prenatal aneuploidy screening using cell free dna · prenatal aneuploidy screening using cell free...

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Prenatal Aneuploidy Screening Using cell Free DNA

Mary E. Norton MDProfessor, Obstetrics,

Gynecology and Reproductive Sciences

University of California, San Francisco

What Does the Evidence Tell Us? Oct 2015

Disclosures

o Research support from Natera and Ariosa

o No lucrative personal financial contracts

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Detection rate of prenatal screening for Down syndrome has improved over time

Det

ectio

n R

ate

(%)

The questions being debated:

o Is cfDNA screening the best option for low risk patients?

o Is cfDNA screening the best choice for primaryscreening for any or all patients?

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Professional Society Opinions: ACOG; ACMG; International Society of Prenatal Diagnosis;

National Society of Genetic CounselorsCommon themes (2012):There are recognized benefits, but…o Not diagnostic

• Needs confirmationo Only detects common trisomieso Requires comprehensive genetic counselingo Should only be used in validated groups (eg high risk)o Need a low risk study before introducing into general

population screening

Rumsfeld on current status of NIPT?“There are known knowns. There are things we know, we know. We also know there are known unknowns. That is to say, we know there are some things we do not know. But there are also unknown unknowns. The ones we don’t know we don’t know.”

-Donald Rumsfeld, 2002

Cell free fetal DNA

o Short segments of fetal DNA (<200 base pairs) circulate in maternal plasma

o Origin is primarily placenta

Maternal DNA

Fetal DNA

Cell free DNA results from apoptosis

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Analysis of cell free DNA

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

Trisomy 21 Non-Trisomy 21

DR: 99.2% (98.5 - 99.6)FPR: 0.09% (0.05 - 0.13)

Trisomy 21 performance cfDNA testing: meta-analysis (Gil et al, Ultrasound Obstet Gynecol, 2015)

Cell free DNA: Biologic ChallengesFalse positives:o Unrecognized or vanishing twino Placental mosaicismo Maternal genetic variation o Maternal malignancyFalse negatives:o Low level of fetal DNA o Placental mosaicismo Maternal genetic variation Failed results: o Increased BMIo Low level of fetal DNAo Fetal aneuploidy

False positive cfDNA results and cancer

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Issues with cfDNA for primary screening

1. Not all abnormalities are detectable• Down syndrome comprises ~50% of aneuploidies

2. The PPV (chance that a positive is a TRUE POSITIVE) depends on maternal age• This is often misunderstood

3. Some tests fail to provide a result• These patients are at HIGH RISK of aneuploidy• Importance of “fetal fraction”

What percentage of chromosome abnormalities will be detected by cfDNAscreening?A. 99%B. 75%C. 50%D. 12%

9 9 % 7 5 % 5 0 % 1 2 %

28%

13%17%

41%

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Spectrum of Genetic Disease

Structural Malformations

Autosomal recessiveAutosomal dominant

X-linked

Chromosomal/karyotype

CNV (microarray)

Spectrum of Genetic Disease

Structural Malformations

Autosomal recessiveAutosomal dominant

X-linked

Chromosomal/karyotype

CNV (microarray)

Aneuploidies Present in HIGH RISK Women

Tri 21: 53.2%Sex chromosomal: 8.2%

Tri 13: 4.6%

Tri 18: 17.0%

Other*16.9%

Norton et al, SMFM, 2014

*Not detected by cfDNA

Aneuploidies Present in LOW RISK Women

Tri 21: 49.2%

Sex chromosomal: 9.9%

Tri 13: 5.5%

Tri 18:12.9%

Other*20.8%

Norton et al, SMFM, 2015

*Not detected by cfDNA

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Rate of abnormalities by maternal age cfDNA Detection Raten=452,901 patients screened in California

Total Cases with Aneuploidy (n=2575)

cfDNADetectable

Not Detected No Result

N=69+560(24.5%)

N=1841(71.4%)

N=105(4.1%)

Not Detectable(False negative+Non-detectable)

In low-risk patients, this is a very accurate test for a rare event.

