Sequencing-Based Tests to Determine Fetal Down Syndrome (Trisomy 21) from Maternal Plasma DNA

Technology Evaluation Center
Sequencing-Based Tests
to Determine Fetal Down
Syndrome (Trisomy 21) from
Maternal Plasma DNA
Assessment
Program
Volume 27, No. 10
April 2013
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) is the most common form of fetal aneuploidy that is associated with survival to birth
and beyond. Trisomy 18 (Edwards syndrome) and trisomy 13 (Patau syndrome) are the next most
common fetal aneuploidy syndromes associated with survival to birth, although the percent of cases
surviving to birth is low and survival beyond birth is limited. The most important risk factor for
trisomy 21, 18, or 13 is maternal age, with an approximate risk of 1/1,600 at age 15 that increases
to 1/28 by age 45.
Current guidelines recommend that all pregnant women be offered noninvasive screening for
trisomy 21 before 20 weeks of gestation, regardless of age. Contemporary screening programs may
also detect trisomy 18 or 13. Combinations of maternal serum markers and fetal ultrasound done at
various stages of pregnancy are used, but there is not one standardized approach. The detection rate
for various combinations of noninvasive tests ranges from 60–96% when the false-positive rate is set
at 5%. Noninvasive screening tests are not sufficiently accurate to diagnose a trisomy syndrome and
confirmatory testing is required. In addition, because of the imperfect parameters of noninvasive
screening strategies, some cases will be missed and the majority of patients who are recommended to
have a confirmatory invasive procedure do not have a fetus with a trisomy syndrome.
Direct karyotyping of fetal tissue obtained by invasive amniocentesis (second trimester) or chorionic
villous sampling (CVS; first trimester) is required to confirm the diagnosis of trisomy. Both amniocentesis and CVS are invasive procedures and have a small but finite risk of miscarriage. A new
screening strategy that reduces unnecessary amniocentesis and CVS procedures (and thus associated
miscarriage) and increases detection of trisomy 21 in particular, and potentially trisomy 18 and 13 as
well, has the potential to improve outcomes.
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Cell-free DNA fragments can be detected in plasma of pregnant women. As early as 8 to 10 weeks
of gestation, fetal DNA fragments (actually derived from the cytotrophoblastic cell layer of the placenta) 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 detection of trisomy 21; the first proof of principle studies were published
in 2008. DNA fragments are first amplified by polymerase chain reaction (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
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.
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Technology Evaluation Center
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 with a normal number of chromosomes (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 trisomy 21 or any other abnormal
chromosome number. 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 trisomy 21 using maternal serum improves outcomes of pregnancies screened for
trisomy 21, compared to traditional serum and ultrasound testing strategies. Additionally, the
evidence supporting similar objectives for trisomy 18 and 13 will be reviewed.
Search Strategy
MEDLINE® (via PubMed) and EMBASE medical literature databases were searched for articles
published in the last 5 years; limited to English-language publication is in human populations.
The search was updated February 26, 2013. 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 trisomy 21, trisomy 18, and trisomy 13; 2) used
the final, ‘locked-down’ version of the sequencing assay that is 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 is
not possible in individual cases), or with phenotype at birth; and, 4) reported information on
sensitivity and specificity, or provided sufficient information to calculate these parameters.
Main Results
The sensitivity and specificity estimates of sequencing-based 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 (pregnant women electing screening) or high-risk (age
>35) populations, were uniformly high, near or at 100% as is desirable for a screening test. Positive
predictive values were 83% and 55% for high- and average-risk populations, respectively, using
point estimates for test 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 three studies reported
sensitivities of 78.6–91.7%, and specificities were 99.1–100% 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 trisomy 21. The strategies tested in the model
include:
1. A traditional screening test followed, if positive, with an invasive procedure (CVS in the first
trimester or amniocentesis in the second trimester) for confirmatory karyotyping; traditional
screening tests chosen for comparison are:
a. Combined screen (first trimester, includes nuchal translucency ultrasound)
b. Integrated screen (first + second trimester serum testing and nuchal translucency ultrasound)
2. Nucleic acid sequencing-based testing in place of traditional serum screening; if positive,
confirm with invasive procedure and karyotyping.
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Sequencing-Based Tests to Determine Fetal Down Syndrome (Trisomy 21) from Maternal Plasma DNA
3. A traditional screening test (first trimester combined screen or first and second trimester
integrated screen); positives followed with sequencing-based testing; subsequent positives
confirmed by invasive procedure and karyotyping.
4. A traditional screening test with performance parameters chosen to allow better case detection
along with an increased false-positive rate; positives followed with sequencing-based testing;
subsequent positives confirmed by invasive procedure and karyotyping.
The outcomes of interest for this decision tree are the number of cases of trisomy 21 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 older, and for 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, according to risk level. 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 3–16% (base case). At
the same time, the number of invasive procedures needed is reduced by as much as 80%. The
number of total miscarriages after an invasive confirmatory procedure in an average risk population is also reduced from, for example, the 22 per 100,000 seen after integrated screening to 4
(an 82% reduction) using base case estimates. Confidence in negative results is high as no more
than 10 of 100,000 (0.01%) screens are false negatives using base case estimates.
When added after a positive traditional screen, a sequencing-based assay does not improve the
trisomy 21 case detection rate. However, fewer invasive procedures are needed than after screening by sequencing alone, due partially to the lower case detection rate. The number of total
miscarriages is reduced to similar or slightly lower numbers than screening by sequencing alone.
These results are seen for both high- and average-risk populations.
Re-interpretation of a first trimester traditional combined test using altered parameters allows an
increased detection rate. Following positive results from such an increased sensitivity combined
assay with a sequencing-based assay could take advantage of the increased detection rate, reduce
the number of sequencing-based tests required, and also reduce invasive procedures and miscarriage rates. This test combination was modeled with final detection rates nearly as good as the
integrated screen alone, but in the first trimester. However, sequencing-based testing alone still
captured the most cases.
Whether sequencing-based screening is used as a replacement for traditional serum screening or
as a follow-on test, there is a large impact on the number of miscarriages of euploid (no trisomy
21) miscarriages following an invasive procedure. With traditional screening in a high-risk population, about 20–30 normal fetuses are lost per 100,000 women screened; using sequencing-based
testing the numbers drop to 2 or fewer. For low-risk women, 10–20 normal fetuses are lost per
100,000 women screened; with the use of sequencing-based testing, the numbers drop to 1 or none.
Another strategy, not shown in the decision model, is also possible: screening by sequencingbased 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.
Authors’ Conclusions and Comment
This Assessment addressed the analytic and clinical validity, and clinical utility of nucleic acid
sequencing-based testing primarily for Down syndrome (trisomy 21) compared to traditional
screening procedures. Detection of Down syndrome cases is the original clinical reason for
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Technology Evaluation Center
testing; standard screening tests may also detect trisomy 18 and 13. Thus, our report is primarily
focused on results for trisomy 21, with discussion of results for trisomy 18 and 13.
There is little information on analytic validity. The available sequencing-based tests have not
been submitted to the U.S. Food and Drug Administration (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. Some were multi-site studies that incorporated specimen collection, transport, and evaluation under conditions simulating real-world clinical testing. 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 companies1 currently offering fetal trisomy screening by sequencing DNA in maternal plasma show good clinical validity, with high sensitivity and specificity for
Down syndrome (trisomy 21) and for trisomy 18. Few studies reported results for trisomy 13 and
few cases were available in those that did, making it difficult to characterize overall performance
for trisomy 13. All calculated negative predictive values for Down syndrome are near or at 100%,
close to ideal for screening. Calculated positive predictive values vary considerably with risk of
trisomy 21 in the tested population. Notably, however, false-positive rates were relatively invariant
across a wide spectrum of T21 prevalence values. 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 for trisomy 21, and published data on traditional 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 and two-thirds of the population
of (~average risk) pregnant women accept screening, then of about 2.8 million screened with the
integrated screen, 74,434 will have an invasive procedure (assuming 50% uptake after a positive
screening test and a recommendation for confirmation), 370 will have a miscarriage, of which 342
will be normal (non-trisomy 21) fetuses, and 3,417 of 3,559 Down syndrome cases will be detected.
Using sequencing-based testing instead of traditional screening reduces the number of invasive
procedures to 6,378 and the number of miscarriages to 32 (after amniocentesis; 14 normal) or
70 (after CVS; 31 normal), while increasing the cases detected to 3,531 of 3,559 possible, using
conservative estimates. False negatives are conservatively estimated at 266 of 2.8 million women
screened (0.01%) and may be lower, indicating that invasive testing after a negative result would
have more risk than benefit.
As this Assessment was in press, a fourth company’s test became clinically available. A supporting “proof of concept”
(author’s words) paper was published, but did not use the final calculation algorithm, so the report was not included as
evidence in this review.
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Sequencing-Based Tests to Determine Fetal Down Syndrome (Trisomy 21) from Maternal Plasma DNA
Another testing strategy is to add sequencing-based testing only after a positive first trimester
traditional combined screen, which in the prior scenario would decrease invasive procedures
further to 3,103, miscarriages would decrease to 34 after CVS (only 1 normal), but only 2,990 of
3,559 cases of Down syndrome/trisomy 21 would be detected. Thus, while this strategy has the
lowest rate of miscarriages of which only one represents a normal fetus, and the lowest rate of
invasive procedures, it detects fewer cases than sequencing-based testing alone. Clearly, a strong
advantage of using sequencing-based assays, either in place of traditional serum screening or as
a follow-on assay, is that miscarriages of normal fetuses during confirmatory invasive procedures
are considerably reduced.
These results 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 strongly recommended for both low- and high-risk populations. Sequencing-based
testing without confirmatory testing carries the risk of misidentifying normal pregnancies as positive for a trisomy syndrome due to the small but finite false-positive rate together with the low
baseline prevalence of trisomy 21, 18, and 13 in all populations.
The decision model discussed in the preceding text was applied only to Down syndrome.
However, based on the assay performance data for trisomy 18, which is very similar to that for
trisomy 21, it is likely the outcome trends would be similar for trisomy 18. The data for trisomy 13
are too sparse for specific conclusions, but there is no biologic reason to suggest outcomes would
be different.
While sequencing-based testing appears most effective as a replacement for traditional screening, it is not a replacement for ultrasound testing. The first trimester ultrasound scan that confirms gestational age and determines whether the pregnancy is multiple also 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.
Limitations of sequencing-based tests include an indeterminate test rate (due either to a low
fetal DNA fraction in the maternal plasma sample, to a deliberately chosen “no call” zone, or to
unexplained assay failure) that may be as low as 1% or as high as about 5%, depending on the
assay. Fetal fraction is determined either in an initial, separate test or as a part of the trisomy test
by all companies. For three companies, the value of the fetal fraction is a quality control criterion.
Below an established cutoff value, the sample is not acceptable for reporting assay results. The
highest indeterminate rates do not reflect poor assay technology, but rather a choice in the case
of one company’s assay to increase the accuracy of positive results by assigning results near the
assay cutoff to a “no call” category. Indeterminate results require follow-up, which could be repeat
testing of a new sample, since fetal fraction increases with time during pregnancy. Currently,
however, there are no data on repeat testing. In addition, repeat testing adds the delay of a new
sample collection in addition to the assay turnaround time for results. Alternatively, patients with
indeterminate results could elect to proceed directly to an invasive procedure and karyotyping.
Each assay is currently specific for certain aneuploidies, and expansion of services is likely in
the near future. For example, two of four companies now report detection of sex chromosome
aneuploidies and a third reports Y chromosome detection for prenatal sex determination (used in
genetic counseling for X-linked disorders). 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
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Technology Evaluation Center
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 non-invasive 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 trisomy 21 in women being screened for fetal trisomy syndromes.
1. The technology must have final approval from the appropriate governmental
regulatory bodies.
None of the commercially available sequencing assays for trisomy 21 has been submitted to or
reviewed by the U.S. 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.
Eight studies reported on the performance of DNA sequencing-based trisomy 21 screening in
singleton high-risk pregnancy populations with invasive confirmatory procedures planned or
completed. A ninth 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. 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 trisomy 21.
3. The technology must improve the net health outcome, and
4. The technology must be as beneficial as any established alternatives.
In a decision model, sequencing-based maternal plasma trisomy 21 testing reduced the number
of invasive confirmatory procedures needed and consequent associated miscarriages, while
improving the number of detected cases of trisomy 21, 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 9 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 trisomy 21 with
confirmatory testing of positive results (as is expected to be performed in a real-world clinical
setting) in both high-risk women and average-risk women being screened for trisomy 21 meets
the TEC criteria.
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Sequencing-Based Tests to Determine Fetal Down Syndrome (Trisomy 21) from Maternal Plasma DNA
Contents
Assessment Objective
8
Background8
Methods14
Formulation of the Assessment
15
Review of Evidence
16
Discussion35
Summary of Application of the
Technology Evaluation Criteria
37
References39
Appendix42
Published in cooperation with Kaiser Foundation Health Plan and
Southern California Permanente Medical Group.
TEC Staff Contributors
Lead Author—Margaret A. Piper, Ph.D., M.P.H.; Co-Authors—Diane Civic, Ph.D., M.P.H.; Mark D. Grant, M.D., M.P.H.;
Frank Lefevre, M.D.; TEC Executive Director—Naomi Aronson, Ph.D.; TEC Director, Technology Assessments—Mark D. Grant,
M.D., M.P.H.; Director, Clinical Science Services—Kathleen M. Ziegler, Pharm.D.; Research/Editorial Staff—Claudia J. Bonnell,
B.S.N., M.L.S.; Kimberly L. Hines, M.S.
