Noninvasive Prenatal Testing (NIPT): The Next Best Aneuploidy Screen?

Noninvasive Prenatal Testing (NIPT): The Next Best Aneuploidy Screen? Becky Crosetto, MGC, LCGC Maureen Cantwell, MS, LCGC Regional Maternal-Fetal Medicine

If you currently do not know a lot about this topic, you are not alone Survey of OB providers (87% MDs & Dos; 11% NPs, RNs, & CNMs) Knowledge level not high 85% Sayers et al., 2011

Prevalence of Aneuploidy Overall (live birth, IUFD, termination): 1 in 228 (0.4%) 16% 5% 8% 5% 13% 53% Down syndrome Trisomy 18 Trisomy 13 45,X XXX, XXY, XYY Other Wellesley et al., 2012

Screening and invasive diagnostic testing for aneuploidy should be available to all women who present for prenatal care before 20 weeks of gestation regardless of maternal age.

Commonly Utilized Screens Test 1 st Trimester 2 nd Trimester 10 11 12 13 14 15 16 17 18 19 20 Sequential Screen Serum Integrated Screen Quad Screen NT, PAPP-A, hcg PAPP-A AFP, hcg, ue3, DIA AFP, hcg, ue3, DIA AFP, hcg, ue3, DIA

Pregnancy Risk Screens Estimate Risks Based Largely on Phenotypic Factors hcg AFP ue3 Inhibin PAPP-A NT Verinata

Noninvasive Prenatal Tests Estimate Risks Based on DNA Verinata

Sources of Fetal DNA in Maternal Blood Cells (~1 fetal cell per 1 billion total cells) Persist between pregnancies Cell-free DNA (Not fetal per se) 150-200 base pairs in length By 10 weeks gestation ~90% of total is maternal o primarily from apoptosis of blood cells ~10% is from the pregnancy o primarily from apoptosis of placental cells Half life < 20 minutes Undetectable < 2 hours postpartum Has been used for years for fetal RhD genotyping

Reduced risk to fetus (Available to all women) Goals of NIPT Reduced anxiety Reduced false positives Increased detection Verinata

Performance Comparison CVS Amnio Sequential NIPT Timing 11-13 weeks 16 weeks 10-22 weeks 10 weeks Procedure Risk Sensitivity Specificity Invasive Invasive Ultrasound and maternal blood 1% miscarriage >99% for aneuploidy >98% for aneuploidy (FPR <2%) 0.1% miscarriage >99% for aneuploidy >99% for aneuploidy (FPR <1%) None to pregnancy 90% for Down syndrome 95% for Down syndrome (FPR 5%) Maternal blood None to pregnancy >98% for Down syndrome >99% For Down syndrome (FPR <0.5%) Turn around <2 weeks <2 weeks <1 week <2 weeks

Sequencing Technology Explodes < 10 per 1 million base pairs by January 2012 Method Base pairs sequenced per run Time Sanger Tens of thousands < 1 day Next Generation Hundreds of billions (our genome is 3 billion bp in length) days

1998 2008 2011 NIPT Timeline

2012 Noninvasive prenatal testing for fetal trisomies in a routinely screened first-trimester population KJ Nicholaides et al.

Companies Offering NIPT Launching early 2013

NIPT Technology Library Preparation Cluster Generation Massively Parallel Sequencing Mapping Reads Quantifying Reads Interpreting Data Reporting

Library Preparation PCR primers and indexes added to DNA sample Several libraries will be pooled (run in parallel), so each given unique index Pooled libraries loaded into flow cell cfdna PCR primer Index www.streck.com/product www.technologyreview.com www.dkfz.de/gpcf/illumina_hiseq_technology.html

Types of Libraries Shotgun sequencing (Sequenom, Verinata) All fragments are run (several million total) Targeted sequencing Fragments containing specific sequences are selected before run via PCR using specific primers Selection of nonpolymorphic loci (Ariosa) ~400 loci per chromosome of interest Each 56 base pairs in length Selection of polymorphic SNPs (Natera) SNP = single nucleotide polymorphism o characterized, common type of genetic variation among people > 10,000 total over all chromosomes of interest

Cluster Generation Single strand library DNA hybridizes to complementary oligonucleotides anchored to the flow cell s surface PCR bridge amplification results in clusters of DNA Reverse strands are cleaved and washed away Sequencing primer is hybridized to the DNA Denature Bridge Amplification Denature Cleave Illumina

