The Verifi® prenatal test – Making a difference in patient safety

The Verifi® prenatal test – Making a difference in patient safety
Lawrence D. Platt, MD Prof Ob Gyn UCLA School of Medicine Peres center for Peace Tel Aviv Yafo July 1,2013

The Verifi® prenatal test: nipt technology overview
Prenatal Prevalence of Chromosomal Abnormalities 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.

Prenatal Screening Options – Risk Score
Detection Rate (%) Down Syndrome Testing with 5% Screen Positive Rate 1st Trimester Blood Screen NT Ultrasound 2nd Trimester Blood Screen Integrated Screen 1st Trimester Triple Screen 2nd Trimester Quadruple Screen Serum Integrated 64-70 82-87 69 81 94-96 85-88 Don’t use the word combined 2nd trimester – this means 1st trim NT + serum analytes Other options are available: look in ACOG bulleting 1st trim blood + NT Integrated screen: 1st trim blood, 2nd trim blood, NT (answer at end of 2nd) Sequential screen: 1st trim blood, interim result (could act), 2nd trim blood (fulll results), NT 2nd Trim test alone Practice bulletin has sensitivities with 5% FP ACOG Practice Bulletin No. 77, January 2007 .

Current Diagnostic Options - Karyotype
Trimester - Test Sensitivity Specificity 1st – CVS 99.25%1 98.65%1 2nd - Amniocentesis 99.4%2 99.5%2 Even the gold standard isn’t currently at 100% sens/spec. Definitive answers, but are invasive and come with risk to the patient Most are unnecessary due to the high rate of false positives in screening** 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 Prenat Diagn Sep;17(9): Mid-trimester amniocentesis for prenatal diagnosis. Safety and accuracy. JAMA Sep 27; 236(13): .

Spectrum of Prenatal Testing*
SCREENING Risk scores are generated and modified based on biochemical analysis and population statistics DIAGNOSTIC Results are based entirely on genetic factors Serum Screening CVS Combined Serum Screens, NT, Ultra-sound NIPT Amnio SCREENING DIAGNOSTIC *Not meant to represent percentage of accuracy .

What Are the Goals of NIPT?
Reduce exposure of fetus to risk Reduce false positives Enable a high detection rate Testing that can easily be offered to all pregnant women .

Two Sources of Fetal DNA in Maternal Blood
Fetal cells 1 in a billion of total cell population Require isolation via mechanical and/or biochemical means Cell-free DNA (cfDNA) Maternal blood contains both maternal and fetal cfDNA 2–20% of total cfDNA is fetal There are two possible sources of fetal DNA in maternal blood, either from whole fetal cells or from cell-free DNA. Fetal cells are very rare, representing about 1 in a billion cells in maternal circulation, and require laborious cell isolation techniques to capture. Cell-free DNA from the fetus is actually much more common, with 2 to 20 percent of total cell-free DNA in maternal blood coming from the fetus. This cell-free DNA requires isolation and counting to analyze for fetal aneuploidies, which was invented by Dr. Steven Quake from Stanford University. Dr. Quake is a scientific co-founder of Verinata. [Transition to Next Slide]: So what is cell-free DNA and where does it come from? Fetal cell image from: (purchased) .

Fetal Cell-free DNA in Maternal Blood
A Reliable Analyte During Pregnancy Released through apoptosis Fetal cfDNA likely arises from cytotrophoblastic cells of placenta Released into bloodstream as small DNA fragments ( bp) Reliably detected after 7+ weeks gestation Undetectable within hours postpartum .

DNA Sequencing using cell free DNA
Fetal DNA fragments in maternal blood. Cell free DNA fragments are then sequenced. Cutting Edge Technology The verifi™ prenatal test utilizes cell-free DNA (cfDNA) from both the patient and fetus. Patient plasma contains more than enough fetal fragments for our test. Each sample is sequenced with millions of short reads. The reads are aligned to the human genome and counted using our proprietary SAFeR™ (Selective Algorithm for Fetal Results) method. Our CLIA lab then analyzes and classifies the sample. Compare the individual sequenced chromosomes against a reference for analysis. .

Verinata’s Massively Parallel Sequencing (MPS)- A Superior Approach
MPS Provides Precise, Across the Genome Coverage Benefits Lowest Assay Failure Rates <1% Ability to rapidly add new content to test menu. Verinata’s approach allows rapid evolution of product 22.8 Million reads Targeted Sequencing is Limited to Few Chromosomes, Loci There are alternate approaches to detect fetal aneuploidy from cell-free DNA, and one recently developed approach is called targeted sequencing. The verifi® test uses MPS as described, which involves sequencing cfDNA fragments from all chromosomes, and a broad coverage of unique sites within chromosomes. This enables robust data normalization, and provides simple assay expansion to additional chromosomes. In fact, Verinata has demonstrated this promise through 2 product extensions in less than a year since initial launch of the test. Note: While cfDNA from all chromosomes is sequenced as part of the verifi® test, the test currently reports results for chromosomes 21, 18, 13, X and Y only. Aneuploidy analysis is not performed on the other chromosomes, and no Verinata personnel is ever aware of aneuploidy status on other chromosomes. Drawbacks High Assay Failure Rates of 4-12% Limited ability to add new content leads to static product profile 500K to 6.4M reads © 2013 Verinata. Content is proprietary and confidential.

