Prenatal Aneuploidy Screening Using cell Free DNA. Detection rate of prenatal screening for Down syndrome has improved over time

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1 Prenatal Aneuploidy Screening Using cell Free DNA Disclosures o Research support from Natera and Ariosa Mary E. Norton MD Professor, Obstetrics, Gynecology and Reproductive Sciences University of California, San Francisco o No lucrative personal financial contracts What Does the Evidence Tell Us? Oct 2015 Detection rate of prenatal screening for Down syndrome has improved over time The questions being debated: Detection Rate (%) o Is cfdna screening the best option for low risk patients? o Is cfdna screening the best choice for primary screening for any or all patients? 1

2 Professional Society Opinions: ACOG; ACMG; International Society of Prenatal Diagnosis; National Society of Genetic Counselors Common themes (2012): There are recognized benefits, but o Not diagnostic Needs confirmation o Only detects common trisomies o Requires comprehensive genetic counseling o Should only be used in validated groups (eg high risk) o Need a low risk study before introducing into general population screening Rumsfeld on current status of NIPT? There are known knowns. There are things we know, we know. We also know there are known unknowns. That is to say, we know there are some things we do not know. But there are also unknown unknowns. The ones we don t know we don t know. -Donald Rumsfeld, 2002 Cell free fetal DNA Cell free DNA results from apoptosis o Short segments of fetal DNA (<200 base pairs) circulate in maternal plasma o Origin is primarily placenta Maternal DNA Fetal DNA 2

3 Analysis of cell free DNA Trisomy 21 performance cfdna testing: meta-analysis (Gil et al, Ultrasound Obstet Gynecol, 2015) Trisomy 21 Non-Trisomy 21 DR: 99.2% ( ) FPR: 0.09% ( ) Zhong, X, Holzgreve, W, Glob. libr. women's med 2009 Cell free DNA: Biologic Challenges False positives: o Unrecognized or vanishing twin o Placental mosaicism o Maternal genetic variation o Maternal malignancy False negatives: o Low level of fetal DNA o Placental mosaicism o Maternal genetic variation Failed results: o Increased BMI o Low level of fetal DNA o Fetal aneuploidy False positive cfdna results and cancer 3

4 Issues with cfdna for primary screening 1. Not all abnormalities are detectable What percentage of chromosome abnormalities will be detected by cfdna screening? Down syndrome comprises ~50% of aneuploidies 2. The PPV (chance that a positive is a TRUE POSITIVE) depends on maternal age This is often misunderstood 3. Some tests fail to provide a result A. 99% B. 75% C. 50% D. 12% 28% 41% 17% 13% These patients are at HIGH RISK of aneuploidy Importance of fetal fraction 9 9 % 7 5 % 5 0 % 1 2 % 4

5 Spectrum of Genetic Disease Spectrum of Genetic Disease Autosomal recessive Autosomal recessive CNV (microarray) Autosomal dominant X-linked CNV (microarray) Autosomal dominant X-linked Chromosomal/ karyotype Chromosomal/ karyotype Structural Malformations Structural Malformations Aneuploidies Present in HIGH RISK Women Aneuploidies Present in LOW RISK Women *Not detected by cfdna *Not detected by cfdna Sex chromosomal: 8.2% Other* 16.9% Tri 21: 53.2% Other* 20.8% Tri 21: 49.2% Tri 13: 4.6% Sex chromosomal: 9.9% Tri 18: 17.0% Tri 13: 5.5% Tri 18:12.9% Norton et al, SMFM, 2014 Norton et al, SMFM,

6 Rate of abnormalities by maternal age cfdna Detection Rate n=452,901 patients screened in California Total Cases with Aneuploidy (n=2575) cfdna Detectable N=1841 (71.4%) Not Detectable Detected (False negative+ Non-detectable) N= (24.5%) No Result N=105 (4.1%) In low-risk patients, this is a very accurate test for a rare event. 6

7 NIPT and diagnostic testing with chromosomal microarray (CMA) NIPT and diagnostic testing with chromosomal microarray (CMA) Microarray detects an abnormality in 1.7% of cases (about 1/60) AND: NIPT detects T13,18, 21 about 1/500 pregnancies THEN: If NIPT is the routine screening test, it will detect about 12% of diagnosable chromosomal abnormalities Microarray detects an abnormality in 1.7% of cases (about 1/60) AND: NIPT detects T13,18, 21 about 1/500 pregnancies THEN: If NIPT is the routine screening test, it will detect about 12% of diagnosable chromosomal abnormalities NIPT and diagnostic testing with chromosomal microarray (CMA) Microarray detects an abnormality in 1.7% of cases (about 1/60) AND: NIPT detects T13,18, 21 about 1/500 pregnancies THEN: If NIPT is the routine screening test, it will detect about 12% of diagnosable chromosomal abnormalities Your 25 yo patient has cfdna screening and the result is positive for trisomy 13. What is the chance that the fetus actually has trisomy 13? A. >99% B. 75% C. 50% D. <10% 34% 21% 17% 28% > 9 9 % 7 5 % 5 0 % < 1 0 % 7

