non-invasive prenatal testing – where does it fit in?...5.80-90% detection for t18, t13 and other...
TRANSCRIPT
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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
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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
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Risk Trisomy 21 is approximately 1% at age 40
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2.5x less common 7x less common
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Combined 1st Trimester Screening (cFTS)• AKA “nuchal translucency” test
• Mainstay of screening since 1990s
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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”)
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…remember, “does my baby have
Down syndrome?” is often the patient
asking “is my baby healthy?”
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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 β-hCG Free β-hCG Free β-hCG Free β-hCG
PAPP-A PAPP-A PAPP-A PAPP-A
Nasal bone Nasal bone Nasal boneDV, TR, face DV, TR, face
PlGF, αFPDetection T21 85% 93% 94% 95%False (+) rate 3% 2.5% 2.5% 1.5%
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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
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What do the bloods mean?
T21 T18 T13 45X
β-hCG
PAPP-A
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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
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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
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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
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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
T21
T18/13
45X
Translocation
Other
56%
24%
Chromosomal abnormality (n=68)
38
16
3
4
7
Sheet1
Chromosomal abnormality (n=68)
T2138
T18/1316
45X3
Translocation4
Other7
To resize chart data range, drag lower right corner of range.
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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
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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
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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
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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
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11-14 week fetal anatomy scan
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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
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• 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 (
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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)
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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
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Non-InvasivePrenatal Testing
(NIPT)
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• Cell-free fetal DNA testing
• In pregnancy, woman’s bloodcontains maternal DNA and“fetal” 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)
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NIPT (cfDNA) – the background
• Fetal DNA extracted from 43 women in Oxford, UK• 24/30 male fetuses correctly Dx from maternal plasma
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• 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…
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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
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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
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cfDNAtest
performance
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Sensitivity False positive Sensitivity False positive
Sensitivity False positive Sensitivity False positive
Down syndrome Edwards syndrome
Patau syndrome Turner syndrome
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How does cfDNA testing perform
in “low-risk” pregnant women?
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• 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
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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
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NEXT study (n=15,581)
99.6%
4%15%
1 in 236 pregnancies had chromosomal abnormality
Chart1
Euploid
Aneuploid (n=68)
Total (n=15,841)
15773
68
Sheet1
Total (n=15,841)
Euploid15773
Aneuploid (n=68)68
To resize chart data range, drag lower right corner of range.
Chart1
T21
T18/13
45X
Translocation
Other
56%
24%
Chromosomal abnormality (n=68)
38
16
3
4
7
Sheet1
Chromosomal abnormality (n=68)
T2138
T18/1316
45X3
Translocation4
Other7
To resize chart data range, drag lower right corner of range.
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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
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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
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…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
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“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
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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
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Cell-free DNA testing
Invasive fetal testing
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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)
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• 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)
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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
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NIPT – special situations
1. “The test has failed”
2. “I’m expecting twins…”
3. “I’m expecting triplets….gulp”
4. “What about sex chromosome aneuploidy?”
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What to do when NIPT fails
• 1-5% test failure rate
• Critical fetal fraction is 4% (hence ≥10 weeks)
• ↑ BMI = ↓ fetal fraction
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What to do when NIPT fails• NEXT study NEJM 2015
• 488 / 16,329 (3%) 1.2% fetal fraction
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Multifetal Pregnancies
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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
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Prenatal screening in twins – the options
Combined FTS• Allows ultrasound confirmation of chorionicity
• Provides a “fetus-specific” aneuploidy risk
• Serum β-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”
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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”
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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
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Sex chromosome aneuploidy and NIPT
• Includes 45X, 47XXY (Klinefelter’s), 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?
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• Sex aneuploidies (n=59) v 46XX (n=59) v 46XY (n=59)
• Harmony™ cfDNA testing performed
• Sensitivity for 45X was 92% (no false positives)
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Managing a
“positive”
NIPT result
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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)
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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 patient’s risk
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PPV of a screening test differs significantly in general obstetric and “high-risk” populations
27yo 40yo
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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
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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
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Clinical Scenario #1 - PS37yo P1+1 (1 previous miscarriage) had NIPT at 11/40
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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
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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
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Clinical Scenario #2 - BE
• 34yo P0+0 with 12 previous failed IVF cycles
• IVF conception, DET DCDA twin pregnancy
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Clinical Scenario #2 - BE
Harmony NIPT – “high-risk” for Trisomy 21 (>99%)
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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
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Clinical Scenario #2 - BE
Normal 46XXkaryotype forboth twins
UncomplicatedPregnancy
Elective CS at37 weeks
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• 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
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Clinical Scenario #3 - JC
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• 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 Turner’s mosaicism (46XX / 45X)-Mother has Turner’s mosaicism (46XX / 45X)
• Patient declined invasive testing
Clinical Scenario #3 - JC
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• Maternal karyotype sent for completeness
• Maternal mosaic Turner’s in 20% of cells• Genetic Counsellor – cardiac, thyroid, POF
Clinical Scenario #3 - JC
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How should cfDNA
(NIPT) testing be
integrated into
current prenatal
screening model?
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• Conventional screening remains the mostappropriate choice in most populations
• Parallel screening with multiple screeningmodalities not recommended
• cfDNA testing not recommended in twins
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• 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
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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
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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
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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)
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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
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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 �test�performanceSlide Number 31Slide Number 32Slide Number 33NEXT studyNEXT study (n=15,581)Slide Number 36Cell-free DNA testing – initial conclusionsSlide Number 38Other benefits of combined FTS“Chromosome 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