(Bio)marking the spot – utilizing droplet digital PCR for risk biomarker identification in colorectal cancer

From the Grady Lab, Translational Science and Therapeutics

Regular screening practices can identify malignant disease at an early stage, which may provide promising therapeutic options for patients, resulting in improved long-term outcomes. However, for some cancer types, including colorectal cancer (CRC), screening procedures can be invasive and potentially stressful for patients. “The majority of CRC arises from pre-cancerous adenomas. Individuals with adenomas, especially those with advanced adenomas, are at heightened long-term risk of subsequent CRC compared to those with no personal history of adenoma. As such, individuals with a history of colon adenomas are advised to undergo more frequent surveillance colonoscopy exams,” explained Dr. Ming Yu, Principal Staff Scientist in the Grady Lab, part of Fred Hutch’s Translational Science and Therapeutics Division. “However, with only an approximate 10% recurrence rate within 3-5 years, more frequent surveillance colonoscopy in all of these individuals is an overly expensive, inefficient approach to decrease CRC incidence and an unnecessary burden on patients,” she continued.

Discovery of biomarkers to identify those most at risk of developing or experiencing recurrent CRC could aid in reducing this burden for patients. A distinct molecular feature of colon adenomas is the prominent presence of aberrant gene methylation, one of the epigenetic events occurring during cancer formation. Previous research from Dr. Grady’s group described how aberrant methylation of one specific gene EVL is detected at higher frequency in the normal colon of people with CRC compared to average risk individuals. In addition, several independent genome-wide studies also identified methylated EVL as a potential prognostic biomarker for CRC,” said Dr. Yu. These findings formed the basis of their recently published study in Cancer Epidemiology, Biomarkers and Prevention, where they investigated the utility of EVL methylation as a risk biomarker in CRC. Emphasizing the importance of this research, Dr. Yu noted how “use of molecular markers such as methylated EVL has the potential to be able to accurately predict an individual’s risk of metachronous adenomas or cancer during surveillance colonoscopy and accurately select the optimal surveillance interval for those individuals. This will result in fewer CRC related deaths from interval CRCs and in cost and time savings for patients.”

Methylation profiling of patients with and without colorectal cancer identifies EVL as a potential biomarker for risk stratification.
Methylation profiling of patients with and without colorectal cancer identifies EVL as a potential biomarker for risk stratification. Figure provided by Dr. Ming Yu.

To determine the utility of EVL methylation in predicting risk of CRC, the authors undertook a specialized methylation-specific droplet digital PCR assay, a highly specific and sensitive assay that enabled them to accurately detect methylated EVL levels in normal colon mucosa. With a retrospective patient cohort from the University of Washington Medical Center, they derived three comparison groups to assess the link between EVL levels and detecting adenomas, and subsequently developing CRC: Comparison group 1 – baseline visit no adenoma detected vs. adenoma detected, Comparison group 2 – a more restricted analysis, including only patients with at least one follow-up colonoscopy (no adenoma detected vs. adenoma detected), and Comparison group 3 – the control group (no adenoma detected), who must have had at least four years of follow up with no detection of adenoma or cancer. After droplet digital PCR profiling, the authors performed univariate analyses and found that methylated EVL levels were significantly higher in those patients with adenomas in Comparison group 1. For Comparison group 2, their analysis determined an increased risk of adenoma detection with elevated EVL methylation, a finding that remained consistent in the stricter parameters of Comparison group 3. The authors also derived multivariate models adjusted for confounding factors that determined increased likelihood of adenoma detection with elevated methylated EVL, including in their multivariate models that excluded patients with cancer. Taken together, these data suggest a strong association between methylated EVL levels and adenoma detection.

Discussing the impact of their study, Dr. Yu said “the potential of methylated genes to be used as CRC risk biomarkers has not been evaluated for predicting the risk of metachronous adenomas. This is, partly, due to the lack of sensitive and precise detection methods for rare events like methylated EVL. Thanks to the recent development of sensitive and precise detection methods for methylated genes based on droplet digital PCR technology [by Drs. Yu and Grady], we are able to more accurately determine whether methylated genes can be used as cancer risk markers.”

The results from the present work “support future studies using an independent sample set with a prospective design to evaluate the potential of EVL methylation as a CRC risk biomarker, which could be used to individualize clinical colorectal cancer prevention programs,” according to Dr. Yu. Additionally, EVL methylation has potential for use as a “surrogate marker for chemoprevention studies,” she continued.  Importantly, this study would not have been possible without collaboration from ongoing research initiatives. “We feel indebted to patients and staff of GICaRes and ColoCare study teams who have provided precious samples needed to complete this study,” concluded Dr. Yu. 


This work was supported by funding from the National Cancer Institute, National Institutes of Health, Listwin Family Foundation, Cottrell Family Fund, R.A.C.E. Charities, National Institutes of Health Early Detection Research Network and the Huntsman Cancer Foundation.

Fred Hutch/University of Washington/Seattle Children's Cancer Consortium members Mary Redman, Christopher Li, Stacey Cohen, and William Grady contributed to this work.

Yu M, Carter KT, Baker KK, Redman MW, Wang T, Vickers K, Li CI, Cohen SA, Krane M, Ose J, Gigic B, Figueiredo JC, Toriola AT, Siegel EM, Shibata D, Schneider M, Ulrich CM, Dzubinski LA, Schoen RE, Grady WM. Elevated EVL methylation level in the normal colon mucosa is a potential risk biomarker for developing recurrent adenomas. Cancer Epidemiol Biomarkers Prev. 2023 Jun 9:EPI-22-1020. doi: 10.1158/1055-9965.EPI-22-1020. Epub ahead of print. PMID: 37294695.