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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.