Calise K. Berger

Esophageal squamous cell carcinoma (ESCC) remains a leading cause of cancer-related mortality worldwide, and there are limited molecular screening options. Following a prior discovery and validation studies in subjects from the United States, China, and Iran, we aimed to validate methylated DNA markers (MDM) for ESCC detection in Malawi, a country with one of the highest ESCC incidence. Fourteen MDMs were tested on 177 tissue samples. Ten MDMs showed excellent performance (AUCs ≥ 0.85); a random forest model achieved an AUC of 0.97. The consistent cross-population performance suggests potential for universal application of these MDMs and further justifies exploring nonendoscopic sampling methods, such as swallowed cell collection devices in combination with MDMs, for efficient and cost-effective screening in high-incidence populations. Prevention Relevance: This study validates MDMs for early detection of ESCC in Malawi, supporting their potential role in noninvasive, population-based screening strategies in Africa to enable early detection and timely intervention for cancer interception, potentially reducing cancer burden in high-incidence, resource-limited regions.

Multistage study design from marker discovery to validation. Workflow overview of initial marker discovery using WGMS on DNA extracted from endoscopic brushing samples with cross-reference to an archival RRBS dataset from DNA extracted from independent tissue samples, and independent validation comparing paired endoscopic brushings and sponge-on-string samples. EAC, esophageal adenocarcinoma; EM-seq, enzymatic methyl sequencing; methyl-seq, methyl sequencing; WGBS, whole genome bisulfite sequencing.

AUC of the 52-MDM model in cytology brush validation data comparing NDBE vs. HGD/EAC, stratified by covariates.

ROC curves for MDM performance in paired endoscopic brush and sponge samples. A, AUC analysis demonstrating discrimination of HGD/esophageal adenocarcinoma (EAC) from NDBE using the MDM panel, AS, and the combination in endoscopic brush samples. B, Corresponding AUC analysis showing marker performance in paired available (Table 1) sponge-on-string samples from the same patients.

Baseline demographic characteristics of patients whose samples were included in the experiments.

Methylation and copy-number profiles in discovery and validation case and control sample sets. For methylation, each row is a marker from the filtered set of 52 MDMs and each column a patient sample. For copy number, each row is a chromosomal arm. Methylation marker levels measured in deciles above the 95th percentile value in NDBE are denoted by the increasing intensity of yellow to red color spectrum. Copy-number analysis shows amplifications (red) or deletions (blue) referent to neutral (white) as estimated by the AS method. NDBE referent to NE (gray, neutral by definition). EAC, esophageal adenocarcinoma.

Aberrant DNA methylation and copy-number alterations (CNA) drive Barrett’s esophagus progression to esophageal adenocarcinoma; however, their combined utility for early detection is unclear. We aimed to identify and validate methylated DNA markers (MDM) and CNAs to distinguish esophageal adenocarcinoma/high-grade dysplasia (HGD) from nondysplastic Barrett’s esophagus (NDBE). In this multiphase, multicenter study, we discovered and validated MDMs and quantified CNAs utilizing whole-genome methylation sequencing of esophageal brushings. DNA biomarkers identified from discovery were further validated in independent patients with paired esophageal brushing and swallowed capsule sponge samples. MDMs were filtered against a reduced representation bisulfite sequencing dataset obtained from independent tissue samples to advance only concordant candidates. CNA burden was quantified using ichorCNA-derived aneuploidy scores (AS). Two hundred MDMs discovered in HGD (N = 18) and esophageal adenocarcinoma (N = 18) versus NDBE brushing samples (N = 18) were tested in independent samples (N = 146). A 52-MDM panel achieved a cross-validated AUC of 0.88 [95% confidence interval (CI), 0.82–0.95]; the addition of AS improved discrimination of HGD/esophageal adenocarcinoma from NDBE to 0.91 (95% CI, 0.86–0.97) AUC. At 80% specificity, the combined model detected 93% of esophageal adenocarcinoma and 88% of HGD cases. In paired capsule sponge samples, a 58-MDM panel achieved a cross-validated AUC of 0.77 (95% CI, 0.66–0.88); a combined 58-MDM and AS model achieved AUC 0.80 (95% CI, 0.7–0.9). MDMs and AS discerned HGD/esophageal adenocarcinoma from normal esophagus/NDBE in endoscopic brushing and capsule sponge samples. This approach may improve Barrett’s esophagus surveillance.Prevention Relevance:This study demonstrates that combining epigenetic and genomic biomarkers across minimally invasive sampling methods can accurately distinguish HGD/esophageal adenocarcinoma from nondysplastic Barrett’s esophagus, offering promising, less invasive strategies to improve BE surveillance and enable endoscopic therapy for esophageal adenocarcinoma prevention and treatment.

Supplemental Table 1: Study adherence to the STARD (Standards for Reporting Diagnostic Accuracy Studies) 2015 checklist.

Supplemental Figure 2: Distributions of all methylated DNA markers tested for endometrial cancers and controls, stratified by Lynch syndrome. Log10 strand counts are adjusted based on the level of the control gene, ACTB, in all controls (SPOR-EC-CTRL and LS-EC-CTRL). P-value provided to show statistical significance of difference of medians between CASE and CTRL samples within SPOR and LS subgroups.

Supplemental Figure 1: Distributions of all methylated DNA markers tested for colorectal cancers and controls, stratified by Lynch syndrome. Log10 strand counts are adjusted based on the level of the control gene, ACTB, in all controls (SPOR-CRC-CTRL and LS-CRC-CTRL). P-value provided to show statistical significance of difference of medians between CASE and CTRL samples within SPOR and LS subgroups.