Detection of Fetal Aneuploidy
MPS Enables Precise Molecular Counting Fetal cfDNA (20%) NOT TO SCALE Counting 10% more Chr21 cfDNA in T21 Maternal cfDNA VS …… Chromosomes: 1 2 3 21 Trisomy 21 © 2013 Verinata. Content is proprietary and confidential.

Dual Threshold Classification
Verinata Veritas, College Station, TX February 27, 2013 Dual Threshold Classification Indicates Borderline Results Trisomy Diploid VS. 0.2 to 0.6 % verifi® prenatal test Dual Threshold Single Threshold Method © 2013 Verinata. Content is proprietary and confidential.

Publications in Most Prestigious Journals in Field
Large-scale, prospective and blinded clinical trials Only study in the industry that represents real-world clinical use High-risk patient population Singleton gestations analyzed Over 60 U.S. centers enrolled Verinata’s MELISSA study was a large-scale, truly prospective and blinded clinical validation study which demonstrated the excellent clinical performance of the verifi® prenatal test. Over 60 U.S. centers participated in enrolling ‘high-risk’ patients with 1 or more indications of a high-risk pregnancy, such as AMA, ultrasound findings, positive serum screens, or family genetic history. A defining and unique characteristic of the MELISSA study is that all abnormal karyotyped samples were analyzed. This emulates real world testing where the fetal chromosome status is not known before testing. Also, the MELISSA study is the first and only study to date to perform sequencing analysis across all chromosomes at once. This demonstrates the true potential of the technology underpinning the verifi® test. [Transition to Next Slide]: Next we will dive into the details of the study .

Which Patients Should Be Offered NIPT?
Patients Wanting Early, Accurate Test And Are At High Risk Of Aneuploidy Due To: Maternal age-related risks Positive results on maternal serum screening Abnormal ultrasound finding(s) Prior pregnancy with aneuploidy Parental Robertsonian translocation involving one of the tested chromosomes

verifi® prenatal test Options
Verinata Veritas, College Station, TX February 27, 2013 verifi® prenatal test Options Provides testing for chromosomes 21,13,18 Provides option for sex chromosome testing Monosomy X, XXX, XXY, XYY If no aneuploidy detected, fetal sex (XX, XY) reported Option to select on test requisition form No change to price, turnaround time © 2013 Verinata. Content is proprietary and confidential.

verifi® prenatal test Performance
Sensitivity 95% CI Specificity Trisomy 21 >99.9% 96.0 – 100.0 99.8% 98.7 – 100.0 Trisomy 18 97.4% 86.2 – 99.9 99.6% 98.5 – 100.0 Trisomy 13 87.5% 61.7 – 98.5 99.2 – 100.0 Sensitivity 95% CI Specificity Monosomy X 95.0% 75.1 – 99.9 99.0% 97.6 – 99.7 XX 97.6% 94.8 – 99.1 99.2% 97.2 – 99.9 XY 99.1% 96.9 – 99.9 98.9% 96.9 – 99.8 XXX, XXY, XYY Limited data of these more rare aneuploidies preclude performance calculations. Verinata Health, Inc. Data on file. Original Publication: *Bianchi et al. Obstetrics and Gynecology, Vol 119, No. 5, May 2012

Large Clinical Trials Source: ISPD Position Statement April 2013

Sex Chromosome Analysis

Sex Chromosomes Aneuploidy Why?

Consideration of NIPT for Cystic Hygroma
Highly associated with common aneuploidies including monosomy X (Turner syndrome) that are associated with pregnancy loss and are medically significant at birth. Prevalence ~1:285 To study, we examined performance of NIPT for patients with cystic hygroma in the MELISSA study Prevalence is ~1:285 Bianchi, et al, Obstetrics and Gynecology, Vol 121, No. 5, May 2013 Cystic hygroma

MPS of Maternal Plasma DNA in Cystic Hygroma
Electronic clinical database of the MELISSA study was searched to identify women carrying singleton fetuses with cystic hygroma 113 cases were identified—69 (61%) had chromosome abnormalities, but 4 had abnormalities other than T21, T18, T13, or monosomy X Archived plasma samples were sequenced using updated chemistry Samples were classified for aneuploidy status of chromosomes 21, 18, 13, and presence or absence of monosomy X Bianchi, et al, Obstetrics and Gynecology, Vol 121, No. 5, May 2013

MPS and Fetal Nuchal Cystic Hygroma
Obstet Gynecol 2013;121:1057–62

Cystic Hygroma: Sequencing Results
113 cases 29/30 cases of T21 detected, 1 suspected 20/21 cases of monosomy X detected 10/10 cases of T18 detected 2/4 cases of T13 detected, 1 suspected None of the 44 euploid cases was called positive Review notes on low NCV case Bianchi, et al, Obstetrics and Gynecology, Vol 121, No. 5, May 2013

Conclusions and Practice Implications
Using sequencing of 4 chromosomes, 61/65 (94%) of aneuploid cases were detected and two more were suspected Overall, 107/113 (95%) of cases were correctly classified 4 cases were “other” and 2 aneuploidies were not detected NIPT for aneuploidy can be considered as an immediate noninvasive point of care test at time of sonographic diagnosis of cystic hygroma. Allow management to move forward for women who decline, do not have access, or have a contraindication to an invasive procedure.

