A paradigm shift for ovarian cancer biomarkers: Utilizing routine Pap tests as liquid biopsies for the development of targeted mass spectrometry-based proteomic assays for early detection

Ovarian cancer is the 5th leading cause of cancer deaths in women in the U.S. Earlier detection is the key to increased survival for women with ovarian cancer, yet a screening tool that is both sensitive and specific enough for use in the general population has yet to be developed. In contrast, screening for cervical cancer by Pap tests has been routinely performed for over 50 years. In the liquid-based Pap test, cells are collected from the cervical opening and placed into an alcohol-based fixative and then examined for abnormal cells. Since ovarian cancer cells have been observed in Pap tests, ovarian cancer peptide biomarkers may also be present; yet Pap samples have not been rigorously examined for diagnostic peptides. Our central hypothesis is that proteins shed by ovarian cancer cells can be detected in the Pap test fixative and on cervical-vaginal swabs by Mass Spectrometry (MS)-based proteomics. The Pap test fixative and swabs are ideal for biomarker discovery since they are derived from a site proximal to the ovarian cancer (i.e. proteins may be secreted or shed from the tumor and flow through the fallopian tube into the uterus and out the cervical opening). Recently, ovarian cancer precursor lesions have been identified in the fimbria of the fallopian tube, strengthening the hypothesis that ovarian cancer proteins will be found in the lower genital tract, perhaps even at early stages. In preliminary studies, candidate biomarkers were successfully identified by MS-based proteomics in Pap test fixatives from women with ovarian cancer, and the levels of biomarker peptides were quantified in cases vs. benign and healthy controls, demonstrating the feasibility of this approach. The longterm goal of this project is to develop a noninvasive screening test that can be incorporated into a routine Pap test or a self-administered home test, so that women can be screened simultaneously for cervical and ovarian cancer. The objective of this study is to verify ovarian cancer biomarker peptides identified in Pap test samples and extend the results to cervical swabs, using our collection of biospecimens from over 600 patients and controls. Aim #1 will build on our preliminary studies to prioritize ovarian cancer biomarker candidates found in liquid-based Pap test samples by performing Tandem Mass Tag™ 11-plex isobaric labeling, 2D LC-MS/MS, and bioinformatics integration. In Aim #2, the most robust candidate biomarkers will be quantified by developing a targeted Selected Reaction Monitoring (SRM)-MS assay coupled with a multi-protein classifier, which will then be used to test hundreds of Pap test samples from women with ovarian cancer, other gynecologic cancers, and benign gynecologic conditions, as well as healthy women. In Aim #3, proteins eluted from cervical swabs will be tested to determine whether they are also detected by our SRM-MS assay. Results from this study may be extended to the early detection of ovarian cancer in women in high risk groups and eventually for screening the general population, thereby shifting the paradigm for how women are screened for ovarian cancer and improving survival rates for the >22,000 women diagnosed with ovarian cancer each year.