Category Archives: Chromosome 21

Chromosome 21

Cell-free DNA screening tests in the general obstetrical population


DNAIt has been several years since cell-free DNA (cfDNA) tests for the detection of fetal aneuploidies became available. The first clinical studies of these tests were reported in women who, because of age or other reasons, were already at increased risk of having an affected pregnancy (i.e. “high risk” women). While these studies demonstrated the superior performance of cfDNA tests compared to traditional biochemical tests, their application to women at low risk was not encouraged because of lack of evidence regarding how well they would work in that population. A recent report on cfDNA screening tests in the general obstetrical population now provides much needed evidence.

Investigators at Brown University described several clinical utility aspects of cfDNA screening for common aneuploidies through the implementation of a statewide program called DNAFirst that offered cfDNA screening tests to the general pregnancy population in the state of Rhode Island. The clinical utility aspects that were investigated were a comparison of screening uptake rates before and after the DNAFirst program, an evaluation of a reflexive serum testing protocol for cfDNA tests that failed to produce a result, and explored women’s decision-making.

Over 11 months, 2,681 women agreed to undergo screening through 72 providers. Prior to undergoing testing, the women received information about cfDNA testing by primary obstetrical care providers. The median maternal age was 31 years and 79% of the women were younger than 35 years of age. There were 16 positive (i.e. abnormal for trisomy 21, 18, or 13) cfDNA results, 12 of which were confirmed as true positive and 4 of which were false-positive. 2,515 women had a negative screening result and all were true-negatives. 150 tests failed to produce a result (none of which were known to have trisomy). Collectively, these data produced a sensitivity of 100%, a positive predictive value of 75% and a false-positive rate of 0.15%. By comparison, the most effective biochemical screening test (the Integrated test) has a 90% detection rate, a 3% false-positive rate, and a positive predictive value of only 6%.

A small number of women who participated in the study (113) completed a survey asking them about their understanding of cfDNA testing. Women reported receiving information from their care provider in 9 minutes or less. While 85% understood that the test identified Down syndrome, 15% incorrectly thought it identified all genetic problems. 79% understood that a negative result did not rule out Down syndrome but 13% thought it did. These survey results suggest that most women do understand the basic concepts of cfDNA screening.

The study’s authors concluded that cfDNA screening tests perform very well in the general pregnancy population and that women understand the basic concepts of screening. Further, the tests were easily incorporated into routine practices. They encouraged clinical laboratories to offer cfDNA screening tests to improve access to better aneuploidy screening for the more than 2 million pregnant women in the United States who choose to undergo such testing each year.

Conventional aneuploidy screening remains “most appropriate” choice for general population


OpinionThe American Congress of Obstetricians and Gynecologists (ACOG) have updated their guidance on cell-free DNA (cfDNA) screening tests for fetal aneuploidy. In it, they state that any patient (i.e. women at high-risk OR low-risk for having an affected pregnancy) may choose cfDNA testing but they caution that conventional screening tests are more appropriate. This document replaces an earlier opinion, published in 2012, which clearly stated that cfDNA screening tests should not be offered to the general obstetrical population because they are considered to be at low-risk.

So ACOG went from recommending that cfDNA testing not be performed on low-risk women to say that they may choose cfDNA testing. Why the subtle change? Well, as ACOG correctly notes, the landscape of cfDNA is changing rapidly. New studies are published frequently and those that have examined the performance of cfDNA tests in  low-risk women have reported that the test performs just as well in them as it does in high-risk women.

However, they make an important point about a metric that doesn't get the attention it deserves. The positive predictive value (PPV). See here for background. Because the prevalence of fetal aneuploidy in low-risk women is lower than it is in high-risk women, a "positive" or "abnormal" test result in low-risk women is more likely to be a false-positive result. For example, a positive result in a 25-year-old woman gives a 33% chance that the fetus is affected but that chance increases to 87% in a high-risk woman.

The report also calls out the "no result" problem. cfDNA tests fail to produce a result in 1-8% of samples tested, usually due to a low amount of fetal DNA in the blood sample. It's becoming clear that women with samples that fail to produce a result are at increased risk of having an affected fetus. According to ACOG, these women she be offered diagnostic testing such as fetal karyotyping using amniotic fluid obtained by amniocentesis.

