American Association for Clinical Chemistry
Better health through laboratory medicine
May 2007 Clinical Laboratory News: The Road to Better Down Syndrome Screening

May 2007: Volume 33, Number 5

The Road to Better Down Syndrome Screening
Will Fetal Nucleic Acids Someday Provide Safer Answers?
By Deborah Levenson

While first and second trimester prenatal screening tests for Down syndrome and other chromosomal abnormalities are well established and easy to perform, their less than ideal false-positive rates still spur thousands of unnecessary invasive diagnostic procedures each year. About 5% of women who get screened for the fetal genetic abnormality receive false-positive results, and many go on to have diagnostic procedures that carry a 1 in 200 chance of miscarriage. Yet these tests are likely to become even more common. Today, more women give birth after age 35, when risk of Down syndrome increases, and a new American College of Obstetricians and Gynecologists (ACOG) recommendation (Obstetrics & Gynecology; 2007; 109: 217–228) urges screening regardless of a pregnant woman’s age. In the future, however, noninvasive techniques that identify a Down syndrome pregnancy by genetic markers in cell-free, fetal nucleic acids circulating in maternal blood could offer a safer, more accurate way to inform pregnant women about their fetuses’ health status in the first trimester, thereby diminishing worries for obstetricians and expectant mothers.

“The new goal is a definitive diagnosis for everyone who wants one. We’re now aiming for a 100% detection rate, not just a sensitivity of 80% or 90% in screening,” explained Joe Leigh Simpson, MD, Professor of Obstetrics, Gynecology, and Human and Molecular Genetics at Florida International University College of Medicine (Miami), who conducts research in prenatal genetic diagnosis. Simpson and other investigators are interested in early studies of the fetal nucleic acids testing scheme, but before any such technology can become part of clinical practice, larger trials must validate and expand the current findings. Researchers must also overcome several problems to make their technologies easier to use, and therefore commercially viable.

Common Problem, Different Strategies

Since the 1997 discovery of fetal cell-free DNA in the blood of pregnant women, researchers have been looking for ways to distinguish the fetus’ DNA from the mother’s. The problem has been interference: the fetus’ DNA comprises only about 3% to 6% of cell-free nucleic acids in maternal blood. To date, studies have shown that certain methods using cell-free fetal nucleic acid are safe and accurate enough to detect fetal genes that aren’t in the mother’s genome, including those on the Y chromosome and for Rhesus D. But detecting those genes among the vast amount of circulating maternal nucleic acids make the diagnosis of trisomy 21 much more complex.

In two recently published studies, researchers from the Chinese University of Hong Kong (CUHK) and Ravgen, Inc. (Columbia, Md.) describe very different strategies for dealing with the challenges of finding fetal nucleic acids in maternal blood.

The Hong Kong team’s strategy involved circumventing the maternal DNA by using cell-free RNA (Nature Medicine 2007, 13: 218–233). “Our approach was that if fetal DNA is so low, let’s work on an alternative,” explained Dennis Lo, MD, PhD, who first discovered free fetal DNA and is CUHK’s Dr. Li Ka Shing Professor of Medicine and the 2006 recipient of the National Academy for Clinical Biochemistry’s Distinguished Scientist Award for outstanding research advances. Lo holds and has filed patents for fetal nucleic acid technologies licensed to a number of companies. “A key characteristic of RNA is that not all genes are switched on in all tissues, and therefore fetal RNA may not be available. So we looked at a gene that is switched on only in the placenta, an organ which only the fetus, and not the mother, has. Such RNA is virtually 100% fetal-specific.”

The CUHK team used mass spectrometry to detect and quantify one fetal-specific gene, called PLAC4, located on chromosome 21, and then determined how many copies the fetus had by assessing the ratio between alleles of a single nucleotide polymorphism (SNP) (See Figure 1). To prove that the maternal plasma PLAC4 was derived solely from the fetus, the CHUK team studied 11 fetus-mother pairs in which the fetuses and mothers had different PLAC4 genotypes. In each pair, the PLAC4 SNP genotype in the plasma corresponded to the placenta’s genotype, confirming that PLAC4 RNA was derived from the fetus with no contribution from the mother. After studying 57 normal and 10 trisomy 21 pregnancies, the team reported the technology had diagnostic sensitivity and specificity of 90% and 96.5%, respectively. However, the method doesn’t work if the fetus is homozygous for PLAC4, Lo noted.