Acknowledgments
The authors would like to thank William A. Grobman, M.D., M.B.A., Professor, Obstetrics and Gynecology, Feinberg School of Medicine,
Northwestern University, for his contributions to the research and development of this Assessment.
Blue Cross and Blue Shield Association Medical Advisory Panel
Allan M. Korn, M.D., F.A.C.P.—Chairman, Senior Vice President, Clinical Affairs/Medical Director, Blue Cross and Blue Shield
Association; Steven N. Goodman, M.D., M.H.S., Ph.D.—Scientific Advisor, Dean for Clinical and Translational Research, Stanford
University School of Medicine, Professor, Departments of Medicine, Health Research and Policy; Mark A. Hlatky, M.D.—Scientific
Advisor, Professor of Health Research and Policy and of Medicine (Cardiovascular Medicine), Stanford University School of Medicine.
 Panel Members Peter C. Albertsen, M.D., Professor, Chief of Urology, and Residency Program Director, University of
Connecticut Health Center; Sarah T. Corley, M.D., F.A.C.P., Chief Medical Officer, NexGen Healthcare Information Systems,
Inc.—American College of Physicians Appointee; Helen Darling, M.A., President, National Business Group on Health;
Josef E. Fischer, M.D., F.A.C.S., William V. McDermott Professor of Surgery, Harvard Medical School—American College
of Surgeons Appointee; I. Craig Henderson, M.D., Adjunct Professor of Medicine, University of California, San Francisco;
Jo Carol Hiatt, M.D., M.B.A., F.A.C.S., Chair, Inter-Regional New Technology Committee, Kaiser Permanente; Saira A. Jan,
M.S., Pharm.D., Associate Clinical Professor, Ernest Mario School of Pharmacy, Rutgers, The State University
of New Jersey, Residency Director and Director of Clinical Programs Pharmacy Management, Horizon Blue Cross and
Blue Shield of New Jersey; Thomas Kowalski, R.Ph., Clinical Pharmacy Director, Blue Cross Blue Shield of Massachusetts;
Bernard Lo, M.D., Professor of Medicine and Director, Program in Medical Ethics, University of California, San Francisco;
Randall E. Marcus, M.D., Charles H. Herndon Professor and Chairman, Department of Orthopaedic Surgery, Case Western
Reserve University School of Medicine; Barbara J. McNeil, M.D., Ph.D., Ridley Watts Professor and Head of Health Care
Policy, Harvard Medical School, Professor of Radiology, Brigham and Women’s Hospital; William R. Phillips, M.D., M.P.H.,
Clinical Professor of Family Medicine, University of Washington—American Academy of Family Physicians’ Appointee;
Richard Rainey, M.D., Medical Director, Regence BlueShield of Idaho; Rita F. Redberg, M.D., M.Sc., F.A.C.C., Professor of
Medicine and Director, Women’s Cardiovascular Services, University of California San Francisco; Alan B. Rosenberg, M.D., Vice
President, Medical Policy, Technology Assessment and Credentialing Programs, WellPoint, Inc.; Maren T. Scheuner, M.D., M.P.H.,
F.A.C.M.G., Clinical Genetics and Principal Investigator, Health Services Genomics Program, VA Greater Los Angeles Healthcare
System; Health Sciences Associate Clinical Professor, Department of Medicine, David Geffen School of Medicine at UCLA; Natural
Scientist, RAND Corporation; J. Sanford Schwartz, M.D., F.A.C.P., Leon Hess Professor of Medicine and Health Management &
Economics, School of Medicine and The Wharton School, University of Pennsylvania; Earl P. Steinberg, M.D., M.P.P., Executive Vice
President, Innovation and Dissemination & Chief, Geisinger Healthcare Solutions Enterprise.
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and other subscribers to the TEC Program. The contents of this document are not to be provided in any manner to any other
parties without the express written consent of the Blue Cross and Blue Shield Association.
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Technology Evaluation Center
Assessment Objective
The overall objective of this Assessment is to
determine whether nucleic acid sequencingbased testing for Down syndrome (trisomy 21)
using maternal serum improves outcomes of
pregnancies screened for trisomy 21, compared
to traditional serum and ultrasound testing
strategies. Additionally, the evidence supporting
similar objectives for trisomy 18 and 13 will
be reviewed.
Commercial, noninvasive, sequencing-based
testing of maternal serum for trisomy 21 has
recently become available and has the potential to substantially alter the current approach
to screening for trisomy 21. The current
noninvasive testing strategies are relatively
cumbersome and have suboptimal accuracy.
The imperfect specificity, together with a low
baseline rate of trisomy 21, results in low positive predictive values. As a result, many invasive procedures are required to identify a small
number of pregnancies with trisomy 21. More
accurate tests have the potential to improve
the efficiency and accuracy of screening and
reduce unnecessary invasive procedures.
Sequencing-based testing will be compared
to current alternative strategies for screening, which include various combinations of
noninvasive maternal serum biomarkers and
fetal ultrasound examination. The role of
sequencing-based testing will be compared to
traditional screening in 2 scenarios: 1) In place
of traditional noninvasive screening to recommend patients for invasive procedures;
or 2) As a follow-up test for patients with a positive result on traditional noninvasive screening
to recommend patients for invasive procedures.
For all screening scenarios, both first and
second trimester testing will be evaluated.
The relevant clinical outcomes include: detection rates for trisomy 21, number of cases of
trisomy 21 missed, number of invasive procedures that are performed, number of invasive
procedures that are performed with normal
results, and number of avoidable fetal losses.
To estimate these rates, test sensitivity and
specificity must first be established in com­
parison to the gold standard of invasive tissue
sampling and karyotyping. If the sensitivity
and specificity with accompanying uncertainties can be defined, then a decision model
incorporating other necessary existing data can
estimate rates of the relevant clinical outcomes
for each comparative strategy.
Background
Down Syndrome and Other Fetal
Trisomy Syndromes
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.2 The trisomy syndromes
are aneuploidies involving 3 copies of one
chromosome. Trisomy 21 (Down syndrome)
is the most common form of fetal aneuploidy
that is associated with survival to birth and
beyond. Trisomy 18 (Edwards syndrome) and
trisomy 13 (Patau syndrome) are the next most
common fetal aneuploidy syndromes associated
with survival to birth, although the percent of
cases surviving to birth is low and for these
survival beyond birth is limited. (Driscoll and
Gross 2009). Trisomy of sex chromosomes also
occurs, e.g. 2 X plus one Y chromosome results
in Klinefelter’s syndrome. Trisomy may occur
de novo as a result of failure of chromosomal
pairs to separate during meiosis (“nondisjunction”) or less often as a result of a Robertsonian
translocation3; either way the result is 3 copies
of a specific chromosome rather than 2 after
fertilization. Mosaic forms of trisomy may also
occur, in which only some of the cells in the
body show trisomy and other cells are normal.
The severity of the mosaic trisomy phenotype
depends on the type and number of cells that
have the extra chromosome.
Maternal age remains the most important risk
factor for a trisomy syndrome, with a risk of
1/1,600 at age 15 that increases to 1/28 by age
45 (Cuckle et al. 1987). Together with this,
A euploid individual or cell has the normal number of chromosomes for that species. Humans have 46 chromosomes, 2 copies of
each of 23 chromosomes except for unfertilized egg and sperm cells, which have only 23 chromosomes or one copy of each.
3
A Robertsonian translocation is a type of nonreciprocal translocation that can occur in one of the acrocentric chromosomes,
including chromosomes 13 and 21. During a Robertsonian translocation, the participating chromosomes break at their
centromeres and the long arms fuse to form a single chromosome with a single centromere. The short arms may also fuse
but are usually lost in subsequent cell divisions. A carrier of a Robertsonian translocation involving chromosome 13 or 21 is
phenotypically normal, but the carrier’s progeny may inherit an unbalanced trisomy 13 or trisomy 21. Inherited translocations
result in genetic counseling that is different from typical de novo cases of Down syndrome. Robertsonian translocations can also
occur de novo and in total account for about 4% of all Down syndrome cases (Noble 1998).
2
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Sequencing-Based Tests to Determine Fetal Down Syndrome (Trisomy 21) from Maternal Plasma DNA
the percentage of pregnancies that occur with
“advanced maternal age” has tripled over the
last 30 years, to 14% of all pregnancies in the
U.S. (Bornstein et al. 2009). Prenatal screening
for trisomy 21 became an option in the 1970s,
when amniocentesis was first used to obtain
fetal tissue for karyotyping from the amniotic
fluid of mothers determined to be at high risk
for trisomy 21 based on maternal age. Current
guidelines recommend that all pregnant
women should be offered noninvasive screening for trisomy 21 before 20 weeks of gestation,
regardless of age (ACOG 2007). Contemporary
screening programs may also detect trisomy 18
or 13.
Down syndrome, or trisomy 21, is the most
common cause of human birth defects, occurring in 1 in 800 live births (Driscoll and Gross
2009) and 1 in 654 pregnancies (Siffel et al.
2004). Trisomy 21 is associated with a variety of
clinical abnormalities, including mild-to-moderate mental retardation, cardiac defects, thyroid
problems, seizures, hearing loss, and duodenal
atresia. The majority of individuals with Down
syndrome are currently cared for in the home
by family members, in contrast to the high rate
of institutionalization in past eras. Individuals
with Down syndrome individuals require a high
degree of medical surveillance and treatment
for associated medical conditions.
Life expectancy is reduced for individuals
with trisomy 21, but has improved with current
medical care. The average age of death in
1983 was 25 years, which increased to 49 by
1997. In contrast, trisomy 18 and 13 are not
compatible with life in most instances, with the
majority of fetuses dying in utero or within the
first few weeks after birth, and only approximately 10% of infants reaching 1 year of age
(Driscoll and Gross 2009).
Prenatal screening and diagnosis for trisomy
21 has decreased the prevalence of Down
syndrome as a percentage of live births. The
majority of women who agree to screening
elect to terminate the pregnancy if trisomy 21
(or trisomy 18 or 13) is diagnosed. A systematic review published in 2012 that included
24 U.S. studies reported a range of pregnancy
termination of 61-93% (weighted mean average
of 67%) after a prenatal diagnosis of Down
syndrome had been made (Natoli et al. 2012).
Due to this high rate of termination, the prevalence of Down syndrome among all live births
decreased by 80% between the years 1979–1999
(Stoll et al. 2002).
Screening for Trisomy 21
As guidelines note, all pregnant women should
be offered noninvasive screening for trisomy
21. Contemporary screening programs may also
detect trisomy 18 or 13. Screening pregnant
women usually starts with noninvasive testing,
followed by invasive tests to confirm the diagnosis, if needed.
Noninvasive screening involves combinations of
maternal serum markers and fetal ultrasound
performed at various stages of pregnancy, but
there is not one standardized approach. The
most common noninvasive screening test consists of a panel of maternal serum markers.
Maternal serum markers are alpha-fetoprotein
(MSAFP), the free beta subunit of human chorionic gonadotropin (free beta hCG), unconjugated estriol, and inhibin A (not used in the
risk calculation for trisomy 18). Some screening
strategies employ 3 of these markers (“triple
screen,” not including inhibin A) while others
incorporate all 4 (“quad screen”). These marker
combinations are used for screening during the
second trimester, when, for example, the detection rate of the quad screen for Down syndrome
is 81% if the false-positive rate is set to 5%,
corresponding to a sensitivity and specificity
of 81% and 95%, respectively (ACOG 2007).
First trimester screening became available after
second trimester screening became common,
incorporating free beta hCG with pregnancyassociated plasma protein A (PAPP-A), also
strongly associated with Down syndrome.
Fetal ultrasound measures are used to supplement the maternal serum markers and increase
the performance characteristics of the screening test panels. There is a strong association
between Down syndrome and fetal “nuchal
translucency” (ACOG 2007), which is defined
as the size of fluid collection at the back of the
neck during the first trimester. Another fetal
ultrasound marker is nasal bone examination,
since abnormalities of nasal bone growth are
associated with Down syndrome. Specialized
training is required to accurately determine
nuchal translucency, and as a result, this
screening technique is not available in all areas.
The detection rate for various combinations of
these measures ranges from 60–96% in various
studies (Malone et al. 2005; Benn et al. 2011)
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Technology Evaluation Center
when the false-positive rate is set at 5%. The
sensitivity is highest when testing involves a
“sequential” or “integrated” approach. These
strategies offer both first and second trimester
screening, recommending invasive, confirmatory testing when either returns a result indicating a high risk of trisomy.
Noninvasive tests are not sufficiently accurate
to confirm the diagnosis of trisomy 21 or other
trisomy syndromes. Direct karyotyping of fetal
tissue obtained by amniocentesis or chorionic
villous sampling (CVS) is required to confirm
that a chromosomal abnormality is present.
