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Yale Team Developing Novel ctDNA Sequencing Method for Early Cancer Detection

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NEW YORK (GenomeWeb) — Researchers from Yale University are working to advance their novel method for targeted sequencing of circulating tumor DNA as a tool for early detection and diagnosis of cancer.

The researchers published an initial description of their sequencing strategy, called error-suppressed multiplexed deep sequencing, in Cancer Research in 2012. Last month, the group shared two studies at the annual meeting of the American Society of Clinical Oncology validating their initial findings.

Following on these successful studies, the team is now preparing a larger analysis of samples collected over the last several years, including early-stage cancers, to develop the method as an early detection tool, the group's leader Abhijit Patel told Clinical Sequencing News.

"One of the nice things about our assay is that it's very sensitive and it's designed to be able to assess mutations without prior knowledge, which would be important in early detection and screening, since you don't know what you are looking for," Patel said.

"The specificity of ctDNA is also very high," Patel explained, "so it offers some unique advantages compared to protein biomarkers. I always tell people, proteins have a [significant] physiological background, so to detect a small early-stage tumor you have to be well above that background to make that call."

"But with ctDNA," he said. "There is no physiologic background so if you can decrease the technical background of your assay low enough you have a really powerful method for screening patients."

Patel and his colleagues have since been focused on this task, decreasing technological background, and reducing errors from sequencing or PCR amplification, as well as running the method through its paces using clinical samples.

Since the team's initial paper in 2012, Patel said it has been working to expand the panel of genes that the assay can cover.

"We realized that what we had developed initially was not so scalable … in practice it would require complex microfluidics and complex automation to expand it," Patel said. "So we went back to the drawing board … and basically … reengineered the system to be able to multiplex more genes by upfront barcoding."

He said the group has also come up with other ways to suppress errors from sequencing and PCR. "The combination has allowed us to push the limits of sensitivity further and also expand the range of what we can cover in terms of mutation coverage," he explained.

In their 2012 Cancer Research paper, Patel and his colleagues reported that by using layers of sequence redundancy designed to distinguish true mutations from sequencer misreads and PCR misincorporations, they could achieve a detection sensitivity of approximately one variant in 5,000 molecules. And by attaching modular barcode tags, they enabled simultaneous analysis of more than 100 patient samples, the authors wrote.

In their presentations at ASCO last month, the researchers shared data from two studies using the approach. In one, they tested 75 plasma samples from 17 patients with gastric cancers for the presence of cancer-associated mutations.

Among the eight patients whose tumors were profiled by an independent clinical lab, the team's plasma results "showed perfect concordance of mutation status and type," the authors wrote. Testing of serial samples revealed that ctDNA levels "changed appropriately with therapy or with disease progression," the group reported, including one case in which ctDNA levels dramatically decreased after initiation of first-line chemotherapy, a second case in which they rose upon development of liver metastases, and a third case in which rising levels reversed upon switching to a different chemotherapeutic regimen.

In a second study, the team looked at single and serial plasma samples from 52 lung adenocarcinoma patients with known activating EGFR mutations, and found that the ctDNA sequencing approach showed a high concordance with results from tumor biopsy.

While the implications of these studies speak mostly to the method's potential for tumor mutation status assessment, prognostic testing, or therapeutic monitoring, the team's main intention is to show that the approach could work for early cancer detection, Patel said.

"If you're just interested in assessing whether a patient is developing resistance to erlotinib and you just want to look for the T790M mutation you don't need to be broad with your assay," Patel said, so other simpler and cheaper approaches using limited panels or nucleic acid amplification may be a better choice.

But for early detection, "other approaches to circulating tumor DNA tend to be limited in the mutation coverage they can achieve," he added. "For example with something like droplet digital PCR you have to have specific probes for specific mutations. You can't query broadly and cover large regions," Patel said.

"Or with other [sequencing] approaches you can query very broadly but then the detection sensitivity is not as good," he added. "It's a very challenging problem, so I think there is a ways to go. But we've already been looking at [the ability of our method] by examining samples from early stage patients with lung cancer and other malignancies like ovarian cancer, and so far we've got some good preliminary results."

The next step will be for the team to expand its study to a much larger and broader set of retrospective samples, which have been collected and stored as the sequencing approach was being refined over the last few years, said Patel.

If things go well, he said the group hopes to be able to commercialize an early detection assay for different malignancies using error-suppressed multiplex ctDNA sequencing.

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