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Advanced Cell Diagnostics Preps HPV Assay for Launch, Seeks Entry into Clinical Dx Market

NEW YORK (GenomeWeb) – After gaining traction in the research market, Advanced Cell Diagnostics is poised to bring its RNA detection technology to the clinical space.

For the past three years, the Hayward, Calif.-based firm has made its technology called RNAscope available to the research-use only market. Based on the reception the technology has received and continued improvements to it, ACD now plans to plans to pursue CE marking then enter the clinical diagnostic space with a small-scale launch of an assay for human papillomavirus and head-and-neck cancer by the end of the year.

Since launching RNAscope, ACD has signed on about 800 accounts in the pharmaceutical space, which has used RNAscope for new drug discovery purposes, and with academic/government researchers, who have used the technology for translational research, stem cell research, and infectious disease studies.

In August, the company received ISO 13485:2003 certification, setting the table for its diagnostics ambitions. In addition to the HPV test, ACD is developing a test for B-cell lymphoma and proliferative diseases.

In approaching the clinical diagnostic space, ACD is taking a three-pronged strategy, Co-founder and CEO Yuling Luo told GenomeWeb Daily News recently.
First, the company is developing its own tests, such as the HPV and B-cell lymphoma assays. Secondly, ACD will partner with pharma and other companies to develop companion diagnostic tests. And lastly, it plans to license the RNAscope technology to customers who want to develop their own assays.

For now, the company is focused on preparing for the launch of the HPV assay. RNAscope assays are designed to be platform-agnostic, and ACD has deals with Roche's Ventana and Danaher's Leica Biosystems to co-market its tests on their automated in situ hybridization platforms. However, the HPV assay will be initially made available in a manual format as that is the quickest way to market, Luo said.

While others have tried to develop RNA-based diagnostics in the past, "no robust RNA ISH technology [has been available] until RNAscope came along," he told GWDN. "We have always set our sight on the diagnostic market, but there is a strong demand for our technology in the research market, as well. Like PCR, going into the research market first allowed [the] natural evolution of a novel technology."

Having achieved that, "we are at the stage where the technology is ready to move into diagnostics," Luo said.

Solving the heterogeneity puzzle

Luo started developing the RNAscope technology while he was at Panomics, a company that he co-founded and which Affymetrix acquired in 2008. The work he did developing RNAscope was independent of Panomics, though it had access to the technology. As a result of its Panomics buy, Affy has a non-exclusive license to the early version of the technology, but not the current iteration of it.

The goal in developing RNAscope was to create a technology that could address on the molecular level intra-tumor heterogeneity, an underlying factor in many instances of drug resistance in cancer patients and overall drug ineffectiveness, Luo explained.

RNA in situ detection technology has been around for about 40 years, but its performance has been disappointing, Luo said, because development efforts have focused on amplifying the signal.

"The fundamental problem is that the probe that [binds] to your target gene also has a propensity to bind with non-specific sequences because you have billions of bases out there," he said. "So when you amplify [your] signal, you basically also amplify the background."

RNAscope seeks to solve this through the use of independent oligonucleotides, which are placed next to each other and bind to the same target sequence, resulting in the amplification of the signal but not the background.

"As you can imagine, the chances of two independent oligos [binding] to a non-specific target and being next to each other … is close to zero," Luo said, adding this design results in practically no background noise.

The oligos are generic, being 20 to 25 bases in length, but ACD has developed an algorithm that determines suitable sequences for the assay while ensuring that hybridization to non-target sequences don't occur. "They don't get into high [guanine-cytosine] content or low [guanine-cytosine] content," Luo said.

Meanwhile, the other parts of the probe will bind to the signal amplifier.

The technology works for any gene and species, and can be used on formalin-fixed, paraffin-embedded tissue, a tissue type routinely used in clinical applications. As it prepared to move RNAscope to clinical applications, ACD has been further developing and improving its performance for FFPE.

ACD's clinical ambitions for the technology are driven by what Luo said are unmet needs. Specifically, while there are HPV tests already on the market, he said that most are for screening, while ACD is developing its assay to detect the two gold standard biomarkers, E6 and E7 mRNA, for transcriptionally active HPV.

Unlike ACD's planned test, "[t]he HPV screening tests out there use cytology specimens and are not designed to diagnose a specific lesion," Luo said.

