Non-Invasive Prenatal Testing where does it fit in?
Colin WalshMB BCh BAO MRCOG MRCPI FRANZCOG CCT-MFM PhD
Obstetrician, Gynaecologist and Maternal-Fetal Medicine Specialist
SHORE for Women, Suite G6, 460 Pacific Highway, St. Leonards
www.shoreforwomen.com.au Tel: 1300 460 111 Fax: 1300 460 222
The Evolution of Prenatal Screening1980 1990
2006 2016
Amniocentesis
No amniocentesis
Serum screening(Triple/Quad test)
CVS/Amniocentesis
No screening
1st trimester (NT)screening
CVS/Amniocentesis
No screening
1st trimester (NT)screening
NIPT +/- ultrasound
CVS/Amniocentesis
No screening
http://www.google.com.au/url?url=http://www.mirror.co.uk/lifestyle/sex-relationships/relationships/dear-coleen-im-thinking-leaving-7664512&rct=j&frm=1&q=&esrc=s&sa=U&ved=0ahUKEwiSkOrX8uTMAhWEo5QKHQ4lDJwQwW4IJDAH&usg=AFQjCNG-Wd6LDZ8OoHW46kYtkU1aizJNcw
Risk Trisomy 21 is approximately 1% at age 40
2.5x less common 7x less common
Combined 1st Trimester Screening (cFTS) AKA nuchal translucency test
Mainstay of screening since 1990s
Combined 1st Trimester Screening (cFTS) Maternal age combined with NT (mm) and serum biochemistry A priori risk [age, history, GA] adjusted using likelihood ratios
Gives numerical risk for:
-Trisomy 21
-Trisomy 18
-Trisomy 13
In an unselected pregnant population, 5% of women screen positive on NT scan
(i.e. high-risk result)
remember, does my baby have
Down syndrome? is often the patient
asking is my baby healthy?
Combined 1st Trimester Screening (cFTS) Models
11-13+6 week Standard + Nasal Bone + New Markers + EnhancedAnalytesAge Age Age Age
NT (mm) NT (mm) NT (mm) NT (mm)
Fetal heart rate Fetal heart rate Fetal heart rate Fetal heart rate
Free -hCG Free -hCG Free -hCG Free -hCG
PAPP-A PAPP-A PAPP-A PAPP-A
Nasal bone Nasal bone Nasal boneDV, TR, face DV, TR, face
PlGF, FPDetection T21 85% 93% 94% 95%False (+) rate 3% 2.5% 2.5% 1.5%
Combined 1st Trimester Screening (cFTS)
Performance of NT screening for 3 main trisomies:
Detection Rate False Positive Rate
Trisomy 21 90-95% 2-3%
Trisomy 18 82% 6%
Trisomy 13 66% 6%
Turner syndrome (45X) 73% (cystic hygroma) N/A
What do the bloods mean?
T21 T18 T13 45X
-hCG
PAPP-A
Other benefits of combined FTS?
1. Enlarged NT is associated with otherchromosomal anomalies (not T21/18/13)
2. Enlarged NT is associated with increasedrisk of congenital cardiac defects
3. Allows for an early fetal anatomy survey
4. PAPP-A level can identify fetuses at highrisk for growth restriction
Other benefits of combined FTS?
1. Enlarged NT is associated with otherchromosomal anomalies (not T21/18/13)
2. Enlarged NT is associated with increasedrisk of congenital cardiac defects
3. Allows for an early fetal anatomy survey
4. PAPP-A level can identify fetuses at highrisk for growth restriction
Other benefits of combined FTS?
1. Enlarged NT is associated with otherchromosomal anomalies (not T21/18/13)
12-month NEXT study, 6 countries
Harmony NIPT used
Standard cFTS in singletons at 10-14 weeks (excluded twins)
15,800 women had cFTS
6% (884) had a high-risk result
0.4% (68) had aneuploidy
Other benefits of combined FTS?