Discordant Results Second Trimester Ultrasound Male Genitalia Case
VERIFY …… XY Amniocentesis ………46,XX WHAT NEXT???????????

Test Performance in “ Real World Population”
May 2013 Initial clinical laboratory experience in noninvasive prenatal testing for fetal aneuploidy from maternal plasma DNA samples Tracy Futch1, John Spinosa2,3, Sucheta Bhatt1, Eileen De Feo2, Richard P. Rava4 and Amy J. Sehnert5* DOI: /pd.4123 5, 974 patients Positive Predictive value of 99.8% Negative Predictive Value of 99.92% Turn around time 5.1 days Assay failure rate of 0.7%

Rolling 6 month window, ending May 2013
Quantity Not Sufficient 0.53% Cancelled by Phs/Pat 0.23% Sample received beyond stability 0.22% Gestational Age less than 10 Weeks 0.15% Sample Improperly Labeled 0.13% Test Valid for Singletons Only 0.09% Interfering Substances (too much cfDNA) 0.08% Duplicate Specimen 0.04% Improper sample type 0.03% NY State Permit Denied Internal Processing Error 0.01% Unable to isolate sufficient cfDNA Sample integrity compromised Sample lost or destroyed during shipping Expired Tube Test Cancellations: Rolling 6 month window, ending May 2013 Red text indicates a technical test failure © 2013 Verinata. Content is proprietary and confidential.

Key Differentiators – Clinical Test
Low assay failure rate and need for re-draw (<1%) Low TAT (Average TAT 4 days, 95% 8 days) Sample requirement – 1 tube (7-10 mL) maternal blood only Weeks Matter! Broadest validated test menu including sex chromosomes Accept samples from ART pregnancies including egg donors Result type – Clear and descriptive for physicians Non risk score Independent of other pre-test factors (i.e., gestational age, maternal age) © 2013 Verinata. Content is proprietary and confidential.

NIPT Test Comparison verifi® Verinata Harmony Ariosa MaterniT21
Sequenom NIPT Natera Result Types Aneuploidy Detected Aneuploidy Suspected No Aneuploidy Detected Risk score incorporating maternal, gestational age Positive Negative Assay Failure Rate <0.7% 4.6 – 4.9% 1% 5.9 – 12.6% Sample 1 tube maternal blood 2 tubes maternal blood 2-4 tubes maternal blood (best with paternal sample) Egg Donors Yes (with data) No Yes Test Menu T21, T18, T13 Optional sex chromosome aneuploidies (Published data) Y chromosome (optional)(not published) Mandatory sex chromosome aneuploidies (not published) Sex chromosome aneuploidies (only MX published) Published Clinical Validation Large-scale, blinded clinical validation Small, blinded clinical validation

NIPT is A GAME CHANGER

Effect of NIPT on Invasive Testing: EVMS

Invasive Procedure Update by Month (7/2010 through 3/2013)

Impact of invasive testing in Patients with a positive First or second trimester screen

NIpT Results from CFFM 6/1/2010 through 3/31/2013 Positive First and second Trimester Screen
Total patients seen 6/1/2010 – 3/31/2013: 808 Total procedures performed 6/1/2010 – 3/31/2013: 539 Total singleton, non-ONTD patients seen: 728 Total singleton, non-ONTD procedures performed: 500 6/2010 – 10/2011 10/2011 – 3/2013 Total patients seen 362 446 Total singleton, non-ONTD, patients seen 323 405 Total procedures performed 283 256 Total singleton, non-ONTD procedures performed 262 238 .

NIPT Status and Declining Invasive Testing

Changing trends in prenatal diagnosis the platt experience
2010 (%) 2011 (%) 2012 (%) 2013 (%) Amnio 8.0 6.0 4.2 Next slide CVS 4.0 3.9 3.0 Total 12.0 9.9 7.2

Changing trends in prenatal diagnosis the platt experience January through may by year
2010 (%) 2011 (%) 2012 (%) 2013 (%) Amnio 7.5 5.3 2.7 CVS 4.4 4.6 3.5 3.4 Total 11.9 12.1 8.8 6.1

