second trimester inhibin a and new approaches in prenatal screening

J. Canick, 2003

Second Trimester Inhibin A andNew Approaches in Prenatal Screening

Jacob A. Canick, Ph.D.

Women and Infants Hospital and Brown UniversityProvidence, Rhode Island, USA

J. Canick, 2003


• Goals in prenatal screening

• Where are we now?

• New approaches: Addition of inhibin A

• New approaches: The Integrated Test

• New approaches: Fetal cells and DNA

J. Canick, 2003

Goals in Prenatal Screening

• Improved test performance (make screening safer)

• Optimal timing of test

• Offer the best, safest diagnostic test

• Make screening available to the largest number of women

J. Canick, 2003

detection rate percentage of affecteds calledscreen positive by the test

The higher the better!

false positive rate percentage of unaffecteds called screen positive by the test

The lower the better!

Improving Screening Performance

The challenge in screening is to have a test that has a high detection rate and low false positive rate.

J. Canick, 2003

Optimal Timing of Screening Test

• The earlier the better – only if it is also a better test!

• How early do most women present for prenatal care?

• What diagnostic test will be offered if a woman is screen positive?

• Safer options if a serious fetal abnormality is identified

J. Canick, 2003

Offer the best, safest diagnostic test

• For prenatal chromosome analysis, the choices are CVS or amniocentesis.

• CVS is done no earlier than 10.5 weeks and usually not later than 13-14 weeks.

• Amniocentesis is usually done no earlier than 14-15 weeks.

• Is CVS easily available in your geographic area?

• Which procedure is safer?

• Which procedure has more errors?

J. Canick, 2003

Make prenatal screening available to the largest number of women

• Ability to pay for test (government, insurance, self-pay?)

• Geographic dispersion (in city, suburban area, rural area)?

• Availability of quality laboratory services?

• Availability of quality obstetrical ultrasound?

• Availability of genetic counseling?

• Availability of appropriate follow-up diagnostic procedures and tests?

J. Canick, 2003

Where are we now?

J. Canick, 2003

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

Background

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

1984 Maternal serum AFP is low in Down syndrome pregnancy.

1987 Maternal serum uE3 is low in Down syndrome pregnancy.

1987 Maternal serum hCG is elevated in Down syndrome pregnancy.

1988 Triple marker screening for Down syndrome in the second trimester is introduced.

J. Canick, 2003

100

80

60

40

20

0

Serum Screening Test Performanceat a fixed 5% False Positive Rate

(Dating by Ultrasound)

AGE +AFP +hCG +uE3

DR

at

5% F

PR

2nd trimester

single double triple

30%37%

59%

69%

Wald et al. 2000

J. Canick, 2003

New Approaches:

Addition of Inhibin A

J. Canick, 2003

Background

1992 Maternal serum total inhibin is elevated in Down syndrome pregnancy

1994 Maternal serum inhibin A is elevated in Down syndrome pregnancy

1996 Inhibin A added to the triple test to make a quad marker test in the second trimester. Introduced in the UK at Barts.

1998 Introduction of Quad Test in U.S. at Women & Infants’

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

J. Canick, 2003

What is Inhibin A?

• alpha-beta subunit glycoprotein hormone

• inhibits the secretion of FSH from the anterior pituitary

• synthesized in ovary and placenta (Inhibin B is synthesized in ovary and testis)

• molecular weight approx. 32,000 daltons

• inhibin-A = alpha subunit + betaA subunitPro- subunit

subunit

subunit

Inhibin A

Inhibin B

J. Canick, 2003

Maternal Serum Inhibin A:

Like hCG, it is, on

average, about two times

higher in Down syndrome

than in unaffected

pregnancies.

