6/5/2014
Cell free DNA:
The Popular Press vs The Evidence
Mary E Norton, MD
Professor of Obstetrics, Gynecology & Reproductive Sciences; UCSF
Antepartum and Intrapartum Management
Disclosures
• Principal Investigator of ongoing clinical trial
on cfDNA supported by Ariosa Diagnostics
• Unpaid clinical consultant for CellScape
and Natera
• Research support from Natera
• No personal financial involvement in any of
the cfDNA companies
1
6/5/2014
Detection rate of prenatal
screening for Down syndrome has
improved over time
Detection Rate (%)
120
Cell free fetal DNA
• Cell free fetal DNA (cffDNA) is made up of short
segments of fetal DNA (<200 base pairs) that
circulate in maternal plasma
100
80
60
40
20
0
Cell free DNA results from
apoptosis
• Origin of these fragments is thought to be primarily
placenta
Maternal DNA
Fetal DNA
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6/5/2014
Companies currently offering cfDNA
screening (in order of appearance):
Noninvasive Prenatal
Testing (NIPT)
• Detection requires accurate quantification of
DNA from a specific chromosome
• Somewhat different methods are utilized by
different laboratories
Analysis of fetal DNA
Trisomy 21 performance cfDNA testing:
meta-analysis (Gil et al, Fetal Diagn Ther, 2014)
Author
Chiu et al., 2011 [86]
Ehrich et al., 2011 [39]
Palomaki et al., 2011 [212]
Sehnert et al., 2011 [13]
Ashoor et al., 2012 [50]
Bianchi et al., 2012 [89]
Jiang et al., 2012 [16]
Lau et al., 2012 [11]
Nicolaides et al., 2012 [8]
Norton et al., 2012 [81]
Sparks et al., 2012 [36]
Zimmerman et al., 2012 [11]
Guex et al., 2013 [30]
Nicolaides et al., 2013 [25]
Song et al., 2013 [8]
Verweij et al., 2013 [18]
DR (95% CI)
Wts (%)
100 (95.8 - 100)
11.6
100 (91.0 - 100)
5.3
98.6 (95.9 - 99.7)
28.4
100 (75.3 - 100)
1.9
100 (92.9 - 100)
6.8
100 (95.9 - 100)
12.0
100 (79.4 - 100)
2.3
100 (71.5 - 100)
1.6
100 (63.1 - 100)
1.2
100 (95.6 - 100)
11.0
100 (90.3 - 100)
4.9
100 (71.5 - 100)
1.6
100 (88.4 - 100)
4.1
100 (86.3 - 100)
3.5
100 (63.1 - 100)
1.2
94.4 (72.7 - 100)
2.5
2.06 (0.4 - 5.9)
0.24 (0.0 - 1.4)
0.20 (0.0 - 0.6)
0.00 (0.0 - 10.3)
0.00 (0.0 - 1.1)
0.00 (0.0 - 0.9)
0.00 (0.0 - 0.4)
0.00 (0.0 - 3.7)
0.00 (0.0 - 0.2)
0.04 (0 - 0.2)
0.00 (0.0 - 2.8)
0.00 (0.0 - 2.7)
0.00 (0.0 - 2.5)
0.00 (0.0 - 1.8)
0.00 (0.0 - 0.2)
0.00 (0.0 - 0.7)
99.1 (98.3 - 99.6) 100
Pooled analysis
50 60 70 80 90100 DR % (95% CI) (%)
Zhong, X, Holzgreve, W, Glob. libr. women's med 2009
FPR (95% CI)
T21: n=733
Wts (%)
2.0
4.9
12.6
0.5
4.3
4.9
9.0
1.3
14.8
18.0
1.8
1.8
2.0
2.7
13.9
5.8
0.08 (0.03 - 0.17) 100
0
3
6
9 12
FPR % (95% CI) (%)
11,475 non-T21
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6/5/2014
Trisomy 21 performance cfDNA testing:
meta-analysis (Gil et al, Fetal Diagn Ther, 2014)
Author
Chiu et al., 2011 [86]
Ehrich et al., 2011 [39]
Palomaki et al., 2011 [212]
Sehnert et al., 2011 [13]
Ashoor et al., 2012 [50]
Bianchi et al., 2012 [89]
Jiang et al., 2012 [16]
Lau et al., 2012 [11]
Nicolaides et al., 2012 [8]
Norton et al., 2012 [81]
Sparks et al., 2012 [36]
Zimmerman et al., 2012 [11]
Guex et al., 2013 [30]
Nicolaides et al., 2013 [25]
Song et al., 2013 [8]
Verweij et al., 2013 [18]
DR (95% CI)
Wts (%)
100 (95.8 - 100)
11.6
100 (91.0 - 100)
5.3
98.6 (95.9 - 99.7)
28.4
100 (75.3 - 100)
1.9
100 (92.9 - 100)
6.8
100 (95.9 - 100)
12.0
100 (79.4 - 100)
2.3
