Noninvasive prenatal testing_for_fetal_aneuploidy

1. Noninvasive Prenatal Testing for
Fetal Aneuploidy
Clinical Assessment and a Plea for Restraint
Mary E. Norton, MD, Nancy C. Rose, MD, and Peter Benn, PhD
The recent introduction of clinical tests to detect fetal
aneuploidy by analysis of cell-free DNA in maternal
plasma represents a tremendous advance in prenatal
diagnosis and the culmination of many years of effort by
researchers in the field. The development of noninvasive
prenatal testing for clinical application by commercial
industry has allowed much faster introduction into
clinical care, yet also presents some challenges regarding
education of patients and health care providers strug-
gling to keep up with developments in this rapidly
evolving area. It is important that health care providers
recognize that the test is not diagnostic; rather, it
represents a highly sensitive and specific screening test
that should be expected to result in some false-positive
and false-negative diagnoses. Although currently being
integrated in some settings as a primary screening test
for women at high risk of fetal aneuploidy, from
a population perspective, a better option for noninvasive
prenatal testing may be as a second-tier test for those
patients who screen positive by conventional aneuploidy
screening. How noninvasive prenatal testing will ulti-
mately fit with the current prenatal testing algorithms
remains to be determined. True cost–utility analyses will
be needed to determine the actual clinical efficacy of this
approach in the general prenatal population.
(Obstet Gynecol 2013;121:847–50)
DOI: http://10.1097/AOG.0b013e31828642c6
The potential to obtain and analyze fetal genetic
material from maternal plasma has stimulated vig-
orous research for more than three decades. In October
2011, these efforts resulted in the first commercially
available test to detect fetal aneuploidy by analysis of
cell-free DNA in maternal plasma. Over time, this
technology is likely to be applied to the prenatal detec-
tion of an ever-increasing range of genetic disorders.
This commentary presents an overview of the devel-
opment, clinical application, and limitations of nonin-
vasive prenatal testing as currently available in clinical
practice.
DEVELOPMENT OF NONINVASIVE
PRENATAL TESTING
The development of cell-free DNA testing differs in
important ways from that of other methods of prenatal
diagnosis and screening. Amniocentesis, chorionic
villous sampling, and serum screening were largely
developed by academic investigators who were sup-
ported by public funding. After the conduct of
independent clinical trials, with results presented at
scientific meetings and published in peer-reviewed
journals, clinical tests were introduced and supported
by national committee guidelines. This process was
slow but relied on the unbiased external review of
data before widespread introduction of tests and
changes in the standards of clinical care. The techni-
ques to analyze cell-free DNA in maternal plasma
were also originally developed in academic settings
but were rapidly licensed to commercial companies
typically supported by venture capital or other private
funding. The tests that were developed are now
publicized through marketing directly to the clinician,
From the Department of Obstetrics and Gynecology, Stanford University School
of Medicine/Lucile Packard Children’s Hospital, Stanford, California; Inter-
mountain Healthcare, University of Utah School of Medicine, Intermountain
Medical Center, Maternal Fetal Medicine, Salt Lake City, Utah; and the Depart-
ment of Genetics and Developmental Biology, University of Connecticut Health
Center, Farmington, Connecticut.
Corresponding author: Mary E. Norton, MD, 300 Pasteur Drive, HH333,
Stanford University/Lucile Packard Children’s Hospital, Stanford, CA 94305;
e-mail: menorton@stanford.edu.
Financial Disclosure
Dr. Norton is a coprincipal investigator on clinical trial NCT0145167
sponsored by Ariosa Diagnostics. The other authors did not report any potential
conflicts of interest.
© 2013 by The American College of Obstetricians and Gynecologists. Published
by Lippincott Williams & Wilkins.
ISSN: 0029-7844/13
VOL. 121, NO. 4, APRIL 2013 OBSTETRICS & GYNECOLOGY 847

2. and scientific presentations at times appear geared
toward a specific product. Although such commercial
development may allow for faster introduction into
clinical practice, to some degree, it bypasses some
components of a rigorous, unbiased vetting process,
which helps to provide quality assurance.
The cell-free DNA field is still evolving, and the
clinical tests have limitations that have not been
highlighted in the rush to capture market share.
