non-invasive prenatal testing – where does it fit...

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


NIPT +/- ultrasound

CVS/Amniocentesis

No screening

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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



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



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


T2138

T18/1316

45X3

Translocation4

Other7

To resize chart data range, drag lower right corner of range.



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



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

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


CVS/Amniocentesis

No 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


Chart1

38

16

3

4

7

56%

24%


Sheet1


T2138

T18/1316

45X3

Translocation4

Other7


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





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


Harmony NIPT high-risk for Trisomy 21 (>99%)


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


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

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


Maternal karyotype sent for completeness

Maternal mosaic Turners in 20% of cells Genetic Counsellor cardiac, thyroid, POF


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

non-invasive prenatal testing – where does it fit...

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