Wald NJ et al, J Med Screen, 1997

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Similarity ofhCG and inhA distributions in

Down syndrome and unaffected

pregnancy

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.1

1

10

Mo

M

AFP inhAuE3

0.74

2.00

Second Trimester Markersin 73 Down Syndrome Cases

hCG

0.61

1.91

J. Canick, 2003

Prenatal Screening Performance

with Inhibin A

J. Canick, 2003

AGE +AFP +hCG +uE3 +InhA

DR

at

5% F

PR

100

80

60

40

20

0

2nd trimester

single double triple quadruple


(Dating by Ultrasound)

76%

30%37%

59%

69%

Wald et al. 2000

J. Canick, 2003

Performance of the 2nd Trimester Quad Marker Testin Practice

no. inhA DR FPRStudy of DS med.MoM (%)

(%)

Barts program* 111 2.18 81 6.9

WIH program** 61 1.93 83 7.1

* 46,193 pregnancies screened over 5 years; 1 in 300 term risk cut-off used; Wald et al., Lancet 2003

** 40,450 pregnancies screened over 5 years; 1 in 380 term risk cut-off used; median MA = 28 years.; mss in preparation

J. Canick, 2003

Results of SURUSS(Serum, Urine, Ultrasound Study)

PI: Nicholas Wald, FRCP, Wolfson Institute, London

Objective: To determine the most effective, safe and cost-effective method of antenatal screening

for Down’s syndrome using maternal age, nuchal translucency, and maternal serum and urine markers.

Setting: 25 maternity units (24 in the UK) offering 2nd trimester serum screening that agreed to

collect observational data in the 1st trimester.

Size: results on 47,053 singleton pregnancies, including 101 with Down’s syndrome.

J. Canick, 2003


Study Design:

Serum and Urine Markers:• 101 Downs, 505 controls• matched for gestational age and duration of

sample storage• markers measured at 9-13 weeks gestation

and at 14-22 weeks

Nuchal translucency:• All pregnancies

Intervention:• Based on on 2nd trimester screening• 1st trimester data observational

J. Canick, 2003


Ability to compare 1st and 2nd trimester results:

• Currently, the only study in the literature that can compare 1st and 2nd trimester screening fairly

• The FASTER Trial, an NIH-sponsor study in the U.S. will also be able to compare 1st and 2nd trimester screening fairly

• This is because there are two biases that must be taken into account in comparing the two time periods:

- Natural fetal loss in Down’s syndrome- Marker related spontaneous fetal loss

J. Canick, 2003


Wald et al. 2000

Prediction

SURUSS

AGE +AFP +hCG +uE3 +InhA

DR

at

5% F

PR

100

80

60

40

20

0

2nd trimester

single double triple quadruple

76%

30%37%

59%

69%

42%

66%

74%81%

J. Canick, 2003

Mechanism?

• Genes are not located on chromosome 21

• Tissue source?– Placenta– Fetal membranes – Fetus

J. Canick, 2003

Chromosome 21:Not the source of genes for serum markers identified so

far.

Marker chromosome

AFP 4qhCG 6qhCG 19qinhibin 2qinhibin A 7p

• 300 genes

• 130 known human genes

• Down syndrome critical region is 2-20 Mb in q22.

• Likely that Down syndrome is a contiguous gene syndrome; unlikely that a single gene region is responsible.

• Transcription factors, GF receptors, SOD1 on 21.

Chromosome 21

J. Canick, 2003

Down Syndrome PregnancyFetal Underproduction / Placental

overproduction ?Fetal origin: Placental origin:

AFP hCGDHEAS free -subunit of hCG16OHDHEAS free -subunit of hCG

hPLFetoplacental origin: SP1

estriol (unconj) inhibin-A (also total inhibin)estriol (conj) progesterone

PLGFplacental GHpro-MBPNAP (neutrophil alk phos)PAPP-A

J. Canick, 2003

Placental Inhibin A Subunit mRNA Levels

0

0.02

0.04

0.06

0.08

0.1

0.12

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

ControlDown syndrome

Alpha Beta A

Lambert-Messerlian et al., 1998

Alp

ha

: G

AP

DH

Bet

a A

: G

AP

DH

J. Canick, 2003

New Approaches:

The Integrated Test

J. Canick, 2003

Prenatal Screening for Down SyndromeA New Approach: The Integrated Test

• Developed by Nicholas Wald in 1999.

