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Non-Invasive Prenatal Testing Karlene Coleman, RN, MN, CGC Certified Genetics Counselor Children’s Healthcare of Atlanta, Atlanta, GA Clinical Instructor Nell Hodgson Woodruff School of Nursing, Emory University, Atlanta, GA The speaker has signed a disclosure form and indicated she has no significant financial interest or relationship with the companies or the manufacturer(s) of any commercial product and/or service that will be discussed as part of this presentation. Session Summary This new technology is non-invasive and detects fetal trisomies 21, 18, and 13 in pregnancies of 10 weeks or more. The test analyzes maternal blood and provides a risk assessment for pregnant women with singleton and naturally conceived or self-egg donor twin pregnancies. Session Objectives Upon completion of this presentation, the participant will be able to: define the difference between prenatal screening versus diagnostic testing; state the approximate sensitivity of CVS and amniocentesis for Down syndrome; state what type of fetal DNA is used in NIPT; know what percentage of cfDNA comes from the fetus in the maternal blood; state the week of gestation when cfDNA can be reliably detected and the half life of cfDNA post partum; list the test accuracy for conventional screening and the false positive rate; list the ACOG Genetics Committee Opinion on test accuracy for cfDNA and the false positive rate; know how many women would need an amniocentesis or CVS after conventional screening versus cfDNA; know what test to order to resolve the discrepancy if the NIPT was normal but the infant has features of Down syndrome. References Gene Tests: www.genetests.org Nussbaum, Robert L. et al. (2007). Thompson &Thompson genetics in medicine (7th ed.). Philadelphia: Elsevier Sanders. OMIM -Online Mendelian Inheritance in Man: www.omim.org/ Session Outline See presentation handout on the following pages. A1b FANNP 24TH NATIONAL NNP SYMPOSIUM: CLINICAL UPDATE AND REVIEW A2b: NON-INVASIVE PRENATAL TESTING Page 1 of 7

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Non-Invasive Prenatal Testing Karlene Coleman, RN, MN, CGC Certified Genetics Counselor Children’s Healthcare of Atlanta, Atlanta, GA Clinical Instructor Nell Hodgson Woodruff School of Nursing, Emory University, Atlanta, GA

The speaker has signed a disclosure form and indicated she has no significant financial interest or relationship with the companies or the manufacturer(s) of any commercial product and/or service that will be discussed as part of this presentation.

Session Summary

This new technology is non-invasive and detects fetal trisomies 21, 18, and 13 in pregnancies of 10 weeks or more. The test analyzes maternal blood and provides a risk assessment for pregnant women with singleton and naturally conceived or self-egg donor twin pregnancies.

Session Objectives

Upon completion of this presentation, the participant will be able to:

define the difference between prenatal screening versus diagnostic testing;

state the approximate sensitivity of CVS and amniocentesis for Down syndrome;

state what type of fetal DNA is used in NIPT;

know what percentage of cfDNA comes from the fetus in the maternal blood; state the week of gestation when cfDNA can be reliably detected and the half life of cfDNA post

partum;

list the test accuracy for conventional screening and the false positive rate;

list the ACOG Genetics Committee Opinion on test accuracy for cfDNA and the false positive rate; know how many women would need an amniocentesis or CVS after conventional screening versus

cfDNA; know what test to order to resolve the discrepancy if the NIPT was normal but the infant has

features of Down syndrome.

References

Gene Tests: www.genetests.org

Nussbaum, Robert L. et al. (2007). Thompson &Thompson genetics in medicine (7th ed.). Philadelphia: Elsevier Sanders.

OMIM -Online Mendelian Inheritance in Man: www.omim.org/

Session Outline

See presentation handout on the following pages.

