Sequencing-Based Tests to Determine Fetal Trisomy 21 from
EXECUTIVE SUMMARY
Background. Fetal chromosomal abnormalities occur in approximately 1 in 160 live births. The majority of fetal chromosomal abnormalities are aneuploidies, defined as an abnormal number of chromosomes. The trisomy syndromes are aneuploidies involving 3 copies of one chromosome. Trisomy 21 (Down syndrome), trisomy 18 (Edwards syndrome), and trisomy 13 (Patau syndrome) are the most common forms of fetal aneuploidy that survive to birth. The most important risk factor for fetal aneuploidy is maternal age, with an approximate risk of 1/1600 at age 15 that increases to 1/28 by age 45.
Current guidelines recommend that all pregnant women be offered screening for aneuploidy before 20 weeks of gestation, regardless of age. Combinations of maternal serum markers and fetal ultrasound done at various stages of pregnancy are used, but there is not a standardized approach. The detection rate for various combinations of noninvasive testing ranges from 60-96% when the false-positive rate is set at 5%. Noninvasive tests are not sufficiently accurate to confirm the diagnosis of aneuploidy. Because of the imperfect sensitivity of noninvasive screening strategies, some cases of aneuploidy will be missed. In addition, the suboptimal specificity of testing, combined with a low baseline rate for aneuploidy, means that the majority of patients who have an invasive procedure do not have aneuploidy.
Direct karyotyping of fetal tissue obtained by amniocentesis or chorionic villous sampling (CVS) is therefore required to confirm that aneuploidy is present. Both amniocentesis and CVS are invasive procedures and have an associated risk of miscarriage. A new screening strategy that reduces unnecessary amniocentesis and CVS procedures and increases detection of aneuploidy has the potential to improve outcomes.
Fetal cell-free DNA fragments can be detected in plasma of pregnant women. As early as 8 to 10 weeks of gestation, these fetal DNA fragments comprise 6 to 10% or more of the total cell-free DNA in a maternal plasma sample. Massively parallel sequencing (MPS; also known as next generation or “next-gen” sequencing) can be used to design assays for prenatal diagnosis of fetal aneuploidy; the first proof of principles studies were published in 2008. DNA fragments are first amplified by PCR; during the sequencing process, the amplified fragments are spatially segregated and sequenced simultaneously in a massively parallel fashion. Sequenced fragments can be mapped to the reference human genome in order to obtain numbers of fragment counts per chromosome,. Alternatively, chromosome-targeted sequencing can be used, which obviates the need for mapping to the reference human genome.
The sequencing-derived percent of fragments from the chromosome of interest reflects the chromosomal representation of the maternal and fetal DNA fragments in the original maternal plasma sample. Additionally, in a euploid individual (e.g. the woman from whom the plasma sample was taken), the proportional contribution of DNA sequences per chromosome correlates with the relative size of each chromosome in the human genome. Any detectable difference from the euploid mean for each chromosome of interest is determined for the sample. A predetermined cutoff identifies aneuploid samples by chromosome. Thus, the technology must be sensitive enough to detect a slight shift in DNA fragment counts among the small fetal fragment representation of an aneuploid chromosome against a large euploid maternal background.
Objective. The overall objective of this Assessment is to determine whether nucleic acid sequencing-based testing for fetal aneuploidy using maternal serum improves outcomes of pregnancies screened for aneuploidy, compared to current testing strategies.
Search strategy. PubMed and EMBASE medical literature databases were searched for articles published in the last 5 years; limited to English language publication in human populations. The search was updated August 15, 2012. Several search terms were combined, such as ‘trisomy, ‘aneuploidy,’ ‘sequencing,’ ‘prenatal diagnosis,’ ‘chromosome 21’ [or 18 or 13], ‘cell-free DNA,’ etc.