1. Enlarged NT is associated with otherchromosomal anomalies (not T21/18/13)
15%0.4% (68 / 15,800) had
fetal aneuploidy
20% of fetal aneuploidy in this large cohort was
not T21/18/13
5%
Chart1
38
16
3
4
7
56%
24%
Chromosomal abnormality (n=68)
Sheet1
Chromosomal abnormality (n=68)
T2138
T18/1316
45X3
Translocation4
Other7
To resize chart data range, drag lower right corner of range.
Other benefits of combined FTS?
1. Enlarged NT is associated with otherchromosomal anomalies (not T21/18/13)
2. Enlarged NT is associated with increasedrisk of congenital cardiac defects
3. Allows for an early fetal anatomy survey
4. PAPP-A level can identify fetuses at highrisk for growth restriction
Other benefits of combined FTS?
2. Enlarged NT is associated with increasedrisk of congenital cardiac defects
What is enlarged NT?-2% population 3.0mm-1% population 3.5mm
http://www.google.com.au/url?url=http://www3.imperial.ac.uk/portal/page/portallive/DB907CF3DD24D872E040C69B473966E1&rct=j&frm=1&q=&esrc=s&sa=U&ei=KyORU7fIAcellAWiiICQCA&ved=0CD4Q9QEwDjgU&sig2=ppmSE0aregD9GHQYMQ7hWg&usg=AFQjCNHTtDzVoBnzRrFVB2fpKOgyWCLlbA
Other benefits of combined FTS?
1. Enlarged NT is associated with otherchromosomal anomalies (not T21/18/13)
2. Enlarged NT is associated with increasedrisk of congenital cardiac defects
3. Allows for an early fetal anatomy survey
4. PAPP-A level can identify fetuses at highrisk for growth restriction
Other benefits of combined FTS?
3. Allows for an early fetal anatomy survey
At a 13-14 week fetal anatomy scan:
2/3s major defects detected
Heart and kidneys most challenging
15/17 CHD detected at 13-16 weeks
11-14 week fetal anatomy scan
Other benefits of combined FTS?
1. Enlarged NT is associated with otherchromosomal anomalies (not T21/18/13)
2. Enlarged NT is associated with increasedrisk of congenital cardiac defects
3. Allows for an early fetal anatomy survey
4. PAPP-A level can identify fetuses at highrisk for growth restriction
cFTS performed 10+3 13+6 in 34,271 pregnancies High PAPP-A does not complications
Low PAPP-A ( 5%ile): PPV
-Loss before 24 weeks 2.2%-IUFD after 24 weeks 0.5%-Preterm (
cFTS screening at 11-14 weeks: summary
1.90-95% detection rate for Trisomy 21 (misses 5-10%)
2.Early anatomy scan detects 2/3s major congenital anomalies
3.More than 50% cases CHD associated with abnormal NT
But
4.False positive rate is 1.5 - 3% (unnecessary invasive testing)
5.80-90% detection for T18, T13 and other aneuploidies
6.Does not provide information on fetal gender or 45X
7.Cannot be performed after 14+0 weeks (CRL 85mm)
The Evolution of Prenatal Screening1980 1990
2006 2016
Amniocentesis
No amniocentesis
Serum screening(Triple/Quad test)
CVS/Amniocentesis
No screening
1st trimester (NT)screening
CVS/Amniocentesis
No screening
1st trimester (NT)screening
NIPT +/- ultrasound
CVS/Amniocentesis
No screening
http://www.google.com.au/url?url=http://www.mirror.co.uk/lifestyle/sex-relationships/relationships/dear-coleen-im-thinking-leaving-7664512&rct=j&frm=1&q=&esrc=s&sa=U&ved=0ahUKEwiSkOrX8uTMAhWEo5QKHQ4lDJwQwW4IJDAH&usg=AFQjCNG-Wd6LDZ8OoHW46kYtkU1aizJNcw
Non-InvasivePrenatal Testing
(NIPT)
Cell-free fetal DNA testing
In pregnancy, womans bloodcontains maternal DNA andfetal DNA
Fetal DNA actually placentalin origin
At 10 weeks, 4-5% of totalDNA is fetal (minimumneeded to detect anomaly)
Fetal DNA rapidly clearedafter pregnancy (i.e. unique tocurrent baby)
NIPT (cfDNA) the background
Fetal DNA extracted from 43 women in Oxford, UK 24/30 male fetuses correctly Dx from maternal plasma
Used for many years for fetal Rh genotyping / sexing (X-linked)
cfDNA testing for aneuploidy screening made available in 2011
Initial studies analysed performance of cfDNA testing in groups of high-risk pregnant women
Emerging data on test performance in medium- and low-risk populations
cfDNA testing 2 decades on
Are all NIPT tests created equal?
