Noninvasive Prenatal Cell-Free Fetal DNA Based Screening for Aneuploidies Other Than Trisomy 21
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Noninvasive Prenatal Cell-Free Fetal DNABased
Screening for Aneuploidies Other Than Trisomy 21
EXECUTIVE SUMMARY
Background
Fetal chromosomal abnormalities occur in approximately 1 in 160 live births. The majority of fetal
chromosomal abnormalities are autosomal aneuploidies, defined as an abnormal number of autosomes.
The trisomy syndromes are aneuploidies involving 3 copies of 1 chromosome. Trisomy 21 (T21; Down
syndrome) is the most common fetal aneuploidy that is associated with survival to birth and beyond.
Trisomy 18 (T18; Edwards syndrome) and trisomy 13 (T13; Patau syndrome) are the next most common
fetal aneuploidy syndromes associated with survival to birth, although the percentage of cases surviving
to birth is low and survival beyond birth is limited. Maternal age is the most important risk factor for T21,
T18, and T13, with an approximate risk of 1:1600 at age 15 and1:28 by age 45.
Sex chromosome aneuploidies (SCA; eg, 45, X; 47, XXY; 47, XYY) may occur in 1 of 400 live births,
which makes them more common than individual autosomal aneuploidies. Turner syndrome (45, X) is a
relatively common SCA that may be suspected prenatally based on fetal ultrasound findings, which may
include nuchal translucency, and cardiac abnormalities plus abnormal serum biomarker levels (elevated
human chorionic gonadotropin and inhibin, decreased alpha-fetoprotein, and unconjugated estriol levels).
However, well-defined maternal serum biomarkers and algorithms for a prenatal screen for specific SCA,
as for T21 or other autosomal aneuploidies, are not reported. SCA are usually diagnosed postnatally, in
association with specific health problems, including diminished fertility or infertility. They also may be
detected by karyotyping fetal cells obtained via amniocentesis or chorionic villus sampling (CVS) in
pregnant women. However, the clinical significance of diagnosing an SCA is not entirely clear.
Current guidelines recommend that all pregnant women be offered screening for T21 before 20 weeks of
gestation, regardless of age. Screening programs may also detect T18 or T13. Noninvasive screening
typically involves combinations of maternal serum markers and fetal ultrasound at various stages of
pregnancy, but a standard approach is not defined. The detection rate for T21 using various combinations
of noninvasive tests ranges from 60% to 96% when the false-positive rate is set at 5%, although the false-
positive rate may be set lower for the rarer aneuploidies. Currently, noninvasive screening tests are not
sufficiently specific to diagnose a trisomy syndrome, so confirmatory karyotyping is required. Given the
low prevalence of trisomy syndromes, most patients who are recommended for confirmatory, invasive
prenatal diagnostic procedures (eg, amniocentesis and CVS) have normal results.
Direct karyotyping of fetal tissue obtained by amniocentesis (second trimester) or CVS (first trimester) is
required to confirm the diagnosis of trisomy. Both amniocentesis and CVS are invasive procedures and
have a small risk of miscarriage. A screening strategy minimizing amniocentesis and CVS procedures
(and thus associated miscarriage) and increasing detection of aneuploidies has potential to improve
outcomes.
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Notice of Purpose: TEC Assessments and Special Reports are scientific opinions, provided solely for informational purposes. TEC Assessments and
Special Reports should not be construed to suggest that Blue Cross Blue Shield Association 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 nonpayment of the technology or technologies evaluated.
Blue Cross Blue Shield Association is an association of independent Blue Cross and Blue Shield companies.
© 2014 Blue Cross Blue Shield Association. Reproduction without prior authorization is prohibited.TEC ASSESSMENT
Noninvasive Prenatal Cell-Free Fetal DNABased Screening for Aneuploidies Other Than Trisomy 21
As early as 8 to 10 weeks of gestation, cell-free fetal DNA fragments (derived from the cytotrophoblastic
cell layer of the placenta) may comprise 6% to 10% or more of the total cell-free DNA in a maternal
plasma sample. As detailed in a 2012 TEC Assessment, DNA sequencing, particularly massively parallel
sequencing (MPS; also known as next-generation or “next-gen” sequencing) is highly accurate for
prenatal detection of T21. The methods used to screen for T21 using MPS have been extended to other
less common aneuploidies (primarily T13 and T18), as well as the most common SCA (45, X; 47, XXY;
47, XYY).
Objective
To determine whether DNA sequencingbased testing for T13, T18, and SCA using cell-free fetal DNA
improves net health outcomes compared with a traditional combined serum- and ultrasound-based
screening strategy. In the analysis, we presume that a decision to screen for fetal aneuploidy would
initially focus on T21 and that downstream events related to T21 screening will hold for screening for T13,
T18, or SCA.
Search Strategy
PubMed and EMBASE databases were searched for articles published between January 1, 2009, and
June 23, 2014, limited to English-language publication in human populations. Several search terms were
combined, such as “trisomy,” “aneuploidy,” “sequencing,” “prenatal diagnosis,” “chromosome 18 or 13,”
and “cell-free DNA.”
