Development of a multi-analyte diagnostic assay to improve the risk stratification of indeterminate thyroid nodules

摘要 在临床实践中广泛使用的敏感成像导致了偶发性甲状腺结节 被发现的频率越来越高，创造了一个独特的机会，以检测和删除 恶性肿瘤早期病变。大多数偶发结节仍为良性， 无症状，但10-20%被发现是恶性手术切除，导致急需 为了改善诊断，目前75%的患者接受不必要的甲状腺切除术， 切除良性结节。目前的护理标准包括通过以下方式收集组织活检： 超声引导细针穿刺：这些样本通过细胞学进行检测， 异常细胞和遗传风险标记。这些诊断测试的特点是穷人 特异性，三分之一的测试结核仍然被归类为不确定的，需要 切除以进行病理诊断。我们最初假设蛋白水解失调， 一种酶活性是侵袭性癌症的标志，可能会产生不同水平的 良性和恶性结节的FNA组织中的活性。在第一阶段的工作中，我们利用了Allaunus Biosciences，Inc.诊断管道，一个平台，允许创新的基于蛋白质组学的生物标志物 发现，以鉴定和表征一组蛋白酶活性（功能标记物）， 恶性结节显著增加。然后，我们开发了荧光底物测定， 使用少量生物样品监测这些活动。同时，我们还发现 可以用标准的基于抗体的测定法测量的另外的基于质量的标记物。我们 研究性多分析物诊断测定具有区分恶性与良性的潜力 甲状腺组织的敏感性和特异性> 90%。在第二阶段的提案中，我们的目标是执行 技术和临床验证研究，以将检测试剂盒推向临床使用， 甲状腺结节的早期诊断在目标1中，我们将对 我们在手术组织中选择的蛋白质和功能性生物标志物来评估评分， 诊断阈值，并报告关键检测参数。在目标2中，我们将执行 对检测试剂盒的实验室性能进行全面的技术评估，以满足行业要求 标准在目标3中，我们将通过我们的IRB创建一个独立的FNA组织样本队列- 在UCSF、UCLA和IU批准的生物库工作，用于对以下内容进行稳健的前瞻性验证： 分析的临床实用性。我们的目标是开发一种快速检测方法， 对当前标准诊断标志物进行分层，指导临床决策，以避免 不必要的手术干预，并改变这些具有挑战性的临床管理 病变

Abstract Widespread use of sensitive imaging in clinical practice has resulted in incidental thyroid nodules being discovered with increasing frequency, creating a unique opportunity to detect and remove lesions at early stages of malignancy. The majority of these incidental nodules remain benign and asymptomatic, but 10-20% are found to be malignant on surgical excision, resulting in a critical need for improved diagnostics, as 75% of patients currently undergo unnecessary thyroidectomies to remove benign nodules. The 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 are characterized by poor specificity, and a third of all tested nodules are still classified as indeterminate and need to be resected for a pathological diagnosis. We originally 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. In Phase I work, we leveraged the Alaunus Biosciences, Inc. diagnostic pipeline, a platform that allows innovative proteomics-based biomarker discovery, to identify and characterize a set of protease activities (functional markers) that are significantly increased in malignant nodules. We then developed fluorogenic substrate assays to monitor these activities using small volumes of biological samples. In parallel, we also discovered additional mass-based markers that can be measured with standard antibody-based assays. Our investigational multi-analyte diagnostic assay has the potential to differentiate malignant from benign thyroid tissue with sensitivity and specificity >90%. In this Phase II proposal, we aim to perform technical and clinical validation studies required to advance the assay to readiness for clinical use in the early diagnosis of indeterminate thyroid nodules. In Aim 1, we will benchmark the clinical utility of our selected protein and functional biomarkers in surgical tissue to evaluate scoring, establish diagnostic thresholds, and report on key assay parameters. In Aim 2, we will perform a comprehensive technical assessment of the laboratory performance of the assay to satisfy industry standards. In Aim 3, we will create an independent cohort of FNA tissue samples, through our IRB- approved biobanking efforts at UCSF, UCLA and IU, to be used for a robust prospective validation of the clinical usefulness of the assay. Our goal is to develop a rapid assay that improves patient stratification over current standard diagnostic markers, guides clinical decision-making to avoid unnecessary surgical intervention, and transforms the clinical management of these challenging lesions.