Massively Parallel Sequencing Clusters are sequenced simultaneously One by one, fluorescently-labeled reversible terminator nucleotides are incorporated and fluoresced by laser Each base (A, T, C, G) has its own color Each cycle is captured digitally Illumina

Factors Influencing Sequencing Depth of sequencing coverage Average number of times each base pair is sequenced GC content G-C nucleotide pairs held together by 3 hydrogen bonds and A-T pairs by two When GC content high or low, DNA forms hairpins and polymerase does not bind well In some types of data analysis a correction factor is needed 50 45 40 35 % GC Content by Chromosome 4 13 5 6 3 18 8 2 7 12 21 14 9 11 10 1 15 20 16 17 22 19 Natera

Mapping Reads 1 read = 1 fragment of contiguous nucleotide sequence Chromosomal origin of each fragment is determined by comparison to genome map DNA of known origin

Quantifying Fragments (8%) (1.5% of total genome) 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 X Y

cfdna Maternal + Fetal Reference Chromosome(s) If expected amount: disomy If extra amount: trisomy = Thousands of chromosome 21 cfdna fragments

) Fetal Fraction (%) Fetal Fraction (%) Importance of Fetal Fraction Relatively constant 10 weeks gestation 70 50 30 20 10 5 Norton et al., 2011 Maternal obesity lowers fraction 1 Likely due to apoptosis of adipose tissue and larger blood volume Correction factor or cut-off needed? 70 50 30 20 10 5 1 70 50 Euploid Down syndrome Euploid 100 150 200 250 300 350 Maternal weight (lbs) Palomaki et al., 2011

Trisomy increases total DNA by only a small amount Accurate analysis requires 4% (currently) Fetal fraction Fetal Karyotype Chromosome 21 DNA as % of total DNA Any (0-100%) euploid 1.5 0% trisomy 21 1.5 4% trisomy 21 1.53 10% (typical) trisomy 21 1.58 1.5% of our genome is chromosome 21 DNA When a pregnancy has trisomy 21 and the fetal fraction is 10%, the amount of chromosome 21 DNA increases to 1.6% of the total DNA: = (%maternal)(%chr21)(#chr21 to #expected) + (%fetal)(%chr21)(#chr21 to #expected) = (90%)(1.5%)(2/2) + (10%)(1.5%)(3/2) = 0.0135 + 0.00225 = 0.01575 = 1.6%

Normalizing Quantified Fragments Intra- & inter-run variation in the chromosomal distribution of reads can obscure aneuploidy Therefore, # reads mapping to chromosome of interest is normalized to # reads mapping to reference chromosomes # of standard deviations from the mean is calculated If reference chromosomes = all chromosomes: o Called Z-score o Correction factor for GC content needed If reference chromosomes = chromosomes with biochemical behaviors similar to chromosome of interest: o Called Normalized Chromosome Value (NCV) o If GC content similar, correction factor not needed

Normalized Chromosome Value (Verinata) Z-Score (Sequenom, Ariosa) Bianchi et al., 2012 Palomaki et al., 2011

Analyzing Allelic Distribution Maternal + fetal cfdna Buffer separates cells (virtually all maternal) from cfdna Maternal cellular DNA Targeted SNP sequencing Targeted SNP sequencing Multiple hypotheses are generated for each chromosome of interest, taking possible gamete crossover events into account Natera Algorithm subtracts out maternal allelic distribution (and paternal if available), taking possible crossovers into account, and selects result with highest significant likelihood Quantification of reads not needed Mapping not needed because location of SNPs known

