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Cell Free DNA Screening
Mary E. Norton MDProfessor; Obstetrics, Gynecology, and Reproductive SciencesUniversity of California, San Francisco
61st Annual OBGYN UpdatePark City UTFebruary 2020
Disclosures
o Research support: Naterao Consultant, Scientific Advisory Board: Invitae
Prenatal testing is increasingly complex…
The Prenatal Testing Paradigm
Down syndrome
No Yes
Terminate pregnancy
No Yes
0
20
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60
80
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120
Detection rate of prenatal screening for Down syndrome has improved over time
Det
ectio
n R
ate
(%)
Why all the focus on Down syndrome?
Lejeune, 1959
1979: NICHD Consensus Panel on Amniocentesis
1979 Cost Benefit Analysis
Amniocentesis Care for child with Down syndrome
Everyone would want it
Everyone would terminate
The first prenatal screening test:
How old are you?
Traditional Serum Screening
10-14 weeks 15-20 weeks
1st trimester biochemistry 2nd trimester biochemistry
Nuchal translucency
Down Syndrome: 93% detection, 4.5% screen positive rate
Detection Rates: Sequential Screening
Chromosomalabnormality
# of cases screened
Detection rate
Trisomy 21 1276 93%Trisomy 18 336 93%Trisomy 13 143 80% 45,X 161 80% 47,XXY 36 53%Other SCA 59 63%
All common aneuploidies 2,015 89%
Norton et al, 2015
1997
Noninvasive Prenatal Testing (NIPT) for aneuploidy using cell free DNA (cfDNA)
o Detection requires accurate quantification of DNA from a specific chromosome
o Somewhat different methods are utilized by different laboratories
Analysis of cell free DNA
DR: 99.2% (98.5 - 99.6) FPR: 0.09% (0.05 - 0.14)False Positive Rate: 1/2500
cfDNA screening for T21: meta-analysis(Gil et al, Ultrasound Obstet Gynecol, 2017)
Detection Rate: 99.7%
Other aneuploidies
Trisomy Detection Rate False Positive Rate
Trisomy 21 99% 0.1%Trisomy 18 97% 0.3%Trisomy 13 87% 0.6%Sex chromosomes 86% 0.6%Total 1.6%
The performance of NIPT for other aneuploidies is not as good as for trisomy 21
Gil et al, 2017
Ultrasound in Obstetrics & Gynecology; 2016
First trimester screening
cfDNA
amnio
CVS
Amniocentesis loss rate now estimated at 0.11% (1/900)
Yearly volume of prenatal diagnostic procedures
Down syndrome is uncommon in young women
Increasing maternal age
35 yo
o 28% decrease in referrals to genetic counseling for families affected by single-gene disorders because some practitioners mistakenly think that cfDNA screening tests for all genetic conditions.
Williams J, AJOG 2015
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=0.008False positive rate
0.06% 5.4% P<0.0001
Positive predictivevalue
81% 3.4% P<0.0001
Norton et al, NEJM, 2015
Risk Group Positive predictive valueEntire cohort (mean age 30.7 yrs
81%
Maternal age <35 yo 76%Low risk serum FTS (<1/270) 50%
Wang et al, 2014
Wang et al, Genetics in Medicine, 2014
Aneuploidy Number of positives
Number (%)confirmed
T21 41 38/41 (93%)T18 25 16/25 (64%)T13 16 7/16 (44%)45,X 16 6/16 (38%)Total 98 67 (67%)
High Risk, Low Risk, and Positive Predictive Value
The poorly understood PPV
o 6.2% terminated the pregnancy without karyotype confirmation
o N=116 abnormal cfDNA resultso 19.6% terminated without confirmationo Disconcerting if only 50% are true positives
2016
PPV Calculator: www.perinatalquality.org
“How did this happen? I had the non-invasive amnio…”
Spectrum of Congenital Disease
Structural Malformations
Autosomal recessive
Autosomal dominant
X-linked
Chromosomal/karyotype
Conditions found by microarray
• 1/300 pregnancies
• 20% of infant deaths
Half of these are Down syndrome
Copy numbervariants
Rate of abnormalities by maternal age
Increasing maternal age
Microdeletions are genomic imbalances detected by microarray but not karyotype
Miller et al, 2010, AJHG
Wolf Hirschhorn syndrome:Learning disabilities, heart defects, CL/CP
Prader-Willi:Hypotonia, significant learning disabilities, obesity
