Prenatal Aneuploidy Screening Disclosures Using cell Free DNA o - - PowerPoint PPT Presentation

10/15/2015 1

Prenatal Aneuploidy Screening Using cell Free DNA

Mary E. Norton MD Professor, Obstetrics, Gynecology and Reproductive Sciences University of California, San Francisco

What Does the Evidence Tell Us? Oct 2015

Disclosures

• Research support from Natera and Ariosa
• No lucrative personal financial contracts

20 40 60 80 100 120

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

Detection Rate (%)

The questions being debated:

• Is cfDNA screening the best option for low risk

patients?

• Is cfDNA screening the best choice for primary

screening for any or all patients?

10/15/2015 2 Professional Society Opinions: ACOG; ACMG; International Society of Prenatal Diagnosis; National Society of Genetic Counselors

Common themes (2012): There are recognized benefits, but…

• Not diagnostic
• Needs confirmation
• Only detects common trisomies
• Requires comprehensive genetic counseling
• Should only be used in validated groups (eg high risk)
• 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

• Short segments of fetal DNA (<200 base pairs) circulate in

maternal plasma

• 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 Challenges

False positives:

• Unrecognized or vanishing twin
• Placental mosaicism
• Maternal genetic variation
• Maternal malignancy

False negatives:

• Low level of fetal DNA
• Placental mosaicism
• Maternal genetic variation

Failed results:

• Increased BMI
• Low level of fetal DNA
• 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 cfDNA screening?

• A. 99%
• B. 75%
• C. 50%
• D. 12%

9 9 % 7 5 % 5 % 1 2 %

28% 13% 17% 41%

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Spectrum of Genetic Disease

Structural Malformations

Autosomal recessive Autosomal dominant X-linked Chromosomal/ karyotype

CNV (microarray)

Spectrum of Genetic Disease

Structural Malformations

Autosomal recessive Autosomal 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 Rate n=452,901 patients screened in California

Total Cases with Aneuploidy (n=2575) cfDNA Detectable 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.

10/15/2015 7 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 pregnancies THEN: If NIPT is the routine screening test, it will detect about 12% of diagnosable chromosomal abnormalities

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

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 pregnancies THEN: 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 % < 1 %

34% 28% 17% 21%

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• N=1914 women undergoing standard screening
• Mean maternal age = 29.6 yrs
• Primary outcome = false positive rates for T18 and T21

cfDNA vs Standard Screening

Bianchi et al, NEJM, 2014 FPR PPV cfDNA 0.3% 45.5% p<.001 Standard 3.6% 4.2%

• Only 8 aneuploidy cases in the cohort (5: T21, 2:

T18, and 1: T13)

• All were detected
• 15,841 women had cfDNA and first trimester

screening

• 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 predictive value 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%)

• 6.2% had termination without karyotype

confirmation

• Disconcerting if only 67% are true positives

10/15/2015 10 Consequences of false positive results

N=100,000 1% false positives 1000 abnormal results 6.2% TAB w/o confirmation 62 TAB 42 TP 21 FP 21 TAB of normal fetuses

NIPT

67% PPV

Consequences of false positive results

N=100,000 5% false positives 5000 abnormal results 0.2% loss rate (amnio) 10 losses of normal fetuses N=100,000 1% false positives 1000 abnormal results 6.2% 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

10/15/2015 11

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 obesity
• B. The fetus has a chromosome

abnormality

• C. The blood was drawn too early
• D. All of the above

M a t e r n a l

• b

e s i t y T h e f e t u s h a s a c h r

• m
• .