Ariosa Illumina Natera Genea
Douglass Hanly Moir ? IVF Australia Genea
Low risk (99%)
Aneuploidy detected
Aneuploidy suspected
No aneuploidy
Low risk
High risk
Aneuploidy detected
No aneuploidy
$450 N/A $585 - $825 $445
Processing 1 week Processing 2-3 weeks Processing 1 week
Supported byNEJM study data
Optional testing for 5 microdeletions
22q / 1p36 / 5pPWS / Angelman
Analysed locally
Turnaround time
Cell-free DNA fetal testing in practice
EDD must be confirmed by ultrasound
Gestation must be 10 weeks (9 weeks for Panorama)
No MBS rebate. Only available in private sector.
10ml maternal blood sample (20ml for Panorama)
Turnaround time 1 week (Genesyte, Harmony)
Gender routinely available (accuracy 99.5%)
Caution: IVF, twins, previous organ donor, BMI
cfDNAtest
performance
Sensitivity False positive Sensitivity False positive
Sensitivity False positive Sensitivity False positive
Down syndrome Edwards syndrome
Patau syndrome Turner syndrome
How does cfDNA testing perform
in low-risk pregnant women?
12-month NEXT study
6 countries, 35 centres
Sponsored by Ariosa Dx
Harmony NIPT used
Blinded analysis of: cfDNA Standard cFTS
in singletons, 10-14 weeks
Excluded: Twins / empty IUGS Donor egg Maternal cancer
NEXT study N=15,841 women total (884 [6%] had high-risk cFTS)
cFTS high-risk if 1:270 (T21) or 1:150 (T13 / 18)
cfDNA samples with fetal fraction
NEXT study (n=15,581)
99.6%
4%15%
1 in 236 pregnancies had chromosomal abnormality
Chart1
15773
68
Total (n=15,841)
Sheet1
Total (n=15,841)
Euploid15773
Aneuploid (n=68)68
To resize chart data range, drag lower right corner of range.
Chart1
38
16
3
4
7
56%
24%
Chromosomal abnormality (n=68)
Sheet1
Chromosomal abnormality (n=68)
T2138
T18/1316
45X3
Translocation4
Other7
To resize chart data range, drag lower right corner of range.
NEXT study performance in low risk women
Trisomy 21(n=38)
Trisomy 18 (n=10)
Trisomy 13 (n=6)
cFTS NIPT cFTS NIPT cFTS NIPT
N= 15,841 15,841 15,841 15,841 11,185 11,185
Sensitivity 79% 100% 80% 90% 50% 100%
False positive 5% 0.1% 0.3% 0% 0.3% 0%
PPV 3.5% 81% 14% 90% 3.5% 50%
NPV 99.9% 100% 100% 100% 100% 100%
3% of cfDNA tests failed
Cell-free DNA testing initial conclusions
cfDNA is superior to nuchal translucency screening for detecting Down syndrome (99.5% v 90%)
cfDNA is superior to nuchal translucency screening for detecting Edwards syndrome (95% v 80%)
cfDNA has lower false positive rate than nuchaltranslucency screening (0.1% v 3-5%)
cfDNA can be performed any time after 10 weeksand provides accurate prediction of gender
remember, does my baby have
Down syndrome? is often the patient
asking is my baby healthy?
