Abstract Background: Pancreatic cancer is one of the incurable cancers among solid malignancies. Basically, R0 tumor resection could be one of the critical factors for their prognosis. However, pathological diagnosis of surgical margins is sometimes unreliable due to tissue shrinkage and skipped lesion like tumor budding. We applied our ‘molecular surgical margin analysis' using tissue imprinting procedure to evaluate the precise surgical margin status of the surgical specimens after pancreatoduodenectomy. Methods: Quantitative methylation specific PCR (QMSP) assay for 3 representative tumor-specific methylation markers (CD1D, KCNK12, PAX5) were established and validated by paired normal and tumor tissues from pancreatic ductal carcinoma cases (n=48). Then, these markers were applied to prospectively collected peritoneal lavage samples (n=16), drainage fluid samples (n=18) and surgical margin imprinting samples (n=11). Results: QMSP values of tumor tissues were significantly higher than adjacent normal tissues and relatively high specificity to cancer cells in all three markers with optimal cut-off values (CD1D: P<0.001, 94%, KCNK12: P=0.001, 75%, PAX5: P<0.001, 87%). These marker positive cases also showed marginally or significantly poor overall survival in univariate analysis (CD1D: HR=1.78, P=0.083, KCNK12: HR=2.57, P=0.005, PAX5: HR=2.64, P=0.003). Since these markers were revealed to be very cancer-specific, we tried to use them as cancer detection markers in clinical samples. 1) CD1D methylation was positive in 6 cases among 16 cases. Although 4 cases showed histopathologically positive, 2 cases were negative. These cases might have subclinically positive cancer cells in the fluid. Actually, one of them relapsed 5 months after the surgery. 2) CD1D methylation was positive in 4 cases among 18 postoperative cases. These cases were all treated with surgery first, while neoadjuvant cases showed no methylation in postoperative drainage fluid. 3) Molecular surgical margin analysis revealed 4 positive cases and 7 negative cases. To find the meaning of molecular margin positive, we still following-up these cases. Conclusion: Very cancer-specific methylation detection assay was established. Although we still need to follow-up the cases for a year or more, invisible cancer cells in the fluid or on the surgical margin might have an association with tumor recurrence after the surgery. Citation Format: Masamichi Hayashi, Suguru Yamada, Hiroshi Tanabe, Yoshiyasu Kato, Katsuhito Tanaka, MItsuru Tashiro, Haruyoshi Tanaka, Tomonari Asano, Hideki Takami, Masaya Suenaga, Yukiko Niwa, Yasuhiro Kodera. Molecular surgical margin analysis of pancreatic cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3330.