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Cell-free DNA screening blooms in expansion to low-risk pregnancies

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Anne Paxton

March 2017—Something about having the letters “DNA” in a test’s name may make the test seem like the last word, the key to a black-and-white, definitive diagnosis. That connotation has been problematic for cell-free DNA sequencing used for noninvasive prenatal testing, because the test is not intended or designed for diagnosis, but only for screening. It’s for that reason, in fact, that some maternal-fetal medicine specialists and clinical geneticists prefer to use the term “noninvasive prenatal screening,” with the acronym NIPS.

By either name, NIPS is on a roll. The cfDNA test, which detects common aneuploidies in chromosomes 13, 18, and 21 (Patau, Edwards, and Down syndromes), as well as sex chromosome aneuploidies, first became clinically and commercially available in the U.S. in 2011, says Diana W. Bianchi, MD, director of the Eunice Kennedy Shriver National Institute of Child Health and Human Development. “Since then, it has been the fastest-growing genetic test in medical history. Well over 2 million women have undergone noninvasive prenatal screening worldwide—and that’s despite the fact that it’s not really considered standard of care yet; it’s considered an alternative option.”

Ted Snelgrove, chief business officer of the health technology company Counsyl, which offers a NIPS test, says the current growth in that testing is about 25 percent per year. “We anticipate the market in the U.S. to be between 1 million and 1.2 million tests in our sector.” He explains that projection by noting that of the 4.1 million pregnancies each year in the U.S. that come to term, about 3.3 million will be classified as average risk, while 800,000 will be classified as high risk. “Of the high-risk cohort, approximately 60 percent of them will receive NIPS. Of the average-risk cohort, we’ve estimated it’s closer to 18 percent.”

Snelgrove

Snelgrove

The momentum for this kind of test volume is unusual, in Snelgrove’s view. “Usually, innovative tests like NIPS are introduced to physicians, who then offer it to patients, but in this case, patients seeking NIPS also drove rapid adoption. Doctors and insurers are just starting to come online with the idea of this screening for average-risk women. But the women themselves have been pursuing this.”

Counsyl was founded in 2007 to offer “clinically actionable genetic testing,” Snelgrove says, and added NIPS in late 2014 when it partnered with Illumina. “By and large, we’ve taken over their noninvasive prenatal testing. We’ve tweaked it to be a little more precise and to bring the cost down,” Snelgrove says.

Although NIPS-positive results must be confirmed by a diagnostic test like chorionic villus sampling or amniocentesis, retrospective studies have shown a significant decline in the number of these invasive procedures, particularly amniocentesis, during the period that NIPS has been available (Khalifeh A, et al. Fetal Diagn Ther. 2016;39[4]:292–296).

At the same time, the American College of Medical Genetics and Genomics notes, “Health care providers and patients have experienced marketing pressure, rapidly evolving professional practice guidelines, and confusion regarding the appropriate role of NIPS in prenatal practice.” Those forces helped spur the ACMG’s recent publication (Gregg AR, et al. Genet Med. 2016;18[10]:1056–1065) of new standards for use of NIPS in the general obstetrical population (updating a 2013 statement on NIPS for high-risk pregnancies).

Dr. Bianchi’s research is part of what has helped expand the use of NIPS in the general obstetrical population. She carried out a study in collaboration with Illumina (Bianchi DW, et al. N Engl J Med. 2014;370[9]:799–808) to address how sequencing of maternal plasma DNA compares with the standard of care, which is currently some variation of serum analyte or serum biochemical testing and nuchal translucency measurement through ultrasonography.

“We were asking: How does cfDNA compare to the standard of care in the same pregnant women? And what’s the performance in a general obstetrical risk population? Our study showed that cfDNA performed significantly better than the standard of care in terms of positive predictive values: 63 percent in a low-risk population, compared with only 4.2 percent using current standard screening for all pregnant women,” Dr. Bianchi says.

But the spike in orders for a test that has been available for barely five years came as a bit of a surprise to Dr. Bianchi. She credits social media, in part, for the increase. “It was almost a perfect storm. The test became available at more or less the same time that social media became active, so pregnant women were talking about it. In addition, this test, unlike prior genetic tests largely developed by academia and then transferred to industry, was intended for clinical use by the commercial sector from the very start. So there was the whole industry marketing piece as well.”

