Next-gen arrives for next (prenatal) generation

“Those specialists are not used to conducting their practices on the technical details of controlled trials and validation studies. But that’s all in the wheelhouse of pathologists who are actually involved in the laboratory.”

He argues that pathologists need to represent the interests of the health care system in evaluating the usefulness of NIPS assays. For example, although spending on the assays might increase the pathology budget marginally, the total cost to the health care system might drop significantly because of other costs that are avoided as a result of using NIPS.

For Genevieve L. Fairbrother, MD, chief of staff at Northside Hospital in Atlanta, adopting NIPS as a screening test for the general population was a no-brainer. “Nuchal translucency ultrasound evaluation along with a blood draw is not performed until 11 weeks and it provides a 92 percent to 95 percent trisomy 21 pickup rate, whereas the Ariosa Harmony test can be performed at 10 weeks with a 99.9 percent pickup rate and less than .1 percent false-positive rate.”

“Before, if I had a thousand pregnant patients 30 years old, I could anticipate that three would be carrying a Down syndrome-affected pregnancy, but current aneuploidy screening would produce an additional 50 false-positives. From the start, I was on board with the noninvasive screening test and its one in a thousand false-positive rate.”

In her view, it doesn’t make sense that the test would be somehow better for a high-risk person and less valuable to a patient at low risk. “Why would a 22-year-old not be offered as accurate a test as a 35-year-old, especially if there was no risk to the pregnancy?”

Northside, a community hospital system that does 18,000 deliveries per year, was a beta site for the screening test, and the high sensitivity/high specificity results found in clinical trials were borne out in her patient population, Dr. Fairbrother says. “I think noninvasive prenatal screening is a game changer. It will become the standard of care, because it doesn’t require any special expertise.”

“It’s a simple blood draw with a low false-positive rate, and you get results in the first trimester—you’re not waiting on an ultrasound and more blood work before you come up with something.”

NIPS is expensive, Dr. Fairbrother admits. “But it’s not as expensive on a population level when you consider all those under 35-year-olds with false-positive results who would be offered unnecessary genetic counseling, referral to high-risk doctors, extra ultrasound, and invasive testing.”

“It always takes a while to adopt new technology,” she adds. “But I think once patients understand the test, they’re going to demand it, and it will become universally accepted.” Easing the path will be the fact that the test involves a simple blood draw.

“The standard of care tends to be connected to access. If you’re in a rural or underserved part of the country where you don’t have access to specialized ultrasound, that is a barrier to health care. And the standard tends to the lowest common denominator, because you can’t hold physicians to a standard that is unachievable. But there’s no place in the country that cannot draw blood.”

The test also works for almost all pregnancies including twins and donor egg singleton pregnancies, she notes; the only exception is donor egg twin pregnancies.

Has the availability of NIPS affected genetic counseling in her practice? “We explain to all our patients what screening and invasive tests are available and what it can and can’t tell them. We continue to offer first-trimester ultrasound and maternal serum alpha fetoprotein at 15 to 20 weeks, and a mid-trimester ultrasound as well.”

“We encourage our patients to take advantage of aneuploidy testing, but it is a personal choice.” She is seeing fewer patients who need an amniocentesis or chorionic villus sampling. A high-specificity test is a great boon to patients, she says. “I can tell you fear of a false-positive sends people into a tailspin.”

She particularly likes the Harmony results reporting. “They incorporate in their algorithm the fetal fraction of cell-free DNA along with maternal and gestational age, so by knowing how much of the cell-free DNA belongs to the fetus in maternal bloodstream, it allows them to do what I like to call a binary approach.”

“It’s a very clear ‘It is or it isn’t.’ For example, if you know the cell-free fetal fraction is four percent by analyzing the reference chromosomes, then when you find that the fetal fraction of chromosome 21 is 50 percent increased to six percent, that’s clear. You are more confident it’s an affected pregnancy. It’s not a random cutoff that determines an affected pregnancy.”