Selection Criteria
Included studies had the following characteristics: (1) performed maternal cell-free fetal DNA testing of
pregnant women being screened for T18, T13, and SCA; (2) used a sequencing assay that is clinically
available or applied clinical laboratory quality control measures; (3) compared the results of cell-free fetal
DNA testing with the results of karyotype analysis (or fluorescence in situ hybridization 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
We sought to determine the analytical and clinical validity and clinical utility of cell-free fetal DNAbased
screening for fetal aneuploidies T13, T18, and SCA. We identified 29 published articles that described
results of cell-free fetal DNA sequencingbased screening using one of several tests. Studies reported
results for T13 (N=16,927 patients screened), T18 (N=32,554 patients screened), monosomy X (N=8994
patients screened), and other SCA (N=6449 patients screened) in high-risk (age >35) and average-risk
(pregnant women who elect screening) cohorts. Maternal study populations were described or we inferred
them to be: high risk for fetal aneuploidies in 21 of 29 (72%) studies; average risk in 6 (21%); mixed risk
in 1 (3.5%); and not reported in 1 (3.5%). Among studies of T13 screening, 15 reported results in women
deemed high risk for fetal aneuploidy (n=13,680) and 3 reported results in average-risk women (n=2144).
For T18, 16 studies included women at high risk (n=16,694), and 6 included average-risk women
(n=14,757).
After assessing the quality of individual published studies using the QUADAS-2 tool, we pooled sensitivity
and specificity estimates of fetal DNA‒based testing (all tests compiled), stratified according to maternal
risk level (high or average). To address statistical issues in pooling studies with 1 or just a few cases, we
pooled studies that included 5 or more cases (see Table A). The approach removed most studies in the
average-risk population; however, it had little effect on the overall pooled estimates. The detection rates
for T13 ranged from 76% to 92%; for T18, they ranged from 91% to 97%. The pooled specificity for either
T13 or T18 was nearly 100%. The detection rates for the SCA ranged from 77% to 91%, with specificity
nearly 100%.
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Noninvasive Prenatal Cell-Free Fetal DNABased Screening for Aneuploidies Other Than Trisomy 21
Table A. Meta-Analysis Results of Cell-Free Fetal DNA‒Based Test Screen Performance
Sensitivity Specificity
Pooled Estimate Normal Pooled Estimate
2 2
Aneuploidy Studies Cases FN (95% CI) I,% (N) FP (95% CI) I,%
T13 7 95 8 0.86 (0.76 to 0.92) 0 8724 20 0.99 (0.99 to 1.00) 0
T18 15 392 10 0.95 (0.91 to 0.97) 0 22,754 15 1.00 (0.99 to 1.00) 68
Monosomy X 14 137 14 0.86 (0.79 to 0.92) 0 5286 15 1.00 (0.99 to 1.00) 0
Other SCA 5 37 1 0.91 (0.77 to 0.97) 0 4643 3 1.00 (0.99 to 1.00) 39
2
CI: confidence interval; FN: false negatives; FP: false positives; I : measure of statistical heterogeneity; SCA: sex
chromosome aneuploidies.
Table B shows posttest probabilities calculated for cell-free fetal DNA‒based testing for T13 and T18.
These calculations show that a screen-positive result will require karyotype analysis to confirm or rule out
the presence of T13 or T18. A negative finding reflects an exceedingly low probability for the presence of
aneuploidy, sufficient to preclude the need for a diagnostic karyotype.
Table B. Posttest Probabilities of Aneuploidy Following Cell-Free Fetal DNA‒Based Screening
Test
Prevalence/ Prevalence Positive Probability After Negative Probability After
Risk Per 100,000 Likelihood Ratio Positive Test Likelihood Ratio Negative Test
High
T13 60 86 0.049 0.14 0.00008
T18 170 95 0.14 0.05 0.00009
Average
T13 13 86 0.011 0.14 0.00002
T18 40 95 0.037 0.05 0.00002
We constructed a simple decision model to assess cell-free fetal DNA‒based screening compared with a
standard integrated screen for T13 and T18. The model presumes that screening for T21 is performed
concurrently and that amniocentesis or CVS with karyotype analysis subsequent to non-sequencing-
based screening is prompted by the desire to detect T21. This model assumes that invasive procedures
of amniocentesis or CVS and any resulting miscarriages are not a consequence of T13 or T18 screening.
We did not model case detection rates for SCA owing to difficulty defining health outcomes for SCA
identification.
The strategies examined in the model include:
a. a traditional integrated screen (first plus second trimester serum testing and nuchal translucency
ultrasound) test followed, if positive, with an invasive procedure (CVS in the first trimester or
amniocentesis in the second trimester) for confirmatory karyotyping; and
b. cell-free fetal DNAbased testing in place of traditional serum screening; if positive, confirm with
invasive procedure and karyotyping.
The outcomes of interest are the number of cases of T13 or T18 correctly identified and the number of
cases missed. 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 who tested
positive on initial screen and were offered an invasive confirmatory procedure, we assumed 75% of high-
risk and 50% of average-risk women would proceed to an invasive test after a positive screen. We varied
sensitivities and specificities for both standard and cell-free fetal DNA‒based screening tests to represent
a plausible range of possible values.
For either high- or average-risk women, our results suggest that a strategy of screening by sequencing-
based assay followed by confirmatory diagnostic karyotype testing generally detects at least the same if
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Noninvasive Prenatal Cell-Free Fetal DNABased Screening for Aneuploidies Other Than Trisomy 21
not more T13 and T18 cases and misses fewer than does a traditional integrated screen followed by a
diagnostic invasive karyotype analysis. This relationship held whether we obtained estimates using
pooled sequencing performance parameters specific to T13 and T18 or performance parameters similar
to the more prevalent T21 (not shown).
Although fewer T13 and T18 cases are detected than T21 because prevalences of the former are lower
than T21, base case estimates show detection of high proportions of cases for both populations (98%,
respectively) using the sequencing to invasive testing strategy. In high-risk women, eg, for T13, T18, and
T21 combined, the number of cases missed with integrated screening followed by invasive testing and
karyotyping (40/884 [4.5%] maximum cases) falls to 19 of 884 (2.1%) (see Table 6 in the body of the
Assessment). It follows that the number of invasive procedures needed and the number of total
miscarriages after an invasive confirmatory karyotype procedure in this population would be reduced
commensurately. The numbers derived for the average-risk population would be expected to be
consistent in direction and magnitude of change with those for high-risk women.
