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Trovagene's Dual-Sample Liquid Biopsy Performs Well in First Peer-Reviewed Study

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NEW YORK (GenomeWeb) – A new study this week by Trovagene and its academic collaborators is the first to demonstrate the validity of the company's liquid biopsy strategy for detecting EGFR mutations in both blood and urine of patients with lung cancer.

The paper, published online in the Journal of Thoracic Oncology this week, is an important step for Trovagene toward broadening adoption if its tests beyond what is currently a relatively small corps of early adopters, company officials told GenomeWeb.

"Currently, the majority of the customers adopting our test are [key opinion leaders] who are early adopters because they have access to clinical trials and unpublished data, so this is a game changer for us because it's the first published data showing that urine and or blood are equivalent to tissue," Trovagene vice president of marketing Rob Kelley said.

"For community oncologists who really wait for published data to start adopting these types of tests, this is the data that they really have been waiting for," he argued.

The study results demonstrate that Trovagene's Trovera EGFR assay can successfully identify EGFR mutations in both urine and blood of patients with metastatic non-small cell lung cancer with high sensitivity and with high concordance to tumor tissue.

Additionally, the data speaks to the ability of Trovagene's unique dual-sample capability to potentially offer increased sensitivity over other tests on the market or in development that look for circulating tumor DNA in blood alone.

In the study, researchers conducted a blinded analysis of matched urine and plasma specimens collected from the first 63 patients enrolled in the Phase I/II TIGER-X trial of Clovis Oncology's investigational EGFR inhibitor rociletinib.

Of the 63 patients in the study, 60 also had evaluable tissue specimens. Using the tissue results as a reference, the investigators found that the sensitivity of EGFR mutation detection in urine was 72 percent for T790M, 75 percent for L858R, and 67 percent for exon 19 deletions.

However, amongst urine samples meeting the company's recommended volume, that sensitivity rose to 93 percent for T790M, and was 71 percent for L858R and 83 percent for exon 19 deletions, suggesting that attention to this detail in clinical practice would be crucial for achieving the highest sensitivity possible.

In plasma the results were smilar: 93 percent sensitivity again for T790M, 100 percent for L858R, and 87 percent for exon 19 deletions.

According to the authors, these results, though from a relatively small sample set, hint that Trovagene's hotspot amplification plus next-gen sequencing strategy may have a bit of an advantage in sensitivity for T790M resistance mutations compared to some of the other PCR methods being advanced for single-gene liquid biopsy testing.

For example, Roche's recently US Food and Drug Administration-approved cobas EGFR Mutation Test v2 plasma test has demonstrated relatively lower sensitivity for T790 — 73 percent in one head-to-head study.

The Roche test's approval for blood-based mutation detection is specific to alterations associated with response to AstraZeneca's Tarceva (erlotinib). This does not include T790M, which is useful in identifying patients who have developed resistance to Tarceva or similar drugs, but could respond to another newer-generation EGFR therapy.

Currently, no liquid biopsy assays are FDA-approved for guiding therapy for patients with T790M mutations, and Trovagene's data, though limited, suggests it may have sensitivity advantages in this vein over other competitors.

Clovis recently ended development of rociletinib, but there remain several other third-generation EGFR TKIs in development, as well as AstraZeneca's already approved TAGRISSO (osimertinib).

According to Trovagene's Chief Scientific Officer Mark Erlander, the company is reaching out to drugmakers advancing these therapies with the message that its platform may be the more sensitive choice for non-invasive detection of T790M.

The company is also engaging with the FDA, he said. However, Trovagene isn't sure if the future holds a path for its EGFR assay as a traditional companion diagnostic in a one test-one drug model, the way the Roche' cobas test was approved alongside Tarceva.

"There are now five different companies with third-generation TKIs, even without Clovis," Erlander said. "So I don't think we are the only ones wondering, if there is going to be a companion diagnostic to only one of those then what about the other five?"

Another lingering debate around liquid biopsy approaches has been how to best understand the sensitivity of these technologies compared to tumor tissue testing, which itself may not be yielding accurate results due to sampling issues or tumor heterogeneity.

