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

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80

100

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!