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AltheaDx Explores Desktop NGS Platforms for Cancer Gene Panels


By Julia Karow

Clinical molecular pathology testing laboratory AltheaDx is adding next-generation sequencing to help it discover genetic signatures of cancer subtypes that can predict response to therapy.

Last week, the privately held company said it is now offering next-generation sequencing services for applications in biomarker discovery and clinical trial testing (CSN 6/29/2011). So far, the company has been using Roche's 454 GS Junior for its work but it has ordered an Ion Torrent PGM from Life Technologies and is also interested in the Illumina MiSeq that is scheduled to launch later this year.

AltheaDx, which has about 40 employees and operates a CLIA- and GLP-certified laboratory in San Diego, provides clinical biomarker discovery, assay development, and validation services to pharmaceutical companies as part of clinical trials. The company counts more than 50 pharma and biotechnology firms as its customers, including Pfizer and Sanofi-Aventis.

"We are deeply embedded, especially in the oncology space," said Joseph Monforte, the firm's CSO. The aim is to discover genetic signatures — both at the RNA and at the DNA level — that correlate with therapeutic response, with the ultimate goal of developing companion diagnostics for drugs.

Under its OncoScore initiative, the company is developing molecular subtyping panels for about 15 cancer types, based on two platforms: RNA profiling by TaqMan real-time PCR, and, more recently, DNA profiling by next-gen sequencing. Late last year, it launched its OncoScore Breast Cancer Segregation Panel, which uses RT-PCR to measure the expression of 96 genes. The company plans to make a breast cancer DNA panel available towards the end of summer and is also working on RNA and DNA panels for ovarian, lung, and colorectal cancer.

AltheaDx selects the genes for these panels through a collaboration with Compendia Bioscience, a cancer bioinformatics company based in Ann Arbor, Mich., that aggregates and mines publicly available cancer genomics data. According to its website, Compendia's Oncomine database contains about 550 curated cancer genomic datasets from more than 47,000 samples, including sequencing data from the Cancer Genome Atlas, microarray data, and copy number data.

Mining these data allows AltheaDx to identify, for each cancer type, key gene expression profiles that reflect different molecular subtypes, as well as genes that are mutated in at least 2 percent of samples. Genes mutated at lower frequencies are generally not included because it would be difficult to correlate them with therapy response with statistical significance, Monforte said.

"We are actually taking more of a population genetics approach to identifying what the key variables are in each of these cancer types," he said. For breast cancer, for example, TCGA data shows that 19 genes are mutated in at least 2 percent of breast cancer samples, he explained.

Genes mutated at this frequency — along with some others that are interesting because of their function — then become part of AltheaDx's testing panels.

For the last several months, the company has been using Roche's 454 GS Junior for its next-gen sequencing work but it has ordered an Ion Torrent PGM, which it plans "to be very aggressively working on" starting this month. Also, "we certainly will take a look at the [Illumina] MiSeq when it's launched," Monforte added.

Regarding the PGM, he said, the company decided to wait until the Ion OneTouch sample prep system, which largely automates the emulsion PCR process, and the 316 chip, which has a higher capacity than the current 314 chip, become available. Both are scheduled to launch commercially this month. "We also wanted the read length solidly over 100 [base pairs]," he explained.

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"We are being fairly brave in terms of adopting those clinically focused platforms," Monforte said. "The moment we believe they are robust enough, we are pulling them into the lab and trying them out."

Dealing with the amount of data generated by next-gen sequencing has been a challenge, he said, and the firm had to invest "significantly more" into enhancing its bioinformatics capabilities than originally planned.

But its expertise with microarrays and PCR is helping it with sequencing as well. "We are launching straight ahead with sequencing analysis in formalin-fixed paraffin-embedded tissue," Monforte said, "whereas others have taken their time to try to explore that."

Other firms are also addressing the challenges of sequencing FFPE samples. Foundation Medicine, for example, presented data at last month's American Society of Clinical Oncology meeting demonstrating the performance of its sequencing-based test on 75 FFPE tissue samples from non-small cell lung cancer, melanoma, and colon cancer (CSN 6/8/2011).

Foundation Medicine demonstrated its test on the Illumina HiSeq, but is considering the Ion Torrent PGM and other platforms for the commercial version of its test — a choice that AltheaDx has already weighed. High-throughput sequencing platforms such as the HiSeq or Life Tech's SOLiD are "not compatible with the clinical setting," Monforte said. "They are too costly per run, and you have to pool way too many patient samples."

For example, he said, more than 100 barcoded patient samples would need to be pooled per HiSeq run to make the assay cost-effective, but only "handfuls" of patients are recruited to clinical trials each week, and clients "want the data quickly." In addition, he said, barcoding and keeping the data from different samples separate is "challenging."

AltheaDx is currently validating a colorectal RNA test technically, which is scheduled to be launched in September. Designs for an RNA test for ovarian and lung cancer are currently being finalized, with the goal to launch them by the end of the year. In parallel, the firm is working on DNA panels for those.

For the first few cancer types, the company has been developing the RNA and DNA panels largely on its own, but is now beginning to see pharmaceutical clients underwrite assay development for some additional cancer types, Monforte said.

AltheaDx is already applying its existing panels in a number of collaborative projects. For example, it is working with an undisclosed European university to study colorectal cancer as part of three clinical trials in order to correlate its data with clinical response, and with an undisclosed US university to study about 500 breast cancer patients, looking "at a number of different clinical questions within that population," according to Monforte.

In addition, the company is conducting retrospective analyses of data from existing clinical trials for several pharmaceutical companies and is integrating RNA and DNA assays into upcoming trials.

While AltheaDx plans to eventually develop its RNA panels into in vitro diagnostics, working with undisclosed pharmaceutical partners to launch them as companion diagnostics, it likely won't follow the same path for its DNA panels.

"On the RNA side, it's very easy for us to map out these panels, identify the PCR, do the design work, and for the breast panel, we are already very actively in the true diagnostic development on it," Monforte said. "[On] the DNA side, on the other hand, it's non-trivial at this point," for example because the FDA has not yet come to grips with the complexities of next-gen sequencing.

Over the next two to three years, he said, AltheaDx is thus likely to translate any discoveries it makes using its sequencing panels into PCR genotyping assays. There may be exceptions, though, for example for tumor suppressor genes that are of key importance but have a broad mutation spectrum. "In those cases, we are going to have to join the brave new world and try to address that with the FDA using a sequencing platform," he said.

Eventually, several years from now, focused gene panels may be superseded by exome or whole-genome sequencing, he said, though researchers would likely still focus on mutations occurring with modest or high frequency, "just because that's all you can work with statistically."

Have topics you'd like to see covered by Clinical Sequencing News? Contact the editor at jkarow [at] genomeweb [.] com.

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