100 (71.5 - 100)
1.6
100 (63.1 - 100)
1.2
100 (95.6 - 100)
11.0
100 (90.3 - 100)
4.9
100 (71.5 - 100)
1.6
100 (88.4 - 100)
4.1
100 (86.3 - 100)
3.5
100 (63.1 - 100)
1.2
94.4 (72.7 - 100)
2.5
2.0
4.9
12.6
0.5
4.3
4.9
9.0
1.3
14.8
18.0
1.8
1.8
2.0
2.7
13.9
5.8
Author
Chiu et al., 2011 [86]
Ehrich et al., 2011 [39]
Palomaki et al., 2011 [212]
Sehnert et al., 2011 [13]
Ashoor et al., 2012 [50]
Bianchi et al., 2012 [89]
Jiang et al., 2012 [16]
Lau et al., 2012 [11]
Nicolaides et al., 2012 [8]
Norton et al., 2012 [81]
Sparks et al., 2012 [36]
Zimmerman et al., 2012 [11]
Guex et al., 2013 [30]
Nicolaides et al., 2013 [25]
Song et al., 2013 [8]
Verweij et al., 2013 [18]
0.08 (0.03 - 0.17) 100
Pooled analysis
FPR (95% CI)
2.06 (0.4 - 5.9)
0.24 (0.0 - 1.4)
0.20 (0.0 - 0.6)
0.00 (0.0 - 10.3)
0.00 (0.0 - 1.1)
0.00 (0.0 - 0.9)
0.00 (0.0 - 0.4)
0.00 (0.0 - 3.7)
0.00 (0.0 - 0.2)
0.04 (0 - 0.2)
0.00 (0.0 - 2.8)
0.00 (0.0 - 2.7)
0.00 (0.0 - 2.5)
0.00 (0.0 - 1.8)
0.00 (0.0 - 0.2)
0.00 (0.0 - 0.7)
DR: 99.1% (98.3 - 99.6)
99.1 (98.3 - 99.6) 100
Pooled analysis
50 60 70 80 90100 DR % (95% CI) (%)
T21: n=733
0
3
6
Trisomy 21 performance cfDNA testing:
meta-analysis (Gil et al, Fetal Diagn Ther, 2014)
9 12
Wts (%)
FPR % (95% CI) (%)
11,475 non-T21
DR (95% CI)
Wts (%)
100 (95.8 - 100)
11.6
100 (91.0 - 100)
5.3
98.6 (95.9 - 99.7)
28.4
100 (75.3 - 100)
1.9
100 (92.9 - 100)
6.8
100 (95.9 - 100)
12.0
100 (79.4 - 100)
2.3
100 (71.5 - 100)
1.6
100 (63.1 - 100)
1.2
100 (95.6 - 100)
11.0
100 (90.3 - 100)
4.9
100 (71.5 - 100)
1.6
100 (88.4 - 100)
4.1
100 (86.3 - 100)
3.5
100 (63.1 - 100)
1.2
94.4 (72.7 - 100)
2.5
FPR (95% CI)
2.06 (0.4 - 5.9)
0.24 (0.0 - 1.4)
0.20 (0.0 - 0.6)
0.00 (0.0 - 10.3)
0.00 (0.0 - 1.1)
0.00 (0.0 - 0.9)
0.00 (0.0 - 0.4)
0.00 (0.0 - 3.7)
0.00 (0.0 - 0.2)
0.04 (0 - 0.2)
0.00 (0.0 - 2.8)
0.00 (0.0 - 2.7)
0.00 (0.0 - 2.5)
0.00 (0.0 - 1.8)
0.00 (0.0 - 0.2)
0.00 (0.0 - 0.7)
Wts (%)
2.0
4.9
12.6
0.5
4.3
4.9
9.0
1.3
14.8
18.0
1.8
1.8
2.0
2.7
13.9
5.8
DR: 99.1% (98.3 - 99.6) FPR: 0.08% (0.03 - 0.17)
0.08 (0.03 - 0.17) 100
99.1 (98.3 - 99.6) 100
50 60 70 80 90100 DR % (95% CI) (%)
T21: n=733
0
3
6
9 12
FPR % (95% CI) (%)
11,475 non-T21
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6/5/2014
Published Trials of NIPT:
failure rates
NIPT: Clinical Challenges
False positives:
• Unrecognized or vanishing twin
• Placental mosaicism
• Low level maternal mosaicism, esp sex chromosomal
• Maternal malignancy
False negatives:
• Genetic variants
• Placental mosaicism
Failed results:
• Increased BMI
• Failure to extract adequate material
• Individual variation in amount of cell free fetal DNA
• Fetal aneuploidy
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%)
All
241/6853 (3.5%)
Detection
False positive rate
86/86
39/39
209/212
89/89
81/81
11/11
424/427 (99.3%)
3/146
1/410
3/1471
0/404
1/2888
0/145
8/5319 (0.15%)
Fetal fraction of DNA and
test failure
Up to 5% of samples do not provide a result
o Low fraction fetal DNA, failed sequencing, high
variability in counts
o Some association with gestational age (<10 wks)
o Low fetal fraction associated with maternal BMI
• 20% at >250 lbs
• 50% at >350 lbs
Low fetal fraction appears to be associated with
aneuploidy
Repeating test will provide a result in some cases
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6/5/2014
Why is NIPT not diagnostic?