Author Conclusions and Comment
This Assessment addressed the analytic and clinical validity and clinical utility of cell-free fetal DNA-based
testing for T13, T18, and SCA compared with traditional screening procedures. Little direct evidence
exists on analytic validity. The commercially available tests are offered as laboratory-developed tests
subject to laboratory operational oversight under the Clinical Laboratory Improvement Act (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 is becoming more common in the clinical laboratory, and regulatory and
professional organizations are addressing important issues of methods standardization.
Numerous studies of cell-free fetal DNA‒based assay performance relative to the reference standard of
karyotyping in high- and average-risk populations are available. Some are multisite studies that have
incorporated specimen collection, transport, and evaluation under conditions simulating real-world clinical
testing. Our assessment of overall study quality indicated a low risk of bias, except in the domain of
patient selection.
In general, assays from all companies currently offering fetal trisomy screening by sequencing cell-free
fetal DNA in maternal plasma show high sensitivity and specificity for T13, T18, and SCA. False-positive
rates were relatively consistent across the prevalence rates for the aneuploidies. Calculated posttest
probabilities for a negative T13 or T18 test were exceedingly small. Thus, the clinical validity of
sequencing-based cell-free fetal DNA screening appears to be well defined for T13, T18, and SCA.
To examine clinical utility requires a comparison with current screening practices and evaluation of the
impact of screening on health outcomes. We did not identify direct comparative evidence of outcomes, so
we performed a decision analysis model exercise to estimate the number of cases detected and cases
missed using prevalence data from the literature; summarized data on cell-free fetal DNA assay
performance for T13 and T18; and published data on traditional screening performance and patient
uptake of confirmatory test procedures.
Our findings indicate that for pregnant women undergoing aneuploidy screening, a strategy of using a
cell-free fetal DNA‒based screening test followed by confirmation of positive test results with an invasive
procedure (amniocentesis or CVS) to determine fetal karyotype detected an equivalent or larger
proportion of fetal T13 or T18 and missed fewer cases than a strategy employing the traditional integrated
screen followed by amniocentesis or CVS diagnosis. Given that T13 and T18 cell-free fetal DNA‒based
tests will be performed along with T21 testing, the number of invasive procedures and miscarriages
secondary to an invasive diagnostic procedure will be reduced with the cell-free fetal DNA‒based strategy
(based on the conclusions of the 2012 TEC Assessment examining T21). The nearly null posttest
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Noninvasive Prenatal Cell-Free Fetal DNABased Screening for Aneuploidies Other Than Trisomy 21
probability of a negative sequencing-based screen T18 and T13 provides assurance to pregnant women
in any risk category that a negative screen minimizes any need (except as indicated by abnormal
ultrasound—eg, nuchal cord translucency, cystic hygroma) for a subsequent invasive diagnostic
karyotype test and negates accompanying risks for fetal loss.
Cell-free fetal DNA‒based testing without confirmatory karyotype analysis carries a risk of misidentifying
normal pregnancies as positive for a trisomy syndrome due to the small false-positive rate together with
the low baseline prevalence of T13, T18, and SCA in all populations. The false-positive rates of
sequencing-based tests appear lower than those of traditional serum- and ultrasound-based screening
tests. However, cell-free fetal DNA‒based testing does not eliminate the necessity of ultrasound studies.
The first trimester ultrasound scan is required to confirm gestational age and to determine whether the
pregnancy is multiple, findings that provide necessary information for sequencing-based testing of cell-
free fetal DNA. Ultrasound examination that details fetal anatomy in the second trimester is important for
fetal risk assessment, and may provide indications of chromosomal abnormalities not currently detected
by cell-free fetal DNA sequencingbased tests. Cell-free fetal DNAbased testing is also not a
replacement for second trimester maternal screening for risk of neural tube defects.
Finally, we did not compare all potential screening strategies such as serum screening and ultrasound
followed by cell-free fetal DNA or cell-free fetal DNA plus subsequent serum screening and ultrasound to
examine which might be optimal.
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 DNA sequencingbased testing of cell-free fetal DNA
meets the Blue Cross and Blue Shield Association Technology Evaluation Center (TEC) criteria to screen
expecting women for fetal trisomy syndromes 13 (T13), 18 (T18), and sex chromosome aneuploidies
(SCA).
1. The technology must have final approval from the appropriate governmental regulatory
bodies.
None of the commercially available sequencing assays for T13, T18, or SCA 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 [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.
Although we identified no direct evidence for the analytical validity of the massively parallel cell-free fetal
DNA sequencing assays that are commercially available, they are subject to laboratory operational
oversight under CLIA. Recommendations for good laboratory practices to ensure the quality of molecular
genetic testing for heritable diseases and conditions under CLIA have been published. Furthermore, next-
generation sequencing technology is becoming more common in the clinical laboratory, and regulatory
and professional organizations are addressing important issues of methods to standardize the use of this
technology. Therefore we have reason to conclude the analytical validity is sufficient to support use of the
available tests for the purposes described in this Assessment.