Selection criteria. Included studies had the following characteristics: 1) performed maternal plasma fetal DNA testing of pregnant women being screened for aneuploidy (trisomy 21, trisomy 18, trisomy 13); 2) used the final, ‘locked-down’ version of the sequencing assay that is or is soon to be clinically available and applied all clinical laboratory quality control measures; 3) compared the results of plasma fetal DNA testing with the results of karyotype analysis (or fluorescence in situ hybridization [FISH] if karyotype not possible); and, 4) reported information on sensitivity and specificity, or provided sufficient information to calculate these parameters.
Main results. The sensitivity and specificity estimates of testing for trisomy 21 were uniformly high, ranging from 99.1% to 100%, and from 99.7% to 100%, respectively. Negative predictive values, whether calculated for average or high risk populations, are uniformly high, near or at 100% as is desirable for a screening test. Positive predictive values are 83% and 55% for high and average risk populations, respectively, using test point estimates for sensitivity and specificity. For trisomy 18, the sensitivity ranged from 97.2% to 100% and the specificity ranged from 99.7% to 100%. For trisomy 13 two studies reported sensitivities of 91.7% and 78.6%, specificities were 99.1% and 100% based on a small number of cases.
A simple decision model was constructed to compare the health outcomes of nucleic acid sequencing-based testing with standard testing for fetal aneuploidy. The strategies tested in the model include:
1. A standard screening test followed, if positive, with an invasive procedure (CVS in the first trimester or amniocentesis in the second trimester) for confirmatory karyotyping; standard screening tests chosen for comparison are:
a. Combined screen (first trimester)
b. Stepwise sequential screen
2. Nucleic acid sequencing-based testing as an alternative to current serum screen; if positive, confirm with invasive procedure and karyotyping.
3. A standard screening test; positives followed with sequencing-based testing; positives confirmed by invasive procedure and karyotyping.
The outcomes of interest for this tree are the number of cases of aneuploidy correctly identified, the number of cases missed, the number of invasive procedures potentially avoided (because of normal DNA test results) and the number of miscarriages potentially avoided as a result. The results were calculated for a high-risk population of women age 35 or more, and an average risk population including women of all ages electing an initial screen. For women testing positive on initial screen and offered an invasive, confirmatory procedure, it was assumed that only a proportion would accept. Sensitivities and specificities for both standard and sequencing-based screening tests were varied to represent the range of possible values.
For either high or average risk populations, the second strategy, screening by sequencing-based assay followed by confirmatory testing, detects the most cases. Base case estimates show detection of nearly the maximum possible number of cases for both populations. The improvement over first or second trimester standard screening assays is approximately 5-15% (base case). At the same time, the number of invasive procedures needed is reduced by as much as 90%. The number of miscarriages after an invasive confirmatory procedure in an average risk population is also reduced from, for example, the 16 per 100,000 seen after stepwise standard screening to 1 (a 94% reduction) using base case estimates.
When added after a positive standard screen, a sequencing-based assay also improves the detection rate, although by a much lesser amount. However, combining tests in this way reduces the number of invasive procedures to slightly below the number needed after screening by sequencing alone, and also reduces the number of miscarriages to similar or slightly lower numbers than screening by sequencing alone. These results are seen for both high and average risk populations.
A fourth strategy, not shown in the decision model, is also possible: screening by sequencing-based testing without confirmatory testing. This would take advantage of the high detection rate and avoid the disadvantages of an invasive procedure and consequent risk of miscarriage. For this strategy, the most important information is the false positive rate, which should ideally be zero. However, studies to date report rare but occasional false positives.
Author’s Conclusions and Comment. This Assessment addressed the analytic and clinical validity, and clinical utility of nucleic acid sequencing-based testing for fetal aneuploidy compared to standard screening procedures. There is little information on analytic validity. The available sequencing-based tests have not been submitted to the FDA for regulatory review, and are offered as laboratory-developed tests subject only to laboratory operational oversight under CLIA. In recent years, recommendations for good laboratory practices for ensuring the quality of molecular genetic testing for heritable diseases and conditions under CLIA have been published. However, next-generation sequencing technology in general is new to the clinical laboratory, and regulatory and professional organizations are only beginning to address important issues of methods standardization.
