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.

We propose an ESCC diagnostic strategy comprising a single-use swallowed retrievable sponge-capsule to collect esophageal cells for biomarker analysis, coupled with a portable microfluidic chip device for automated POC sample processing and DNA methylation detection. This strategy does not require endoscopy (EGD), can be administered in rural settings without the need for electricity or persons with medical training, and uses a portable analytic chip. The sponge is cheaper, less invasive, safer, and faster than EGD; the microchip integrates DNA extraction, bisulfite DNA conversion, and methylation analysis into a single POC device.