The four leading U.S. companies offering NIPS use slightly different technologies for their tests, all of which are laboratory developed; none are approved by the Food and Drug Administration. “Two are similar: Both Illumina and Sequenom use whole genome sequencing. Ariosa Diagnostics, which offers the Harmony test, uses a targeted approach on a microarray, and Natera’s Panorama test uses targeted sequencing employing single nucleotide polymorphisms. But there are dramatic differences in test failure rates among the different technologies,” she says.

Some NIPS laboratories now offer screening for microdeletions, which has much lower positive predictive values, Dr. Bianchi points out. “The PPVs are generally somewhere between zero to five percent for many of the conditions being tested. So there is a concern that all of the gains we’ve made with testing for the common whole chromosome aneuploidies might be lost with the low PPVs for copy-number variant testing.” Nevertheless, she expects that NIPS will eventually become a first-tier screen for common chromosome aneuploidies, if costs come down and if insurance will cover it.

When NIPS first came on the scene, it was primarily offered to high-risk patients—women age 35 or older at time of delivery, who had a positive maternal serum screening result, an ultrasound examination suggestive of trisomy, or who had a previous child or fetus with a chromosomal abnormality. “That was the bulk of the patients, and certain test providers would even refuse a sample if it did not come from a high-risk patient,” says Charles M. Strom, MD, PhD, vice president of genetics and genomics at Quest Diagnostics.

But about two years ago, “OB/GYNs began to demand the test become available for all patients because it’s so much better than maternal serum screening in terms of its PPV. They had to deal with maternal anxiety that surrounds an abnormal serum screening result, which occurs in about five percent of women. Among these women, the PPV was about 10 percent,” Dr. Strom says. “You end up doing a lot of amnios, and the amnios are mostly normal.” With NIPS, the PPV can be up to 80 percent, he says, “so instead of doing 100 amnios and having only 10 positives, you do 100 and have 80 positives. Clinicians were much happier with that [DNA-based] test.”

Much confusion surrounds the PPV numbers, however, and Dr. Strom decided he needed to initiate a study. “I felt that the public and physicians in general needed to be educated on the difference between false-positives and PPV so they wouldn’t over-interpret the test, and I also wanted to see how the test was performing in the real world.”

As use of NIPS expands to the low-risk population, that will change the PPV of the test, because PPV depends on test performance and the prevalence of the disorder, he explains. “So if you’re doing the test on a high-risk population, the prevalence will be highest; it will be lower in low risk. So you’d expect the PPV to decrease.” Clinicians need to be aware of what PPV is and how it’s calculated, he notes, so they can inform their patients.

Dr. Strom’s study was published in 2015 as “Discordant noninvasive prenatal testing and cytogenetic results: a study of 109 consecutive cases” (Wang JC, et al. Genet Med. 2015;17[3]:234–236). He reported finding significant discordance between conventional cytogenetics as a reference standard and positive NIPS findings. “I was not surprised at all by that finding, given the sensitivities and specificities of the assays. I have to say: This is a phenomenal test. It is performing at a very high level. It’s just looking at things with a very low prevalence. And when you’re doing that, even if you have specificities of more than 99.9 percent, your PPV is still not that good.”

There are also biological reasons for the PPV to be low—in particular with trisomy 13. “There’s a phenomenon called confined placental mosaicism where the placenta has trisomy 13 but the fetus is normal, so you will get a false-positive for trisomy 13 with NIPS, but it’s not the fault of the test.” This occurs fairly commonly, Dr. Strom says. “One thing we’ve learned over time with this new technology is that maternal variants can lead to false-positives.”

Maternal microduplications can also affect test results. “We didn’t know about them before we started doing NIPS,” Dr. Strom says. “It turns out that women [as well as men] have duplications of parts of their chromosomes that have no effect on their health; they are just there as excess baggage, if you will—until they have NIPS, and if they have these duplications on chromosome 13, 18, or 21, it has the potential to create a false-positive. Ninety percent of circulating DNA is maternal, so if you just have a little bit of the chromosome mutated, it can be enough to skew the whole test to make it appear that she’s carrying a fetus with a trisomy.” But knowing about these maternal microduplications “allows you to increase the sensitivity/specificity of the test,” he says.

These factors are all the more reason, he adds, why clinicians should take a cautious look at NIPS datasets and their sources to appreciate the subtleties of the testing. “Noninvasive prenatal testing is a wonderful test, but it is not perfect yet. It is screening, and the PPV is getting up there, but even at 98 percent, that means there is a two percent chance the fetus won’t be affected, and that’s what we need to emphasize.”

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