Cell free DNA: Disclosures The Popular Press vs The Evidence Mary - PowerPoint PPT Presentation

6/5/2014 Cell free DNA: Disclosures The Popular Press vs The Evidence Mary E Norton, MD Professor of Obstetrics, Gynecology & Reproductive Sciences; UCSF • Principal Investigator of ongoing clinical trial Antepartum and Intrapartum Management on cfDNA supported by Ariosa Diagnostics • Unpaid clinical consultant for CellScape and Natera • Research support from Natera • No personal financial involvement in any of the cfDNA companies 1

6/5/2014 Detection rate of prenatal screening for Down syndrome has improved over time 120 Detection Rate (%) 100 80 60 40 20 0 Cell free DNA results from Cell free fetal DNA apoptosis • Cell free fetal DNA (cffDNA) is made up of short segments of fetal DNA (<200 base pairs) that circulate in maternal plasma • Origin of these fragments is thought to be primarily placenta Maternal DNA Fetal DNA 2

6/5/2014 Noninvasive Prenatal Companies currently offering cfDNA Testing (NIPT) screening (in order of appearance): • Detection requires accurate quantification of DNA from a specific chromosome • Somewhat different methods are utilized by different laboratories Trisomy 21 performance cfDNA testing: meta-analysis ( Gil et al, Fetal Diagn Ther, 2014) Analysis of fetal DNA Author DR (95% CI) Wts (%) FPR (95% CI) Wts (%) Chiu et al., 2011 [86] 100 (95.8 - 100) 11.6 2.06 (0.4 - 5.9) 2.0 Ehrich et al., 2011 [39] 100 (91.0 - 100) 5.3 0.24 (0.0 - 1.4) 4.9 Palomaki et al., 2011 [212] 98.6 (95.9 - 99.7) 28.4 0.20 (0.0 - 0.6) 12.6 Sehnert et al., 2011 [13] 100 (75.3 - 100) 1.9 0.00 (0.0 - 10.3) 0.5 Ashoor et al., 2012 [50] 100 (92.9 - 100) 6.8 0.00 (0.0 - 1.1) 4.3 Bianchi et al., 2012 [89] 100 (95.9 - 100) 12.0 0.00 (0.0 - 0.9) 4.9 Jiang et al., 2012 [16] 100 (79.4 - 100) 2.3 0.00 (0.0 - 0.4) 9.0 Lau et al., 2012 [11] 100 (71.5 - 100) 1.6 0.00 (0.0 - 3.7) 1.3 Nicolaides et al., 2012 [8] 100 (63.1 - 100) 1.2 0.00 (0.0 - 0.2) 14.8 Norton et al., 2012 [81] 100 (95.6 - 100) 11.0 0.04 (0 - 0.2) 18.0 Sparks et al., 2012 [36] 100 (90.3 - 100) 4.9 0.00 (0.0 - 2.8) 1.8 Zimmerman et al., 2012 [11] 100 (71.5 - 100) 1.6 0.00 (0.0 - 2.7) 1.8 Guex et al., 2013 [30] 100 (88.4 - 100) 4.1 0.00 (0.0 - 2.5) 2.0 Nicolaides et al., 2013 [25] 100 (86.3 - 100) 3.5 0.00 (0.0 - 1.8) 2.7 Song et al., 2013 [8] 100 (63.1 - 100) 1.2 0.00 (0.0 - 0.2) 13.9 Verweij et al., 2013 [18] 94.4 (72.7 - 100) 2.5 0.00 (0.0 - 0.7) 5.8 99.1 (98.3 - 99.6) Pooled analysis 0.08 (0.03 - 0.17) 100 100 50 60 70 80 90100 DR % (95% CI) (%) 0 3 6 9 12 FPR % (95% CI) (%) Zhong, X, Holzgreve, W, Glob. libr. women's med 2009 T21: n=733 11,475 non-T21 3

