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

项目摘要/摘要 卵巢癌是美国女性癌症死亡的第五大原因。早期发现是关键 提高卵巢癌患者的生存率，但这是一种既敏感又特异的筛查工具 还没有开发出足够在普通人群中使用的药物。相比之下，通过以下方式筛查宫颈癌 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，细胞从 切开宫颈，放入含酒精的固定剂中，然后检查异常细胞。自.以来 卵巢癌细胞已在巴氏试验中观察到，卵巢癌多肽生物标志物也可能 目前；然而巴氏样本还没有经过严格的诊断多肽检查。我们的中心假设 卵巢癌细胞脱落的蛋白质可以在巴氏试验固定剂和宫颈阴道上检测到 拭子用质谱仪(MS)进行蛋白质组学研究。巴氏试验固定剂和棉签是生物标志物的理想选择 发现，因为它们来自卵巢癌近端的部位(即蛋白质可能分泌或 从肿瘤脱落并通过输卵管流入子宫并从宫颈开口流出)。 最近，在输卵管伞端发现了卵巢癌前驱病变， 强化了卵巢癌蛋白将在下生殖道被发现的假设，甚至可能 在早期阶段。在初步研究中，基于MS的方法成功地确定了候选生物标志物 来自卵巢癌妇女的巴氏试验固定物中的蛋白质组学，生物标志物多肽的水平 在病例与良性和健康对照中进行量化，证明了这种方法的可行性。长的- 该项目的学期目标是开发一种可以合并到常规巴氏检查中的非侵入性筛查测试 测试或自行进行家庭测试，以便妇女可以同时进行宫颈和卵巢筛查 癌症。本研究的目的是验证在巴氏试验样本中发现的卵巢癌生物标志物多肽 并将结果扩展到宫颈拭子，使用我们从600多名患者和 控制。目标1将建立在我们的初步研究的基础上，以确定卵巢癌生物标志物候选的优先顺序 通过执行串联质量标签™11-plex等压标记，2D LC-MS/MS， 和生物信息学的整合。在Aim#2中，最可靠的候选生物标记物将通过 开发与多蛋白质分类器耦合的靶向选择反应监测(SRM)-MS分析， 然后将被用来检测数百个来自卵巢癌女性的巴氏检测样本，其他 妇科癌症、妇科良性疾病以及健康女性。在AIM#3中，蛋白质洗脱 将对宫颈拭子进行检测，以确定我们的SRM-MS分析是否也检测到它们。结果 这项研究可能扩展到高危人群中卵巢癌的早期发现和 最终用于对普通人群进行筛查，从而改变对妇女进行筛查的方式 卵巢癌和提高每年22,000名被诊断为卵巢癌的妇女的存活率。

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.