Point-of-Care Diagnosis of Esophageal Cancer in LMICs

摘要：尽管食管鳞状细胞癌（ESCC）的临床方法取得了实质性进展， 这种致命的肿瘤通常发生在疾病晚期， 阶段，生存率极低。内镜检查（EGD）的可用性有限，沿着罕见和延迟， 组织学上，损害了LMIC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，在中低收入国家，廉价，安全，当地可执行的战略，用于检测食管鳞癌是必要的，以确定 高风险患者，并将他们迅速转介到合适的诊断和治疗方案。 因此，一种诊断方法，其特征在于可回收的吞咽海绵串，以收集食管 用于分子检测的样品，结合用于样品处理的即时护理（POC）磁流体芯片 和DNA甲基化检测。线海绵更便宜，更无创， 比EGD+活检更方便、快捷。磁流体芯片简化了DNA纯化， 亚硫酸氢盐处理和甲基化标志物的PCR检测到单个POC装置中。这种方法 不需要EGD，可以在偏远地区使用便携式能源供应进行，并且不需要 广泛的医疗培训，因此适合在中低收入国家实施。 我们的具体目标是：1：使用海绵胶囊吞咽/栓系收集装置，构建一个 基于甲基化标记的策略来检测ESCC。在100例ESCC和100例良性对照患者中，我们 建议（1）建立包含生物标志物候选物的多变量模型;（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会面，以微调最终的POC 用途; 2：为了实现样品到答案的测定，实施DNA提取，亚硫酸氢盐处理， 和甲基化特异性PCR到具有干燥试剂的磁流体芯片中。我们会用磁流体 将细胞裂解、DNA提取、亚硫酸氢盐处理和甲基化特异性PCR技术简化为一种 由廉价热塑性材料制成的紧凑芯片。另外，我们会冷冻试剂并加热- 部署蜡密封塞，以允许在室温下储存。这样，我们就可以做一个样本-- 回答分析，易于使用，价格低廉，无冷链步骤; 3：为了充分实现 自动高速生物标志物分析，以设计一个小而轻的装置。我们会设计一种仪器 包含可编程磁致动、温度控制和荧光检测，以执行 在芯片中进行测试，只需很少的用户输入。我们还将把仪器设计得小巧、轻便、易于操作、便于携带 电动和移动的电话控制，以便于与基于LMIC的临床任务整合;以及4： 使用我们的POC方法在乌干达进行ESCC的诊断性试点研究。在应用 目标2和目标3中使用的磁流体芯片和装置，我们将进行一项试验来测量特异性， 在乌干达坎帕拉的120例EGD证实的ESCC病例和360例良性疾病对照患者中进行了敏感性研究。

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.