Second Trimester Inhibin A and
New Approaches in Prenatal Screening
Jacob A. Canick, Ph.D.
Women and Infants Hospital and Brown University
Providence, Rhode Island, USA
J. Canick, 2003
Second Trimester Inhibin A and
New Approaches in Prenatal Screening
• 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
Improving Screening Performance
The challenge in screening is to have a test that has a high
detection rate and low false positive rate.
detection rate
percentage of affecteds called
screen positive by the test
The higher the better!
false positive rate
percentage of unaffecteds called
screen positive by the test
The lower the better!
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
Serum Screening Test Performance
at a fixed 5% False Positive Rate
(Dating by Ultrasound)
100
DR at 5% FPR
80
69%
59%
60
40
37%
30%
20
0
AGE
Wald et al. 2000
J. Canick, 2003
+AFP
+hCG
+uE3
single
double
triple
2nd trimester
New Approaches:
Addition of Inhibin A
J. Canick, 2003
Maternal Serum Markers in the 2nd Trimester:
Further improvement in screening for Down syndrome
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’
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 subunit
Pro-a subunit
Inhibin A
bA subunit
bB subunit
J. Canick, 2003
Inhibin B
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
J. Canick, 2003
Similarity of
hCG and inhA
distributions in
Down syndrome and
unaffected
pregnancy
J. Canick, 2003
Second Trimester Markers
in 73 Down Syndrome Cases
10
MoM
1.91
1
.1
J. Canick, 2003
0.74
AFP
2.00
0.61
uE3
hCG
inhA
Prenatal Screening Performance
with Inhibin A
J. Canick, 2003
Serum Screening Test Performance
at a fixed 5% False Positive Rate
(Dating by Ultrasound)
100
76%
DR at 5% FPR
80
69%
59%
60
40
30%
37%
20
0
AGE
Wald et al. 2000
J. Canick, 2003
+AFP
+hCG
+uE3
single
double
triple
2nd trimester
+InhA
quadruple
Performance of the 2nd Trimester Quad Marker Test
in Practice
no.
of DS
inhA
med.MoM
DR
(%)
FPR
(%)
Barts program*
111
2.18
81
6.9
WIH program**
61
1.93
83
7.1
Study
* 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 costeffective 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
Results of SURUSS
(Serum, Urine, Ultrasound Study)
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
Results of SURUSS
(Serum, Urine, Ultrasound Study)
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
Serum Screening Test Performance
at a fixed 5% False Positive Rate
Prediction
SURUSS
100
74%
DR at 5% FPR
80
66%
81%
76%
69%
60
42%
59%
40
37%
20
30%
0
AGE
Wald et al. 2000
J. Canick, 2003
+AFP
+hCG
+uE3
single
double
triple
2nd trimester
+InhA
quadruple
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
hCG a
hCG b
inhibin a
inhibin bA
J. Canick, 2003
4q
6q
19q
2q
7p
Chromosome 21
• 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.
Down Syndrome Pregnancy
Fetal Underproduction / Placental overproduction ?