Karyotyping is the microscopic analysis of
chromosomes prepared from cultured fetal
cells at a stage when chromosomes are highly
condensed. Large gains, losses, or exchanges
(translocations) of chromosomal material can
be detected. Both amniocentesis and CVS are
invasive and have an associated risk of miscarriage. Amniocentesis is safest when performed
between 15 and 20 weeks of gestation. The risk
of pregnancy loss after mid-term amniocentesis
is less than 1%, and has been estimated to
be in the range of 1 in 300–500 procedures
at experienced centers (ACOG 2007). Other
complications of amniocentesis include vaginal
spotting and/or chorioamniotic leakage in
1–2% of cases and chorioamnionitis in 0.1%
of cases (ACOG 2007).
for invasive testing based upon the results on
screening tests (Driscoll and Gross 2009). Some
women who are at particularly high risk, and/
or who wish to rule out chromosomal abnormalities with certainty, may elect to proceed
directly to invasive testing. Other women
decline testing for trisomy altogether if they
wish to avoid the risks of invasive testing (see
following), or are certain that they would not
alter decisions regarding their pregnancy based
on results. There is also variability among clinicians as to the specific screening strategy that
is preferred.
Unmet Needs
There are numerous limitations to the current
screening strategies. Most importantly, current
testing strategies have a low positive predictive
value due to the suboptimal specificity and low
prevalence of trisomy syndromes. As a result,
the majority of patients who have an invasive
procedure are not found to have a fetal trisomy
syndrome. The largest potential benefit of a
new screening strategy would therefore be
in reducing unnecessary amniocentesis and
CVS procedures. The sensitivity of noninvasive
screening strategies is also imperfect, and some
trisomy cases are not detected. A noninvasive
test with improved sensitivity will therefore
reduce the number of cases that are missed.
Chorionic villous sampling can be done earlier
in pregnancy, most commonly after 9 weeks
gestation. This advantage of CVS may be offset
by a higher rate of adverse events. The rate
of pregnancy loss due to chorionic villous
sampling is in the range of that for mid-term
amniocentesis, but may be slightly higher
(ACOG 2007). There may be an increased
rate of limb defects following CVS, but this
association is controversial. Vaginal spotting or
bleeding is common after the procedure, occurring in up to a third of cases. Amniotic fluid
leakage and/or chorioamnionitis occurs at a
rate of less than 0.5%.
Another limitation of current testing is the
narrow gestational window of applicability and
a need to combine multiple markers, sometimes from different time points, to arrive at
a clinically useful sensitivity and specificity
profile. For example, one of the best case standard screening scenarios, termed “integrated”
screening, combines results from first trimester
nuchal translucency and PAPP-A test results
with second trimester quad screen results for
an overall interpretation of risk. Sensitivity for
integrated screening is 94–96% when specificity is 5% (ACOG 2007). Therefore, alternate
screening methods would offer the opportunity
for improved efficiency and convenience for
both patients and providers.
The choice of screening strategy depends
on numerous factors such as maternal age,
gestational age at first prenatal visit, previous
obstetrical history, family history, availability of
fetal ultrasound, risk tolerance of the parents,
and desire to undergo pregnancy termination if a trisomy syndrome is detected (ACOG
2007). The majority of women elect to have
noninvasive testing performed, with a decision
Ultimately, a noninvasive test could replace
invasive testing if the accuracy of both were
found to be equivalent, thus entirely eliminating
the risk of invasive testing. This has the potential to greatly simplify screening, reduce risk,
and eliminate the anxiety and inconvenience
of protracted testing sequences.
10
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Sequencing-Based Tests to Determine Fetal Down Syndrome (Trisomy 21) from Maternal Plasma DNA
Detecting Fetal DNA in Maternal Serum
and Plasma
In 1997, Lo et al. reported that fetal cell-free
DNA could be detected in the serum and
plasma of pregnant women (Lo et al. 1997), and
that this fetal DNA comprised approximately
3–6% of total cell-free DNA in a maternal blood
sample (Lo et al. 1998). This raised the possibility of noninvasive prenatal diagnosis by
fetal DNA analysis with a number of potential
clinical applications. These include fetal RhD
genotyping, fetal trisomy syndrome detection,
gender determination, and prenatal diagnosis
of numerous inherited genetic diseases.
Studies determined that the cytotrophoblastic
cell layer of the placenta is actually the source
of the fetal DNA rather than circulating fetal
cells (Hahn et al. 2011), that fetal DNA is
entirely present in short fragments (majority
<200bp; Chan et al. 2004; Fan et al. 2008), and
that maternal DNA may have a broader fragment size distribution (majority <800bp; Chan
et al. 2004), although this last may be in contention (Fan et al. 2008; Hahn et al. 2011).
Detection of Trisomy 21
by Sequencing-Based Testing
Early attempts to detect trisomy 21 focused
on the use of methods to detect allele-specific
variation between the mother and fetus and
imbalances in allelic ratios, typically based on
quantitative polymerase chain reaction (PCR)
(Lo et al. 1998; Tong et al. 2006; Dhallan et
al. 2007). Although successful in principle,
these methods had poor sensitivity. Digital
PCR improved sensitivity to a large degree. In
this method, the nucleic acid sample is highly
diluted into separate PCR reactions. This allows
a “digital” readout to be obtained, since any
of these multiple PCR analyses will be either
positive or negative, corresponding to the presence or absence of the target molecule. Thus,
chromosome-specific target sequences can
be counted without the need for allelic differentiation between maternal and fetal DNA
sequences. This method is also limited in sensitivity by samples that have a low fraction of
fetal DNA (Fan et al. 2008; Evans et al. 2012).
Locus-specific PCR-based methods interrogate
a very limited sample of the DNA fragments
present in the maternal sample that represent
the chromosome of interest. In 2007 Lo et
al. (Lo et al. 2007) suggested that massively
parallel sequencing (MPS; also known as nextgeneration or “next-gen” sequencing) could
be used to enhance noninvasive methods for
prenatal diagnosis, and in 2008, two groups
published proof of principle papers (Chiu et al.
2008; Fan et al. 2008). To prepare for sequencing, the DNA fragments in maternal plasma are
first amplified by PCR. During the sequencing
process, the amplified fragments are spatially
segregated and approximately 36 initial bases
of each fragment are simultaneously sequenced
in a massively parallel fashion. Sequenced fragments are then mapped to the reference human
genome in order to obtain fragment counts
per chromosome.
The determination of trisomy (or any variation
from the normal chromosome number) for a
particular chromosome depends upon the following (Chiu et al. 2008):
n
MPS captures and generates fragment
sequence reads for the small fraction of
fetal DNA in maternal plasma alongside the
background maternal DNA (maternal and
fetal fragments are indistinguishable).
n The pool of plasma DNA fragments prepared
for sequencing is representative of the
original maternal plasma.
n There is no major bias in the ability to
sequence DNA fragments originating from
each chromosome.
When the above conditions are met, the
sequencing-derived percent of fragments from
the chromosome of interest should reflect the
chromosomal representation of the maternal
and fetal DNA fragments in the original maternal plasma sample. Additionally, in a euploid
individual with a normal number of chromosomes, the proportional contribution of DNA
sequences per chromosome should correlate
with the relative size of each chromosome in
the human genome.
Using MPS results for plasma samples from
known euploid pregnant women as a reference,
the distance from the euploid mean for the
chromosome of interest is determined for
the sample. A predetermined cutoff (e.g., a
z-statistic of +3, which includes 99.9% of a
euploid distribution below the cutoff) identifies
samples with abnormal numbers of chromosomes by values above the cutoff.
Thus, the determination of trisomy 21 or any
other abnormal chromosome number is essentially a counting exercise. However, the technology must be sensitive enough to detect a slight
©2013 Blue Cross and Blue Shield Association. Reproduction without prior authorization is prohibited.11
Technology Evaluation Center
shift in counts among the small fetal fragment
representation of 3 copies of chromosome 21
against a large euploid maternal background.
For example, if the fetal fraction is 10%, then
the total number of chromosome 21 is only
increased by a factor of 1.05. This detection
problem is further compounded by the fact that
chromosome 21 is the smallest of all the human
chromosomes, accounting for only 1.5% of the
entire genome. However, once the sequencing
assay is optimized, the desired sensitivity can
be obtained by counting a sufficient number
of fragments (Fan and Quake 2010). Counting
a large number of fragments also minimizes
imprecision (Chiu et al. 2008). An advantage of
sequencing all fragments across the genome is
that the method should be capable of detecting
any chromosomal aneuploidy or other copy
number variation including insertions and deletions. A disadvantage is that beyond the most
common aneuploidies (trisomy 21, 18, and 13,
sometimes abbreviated T21, T18, and T13),
these are rare events and sequencing fragments
from all chromosomes may be less desirable
in terms of cost and time. Targeted sequencing
assays have been designed to take advantage
of the sensitivity of sequencing, but target the
chromosomes of interest to interrogate. In these
assays, fragments from nontargeted chromosomes are not sequenced.
MPS assays for detecting fetal trisomy syndromes
in maternal plasma are commercially available
in the U.S. and are summarized in Table 1.
Whether sequencing-based assays require confirmation by invasive procedures and karyotyping depends on assay performance. However,
discrepancies between sequencing and invasive
test results that may occur for biological reasons
could make confirmation by invasive testing
necessary at least in some cases, regardless of
sequencing test performance characteristics:
n False-positive results due to a vanished,
nonviable twin.
n Known discrepancies between the
cytotrophoblastic cell layer (from which
the fetal DNA in maternal plasma arises)
and the fetus itself for certain trisomies
(e.g., trisomy 18).
Additionally, sequencing-based assays would
not differentiate de novo trisomy 21 from
trisomy 21 by Robertsonian translocation. While
this would not affect interpretation for purposes
of the pregnancy, eventual karyotyping of a
trisomy 21-positive fetus or newborn would be
12
helpful for future family genetic counseling.
Sequencing assays may not detect all cases of
mosaicism (not 100% detectable by karyotyping).
Statements from Professional Societies
The International Society for Prenatal Diagnosis
(ISPD) published a rapid response statement on
October 24, 2011 (www.ispdhome.org) regarding noninvasive tests based on the presence of
cell-free fetal nucleic acids in maternal plasma.
ISPD considers these tests to be advanced
screening tests, requiring confirmation through
invasive testing. They further suggest that trials
are needed in low-risk populations and in subpopulations such as twin pregnancies and in
vitro fertilization donor pregnancies.
The National Society of Genetic Counselors
(NSGC) published a position statement on
their website (www.nsgc.org/Advocacy/
PositionStatements/tabid/107/Default.aspx)
regarding noninvasive prenatal testing of
cell-free DNA in maternal plasma. The NSGC
supports this testing “as an option for patients
whose pregnancies are considered to be at
an increased risk for certain chromosome
abnormalities.” They recommend that the
test be offered in the context of informed
consent, and that patients whose results are
abnormal be offered standard confirmatory
(i.e., invasive) testing.
In December, 2012 The American College of
Obstetricians and Gynecologists Committee on
Genetics and The Society for Maternal-Fetal
Medicine Publications Committee published
a Committee Opinion on noninvasive prenatal testing for fetal aneuploidy (ACOG 2012).
The Opinion states that pregnant women at
increased risk of aneuploidy, but not those at
low risk, can be offered cell-free (i.e., sequencing-based) fetal DNA testing after pretest counseling. The Opinion further states that women
with positive test results should be offered
counseling and invasive prenatal diagnosis for
confirmatory testing, noting that cell-free fetal
DNA testing is not a replacement for invasive
prenatal diagnosis.
FDA Status. None of the commercially available sequencing assays for trisomy syndromes
has been submitted to or reviewed by the U.S.
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
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Table 1. Clinically Available Sequencing Assays for Detecting Trisomy Syndromes (U.S.)
Sequenom
MaterniT21 PLUS 3
Testing Method
MPS of first 36 base pairs of each serum DNA fragment with alignment to reference
genome to determine chromosome assignment; relative number of reads for chromosome
of interest compared to predetermined cutoff obtained using euploid reference samples
(Z score >3 = positive)
Test
Development
(Training Set/Test
Set) Published
Retrospective1
Clinical Study
Published
T21 ✓
T21, T13, T18 ✓
NCT01597063
(low-risk pop.)
NCT01555346
(high-risk pop.)
Fetal fraction: separate test, differential methylation markers, QC criterion, 4–50%
Ariosa Diagnostics
Harmony
Sequencing of 384 selected nonpolymorphic loci on each of chromosomes 18 and 21;
algorithm incorporates fetal fraction, risk according to maternal age, and sequencing results
to estimate risks of T18 and T21
Prospective1 Clinical
Study Published/
Trials in progress2
T21, T18, T13 ✓
T21, T18 ✓
T21, T18 ✓
NCT01511458, NEXT
(any risk)
LabCorp
Fetal fraction: part of same test, uses differential 192 SNP loci; QC criterion, not <4%
Verinata
verifi 4
PerkinElmer
MPS of first 36 base pairs of each serum DNA fragment with alignment to reference genome
to determine chromosome assignment; relative number of reads for chromosome of interest
compared to pre-determined cutoff obtained using selected chromosomes from euploid
training samples (Z score equivalent >4 = positive; Z score between 2.5 and 4 = “no call”)
T21, T18 ✓
T21, T18, T13 ✓
Fetal fraction: separate test(s), determined by allele-specific methods, NOT a QC criterion
Natera
Panorama 4
Quest
SNP-targeted sequencing to obtain allele frequency data from >10,000 loci on chromosomes
13, 18, 21, X and Y; different probability distributions are expected for each of two possible
alleles for the set of SNPs on the target chromosome depending on parental genotypes,
fetal fraction, and fetal chromosome copy number; comparison of observed to expected
allele distributions for each of the possible scenarios allows determination of the most likely
scenario
T21, T18, T13 ✓
NCT01574781
(any risk)
NCT01545674,
PreNATUS
(high-risk pop.)
