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RAPIDLY CHANGING TRENDS IN ANEUPLOIDY SCREENING

Mary E. Norton MDProfessor, Obstetrics, Gynecology and Reproductive SciencesUniversity of California, San Francisco

Conflict of Interest Disclosure

• I have no personal financial stake in any commercial entity related to this talk

• I am co-PI of a clinical trial supported by AriosaDiagnostics and site PI of clinical studies and unpaid clinical advisor for CellScape.

History of Prenatal Screening

• Maternal age 35 yo 1960’s• MSAFP for NTD 1977• Multiple marker screening 1988

• First trimester markers/NT 1994• ACOG “all tests available” 2007• Universal screening (multiplex panels) 2010• Noninvasive prenatal testing (NIPT) 2011• Fetal whole genome sequencing

2012

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Detection Rate of Prenatal Screening for Down syndrome has improved over time

Det

ectio

n R

ate

(%)

Multiple marker screening

• Uses a combination of first and second trimester serum analytes and ultrasound markers

• Primary purpose is screening for Down syndrome and neural tube defects

• Can also detect other birth defects and abnormalities

• In absence of fetal anomalies, abnormal levels of some analytes are associated with adverse obstetric outcomes

Fetal Anomalies Associated with Elevated MSAFP

• Open fetal anomalies• Neural tube defects (spina bifida/anencephaly)Ventral wall defects (omphalocele/gastroschisis)

Chromosome abnormalitiesTriploidy

Severe renal abnormalitiesBilateral renal agenesis

Autosomal recessive polycystic kidney disease

Congenital skin disordersUpper GI tract obstruction

Fetal Anomalies Associated with Elevated MSAFP

• Open fetal anomalies• Neural tube defects (spina bifida/anencephaly)• Ventral wall defects (omphalocele/gastroschisis)

• Chromosome abnormalities• Triploidy

• Severe renal abnormalities• Bilateral renal agenesis

• Autosomal recessive polycystic kidney disease

• Congenital skin disorders• Upper GI tract obstruction

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UpToDate, 2008

Analyte PatternsNuchal translucency

• Increased NT highly associated with aneuploidy

• Also with several other abnormalities

Nonchromosomal Defects and Increased Nuchal Translucency

• Major cardiac defects • Diaphragmatic hernia• Omphalocele

• Limb body wall defect• Fetal akinesia• Noonan syndrome• Skeletal dysplasias• Other structural and genetic disorders

Euploidfetuses (n)

NT (mm) Genetic disorders and neurodevelopmental delay

Mangione et al., 2001 202 > 3mm 1/202 (0.5%)

Souka et al., 2001 1320 > 3.5 mm 44/1320 (3.3%)

Senat et al., 2002 89 > 4 mm 4/62 (6.4%)

Bilardo et al., 2007 425 > 95th % 23/425 (5.4%)

Total 2271 72/1629 (4.4%) (range 0.5-6.4%)

Genetic Disorders Detected In Fetuses With Enlarged Nuchal Translucency

Bilardo Prenatal Diagnosis 2010

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Fetal cardiac defects and enlarged NT

NT Incidence CHD

<2.5mm 0.16%2.4-3.4mm 1%3.5-4.5mm 3%4.5-5.4mm 7%5.4-6.4mm 20%>6.5mm 30%

Souka, 2005

Abnormal Analytes and 3d Trimester Pregnancy Complications

• Several patterns of abnormal analytes have been associated with placental dysfunction

• Low PAPP-A, low uE3, high AFP, hCG, inhibin• 3d trimester pregnancy complications

• Preeclampsia, early fetal loss, late fetal loss, preterm delivery, IUGR

• Risk of adverse outcomes increases with higher levels or with multiple abnormal analytes

Odds Ratios for Outcomes Associated with Abnormal Analytes

Analyte IUFD PTB IUGR PreE

PAPP-A <0.29 MoM 3.0 3.3 4.64 1.79

PAPP-A <0.42 MoM 2.15 1.9 2.81 1.54

hCG >2.0 MoM 1.5-4.7 1.7-2.8 1.8-4.8 2.4

AFP >2.5 MoM 4.4-9.8 1.8-4.8 1.6-4.0 3.8

Inh A >2.0 MoM 2.4 2.4 2.4 2.4

uE3 <0.5 MoM 3.3 2.3

Association between Adverse Outcomes and Serum Analytes

Dugoff, Obstet Gynecol, 2010

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Is detection of abnormal analytes helpful?