A body of 29 individual publications provides sufficient evidence to establish the clinical validity (sensitivity
and specificity vs criterion reference of karyotype analysis) of the available screening tests for T13, T18,
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© 2014 Blue Cross Blue Shield Association. Reproduction without prior authorization is prohibited.TEC ASSESSMENT Noninvasive Prenatal Cell-Free Fetal DNABased Screening for Aneuploidies Other Than Trisomy 21 and SCA. Among studies of T13 screening, 15 reported results in women deemed high risk (age >35) for fetal aneuploidy (n=13,680) and 3 reported results in average-risk (pregnant women who elect screening) (n=2144) populations. For T18, 16 studies included women at high risk (n=16,694), and 6 included average-risk women (n=14,757). We identified no direct comparative evidence that use of any of the cell-free fetal DNA‒based tests alters clinical management compared with other screening strategies. To address clinical utility, we conducted a decision-modeling exercise to compare T13 and T18 case detection rates using either a cell-free fetal DNA‒based screen followed by invasive diagnostic karyotype confirmation of screen-positives or a traditional integrated maternal serum- and ultrasound-based screen followed by the same invasive diagnostic karyotype analysis. The decision model showed that the cell-free fetal DNA‒based strategy was at least equivalent, but generally superior to, a traditional screening strategy, using 2 different sets of cell-free fetal DNA test sensitivity and specificity estimates, thus indirectly supporting the clinical utility of the tests for T13 and T18. We did not model SCA in our analysis because the balance of benefits and harms of a positive cell-free fetal DNA‒based prenatal screen and subsequent karyotype diagnosis of an SCA—each of which has variable and uncertain prognosis and management—is unclear. Although evidence supports the accuracy of cell-free fetal DNA‒based test performance for SCA, we could not determine an effect of cell-free fetal DNA‒based screening on net health outcomes. Therefore, evidence on clinical utility is insufficient to draw conclusions for these aneuploidies. 3. The technology must improve the net health outcome. Noninvasive cell-free fetal DNA sequencing‒based screening for T13 or T18 will improve the net health outcome when used in a strategy that includes a sequencing-based screen followed by invasive diagnostic karyotype analysis in those who are screen-positive. Thus, compared with a standard integrated screening strategy, the number of invasive procedures, possible miscarriages secondary to an invasive diagnostic procedure, and the number of affected births will be reduced with the cell-free fetal DNA‒based strategy for T13 and T18. Furthermore, the nearly null posttest probability of a negative cell- free fetal DNA‒based screening test result provides assurance to pregnant women, in any risk category, that a negative screen obviates the need for a subsequent invasive diagnostic test and negates associated downstream risks. We did not include SCA in the decision analysis, because the implications of a screen-positive finding and diagnostic confirmation differ significantly from those of T13 and T18. The balance of benefits and harms of a positive cell-free fetal DNA‒based prenatal screen and subsequent diagnosis of SCA, each of which has variable and uncertain prognosis, is unclear. 4. The technology must be as beneficial as any established alternatives. A decision model showed that the use of sequencing-based cell-free fetal DNA screening increased the number of detected cases of T13 and T18, with commensurate reduction of missed cases, compared with standard integrated screening procedures for those parameters, in high- and average-risk (general obstetric) populations of pregnant women. Evidence is insufficient to determine the clinical benefit of cell-free fetal DNA‒based screening for SCA compared with traditional tests. 5. The improvement must be attainable outside the investigational settings. A number of studies were conducted by third-party investigators at multiple clinical locations (13-60 sites) in the United States and other countries; all companies’ assays were represented and samples were sent Volume 29, No. 7 Publication Date: December 2014 Page: 6 http://www.bcbs.com/blueresources/tec/vols/ © 2014 Blue Cross Blue Shield Association. Reproduction without prior authorization is prohibited.
TEC ASSESSMENT
Noninvasive Prenatal Cell-Free Fetal DNABased Screening for Aneuploidies Other Than Trisomy 21
to company laboratories for cell-free fetal DNA 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,
Sequencing-based analysis of cell-free fetal DNA obtained from maternal plasma to screen for
the presence of fetal T13 or T18—followed by diagnostic karyotype analysis of screen-positive
results—in either high-risk or average-risk pregnant women being screened for fetal autosomal
aneuploidies meets the Blue Cross and Blue Shield Association Technology Evaluation Center
(TEC) criteria.
Sequencing-based analysis of cell-free fetal DNA obtained from maternal plasma to screen for
the presence of fetal sex chromosome aneuploidies in pregnant women being screened for fetal
aneuploidies does not meet the Blue Cross and Blue Shield Association Technology Evaluation
Center (TEC) criteria.
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Noninvasive Prenatal Cell-Free Fetal DNABased Screening for Aneuploidies Other Than Trisomy 21
AUTHORS, STAFF, AND MEDICAL ADVISORY PANEL
TEC Staff Contributors
Lead Author: Thomas A. Ratko, Ph.D.; Co-Author: Ryan D. Chopra, M.P.H.
Executive Director, Center for Clinical Effectiveness: Suzanne E. Belinson, Ph.D., M.P.H.
Executive Director, Clinical Evaluation, Innovation, and Policy: Naomi Aronson, Ph.D.
Director, Technology Assessment: Mark D. Grant, M.D., Ph.D.
Research/Editorial Staff: Claudia Bonnell, R.N., M.L.S., Kimberly Hines, M.S., Michael Vasko, M.A.
Blue Cross Blue Shield Association Medical Advisory Panel
Chair
Trent T. Haywood, M.D., J.D., Senior Vice President, Clinical Affairs/Medical Director, Blue Cross Blue Shield Association
Vice Chair
Suzanne E. Belinson, Ph.D., M.P.H., Executive Director, Center for Clinical Effectiveness, Blue Cross Blue Shield Association
Scientific Advisors
Steven N. Goodman, M.D., M.H.S., Ph.D., Dean for Clinical and Translational Research, Stanford University School of Medicine, and
Professor, Departments of Medicine, Health Research and Policy
Mark A. Hlatky, M.D., Professor of Health Research and Policy and of Medicine (Cardiovascular Medicine), Stanford University School of
Medicine; American College of Cardiology Appointee
Panel Members
Peter C. Albertsen, M.D., Professor, Chief of Urology, and Residency Program Director, University of Connecticut Health Center
Ann Boynton, Deputy Executive Officer, Benefits Programs Policy and Planning, CalPERS
Virginia Calega, M.D., M.B.A., F.A.C.P., Vice President, Medical Management and Policy, Highmark Inc.