Other benefits of combined FTS
1. Enlarged NT is associated with otherchromosomal anomalies (not T21/18/13)
2. Enlarged NT is associated with increasedrisk of congenital cardiac defects
3. Allows for an early fetal anatomy survey
4. PAPP-A level can identify fetuses at highrisk for growth restriction
Chromosome problem just Down syndrome
Unbalanced translocations, deletions and duplicationswill not be detected on NIPT
Overall, 50% of aneuploidies found on amniocentesiswill be missed on cfDNA testing (i.e. not T21/18/13)
Detection rate from invasive testing even higher (5-10%higher yield) using array-CGH
1,300,000 women had cFTS
69,000 screen positive (5%)
26,000 invasive testing (38%)
3,000 abnormal result (12%)
500 not detectable on NIPT
17% abnormal resultsor
1% screen-positives
Cell-free DNA testing
Invasive fetal testing
Invasive Fetal Testing in 2016
CVS at 11+ weeks 0.5% miscarriage
Amniocentesis at 15+ weeks 0.5% miscarriage
CVS samples placenta; Amnio samples skin cells
Small risk of confined placental mosaicism w/ CVS
Specimen sent for:-FISH 13/18/21/XY (24-48 hours)
PLUS
-Conventional karyotype (2-2.5 weeks)OR
-Prenatal microarray (1-2 weeks)
4,400 women undergoing invasive testing Microarray detected all aneuploidies and the
unbalanced rearrangements Microarray does not detect balanced translocations
In samples with normal karyotype, array detectedclinically-relevant abnormalities in 6% of thosewith structural anomalies (2% others)
Concern
cfDNA testing is excellent at detecting cases of Trisomy 21, but misses
chromosomal abnormalities which might be detected on NT screening and
subsequent invasive fetal testing
NIPT special situations
1. The test has failed
2. Im expecting twins
3. Im expecting triplets.gulp
4. What about sex chromosome aneuploidy?
What to do when NIPT fails
1-5% test failure rate
Critical fetal fraction is 4% (hence 10 weeks)
BMI = fetal fraction
What to do when NIPT fails NEXT study NEJM 2015
488 / 16,329 (3%) 1.2% fetal fraction
Multifetal Pregnancies
Prenatal screening in twins the numbers
In DZ twins, either 0,1 or 2fetuses are affected
each twin carries samerisk as a singleton
risk of any affected fetusis twice risk of singleton
In MZ twins, either 0 or 2fetuses are affected
risk of both affectedfetuses is same as singleton
Prenatal screening in twins the options
Combined FTS Allows ultrasound confirmation of chorionicity
Provides a fetus-specific aneuploidy risk
Serum -hCG and PAPP-A are validated in twins
Must disclose IVF conception +/- age of egg donor
DC 90% sensitivity, 6% FPR (3% per twin)
MC 90% sensitivity, 8% FPR (TTTS)
MC average the 2 risks for the pregnancy risk
Prenatal screening in twins the options
Non-Invasive Prenatal Testing (cfDNA)
438 twin pregnancies v 10,700 singletons
100% Trisomy 21 detected in twins (n=8)
60% of Trisomy 18/13 detected in twins (n=5)
Higher failure rate in twins
Risk generated is not fetus-specific
Triplets and higher-order multiples
NIPT cannot be used
Only available option is NTscreening
Serum biochemistry is notvalidated in triplets
combine maternal age + NT(mm) to obtain risk for eachfetus
Sex chromosome aneuploidy and NIPT
Includes 45X, 47XXY (Klinefelters), 47XXX, 47XYY
Combined prevalence 1:500 (> major trisomies)
Not detected with traditional (cFTS) screening
Not usually properly consented for..
What is the chance of a false positive?
Sex aneuploidies (n=59) v 46XX (n=59) v 46XY (n=59)
Harmony cfDNA testing performed
Sensitivity for 45X was 92% (no false positives)
Managing a
positive
NIPT result
Q1: If our baby has Down syndrome, what is the chance the test will detect it?
