S H O B A N A K U B E N D R A N , M B B S , M S , C G C

G E N E T I C C O U N S E L O R

A S S T . P R O F E S S O R

D E P T O F P E D I A T R I C S

K U S M W I C H I T A

M A R C H 3 0 , 2 0 1 6

Prenatal Cell Free DNA Screening

Nothing to Disclose!

Not affiliated with any genetic testing laboratory

Acknowledgement

Slides from

NSGC

ACOG

SMFM

Images of technology from laboratory websites

Objectives

Compare cfDNA screening technologies versus conventional screening

Performance and limitations of cfDNA for Common aneuploidy

Microdeletion syndrome

Whole genome

Pre and post test counseling for cfDNA

Educational resources for providers and patients

Terminology

Prenatal cell-free DNA (cfDNA) Screening

Non-invasive Prenatal Screening (NIPS)

Non-invasive Prenatal Testing (NIPT)

FISH – fluorescent insitu hybridization

SNP – single nucleotide polymorphism

Sequencing – reading nucleotide sequence

Where does cell free fetal DNA originate from

1. 2. 3.

18%14%

68%1. Fetus

2. Trophoblast

3. Mesenchyme

In the past 6 months which is your preferred method of prenatal aneuploidy screening for low risk

1. 2. 3. 4.

95%

0%0%5%

1. Quad screen

2. Integrated screen

3. Other analyte screen

4. Cell free fetal DNA

In the past 6 months which is your preferred method of prenatal aneuploidy screening for high risk

1. 2. 3. 4.

6%

67%

0%

28%

1. Quad screen

2. Integrated screen

3. Other analyte screen

4. Cell free fetal DNA

When you order NIPT have you included screening for microdeletion syndromes?

1. 2. 3.

18%

47%

35%

1. Yes

2. No

3. I have not ordered NIPT

I am knowledgeable about microdeletion conditions on the NIPT panel and can explain the pros and cons of screening for those conditions to a patient

1. 2. 3.

0%

13%

87%1. Yes

2. No

3. Somewhat knowledgeable

27 year old has fetal DNA test and it detects T21. The test is 99% sensitive and specific. She is 16 weeks GA and no apparent anomalies on ultrasound. The chance for her to have a

child with Down syndrome is

1. 2. 3. 4.

12%

47%

29%

12%

1. 25%

2. 50%

3. 75%

4. 99%

How is cfDNA screening different than conventional screening?

Uses a DNA-based technology

Can be performed anytime after 9-10 weeks gestation

Differences in conditions screened for

Has higher sensitivity/specificity

Implications of failed screening

Results reporting

Fetal DNA – originates from trophoblast cells of placenta

Opstal et alFalse Negative NIPT Results: Risk Figures for Chromosomes 13, 18 and 21 Based on Chorionic Villi Results in 5967 Cases and Literature Review

Circulating cell free DNA (cfDNA)

Originates from cells of the trophoblast (placenta)

Once in maternal serum, cells lyse and the chromosomes degrade into small DNA fragments

3-13% of total cell free DNA in maternal serum

Reliably detected >9-10 weeks gestation

Clears from maternal system after birth

Diagrams by Ariosa Diagnostics- http://www.ariosadx.com

http://www.edgc.com/wp-content/uploads/2013/10/be_img1.jpg

“Counting” Method

Genotyping Method

Natera/Panorama/Quest

NIPT Methods and Laboratories

Counting Genotyping

Shotgun MPSS Targeted MPSS SNP

Sequenom (MaterniT21) Ariosa (Harmony) Labcorp (Informaseq)

Natera (Panorama)

Illumina (Verifi) Progenity

Smith et al 2013 Noninvasive screening tools for Down syndrome: a review

Smith et al 2013 Noninvasive screening tools for Down syndrome: a review

Performance Comparison

All tests have a high sensitivity and specificity for trisomy 18 and 21 regardless of which molecular technique is used

NIPT Methods and Laboratories

Counting Genotyping

Shotgun MPSS Targeted MPSS SNP

Sequenom (MaterniT21) (multifetal gestation, egg donor, IVF). Fetal fraction reported

Illumina platform: Ariosa (Harmony); Labcorp (Informaseq) (twin)