Sarah T. Corley, M.D., F.A.C.P., Chief Medical Officer, NextGen Healthcare Information Systems Inc.; American College of Physicians
Appointee
Helen Darling, M.A., Strategic Advisor, 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
Lee A. Fleisher, M.D., Professor and Chair, Department of Anesthesiology and Critical Care, University of Pennsylvania Perelman School of
Medicine, Senior Fellow, Leonard Davis Institute of Health Economics
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
Lawrence Hong Lee, M.D., M.B.A., F.A.C.P., Vice President and Executive Medical Director for Quality and Provider relations, Blue Cross
and Blue Shield of Minnesota
Bernard Lo, M.D., President, The Greenwall Foundation
Randall E. Marcus, M.D., Charles H. Herndon Professor and Chairman, Department of Orthopaedics, Case Western Reserve University
School of Medicine and University Hospitals Case Medical Center, Cleveland, Ohio
Barbara J. McNeil, M.D., Ph.D., Ridley Watts Professor and Head, Department of Health Care Policy, Harvard Medical School; Professor of
Radiology, Brigham and Women's Hospital
William R. Phillips, M.D., M.P.H., T.J. Phillips Endowed Professor in Family Medicine, University of Washington; American Academy of
Family Physicians Appointee
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
Maren T. Scheuner, M.D., M.P.H., F.A.C.M.G., Chief, Medical Genetics, VA Greater Los Angeles Healthcare System; Professor,
Department of Medicine, David Geffen School of Medicine at UCLA, Affiliate Natural Scientist, RAND Corporation; American College of
Medical Genetics and Genomics Appointee
Leslie Robert Schlaegel, M.S., Associate Vice President of Human Resources, Stanford University
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
John B. Watkins, Pharm.D., M.P.H., B.C.P.S., Pharmacy Manager, Formulary Development, Premera Blue Cross
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© 2014 Blue Cross Blue Shield Association. Reproduction without prior authorization is prohibited.TEC ASSESSMENT
Noninvasive Prenatal Cell-Free Fetal DNABased Screening for Aneuploidies Other Than Trisomy 21
Noninvasive Prenatal Cell-Free Fetal DNABased
Screening for Aneuploidies Other Than Trisomy 21
ASSESSMENT OBJECTIVE
The overall objective of this Assessment is to determine whether DNA sequencingbased testing to
screen for trisomy 13 (T13), trisomy 18 (T18), and sex chromosome aneuploidies (SCA) using cell-free
fetal DNA improves net health outcomes compared with a traditional serum- and ultrasound-based
integrated screening strategy.
Commercial, noninvasive, cell-free fetal DNA‒based testing of maternal plasma for T13, T18, and SCA
has recently become available and has the potential to substantially alter screening strategies for these
anomalies. Current noninvasive testing strategies have suboptimal accuracy and imperfect specificity, the
latter of which results in low positive predictive values. As a result, many invasive procedures are required
to identify a small number of pregnancies with T13, T18, and SCA. More accurate screening tests could
improve the efficiency and accuracy of screening and reduce unnecessary invasive procedures.
In this Assessment, cell-free fetal DNA‒based testing for T13 and T18 is compared with a current
integrated strategy for screening, which comprises noninvasive maternal serum biomarkers and fetal
ultrasound examination. We assumed that screening for trisomy 21 (T21) is performed as the priority and
that amniocentesis or chorionic villous sampling (CVS) subsequent to non-sequencing-based screening is
performed to detect T21. Based on these assumptions, we did not consider amniocentesis or CVS and
any resulting miscarriages to be a consequence of T13 or T18 screening.
Relevant clinical outcomes will include detection rates for T13 or T18, and number of cases missed using
each strategy. To estimate these rates, we estimated test sensitivity and specificity using meta-analysis
and the reference standard of invasive tissue sampling and karyotyping. The sensitivity and specificity will
be used in a decision model incorporating existing data to estimate rates of detected and missed cases.
BACKGROUND
Fetal Trisomy Syndromes
Fetal chromosomal abnormalities occur in approximately 1 in 160 live births. A majority of fetal
chromosomal abnormalities are aneuploidies, defined as an abnormal number of chromosomes
a
(autosomes or sex chromosomes). Trisomy syndromes are autosomal aneuploidies involving 3 copies of
1 chromosome. T21 (Down syndrome) is the most common form of fetal aneuploidy associated with
survival to birth and beyond. T18 (Edwards syndrome) and T13 (Patau syndrome) are the next most
common fetal aneuploidy syndromes associated with survival to birth, although the percentage of cases
1
surviving to birth is low and survival beyond birth is limited.
Trisomy may result from failure of chromosomal pairs to separate during meiosis (“nondisjunction”) or less
b
often from a Robertsonian translocation either way. The result is 3 copies of a chromosome rather than 2
a
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 1 copy of each.
b
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
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Noninvasive Prenatal Cell-Free Fetal DNABased Screening for Aneuploidies Other Than Trisomy 21
after fertilization. Mosaic forms of trisomy may also occur, in which only some cells 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 is the most important risk factor for a trisomy syndrome. The proportion of pregnancies that
occur with “advanced maternal age” has tripled over the last 30 years, to 14% of all pregnancies in the
2
United States. Prenatal screening for an autosomal trisomy began 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 T21 based on maternal age.