• The integration of the best tests performed at different times in pregnancy into a single test.

• This will be more effective than current tests performed at any one time.

0 13 26 40 PAPP-A quad test = SERUM INTEGRATED NT+PAPP-A quad test = FULL INTEGRATED

(weeks)

Integrate results into a single risk

J. Canick, 2003

0

10

20

30

40

50

60

70

80

90

100

De

tect

ion

rat

e (

%)

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

Detection rate at a fixed 5% false positive rate

according to method of screening:Performance estimates based on modeling

Wald et al, 1997, 1999, 2001

PPV: 1:140 1:110 1:60 1:50 1:45 1:45 1:40

30% 37% 69% 76% 85% 85% 94%

J. Canick, 2003

0

10

20

30

40

50

60

70

80

90

100

De

tect

ion

rat

e (

%)

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

Detection rate at a fixed 5% false positive rateaccording to method of screening:

Modeling vs SURUSS ( )

PPV: 1:140 1:110 1:60 1:50 1:45 1:45 1:40

30% 37% 69% 76% 85% 85% 94%

Wald et al, 1997, 1999, 2001, 2003

74%81% 83%

89% 93%

J. Canick, 2003

0

5

10

15

20

Fa

sle

po

sit

ive

rat

e (

%)

9.0%

5.0% 5.0%

1.0%

triple quad combined serum full---- 2nd trim ---- 1st trim -- integrated --

False positive rate to achieve an 85% detection rateaccording to method of screening:

Performance estimates based on modeling

Wald et al, 1997, 1999, 2001

PPV: 1:130 1:85 1:45 1:45 1:9

14%

J. Canick, 2003

0

5

10

15

20

Fa

sle

po

sit

ive

rat

e (

%)

14%

9.0%

5.0% 5.0%

1.0%



Performance estimates based on modeling

Wald et al, 1997, 1999, 2001

PPV: 1:130 1:85 1:45 1:45 1:9

J. Canick, 2003

0

5

10

15

20

Fa

sle

po

sit

ive

rat

e (

%)



Modeling vs SURUSS ( )

PPV: 1:130 1:85 1:45 1:45 1:9

14% 9.0% 5.0% 5.0% 1.0%

Wald et al, 1997, 1999, 2001, 2003

1.3%3.0%

6.1%7.1%

10.9%

J. Canick, 2003

0

5

10

15

20

21.5% 15.2% 9.9% 8.8% 2.2%


Fa

sle

po

sit

ive

rat

e (

%)

False positive rate to achieve a 90% detection rateaccording to method of screening:

modeling vs SURUSS ( )

Wald et al, 1997, 1999, 2001, 2003

PPV: 1:190 1:135 1:90 1:90 1:20

2.8%

5.8%

10.8%11.7%

17.0%

J. Canick, 2003

Advantages:• Safest and most effective screening policy• Substantially reduces the number of amnios

needed• Preserves AFP screening for open NTD’s• Avoids confusion from several results

Stated Concerns:• Antenatal diagnosis and termination, if chosen, at

about 16-17 instead of 13-14 weeks of pregnancy• 2-5 week waiting period before results are

reported

The Integrated Test

J. Canick, 2003

New Approaches:

Fetal Cells and Fetal DNA in the

Maternal Circulation:

Are They Useful in Prenatal Screening

for Down Syndrome?

J. Canick, 2003

Gamma positive fetal cell pre-FISH

Same cell post-FISHshowing X and Y

Fetal Cells Sorted from Maternal Blood Using Anti-Gamma Globin

Courtesy of Dr. Diana Bianchi

J. Canick, 2003

1979 fetal lymphocytes first fetal cells to be successfully enriched lymphocytes

from maternal blood by the use of FACS (Herzenberg et al, PNAS 76:1453-5)

1990 nucleated RBCs first study to enrich for fetal nucleated erythrocytes (Bianchi

et al, PNAS USA 87: 3279-83)

1991 nucleated RBCs first correct determination of fetal aneuploidy (Price et al, Am J Obstet Gynecol 165:1731-7)

1997 nucleated RBCs increased number of fetal cells in blood of women with trisomy 21 fetuses (Bianchi et al, Am J Hum Genet 61:822-9)

Fetal Cells in the Maternal Circulation: History

J. Canick, 2003

test DR at 1.5% FPR

fetal cells ~ 45%

double screen ~ 40%

triple screen ~ 50%

quad screen ~ 60%

Fetal cell data from Bianchi DW et al, Prenat Diagn 19: 993-7, 1999.

