Colitis-associated cancer (CAC) is a common complication among patients with a long history of ulcerative colitis (UC). Previous studies revealed that dysplasia precedes CAC development, and more than 20% of CACs are multicentric, suggesting the presence of a field effect on colorectal carcinogenesis in UC patients. However, the genetic basis of the field effect in UC patients is poorly understood.To address this issue, we isolated single crypts from non-tumorous colon mucosa from 6 UC patients, as well as those from apparently normal mucosa from non-UC patients, and “single crypt-derived DNA” was analyzed for somatic mutations using whole-exome sequencing (WES). In total, 19 crypts from histologically non-dysplastic, but inflamed mucosa, and 2 from apparently normal mucosa were isolated from 6 UC patients, including 4 with pancolitis, 1 with left-sided colitis and 1 with proctitis, while 32 crypts were obtained from normal mucosa from 20 non-UC patients, including 10 with colorectal cancer, 6 with adenoma and 4 healthy volunteers. Regardless of the disease status, mutations were detected in all crypts analyzed with the median of 43/crypt (3 − 121), showing high variant allele frequencies (VAFs) (~0.5), suggesting that these mutations were shared by all cells within single crypts and therefore by crypt-maintaining stem cells. The number of mutations increased with age, in crypts from both UC and non-UC patients (P < 10-9), showing clear age-related C to T transition in both normal and UC-inflamed crypts. Strikingly, however, the age-adjusted number of mutations was significantly higher in UC-derived crypts than in those from non-UC individuals (P < 10-3). KRAS and TP53 mutations were reported to be the most prevalent alterations in UC-associated CAC, but only rarely seen in UC-derived normal crypts. Next, to evaluate clonal expansion of mutated clones, we performed WES for the bulk specimens composed of 20 - 30 mutually adjacent crypts. However, this time, mutations and copy number alterations (CNAs) with high VAFs were detected only in UC-inflamed mucosa, suggesting that clonal crypt expansion occurred only in UC-derived mucosa. WES was also performed for 14 CACs. The number of non-synonymous mutations (101/sample) in CAC were significantly higher than that in UC-derived normal crypts (38/sample) and recurrently involved known driver genes, such as TP53 (71%) and KRAS (36%). CNAs were also more frequent in CAC than UC-derived, normal crypts. In conclusion, our study revealed that somatic mutations accumulated in normal mucosa of both UC and non-UC cases, but the mutational burden increased in UC-inflamed mucosa. Chronic inflammation caused clonal expansion of crypts even in histologically non-dysplastic mucosa in UC case, accompanied by acquisition of driver mutations and CNAs, which could trigger the development of CAC. Our findings provide novel insights into the carcinogenic sequence in UC-derived CAC.

Citation Format: Nobuyuki Kakiuchi, Kenichi Yoshida, Yusuke Shiozawa, Akira Yokoyama, Keisuke Kataoka, Yoshikage Inoue, Yasuhide Takeuchi, Yasunori Kogure, Ayana Kon, Masahiro Nakagawa, Kenichi Chiba, Hiroko Tanaka, Yuichi Shiraishi, Satoru Miyano, Kenji Kawada, Hideaki Okajima, Yoshiharu Sakai, Takaki Sakurai, Hironori Haga, Hiroshi Nakase, Motoi Uchino, Hiroki Ikeuchi, Takako Kihara, Seiichi Hirota, Takahiro Horimatsu, Minoru Matsuura, Hiroyuki Marusawa, Hiroshi Seno, Seishi Ogawa. The genetic aberrations in carcinogenic sequence of colitis-associated cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5754. doi:10.1158/1538-7445.AM2017-5754