Other notable points contained within the updated guidance include:

  • Caution about not routinely performing microdeletion screening (offered by some labs) because it has not been fully validated in clinical studies.
  • Clearly indicating that a negative or normal result does not rule out the possibility of an affected fetus.
  • Providing genetic counseling to patients about test limitations and that decisions such as pregnancy termination should not be based on these screening tests.
  • A reminder that cfDNA tests do not screen for neural tube or ventral wall defects

This certainly won't be the final say that ACOG has on cfDNA aneuploidy screening tests. Indeed, they state that "It will be critical to remain abreast of this rapidly changing technology to provide patients with the most effective, accurate, and cost-conscious methods for aneuploidy screening."

Momentum grows for use of cell-free DNA Down syndrome screening tests in all pregnant women


Low risk

The use of cell-free DNA (cfDNA) testing to screen for fetal aneuploidies has been the topic of several posts on this blog. Large clinical studies that have evaluated the performance of cfDNA tests have all arrived at the same conclusion: cfDNA testing is superior to traditional biochemical screening tests for the detection of trisomy 21 (Down syndrome) and other trisomies. However, most of these studies have tested women who are considered to be at high risk (e.g. over 35 years of age or who have had an abnormal biochemical screening test) of having an affected fetus. Fewer studies have evaluated test performance in women considered to be at low risk. Because of limited data in low-risk women, the majority of professional societies recommend restricting the use of cfDNA screening tests to only high-risk women. 

This is certainly going to change, and sooner rather than later.

The New England Journal of Medicine recently published a very large, well-designed study that compared the performance of a cfDNA screening test to a biochemical screening test (the first trimester combined test) in an unselected population of almost 15,841 women.

The results were rather unsurprising. There were 38 pregnancies affected by Down syndrome. All 38 (100%) were identified by cfDNA testing but only 30 (79%) were identified by biochemical testing. While that was a significant difference in the detection rate there was a greater significant difference in the false-positive rates. There were 854 false-positive results from biochemical screening and only 9 from cfDNA screening. These numbers translate into a false-positive rate of 5.4% and 0.06% for biochemical and cfDNA screening, respectively.

As the proportion of true positive results divided by the number of all positive results, the positive predictive value answers the question: "What is the probability of an affected fetus given a positive result?” In this study, these predictive values were 3.4% for biochemical screening and 80.9% for cfDNA screening. Clearly, cfDNA offers a huge improvement.

I must stress (as I’ve done several times before) that cfDNA tests are screening tests. The better performance of cfDNA tests has, unfortunately, created the perception that cfDNA tests produce conclusive results and, as such, are diagnostic tests. This could not be further from the truth. Just as with a positive biochemical screening test, a positive result from cfDNA testing should be followed by invasive diagnostic testing. Consider, for example, that the positive predictive value of the cfDNA test that was reported in this study for the 14,947 low-risk women was 50%. That’s a coin toss! Without a doubt it is vastly better than biochemical screening but no woman should make a decision to terminate her pregnancy based on cfDNA testing alone.

So is cfDNA testing an appropriate Down syndrome screening strategy for low-risk women? Yes, it is. It’s just a matter of time before professional societies recognize that fact. Indeed, the International Society for Prenatal Diagnosis did just that in their new position statement

Stay tuned…

Confusion over NIPT invites catastrophe


Timing is everything. A week after I wrote about false-positive NIPT results, the Boston Globe published an article titled "Oversold prenatal tests spur some to choose abortions" written by Beth Daley of the New England Center for Investigative Reporting. The article describes non-invasive prenatal testing (NIPT) using relatively new cell free DNA tests with a focus on women who have experienced receiving incorrect results.

The article focuses on one woman who had Sequenom's MaterniT21 PLUS test that indicated her fetus had trisomy 18 or Edwards syndrome. She initially considered immediately terminating the pregnancy and her doctor helped her locate a physician who could perform the procedure the next day. Only hours later did her doctor caution her to consider diagnostic testing which confirmed the fetus did not have trisomy 18. Additional cases in the article tell of one woman who experienced a false-positive result (confirmed by diagnostic testing) but so trusted the results of the DNA test that she aborted her pregnancy anyway and a woman who experience the trauma of a false-negative result.

