Abstract. The primary goal of this proposal is to develop novel molecular approaches for early detection of esophageal adenocarcinoma (EAC). EAC arises in the setting of gastroesophageal reflux disease (GERD) and Barrett's esophagus (BE); a metaplastic change resulting from GERD. This knowledge has driven endoscopic screening and surveillance recommendations for individuals with GERD and BE but compliance is low and the vast majority of new EAC cases are still diagnosed at late stage, when the tumor is inoperable and prognosis is extremely poor. Over the past 6-8 years, we have developed a highly collaborative, multi-institutional and multi- disciplinary team focused on the development of clinically useful biomarkers for EAC. This proposal will build on our previously funded projects and the clinical and technical resources available to us through these studies, in order to evaluate two different approaches to early detection of EAC. The first approach is based on detection of somatic mutation and DNA copy number abnormality signatures in esophageal cytology samples. Published studies and our own ongoing work strongly indicate that accumulation of somatic mutations and major chromosomal rearrangements drive the progression from BE to EAC. We believe that these changes can be leveraged to identify a genomic signature for progression that can be detected in cytology samples using a targeted next-generation sequencing (NGS) test. Identification of the genomic signature is ongoing in our group through large-scale comparative sequencing of EAC and pre-neoplastic tissue samples. To evaluate the effectiveness of this signature in identifying progression, we are currently collecting ~350 esophageal cytology samples using an encapsulated sponge called the EsophaCap™. This device is not yet commercially available, but has been provided to us under arrangement with CapNostics LLC and is currently approved for investigational testing in the USA and Canada. These unique data and tissue resources will be used in Specific Aim 1 to evaluate the sensitivity and specificity of a targeted NGS analysis of esophageal cytology samples to detect dysplasia and early EAC. The second approach also leverages our large-scale sequencing data and targeted sequencing assays, but in this case we will evaluate the detection of mutations in circulating, cell-free DNA isolated from plasma as a biomarker for EAC. We are currently collecting plasma from all patients who swallow the EsophaCap™ device and this provides an excellent tissue resource in which to test detection of circulating tumor DNA (ctDNA). To aid in the detection of ctDNA, we have developed an ultra-sensitive NGS- based assay that can detect mutations at multiple loci with sensitivity as low as 0.05% (1/2000) mutant allele fraction. These resources will be used in Specific Aim 2 in order to evaluate the sensitivity and specificity of ctDNA for detection of stage I and II EAC. Finally, studies indicate that ctDNA dete...