Uptake of NIPT in women following Positive aneuploidy screening Chetty S, garabedian MJ, Norton ME. Prenat Diagn 2013; 33: Norton et al observed an increased uptake of NIPT following abnormal 1st ∆ screening compared with abnormal 2nd ∆ screening (56% vs 37%) This study revealed that women with a positive aneuploidy screening result are influenced by NIPT for their follow-up testing When the procedure-associated risk is eliminated, women may be less likely to decline testing

Uptake of NIPT in women following Positive aneuploidy screening Chetty S, garabedian MJ, Norton ME. Prenat Diagn 2013; 33: Rates of accepting/declining invasive PND vy socioeconomic characteristics Pre-NIPT (n=638) Post-NIPT (n=398) P value TOTAL ACCEPTING INVASIVE PND, N (%) Maternal age >35 yrs 301/638 (47.2) 185/349 (53.0) 156/398 (39.2) 94/234 (40.2) 0.012 0.002 Race/ethnicity, n (%) Caucasian 97/146 (66.4) 53/110 (48.2) 0.003 Hispanic 80/289 (27/7) 45/177 (25/4) 0.593 Asian/Pacific Islander 114/182 (62/6) 56/106 (52/8) 0.103 Other 10/21 (47/6) 2/5 (40.0) 0.759 Insurance Medicaid 71/258 (27.5) 42/158 (26/6) 0.835 Private 230/380 (60.5) 114/240 (47/5)

Uptake of NIPT in women following Positive aneuploidy screening Chetty S, garabedian MJ, Norton ME. Prenat Diagn 2013; 33: Rates of accepting/declining invasive PND vy socioeconomic characteristics Pre-NIPT (n=638) Post-NIPT (n=398) P value TOTAL DECLINING FURTHER TESTING, N (%) Maternal age >35 yrs 337/638 (52.8) 164/349 (47.0) 84/398 (21.2) 46/234 (19.7)) <0.001 Race/ethnicity, n (%) Caucasian 49/146 (33.6) 15/110 (13.6) Hispanic 209/289 (72.3) 56/177 (31.6) Asian/Pacific Islander 68/182 (37.40 13/106 (12.3) Other 11/21 (52.4) 0/5 (0.0) 0.033 Insurance Medicaid 187/258 (72.5) 42/158 (26.6) Private 150/380 (39,5) 41/240 (17)

California pdc experience with nipt
Data for patients with initial PDC visit (January – March 2013) Totals # of pts offered NIPT at PDC 3,400 # of PNS screen positive pts offered/requested NIPT 1,063 PNS screen positive patients accepting NIPT 454 PNS screen positive patients declining NIPT 609 # of patients offered/requesting NIPT for other indications (i.e., maternal age, US abnormality, US marker, etc.) 2337 Pts with other indications accepting NIPT 661 Pts with other indications declining NIPT 1676 # of patients coming to PDC with prior NIPT results through OB 147 TOTAL PATIENTS HAVING NIPT PERFORMED 1,262

Data for patients with initial PDC visit (January – March 2013) Totals Total # of NIPT positive results 53 Number of NIPT positive patients having diagnostic procedures: 29 T21 confirmed by CVS/amnio/other 16 T18 confirmed by CVS/amnio/other 3 T13 confirmed by CVS/amnio/other 45,X/other confirmed by CVS/amino/other 2 Normal karyotype (false positive) confirmed by CVS/amnio/other 5 SUB-TOTAL OF NIPT POSITIVE RESULTS W/ DIAGNOSIS

Data for patients with initial PDC visit (January – March 2013) Totals Total # of NIPT indeterminate results 13 Number of NIPT indeterminate patients having diagnostic procedures: 4 T21 confirmed by CVS/amnio/other T18 confirmed by CVS/amnio/other 1 T13 confirmed by CVS/amnio/other 45,X/other confirmed by CVS/amino/other Normal karyotype confirmed by CVS/amnio/other 2 SUB-TOTAL OF NIPT INDETERMINATE RESULTS W/ DIAGNOSIS

Official Society Statements

The National Society of Genetic Counselors (NSGC) currently supports
Noninvasive Prenatal Testing/Noninvasive Prenatal Diagnosis (NIPT/NIPD) as an option for patients whose pregnancies are considered to be at an increased risk for certain chromosome abnormalities. NSGC urges that NIPT/NIPD only be offered in the context of informed consent, education, and counseling by a qualified provider, such as a certified genetic counselor. Patients whose NIPT/NIPD results are abnormal, or who have other factors suggestive of a chromosome abnormality, should receive genetic counseling and be given the option of standard confirmatory diagnostic testing

cfDNA should not be part of routine prenatal lab assessment, but should be an informed patient choice after pretest counseling cfDNA should not be offered to low-risk women or women with multiple gestations – not yet sufficiently evaluated Negative cfDNA test result does not ensure an unaffected pregnancy Patient with a positive test should be referred for genetic counseling and offered invasive PND for confirmation cfDNA does not replace the accuracy and diagnostic precision of PND w/ CVS or amnio