Several studies of assay performance relative to the gold standard of karyotyping in high-risk populations were available. Review of study quality overall found low risk of bias except in the domain of patient selection. A majority of studies reported insufficient information on how patients were enrolled, and/or on reasons for exclusion prior to testing. Risk of bias in this domain was largely unclear due to lack of information. However, the impact on performance characteristics of the assay and ultimately on pregnancy outcomes is likely to be low, with one exception. The single study in an average screening population was judged to have a high risk of bias due to exclusions (some unavoidable) likely to affect case detection. In this study, cases were verified primarily by phenotype at birth from medical records, a poor standard compared to karyotyping.
In general, assays from all three companies currently offering fetal aneuploidy screening by sequencing DNA in maternal plasma show good clinical validity, with high sensitivity and specificity for Down syndrome (T21) and for T18. Few studies reported results for T13 and few cases were available in those that did, making it difficult to characterize overall performance for T13. All calculated negative predictive values for Down syndrome are nearly 100%, close to ideal for screening. Calculated positive predictive values vary considerably. As more experience is gained with testing, it will be necessary to carefully document the false positive rate for each assay.
Determination of clinical utility depends on a comparison with current screening practices and evaluation of impact on the outcomes of case detection, invasive confirmatory procedures required, and miscarriages resulting from invasive procedures. Actual comparative outcomes were not available, but instead were calculated from the summarized data on sequencing-based assay performance, and published data on standard screening performance, patient uptake of confirmatory testing, and miscarriage rates associated with invasive procedures to acquire confirmatory samples.
For each comparison and in each risk population, sequencing-based testing improved outcomes. As an example, if there are 4.25 million births in the U.S. per year (Palomaki et al. 2012) and two-thirds of the population of (~average risk) pregnant women accept screening, then of 2.8 million screened with the stepwise sequential screen, 87,780 will have an invasive procedure (assuming 60% uptake after a positive screening test and a recommendation for confirmation), 448 will have a miscarriage, and 3,976 of 4,200 Down syndrome cases will be detected. Using sequencing-based testing instead of standard screening reduces the number of invasive procedures to 7,504 and the number of miscarriages to 28, while increasing the cases detected to 4,144 of 4,200 possible, using conservative estimates. Another testing strategy is to add sequencing-based testing only after a positive standard screen, which in the prior scenario would decrease invasive procedures further to 4,116, miscarriages would remain the same at 28, but only 3,948 of 4,200 cases would be detected. Thus, while this strategy has the lowest rate of miscarriages and invasive procedures it detects fewer cases than sequencing-based testing alone.
These are likely to apply to lower risk/prevalence populations because negative predictive value changes very little. Positive predictive value changes considerably, however, and confirmatory testing is required for both low and high risk populations. Sequencing-based testing without confirmatory testing carries the risk of misidentifying normal pregnancies as positive for trisomy due to the small but finite false positive rate together with the low baseline prevalence of trisomy in all populations.
While sequencing-based testing appears most effective as a replacement for standard screening, it is not a replacement for standard ultrasound testing. In fact, the first trimester ultrasound scan that confirms gestational age and determines whether the pregnancy is multiple provides necessary information for sequencing-based testing. The ultrasound exam that details the fetal anatomy in the second trimester is important for fetal risk assessment and may detect indications of chromosomal abnormalities in addition to those tested by currently available sequencing-based tests. Sequencing-based testing is also not a replacement for second trimester maternal serum AFP screening for risk of neural tube defects. The replacement of current maternal serum screening with sequencing-based testing would likely be accompanied by operational changes in screening programs and procedures and the need for provider education. Guidance from professional organizations is currently lacking.
Limitations of sequencing-based tests include a test failure rate (due either to a low fetal DNA fraction in the maternal plasma sample or to unexplained assay failure) that may be as low as 1% or as high as about 5% depending on the assay. Test failures require collection and delivery of a new sample, causing delay. In addition, each test (for each company) has a 7-10 day turnaround time for results. Each assay is currently specific for certain aneuploidies, and expansion of services is likely in the near future. For example, Verinata has already added detection of Turner syndrome (monosomy X) to their assay. Published data were too few to evaluate this indication for this Assessment.