6/5/2014 Trisomy 21 performance cfDNA testing: Trisomy 21 performance cfDNA testing: meta-analysis ( Gil et al, Fetal Diagn Ther, 2014) meta-analysis ( Gil et al, Fetal Diagn Ther, 2014) Author DR (95% CI) Author DR (95% CI) Wts (%) FPR (95% CI) Wts (%) Wts (%) FPR (95% CI) Wts (%) Chiu et al., 2011 [86] 100 (95.8 - 100) 11.6 2.06 (0.4 - 5.9) 2.0 Chiu et al., 2011 [86] 100 (95.8 - 100) 11.6 2.06 (0.4 - 5.9) 2.0 Ehrich et al., 2011 [39] 100 (91.0 - 100) 5.3 0.24 (0.0 - 1.4) 4.9 Ehrich et al., 2011 [39] 100 (91.0 - 100) 5.3 0.24 (0.0 - 1.4) 4.9 Palomaki et al., 2011 [212] 98.6 (95.9 - 99.7) 28.4 0.20 (0.0 - 0.6) 12.6 Palomaki et al., 2011 [212] 98.6 (95.9 - 99.7) 28.4 0.20 (0.0 - 0.6) 12.6 Sehnert et al., 2011 [13] 100 (75.3 - 100) 1.9 0.00 (0.0 - 10.3) 0.5 Sehnert et al., 2011 [13] 100 (75.3 - 100) 1.9 0.00 (0.0 - 10.3) 0.5 Ashoor et al., 2012 [50] 100 (92.9 - 100) 6.8 Ashoor et al., 2012 [50] 100 (92.9 - 100) 6.8 0.00 (0.0 - 1.1) 4.3 0.00 (0.0 - 1.1) 4.3 Bianchi et al., 2012 [89] 100 (95.9 - 100) 12.0 Bianchi et al., 2012 [89] 100 (95.9 - 100) 12.0 0.00 (0.0 - 0.9) 4.9 0.00 (0.0 - 0.9) 4.9 Jiang et al., 2012 [16] 100 (79.4 - 100) 2.3 Jiang et al., 2012 [16] 100 (79.4 - 100) 2.3 0.00 (0.0 - 0.4) 9.0 0.00 (0.0 - 0.4) 9.0 Lau et al., 2012 [11] 100 (71.5 - 100) 1.6 0.00 (0.0 - 3.7) 1.3 Lau et al., 2012 [11] 100 (71.5 - 100) 1.6 0.00 (0.0 - 3.7) 1.3 Nicolaides et al., 2012 [8] 100 (63.1 - 100) 1.2 0.00 (0.0 - 0.2) 14.8 Nicolaides et al., 2012 [8] 100 (63.1 - 100) 1.2 0.00 (0.0 - 0.2) 14.8 DR: 99.1% (98.3 - 99.6) Norton et al., 2012 [81] 100 (95.6 - 100) 11.0 0.04 (0 - 0.2) 18.0 DR: 99.1% (98.3 - 99.6)FPR: 0.08% (0.03 - 0.17) Norton et al., 2012 [81] 100 (95.6 - 100) 11.0 0.04 (0 - 0.2) 18.0 Sparks et al., 2012 [36] 100 (90.3 - 100) 4.9 0.00 (0.0 - 2.8) 1.8 Sparks et al., 2012 [36] 100 (90.3 - 100) 4.9 0.00 (0.0 - 2.8) 1.8 Zimmerman et al., 2012 [11] 100 (71.5 - 100) 1.6 0.00 (0.0 - 2.7) 1.8 Zimmerman et al., 2012 [11] 100 (71.5 - 100) 1.6 0.00 (0.0 - 2.7) 1.8 0.00 (0.0 - 2.5) 2.0 0.00 (0.0 - 2.5) 2.0 Guex et al., 2013 [30] 100 (88.4 - 100) 4.1 Guex et al., 2013 [30] 100 (88.4 - 100) 4.1 0.00 (0.0 - 1.8) 2.7 0.00 (0.0 - 1.8) 2.7 Nicolaides et al., 2013 [25] 100 (86.3 - 100) 3.5 Nicolaides et al., 2013 [25] 100 (86.3 - 100) 3.5 Song et al., 2013 [8] 100 (63.1 - 100) 1.2 0.00 (0.0 - 0.2) 13.9 Song et al., 2013 [8] 100 (63.1 - 100) 1.2 0.00 (0.0 - 0.2) 13.9 Verweij et al., 2013 [18] 94.4 (72.7 - 100) 2.5 0.00 (0.0 - 0.7) 5.8 Verweij et al., 2013 [18] 94.4 (72.7 - 100) 2.5 0.00 (0.0 - 0.7) 5.8 0.08 (0.03 - 0.17) 0.08 (0.03 - 0.17) Pooled analysis 99.1 (98.3 - 99.6 ) Pooled analysis 99.1 (98.3 - 99.6 ) 100 100 100 100 50 60 70 80 90100 DR % (95% CI) (%) 50 60 70 80 90100 DR % (95% CI) (%) 0 3 6 9 12 FPR % (95% CI) (%) 0 3 6 9 12 FPR % (95% CI) (%) T21: n=733 T21: n=733 11,475 non-T21 11,475 non-T21 4

