Prenatal screening using circulating cell

/6/4 Prenatal screening using circulating cell free DNA in maternal plasma 3 rd Annual Symium on Molecular Pathology Sept 7, 4 Glenn E. Palomaki, PhD Associate Professor Department of Pathology and Laboratory Medicine Women & Infants Hospital Alpert Medical School of Brown University Providence, Rhode Island (gpalomaki@ipmms.org) Disclosures Employee of Women & Infants Hospital of Rhode Island, with all grants/contracts, honorarium and consultant fees paid to WIH Grant / Research Support: 8/: Co PI of InFANet Study Sequenom 3/: PI of DNAFirst Study Natera Salary / Consultant Fees: Beckman Coulter, PerkinElmer, Ansh Labs Honorarium / Expenses: Beckman Coulter, Ansh Labs, Natera, Celula Stocks / Bonds: None IP / Royalty: None Women & Infants BROWN Outline History of prenatal screening for syndrome Review of testing cell free DNA in maternal circulation for syndrome Testing maternal plasma DNA for other disorders Trisomy 8 / 3 syndrome in twins Fetal sex and sex aneuploidies microdeletions Professional recommendation for high risk testing DNA testing for aneuploidy in the general population Risk of aneuploidy in pregnancies with a no call syndrome screening: Maternal age Screening test: How old are you? Simple, inexpensive, reliable, early but not so good! At a 3 yo cut off 3% to % DR % to % FPR Syndrome 3 3 4 4 Maternal age (completed years) syndrome screening: Integrated Prenatal screening in the US in combined + quadruple = integrated test At a : risk cut off 9% DR % FPR Syndrome Type of test Labs Median N Number (%) st trimester 34 3, 6,69 ( 9) nd trimester,38,77,4 ( 6) st & nd trim 3 4,76 83,46 ( ) All 3 3,66,963,9 () -3 - - 3 4 6 7 's syndrome risk (:n) Palomaki GE et al., Archives Path Lab Med 3 Represents about 7% of all US pregnancies

/6/4 Original finding: circulating cell free DNA Placenta Maternal plasma Maternal blood cells Lancet Aug 997 Both cell-free fetal and cell-free maternal DNA circulate in maternal plasma. Fetal DNA comes primarily from the placenta. Maternal DNA comes primarily from maternal blood cells. Cell-free fetal and maternal DNA circulate in maternal plasma as relatively short fragments (- base pairs) and represent the entire genome. Fetal DNA is about % (usually -%) of the total circulating cell-free DNA in maternal plasma. Chromosome contribution to the genome Schematic illustration: Detecting syndrome 3 8% Size (MBases).% 8 6 4 Size (%) 3 4 6 7 8 9 346789 X Y Chromosome Chiu R W K et al. PNAS 8;:48 Schematic illustration (cont) Schematic illustration (cont) To test an individual: Determine the % of sequenced fragments that map to chromosome for that person and compare that to the mean %, as a z score. Chiu R W K et al. PNAS 8;:48 %chr = / = % % sequences mapped to chromosome in euploid in test sample..49.48..9.3.49. ±. z = z = z = z score calculation (.9.)..9. +4.

/6/4 Schematic illustration (cont) Sequenom Ctr for Mol Med San Diego, California Commercial companies offering clinical testing euploids trisomy cases Verinata Health Redwood City, California Ariosa Diagnostics San Jose, California Lifecodexx AG Konstanz, Germany PrenaTest Beijing Genomics Institute Shenzhen, China NIFTY Test Chiu R W K et al. PNAS 8;:48 Natera Inc. San Carlos, California Berry Genomics Beijing, China BambniTest Commercial methodologies syndrome screening: ccf DNA Company (Country) Sequencing Interpretation Berry Genomics (China) Shotgun Counting BGI (China) Shotgun Counting LifeCodexx (Germany) Shotgun Counting Sequenom (US) Shotgun Counting Verinata (US) Shotgun Counting Ariosa (US) Targeted Counting Natera (US) Targeted Pattern matching ccf DNA testing of maternal plasma Tests involve next generation sequencing (NGS) Very high performance 98 99% DR <.% FPR Syndrome Palomaki GE, GIM.3.3.4.4.. Chromosome % Detection of syndrome (T): All T Study FPR (%) No calls DR (%) No call Palomaki 3/,47 (.) 3/,697 (.8) 9/ (98.6) Ashoor / 3 ( ) / 4 (.7) / () Bianchi / 3 ( ) 3/ 3 (4.3) 89/ 89 () Norton /,887 (.) 48/3,8 (4.6) 8/ 8 () 3 Nicolaides 3 / 4 ( ) 3/ 4 (.4) / () Pergament 4 / 86 ( ) 93/, (8.8)* 8/ 8 () 9* All 4/,34 (.) 9/7, (4) / (99.) * A second Accounting sample and/or for no-calls paternal sample DR = may /3 resolve or many 96.6% no calls Conclusions Detection rate is about 98% 98 of syndrome fetuses tested will have a itive result; one will be missed and another will be a no-call. False itive rate is about.% or less Only in normal fetuses will have a itive DNA test for syndrome. Failure rate ranges from < % to % Depending on the test, between and of every women will have a test result that does not provide useful information about the woman s syndrome risk 3

