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， 在恶性肿瘤的早期阶段切除病变。这也产生了对改进的关键需求。 诊断，因为80%的患者目前接受不必要的甲状腺切除术，因为他们 结节是良性的。目前的护理标准包括通过超声收集组织活检- 引导细针抽吸：通过细胞学检测这些样本，寻找异常细胞， 和遗传风险标记。这些诊断测试表现不佳，因此三分之一的 结节仍然被分类为不确定的，并且被移除用于病理诊断。我们 假设失调的蛋白水解，一种酶活性，是侵袭性的标志， 癌症，可能产生区分FNA组织中良性和恶性结节的活性水平。 Allaunus Biosciences，Inc.诊断流水线利用衬底轮廓技术 在加州大学旧金山分校的Craik实验室开发，这是一个允许基于蛋白质组学的创新平台。 生物标志物发现利用这项技术，我们以前已经确定和表征了一种 专有的一套蛋白酶活性，在癌前胰腺囊肿中显著增加; 然后，我们开发了荧光底物测定法，使用小体积的 液体活检（囊液）。在第一阶段的提案中，我们的目标是将我们的发现管道应用于 病理证实的甲状腺癌和匹配的正常组织的回顾性队列，以确定 恶性组织特异性的蛋白酶活性。在目标1中，我们将描述甲状腺肿瘤特异性 蛋白水解签名，并确定候选酶标记负责观察到的 蛋白水解活性在目标2中，我们将设计和合理优化特定的荧光底物， 这些蛋白酶可用于快速检测FNA样本中的恶性结节。铅 候选底物将通过以下方式进行其作为诊断工具的临床效用的稳健验证： 对照当前临床标准指南对原型测定进行基准测试。 我们的目标是开发一种快速的检测方法，改善患者分层超过目前的标准诊断 标记并指导临床决策，以避免不必要的手术干预。 如果成功，该提案将为可行的诊断测试奠定基础， 改善不确定甲状腺结节的风险分层，并改变临床管理， 这些具有挑战性的病变。

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.