A1b

FANNP 24TH NATIONAL NNP SYMPOSIUM: CLINICAL UPDATE AND REVIEW

A2b: NON-INVASIVE PRENATAL TESTING Page 1 of 7

FANNPNon‐Invasive Prenatal 

Testing

Karlene Coleman, RN, MN, CGCCertified Genetic Counselor

Children’s Healthcare of Atlanta 

[email protected]

Some slides courtesy of Verinata and Integrated Genetics

Glossary of Abbreviations

• NGS – Next Generation Sequencing

• MPS – Massively Parallel Sequencing

• NCV – Normalized Chromosome Value

• NIPT – Noninvasive Prenatal Testing

• HEBIC – Health Economic Budget Impact Calculator

• cfDNA – Cell Free DNA

• ART – Assisted Reproductive Technology

• SAFeR™ ‐ Selective Algorithm for Fetal Results

• CPM – Confined Placental Mosaicism

2

Current Prenatal Screening and Diagnosis Landscape

Prenatal Prevalence of Chromosomal Abnormalities

Four major

4

Data adapted from Wellesley, D, et al., Rare chromosome abnormalities, prevalence and prenatal diagnosis rates from population‐based congenital anomaly registers in Europe. Eur J of Hum Gen, 11 January 2012.

fetal trisomies

Prenatal Screening and Diagnostic Testing Today

1st Trimester 2nd Trimester 3rd Trimester

1st trimester Screen

serum + U/S

2nd trimester Screenserum

5

First day of LMP

40 wksTerm

13 wks 27 wks

amnio16‐22 wks

18 wks

CVS10‐14 wks

12 wks

NT measurement(limited anatomy)

Full anatomy

Screening Tests

• Definition – a test applied to an asymptomaticpopulation in order to classify them with respect to their likelihood of having a specific condition

• The difference between screening and diagnostic g gtests:– Screening tests give a risk for a condition

• MSAFP, Multiple Marker Screen, Ultrasound for Down Syndrome

– Diagnostic tests give a definitive result as to the presence or absence of a condition

• Amniocentesis, CVS, Ultrasound for spina bifida

FANNP 24TH NATIONAL NNP SYMPOSIUM: CLINICAL UPDATE AND REVIEW

A2b: NON-INVASIVE PRENATAL TESTING Page 2 of 7

Numerous Options with Variable Performance

FASTER Trial Malone et al, NEJM, 2005

Reference: ACOG Practice Bulletin, Number 77, Jan 2007

7

Modeled predicted performanceCuckle et al, Semin Perinatol, 2005

What Do Screening Results Tell Us Today?

• Screening results offer estimates of risk.

– A ‘negative’ or low‐risk result may be 

hCG

3

AFPy

misinterpreted to mean the fetus is normal.

– Positive screens, based on risk can lead to unnecessary invasive procedures.

8

uE3

Inhibin

PAPP‐A

Invasive Diagnostic Options

Trimester ‐ Test Sensitivity Specificity

1st – CVS 99.25%1 98.65%1

9

Even the gold standard is not 100% sensitive and specific.

2nd ‐ Amniocentesis 99.4%2 99.5%2

1. Hahnemann JM, Vejerslev LO. Accuracy of cytogenetic findings on chorionic villus sampling (CVS)‐‐diagnostic consequences of CVS mosaicism and non‐mosaic discrepancy in centres contributing to EUCROMIC 1986‐1992. Prenat Diagn. 1997 Sep;17(9):801‐20.

2. Mid‐trimester amniocentesis for prenatal diagnosis. Safety and accuracy. JAMA. 1976 Sep 27; 236(13): 1471‐6.

Noninvasive Prenatal Testing (NIPT)

What is NIPTTests that utilize the presence of cff‐DNA in the maternal circulation to screen for the risk of fetal aneuploidy and other chromosomal aberrations

Serum Screening – What is Needed to Measure?

Account for These Factors:• Age

hCG

11

• History• Multiple Gestation• Ethnicity• Smoking• Diabetes• Weight• Gestational Age

hCG

AFP

uE3

PAPP‐A

Inhibin

Measuring with NIPT – Eliminate External Factors

Factors Needed:• Age

12

• History• Multiple Gestation

• Ethnicity• Smoking• Diabetes• Weight

• Gestational Age

FANNP 24TH NATIONAL NNP SYMPOSIUM: CLINICAL UPDATE AND REVIEW

A2b: NON-INVASIVE PRENATAL TESTING Page 3 of 7

What are the Goals of NIPT?

