Impact of preanalytic procurement and processing variables on the detection of HCC DNA in urine

分析前采集和处理变量对尿液中HCC DNA检测的影响 肝细胞癌（HCC）是世界上第二大癌症相关死亡原因，也是世界上最严重的癌症之一。 美国增长最快的癌症; 85%的患者在5年内死亡，主要是由于晚期发现， 治疗选择和高复发率。目前，监测HCC复发和治疗反应 通过血清AFP和序列成像。不幸的是，血清AFP只能用于~50%的HCC病例， 低敏感性。虽然MRI/CT成像是诊断的黄金标准，但它昂贵且不易获得。 它在检测小肿瘤（< 2 cm）方面的效用也有限，并且在存在以下情况下具有挑战性： 发育不良结节复发性HCC的早期检测与目前可用的方法是无效的。遗传 作为国家癌症研究计划的一部分，液体活检已被常规用于几乎所有癌症类型的诊所。 综合癌症网络（NCCN）指南。然而，其在HCC中的使用是有限的，即使其 在文献中已经有很好的记载，大多数是血液，有些是尿液。我们的假设 通过建立一个基于证据的尿液收集，储存和经肾的生物标本实践， DNA（trDNA）分离，尿HCC DNA分析与定制的敏感性测定可以提供HCC遗传学， 与当前标准治疗相比，更频繁的肿瘤监测和治疗反应评估 用于预后。作为发展尿液癌液体活检的先驱，我们建立了标准， 结肠癌早期检测研究网络尿液采集的操作规程（SOP） 验证研究（方案ID：320）和其他非尿路癌症。我们已经确定了预分析 在开发HCC尿液DNA的过程中影响尿液HCC DNA测定性能的变量 检测HCC液体活检。在本申请中，我们建议使用肝脏特异性trDNA标记物，即B型肝炎 病毒（HBV）DNA，这是在尿液中发现的慢性HBV感染患者，以验证和减轻 这些分析前的变量。目标1和2将识别、验证和减轻分析前因素， 尿液收集、储存和处理用于trDNA分离，并表征trDNA的大小， 影响临床测定结果和再现性。因此，基于证据的尿液生物样本 可以建立实践来帮助支持尿液HCC DNA检测开发，验证HCC遗传学 液体活检和治疗反应评估。在目标3中，我们将证明再现性 以及临床验证尿HCC DNA在评估对HCC治疗的反应中的效用。发展中 用于样本采集、处理和长期储存的可靠循证尿液生物标本实践 将导致尿液检测在临床试验中用于评估HCC治疗的有效性。

Impact of pre-analytic procurement and processing variables on the detection of HCC DNA in urine Hepatocellular carcinoma (HCC) is the world’s 2nd leading cause of cancer-related death and one of the fastest-growing cancers in the US; 85% of patients die within 5 years, mainly due to late detection, limited treatment options, and high recurrence. Currently, HCC recurrence and treatment responses are monitored by serum AFP and serial imaging. Unfortunately, serum AFP can only be used for ~50% of HCC cases due to low sensitivity. While MRI/CT imaging is the gold standard for diagnosis, it is expensive and less accessible. It also has limited utility in the detection of small tumors (< 2 cm) and is challenging in the presence of dysplastic nodules. Early detection of recurrent HCC is ineffective with the current available methods. Genetic liquid biopsies have been routinely used in clinics for almost all cancer types as part of the National Comprehensive Cancer Network (NCCN) guideline. However, its usage in HCC is limited even though its promises have been well documented in the literature, mostly for blood and some for urine. Our hypothesis is that by establishing an evidence-based biospecimen practice for urine collection, storage, and transrenal DNA (trDNA) isolation, urine HCC DNA analysis with tailored sensitive assays can provide HCC genetics with more frequent tumor surveillance and treatment response assessment compared to current standard of care for prognosis. As a pioneer in developing urine cancer liquid biopsies, we have established the standard operating procedure (SOP) for urine collection for the Early Detection Research Network colon cancer validation study (protocol ID: 320) and other non-urinary tract cancers. We have identified pre-analytic variables that influence urine HCC DNA assay performance in the process of developing an HCC urine DNA test for HCC liquid biopsies. In this application, we propose to use a liver-specific trDNA marker, hepatitis B virus (HBV) DNA, which is found in urine, from patients with chronic HBV infection to validate and mitigate these pre-analytic variables. Aims 1 and 2, will identify, validate, and mitigate pre-analytic factors, associated with urine collection, storage and processing for trDNA isolation, and characterize of the size of trDNA that influences clinical assay results and reproducibility. As a result, an evidence-based urine biospecimen practice can be established to help support urine HCC DNA assay development, validation for HCC genetic liquid biopsy and assessment of treatment response. In Aim 3, we will then demonstrate the reproducibility and clinical validation of the utility of urine HCC DNA in assessing responses to HCC treatment. Developing a robust evidence-based urine biospecimen practice for sample collection, processing, and long-term storage will lead to the validation of urine tests for assessing HCC treatment in clinical trials.