Angelmansyndrome:Severe intellectual disability and speech impairment
Some Microdeletion Syndromes
Ch 4
Ch 15
Ch 5Cri du chat syndrome:Poor feeding and growth, microcephaly, severe learning difficulties
Chromosomal Microarray (CMA) for Prenatal Diagnosis
Diagnostic Yield of Chromosomal Microarray in Cases with Normal Karyotype
Indication for Testing Clinically Relevant (N=96)
U/S AnomalyN=755
6.0%
Maternal age>35yoN=1,966
1.7%
Positive ScreenN=729
1.7%
OtherN=372
1.3%
Cell free DNA: Expanded panels
Laboratories have added other trisomies and microdeletionso Trisomies 9, 16, and 22o Microdeletion syndromes
• 22q (diGeorge)• 5p (cri-du-chat)• 1p36• 15q (Prader Willi)• 4p (Wolf-Hirshhorn)
Microdeletion syndromes on cfDNA panels
Syndrome 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
Total 1/2500
MaterniTGenome: “Non-invasive Genome”
Screen positive rate: 5.4%December, 2017
Diagnostic confirmation of cfDNAfor microdeletions
Abnormality Total # of cases # confirmed Positive Predictive Value
1p 21 1 4.8%
4p 6 1 16.7%
5p 45 6 13.3%
15q 80 5 6.3%
22q 183 12 6.6%
Total 335 25 7.4%
Schwartz et al, Prenatal Diagn, 2018
Cell free DNA screening: Biologic ChallengesFalse positives:o Unrecognized or “vanishing” twino Placental mosaicismo Maternal genetic variationo Maternal malignancyFalse negatives:o Inadequate fetal DNA o Placental mosaicismo Maternal genetic variationFailed results: o Increased BMIo Inadequate fetal DNAo Fetal aneuploidy
Maternal genetic variants and cfDNA
Snyder et al, NEJM, 2015
Traditional screening Trisomy 18, 21, +/-13 Other chromosomal Fetal anomalies, esp cardiac
(NT) Spina bifida and ventral wall
defects (MSAFP) Adverse obstetric outcomes Preeclampsia, preterm birth,
fetal growth restriction
cfDNA screening Trisomy 13, 18, 21 Sex chromosomes Maternal genetic
disorders Maternal malignancy Maternal
chromosomal abnormality
NEXT trial: 15,841 women had cfDNA and first trimester screeningo 488 (3%) had no result
• Low fetal fraction, failed sequencing, high variance in sequencing
Fraction of cell free DNA that is fetal in origin: “Fetal Fraction” varies
Fetal fraction and maternal weight
Hudecova I et al, PLoS One, 2014
Obesity in US Adults
The less fetal DNA, the harder to tell normal from abnormal
Fetal Fraction
Fetal Fraction
Fetal Fraction
NEXT trial: 15,841 women had cfDNA and first trimester screeningo 488 (3%) women had no result
• Low fetal fraction, failed sequencing, high variance in sequencingRisk of aneuploidy was 1/38 (2.7%)Much higher than 1/236 (0.4%) in cohort
Norton et al, NEJM, 2015
42 yo G2P0 at 11 weeks
o Cell free x 2 with no result
o CVS at 13 weeks• mosaic trisomy 18
o Amniocentesis: trisomy 18
Non-Invasive Single Gene Tests
o Maternal and fetal cell free DNA are not easily distinguished
o However, can identify de novo or paternal gene mutation
De Novo Variants
• Relatively common• Associated with
increased paternal age
• Can be identified with cfDNA
• De novo disorders are associated with advanced paternal age
• Who should be offered this test?
37 yo patient at 27+ wks’ gestation
Shortened long bones by ultrasound
o Most often normal o Could be
achondroplasia
o N=47 caseso Correct in 46 (96.2%)o Useful tool in 3rd trimester to distinguish FGR
from achondroplasia
Chitty LS, et al. Prenat Diagn, 2015
Whole Genome and Exome Sequencing
o Whole Genome Sequencing• Obtaining the complete sequence of all 3 billion
base pairs of DNA in any individual
o Exome Sequencing• Obtaining the complete sequence of the ~2% of
the genome containing the exons that encode proteins
The future: Whole genome sequencing
Conclusions
o Patients need to understand the benefits and limitations of test options• The difference between screening and diagnostic
tests• What the tests can and cannot detect• The potential for unexpected findings• What can be done with the informationAll of this is changing very rapidly
What is the future?
Thank you!