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Next-gen arrives for next (prenatal) generation

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Anne Paxton

July 2013—In his 25-year practice career, Texas obstetrician James Maher, MD, has performed several thousand amniocenteses, using a long needle to draw amniotic fluid from the uterus of higher-risk pregnant women to rule out certain fetal chromosomal abnormalities—trisomy 21, or Down syndrome, in particular.

Even in good hands, the procedure can lead to complications, says Dr. Maher, who supervises residents at Texas Tech University.

But with the new generation of noninvasive prenatal screening (NIPS), made possible by next-generation DNA sequencing, there will likely be a lot fewer of the invasive procedures in Dr. Maher’s future, and that of OB/GYNs across the country.

By ordering one of the four screening tests now available in all 50 states, OB/GYNs are finding they can confidently rule out the most common autosomal aneuploidies, trisomy 21, 18, and 13, with a cell-free fetal DNA test of maternal serum as early as 10 weeks of pregnancy—as opposed to the 16 weeks required for an amniocentesis.

“For prenatal aneuploidy screening and detection, noninvasive genetic testing is the most dramatic technological advance in the past couple of decades,” Dr. Maher says, noting that NIPS has a sensitivity of 99.98 percent and is extraordinarily specific as well.

“Instead of saying to a mom, you can either do a non-sensitive, non-specific blood test, the maternal serum test, or do an amniocentesis, now we have something in the middle that is going to give you very precise information without putting the baby at risk. It’s an exponential improvement.”

The sharp rise in the volume of NIPS testing suggests the OB/GYN world shares that view. The culmination of 30 years of research, NIPS has been available only since late 2011, yet tens of thousands of the tests have been performed, and some hospitals report that their amniocentesis rate has already dropped by 40 to 50 percent.

However, genetics experts warn that like all laboratory tests, NIPS must be used with appropriate caution. “While NIPS is a fantastic technology compared to our old methods, the limitations should not be overlooked,” says Martha Dudek, MS, genetic counselor with the Division of Maternal Fetal Medicine at Children’s Hospital Vanderbilt in Tennessee.

As a genetic test, NIPS requires pretest and posttest counseling, and presents clinicians, genetic counselors, and patients with new challenges in understanding test results and weighing them to decide how to manage pregnancies. Cost, turnaround time, negative and positive predictive value, reporting format, informed consent, and confirmation testing throw additional factors into the equation.

In interviews with CAP TODAY, laboratory experts and clinicians explain how they are taking advantage of this prenatal screening breakthrough—and steering through the web of complex medical and ethical issues associated with it.

Four companies currently share the market for noninvasive prenatal tests, which are all laboratory-developed tests for screening and do not have approval of the Food and Drug Administration as diagnostic tests. Sequenom makes MaterniT21 PLUS, which detects X and Y chromosomal material in addition to trisomy 21, 18 (Edwards syndrome), and 13 (Patau syndrome), and was performed more than 61,000 times in its first year, 2012.

Panorama, made by Natera and the most recently launched test (March 2013), uses a proprietary bioinformatics technology called NATUS to test for the same abnormalities. Verinata Health, owned by gene-sequencing company Illumina, makes Verifi, while Ariosa Diagnostics offers the Harmony test.

These platforms vary according to the targets for sequencing: amplified regions throughout the genome, chromosome-specific regions, or single-nucleotide regions, according to the American College of Medical Genetics and Genomics. List prices range from $2,700 for Sequenom’s test to $795 for Ariosa’s Harmony.

Since the 1980s, the most common prenatal screening regimen has combined maternal age, sonographic measurement of the fetal nuchal translucency, and measurement of maternal serum screening markers in the first and second trimesters. And for the vast majority of maternal patients, who are considered average risk, the maternal serum test—testing alpha fetoprotein, human chorionic gonadotropin, unconjugated estriol, and, if it is a quadruple test, inhibin A—continues to be offered routinely.

Dr. Wick

“The NIPS test is validated for high-risk patients—those who are already over 35, who have a positive first-trimester aneuploidy screen or ultrasound abnormalities, or who have a previous pregnancy affected with aneuploidy for chromosomes 13, 18, or 21. At Mayo, we are offering the test to that population,” says Myra Wick, MD, PhD, geneticist and OB/GYN at Mayo Clinic, Rochester, Minn.