8 cfdna vs Standard Screening Bianchi et al, NEJM, 2014 o N=1914 women undergoing standard screening o Mean maternal age = 29.6 yrs o Primary outcome = false positive rates for T18 and T21 FPR PPV cfdna 0.3% 45.5% p<.001 Standard 3.6% 4.2% o Only 8 aneuploidy cases in the cohort (5: T21, 2: T18, and 1: T13) o All were detected NEXT study: 15,841 average risk women o 15,841 women had cfdna and first trimester screening o Mean maternal age = 30.7 yrs Cell free DNA screening Detection rate 38/38 (100%) False positive rate Positive predictive value First trimester screening 30/38 (79%) P= % 5.4% P< % 3.4% P< Norton et al, NEJM,

9 Wang et al, Genetics in Medicine, 2014 Aneuploidy No. of positives No (%) confirmed T /41 (93%) T /25 (64%) T /16 (44%) 45X 16 6/16 (38%) Total (67%) o 6.2% had termination without karyotype confirmation o Disconcerting if only 67% are true positives 9

10 Consequences of false positive results NIPT N=100,000 1% false positives 1000 abnormal results 6.2% TAB w/o confirmation 62 TAB 67% PPV 42 TP 21 FP 21 TAB of normal fetuses Consequences of false positive results NIPT N=100,000 1% false positives 1000 abnormal results 6.2% TAB w/o confirmation 62 TAB 67% PPV 42 TP 21 FP 21 TAB of normal fetuses Serum Screening N=100,000 5% false positives 5000 abnormal results 0.2% loss rate (amnio) 10 losses of normal fetuses If your patient has a positive result: 10

11 The poorly understood PPV Your patient has cfdna screening, and the lab calls to tell you the test failed to provide a result. What are possible reasons for this? A. Maternal obesity B. The fetus has a chromosome abnormality C. The blood was drawn too early D. All of the above M a t e r n a l o b e s i t y 16% T h e f e t u s h a s a c h r o m o... 1% T h e b l o o d w a s d r a w n t.. 12% A l l o f t h e a b o v e 71% 11

12 Published Trials of NIPT: failure rates Trial Failure rate Chiu et al (2011) 11/764 (1.4%) Ehrich et al. (2011) 18/467 (3.8%) Palomaki et al. (2011) 13/1696 (0.8%) Bianchi et al. (2012) 30/532 (3.0%) Norton et al (2012) 148/3228 (4.6%) Zimmermann et al (2012) 21/166 (12.6%) Pergament et al (2014) 85/1051 (8%) Norton et al (2015) 488/16,329 (3.0%) All 729/23,182 (3.1%) Fetal fraction of DNA and test failure 3-5% of samples do not provide a result Low fraction fetal DNA, failed sequencing, high variability in counts Some association with gestational age (<10 wks) Low fetal fraction associated with maternal BMI - 20% at >250 lbs - 50% at >350 lbs Low fetal fraction is associated with aneuploidy Repeating test will provide a result in SOME cases Obesity in US Adults Pergament et al, 2014 o N=1051 samples were analyzed o N=85/1051 (8%) samples failed to obtain a result o 20/85 (22%) were aneuploid No call cases represent a very high risk group Obstet Gynecol

13 Kaiser cfdna Experience: No Results Cases Total Pregnancies Sampled N = 4446 Kaiser cfdna Experience: No Results Cases Total Pregnancies Sampled N = 4446 Low Risk N = 4187 (94.2%) High Risk N = 157 (3.5%) No Results N = 102 (2.3%) Low Risk N = 4187 (94.2%) High Risk N = 157 (3.5%) No Results N = 102 (2.3%) Redrawn N = 63 Redraw Declined N=39 Redrawn N = 63 Redraw Declined N=39 Low Risk N=32 (50.8%) High Risk N=5 (7.9%) No Result N=26(41.3%) Low Risk N=32 (50.8%) High Risk N=5 (7.9%) No Result N=26(41.3%) NO FINAL RESULT N = 65 (1.5%) NO FINAL RESULT N = 65 (1.5%) Kaiser cfdna Experience: No Results Cases Low Risk N = 4187 (94.2%) Total Pregnancies Sampled N = 4446 High Risk N = 157 (3.5%) No Results N = 102 (2.3%) Kaiser cfdna: No Results KPNC 10/29/12 6/30/14 Total pregnancies sampled = 4446 NO FINAL RESULT N = 65 (1.5%) Redrawn N = 63 Redraw Declined N=39 Low Risk N=32 (50.8%) High Risk N=5 (7.9%) No Result N=26(41.3%) Chromosomes not done 43/65 (66%) Normal chromosomes 13/65 (20%) Abnormal chromosomes 9/65 (14%) NO FINAL RESULT N = 65 (1.5%) 13