Fetal fraction: part of same test, uses parent genotypes (paternal sample also required); QC
criterion, not <4%
PCR, polymerase chain reaction; MPS, massively parallel sequencing; SNP, single nucleotide polymorphism; QC, quality control; T21, 18, 13, trisomy 21, 18, 13.
1
For purposes of this review, retrospective refers to studies that evaluate previously collected, archived samples or those collected later than they would normally be collected for clinical use; prospective refers to studies
that prospectively enroll patients and collect samples during the intended clinical time frame for testing, although only a subset of samples (e.g. selected cases and controls) may actually be evaluated.
2
Trials in progress are identified from listings in ClinicalTrials.gov and are specified as enrolling populations (pop.) at either low or high risk for trisomy 21 based on e.g. maternal age, family history or a positive serum
and/or sonographic screening test.
3
Test includes Y chromosome detection results for prenatal sex determination
4
Test results also include aneuploidies of sex chromosomes; not evaluated in this Assessment.
Sequencing-Based Tests to Determine Fetal Down Syndrome (Trisomy 21) from Maternal Plasma DNA
©2013 Blue Cross and Blue Shield Association. Reproduction without prior authorization is prohibited.13
Company/
Assay Name/
Clinical Service
Partner (if any)
Technology Evaluation Center
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.
Methods
Search Methods
The MEDLINE® biomedical literature database
was searched in June 2012 and again on August
15, 2012 (via PubMed). The search strategy and
search results for August are shown in Table 2.
The EMBASE database was also searched
using the strategy below with the result of 151
hits (MEDLINE® deselected, duplications not
removed) in August.
‘down syndrome’/exp OR ‘trisomy’/exp
OR ‘aneuploidy’/exp AND (‘dna’/exp AND
(sequencing OR ‘selective analysis’) OR ‘cellfree dna’ OR noninvasive OR ‘non invasive’)
AND (fetal OR maternal) AND (testing OR
sequencing OR detection) AND [humans]/lim
AND [english]/lim AND [embase]/lim AND
[2008-2013]/py
Study Selection
The following selection criteria were applied to
select studies for inclusion:
1. Performed maternal plasma fetal DNA testing of pregnant women being screened for
trisomy 21, trisomy 18, and trisomy 13.
2. Used the final, “locked-down” version of the
sequencing assay that is 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 in individual cases),
or with phenotype at birth.
4. Reported information on sensitivity and
specificity, or provided sufficient information
to calculate these parameters.
Data Abstraction, Calculations
Data Abstraction. Data were abstracted from
each study on the following elements:
nStudy/authors/year
n Manufacturer of test and degree
of industry involvement
n Number of participants and selection
process for testing (e.g., consecutive
patients; archived samples)
n Stage of pregnancy
n Rates of indeterminate tests
(i.e. samples without test results due
to quality control or assay failures)
n Sensitivity, specificity
n Prevalence of trisomy in
the population studied
Because reported sensitivity and specificity
results were not always accompanied by 95%
confidence intervals (CI), and any reported
CIs may not have been calculated by the
same method, we (re-)calculated all CIs
Table 2. Medical Literature Search Strategy and PubMed Result for August 15, 2012
Step
Action
Search Strategy
No. Hits
#20
Add
Search #14 OR #11 Filters: published in the last 5 years; English
878
#19
Add
Search #14 OR #11
2,469
#14
Add
Search ((«down syndrome» OR trisomy OR aneuploidy)) AND (dna AND
(sequencing OR «selective analysis»)) OR «cell-free DNA» OR ((noninvasive OR
non-invasive) AND (fetal OR maternal) AND (testing OR sequencing OR detection))
1,755
#11
Add
Search #7 AND #8 Filters: Humans; English
1,745
#10
Add
Search #7 AND #8 Filters: English
1,756
#9
Add
Search #7 AND #8
1,948
#8
Add
Search «Down Syndrome»[Mesh] OR «Chromosomes, Human, Pair 13»[Mesh]
OR «Chromosomes, Human, Pair 18»[Mesh] OR «Chromosomes, Human, Pair
21»[Mesh] OR «Prenatal Diagnosis»[Mesh] OR «Trisomy/diagnosis»[Mesh]
80,444
#7
Add
Search «DNA/blood»[Mesh] OR «DNA Mutational Analysis»[Mesh] OR (dna AND
(sequencing OR «selective analysis»)) OR «cell-free DNA» OR ((noninvasive OR
non-invasive) AND (fetal OR maternal) AND (testing OR sequencing OR detection))
115,274
14
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Sequencing-Based Tests to Determine Fetal Down Syndrome (Trisomy 21) from Maternal Plasma DNA
using the exact method and the R statistical
software package.
Study Quality Assessment. Formal quality
assessment for individual studies was performed using the Quality Assessment of
Diagnostic Accuracy Studies (QUADAS) 2
instrument (Whiting et al. 2011). This instrument asks questions within four domains on
the risk of bias, and three questions on the
applicability of the studies with respect to the
specific review key questions. The domain
summary questions and the applicability questions are each assigned a rating of “low-risk,”
“high-risk,” or “unclear”. Two reviewers independently rated study quality. Disagreements
were resolved by discussion and consensus.
Decision Analysis. We constructed four possible prenatal screening and confirmatory
strategies for comparison in a decision tree. For
each of the four testing strategies and for each
of two risk populations we calculated detected
cases, invasive procedures, and miscarriages
per 100,000 pregnancies. Strategies could be
applied in the first or second trimesters with
relevant fetal loss rates and test performance
characteristics applied. Estimates were obtained
using R (R_Core_Team 2012) (code to replicate
exact calculations available upon request).
Medical Advisory Panel Review
This Assessment was initially reviewed by the
Blue Cross and Blue Shield Association Medical
Advisory Panel (MAP) on September 28, 2012.
In order to maintain the timeliness of the scientific information in this Assessment, literature
searches were performed subsequent to the
Panel’s review (see “Search Methods”). If the
search updates identified any additional studies
that met the criteria for detailed review, the
results of these studies were included in the
tables and text where appropriate. There were
no studies that would change the conclusions of
this Assessment.
Formulation of the Assessment
Patient Indications
The overall patient indications are pregnant
women who are being screened for trisomy
21. Specific subsets of interest include stratified
patients based on risk of trisomy 21 and
pregnancy stage:
n
Pregnant women
Pregnant women
n Pregnant women
of pregnancy;
n Pregnant women
of pregnancy
n
at high risk for trisomy 21;
at low risk for trisomy 21;
in their first trimester
in their second trimester
Technologies to be Evaluated and Compared
The technologies for comparison are the
current screening and confirmatory tests for
trisomy 21, including:
n Noninvasive testing
– First trimester
– Maternal serum screening for free
beta hCG and PAPP-A
– Ultrasound: specific measures
of nuchal translucency (NT) and
nasal bone
– Second trimester
– Maternal serum screening for AFP,
unconjugated estriol, free beta hCG,
and inhibin A
– Ultrasound: basic fetal anatomy survey
n Invasive testing (confirmatory testing after a
positive screen, or primary testing when risk
is already known to be very high) to determine karyotype (or conduct FISH analysis)
– First trimester
– Chorionic villus sampling (CVS)
– Second trimester
–Amniocentesis
In the first trimester, use of free beta hCG,
PAPP-A, and NT together may be referred to
as the “combined screen”; we use this as one
comparison test in this review since sequencebased testing may be offered as early as 10–12
weeks. In the second trimester, various of the
available tests may be combined as a screen,
and may also include the results of first trimester testing in order to improve risk estimates.
Rather than explore all second trimester
screens in this review, we compare to one of
the best case scenario screens, the “integrated
screen” (ACOG 2007).
Health Outcomes
The primary health outcomes of this review are:
n Assay performance characteristics regarding
trisomy 21 prediction (intermediate
outcome); including correctly identified
trisomy 21 cases (true-positive rate) and
number of cases of trisomy 21 missed
(false-negative rate).
n Invasive procedures avoided (because of
negative sequencing assay).
n Fetal loss averted by avoiding invasive
procedure.
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Technology Evaluation Center
Specific Assessment Questions
Overarching Question. Do maternal plasma
DNA sequencing-based assays improve outcomes of pregnancies being screened for
trisomy 21? Assessment Questions.
1. What is the analytic validity of the available
maternal plasma DNA sequencing-based
tests for trisomy 21?
2. What is the clinical validity of the available
maternal plasma DNA sequencing-based
tests for trisomy 21 compared to standard
confirmatory tests as measured by sensitivity,
specificity, and predictive value? (This question is also addressed for trisomy 18 and 13.)
3. What is the clinical utility of the available
maternal plasma DNA sequencing-based tests
for trisomy 21? How are pregnancy outcomes
improved compared to current screening
methods (first and second trimester combinations tests, see Technologies to be Evaluated
and Compared) followed by invasive testing,
for the following potential uses:
a. As a replacement test for current noninvasive screening tests, with positive results
confirmed by invasive testing.
b. As a follow-up test for pregnancies with a
positive noninvasive screen, with positive
results confirmed by invasive testing.
c. As a follow-up test for pregnancies with a
positive enhanced sensitivity noninvasive
screen, with positive results confirmed by
invasive testing.
Review of Evidence
Description of Included Studies
There were 9 studies reported in 11 publications included for this Assessment; all were
published in 2011–2013. The characteristics
of each of these studies are shown in Table 3.
Six studies were entirely prospective and
the rest retrospectively evaluated archived
samples. Tests from 3 commercial sources were
identified: 2 studies (4 publications) used the
Sequenom test (Ehrich et al. 2011; Palomaki
et al. 2011; Canick et al. 2012; Palomaki et al.
2012), 2 studies used the Verinata test (Sehnert
et al. 2011; Bianchi et al. 2012) and 5 studies
used the Ariosa Diagnostics test (Ashoor et al.
2012; Nicolaides et al. 2012; Norton et al. 2012;
16
Sparks et al. 2012; Ashoor et al. 2013). A fourth
manufacturer, Natera, published one study
(Zimmermann et al. 2012); the authors noted
that it was a “proof of principle” study and refer
to unpublished new data from an improved
method and an updated calculation algorithm.
Because the published study was not conducted
with the final version of the assay, the results
were not included as evidence.
With one exception, the enrolled study populations included women at increased risk for
trisomy 21 due to increased age and/or standard screening results or assumed increased
risk because they were already scheduled for
amniocentesis or CVS. Nicolaides et al. evaluated archived samples from women attending
their routine first pregnancy visit at 11–14
weeks gestation (Nicolaides et al. 2012). Study
enrollees had singleton pregnancies except in
the Canick et al. (Canick et al. 2012) sub-study,
which evaluated the sequencing assay in twin
pregnancies.
Studies generally included women at a wide
range of gestational ages (e.g., 8–36 weeks
or 11–20 weeks) spanning first and second
trimesters. Two studies approached limiting
enrollment to women in the first trimester of
pregnancy by enrolling women at 11–13/14
weeks’ gestation (Ashoor et al. 2012; Nicolaides
et al. 2012). The earliest gestational age in any
trial was 6 weeks (Sehnert et al. 2011) and the
latest was 38 weeks (Sehnert et al. 2011; Norton
et al. 2012).
Sample sizes ranged from 119 to 1988 patients.
These numbers represent the samples analyzed, including euploid controls; in some
studies samples were drawn from larger available cohorts of collected samples. All studies
but one included fewer than 100 cases of
trisomy 21; Palomaki et al. (Palomaki et al. 2011;
Palomaki et al. 2012) included 212 trisomy 21
cases. The approach to analysis varied. Some
studies analyzed samples from all enrolled
women and others analyzed samples from all
women with trisomy 21, 18, or 13 pregnancies
and selected controls (i.e. nested case-control
analysis within cohort).
All studies but one evaluated the results of
maternal fetal DNA testing in comparison
to the gold standards of karyotyping or, in
individual cases when a sample did not allow
karyotyping, fluorescence in situ hybridization
©2013 Blue Cross and Blue Shield Association. Reproduction without prior authorization is prohibited.
Study/year
Country(ies)
Role of Study
Sponsor
Trisomy
Syndromes
Included
Study Sample
Patient Population(s)
4,664 enrolled
4,385 (94%) viable singleton
pregnancies
77 (2%) twin pregnancies
Pregnant women at
high risk for Down
syndrome based on
maternal age, family
history, or a positive
serum and/or ultrasound
screening test. Singleton
pregnancy.
Gestational
Age
Study Methods
11–20 weeks
Prospective
Sequenom (MaterniT21 PLUS)
Palomaki et al.
2011; Canick et al.
2012; Palomaki
et al. 2012
27 sites
worldwide
(12 in U.S.)
Sequenom
developed the
test and tested
the samples
during the formal
testing period, but
did not control
study design,
communicate with
enrollment sites,
have prior access to
patient information,
analyze study
results, prepare
or have final
decisions on the
manuscript.
Singleton
Trisomy 21
(2011)
Trisomy 13 and
Trisomy 18
(2012)
Twin
Trisomy 21, 13
and 18 (2012)
Singletons:
Analyzed: n=1,988
Trisomy 21 analysis
Cases: n=212
Controls: n=1,483 euploid
Trisomy 13 analysis:
Cases: n=12
Controls: n=36
Trisomy 18 analysis:
Cases: n=62
Controls: n=183 euploid.
