1. BROWN Prenatal Screening for Trisomy 21: Recent Advances and Guidelines Jacob Canick, PhD Alpert Medical School of Brown University Women & Infants Hospital Providence, RI, USA Department of Pathology Montefiore Medical Center Bronx, NY March 17, 2011 Women & Infants’

2. Grant & Research Support: -Beckman Coulter, Inc., Brea, CA -Beckman Coulter Foundation, Brea, CA -Sequenom, Inc., San Diego, CA Other: -Patent Holder – uE3 in prenatal screening Disclosure Information

3. Prenatal Screening for Trisomy 21: Recent Advances and Guidelines 1.Brief history of prenatal screening 2.How is screening performance measured? 3.Performance of second trimester, first trimester, integrated, and sequential tests for Down syndrome 4.Guidelines: standard of practice in EUROCAT countries and the United States 5.Trisomy 18 and 13 6.Laboratory issues in prenatal screening

4. Brief history of prenatal screening

5. Spina bifida Anencephaly The birth prevalence of spina bifida and anencephaly in England and Wales between 1964 and 1975. (N Wald) Prenatal Screening History Begins with Prenatal Screening for Open Neural Tube Defects using MSAFP 1970s Maternal Serum AFP screening 1980s acceptance in U.S. (ACOG liability alert) 1980s primary prevention with folic acid 2000s move to ultrasound screening? 4 per 1000 births 1970s AFP in maternal serum elevated in NTDs

6. The birth prevalence of spina bifida and anencephaly in England and Wales between 1964 and 1993. (N Wald) 0.3 per 1000 births Effect of primary prevention (folic acid) and prenatal screening (MSAFP) Prenatal Screening History Begins with Prenatal Screening for Open Neural Tube Defects using MSAFP

7. Fetal nucleic acids in maternal plasma? 2011 Integrated 1 st and 2 nd trim Sequential 1 st and 2 nd trim 2000 1 st trim nuchal translucency (NT) 1 st trim NT + PAPP-A + free  hCG 1990 1980 Screening using maternal age, offer of amnio 1970 1960 Down syndrome caused by chromosome trisomy (2009, the 50 th Anniversary of discovery by LeJeune) 1950 1940 Down syndrome – maternal age association 1930 Prenatal Screening for Aneuploidies: History 2 nd trim AFP (with Mat Age) * 2 nd trim Multiple markers (Double, Triple, Quad)

8. Maternal Serum Markers in the 2nd Trimester: Major improvement in screening for Down syndrome Background 1980sMaternal age as a reason for amniocentesis. 1980s Maternal serum AFP already in use in screening for open NTDs. 1984Maternal serum AFP is low in Down syndrome pregnancy. Irwin Merkatz, MD Albert Einstein Col. Med.

9. How is screening performance measured?

10. Detection Ratepercentage of affecteds called (sensitivity) screen positive by the test The higher the better! False Positive Ratepercentage of unaffecteds called (1 – specificity) screen positive by the test The lower the better! Screening Performance: The challenge in screening is to have a test that has a high detection rate and low false positive rate.

11. Performance of second trimester, first trimester, integrated, and sequential tests for Down syndrome

12. Advantages: Simplest screening method (all serum at one time) Includes screening for open NTDs using MSAFP Will include a larger proportion of pregnant women Follow-up diagnostic test is amniocentesis 0 13 26 40 weeks Screening in the Early 2 nd Trimester (15-20 weeks) with Multiple Serum Markers

13. .1 1 10 MoM AFPinhAuE3 0.74 2.00 hCG 0.61 1.91 2 nd Trimester Serum Markers in Down Syndrome Pregnancies Data from FASTER

14. Prenatal Screening for Down Syndrome in the Early 2 nd Trimester (15 -20 weeks) 0 10 20 30 40 50 60 70 80 90 100 Detection Rate (%) MA + double triple quad ---2 nd trimester----- 30% 69%79% at 5% false positive rate 59% 81% 79% 74% 72% SURUSS FASTER 66%

15. Advantages: Patient privacy Earlier diagnosis (if CVS is available) Greater availability of pregnancy termination Earlier, safer pregnancy termination 0 13 26 40 weeks Screening in the Late 1 st Trimester (11-13 weeks)

16. F Malone, with permission First Trimester Combined Test: NT ultrasound serum PAPP-A serum  -hCG Data from FASTER

17. 0 10 20 30 40 50 60 70 80 90 100 Detection Rate (%) MA + triple quad NT combined 2 nd trimester 1 st trimester 30%69%79% 85% at 5% false positive rate 64% Prenatal Screening for Down Syndrome in the Late 1 st Trimester (11-13 weeks) SURUSS FASTER BUN 85% 79% 69% 68% 62%

18. Combination of the best markers measured at different times in pregnancy into a single test result. This will be more effective than current tests performed at any one time. PAPP-A + quad markers = SERUM INTEGRATED NT + PAPP-A + quad markers = FULL INTEGRATED Integrate results into a single risk 0 13 26 40 weeks The Integrated Test: Two Stages to Reach a Single Risk Assessment Wald NJ et al. N Eng J Med 1999

