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© 2014 Illumina, Inc. All rights reserved. 24sure, Advanced Liquid Logic, Ampligase, Array of Arrays, BaseSpace, BeadArray, BeadChip, BeadStudio, BlueFish, BlueFuse, BlueGnome, CASAVA, cBot, CSPro, CircLigase, ClearHyb, ClearLab, ClearPack Lite, ClearScan, CytoChip, DASL, DecisionTrack, DesignStudio, DuraScribe, DuraScript, Epicentre, EpiGnome, FastTrack, ForenSeq, Genetic Energy, GenomeStudio, GoldenGate, HiScan, HiScanSQ, HiSeq, HiSeqDx, HiSeq X, HumanCytoSNP, HumanOmni, HumanHap, iCommunity, iControlDB, iGenome, ign, Infinium, Infinium Dx, IntelliHyb, Isaac, iScan, iSelect, KaryoStudio, KaryoStudioDx, MiSeq, MiSeqDx, MiSeq FGx, MyGenome, NeoPrep, Nextera, NextBio, NextSeq, Powered by Digital Microfluidics, Powered by Illumina, Rapid WGS Service, RapidTrack, Ribo-Zero, ScriptSeq, SeqMonitor, SureMDA, SurePlex, TruGenome, TruSeq, TruSight, Understand Your Genome, UYG, VeraCode, VeraScan, VeriSeq, the pumpkin orange color, and the streaming bases design are trademarks of Illumina, Inc. and/or its affiliate(s) in the U.S. and/or other countries. All other names, logos, and other trademarks are the property of their respective owners.
Christin Coffeen, MS, LCGC Senior Genetic Counselor Illumina
Non-Invasive Prenatal Testing (NIPT): Introduction and Technology Overview
2
Birth Defects: Rates and Causes
Adapted from Stevenson, RE and Hall, J. Human Malformations and Related Anomalies, 2nd ed. 2006
Normal (97%)
Defects (3%)
Unknown (40–60%)
Chromosomal (10–15%)
Prenatal Exposure (8–12%)
Single Gene (2–10%)
Multifactorial (20–25%)
3
Prenatal Prevalence of Reported Chromosomal Abnormalities
T21 53%
T18 13%
T13 5%
45,X 8%
Sex trisomy 5%
Other rare 16% T21
T18T1345,XSex trisomyOther rare
Major fetal aneuploidies
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.
4
Prenatal Screening and Diagnostic Testing Prior to NIPT
1ST TRIMESTER 2ND TRIMESTER 3RD TRIMESTER
First day of LMP
40 wks Term 13 wks 27 wks
1st trimester Screen
serum + U/S
2nd trimester Screen serum
amnio 16-22 wks
18 wks
CVS 10-14 wks
12 wks
NT MEASUREMENT (LIMITED ANATOMY)
5
Conventional Prenatal Screening Options Detection Rates for Trisomy 21
ACOG Practice Bulletin No. 77, January 2007 Actual detection rates and false positive rates will vary slightly based on the laboratory used.
1st Trimester Blood Screen NT Ultrasound 2nd Trimester
Blood Screen Integrated Screen
NT Ultrasound 1st Trimester
1st Trimester Blood Screen NT Ultrasound 1st Trimester
Triple Screen 2nd Trimester
Quadruple Screen 2nd Trimester
1st Trimester Blood Screen 2nd Trimester Blood Screen
Serum Integrated
64-70
82-87
69
81
94-96
85-88
Detection Rate (%)
False Positive Rate: 5%
6
Gold-standard diagnostic tests – Chorionic Villus Sampling (CVS) at 11-13
weeks – Amniocentesis at 15-20 weeks
Present risk to patient and fetus – 0.4% risk of miscarriage with amniocentesis* – Risk of maternal bleeding, infection, leaking
Invasive Prenatal Testing
*Ultraschall Med. 2012 Dec;33(7):E75-9. doi: 10.1055/s-0031-1299388
7
Useful because fetuses affected with aneuploidy often have anatomic changes or anomalies.
A genetic sonogram uses ultrasound to assess the fetus for both structural anomalies and soft markers suggestive of aneuploidy
Invasive testing still is required to obtain a definitive diagnosis.
