Project Summary There is an unmet need for innovative molecular tools that can assist in the clinical management of high-grade serous ovarian cancer, given that more than 220,000 women in the U.S. are living with ovarian cancer and are at risk of recurrence. Improving the performance of validated biomarkers by reinventing their detection strategy is a compelling and underused approach to meet this need. The serum marker CA125 plays a crucial role in the clinical management of ovarian cancer, but, despite its importance, it remains poorly understood. The iden- tity and location of the CA125 epitopes—detected in the clinical test—are not known. In other words, the test that underlies vital decisions in ovarian cancer care employs a mechanism that is not understood. Prior re- search has located the CA125 epitopes somewhere within the tandem-repeat domain of a mucin (MUC16) that contains 19 similar but non-identical subdomains. Glycan modifications, alternative splicing of mRNA, and pro- teolytic cleavage generate many MUC16 proteoforms. Only a subset of these proteoforms contain detectable CA125 epitopes, whereas, in others, the epitope is masked or eliminated. As a result, only a subset of the total pool of MUC16 molecules is detectable by the serum CA125 assay. This undercounting has potentially serious clinical implications, including mistaken decisions about whether a patient should be treated by a gynecologic oncologist or failure to detect early evidence of possibly treatable cancer recurrence. Without knowledge of the CA125 epitopes, it is impossible to characterize individual variation in MUC16 proteoforms or to determine whether MUC16 expression changes during cancer development, in response to treatment, or during recur- rence. Importantly, serum CA125 levels in Black women have been shown to be lower than levels in white women, but the molecular-level reasons for these racial/ethnic differences in serum CA125 are unknown. The tests and algorithms currently in use employ a single CA125 cut-off value and do not consider racial/ethnic dif- ference, which may result in higher rates of false negative diagnoses among Black patients. To improve the long-term survival of ovarian cancer patients of all backgrounds, there is a vital need to improve the diagnostic value of CA125. The central hypothesis that will be tested is that CA125 epitopes are unevenly distributed over tandem-repeat domains of MUC16, are unaffected by glycosylation, and display differences in abundance de- pending on the cell type that expressed the MUC16. We will pursue two specific aims: (1) Map the locations of OC125 and M11 binding on individual tandem repeats of MUC16, and (2) Characterize the binding of OC125 and M11 to intact MUC16 from diverse sources. Successful completion of this research will identify the CA125 epitopes on MUC16 at the amino-acid level. This accomplishment will represent an unprecedented level of understanding of the CA125 test. This contribution wil...