BROWN

Prenatal Screening for Trisomy 21:Recent Advances and Guidelines

Jacob Canick, PhD

Alpert Medical School of Brown UniversityWomen & Infants Hospital

Providence, RI, USA

Department of PathologyMontefiore Medical Center

Bronx, NYMarch 17, 2011

Women & Infants’

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

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

Brief history of prenatal screening

Spina bifidaAnencephaly

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

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)

andprenatal screening

(MSAFP)

Prenatal Screening History Begins with Prenatal Screening for Open Neural Tube Defects using MSAFP

Fetal nucleic acids in maternal plasma?

2011

Integrated 1st and 2nd trim Sequential 1st and 2nd trim

2000

1st trim nuchal translucency (NT) 1st 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 50th Anniversary of discovery by LeJeune)

1950 1940

Down syndrome – maternal age association1930

Prenatal Screening for Aneuploidies:History2nd trim AFP (with Mat Age)*

2nd trim Multiple markers (Double, Triple, Quad)

Maternal Serum Markers in the 2nd Trimester:Major improvement in screening for Down syndrome

Background

1980s Maternal age as a reason for amniocentesis.

1980s Maternal serum AFP already in use in screening for open NTDs.

1984 Maternal serum AFP is low in Down syndrome pregnancy.

Irwin Merkatz, MDAlbert Einstein Col. Med.

How is screening performancemeasured?

Detection Rate percentage of affecteds called (sensitivity) screen positive by the test

The higher the better!

False Positive Rate percentage 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.

Performance of second trimester,first trimester, integrated, and

sequential tests for Down syndrome

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 40weeks

Screening in the Early 2nd Trimester (15-20 weeks)with Multiple Serum Markers

.1

1

10

MoM

AFP inhAuE3

0.74

2.00

hCG

0.61

1.91

2nd Trimester Serum Markers in Down Syndrome Pregnancies

Data from FASTER

Prenatal Screening for Down Syndrome in the Early 2nd Trimester (15 -20 weeks)

0102030405060708090

100

Det

ectio

n R

ate

(%)

MA + double triple quad ---2nd trimester-----

30%

69% 79%

at 5% false positive rate

59%

81%79%74%

72%

SURUSSFASTER

66%

Advantages:• Patient privacy

• Earlier diagnosis (if CVS is available)

• Greater availability of pregnancy termination

• Earlier, safer pregnancy termination

0 13 26 40weeks

Screening in the Late 1st Trimester (11-13 weeks)

.1

1

10

MoM

NT f-hCG PAPP-A

1.99 2.29

0.50

F M

alon

e, w

ith p

erm

issi

on

First TrimesterCombined Test:• NT ultrasound• serum PAPP-A• serum -hCG

Data from FASTER

0102030405060708090

100

Det

ectio

n R

ate

(%)

MA + triple quad NT combined 2nd trimester 1st trimester

30% 69% 79% 85%

at 5% false positive rate

64%

Prenatal Screening for Down Syndrome in the Late 1st Trimester (11-13 weeks)

SURUSSFASTERBUN

85% 79%

69% 68% 62%

• 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 40weeks

The Integrated Test:Two Stages to Reach a Single Risk Assessment

Wald NJ et al. N Eng J Med 1999

0102030405060708090

100

Dete

ctio

n Ra

te (%

)

MA + triple quad combined serum full 2nd trimester 1st trimester -integrated-

30%

69%79%

85% 85%94%

89%86%

95%93%

Performance of the Integrated Test

at 5% false positive rate

85%

SURUSSFASTER

0102030405060708090

100

12%

50%60% 66% 66%

85%

Det

ectio

n R

ate

(%)

at 1% false positive rate

MA + triple quad combined serum full 2nd trimester 1st trimester -integrated-

85% DR at 1%

73%70%

87%86%

72%

SURUSSFASTER

Performance of the Integrated Test

Relative Screening Performance of1st Trimester Combined and Integrated Tests

If 1st Trimester Combined Test Performance is:

DR FPR

Then Full Integrated TestPerformance 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%

Is there a way to have both:

earlier screening and diagnosis – as in the 1st trimester test

and

much lower screen positive rate – as in the Integrated Test

YES

Sequential protocols

Sequential Screening: Towards 1st trimester combined or towards integrated, depending on the 1st trimester risk cut-off

2 3 4 570

80

90

100

Full Integrated

First Trimester CombinedSequential protocols

False positive rate (%)

Det

ectio

n ra

te (%

)

Example of Sequential ScreeningPalomaki et al. Obstet Gynecol 2006;107:367-75.

DR FPRStep 1. 1st trimester markers

measured on all women at 10-13 wk. Risk cut-off set at ≥ 1:63 71.8% 1.5%

Step 2. Screen negatives (98.5% of population) have quad markers measured at 15-18 wk. Risk cut-off set at ≥ 1:65 12.5% 0.5%

Overall 84.3% 2.0%

Assumes 1:600 prevalence

Comparing Sequential Screening to 1st Trimester Combined Test and Integrated Test

Palomaki et al. Obstet Gynecol 2006;107:367-75.