14 Failed cfdna screens indicate increased risk for aneuploidy The questions being debated: Failed tests increase aneuploidy risk: Author OR for aneuploidy o Norton et al, NEJM 2015: 6.2 o Pergament et al, Obstet Gynecol 2014: 2.5 o Turocy et al, SMFM 2015: 5.7 o Is cfdna screening the best option for low risk patients? o Is cfdna screening the best choice for primary screening for any or all patients? NIPT is more precise for T13, 18, 21 NIPT is more precise for T13, 18, 21 Other abnormalities cfdna Current NT + serum screen cfdna Current NT + serum screen 14

15 cfdna vs Sequential Screening: Detection and False Positive Rates Sequential screening cfdna if no results cases = high risk cfdna if no results have no follow up Cohort Detection Rate 81.6% 4.5% 77.1% 3.7% 70.7% 0.7% False Positive Rate cfdna and Ultrasound Abnormalities Normal U/S Abnormal U/S T13, 18, (4.9%) 88 (23.4%) Other chromosomal 13 (2.5%) 29 (7.7%) abnormalities Total detectable cfdna 25/38 (66%) 88/117 (75%) Benachi et al, Obstet Gynecol, 2015 NIPT: Expanded panels NIPT: Expanded panels Laboratories have added other trisomies and microdeletions o Trisomies 16 and 22 o Microdeletion syndromes 22q (digeorge) 5p (cri-du-chat) 1p36 15q (Prader Willi) 4p (Wolf-Hirshhorn) o Trisomies 16 and 22 Rarely seen in viable pregnancies except as mosaics Common causes of confined placental mosaicism - Much more common in CVS samples than amniocentesis Even complete trisomy in the placenta often associated with a normal fetus With both, confined placental mosaicism can be associated with IUGR, so false positive cases should be followed for fetal growth (eg ultrasound at wks) 15

16 Microdeletion syndromes are rare Syndrome Frequency Features 22q11.2 (DiGeorge) 1/4,000 Varies: cardiac, palatal, immune, intellectual disability 1q36 1/10,000 Severe intellectual disability (ID), +/- obvious structural anomalies Angelman 1/20,000 Severe ID, seizures, speech delay Prader-Willi 1/30,000 Obesity, ID, behavioral problems Cri-du-chat 1/50,000 Microcephaly, ID, +/- CHD Wolf-Hirshhorn 1/50,000 ID, seizures, +/- CL/CP Testing for Rare Disorders (Wolf-Hirschhorn, 4p-: Assume 99% sensitivity and 99.2% specificity) Population Risk = 1/50,000 N=100,000 2 Wolf-Hirschhorn 99,998 not WHS 2 TP; 0 FN 800 FP; 99,198 TN OAPR = 1/400 ACOG/SMFM September 2015 o Conventional screening is most appropriate first line screen for most patients o Ethically any patient may choose cfdna screening, but should be counseled regarding limitations and benefits o Diagnostic testing is required to confirm abnormal results before irreversible decisions o Testing for microdeletions and in twins should not be performed 16

17 Important counseling points All patients deserve equal access o NIPT is NOT diagnostic o Extremely high sensitivity and specificity for Down syndrome o Somewhat lower for trisomy 13, 18 and sex chromosomes o Approximately 20-30% of chromosome abnormalities identified with invasive testing are NOT detectable with NIPT o ACOG indicates that testing should no longer be stratified by maternal age o We ve spent the past decade trying to abolish advanced maternal age and the 35 yo cutoff o It is unethical to withhold this test All patients deserve equal access o ACOG indicates that testing should no longer be stratified by maternal age o We ve spent the past decade trying to abolish advanced maternal age and the 35 yo cutoff o It is unethical to withhold this test There is a difference between withholding something for an individual patient, and recommending it for all patients as policy All patients should have access to all test options (the Jim Carrey approach) 17

18 Ethics, access and counseling o While it is not ethical to withhold tests from one group o It is necessary to provide fair and balanced counseling regarding the pros and cons of ALL test options cfdna is very good for the common aneuploidies, but doesn t detect other serious chromosome abnormalities In low risk patients, a positive result is more likely to be a false positive In patients at low risk for the common aneuploidies, other screening options provide broader coverage at lower cost Appropriate counseling o While it is not ethical to withhold tests from one group o It is necessary to provide fair and balanced counseling regarding the pros and cons of ALL test options cfdna is very good for the common aneuploidies, but doesn t detect other serious chromosome abnormalities In low risk patients, a positive result is more likely to be a false positive In patients at low risk for the common aneuploidies, other screening options provide broader coverage at lower cost Appropriate counseling o While it is not ethical to withhold tests from one group o It is necessary to provide fair and balanced counseling regarding the pros and cons of ALL test options cfdna is very good for the common aneuploidies, but doesn t detect other serious chromosome abnormalities In low risk patients, a positive result is more likely to be a false positive In patients at low risk for the common aneuploidies, other screening options provide broader coverage at lower cost Appropriate counseling o While it is not ethical to withhold tests from one group o It is necessary to provide fair and balanced counseling regarding the pros and cons of ALL test options cfdna is very good for the common aneuploidies, but doesn t detect other serious chromosome abnormalities In low risk patients, a positive result is more likely to be a false positive In patients at low risk for the common aneuploidies, other screening options provide broader coverage for more conditions 18

19 Thank You! 19