Detection of esophageal cancer via assay of novel methylated DNA markers in plasma

Feb. 14, 2020

Currently the sixth most common cause of cancer death worldwide, esophageal cancer has a five-year survival rate of less than 20% in developed countries and less than 5% in many developing countries. The two main subtypes of esophageal cancer, esophageal adenocarcinoma (EAC) and esophageal squamous cell carcinoma (ESCC), are both increasing in incidence. Because these cancers are often asymptomatic in their early stages, patients typically don't seek medical care until they have advanced-stage disease, when the potential for survival is more limited.

As the burden of esophageal cancer rises, the development of noninvasive screening tools has become a priority. Methylated DNA markers (MDMs) assayed from plasma have been used to detect other cancers. Building on that knowledge, Mayo Clinic researchers recently conducted a study to evaluate whether plasma assay of MDMs using optimized markers and analytically sensitive assays will detect early-stage esophageal cancer at high specificity. The results of that study were published in Clinical Cancer Research in December 2019.

One of that article's lead authors, John B. Kisiel, M.D., notes that the research team's goals included discovering and validating MDMs in tissue and determining their feasibility when assayed from plasma. Dr. Kisiel is a gastroenterologist and researcher at Mayo Clinic's campus in Rochester, Minnesota.

"Esophageal cancer is a highly lethal disease for which an effective screening tool at the population level does not exist," explains Dr. Kisiel. "Development of an inexpensive, automatable and noninvasive screening tool such as a blood-based biomarker test could be transformative, leading to early diagnosis in a wider population, as well as have implications for prognosis and surveillance."


Researchers performed whole-methylome sequencing on DNA extracted from 37 tissue samples, including 28 esophageal cancer (EC) samples, nine normal esophagus samples and eight buffy coat samples. Top MDMs were validated with methylation-specific polymerase chain reaction (PCR) in 76 tissue samples with EC (41 EAC, 35 ESCC) and 17 normal esophagus samples. Researchers used quantitative allele-specific real-time target and signal amplification to assay MDMs in plasma from 183 patients (85 with EC and 98 controls). Recursive partitioning (rPART) identified MDM combinations predictive of esophageal cancer. Researchers then validated the performance of these MDM combinations in silico by bootstrapping.


  • From discovery, researchers selected 23 candidate MDMs for independent tissue validation; the median area under the receiver operating curve (AUC) for individual MDMs was 0.93.
  • Among the 12 MDMs advanced to plasma testing, rPART modeling selected a panel of five MDMs that achieved AUCs of 0.93, with a 95% confidence interval (CI) of 0.89 to 0.96 on best-fit, and 0.81 (95% CI, 0.75 to 0.88) on cross-validation. This panel included the following MDMs: FER1L4, ZNF671, ST8SIA1, TBX15, ARHGEF4.
  • At 91% specificity, the panel detected 74% of esophageal cancers overall, and 43%, 64%, 77% and 92% of stage I, II, III and IV cancers, respectively. Researchers determined that discrimination was not affected by age, sex, smoking or body mass index.


According to Dr. Kisiel, the study data demonstrate that novel MDMs assayed from plasma detect esophageal cancer with moderate accuracy.

"Through an unbiased whole-methylome discovery effort, we discovered novel methylated DNA markers for esophageal adenocarcinoma and squamous cell carcinoma," explains Dr. Kisiel. "Plasma assays for candidate markers were developed, and a five-marker panel achieved high discrimination for cancer versus normal in plasma. These results open the door for using a noninvasive test for detection, prognosis and surveillance of esophageal cancer."

Dr. Kisiel acknowledges that further optimization and clinical testing are necessary. "Although our study is the largest plasma-based pilot of esophageal cancer biomarkers to date, it is limited by the retrospective design and the archival nature of the samples. To overcome this, we are recruiting and enrolling patients into a phase two of this study, with prospectively collected samples using optimized processing and storage methods," explains Dr. Kisiel.

Additional research involving an international population is also underway. "Although esophageal squamous cell carcinoma is a relatively rare cancer in the U.S., it is the most common type of esophageal cancer worldwide," says Dr. Kisiel. "In collaboration with the National Cancer Institute, we have conducted a multinational study validating the role of our methylated DNA markers for detection of esophageal cancer in China and Iran. We also have ongoing efforts with prospective collection in remote areas in China where esophageal cancer has a very high prevalence."

For more information

Qin Y, et al. Discovery, validation, and application of novel methylated DNA markers for detection of esophageal cancer in plasma. Clinical Cancer Research. 2019;24:7396.