NIPT offers greater sensitivity than traditional blood serum screening methods for genetic disorders involving extra copies of chromosomes, most notably Down syndrome.
A dichotomy of strongly held opinions has emerged on how quickly the tests should be adopted and which populations of pregnant women should receive them based on current evidence.
The companies point out that their tests are more accurate and sensitive than traditional screening methods and therefore should be available to all who want them.
Genetic counselors, meanwhile, argue for caution. They say overuse of the more expensive NIPT – especially as a primary screening tool for women at average risk for genetic abnormalities – is shifting the focus away from identification of other, more common pregnancy risks.
NIPT has the advantage of being one of the only large-market areas in clinical genomics where the correlation between a single marker (in this case, chromosomal copy number) and disease is absolute. But for the current players and their technologies, that advantage may not be sustaining.

Since the commercial introduction of Sequenom Inc.’s MaterniT21 test in October 2011, noninvasive prenatal testing (NIPT) has become the hottest – and most controversial – area of clinical genomics.

The drive toward NIPT was rooted in the bold dream of replacing invasive diagnostic testing for genetic disorders, which entails karyotyping (chromosome counting) using chorionic villus sampling (CVS) or amniocentesis, with noninvasive tests using a simple blood draw from the mother.

A few years ago, the challenges seemed squarely technological – and daunting: could sequencing methods and bioinformatics accurately and reliably measure the presence of aneuploidy (extra copies of chromosomes), alterations in sex chromosomes, and even microdeletion and microduplication syndromes too small to be detected by standard cytogenetic chromosome analysis. The sample would come from cell-free fetal DNA taken from maternal blood or alternatively, from the capture of whole fetal cells in maternal circulation. (Pursuit of the latter technology has been abandoned by almost all companies.)

In rapid succession, four NIPT offerings have come to market. MaterniT21 has now been joined by products from three other companies: Verinata Health Inc.’s verifi test launched in March 2012 (the firm was subsequently acquired by Illumina Inc. in January 2013 [See Deal]); ’s Harmony test, which followed two months later; and Natera Inc.’s Panorama test, which hit in December 2012. All four screen for genetic abnormalities caused by extra copies of chromosomes: trismony 21 (Down syndrome), trisomy 18 (Edwards syndrome), and trisomy 13 (Patau syndrome), plus sex chromosome aneuploidies. They all interrogate cell-free fetal DNA from maternal blood. Natera and Ariosa have adopted chromosome-targeted sequencing approaches, whereas Sequenom and Verinata use forms of massively parallel (shotgun) sequencing.

Currently, NIPT is focused on the high-risk population of expectant mothers. High-risk pregnancies are defined by a number of factors including advanced maternal age (over 35), family history, and the results of ultrasound or serum screening. They account for roughly three-quarters of a million pregnancies per year in the US – a market an order of magnitude higher than that of many complex molecular diagnostic tests. NIPT is further differentiated from other areas of molecular diagnostics and screening because pregnant women are not normally under a physician’s care.

According to William Quirk, analyst at Piper Jaffray, aggregated data from the NIPT companies in the first quarter of 2013 suggest, on an annualized basis, a 40% penetration of the high-risk market. “A little over a year ago, that was nothing,” he says. “You’ve seen rapid adoption in the high-risk group. And I would anticipate that all four companies expect to move beyond that group.”

But these first NIPT offerings are partial solutions. Rather than serve as replacements for amnio or CVS, they are screening tests intended to winnow down the number of women believed to carry babies with trisomic disorders who then go on to invasive diagnostic testing. To some, that suggests an even greater market potential; to others, a potential for misuse.

Even when companies emphasize use of NIPT in the high-risk population (and some would say they do so with a wink and a nod, like drug companies dancing around off-label use), that message is not being taken. Investors are arguing for broad use – backed by the companies saying there is no technical reason to argue their tests perform differently in different populations.

Who To Test, When To Test, And What To Look For

A few years ago, the NIPT discussion would have been around the relative merits of one sequence-based technology over another, the cutoff level for the amount of fetal DNA that is present in a sample (which could affect test quality and predictive value), how companies use different bioinformatics packages to develop their results, and test efficiency and cost. These remain real considerations. But the major issues of NIPT adoption lie elsewhere.