Sex Chromosome Aneuploidy
Trisomy of sex chromosomes also occurs; eg, 46 XXY (2 X plus 1 Y chromosomes) results in Klinefelter
syndrome. SCA (eg, 45, X; 47, XXY; 47, XYY) may occur in 1 of 400 live births, which make them more
common than individual autosomal aneuploidies.
Turner syndrome (45, X) is a relatively common SCA that may be suspected prenatally based on fetal
ultrasound findings, which may include nuchal translucency, cystic hygroma, and cardiac abnormalities,
plus abnormal serum biomarker levels (elevated human chorionic gonadotropin and inhibin, decreased
3,4
alpha-fetoprotein and unconjugated estriol levels). However, well-defined algorithms screening for other
specific SCA, as for T21 or other autosomal aneuploidies, are not reported. Because no maternal
biomarkers exist to screen for specific SCA, they are usually diagnosed postnatally, in association with
4,5
specific health problems including diminished fertility or infertility. When SCA are diagnosed prenatally,
they are often found incidentally, during invasive karyotype testing of pregnant women who are deemed
at high risk for an autosomal aneuploidy. Because most women are screened and tested to exclude fetal
T21-associated Down syndrome, diagnosis of an incidental SCA in an otherwise karyotypically normal
fetus may present unanticipated findings to parents This is a key consideration because the clinical
significance of an SCA may be unclear and ill-defined; many affected individuals in the end may not
exhibit any relevant symptoms or will have only mild symptoms, including infertility, whereas others will be
5-7
affected to a greater, unpredictable extent.
Unlike prenatal diagnosis of T13 or T18, prenatal diagnosis of an SCA may offer opportunity for early
prevention and management of SCA-related disease, psychological issues, and fertility problems in the
live-born child. Given that fetuses that carry an SCA typically survive to term—unlike those with T13 or
T18—the considerations for use of sequencing-based prenatal screening findings are not necessarily the
same.
Screening for Autosomal Trisomies Other Than T21
Guidelines from the American Congress of Obstetricians and Gynecologists (ACOG) recommend that all
8
pregnant women should be offered noninvasive screening for aneuploidies, particularly T21. Traditional
noninvasive screening has involved combinations of maternal serum markers and fetal ultrasound
performed at various stages of pregnancy, but a standard approach has not been defined. The most
common serum T21 screening test consists of a panel of maternal markers. Maternal serum markers
include alpha-fetoprotein, the free beta subunit of human chorionic gonadotropin (hCG), unconjugated
estriol, and inhibin A (not used in the risk calculation for T18). 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, in part, because first
trimester screening became available after second trimester screening became common.
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.
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Noninvasive Prenatal Cell-Free Fetal DNABased Screening for Aneuploidies Other Than Trisomy 21
Fetal ultrasound measures (nuchal cord translucency) are used to supplement the traditional maternal
serum markers and to increase the performance characteristics of the screening test panels. However,
specialized training is required to accurately determine nuchal translucency, and, as a result, the
performance of this screening technique may vary. Review of observational studies, evaluating nuchal
translucency screening in the general population, has revealed variation in reported Down syndrome
9
detection rates ranging from 29% to 91%. Other investigators have suggested that studies may be
limited because scans were often carried by sonographers who were either inadequately trained or not
10
sufficiently motivated to measure nuchal translucency. This wide rate detection variation emphasizes
the importance of training, measurement standardization, and ongoing quality control in screening
programs.
11
Contemporary T21 screening programs may also be used to detect T13, T18, and SCA. In a large
(N=36,171) study (FASTER), first trimester screening by cystic hygroma (present in ≥40% of fetuses with
T13 and T18) or combined screening had a 78% detection rate for all non-Down syndrome aneuploidies,
11
with an overall false-positive rate of 6.0%. In the same study, the second trimester quadruple screen
identified 69% of non-Down syndrome aneuploidies, at a false-positive rate of 8.9%. With the combined
screen, the use of T18 risk factors did not detect any additional non-Down syndrome aneuploidies
compared with the Down syndrome risk alone. In a position statement from the International Society for
Prenatal Diagnosis (ISPD), the detection rate for various combinations of these measures for T13 and
12
T18 ranges from 60% to 96% in various studies when the false-positive rate is set at 5%. The sensitivity
is highest when testing involves a “sequential” or “integrated” approach.
Although noninvasive tests have improved since they first became available, they are not sufficiently
sensitive or specific to diagnose any trisomy syndromes. Direct karyotyping of fetal tissue obtained by
amniocentesis or 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
13
of 1 in 300 to 500 procedures at experienced centers. Other complications of amniocentesis include
vaginal spotting with or without chorioamniotic leakage in 1% to 2% of cases and chorioamnionitis in
13
0.1% of cases.
CVS can be performed earlier in pregnancy, most commonly at 10 to 12 weeks of gestation. This
advantage of CVS may be offset by a higher rate of adverse events. The rate of pregnancy loss due to
CVS is in the range of that for mid-term amniocentesis, though some estimates put the rate slightly
13
higher. An increased rate of limb defects may exist after 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 with or without chorioamnionitis occur at a rate of less than 0.5%.
The choice of screening strategy depends on factors such as maternal age, gestational age at first
prenatal visit, previous obstetrical history, family history, availability of fetal ultrasound, parents’ risk
13
tolerance, and desire to undergo pregnancy termination if a trisomy syndrome is detected. A majority of
women elect to have noninvasive testing performed, with a decision for invasive testing based on
1
screening test results. Some women who are at particularly high risk due to factors outlined above, or
who want to rule out chromosomal abnormalities with certainty, may proceed directly to invasive testing.
Other women decline testing for trisomy altogether if they want to avoid the risks of invasive testing (see
following), or are certain that they would not alter decisions regarding their pregnancy based on results.
