Testing Fetal DNA

Courtesy of Sam Shum, Rossa Chui, and Dennis Lo When Charles Cantor and Dennis Lo flew to Pattaya, Thailand in late 2002 to attend a conference, neither man knew they would end up collaborating on a blood test that could one day reduce reliance on invasive prenatal diagnostic methods such as amniocentesis and chorionic villus sampling (CVS) Cantor, chief scientific officer at Sequenom in San Diego, delivered a presentation on the company's MassARRAY system, which uses mass spectrometry and analyte specific reagents (ASRs) to measure disease biomarkers. Sequenom was looking for potential diagnostic applications of the technology. Lo, professor of chemical pathology at the Chinese University of Hong Kong (CUHK), had discovered that fetal DNA makes up about 5% of the cell-free DNA in maternal plasma. In contrast, fetal cells are present in about one in a million cells. Lo needed a technology that would minimize interference from the large amounts of maternal DNA present in the sample. Two halves of a puzzle clicked into place as Lo listened to Cantor's presentation. "I had heard of the Sequenom technology but didn't know about it," says Lo. "As I listened, I knew immediately that this system could solve the qualitative problem I was having." The result was an international consortium led by CUHK, Boston University (BU), where Cantor is head of the Center for Advanced Biotechnology, and Sequenom to develop a test that could be used in the first trimester of pregnancy to determine cell-free fetal DNA. So far, the team can detect gender, paternally inherited genetic characteristics such as fetal rhesus-D genotyping, and certain autosomal genetic disorders such as beta-thalassemia. 1 Marketing of the first commercially available tests that result from the collaboration is expected to occur in the United States and Europe. Possible candidates are a test for rhesus-D or ASRs for tests already available, such as for cystic fibrosis. When any of the tests are due for FDA approval depends on the eventual licensee. Lo is currently working on detecting Down syndrome. "We are cautiously optimistic. We are closer than ever, but detecting Down syndrome is different to [detecting] gender, rhesus-D blood-type, or thalassemia, because they all stem from qualitative differences caused by mutations inherited from the father," says Lo. "With Down syndrome there is a quantitative difference, an extra chromosome 21. We need to develop the work further in order to differentiate quantitatively." If this problem can be solved, the market potential is huge. In the United States alone, approximately 200,000 invasive prenatal testing procedures such as amniocentesis and CVS are performed each year, at a cost of approximately $200 million. Such tests carry a small risk of miscarriage. Moreover, amniocentesis cannot be conducted until the second trimester of pregnancy. Noninvasive tests that can be used during the first trimester of pregnancy would be ideal, especially for detecting chromosome abnormalities such as Down syndrome, and neural tube defects such spina bifida. These genetic disorders currently can be detected only by invasive tests. THE HOLY GRAIL OF PRENATAL SCREENING The CUHK/BU/Sequenom consortium is not alone in trying to tap the market for noninvasive prenatal tests. "Noninvasive tests are the Holy Grail for prenatal screening," says Ian Findlay, chief scientist at Gribbles Molecular Science in Australia. Findlay is developing a pap smear-based prenatal test for Down syndrome and other genetically inherited conditions. "Cells are forensically DNA fingerprinted to ensure that they come from the baby," says Findlay. Gribbles anticipates having a commercially available test on the market in Australia in late 2005 or early 2006. Scientists in this field are also working on improving the sensitivity of testing using PCR alone, rather than the combination of PCR and mass spectrometry that the Sequenom platform employs.2 PCR technology alone still poses challenges. "Using PCR might have limitations because of the false-negative and false-positive results," says K.Y. Leung, honorary clinical associate professor in the obstetrics and gynecology department at the University of Hong Kong Faculty of Medicine. "It's too early to say if tests using fetal DNA in maternal plasma will be able to replace amniocentesis." Fetal circulatory nucleic acids are one of the key areas of investigation for the Special Non-Invasive Advances in Fetal and Neonatal Evaluation Network. (SAFE). The network is a €12 million project with the aim of implementing routine noninvasive prenatal diagnosis and cost-effective neonatal screening across the European Union. "Free fetal DNA is of paramount importance," says Maj Hulten, professor of medical genetics at the University of Warwick in Coventry, UK, and also the project coordinator for SAFE. "There are fetal cells, too, but they are difficult to hunt, and it's difficult to get at their special characteristics. ... Dennis Lo is an undoubted authority on this. He was the first person to show convincingly that there was such a lot of free fetal DNA circulating in the mother's plasma," says Hulten. Whether cell-free fetal DNA is examined using PCR alone or with Sequenom's additional firepower of mass spectrometry, the initial hurdle remains the same: identifying the markers. "Today it is very clear that one can do paternal DNA sequencing, and in good labs that [sequencing] can be done with high efficacy," says Hulten. "The main problem is to find the markers that are fetal-specific." PROFIT POTENTIAL In August 2004 a Sequenom MassARRAY Compact DNA analysis system was installed in the chemical pathology department at CUHK. "Dennis and his group were the first people to get it under our technical access program, whereby we give people the sequencing facilities before they buy them. We want people to use the instruments, get good results and publish, and also make a good faith effort to buy the instrument," says Cantor. A MassARRAY unit costs approximately $250,000 depending on the user's software and application needs, and Sequenom is targeting to sell units to testing laboratories, hospitals, and medical schools. "CLIA [Clinical Laboratory Improvement Amendments] research labs are targeted, as they conduct most of the ASR testing," says a Sequenom spokesperson. The technology can be used for detection of point mutations causing cystic fibrosis, or anything that is caused by a single point mutation inherited from the father. "With the use of epigenetic markers this approach can also be used to detect sequences that are maternally inherited. The research challenge is to discover more of these epigenetic markers," says Lo. Sequenom has not disclosed how many MassARRAY units it hopes to sell as a result of this collaboration, nor has it disclosed the likely cost of future tests, but it expects the work to generate revenues of $25–$28 million. The profit potential for Sequenom is two-fold: reagents for specific tests, and sales of the MassARRAY hardware. "The value in diagnostics is the markers, because they can be patented, and the technology, which can be patented if there is no competing technology," says Cantor. "Today there is no competing technology with the same specificity. We completely own the intellectual property [IP] space of mass spectrometry of RNA and DNA, and we have a totally dominant IP position in this field," says Cantor. For this new technology to challenge the dominance of invasive screening, it must be able to detect Down syndrome with the same reliability as amniocentesis. "That will be our main goal in the next few years," says Lo.

Testing Fetal DNA

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Jane Parry

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June 2005

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