First and Second trimester ultrasound – Can detect many other abnormalities that can be associated with
other chromosomal/genetic syndromes – Can detect structural abnormalities not associated with genetic
syndrome
Ultrasound Examination
8
What are the Goals of NIPT?
Reduce exposure of risk to fetus
Reduce false positives
Enable a high detection rate
Testing that can easily
be offered to pregnant
women
9
NIPT Technology Overview
10
Detect fetal aneuploidy using cell-free DNA from maternal blood
– Analyzed by next-gen DNA sequencing
Other used nomenclature: – NIPD: Noninvasive Prenatal Diagnosis – NIPS: Noninvasive Prenatal Screening – cfDNA: Cell-free DNA – cffDNA: Cell-free fetal DNA – DNA-based noninvasive prenatal screening
Non-Invasive Prenatal Testing (NIPT) A new category of prenatal testing
11
Released through apoptosis
– Fetal cfDNA likely arises from cytotrophoblastic cells of placenta
Released into bloodstream as small DNA fragments (150–200 bp)
Maternal blood contains both fetal, maternal cfDNA
– 2–20% of total cfDNA is fetal
Fetal cfDNA reliably detected after 7+ weeks gestation
Fetal cfDNA undetectable within hours postpartum
Cell-Free DNA (cfDNA) A reliable analyte during pregnancy
1. Barrett, A. et al. Implementing prenatal diagnosis based on cell-free fetal DNA: Accurate identification of factors affecting fetal DNA yield. PLoS One 6; (2011);e25202. 2. Nigam, A. et al. Detection of fetal nucleic acid in maternal plasma: A novel noninvasive prenatal diagnostic technique. JIMSA 25:119–200(2012).
12
Massively Parallel Sequencing (MPS)
Fetal DNA fragments in
maternal blood.
Cell free DNA fragments are
then sequenced.
Compare the individual
sequenced chromosomes
against a reference for analysis.
13
Benefits – Low assay failure rates – Ability to add new content to test menu
Genome-Wide MPS Provides precise, across-the-genome coverage
1 2 3 4 5 6 7 8 9 10
11 12 13 14 15 16 17 18 19 20
21 22 X Y Chromosome-Wide Coverage
NOT TO SCALE
14
Drawbacks – High assay failure rates – Limited ability to add new content without changing assay
Targeted MPS Limited to few chromosomes, loci
1 2 3 4 5 6 7 8 9 10
11 12 13 14 15 16 17 18 19 20
21 22 X Y Chromosome-Wide Coverage
NOT TO SCALE
15
Drawbacks – High Assay Failure Rates – Difficult to analyze egg-donation, surrogacy, consanguinity,
maternal transplant, multiple gestation samples
Targeted MPS (SNP-Based Method) Complex, failure-prone method
Maternal Blood
Plasma = Maternal +
Fetal cfDNA
Maternal + Fetal
Genotype
Buffy Coat = Maternal DNA
Maternal Genotype
Deduce Fetal
Genotype
Risk Result
SNP Sequencing
16
Updated Meta-analysis: To review the clinical validation of cfDNA screening for fetal aneuploidies
37 publications on NIPT for detection of aneuploidies between 2011-2015
Evidence for NIPT Performance with MPS
DR (%) 95% CI FPR (%) 95% CI
Trisomy 21 99.2 98.5-99.6 0.09 0.05-0.14
Trisomy 18 96.3 94.3-97.9 0.13 0.07-0.20
Trisomy 13 91.0 85.0-95.6 0.13 0.05-0.26
Monosomy X 90.3 85.7-94.2 0.23 0.14-0.34
Other sex aneuploidies
93.0 85.8-97.8 0.14 0.06-0.24
Twins T21 93.7 83.6-99.2 0.23 0.00-0.92
Gil et al (2015). Ultrasound Obstet Gynecol, 45: 249-266
17
NIPT with Arrays? An Unknown Limit of Detection
Drawbacks Array NIPT unproven High samples failure
Likely requires high FF call Late term
High FF means late term testing Juneau et al (2014). Fetal Diagn Ther, DOI: 10.1159/000367626. Stokowski R, Wang E, White K, et al (2015) Prenat Diagn, DOI: 10.1002/pd.4686
18
382.996
37.206 878 32.916 -
100.000
200.000
300.000
400.000
500.000
WGS TargetedSequencing
ArrayTechnology
Targeted SNPSequencing
Published and presented samples1
Proof is in the Data
1 A PubMed search for “cell-free, DNA, prenatal”, “noninvasive prenatal testing”, and “noninvasive prenatal screening” was performed on April 30, 2015. All validation and clinical studies using unique samples were included, where a current clinical NIPT provider performed sample analysis. Case studies and studies published in a language other than English were excluded. Data from a 2015 ESHG conference abstract was also included. A total of 45 published studies were surveyed. Data calculations on file. Illumina, Inc. 2015. NGS = next-generation sequencing; either whole-genome or targeted.
