Identification of Ovarian Cancer Plasma Biomarkers

描述(申请人提供)：上皮性卵巢癌(EOC)是美国最致命的妇科癌症，急需新的血液生物标志物。CA125是FDA批准的最常用的卵巢癌生物标志物，但由于敏感性和特异性不足，未被批准用于早期诊断。此外，尽管CA125通常用于辅助诊断后的疾病的临床治疗，但它不能用于大部分卵巢癌患者，因为在诊断时CA125没有显著升高，包括透明细胞和粘液亚型。我们的长期目标是确定新的基于EOC血液和肿瘤组织的生物标记物，并进行实验室规模的验证，以确定哪些生物标记物可以改善这种疾病的早期诊断和/或临床治疗。我们的总体工作假设是，最具特异性的EOC血液生物标志物将是卵巢肿瘤进入血液的蛋白质，这些蛋白质在未患癌症的人的血液中处于非常低的丰度水平。在过去的拨款年度内，我们达到了原来建议的所有目标。具体地说，我们使用一种新的深入蛋白质组分析方法确定了29个低丰度的新生物标记物，以识别含人类肿瘤的异种移植小鼠血清或血浆中的人类蛋白。然后，通过无标记多重反应监测(MRM)质谱仪(MS)分析显示，与非癌症对照组相比，卵巢癌患者血液中的这些蛋白质水平升高。我们现在已经使用生物学和功能标准将这一列表缩小到12个高优先级生物标记物。我们下一个资助期的具体目标是在几个更大的患者队列中对这12个生物标记物进行实验室规模的验证，以确定最好的生物标记物用于早期诊断。 EoC和改善这种疾病的临床治疗。我们亦会初步评估这些生物标志物区分主要的EoC亚型的能力，因为这些亚型在遗传上是不同的疾病。为了确定某些生物标志物是否可以补充CA125，我们将对透明细胞和粘液性卵巢肿瘤的分泌体进行重点评估。我们还将单独评估我们的生物标记物在我们新的更大的患者队列中表现出低CA125水平的患者血浆样本子集中的预测价值。将在5年内实现的两个目标是：1)为我们的12个新的EOC生物标记物开发组织和高通量临床前血浆分析；2)验证我们的12个新的EOC生物标记物水平在多个独立患者队列的血浆或血清中的敏感性和特异性。生物标志物将在患者和对照血浆或血清中使用一种新的免疫核磁共振方法进行量化，该方法使用稳定的同位素标记的完整蛋白作为内部标准，通过商业上可获得的抗体对目标完整蛋白的下拉进行多路复用。预计我们的12个生物标志物中被证明对EOC的早期诊断和/或临床管理有用的子集将在未来在CLIA认证的实验室中推进到更大规模的多中心临床前测试。

DESCRIPTION (provided by applicant): There is an urgent need for new blood biomarkers for epithelial ovarian cancer (EOC), which is the most lethal gynecological cancer in the United States. CA125 is the most commonly used FDA approved biomarker for EOC, but it is not approved for early diagnosis due to inadequate sensitivity and specificity. Furthermore, although CA125 is commonly used to assist in clinical management of the disease after diagnosis, it cannot be used for the substantial portion of EOC patients where CA125 is not substantially elevated at time of diagnosis, which includes the clear cell and mucinous subtypes. Our long-term goal is to identify novel EOC blood-based and tumor tissue biomarkers and conduct laboratory-scale validation to determine which biomarkers can improve early diagnosis and/or clinical management of this disease. Our overall working hypothesis is that the most specific EOC blood biomarkers will be proteins shed by the ovarian tumor into the blood and that are at very low abundance levels in the blood of cancer-free individuals. During the past grant year, we achieved all goals of the original proposal. Specifically, we identified 29 low-abundance, novel biomarkers using a novel in-depth proteome analysis method to identify human proteins in serum or plasma of xenografted mice containing human tumors. These proteins were then shown to be elevated in blood from EOC patients compared with non-cancer controls using a label-free multiplexed multiple reaction monitoring (MRM) mass spectrometry (MS) assay. We have now narrowed this list to 12 high priority biomarkers using biological and functional criteria Our specific goal for the next grant period is to conduct laboratory-scale validation of these 12 biomarkers in several larger patient cohorts to identify the best biomarkers for early diagnosis of EOC and for improved clinical management of this disease. We will also conduct preliminary evaluations of the capacities of these biomarkers to distinguish among major EOC subtypes, as these are genetically different diseases. To determine whether some biomarkers could complement CA125, we will conduct a focused evaluation of secretomes of clear cell and mucinous ovarian tumors. We will also separately evaluate the predictive value of our biomarkers in the subset of patient plasma samples that exhibit low CA125 levels in our new larger patient cohorts. The two aims that will be pursued over a 5- year period are: 1) Develop tissue and high-throughput preclinical plasma assays for our 12 novel EOC biomarkers, and 2) Validate sensitivity and specificity of our 12 novel EOC biomarker levels in plasma or serum from multiple independent patient cohorts. The biomarkers will be quantified in patient and control plasma or serum using a novel ImmunoMRM method that multiplexes pull-down of targeted intact proteins by commercially-available antibodies using stable isotope-labeled intact proteins as internal standards. It is expected that the subset of our 12 biomarkers that are shown to be useful for early diagnosis and/or clinical management of EOC will advance to larger-scale multi-center, preclinical testing in CLIA certified laboratories in the future.