Biomarker discovery for the early detection of esophageal adenocarcinoma and high-grade dysplasia

From the Grady Lab, Clinical Research Division

Rising cases of esophageal adenocarcinoma in the United States reflect a critical need for the development of early intervention strategies that can aid in improving poor survival rates for this patient population. Esophageal adenocarcinoma is unusual in its onset in that, in nearly all cases, it follows a specific developmental pattern from pre-cancerous Barrett’s esophagus to high-grade dysplasia to the onset of esophageal adenocarcinoma. “The high and rising death rate from esophageal adenocarcinoma is a major health problem. Patients with Barrett’s esophagus are at risk for esophageal adenocarcinoma and are placed in surveillance programs requiring regular upper endoscopy procedures. The current screening and surveillance program for Barrett’s esophagus and esophageal adenocarcinoma is suboptimal because it uses an expensive, invasive surveillance method with modest accuracy and limited efficacy,” explained Dr. Ming Yu, principal staff scientist working with Dr. William Grady, a member of Fred Hutch’s Clinical Research Division. Elaborating further, Dr. Yu stated “with recent advances in the management of high-grade dysplasia and early esophageal adenocarcinoma with non-endoscopic therapies, there is a need for more convenient and sensitive non-endoscopic surveillance methods that can save costs, avoid harm, and reduce cancer burden. Newer methods that use swallowed esophageal cytology collection devices and molecular biomarkers have the potential for non-invasive detection of high-grade dysplasia and early esophageal adenocarcinoma.” To this end, investigations into the pre-cursor for adenocarcinoma, Barrett’s esophagus, have revealed irregular DNA methylation profiles that, although their molecular significance remains unknown, demonstrated great potential as biomarkers for successfully diagnosing Barrett’s esophagus and probable progression to high-grade dysplasia and adenocarcinoma.

Building on these previous findings, Dr. Yu, Dr. Grady, and colleagues sought to identify more specific biomarkers for the detection of high-grade dysplasia and esophageal adenocarcinoma by undertaking whole genome methylation analysis, with a successful diagnosis potentially allowing for the use of early intervention strategies that are effective in reducing disease related mortality. In their study, recently published in Clinical Cancer Research, they “have discovered and validated novel methylated DNA biomarkers for the detection of high-grade dysplasia and esophageal adenocarcinoma. We envision that the biomarker panel we developed could be implemented in the clinic through a non-invasive procedure, which can aid screening for esophageal adenocarcinoma and its precancerous lesion high-grade dysplasia to reduce deaths from esophageal adenocarcinoma,” detailed Dr. Yu. 

Interrogation of DNA methylation profiles from esophageal brushing samples enables early detection of esophageal adenocarcinoma and high-grade dysplasia.
Interrogation of DNA methylation profiles from esophageal brushing samples enables early detection of esophageal adenocarcinoma and high-grade dysplasia. Figure provided by Dr. Yu.

The authors collected samples for their study by routine esophageal brushing – brushing the lining of the esophagus during endoscopy to collect cells for analysis – and compiled samples from a patient population with individuals with either normal squamous tissue, Barrett’s esophagus, high-grade dysplasia, or esophageal adenocarcinoma, which could inform any potential differences in methylation patterns. The authors undertook a two-pronged approach to aid in identifying potential methylation markers. First, they performed whole genome DNA methylation profiling and identified 24 CpG islands that were hypermethylated in high-grade dysplasia and esophageal adenocarcinoma when compared to the normal tissue or non-dysplastic samples. To further investigate candidate CpGs, the authors designed a digital droplet PCR assay (a sensitive assay for detecting rare variants) and identified three markers, Cg6522, YPEL3 and POU3F1, that were significantly differentially methylated in esophageal adenocarcinoma and high grade dysplasia. In parallel, as an alternative approach, the authors performed reduced-representation bisulfite sequencing (RRBS) that allowed for the detection of CpG patches, groups, or clusters of differentially methylated CpG islands present in close proximity to each other. Of note, two patches were observed to distinguish non-dysplastic Barrett’s esophagus from high grade dysplasia and esophageal adenocarcinoma, Up10 and Up35.

Having identified promising markers for the detection of cancerous samples, the authors assessed their validity in a training set both as single markers and in combination on a panel. Each marker performed well at an individual level, with the authors observing increased methylation in brushing samples from both adenocarcinoma and high-grade dysplasia samples. The authors designed two panels to test the sensitivity and specificity of using multiple markers (Model 1: Up10, UP35, Cg6522 and YPEL3, Model 2: Cg6522 and POU3F1). Promisingly, Model 1 demonstrated high specificity and sensitivity in the detection of adenocarcinoma and high dysplasia, as did Model 2.  Lastly, in a final independent validation cohort of brushing samples, both biomarker panels performed robustly, with strong sensitivity and specificity observed in the detection of esophageal adenocarcinoma and high-grade dysplasia.

These findings lay the foundation for the utilization of DNA methylation markers in the early detection of esophageal adenocarcinoma, moving away from more invasive procedures such as esophageal brushing, with the potential for improving outcomes for this patient population. As Dr. Yu concluded “the biomarker panel we have put forward needs further validation and optimization to be implemented in the clinical setting. We will further validate this panel using samples collected via the Esocheck device, a non-endoscopic sampling device. Successful validation of the Esochek-based biomarker panel will support the development of a commercial DNA methylation-based test for the detection of high-grade dysplasia and esophageal adenocarcinoma.”

This work was supported by funding from the National Cancer Institute, the National Institutes of Health, the Prevent Cancer Foundation, Exact Sciences, Pentax Medical, Cernostics, the Cottrell Family Fund, Evergreen Fund, and the Listwin Foundation.

Fred Hutch/University of Washington/Seattle Children's Cancer Consortium members Dr. Andrew Kaz, Dr. Georg Luebeck and Dr. William Grady contributed to this research.

Yu M, Moinova HR, Willbanks A, Cannon VK, Wang T, Carter K, Kaz A, Reddi D, Inadomi J, Luebeck G, Iyer PG, Canto MI, Wang JS, Shaheen NJ, Thota PN, Willis JE, LaFramboise T, Chak A, Markowitz SD, Grady WM. Novel DNA Methylation Biomarker Panel for Detection of Esophageal Adenocarcinoma and High-Grade Dysplasia. Clin Cancer Res. 2022 Sep 1;28(17):3761-3769. doi: 10.1158/1078-0432.CCR-22-0445. PMID: 35705525; PMCID: PMC9444948.