Colorectal Cancer (CRC) incidence is rapidly rising in patients younger than fifty with no prior family history. Recent research has revealed that very low frequency somatic mutations accumulate in normal tissue with aging. Many of these mutations occur in common cancer genes and are positively selected, producing clonal expansions, that might be linked to cancer risk. I hypothesize that the normal colon of patients with early onset CRC might carry clonal expansions driven by mutations in CRC genes, which could be detected with ultra deep duplex sequencing. Duplex sequencing is an error correction method that improves sequencing accuracy through double stranded molecular tagging. I used duplex sequencing to perform ultra deep sequencing (~3000x) of the main driver genes of colorectal cancer (CRC) which includes: BRAF, APC, FBXW7, KRAS, PIK3CA, SMAD4, and TP53. First, I performed adjustments of gene probes by increasing or decreasing the proportion of each probe in each gene pool to achieve comparable depth of sequencing across genes. Then, I sequenced normal colon tissue of two patients with and without CRC. Preliminary data showed deleterious mutations in tumor suppressor genes: APC, FBXW7, and TP53, which occurred at higher frequency in the patients with cancer. The next steps of this research involve analyzing the normal colon from a larger cohort of patients with and without CRC. Our results have demonstrated that clonal expansions are visible in early onset CRC with these sequencing methods, and I anticipate that I will identify more clonal expansions in patients with early onset CRC than in those without CRC. In conclusion, duplex sequencing allows for the detection of cancer driver mutations in normal tissue, allowing the discovery of early somatic events in CRC and offering potential for early CRC detection, prevention, and prediction.

High-grade serous carcinoma (HGSC), the most common subtype of ovarian cancer, originates in the fallopian tube epithelium from precursor lesions carrying somatic TP53 mutations. Individuals with germline mutations in DNA repair genes are at high risk of HGSC but the reason is unknown. We hypothesize that individuals at high risk of HGSC carry an excess of pathogenic TP53 mutations in fallopian tube epithelium, which predisposes them to cancer. Preliminary data suggests that individuals with germline mutations in BRCA1 and BRCA2 (lifetime risk of HGSC 45% and 21%, respectively) have more TP53 mutations in fallopian tube than individuals without germline mutations, supporting our hypothesis. However, TP53 mutations have not yet been characterized in individuals with germline mutations in RAD51C/RAD51D, BRIP1 and PALB (lifetime risks of HGSC 10%, 6% and 5%, respectively). We aimed to conduct an ultra-sensitive characterization of TP53 mutations in patients with germline mutations in RAD51C/RAD51D, BRIP1, and PALB2, and compare their mutational profile with those of individuals without germline mutations in HGSC risk genes and those with BRCA1 or BRCA2 germline mutations. Right and left fallopian tube biopsies were collected, frozen, and macrodissected using a 1mm biopsy punch. DNA was extracted and sequenced for TP53 using ultra-deep (15,000x) duplex sequencing. Data from 6 patients revealed varying degrees of pathogenic mutations in individuals with germline mutations. BRIP1 and PALB2 patients showed low and moderate levels of TP53 pathogenic mutations (11% and 41%, respectively), while RAD51C patients showed the highest percentage of pathogenic mutations (67%), matching their higher HGSC risk. We plan to sequence 6 additional patients to get more comprehensive data. By showing the differences in TP53 mutation patterns among these distinct populations, our research seeks to enhance our understanding of the underlying mechanisms of ovarian cancer predisposition and design better tools for early cancer detection, prediction, and risk assessment.