Refers to test sensitivity (true positive rate)
= True positive tests / Total positives (TP + FN)
The likelihood that the test will be positive if thecondition is present
Reciprocal = false negative (failure to detect)
Q2: If the test is positive, what is the chance that our baby has Down syndrome?
Refers to positive predictive value (PPV)
= True positives / Positive calls (TP + FP)
The likelihood that the fetus is truly affected if thetest is positive. >99% sensitivity >99% PPV
PPV is highly dependent on disease prevalence
PPV refers to population data; post-testprobability refers to an individual patients risk
PPV of a screening test differs significantly in general obstetric and high-risk populations
27yo 40yo
NEXT study performance in low risk women
Trisomy 21(n=38)
Trisomy 18 (n=10)
Trisomy 13 (n=6)
cFTS NIPT cFTS NIPT cFTS NIPT
N= 15,841 15,841 15,841 15,841 11,185 11,185
Sensitivity 79% 100% 80% 90% 50% 100%
False positive 5% 0.1% 0.3% 0% 0.3% 0%
PPV 3.5% 81% 14% 90% 3.5% 50%
NPV 99.9% 100% 100% 100% 100% 100%
3% of cfDNA tests failed
cfDNA test returns high-risk result
Possibilities:
1. True positive-Full fetal aneuploidy-Fetal chromosomal mosaicism
2. False positive-Confined placental mosaicism-Maternal aneuploidy-Vanishing twin-Artifactual
true false positives
Clinical Scenario #1 - PS37yo P1+1 (1 previous miscarriage) had NIPT at 11/40
Clinical Scenario #1 - PS
cfDNA high risk for Trisomy 21 in 37 year old
Risk fetal Trisomy 21 in this case 70%
Do NOT permit TOP based on cfDNA result
Options for fetal karyotyping CVS v Amnio
-High % placental mosaicism 45X and T13-Low % placental mosaicism T21 and T18
Clinical Scenario #1 - PS
Patient requested CVS
Discussed 2% chance of needing amniocentesis due to confined placental mosaicism
Uncomplicated TA-CVS
FISH available in 36 hours confirmed fetal Trisomy 21 patient opted for termination check parental karyotypes
Clinical Scenario #2 - BE
34yo P0+0 with 12 previous failed IVF cycles
IVF conception, DET DCDA twin pregnancy
Clinical Scenario #2 - BE
Harmony NIPT high-risk for Trisomy 21 (>99%)
Clinical Scenario #2 - BE
Counselled patient explained risk of Trisomy 21approximately 50-60%
Already 15+6 weeks amniocentesis clear choice
Option of amniocentesis for 1 or both twins?
Amniocentesis (x2) performed FISH / karyotype
Clinical Scenario #2 - BE
Normal 46XXkaryotype forboth twins
UncomplicatedPregnancy
Elective CS at37 weeks
23yo P1 (emergency CS)
1st son has Down syndrome
Spontaneous DCDA twin pregnancy
cFTS gave high-risk T21 in Twin 1 (1:46)
Further testing options discussed:-cfDNA testing-Invasive testing with CVS or amniocentesis
Clinical Scenario #3 - JC
Clinical Scenario #3 - JC
DCDA twins high risk NIPT for 45X
Possible explanations:-1 twin has Turner syndrome-Both twins have Turner syndrome-Neither twin has Turner syndrome-1 Twin has Turners mosaicism (46XX / 45X)-Mother has Turners mosaicism (46XX / 45X)
Patient declined invasive testing
Clinical Scenario #3 - JC
Maternal karyotype sent for completeness
Maternal mosaic Turners in 20% of cells Genetic Counsellor cardiac, thyroid, POF
Clinical Scenario #3 - JC
How should cfDNA
(NIPT) testing be
integrated into
current prenatal
screening model?