“I think the social and ethical issues involved with NIPT have only been a small part of the conversation, and I am concerned that they are being minimized by the companies,” says Katie Stoll, a genetic counselor in Washington State.

“Because we are able to do something doesn’t always mean we know what to do with the information,” says Mary Norton, MD, of the Stanford University School of Medicine. Moreover, in the case of prenatal screening and diagnosis, “what we as highly educated knowledgeable experts think should be available or should be done is often really different from what the average man or woman on the street might think,” she says.

The primary controversy surrounding NIPT arises out of concerns that pregnant women and their Ob-Gyns do not understand how to position NIPT in the context of its overall importance in evaluating potential birth problems.

“The media and the companies generally are not saying this is a better screening test than current integrated screening,” says Norton. “They say this is an alternative to amnio. But amnio is a diagnostic test and not a screening test.” In their reports, two of the companies (Sequenom and Verinata) say aneuploidy is “detected,” she points out. “That sure sounds diagnostic to me.”

Theoretically, NIPT may be used before, after, or in place of quad screening, which measures the levels of four substances in maternal blood, including AFP (alpha-fetoprotein), hCG (human chorionic gonadotropin), estriol, and inhibin-A: low levels of AFP and abnormal levels of hCG and estriol may indicate Down syndrome or Edwards syndrome; measuring inhibin-A also informs about Down syndrome and reduces the rate of false-positives. Quad screening may be combined with nuccal translucency, an ultrasound technique performed in the first trimester, to give further information about potential birth defects (integrated screening).

Integrated screening provides “very good information about Down syndrome,” says Norton. Although the detection rate difference between integrated screening and NIPT is a few percent, that difference is minimal for women at low risk of Down syndrome. “For young women having children, that is a relatively small concern in comparison to many other things that could potentially affect their baby,” she says.

“There are fewer false-positives, but [integrated] screening gives you lots of information about preeclampsia, preterm birth, fetal growth, and other structural birth defects and heart defects,” Norton says. “NIPT is kind of a one-trick pony.”

Norton and others have expressed concern that NIPT is currently being integrated in some settings as a primary screening test for women at high risk for fetal aneuploidy. “From a population perspective, a better option for noninvasive prenatal testing may be as a second-tier test for those patients who screen positive by conventional aneuploidy screening,” she and co-authors wrote in the April 2013 issue of Obstetrics & Gynecology.

That’s an important distinction, and one that genetic counselors claim is being lost. “I think it’s been hyped as a near diagnostic and that doctors are accepting this as a replacement for diagnostics like amniocentesis,” says Stoll. “There’s a lot of misunderstanding both in the genetics community and the obstetrics community, which is trickling down to patients in terms of appreciating the limitations of this testing.”

The reliability of NIPT depends on the incidence of the trisomies in a given patient population. Even in the high-risk population, “the likelihood of a false-positive can be higher than the likelihood of a true-positive in some cases,” Stoll says. NIPT is valuable in some situations, she says. “But it is imperative that patients be counseled about the limitations and possible outcomes so they are able to make informed decisions about all testing outcomes.”

The problem is that for a 25-year-old of average risk, the prevalence of having a trisomy is different than for a woman at higher risk. “You have to understand what the positive and negative predictive values are using NIPT,” says Thomas Musci, VP, clinical and medical affairs at Ariosa Diagnostics. There may be sufficient fetal DNA to generate a result (another issue, as overweight women, for example, will have a lower proportion of fetal DNA in circulation), and the performance of the assay is the same regardless, but the clinical determination and the use of it depends on the age and risk factors of the individual subject.

“The positive predictive value [PPV] is an important metric and one that has not been promoted by the cell-free companies,” says Karen Drexler, founder of CellScape Corp., the only company currently pursuing whole fetal cell technology. “In full disclosure, that is information that should be provided.”

Stoll has looked at how PPV changes when looking beyond the high-risk group. In one calculation, posted on The DNA Exchange web site, she used a sensitivity of 98% and a specificity of 99.6% for trisomy 18, and for trisomy 13, a sensitivity of 99.0% and a specificity of 99.8%. “This is in the range of what the labs are reporting,” she says, and results in a PPV for a 35-year-old woman of about 11% for trisomy 18 and 8% for trisomy 13. “Most of the positive results using NIPT for trisomy 18 and 13, by my calculations, would be false. But either of these would be a devastating diagnosis to receive. I could see women really being stuck with this information and waiting without having any realistic option in small communities where this is being marketed heavily.”

