C HIEF E DITOR ’S N OTE :This article is part of a ries of continuing education activities in this Journal through which a total of 36AMA PRA Category 1Credits i can be earned in 2014.Instructions for how CME credits can be earned appear on the last page of the Table of Contents.
Noninvasive Prenatal Testing
Jamie O.Lo,MD (*Cori D.Feist,MS,CGC (†Mary E.Norton,MD (‡
establishing
and Aaron B.Caughey,MD,PhD ‡
*Maternal Fetal Medicine Fellow,†Genetic Counlor,and ‡Professor,Department of Obstetrics and Gynecology,Oregon
Health &Science University,Portland,OR Noninvasive prenatal testing (NIPT)refers to recently developed genetic tests of the maternal rum that allow higher detection rates of trisomy 21and other chromosomal aneuploidies in high-risk pregnancies.Noninvasive prenatal test analyzes cell-free DNA (cfDNA)in the maternal rum.Ap-proximately 3%to 15%of cfDNA in the maternal blood is of fetal origin.Analysis of cfDNA can help identify fetus affected with trisomy 21and veral other fetal aneuploidies.Testing can be per-formed after 9to 10weeks’gestation and has a higher nsitivity
and specificity for trisomy 21than other aneuploidy screening test.Noninvasive prenatal test has been studied and validated in singleton pregnancies at risk for trisomy 21condary to advanced maternal age,an abnormal rum screen,personal or family history of aneuploidy,or abnormal ultrasound findings,if the are suggestive of trisomy 13,18,or 21.The utilization of NIPT for genetic screening has incread rapidly since intro-duction of the first clinical test in October 2011.Currently,there are limitations to NIPT including the possibility of test failure (2.6%Y 5.4%)and the focus on only the common trisomies.Noninvasive prenatal test is a screening test,and both fal-positive (0.2%Y 1%)and fal-negative results can occur.As the technology for NIPT is further evaluated,this test is likely to be increasingly ud for prenatal screening.This review provides the obstetric clinician with an update of the current issues concerning NIPT.
Target Audience:Obstetricians and gynecologists,family physicians
Learning Objectives:After completing this CME activity,physicians should be better able to compare the various noninvasive screening options available and describe the indications and proper utilization of NIPT for prenatal screening.
Historically,women of advanced maternal age (Q 35years)were identified as having an incread risk
of having a fetus with trisomy 21and were offered genetic counling and amniocentesis or chorionic villus sampling (CVS).Invasive prenatal diagnosis was offered only to tho at incread risk of aneu-ploidy,in part becau it is associated with preg-nancy loss.The threshold of maternal age 35years
was established in 1979at a National Institutes of Health connsus conference 1bad on the incread aneuploidy risk of women at this age or older.In 1984,rum screening for trisomy 21was introduced when a low maternal rum >-fetoprotein (AFP)level was discovered to be associated with trisomy 21.2In the 1990s,detection rates improved when human chori-onic gonadotropin (hCG)and unconjugated estriol were utilized in addition to AFP,and the so-called ‘‘triple screen’’was introduced.However,the n-sitivity of the triple test was only 60%overall and only 50%in women younger than 35years.3Sub-quently,other rum analytes were introduced in combination with ultrasound markers including the cond trimester ‘‘genetic sonogram’’and more im-portantly nuchal translucency (NT)screening in the first trimester.This eventually led to the development
|89
Volume 69,Number 2
O BSTETRICAL AND G YNECOLOGICAL S URVEY Copyright *2014
dannyby Lippincott Williams &Wilkins
CME REVIEWARTICLE
4
All authors and staff in a position to control the content of this CME activity and their spous/life partners (if any)have disclod that they have no financial relationships with,or financial in-terests in,any commercial organizations pertaining to this edu-cational activity.