19
Illumina Technology Has Enabled NIPT
Company Approach Sequencing Platform
Whole Genome
Whole Genome
Targeted SNP Sequencing
Whole Genome
Whole Genome
Illumina NGS platform clinically validated for NIPT on over 35,000 patients; over 1,000,000 clinical reports issued
20
Clinical Implementation and Counseling Considerations
21
Professional Society Guidelines Endorsements
22
ISPD Position Statement April 2013
“Non-invasive prenatal testing based on massively parallel sequencing of circulating free fetal DNA (cfDNA) in maternal plasma has been shown to be highly effective for aneuploidy detection”
“[NIPT] would appear to be the most effective method for screening for fetal trisomy 21 and trisomy 18”
“The tests should not be considered to be fully diagnostic and therefore are not a replacement for amniocentesis and CVS”
“Laboratory providers should also be prepared to provide ongoing specifics on accuracy, test failure rates and turn-around time”
Also supporting NIPT for high risk pregnancies:
23
ISPD Position Statement on Chromosome Abnormality Screening: April 2015
Some important points from position statement: – cfDNA screening as a primary test offered to all pregnant
women – cfDNA secondary to a high risk assessment based on serum
and ultrasound screening – cfDNA contingently offered to a broader group of women
ascertained as having high or intermediate risks by conventional screening Contingent provision of cfDNA, could also include a protocol in
which women with very high risks are offered invasive prenatal diagnosis while those with intermediate risk are offered cfDNA
24
Patients wanting early, accurate testing and are at high risk of aneuploidy due to:
Maternal age-related risks
Positive results on maternal-serum screening
Abnormal ultrasound finding(s)
History suggestive of increased risk for T21, T18,T13 or sex chromosome aneuploidy
Parental translocation involving one of the tested chromosomes
Patients wanting early, accurate testing and are at average risk of aneuploidy
Which Patients Should Be Offered NIPT?
25
NIPT Failure Rates by Technology & Company
0.1% 1,3%
3,0%
6,4%
0%
1%
2%
3%
4%
5%
6%
7%
1Taneja et al. Abstract presented at ESHG, 2015 2McCullough RM et al. PLoS One. 2014 3Norton ME, et al. New Engl J Med 2015 4Dar, et al. Am J Obstet Gynecol. 2014
verifi1 MaterniT212 *Harmony3 Panorama4
Whole Genome Sequencing
Targeted Sequencing
*Very limited data published using array technology, no clinical experience available
26
Actual sensitivity is less than claimed sensitivity
Studies have shown a high rate of aneuploidy in test failures
Redraw for NIPT is usually ineffective – High published redraw failure rates
– Leads to increased turnaround time, office visits, patient/physician frustration
Why Do Test Failures Matter in NIPT?
1 Pergament E, et al. Obstet Gynecol. 2014 Aug;123(2 Pt 1):210–8 2 ACOG Committee Opinion Number 640, Sept 2015
Up to
22% Aneuploidy
Rate1,2 in NIPT test failures
Up to
65% NIPT Redraw
Failure Rate1,2
27
Women whose results are not reported, indeterminate, or uninterpretable (a “no call” test result) from cell-free DNA screening should receive genetic counseling and be offered comprehensive ultrasound evaluation and diagnostic testing because of an increased risk of aneuploidy.
