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Chronix Team Publishes Validation Study of NGS-based Liquid Biopsy Test


NEW YORK (GenomeWeb) – Chronix Biomedical has published a clinical validation study of a next-generation sequencing-based test that analyzes circulating cell-free DNA to gauge whether the patient is at high risk for prostate cancer and should go on to receive a biopsy.

The company is now offering the test out of its clinical laboratory in Göttingen, Germany and plans to launch it in the UK, other European countries, and the US.

Chronix CEO Howard Urnovitz told GenomeWeb that the test is aimed at patients whose prostate specific antigen levels are "in the gray zone," which he defined as a PSA level of between 4 ng/ml and 30 ng/ml, and is designed to serve as a supplementary PSA test. The goal is to provide a clearer guide of who is at high risk for developing prostate cancer and should receive further testing, reducing the numbers of unnecessary biopsies.

"That's the market we're laser focused on now," Urnovitz said. Currently, PSA testing has a false positive rate of nearly 75 percent, he said, leading to many unnecessary, invasive, and costly biopsies. While very high levels of PSA are typically indicative of prostate cancer, most men have PSA levels in the intermediate zone, Urnovitz said. Offering a noninvasive NGS-based test for those patients should help clarify who should go on to receive biopsies. And at a price of $600, Urnovitz said that the test should help reduce overall healthcare costs.

In its validation study, which was published this month in Clinical Chemistry, Chronix collaborated with researchers at Vanderbilt University. They ran the test in more than 400 patients, including 204 patients with confirmed prostate cancer and 207 controls. In addition, they evaluated 20 patients with either benign prostate hypertrophy or prostatitis, conditions known to elicit a false positive result with standard PSA testing.

To design their test, the researchers first performed whole-genome amplification on the cell-free DNA using random primers tagged with molecular identifiers. Next, they did whole-genome sequencing and aligned the reads to the human genome. Sequence reads were evaluated in 100-kbp bins and chromosomal variations were evaluated for their ability to distinguish patients from matched controls.

The Chronix researchers first used a subset of 82 prostate cancer samples and 95 controls to determine which copy number variants differed between the two sets and could be used as markers of genomic instability for the test.

For each sample, they generated a copy number instability score, defined by the number of bins that had higher- or lower-than-expected read counts, which could correlate with a CNV. After several rounds of testing and validation the group identified 20 regions on which to validate its final test.

For a commercial test, Chronix will use a targeted approach, and the firm holds a patent on its hybridization method for detecting disease-specific cell-free nucleic acids.

The team used ROC curves to assess diagnostic performance, showing that its test could discriminate prostate cancer from controls with an area under the curve of .92, for a diagnostic accuracy of 83 percent. The test performed slightly better when the control set was limited to patients with a Gleason score of less than 7, and was good at distinguishing prostate cancer cases from both benign prostatitic hypertrophy and prostatitis, demonstrating an accuracy of 90 percent.

The Chronix test is "a heck of a lot better than PSA testing," Bill Mitchell, a professor of pathology, microbiology and immunology at Vanderbilt University and corresponding author of the paper, told GenomeWeb. "The initial thing this can be used for is as a screening assay for positive PSA tests or a PSA test in an intermediate zone."

Urnovitz noted that the test's accuracy is not high enough to serve as a definitive diagnostic test, which is one reason why Chronix is positioning it as either a confirmatory or supplementary test to PSA testing for patients with intermediate scores. Urnovitz said the company is also working on improving the test's accuracy and diagnostic capabilities. "That's on our radar," he said, "but won't be the first application."

In the study, the Chronix team used Thermo Fisher's SOLiD platform to do the sequencing, but Urnovitz said that the test would be launched commercially on a different, undisclosed platform.

Urnovitz noted that Chronix is also considering partnering with other companies in order to commercialize the test in the US and he said he would like to eventually bring the test through US Food and Drug Administration clearance.

"We're developing our US commercialization strategy over the next three to six months," he said.

In 2011, Myriad licensed technology from Chronix, but returned the licenses in 2012. Urnovitz said that Myriad had been considering the technology as a screening test, rather than as a PSA supplemental test.

Chronix also has a similar test in the works for breast cancer, which Urnovitz said would be its next product. At the 2013 American Society for Clinical Oncology meeting, Chronix researchers presented a poster that showed that NGS of circulating cell-free DNA could monitor breast cancer patients for minimal residual disease following surgery.

Urnovitz said that the company plans to publish the results of its breast cancer test. Similar to its prostate cancer test, Chronix plans to position its breast cancer test as a cost-effective way to determine which patients who have received an indeterminate mammogram result should go on to receive a biopsy. 

Chronix is one of several companies looking to tap into the so-called liquid biopsy space. Sequenta currently markets its ClonoSight test for MRD monitoring in blood-based cancers; Guardant Health has a sequencing-based test that analyzes circulating cell-free DNA to monitor cancer; Personal Genome Diagnostics this week launched a test called Plasma Select-R, a targeted sequencing-based test for circulating tumor DNA that is based on its proprietary PARE method; and Trovagene markets a KRAS test that analyzes cell-free DNA in urine, to name a few.

Urnovitz said that Chronix aims to differentiate itself from these potential competitors by offering its test at a low price point, something it is able to do because it is targeted and evaluates copy numbers, which doesn't require as much sequencing depth as looking at point mutations. In addition, he said that the company is focused initially on a very specific market to which it can add value and cost savings — the indeterminate risk market. Rather than try and replace the existing reimbursable PSA test, Urnovitz said Chronix's test will supplement it in cases where it is needed to avoid unnecessary biopsies, which he said cost an estimated $12 billion annually.

Vanderbilt's Mitchell added that NGS-based testing of circulating tumor DNA holds great clinical potential not just in prostate cancer, but likely many other areas. He said that in collaboration with Chronix, Vanderbilt researchers have shown the potential for such testing in breast and head and neck cancers.

"We now know, from numerous studies, that what you see in [the tumor DNA] in plasma or serum, recapitulates what the tumor genome actually is," Mitchell said. "That has enormous ramifications and I think in the next two to five years, we'll know what can be done and what the limitations are."