Correspondence requests to:Jamie O.Lo,MD,3181SW Sam Jackson Park Rd,Mail Code L466,Portland,OR 97239.E-mail:Loj@ohsu.edu.
of different first-and cond-trimester genetic screen-ing options,including the quadruple screen,combined screen,integrated screen,and quential screen with detection rates of80%to95%.4,5
In addition to the development of screening tests, the1990s also saw noninvasive efforts to prenatally diagno fetal trisomy21through identification of fetal cells in the maternal circulation.Early efforts focud on noninvasively obtaining intact fetal cells, but to date,the efforts have not been successful. In2002,a large,prospective National Institute of Child Health and Human Development Y sponsored Fetal Cell Isolation Study(NIFTY)reported that an-euploidy detection using fetal cell analysis from maternal blood was possible,but with a nsitivity (74%detection rate)and specificity(fal-positive rate0.6%Y4%)that was not diagnostic and was in-ferior to existing screening methods.6In1997,the identification of free fetal DNA in maternal plasma was described,launching rearch efforts to develop a prenatal diagnostic test.7
Subquently,it was determined that approximately 3%to15%of cell-free DNA(cfDNA)circulating in the maternal blood is of fetal origin.7Y9Becau cfDNA degrades rapidly,fetal DNA detected during pregnancy reprents DNA from the current fetus; this is in contrast to intact fetal cells,which can persist for many years in the maternal circulation.By com-paring the relative amounts of DNA prent from each chromosome,it is now possible to identify nu-merical chromosomal differences,which are prent in aneuploidy.In2011,this work led to the clinical introduction of noninvasive prenatal tests(NIPTs) that utilized cfDNA in the maternal plasma as a highly nsitive an
d specific noninvasive screening tool for fetal aneuploidy of chromosome21.Since then,tests have expanded to evaluate trisomies13and 18,as well as various x chromosomal aneuploidies. The major advantage of NIPT over existing screen-ing tests is the improved nsitivity and specificity,significantly decreasing both fal-negative and fal-
positive results.Noninvasive prenatal testing involves
only a maternal blood draw,so geographic availabil-
ity is not limited to practices with access to maternal-
fetal medicine providers.
As the technology for NIPT continues to evolve,
and there are better insurance coverage and further
asssment of clinical validity and more importantly
of clinical utility,it appears that NIPT will become
widely ud for prenatal screening in the future.The
intent of this review article was to discuss noninva-
sive screening for aneuploidy,the specific charac-
teristics of the cfDNA screening tests,and potential issues related to the u of the tests.
OTHER ANEUPLOIDY SCREENING
OPTIONS
Historically,aneuploidy screening tests have uti-
acomelized the interpretation of maternal rum analyte
levels with or without NT measurement in the context of maternal age Y related risk for chromosomal aneu-
ploidy,primarily trisomies18and21(Table1).Such
screening can be done in the first and/or cond tri-
mester;optimal performance is achieved utilizing a
combination of first-and cond-trimester rum an-
alytes in combination with ultrasound.
First-Trimester Combined Test
Thefirst-trimester combined test involves sono-
graphic determination of NT and gestational age(by
crown-rump length)combined with the rum markers
pregnancy-associated plasma protein A(PAPP-A)and A-hCG(either total or the free A-subunit).Nuchal translucency screening measures the fluid accumula-
tion behind the neck of the fetus;extra fluid,an in-cread NT,is associated with trisomy21,18,or13. Depending on the performing laboratory,screening can be performed between9weeks and13weeks6/7days’
TABLE1
Prenatal Screening Options4
Test When,wk Detection Rate,%Fal-Positive Rate,%Turnaround Time,d First-trimester screen11Y1480Y9055Y7 Integrated screen11Y14and15Y2190Y94 3.5Y5.55Y7 Sequential screen11Y14and15Y2190Y94 3.5Y5.55Y7 Quadruple screen*15Y248055Y7 Noninvasive prenatal testing Q10V VÈ10 Trisomy21999G1V Trisomy18999G1V Trisomy1370Y100N/A†V *Test characteristics of the Penta screen are the same.
†Some laboratories will report risk as same detection rate/fal-positive rate as trisomy18.