The first-trimester maternal serum screening has a relatively high number of false-positive results, especially for women over 40. “So if the woman is 36, I’ll offer her the noninvasive testing, rather than the first-trimester screening.”

The extent of first-trimester screening differs substantially from state to state. In California, for example, under state law, all pregnant women must be offered a first-trimester screening, with the assurance that if an invasive test is indicated later, costs beyond $200 will be covered.

For the population served in Rochester, Minn., on the other hand, “only 25 to 30 percent of our pregnant patients elect for first-trimester screening, while in other parts of the country, that percentage is much higher,” Dr. Wick says.

The response of pregnant women to the noninvasive testing has been enthusiastic. “Patients are very happy with the noninvasive option, and several patients have asked for noninvasive testing at their first OB visit,” Dr. Wick says. Although the test hasn’t been clearly validated in lower-risk populations, she believes the data will come out shortly and probably show that the noninvasive testing has much better sensitivity and specificity than the first-trimester screening test.

Mayo has the capability to do next-generation sequencing, but not in the context of prenatal testing. “There are intellectual property and patent issues associated with NIPS testing, and we are not performing the test at Mayo. We collect the samples on site and send them out for testing. However, the laboratories currently offering clinical NIPS testing may partner with other labs in the future, allowing for ‘in-house’ testing.”

While insurers such as Aetna and UnitedHealth Group are willing to cover the test for higher-risk mothers, others do not, and coverage depends on which state you’re in, Dr. Maher says. “Some states will allow companies to say we will guarantee your out-of-pocket expense is no more than $200; we will work with your insurance.”

“But in other states, and Texas is one, that’s not legal. The insurance company would say you’re giving the patient a better discount than it is getting. So in different states, mothers have to make different calculations.”

Dr. Maher

Despite the tests’ expense, some OB/GYNs who have started employing the tests are confident that noninvasive prenatal testing could soon become a standard part of the test options for all of the nation’s several million annual pregnancies, not just those classified as high risk. The reason is that in the trials completed so far, the NIPS tests are so much more accurate.

“I’m hoping we can offer the test to the general obstetric population in the next year or so,” says Dr. Wick. Dr. Maher also supports widening the target market for the test.

Historically, he notes, “The way of guidelines has been to pick on moms when they turned 35. It’s like the warranty expired and you’re old; you need an amniocentesis.” In 2007, however, the American College of Obstetricians and Gynecologists came out with revised guidelines saying every woman is entitled to be offered prenatal screening.

Saying that every woman should be offered aneuploidy screening doesn’t mean they have to have it, Dr. Maher adds. “Your job is to basically be the waiter and present the menu; it’s up to the woman to decide if any of those options meet her needs.”

Next-generation sequencing is what allows NIPS tests to detect chromosome abnormalities. At Illumina, for example, “They have this million dollar machine called an autosequencer, which does DNA sequencing through synthesis,” Dr. Maher says.

“NGS is a variation on the old Sanger sequencing where you have a strand of DNA that you want to sequence. You get this soup of cell-free DNA from the maternal serum and you take and anneal it or staple it down to the substrate, so it has fragments of DNA that are 150 to 170 base pairs long, and the machine automatically mixes in these reagents and can sequence these little strands of DNA one bit at a time.”

“After 25 or 35 cycles of sequencing, they have this humongous terabyte of information, and using very fast computers and very clever algorithms and having the DNA code, which was cracked with the human genome project, they can say here’s what the sequence is from the start of chromosome 1 all the way to the very end of the XY chromosome.”

“So this magical machine takes a 30-million-piece jigsaw puzzle and puts it back together into a complete replication of the DNA pool you’re looking at, the sample from the mom, through NGS.”

Down syndrome, chromosome 21, makes up slightly more than half of all the chromosome abnormalities. “That’s why most of the tests focus on the sensitivity and specificity of Down syndrome—because that’s where the bang is,” Dr. Maher says. “Then if you add 13, 18, and the sex chromosome aneuploidies, you get up to about 85 percent, and the other 19 chromosomes combined make up 15 percent of chromosome abnormalities, which are rare and almost always lethal.”