Clinicians disagree as to the specific screening strategy that is preferred.
Current screening strategies have a number of limitations. Foremost, current testing strategies have a low
positive predictive value due to the suboptimal specificity and low prevalence of trisomy syndromes. As a
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Noninvasive Prenatal Cell-Free Fetal DNABased Screening for Aneuploidies Other Than Trisomy 21
result, a majority of fetuses that are diagnosed using an invasive procedure are not found to have a
trisomy syndrome. The largest potential benefit of a sequencing-based screening strategy would
therefore be reducing amniocentesis and CVS procedures and their associated risks including fetal loss.
The sensitivity of noninvasive screening strategies is also imperfect, and some trisomies are not detected.
A noninvasive test with improved sensitivity would therefore reduce the number of cases that are missed.
Another limitation of current testing is the narrow gestational window of applicability and a need to
combine multiple markers, sometimes at different time points, to arrive at a clinically useful sensitivity and
specificity profile. For example, one of the best-case standard screening scenarios, called “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 T21 integrated screening
8
is 94% to 96% when specificity is 5%. Alternative screening methods would offer the opportunity for
improved efficiency.
Sequencing-Based Testing of Cell-Free Fetal DNA to Screen for Aneuploidies
14
In 1997, Lo et al reported that fetal cell-free DNA could be detected in the plasma of pregnant women,
and that this fetal DNA comprised approximately 3% to 6% of total cell-free DNA in a maternal blood
15
sample. The cytotrophoblastic cell layer of the placenta is the source of the fetal DNA rather than
16
circulating fetal cells. Fetal DNA is entirely present in short fragments, with a majority less than 200
17,18 17
base pairs (bp). Maternal DNA may have a broader fragment size distribution (majorityTEC ASSESSMENT
Noninvasive Prenatal Cell-Free Fetal DNABased Screening for Aneuploidies Other Than Trisomy 21
syndromes
1p36 deletion
Panorama (Natera) ● ● ● ● ●
Verifi (Verinata Health) ● ● ● ● ● ● ●
T: trisomy.
Statements From Professional Societies
ISPD published a rapid response statement (http://www.ispdhome.org) on 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 studies are
needed in average-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 its website
(http://nsgc.org/p/bl/et/blogid=47&blogaid=33). NSGC currently supports noninvasive prenatal
testing/noninvasive prenatal diagnosis (NIPT/NIPD) as an option for patients whose pregnancies are
considered at an increased risk for certain chromosome abnormalities. NSGC recommends that
NIPT/NIPD only be offered after informed consent, education, and counseling by a qualified provider (eg,
a certified genetic counselor). Patients whose NIPT/NIPD results are abnormal, or who have other factors
suggestive of a chromosome abnormality, should receive genetic counseling and be given the option of
standard confirmatory diagnostic testing.
The ACOG Committee on Genetics and the Society for Maternal-Fetal Medicine’s Publications Committee
21
published a Committee Opinion on noninvasive prenatal testing for fetal aneuploidy. The Opinion states
that pregnant women at increased risk of aneuploidy, but not those at average risk, can be offered
sequencing-based cell-free fetal DNA screen testing after pretest counseling. The Opinion further states
that women with positive screen 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.
The American College of Medical Genetics and Genomics (ACMG) has recently published a statement on
22
noninvasive prenatal screening for aneuploidy. The statement supports a conclusion that cell-free fetal
DNA‒based tests to screen for aneuploidy have high sensitivity and specificity, but ACMG highlighted
several limitations of the testing strategy, including:
Approximately 50% of cytogenic abnormalities identified by amniocentesis will not be detected
Chromosomal abnormalities such as unbalanced translocations, deletions, or duplications will not
be detected
Cell-free fetal DNA‒based tests cannot distinguish between specific types of aneuploidy (eg, an
extra copy of chromosome 21, a Robertsonian translocation of chromosome 21, high-level
mosasicim)
No screening for single-gene mutations
Delayed diagnosis if insufficient cell-free fetal DNA is isolated
Longer turn-around time for cell-free fetal DNA sequencing than for maternal serum testing
No testing for open neural tube defects
Limited data on nonsingleton pregnancies
No role in predicting late-pregnancy complications.
ACMG recommends that before a mother undergoes cell-free fetal DNA‒based testing for aneuploidy,
she should receive information on the purpose of the testing, advantages and disadvantages compared
with standard screening, testing limitations, and considerations for posttest counselling in the event of a
positive test.
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Noninvasive Prenatal Cell-Free Fetal DNABased Screening for Aneuploidies Other Than Trisomy 21
FDA Status
None of the commercially available sequencing assays for trisomy syndromes has been reviewed by the
Food and Drug Administration (FDA). Clinical laboratories may develop and validate tests in-house
(laboratory-developed tests [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.
METHODS
Search Strategy
We searched the National Library of Medicine PubMed and EMBASE biomedical literature databases in
April 2014, updated June 23 2014, after the MAP review, and show the search strategies and results in
Appendix Table 2.
Study Selection
The following selection criteria were applied to select studies for inclusion:
1. Performed maternal plasma cell-free fetal DNA testing of pregnant women being screened for
T13, T18, and SCA.
2. Used a sequencing assay that is clinically available and has reported application of clinical
laboratory quality control measures.
3. Compared the results of plasma cell-free 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
Data were abstracted from each study including the following elements:
Study/authors/year
Manufacturer of test
Number of participants and selection process for testing
Stage of pregnancy
Maternal risk for aneuploidy
Rates of indeterminate tests
Sensitivity, specificity
Because reported sensitivity and specificity results were not always accompanied by 95% confidence
intervals (CIs), and any reported CIs may not have been calculated by the same method, we (re-)
calculated all CIs using the exact method.