According to a presentation by Norton at a meeting on antepartum and intrapartum management sponsored by the University of California, San Francisco, using traditional serum screening, for every eighteen amniocenteses performed, one case of Down syndrome is detected. With NIPT as a secondary screen (after integrated testing), one of every two amniocenteses would detect a case of Down syndrome. But when NIPT is used as a primary screening, the number of amnios performed to detect a case of Down syndrome goes up to 13. Cost is also a factor: “NIPT costs 10 times more than traditional screening,” says Stoll. However, using NIPT as a second-tier test “could dramatically decrease the number of invasive procedures,” she says.

That said, “the companies are certainly trying to get NIPT to be the standard of care for everybody,” says Norton. “They are not directly doing it right now because it is against what ACOG [the American Congress of Obstetricians and Gynecologists] and other professional societies are claiming. But they are publishing papers that conclude this is a good test for all women and there is no reason they can’t be applied to a low-risk population.”

Industry Counters

Balanced against those concerns is the fact that physicians in the prenatal screening/testing space have been doing testing for a long time, says Musci, a former chair of ACOG. “We communicate risk to patients, false-positives, false-negatives, and we have been doing this for years with maternal serum screening. This technology is no different in those things but does it better. What’s been portrayed a lot of times by the media is that this represents something new and radically different in terms of our approach to the patient, and it really isn’t.”

Unfortunately, however, as the media has jumped on the NIPT story, “you can get to a point where it’s less about educating physicians and more about the commerce,” says Musci. “That’s where the message can get mixed up.” More than 98% of women getting testing results from NIPT are getting accurate information that increases their confidence in their pregnancy; it has a lot of beneficial effects on their outlook.

“Nobody is capturing the story of the upside of having good information,” says Ted Snelgrove, CEO of CellScape. “Everybody is looking at the 2% and forgetting about the 98%.”

“If you look at first trimester screening as a common standard-of-care technology for women at that stage, the quality of that information is pretty weak,” he says. But people focus almost obsessively on the gap between the delivery of test information and the concept of perfect information and will spend 90% of the time in their assessment focusing on the potential harm that comes from that gap, he says. “What they fail to address often at all or in passing is the huge advance that testing information may represent compared to status quo, which is in fact the relevant medical adoption question.”

Snelgrove has seen this before. While at Genomic Health Inc. in the early 2000s, he witnessed the commercialization of the company’s OncotypeDX breast cancer assay. “Everybody was saying it was being adopted too fast,” he says. Later, at Crescendo Bioscience Inc., he heard similar criticism with respect to that firm’s protein-based Vectra DA blood-based test for rheumatoid arthritis disease activity. “Anything that is disruptive will encounter that kind of criticism,” he says.

Expanding The Menu

Even as NIPT is focused commercially on the trisomies, companies are contemplating whether to expand their menus.

A landmark study from the National Institute of Child Health and Human Development (NICHD) headed by Columbia University Medical Center’s Ron Wapner, MD, and published late last year in the New England Journal of Medicine, used microarray techniques and showed a higher frequency of detecting genetic abnormalities (microdeletions and duplications) than with karyotyping. It also established that these disorders, while rare, are more prevalent in the average-risk population of pregnant women than had been previously thought. That means that viewing risk through the lens of the three aneuploidies and the concept of risk for women of genetic abnormalities more generally is just not the same. “The concept of risk needs to change in the whole NIPT market,” says CellScape’s Drexler.

Although Wapner’s work showed that copy number variants are probably more common than were thought in the otherwise general population of pregnant women, the question becomes whether any single one or group of them could comprise a menu and could be validated by sampling cell-free fetal DNA.

