Risques, Rosa AnaBaker, Kathryn Terese2019-02-222019-02-222019-02-222018Baker_washington_0250E_19537.pdfhttp://hdl.handle.net/1773/43436Thesis (Ph.D.)--University of Washington, 2018Humans have attempted to fight cancer since the beginning of recorded history, but advances in the early detection of cancer have begun to emerge only in the last several decades. Patient survival increases with earlier detection and cancer incidence decreases with efficacious screening tests, such as colonoscopy or the pap test. Early detection has been difficult for many cancer types, however, because of non-unique symptoms, inaccessibility of certain tissues for biopsy, and false-positives from tests that lack acceptable levels of specificity. One method to overcome these issues is to analyze the genetic mutations of precancerous tissue. Cancer is considered a genetic disease, and molecular alterations may be specific to each cancer type. Despite the great utility of next-generation sequencing technology, many techniques are still plagued by significant error rates. Error rates range from 1/100 to 1/1000 for standard NGS, which precludes the identification of rare or very low frequency events that may signal early tumorigenic processes. Ultra-accurate technology is necessary to confidently call these mutations. Duplex sequencing is an NGS method with unprecedented accuracy and sensitivity, with a detection rate of 1 in 10^7 reads. Our use of this technology facilitated several applications for characterizing precancer and early cancer detection. The first is in the characterization of mitochondrial DNA mutations in colorectal cancer associated with ulcerative colitis, a preneoplastic inflammatory bowel disease that increases patient risk for tumorigenesis. Using DS to comprehensively catalogue these mutations, we found that mitochondrial DNA mutations increase in frequency and pathogenicity and appear to be positively selected in early dysplasia, but are removed in late dysplasia in cancer, implying that functional mitochondria are necessary for tumorigenic progression. Additionally, we posit that the presence of clonally expanded fields may serve as a predictive biomarker in this disease. The second application presented is for the early detection of TP53 mutations in high-grade serous ovarian cancer, a disease for which there is currently not a reliable biomarker. Through the analysis of DNA collected from uterine lavage we were able to identify TP53 tumor mutations in 80% of HGSOC patients. We also found a low level of background TP53 mutations in all patients, including healthy controls, that increased with patient age. These results demonstrate that careful calibration based on the accumulation of somatic mutations with age is necessary for an accurate biomarker. Finally, with the important modification of using CRISPR/Cas9 digestion as a target enrichment tool, CRISPR-DS is able to leverage the same accuracy of DS with the ability to use as little as 10ng of input DNA, opening the technology to further clinical applicability, as well as to use in any setting that requires low amounts of starting genetic material.application/pdfen-USCC BY-NC-NDbiomarkercolorectal cancerearly cancer detectionovarian cancerprecancerulcerative colitisPathologyMolecular biologyPathologyThesis