screening method is most effective.
Reliable cfDNA screening methods have only been reported for trisomy 21 and 18. cfDNA screening results have been reported for trisomy 13 but the numbers are not large and efficacy appears to be less than for trisomies 21 and 18. cfDNA screening results have also been reported for sex chromosome aneuploidy and the efficacy is unacceptably low. There are insufficient data available to judge whether any specific cfDNA screening method is most effective. The tests should not be considered to be fully diagnostic and therefore are not a replacement for amniocentesis and CVS. Analytic validity trials have been mostly focused on patients who are at high risk. Efficacy in low risk populations has not yet been fully demonstrated. There is insufficient information to know how well the test will perform in multiple gestation pregnancies ISPD Position Statement April 2013

plasma specimen or there is test failure for other reasons.
In a proportion of cases there is insufficient fetal cfDNA in the maternal plasma specimen or there is test failure for other reasons. Specific independently developed laboratory minimum standards, quality control, proficiency testing and inspection requirements have not yet been developed for this testing. It has not been demonstrated that the test can be provided in a cost-effective, timely, and equitable manner to total populations ISPD Position Statement April 2013

Future Horizons

Future Horizons Large studies in low-risk populations
Studies in multiple gestations Detection of sub-chromosomal abnormalities cfDNA and pregnancy complications Treatment of genetic syndromes (Downs)

Cell-free Fetal DNA in Multiples
Presented at SMFM, Feb. 2013 Study Objective: Effects of Multiple Gestation on Aneuploidy Detection and the Relative Cell-free Fetal DNA (cffDNA) per Fetus About.com

Results: FF/Fetus Distribution for Twin and Singleton Pregnancies
© 2013 Verinata. Content is proprietary and confidential.

Results: Calculated FFs for Twins
Chorionicity Karyotype Total FF FF/Fetus Dichorionic (n=19) (Both male) 46,XY/46,XY 47,XY+21/46,XY 0.124 0.082 0.062 0.041 Dichorionic ([n=26) (different genders) 46,XY/46,XX 0.068 Monochorionic (n=3) 47, XY+18/47,XY+18 0.113 0.073 0.057 0.037 Singleton (n=160) 46,XY 0.126

Results: Deeper Sequencing (16 Twins)
NCVs for Trisomy 21 and 18 Increase with Deeper Sequencing Sequencing with more sequence tags/sample (180M versus 25M) reduces the standard deviation of the measurement and increases the NCV The measurement leads to more confidence at the same FF per fetus © 2013 Verinata. Content is proprietary and confidential.

Conclusions From the Study
Autosomal Aneuploidies in 2 Twin Cases Here Were Correctly Classified However, Deeper Sequencing is Required to Classify Samples from Multiple Gestations with High Confidence due to the Lower Fetal Fraction per Fetus The Deeper Sequencing Approach Provides a “Safety Net”, Thereby Reducing the Potential Number of False Negative Cases © 2013 Verinata. Content is proprietary and confidential.

The performance of screening for trisomy 21 and trisomy 18 by NIPT using chromosome-selective sequencing in a routine population is as effective as previously reported in high-risk groups with DR 99% and FPR 1%. Am J Obstet Gynecol 2012;207:374.e1-6.

On the basis of existing data, NIPT can potentially be used in universal screening for trisomies 21 and 18 in all singleton pregnancies and the main limiting factor for such widespread application of the test at present is the associated cost. Am J Obstet Gynecol 2012;207:374.e1-6.

Chromosomal Microarray for PND
N Engl J Med 2012; 367 (23):

Non-Invasive Subchromosomal Analysis
Noninvasive Detection of Fetal Subchromosome Abnormalities via Deep Sequencing of Maternal Plasma Anupama Srinivasan1, Diana W. Bianchi2, Hui Huang1, Amy J. Sehnert1 and Richard P. Rava1 The American Journal of Human Genetics, Volume 92, Issue 2, , 10 Jan 2013 Verinata’s MELISSA study was a large-scale, truly prospective and blinded clinical validation study which demonstrated the excellent clinical performance of the verifi® prenatal test. Over 60 U.S. centers participated in enrolling ‘high-risk’ patients with 1 or more indications of a high-risk pregnancy, such as AMA, ultrasound findings, positive serum screens, or family genetic history. A defining and unique characteristic of the MELISSA study is that all abnormal karyotyped samples were analyzed. This emulates real world testing where the fetal chromosome status is not known before testing. Also, the MELISSA study is the first and only study to date to perform sequencing analysis across all chromosomes at once. This demonstrates the true potential of the technology underpinning the verifi® test. [Transition to Next Slide]: Next we will dive into the details of the study

cfDNA and detection of 22q11
Clin chem. 2012; 58:

This work shows that in nonmosaic cases, it is possible to obtain a fetal molecular karyotype by MPS of maternal plasma cfDNA that is equivalent to a chromosome microarray and in some cases is better than a metaphase karyotype Am J Hum Genet 2013;92:167-76

Exciting Time Advancement in ultrasound technology Early & accurate diagnosis of fetal congenital abnormalities NIPT on cfDNA in maternal plasma Early screening of fetal karyotypic abnormalities Expand access for fetal intervention © 2013 Verinata. Content is proprietary and confidential.