Disorder ASA No ASA RR (CI)

PreE 9.3% 21.3% 0.47 (0.34-.65)

Severe PreE 7% 15% 0.09 (0.02-.37)

IUGR 7% 16.3% 0.44 (0.30-.65)

Bujold et al, 2010, Obstet GynecolMeta-analysis of studies of aspirin in high risk women

ASA decreased risks only when started at <16 weeks gestation

Is detection of abnormal analytes helpful?

Roberge et al, Ultrasound Obstet Gynecol 2013Meta-analysis of studies of aspirin in high risk women

ASA decreased risks only when started at <16 weeks gestation

Disorder ASA No ASA RR (CI)

Perinatal death 1.1% 4.0% 0.41 (0.19-0.92)

Preeclampsia 7.6% 17.9% 0.47 (0.36-0.62)

Severe PreE 1.5% 12.3% 0.18 (0.08-0.41)

IUGR 8.0% 17.6% 0.46 (0.33-0.64)

Preterm birth 4.8% 13.4% 0.35 (0.22-0.57)

Noninvasive Prenatal Testing (NIPT) using Cell Free DNA

• Tests for aneuploidy by directly sequencing of fetal DNA, largely derived from placenta

• Compared with current screening which uses indirect measurements of protein products

• NIPT for detection of trisomy 21 has greater sensitivity and much greater specificity than multiple marker screening

Analysis of cell free DNA

Zhong, X, Holzgreve, W, Glob. libr. women's med 2009

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Non-invasive Prenatal Testing (NIPT) vs First Trimester Screening (FTS)

Disorder Test Detection rate False positive rate

Trisomy 21 NIPT 99.4% 0.15%

FTS 94% 5.8%

Trisomy 18 NIPT 98.5% 0.2%

FTS 100% 0.3%Trisomy 13 NIPT 86% 0.7%

FTS 100% 0.3%

Non-invasive Prenatal Testing (NIPT) vs First Trimester Screening (FTS)

Disorder Test Detection rate False positive rate

Trisomy 21 NIPT 99.4% 0.15%

FTS 94% 5.8%

Trisomy 18 NIPT 98.5% 0.2%

FTS 100% 0.3%Trisomy 13 NIPT 86% 0.7%

FTS 100% 0.3%

Alamillo et al. 2013

False positive rate of NIPT

As you add additional tests, the false positive rate increases

T21 0.1%T18 0.5%T13 0.7%45X ~1.0%

Other SCA ? Total >2.3%

• Survival rates of T13, 18 and 21 are relatively low, but vary

• Study of karyotype of cytotrophoblast, villous stroma, chorion, amnion, and cord blood w/T13 and T18 infants

Fetal trisomy

% cells trisomic in cytotrophoblast

% cells trisomic in all other tissues

T13, T18(n=14) 30% (average) 100%

T21 (n=12) 100% 100%

NIPT false negatives and positives more likely in T13 and T18 due to underlying biology of fetal and placental development

Placental mosaicism and T13 and 18

Kalousek et al, 1989

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Natural X Chromosome Loss

• 665 women (0-80yrs)

• Lymphocyte cultures on 19,650 cells

• G-banding analysis for presence of 1 or 2 X chromosomes

Russell et al, 2007

NIPT is more precise for T21, T18

NIPT Current NT + serum screen

NIPT is more precise for T21, T18

NIPT Current NT + serum screen

Other abnormalities

NIPT is more precise for T21, T18

NIPT FTSOther abnormalities

8/8 T212/3 T18; 1/3 no result

8/8 T213/3 T187/7 others (45X; triploidy; deletions and duplications)Nicolaides et al, 2012

