APPLICATION OF A MULTIPLEXED IMMUNOASSAY FOR THE DETECTION OF BLADDER CANCER

项目总结/摘要 背景膀胱癌的无创性诊断以血尿患者最常见 膀胱癌临床表现，或肿瘤监测中有膀胱癌病史的受试者 仍然是一个挑战，因此，这些患者需要侵入性测试。目前基于尿液的测定不是 因此不能用于“排除”不需要这些侵入性测试的患者。 先前，我们a）鉴定了膀胱癌相关的诊断蛋白质“指纹”，其由10个 生物标志物，B）开发了一种多重免疫测定法以查询排泄尿液样品中的这10种生物标志物， c）进行多重免疫测定的分析验证。使用多重免疫测定，我们有 在326例受试者（46例癌症）中产生了令人鼓舞的初步数据，灵敏度和特异性为93%。 因此，我们第一次拥有了一种准确的检测方法，可以用来“排除”不需要治疗的患者。 侵入性测试此外，早期检测（即，在临床表现之前检测）是重要的目标 膀胱癌的风险。在介绍，超过70%的膀胱癌病例是非- 肌肉浸润性膀胱癌（NMIBC），而其余30%是肌肉浸润性膀胱癌 （MIBC）或转移性。当早期检测到（即，NMIBC），5年生存率约为94%， 相比之下，当疾病被检测为肌肉浸润性膀胱癌时，5年生存率约为50 （MIBC），并且当疾病是转移性时<20%。然而，到目前为止，还没有早期检测方法。中 来自我们正在进行的R 01研究的20名膀胱癌患者和20名匹配对照的小队列，我们 早在18个月前， 膀胱癌的临床诊断和所有膀胱癌的实际阳性多重免疫测定 在临床诊断膀胱癌之前12个月的患者。假设：膀胱癌相关 诊断蛋白质“指纹”的存在，可以用来指示膀胱癌的存在， 不仅在诊断时，而且在临床表现之前侵入性获得尿液样本， 膀胱癌的诊断具体目的：1）验证膀胱癌多重免疫检测方法 在一项大型巢式病例对照研究（n=800）中进行检测; 2）评价多重免疫测定用于早期 在一项大型巢式病例对照研究中检测（n=600）。这项研究将打开大门， 改善非侵入性方法检测膀胱癌，因此，它将有一个显着的 对病人护理的影响。以前，我们的团队已经发现并进行了早期验证， 膀胱癌相关的诊断蛋白质“指纹”，结果非常令人鼓舞。在当前 建议，我们现在试图在两个大型的巢式病例对照研究中测试多重免疫测定， 将使我们能够开发和锁定体外诊断多变量指数测定（IVDMIA）算法， 目标中列出的两种不同适应症。这样的算法可以部署在未来的前瞻性研究。 存在对可靠生物标志物的未满足的临床需求a）"排除“哪些患者 血尿或膀胱癌监测不需要进一步评估和B）早期检测 膀胱癌更容易治疗，生存率更高。实施这种稳健的测定 可能会产生深远的影响，从而改善护理并降低医疗成本。

Project Summary/Abstract Background The non-invasive diagnosing of bladder cancer in patients with hematuria, the most common clinical presentation of bladder cancer, or in subjects with a history of bladder cancer on tumor surveillance remains a challenge, and as such, these patients require invasive testing. Current urine-based assays are not robust and therefore cannot be used to ‘rule out’ patients who do not require these invasive testing. Previously, we a) identified a bladder cancer-associated diagnostic protein “fingerprint” comprised of 10 biomarkers, b) developed a multiplex immunoassay to query these 10 biomarkers in voided urine samples and c) performed analytical validation of the multiplex immunoassay. Using the multiplex immunoassay, we have generated encouraging preliminary data in 326 subjects (46 cancer) noting a sensitivity and specificity of 93%. Thus, for the first time, we possess an accurate assay that can be used to ‘rule out’ patients who do not require invasive testing. Furthermore, early detection (i.e., detection prior to clinical manifestation) is an important goal for patients at risk for bladder cancer. At presentation, more than 70% of bladder cancer cases are non- muscle invasive bladder cancer (NMIBC), whilst the remaining 30% are muscle invasive bladder cancer (MIBC) or metastatic. When detected early (i.e., NMIBC), the 5-year survival rate is approximately 94%, compared to a 5-year survival rate of ~50% when the disease is detected as muscle invasive bladder cancer (MIBC), and <20% when the disease is metastatic. However, to date, no early detection assay is available. In a small cohort of 20 bladder cancer patients and 20 matched controls from our ongoing R01 studies, we have noted an elevation in our bladder cancer-associated diagnostic protein “fingerprint” as early as 18 months prior to the clinical diagnosis of bladder cancer and an actual positive multiplex immunoassay in all bladder cancer patients 12 months prior to the clinical diagnosis of bladder cancer. Hypothesis: A bladder cancer-associated diagnostic protein “fingerprint” exists that can be leveraged to indicate the presence of bladder cancer in non- invasively obtained urine samples not only at the time of diagnosis, but prior to the clinical presentation and diagnosis of bladder cancer. Specific Aims: 1) To validate the multiplex immunoassay for bladder cancer detection in a large, nested case-control study (n=800) and 2) To evaluate the multiplex immunoassay for early detection in a large, nested case-control study (n=600). Significance This research will open the door for improving on the non-invasive methods for detecting bladder cancer and as such it will have a marked impact on patient care. Methodology Previously, our group has discovered and performed early validation of bladder cancer-associated diagnostic protein” fingerprint’ with extremely encouraging results. In the current proposal, we now seek to test the multiplex immunoassay in two large, nested case control studies, which would allow us to develop and lockdown In Vitro Diagnostic Multivariate Index Assay (IVDMIA) algorithms for two distinct indications listed in the aims. Such algorithms can be deployed in future prospective studies. Expected Results There exists an unmet clinical need for reliable biomarkers a) to ‘rule out’ which patients with hematuria or on bladder cancer surveillance do not require further evaluation and b) to early detect bladder cancer when its more treatable with improved survival rates. Implementation of such a robust assay could have a profound impact leading to improved care and reduced healthcare costs.