Since cell-free DNA sequencing produces 30 million copies of the sequence, “if you find that the number of copies that map to chromosome 1, 2, 3, 4, 5, and on up are all coming up with a certain number and when you get down to chromosome 21 you’re seeing 10 percent more of that than all the other ones, you can say this is statistically significantly unlikely.”

“There’s less than a one in 10,000 chance that this 10 percent excess in DNA at chromosome 21 is just a statistical fluke. The much more likely answer is there’s too much DNA there because the baby, who is the contributing factor for the extra DNA, has an extra copy of chromosome 21, and this baby has Down syndrome.”

As the clinical trials have revealed, there are interfering factors that can throw off results. “It’s an overwhelmingly reasonable assumption that most moms are normal, but it’s not impossible for the mother to carry an abnormal quantity of certain chromosomes from tumors, or from what’s called mosaicism.” Mosaicism is where either the mother, baby, or placenta carries some cells with a different chromosome number than the majority of the cells. “For instance,” Dr. Maher says, “if 99 percent of the cells sampled carried 46 chromosomes, but one percent of the cells had an extra copy of a chromosome, the tissue would be mosaic. The cell-free DNA test could pick up a mosaic and report as ‘aneuploid’ as a result of the low quantity of abnormal cells.”

The research arm of ARUP Laboratories has been involved for several years with researching cell-free fetal DNA, Elaine Lyon, PhD, medical director of molecular genetics, says. “We’ve been watching the field unfold, and we were involved with contributing samples to a large prospective study of high-risk pregnancies, although we haven’t brought the test in clinically yet.”

Dr. Lyon

NGS has been necessary to develop noninvasive aneuploidy screening, Dr. Lyon believes. “Scientists have tried several methods that could work, but they didn’t fit well in the laboratory workflow. Even though NGS is still relatively new within the clinical laboratory, it is fitting within a workflow.” Eventually, she thinks ARUP Labs will want to bring NIPS in-house, but for the moment it seems to make better sense to let the technology mature a little in the field.

“I don’t think NIPS is replacing the serum testing for the moment. Right now it’s being used in conjunction with serum testing, so the results showing a high risk typically would have amniocentesis. NIPS is helping to reduce the number of amniocenteses, which I think is one of the reasons for using it.”

“But the results so far from this noninvasive testing have a very high sensitivity and specificity, even though it’s still considered a screening test.” She expects that in a few years NIPS will become diagnostic and receive approval from the Food and Drug Administration.

“I think NIPS is a phenomenal development in terms of patient care in maternal fetal medicine,” says John C. Spinosa, MD, PhD, medical director of Scripps Medical Laboratories in San Diego and former medical director of Verinata Health. “It gives an answer that patients can actually use in pregnancy determinations, but also, by letting a whole population of high-risk mothers stop being exposed to a marginally risky procedure, amniocentesis, it heralds the need we talk about in medicine, of using our resources to take care of patients in the best and safest way possible while still paying attention to resources.”

He believes Verinata, for example, has done a good job by emphasizing that clinical data about its test Verifi so far only relates to high-risk patients. “I’m pretty confident Verifi will end up working well as a general tool, but you have to pay attention to how it’s brought out and performance parameters.”

Sensitivity and specificity of a test are independent of the prevalence of a disease or condition, so they can be high while positive or negative predictive value may not be. “You can think that a test is performing well when it’s just a matter of the prevalence of disease in a population, so it’s important to roll out the test conservatively where you can actually see the studies to back it up.”

On this score, the Verifi test has so far been performing with extremely high accuracy. “The performance is not at the level of karyotyping, but it’s very close to that level and will reach it over time as the test evolves,” Dr. Spinosa says. That there are already four companies offering NIPS, he adds, “probably indicates there’s a real need for safer prenatal testing that’s more specific. It’s not an artifactual market.”

Dr. Spinosa

Pathologists need to be much more aware of NIPS, in his view, even if they are not performing the tests now. “One reason is that it makes sense that the initial marketing has been geared to maternal fetal medicine, but the differences among the tests and the reasons why you might argue one is better or not are entirely of non-interest to maternal fetal medicine.”

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