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NIPT and diagnostic testing with chromosomal microarray (CMA)

Microarray detects an abnormality in 1.7% of cases (about 1/60)AND:

NIPT detects T13,18, 21 – about 1/500 pregnanciesTHEN:

� If NIPT is the routine screening test, it will detect about 12% of diagnosable chromosomal abnormalities









Your 25 yo patient has cfDNA screening and the result is positive for trisomy 13. What is the chance that the fetus actually has trisomy 13?A. >99%B. 75%C. 50%D. <10%

> 9 9%

7 5%

5 0%

< 1 0%

34%

28%

17%21%

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o N=1914 women undergoing standard screeningo Mean maternal age = 29.6 yrso Primary outcome = false positive rates for T18 and T21

cfDNA vs Standard Screening

Bianchi et al, NEJM, 2014

FPR PPVcfDNA 0.3% 45.5% p<.001Standard 3.6% 4.2%

o Only 8 aneuploidy cases in the cohort (5: T21, 2: T18, and 1: T13)

o All were detected

o 15,841 women had cfDNA and first trimester screening

o Mean maternal age = 30.7 yrs

“NEXT” study: 15,841 average risk women

Cell free DNA

screening

First trimester screening

Detection rate 38/38 (100%)

30/38 (79%) P=0.008

False positive rate

0.06% 5.4% P<0.0001

Positive predictivevalue

81% 3.4% P<0.0001

Norton et al, NEJM, 2015

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Wang et al, Genetics in Medicine, 2014

Aneuploidy No. of positives No (%)confirmed

T21 41 38/41 (93%)T18 25 16/25 (64%)T13 16 7/16 (44%)45X 16 6/16 (38%)

Total 98 67 (67%)

o 6.2% had termination without karyotype confirmation

o Disconcerting if only 67% are true positives

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Consequences of false positive results

N=100,000 1% false positives

1000 abnormal results6.2% TAB w/o confirmation

62 TAB

42 TP 21 FP

21 TAB of normal fetuses

NIPT

67% PPV

Consequences of false positive results

N=100,0005% false positives

5000 abnormal results

0.2% loss rate (amnio)

10 losses of normal fetuses

N=100,000 1% false positives

1000 abnormal results6.2% TAB w/o confirmation

62 TAB

42 TP 21 FP

21 TAB of normal fetuses

NIPT Serum Screening

67% PPV

If your patient has a positive result:www.perinatalquality.org

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The poorly understood PPV Your patient has cfDNA screening, and the lab calls to tell you the test failed to provide a result. What are possible reasons for this?

A. Maternal obesityB. The fetus has a chromosome

abnormalityC. The blood was drawn too earlyD. All of the above

M at e r n

a l ob e s

i t y

T h e f e t

u s ha s a

c h ro m

o . . .T h e

b l oo d

w a s d r a

w n t . .

A l l o f t

h e a b o

v e

16%

71%

12%1%

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Published Trials of NIPT: failure ratesTrial Failure rate

Chiu et al (2011) 11/764 (1.4%)Ehrich et al. (2011) 18/467 (3.8%)Palomaki et al. (2011) 13/1696 (0.8%)Bianchi et al. (2012) 30/532 (3.0%)Norton et al (2012) 148/3228 (4.6%)Zimmermann et al (2012) 21/166 (12.6%)Pergament et al (2014) 85/1051 (8%)Norton et al (2015) 488/16,329 (3.0%)

All 729/23,182 (3.1%)

Fetal fraction of DNA and test failure

3-5% of samples do not provide a result

• Low fraction fetal DNA, failed sequencing, high variability in counts

• Some association with gestational age (<10 wks) • Low fetal fraction associated with maternal BMI

- 20% at >250 lbs- 50% at >350 lbs

� Low fetal fraction is associated with aneuploidy�Repeating test will provide a result in SOME cases

Obesity in US Adults Pergament et al, 2014

o N=1051 samples were analyzed

o N=85/1051 (8%) samples failed to obtain a result

o 20/85 (22%) were aneuploid

�“No call” cases represent a very high risk group

Obstet Gynecol 2014

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No ResultsN = 102(2.3%)

Total Pregnancies SampledN = 4446

Redraw Declined N=39

High Risk N = 157(3.5%)

RedrawnN = 63

Low RiskN = 4187(94.2%)

Low RiskN=32 (50.8%)

High Risk N=5 (7.9%)

No ResultN=26(41.3%)

NO FINAL RESULTN = 65 (1.5%)

Kaiser cfDNA Experience: No Results Cases




High Risk N = 157(3.5%)

RedrawnN = 63

Low RiskN = 4187(94.2%)









High Risk N = 157(3.5%)

RedrawnN = 63

Low RiskN = 4187(94.2%)






KPNC 10/29/12 – 6/30/14Total pregnancies sampled = 4446

Abnormal chromosomes 9/65 (14%)

Normal chromosomes13/65 (20%)

Chromosomes not done43/65 (66%)

Kaiser cfDNA: No Results


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Failed cfDNA screens indicate increased risk for aneuploidy

Failed tests increase aneuploidy risk:

Author OR for aneuploidyo Norton et al, NEJM 2015: 6.2o Pergament et al, Obstet Gynecol 2014: 2.5o Turocy et al, SMFM 2015: 5.7

The questions being debated:

o Is cfDNA screening the best option for low risk patients?

o Is cfDNA screening the best choice for primaryscreening for any or all patients?