Natera (Panorama) (cannot use for multifetal gestations, donor egg). Detects triploidy, vanishing twin

Illumina platform:(Verifi) Progenity (twin) Fetal fraction not reported

Comparison of prenatal screening and diagnostic test options

Test Detection rate for DS

(%)

Detection rate for all

aneuploidies (%)

Screen positive rate (%)

First trimester screen 80 69 5

Sequential/Integrated screen

93 82 5

Cell-free DNA screen 99 72 1-9

(includes no call/test failures)

Chorionic Villus Sampling

>99 >99 1 (includes mosaicism)

Amniocentesis >99 >99 0.2 (includes mosaicism)

SMFM. Prenatal aneuploidy screening with cfDNA. Am J Obstet Gynecol 2015.

Screening for Fetal Aneuploidy

No one screening test is superior to other screening tests in all test characteristics

A discussion of the risks, benefits, and alternatives of various

methods of prenatal screening and diagnostic testing should occur with all patients.

Parallel or simultaneous testing with multiple screening methodologies for aneuploidy is not cost-effective and should not be performed.

Patients may decline all screening or diagnostic testing for

aneuploidy.

How is cfDNA screening different than conventional screening?

Uses a DNA-based technology

Can be performed anytime after 9-10 weeks gestation

Differences in conditions screened for Microdeletions

Whole genome

Has higher sensitivity/specificity particularly for T21

Implications of failed screening

Results reporting

What can cfDNA screen for?

All labs Trisomy 21 (Down syndrome)

Trisomy 18 (Edwards syndrome)

Trisomy 13 (Patau syndrome)

What can cfDNA screen for?

X & Y chromosome variation in singleton pregnancies

Monosomy X (Turner syndrome)

XXY (Klinefelter syndrome)

XXX

XYY

What can cfDNA screen for?

Some labs offer screening for: Triploidy

Trisomy 16

Trisomy 22

Trisomy 9

Specific microdeletion syndromes

22q, 1p36, 4p, 5p, 8q, 11q, 15q

Microdeletion Conditions

Syndrome Incidence Features

22q11.2 1 in 2000 Mild to moderate ID, heart defect, hypocalcemia, immune problems,

Prader Willi 1 in 10,000 Mild to severe ID, behavior problems, hypotonia, obesity

Angelman syndrome 1 in 12,000 Severe ID, no speech, seizures

Cri du chat 1 in 20,000 Moderate to severe ID, behavior problems

1p36 1 in 5000 Severe ID, hearing loss, seizures,

2015 ACOG/SMFM Committee Opinion: Cell-free DNA screening for fetal aneuploidy

Rare conditions Low Positive Predictive Value

Routine cfDNA screening for microdeletion syndromes should not be performed.

cfDNA screening is not recommended for women with multiple gestations.

How is cfDNA screening different than conventional screening?

Uses a DNA-based technology

Can be performed anytime after 9-10 weeks gestation

Differences in conditions screened for Microdeletions

Whole genome

Has higher sensitivity/specificity particularly for T21

Implications of failed screening

Results reporting

How accurate is cfDNA screening?

Gil et al., Ultrasound in Obstetrics & Gynecology 2015; 45: 249-266 * Data from high risk pregnancies. In many studies, cases with mosaicism, complex karyotypes and maternal samples with low fetal fraction were excluded.

Accuracy

Accuracy describes the proportion of all tests that are correctly called. For rare conditions, as is often the case for conditions screened with NIPT/cfDNA, the majority of individuals screened will be correctly called as “negative” and therefore NIPT/cfDNA is described as “highly accurate”. However, the chance that any positive result is a true positive result depends on the Positive Predictive Value (PPV) of the test (see above). The table below illustrates components of accuracy:

http://www.perinatalquality.org/Vendors/NSGC/NIPT/

Sensitivity and Specificity

Sensitivity refers to the detection rate - the proportion of all individuals with a condition who are correctly identified as “positive” by a screening test. If 1,000 tested individuals have a given condition and 990 of them

test “positive“ for that condition, the test’s sensitivity is 99%. Sensitivity = Detection Rate = 990/1,000 = 99%