Goals

Reduce exposure of fetus to 

risk

ReduceTesting that can easily be

13

Goals of NIPT

Reduce false 

positives

Enable a high 

detection rate

can easily be offered to all pregnant women*

*When data supports testing in all patients, instead of only high risk patients.

Technology Behind NIPT

Two Sources of Fetal DNA in Maternal Blood

• Fetal cells (intact)– 1 in a billion of total cell population

– Requires fetal cell isolation via mechanical and/or biochemical means

• Cell‐free DNA (cfDNA) – 2–20% of total cfDNA is fetal

– Requires DNA isolation and counting

– Counting method developed by Dr. Steven Quake, Stanford University

15

• Released through cellular death (apoptosis)

– Cleaves DNA into small fragments (150-200bp)

– Released into

What is “cell‐free DNA”?

bloodstream as cell-free DNA (cfDNA)

• Fetal cells also release cfDNA

– Maternal blood contains both fetal and maternalcfDNA

16

Fetal cfDNA Can Be Measured

• Fetal cfDNA muchmore common than intact fetal cells 

• Reliably detected after 7 weeks gestational age

• Short half life (16 min), undetectable after 2 hours postpartum 

17

How is this addressed by NIPT today?

2 ways to sequence using cfDNA

Massively Parallel Sequencing

Targeted Sequencing

18

Sequencing

3 ways to analyze

Normalized Chromosome Value (NCV)

Estimate risk using combination of sequence data and other factors

Z‐Score

FANNP 24TH NATIONAL NNP SYMPOSIUM: CLINICAL UPDATE AND REVIEW

A2b: NON-INVASIVE PRENATAL TESTING Page 4 of 7

Massively Parallel Sequencing (MPS)Method of Analysis for MPS

cfDNADNA 

SequencingAlignment

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CGATTTAACT…CGATTTAACT…

> 5,000,000 “counts” per blood sample

Precise Molecular Counting Can Detect Fetal Aneuploidy

• Fetus with trisomy contributes ~50% more cfDNA from the trisomic chromosome

Example: 20% fetal fraction

EuploidChr 21 cfDNA

• Millions of “counts” allows detection of relative cfDNA increase

• No need to distinguish fetal from maternal cfDNA

20

T21

Fetal Trisomy Detection With cfDNA

Fetal cfDNA

Maternal

Extra fragments derived from fetal trisomy 21

Chromosome 21 fragments

Referencechromosome

Each bar represents thousands of cfDNA fragmentsThe overabundance of chromosome 21 cfDNA fragments in trisomy 21, although small, can be measured with DNA sequencing

MaternalcfDNA

21

cfDNA Analysis – Not Diagnostic

• Positive results need to be taken in the context of disease prevalence

• The less prevalent a disease, the more likely a positive test may be a false positive 

Test accuracy: 

1,000 women

Example:

99% detection

0.2% false positive

T21 prevalence: 

1 in 750

1 T21 999 non‐T21

1 2 9970

Test + Test +Test ‐ Test ‐

T21 prevalence at mid‐trimesterTest accuracy rates are derived from  published data. Palomaki GE, Kloza EM, Lambert‐Messerlian GM, et al. DNA sequencing of maternal plasma to detect Down syndrome: an international clinical validation study. Genet Med 2011 Nov;13(11):913–20. 

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Conventional screening: detection 64‐94%;  False positive: 5%

Invasive Procedures Needed Because of False Positives

• Conventional Maternal Serum Screen and Ultrasound 

– False positive rate is 5%

50 of 1000 women screened will need invasive– 50 of 1000 women screened will need invasive procedure like CVS or amniocentesis

• NIPT Non Invasive Prenatal Testing (cfDNA)

– False positive rate is <0.5%

– 5 of 1000 women screened with need invasive procedure like CVS or amniocentesis

Clinical Data

FANNP 24TH NATIONAL NNP SYMPOSIUM: CLINICAL UPDATE AND REVIEW

A2b: NON-INVASIVE PRENATAL TESTING Page 5 of 7

MELISSA Study

• 2,882 samples collected

• 534 selected for analysis

– Including all abnormal karyotypes (N 221)