How does Verifi® make pregnancy safer?

Nipt Making Prenatal Diagnosis Safer
1. NIPT broadens the available prenatal testing options for women 2. NIPT offers an alternative to invasive diagnostic testing for women with screen positive fetuses With high sensitivity and specificity for T18 and T21 (T13 performance somewhat poorer comparatively)

Nipt Making Prenatal Diagnosis Safer
3. NIPT offers reassurance to those women for whom invasive diagnostic testing was not an acceptable option 4. NIPT removes the risk of procedure-related risks and women may be less likely to decline testing

THANK YOU תודה

1st trimester contingent screening for T21 by biomarkers and maternal blood cell-free dna testing Nicolaides KH, et al. Ultrasound Obstet Gynecol 2013; doi: /uog (ahead of publication) Objective of study is to define cut-offs w/DRs and FPRs in T21 screening using maternal age and combinations of 1st ∆ biomarkers to determine which women should undergo contingent maternal blood cell-free DNA (cfDNA) testing March 2006 through May 2012, prospective analysis using biomarkers (NT, DV-PIV at 11+0 to 13+6 weeks, and ß-hCG, PAPP-A, PIGF, and AFP at 8+0 to 13+6 weeks

1st trimester contingent screening for T21 by biomarkers and maternal blood cell-free dna testing Nicolaides KH, et al. Ultrasound Obstet Gynecol 2013; doi: /uog (ahead of publication) Contingent screening offers 98% DR of T21 fetuses; overall invasive testing rate <0.5% can be potentially achieved through cfDNA testing to 36% combined test alone 21% using combined test + PIGF and AFP 11% using combined test + PIGF, AFP and DV-PIV Conclusion: Effective 1st trimester screening for T21 with DR of 98% and invasive testing rate <05% can be potentially achieved by contingent screening incorporating biomarkers and cfDNA.

Potential Reasons for Discordant Results
Technology Biology MPS counting statistics follow a normal distribution Classification cut-off sets likely false positive rate 3 standard deviations above mean cut-off yields 0.13% FP 4 standard deviations above mean cut-off yields 0.003% FP Placental mosaicism reflected in cfDNA (similar to CVS) Vanishing twin may be reflected in cfDNA Maternal Fetal fraction

Discordant Results Maternal aneuploidy Co-twin demise
Case Discussion Maternal aneuploidy NIPT: Detected XXX CVS: 46,XX Maternal karyotype: Mosaic 46,XX[44]/45,X[5]/47,XXX[1] Co-twin demise NIPT: Detected 13 Amnio: normal karyotype Co-twin demise reported in first trimester Low fetal fraction False negative T21

Discordant Results Confined placental mosaicism Maternal malignancy
Case Discussion Confined placental mosaicism NIPT: Detected 13 CVS FISH: mosaicism for trisomy 13 Cultured CVS: normal karyotype Amnio (FISH and cultured cells): normal karyotype Maternal malignancy NIPT: Double detected 13 and 18 (confirmed on repeat testing) Amnio: normal karyotype Patient diagnosed with cancer postpartum

Discordant Results Maternal aneuploidy Co-twin demise
Case Discussion Maternal aneuploidy NIPT: Detected XXX CVS: 46,XX Maternal karyotype: Mosaic 46,XX[44]/45,X[5]/47,XXX[1] Co-twin demise NIPT: Detected 13 Amnio: normal karyotype Co-twin demise reported in first trimester Low fetal fraction False negative T21

Summary verifi® Test Low assay failure rate and need for re-draw (<1%) Lowest TAT in industry (Average TAT 4 days, 95% 8 days) Sample requirement – 1 tube (7-10 mL) maternal blood only Weeks Matter! Broadest validated test menu including sex chromosomes Accept samples from ART pregnancies including egg donors Result type – Clear and descriptive for physicians Non risk score Independent of other pre-test factors (i.e., gestational age, maternal age) © 2013 Verinata. Content is proprietary and confidential.

Appendix

Product Profile Chromosomes Analyzed
21, 18, 13, and (Optional) X and Y Blood draw requirement 1 blood tube (7-10mL) Patient Eligibility Validated in high risk pregnancies Singletons at ≥10 weeks gestation Sample collection On-site collection kits, ambient shipping Turn-around time Average of 4 days with 95% within 8 days⃰ Clinical Support In-house genetic counselors for consultation with healthcare providers Cancellation Rate <1% (0.14% current rate) TAT and cancellation: emphasize strength re what’s important to OBs – TIME and patient & physician convenience

verifi® prenatal test Test Report
Red alert at top to highlight abnormal results Abnormal results are highlighted in red Comments included to provide additional guidance Test claims restated as reference Here is what the test report from the verifi® prenatal test looks like. The test provides a clear result and interpretation for each chromosome 21, 18, 13 and if ordered, sex chromosome analysis. The possible results for chromosomes 21, 18, and 13 are aneuploidy detected, no aneuploidy detected, or aneuploidy suspected (borderline). The possible results for sex chromosomes are detected or not detected (XX or XY). Any result with an aneuploidy detected or suspected will be flagged by a red alert at the top of the report. Abnormal results are highlighted in red, and comments are included to provide additional guidance. The test claims are also restated as reference on the report. All detected/suspected results will be called to the healthcare provider by a Verinata genetic counselor.