Twins:
Trisomy 21 analysis
Cases: n=7
Controls: n=17
Trisomy 13 analysis
Cases: n=1
Controls: n=17
(There were 0 T18 twin
pregnancies)
Cases and controls matched on
gestational age (nearest week, same
trimester), enrollment site, race, and
time in freezer (within 1 month).
Singleton pregnancies: Matched 1:7
for trisomy 21 and 1:3 for trisomy 13
and 18.
Twin pregnancies: Included all
affected pregnancies and random
sample of euploid twin pregnancies.
Reference standard: fetal karyotype
on CVS or amniocentesis.
Blinded evaluation of samples.
Sequencing-Based Tests to Determine Fetal Down Syndrome (Trisomy 21) from Maternal Plasma DNA
©2013 Blue Cross and Blue Shield Association. Reproduction without prior authorization is prohibited.17
Table 3. Characteristics of Included Studies
©2013 Blue Cross and Blue Shield Association. Reproduction without prior authorization is prohibited.
Study/year
Country(ies)
Role of Study
Sponsor
Trisomy
Syndromes
Included
Study Sample
Patient Population(s)
Trisomy 21
n=480 enrolled
Gestational
Age
Study Methods
Pregnant women at
increased risk for
trisomy 21. Risks
included positive serum
biochemical screening
test or ultrasound
suggestive of Down
syndrome; age 35
or older, personal/
family history of Down
syndrome.
8–36 weeks
Prospective
Adult pregnant women at
increased risk of trisomy
21. Risk factors included
age 38 or older, positive
screening result for
fetal trisomy syndrome,
presence of ultrasound
markers associated with
increased risk, prior fetus
with a trisomy syndrome.
Eligible
patients:
8–22 weeks
Sequenom (MaterniT21 PLUS)
Ehrich et al. 2011
U.S.
In–house
Funded by
Sequenom; all
authors employees
and shareholders of
Sequenom
n=13 samples insufficient
quality or quantity
n=467 samples analyzed.
Reference standard: fetal karyotype
or quantitative fluorescent PCR on
either CVS or amniocentesis
Blinded evaluation of samples.
Verinata (verifi)
Bianchi et al. 2012
60 sites, all in U.S.
Role of the sponsor
(Verinata) vs. the
authors is not
described.
Trisomy 13
Trisomy 18
Trisomy 21
Sex chromosome
aneupolidies
Of n=2,882 enrolled, selected
n=534 (all singleton trisomy
syndrome pregnancies and
random sample of singleton
euploid pregnancies)
n=2 failed sample QC
n=532 analyzed
Cases: All singleton.
Analyzed
patients:
10–23 weeks
Prospective
Reference standard: fetal karyotype
on CVS, amniocentesis or products
of conception. If patients underwent
both tests, amniocentesis findings
were used.
Blinded evaluation of samples.
Technology Evaluation Center
18
Table 3. Characteristics of Included Studies (cont’d)
Study/year
Country(ies)
Role of Study
Sponsor
Trisomy
Syndromes
Included
Gestational
Age
Study Sample
Patient Population(s)
1,014 patients enrolled.
Pregnant women at
increased risk for
aneupoidy. Risks include
age at least 35 years,
positive screening test or
other risk factor.
6 weeks,
1 day—
38 weeks,
1 day
Women with singleton
pregnancies attending
their routine first
hospital visit at 11–14
weeks gestation; visit
included first trimester
combined screening for
aneuploidies.
11–14 weeks
Singleton pregnancies
at least 10 weeks’
gestational age at
increased risk of
trisomy 21.
10.0–38.7
weeks
Study Methods
Verinata (verifi)
Sehnert et al. 2011
13 U.S. clinic
locations
Verinata played a
direct role in study
design, patient
enrollment, data
review and
interpretation,
and manuscript
preparation and
approval.
Trisomy 18
Trisomy 21
119 samples analyzed: n=71
training set, n=47 singleton
samples in test set
Prospective
Reference standard: fetal karyotype
on CVS or amniocentesis.
Blinded evaluation of samples in
test set.
Ariosa Diagnostics (Harmony)
Nicolaides et al.
2012
Single U.K.
hospital
Norton et al. 2012
48 sites in 3
countries, most
in U.S.
Supported by a
grant from the
Fetal Medicine
Foundation. Cost
of sample
collection and
analysis was
covered by Ariosa
Diagnostics.
Trisomy 18
Trisomy 21
Role of the sponsor
(Ariosa) vs. the
authors is not
described.
Trisomy 18
Trisomy 21
n=2,230 available samples in
designated time period
n=181 exclusions
n=46 fetal fraction failure
n=54 assay failure
n=1,949 analyzed
n=3,228 from cohort of 4,002
(other samples from cohort
included in Sparks 2012
analysis)
Retrospective (stored samples)
Reference standard: fetal karyotype
on CVS or amniocentesis sample in
86 cases; in 1,963 cases phenotype at
birth was assumed euploid
Prospective
Reference standard: FISH, QF-PCR or
karyotype analysis
Blinded analysis of samples
Sequencing-Based Tests to Determine Fetal Down Syndrome (Trisomy 21) from Maternal Plasma DNA
©2013 Blue Cross and Blue Shield Association. Reproduction without prior authorization is prohibited.19
Table 3. Characteristics of Included Studies (cont’d)
©2013 Blue Cross and Blue Shield Association. Reproduction without prior authorization is prohibited.
Study/year
Country(ies)
Role of Study
Sponsor
Trisomy
Syndromes
Included
Study Sample
Patient Population(s)
Cases: n=50 trisomy 21 and
n=50 trisomy 18 selected
from database
Controls: 300 euploid cases.
Singleton pregnancies
between 11–13 weeks’
gestation at increased
risk of trisomy 21.
Gestational
Age
Study Methods
Ariosa Diagnostics (Harmony)
Ashoor et al. 2012
Single U.K.
hospital
Sparks et al. 2012
U.S.
In-house
Funded by a
grant from the
Fetal Medicine
Foundation. All
authors from the
King’s College
Hospital and the
University College
London Hospital.
Sample analysis
provided gratis by
Ariosa Diagnostics
Trisomy 18
Trisomy 21
Funded by Ariosa
Diagnostics; all
authors employees
of Ariosa
Trisomy 18
Trisomy 21
11–13 weeks
Retrospective (fetal medicine center
database)
Controls matched for length and
storage of blood samples
Used stored maternal plasma
collected prior to CVS
Reference standard: CVS
Blinded evaluation of samples.
A subset of enrolled subjects
were included:
Trisomy 18: n=16
Trisomy 21: n=72
Disomic pregnancies: n=250
(Total n enrolled not reported)
Training set: n=36, Trisomy
21, n=8 Trisomy 18, and
n=127 disomic pregnancies)
Validation set: n=36 Trisomy
21, n=8 Trisomy 18 and n=123
disomic pregnancies
Singleton pregnancies
at least 10 weeks’
gestational age at
increased risk of
trisomy 21.
Training set:
10.3–33 weeks
Validation set:
11–36.1 weeks
Prospective
Randomized to training set and
validation set
Reference standard: FISH or
karyotype analysis
Blinded analysis of samples in
validation set
Technology Evaluation Center
20
Table 3. Characteristics of Included Studies (cont’d)
Study/year
Country(ies)
Role of Study
Sponsor
Trisomy
Syndromes
Included
Study Sample
Patient Population(s)
Training set: n=156
Trisomy 13, n=11
Training set: high-risk
pregnancies defined
by positive traditional
maternal serum screening
Gestational
Age
Study Methods
Ariosa Diagnostics (Harmony)
Ashoor et al. 2013
Single U.K.
hospital
Funded by a
grant from the
Fetal Medicine
Foundation. Authors
from the King’s
College Hospital, the
University College
London Hospital, and
Ariosa Diagnostics.
Sample analysis
provided gratis by
Ariosa Diagnostics
Trisomy 13
Validation set: n=1,949
Trisomy 13, n=10
Validation set: banked
euploid samples based
on combined screening
results and normal
neonates at delivery, and
10 verified trisomy 13
samples from the U.S.
Training set:
11–13 weeks
Retrospective
Reference standard: karyotype
Validation set:
13–26 weeks
Sample analysis blinded to trisomy
status
Pre-defined cutoff for classifying
sample as high or low risk
Abbreviations: T13, trisomy 13; T18, Trisomy 18; T21, Trisomy 21; N, number of patients
1
Other than Ashoor 2012, all studies had industry-funding and additionally, at least some authors are company employees and/or shareholders.
2
Retrospective refers to studies that evaluate previously collected, archived samples; prospective refers to studies that prospectively enroll patients and collect samples, although only a subset of samples
(e.g. selected cases and controls) may actually be evaluated.
Sequencing-Based Tests to Determine Fetal Down Syndrome (Trisomy 21) from Maternal Plasma DNA
©2013 Blue Cross and Blue Shield Association. Reproduction without prior authorization is prohibited.21
Table 3. Characteristics of Included Studies (cont’d)
Technology Evaluation Center
(FISH). Because they were evaluating an
average-risk population, Nicolaides et al. had
karyotyping results for only a small percentage of women in their study; for the rest of the
enrollees, ploidy was imputed by phenotype at
birth obtained from medical records. All studies
included testing for trisomy 21. Eight studies
additionally tested for trisomy 18 (Sehnert
et al. 2011; Ashoor et al. 2012; Bianchi et al.
2012; Canick et al. 2012; Nicolaides et al. 2012;
Norton et al. 2012; Palomaki et al. 2012; Sparks
et al. 2012) and 5 studies additionally tested for
trisomy 13 (Sehnert et al. 2011; Bianchi et al.
2012; Canick et al. 2012; Palomaki et al. 2012;
Ashoor et al. 2013).
All studies had partial or total funding
from sequencing-based testing companies.
Additionally, for all studies, at least some of
the authors were company employees, and/or
patent holders or shareholders.
Study Quality Assessment. A formal quality
rating of included studies was performed
according to the QUADAS-2 review instrument for diagnostic studies (Whiting et al.
2011); results are summarized in the Appendix
Table. Three studies (5 publications) met all
quality criteria (low risk of bias for all items)
(Palomaki et al. 2011; Ashoor et al. 2012;
Canick et al. 2012; Norton et al. 2012; Palomaki
et al. 2012). Five studies did not meet all quality
criteria (Ehrich et al. 2011; Sehnert et al. 2011;
Bianchi et al. 2012; Nicolaides et al. 2012;
Sparks et al. 2012), specifically in the domain
of patient selection. Most commonly, authors
did not report whether a consecutive or random
sample of patients was enrolled, or whether
patients were excluded appropriately. For
those studies using a subsequent case-control
design, the method of selecting controls was
also not well-described. The deficiencies of
information regarding patient selection is
unlikely to have actually biased study results,
with one exception.
Nicolaides et al. was the only study of an
average screening population and was judged
to have a high risk of bias due to exclusions
(Nicolaides et al. 2012). Twenty-eight pregnancies ending in stillbirth or miscarriage
were excluded for lack of karyotype; while
unavoidable, these exclusions likely affect the
case detection rate. Additional exclusions for
chromosomal abnormalities other than trisomy
21, 18, or 13, for lack of followup, for laboratory
procedure errors, and for inadequate sample
volume totaled 153 out of ,2230 pregnancies
originally enrolled. In this study, cases were
verified primarily by phenotype at birth from
medical records, a poor standard compared to
karyotyping.
Assessment Question 1: What is the analytic
validity of the available maternal plasma
DNA tests for trisomy 21?
None of the included publications provided any
direct evidence of analytic validity. The only
result discussed in common across studies that
is somewhat related to analytic validity was the
indeterminate sample rate, comprised of all
samples that failed test quality control requirements including fetal fraction if applicable, “no
call” results for one company’s assay, and those
samples for which a test result could otherwise
not be obtained for unspecified reasons (see
Table 4). There was variability in this result,
which ranged from less than 1% to nearly 5%.
Fetal fraction is determined either in an initial,
separate test or as a part of the trisomy test
by all companies. For two of three companies
for which evidence is reviewed4, the value of
the fetal fraction is a quality control criterion.
Below an established cutoff value, the sample
is not acceptable for reporting assay results.
The highest indeterminate rates do not reflect
poor assay technology, but rather a choice in
the case of one company’s assay to increase the
accuracy of positive results by assigning results
near the assay cutoff to a “no call” category.
Indeterminate samples were not included in
test performance calculations in this report.
Because indeterminate samples would likely
result in a request for a new sample from the
patient, causing delay at a critical time point
in pregnancy, a low failure rate is highly desirable. A new sample collected at a later date
after a rejection for a low fetal DNA fraction
would likely have an increased value as fetal
fraction increases with time during pregnancy;
with experience, laboratories may be able to
predict an interval between sample collections
that would result in a successful test. No information was provided regarding failed samples
for reasons other than fetal fraction or no
call, or whether a new sample would likely
be successful.
As already noted, the sole publication reporting results for the Panorama assay from Natera was not included as evidence
because the study did not use the final version of the assay. Fetal fraction is included as a quality control criterion, see Table 1.
4
22
©2013 Blue Cross and Blue Shield Association. Reproduction without prior authorization is prohibited.