Sponsor Sequenom Verinata Ariosa Natera Publication Palomaki et al., 2012, 2011 Bianchi et al., 2012 Norton et al., 2012 Zimmerman et al., 2012 Blinded nested casecontrol study Prospective blinded nested case-control study Prospective blinded cohort study Proof-of-principle study, algorithm blinded to karyotypes Eligibility High risk, 9-22 wks, singleton, undergoing invasive procedure High risk, 8-22 wks, singleton, undergoing invasive procedure High (primarily) and general risk, 10-39 wks, singleton, undergoing invasive procedure Singleton, 9wks, aneuploids confirmed by invasive procedure Method Shotgun MPS (4-plex, 36-bp reads, 5 million reads) Shotgun MPS (6-plex, 36-bp reads, 2-14 million reads) Targeted MPS (oligonucleotides) (56-bp reads) Targeted MPS (SNPs) (10 million reads, 6.5 million mapping to targeted SNPs) Analyzed cohort 1,971, 286 aneuploids 532, >155 aneuploids 3,007, >119 aneuploids 145, 20 aneuploids # True positives T21: 209/212 T18: 59/59 T13: 11 /12 Sensitivity % (95% CI) Specificity % (95% CI) T21: 98.6% (95.9-99.7) T18: 100% (93.9-100) T13: 91.7% (61-99) T21: 99.8% (99.4-99.9) T18: 99.7% (99.3-99.9) T13: 99.0% (98.5-99.5) T21: 89/89 T18: 35/36 T13: 11/14 45X: 15/16 T21: 100.0% (95.9-100) T18: 97.2% (85.5-99.9) T13: 78.6% (49.2-99.9) 45X: 93.8% (69.8-99.8) T21: 100.0% (99.1-100) T18: 100.0% (99.2-100) T13: 100.0% (99.2-100) 45X: 99.8% (93.7-99.9) T21: 81/81 T18: 37/38 T21: 100% (95.5-100) T18: 97.4% (86.5-99.9) T21: 99.97% (99.8-100) T18: 99.93% (99.75-99.98) T21: 11/11 T18: 3/3 T13: 2/2 45X: 2/2 XXY: 2/2 No call rate 0.8% 2.8% 0% 12.6% > 99% for T21, T18, T13, 45X, XXY > 99% for T21, T18, T13, 45X, XXY Failure rate 5.3% 3.0% 4.6% Majority of no calls

Z-score / NCV Z-score / NCV Challenge of Threshold Results Unaffected Affected Verinata VS. Trisomy Diploid Dual Threshold Method (Verinata) Single Threshold Method (Sequenom)

Eligibility High risk High risk Method Shotgun MPS Shotgun MPS High and general risk Oligo targeted MPS? SNP targeted MPS Analysis Quantification, Z-score Quantification, NCV Quantification, Z-score Allelic distribution Report Positive (if Z-score >3) Negative (if Z-score <3) Aneuploidy Detected (if NCV >4) Aneuploidy Not Detected (if NCV <2.5) Aneuploidy Suspected (if NCV 2.5-4) High Risk >99% Low Risk <0.1% In Between?

Sample Lab Reports

Some Limitations Diagnostic testing, = Advanced screening Diagnostic testing is recommended following all positive results Chance pregnancy affected 50% Negative result does not ensure unaffected pregnancy Placental mosaicism (such as seen with CVS) Maternal mosaicism Unrecognized or vanishing twin Some screens not appropriate if egg donated Generating post-screen risks (continued )

Unaffected Affected Care must be taken not to generate unrealistic risks Risks only relevant in area of overlap May be reasonable to generate likelihood ratios in this area Not clear at this time whether post-test risk calculation is appropriate e.g. not yet determined whether NIPT behaves similarly in highrisk and general-risk populations

Ethical Considerations Informed consent for a complex test without fetal risk Just a tube of blood Will NIPT be lumped into other blood draws? Will ramifications be adequately presented by providers? Will ramifications be adequately considered by patients?

Future Directions Where will NIPT fit in? Replace or accompany serum screening? NT screening? Validation in the general population ACOG states NIPT should not be offered to general-risk patients at this time Validation in multiple gestations ACOG states NIPT should not be offered to patients with multiples at this time Determine whether feasible technology for detection of chromosomal mosaicism, subchromosomal anomalies (microdeletions and duplications)

Published Guidelines International Society for Prenatal Diagnosis (2011) With suitable genetic counseling MPS can be helpful for women who may have been determined to be high risk. Before routine MPS-based population screening for fetal Down syndrome is introduced additional trials are needed. This test is not fully diagnostic and therefore constitutes an advanced screening test. Accordingly, confirmation of MPS positive results through invasive testing would still be required.

NSGC currently supports NIPT 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 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. (2012)

Patients at increased risk of aneuploidy can be offered testing (AMA, associated u/s anomaly, prior affected pregnancy, positive risk screen, parent carries robertsonian translocation involving chromosome 13 or 21) Should not be part of routine prenatal laboratory assessment, but an informed patient choice after appropriate pretest counseling. Should not be offered to low-risk women or women with multiple gestations. If a fetal structural anomaly is identified on ultrasound, invasive prenatal diagnosis should be offered. A patient with a positive test result should be referred for genetic counseling and offered invasive prenatal diagnosis for confirmation of test results. Does not replace the accuracy and diagnostic precision of CVS or amniocentesis, which remain an option for women.

Thank you! Becky Crosetto, MGC Licensed & Certified Genetic Counselor Maureen Cantwell, MS Licensed & Certified Genetic Counselor Regional Maternal-Fetal Medicine 253-403-9200

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