Specificity refers to the proportion of all individuals without a condition who are correctly identified as “negative” by a screening test. If 99,000 out of 100,000 unaffected individuals do not have a

condition and 98,000 test “negative”, the test’s specificity is 99%. Specificity = 98,000/99,000 = 99%

http://www.perinatalquality.org/Vendors/NSGC/NIPT/

PPV and NPV

The Positive Predictive Value (PPV) represents the proportion of positive test results that are truly positive. It answers the question: “If my test is positive, what is the chance my baby is affected?” For example, a PPV of 50% indicates that half of the cases in a given population

who have a positive test result are predicted to actually be affected with the condition. Although test sensitivity and specificity are expected to remain the same across a broad population, the PPV of a test varies based on the prevalence of the condition in a given population. The rarer the condition in a given population, the lower the PPV when sensitivity and specificity remain unchanged.

The Negative Predictive Value (NPV) represents the proportion of

negative test results that are truly negative. It answers the question: “If my test is negative, what is the chance that my baby is unaffected?” For example, if the NPV is 99% in a given population, then approximately 99%

of individuals who have a negative test result in that population would be expected to have an unaffected pregnancy. In this example, 1% of women receiving a negative result will have an affected pregnancy (false negative result).

False Positive and False Negative

The False Positive Rate (FPR) reflects the percentage of unaffected cases which test positive. If 1,000 of 99,000 unaffected individuals have a positive test result, the false positive rate is 1,000/99,000 = .01 or 1%.

The False Negative Rate (FNR) reflects the percentage of affected cases which test negative. If 100 of 1,000 affected individuals have a negative test result, the false negative rate in is 100/1,000 = .01 or 1%.

http://www.perinatalquality.org/Vendors/NSGC/NIPT/

Positive and Negative Predictive Value (PPV and NPV)

PPV: the likelihood that a positive test is a true positive

NPV: the likelihood that a negative test is a true negative

Depends on:

the prevalence of the condition

a priori risk age, family history, maternal serum screen results, ultrasound findings, etc.

As the prevalence of disease decreases, so will the PPV

Noninvasive prenatal testing in the general obstetric population: clinical performance and counseling considerations in over 85 000 cases

Prenatal Diagnosis Volume 36, Issue 3, pages 237-243, 27 JAN 2016 DOI: 10.1002/pd.4766 http://onlinelibrary.wiley.com/doi/10.1002/pd.4766/full#pd4766-fig-0003

Positive Predictive Value of cfDNA screening

Need Help Calculating PPV?

http://nsgc.org/page/nipt-calculator http://www.perinatalquality.org/Vendors/NSGC/NIPT/

Calculate PPV

https://www.perinatalquality.org/Vendors/NSGC/NIPT/

How is cfDNA screening different than conventional screening?

Uses a DNA-based technology

Can be performed anytime after 9-10 weeks gestation

Differences in conditions screened for Microdeletions

Whole genome

Has higher sensitivity/specificity particularly for T21

Implications of failed screening

Results reporting

“No call” results (screening failure)

Not reported, indeterminate, or uninterpretable results Occurs in 1-8% of patients 50-60% of repeat screens will provide a result Repeat screening may delay diagnosis of aneuploidy

Can be due to: Low fetal fraction Maternal conditions Aneuploidy

Call the performing laboratory for more information

Fetal fraction (the amount of cfDNA coming from the pregnancy)

Lower earlier in gestation

Lower in obese women

Lower in placentas with certain aneuploidies Particularly trisomy 18 and triploidy

Can lead to false negatives

2015 ACOG/SMFM Committee Opinion: Cell-free DNA screening for fetal aneuploidy

Women with a “no call” test result should receive further genetic counseling and be offered comprehensive ultrasound evaluation and diagnostic testing because of an increased risk of aneuploidy.

Patients should be counseled that a negative cfDNA test result does not ensure an unaffected pregnancy.

How is cfDNA screening different than conventional screening?

Uses a DNA-based technology

Can be performed anytime after 9-10 weeks gestation

Differences in conditions screened for

Has higher sensitivity/specificity

Implications of failed screening

Results reporting

Results reporting

Normal

Negative

Low risk (may give specific probability)

No aneuploidy detected

Results reporting

Results reporting

“No call”

No results available

Inconclusive

Unable to interpret

How is cfDNA screening similar to conventional screening?