• Confined Placental Mosaicism
• THIS IS A PLACENTAL, AND NOT A FETAL TEST
False positive
NIPT: Trisomy 13 and Trisomy 18
Author
T13
DR
Palomaki ’11
11/12 (92%)
Bianchi ’12
11/14 (79%)
Norton ’12
---
Zimmermann
2/2 (100%)
Porreco ’14
14/16 (92%)
‘12
Bianchi ’14
TOTAL
1/1 (100%)
39/45 (87%)
T18
FPR
16/1688 (0.97%)
DR
FPR
59/59 (100%)
5/1688 (0.28%)
35/36 (99%)
0/460 (0%)
---
37/38 (97.4%)
2/2888(0.07%)
0/145
3/3 (100%)
0/488
0/3322
1/899 (0.1%)
17/6542 (.03%)
36/39 (92%)
2/2 (100%)
172/177 (97%)
0/145
0/3322
3/1905 (0.2%)
False negative
Sex Chromosomal Aneuploidy
Author
SamangoSprouse
Bianchi
Nicolaides
Total
Cases:
Controls
16:185
20:532
59:118
95:835
DR
FPR
No result
92%
0
7%
75%
88%
86%
0.2%
0.8%
0.6%
20%
2.7%
10%
10/10,408 (0.1%)
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6/5/2014
Professional Society Opinions: ACOG; ACMG;
International Society of Prenatal Diagnosis;
National Society of Genetic Counselors
Common themes:
There are recognized benefits, but…
• Not diagnostic
o Needs confirmation
o “Advanced screening test”
•
•
•
•
Only detects common trisomies (vs invasive testing)
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
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6/5/2014
How Does Test Performance
>99% sensitivity and
Differ with Risk? (Assume
99.8% specificity)
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
How Does Test Performance
Assume 99% sensitivity and
Differ with Risk? (T13:
99% specificity)
Low Risk (age 25; 1/8,000)
High Risk (age 38; 1/1000)
1 T13
10 T13
N=10,000
9,999 not T13
1 TP, 0 FN 100 FP, 9900 TN
OAPR = 1/100
N=10,000
9 TP, 1 FN
9,990 not T13
100 FP, 9890 TN
• N=1914 women undergoing standard screening
• Mean maternal age = 29.6 yrs
• Primary outcome = false positive rates for T18 and T21
OAPR = 9/100
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6/5/2014
cfDNA vs Standard
Screening
Bianchi et al, NEJM, 2014
cfDNA
Standard
FPR
0.3%
3.6%
PPV
45.5%
4.2%
Where does cfDNA fit?
Is this an outstanding screening test or an imperfect
diagnostic test?
p<.001
• Only 8 aneuploidy cases in the cohort (5: T21, 2: T18,
and 1: T13)
• All were detected
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 cfDNA?