19. 0 10 20 30 40 50 60 70 80 90 100 Detection Rate (%) MA + triple quad combined serum full 2 nd trimester 1 st trimester -integrated- 30% 69% 79% 85% 94% 89% 86% 95% 93% Performance of the Integrated Test at 5% false positive rate 85% SURUSS FASTER

20. 0 10 20 30 40 50 60 70 80 90 100 12% 50% 60% 66% 85% Detection Rate (%) at 1% false positive rate MA + triple quad combined serum full 2 nd trimester 1 st trimester -integrated- 85% DR at 1% 73% 70% 87% 86% 72% SURUSS FASTER Performance of the Integrated Test

21. Relative Screening Performance of 1 st Trimester Combined and Integrated Tests If 1 st Trimester Combined Test Performance is: DR FPR Then Full Integrated Test Performance will be: DR FPR 85% 5% 94% 5% 90% 2% 85% 1% 90% 5% 96% 5% 93% 2% 90% 1% 90% 2% 96% 2% 93% 1% 90% 0.4%

22. Is there a way to have both: earlier screening and diagnosis – as in the 1 st trimester test and much lower screen positive rate – as in the Integrated Test YES Sequential protocols

23. Sequential Screening: Towards 1 st trimester combined or towards integrated, depending on the 1 st trimester risk cut-off 2345 70 80 90 100 Full Integrated First Trimester Combined Sequential protocols False positive rate (%) Detection rate (%)

24. Example of Sequential Screening Palomaki et al. Obstet Gynecol 2006;107:367-75. DR FPR Step 1. 1 st trimester markers measured on all women at 10-13 wk. Risk cut-off set at ≥ 1:6371.8% 1.5% Step 2. Screen negatives (98.5% of population) have quad markers measured at 15-18 wk. Risk cut-off set at ≥ 1:6512.5% 0.5% Overall84.3% 2.0% Assumes 1:600 prevalence

25. Comparing Sequential Screening to 1 st Trimester Combined Test and Integrated Test Palomaki et al. Obstet Gynecol 2006;107:367-75. Test 1 st Trimester Combined Sequential example Full Integrated FPR to get 84.3% DR 6.0% 2.0% 1.2%

26. Sequential Integrated Screening at Women & Infants ( Expected Performance) Stage 2: Quad markers Full Integrated Risk 1:110 risk cut-off 31% DR, 1.6% FPR Stage 1: 1 st trimester markers PAPP-A and NT 1:25 risk cut-off 59% DR, 0.9% FPR Compare to Integrated Test: 90% DR, 2.0% FPR Compare to 1 st TrimesterTest: 90% DR, 8.0% FPR Sequential Integrated Test: 90% DR, 2.5% FPR

27. Prenatal Screening Guidelines: Practice Standards in the United States

28. American College of Obstetricians and Gynecologists (ACOG) January 2007

29. ACOG 2007 1.“… all women should be offered aneuploidy screening before 20 weeks of gestation, regardless of maternal age.” 2.“… patients seen early in pregnancy should be offered aneuploidy screening that combines first- and second-trimester testing (integrated or sequential).” 3.“The screening strategy chosen will depend on availability of CVS and of personnel trained in NT measurement …” ACOG Practice Bulletin, No. 77, January 2007

30. ACOG 2007 4. “When CVS is not available, … offer integrated screening to patients who present in the first trimester … to take advantage of the improved detection rate and low false-positive rate.” 5.“If NT measurement is not available or cannot be obtained …, offer serum integrated screening to patients who present early and second-trimester screening to those who present later.” ACOG Practice Bulletin, No. 77, January 2007

31. Trisomy 18 (Edwards Syndrome) and Trisomy 13 (Patau Syndrome)

32. 1 st trim. marker Median MoM NT2.17 PAPP-A0.20 Free  -hCG 0.25 Trisomy 18: Marker Levels GE Palomaki 2011, analysis of published literature 2 nd trim. marker Median MoM AFP0.66 uE30.36 hCG0.39 inhA0.88 PAPP-A 0.10

33. Modeled Performance Test DR for 0.1% FPR DR for 0.5% FPR 2 nd trim. triple7282 1 st trim. combined8090 Serum integrated8089 Full integrated9095 Trisomy 18: Screening Performance GE Palomaki 2011, unpublished Actual Performance Based on clinical trial data, the observed screen positive rate in practice will be higher than the modeling suggests.

34. Trisomy 13 2 nd trimester markers are not informative. 1 st trimester markers are informative.  Their pattern is similar to that found in trisomy 18.  But, published data on trisomy 13 marker levels are biased and too small to estimate their levels with accuracy. It is reasonable to assume that the majority of trisomy 13 cases will be identified as part of current trisomy 18 screening protocols that include 1 st trimester markers.

