7/1/2016 Non-invasive prenatal screening (NIPS): Test Validation, Performance, Interesting Cases Presented by: Jim Waurin, MS, CG(ASCP)CM June 24, 2016 Disclosures I am an employee of Quest Diagnostics. I have stock in Quest Diagnostics. 2 Agenda Overview QNatal™ Advanced Verification and Validation QNatal™ Advanced Performance Interesting Cases 3 1 7/1/2016 Chromosomal aneuploidy Common autosome • Trisomy 21 (Down syndrome): 47, XX,+21 • Trisomy 18 (Edwards syndrome): 47, XX,+18 • Trisomy 13 (Patau syndrome): 47, XX,+13 Common sex chromosome • Monosomy X (Turner syndrome): 45, X • Triple X (Triple X syndrome): 47, XXX • Klinefelter (Klinefelter syndrome): 47, XXY • XYY (XYY syndrome): 47, XYY Fetal sex can be opted out. 4 Chromosomal microdeletions Microdeletion syndrome - Characteristics vary based on the size and location of the deletion • DiGeorge syndrome: 22q • 1p36 syndrome: 1p • Angelman syndrome: 15q • Prader-Willi syndrome: 15q • Cri-du-chat syndrome: 5p • Wolf-Hirschhorn syndrome: 4p • Jacobsen syndrome: 11q • Langer-Giedion syndrome: 8q Microdeletion syndromes can be opted out. 5 Chromosomal aneuploidy and microdeletions: Frequency Location Syndrome Name Estimated Frequency of Live Birth Chromosome 21 Down Chromosome 18 Edwards 1/6,000 Chromosome 13 Patau 1/7,000-33,000 Chromosome X,Y X, XXX, XXY, XYY 1/1700 22q* DiGeorge 1/4,000 1p* 1/800 1p36 1/10,000 15q* Angelman 1/20,000 15q* Prader-Willi 1/20,000 5p* Cri-du-chat 4p* Wolf-Hirschhorn 1/50,000 11q* Jacobsen 1/100,000 8q* Langer-Giedion Rare 1/50,000 *Microdeletion National Institutes of Health. February 2014. http://ghr.nlm.nih.gov. Accessed February 11, 2015. 6 2 7/1/2016 QNatal® Advanced: Platform Discussion Massively Parallel Shotgun Sequencing (MPSS) platform is used to detect maternal and fetal cfDNA MPSS can eludicate the chromosome of origin for a particular DNA fragment (see next slide) MPSS cannot differentiate between maternal and fetal DNA fragments Bioinformatic analysis is used to determine the representation of fetal cfDNA GC normalization: routine correction to account for suboptimal amplification of GC-rich regions Bianchi DW. Nat Med. 2012;18(7):1041-1051.; Bianchi DW et al. Obstet Gynecol. 2012;119(5):890-901.; Benn P. Prenat Diagn. 2012;32(1):1-2.; Zhao C et al. PloS One. In press 7 QNatal® Advanced summary Our laboratory-developed NIPS Can be performed as early as 10 weeks’ gestation At any gestational age thereafter without restriction Whole genome massively parallel shotgun sequencing (MPSS) Measures and reports fetal fraction as either sufficient or insufficient for analysis using a 5% cut-off (Soon to change to reporting an actual numeric value.) Binary positive/negative result for core trisomies (T21, T18, T13) Clear reporting of sex chromosome aneuploidy and microdeletions as additional, incidental findings Opt out selections for microdeletions and fetal sex 7-day turn-around-time (TAT) ~1% redraw rate based on data from 10,000 specimens 8 QNatal® Advanced validation and verification Assay Development • 1,288 total blinded samples • All correctly identified as unaffected Validation and Verification Study Design • • 2,752 total samples comprised of Trisomy 21 (n=100), Trisomy 18 (n=34), Trisomy 13 (n=15), and Turner syndrome (n=1) Verification (n=2,085) Trisomy 21: 69 Trisomy 18: 20 Trisomy 13: 17 • Detection (sensitivity) rate = 100% 9 3 7/1/2016 QNatal® Advanced verification and validation Validation and Verification Study Design Cont’d • Singleton validation (n=552) • Trisomy 21: 21 Trisomy 18: 10 Trisomy 13: 1 45,X: 1 Twin validation (n=115) Trisomy 21: 10 Trisomy 18: 4 Trisomy 13: 1 • Detection (sensitivity) rate = 100% 10 QNatal® Advanced validation and verification: Data summary Complete discrimination between affected and unaffected pregnancies In both singleton and twin gestations, all trisomies had Zscores >8 All unaffected pregnancies had Z-scores <4 Single 45,X sample was also correctly identified Singletons # Positives/ # Specimens Trisomy 18 Trisomy 13 45,X 90/2637 30/2637 20/2637 1/2637 90/90 30/30 20/20 1/1 Sensitivity (%) >99.9 >99.9 >99.9 >99.9 Specificity (%) >99.9 >99.9 >99.9 >99.9 4 Positives Detected Trisomy 21 Z-Score: Number of standard deviations from the mean (average) Z-Score (chromosome 21) = (chromosome 21% - mean chromosome 21%) (standard deviation chromosome 21%) 11 QNatal® Advanced positive rate: 1st 10,000 Specimens Data presented at FIGO on October 9, 2015 Positive Test Result Population Prevalence Positive Findings Observed Prevalence Singleton Twin Trisomy 21 0.54% 98 5 1.03% Trisomy 18 0.21% 35 1 0.36% Trisomy 13 0.07% 20 1 Sex Chromosome Aneuploidy 0.10% 17 0 0.17% Microdeletions (DiGeorge) 0.30% 1 0 0.01% Cumulative positive rate 178 0.21% 1.78% Anderson B, K Zhang K, Nguyen Q, et al. An automated, non-invasive prenatal screening assay (NIPS) for trisomy 21,18,13 in singleton and twin gestations [FIGO abstract FCS79.3.]