Study1
n in Final Analysis
(after indeterminate
samples removed)
Indeterminate
Samples
Sensitivity2 (%)
(95% CI)
Specificity2 (%)
(95% CI)
T21
T13
T18
T21
T13
T18
91.7
(61.5–99.8)
100
(93.9–100)
99.9
(99.7–99.9)
99.1
(98.5–99.5)
99.7
(99.3–99.9)
100
(99.2–100)
100
(99.2–100)
Sequenom (MaterniT21 PLUS)
Palomaki et al. 2012 3
3rd-party
Ehrich et al. 2011
Total n=1,971
Trisomy 21: n=212
Trisomy 18: n=59
Trisomy 13: n=12
17/1,988 (0.9%)
Test failure including
fetal fraction QC
99.1
(96.6–99.9)
Total n=449
Trisomy 21: n=39
18/467 (3.8%)
Failed test QC,
including fetal fraction
100
(91.0–100)
Total n=5,164
Trisomy 21: n=89
Trisomy 18: n=36
Trisomy 13: n=14
16/532 (3%)
Low fetal DNA
100
(95.9–100)
Total test set=46
Trisomy 21: n=13
Trisomy 18: n=8
Trisomy 13: n=1
1/47 (2%)
T13 classified as “no
call”
Total n=2,049
Trisomy 21: n=8
Trisomy 18: n=3 (2)
(1 T18 sample was
a test failure)
n=46/2,049 (2.2%)
Low fetal DNA
54/2049 (2.6%)
Test failure
Total (4.9%)
In-house
99.7
(98.6–99.9)
Verinata (verifi)
Bianchi et al. 2012
3rd-party
Sehnert et al. 2011
In-house
78.6
(49.2–95.3)
97.2
(85.5–99.9)
100
(99.1–100)
100
(75.3–100)
100
(63.1–100)
100
(89.7–100)
100
(91.0–100)
100
(63.1–100)
100
(15.8–100)
99.9
(99.6–99.9)
99.9
(99.6–99.9)
Ariosa (Harmony)
Nicolaides et al. 2012
3rd-party
Sequencing-Based Tests to Determine Fetal Down Syndrome (Trisomy 21) from Maternal Plasma DNA
©2013 Blue Cross and Blue Shield Association. Reproduction without prior authorization is prohibited.23
Table 4. Trisomy Syndrome Detection by Sequencing in Singleton Pregnancies: Test Performance
©2013 Blue Cross and Blue Shield Association. Reproduction without prior authorization is prohibited.
Study1
n in Final Analysis
(after indeterminate
samples removed)
Indeterminate
Samples
Total n=3,080
Trisomy 21: n=81
Trisomy 18: n=38
[73 =’other’ based on
invasive testing]
n=57/3,228 (1.8%)
Low fetal DNA
91/3,228 (2.8%)
Test failure
Total (4.6%)
Total n=397
Trisomy 21: n=50
Trisomy 18: n=50
Sensitivity2 (%)
(95% CI)
T21
T13
Specificity2 (%)
(95% CI)
T18
T21
T13
T18
100
(95.5–100)
97.4
(86.2–99.9)
99.97
(99.8–99.9)
99.93
(99.7–99.9)
3/400 (0.75%)
Test failure
100
(92.9–100)
98
(89.4–99.9)
100
(98.8–100)
100
(98.8–100)
Validation set
Total n=167
Trisomy 21: n=36
Trisomy 18: n=8
n=0
No failures in test set
100
(90.3–100)
100
(63.1–100)
100
(97.0–100)
100
(97.0–100)
Validation set
Total n=2,002
Trisomy 13: n=10
n=53 (2.6%)
(Test set, 9 of 165
samples failed, 5%)
Ariosa (Harmony)
Norton et al. 2012
3rd-party
Ashoor et al. 2012
3rd-party
Sparks et al. 2012
In-house
Ashoor et al. 2013
3rd-party
80
(44.4–97.5)
99.9
(99.7–100)
Abbreviations: T13, trisomy 13; T18, Trisomy 18; T21, Trisomy 21; N, number of patients
1
Other than Ashoor 2012, all studies had industry-funding and additionally, at least some authors were company employees and/or shareholders. ‘In-house’ indicates that all study authors were employees of the
company at the time of the study. ‘3rd-party’ indicates that the first author and at least some of the other authors were not employees of the company.
2
All 95% confidence intervals were calculated by exact methods, see Methods, Data Abstraction, Calculations.
3
Results for T21 were abstracted from Palomaki 2012, rather than Palomaki 2011, because of data corrections for GC content and use of repeat masking, part of the current test procedure.
4
Patients with complex karyotypes were censored from the total population for the analysis of each trisomy; the exact number was dependent on the trisomy being analyzed.
Technology Evaluation Center
24
Table 4. Trisomy Syndrome Detection by Sequencing in Singleton Pregnancies: Test Performance (cont’d)
Sequencing-Based Tests to Determine Fetal Down Syndrome (Trisomy 21) from Maternal Plasma DNA
Each of the various tests uses massively parallel sequencing (MPS; also called next-generation sequencing), a relatively new technology
but not an entirely new concept for the clinical
laboratory. Traditional Sanger sequencing has
been used for some time to sequence short,
targeted genomic regions in search of variants relevant to specific medical conditions.
Although offered as a laboratory-developed
test (LDT) subject only to regulation of laboratory operations under the Clinical Laboratory
Improvement Amendments (CLIA), Sanger
sequencing has been widely accepted as a
gold standard. In contrast, MPS is a research
laboratory sequencing technology that has only
recently been adopted by a relatively small but
rapidly growing number of molecular genetic
clinical laboratories nationwide.
Currently, there are no recognized standards
for conducting clinical sequencing by MPS. The
College of American Pathologists Laboratory
Accreditation Program, a deemed accreditor
for CLIA, has revised their molecular pathology checklist to include a dedicated section on
MPS as of July 31, 20125. The CDC Laboratory
Science, Policy and Practice Program Office has
developed evidence- and quality managementbased recommendations for analytic validity and regulatory and professional practice
standards for MPS (Gargis et al. 2012); and is
coordinating the development of reference
materials for clinical MPS applications.6 On
June 23, 2011, the FDA held an exploratory,
public meeting on the topic of MPS, in preparation for an eventual goal of developing “a transparent evidence-based regulatory pathway for
evaluating medical devices/products based on
NGS that would assure safety and effectiveness
of devices marketed for clinical diagnostics.”7
More recently the FDA Genomics Working
Group reported to the FDA Science Board on
the next-gen sequencing explosion, noting
regulatory implications for several centers at
the FDA and the need to coordinate both internally and externally to develop data standards
for bioinformatics needs.8
Each of the companies offering a maternal
plasma DNA sequencing test for fetal trisomy
has developed a specific procedure for its
private, CLIA-licensed laboratory where all
testing takes place. The Sequenom and Verinata
tests are similar in that both sequence the
first 36 bases of a large sampling of all DNA
fragments in the plasma sample, determine
chromosome assignments of the fragments,
and use z-scores (with different thresholds for
each company) to classify results as normal or
positive for a specific trisomy (see Table 1). In
order to calculate z-scores, a euploid reference
is required. Verinata used an “unaffected subset
of the training data” (Sehnert et al. 2011) to first
calculate sequencing platform-specific chromosome ratios that minimized inter- and intra-run
sequencing variation, then to calculate z-score
equivalents and determine classification thresholds that were then applied to subsequent
studies. Sequenom used a set of “known euploid
reference samples . . . to calculate the mean
and standard deviation (SD) of the [fragment]
representation of chromosome 21,” prior to
calculating z-scores (Ehrich et al. 2011). This
same procedure was applied to subsequent
studies and other chromosomal trisomies
(Palomaki et al. 2011; Palomaki et al. 2012).
The Sequenom test procedure also adjusts
for differential guanine (G) and cytosine (C)
content of the DNA and masks simple repeat
sequences during alignment to the human
reference genome, both of which improved
z-score classification (Palomaki et al. 2012).
The Ariosa sequencing procedure depends
on detecting only those plasma DNA fragment sequences specific to chromosomes 18
and 21. To accomplish this, they created short
sequencing assays for many nonpolymorphic
loci on each of these chromosomes. Additional
assays targeting loci where fetal alleles differ
from maternal alleles are used to determine
fetal fraction in the same overall procedure.
For interpretation, external euploid reference
samples are not used; rather, sequence counts
are first normalized “by systematically removing sample and assay biases” within each
sequencing run. Risk of a trisomy syndrome
See CAP press release available at http://www.cap.org/apps/cap.portal?_nfpb=true&cntvwrPtlt_actionOverride=
%2Fportlets%2FcontentViewer%2Fshow&_windowLabel=cntvwrPtlt&cntvwrPtlt%7BactionForm.contentReference%7D=media_
resources%2Fnewsrel_checklist_next_gene.html&_state=maximized&_pageLabel=cntvwr
6
See information at http://www.cdc.gov/osels/lspppo/Genetic_Testing_Quality_Practices/Nex-StoCT.html
7
Documents related to the meeting available at http://www.fda.gov/MedicalDevices/NewsEvents/WorkshopsConferences/
ucm255327.htm
8
See slide presentation at http://www.fda.gov/downloads/AdvisoryCommittees/CommitteesMeetingMaterials/
ScienceBoardtotheFoodandDrugAdministration/UCM341042.pdf
5
©2013 Blue Cross and Blue Shield Association. Reproduction without prior authorization is prohibited.25
Technology Evaluation Center
is determined by an algorithm that compares
the probabilities of disomic vs. trisomic explanatory models of the data. The algorithm also
takes fetal fraction and prior risk due to maternal
age into account.
In summary, although all 3 tests use MPS,
actual procedures vary considerably, clinical
sequencing in general is not standardized or
regulated by the FDA, and neither the routine
quality control procedures used for each of
these tests, nor the analytic performance
metrics have been published.
Assessment Question 2. What is the clinical
validity of the available maternal plasma
DNA tests for trisomy 21 compared to standard confirmatory tests as measured by
sensitivity, specificity, and predictive value?
(This question is also addressed for trisomy
18 and 13.)
The performance characteristics of the fetal
DNA sequencing tests in studies of singleton
pregnancies are summarized in Tables 4 and
5. 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. Predictive values depend
on the prevalence of the condition being
detected in the population, as well as on performance characteristics of the test; estimates
are summarized for a high-risk population
with an in utero T21 prevalence of 950 per
100,000 (0.95%) (Snijders et al. 1999) and for
an average risk population with an in utero T21
prevalence of 250 per 100,000 (0.25%) (Spencer
et al. 2003; Malone et al. 2005). Predictive
values were calculated for 3 pairs of sensitivity
and specificity values representing sequencing
assays in general for trisomy 21 results (point
estimates, high values, low values, see Table 5).
Negative predictive values are uniformly high,
near or at 100% as is desirable for a screening
test. Positive predictive values are high for the
high end sensitivity and specificity estimates
of nearly 100% (i.e. a nearly perfect test), but
much lower otherwise, e.g. 83% and 55% for
high and average risk populations, respectively,
using test point estimates for sensitivity and
specificity. These calculations assume that this
testing technology will vary as any other by
prevalence of the condition; actual performance
will be determined in ongoing trials.
There are indications, however, that trisomy 21
prevalence may be less influential with regard
to the results of sequencing-based testing.
Table 6 summarizes trisomy 21 prevalence for
all included studies along with false positive
rates (1-specificity), accompanied by a graphical presentation of these results (Figure 1).
While prevalence in these studies spans a wide
spectrum from the general population prevalence of the Nicolaides et al. study (Nicolaides
et al. 2012) to enrolled high-risk clinical populations and artificially created study populations,
the false positive rates are relatively invariant.
In general and most importantly, there were no
major increases in the false-positive rate across
considerable decreases in prevalence.
For trisomy 18, the sensitivity ranged from
97.2% to 100% and the specificity ranged from
99.7% to 100%. These estimates tended to be
based on a small number of cases, e.g., as low
as 8 cases of trisomy 18 in 2 studies and only
2 cases in 1 study (plus 1 test failure), and as
high as 59 cases (plus 3 test failures).
Only 4 studies reported results for trisomy
13 (Sehnert et al. 2011; Bianchi et al. 2012;
Table 5. Predictive Value Estimates for Down Syndrome (T21)
Sensitivity, Specificity:
high, point estimate,
and low values
Sensitivity = 99.999%
Antenatal prevalence=0.95%
(high-risk, age >35 years)
Antenatal prevalence=0.25%
(average-risk population)
PPV (%)
NPV (%)
PPV (%)
NPV (%)
99.9
100
99.6
100
82.6
99.99
55.4
100
31.3
99.95
10.6
99.99
Specificity = 99.999%
Sensitivity = 99.0%
Specificity = 99.8%
Sensitivity = 95.0%
Specificity = 98.0%
26
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Sequencing-Based Tests to Determine Fetal Down Syndrome (Trisomy 21) from Maternal Plasma DNA
Table 6. Trisomy 21 Prevalence Values and Assay False-Positive Rates from Included Studies
Study
n (T21 cases / total
trisomy cases +
controls)
T21 Prevalence
(%)
T21 Prevalence
(1 in … )
False-positive
rate (%)
Sehnert et al. 2011
13/46
28.3
3.5
0
36/167
21.6
4.6
0
Bianchi 2012
89/516
17.3
5.8
0
Ashoor 2012
50/397
12.6
7.9
0
Palomaki 2012
212/1,971
10.8
9.3
0.1
Ehrich 2011
39/449
8.7
11.5
0.3
Norton 2012
81/3,080
2.6
38.5
0.03
Nicolaides 2012
10/2,049
0.4
250
0.1
Figure 1. Plot of Trisomy 21 Prevalence vs. Assay False-Positive Rate for Included Studies
0.5
False Positive Rate, by Study (%)
0.4
0.3
0.2
0.1
0
1,000
100
10
1
Down Syndrome Prevalence (log scale; 1 in x)
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Technology Evaluation Center
Palomaki et al. 2012; Ashoor et al. 2013); one
of these reported on only one case that gave a
result of “no call,” which is neither a clear positive nor a clear negative according to the assay
cutoff values (Sehnert et al. 2011). For the other
2 studies, sensitivities were 91.7% and 78.6%,
specificities were 99.1% and 100%. These estimates were based on 12 cases in one study, and
14 cases in the other (see Table 3).
