Methylation Patterns Are New Target for ctDNA Analysis

Tests that measure circulating DNA are not entirely new. Yet, until now such tests have been unable to determine the tissue of origin (other than in cases of noninvasive prenatal testing, in which DNA can be determined to be from fetal or maternal origin).

But, two recent studies highlight the potential for assessment of methylation patterns. One study found a common methylation signature across cancers and another study found that methylation patterns can trace circulating DNA back to the tissue of origin. In both cases, these discoveries may make noninvasive diagnostics possible for detection of a wide number of conditions.

Methylation Patterns May Lead to Pan-Cancer Test
Hypermethylation of the ZNF154 CpG island is common across tumors and may have utility as a generalizable marker for circulating tumor DNA (ctDNA), according to a study published in the March issue of the Journal of Molecular Diagnostics.

DNA methylation has been known to control gene expression. Previously, researchers at the National Human Genome Research Institute (NHGRI) identified pan-cancer hypermethylation at the ZNF154 CpG island in 15 solid epithelial tumor types from 13 different organs. In the present study they measured the magnitude and pattern of differential methylation of this region across colon, lung, breast, stomach, and endometrial tumor samples using next-generation bisulfite amplicon sequencing.

The authors previously validated the marker on an Illumina methylation array. But, the authors say bisulfite amplicon sequencing holds the advantage of being time efficient and cost-effective due to the ability to conduct multi-sample sequencing in parallel. Additionally, the approach offers greater resolution of a target region than array methods, showing patterns of methylation.

The researchers confirmed that the ZNF154 amplicon region was significantly hypermethylated in all of these tumor types (n=184 samples), compared to normal samples (n=34). The marker performed best for endometrial and colon tumors. The hypermethylation occurred regardless of subtype, stage of differentiation, age, or sex. In a computational simulation to predict a threshold of ctDNA detection, limited amounts of ctDNA (1 percent tumor to 99 percent normal) yielded areas under the curve of up to 0.79 for detection of cancer-related methylation markers, and led the authors to conclude that the ZNF154 amplicon can distinguish ctDNA in a blood-based test.

“We have laid the groundwork for developing a diagnostic test, which offers the hope of catching cancer earlier and dramatically improving the survival rate of people with many types of cancer,” senior author Laura Elnitski, Ph.D., from NHGRI, said in a statement.

Methylation May Identify Multiple Diseases
Tissue-specific methylation patterns in circulating DNA that is released by dying cells can implicate the tissue source involved in cell death, according to a proof-of-concept study published March 14 in Proceedings of the National Academy of Sciences. A blood test, based on methylation, can detect multiple pathologies, including diabetes, cancer, traumatic injury and neurodegeneration.

“In the long run, we envision a new type of blood test aimed at the sensitive detection of tissue damage, even without a-priori suspicion of disease in a specific organ,” said co-lead author Benjamin Glaser, from Hadassah Medical Center in Israel. “We believe that such a tool will have broad utility in diagnostic medicine and in the study of human biology.”

Since the DNA sequence is identical between all normal cells in a body, it has not been possible to determine the tissue of origin of circulating DNA. But DNA contains methylation modifications that are unique to each cell type and are stable in both healthy and disease conditions.

The researchers identified tissue-specific DNA methylation markers and developed a method for sensitive detection of these markers in plasma or serum. They isolated cfDNA from plasma or serum of donors, treated the cfDNA with bisulfite, PCR amplified the cfDNA, and sequenced it to quantify cfDNA carrying the methylation markers of the cell type of interest. They then demonstrated the utility of the method for identification of pancreatic β-cell death in type 1 diabetes, oligodendrocyte death in relapsing multiple sclerosis, neuronal/glial cell death in patients after traumatic or ischemic brain damage, and exocrine pancreas cell death in pancreatic cancer or pancreatitis.

“Methylation patterns are unique to each cell type, are conserved among cells of the same type in the same individual and among individuals, and are highly stable under physiologic or pathologic conditions,” the authors write. “Therefore, it is possible to use the DNA methylation pattern of cfDNA to determine its tissue of origin and hence to infer cell death in the source organ.”

Takeaway: While it has long been understood that dying cells release DNA fragments into the blood and that methylation patterns are tissue-specific, emerging evidence shows that it is possible to use these methylation patterns to screen for and diagnose a variety of diseases.


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