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NIPT is more precise for T21, T18

NIPT FTSOther abnormalities

8/8 T212/3 T18; 1/3 no result

8/8 T213/3 T187/7 others (45X; triploidy; deletions and duplications)

55%(10/18)

100%(18/18)

Nicolaides et al, 2012

“Nearly a third of abnormalities found after first trimester screening are different than expected” Alamillo et al. 2013

• N=23,329 cases of FTS over 10 years• 6.3% screen positive

• 5.7% for T21; 0.4% for T13/18; 0.3% for both

• 97 had a chromosome abnormality (1/240)• 47 Down syndrome (T21) (1/500)• 22 Trisomy 13 or 18

• 29 Other chromosome abnormalities• Range of severity from mild to lethal• Detected by combination of NT and analytes

DS and T18 make up 2/3 of aneuploidies detectable by karyotype

Disorders potentially detectable by serum screening and NIPT

NIPT

• Trisomy 21• Trisomy 18• Trisomy 13

• Some sex chromosomes

Current Screening• Trisomy 21

• Trisomy 18• Trisomy 13• Some sex chromosomes• Triploidy

• Other rare aneuploidies• Congenital heart defects• Noonan syndrome

• Neural tube defects• Ventral wall defects• Congenital adrenal hypoplasia• Smith Lemli Opitz syndrome

• Steroid sulfatase deficiency• Poor OB outcomes (IUGR, PreE, PTB)

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Rate of aneuploidy varies by maternal age

• >35 yo 43% of abnormalities will be missed by NIPT• <35 yo 75% of abnormalities will be missed by NIPT

• This includes only those that are detectable by karyotype• Rate is significantly higher if include those detectable by

chromosomal microarray

ACMG, Statement on Noninvasive Prenatal Screening, 2013

Chromosomal Microarray (CMA) for Prenatal Diagnosis

Diagnostic Yield in Cases with Normal Karyotype

Indication for Testing

Clinically Relevant (N=96)

U/S AnomalyN=755

6.0%

AMAN=1,966

1.7%

Positive ScreenN=729

1.7%

OtherN=372

1.3%

Abnormalities detectable per 1000 births

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15

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25

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35

40

45

20 yo 25yo 30yo 35yo 40yo

NIPT

Amnio/karyotype

Amnio/microarray

Wapner et al 2012; Hook 1983

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Detection rate of FTS: Alamillo et al 2013

N=23,329 patients having FTS• N=50 cases of DS

• DR = 47 (94%)• FPR = 1345 (5.8%)

• N=22 cases of T13 or 18• DR = 22 (100%)• FPR = 134 (0.6%)

NIPT versus First Trimester Screening

Trisomy 21 Detection Rate False Positive Rate

NIPT 100% 0.15%

First Trimester Screen

94% 5.8%

Alamillo et al, 2013

NIPT versus First Trimester Screening

Trisomy 21 Detection Rate False Positive Rate

NIPT 100% 0.15%

First Trimester Screen

94% 5.8%

Trisomy 13/18 Detection Rate False Positive Rate

NIPT 86-98% 0.97%

First Trimester Screen

100% 0.6%

Alamillo et al, 2013

NIPT versus First Trimester Screening

Trisomy 21 Detection Rate False Positive Rate

NIPT 100% 0.15%

First Trimester Screen

94% 5.8%

Trisomy 13/18 Detection Rate False Positive Rate

NIPT 86-98% 0.97%

First Trimester Screen

100% 0.6%

Alamillo et al, 2013

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NIPT versus First Trimester Screening

Trisomy 21 Detection Rate False Positive Rate

NIPT 100% 0.15%

First Trimester Screen

94% 5.8%

Trisomy 13/18 Detection Rate False Positive Rate

NIPT 86-98% 0.97%

First Trimester Screen

100% 0.6%

Alamillo et al, 2013

Disorder Prevalence

Common trisomies(13,18,21)