verifi® prenatal test Limitations
Test designed to detect full chromosomal aneuploidies, and has been validated for chromosomes 21, 18, 13, X and Y. Possibility test results might not reflect the chromosomes of the fetus, but may reflect chromosomal changes to the placenta or of the mother. Does not eliminate the possibility of other chromosomal abnormalities, birth defects, or other genetic disorders. Not currently for use in multiple gestations. © 2013 Verinata. Content is proprietary and confidential.

verifi® prenatal test Disclaimers
If definitive diagnosis desired, invasive procedures are suggested to confirm detected and suspected (borderline) results. Test results should always be used in the context of all available clinical findings. How the test is used is at the discretion of the healthcare provider. © 2013 Verinata. Content is proprietary and confidential.

Follow-up and Outcomes
All “Detected” and “Suspected” results are called out to a client by a Genetic Counselor Outbound Calls Collect relevant pre-test indication when calling result Indicate interest to collect follow-up information if/when available Call (or fax request) for follow-up at or near due date Inbound Calls Record information that is called from sites If discordance, file complaint and follow internal process

verifi® test Advantages
Non-invasive reassurance, available as early 10 weeks Not affected by maternal factors (like serum screen) Can be used in IVF and with egg-donor pregnancies Higher sensitivity and specificity compared to analyte screen Low (<1%) false positive rate Very low false negative rate TAT: Average of 4 days with 95% within 8 days⃰ Failure rate of <1%

Case Examples

Case 1 Primary Screen for High Risk Patients
Patient Profile: 33 year old woman at 10 weeks gestation with a history of a prior miscarriage affected with Down syndrome Genetic Counseling: Screening CVS/amniocentesis verifi® prenatal test Ultrasound Too early for conventional serum screening and detailed ultrasound and concerned about undergoing CVS/amniocentesis due to procedural loss rate verifi® prenatal test Results (option of sex chromosome analysis elected): Chromosome 21 – aneuploidy detected Chromosome 18 – no aneuploidy detected Chromosome 13 – no aneuploidy detected Sex chromosomes – no aneuploidy detected (XY)

Suggestive of trisomy 21 in fetus
Case 1 Counseling considerations verifi® prenatal test – chromosome 21 aneuploidy detected Received results prior to 12 weeks gestation Suggestive of trisomy 21 in fetus Patient now has information earlier in pregnancy and has more time to make informed decisions regarding testing options and pregnancy management.

Case 2 Secondary Screen for Screen Positive Patients
Patient Profile: 40 year old woman with 1 in 32 risk for Down syndrome on serum screening. Genetic Counseling: amniocentesis verifi® prenatal test ultrasound verifi® prenatal test Results: Chromosome 21 – no aneuploidy detected Chromosome 18 – no aneuploidy detected Chromosome 13 – no aneuploidy detected

Case 2 Counseling Considerations
Serum screen - screen positive for Down syndrome (1 in 32) Serum screening can have screen/false positive rate (FPR) in women 40+ of 20% (NOTE: FPR dependent upon type of screening utilized) verifi® prenatal test – no aneuploidy detected for chromosomes 21, 18 and 13 Normal ultrasound - reassuring but need to discuss limitations Patient comfortable declining amniocentesis and avoids risk of procedural loss

Case 3 High Risk Patient Considering CVS/Amniocentesis
Patient Profile: 38 year old woman with history of infertility who conceived via in vitro fertilization (IVF). Genetic Counseling: Screening Declined serum screening due to sub-optimal detection rates for trisomies CVS/Amniocentesis Considering invasive testing but fearful of procedural loss verifi® prenatal test Ultrasound verifi® prenatal test Results (option of sex chromosome analysis elected): Chromosome 21 – no aneuploidy detected Chromosome 18 – no aneuploidy detected Chromosome 13 – no aneuploidy detected Sex chromosomes – no aneuploidy detected (XX)

Case 3 verifi® prenatal test – no aneuploidy detected
Counseling Considerations verifi® prenatal test – no aneuploidy detected Normal ultrasound - reassuring but need to discuss limitations Patient comfortable declining invasive testing due to NIPT results and normal ultrasound. Procedural risks avoided.

