Cell free DNA:

Low Risk, High Risk, Obesity,

Microdeletions, and Other Applications

Nancy C. Rose, MDProfessor, Obstetrics and Gynecology

University of UtahDirector, Reproductive Genetics

Intermountain HealthcareNancy.Rose@hsc.utah.eduFebruary, 2017

Conflict of Interest Disclosure:

2015: Lab services for an IRB-approved research project were provided by Progenity, Inc.

“Cell free DNA, the Inflammatory Cascade, and the Initiation of Term Labor”.

Aneuploidy Screening:What is Aneuploidy?

• Due to meiotic non-disjunction• Each chromosome: hundreds of genes

– Gain or loss disruptive• Most often results in a non viable

pregnancy–Miscarriage or stillbirth

• Not related to race or ethnicity– Incidence: 1:150 live births

How to Counsel Your Patients:What are the Risks for

Fetal Down Syndrome at Term?*

• Age 20 1:1500• Age 30 1:1000• Age 35 1:365• Age 40 1:100• Age 45 1:30

* one third of affected pregnancies miscarry between midtrimester and term

Down Syndrome

• Link to Alzheimer’s Disease.• 75% of adults affectedby age 60

•Median age of death: 59 years

(in industrialized countries)

Down Syndrome: Potential Phenotype• Congenital heart disease (50%)• Bowel obstruction (20%)• Hearing loss (70%)

(conductive, sensorineural, or mixed)• Ophthalmologic disorders (80%)

(refractive, strabismus, nystagmus)• Psychiatric disorders (18%)• Thyroid dysfunction (10%)• Seizure disorder (8%)• Leukemia (1%)

ACOG Practice Bulletin 163:Screening for Aneuploidy (2016)

• All women, regardless of maternal age, should be offered aneuploidy screening.

• All women, regardless of maternal age, should be offered invasive diagnostic testing.– First published in 2007

• (ACOG PB #77)!

Lots of Ways to Screen for Aneuploidy

• Maternal age X• Ultrasound X• Single tests:

– First trimester and Quad • Nuchal translucency screening

– Isolated use for multiples only!• Combined first and second trimester tests:

– Integrated/sequential screening

• cfDNA

Cell-free- What?• cfDNA: Cell Free DNA• cffDNA: Cell free fetal DNA • NIPT: Noninvasive Prenatal Testing • NIPS: Noninvasive Prenatal Screening • Commercial Laboratories:

– Progenity-Innatal– LabCorp - InformaSeq– Counsyl-Informed Pregnancy Screen– Natera - Panorama– Ariosa - Harmony

Cell Free DNA • Short, 200bp DNA fragments in maternal

plasma.• Its source is both “fetal” and maternal

• The fetal component is placental• 3-13% of total cfDNA

• Can sample from 10 weeks until term• Of the three trisomies, the accuracy

is best for Down syndrome.• No risk for miscarriage• Undetectable at about 2 hours postpartum

cfDNA: Problems with Research, Development, and Implementation

• Rapid introduction into clinical practice– Described by Lo in 2008– Introduced into clinical practice 2011

• Industry funded• Direct public marketing• Competition and litigation, not

collaboration, between laboratories– CAPS project: January, 2017

Three Main Lab Techniques forcfDNA Screening

• Massively parallel shotgun sequencing• Selective sequencing of chromosomes

of interest• SNP analysis

Current Clinical Applications for the Prenatal use of cfDNA

• Aneuploidy: 13, 18, 21, X and Y– Screen-positive serum screen

• Sex determination for X-linked disorders – Example: Hemophilia

• RhD status– Alloimmunized Rh-negative mother

• Paternally inherited AD genetic disorder with a known mutation:– Example: Neurofibromatosis

ACOG Committee Opinionon cfDNA Screening

• cfDNA is a screening test– A screen positive test should be

confirmed– No irreversible actions without

diagnostic testing– Do not send analyte screening

with cfDNA screening

ACOG Committee Opinion 640, June 2015

cfDNA Aneuploidy Current Screening Options

AUTOSOMAL TRISOMIES• Trisomy 21 (Down syndrome)• Trisomy 18• Trisomy 13

SEX CHROMOSOME ANEUPLOIDIES

• 45,X• 47,XXY• 47,XYY• 47,XXX

MICRODELETIONS• DiGeorge (22q deletion)• 1p36 deletion syndrome• Angelman/Prader-Willi• Cri-du-Chat syndrome• Jacobsen syndrome• Langer-Giedion syndrome

OTHER AUTOSOMAL TRISOMIES• 9, 16, 22

cfDNA screening and the Use of Ultrasound

• Confirms gestational dating, viability,empty sac, and singleton gestation

• Nuchal measurement not essential• If fetal abnomalitiesare seen, cfDNAis not the right test.

What To Do with a Screen Positive cfDNA Result?

• Offer Diagnostic Testing– Confirmation of screening test– Confirm type of aneuploidy:

• Trisomy vs. Translocation– Recurrence risk issues

• Don’t repeat the test!

What Are Some Reasons for a False Positive Test Result?

• For Autosomal Aneuploidies:– Screening variability– Twin demise– Vanishing twin– Placental mosaicism– Maternal malignancies

• For Sex chromosome aneuploidies:– Could be the mother

Need Diagnostic Confirmation!!!• Study of cfDNA screening over

six months:– 356 high risk results

• 184 true positives (51.7%)• 38 false positives (10.8%)• 22 (6.2%) pregnancies were

terminated without confirmatory testing

Dar et al. Am J Obstet Gynecol, 2014

ACOG Committee Opinionon cfDNA Screening

• Can be used in all women– Provides less information in

younger women than conventional screening

• Should not be used:– In multiple gestations– To screen for microdeletions

ACOG Committee Opinion 640, June 2015

Why Does a cfDNA Screen Fail?