28
NIPT is now part of prenatal screening options.
Important to remember the benefits and limitations of the various prenatal tests
– NIPT does not test for all chromosome abnormalities, birth defects, genetic disorders or other pregnancy complications.
– No testing is 100%.
Labs have varying restrictions regarding gestational age, multiples, consanguinity and pregnancies conceived through the use of donor eggs/surrogacy.
Labs have varying test failure rates (some of which may include an increased risk of aneuploidy).
Possibility test results might not reflect the chromosomes of the fetus, but may reflect chromosomal changes to the placenta or of the mother.
Co-twin demise
Key Points to Remember
29
Thank You
30
Appendix
31
Whole Genome Sequencing Has Benefits Over Targeted Sequencing & Arrays
Benefits • Low assay failure rates (<1%) • Ability to add new content to test menu
Drawbacks • High assay failure rates (up to 12%) • Limited ability to add new content without changing assay
Targeted sequencing is limited to few chromosomes, loci
WGS provides precise counts, across the genome
32
Fetal Fraction in NIPT
Fetal Fraction = amount of fetal cfDNA in total cfDNA
% fetal fraction (FF) affects ability of NIPT to detect fetal aneuploidy
– Very low fetal fraction may lead to false negative results
Several methods currently in use to estimate fetal fraction
– Inaccurate at low fetal fraction (much variation in the measurement)
Threshold for fetal fraction depends on coefficient of variation obtained for an individual chromosome
– May be improved through algorithm improvements
33
Assay Quality – Lowers the limit of detection (LOD) – Based on sequencing methodology and analysis method
Fetal Fraction – Lower fetal fraction demands a lower LOD
Finding the Fetal Fraction
Limit of Detection
Fetal Fraction
Assay Quality
34
Why does anyone measure Fetal Fraction?
NIPT assays with lower quality use Fetal Fraction to eliminate difficult samples
– Eliminating samples with low fetal fraction increase sensitivity and specificity
Some labs do not measure fetal fraction and do not eliminate samples from analysis
Laboratory Clinical experiences – Assay failure rates – Negative Predictive Value (NPV)
Lowest limit of detection – Combination of accurate sequencing and data analysis algorithms
35
Clinical Factors Affecting Fetal Fraction Significant correlation with aneuploidy
– FF higher for trisomy 21 – FF lower for trisomy 18, trisomy 13, monosomy X
Correlation with gestational age – Slight increase from 10-21 weeks gestation – Significant increase after 21 weeks gestation
Weak correlation with maternal BMI – Slight decrease in FF with maternal BMI – No specific threshold has been established where results
cannot be obtained relative to maternal weight
Not affected by maternal age, ethnicity, a priori trisomy risk
Rava et. al, Clin Chem 2013 Jan;60(1):243-50.; Wang, E. et al. Prenat Diagn 2013 Jul;33(7):662-6. Galbiatia, D. et al. Hum Genet 2005,117(2-3):243-8.; Bianchi, D., et al. N Engl J Med 2014 Feb 27;370(9):799-808.
36
Fetal Fraction in NIPT Vast majority of samples have FF levels well above lower threshold
From Wang et al. Prenatal Diagnosis 2013 33, 1-5.
