DNA Methylation Tests May Be the Next Game Changer in Low Cost, Noninvasive Cancer Testing

As liquid biopsies that analyze cell-free DNA (cfDNA) continue to gain traction in oncology, several companies are stepping up efforts to develop other noninvasive early cancer detection tests using similar biomarkers. One of the most promising approaches are tests that can detect abnormal DNA methylation associated with early-stage cancers. Here are some of the DNA methylation techniques in the pipeline based on the presentation of companies at the American Society of Clinical Oncology’s (ASCO) virtual annual meeting in early June.

The Diagnostic Promise

Blood plasma contains cfDNA composed of fragmented DNA released by cells into the circulation, typically after the cell dies. If a patients have cancer, their plasma will include cfDNA released by tumor cells, aka, circulating tumor DNA (ctDNA). Methylation analysis involves chemically treating cfDNA to convert unmethylated cystosine to uracil and identifying changes in next generation sequencing.

Methylation patterns of ctDNA are consistent with the cells or tissues from whence they originate. Accordingly, ctDNA is a biomarker exhibiting cancer-specific and epigenetic aberrations which can be used as a surrogate source of tumor DNA in cancer diagnosis. And because methylation happens early in cancer development, ctDNA methylation patterns can be used to detect early-stage cancer.

Bluestar Genomics

During the ASCO meeting, San Diego-based Bluestar Genomics new data on a liquid biopsy assay it is developing for breast, colorectal, lung, ovarian and pancreatic cancer by tracking 5-hydroxymethylcytosine (5hmC) DNA modifications in a blood sample. Bluestar researchers isolated DNA from 176 fresh frozen tissues from stage I to IV cancer patients and collected cfDNA from the plasma of 783 non-cancer controls and 567 cancer patients. It then enriched for 5hmC using chemical labeling and sequenced the samples in normal and tumor tissues. After aligning the results to a reference genome to build feature sets of 5hmC patterns, the researchers identified specific and discrete gene-based features for both tumor and normal tissue. Finally, they applied a machine learning algorithm to the cfDNA dataset to spot a signature allowing for classification between non-cancer and cancer patients.

On a cancer-specific level with a specificity of 99 percent, the assay had a sensitivity of:

  • 30 percent for breast cancer;
  • 43 percent for colorectal cancer;
  • 52 percent for lung cancer
  • 57 percent for pancreatic cancer; and
  • 75 percent for ovarian cancer.

Based on these results, Bluestar believes that the combined epigenomic and genomic profiles can distinguish between cancer and normal tissues and between different cancer types in asymptomatic high-risk individuals.

Avida Biomed

Fremont, California-based Avida Biomed, which has already developed a targeted methylation sequencing, or TMS, dual analysis assay called Point-N-Seq, presented data validation data for an updated version of the assay that incorporates both genomic and epigenomic analysis without sample splitting. The Avida research team used spike-in titrations of cancer cell line genomic DNA with known mutations and methylation profiles and achieved a detection level down to .003 percent of tumor DNA.

First, the team integrated a colorectal adenocarcinoma TMS panel covering 560 methylation markers with a mutation panel with more than 350 hotspot mutations in 22 genes into the assay. Using plasma from a cohort of colorectal cancer patients, it detected cancer-specific methylation signals and oncogenic mutations in all samples.

In a cohort of stage I/II CRC patients, the team compared tumor-informed, personalized-mutation panels (100 SNVs) and tumor-independent colorectal cancer methylation panels for each patient. Result: The integrated assay achieved similar detection as the personalized tumor-informed approach, with an Area Under the Curve (AUC) factor of 0.91. In the pilot study performed on plasma of cancer patients at different stages, at 91 percent specificity, the assay had sensitivity of:

  • 70 percent for stage I (75 patients);
  • 94 percent for stage II (46 patients);
  • 96 percent for stage III (24 patients); and
  • 96 percent for stage IV (23 patients).

Based on these results, Avida believes the new TMS dual assay will have clinical and research application for early cancer detection, colorectal minimal residual disease (MRD) detection and patient monitoring.

  1. Guardant Health

Guardant Health, which is challenging Exact Sciences for share of the early colorectal cancer and MRD detection market (See DTET, March 21, 2021), presented data on LUNAR-2, its developmental blood-based colorectal neoplasia diagnostic assay that assesses somatic mutations and methylation and fragmentation patterns in ctDNA to improve sensitivity for early-stage colorectal cancer detection. Guardant researcher used the LUNAR-2 assay to analyze plasma samples from 434 colorectal cancer patients with CRC prior to resection and 271 aged-matched controls. Training the assay on a separate 614-sample set, the Redwood, Ca., firm found:

  • A 94 percent overall colorectal cancer specificity;
  • An 88 percent sensitivity in stage I-II patients; and
  • A 93 percent sensitivity in stage III patients.

There were no differences in sensitivity between asymptomatic and symptomatic colorectal cancer cohorts; higher cfDNA tumor fractions were found in the latter.

Guardant says it expects LUNAR-2 to achieve “meaningful performance in an average risk screening population.” To prove its hypothesis, the firm has launched a prospective registration study to evaluate the assay in an average colorectal cancer risk screening cohort.


Liquid biopsy was a significant step forward for cancer treatment and the diagnostics industry. Now firms in that space are doubling down on developing new cfDNA-based technologies to further advance noninvasive early cancer detection. Tests using DNA methylation to detect ctDNA associated with specific tumors appears to be an extremely promising and exciting path forward.


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