The Pregnancy Lab

More on noninvasive prenatal testing for fetal aneuploidy

We have written about nonivasive prenatal testing (NIPT) on this blog several times.  Because they are so new, the landscape around these tests is continually evolving.  The American College of Obstetricians and Gynecologists (ACOG) published guidelines on these tests in December of last year.  Just this week, the American College of Medical Genetics and Genomics (ACMG) released its policy statement on the same topic.  Note that the ACMG refers to these tests as "noninvasive prenatal screening" (NIPS) tests to emphasize that this is what they are: screening, not diagnostic tests.

The ACMG calls for caution before these tests become widely integrated into prenatal care due to the current lack of data obtained from prospective clinical trials.  While they acknowledge that NIPS tests have high sensitivity and specificity there are limitations to the technology and false-positive and false-negative results do occur.

A particular concern, and one that doesn't get as much attention as it should, is that most of the fetal DNA in the mother's blood sample originates from the placenta and not the fetus and it may not accurately reflect the fetal karyotype.  They emphasize (as have others), that abnormal NIPS test results must be confirmed by invasive diagnostic tests such as amniocentesis.

The policy statement also lists several limitations to NIPS tests.  Among them:

• They only detect aneuploidies (and some detect sex chromosome abnormalities).
• Certain chromosome abnormalities are not detected.
• The tests take longer to perform and result than more well-established tests.
• Data on the performance of the tests in twin and triplet pregnancies is not well established.

A recent paper published in the journal Obstetrics and Gynecology has a similar ring to it.  The authors make several interesting observations:

• First, they point out that well-established tests were developed in academic settings and came into use gradually and only after independent clinical studies generated data to support their use.  In contrast, NIPT (also developed in academic settings) was quickly licensed to commercial enterprises that have brought them to market without FDA review (as these are "lab-developed tests," FDA appoval is not required).
• From the analytical perspective, there are currently no guidelines regarding quality control and quality assurance for NIPT; a vital component of any lab test.
• The performance of NIPT in actual clinical practice settings (i.e. not a clinical study) is currently not well known or documented.  This is especially true for populations of women that have not been represented in the clinical studies (e.g. woman at low risk for having a fetus with an aneuploidy).
• The more well-established tests are able to detect fetal anomalies besides aneuploidy (e.g. open neural tube defects).

The authors also reflect on how NIPT should be incorporated into clinical care.  They agree with the ACOG recommendations that the tests should not be offered to low-risk women but they go a bit further and state that the most appropriate use of NIPT is as a second screening test used for those who have an abnormal result from convential, more well-established screening tests.  The latter point is something I have commented on before and I could not be in more complete agreement.

Should DNA-based tests for Down syndrome screening replace biochemical tests?

In a previous post I described the clinical performance of DNA-based screening tests for fetal aneuploidies like Down syndrome.  Overall, these tests have excellent detection rates (~99%) with very low false-positive rates (~0.2%).  In other words, these tests are about 99.0% sensitive and 99.8% specific.

With performance like that one might expect these to be considered diagnostic tests.  They are not! Although quite good, test results must not be interpreted as definitive evidence that a fetus does or does not have an aneuploidy.  Recent recommendations from the American College of Obstetricians and Gynecologists (ACOG) are quite clear on that issue.

In those same recommendations, ACOG also states that DNA-based screening tests may be performed only on women who are at increased risk of having a fetus with aneuloidy.  Among the indications listed for women considered to be at increase risk are:

• Maternal age 35 years or older at delivery
• Fetal ultrasound findings suggesting aneuploidy
• A previous aneuploid pregnancy
• Abnormal biochemical screening test results

The ACOG is right to avoid recommending that DNA-based screening tests are acceptable to use regardless of risk factors.  Unfortunately, many women who are not at increased risk are using these new tests as a primary screening test and that's not a good idea.

To understand why, considered a population of 100,000 pregnant women from the general population and assume that the prevalence of Down syndrome is 1 in 500 pregnancies.  That means that there would be 99,800 unaffected pregnancies and 200 pregnancies with Down syndrome.  The table below compares the results of the most commonly used biochemical screening test (the Quad test) to a DNA-based screening test.