37
Illumina verifi
MaterniT21 Sequenom
Harmony Ariosa
Panorama Natera
Method Whole Genome Whole Genome Targeted/Array Targeted
Limit of Detection 1.4–2.71 4%3 4%5
(unknown with microarray)
3.8–8.08
Specimen 1 tube maternal blood 2 tubes maternal blood
2 tubes maternal blood
2 tubes maternal blood, paternal sample optional
Failure Rate 0.1%2 1.9%4 4.6–4.9%5,6
(unknown with microarray)
6.4–8.1%8,9
Time to Report 3–5 business days
5 business days
7–10 business days
9.29 calendar days
Egg Donors & Twins Yes Yes Yes
(13% failure rate7) No
Microdeletions Offered Yes Yes No Yes
Comparison of NIPT Service Providers iIlumina verifi® prenatal test leads in performance
• Rabinowitz, et al. ASHG Abstract 2012.; Presented data at NSGC AEC 2012
• Norton ME, et al. Am J Obstet Gynecol. 2012 doi:10.1016/j.ajog.2012.05.021 • Palomaki GE, et al. Genet Med. 2012 Mar;14(3):296-305; M. Ehrich communication • Futch et al., Prenat Diagn 2013 Apr [Epub ahead of print]
1. Rava, et al., Clin Chem 2014;60(1):243-50 2. Bhatt et al. Poster presented at ISPD, 2014 3. Jensen TJ, et al. PLoS ONE 2013:8(3): e57381. doi:10.1371/journal.pone.0057381 4. McCullough RM, et al., PLoS ONE 9(10): e109173. doi:10.1371/journal.pone.0109173 5. Norton ME, et al. Am J Obstet Gynecol. 2012 doi:10.1016/j.ajog.2012.05.021 6. Nicolaides, et al., Am J Obstet Gynecol 2012;207(5):374.e1-6 7. Gil, et al., Fetal Diagn Ther 2013 8. Pergament E, et al. Obstet Gynecol. 2014 Aug;123(2 Pt 1):210-8 9. Dar P, et al., Am J Obstet Gynecol (2014), doi: 10.1016/j.ajog.2014.08.006.
38
Illumina verifi
NIFTY BGI
Method Whole Genome Whole Genome
Failure Rate 0.1% ~2% before redraw1,3
Turn Around Time 3–5 business days
10–15 business days2,3,4
Sample (blood) 1 tube maternal blood 2 tubes maternal
Published Laboratory Clinical Experience Yes Yes
Laboratory CLIA/CAP-certified laboratory
Non CLIA/CAP-certified laboratory
Comparison of NIPT Service Providers illumina verifi Prenatal Test Leads in Performance
1. Zhang et al Ultrasound Obstet Gynecol. 2015 Jan 19. doi: 10.1002/uog.14792. 2. http://www.niftytest.com/wp-content/uploads/2014/09/BGIDX_NIFTY_Leaflet_24.06.2014_New_Code.pdf 3. http://www.thisismy.co.uk/non-invasive-prenatal-testing-nipt/ 4. http://igenescreen.com/for-doctors/
39
5,5%
0.1% 1.9%
NIPT Test Failure Rates
8,1%
4,6% ~5.0%
13.2% With paternal sample
Natera1,2 Sequenom5,6 Ariosa3,4 7
Whole Genome Sequencing Targeted Sequencing
Requires retest of sample
NIP
T Fa
ilure
Rat
e
1. Pergament E, et al. Obstet Gynecol. 2014 Aug;123(2 Pt 1):210-8 2. Dar P, et al. Am J Obstet Gynecol. 2014 Aug 8. doi: 10.1016/j.ajog.2014.08.006 3. Norton ME, et al. Am J Obstet Gynecol. 2012 doi:10.1016/j.ajog.2012.05.021 4. Gil M, et al. Fetal Diagn Ther. 2014;35(3):204-11 5. Palomaki GE, et al. Genet Med. 2012 Mar;14(3):296-305 6. McCullough RM, et al., PLoS ONE 9(10): e109173. doi:10.1371/journal.pone.0109173 7. Bhatt et al. Poster presented at ISPD, 2014
Failure rate in twins
40
10 pages with illustrations to help patients understand the NIPT testing process, conditions tested, result interpretation
Used by healthcare providers prior to NIPT
verifi prenatal test specific
Available in 9 languages: English, Spanish, Portuguese, French, German, Italian, Korean, Japanese, Chinese
Download at verifitest.com Tools For Your Practice
Genetic Counseling NIPT Flipbook
41
12-minute video providing an overview of the benefits and limitations of various prenatal testing options – Prenatal screening (e.g. first trimester combined screen) – CVS/Amniocentesis – NIPT (verifi prenatal test specific)
Includes description of conditions tested