Stories like this indicate a clear lack of understanding regarding the limitations of NIPT and demonstrate that physicians and consumers don't always appreciate the fact that these are screening tests. In a post on its blog about the Globe article, the Society for Maternal Fetal Medicine emphasizes just that by stating "It is important for providers to remember that cell free DNA is a screening test, and does not have the diagnostic accuracy of amniocentesis." They also point out that doctors who order DNA-based screening tests need to understand the test characteristics and they emphasize the role of genetic counseling for women who undergo screening for aneuploidy. The Society's statement was the focus of a follow-up piece by the New England Center for Investigative Reporting.

Whether aneuploidy screening is performed using DNA-based tests or by traditional biochemical screening, it is a screening test. Neither are diagnostic tests. Abnormal results from any screening test must be followed up by diagnostic testing to confirm (or not) the results of the screening test. To be misinformed on this basic fact of laboratory medicine is to flirt with disaster.

The ugly stepsister: false positive NIPT test results


Positive Negative

© Stuart Miles – Fotolia.com

NIPT (non-invasive prenatal testing) continues to get lots of attention lately. Indeed, we've written about it extensively on this blog. None of this is suprising because NIPT is a new technology that is continually evolving. Two years ago, I wrote about NIPT here and provided information showing it's excellent diagnostic sensitivity and specificity. To be clear: these tests are more accurate than traditional biochemical screening for detecting fetal aneuploides but they are still screening tests, meaning that positive (or abnormal) test results must be confirmed with diagnostic testing.

As is commonplace, with time comes experience and the lens of scruitiny has recently been focused on the positive predicitive value (PPV) of NIPT. What's a PPV? It's the proportion of true positive results divided by the number of all positive results. For NIPT testing, it answers the question: "What is the probability that a positive result means that the fetus is affected?" It is very important to stress that the PPV of any test is not intrinsic to the test. The PPV is also dependent on the prevalence of the condition in the tested population. If the condition is very rare in the tested population, then the PPV will likely be low, meaning that a positive result is more likely to be a false positive. The opposite is also true (positive test results are more likely to be "true" when the condition is highly prevalent).

NIPT is done to screen for fetal aneuploidies (extra copies of specific chromosomes) such as Down syndrome (trisomy 21), Edwards syndrome (trisomy 18), and Patau syndrome (trisomy 13). The prevelance of each of these disorders is influenced by the woman's age. As examples, the prevalence of each in a 35-year-old woman with a fetus at 10 weeks’ gestational age is 1:185, 1:470, and 1:1,500, respectively. As you might expect, the less prevalent a condition is, the more likley a positive result will be falsely positive.

This has been demonstrated for NIPT. A study published earlier this year evaluated the concordance of NIPT and cytogenetic results among cases with positive or negative NIPT results. The study examined test results from 109 consecutive specimens that were either prenatally and/or postnatally studied by fluorescence in situ hybridization, karyotyping, and/or oligo–single-nucleotide polymorphism microarray (as the definitive, or diagnostic, test). NIPT testing was performed with the Panorama (Natera, San Carlos, CA), Harmony (Ariosa Diagnostics, San Jose, CA), MaterniT21 (Sequenom, San Diego, CA), or Verifi (Illumina, Redwood City, CA).

The PPV for T21 was highest at 93% followed by a 64% PPV for T18.  The PPV for T13 was only 44%. Given the prevalence of each of these conditions, these data aren't all that surprising but they are still rather alarming. Why? Because several studies have claimed NIPT tests are >99% specific (e.g. ~1% false-positive rate). As the authors of the study described here state: "To an average clinician, the claim that a test is >99% specific leads him or her to expect that the false-positive rate will be <1%."

As I stated this above and in several other posts on this blog (but is worth emphasizing again): NIPT is a screening test, not a diagnostic test and it cannot be considered a replacement for diagnostic testing.

Screening for Down syndrome in the United States


This year marks the 30th anniversary of the use of laboratory tests to screen pregnancies for Down syndrome. The tests have evolved over the last three decades and as have their ability to detect affected fetuses. Currently there are several different lab tests available to screen for Down syndrome during pregnancy, including the recently developed cell-free DNA tests (commonly referred to as non-invasive prenatal testing, or NIPT).

How has the landscape of Down syndrome screening evolved over the last few decades? That was the question considered by investigators of a recent report that sought to determine the number of women screened for Down syndrome in the United States in 2011 and 2012, along with the type of test they received.