Clearly, the DNA-based test has several advantages over the Quad test.  Its positive predictive value is nearly 17 times greater than the Quad's and a positive DNA-based test result substantially increases the odds of having an affected fetus.  So why not use the DNA-based test as a primary screening test?  For the following reasons:

• No studies have been published that have evaluated the performance of DNA-based tests in women who are not at increased risk of having a fetus with an aneuploidy
• DNA-based tests are not widely available
• The time it takes to report results of DNA-based testing is about 3 times greater than it is with biochemical testing
• DNA-based tests are considerably more expensive than biochemical tests
• Relative lack of insurance coverage for DNA-based tests

Until these these limitations can be resolved, it makes more sense to use DNA-based testing as a secondary screening test.  In other words, it is only done after one of the risk factors described by ACOG (above) are met.  Doing so greatly improves the performance of both tests (see figure below).  A limitation of this approach is that the detection rate is that of the biochemical test which is not as high as it is with the DNA-based test.  Still, given the current limitations of DNA-based testing, this 2-step testing approach makes the most sense.

DNA-based tests for Down syndrome screening show excellent clinical performance

The use of biochemical screening tests to identify pregnant women who are at high risk of having a fetus with Down syndrome is well established.  Biochemical screening began nearly 30 years ago and, over the years, the tests have evolved and improved.  Now there’s a new kid on the screening test block and it’s name is DNA.

The discovery of cell-free fetal DNA in maternal plasma in 1997 opened up new possibilities for Down syndrome and other aneuploidy screening protocols.  Rather than rely on biochemical testing to determine a biochemical phenotype, DNA-based tests have been developed that can detect the molecular pathology of aneuploidies (e.g. a fetus that has more than the expected 2 copies of chromosomes 21, 18, or 13; the cause of Down syndrome, Edwards syndrome, and Patau syndrome, respectively).

We’ve written about DNA-based screening tests before (here and here) and described the clinical performance of the Sequenom test.  Now, other clinical performance studies have been published for 3 of the 4 tests that are (or will be) commercially available.  As expected, all of them show excellent clinical performance.  As shown in the table below, the detection rates for trisomy 21 are greater than or equal to 99% with very low false-positive results.  Similar performance has been reported for trisomy 18 and 13.

Table References: Genet Med 2011;13:913-920; Genet Med 2012;14:296-305; Obstet Gynecol 2012;119:890-901

By comparison, the detection rate of the best biochemical Down syndrome screening test (the Integrated test) is very good at 93%.  However, about 5% of all Integrated test results are false-positive.  A 5% false-positive rate may not seem very high but it is.  For example, consider a population of 100,000 pregnant women who choose Integrated testing in the second trimester.  The prevalence of Down syndrome in the second trimester is about 1 in 500 pregnancies so 200 of those 100,000 women will have a fetus with Down syndrome and 99,800 women (100,000 – 200) will have unaffected fetuses.  Of those 99,800 women with unaffected fetuses, 4,900 will have a false-positive Integrated test result.

Because the false-positive rate of the DNA-based tests is so low (about <0.2%), then if those same 100,000 women were screened there would be only 200 false-positive results, a 96% decrease!

Does this mean that DNA-based tests should replace biochemical screening tests?  Probably not but I’ll leave the explanation as to why for my next post.

Molecular testing for Down syndrome: proceed with caution

Ann recently posted about massively parallel genomic sequencing using maternal blood as a
screening test for Down syndrome.  In it, she described a recently published, multi-center clinical study that validated this molecular test in just over 1,600 women.  The test detected 98.6% of the Down syndrome fetuses and had a false-positive rate of 0.2%.  Notably, this performance is considerably better than current biochemical marker Down syndrome screening tests.

However, there are some important limitations to this new test.

