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Trovagene Bets on Digital PCR as Detection Technology for Urine-Based MDx Testing Platform

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This article has been updated from a previous version to include information about the current status of Fluidigm's collaboration with Novartis.

Trovagene, a San Diego-based company that has spent several years attempting to commercialize molecular diagnostic tests based on detecting cell-free nucleic acids in urine, may have finally found its ideal detection technology: digital PCR.

The company said this week that it has successfully completed the analytical development of digital PCR assays to detect the most prevalent cancer-related KRAS mutations, and now intends to establish and validate the performance of these assays to detect such mutations in the urine of pancreatic cancer patients.

If successful, the test could potentially help doctors non-invasively diagnose and monitor pancreatic cancer progression. It could also be one of the earliest examples of the molecular diagnostic potential of digital PCR technology, which has thus far been primarily used for research applications such as rare mutation detection and copy number variation.

Trovagene was founded in 1999 and currently trades as an over-the-counter stock but is working on requirements necessary for relisting on a larger exchange, according to the company's website.

Its founding scientists were the first to report that cell-free fragments of DNA from normal cell death that circulate in the blood can cross the kidney barrier and be detected in urine.

These fragments, called transrenal DNA, or Tr-DNA, can also be used as genetic markers of disease, setting up the possibility of non-invasive molecular diagnostic tests, particularly for cancer.

Trovagene said this week that earlier published work by the company and its scientific collaborators has demonstrated that KRAS mutations — which have been implicated in more than 90 percent of pancreatic cancers and in 23 percent of solid tumors — can be more reliably detected in urine as compared to plasma or biopsy material.

The company also said that next-generation digital PCR platforms now enable the design of oncogene mutation assays that are compatible with the throughput and reliability requirements of a clinical diagnostic laboratory at acceptable cost levels.

Elaborating on this point, Trovagene CEO Antonius Schuh, who joined the company in October, told PCR Insider via e-mail that the company is exploring the use of a commercial digital PCR platform for its KRAS mutation assay, but declined to identify which commercial platform it is working with.

He also noted that the ability of digital PCR to potentially detect a mutation in a sample with a large amount of wild-type background makes it particularly attractive for picking up mutations in cell-free nucleic acids in a complex matrix like urine.

And, Schuh said, like next-generation sequencing, digital PCR has over the past few years become affordable enough that it becomes feasible for routine use in clinical labs.

There are currently only a handful of commercial digital PCR platforms on the market, and none is cleared by the US Food and Drug Administration for clinical diagnostic use.

Fluidigm was the first to launch such a platform, which combines its integrated fluidic circuit chips and BioMark HD system. The system is somewhat limited by its physical footprint and thus may not be able to detect mutations at the level of sensitivity required by a test like Trovagene's — possibly as low as one in 100,000. Fluidigm had a collaboration in place with Novartis Vaccines & Diagnostics to develop non-invasive tests for fetal aneuploidies and other genetic abnormalities in pregnant women (PCR Insider, 12/9/2010); however, after this article was originally published the company said that partnership would not move forward.

Another system whose sensitivity most closely aligns with that of Fluidigm's product is Life Tech's OpenArray platform. In addition, Life Tech offers a panel of mutation-detection assays based on its competitive allele-specific TaqMan technology for 241 of the most common mutations in 21 important cancer genes, including KRAS (PCR Insider, 4/5/2012).

Two newer entries on the market — Bio-Rad's QX100 Droplet Digital PCR system, which the company acquired last year along with biotech firm QuantaLife; and RainDance Technologies' RainDrop digital PCR system — use similar means of generating millions of individual droplet reaction volumes to achieve similar ends of sensitivities of one in 100,000 or even 1,000,000, depending on experimental conditions.

In fact, early-access users of RainDance's platform at Université Paris Descartes in France are exploring the use of Life Tech's CAST-PCR assays with the RainDrop digital PCR system to potentially detect KRAS, BRAF, and EGFR mutations from clinical samples. Preclinical work by the group has shown that the combined technologies can detect single mutant alleles in a background of 200,000 wild-type alleles.

Bio-Rad has not made much noise about the clinical diagnostic potential of the QX100 Droplet Digital platform, as it is still integrating the product and QuantaLife into its business. However, in 2010 QuantaLife was awarded a five-year grant from the National Institutes of Health to explore the use of its platform to detect methicillin-resistant Staphylococcus aureus for prevention and surveillance purposes (PCR Insider, 8/26/2010).

Whatever the case, using one of these technologies, Trovagene might be able to detect oncogene mutations from urine, which could "eventually lead to a comprehensive platform for monitoring minimal residual disease and progression of disease in oncology, and also for the early detection of cancer," Schuh noted in a statement.

The company said that it plans to enter into collaborative agreements, providing access to patient samples, with "leading endocrine oncology clinical sites" in the US by June.

In addition, in a company presentation filed with the US Securities and Exchange Commission earlier this year, Trovagene said that by the third quarter of this year it expects to establish concordance of tissue- and urine-based diagnostics, with the hopes of launching a urine-based KRAS monitoring test by the end of the year.

The company also disclosed this week that it plans to commercialize another diagnostic test to determine the presence of high-risk human papillomavirus subtypes in urine samples by amplifying the E1 region of the viral genome. The company said that the US patent application related to this test is currently pending.

In a study, Trovagene compared the analytical validity of its urine-based diagnostic to a marketed HPV test and resolved the discordant results by Sanger sequencing. The data show that Trovagene's assay had a sensitivity of 93 percent and a specificity of 96 percent, while the commercially available liquid-based cytology test had a sensitivity of 78 percent and a specificity of 86 percent.

Trovagene said that the test would be simple to perform, requiring only one PCR reaction to identify all high-risk HPV subtypes; and that it would be applicable to all specimen types, including cervical swabs.