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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
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
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Current Prenatal Screening and Diagnosis Landscape
Prenatal Prevalence of Chromosomal Abnormalities
Four major
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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
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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
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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
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AFPy
misinterpreted to mean the fetus is normal.
– Positive screens, based on risk can lead to unnecessary invasive procedures.
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uE3
Inhibin
PAPP‐A
Invasive Diagnostic Options
Trimester ‐ Test Sensitivity Specificity
1st – CVS 99.25%1 98.65%1
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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
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• History• Multiple Gestation• Ethnicity• Smoking• Diabetes• Weight• Gestational Age
hCG
AFP
uE3
PAPP‐A
Inhibin
Measuring with NIPT – Eliminate External Factors
Factors Needed:• Age
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• 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
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• 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
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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
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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
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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
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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
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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
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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)
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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
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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
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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
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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.
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Available Tests
• Harmony (Ariosa)
• Panorama (Natera)
• Verifi (Verinata)
• MaterniT21 Plus (Sequonom)
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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
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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
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