. . T h e b l

• d

w a s d r a w n t . . A l l

• f

t h e a b

• v

e

16% 71% 12% 1%

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Published Trials of NIPT: failure rates

Trial 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

• N=1051 samples were analyzed
• N=85/1051 (8%) samples failed to obtain a result
• 20/85 (22%) were aneuploid

“No call” cases represent a very high risk group

Obstet Gynecol 2014

10/15/2015 13

No Results N = 102 (2.3%) Total Pregnancies Sampled N = 4446 Redraw Declined N=39 High Risk N = 157 (3.5%) Redrawn N = 63 Low Risk N = 4187 (94.2%) Low Risk N=32 (50.8%) High Risk N=5 (7.9%) No Result N=26(41.3%) NO FINAL RESULT N = 65 (1.5%)

Kaiser cfDNA Experience: No Results Cases

No Results N = 102 (2.3%) Total Pregnancies Sampled N = 4446 Redraw Declined N=39 High Risk N = 157 (3.5%) Redrawn N = 63 Low Risk N = 4187 (94.2%) Low Risk N=32 (50.8%) High Risk N=5 (7.9%) No Result N=26(41.3%) NO FINAL RESULT N = 65 (1.5%)

Kaiser cfDNA Experience: No Results Cases

No Results N = 102 (2.3%) Total Pregnancies Sampled N = 4446 Redraw Declined N=39 High Risk N = 157 (3.5%) Redrawn N = 63 Low Risk N = 4187 (94.2%) Low Risk N=32 (50.8%) High Risk N=5 (7.9%) No Result N=26(41.3%) NO FINAL RESULT N = 65 (1.5%)

Kaiser cfDNA Experience: No Results Cases

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

Abnormal chromosomes 9/65 (14%)

Normal chromosomes 13/65 (20%) Chromosomes not done 43/65 (66%)

Kaiser cfDNA: No Results

NO FINAL RESULT N = 65 (1.5%)

10/15/2015 14 Failed cfDNA screens indicate increased risk for aneuploidy

Failed tests increase aneuploidy risk:

Author OR for aneuploidy

• Norton et al, NEJM 2015:

6.2

• Pergament et al, Obstet Gynecol 2014:

2.5

• Turocy et al, SMFM 2015:

5.7

The questions being debated:

• Is cfDNA screening the best option for low risk

patients?

• Is cfDNA screening the best choice for primary

screening 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

10/15/2015 15

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/S T13, 18, 21 25 (4.9%) 88 (23.4%) Other chromosomal abnormalities 13 (2.5%) 29 (7.7%) Total detectable cfDNA 25/38 (66%) 88/117 (75%)

Benachi et al, Obstet Gynecol, 2015

NIPT: Expanded panels

Laboratories have added other trisomies and microdeletions

• Trisomies 16 and 22
• Microdeletion syndromes
• 22q (diGeorge)
• 5p (cri-du-chat)
• 1p36
• 15q (Prader Willi)
• 4p (Wolf-Hirshhorn)

NIPT: Expanded panels

• 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 rare

Syndrome Frequency Features

22q11.2 (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, +/- CHD Wolf-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 2015

• Conventional screening is most appropriate

first line screen for most patients

• Ethically any patient may choose cfDNA

screening, but should be counseled regarding limitations and benefits

• Diagnostic testing is required to confirm

abnormal results before irreversible decisions

• Testing for microdeletions and in twins should

not be performed

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Important counseling points

• NIPT is NOT diagnostic
• Extremely high sensitivity and specificity for

Down syndrome

• Somewhat lower for trisomy 13, 18 and sex

chromosomes

• Approximately 20-30% of chromosome

abnormalities identified with invasive testing are NOT detectable with NIPT

All patients deserve equal access

• ACOG indicates that testing should no longer

be stratified by maternal age

• We’ve spent the past decade trying to abolish

“advanced maternal age” and the 35 yo cutoff

• It is unethical to withhold this test

All patients deserve equal access

• ACOG indicates that testing should no longer

be stratified by maternal age

• We’ve spent the past decade trying to abolish

“advanced maternal age” and the 35 yo cutoff

• 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

• ptions…(the Jim Carrey approach)

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Ethics, access and counseling

• While it is not ethical to withhold tests from one

group

• 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,
• ther screening options provide broader coverage at

lower cost

Appropriate counseling

• While it is not ethical to withhold tests from one

group

• 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,
• ther screening options provide broader coverage at

lower cost

Appropriate counseling

• While it is not ethical to withhold tests from one

group

• 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,
• ther screening options provide broader coverage at

lower cost

Appropriate counseling

• While it is not ethical to withhold tests from one

group

• 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,
• ther screening options provide broader coverage for

more conditions

10/15/2015 19

Thank You!