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Introduction: In patients with colorectal cancer, the results of intraoperative peritoneal cytology do not affect the prognosis. One of the reasons is the low sensitivity of peritoneal cytology. Thus, improving the sensitivity of peritoneal cytology may improve the accuracy of prognostic prediction. Digital cytology (dCytology), which detects genetic mutations in ascites or abdominal lavage fluid using digital polymerase chain reaction (PCR), is a new method by which to evaluate peritoneal cytology. In this study, we evaluated the sensitivity and clinical significance of dCytology. Methods: We included patients with clinical T3-T4 colorectal cancer who underwent surgery from November 2018 to November 2019 in our department. In patients with ascites, ascitic fluid was collected at the start of surgery. In patients without ascites, lavage fluid was collected using 50 mL of physiological saline. Half of the liquid was used for conventional cytology, and the other half was used for dCytology. DNA was extracted from the supernatant and intraperitoneal free cells and from the cancer tissue. Circulating tumor DNA was extracted from the preoperative plasma. Digital PCR was performed on cancer tissue, supernatant DNA, intraperitoneal free cell DNA, and circulating free DNA (cfDNA) using a KRAS multi-probe. Results: This study included 150 patients, 75 of whom had KRAS mutation in their tissues. Among them, dCytology was positive in 45 patients (60%, pStage I/II/III/IV/recurrence/unresectable: 6/22/23/16/4/4). Thirty patients had right-sided colon cancer, 20 had left-sided colon or upper rectal cancer, 7 had lower rectal cancer, and 4 had recurrent disease. Conventional cytology was positive in 11 of the 45 patients (24%), and dCytology was positive in 7 of the 11 patients. cfDNA was positive in 19 patients, and dCytology was positive in 10 of the 19. Four patients, including two patients with positive dCytology but negative conventional cytology, developed recurrence disease. Two of the four patients developed liver metastasis but no peritoneal dissemination. Discussion: The sensitivity of dCytology was higher than that of conventional cytology. Our results suggest that tumor cells can exist in the abdominal cavity of patients with colorectal cancer. Interestingly, dCytology was positive even for patients in whom the tumor was not exposed on the outside of the colon wall (T3) and in whom the tumor was not located in the abdominal cavity (Rb). Citation Format: Koji Ueda, Takeshi Yamada, Michihiro Koizumi, Seiichi Shinji, Akihisa Matsuda, Ryo Ohta, Goro Takahashi, Masahiro Hotta, Kohki Takeda, Keisuke Hara, Sho Kuriyama. Possibility of digital cytology of intraoperative ascites and lavage fluid in patients with colorectal cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6470.