Clearly, the sensitivity and specificity are very high,
and this is likely to result in less patient anxiety as
a result of false-positive results and a decrease in
invasive procedures. However, both false-positive and
false-negative results do occur, and, therefore, these
should be considered screening tests. The positive
predictive value varies substantially with other risk
factors and is relatively low in low-risk patients. For
example, assuming a detection rate of 99% and a 0.5%
false-positive rate, in a woman with a prior risk of one
in 1,000 for Down syndrome, the likelihood that
a positive result is a true-positive is only approxi-
mately one in six. Even for a higher risk woman with
a prior risk of one in 100, the chance of Down
syndrome with a positive result is two in three; one
third of positive results will be false positives. Online
conversations among patients, health care providers,
and even geneticists regarding false-positive results
and other test characteristics demonstrate a lack of
understanding of some of these features of current
cell-free DNA tests. Further education is critically
important to assure appropriate interpretation of
positive test results.
The three clinically available tests offered by
Sequenom, Inc., Verinata Health, Inc., and Ariosa
Diagnostics, Inc. have quite different characteristics in
terms of how the sequencing data are analyzed and
results are reported. Sequenom presents results as
dichotomous outcomes, in which they indicate that
increased chromosome 21 material is either present or
not, implying to some health care providers (although
not explicitly stating) diagnostic accuracy. Ariosa
provides results as a risk, more typical of a screening
test. Verinata reports three categories of results, includ-
ing “aneuploidy detected,” “aneuploidy suspected,” or
“no aneuploidy detected.” Another approach that pro-
vides a case-specific estimate of risk is being developed
by Natera Inc. This heterogeneity in approach contrasts
with current serum screening tests, whose reporting and
laboratory standardization are much more consistent
between laboratories.
The ease of obtaining blood samples for cell-free
DNA testing, together with the promise of early and
accurate clinical information and the intense marketing,
make it tempting to use these tests even in the absence
of comprehensive validation or understanding. From
a clinical provider’s standpoint, it is difficult and time-
consuming enough to review standard information in
established prenatal care without also having to explain
to patients the limitations of these screening tests and
the differences between clinical validity and clinical
use. However, given that pregnancy termination is
a potential result of a positive test, and that cell-free
DNA tests will now be provided through general pre-
natal practices rather than specialized prenatal diagno-
sis centers, obstetric providers will absorb more of the
burden of discussing these complex results.
QUALITY CONTROL AND REGULATION OF
GENETIC TESTING
Unlike many European countries, the United States has
limited direct regulation of genetic testing. As a result,
large professional societies, private medical insurers,
and for-profit companies largely govern the uptake and
integration of new technologies into prenatal practice,
and there is currently no legal obligation to demon-
strate safety and effectiveness of cell-free DNA tests. As
laboratory-developed tests, there is no requirement for
premarket approval by the U.S. Food and Drug
Administration.1
Although the laboratories providing
noninvasive prenatal testing in the United States are
compliant with the Clinical Laboratory Improvement
Amendments of 1988, Clinical Laboratory Improve-
ment Amendments regulations are designed to oversee
compliance at a laboratory operational level and do not
extend to validation of specific tests.
The absence of guidelines for quality control and
quality assurance for the laboratory testing is a signif-
icant concern. The methodologies are highly complex
and the testing is subject to intense commercial
competitive pressures. Such competition has led to
a series of patent infringement lawsuits between the
different companies; how these legal challenges will be
decided, and whether they will affect access to tests and
viability of some of the companies, remains uncertain.
The Secretary’s Advisory Committee on Genet-
ics, Health and Society has noted gaps in five main
areas of oversight that directly affect optimal use of
genetic testing, including clinical laboratory quality
assurance, transparency of genetic testing, oversight
of clinical validity of genetic tests, level of knowledge
regarding clinical use of genetic tests, and meeting the
informational needs of health professionals, the public
health community, patients, and consumers. This
group has noted that “.the growing use of genetic
testing will require significant investment in evidence-
based assessments to understand the validity and
848 Norton et al Noninvasive Prenatal Testing for Aneuploidy OBSTETRICS & GYNECOLOGY

3. utility of these tests in clinical and personal decision-
making.”1
In the absence of direct regulation, other options
have been suggested for oversight of these, and other,
genetic tests. Professional societies could encourage
voluntary registration of new prenatal tests with the
National Institute of Health’s Genetic Test Registry,
which would promote the documentation of clinical
and analytic validity of new tests before active use. A
model that could be useful to evaluate new prenatal
genetic testing is one similar to the Secretary’s Advi-
sory Committee for Heritable Diseases in Newborns
and Children. Chartered in 2003, this multidisciplinary
working group is designed to report to the Secretary of
Health and Human Services on “.the most appropri-
ate application of universal newborn screening tests,
technologies, policies, guidelines and standards” to
effectively reduce morbidity and mortality in newborns
and children having, or at risk for, heritable disorders.2
A similar multidisciplinary committee to oversee pre-
natal genetic testing, which is likely to increase greatly
in scope and complexity in coming years, could be an
appropriate venue for test evaluation and consider-
ation. In the meantime, the Secretary’s Advisory Com-
mittee for Heritable Diseases in Newborns and
Children suggests that professional societies should
issue guidelines that encourage physicians to review
validity data before ordering tests and likewise should
recommend minimum levels of clinical use.1
APPROPRIATE CLINICAL APPLICATION
Despite the complex background of cell-free DNA
testing, benefits for implementation into patient care
clearly exist. However, the aggressive advertising, high
visibility in the press, and the initial introduction of
these tests before publication of professional society
guidelines has left the general obstetrician with some
confusion on how to best incorporate this new tool into
clinical practice. To critically review the scientific
publications and noninvasive prenatal testing clinical
trial reports requires an in-depth understanding of
genetics, molecular biology, and statistics. Moreover,
there are limited data on how well the tests will perform
in actual clinical practice, particularly for specific
subgroups of women in which noninvasive prenatal
testing has not been adequately validated. The tests
have primarily been validated on archived samples in
carefully selected groups of high-risk women; such
studies do not answer the question of clinical use in
the general population. Nevertheless, obstetricians may
be concerned about their liability if they do not offer
cell-free DNA tests as an available option. Conversely,
given that pregnancy termination is a potential result of
cell-free DNA testing, the obstetrician bears the
burden for accurate counseling and interpretation
of test results.