The results of this report were based on surveys completed by 168 laboratories that offered Down syndrome screening tests in 2011 and/or 2012 and are rather interesting:

  • Of the 4.13 million pregnancies that occur each year in the United States, approximately 72% are screened for Down syndrome.
  • The most common screening test is the quadruple test (50%) followed by the first-trimester combined test (18%), and the sequential test (14%). The other types of tests (e.g. triple test, full integrated test, and serum integrated test) were less commonly performed.
  • The 6 largest laboratories (those that screened more than 100,000 pregnancies each year) performed 61% of all screening tests.
  • The 32 smallest laboratories (those that screened fewer than 1,500 pregnancies each year) performed only 1% of all screening tests.
  • Between 2011 and 2012, the use of the first trimester and integrated testing increased by 27% and 6% and the use of the quad test decreased by 1.2%.
  • As shown in the figure below, there was an inverse relationship between the percent of laboratories that offered testing only in the second trimester (e.g. triple or quadruple tests) and the number of pregnancies screened each year.

Labs offering only 2nd trimester tests

That last bullet point is an important one because nearly 70% of women have their first prenatal visit in the first trimester and it is recommended that integrated screening be offered at this visit. However, because the number of laboratories offering only second trimester testing is much greater than the number of labs that offer all types of screening tests, this recommendation may not be easily adopted.

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.

Quad vs DNA performance
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 as secondary test

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.

DNA test performance

Table References: Genet Med 2011;13:913-920Genet Med 2012;14:296-305Obstet 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.

Caution
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.

Choosing a screening test for Down syndrome


Last month I wrote about the many different tests used for Down syndrome screening.  Today, there are 6 different screening tests that a woman can choose from should she opt for any testing at all.  Selecting one can't be an easy task for most women and, given the relatively short time that most physicians have to spend with patients today, many simply follow their doctor's advice and don't truly understand their options.

The choice of which Down syndrome screening test to have depends on the answers to a few questions:

  • When during pregnancy does the patient first seek obstetrical care for her pregnancy?

Some screening tests are performed in the first or the second trimester while other tests are performed in both first and second trimesters.  If a woman first seeks obstetrical care after the 14th week of pregnancy then only the second trimester tests can be offered.  That means either the Triple or the Quad tests.  If she presents earlier then all screening tests can be considered.

Nuchal translucency (NT) is an ultrasound measurement of space under the skin behind the fetal neck and is performed only during the first trimester.  An increased NT is strongly associated with Down syndrome and other chromosomal abnormalities.  It's a component of the Combined test, the Integrated test, and the Sequential test.

Chorionic villus sampling (CVS) is the removal of a small piece of placental tissue in order to obtain fetal cells in order to obtain the fetal karyotype.  The karyotype will identify the number of chromosomes the fetus has inherited and is usually only performed after an abnormal Down syndrome screening test.  This procedure is performed in the first trimester because amniocentesis can't be performed this early in pregnancy.

If CVS is not available then options include the Integrated, the Triple, or the Quad test.  If the NT is not available then the choice of screening tests is limited to the Serum Integrated test, the Triple test, or the Quad test (the same is true if both NT and CVS are not available).

  • When does the mother want the results of the screening test?

If the woman wants the earliest assessment of the risk of carrying a fetus with Down syndrome then she should opt for the Combined test.  The test is performed only in the first trimester and results are available sooner than with any of the other test options.  However, the test doesn't perform as well as some of the other tests.  On average, the Combined test detects about 85% of all Down syndrome fetuses at a 5% false-positive rate.

If she's willing to wait until the second trimester for the test results then the best screening test is the Integrated test.  This test gives the highest Down syndrome detection rate (about 95%).  The test can also be performed without an NT (the Serum Integrated test) and will provide the same detection rate with a slightly higher false-positive rate.

If the patient desires early risk assessment but is willing to wait for test results if needed, then the Sequential test may be appropriate.  The Sequential test reports results in the first trimester only if the risk of having a Down syndrome fetus is very high.  If the risk is not high, then results are not reported until the second trimester test is completed.

The table below summarizes the performance of the 6 different Down syndrome screening tests.  The "detection rate" (DR) is the percentage of Down syndrome pregnancies that are correctly identified by the test and the "false positive rate" (FP) is the percentage of unaffected pregnancies identified as abnormal.

Performance of maternal serum screening tests

Selecting a test for Down syndrome screening doesn't have to be complicated.  Patient's should be informed about the pros and cons of each test (and screening tests in general).  It's often said that information is power and, when it comes to Down syndrome screening, women should be empowered to choose the test that is right for them.