Looking toward future developments, there are broader implications for sequencing-based evaluation of fetal DNA in maternal plasma. Currently available tests include RhD blood type, fetal sex determination (clinically useful if, for example, a woman is a carrier of an X-linked condition such that a male fetus would be at risk), and detection of the aneuploidies discussed in this Assessment. However, it may be possible to use the technology to detect microdeletions and single-gene disorders. Moreover, the feasibility of mapping an entire fetal genome using this technology has been demonstrated. In short, an excess of information may be possible. Thus, some have called for “standardized regulations and guidelines that can harness the potential benefits and minimize the risks of noninvasive prenatal testing.”
Based on the available evidence, the Blue Cross and Blue Shield Association Medical Advisory Panel (MAP) made the following judgments about whether nucleic acid sequencing-based testing of maternal plasma meets the Blue Cross and Blue Shield Association Technology Evaluation Center (TEC) criteria to detect chromosomal trisomies 21, 18, and 13 in women being screened for fetal aneuploidy.
1. The technology must have final approval from the appropriate governmental regulatory bodies.
None of the commercially available sequencing assays for aneuploidy has been submitted to or reviewed by the Food and Drug Administration (FDA). Clinical laboratories may develop and validate tests in-house (laboratory-developed tests or LDTs; previously called “home-brew”) and market them as a laboratory service; LDTs must meet the general regulatory standards of the Clinical Laboratory Improvement Act (CLIA). Laboratories offering LDTs must be licensed by CLIA for high-complexity testing.
2. The scientific evidence must permit conclusions concerning the effect of the technology on health outcomes.
Seven studies reported on the performance of DNA sequencing-based aneuploidy screening in singleton high-risk pregnancy populations with invasive confirmatory procedures planned or completed. An eighth study in an average risk singleton pregnancy population primarily compared DNA sequencing-based testing to a less accurate standard, phenotype at birth. The results of these studies provided strong estimates of assay performance characteristics for trisomy 21 and 18, less so for trisomy 13 due to fewer cases. Results for assay performance characteristics compared to the gold standard of karyotyping along with already available evidence on the performance of standard screening panels and confirmatory testing allowed the construction of a simple decision model to compare the health outcomes of nucleic acid sequencing-based testing with standard testing for fetal aneuploidy.
3. The technology must improve the net health outcome, and
4. The technology must be as beneficial as any established alternatives.
Sequencing-based maternal plasma fetal aneuploidy testing reduces the number of invasive confirmatory procedures needed and consequent associated miscarriages, while improving the number of detected cases of aneuploidy compared to standard screening procedures in either high- or average-risk populations of pregnant women.
5. The improvement must be attainable outside the investigational settings.
Four of eight studies were conducted by third-party investigators at multiple clinical locations (13-60 sites) in the U.S. and other countries; all companies’ assays were represented and samples were sent to company laboratories for sequencing-based testing, as would occur for routine clinical test orders. Thus, the test performance leading to improved overall screening outcomes should be attainable outside the investigational settings.
Based on the above, nucleic acid sequencing-based testing of maternal plasma for fetal aneuploidy with confirmatory testing of positive results in both high risk women and average risk women being screened for aneuploidy meets the TEC criteria. Nucleic acid sequencing-based testing of maternal plasma for fetal aneuploidy without confirmatory testing of positive results does not meet the TEC criteria.
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NOTICE OF PURPOSE: TEC Assessments are scientific opinions, provided solely for informational purposes. TEC Assessments should not be construed to suggest that the Blue Cross Blue Shield Association, Kaiser Permanente Medical Care Program or the TEC Program recommends, advocates, requires, encourages, or discourages any particular treatment, procedure, or service; any particular course of treatment, procedure, or service; or the payment or non-payment of the technology or technologies evaluated.