Study Quality Assessment
We used the Quality Assessment of Diagnostic Accuracy Studies (QUADAS)‒2 instrument to perform a
23
formal quality assessment for individual studies. This instrument includes questions within 4 domains on
the risk of bias, and 3 questions on the applicability of the studies with respect to the specific Assessment
key questions. The domain summary questions and the applicability questions are each assigned a rating
of average risk, high risk, or unclear.
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Noninvasive Prenatal Cell-Free Fetal DNABased Screening for Aneuploidies Other Than Trisomy 21
Meta-Analysis
We conducted a meta-analysis of the sensitivity and specificity of studies reporting cell-free fetal DNA
analysis for T18, T13, monosomy X (Turner syndrome), and other SCA. In the main analysis, studies with
fewer than 5 cases were excluded due to unreliability of the estimates. A pooled summary estimate was
calculated using a random-effects (DerSimonian-Laird) model. Due to the small number of studies in
“average-risk” mothers and small numbers of cases, a subgroup analysis of “average-risk” and “high-risk”
populations was undertaken, was deemed relatively unreliable, and is reported in Appendix Table 9. All
analyses were obtained using Open Meta Analyst (Center for Evidence-Based Medicine, Brown
University).
Decision Analysis
We constructed 2 prenatal screening and confirmatory strategies for comparing detected and missed
cases for T13 and T18 in average-risk and high-risk populations.
Medical Advisory Panel Review
The Blue Cross and Blue Shield Association Medical Advisory Panel (MAP) reviewed this assessment on
June 10, 2014. To maintain the timeliness of the scientific information in this Assessment, we performed
literature search updates subsequent to the Panel's review (see Search Strategy section above). If we
identified any additional studies that met the criteria for detailed review, we included those results in the
tables and text where appropriate. No additional studies were identified that would change the
conclusions of this Assessment.
MAP reviewed a previous TEC Assessment on cell-free fetal DNAbased tests for T21 in December
2012. At that review, the MAP found that the evidence for noninvasive, cell-free fetal DNA‒based testing
to screen for T21 met TEC criteria compared with standard noninvasive screening methods in both high-
and average-risk pregnant women. MAP deemed the screening evidence for T13 and T18 insufficient to
draw conclusions.
FORMULATION OF THE ASSESSMENT
Patient Indications
The patient indications are pregnant women who are being screened for T13, T18, and SCA. Specific
subgroups of interest include:
pregnant women at high risk; and
pregnant women at average risk.
We did not discern women in the first or second trimester because the sequencing-based evidence was
not necessarily specific to the trimester.
Technologies to Be Evaluated and Compared
The technologies for comparison are the current traditional screening and confirmatory tests for T13, T18,
and SCA:
Noninvasive traditional testing
o First trimester
Maternal serum screening for free beta hCG and PAPP-A
Ultrasound: specific measures of nuchal translucency and nasal bone
o Second trimester
Maternal serum screening for alpha-fetoprotein, unconjugated estriol, free beta hCG, and
inhibin A
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Noninvasive Prenatal Cell-Free Fetal DNABased Screening for Aneuploidies Other Than Trisomy 21
Ultrasound: basic fetal anatomy survey
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)
o First trimester
CVS
o Second trimester
Amniocentesis
In the first trimester, use of free beta hCG, PAPP-A, and nuchal translucency together may be referred to
as the “combined screen.” In the second trimester, a number 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
8
than explore all second trimester screens in this Assessment, we used the “integrated screen” (one of
the best-case scenario screens) as our comparator.
Health Outcomes
We evaluated cell-free fetal DNA‒based assay performance characteristics for T13, T18, and SCA
prediction. Next, we estimated correctly identified trisomy cases (true positive rate) and number of cases
of trisomy cases missed (false negative rate). We did not carry the analysis further based on the rationale
that prenatal screening would include T21. In the previous TEC Assessment, we determined the impact of
T21 cell-free fetal DNA‒based screening on the numbers of invasive procedures performed and avoided,
and the numbers of miscarriages averted by avoiding an invasive diagnostic procedure triggered by a
false-positive initial screen. For this Assessment, we assumed that decisions and actions after a positive
T13 or T18 result with a cell-free fetal DNA‒based test would be identical to those for T21. Thus,
downstream events would be the same for T13 or T18 as for T21.
The implications of an SCA-positive initial screen may differ substantially from those of the autosomal
aneuploidies and so the former were not assessed using a similar methodology. However, because most
available cell-free fetal DNA screening tests can identify common SCA, if an identified SCA would impact
decisions, logic similar to that of the autosomal conditions would apply.
Analytic Framework
See Figure 1 (below) for a decision tree comparing screening.
Specific Assessment Questions
1. What is the analytic validity of cell-free fetal DNA sequencingbased screening tests for T13, T18,
and SCA?
2. What is the clinical validity of cell-free fetal DNA sequencingbased screening tests for T13, T18,
and SCA as measured by sensitivity, specificity, and predictive value?
3. What is the clinical utility of cell-free fetal DNA sequencingbased screening tests for T13, T18,
and SCA (detected and missed cases) for:
a. a positive standard integrated noninvasive screen followed by diagnostic invasive karyotype
analysis; and
b. a positive cell-free fetal DNA‒based screen followed by diagnostic invasive karyotype
analysis.
REVIEW OF EVIDENCE
Overview of Included Evidence
We identified 29 original publications that met the inclusion criteria for this Assessment, which included 9
from the 2012 TEC Assessment. As shown in Table 2, 17 of 29 publications (59%) were described as
prospective cohort studies; 10 (34%) were case-control studies; and 2 (7%) were retrospective cohort
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