Healthcare providers can direct their patients to watch this video in the clinic or at home prior to their OB appointment
Available in 9 languages: English, Spanish, Portuguese, French, German, Italian, Korean, Japanese, Chinese
Can be viewed at verifitest.com Tools for Your Practice – Downloadable to other Practices’ websites
Patient Education Video
42
NIPT Test Failures Not Only Due to Fetal Fraction Cutoffs Comparison of test providers
6,1%
1,8% 0,9%
2,0%
2,8%
1,0%
Natera Ariosa Sequenom verifi
Other Technical Failures*Fetal Fraction
1 2 3 4
Test Failure Rates Depend on Assay Reliability, Limit of Detection (LOD) * Reasons include insufficient cfDNA, inability to measure fetal fraction, lab error, contamination, bad statistical fit,
highly variable cfDNA counts, or other sequencing failure
1. Pergament E, et al. Obstet Gynecol. 2014 Aug;123(2 Pt 1):210-8 2. Norton ME, et al. Am J Obstet Gynecol. 2012 doi:10.1016/j.ajog.2012.05.021 3. McCullough RM, et al., PLoS ONE 9(10): e109173. doi:10.1371/journal.pone.0109173 4. Bhatt et al. Poster presented at ISPD, 2014 5. Rava, et al., Clin Chem 2014;60(1):243-50
Test
Fai
lure
Rat
e
3.8–8.0%1 4%2 4%3 1.4–2.7%5 LOD:
8.1%
4.6%
1.9% 0.1%
43
Screening Method
Detection Rate
Cases Detected
False Positive
Rate
Failure Rate
Invasive Tests
Procedure Related
Loss
T21 (n=200) Normal (n=99,800)
Theoretical population 100,000 Pregnancies, T21 Prevalence 1:500
Maternal age 30% 60 5% - 4,990 10
Integrated Screen 95% 190 5% - 4,990 10
Natera >99.9% >199 <0.1% 6.3% 6,287 12
Ariosa >99.9% >199 <0.1% 3% 3,094 6
Sequenom 98.6% 197 <0.1% 1.9% 1,996 4
Verifi >99.9% >199 <0.1% 0.1% <100 <1
Implications of Test Failure
44
NIPT as a Primary Screen
NIPT CVS/Amniocentesis (Invasive)
Aneuploidy detected or suspected?
Continue with pregnancy management according to your
practice’s protocols
YES
(Genetic counseling is recommended)
NO
Continue with pregnancy management according to your
practice’s protocols
• Maternal age-related risks
• Abnormal ultrasound finding(s)
• Hx suggestive of increased risk for T21, T18,T13 or SCA
• Parental translocation involving one of the tested chromosomes
START HERE
45
NIPT as a Secondary Screen (following a positive serum screen)
NIPT CVS/Amniocentesis (Invasive)
Aneuploidy detected or suspected?
YES
(Genetic counseling is recommended)
NO
Serum Screening (per practice’s protocol)
Is the serum screen result
positive?
Continue with pregnancy management according to your
practice’s protocols
NO
Counsel the patient about the verifi prenatal test and invasive
test options
YES
(START HERE)
46
Clinical Methods in Published Series
Test (Company) Current Clinical NIPT Method
No. of Published NIPT Samples
Bambni™ Assay (Berry Genomics) Illumina NGS 2,351
MaterniT21 PLUS™ Test (Sequenom) Illumina NGS 108,665
NIFTY™ Test (BGI) Illumina NGS 160,667
Panorama™ Prenatal Screen (Natera) Illumina NGS 32,916 PrenaTest (LifeCodexx AG/GATC Biotech AG) Illumina NGS 504
verifi® Prenatal Test (Illumina) Illumina NGS 113,367
Harmony™ Prenatal Test (Ariosa) Illumina NGS 37,206
Harmony™ Prenatal Test (Ariosa) Affymetrix Array 878 A survey of 45 published studies* revealed that 99.8% of reported NIPT samples
run on Illumina NGS systems * A Pubmed search for “cell-free, DNA, prenatal”, “noninvasive prenatal testing”, and “noninvasive prenatal screening” was performed on April 30, 2015. All validation and clinical studies using unique samples were included, where sample analysis was performed by a current clinical NIPT provider. Case studies and studies published in a language other than English were excluded. Also included data from a 2015 ESHG conference abstract.