90Obstetrical and Gynecological Survey
gestation.At a5%fal-positive rate,the rate of de-tection of trisomy21is79%to87%.5,10
Quadruple and Penta Screen
The cond-trimester screening tests are available from15to22weeks and ud for prenatal risk as-ssment for neural tube defects,trisomy21,and trisomy18.The quadruple screen measures AFP,
uE3, hCG,and inhibin A in the maternal rum,whereas the penta screen measures the same4analytes with the addition of hyperglycosylated hCG.For both tests, at a5%fal-positive rate,the rate of detection of trisomy21is81%to83%.4,11
shankIntegrated Screening Test
The integrated test typically refers to measurement of NT and PAPP-A between10to13weeks and the rum markers unconjugated estriol,hCG,AFP,and inhibin A between15to18weeks;a single integrated result is provided to the patient in the cond trimester after all results are available and analyzed in a single risk calculation.This test includes screening for open neural tube defects and has the highest detection for trisomy21.At a5%fal-positive rate,the rate of detection of trisomy21is96%.4
Sequential Screening Test
(Stepwi vs Contingent)
There are2types of quential screening:stepwi and contingent.Sequential tests combine analyte data from2specimens collected at different times during the pregnancy.In thefirst trimester specimen,
rum levels of PAPP-A and total or free A-hCG are mea-sured,and the results evaluated in combination with the fetal NT ultrasound measurement to determine the fetal risk for DS and trisomy18.With stepwi screening,patients are provided a first trimester risk bad on a first-trimester combined test.High-risk patients receive genetic counling and can elect ei-ther diagnostic testing or the option of completing the cond-trimester screening component before making a final decision about diagnostic testing.Low-risk patients can continue with cond-trimester screen-ing or choo to decline any further testing.With contingent screening,a pregnancy is typically clas-sified as low,intermediate,or high risk bad on first-trimester screening results.Women at high risk are offered invasive testing,women at intermediate risk are offered continued screening in the cond tri-mester,and women at low risk are advid that they are unlikely to convert to high risk with further screening,and typically no further testing is per-formed.It is important to remember that with any of the protocols,a patient has the option to decline further testing or follow-up.
In the cond-trimester specimen,rum levels of AFP,hCG,unconjugated estriol,and dimeric in-hibin A are measured,and the results combined with the PAPP-A and NT measurements from thefirst-trimester specimen.The patient’sfinal risk for tri-somy21,trisomy18,and open neural tube defect is evaluated and reported.At a5%fal-positive rate, the rate of detection of trisomy21is95%in the step
wi quential screening test and88%to94% in the contingent quential screening test.4,12The first-trimester screen alone,when not combined with cond-trimester testing,has a higher fal-positive rate of9.4%and a rate of detection of trisomy21% of85%.10
TECHNOLOGY BEHIND NONINVASIVE PRENATAL TESTING USING
CELL-FREE DNA
Cell-free fetal DNA is compod of short gments of fetal DNA(G200ba pairs),believed to be pres-ent as a result of apoptosis of placental cells with relea of very small DNA fragments into the ma-ternal plasma.Such fragments are detectable after 7weeks’gestation.13,14Unlike intact fetal cells in maternal blood that can persist for years after a preg-nancy,cfDNA clears from the maternal system within 2hours of delivery.15Approximately3%to15% of cfDNA circulating in the maternal blood is of pla-cental or fetal origin(fetal fraction).7Y9Bad on the characteristics,veral approaches to analyzing and quantifying the cfDNA to predict fetal aneuploidy have been utilized.Of the different NIPT options,some tests provide a‘‘positive’’or‘‘negative’’result,whereas others provide a numerical risk,similar to current genetic screening options.
Two of the current NIPTs utilize the massively parallel shotgun quencing(MPSS)approach,which si
multaneously analyzes a random subt of millions of small cfDNA fragments sampled from maternal plasma and maps them to the human genome to de-termine frequency and density along each chromo-some.The ratio of cfDNA fragments from particular chromosomes of interest is compared with the ex-pected;a deviation greater than a prespecified cut-off(typically a z score of3Y4is ud)suggests a high likelihood of aneuploidy.With high-throughput
91
Prenatal Testing and Prenatal Diagnosis&CME Review Article
next-generation quencing strategies,a total of4to 8patient samples can be analyzed concurrently. Another clinically available NIPT test utilizes a targeted quencing approach.This test characterizes the prence of aneuploidy using a technique referred to as directed analysis of lected regions.16This technique is similar to MPSS,but instead of a random analysis of cfDNA,it involves lected quencing of fewer chromosome regions,approximately1/10 the number required for MPSS.This lective -quencing strategy allows for greater efficiency and decread cost.