. Int J Gynaecol Obstet. 2015;131(suppl 5): E264 12 4 7/1/2016 QNatal® Advanced PPV: Current to Date (>25,000 Specimens) Singleton gestation Aneuploidy Trisomy 21 Trisomy 18 Trisomy 13 Sex chromosome Microdeletions PPV 99% 89% 71% 93% 100% n 80 36 21 14 2* PPV 100% n/a n/a n/a n/a n 3 3** 2** n/a*** 0 PPV 100% 67% 100% n/a n 10 3 1 0 Twin gestation Aneuploidy Trisomy 21 Trisomy 18 Trisomy 13 Sex chromosome Microdeletions Sex Chromosome Aneuploidy Aneuploidy 45,X 47,XXX 47,XXY 47,XYY *22q11.2 (DiGeorge) syndrome **pending outcome ***not performed for multiple gestation 13 Positive and Negative Predictive Value (PPV and NPV) Unlike sensitivity and specificity, PPV and NPV are dependent upon condition prevalence PPV: percentage of patients with a positive test who truly have the condition PPV (%) = (true positive) (true positive + false positive) x 1000 PPV for NIPS is lower in the general pregnancy population, as the incidence of chromosomal aneuploidy is lower in these patients: In a test with 99.9% sensitivity and specificity 20 years • 1/800 risk for T21 at 10 weeks • PPV = 55.6% 25 years • 1/710 risk for T21 at 10 weeks • PPV = 58.5% 35 years • 1/470 risk for T21 at 10 weeks • PPV = 84.4% 40 years • 1/50 risk for T21 at 10 weeks • PPV = 95.3% Wang JC. Genet Med. 2014. doi: 10.1038/gim.2014.92. 14 Clinical complications of NIPS: False positives and negatives Confined placental mosaicism (CPM) Co-twin demise/vanishing twin Fetal mosaicism Maternal mosaicism Maternal malignancy Maternal benign masses (e.g., uterine fibroids) Collection/laboratory error Directly related to prevalence (positive and negative predictive value) 15 5 7/1/2016 Improving performance of QNatal® Advanced Laboratory-developed test License agreement with Sequenom Laboratories Validated on Illumina HiSeq v4 platform Increased capacity Improved MPSS reagents Totally automated (manual procedures removed) Consistent Performance Plasma separation Extraction Library quantitation and dilution Advanced, in-house biostatistics and bioinformatics 16 Case 1 QNatal® Advanced order received on 07/15/2015 Indication: Advanced Maternal Age Patient Demographics – 36 year old 11w 6d gestation 1 fetus listed Reported on 07/28/15 – Positive, T21 Positive, T13 Our assumption at this point was that a concurrent aneuploidy would have been embryonically lethal 17 Case 1 (cont.) Z-scores – Trisomy 21: 11.8 Trisomy 13: 5.9 Fetal Fraction: 7% All other autosome and sex chrom Z scores within normal range Clinical Correlation – Originally a twin gestation Co-twin demise at about 9 weeks gestation Follow-up Spontaneous abortion of surviving twin. Unfortunately, cytogenetics studies were not performed 18 6 7/1/2016 Hypotheses The lower Z-score for chromosome 13 (5.9) could be attributed to trisomy 13 in the co-demise twin, which had occurred about 3 weeks earlier The higher Z-score for chromosome 21 (11.8) could be attributed to trisomy 21 in the surviving twin, which was still viable at the time of draw 19 What do we know about twins? Vanishing twins – Associated with Fertility procedures and Advanced Maternal Age (AMA) Fetuses at higher risk for aneuploidy Extrapolating from aneuploidy observed in POC specimens (~60%), approximately 0.11% of NIPS involve a chromosomally abnormal vanishing twin.* This is reported to account for 15% of false positive NIPS** Fetal fraction indistinguishable from viable twin gestations *Curnow, K. et.al, Detection of triploid, molar, and vanishing twin pregnancies by a single-nucleotide polymorphism-based noninvasive prenatal test. AJObGyn, Nov 7, 2014 **Futch T, Spinosa J, Bhatt S, de Feo E, Rava RP, Sehnert AJ. Initial clinical laboratory experience in noninvasive prenatal testing for fetal aneuploidy from maternal plasma DNA samples. Prenatal Diagn 2013;33:589-74. 20 Case 2 QNatal® Advanced order received on 11/04/2015 Indication: Abnormal US Patient Demographics – 26 year old 25w 3d gestation 1 fetus listed Reported on 11/18/15 – Positive, T13 (Z-score 54.59) Abnormally high Z-score for chromosome 7 (Z-score 63.67) Fetal Fraction – 22% 21 7 7/1/2016 Case 2 (cont.) Patient had amnio. Results were 46,XX Maternal microarray, normal Patient delivered at 37 weeks Baby small, but otherwise healthy. Spent 1 week in NICU. Placental pathology – 35% infarcted tissue and fibrin deposits-slightly small 22 Questions?? 23 Above all: It’s a Screening Test 24 8
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