Conventional screening remains the mostappropriate choice in most populations
Parallel screening with multiple screeningmodalities not recommended
cfDNA testing not recommended in twins
If a woman has received a normal/low risk resultfrom a cfDNA testing test, an additional riskcalculation for aneuploidy is not recommended
The presence of a fetal structural anomaly remainsan important indication for invasive prenatal testing,even in the presence of a prior normal/low riskcfDNA result
Prenatal Screening: my 2 centsA: Low a priori risk of aneuploidy
Combined FTSat 11-14 weeks
Low-risk(1:50)
orNT 3.5mm
orFetal anomaly
Reassure
Discuss NIPTConsider maternal age?
Recommend invasive testingwith CGH array
Prenatal Screening: my 2 centsB: High a priori risk of aneuploidy
Offer NIPT from 10 weeks
Low-risk
No result
High-risk
Consider early fetalanatomy scan (withNT) + routine mid-trimester morphology
Recommend invasivetesting (or repeat NIPT)
Invasive testing ismandatory (ideally anamniocentesis)
-Advanced maternal age -Intermediate/high risk result on cFTS-Previous aneuploidy -Too late for cFTS (>14 weeks)-Patient request -Parental Robertsonian translocation
Conclusions1. cfDNA has better detection for Trisomy 21 than
traditional combined 1st trimester screening
2. cfDNA should be performed after 10 weeks tominimise failed draws. Increased BMI andtwins increase the failure rate
3. cFTS offers the advantages of an early fetalanatomical survey and 1% of high risk NTresults will have an atypical aneuploidy.cfDNA testing (10-11 weeks) may be combinedwith an early anatomy scan (12-16 weeks)
Conclusions4. Women with low risk cfDNA result should not have
further screening (i.e. NT + bloods)
5. Fetuses with structural anomalies (including largeNT) should be offered invasive test
6. cfDNA offers information on gender but alsopopulation screening for sex chromosomeanomalies, which is unprecedented
7. cFTS recommended 1st line screening in twins
8. Most current guidelines support cfDNA as 1st linescreening in high-risk populations only
Questions?
www.shoreforwomen.com.au Tel: 1300 460 111 Fax: 1300 460 222
SHORE for Women, Suite G6, 460 Pacific Highway, St. Leonards
Slide Number 1The Evolution of Prenatal ScreeningSlide Number 3Slide Number 4Combined 1st Trimester Screening (cFTS)Slide Number 6Slide Number 7Slide Number 8Combined 1st Trimester Screening (cFTS)Slide Number 10Other benefits of combined FTS?Other benefits of combined FTS?Other benefits of combined FTS?Other benefits of combined FTS?Other benefits of combined FTS?Other benefits of combined FTS?Other benefits of combined FTS?Other benefits of combined FTS?11-14 week fetal anatomy scanOther benefits of combined FTS?Slide Number 21Slide Number 22The Evolution of Prenatal ScreeningSlide Number 24Slide Number 25Slide Number 26Slide Number 27Slide Number 28Cell-free DNA fetal testing in practicecfDNA testperformanceSlide Number 31Slide Number 32Slide Number 33NEXT studyNEXT study (n=15,581)Slide Number 36Cell-free DNA testing initial conclusionsSlide Number 38Other benefits of combined FTSChromosome problem just Down syndromeSlide Number 41Slide Number 42Invasive Fetal Testing in 2016Slide Number 44ConcernNIPT special situationsWhat to do when NIPT failsWhat to do when NIPT failsSlide Number 49Prenatal screening in twins the numbersPrenatal screening in twins the optionsPrenatal screening in twins the optionsTriplets and higher-order multiplesSex chromosome aneuploidy and NIPTSlide Number 55Slide Number 56Slide Number 57Slide Number 58Slide Number 59Slide Number 60cfDNA test returns high-risk resultClinical Scenario #1 - PSClinical Scenario #1 - PSClinical Scenario #1 - PSClinical Scenario #2 - BEClinical Scenario #2 - BEClinical Scenario #2 - BESlide Number 68Clinical Scenario #3 - JCClinical Scenario #3 - JCClinical Scenario #3 - JCClinical Scenario #3 - JCSlide Number 73Slide Number 74Slide Number 75Prenatal Screening: my 2 centsPrenatal Screening: my 2 centsConclusionsConclusionsSlide Number 80