0.2%

Other chromosomeabnormalities

0.4%

Microdeletions and duplications

1.5%

Mendelian GeneticDisorders

0.4%

Congenital heart defects

0.3%

Other structural defects

3%

Adverse OB outcomes 15-20%

Total ~25%

Causes of Birth Defects and

Other Adverse Perinatal Outcomes:

It’s Not All Down Syndrome

Disorder Prevalence NIPT Integrated screening

CVS/amnio

Common trisomies(13,18,21)

0.2% Yes Yes Yes

Other major chromosomeabnormalities

0.4% No Some Yes

Microdeletions and duplications

1.5% No Some Yes (array)

Mendelian GeneticDisorders

0.4% No Some If requested

Congenital heart defects 0.3% No Yes (NT) No

Other structural defects 3% No Yes (NT) No

Adverse OB outcomes 15-20% No Yes (serum analytes)

No

Total ~25% 0.2% 25% 2%

• N=611 pregnancies• Rates of preeclampsia, preterm birth, and SGA were not

associated with cell free fetal DNA concentration

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Prenatal Genetic Screening Comparison

Integrated Screening

- 2 blood tests + NT ultrasound- With sequential screen, patients

get two results over 6+ weeks- Complex counseling

- Stringent GA criteria- Relatively high screen positive

rate and good, not great, detection rate

- NT requires training and on-going QA

- NT very operator dependent+ Detects additional disorders+ Adjusts results by a priori risk

Noninvasive Prenatal Testing

+ Single blood test anytime after 10 weeks GA

+ Excellent performance = more straightforward counseling

+ Fewer invasive procedures+ One result that is highly

reliable- Tests for few conditions- Expensive- High test failure rate, esp.

with high BMI- Not validated in all subgroups- Doesn’t adjust for a priori risk

How Does Test Performance Differ with Risk?

Low Risk (age 25; 1/1000)

N=1000

1 T21 999 not T21

1 TP, 0 FN 2 FP, 998 TN

OAPR = 1/3

High Risk (age 38; 1/100)

N=1000

10 T21 990 not T21

10 TP, 0 FN 2 FP, 988 TN

OAPR = 10/12 or 5/6

(T21: Assume >99% sensitivity and 99.8% specificity)

How Does Test Performance Differ with Risk?

Low Risk (age 25; 1/8,000)

N=10,000

1 T13 9,999 not T13

1 TP, 0 FN 100 FP, 9900 TN

OAPR = 1/100

High Risk (age 38; 1/1000)

N=10,000

10 T13 9,990 not T13

10 TP, 0 FN 100 FP, 9890 TN

OAPR = 10/100 = 1/10

(T13: Assume 99% sensitivity and 99% specificity)

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Where does NIPT fit?

�Is this an outstanding screening test or an imperfect diagnostic test?

�Is this best used as a secondary screening test, or as a first tier screening test?

�Are we ready to abandon current screening in favor of NIPT?

Let’s not throw out the baby with the bathwater….. Hype cycle of new technology

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• Report on 8 false positive cases of T13 or T18• It is estimated that over 100,000 cases were done last year• Inflated expectations, lack of understanding, aggressive

marketing and competition are problematic

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Secretary’s Advisory Committee on Genetics, Health and Society

�Analytic validity: ability of test to measure particular genetic characteristics (eg DNA sequence) accurately and reliably in a given specimen

�Clinical validity: test’s accuracy in detecting the presence of, or predicting risk for, a health condition or phenotype

�Clinical utility: balance between health related benefits and harms that can ensue from a genetic test

Personalized Medicine 2008

Summary• NIPT has great value in detection of common trisomies.

• Multiple marker screening can play a much broader role in evaluating all pregnancies than NIPT.

• First trimester screening addresses not only fetal genetic risk, but also fetal structural abnormalities and pregnancy complications.

• Relative benefits of each test remain unclear, but may vary by patient risk category.

• Further study urgently needed before standard of care is completely changed.

Thank you!