/6/4 Will plasma DNA testing ever be diagnostic? The circulating cell free DNA is from the mother and the placenta, not the fetus and, some no calls will also occur. A recent study compared CVS with subsequent amniocentesis results in,673 cases (Grati FR, GIM 4) False atives occur when the defect is confined to the fetus; the placenta is normal :3 (.7%) for syndrome (DR < 99.3%) :7 (.%) for syndrome plus T8 and T3 : 68 (.4%) with the addition of monosomy X False itives occur when the defect is confined to the placenta; the fetus is normal. Depends on % abnormal cells : to :7 for syndrome : to :4 for Done syndrome plus T8 and T3 : to : with the addition of monosomy X Detection of Edwards syndrome (T8): T8 Study FPR (%) DR (%) No call Palomaki /,688 (.3) 9/9 () 3 Ashoor / 3 ( ) 48/ ( 96) Bianchi / 46 (.) 3/37 ( 9) Norton /,888 (.) 37/38 ( 97) 4 Nicolaides 3 / 9 ( ) 3/ 3 () Pergamant 4 / 96 ( ) / () 7 All 9/,9 (.) 7/ (97.6) 6 Accounting for no-calls DR = 7/8 or 9% Detection of Patau syndrome (T3): T8 Study FPR (%) DR (%) No call Palomaki 6/,688 (.9) / ( 9) Bianchi 3/ 488 (.) /4 ( 79) Ashoor 3 /,838 (.) 8/ ( 8) Nicolaides 3 / 9 ( ) / () Pergament 4 / 86 ( ) / () All /4,37 (.) 43/49 ( 88) 4 Accounting for no-calls DR = 43/3 or 8% Professional Practice guidelines The guidelines generally agree that Sequencing of cell free DNA is sensitive and specific for trisomies of chromosome, 8 and 3 Testing should be offered to high risk pregnancies Patient and provider education is important Insufficient data for twins Positive results followed up by offer of invasive testing Testing should not be offered to the general pregnancy population ( low risk ) until more information is available ACMG allows for such testing in the general population Includes: ACOG, NSGC, ISPD, ACMG, SOGC ccfdna as a secondary screening test Routine prenatal screening test Low risk High risk Offer invasive testing and karyotype Positive or Test ed syndrome Other outcome 98 syndrome testing: High risk setting (DR=99%, FPR=.%, No call=%) DS, High risk (:49) 4,9 4,84 49 Routine prenatal care Neg ccfdna testing (high detection, few false itives) 8 (.%) Offer Dx testing : (98:) (.%) Offer Dx testing :49 (:8) 4,84 (96.9%) Routine care :4,8 4

/6/4 Chromosome (GCRM z-score) syndrome in twin pregnancies - A Canick JA. Prenat Diagn concordant Most methods WILL NOT KNOW they are testing twins! discordant 4 6 Fetal Fraction (%) Zygosity and fetal fraction in twins Most twins are now dizygous, many due to IVF Detection in monozygotic twins is not in question Several groups have demonstrated methods to estimate fetal fraction in dizygotic pregnancies Zheng, PLoS One, 3; Leung Prenat Diagn 3; Qu, Clin Chem 3; Gil, Fetal Diagn Ther 4 Most methods require deeper shotgun sequencing, or use of multiple polymorphic SNPs If lower threshold is FF>4% for each twin, ure/nocall rates will be higher Generally, no more than a : ratio of FF (e.g., %,6%) Shotgun sequencing may be OK, if FF > % (e.g., 8%, 4%) Detection of aneuploidy in twins: All Comment Study FPR (%) No calls DR (%) No call Sehnert / 3 ( ) / ( ) / () Canick / 7 ( ) / ( ) 8/ 8 () Lau 3 / ( ) / ( ) / () Huang 4 /78 ( ) /89 ( ) / ( 9) ( T8 missed) Gil 4 /8 ( ) /7 (7.) / ( 9) ( T8 missed) ( T8/ T3) All /39 ( ) 3/33 ( 94) Detection of fetal sex and associated no call rates: Study N DR ( ) Males (%) Females (%) No call FPR ( /Ab) N DR ( ) FPR ( /Ab) rate Bianchi 84. 33 99.6.4. Nicolaides 3 9. 9.7.7 Mazloom 3 94 99.. 9 98.6.4.6 Pergament 4 48. 38.3 9. All 7 99.7 v low 99. low high Unpublished data also available on Verinata/Illumina website. Unpublished data also available on Verinata/Illumina website. Detection of sex aneuploidies: Deletion syndromes identified Turner (4,X) Triple X (47,XXX) Klinefelter (47,XXY) (47,XYY) Study DR (%) Fail DR (%) Fail DR (%) Fail DR (%) Fail Bianchi /6 4 3/4 / 3/3 Nicolaides 3 / Mazloom 3 /6 4 / / 3/3 Samango 3 / / / Pergament 4 9/ / / / All (%) (% corr) 6/66 (94) (77) (4) 9/ (9) (8) (9) 6/6 () ( 89) () 8/8 () ( 8) () Sequenom q deletion (DiGeorge) p (Cri du chat) q (Prader Willi/Angleman) p36 deletion syndrome trisomy 6 trisomy q (Jacobsen) 8q (Langer Giedion) 4p (Wolf Hirschorn) Natera q deletion (DiGeorge) p (Cri du chat) q (Prader Willi/Angleman) p36 deletion syndrome