Study Design, Demographics

karyotypes (N=221)

– Gestational age: 10 – 23 weeks

– BMI (kg/m2): 17 – 59 

– Includes 38 ART pregnancies

– Diverse (27.3% non‐white)

25

MELISSA Study

MPS Performance (Autosomes)

Classified Sensitivity 

(%)

95% CI Specificity

(%)

95% CI

Chromosome 21 100∙0 95∙9 100∙0 100∙0 99∙1 100∙0

<1% unclassifiable per  0% False Positiveschromosome 

26

Chromosome 21

(n=493)

100∙0

(89/89)

95∙9 ‐ 100∙0 100∙0

(404/404)

99∙1 ‐ 100∙0

Chromosome 18

(n=496)

97∙2

(35/36)

85∙5 ‐ 99∙9 100

(460/460)

99∙2 ‐ 100∙0

Chromosome 13

(n=499)

78∙6

(11/14)

49∙2 – 95.3 100∙0

(485/485)

99∙2 ‐ 100∙0

Other Abnormal Karyotypes

• Mosaicism– 4 / 4 detected (T21, T18)

• Robertsonian Translocations

MELISSA Study Demonstrated Other Aneuploidy Detection

• Robertsonian Translocations– 3 / 3 detected (T21, T18)

• Rare autosomal aneuploidies– Detected T16, T20

• Sex aneuploidies– Monosomy X, XXX, XXY, XYY

27

Monosomy X Analysis1

Analyzed MELISSA Samples with Cystic Hygroma

Classified Sensitivity 

(%)

95% CI Specificity

(%)

95% CI

Monosomy X 95∙0 75∙1 ‐ 99∙9 100∙0 93∙4 ‐ 100∙0

0% False Positives

28

Monosomy X

(n=74)

95 0

(19/20)

75 1  99 9 100 0

(54/54)

93 4  100 0

Clinical Recommendations

Invasive procedures are suggested to confirm a positive or unclassifiable NIPT result.

l h ld l b d h f llTest results should always be used in the context of all available clinical findings.

It is recommended that the healthcare provider determine the utilization of the test, including the need for genetic counseling.

29

Available Tests

• Harmony  (Ariosa)

• Panorama  (Natera)

• Verifi (Verinata)

• MaterniT21 Plus (Sequonom)

FANNP 24TH NATIONAL NNP SYMPOSIUM: CLINICAL UPDATE AND REVIEW

A2b: NON-INVASIVE PRENATAL TESTING Page 6 of 7

Product Profile

Chromosomes Analyzed 21, 18, 13Monosomy X Option

Blood draw requirement 1 blood tube (7‐10mL)

Patient Eligibility 21, 18, 13: High risk pregnanciesMonosomy X: Cystic hygroma

31

Monosomy X: Cystic hygroma

Gestational Age  10 or more weeks estimated gestational age

Sample collection On‐site collection kits, ambient shipping

Turn‐around time 8‐10 business days

Clinical Support Full‐time genetic counselors on staff forprovider phone support

verifi® prenatal test – Test Report

• Abnormal results are flagged at top to alert the clinician

• Abnormal results are 

Test Report

highlighted in red

• Comments included to provide additional guidance

• Test claims restated as reference 

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ACOG Genetics Committee Opinion

ACOG ‐ Noninvasive Prenatal Testing for Fetal Aneuploidy

Patients at increased risk of aneuploidy can be offered testing with cell free fetal DNA.  This technology can be expected to identify approximately 98% of cases of Down syndrome with a false positive rate of less than 0.5%

Parents tell you they have a Trisomy 21 diagnosis by NIPT – what to do?

• If they did not have amniocentesis to confirm:– Order chromosomes

• If they did have an amniocentesis and it• If they did have an amniocentesis and it confirmed the NIPT: – Do not need to repeat postnatal chromosomes

• If they had NIPT that was negative but infant has features of major trisomy:– Order chromosomes 

FANNP 24TH NATIONAL NNP SYMPOSIUM: CLINICAL UPDATE AND REVIEW

A2b: NON-INVASIVE PRENATAL TESTING Page 7 of 7