The fourth clinically available test us NATUS (next-generation aneuploidy test using single-nucleotide polymorphisms[SNPs])technology,a different ap-proach that us targeted quencing to
evaluate ap-proximately20,000SNPs from maternal plasma.14 Cell-free DNA from maternal plasma is amplified and analyzed by high-throughput quencing,gener-ating millions of pieces of data for each sample. The NATUS algorithm identifies patterns in the data caud by the fetal DNA signal,distinguishing it from the maternal cfDNA signal.This algorithm models expected allele frequency distributions to es-tablish the maximum likelihood for the fetal chromo-somal copy number(euploid or aneuploid)and assigns a personalized risk score for each abnormality.
community responsibility
Clinical Application
In addition to aneuploidy screening,clinical appli-cations for NIPT also include fetal x determina-tion,detection of single-gene disorders(bad on detection of a paternally inherited allele),and detec-tion of fetal Rh type.Aneuploidy testing is currently the area of greatest focus becau of the large po-tential market for the tests.As oppod to detection of a single fetal gene,detection of a chromosomal abnormality requires accurate quantification of DNAx from a specific chromosome.Currently veral lab-oratories,utilizing the different detection methods described above,are offering cfDNA testing for tri-somies21,18,and13as well as x chromosome aneuploidy.Laboratories are also considering the addition of other chromosomal trisomies or clinically significant microdeletions.
Possible NIPT results can include abnormal,nor-mal,aneuploidy suspected,and no-call.Some labo-ratories report a result as‘‘aneuploidy suspected’’if value falls near the z-score cutoff.Both affected and unaffected cas may occur in this zone.The results should be treated as a screen positive,with the chance for aneuploidy most likely between40%and80%; the patients should be offered confirmatory inva-sive diagnostic testing.Becau existing studies are relatively small,even for patients with an abnormal test,subquent invasive diagnostic testing is recom-mended as well.17No-call results refer to the possi-bility of test failure and the inability to determine a result bad on veral factors including low fetal fraction.Abnormal NIPT results can also occur in women with malignancy,as fal-positive results have been previously reported.In addition,maternal con-ditions,such as chromosomal mosaicism,can affect NIPT results especially if a gene deletion or duplica-tion on a reference chromosome is prent.
CLINICAL STUDIES
The existing literature comparing the different de-tection methods of NIPT are limited,but results of validation studies indicate relatively comparable per-formance.Several large validation studies have been published,reporting on the analytic and clinical va-lidity of the tests.14,18Y20The studies have been primarily conducted on high-risk patients with sin-gleton pregnancies,before invasive diag
nostic testing; data on performance in low-or average-risk patients are still limited.
A large blinded,nested ca-control study in2011 examined4385pregnancies at high risk for trisomy 21bad on maternal age,family history,positive rum screening,or positive sonographic screening.18 This study excluded twin and in vitro fertilization pregnancies.Of the women studied,212were noted to have trisomy21on diagnostic testing,and1484 women were matched controls with euploid fetus. Massively parallel shotgun quencing was ud for cfDNA analysis,and the trisomy21detection rate was98.6%(209/212)with a fal-positive rate of 0.2%(3/1471).This study noted a0.8%risk of test failure.18
A subquent and similar prospective blinded nested ca-control study published in2012sampled2882 women undergoing prenatal diagnostic procedures and analyzed532of the.19Again,cas and controls were lected from the overall cohort.Chromosome classi-fications were made for each sample using MPSS and compared with fetal karyotype.In this analysis,results were classified as‘‘positive’’if a z score of4or more was reported,‘‘negative’’if a z score of less than2.5 was reported,and as‘‘unclassified’’if the z score was between2.5and4.Using this schema,the detection rate for trisomy21was100%(89/89),trisomy13was
92Obstetrical and Gynecological Survey
78.6%(11/14),monosomy X was93.8%(15/16),and trisomy18was97.2%(35/36).There were no fal-positive results for autosomal aneuploidies.Overall, 2.6%of samples had a chromosome value in the midrange risk(between affected and unaffected cut-offs)and were considered‘‘unclassified’’using MPSS; subquent invasive diagnostic testing diagnod an-euploidy in veral such cas.