Case 4 Patients Who Have Abnormal Ultrasound Findings
Patient Profile: 41 year old woman at 34 weeks gestation diagnosed with ultrasound findings of large echogenic kidneys and ventricular septal defect Negative integrated screen (1/800 DS; 1/5000 T18; negative ONTD) Genetic Counseling: Ultrasound findings suspicious for trisomy 13 Amniocentesis Patient declined as she would not alter course of pregnancy verifi® prenatal test verifi® prenatal test Results: Chromosome 21 – no aneuploidy detected Chromosome 18 – no aneuploidy detected Chromosome 13 – aneuploidy detected

Suggestive of trisomy 13 in fetus
Case 4 Counseling Considerations verifi® prenatal test – chromosome 13 aneuploidy detected Consistent with ultrasound findings Suggestive of trisomy 13 in fetus Patient continues to decline amniocentesis but now pregnancy management decisions can be made regarding delivery.

Potential Future Directions
Testing in low-risk population Further testing in multiple gestations Further study of mosaicism Sub-chromosomal abnormalities Other whole autosomes Twins – WIP, working on deeper sequences, mentioned in our original study and at SMFM in Diana Bianchi presentation. © 2013 Verinata. Content is proprietary and confidential.

Non-Invasive Subchromosomal Analysis
Noninvasive Detection of Fetal Subchromosome Abnormalities via Deep Sequencing of Maternal Plasma Anupama Srinivasan1, Diana W. Bianchi2, Hui Huang1, Amy J. Sehnert1 and Richard P. Rava1 The American Journal of Human Genetics, Volume 92, Issue 2, , 10 Jan 2013 Verinata’s MELISSA study was a large-scale, truly prospective and blinded clinical validation study which demonstrated the excellent clinical performance of the verifi® prenatal test. Over 60 U.S. centers participated in enrolling ‘high-risk’ patients with 1 or more indications of a high-risk pregnancy, such as AMA, ultrasound findings, positive serum screens, or family genetic history. A defining and unique characteristic of the MELISSA study is that all abnormal karyotyped samples were analyzed. This emulates real world testing where the fetal chromosome status is not known before testing. Also, the MELISSA study is the first and only study to date to perform sequencing analysis across all chromosomes at once. This demonstrates the true potential of the technology underpinning the verifi® test. [Transition to Next Slide]: Next we will dive into the details of the study

Sub-Chromosome Publication
Proof-of-Principle Study in AJHG Purpose of the Study: To determine sequencing analysis necessary to detect fetal subchromosomal abnormalities from a maternal blood sample Blinded Analysis of 11 Samples from MELISSA 10 cases with subchromosomal abnormalities Duplications, deletions, derivative chromosomes, mosaic cases Trisomy 20 was used as a control

Results All Subchromosomal Abnormalities Detected in Non-Mosaic Samples 7 Non-Mosaic Samples: All Subchromosomal Abnormalities Detected. For karyotypes with unknown genetic material, deep MPS identified both translocation breakpoint as well as chromosomal origin of unknown material Detected microdeletion as small as 300kb 4 Mosaic Samples: Subchromosomal Abnormalities not Detected. Non-detection due to lack of sequence in human genome, or post-zygotic error

Subchromosome Analysis
Example Sample from the study Multiple subchromosome abnormalities detected in one sample Laboratory personnel were blinded to karyotype

Results Abnormalities as small as 300kb detected
Abnormalities Detected by Deep MPS Concordant with Karyotype Abnormalities as small as 300kb detected Precise location of abnormalities concordant with karyotype

Results Additional Abnormalities Detected by Deep MPS Deep MPS provided additional detection of abnormalities NOT identified by karyotype Provides additional information not seen on karyotype All abnormalities < 1 Mb

Subchromosome Analysis
Proof-of-Principle Study Recently Published in AJHG Study Shows the Ability of Deep MPS Technology to Detect Fetal Subchromosomal Abnormalities Non- Invasively Deep MPS Can Provide Greater Resolution and Can Detect Fetal Subchromosomal Abnormalities at a Resolution of 100kb Across the Genome Given the Cost of the Method, a Deep MPS Test for Sub- Chromosome Abnormalities Could Be Available in the Near Future…

Center for Fetal Medicine Results 6/1/2010 through 3/31/2013
Follow-up patients from the California PDC screening program with positive 1st/2nd trimester screening results Total patients seen 6/1/2010 – 3/31/2013: 808 Total procedures performed 6/1/2010 – 3/31/2013: 539 Total singleton, non-ONTD patients seen: 728 Total singleton, non-ONTD procedures performed: 500 6/2010 – 10/2011 10/2011 – 3/2013 Total patients seen 362 446 Total singleton, non-ONTD, patients seen 323 405 Total procedures performed 283 256 Total singleton, non-ONTD procedures performed 262 238

NIPT Status and Invasive Procedure Uptake

Invasive Procedure Update by Month (7/2010 through 3/2013)

Center for fetal medicine: # of invasive procedures 18 months prior & 18 months after introduction of nipt

January through May each year
Center for fetal medicine: # of invasive procedures 18 months prior & 18 months after introduction of nipt January through May each year

The Verifi® prenatal test – Making a difference in patient safety
Lawrence D. Platt, MD Tel Aviv July 2,2013

The Verifi® prenatal test – Making a difference in patient safety

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The Verifi® prenatal test – Making a difference in patient safety

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