Discovery, Validation, and Application of Novel Methylated DNA Markers for Detection of Esophageal Cancer in Plasma

Abstract

Purpose: The burden of esophageal cancer continues to rise, and noninvasive screening tools are needed. Methylated DNA markers (MDM) assayed from plasma show promise in detection of other cancers. For esophageal cancer detection, we aimed to discover and validate MDMs in tissue, and determine their feasibility when assayed from plasma.

Experimental design: Whole-methylome sequencing was performed on DNA extracted from 37 tissues (28 EC; 9 normal esophagus) and 8 buffy coat samples. Top MDMs were validated by methylation specific PCR on tissue from 76 EC (41 adeno, 35 squamous cell) and 17 normal esophagus. Quantitative allele-specific real-time target and signal amplification was used to assay MDMs in plasma from 183 patients (85 EC, 98 controls). Recursive partitioning (rPART) identified MDM combinations predictive of esophageal cancer. Validation was performed in silico by bootstrapping.

Results: From discovery, 23 candidate MDMs were selected for independent tissue validation; median area under the receiver operating curve (AUC) for individual MDMs was 0.93. Among 12 MDMs advanced to plasma testing, rPART modeling selected a 5 MDM panel (FER1L4, ZNF671, ST8SIA1, TBX15, ARHGEF4) which achieved an AUC of 0.93 (95% CI, 0.89-0.96) on best-fit and 0.81 (95% CI, 0.75-0.88) on cross-validation. At 91% specificity, the panel detected 74% of esophageal cancer overall, and 43%, 64%, 77%, and 92% of stages I, II, III, and IV, respectively. Discrimination was not affected by age, sex, smoking, or body mass index.

Conclusions: Novel MDMs assayed from plasma detect esophageal cancer with moderate accuracy. Further optimization and clinical testing are warranted.

©2019 American Association for Cancer Research.

Figures

Figure 1.

Study flow diagram. EAC, esophageal adenocarcinoma; ESCC, esophageal squamous cell carcinoma; PCR, polymerase chain reaction; QuARTS, quantitative allele-specific real-time target and signal amplification.

Figure 2.

Beta-actin corrected methylation intensity in DNA markers assayed from plasma. Each column on the x-axis represents an individual patient’s sample; each row on the y-axis represents the methylation specific PCR product by QuARTS assay for each marker on a logarithmic scale. A 91% specificity threshold was selected by the rPart model. Increasing yellow-red color spectrum reflects increasing methylation intensity above the 91st percentile value of the control patient samples.

Figure 3.

A. At a specificity cut-off of 91% by the model, the 5-marker panel was able to detect 43% stage I, 64% stage II, 77% stage III, and 92% stage IV disease. The marker panel detected 74% of esophageal cancer overall, 74% of esophageal adenocarcinoma, and 78% of esophageal squamous cell carcinoma. Confidence intervals are shown. B. Overall discrimination of esophageal cancer by methylated DNA marker panel assayed from plasma: areas under the receiver operating characteristic curves for best-fit and cross-validation analyses (and 95% confidence intervals) are shown. AUC, area under the receiver operating characteristics curve.

Figure 3.

A. At a specificity cut-off of 91% by the model, the 5-marker panel was able to detect 43% stage I, 64% stage II, 77% stage III, and 92% stage IV disease. The marker panel detected 74% of esophageal cancer overall, 74% of esophageal adenocarcinoma, and 78% of esophageal squamous cell carcinoma. Confidence intervals are shown. B. Overall discrimination of esophageal cancer by methylated DNA marker panel assayed from plasma: areas under the receiver operating characteristic curves for best-fit and cross-validation analyses (and 95% confidence intervals) are shown. AUC, area under the receiver operating characteristics curve.