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
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6/5/2014
NIPT is more precise for T18, 21
cfDNA
NIPT is more precise for T18, 21
Current NT + serum screen
NIPT is more precise for T18, 21
Other abnormalities
Other abnormalities
FTS
cfDNA
cfDNA
Current NT + serum screen
8/8 T21
2/3 T18; 1/3 no result
Nicolaides et al, 2012
8/8 T21
3/3 T18
7/7 others (45X; triploidy;
deletions and duplications)
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6/5/2014
NIPT is more precise for T18, 21
DS and T18 are 2/3 of aneuploidies
detectable by karyotype
Other abnormalities
FTS
cfDNA
55%
(10/18)
100%
(18/18)
8/8 T21
2/3 T18; 1/3 no result
Nicolaides et al, 2012
8/8 T21
3/3 T18
7/7 others (45X; triploidy;
deletions and duplications)
Aneuploidies Detected by Prenatal
Chromosome Abnormalities by
Maternal Age
Diagnostic Testing
100%
90%
Other
16.9%
Tri 21: 53.2%
Tri 13: 4.6%
Percent Detected
Sex chrom: 8.2%
NIPT Detectable
80%
70%
Yes
60%
50%
No
40%
30%
20%
P<0.01
10%
Tri 18: 17.0%
0%
< 25
Norton et al, SMFM, 2014
25 to 29 30 to 34 35 to 39 40 to 44
Age Group (Years)
≥ 45
Norton et al, SMFM, 2014
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6/5/2014
Disorders potentially detectable by
NIPT Detection Rate
• ~83% of chromosomal abnormalities
detected by current screening can
potentially be identified by NIPT
• This varies by maternal age
o Lower detection in younger women (75-80%)
o Greater detection in older women, but still only
90%
Disorder
Prevalence
Common trisomies
(13,18,21)
0.2%
Other chromosome
abnormalities
Microdeletions and
duplications
0.4%
1.5%
Mendelian Genetic
Disorders
Congenital heart
defects
0.4%
Total
~25%
0.3%
Other structural
3%
defects
Adverse OB outcomes 15-20%
serum screening and NIPT
Current Screening
NIPT
•
•
•
•
Trisomy 21
Trisomy 18
Trisomy 13
Some sex
chromosomes
•
•
•
•
•
•
•
•
•
•
•
•
•
•
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
Adverse OB outcomes (IUGR, PreE, PTB)
Causes of Birth
Defects and
Other Adverse
Perinatal
Outcomes:
It’s Not All Down
Syndrome
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6/5/2014
NIPT: Expanded panels
Laboratories have added other trisomies and
microdeletions
• Trisomies 16 and 22
• Microdeletion syndromes
o
o
o
o
o
What is a microdeletion?
• 1MB (megabase) = 1 million base pairs
• Microdeletions are 100kb to several MB
• Karyotype can usually only visually detect >7-10 MB
Outcome will depend on the size & the genes involved
22q (diGeorge)
5p (cri-du-chat)
1p36
15q (Prader Willi)
4p (Wolf-Hirshhorn)
Microdeletion syndromes
Syndrome
22q11.2
(DiGeorge)
1q36
Frequency Features
1/4K
1/5-10K
Angelman
1/12-20K
Prader-Willi
1/10-30K
Cri-du-chat
1/20-50K
WolfHirshhorn
1/50K
Varies: cardiac, palatal,
immune, intellectual disability
Severe intellectual disability
(ID), +/- obvious structural
anomalies
Severe ID, seizures, speech
delay
Obesity, ID, behavioral
problems
Microcephaly, ID, +/- CHD
ID, seizures, +/- CL/CP
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6/5/2014
Prevalence in 100,000 Live Births
NIPT for Rare Disorders
(Wolf-Hirschhorn, 4p-: Assume 99% sensitivity and 99.2% specificity)
Population Risk = 1/50,000
N=100,000
140
120
100
2 Wolf-Hirschhorn
80
60
99,998 not WHS
40
20
2 TP; 0 FN
0
800 FP; 99,198 TN
OAPR = 1/400
53
Chromosomal Microarray (CMA)
for Prenatal Diagnosis
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6/5/2014
Karyotype
Resolution:
>7-10 Million Base Pairs
(7-10 Mb)
Chromosomal
Microarray
Genomic imbalance detected
by CMA but not karyotype
Resolution:
< 0.5 Million Base Pairs
(< 500 kb)
Miller et al, 2010, AJHG
Diagnostic Yield in Cases with Normal
Karyotype
Indication for
Testing
U/S Anomaly
N=755
AMA
N=1,966
Positive Screen
N=729
Other
N=372
Clinically Relevant
(N=96)
6.0%
1.7%
1.7%
1.3%
In patients with fetal structural abnormalities undergoing
prenatal diagnosis, microarray is recommended.
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6/5/2014
New “menu” in prenatal
testing
NIPT and chromosomal
microarray
IF:
Screening test for common aneuploidies
(1/500)
VS
Invasive diagnostic testing with CMA (1/60)
CMA 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 only
about 12% of diagnosable chromosomal abnormalities
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6/5/2014
Cost sensitivity analysis of NIPT
• Calculated marginal costs of Down syndrome detection
compared to current screening
• If NIPT costs $1000, increased cost per case detected is $3.6
million more than first trimester combined screening
Universal NIPT screening will only become cost effective if
costs drop substantially
Contingent screening of highest risk 10-20% is recommended
Cuckle et al, Prenat Diag 2013
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6/5/2014
Summary
• cfDNA is a better test for Down syndrome than
current screening
o For patients that obtain a result, a positive or negative test is
near diagnostic
o Test failure indicates an increased risk for aneuploidy
• These patients require counseling and follow up
If only it were this
simple…
• The detected disorders are fewer than with
traditional screening or diagnostic testing
• Patients need to be carefully counseled about
the trade-offs of lower false positives but fewer
disorders tested
Thank You!
18