NIPT is more precise for T13, 18, 21

cfDNA Current NT + serum screen

NIPT is more precise for T13, 18, 21

cfDNA Current NT + serum screen

Other abnormalities

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cfDNA vs Sequential Screening:Detection and False Positive Rates

Cohort Detection Rate

False Positive Rate

Sequential screening

81.6% 4.5%

cfDNA if “no results” cases = high risk

77.1% 3.7%

cfDNA if “no results” have no follow up

70.7% 0.7%

cfDNA and Ultrasound Abnormalities

Normal U/S Abnormal U/ST13, 18, 21 25 (4.9%) 88 (23.4%)Other chromosomalabnormalities

13 (2.5%) 29 (7.7%)

Total detectable cfDNA

25/38 (66%) 88/117 (75%)

Benachi et al, Obstet Gynecol, 2015

NIPT: Expanded panelsLaboratories have added other trisomies and microdeletionso Trisomies 16 and 22o Microdeletion syndromes

• 22q (diGeorge)• 5p (cri-du-chat)• 1p36• 15q (Prader Willi)• 4p (Wolf-Hirshhorn)

NIPT: Expanded panels

o Trisomies 16 and 22• Rarely seen in viable pregnancies except as mosaics• Common causes of confined placental mosaicism

- Much more common in CVS samples than amniocentesis• Even complete trisomy in the placenta often

associated with a normal fetus• With both, confined placental mosaicism can be

associated with IUGR, so false positive cases should be followed for fetal growth (eg ultrasound at 30-32 wks)

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Microdeletion syndromes are rareSyndrome Frequency Features22q11.2 (DiGeorge)

1/4,000 Varies: cardiac, palatal, immune, intellectual disability

1q36 1/10,000 Severe intellectual disability (ID), +/- obvious structural anomalies

Angelman 1/20,000 Severe ID, seizures, speech delay

Prader-Willi 1/30,000 Obesity, ID, behavioral problems

Cri-du-chat 1/50,000 Microcephaly, ID, +/- CHDWolf-Hirshhorn 1/50,000 ID, seizures, +/- CL/CP

Testing for Rare Disorders

N=100,000

2 Wolf-Hirschhorn 99,998 not WHS

2 TP; 0 FN 800 FP; 99,198 TN

OAPR = 1/400

Population Risk = 1/50,000(Wolf-Hirschhorn, 4p-: Assume 99% sensitivity and 99.2% specificity)

ACOG/SMFM September 2015o Conventional screening is most appropriate

first line screen for most patientso Ethically any patient may choose cfDNA

screening, but should be counseled regarding limitations and benefits

o Diagnostic testing is required to confirm abnormal results before irreversible decisions

o Testing for microdeletions and in twins should not be performed

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Important counseling points

o NIPT is NOT diagnostico Extremely high sensitivity and specificity for

Down syndrome o Somewhat lower for trisomy 13, 18 and sex

chromosomeso Approximately 20-30% of chromosome

abnormalities identified with invasive testing are NOT detectable with NIPT

All patients deserve equal access

o ACOG indicates that testing should no longer be stratified by maternal age

o We’ve spent the past decade trying to abolish “advanced maternal age” and the 35 yo cutoff

o It is unethical to withhold this test

All patients deserve equal access

o ACOG indicates that testing should no longer be stratified by maternal age

o We’ve spent the past decade trying to abolish “advanced maternal age” and the 35 yo cutoff

o It is unethical to withhold this test�There is a difference between withholding

something for an individual patient, and recommending it for all patients as policy

All patients should have access to all test options…(the Jim Carrey approach)

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Ethics, access and counselingo While it is not ethical to withhold tests from one

groupo It is necessary to provide fair and balanced

counseling regarding the pros and cons of ALL test options• cfDNA is very good for the common aneuploidies, but

doesn’t detect other serious chromosome abnormalities

• In low risk patients, a positive result is more likely to be a false positive

• In patients at low risk for the common aneuploidies, other screening options provide broader coverage at lower cost

Appropriate counselingo While it is not ethical to withhold tests from one

















• In patients at low risk for the common aneuploidies, other screening options provide broader coverage for more conditions

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Thank You!

prenatal aneuploidy screening using cell free dna · prenatal aneuploidy screening using cell free...

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