1. It’s expensive and available from only one company.  The Sequenom Center for Molecular Medicine, located in Grand Rapids, MI, developed the test and is currently the only place in the US that can do it.  It's marketed as the MaterniT21 test and costs $1,900 for the uninsured and ~$235 for those with insurance.  Biochemical screening tests cost much less.
2. After the blood sample arrives in the lab, test results are available in about 10 days.  Results for biochemical screening tests take about 1 day.
3. The test can only be performed in singleton pregnancies (not twins, triplets, etc).  Biochemical screening tests work best in singleton pregnancies, too, although risks can be estimated from a twin pregnancy.
4. The test fails to work in about 2% of cases, usually as a result of there not being enough fetal DNA detected in the mother's blood.  This is more likely to be the case in overweight women who have a higher blood volume which dilutes the amount of the fetal DNA.  The concentrations of the markers used in biochemical screening tests are also effected by maternal weight but there are effective ways to account for that so that the final result is not affected.
5. The test is only validated for the detection of Down syndrome and not other fetal aneuploidies such as trisomy 18 or trisomy 13.  The test does have the capability of detecting these disorders and, if they are detected, will be reported.  However, because the test has not been thoroughly investigated for detecting T18 or T13 a negative result doesn't rule out their presence.  Biochemical screening tests can detect these disorders although the detection rates are lower than they are for Down syndrome.
6. The test does not detect open neural tube defects (e.g. spinal bifida) that are usually screened for using biochemical screening tests.

I have no doubt that as the technology required to do massively parallel genomic sequencing becomes less expensive, tests like the MaterniT21 will become more affordable and mainstream.  Most of the limitations I described above will also be addressed with time and technological improvements.  As Ann indicated, this is the dawn of a new era in screening for fetal disorders.

Screening for Down syndrome: Beginning of a New Era?

David has written previously about the different types of screening tests for Down syndrome (trisomy 21). Although these tests have certainly gotten better over time, even the best among them is subject to less than perfect sensitivity (90%) and a false positive rate of between 2 and 5%. Given the prevalence of Down syndrome, this means that out of 16 women that screen positive for Down syndrome, only one will carry an affected infant. Therefore, 15 women are unnecessarily subject to potentially dangerous amniocentesis procedure. Recently, there have been reports of some molecular testing that may change the way we screen women for Down syndrome forever.

In 1997, researchers discovered that DNA from a fetus actually circulates in the blood of the mother during pregnancy. It goes away very rapidly after the baby is delivered. This amazing observation meant that it might be possible to diagnose certain diseases in the fetus by simply taking a sample of mom's blood. Since then, many studies have been done to try to develop a method to screen for Down syndrome using maternal circulating fetal DNA.

In 2008, two papers were published, from different laboratories, which described the detection of trisomy 21 using "massively parallel genomic sequencing" (MPGS). In this method, DNA fragments are isolated from a sample of mom's blood. These include a mix of mom's DNA and infant DNA. The ends of the DNA fragments get a small piece of adapter DNA attached to it which allows the fragments to hybridize to a surface coated with the complimentary adapter DNA sequence. All the fragments bound to the surface are then simultaneously amplified. After amplification, the DNA is sequenced. With the help of bioinformatics, the researchers are able to determine what chromosome the DNA fragment came from. The number of sequences that originate from a particular chromosome is counted and tabulated for each chromosome. If a fetus has an extra chromosome, then the percentage of chromosome 21 fragments is higher than expected.  Early studies suggested that this method might work with high sensitivity, but the studies were small.

Recently, a very large validation study was published that demonstrated that this method is not only sensitive, but also had a very low false positive rate. 

The study involved women at high risk of delivering an infant with Down syndrome from 27 prenatal diagnostic centers worldwide. The authors compared fetal karyotyping (the gold standard for diagnosing Down syndrome) to MPGS in 212 Down syndrome and 1484 matched normal pregnancies. Their research demonstrated that MPGS was able to detect Down syndrome with 98.6% sensitivity and only a 0.20% false positive rate. This represents a huge improvement over currently available methods.

Currently, MPGS is only available at specialized centers and is quite expensive. However, with the advent of studies such as this, the availability and price of MPGS is bound to come within reach. Hopefully, within the next 10 years, we will see the end of maternal serum screening for Down syndrome as we currently know it.