NIPS is likely to edge out many tests commonly used now, says Philip D. Cotter, PhD, co-founder of ResearchDx and laboratory director of Pacific Diagnostics Clinical Laboratory in Irvine, Calif. “I see that noninvasive prenatal screening will pretty much kill off the prenatal cytogenetics business, to the same extent that microarray testing has significantly impacted constitutional blood karyotyping,” he says, noting that over the last five years he’s seen a dramatic shift in referral patterns to microarray as the first test of choice.

“I know there is a lot of verbiage and community concern around just testing high-risk pregnancies with NIPS, but I think realistically this will ultimately become the first prenatal screen.” As far as the potential market for test orders, he thinks, “from a screening perspective, we’re barely getting started.”

The most likely tradeoff for the test makers, Dr. Cotter believes, is to minimize the NIPS false-negatives at the cost of having some false-positives. “No test is perfect, no matter how much we like to think so, and in doing the least amount of harm you’d want to minimize false-negatives, and the only way to do that is probably to adjust the ratio. So you push a few people into invasive procedures, but quite frankly those people were going to have one anyway.”

As to the differing technologies for the NIPS tests on the market, “More than anything, the choice between technological approaches among the test maker is, I think, a price point issue,” Dr. Cotter says. “The sheer amount of sequencing you have to generate is what drives your costs, so by sequencing less, the price point is much lower.”

Dr. Cotter predicts the market will start diverging as more service providers start doing their own testing, just as academic institutions started running their own chromosome microarrays. “At some point, I wouldn’t be surprised if the companies started producing kits.”

“More and more laboratories have next-generation sequencing capability, and I don’t know if it’s viable to put NIPS on a MySeq, but those are about $150,000 each, or $600,000 to $700,000 for a high-throughput HiSeq model, and the average laboratory is not going to be able to go out and buy one.” But inevitably, he says, the larger laboratories and institutions are inclined to internalize those kinds of tests.

Choices about genetic testing can create complex dilemmas for pregnant women. “NIPS is bringing a lot of ladies in who would not have had an amniocentesis but are still very anxious about the possibility of having a baby with Down syndrome,” says Dr. Maher.

“A lot of times they say, ‘I’m not going to terminate so I’m not doing an amnio, but I will have a lot of sleepless nights staring at the ceiling tiles, hoping the baby is okay.’ This blood test may be a way to give them a window into what’s going on with the baby, because it’s the not knowing for sure that drives people crazy.”

The termination issue is linked to all prenatal diagnostic testing, Dr. Maher emphasizes. “The information doesn’t trigger termination, it triggers a discussion between the doctor and the patient and the family. And by getting more precise information in the hands of patients at a much earlier gestational age if they do decide to terminate, it’s potentially a lot safer for the pregnant mom.”

However, even more than previous prenatal testing, NIPS forces medical providers to “step up our game” on pretest counseling, Dr. Maher adds.

In offering NIPS, Children’s Hospital Vanderbilt in Tennessee has followed most institutions in choosing to focus on high-risk patients, says Martha Dudek. She notes that the Maternal Fetal Medicine Society and the National Society of Genetic Counselors have statements saying the screen is not appropriate for the average-risk population, which as a rule does not receive genetic counseling either.

But there’s been a large uptake by patients who are offered NIPS. It has allowed a lot more Vanderbilt patients the opportunity to have more information in a pregnancy that’s been identified at risk than previously.

“Abortion is not really readily available in Tennessee for post-16-week pregnancies, so for amniocentesis that would pretty much rule out termination, but with NIPS they can get a diagnosis earlier. A lot of patients in this population in the South do not consider interruption of pregnancy an option, but they would still like information to be better prepared for the pregnancy,” she says.

With any test there are always challenges for a genetic counselor, Dudek adds. “I think we’ve been able to implement a good protocol to address the benefits and limitations of all patient options. But one challenge has been patients who have an ultrasound abnormality or other pregnancy anomalies.”

“In those cases, an invasive test like amniocentesis microarray is really a much better option in terms of informativeness, but if the patients are aware of the noninvasive testing, it’s hard to get across the limitations of it and why it is not a good option for them.”

On the other hand, “Patients are seeking reassurance that most likely the pregnancy is healthy, and with this test I have a 99 percent chance of telling them the baby doesn’t have a problem, which can really lessen a lot of stress for them and may even have positive effects on the pregnancy.” Just learning of an abnormality early on can help them be better prepared if they continue the pregnancy, she adds.