Current American College of Obstetricians and
Gynecologists guidelines recognize that prenatal diag-
nosis should be available to all women regardless of
maternal age3
and that prenatal screening tests can
help women decide whether to accept or reject inva-
sive testing.4
First-trimester screening with nuchal
translucency and maternal serum screening carries
added benefits, including identification of aneuploidies
beyond those currently detectable with noninvasive
prenatal testing.5
In a recent publication on noninva-
sive prenatal testing in an average risk population of
women undergoing first-trimester screening, noninva-
sive prenatal testing detected 55% of the total chromo-
somal abnormalities (eight of eight cases of trisomy 21
and two of three cases of trisomy 18), whereas first-
trimester screening detected 100% (all T21 and T18 as
well as seven other deletions, duplications, and other
abnormalities).6
In addition, screening for neural tube
and ventral wall defects is an important component of
current screening protocols. Optimal introduction of
noninvasive prenatal testing would preserve the prin-
ciple of providing prenatal screening and diagnosis in
a way that maximizes women’s reproductive choice
and, in addition, ensure that noninvasive prenatal test-
ing is not performed on women who are unaware of
the purpose and scope of the test.7
Not surprisingly, the major companies providing
this testing in the United States are targeting large groups
of women for testing. Sequenom and Verinata advocate
testing on all high-risk women, which includes all women
of advanced maternal age. However, many older women
undergo other screening tests and most are determined
to be lower risk; currently available and recommended
serum and ultrasonographic protocols are better at
identifying high-risk women than just age alone.8
In gen-
eral, the concept of “advanced maternal age” as a screen-
ing test is now considered arbitrary and outdated.
Offering noninvasive prenatal testing to all preg-
nant women eliminates the difficulties associated with
offering the test to only selected groups but at consider-
able financial cost. Ariosa recommends screening
women of all ages and a complete replacement for all
existing aneuploidy screening but makes this recom-
mendation in the absence of published in-depth
research with regard to efficacy. They have carried
out limited studies on low-risk women and demon-
strated that the test failure rate appears to be no higher
and the false-positive rate comparable with that in
high-risk women. Before concluding that conventional
screening could be replaced by noninvasive prenatal
VOL. 121, NO. 4, APRIL 2013 Norton et al Noninvasive Prenatal Testing for Aneuploidy 849

4. testing, a detailed comparison of all costs and benefits,
both direct and indirect, needs to be carried out.
RECOMMENDATIONS
AND CONSIDERATIONS
So what is the general obstetric provider to do at this
point in time? The American College of Obstetricians
and Gynecologists recent published guidelines indi-
cating that cell-free DNA testing is of benefit in high-
risk women.9
However, they caution that such testing
should not be part of routine care but should only be
provided after pretest counseling. Furthermore, they
note that the test has not yet been adequately evalu-
ated in low-risk women, to whom it should not yet be
offered. In addition, we would suggest considering the
following: 1) Although noninvasive prenatal testing
has high detection rates and low false-positive rates,
it is a screening test and a positive noninvasive pre-
natal testing result must be confirmed by invasive
testing if pregnancy termination is being considered.