Detection of trisomy syndromes in twin pregnancies was systematically evaluated in only
one study (Canick et al. 2012); results are
summarized in Table 7. All 7 cases of twin
pregnancies with Down syndrome were
correctly classified. Five of these were discordant, where one twin had trisomy 21 and the
other did not; 2 were concordant where both
twins had trisomy 21. Also correctly classified
were one twin pregnancy with trisomy 13 and
17 euploid twin pregnancies. While encouraging, these results are insufficient to draw
conclusions regarding the detection of trisomy
syndromes by sequencing-based testing in
multiple gestations.
Of 5 trisomy 21 or trisomy 18 mosaic samples
included in two studies, 4 were correctly classified; one was incorrectly classified as euploid
(Bianchi et al. 2012; Norton et al. 2012). Two
cases of mosaicism confined to the placenta
were correctly classified as euploid (Norton
et al. 2012). Sample numbers are too small
to determine performance characteristics for
mosaic samples.
Bianchi et al. reported correctly classifying
2 of 2 trisomy 21 samples arising from
Robertsonian translocations (Bianchi et al.
2012). Of the trisomy twin cases correctly
detected by Canick et al., one of the concordant Down syndrome cases was due to
a Robertsonian translocation (Canick et al.
2012). Sample numbers are too small to determine performance characteristics for trisomy
samples resulting from translocations.
Assessment Question 3: What is the
clinical utility of the available maternal
plasma DNA tests for trisomy 21? How are
pregnancy outcomes improved compared
to current screening methods (first and
second trimester combinations tests,
see Technologies to be Evaluated and
Compared) followed by invasive testing,
for the following potential uses:
a. As a replacement test for current
noninvasive screening tests, with positive
results confirmed by invasive testing.
b. As a follow-up test for pregnancies with a
positive noninvasive screen, with positive
results confirmed by invasive testing.
c. As a follow-up test for pregnancies with a
positive, enhanced sensitivity noninvasive
screen, with positive results confirmed by
invasive testing.
A simple decision model was constructed to
compare the health outcomes of nucleic acid
sequencing-based testing with standard testing
Table 7. Trisomy Syndrome Detection by Sequencing: Results for Twin Pregnancies
Study
Total n
Canick
2012
25 twin
pregnancies
1
2
Criteria for
trisomy by
sequencing z -score >3
Individual/Median z-score (range) Samples, n
T21
T18
T13
Total confirmed twin
T21, n=7
12.3
(5.3–17.4)
<3
<3
Discordant1 twin
T21, n=5
10.1
(5.3–17.4)
Concordant2 twin
T21, n=2
12.3, 13.2
Total confirmed twin
T13, n=1
0.3
0.1
5.1
Euploid twin, n=17
-0.1
None>3
0.1
None>3
-0.2
None>3
Discordant twins: only one fetus is affected by trisomy.
Concordant twins: both fetuses are affected by trisomy.
28
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Sequencing-Based Tests to Determine Fetal Down Syndrome (Trisomy 21) from Maternal Plasma DNA
for fetal trisomy 21 (see Figure 2). 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): PAPP-A,
free beta-hCG, nuchal translucency;
b. Integrated screen (first + second tri­
mester): PAPP-A, nuchal translucency
in first trimester; quad screen composed
of MSAFP, hCG, unconjugated estriol,
inhibin A in second trimester.
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 (first trimester
combined screen or first and second
trimester integrated screen); positives
followed with sequencing-based testing;
positives confirmed by invasive procedure
and karyotyping.
4. A standard screening test with cutoff values
altered to enhance sensitivity and increase
case detection (Enhanced sensitivity noninvasive screen suggested by Glenn Palomaki,
personal communication); positives followed
with sequencing-based testing; positives confirmed by invasive procedure and karyotyping. For this strategy, only the first trimester,
combined screen was modeled.
The outcomes of interest for this tree are the
number of cases of trisomy 21 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 parameters for the decision model
(Table 8) are specific only for T21, for which
the collected data are the most extensive and
which represents the main clinical reason for
testing. The results were calculated for two
different populations, a high-risk population
of women age 35 or older, 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 and that
the proportion would vary according to risk.
For the sake of simplicity, only 2, somewhat
conservative choices of uptake corresponding
to high risk and low risk women were selected
(Table 8). Sensitivities and specificities for both
standard and sequencing-based screening tests
were varied to represent the range of possible
values. Results for T21 screening outcomes
with comparison of sequencing-based testing
to the standard testing methods are shown in
Table 9 and in Table 10.
For either high- or average-risk populations,
the second strategy, screening by sequencingbased assay followed by confirmatory testing,
detects the most cases regardless of the type of
traditional testing used for comparison (Table
9). 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 3–16% (base case). At
the same time, the number of invasive procedures needed is reduced by as much as 80%
The number of total miscarriages after an invasive confirmatory procedure in an average-risk
population is also reduced from, for example,
the 22 per 100,000 seen after integrated screening to 4 (an 82% reduction) using base case
estimates (Table 10). Confidence in negative
results is high as no more than 10 of 100,000
(0.01%) screens are false negatives using base
case estimates (data not shown).
When added after a positive traditional screen,
a sequencing-based assay does not improve
the detection rate. However, fewer invasive
procedures are needed than after screening by sequencing alone, due partially to the
lower case detection rate. The number of total
miscarriages is reduced to similar or slightly
lower numbers than screening by sequencing
alone. These results are seen for both high- and
average-risk populations.
A first trimester traditional combined test can
be reinterpreted using altered parameters
that allow an increased detection rate. Such
parameters are not normally used because the
false-positive rate would be unacceptably high
for referral to an invasive procedure. However,
following positive results from an increased
sensitivity combined assay with a sequencingbased assay could take advantage of the
increased detection rate, reduce the number
of sequencing-based tests required, and also
reduce invasive procedures and miscarriage
rates. This test combination was modeled with
final detection rates nearly as good as the integrated screen alone, but in the first trimester.
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CVS Miscarriage
Trisomy 21
Positive
1st Trimester
Combined → Invasive
1st Trimester
Combined
CVS Confirmatory Test
False Positive
No Confirmatory Test
Negative
Invasive Confirmatory
Test Miscarriage
Positive
Fully Integrated
Screen → Invasive
Fully Integrated
Screen
Invasive
Confirmatory Test
No Invasive
Confirmatory Test
Trisomy 21
False Positive
Negative
Invasive Confirmatory
Test Miscarriage
Trisomy 21
Positive
Sequencing →
Invasive
Sequencing-Based
Screen
Pregnancy
No Invasive
Confirmatory Test
Negative
Fully Integrated Screen →
Sequencing → Invasive
Fully Integrated
Screen
1st Trimester Sequencing → CVS
1st Trimester
Combined
Invasive
Confirmatory Test
(see next page)
(see next page)
False Positive
Technology Evaluation Center
30
Figure 2. Decision Tree for Possible Prenatal Screening and Confirmation Strategies to Detect Trisomy 21
1st Trimester
Combined → Invasive
1st Trimester
Combined
Fully Integrated
Screen → Invasive
Fully Integrated
Screen
Sequencing →
Invasive
Sequencing-Based
Screen
(see previous page)
(see previous page)
(see previous page)
Invasive Confirmatory
Test Miscarriage
Pregnancy
Positive
Fully Integrated Screen →
Sequencing → Invasive
Fully Integrated
Screen
Positive
Sequencing-Based
Screen
Invasive Confirmatory
Test
No Invasive
Confirmatory Test
Trisomy 21
False Positive
Negative
Negative
CVS Miscarriage
Trisomy 21
Positive
1st Trimester Sequencing → CVS
1st Trimester
Combined
Positive
Negative
CVS
False Positive
Sequencing-Based
Screen
No Confirmatory Test
Negative
Sequencing-Based Tests to Determine Fetal Down Syndrome (Trisomy 21) from Maternal Plasma DNA
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Figure 2. Decision Tree for Possible Prenatal Screening and Confirmation Strategies to Detect Trisomy 21 (cont’d)
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Parameter
Value (%)
Range (%)
Source
Noninvasive aneuploidy testing (1st TRI)
Table 31
Sensitivity T21
99.0
95–100
Specificity T21
99.8
98–100
Malone et al. 2005 (FASTER trial)
Combined screen (1st TRI)
Sensitivity3
85.0
77–92
Specificity
96.2
93–98
Integrated (1st + 2nd TRI)
Sensitivity
96.0
92–97
95
94–96
Maternal serum testing2
Specificity
Fetal loss rate after amniocentesis
0.5
Muller et al. 2002; Caughey et al. 2006;
Eddleman et al. 2006; Mungen et al. 2006;
Mujezinovic and Alfirevic 2007; Odibo et
al. 2008
Fetal loss rate after CVS5
1.1
Caughey et al. 2006
Uptake rate by risk, amniocentesis
High Risk 75%
Low Risk 50%
Uptake rate by risk, CVS
High Risk 75%
Low Risk 50%
5
Abbreviations: TRI, trimester; CVS, chorionic villus sampling; FASTER, First- and Second-Trimester Evaluation of Risk
1
Values were chosen to represent average results from 3rd-party studies of all sequencing assays, not any particular sequencing assay
2
Each in combination with maternal age
3
85% is value at 12 wks; range is max and min for CIs for 11-13 wks
4
Arbitrary; false positive rate was set at 5% to obtain statistics on sensitivity, see Malone et al. 2005
5
Derived from studies that adjusted for rate of spontaneous abortion in a control population
Mueller et al. 2005; Nicolaides et al. 2005;
Palomaki et al. 2012
Technology Evaluation Center
32
Table 8. Parameters for Trisomy 21 Screening Decision Model
Cases Detected, Base Case
(range of best, worst cases)
Standard Screen
Type
Invasive Procedure
Traditional
Screening1
Screening by
Sequencing1
Invasive Procedures, Base Case
(range of best, worst cases)
Traditional then
Sequencing1
Traditional
Screening
Screening by
Sequencing
Traditional then
Sequencing
High-risk population, in utero T21 incidence = 950 per 100,000
Maximum cases detectable at 75% confirmatory (invasive) test uptake = 712
Integrated
Amnio
684
(691–656)
705
(712–677)
677
(691–623)
4,398
(3,627–5,148)
854
(677–2,198)
685
(623–780)
Combined,
Standard
Sensitivity
CVS
606
(656–549)
705
(712–677)
600
(656–521)
3,429
(2,034–5,856)
854
(677–2,198)
605
(521–760)
Combined, High
Sensitivity2
CVS
N/A3
705
(712–677)
670
(698–623)
N/A3
854
(677–2,198)
697
(623–1,114)
Average-risk population, in utero T21 incidence = 250 per 100,000
Maximum cases detectable at 50% confirmatory (invasive) test uptake = 125 Integrated
Amnio
120
(121–115)
124
(125–119)
119
(121–109)
2,614
(2,110–3,114)
224
(119–1,123)
124
(109–181)
Combined,
Standard
Sensitivity
CVS
106
(115–96)
124
(125–119)
105
(115–91)
2,002
(1,094–3,606)
224
(119–1,123)
109
(91–185)
Combined, High
Sensitivity
CVS
N/A
124
(125–119)
118
(122–109)
N/A
224
(119–1,123)
136
(109–402)
All screening strategies followed by recommendation for an invasive procedure (CVS in first trimester, amniocentesis in second trimester) if screening assay indicates high risk for trisomy. Standard screening is either
the combined screen (free beta-hCG, PAPP-A, nuchal translucency) or the integrated screen (PAPP-A, nuchal translucency in first trimester; quad screen [MSAFP, hCG, unconjugated estriol, inhibin A] in second trimester,
amniocentesis recommended if positive). ‘Standard then sequencing’ indicates that the standard screening procedure is done first, then patients with positive results are screened again by a sequencing assay. See Figure 2.
2
Assumes parameters for determining results of the traditional combined test are amended to allow improved case detection even though the false positive rate would be unacceptably increased for recommending an
invasive procedure. Would only be used if followed by sequencing-based testing.
3
Because this ‘high sensitivity’ combined screen is currently hypothetical and intended to be used only in combination with a follow up sequencing-based assay, these data are not reported.