In a study published earlier this year in PLoS One, Luo, along with collaborators from the University of Vermont and Fletcher Allen Healthcare were able to correlate the results of the RNAScope HPV chromogenic in situ hybridization assay with pathological diagnoses, in particular the assay's ability to distinguish cervical intraepithelial neoplasia (CIN) grade 2 and 3.

CIN, the development of squamous cells on the cervix's surface which then may progress to cancer, is categorized into three groups — CIN 1 considered a mild stage of dysplasia and CIN 3 considered severe dysplasia to carcinoma in situ.

Luo also noted that its HPV test would have use as a companion diagnostic. There are two distinct classes of patients in head-and-neck cancer with different prognoses, and clinical trials are currently being conducted by pharma to determine whether the different classes may need to be treated differently.

Once ACD's test is launched as a prognostic assay, Luo said, the company anticipates it will also be used as a companion diagnostic test for clinical trials to determine the efficacy of different treatments. To that end, ACD currently is working with pharma firms, including each of the 12 largest drug makers, to explore RNAscope for this application as well as for biomarker validation and safety and toxicity testing.

While companion diagnostic work has traditionally relied on immunohistochemistry and fluorescent in situ hybridization technologies, fundamental problems exist with each of those technologies. For example, with IHC, about 75 percent of human genes lack robust, validated antibodies, and of the remaining 25 percent, only 5 percent have antibodies that will result in the same staining patterns if they come from different sources, Luo said.

"So there is a significant unmet need to come up with technology that can complement what IHC can do," he said, adding that for many genes, including secreted proteins such as growth factors, cytokines, and chemokines, "you are never going to have good antibodies."

Further, pharma customers are also using RNAscope as a follow-up technology to next-generation sequencing, whereby the customers discover new biomarkers via NGS, then use ACD's technology to translate candidate genes for potential diagnostic purposes, he said.

"That's why pharma is using our technology for companion diagnostics — because they see a clear path to IVD testing that can be much more easily adopted, Luo said.

ACD is currently clinically validating its HPV test, and the company expects to first launch the test with key customers so it can gather feedback and better understand the assay's performance to make improvements, Luo said. A broader commercial launch of the assay is anticipated for 2015.

ACD also plans to eventually launch the test in the US, which would require US Food and Drug Administration approval, he added, though he did not provide details about those plans.

In the hopper

Also in the company's pipeline is an assay for detecting B-cell lymphoma or proliferative disease, which is currently performed using IHC methods. Luo said IHC works only for "a small fraction" of lymphoma cases, typically for cancers that are derived from a plasma cell and where the expression level is "super high."

In the majority of cases, though, the marker is expressed at too low a level for antibodies to work well, he added, estimating IHC-based tests are effective in about 20 to 30 percent of cases.

RNAscope, because it can detect down to the single-molecule level, can detect "basically for any type of B-cell lymphoma case," Luo said. Another key differentiator of the assay is that FFPE can be used as the testing sample. By comparison, flow cytometry-based methods for detecting B-cell lymphoma require fresh tissue, which is not always available.

"The ability to analyze all subtypes of B-cell lymphomas is important because most common subtypes express light chains at low levels that are below the limit of detection of conventional ISH and IHC," Luo said. "IHC performs poorly also because it frequently has a high background staining due to light chain proteins secreted from normal B cells."

While Luo did not have a precise figure on revenues anticipated from ACD's entry into the clinical space, he suggested that it could be significant. Because ACD is privately held, he did not want specifics about ACD's finances to be made public, but told GWDN that the firm has been growing "very fast already" and that once it moves into the diagnostics arena, that growth would further accelerate. "We will have a different growth trajectory," he said.

Luo and Steve Chen, ACD's chief operating officer, founded the company together in 2006. In 2009, ACD raised $5.4 million in Series A financing led by Morningside Ventures. Three years later, a Series B round led by New Leaf Venture Partners raised $12 million. Additionally, the company has received about $5 million in total government funding.

Luo said that ACD does not have plans to do any additional financing. "We actually have the funding to go all the way to profitability" and to launch the CE-marked HPV test, he said. However, he did not completely rule out a new financing round in the near- to mid-term in order to finance new technology development or to accelerate the commercialization process.