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Trovagene Launches Noninvasive NGS-based Test to Monitor KRAS Mutations

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Trovagene has launched a noninvasive next-gen sequencing-based test that assesses seven known mutations in the KRAS gene. The test analyzes cell-free DNA from patients' urine and uses a proprietary enrichment technique for detecting mutations with frequencies as low as between .03 and .05 percent.

The test is the firm's first commercial entry to the clinical next-gen sequencing market, and CEO Toni Schuh told Clinical Sequencing News that the company will continue developing NGS-based tests to monitor known cancer mutations in urine, and plans to expand from single-gene to multi-gene panels.

During 2014, Trovagene will "roll out a portfolio of [NGS-based] tests that target mutations known to have predictive qualities either of [drug] response or resistance," Schuh said.

The test runs on Illumina's MiSeq platform and is offered as a laboratory-developed test from Trovagene's CLIA-certified and CAP-accredited laboratory in San Diego. The firm also plans to publish the results of a clinical validation study of the test in the third quarter of this year. It has not yet set a price for the test.

Schuh said that the company is also considering developing a kit to bring through US Food and Drug Administration clearance as an in vitro diagnostic, although he did not specify a time frame for that.

Last year, the firm launched two PCR-based tests — a human papillomavirus test and a cell-free BRAF v600E mutation detection test — both of which assess nucleic acids from urine samples.

In January, Trovagene announced a partnership with US Oncology Research to assess cell-free DNA from urine using next-gen sequencing to determine whether the technique can be used to monitor KRAS mutations in pancreatic cancer patients and predict tumor burden and therapy response.

The company decided to move from PCR into next-gen sequencing because of its ability to analyze "multiple mutations in a multiplexed manner," Schuh said. In cases where the sample is limited or you are looking at rare events, doing separate PCR reactions to look for the presence of multiple mutations wastes sample.

Schuh said that key to the company's test is its ability to generate "highly enriched, very focused sequencing libraries that rely on a very specific pre-amplification of the mutated region."

Trovagene's pre-amplification assays are composed of short primers with synthetic tails that immediately flank the mutation of interest, so that the fragments of cfDNA with those mutations are preferentially amplified. This proprietary pre-amplification step is what enables the company to detect low-frequency events.

One challenge with using next-gen sequencing, said Schuh, is that it "does not really reach the required limits of detection."

For mutations below around 5 percent frequency, next-gen sequencing begins to lose sensitivity. Currently, KRAS detection assays that assess biopsy samples have limits of detection in the "lower single digit percentage range," Schuh said, which is "absolutely not good enough to monitor a patient."

By contrast, Trovagene's KRAS test has a lower limit of detection of three to five mutations per 10,000 KRAS genes, he said. "From the clinical performance that we have seen with this to date, that is sufficient to determine for example, whether a patient is responding to treatment, or, after going through a phase of stable disease, begins to progress again."

Schuh said that the seven-mutation KRAS test was a first step in the company's move toward developing larger NGS-based panels that assess cancer mutations from cfDNA in urine. The seven KRAS mutations were chosen because they comprise more than 99 percent of the observed KRAS mutations, Schuh said.

The initial key focus with the next-gen sequencing platform was to obtain the acquired sensitivity, the .03 to .05 percent limit of detection, Schuh said. "We believe that's quite comfortable performance for a test. Now, the objective is, how can I expand to broader and broader panels?" he said.

Moving forward, he said the company is planning tests that will include genes such as EGFR, HER2, PIK3CA and others that are known to impact treatment response or resistance.

He added that mutation testing in cancer patients using cfDNA from urine as a specimen is "going to become a standard of care for cancer monitoring."

Analyzing cancer mutations from urine is preferable to biopsy samples, which can be difficult to obtain depending on where in the body the tumor is located, he said. Additionally, using urine as a specimen enables a patient to be evaluated over time.

This type of testing can have both clinical benefits for the patient as well as cost benefits, he said, particularly if it enables a clinician to determine early on that a patient is not responding to a particular targeted therapy. "Oncologists are working with highly targeted therapies, and these are expensive," Schuh said. Analyzing cfDNA from the urine could enable the oncologist to make an earlier determination about whether or not a drug is effective, both saving the patient money and enabling the physician to make clinical management decisions earlier.

Trovagene is not the only company to see the benefits of sequencing for noninvasive monitoring of cancer. Startup Guardant Health is developing an NGS-based test that assesses 54 cancer-related genes from cell-free tumor DNA circulating in a patient's blood.

Earlier this year, Johns Hopkins spinout Personal Genome Diagnostics licensed technology from Johns Hopkins for analyzing cell-free tumor DNA circulating in blood. The firm currently offers ctDNA analysis for research applications only.

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