2) In high-risk women, particularly those identified as
such through traditional screening, noninvasive pre-
natal testing can be very useful but it should be made
clear that it screens for limited fetal aneuploidies at
this time (trisomies 21, 18, and 13). A diagnostic inva-
sive test definitively identifies a much broader range
of chromosome abnormalities (especially if microar-
ray technology is used). 3) If patients are to undergo
noninvasive prenatal testing, they need to understand
the purpose and limitations of the test; this cannot be
routinely added to standard prenatal laboratory test-
ing without adequate counseling. 4) Current serum
screening and ultrasonographic strategies that have
been thoughtfully developed and evaluated and any
replacement should be based on evidence of improved
clinical use, including considerations of the range of
abnormalities detected with each strategy. 5) A strategy
of concurrently ordering both noninvasive prenatal
testing and integrated screening cannot be advocated,
because this seems likely to greatly increase costs with-
out current evidence of incremental benefit.
At this time, the most appropriate use of non-
invasive prenatal testing may be as a second-tier test
for those with screen-positive results from conventional
aneuploidy screening. As noninvasive prenatal testing
expands to screen for more disorders and there are
more data available on its use, it may replace current
screening methods. However, at the present time, it
does not replace all forms of prenatal screening.
Finally, the implications of failed tests need to be
considered. Up to 5% of cases do not generate a result,
often as a result of insufficient fetal cell-free DNA
(especially for women with high body mass index).10
It
is unknown at what body mass index cutoff the test is
no longer worth attempting and in what circumstances
a repeat attempt is worthwhile. Results of noninvasive
prenatal testing can take 1–3 weeks, and the implica-
tions of the turnaround time, especially if a test result is
ultimately not provided, need to be considered, espe-
cially for patients who are later in the second trimester,
for whom it may be too late to pursue other screening
or diagnostic testing options.
In summary, noninvasive prenatal testing using
cell-free DNA is an exciting new technology with
tremendous potential to benefit pregnant women.
Undoubtedly, cell-free DNA technology will expand
over the next few years and dramatically advance the
field of prenatal screening and diagnosis. Developing
reasonable clinical management guidelines and edu-
cation will be essential as the testing becomes more
sophisticated. Like other new technologies, it should
be carefully assessed in an unbiased fashion before it
completely replaces our current standard of care and
before we change prenatal practice for the four million
pregnancies that occur in this country each year.
REFERENCES
1. Ferreira-Gonzalez A, Teutsch S, Williams MS, Au SM,
Fitzgerald KT, Miller PS, et al. US system of oversight for
genetic testing: a report from the Secretary’s Advisory Commit-
tee on Genetics, Health and Society. Per Med 2008;5:521–8.
2. Secretary’s Advisory Committee on Heritable Disorders in
Newborns and Children. Available at: http://www.hrsa.gov/
advisorycommittees/mchbadvisory/heritabledisorders/index.html.
Retrieved October 23, 2012.
3. Screening for fetal chromosomal abnormalities. ACOG Prac-
tice Bulletin No. 77. American College of Obstetricians and
Gynecologists. Obstet Gynecol 2007;109:217–27.
4. Invasive prenatal testing for aneuploidy. ACOG Practice Bulle-
tin No. 88, December 2007. American College of Obstetricians
and Gynecologists. Obstet Gynecol 2007;110:1459–67.
5. Syngelaki A, Chelemen T, Dagklis T, Allan L, Nicolaides KH.
Challenges in the diagnosis of fetal non-chromosomal abnor-
malities at 11–13 weeks. Prenat Diagn 2011;31:90–102.
6. Nicolaides KH, Syngelaki A, Ashoor G, Birdir C, Touzet C.
Noninvasive prenatal testing for fetal trisomies in a routinely
screened first-trimester population. Am J Obstet Gynecol 2012;
207:374.e1–6.
7. Benn PA, Chapman AR. Ethical challenges in providing non-
invasive prenatal diagnosis. Curr Opin Obstet Gynecol 2010;
22:128–34.
8. Malone FD, Canick JA, Ball RH, Nyberg DA, Comstock CH,
Bukowski R, et al. First-trimester or second-trimester screening,
or both, for Down’s syndrome. N Engl J Med 2005;353:2001–11.
9. Noninvasive prenatal testing for fetal aneuploidy. Committee
Opinion No. 545. American College of Obstetricians and
Gynecologists. Obstet Gynecol 2012;120:1532–4.
10. Ashoor G, Poon L, Syngelaki A, Mosimann B, Nicolaides KH.
Fetal fraction in maternal plasma cell-free DNA at 11–13 weeks’
gestation: effect of maternal and fetal factors. Fetal Diagn Ther
2012;31:237–43.
850 Norton et al Noninvasive Prenatal Testing for Aneuploidy OBSTETRICS & GYNECOLOGY