In other words, should a potentially incorrect tumor tissue test result be used as the gold standard against which to measure liquid biopsies? Or should patient outcomes be the ultimate determinate?

In Trovagene's study, urine and plasma testing together identified twelve T790M-positive cases that were undetectable by tissue. In nine of these patients who were monitored while on treatment with rociletinib, the researchers saw a concurrent rapid decrease in urine T790M levels, suggesting that they were responding to the drug.

This seems to support the reliability of blood-based positive results, even in the absence of a tissue test. However, because studies have shown clearly that liquid biopsy doesn't pick up some mutations that do appear in tissue, clinicians are reluctant to consider a blood- or urine-based negative result alone enough to deny a patient treatment.

Along those lines, Roche's blood-based cobas EGFR assay was approved with a reflex indication: If the results of the blood-based test are negative, physicians are recommended to seek out tissue testing, and in blood-negative/tissue-positive cases, the tissue should trump the liquid biopsy in terms of whether a patient is eligible for treatment.

Interestingly for Trovagene, in the TIGER-X study, although 10 of the 12 tissue-negative cases were positive for T790M in both urine and blood, two patients had a positive result in only one of these samples: one in urine only, and one in blood only, supporting an added value of the two non-invasive options, with a potential for a dual-sample testing paradigm to increase clinical sensitivity.

Erlander said that a larger investigation of the Trovagene test — in about 200 TIGER-X subjects — was presented earlier this year at the annual meeting of the American Society of Clinical Oncology. In that analysis, researchers compared EGFR testing using Trovagene's assay, Qiagen's therascreen EGFR test, and Sysmex's BEAMing technology, all of which were platforms Clovis chose to work with in its advancement of rociletinib

At ASCO, Heather Wakelee of Stanford University Medical Center reported that in a head-to-head comparison of urine, plasma, and tissue, all had comparable clinical sensitivity in the 80 percent to 82 percent range and were equally predictive of a patient response.

Importantly though, Erlander told GenomeWeb, Wakelee's analysis also revealed that combining blood and urine testing in the same patient could increase the sensitivity of detecting a T790M mutation as much as combining blood and tissue testing.

In other words, adding tissue results (as is recommended as a reflex for the Roche test, for example) would increase the sensitivity of blood-based results alone by about 12 percent, based on the TIGER-X data. But so would reflexing from blood to urine, or vice versa, Erlander said.

While this doesn't mean that tissue shouldn't still be considered in liquid biopsy-negative cases, it does suggest that a non-invasive dual-sample approach could pick out more patients who could benefit from these drugs without the need to then move to an invasive biopsy.

Urine, Trovagene believes, also offers practical advantages for serial monitoring, something the company is also studying.

Outside of the lung cancer space, Trovagene is also advancing tests for colorectal and pancreatic cancer.

In late, 2015, the company announced that it was establishing a European subsidiary, Trovagene Research Institute, charged with expanding the capabilities and adoption of the company's platform, which would be chaired by University of Torino liquid biopsy researcher Alberto Bardelli.

Erlander said that Trovagene is working with Bardelli on research in colorectal cancer, including looking at the possibility of using early detection of resistance mutations in body fluids to guide innovative therapeutic approaches, for example cycling on and off of a particular therapy or back and forth between different therapies.

The company's partnership with Bardelli is expected to yield future commercial implications for Trovagene's European offshoot, but Kelly and Erlander could not provide any details about that expected move.

Aside from that, Kelley said that Erlander is also leading research at Trovagene into the use of its test to pick up evidence of residual disease in colorectal cancer patients.

A recent study by researchers from Johns Hopkins, published in Science Translation Medicine, provided strong evidence that circulating tumor DNA can be used to identify stage II colorectal cancer patients who are highly likely to recur after a surgical tumor resection, helping to better distinguish which patients may need adjuvant chemotherapy treatment and which may avoid it.

However, the researchers in that effort failed to detect ctDNA in more than half of the patients that eventually went on to recur with the NGS approach they used.

"We think that there is a great opportunity for Trovagene to make a clinical impact in this area given the sensitivity of our assay," Erlander said.

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