The average two-week turnaround for NIPS can be something of a concern, Dudek says. “Anytime you are talking to a patient about a pregnancy, it’s always ideal to give results sooner, ideally the next day, but I don’t think it’s realistic.”

“The sequencing alone takes three days, then there’s logistics of getting the sample to them and interpretation. A week is pretty darn good. With an amniocentesis, on the other hand, if you have FISH, you can have results within 24 to 48 hours.”

It took about a year after NIPS became available for the insurers to start agreeing to reimburse for the test, Dudek notes. “Reimbursement is extremely important to patients, and since then it’s been a lot easier to reassure patients they will have coverage,” since the out-of-pocket cost can be as high as $3,000 for patients whose policies exclude NIPS.

Because of NIPS’ high level of accuracy, she feels even more strongly about the importance of written consent. “While it’s a blood test, patients really need to understand exactly what the test is testing for and be sure they really want that information, because there’s no turning back once the information is there. But it’s completely optional for them to have the test.”

It’s equally important for providers to have a good understanding of the merits and possible drawbacks of the test too. “It’s new and challenging, and it’s changing. That’s why it would be very difficult for a provider to be on top of it at the same level as a specialist in prenatal genetics. Fortunately, a lot of the companies do have medical liaisons that are genetic counselors whom they can call as resources.”

NIPS is likely the first major step toward eventual application of whole fetal genome/whole fetal exome sequencing, according to the American College of Medical Genetics and Genomics. But in the near term, the obvious first improvements in NIPS will be ones of content, Dr. Cotter says.

“As the price point lowers, you can afford to do more sequencing and more targeted approaches, and you could start to add in some of the other less common but still not insignificant aneuploidies. At some point, you could step it up and start thinking of the test as a low-density array. If you had appropriate targets every five megabases across the genome, you could start to replicate what a karyotype gets you.”

“Noninvasive prenatal screening is a surprisingly robust and very viable alternative to some of the current prenatal procedures, depending on the patient’s indications,” Dr. Cotter says. “For more advanced maternal age, it’s probably a better idea to go down the karyotype microarray route because you want to rule out a lot of smaller rearrangements. But for the general population, I think this is an amazing technological advancement, and really an impressive screen.”

“We’re now at the level of information we can get with 25 or 30 million copies of the DNA,” Dr. Maher points out. “But if you move that up to a billion copies of the DNA, now you can tell not only is there a whole extra chromosome or one missing, but you can get what are called sub-chromosomal deletions or duplications, and those may be linked to certain abnormalities.”

“So that’s the next step: basically to do this array CGH [comparative genomic hybridization] on the NIPS specimen. And it’s been done. It just requires a 10-fold increase in the number of sequences you look at to have the mathematical precision to say whether or not there’s something there.”

“But if you have a family history of a previous child with Angelman syndrome or a mom carrying a gene for DiGeorge anomaly, it will probably be technically feasible, in the not-too-distant future, to do deep sequencing to actually detect these submicroscopic copy number variations. And you wouldn’t be able to get that information from a straightforward karyotype from just amniocentesis.” This advance is already technically feasible, and he predicts in five years or sooner, the cost will have dropped enough to make it economically feasible too.

“NIPS is just another tool in our armamentarium. But it’s an extraordinarily sensitive and specific tool,” Dr. Maher emphasizes. And NIPS is evolving rapidly. In just the past 18 months, he notes, citing the Verifi test as an example, new reagents and improvements have substantially reduced the cost of reagents and improved the fidelity of the test. “So I think we will eventually get to the point where if you see an abnormality and your standard test is normal, this test to give you a ‘deeper peek’ will be part of the package.”

Anne Paxton is a writer in Seattle. As part of the molecular pathology checklist, CAP’s Laboratory Accreditation Program has adopted new requirements for maternal blood screening to detect fetal aneuploidy using NGS technologies. The checklist additions address information required on requisitions, quality control, monitoring, and reporting. More details on the new requirements will appear in an upcoming issue of CAP TODAY.