Maternal serum data calculated from Wald NJ et al, J Med Screen 4:181-246, 1997.

Comparison of Down Syndrome Screening Performance of Fetal Cells and Second Trimester

Maternal Serum

J. Canick, 2003

Fetal DNA in the Maternal Circulation: History

1997cell-free DNA* circulating cell-free fetal DNA is also present in maternal blood (Lo et al, Lancet 350:485-7)

Method: Real time PCR analysis of maternal plasma or serum samples from pregnancies using

Y chromosome-specific probes.

1999 cell-free DNA* increased DNA concentrations in the plasma of pregnant women with trisomy 21 fetuses (Lo et al, Clin Chem 45:1747-50)

* Note that only DNA from a male fetus can be detected in maternal blood.

J. Canick, 2003

Fetal DNA in the Maternal CirculationAre the levels higher in Down syndrome pregnancy?

copies of fetal DNA/ml case/plasma or serum (N) control

study controls cases ratio comments

Lo et al, 1999 Hong Kong 16 (18) 48 (6) 3.0 plasma /

median Boston 23 (19) 46 (7) 2.0plasma / median

Zhong et al, 2000 83 (29) 186 (15) 2.2 plasma / mean

Lee et al, 2002 24 (55) 41 (11) 1.7 serum / median

Hromadnikova, 2002 25 (10) 23 (11) 0.9 plasma / median

Spencer et al, 2003 34 (10) 32 (10) 0.9 serum / median

Samura et al, 2001 32 (55) 24 (5) 0.8 serum / mean

OVERALL (65) 1.64

J. Canick, 2003










OVERALL (65) 1.64

Small numbers!


J. Canick, 2003Small effect?











OVERALL (65) 1.64

J. Canick, 2003

15 16 17 18 19Gestational Age (weeks)

10

20

50

100

feta

l D

NA

con

cen

trati

on

(GE/m

l)

Levels of Fetal DNA in Maternal Serum in WomenCarrying Male Euploid and Trisomy 21 Fetuses

(Lee et al, Am J Obstet Gynecol 2002;187:1217-21)

J. Canick, 2003

Fetal Cells in the Maternal Circulation: Summary

• Fetal cells in maternal blood is not yet a diagnostic test.

• Fetal cell analysis is not yet as good as most serum screening methods for Down syndrome.

• We must wait for advances in technology before fetal cells are useful in screening for diagnosis.

• Fetal DNA in maternal blood appears to be elevated in Down syndrome pregnancy.

• However, it is not yet useful in screening.

• Further research is necessary, both to confirm the findings and to discover a method to quantify DNA which comes from female fetuses.

J. Canick, 2003


Summary:

• The goal in prenatal screening should be to provide the safest, most effective test to the greatest number of women.

• Second trimester serum screening (double or triple markers) is effective, but is no longer the most effective screening method.

• The addition of inhibin A improves 2nd trimester serum screening. The quad marker test should be offered to all women having screening beginning at 15 weeks gestation.

J. Canick, 2003


Summary (continued):

• First trimester combined screening (NT and serum markers) is an effective test for women who seek earlier diagnosis.

• The Integrated Test is the safest, most effective test currently available. If NT measurement is not available, the Serum Integrated Test is the most effective method of serum screening.

• Fetal cells or fetal DNA in the maternal circulation should be considered research, not to be applied clinically.

J. Canick, 2003

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second trimester inhibin a and new approaches in prenatal screening

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