Introduction: While somatic mutations in colorectal cancer (CRC) are well characterized, little is known about the accumulation of cancer-associated mutations in normal colon. This knowledge is crucial to understand the origin and evolution of cancer and to improve cancer prevention and prediction. The study of somatic mutation within normal tissue, however, is technically challenging because these mutations exist at extremely low frequency limiting their detection by conventional next-generation sequencing (NGS). Here we used Duplex Sequencing (DS), an error correction NGS approach that enables extreme sensitivity for mutation detection, to characterize the mutational landscape of cancer-associated genes in normal colonic mucosa of patients with and without CRC.

Methods: We used a DS-based method called CRISPR-DS, which employs double stranded molecular tagging for ultra-accurate sequencing and CRISPR-Cas9 target enrichment for efficient library conversion. A panel including the full coding region of TP53 and the hotspot codons of BRAF, KRAS and PIK3CA was designed to sequence at high depth (mean 2,500x) the DNA from normal colon mucosa of 47 patients, 23 of them with CRC cancer (mean age 57 years) and 24 without (mean age 59 years). Normal colon tissue from CRC patients was obtained distant from the tumor (>15cm). Mutation frequency was calculated for all target genes and for TP53 only as the number of mutated reads (overall and separately for coding and non-coding regions) divided by the total nucleotides sequenced.

Results: 168 mutations (124 coding and 44 non-coding) were identified. In nearly all patients (43/47), normal colon carried at least one coding mutation in a cancer associated gene included in the panel. Compared to patients without cancer, the normal colon of patients with CRC showed higher mutation frequency overall (5.5x10-7 vs 3.6x10-7, p=0.009) and in coding regions (7.5x10-7 vs 4.2x10-7, p=0.003), but not in non-coding regions. Similar results were observed for overall TP53 mutations (6.4x10-7 vs 3.7x10-7, p=0.01) and TP53 coding mutations (9.5x10-7 vs 4.9x10-7, p=0.006). Both groups of patients carried cancer-associated TP53 mutations but in patients with CRC the proportion of common CRC mutations and pathogenic mutations was significantly higher (p=0.01 and p=0.02, respectively).

KRAS hotspot mutations (codons 12 and 13) were identified in the normal colon of 30% (7/23) of patients with CRC but in none of the patients without CRC (0/24). TP53 hotspot mutations were observed in 22% (5/23) patients with CRC and in 13% (3/24) of patients without cancer. Overall, 44% (10/23) of patients with CRC carried mutations in hotspots vs 25% (6/24) of patients without cancer and half of the CRC patients with hotspot mutations carried hotspot mutations in more than one gene.

Conclusions: Common CRC mutations are present in the normal colon of adult individuals but at higher frequency and with more pathogenicity in those that have developed CRC. These results indicate that somatic evolution takes place in the normal human colon but this process appears to be increased in patients with CRC. Because the biopsies analyzed were collected more than 15cm away from tumors, these results raise questions about the extension of precarcinogenic fields harboring clonal expansions and prompts to further investigation into the landscape of cancer-associated mutations in non-cancerous colonic mucosa.

Citation Format: Júlia Matas Gironella, Brendan Kohrn, Jeanne Fredickson, Kelly Carter, Ting Wang, Ming Yu, William M Grady, Miguel Angel Peinado, Rosa Ana Risques. Somatic evolution in normal colon of patients with and without cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr LB231.