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Methylation Profile Could Potentially Serve as Biomarker to Stratify Breast Cancer Patients

NEW YORK (GenomeWeb) – Methylation markers might be useful as biomarkers to predict the likelihood of disease recurrence in patients who have been treated for breast cancer, according to researchers at the Translational Genomics Research Institute in Phoenix, Arizona.

In a study published this month in Clinical Epigenetics, the researchers found 21 hotspots of methylation differences in circulating tumor DNA that correlated with disease recurrence. 

"This signature is a potential blood-based biomarker that could be advantageous at the time of surgery and/or after the completion of chemotherapy to indicate patients with micrometastatic disease who are at a high risk of recurrence and who could benefit from additional therapy," the authors wrote.

The TGen team performed whole-genome bisulfite sequencing on plasma-derived circulating DNA from 40 women with metastatic breast cancer, 40 women who had previously been treated for breast cancer and were disease-free, and 40 healthy controls, analyzing pooled samples from each cohort on the Illumina HiSeq 2500.

Of the women with metastatic disease, 23 had metastases in the bone, 12 in the liver, three in the brain, 17 in the lung, and six in soft tissue. All but five women had metastases in more than one site. Both the cohort of women with metastasis and the disease-free survivors had similar Her2-receptor and hormone-receptor status and had gone through similar therapy regimens.

After sequencing, the researchers focused their analyses on CpG sites, comparing the methylation profiles between the cohorts. They found that the overall methylation profile of the healthy cohort closely resembled that of the disease-free survivors, while methylation in the metastatic disease cohort "varied dramatically" from both the healthy group and the disease-free survival group.

The majority of CpG sites in the healthy and disease-free survival groups were methylated, whereas the metastatic disease group had much lower levels of methylation.

After looking at broad patterns, the researchers next analyzed methylation status at the single base pair level. The number of differentially methylated loci between the healthy and disease-free groups was relatively small, at 88,192 loci. By contrast, there were about 5 million and 6.3 million differentially methylated sites when comparing the metastatic disease cohort to the disease-free and healthy cohorts, respectively. In addition, around 90 percent of those sites were hypomethylated in the metastatic group, while 9 percent were hypermethylated.

To better understand the biological impact of these differentially methylated loci, the researchers looked at the genomic context of the sites. Around 70 percent of the hypermethylated sites in the metastatic group occurred in CpG islands, with significant levels of hypermethylation also seen in untranslated regions and transcriptional start site regions. Gene bodies were predominantly hypomethylated.

The researchers then focused on the hypermethylated CpG islands "because they tend to be focal in nature and were identified as the regions that differed most dramatically from normal or disease-free patterns," the authors wrote. Looking specifically for CpG islands with eight or more hypermethylated loci with differential methylation values of 50 or more, the team identified 21 CpG island hotspots on 21 different genes.

Of the 21 genes, DNA methylation alterations in six had not previously been reported in cancer. Hypermethylation in four of the genes had previously been linked to breast cancer, while seven have previously been associated specifically with invasion and metastasis. 

The researchers noted that because of the high cost of performing whole-genome bisulfite sequencing, they pooled samples within each cohort. As such, their results should be validated on individual samples and in larger cohorts. Going forward, a good strategy would be to perform targeted bisulfite amplicon sequencing of individual samples and in samples from early stage breast cancer in order "to further validate the predictive power of this signature" and "further help define its association to varying breast cancer subtypes," they wrote.