6/5/2014 Published Trials of NIPT: NIPT: Clinical Challenges failure rates False positives: • Unrecognized or vanishing twin • Trial Failure rate Detection False positive rate Placental mosaicism • Low level maternal mosaicism, esp sex chromosomal Chiu et al (2011) 11/764 (1.4%) 86/86 3/146 • Maternal malignancy Ehrich et al. (2011) 18/467 (3.8%) 39/39 1/410 False negatives: Palomaki et al. (2011) 13/1696 (0.8%) 209/212 3/1471 • Genetic variants Bianchi et al. (2012) 30/532 (3.0%) 89/89 0/404 • Norton et al (2012) 148/3228 (4.6%) 81/81 1/2888 Placental mosaicism Zimmermann et al (2012) 21/166 (12.6%) 11/11 0/145 Failed results: • Increased BMI All 241/6853 (3.5%) 424/427 (99.3%) 8/5319 (0.15%) • Failure to extract adequate material • Individual variation in amount of cell free fetal DNA • Fetal aneuploidy Fetal fraction of DNA and test failure Up to 5% of samples do not provide a result o Low fraction fetal DNA, failed sequencing, high variability in counts o Some association with gestational age (<10 wks) o Low fetal fraction associated with maternal BMI • 20% at >250 lbs • 50% at >350 lbs � Low fetal fraction appears to be associated with aneuploidy � Repeating test will provide a result in some cases 5

6/5/2014 Why is NIPT not diagnostic? • Confined Placental Mosaicism • THIS IS A PLACENTAL, AND NOT A FETAL TEST False positive False negative NIPT: Trisomy 13 and Trisomy 18 Sex Chromosomal Aneuploidy Author T13 T18 DR FPR DR FPR Author Cases: DR FPR No result Palomaki ’11 11/12 (92%) 16/1688 (0.97%) 59/59 (100%) 5/1688 (0.28%) Controls Bianchi ’12 11/14 (79%) 0/488 35/36 (99%) 0/460 (0%) Samango- 16:185 92% 0 7% Sprouse Norton ’12 --- --- 37/38 (97.4%) 2/2888(0.07%) Bianchi 20:532 75% 0.2% 20% Zimmermann 2/2 (100%) 0/145 3/3 (100%) 0/145 ‘12 Nicolaides 59:118 88% 0.8% 2.7% Porreco ’14 14/16 (92%) 0/3322 36/39 (92%) 0/3322 Total 95:835 86% 0.6% 10% Bianchi ’14 1/1 (100%) 1/899 (0.1%) 2/2 (100%) 3/1905 (0.2%) TOTAL 39/45 (87%) 17/6542 (.03%) 172/177 (97%) 10/10,408 (0.1%) 6

6/5/2014 Professional Society Opinions: ACOG; ACMG; International Society of Prenatal Diagnosis; National Society of Genetic Counselors Common themes: There are recognized benefits, but… • Not diagnostic o Needs confirmation o “Advanced screening test” • Only detects common trisomies (vs invasive testing) • Requires comprehensive genetic counseling • Should only be used in validated groups (eg high risk) • Need a low risk study before introducing into general population screening 7

6/5/2014 How Does Test Performance (Assume >99% sensitivity and Differ with Risk? 99.8% specificity) Low Risk (age 25; 1/1000) High Risk (age 38; 1/100) N=1000 N=1000 1 T21 999 not T21 10 T21 990 not T21 1 TP, 0 FN 2 FP, 998 TN 10 TP, 0 FN 2 FP, 988 TN OAPR = 1/3 OAPR = 10/12 or 5/6 How Does Test Performance (T13: Assume 99% sensitivity and Differ with Risk? 99% specificity) Low Risk (age 25; 1/8,000) High Risk (age 38; 1/1000) N=10,000 N=10,000 10 T13 9,990 not T13 1 T13 9,999 not T13 • N=1914 women undergoing standard screening • Mean maternal age = 29.6 yrs 9 TP, 1 FN 100 FP, 9890 TN • Primary outcome = false positive rates for T18 and T21 1 TP, 0 FN 100 FP, 9900 TN OAPR = 9/100 OAPR = 1/100 8