Another2012cohort study examined3228women using a chromosome lective approach,along with reporting of aneuploidy risk(high vs low risk).20All trisomy21cas were classified as high risk,and the detection rate was100%(81/81)with a fal-positive rate of0.03%(1/2888).Of the trisomy18cas,97% (37/38)were classified as high risk,and the detection rate was97.4%(37/38)with a fal-positive rate of 0.07%(2/2888).There was a test failure in4.6%of cas from either low fraction of fetal DNA or failed quencing.
Another prospective blinded study of242singleton pregnancies undergoing CVS at11to13weeks ex-amined NIPT using the SNP-bad approach.14Cell-free DNA was isolated from maternal plasma,and targeted multiplex PCR amplification followed by quencing of19,488polymorphic loci with focud analysis of chromosomes13,18,21,X,and Y was performed.The quencing data were then analyzed using the NATUS algorithm to determine the copy number and to generate a sample-specific risk as-ssment for each of the5chromosomes tested.Re-sults were provided for229(94.6%)
cas,and32 cas were correctly identified as aneuploid with a detection rate for trisomy21(n=25)of100%.Other aneuploidy cas detected included trisomy18(n=3), trisomy13(n=1),Turner syndrome(n=2),and triploidy(n=1).There were no fal-positive or fal-negative results,and median accuracy was noted to be99.9%.In5.4%of cas,the test did not provide a result.
In terms of x chromosome abnormalities,the performance of available testing is notable for a nsitivity of97.6%for XX,99.1%for XY,and 95%for monosomy X.19Some NIPT tests also in-clude the identification of3other x chromosome aneuploidies:XXX,XXY,and XYY.In a study of 532patients,it was noted that3of4subjects with XXX,2of3subjects with XXY,and3of3subjects with XYY karyotype were correctly classified.19The performance of NIPT for x chromosomal aneu-ploidy is less robust than for other numeric aneu-ploidies and will increa the screen-positive rate of the test,although preci estimates of detection and fal-positive rates are not available.Providers should take this into consideration when electing x chro-mosome testing by this method.
Limitations
There are challenges and limitations to NIPT.It is not clear yet how NIPT will be integrated into well-e
stablished prenatal testing strategies as the timing of testing and the degree to which NIPT will sup-plement or replace existing screening and diagnostic tests are undetermined.21Noninvasive prenatal test-ing is a screening test,and results are not considered diagnostic.22Y24It is therefore recommended that women with a positive test proceed to a diagnostic test for confirmation.It is important for providers to appreciate that a high nsitivity and specificity do not necessarily mean high positive predictive value (PPV)and that PPV is the important consideration for providers when interpreting screening tests.For ex-ample,with a nsitivity and specificity of99%, nearly all of the trisomy21fetus would be detected, and the fal-positive rate would be1%.Assuming a baline risk of trisomy21of1in500,however,only 1in6of positive results will reprent a true positive, whereas5of6will be fal positives;this reprents a PPV of16%.Converly,if the specificity is99.9%, with the same baline risk of1/500,2of3abnormal results will reprent true positives,for a PPV of67%. It is also important to remember that fal-negative results exist,so a negative result cannot rule out an affected fetus.In addition,it is important to rec-ognize that trisomies13and18have lower nsitivity, specificity,and prevalence;thus,the PPVs are lower compared with trisomy21.Additional analysis of a larger number of fal-positive cas is needed to determine whether the detection of less frequent an-euploidies reflect the PPV that should be expected.25 Although the data regarding trisomy21demon-strate consistently high analytic validity,the test per-formance is less robust for othe
r aneuploidies.For example,u of NIPT for trisomy13has been re-ported to be associated with detection rates of79% to92%and a fal-positive rate of1%24;given the relative rarity of this aneuploidy,the data are bad on relatively few cas.In general,fal-positive re-sults are often condary to contamination,an un-recognized or vanishing twin,placental mosaicism,or low levels of maternal mosaicism.This is particularly so for trisomies13and18,which are often associated with placental mosaicism,leading to the possibility of both fal-positive and fal-negative results.26 Additionally,NIPT companies have not reported in-formation about their tests’PPV,which is arguably
93
Prenatal Testing and Prenatal Diagnosis&CME Review Article
important to patients undergoing testing as it indicates the probability that a positive test result indicates a true fetal aneuploidy.27
Another limitation of NIPT is the2.6%to5.4%risk of test failure with no results reported.14,19,20This is particularly problematic for women in whom the delay in testing results in their gestational age pre-cluding them from obtaining other testing options.It appears that there are women at incread risk for such test failures due to a lower fraction of fetal DNA.
