Development of activity-based biomarkers for risk stratification of indeterminate thyroid nodules

摘要 被诊断为偶发性甲状腺结节的患者数量急剧增加 通过不同的诊断成像模式创造了一个独特的机会来检测和 在恶性肿瘤的早期阶段切除病变。这也产生了对改进的迫切需求 诊断，因为80%的患者目前正在接受不必要的甲状腺切除术，因为他们的 结节是良性的。目前的护理标准包括通过超声波收集组织活检- 引导细针抽吸：这些样本通过细胞学进行测试，细胞学寻找异常细胞， 以及遗传风险标记。这些诊断测试的表现很差，因此占所有测试的三分之一 结节仍被归类为不确定结节，并被摘除以进行病理诊断。我们 假设失调的蛋白分解，一种酶的活性，是侵袭性的标志 癌症，可能会在FNA组织中产生区分良恶性结节的活动水平。 ALaunus Biosciences，Inc.的诊断流水线利用了底物分析技术 在加州大学洛杉矶分校的Craik实验室开发的一个平台，允许基于创新的蛋白质组学 生物标志物的发现。使用这项技术，我们以前已经识别和表征了一种 一组专有的蛋白水解酶活性在癌前胰腺囊肿中显著增加； 然后，我们开发了荧光底物分析来监测这些活性，使用少量的 液体活检(囊液)。在此阶段I提案中，我们的目标是将我们的发现管道应用于 回顾经病理证实的甲状腺癌和匹配的正常组织的队列，以确定 恶性组织所特有的蛋白酶活性。在目标1中，我们将描述特定于甲状腺肿瘤的 蛋白质分解特征，并确定负责观察到的候选酶标记 蛋白水解性。在目标2中，我们将设计和合理优化特定的荧光底物 这些蛋白酶可以用来快速检测FNA样本中的恶性结节。铅 候选底物将继续对其作为诊断工具的临床效用进行强有力的验证，通过 对照当前的临床标准指南对原型化验进行基准测试。 我们的目标是开发一种快速检测方法，改善患者的分层情况，超过目前的标准诊断 标记和指导临床决策，以避免不必要的手术干预。 如果成功，这项提议将为可操作的诊断测试奠定基础，该测试将使 改进不确定甲状腺结节的风险分层，并改变临床管理 这些具有挑战性的损伤。

Abstract A dramatic increase in the number of patients being diagnosed with incidental thyroid nodules through different diagnostic imaging modalities has created a unique opportunity to detect and remove lesions at early stages of malignancy. This also creates a critical need for improved diagnostics, as 80% of patients currently undergo unnecessary thyroidectomies because their nodules were benign. Current standard of care includes collecting a tissue biopsy by ultrasound- guided fine needle aspiration: these samples are tested by cytology, which looks for aberrant cells, and for genetic risk markers. These diagnostic tests perform poorly, consequently a third of all nodules are still classified as indeterminate and are removed for a pathological diagnosis. We hypothesized that dysregulated proteolysis, a type of enzyme activity that is a hallmark of invasive cancer, might yield discriminating levels of activity in FNA tissue from benign and malignant nodules. The Alaunus Biosciences, Inc. diagnostic pipeline takes advantage of a substrate profiling technology developed in the Craik Laboratory at UCSF, a platform that allows innovative proteomics-based biomarker discovery. Using this technology, we have previously identified and characterized a proprietary set of protease activities that are significantly increased in pre-malignant pancreatic cysts; we then developed fluorogenic substrate assays to monitor these activities using small volumes of liquid biopsy (cyst fluid). In this Phase I proposal we aim to apply our discovery pipeline to a retrospective cohort of pathologically-confirmed thyroid cancer and matched normal tissue, to identify protease activities that are specific to malignant tissue. In Aim 1, we will profile thyroid tumor-specific proteolytic signatures, and identify candidate enzyme markers responsible for the observed proteolytic activity. In Aim 2, we will design and rationally optimize specific fluorogenic substrates for these proteases that can be used to rapidly detect malignant nodules in FNA samples. Lead candidate substrates will proceed to a robust validation of their clinical utility as diagnostic tools, by benchmarking a prototype assay against the current clinical standard guidelines. Our goal is to develop a rapid assay that improves patient stratification over current standard diagnostic markers and guides clinical decision-making to avoid unnecessary surgical intervention. If successful, this proposal will lay the groundwork for an actionable diagnostic test that will enable improved risk stratification of indeterminate thyroid nodules, and transform the clinical management of these challenging lesions.