This article was originally published May 13.
By Julia Karow
Flush with $51.6 million in new funding from a private placement announced last week, Sequenom is forging ahead to develop its non-invasive prenatal trisomy 21 test on a next-generation sequencing platform, its first diagnostic test to use this technology.
Earlier this month, company officials said they are currently developing the test on an Illumina sequencer, although they are considering Life Technologies' SOLiD as an alternative. Following clinical validation studies, Sequenom wants to launch a laboratory developed test by the end of 2011, and file for premarket approval by the end of the following year.
Sequenom had originally been developing a trisomy 21, or Down syndrome, test using its MassArray platform to measure fetal RNA markers in maternal plasma but put that effort on hold about a year ago after it turned out that employees had mishandled R&D test data and results (GWDN 4/30/09).
In parallel, the company was also developing a DNA-based method that uses next-generation sequencing. In late 2008, the company and its collaborator, Dennis Lo from the Chinese University of Hong Kong, published a proof-of-principle paper on this version of the test (GWDN 12/2/08).
Two weeks ago, during the company's first-quarter earnings call, Sequenom chairman and CEO Harry Hixson said that "following extensive scientific experimentation, the company has decided to proceed with a purely DNA-based method for the detection of [trisomy 21] using massively parallel sequencing."
During the call, company officials also provided a timeline and milestones for developing the test, called T21, as well as information about the methodology and expected costs.
Sequenom plans to finish optimizing the test by the end of September. It analyzes circulating cell-free fetal DNA from maternal blood, a technology the company licenses exclusively from Isis Innovation, the tech transfer company of the University of Oxford, and others. The test uses sequencing to determine whether an excess of fetal DNA from chromosome 21is present in the mother's blood.
The test also has the potential to detect some rare forms of Down syndrome, which arise from chromosomal translocations and mosaicism, according to Ron Lindsay, interim senior vice president of research and development at Sequenom.
In addition, the same test method could in principle be used to diagnose other, rarer trisomies, such as trisomy 13 and 18, though it would be challenging to obtain enough samples to validate such tests, he said.
So far, Lindsay said, the company has been using the Illumina Genome Analyzer II to develop the T21 test, and is considering switching to the recently launched HiSeq 2000 platform, which has a higher throughput.
But the company is also validating Life Tech's ABI SOLiD platform. "We are delighted that there are two commercially available platforms of high quality, with companies that can back the long-term production of them," he said. "On technical, cost, and performance grounds, we will make a decision as to which way we go. Clearly, in the longer term, also having an instrument under design control is important, and that also, we believe, both companies are proceeding with."
The test will require "a relatively modest coverage" of sequencing, between 0.1-fold and 0.5-fold, Lindsay said, and as a consequence, it will cost much less than whole-genome sequencing. Today, the estimated reagent cost per sample is around $200, he said, and "we anticipate that cost will halve within the next 12 to 18 months, before we would launch a test."
At that rate, he added, "we are pretty comfortable that sequencing is no longer an expensive platform to do a test like this."
However, since the instrumentation is expensive, amortization will add an unspecified amount to the cost of the test.
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Running the test would take about a week, he said, including sample preparation, sequencing, data analysis, and reporting of results.
On the GAII, at least two and up to eight samples could be analyzed per flow cell lane, so a single instrument could process up to about 3,000 samples per year, he said. The HiSeq could run about 10,000 samples per year, "and [with] competition from ABI, potentially more than that," he added.
Following test optimization, Sequenom plans to start two blinded clinical validation studies during the fourth quarter, using blood samples from women with high-risk pregnancies. It expects that by the end of the year, its collaborators will have collected enough samples to provide a sufficient number of T21 and control samples for such studies, which "represent the pivotal validation studies to support launch of a noninvasive T21 test," according to Hixson.
Sequenom CMM, the company's Michigan-based CLIA laboratory, will start testing the samples in the fourth quarter. In addition, the company plans to open a second CLIA-certified laboratory at its San Diego headquarters, which it hopes will be operational early in the fourth quarter. That lab will validate and launch the T21 laboratory-developed test, according to Hixson.
The clinical samples have been collected through a collaboration with Brown University. In 2008, Sequenom said that researchers at Brown University Medical School were evaluating its earlier T21 test by obtaining blood samples from up to 10,000 women with high-risk pregnancies in a study involving 30 clinical sites worldwide and three laboratories.
According to Lindsay, fewer than 10,000 samples might be required because the incidence of Down syndrome was higher than anticipated. He said the company is committed to carrying out two validation studies, one with about 80 trisomy 21 samples and "a vary large number of euploids," and another with about 200 trisomy 21 samples. Combined, these will provide enough statistical power "to have a very good read on the sensitivity and specificity of the test," he said.
According to Hixson, preliminary results from an R&D study on a smaller number of samples should be available sometime during the fourth quarter. Also, a publication submitted by Sequenom's collaborator Lo, which the company hopes will be published this summer, could give an indication of the capabilities of the test. It "would give you a comfort level of what could be done in an academic study in a large sample," Lindsay said. "Certainly, our goal is to meet and potentially exceed those kinds of numbers."
Testing of the validation samples is scheduled to be completed during the second quarter of 2011, followed by data analysis and the preparation of a manuscript by Sequenom's academic clinical partners for publication in a peer-reviewed journal. Once the study has been accepted by a journal, Sequenom plans to launch T21 as a laboratory-developed test before the end of 2011, "assuming the sensitivity and specificity gives us comfort that it's commercially suitable," according to Lindsay.
After that, the company plans to complete "appropriate studies and documentation necessary" to file for premarket approval of the T21 test with the Food and Drug Administration by the end of 2012, Hixson said.
The T21 test would be Sequenom's first prenatal diagnostic test to use next-generation sequencing. Earlier this year, it launched a non-invasive Rhesus D genotyping test and a fetal sex determination test, both laboratory-developed tests that use its circulating cell-free fetal DNA technology and MassArray mass spectrometry-based genotyping platform.
The company estimates the total market for non-invasive trisomy 21 screening to be approximately $1.5 billion in the US alone. But it is not the only player planning to apply next-generation sequencing to non-invasive prenatal diagnostics.
For example, Fluidigm and Artemis Health acquired co-exclusive licenses to technology developed by Stephen Quake at Stanford University for analyzing fetal DNA in maternal blood using digital PCR and high-throughput sequencing. Quake's team published a proof-of-principle study in 2008 describing the use of the technology to detect trisomies 13, 18, and 21 (GWDN 10/7/2008).
Also, earlier this month, German sequencing service provider GATC Biotech said that it has founded a new subsidiary, LifeCodexx, that will develop non-invasive prenatal diagnostics tests using next-generation sequencing (IS 5/4/2010).