A reduced fetal fraction has been associated particu-larly with increasing maternal body mass index. Maternal obesity can lead to higher rates of test failure becau it appears to be associated with incread maternal circulating cfDNA,which results in a lower relative percentage of fetal DNA.28,29Most labora-tories require at least4%of the cfDNA to be fetal to provide a test result.Given the obesity epidemic in the United States,there is an increasing proportion of women that might receive a‘‘no results’’outcome from NIPT.
Currently NIPT has been studied and evaluated in high-risk patients,but the data for low-risk patients are much more limited.Although theoretically the technology can be applied to women with a lower prevalence of aneuploidy,it is unclear whether the test characteristics will remain the same.When the an-euploidy prevalence is lower,it is more likely that a positive test is a fal-positive result.There has been a single,relatively small,study asssing NIPT in a low-risk population of2049women.30This2012 cohort study reported performance of NIPT in a low-risk population comparable to that in a high-risk population.The detection rate was100%for triso-my21(8/8),whereas they detected2of3cas of trisomy18and1trisomy18ca received no result becau of test failure.Although this was reported as a100%detection rate for trisomy18(the failed test ca was excluded),arguably the detection rate for trisomy18was only67%.The fal-positive rate was less tha
n1%.30Although NIPT may be applicable to low-risk patients,other studies are needed before expansion to the general public is recommended.In addition to low-risk women,there are also limited data available on twins,with a single study reporting on a small cohort of7twin pregnancies.Further-more,NIPT has also not been validated in triplet or other higher-order multiple pregnancies or in preg-nancies conceived using egg donation.Thus,more information is needed before NIPT can be recom-mended in multiple gestations.31
At this time,NIPT screens only for the most com-mon aneuploidies and will miss20%to30%of chromosome abnormalities that could be detected by prenatal karyotype.Lastly,it is important to re-member that,unlike cond-trimester maternal rum screening,NIPT cannot screen for open neural tube defects or ventral wall defects.Therefore,some pro-viders continue to offer maternal rum AFP screen-ing in addition to anatomic fetal ultrasound in the cond trimester to women undergoing NIPT to detect neural tube defects.
RECOMMENDATIONS FOR USE OF
NONINVASIVE PRENATAL TESTING
As NIPT is a relatively new test,professional so-cieties are beginning to issue statements regarding tbrawny
he appropriate u of NIPT.
American College of Obstetricians
and Gynecologists
ted
In2007,the American College of Obstetricians and Gynecologists(ACOG)recommended offering an-euploidy screening or invasive testing to all women regardless of age.12A recently issued committee opin-ion recommends that NIPT testing should be part of an informed patient choice after pretest counling but should not be a part of routine prenatal laboratory as-ssment.ACOG recommends that NIPT not be offered to low-risk women or women with multiple gestations (ACOG2012).They emphasize that a negative test result does not guarantee an unaffected pregnancy and that women with a positive result should be referred for genetic counling and offered invasive prenatal di-agnosis for confirmation of test results.
互动英文
National Society of Genetic Counlors
The National Society for Genetic Counlors de-scribes NIPT as an option for women who preg-nancies are considered to be at an incread risk for chromosome abnormalities.They encourage pr
oviders to offer NIPT only in the context of informed connt, education,and counling by a qualified provider,such as a certified genetic counlor.Patients with abnormal NIPT results,or who have other risk factors suggestive of a chromosomal abnormality,should receive genetic counling and be offered the option of standard con-firmatory diagnostic testing.24
International Society of Prenatal Diagnosis Like the other societies,the International Society of Prenatal Diagnosis committee opinion states that
richard harmon
北京英孚教育怎么样94Obstetrical and Gynecological Survey
