid next generation sequencing (NGS) using the ultra-sensitive MUTE-Seq assay. Methods: Twenty-two patients who underwent surgical resection for pancreatic cystic lesions at Asan Medical Center, Korea, between November 2019 and July 2025 were prospectively enrolled. Approximately 1 mL of pancreatic cyst fluid was obtained intraoperatively from the resected specimens, from which cell-free DNA (cfDNA) was isolated and subjected to targeted NGS using the MUTE-Seq assay, a CRISPR-based technology that enhances cancer-associated variant detection by selectively suppressing cfDNA from normal cells. Custom primers amplified genomic regions of interest across 14 cancer-associated genes (AKT1, APC, BRAF, EGFR, KRAS, PDGFRA, PIK3CA, TP53, BRCA1, CTNNB1, ERBB2, IDH1, MYCN, and NRAS). Results: Twenty-two patients with a median age of 60.5 years were analyzed. Initial pathologic diagnoses included serous cystic neoplasm (SCN, n = 4), mucinous cystic neoplasm (MCN, n = 4), intraductal papillary mucinous neoplasm-low grade (IPMN-LG, n = 7), IPMN-high grade (IPMN-HG, n = 4), pancreatic adenocarcinoma (PDAC, n = 2), and MCN with invasive carcinoma (n =1). Across the 14-gene panel, mutations in ERBB2, IDH1, PIK3CA, and AKT1 were sporadically detected in low-grade lesions without a discernible relationship to neoplastic grade. One PDAC case harbored a high TP53 variant allele fraction (VAF) of 47.475%. In contrast to these sporadic findings, KRAS VAFs measured by MUTE-Seq demonstrated the strongest pathologic correlation. SCN had a median VAF of 0.14% (IQR 0-0.64), and MCN demonstrated a median VAF of 0% (IQR 0-0.12). High-grade lesions showed consistently elevated values, with IPMN-HG exhibiting a median of 41.04% (IQR 37.35-43.19) and PDAC a median of 31.545%. The case of MCN with invasive carcinoma showed a high KRAS VAF of 29.67%. KRAS VAFs in IPMN-LG were heterogeneous: three cases had low values (median 0.35%, IQR 0.3-0.75), while four cases showed high values (median 41.73%, IQR 36.56-42.63). Notably, one IPMN-LG case with a high KRAS VAF progressed to pancreatic carcinoma within three years, suggesting potential prognostic value. Conclusions: Quantitative assessment of KRAS VAF using MUTE-Seq reliably distinguishes benign from neoplastic pancreatic cystic lesions, sensitively detects lesions with mixed invasive pathology, and may offer early molecular clues to malignant transformation, even in cases with borderline histology. Further study with additional specimens is underway to refine performance metrics and establish a clinically meaningful cutoff value for KRAS VAF.