In contrast, the Ravgen researchers, led by Ravinger Dhallan, MD, PhD, the company’s Chairman and CEO, examined cell-free DNA (The Lancet, 2007, 369: 474–481). By adding formaldehyde to maternal blood samples, the researchers increased the yield of available cell-free fetal DNA by stopping lysis of maternal blood cells and subsequent release of more cell-free maternal DNA (See Figure 2). This strategy boosted the percentage of cell-free DNA in maternal blood to a mean of 34%. After examining 570 SNPs on chromosome 21 and 549 SNPs on chromosome 13 as a reference, Dhallan’s team identified multiple sites where mothers’ and fetuses’ DNA differed.

“If the mom is homozygous and the baby is heterozygous, each difference becomes a unique fetal beacon in the DNA. By identifying many such sites that fit a pattern, we can evaluate fetal DNA across multiple chromosomes,” Dhallan explained. “We take the average fetal signal, the ratio of fetal DNA to the mom’s in all sites, for 13 and 21. If the baby is healthy, we should see the same intensity of fetal DNA signals on all chromosomes. But if the baby has Down’s, the extra copy of chromosome 21 will change the ratio for that chromosome.” Dhallan’s team found that their method correctly confirmed the copy numbers of fetal chromosomes 21 and 13 for 58 of 60 samples and identified 56 of the 57 normal samples, plus two of three trisomy 21 samples. The researchers reported preliminary sensitivity and specificity as 66.7% and 98.2%, respectively.

Problems to Work Out

Both methods have unsolved technical problems, according to the researchers. For Lo’s test, these include how to transport samples and use the technology in a diverse population. Ravgen’s test addresses these issues, but creates other concerns about using formaldehyde.

“Our test will only work if the copies of chromosome 21 are different, because we need to compare them to see if there is a 1 to 1 or a 2 to 1 ratio,” Lo pointed out. “Currently, we are looking at just one polymorphism, so we are only able to use the test in 50% of pregnant women. In the other 50%, their test results are not informative.” To improve their technology so that it works in a more diverse population, Lo’s team is developing new markers intended to surmount these problems. “If we use three genes, and two to three SNPs in each, we should have 90% coverage,” Lo noted. To test the methods, Lo and his colleagues are now setting up the infrastructure for a larger, multi-center trial that will draw subjects from hospitals in China and Western countries.

Finding other definitive markers or genes on other chromosomes could be time-consuming, according to both Dhallan and Lo. Dhallan also noted that RNA could be difficult to handle in routine clinical samples. “RNA was meant to be a temporary signal in a cell that disappears, while DNA is more stable chemically and lasts for millennia,” he explained, noting that the fragility of the RNA could present sample transportation challenges.

But efforts to reproduce Ravgen’s success using formaldehyde to increase the yield of available free fetal DNA have yielded mixed results. Initial results reported by Dhallan (JAMA 2004; 291: 1114–1119) in a two-phase study show that formaldehyde boosts the mean percentage of free fetal DNA. In the first phase, the researchers tested samples (n=20) from a single site and found free fetal DNA increased from 7.7% to 20%. In the study’s second phase, conducted at 27 sites, 59% of treated samples (n=69) contained 25% or greater fetal DNA. A team of researchers at the American Hospital of Paris, Neuilly, France led by Jean-Marc Costa, MD, was also able to increase fetal DNA concentration to useful percentages. (Clinical Chemistry 2005; 51: 242–244). But Lo’s team and another at the University Women’s Hospital in Basel, Switzerland, achieved only small, insignificant increases using the formaldehyde method (Clinical Chemistry 2005; 51: 655–658 and 652–655).

During the National Institute of Child Health Fetal Isolation Study (NIFTY), Simpson and colleagues had success with the formaldehyde method. But Stephen Brown, MD, Associate Professor in the Department of Obstetrics and Gynecology at University of Vermont, consultant to Lenetix, Mineola, N.Y., and holder of a pending patent for a noninvasive genetic test, noted that the method rendered plasma DNA “useless” in his lab.

But for Simpson, there is another problem inherent in both the Lo and Ravgen technologies. “For me, the issue regarding these methods is the difficulty of getting a truly pure sample of either DNA or RNA. The sample must be pure for ratios to be applicable,” he commented. “Cell-free technologies are great for single-gene disorders, but trickier for detection of aneuploidy.” Simpson serves on a medical advisory board for Biocept (San Diego, Calif.), which is developing a first trimester screening test that uses fluorescence in situ hybridization to analyze intact fetal tropoblast cells in maternal blood.

