Point-of-Care Diagnosis of Esophageal Cancer in LMICs

摘要：尽管食道鳞癌(ESCC)的临床治疗方法取得了实质性进展， 引起世界上大多数食道癌，这种致命的肿瘤通常发生在疾病晚期。 阶段，存活率非常低。内窥镜检查(EGD)的可获得性受到限制，以及 组织学，损害了LMICs中ESCC的检测，对我们有效治疗这种疾病的能力产生了不利影响。 因此，在LMIC中，廉价、安全、本地可执行的用于检测ESCC的策略是必要的，以确定 并迅速将他们转诊至适当的诊断和治疗方案。 因此，一种以可回收的吞咽海绵为特征的诊断方法收集食道 用于分子测试的样品，与用于样品处理的POC磁流控芯片相结合 和DNA甲基化检测。线海绵更便宜，更无创，更多 活检比EGD更方便、更快速。磁流控芯片简化了DNA纯化， 亚硫酸氢盐处理和聚合酶链式反应检测甲基化标记进入单一的POC仪器。这种方法 不需要EGD，可以在具有便携式能源供应的偏远地区执行，并且不需要 受过广泛的医疗培训，因此易于在低收入国家实施。 我们的具体目标是：1：使用海绵胶囊吞噬/系留收集装置，构建一种 基于甲基化标记的策略检测ESCC。在100名ESCC患者和100名良性对照患者中，我们 建议(1)建立包含生物标志物候选的多变量模型；(2)进行反馈-可行性 与Makerere大学医院的医疗保健和内窥镜检查人员举行会议，以微调最终的POC 用途：2：为了实现样品到答案的分析，实现DNA提取，亚硫酸氢盐处理， 和甲基化特异的聚合酶链式反应，与干燥的试剂放入磁流控芯片。我们将使用磁流体 简化细胞裂解、DNA提取、亚硫酸氢盐处理和甲基化特异性聚合酶链式反应的技术 由廉价的热塑性材料制成的紧凑型芯片。此外，我们将冷冻干燥试剂，并使用热- 部署了蜡封塞，以允许在室温下存储。以这种方式，我们将制作一个从样品到- 简单易用、价格低廉、无需冷链步骤的答题方法；3：为了充分实现 全自动高速生物标记物检测，设计了一种小型、轻便的仪器。我们将设计一种仪器 包含可编程磁驱动、温度控制和荧光检测，以执行 在用户输入非常少的芯片上进行测试。我们还将设计出体积小、重量轻、操作方便、便于携带的仪器 电力供电和移动电话控制，便于与基于LMIC的临床任务集成；以及4： 使用我们的POC方法在乌干达开展ESCC诊断性试点研究。在应用 目标2和目标3中使用的磁流控芯片和仪器，我们将进行一项试验，以测量特异性和 乌干达坎帕拉120例EGD确诊的ESCC患者和360例良性疾病对照患者的敏感性。

SUMMARY: Despite substantial progress in clinical approaches to squamous cancer of the esophagus (ESCC), which causes most esophageal cancers (EC) in the world, this deadly tumor usually occurs at late disease stages, with very poor survival. Restricted availability of endoscopy (EGD), along with rarity and delays in histology, impairs detection of ESCC in LMICs, adversely impacting our ability to treat this disease effectively. Thus, in LMICs, inexpensive, safe, locally performable strategies for detecting ESCC are necessary to identify high-risk patients and refer them quickly to suitable diagnostic and therapeutic options. Therefore, a diagnostic approach featuring a retrievable swallowed sponge-on-a-string to gather esophageal specimens for molecular testing, combined with a point-of-care (POC) magnetofluidic chip for sample processing and DNA methylation detection, is proposed. The string-sponge is less expensive, more noninvasive, more convenient, and more rapid than EGD with biopsy. The magnetofluidic chip streamlines DNA purification, DNA bisulphite treatment, and PCR detection of methylation markers into a single POC apparatus. This approach does not necessitate EGD, can be performed in remote areas with portable energy supplies and does not require extensive medical training, and is thereby amenable to implementation in LMICs. Our Specific Aims are: 1: Using a sponge-capsule swallowed/tethered collection device, to construct a methylation marker-based strategy to detect ESCC. In 100 ESCC and 100 benign control patients, we propose (1) building a multivariate model containing biomarker candidates; (2) carrying out feedback-feasibility meetings with health care and endoscopy personnel at Makerere University Hospitals to fine-tune eventual POC usage; 2: In order to achieve a sample-to-answer assay, to implement DNA extraction, bisulfite treatment, and methylation-specific PCR into a magnetofluidic chip with dried reagents. We’ll use magnetofluidic techniques to streamline cell lysis, DNA extraction, bisulphite treatment, and methylation-specific PCR into a compact chip built from cheap thermoplastic materials. In addition, we’ll lyophilize reagents and use heat- deployed wax sealant plugs to permit storage at room temperature. In this fashion, we will fashion a sample-to- answer assay that is easy to use, inexpensive, and free of cold-chain steps; 3: In order to achieve fully automatic high-speed biomarker assaying, to design a small, light apparatus. We’ll engineer an instrument containing programmable magnetic actuation, temperature control, and detection of fluorescence to execute the test in a chip with very little user input. We’ll also design the apparatus to be small, light, easy to operate, portable electricity-powered, and mobile phone-controlled to ease integration with LMIC-based clinical tasking; and 4: Using our POC approach to carry out a diagnostic pilot study of ESCC in Uganda. While applying the magnetofluidic chip and apparatus used in Aim 2 and Aim 3, we’ll carry out a trial to measure specificity and sensitivity in 120 EGD-confirmed cases of ESCC and 360 benign disease control patients in Kampala, Uganda.