• Sent too early• Maternal obesity• Aneuploidy positive

Low Fetal Fraction!

Significance of the Fetal Fraction

• Fetal fraction =

• The higher the fetal fraction, the easier to obtain a result

• If low fetal fraction, results are not obtained

• If no result, 20% risk for fetal aneuploidy

FETAL DNAMATERNAL + FETAL DNA

What To Do if The Test Fails to Obtain a Result?

• Review the patient’s weight• Review the patient’s

gestational dating• Consider timing:

– May repeat cfDNA screening– May offer diagnostic testing– Not a candidate for serum

screening now

Obesity and Genetic Screening

• Defined as: BMI ≥ 30• Higher risks for poor outcomes

• stillbirths– Diminished ability to be adequately

screened by all methods. • Higher risk neural tube defects

– No change in aneuploidy risk

What about multiple gestations and screening?

• There is no effective method to screen multiple gestations. – Analyte and cfDNA: one test result.– Limited data– Not very helpful

• Nuchal translucency screening at least reflects the individual fetus.

ACOG PB #163.

cfDNA and the Screening of Younger Women

• Additional serum analyteinformation is not obtained

• Younger women have a lower positive predictive value– (the chance that a positive

test result is a true positive).

Why PPV Matters!

ACOG Committee Opinion 640, June 2015

How to Understand PPV• PPV calculators:

– Easiest one: mombaby.org/nips. • This is the free UNC calculator.

–Easy, one click -slide for info.– SMFM PQF calculator:

https://www.perinatalquality.org/Vendors/NSGC/NIPT/• Thorough but much more

complex and less easy to use.• Also free.

PPV age 40

PPV age 20

What About Testing for Microdeletions with cfDNA Screening?

• MICRODELETIONS:– Submicroscopic deletion leading to

monosomy of a small chromosomal segment too small to be detected by cytogenetic testing• Variable phenotype• Not related to maternal age• Ultrasound findings may not be

present

CFDNA: Microdeletion Panels• 22q11 deletion syndrome • 5p- (Cri-du-chat syndrome) • 15q (Prader-Willi/Angelman

syndromes)• 1p36 deletion syndrome• 4p (Wolf-Hirschhorn syndrome) • 8q (Langer-Giedion syndrome) • 11q (Jacobsen syndrome)

Microdeletions: 22q11

• Most common:– 22q11 deletion syndrome

• 1 in 2,000 - 4,000 live births• Wide range of phenotypes• Associated with heart defects,

facial clefts, learning disabilities• About 10% are inherited

• 90% de novo

Microdeletions: How Are They Being Offered?

• Opt in• Opt out• Cost more• Same price• Different panels of deletions• Some labs not offering at all

Some National Guidelines

• ACOG/SMFM:“Routine cell-free DNA screening for microdeletion syndromes should not be performed.”

• ESHG/ASHG: “…not currently recommended.”

Obstet Gynecol June, 2015EJHG March 15, 2015

Too much genomic information: what is different

about prenatal patients?• We are not usually testing an

individual with a known phenotype as we would with pediatric assessments– Genotype/phenotype issues….– Psychosocial issues– Management issues and costs

Same HPE gene, Somewhat Different Phenotype

Unintended Information from cfDNA

• The test cannot distinguish a maternal from a fetal result!– 45, X results can reflect older

mothers, not fetuses– The microdeletion syndrome you

identify may be maternal– Beware the patient with an organ

transplant– At least 26 cases of maternal

cancer diagnosis

Case Report

• 37 Year old G2P1– cfDNA Screening Result:

47, XX+13 (x 2)– Anatomic survey: normal– Amniocentesis result: 46, XY

• Normal male delivered, 3040 grams• Post partum hip pain:

– Small cell carcinoma, vaginal origin

Osborne, Prenat Diagn, 2013

Presymptomatic Identification of Cancers in Pregnant Women

Using Noninvasive Prenatal Testing• From a series of 4000 cfDNA tests, 3

women were referred for whole-body MRI

• In all 3 cases, a cancer was detected– Ovarian carcinoma– Follicular lymphoma– Hodgkin lymphoma

JAMA Oncol. doi:10.1001/jamaoncol.2015.1883Published online June 5, 2015

Limitations of cfDNA Screening

• Limited to Trisomy 21, 18, 13, and sex chromosome aneuploidies– No detection of open fetal

defects• Send MSAFP and/or obtain

detailed anatomic survey• Cannot differentiate between

placental mosaicism, unbalanced translocation, or full trisomy

Other Important Factors• Cost:

– Depends on insurance contracts– Medicaid contracts– Many patients have a high

out-of-pocket deductible• Time of year matters

– Often, analyte screening is more cost-effective• (and is still a good screening

test!)

Summary

• cfDNA is the best available screening test for the common aneuploidies (trisomies 21, 13, and 18) in high-risk women

• A positive screen result needs confirmatory testing

• A screen-negative test is generally reassuring for these limited disorders but is nondiagnostic

Why is Screening for Aneuploidy Valuable?

• A way to gain more information about the pregnancy– Reassurance– Planning– Decision-making– Education/preparation

• May affect antepartum care and delivery management

Pre- and Post-TestCounseling Guidance

• ACOG:– www.acog.org/More-Info/cfDNA

• NSGC:– http://nsgc.org/page/non-invasive-

prenatal-testing-healthcare-providers– http://nsgc.org/page/abnormal-non-

invasive-prenatal-testing-results

Questions?....