“Perhaps there is room to go there – expanding the menu to go into deletions or duplications – but [only] when having a reason for doing that,” says Ariosa’s Musci. And microarray technology, not the analysis of cell-free fetal DNA, would likely be the technology of choice. “You would want a pure fetal sample to do microarray,” he says. That’s where CellScape could differentiate itself, and perhaps gain leverage in a market that although large, is also bounded. (See sidebar, “Illumina’s Unique Leverage In NIPT.”) Assuming both targeted and massively parallel sequencing technologies can provide good test results, that no one has blocking IP (Sequenom in particular is engaged in patent litigation against Ariosa and Natera, which is not expected to progress until an inter partes review sometime in 2014), and that a test menu in NIPT using cell-free fetal DNA remains limited, the competitive advantage for the cell-free fetal DNA companies may only really lie in their bioinformatics or cost advantages. (See Also see "Molecular Diagnostics Ushers In New Era Of Prenatal Testing" - Medtech Insight, 19 September, 2012..)

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CellScape, however, using whole fetal cells pulled from blood, has developed a custom microarray that it says can target the top 40 prenatally relevant genomic syndromes such as deletion and insertion syndromes.

Microarrays have already been validated for use in invasive testing for prenatal disorders and in pediatrics for failure-to-thrive cases. With that as predicate, CellScape could rely on a lot of existing validation work done previously in the field of maternal medicine. “What’s most important in our validation is to show that we have fetal cells, that we have DNA of sufficient quantity and quality to do the genetic analysis,” says Drexler. That’s unlike the cell-free fetal DNA companies, she says, which would need to run a separate study for each microdeletion syndrome using hundreds or thousands of cases to make a statistical cutoff for what is normal and what is not. Because of the nature of the technology, CellScape would not need to go back and prove what is being projected algorithmically from cell-free fetal DNA fragments. “We are looking at the whole intact genome,” she says.

Eventually, as costs go down, all women may get NIPT – be it for aneuploidy screening or screening for a broader set of abnormalities. However, “there is a tremendous amount we have to learn before that goal is realized and feasible,” says Norton. “Often it takes longer than people think.”

Ten years ago, for example, the talk was that comparative genomics hybridization (CGH), used in in cancer, would be applied in the prenatal setting. “Everybody was saying we will be doing that in five years, and here it is 10 years later and it’s just on the brink,” she says. “There are so many [genetic variations] of uncertain penetrance, uncertain expressivity – including in a fetus,” says Norton. “I think the more we apply these tests the more we realize our view of how it all works is very much an oversimplified view. There’s a need for cautious progress.”

A Combination Of Competition And Confusion

Unlike CGH, NIPT has leapt onto the scene and there’s no putting the genie back in the bottle. Hence the alarms being sounded by some of those closest to the clinical management of pregnancy, who are charged with balancing the benefits of more and better genomic information against any mistaken assumptions that the information will bring absolute clarity. The geneticists also need to remind women that although the NIPT companies are marketing aneuploidy screening, it is not the be-all and end-all of pregnancy concerns: physicians and patients must avoid the “streetlight effect” of focusing on trisomies because that is the area being illuminated – what the technology offers the capability to do.

A combination of factors has emerged that encourage a fast-to-market approach to NIPT, intensifying the competition among the players and perhaps confusing the marketplace. The companies have near-identical test menus, which may not be easily expanded. They attempt to differentiate themselves on the basis of technology, test accuracy, quality, cutoff rates, and the like, but their bottom line message is uniform – that the tests are better than traditional testing. Conversely, while the tests may be good technically and even embraced by professional societies, there’s concern by clinicians over their positioning.

NIPT clearly represents a huge commercial opportunity – it already is, despite the conservative response by some. The companies know that time is of the essence. Microarray technology is fast becoming the gold standard for diagnostic testing for aneuploidies and other genetic disorders, now bolstered by the Wapner NICHD study results. In time, it could even be done noninvasively. And if going beyond aneuploidies to identify other, even more rare chromosomal abnormalities proves feasible via the sequencing of cell-free fetal DNA, running the validation studies to establish clinical benefit would be problematic due to the costs involved to gather patients to power such trials, which must be done disorder by disorder.

Eventually, aneuploidy testing in kit form could make its way to the market, which would completely change distribution and pricing to the detriment of today’s players (except Verinata/Illumina). NIPT has the advantage of being one of the only large-market areas in clinical genomics where the correlation between a single marker (in this case, chromosomal copy number) and disease is absolute. But for the current players and their technologies, that advantage may not be sustaining.

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