1. Still a screening test Just as “non-invasive” as other screening tests

Diagnostic testing and genetic counseling is recommended after a positive cfDNA screen

The decision to terminate the pregnancy should not be made based on cfDNA results alone

6% of women with an abnormal NIPT result elected to terminate pregnancy without diagnostic testing

Case Example #1

35yo G2P1 woman with cfDNA test at 10 weeks showing increased risk for trisomy 13

Ultrasound at 12 weeks shows no abnormalities

Patient elects CVS

Case Example #1

Aneuploidy FISH: Mosaic for trisomy 13

Final karyotype: 46, XX

Confined placental mosaicism (CPM) Occurs in ~1% of CVS

Aneuploid --------------------Euploid---------------------

Placenta Fetus

Nondisjunction

Placenta Fetus

How is cfDNA screening similar to conventional screening?

1. Still a screening test

2. False positives and false negatives occur Residual risk for aneuploidy exists after negative result

2015 ACOG/SMFM Committee Opinion: Cell-free DNA screening for fetal aneuploidy

Conventional screening methods remain the most appropriate choice for first-line screening for most women in the general obstetric population due to Limitations of cfDNA screening performance

Limited data on cost-effectiveness in low-risk obstetric population

How is cfDNA screening similar to conventional screening?

Still a screening test

False positives and false negatives occur

Does not screen for all chromosome abnormalities, genetic conditions or birth defects

What doesn’t cfDNA screen for?

All known chromosome abnormalities 50-75% of aneuploidy in high risk population is Down syndrome, trisomy

18, or trisomy 13

Cannot differentiate between non-disjunction trisomy, translocation, mosaic

2% of pregnancies with an abnormal traditional screen and a normal cfDNA screen are found to have a chromosomal abnormality

All genetic conditions Single gene disorders, such as cystic fibrosis or achondroplasia, cannot be

identified

Family history should be evaluated to determine if patient should be offered other testing options for a particular disorder

What doesn’t cfDNA screen for?

Birth defects Such as neural tube defects, congenital heart defects, etc. cannot be

identified

Ultrasound and MSAFP-only should still be considered

If a fetal structural anomaly is identified on ultrasound examination, diagnostic testing should be offered rather than cfDNA screening

Case Example #2

Positive quad for trisomy 18 @ 17 weeks Low uE3

Low risk cfDNA result

Multiple congenital anomalies noted @ 20 week ultrasound

Case Example #2

Patient undergoes amniocentesis 46,XY

Normal chromosome microarray

Amniotic fluid sent for additional testing confirms diagnosis of Smith-Lemli-Opitz Syndrome (SLOS)

Take home message: cfDNA cannot screen for everything-

If a fetal structural anomaly is identified on ultrasound, diagnostic testing should be offered rather than cell-free

DNA screening 2015 ACOG/SMFM Committee Opinion: Cell-free DNA screening for fetal aneuploidy

Discordant NIPT Case

20 year old – NIPT – trisomy 13. 18 week ultrasound - no anomalies on fetus

20 week amnio – Turner syndrome

Coarctation aorta detected at birth

31 year old – NIPT – triple X 20 week amnio – fetus is normal female

31 year old karyotype – triple X!

2 cases of screen positive for 22q11 deletion One confirmed

Second negative

How do you implement cfDNA screening into your practice?

How do you implement cfDNA screening into your practice?

Pre-screen counseling for all patients is imperative

Can be challenging due to Time constraints

Rapid advances

Patients’ focus on fetal sex

Counseling patients

Talk to your patients: If their pregnancy was affected with a genetic disorder or

chromosome abnormality, would they want to know?

What would the information mean to them?