1
Sequencing-Based Tests to Determine Fetal Down Syndrome (Trisomy 21) from Maternal Plasma DNA
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Table 9. Results of a Decision Model, Comparing Trisomy 21 Testing Outcomes from Three Different Screening Plus Confirmatory Testing Strategies:
Detection Rate and Invasive Procedure Outcomes
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Miscarriages, Base Case
(range of best, worst cases)
Standard Screen
Type
Invasive
Procedure
Traditional
Screening1
Screening by
sequencing1
Euploid Miscarriages, Base Case
(range of best, worst cases)
Traditional then
Sequencing1
Traditional
Screening
Screening by
Sequencing
Traditional then
Sequencing
High-risk population, in utero T21 incidence = 950 per 100,000
Maximum cases detectable at 75% confirmatory (invasive) test uptake = 712
Integrated
Amnio
22
(18–26)
4
(3–11)
3
(3–4)
19
(15–22)
1
(0–7)
0
(0–0)
Combined,
Standard
Sensitivity
CVS
38
(22–64)
9
(7–24)
7
(6–8)
31
(16–57)
2
(0–16)
0
(0–1)
Combined, High
Sensitivity
CVS
N/A
9
(7–24)
8
(7–12)
N/A
2
(0–16)
0
(0–5)
Average-risk population, in utero T21 incidence = 250 per 100,000
Maximum cases detectable at 50% confirmatory (invasive) test uptake = 125 Integrated
Amnio
13
(11–16)
1
(1–6)
1
(1–1)
12
(10–15)
0
(0–5)
0
(0–0)
Combined,
Standard
Sensitivity
CVS
22
(12–40)
2
(1–12)
1
(1–2)
21
(11–38)
1
(0–11)
0
(0–1)
Combined, High
Sensitivity
CVS
N/A
2
(1–12)
1
(1–4)
N/A
1
(0–11)
0
(0–3)
All screening strategies followed by recommendation for an invasive procedure (CVS in first trimester, amniocentesis in second trimester) if screening assay indicates high risk for trisomy. Standard screening is either
the combined screen (free beta-hCG, PAPP-A, nuchal translucency) or the integrated screen (PAPP-A, nuchal translucency in first trimester; quad screen [MSAFP, hCG, unconjugated estriol, inhibin A] in second trimester,
amniocentesis recommended if positive). ‘Standard then sequencing’ indicates that the standard screening procedure is done first, then patients with positive results are screened again by a sequencing assay. See Figure 2.
2
Assumes parameters for determining results of the traditional combined test are amended to allow improved case detection even though the false positive rate would be unacceptably increased for recommending
an invasive procedure. Would only be used if followed by sequencing-based testing.
1
Technology Evaluation Center
34
Table 10. Results of a Decision Model, Comparing Trisomy 21 Testing Outcomes from Three Different Screening Plus Confirmatory Testing Strategies:
Total and Euploid (No Trisomy 21) Miscarriage Outcomes
Sequencing-Based Tests to Determine Fetal Down Syndrome (Trisomy 21) from Maternal Plasma DNA
However, sequencing-based testing alone still
captured the most cases. High-sensitivity combined testing followed by a sequencing based
test also resulted in a similar number of invasive procedures as the integrated screen and
about 20% fewer procedures than screening by
sequencing alone.
Whether sequencing-based screening is used as
a replacement for traditional serum screening
or as a follow-on test, there is a large impact
on the number of miscarriages of euploid (no
trisomy 21) miscarriages following an invasive
procedure (Table 10). With traditional screening
in a high-risk population, about 20–30 normal
fetuses are lost per 100,000 women screened;
using sequencing-based testing, the numbers
drop to 0–2. For low-risk women, 10–20 normal
fetuses are lost per 100,000 women screened;
with the use of sequencing-based testing, the
numbers drop to 0–1.
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 (Ehrich et al. 2011; Palomaki et
al. 2011; Nicolaides et al. 2012; Norton et al.
2012; Palomaki et al. 2012). In these studies,
the actual false-positive test results were not
always borderline; some were clearly above
the assay cutoff value, and no processing or
biological explanations for the false-positive
results were reported. Using an overall estimate for predictive value calculations, even in
a high-risk population, the predictive value of a
positive result is only 83% (see Table 4). Thus,
in the absence of substantial data to confidently
characterize the false-positive rate, a confirmatory test remains a strong recommendation.
Evidence suggests that spectrum bias is
unlikely to be a factor in assay performance
characteristics. For example, Palomaki et al.
reported no significant differences in sequencing z-scores for women of advanced maternal
age versus women with an abnormal first
trimester serum and nuchal translucency
combined test result, although the etiology
of risk for a trisomy syndrome is likely different for these 2 populations (Palomaki et
al. 2012). That sequencing-based assays can
detect trisomy in lower prevalence populations
with equal accuracy is suggested although not
confirmed by the Nicolaides et al. study.
The decision model discussed in the preceding text was applied only to Down syndrome.
However, based on the assay performance data
for trisomy 18, which is very similar to that
for trisomy 21, it is likely the outcome trends
would be similar for trisomy 18. The data for
trisomy 13 are too sparse for specific conclusions, but there is no biologic reason to suggest
outcomes would be different.
Discussion
This Assessment addressed the analytic and
clinical validity, and clinical utility of nucleic
acid sequencing-based testing primarily for
Down syndrome (trisomy 21) compared to
traditional screening procedures. Detection of
Down syndrome cases is the original clinical
reason for testing; standard screening tests may
also detect trisomy 18 and 13. Thus, our report
is primarily focused on results for trisomy 21.
As noted, 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 laboratorydeveloped 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 (Chen et
al. 2009). 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 laboratory 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
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Technology Evaluation Center
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 three companies currently offering fetal trisomy syndrome screening by sequencing DNA in maternal plasma
and with published studies meeting inclusion
criteria for this Assessment show good clinical
validity, with high sensitivity and specificity
for Down syndrome and for trisomy 18. Few
studies reported results for trisomy 13 and few
cases were available in those that did, making
it difficult to characterize overall performance
for trisomy 13. Using estimates for in utero
prevalence of Down syndrome for both a highrisk (maternal age ≥35) and an average-risk
(general screening) population, all calculated
negative predictive values for Down syndrome
were nearly 100%, close to ideal for screening.
Calculated positive predictive values varied
considerably according to prevalence and the
estimates used for specificity. Notably, however,
false-positive rates were relatively invariant
across a wide spectrum of trisomy 21 prevalence values. 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 could
be 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. In a
simple decision model that examined both highrisk and average-risk populations, sequencingbased trisomy 21 screening followed by invasive
confirmatory testing was compared to the first
trimester combined screening test and to the
integrated screening test, both followed by
invasive confirmatory testing.
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
36
et al. 2012) and two-thirds of the population of
(average risk) pregnant women accept screening, then of about 2.8 million screened with the
integrated screen, 74,434 will have an invasive
procedure (assuming 50% uptake after a positive screening test and a recommendation for
confirmation), 370 will have a miscarriage,
of which 342 will be normal (non-trisomy 21)
fetuses and 3,417 of 3,559 Down syndrome
cases will be detected. Using sequencing-based
testing instead of traditional screening reduces
the number of invasive procedures to 6,378
and the number of miscarriages to 32 (after
amniocentesis; 14 normal) or 70 (after CVS; 31
normal), while increasing the cases detected
to 3,531 of 3,559 possible, using conservative
estimates. False negatives are conservatively
estimated at 266 of 2.8 million women screened
(0.01%) and may be lower, indicating that invasive testing after a negative result would have
more risk than benefit.
Another testing strategy is to add sequencingbased testing only after a positive first trimester
traditional combined screen, which in the prior
scenario would decrease invasive procedures
further to 3,103, miscarriages would decrease
to 34 after CVS (only 1 normal), but only 2,990
of 3,559 cases would be detected. Thus, while
this strategy has the lowest rate of miscarriages
of which only 1 represents a normal fetus,
and the lowest rate of invasive procedures it
detects fewer cases than sequencing-based
testing alone. Clearly, a strong advantage of
using sequencing-based assays, either in place
of traditional serum screening or as a follow-on
assay, is that miscarriages of normal fetuses
during confirmatory invasive procedures are
likely to be considerably reduced.
The outcomes represented by the model 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 strongly recommended 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, falsepositive rate together with the low baseline
prevalence of trisomy 21 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. The first trimester ultrasound
©2013 Blue Cross and Blue Shield Association. Reproduction without prior authorization is prohibited.
Sequencing-Based Tests to Determine Fetal Down Syndrome (Trisomy 21) from Maternal Plasma DNA
scan that confirms gestational age and determines whether the pregnancy is multiple
provides necessary information for sequencingbased testing. The fetal anatomy detailed by
ultrasound examination 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. Sequencingbased 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.
Limitations of sequencing-based tests include
a test failure rate (due either to a low fetal
DNA fraction in the maternal plasma sample,
to a deliberately chosen “no-call” zone, or to
unexplained assay failure) that may be as low
as 1% or as high as about 5%, depending on
the assay. Test failures incur the additional time
for collection and delivery of a new sample, in
addition to the assay turnaround time; otherwise, patients may proceed directly to an invasive procedure without screening results. Each
assay is currently specific for certain aneuploidies, and expansion of services is likely in the
near future. For example, some assays detect
aneuploidies of sex chromosomes (Table 1,
see footnote). 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 (Peters et al. 2011) and
single-gene disorders (Sayres and Cho 2011).
Moreover, the feasibility of mapping an entire
fetal genome using this technology has been
demonstrated (Kitzman et al. 2012). 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” (Allyse et al. 2012).
Summary of Application of the
Technology Evaluation Criteria
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
trisomy 21 in women being screened for fetal
trisomy syndromes.
1. The technology must have final
approval from the appropriate
governmental regulatory bodies.
None of the commercially available sequencing
assays for fetal trisomy syndromes has been
submitted to or reviewed by the U.S. 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.
Eight studies reported on the performance
of DNA sequencing-based trisomy syndrome
screening in singleton high-risk pregnancy
populations with invasive confirmatory procedures planned or completed. A ninth 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. 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 trisomy 21.
©2013 Blue Cross and Blue Shield Association. Reproduction without prior authorization is prohibited.37
Technology Evaluation Center
3. The technology must improve
the net health outcome, and
4. The technology must be as beneficial
as any established alternatives.
In a decision model, sequencing-based maternal plasma fetal trisomy 21 testing reduced the
number of invasive confirmatory procedures
needed and consequent associated miscarriages, while improving the number of detected
cases of trisomy 21, 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 9 studies were conducted by third-party
investigators at multiple clinical locations
(13–60 sites) in the U.S. and other countries;
3 of 4 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 sequencingbased testing of maternal plasma for trisomy
21 with confirmatory testing of positive results
(as is expected to be performed in a real-world
clinical setting) in both high risk women and
average-risk women being screened for trisomy
21 meets the TEC criteria.
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.
CONFIDENTIAL: This document contains proprietary information that is intended solely for Blue Cross and Blue Shield Plans
and other subscribers to the TEC Program. The contents of this document are not to be provided in any manner to any other
parties without the express written consent of the Blue Cross and Blue Shield Association.
38
©2013 Blue Cross and Blue Shield Association. Reproduction without prior authorization is prohibited.
Sequencing-Based Tests to Determine Fetal Down Syndrome (Trisomy 21) from Maternal Plasma DNA
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©2013 Blue Cross and Blue Shield Association. Reproduction without prior authorization is prohibited.41
Technology Evaluation Center
Appendix
42
©2013 Blue Cross and Blue Shield Association. Reproduction without prior authorization is prohibited.
Yes
No
Not
Clear
Low
Risk
High
Risk
(% of studies1)
QUADAS domain/item
Comment
Patient selection
– Was a consecutive or random sample of patients enrolled?
22
– Was a case-control design avoided?
33
67
– Did the study avoid inappropriate exclusions?
44
11
Could the selection of patients have introduced bias?
78
Seldom well-described
44
44
44
11
Unlikely but rated unclear when the
method of enrollment was unclear
Index test
– Were the index test results interpreted without knowledge of the reference standard?
100
– If a threshold was used, was it pre-specified?
100
Could the conduct or interpretation of the index test have introduced bias?
100
Reference standard
– Is the reference standard likely to correctly classify the target condition?
89
11
One study in average risk population
primarily used phenotype at birth
–Were the reference standard results interpreted without knowledge of the results of the
index test?
89
11
Likely true in most studies but not
well described; evaluation often
inferential
Could the reference standard, its conduct, or its interpretation have introduced bias?
11
89
Sequencing-Based Tests to Determine Fetal Down Syndrome (Trisomy 21) from Maternal Plasma DNA
©2013 Blue Cross and Blue Shield Association. Reproduction without prior authorization is prohibited.43
Appendix Table. Results of QUADAS-2 Study Quality Evaluation (consensus of 2 reviewers)
©2013 Blue Cross and Blue Shield Association. Reproduction without prior authorization is prohibited.
Yes
No
Not
Clear
Low
Risk
(% of studies1)
QUADAS domain/item
High
Risk
Comment
Flow and timing
– Was there an appropriate interval between index test(s) and reference standard?
89
11
– Did all patients receive a reference standard?
89
11
– Did patients receive an acceptable reference standard?
89
11
– Were all patients included in the analysis?
78
22
Could the patient flow have introduced bias?
11
Primarily karyotype; FISH for specific
trisomies when sample not sufficient
for karyotype; one study in average
risk population used either karyotype
(small percent) or phenotype at birth
89
Concerns regarding applicability
Patient selection: Is there concern that the included patients do not match the review question?
100
QUADAS review addressed the high
risk pregnancy population
Index test: Is there concern that the index test, its conduct, or interpretation differ from the
review question?
100
Reference standard: Is there concern that the target condition as defined by the reference
standard does not match the review question?
100
1
The two studies authored by Palomaki et al. (Palomaki et al. 2011; Palomaki et al. 2012) were counted as a single study for this analysis
Technology Evaluation Center
44
Appendix Table. Results of QUADAS-2 Study Quality Evaluation (consensus of 2 reviewers) (cont’d)
Sequencing-Based Tests to Determine Fetal Down Syndrome (Trisomy 21) from Maternal Plasma DNA
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