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

项目概要/摘要 卵巢癌是美国女性癌症死亡的第五大原因。 提高卵巢癌妇女的生存率，但这是一种既敏感又特异的筛查工具 足够用于一般人群的药物还有待开发。相比之下， 巴氏试验已经常规进行了50多年。在基于液体的巴氏试验中，细胞收集自 将宫颈开口置于酒精基固定液中，然后检查异常细胞。以来 在巴氏试验中观察到卵巢癌细胞，卵巢癌肽生物标志物也可能是 目前;但巴氏涂片样本还没有被严格检查的诊断肽。我们的核心假设 卵巢癌细胞脱落的蛋白质可以在巴氏试验固定液和宫颈阴道涂片中检测到， 拭子通过基于质谱（MS）的蛋白质组学。巴氏试验固定剂和拭子是生物标志物的理想选择 因为它们来源于卵巢癌近端的位点（即蛋白质可以被分泌或分泌）， 从肿瘤流出，通过输卵管流入子宫，从宫颈口流出）。 最近，在输卵管伞部发现了卵巢癌前病变， 强化了卵巢癌蛋白质将在下生殖道中发现的假设，甚至可能是 在早期阶段在初步研究中，候选生物标志物通过基于MS的 蛋白质组学研究，以及来自卵巢癌妇女的巴氏试验固定剂中的生物标志物肽的水平。 在病例与良性和健康对照中进行定量，证明了这种方法的可行性。很长的- 本项目的长期目标是开发一种非侵入性的筛查试验，可纳入常规巴氏检查 测试或自我管理的家庭测试，使妇女可以同时筛查宫颈和卵巢癌 癌本研究的目的是验证在巴氏试验样本中鉴定的卵巢癌生物标志物肽 并将结果扩展到宫颈拭子，使用我们收集的600多名患者的生物标本， 对照目标#1将建立在我们的初步研究的基础上，优先考虑在以下研究中发现的卵巢癌生物标志物候选者： 通过进行Tandem Mass Tag™ 11重同量异位素标记，2D LC-MS/MS， 和生物信息学的整合。在目标#2中，最稳健的候选生物标志物将通过 开发与多蛋白分类器偶联的靶向选择反应监测（SRM）-MS测定， 然后将用于测试数百个来自卵巢癌妇女的巴氏试验样本， 妇科癌症和良性妇科病症，以及健康女性。在目标#3中，蛋白质洗脱 将对来自宫颈拭子的样本进行检测，以确定它们是否也可以通过我们的SRM-MS检测试剂盒检测到。结果 从这项研究可以扩展到早期发现卵巢癌的妇女在高风险群体， 最终用于筛查普通人群，从而改变女性如何筛查的模式， 卵巢癌和提高生存率为> 22，000妇女诊断卵巢癌每年。

PROJECT SUMMARY/ABSTRACT 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 long- term 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.