Test

1st Trimester Combined

Sequential example

Full Integrated

FPR to get84.3% DR

6.0%

2.0%

1.2%

Sequential Integrated Screeningat Women & Infants (Expected Performance)

Stage 2: • Quad markers• Full Integrated Risk• 1:110 risk cut-off• 31% DR, 1.6% FPR

Stage 1: • 1st 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 1st TrimesterTest: 90% DR, 8.0% FPR

Sequential Integrated Test: 90% DR, 2.5% FPR

Prenatal Screening Guidelines:Practice Standards in the United States

American College of Obstetricians and

Gynecologists (ACOG)

January 2007

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

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

Trisomy 18 (Edwards Syndrome)and

Trisomy 13 (Patau Syndrome)

1st trim. marker Median MoM

NT 2.17

PAPP-A 0.20

Free -hCG 0.25

Trisomy 18: Marker LevelsGE Palomaki 2011, analysis of published literature

2nd trim. marker Median MoM

AFP 0.66

uE3 0.36

hCG 0.39

inhA 0.88

PAPP-A 0.10

Modeled Performance

Test DR for0.1% FPR

DR for0.5% FPR

2nd trim. triple 72 821st trim. combined 80 90Serum integrated 80 89Full integrated 90 95

Trisomy 18: Screening PerformanceGE Palomaki 2011, unpublished

Actual PerformanceBased on clinical trial data, the observed screen positive rate in practice will be higher than the modeling suggests.

Trisomy 13

• 2nd trimester markers are not informative.

• 1st 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 1st trimester markers.

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

Prenatal Screening: Serum Assays

Assay type Choices? FDA-approved

2nd trimester:

AFP 2 site immunometric Yes Yes

uE3 competitive immunoassay Yes Yes

-hCG 2 site immunometric Yes Yes

inhibin A 2 site immunometric No Yes

1st trimester:

PAPP-A 2 site immunometric Yes Yes

free -hCG

2 site immunometric Yes/No No

free subunit or hCG itself in the 1st trimester?

Palomaki GE et al. Adv Clin Chem 2007;43:177-210.

free hCG hCG

First Trimester Screening Performance

(NT + serum markers): SURUSS

Wald et al., J Med Screen 2003;10:56-104.

Gest age (weeks)

DR

@ 5

% F

PR

60

65

70

75

80

85

90

95

100

9 10 11 12 13 14

+ PAPP-A

+ PAPP-A + free

+ PAPP-A + hCG

First Trimester Screening Performance

(NT + serum markers): FASTER

Canick et al., Obstet Gynecol 2006;108:1192-9.

Gest age (weeks)

DR

@ 5

% F

PR

60

65

70

75

80

85

90

95

100

10 11 12 13 14

+ PAPP-A

+ PAPP-A + free

+ PAPP-A + hCG

First Trimester Screening Performance(NT + serum markers): Evans meta-

analysis

Evans et al., Am J Obstet Gynecol, March 2007

Gest age (weeks)9 10 11 12

+ PAPP-A

+ PAPP-A + free

+ PAPP-A + hCG

60

65

70

75

80

85

90

95

100

DR

@ 5

% F

PR

• Univariately, free- is a better 1st 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 1st trimester screening.

free subunit or hCG itself in the 1st trimester?

Prenatal Screening Quality Assurance

Applying Serum MarkerQuality Assurance Measuresto Nuchal Translucency (NT)

Marker parameters that are monitored:

Rate of change with may go up or down at a constant increasing gestation rate

Median calculated MoM values should be stable at 1.0

SD of the distribution calculated SD of the log MoM values expected to remain steady

Prenatal Screening MarkersQuality monitoring

log-linear increaseslope = +15% per week Examples of Marker

QA Parameters

MS AFP (MoM)

unaffecteddistribution(x and SD)

(median MoM)

http

://w

ww

.feta

lmed

icin

e.co

m

“normal nuchal translucency” “increased nuchal translucency”

Nuchal Translucency (NT)

Measurement

F M

alon

e, w

ith p

erm

issi

on

NT parameters that are monitored:

Rate of increase with CRL log-linear over 10,3 - 13,6 weeksshould go up by ~ 20% per week

Median calculated MoM values should be stable at 1.0 MoM

SD of the distribution calculated SD of the log MoM values expected to be about 0.1

1st Trimester Nuchal Translucency (NT)Quality monitoring

log-linear increaseslope = +15% per

week

log-linear increaseslope = +20% per week

Maternal Serum AFP Increases with

Increasing Gestation

Nuchal Translucency Increases with

Increasing GestationSchuchter et al, Prenat Diagn 1998

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

Overlapping Distributions: First Trimester Markers

NT

PAPP-A

f-hCG

affected

unaffected

affected

affectedunaffected

unaffected

MS AFP (MoM)

NT (MoM)

NT

AFPSD of log MoM = 0.15

The Distribution of AFP and NT MoM

in Unaffected PregnanciesSD of log MoM = 0.10

MS AFP (MoM)

NT (MoM)

NT

hCG

0.2 10hCG (MoM)

The Distribution of hCG and NT MoM

in Unaffected Pregnancies

SD of log MoM = 0.23

SD of log MoM = 0.10

Why is NT such a good marker?

0.2 0.5 1 2 5 10NT (MoM)

0.2 0.5 1 2 5 10hCG (MoM)

unaffected

unaffected DS

DSNT: 0.11 SD50% DR 1% FPR

hCG: 0.24 SD50% DR 8% FPR

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

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

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.”

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

Nick Wald

University of London Primary collaborators

over many years

JimHaddow

GeorgeKnight

GlennPalomaki

GeralynLambert-Messerlian

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