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Hopkins Team Shows Methylation-Specific ddPCR May Help Predict Head and Neck Cancer Recurrence


NEW YORK (GenomeWeb) – Oncologists probe the margins of surgical sites to detect epigenetic indicators that can anticipate cancer recurrence. But deep surgical margin analysis with biopsy can alter the site making it challenging to return to the exact spot if there is a problem. It also takes only a few rogue cancer cells to cause a recurrence and these may be missed by histological techniques.

Researchers at Johns Hopkins University School of Medicine have now developed a method using Bio-Rad's Droplet Digital PCR platform that is amenable to molecular methods and only requires a tiny sample from the surgical margin.

Specifically, in a study published this week in Cancer Prevention Research, scientists examined an epigenetic signature of PAX5 gene methlyation previously determined to be specific to cancer, and found that it could be used to predict local cancer recurrence after tumor removal for head and neck squamous cell carcinoma, or HNSCC.

In a prospective study of 82 patients, if the tumors had methylated PAX5 then the presence of residual methylated cells in the surgical margins was a predictor of poor locoregional recurrence-free survival. And among patients on subgroup of patients who did not receive radiation treatment after surgery, the ddPCR method increased detection of the PAX5 maker from 29 percent to 71 percent.

Compared to conventional methylation analysis, the ddPCR method also reduced the number of false negatives. Importantly, the authors noted in the study that the method can be performed within three hours by one person. Thus, it might be completed before the reconstruction phase of a typical operation, allowing surgeons to resect the margin of the surgical site if methylated cells are detected.

The authors concluded that future personalized oncology workflows could also employ methylation arrays or methylation sequencing to pre-operatively define a patient's methylome and design a panel of primers and probes that could be used in intraoperative surgical margin assays.

A spokesperson at Bio-Rad noted that an increasing number of researchers are using the firm's digital PCR platform for methylation studies.

"There seems to be increasing success in applying ddPCR to measurements of methylation, as judged from an uptick in recent publications," George Karlin-Neumann, the director of scientific affairs at Bio-Rad's Digital Biology Center, told GenomeWeb in an email.

He cited another recent study that examined so-called "field cancerization," or the presence of clonally-related cells in the mucosal area surrounding a tumor that have malignant potential and carry cancer-associated genetic or epigenetic alterations.

That work, published in Epigenetics in July, looked at colorectal cancer and showed MethyLight ddPCR was able to achieve a significantly lower limit of detection than the same technique using standard PCR. The author of that study told GenomeWeb that ddPCR could help detect the one or two cells with cancerous epigenetic changes out of a field of thousands.

This increased sensitivity over conventional qPCR is "translating into better clinical sensitivity and specificity with methylation biomarkers, bringing us closer to the possibility of their clinical implementation," Karlin-Neumann said.

He further noted another recent study, published in Diabetes, which showed absolute measurements of methylated and non-methylated preproinsulin cell-free DNA in blood could provide a better association with Type 1 diabetes than ratio measures.

This result "plays to the strengths of ddPCR's ability to make absolute measurements, rather than just relative ones."

Raleigh, North Carolina-based biopharmaceutical company Islet Sciences is also using the methylation status of cell-free DNA to track pancreatic beta cell death. That firm licensed a ddPCR-based epigenetic method from a lab at Yale University, and representatives told GenomeWeb last year that Islet is working to commercialize the assay.

Viresh Patel, global marketing director at Bio-Rad's Digital Biology Center, told GenomeWeb in an email that the firm is not currently marketing methylation applications specifically, but Bio-Rad continues to work with customers on assay design, sample compatibility, and data analysis.

"This is an emerging application for ddPCR which we expect to gain momentum as researchers continue to publish their breakthrough research," Patel said.