Counseling patients

Review reasons why patients would choose to accept screening Preparation

Termination

Review reasons why patients would decline screening Would not terminate

Would cause unwanted stress or worry

Counseling patients

“I don’t want any testing

that may cause a miscarriage”

Most women fall

somewhere in the middle

“I would terminate a pregnancy

with any major problem”

Fear of Miscarriage Fear of Abnormality

Informed consent

Patients need to know: They can decline all screening or diagnostic testing

for aneuploidy

cfDNA is a screening test and diagnostic testing is recommended for confirmation of positive results

Accuracy of cfDNA screening

What conditions are (and are not) being screened for

The possibility of a “no call” result

Post-test counseling

Normal results: Should be disclosed by a medical professional designated to

review this information

“Low risk”, not “no risk” for conditions on the panel

Still have option to have diagnostic testing

Still be offered MSAFP screening or ultrasound evaluation

Post-test counseling

Abnormal results or “No Call” results NOT diagnostic

Comprehensive ultrasound and diagnostic testing should be offered

Refer to a genetic counselor or MFM specialist

Dar P, Curnow KJ, Gross SJ, Hall MP, Stosic M, Demko Z, et al. Clinical experience and follow-up with large scale single-nucleotide polymorphism-based noninvasive prenatal aneuploidy testing. Am J Obstet Gynecol 2014;

Agarwal et al 2014 Commercial Landscape of noninvasive prenatal testing in the United States

I found this presentation

1 2 3

33% 33%33%

1. Informative and will enable me to order NIPT appropriately

2. Need more information

3. Too much information

Resources Fact sheets SMFM: https://www.smfm.org/publications/189-prenatal-aneuploidy-screening-using-

cell-free-dna NSGC:

Provider fact sheets: http://nsgc.org/page/non-invasive-prenatal-testing-healthcare-providers http://nsgc.org/page/abnormal-non-invasive-prenatal-testing-results

Patient fact sheet: http://nsgc.org/d/do/4584

Perinatal Quality Foundation/National Society of Genetic Counselors Online Predictive Value Calculator: http://nsgc.org/page/nipt-calculator

Lettercase: Prenatal Screening and Testing for Chromosome Conditions: http://www.lettercase.org/prenataltesting/

Genetics Support Foundation Patient video: https://www.geneticsupportfoundation.org/genetics-and-you/pregnancy-

and-genetics/noninvasive-prenatal-testing-the-real-deal-and-what-you-need-to-know

Society for Maternal-Fetal Medicine (SMFM) Consult Series #36: Prenatal aneuploidy screening using cell-free DNA: http://www.ajog.org/article/S0002-9378(15)00324-5/pdf

ACOG & SMFM Joint Committee Opinion: Noninvasive Prenatal Testing for Fetal Aneuploidy: http://www.acog.org/Resources-And-Publications/Committee-Opinions/Committee-on-Genetics/Cell-free-DNA-Screening-for-Fetal-Aneuploidy

ACOG: More Information: For Committee Opinion #640—Cell-free DNA (cfDNA) Screening for Fetal Aneuploidy: www.acog.org/More-Info/cfDNA

Acknowledgements

NSGC

Cori Feist

Shannon Wieloch

Katie Stoll

ACOG

Joseph Biggio

Britt Rink

SMFM

Mary Norton

Sean Blackwell

References

Lo et al (2010), Maternal plasma DNA sequencing reveals the genome wide genetic and mutational profile of the fetus. Science and Translational Medicine. 2, 61.

Nicolaides et al (2012), Noninvasive prenatal testing for fetal trisomies in a routinely screened first-trimester population. American Journal of Obstetrics and Gynecology. 207, 374.

Wang et al (2013), Gestational age and maternal weight effects on fetal cell-free DNA in maternal plasma. Prenatal Diagnosis. 33, 661-666.

Vora and O’Brien (2014), Noninvasive prenatal testing for microdeletion syndromes and expanded trisomies. Obstetrics and Gynecology, 123, 1097- 1099.

Wang et al (2014), Maternal mosaicism is a significant contributor to discordant sex chromosomal aneuploidies associated with noninvasive prenatal testing. Clinical Chemistry, 60, 251-259.

Gil et al (2015), Analysis of cell free DNA in maternal blood in screening for fetal aneuploidies: an updated meta-analysis. Ultrasound in Obstetrics and Gynecology. 45; 249-266.

Norton et al (2015), Cell-free DNA analysis for noninvasive examination of trisomy. New England Journal of Medicine.

SMFM Publications Committee (2015), Prenatal aneuploidy screening using cell-free DNA. American Journal of Obstetrics & Gynecology

ACOG Committee on Genetics & SMFM, Joint Committee Opinion #640 (2015): Cell-free DNA Screening for Fetal Aneuploidy. Published ahead of print.

Questions?