/6/4 Approaches differ Predictive values for microdeletions Sequenom Shotgun NGS data Bioinformatic analysis of existing sequences Likely to have poorer performance than for complete chromosome deletions/duplications Would need to minimize FPR by reducing DR of smaller deletions No additional sequencing expense Natera New mix of K+ SNPs targeted specific deletions Likely to have similar performance compared to whole chromosomes Probably higher detection, but might miss % of DiGeorge Would need to charge for additional sequencing Even assuming very high DR (99.%) and low FPR (.%), the OAPR for rarer disorders (:,) will be relatively low. Cri du chat, pregnancies 99,99 No deletion Pos Neg 98 Pos ~99K Neg OAPR :98 or : Professional Practice guidelines Generally agree that Sequencing of cell free DNA is sensitive and specific for trisomies of chromosome, 8 and 3 Testing should be offered to high risk pregnancies Patient and provider education is important Insufficient data for twins Positive results followed up by offer of invasive testing Testing should not be offered to the general pregnancy population ( low risk ) until more information is available ACMG allows for such testing in the general population Includes: ACOG, NSGC, ISPD, ACMG, SOGC Why not a large low risk observational study? Excluding maternal age > 3 reduces population DS risk to : (even lower if other high risk pregnancies were also removed) Identifying DS would require 6, women tested Identifying DS would require 4, women tested Study design would need to be observational Samples would be frozen and tested after delivery Women could not be screened (serum/us)? Outcomes collected on all enrolled women (6K K) Many missing outcomes (SAb, especially among cases) Some test results ( / ) would not have an associated outcome so DR/FPR would be incomplete Observational study Review study designs Efficient for determining detection / false itive rates Validated outcomes, high prevalence = confidence Demonstration study Develop and validate patient materials, document knowledge Uptake rates (screening, referral, dx testing) Laboratory issues related to transitioning from research to practice (long term performance of assays) Timing (between sampling, reporting results, dx testing) Predictive values, and confirmation of test performance Other defects identified during the process Barriers to participation (travel, costs, availability) If, and when, termination occurred Chromosome (z-score) Chromosome (z-score) - - st Trimester Screen nd Trimester Screen Integrated Screen Ultrasound Abnormality Maternal Age Two or more p =. syndrome p =.9 Family Hx of Aneuploidy Other/Not indicated DNA Test C z score by indication for Dx testing No study has provided evidence that fetal fraction or the DR/FP will differ in a general pregnancy ( low risk ) population. 6

/6/4 syndrome testing: general population (DR 98%, FPR.%, No call.%) 94 DS 3 (.4%) Offer Dx testing : (94:9) 6, background (:99) 9,9 9 3, (.%)???? :99 (:99) 6,786,99 6,787 (93.6%) Routine care :6, (:6,786) Three ways testing differ in the general vs. high risk population Counseling/education High risk often has genetic counseling while the general population is too large for this activity, so education would need to come from primary care providers Positive (Negative) predictive values High risk population has few false itives compared to true itives, while in the general population, half of itives are false itives (even lower rates for other trisomies and sex aneuploidies) Impact on test ures / no calls High risk can still be offered diagnostic testing, while the general population would need other options (e.g., serum/us) General population studies? Defined as: ccf DNA testing as the primary screening test in pregnancy Results returned for clinical use in real time Designed to collect information about Provider uptake and education Patient decision making, knowledge and satisfaction One identified via internet search and clinical trials.gov The DNAFirst project in Rhode Island (NCT96699) 3 women screened in months (now enrolling) Documents women s decision making (uptake, test options) Telephone interview with about participants Survey of practitioners knowledge and satisfaction Documentation of practice parameters (uptake, PPV, ures) Colleagues at Women & Infants Hospital Geralyn Messerlian, PhD Laboratory Director James Haddow, MD Medical Director Ed Kloza, MS, CGC Study Coordinator Jacob Canick, PhD 94 To 3 7