35. Laboratory Issues in Prenatal Screening Serum marker assays Free  -hCG or total  -hCG in the 1st trimester Screening marker quality assurance Nuchal Translucency ultrasound and the laboratory

36. Prenatal Screening: Serum Assays Assay typeChoices? FDA-approved 2 nd trimester: AFP 2 site immunometric Yes uE3 competitive immunoassay Yes  -hCG 2 site immunometric Yes inhibin A 2 site immunometric NoYes 1 st trimester: PAPP-A 2 site immunometric Yes  free  -hCG 2 site immunometric Yes/NoNo

37. free  subunit or hCG itself in the 1 st trimester? Palomaki GE et al. Adv Clin Chem 2007;43:177-210. free  hCG hCG

38. First Trimester Screening Performance (NT + serum markers): SURUSS Wald et al., J Med Screen 2003;10:56-104. Gest age (weeks) DR @ 5% FPR 60 65 70 75 80 85 90 95 100 91011121314 + PAPP-A + PAPP-A + free  + PAPP-A + hCG

39. First Trimester Screening Performance (NT + serum markers): FASTER Canick et al., Obstet Gynecol 2006;108:1192-9. Gest age (weeks) DR @ 5% FPR 60 65 70 75 80 85 90 95 100 1011121314 + PAPP-A + PAPP-A + free  + PAPP-A + hCG

40. First Trimester Screening Performance (NT + serum markers): Evans meta-analysis Evans et al., Am J Obstet Gynecol, March 2007 Gest age (weeks) 9101112 + PAPP-A + PAPP-A + free  + PAPP-A + hCG 60 65 70 75 80 85 90 95 100 DR @ 5% FPR

41. Univariately, free-  is a better 1 st trimester marker than hCG itself. Multivariately, between 11 and 13 gestational weeks, it makes little difference whether free-  or hCG is used. Because of patent issues in the U.S., most labs currently use hCG in 1 st trimester screening. free  subunit or hCG itself in the 1 st trimester?

42. Prenatal Screening Quality Assurance Applying Serum Marker Quality Assurance Measures to Nuchal Translucency (NT)

43. Marker parameters that are monitored: Rate of change with may go up or down at a constant increasing gestation rate Mediancalculated MoM values should be stable at 1.0 SD of the distributioncalculated SD of the log MoM values expected to remain steady Prenatal Screening Markers Quality monitoring

44. log-linear increase slope = +15% per week Examples of Marker QA Parameters MS AFP (MoM) unaffected distribution (x and SD) (median MoM)

45. http://www.fetalmedicine.com “normal nuchal translucency” “increased nuchal translucency” Nuchal Translucency (NT) Measurement F Malone, with permission

46. NT parameters that are monitored: Rate of increase with CRLlog-linear over 10,3 - 13,6 weeks should go up by ~ 20% per week Mediancalculated MoM values should be stable at 1.0 MoM SD of the distributioncalculated SD of the log MoM values expected to be about 0.1 1 st Trimester Nuchal Translucency (NT) Quality monitoring

47. log-linear increase slope = +15% per week log-linear increase slope = +20% per week Maternal Serum AFP Increases with Increasing Gestation Nuchal Translucency Increases with Increasing Gestation Schuchter et al, Prenat Diagn 1998

48. UK SURUSS Trial: NT measurement (mm) in 75 pregnancies with Down syndrome according to CRL Wald NJ et al, Health Technology Assessment 2003; Vol. 7: No. 11 NT in Down Syndrome

49. Overlapping Distributions: First Trimester Markers NT PAPP-A f  -hCG affected unaffected affected unaffected

50. MS AFP (MoM) NT (MoM) NT AFP SD of log MoM = 0.15 The Distribution of AFP and NT MoM in Unaffected Pregnancies SD of log MoM = 0.10

51. MS AFP (MoM) NT (MoM) NT hCG 0.210 hCG (MoM) The Distribution of hCG and NT MoM in Unaffected Pregnancies SD of log MoM = 0.23 SD of log MoM = 0.10

52. Why is NT such a good marker? NT: 0.11 SD 50% DR 1% FPR hCG: 0.24 SD 50% DR 8% FPR

53. Crossley JA et al. BJOG 2002;109:667-76. Inter-operator variation at one hospital Range of NT measurements (in MoM) between hospitals Published Literature: Variation in NT median measurement

54. Schielen PC et al., Prenat Diagn 2006;26:711-8 Published Literature: Variation in NT median measurement FMF median equation FMF-certified centers Non-FMF certified

55. Palomaki GE et al. Genet Med 2008;10(2):131-138 “Laboratories should routinely monitor the quality of nuchal translucency measurements… When possible, instituting sonographer-specific medians and providing individualized feedback about performance and numbers of women tested offer the potential to yield more consistent and improved performance.”

56. NT monitoring: when to make changes  Use objective criteria as guide  Partially subjective process  Look for trends  Sample volume must be considered  What to do with very small volume sonographers?  Sonographer feedback has been minimally useful

57. Nick Wald University of London Primary collaborators over many years Jim Haddow George Knight Glenn Palomaki Geralyn Lambert-Messerlian ---------Women & Infants Hospital - Brown University---------