Another Technology On the Horizon?

Preliminary research presented at the February meeting of the Society for Maternal-Fetal Medicine highlighted another cell-free, fetal DNA technology that could someday be used in a noninvasive test for Down syndrome. While the study by researchers at Sequenom Inc. (San Diego, Calif.), which develops and markets products for genetic analysis, did not aim to show the technology’s diagnostic ability, the data did confirm the test’s ability to identify useful amounts of cell-free fetal DNA.

Researchers purified cell-free fetal DNA from maternal blood samples using Qiagen (Germantown, Md.) MinElute columns and estimated the amount of fetal versus maternal DNA. To arrive at estimates, they used Sequenom’s MassARRAY spectrometry, a multiplexed set of 16 single nucleotide polymorphisms, and an assay that examined AMG, a gene located on both the X and Y chromosomes. The strategy “was geared toward detection of fetal DNA independent of gender,” noted Dirk van den Boom, Director of Research and Development for Application Development. Previously, researchers have had difficulty confirming the presence of cell-free fetal DNA from female fetuses.

Sequenom intends to further develop the technology as part of a noninvasive, prenatal diagnostic approach for a variety of genetic disorders that are currently identified by amniocentesis, including cystic fibrosis and Tay-Sachs disease, and ultimately, Down syndrome.

A Possible Diagnostic Test?

While both the Ravgen and Hong Kong teams noted they currently view their technologies as screening tests, Jacob Canick, PhD, Professor in the Department of Pathology at Brown Medical School (Providence, R.I.) was optimistic that research can overcome the technical challenges of working with cell-free nucleic acids, and even achieve a viable diagnostic technology. “If either one approaches 99 percent sensitivity and specificity, that would rival amniocentesis or CVS. The best we can do now in screening is the integrated test, which involves nuchal translucency and one serum marker at 11 to 13 weeks, followed by a second blood draw for four more markers at 15 to 16 weeks. It has the single best performance for any single screening test for Down syndrome, with about 90 percent sensitivity and 98 percent specificity. Lo’s PLAC4 marker appears to be almost as good as that now. That’s why I’m optimistic that as he adds some more SNPs to his study, he’ll get better and better with it.”

Either technology, “with major refinements,” could complement current screening for Down syndrome, predicted Brown, who noted that not all women have access to the nuchal translucency test, and emphasized his view that a better screening test would be more useful than a new diagnostic one. “What the world needs isn’t a diagnostic test. We already have a diagnostic test that works. What we need in the trenches is a screening test with a very high degree of both sensitivity and specificity, that gives a very high degree of assurance that the fetus doesn’t have Down syndrome,” he asserted. “If you could come up with a test that offers very good exclusion of Down’s syndrome and dramatically reduces the false-positive problem from its current state, that would be something.”

While CVS and amniocentesis provide solid diagnostic information, they do have significant problems, Canick pointed out. “Loss of pregnancy is still a major risk, and the tests are unpleasant for women.” He predicted, however, that a screening test employing cell-free fetal nucleic acids will come sooner than a diagnostic one.

Use in the Real World

Future use of either technology will depend on practical issues, including cost and ease of use, according to Canick and Brown. In order to become commercially viable, any new technology cannot cost much more than the current prenatal diagnostic tests, which run about $800 to $1,000, they agreed.

In their current preliminary forms, “both (the Lo and Ravgen) technologies are a struggle with respect to the complexity of the assay, the degree of meticulous detail, and how robust the data are,” Brown opined. Greater simplicity would confer an advantage for routine clinical use, he asserted. “I don’t doubt at all the principle of either test. It’s all intellectually acceptable, but the commercial viability of both products is a huge outstanding issue,” added Brown, who emphasized that he read both preliminary studies as a practicing obstetrician looking for a test that is both easy to use and informative.

Canick, who identified stability of RNA in Lo’s technology as a potential challenge in transporting samples, was more optimistic. He called RNA instability in samples a “relatively straightforward technical issue that can be solved.”

“The technology that’s destined to become dominant will achieve the right balance between perfection—100 percent sensitivity and specificity—and price,” Brown predicted. Canick expressed hope that such a test is 5 years away, although he added, “Claims that similar methods are just around the corner have been made since the 1990s.” Lo, though, took a longer view. “It was 8 years from the first report on fetal DNA to widespread use of fetal DNA to detect Rhesus D in the U.K. Down’s is more complicated technically, so it could be quite a few more years.”