cfDNA Methylation Assay Allows for Early Lung Cancer Detection


February 1, 2021 — Targeted circulating tumor DNA methylation marker panels possess the potential for early blood-based detection of lung cancer with a high sensitivity and specificity.

Targeted cell-free DNA (ctDNA) methylation marker panels possess the potential for early blood-based detection of lung cancer with a high sensitivity and specificity, according to data from a poster presentation delivered during the International Association for the Study of Lung Cancer 2020 World Conference on Lung Cancer.1

Results showed that the 10-methylation marker panel showcased an overall sensitivity of 73% at a specificity of 90%. Moreover, the panel exhibited a stage I sensitivity of 73%, with good sensitivity for the detection of main histological disease subtypes.

Additionally, Pearson’s correlation test panel prediction was not found to be correlated with main clinical or physiological features (P >.05), such as sex (P = .70), smoking history (P = .51), histology (P = .64), and age (P = .32). Certain single markers were found to have significant discriminative power (P <.05). 

“Methylation panel performance is not connected to the smoking history of the patient,” Kristi Kruusmaa, of Universal Diagnostic SL, in Spain, stated in the poster. “This method could serve as the basis for further development of a highly accurate and minimally invasive blood-based screening test for a wider population.”

Patients with lung cancer whose disease has spread to distant sites have a survival rate of 6% at 5 years, while those with localized disease have a 5-year survival rate of 59%. “Early detection saves lives,” stressed Kruusmaa, who added that current screening approaches often have low accuracy. Additionally, screening is currently only being done for patients who are high risk, according to Kruusmaa, while the number of low-risk cases continues to climb.

Epigenetic mechanisms can be impacted by several factors and processes, including development, environmental chemicals, drugs, aging, and diet. When methyl groups tag DNA and trigger or inhibit genes, DNA methylation occurs.2 DNA is able to wind around histones for both compaction and gene regulation. When epigenetic factors bind to histone “tails,” histone modification occurs; this process impacts the extensiveness of DNA wrapping around histones and the accessibility of genes in the DNA that can be triggered. All these factors and processes possess the power to impact a patient’s health.

DNA methylation may be an “ideal” biomarker, as it can serve as an early indicator for cancerogenesis, according to Kruusmaa.

The analysis presented during the meeting included a total of 108 patients who attended a general check-up or oncology units in Spain and the United States. The median age of patients in the lung cancer cohort was 59 years versus 50 years in the control cohort; 25% versus 35%, respectively, were male.

In the lung cancer cohort, 11 patients had stage I disease, 4 had stage II disease, and 7 had stage III disease. Additionally, 11 had adenocarcinoma, 11 had squamous cell carcinoma, 9 had small cell lung cancer (SCLC), and 6 had a different histology. Regarding smoking history, 14 patients in the lung cancer cohort were current or ex-smokers, 13 were never smokers, and 10 were unknown.

To perform the analysis, investigators collected 8 mL of plasma from the 108 patients. To be eligible for inclusion, patients with lung cancer had to have a confirmed diagnosis with histology and stage information included. The control cohort was comprised of asymptomatic patients with no known cancer diagnoses or history.

For the initial biomarker selection, investigators utilized Human Methylation 450K data collected from The Cancer Genome Atlas consortium.3 To target regions of interest within the plasma cfDNA, investigators used a methylation-sensitive restriction enzyme–quantitative polymerase chain reaction strategy. For additional analysis, delta cycle threshold was utilized. 

Moreover, to build and test the marker panel, investigators used a random forest feature selection algorithm that comprised Monte-Carlo cross-validation of over 50 sub-settings. To measure the accuracy of the panel, investigators looked at the fraction of correct calls.

Additional results revealed that the 10-methylation marker panel had stage II sensitivity of 75% and stage III sensitivity of 77%. The panel had an 82% sensitivity for the adenocarcinoma subtype, an 80% sensitivity for squamous cell histology, a 73% sensitivity for SCLC, and a 40% sensitivity for other histologies.

“The biological relevance of the 10 markers were checked,” concluded Kruusmaa. “For example, THBD promotes angiogenesis by enhancing cell adhesion, migration, and FAK activation through interaction with fibronectin.”


  1. Kruusmaa K. Cell-free DNA (cfDNA) methylation assay allows for early detection and identification of lung cancer. Presented at: International Association for the Study of Lung Cancer 2020 World Conference on Lung Cancer; January 28-31, 2021; Virtual. Poster P46.06.
  2. A scientific illustration of how epigenetic mechanisms can affect health. National Institutes of Health. March 5, 2018. Accessed February 1, 2021.
  3. The Cancer Genome Atlas Program. National Cancer Institute. Accessed February 1, 2021.
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