Development of an automated, point of care DNA methylation cartridge blood test for colorectal cancer detection in LMICs- an academic-industrial partnership

摘要 在许多低收入和中等收入国家(LMIC)，结直肠癌(CRC)被诊断为晚期。 患者和社区卫生从业人员对结直肠癌体征和症状缺乏了解 导致3-4期疾病的延迟表现。最初的延误，加上有限的结肠镜检查设备， 导致诊断延迟时间延长。结果是LMIC的5年死亡率高出5倍。 在美国。这一问题的创新解决方案可能是一种负担得起、易于部署的“护理点”分子 用于快速结肠镜检查和病理检查的用于识别和优先处理可能患有恶性肿瘤的患者的测试 带来更好的结果。继续与我们的工业合作伙伴Cepheid建立合作关系，我们 建议在我们公布的关于结直肠癌高甲基化标记的强大数据的基础上，开发出一种负担得起的、 小时，自动CRC甲基化检测血液测试，分析一组五个超甲基化基因在 从1毫升血浆中提取游离DNA。拟议的创新可能导致快速检测的单试剂盒。 CRC检测，成本降低3倍。在目标1a中，我们将优化一种无盒的亚硫酸氢盐DNA 血浆转换法，检测患者组1血浆(恶性组20例，正常组20例)的血浆转换率。在……里面 目标1b我们将使用来自FFPE样本的DNA从20个CRC标记中选择一个最佳的5标记组合 美国和尼日利亚(N=30个恶性，30个良性)和一个最佳的“PAN”集合将在血浆中被确认使用 美国患者组2例，尼日利亚组3例(恶性35例，良性35例)。在目标1c中，我们将评估分析 CRC-MD检测的性能。将在美国的多个等量样品上评估试剂盒内的重复性 患者组血浆4例(恶性35例，良性35例)。操作员间的重复性将使用 复制患者组4的等量血浆(N=35例恶性，35例良性)。目标2a的目标是在技术上 使用尼日利亚预期收集的样本验证CRC-MD检测。我们将首先在 来自患者组4(N=90名恶性患者，90名良性患者)的血浆训练集，以最佳平衡敏感性和 在来自患者组5(N=90)的血浆测试集中所选阈值的特异性和验证性能 恶性，良性90例)。CRC的准确性(敏感性、特异性、阳性预测值和阴性预测值) 基于MD的诊断将通过组织病理学来衡量，以区分良恶性疾病 以病变的诊断为金标准。最后，在目标2b中，确定 CRC-MD检测是根据特定患者的特征而改变的，我们将在特定患者中测试其临床准确性 按年龄、性别、体重指数和肿瘤特征分类的亚组。我们之前在自动化开发方面的成功 Cepheid的细胞/液体活组织检查为确保准确和可靠的测试奠定了基础。 这种干预可以通过为迫切需要的人加速结肠镜检查来节省成本，从而加快 LMICs中CRC的检测和治疗。这每年将拯救数以千计的生命。这项研究也将有助于 CRC-MD分析的进一步发展走向未来的商业化和全球接入。

ABSTRACT Colorectal cancer (CRC) is diagnosed at advanced stages in many low- and middle-income countries (LMICs). The lack of knowledge of CRC signs and symptoms by patients and community health practitioners frequently leads to delayed presentation with Stage 3-4 disease. This initial delay, paired with limited colonoscopy facilities, leads to prolonged diagnostic delays. The result is a 5-year mortality rate in LMIC up to 5 times higher than that in USA. An innovative solution to this problem could be an affordable, easily deployable, “point of care” molecular test to identify and prioritize patients likely to have a malignancy for expedited colonoscopy and pathology review leading to better outcomes. Continuing our established collaboration with our industrial partner, Cepheid, we propose to build on our strong published data on hypermethylated markers in CRC to develop an affordable, <3- hour, automated CRC-methylation detection blood test that analyzes a panel of five hypermethylated genes in cell free DNA from 1 ml of plasma. The proposed innovations could lead to a single-cartridge assay for quick CRC detection with a 3-fold reduction in cost. In Aim 1a, we will optimize a cartridge-less bisulfite DNA conversion method for plasma and test its efficiency in Patient Set 1 plasma (N= 20 malignant, 20 normal). In Aim 1b we will select one optimal 5-marker panel out of 20 CRC markers using DNA from FFPE samples from the U.S and Nigeria (N= 30 malignant, 30 benign), and one optimal “pan” set will be confirmed in plasma using U.S Patient Set 2 and Nigeria Set 3 (N=35 malignant, 35 benign). In Aim 1c, we will evaluate analytical performance of the CRC-MD assay. Intra-assay reproducibility will be assessed on multiple aliquots of U.S Patient Set 4 plasma (N=35 malignant, 35 benign). Inter-operator reproducibility will be determined using replicate aliquots of plasma from Patient Set 4 (N= 35 malignant, 35 benign). The goal of Aim 2a is to technically validate the CRC-MD assay using prospectively collected samples in Nigeria. We will first select a threshold in a Training set of plasma from Patient Set 4 (N=90 malignant, 90 benign) to optimally balance sensitivity and specificity, and validate performance of the selected threshold in a Test set of plasma from Patient Set 5 (N= 90 malignant, 90 benign). Accuracy (sensitivity, specificity, and positive- and negative-predictive value) of CRC- MD-based diagnosis to distinguish benign versus malignant disease will be measured using histopathological diagnosis of the lesion as the gold standard. Lastly, in Aim 2b, to determine whether the performance of the CRC-MD assay is altered by select patient characteristics, we will test its clinical accuracy among specific patient subgroups classified by age, sex, BMI, and tumor characteristics. Our prior success in developing automated cell-based/liquid biopsy assays with Cepheid has established the path ensuring an accurate and reliable test. This intervention could be cost saving by hastening colonoscopy for those who need it urgently, thus expediting detection and treatment of CRC in LMICs. This will save thousands of lives yearly. This study will also facilitate further development of the CRC-MD assay moving toward future commercialization and access globally.