Fetal origin:
AFP
DHEAS
16aOHDHEAS
Fetoplacental origin:
estriol (unconj)
estriol (conj)
J. Canick, 2003
Placental origin:
hCG
free a-subunit of hCG
free b-subunit of hCG
hPL
SP1
inhibin-A (also total inhibin)
progesterone
PLGF
placental GH
pro-MBP
NAP (neutrophil alk phos)
PAPP-A
Placental Inhibin A Subunit mRNA Levels
0.7
0.12
0.6
Beta A : GAPDH
Alpha : GAPDH
0.1
0.08
0.06
0.04
0.02
0
0.4
0.3
0.2
0.1
0
Alpha
Lambert-Messerlian et al., 1998
J. Canick, 2003
0.5
Beta A
Control
Down syndrome
New Approaches:
The Integrated Test
J. Canick, 2003
Prenatal Screening for Down Syndrome
A 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
PAPP-A
NT+PAPP-A
quad test = SERUM INTEGRATED
quad test = FULL INTEGRATED
Integrate results into a single risk
J. Canick, 2003
26
40
(weeks)
Detection rate at a fixed 5% false positive rate
according to method of screening:
Performance estimates based on modeling
Detection rate (%)
100
90
80
70
60
50
40
30
20
10
0
PPV:
30%
37%
MA +
AFP
triple
quad combined serum
full
------ 2nd trimester ------ 1st trim
-- integrated --
1:140
1:110
69%
1:60
76%
1:50
85%
1:45
85%
1:45
94%
1:40
Wald et al, 1997, 1999, 2001
J. Canick, 2003
Detection rate at a fixed 5% false positive rate
according to method of screening:
Modeling vs SURUSS (
)
Detection rate (%)
100
89%
90
80
81%
93%
83%
74%
70
60
50
40
30
20
10
0
PPV:
30%
37%
MA +
AFP
triple
quad combined serum
full
------ 2nd trimester ------ 1st trim
-- integrated --
1:140
1:110
69%
1:60
76%
1:50
85%
1:45
85%
1:45
94%
1:40
Wald et al, 1997, 1999, 2001, 2003
J. Canick, 2003
False positive rate to achieve an 85% detection rate
according to method of screening:
Performance estimates based on modeling
Fasle positive rate (%)
20
14%
15
9.0%
10
5.0%
5.0%
5
1.0%
0
triple
quad
---- 2nd trim ---PPV:
1:130
1:85
combined
1st trim
1:45
serum
full
-- integrated -1:45
1:9
Wald et al, 1997, 1999, 2001
J. Canick, 2003
False positive rate to achieve an 85% detection rate
according to method of screening:
Performance estimates based on modeling
Fasle positive rate (%)
20
14%
15
9.0%
10
5.0%
5.0%
5
1.0%
0
triple
quad
---- 2nd trim ---PPV:
1:130
1:85
combined
1st trim
1:45
serum
full
-- integrated -1:45
1:9
Wald et al, 1997, 1999, 2001
J. Canick, 2003
False positive rate to achieve an 85% detection rate
according to method of screening:
Modeling vs SURUSS (
)
Fasle positive rate (%)
20
15
10.9%
10
7.1%
6.1%
5
3.0%
14%
0
9.0%
triple
quad
---- 2nd trim ---PPV:
1:130
1:85
5.0%
combined
1st trim
1:45
5.0%
1.3%
1.0%
serum
full
-- integrated -1:45
1:9
Wald et al, 1997, 1999, 2001, 2003
J. Canick, 2003
False positive rate to achieve a 90% detection rate
according to method of screening:
modeling vs SURUSS (
Fasle positive rate (%)
20
)
17.0%
15
11.7%
10.8%
10
5.8%
5
2.8%
21.5%
0
15.2%
triple
quad
---- 2nd trim ---PPV:
1:190
1:135
9.9%
combined
1st trim
1:90
8.8%
2.2%
serum
full
-- integrated -1:90
1:20
Wald et al, 1997, 1999, 2001, 2003
J. Canick, 2003
The Integrated Test
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
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
Fetal Cells Sorted from Maternal Blood Using
Anti-Gamma Globin
Gamma positive
fetal cell pre-FISH
Same cell post-FISH
showing X and Y
Courtesy of Dr. Diana Bianchi
J. Canick, 2003
Fetal Cells in the Maternal Circulation: History
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
first correct determination of fetal
aneuploidy (Price et al, Am J Obstet
Gynecol 165:1731-7)
nucleated RBCs
1997 nucleated RBCs
J. Canick, 2003
increased number of fetal cells in
blood of women with trisomy 21
fetuses (Bianchi et al, Am J Hum
Genet 61:822-9)
Comparison of Down Syndrome Screening Performance
of Fetal Cells and Second Trimester Maternal Serum
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.
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 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 Circulation
Are the levels higher in Down syndrome pregnancy?
study
copies of fetal DNA/ml
plasma or serum (N)
controls
cases
case/
control
ratio
comments
Lo et al, 1999
Hong Kong
Boston
16 (18)
23 (19)
48 (6)
46 (7)
3.0
2.0
plasma / median
plasma / 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
(65)
1.64
OVERALL
J. Canick, 2003
Fetal DNA in the Maternal Circulation
Are the levels higher in Down syndrome pregnancy?
study
copies of fetal DNA/ml
plasma or serum (N)
controls
cases
case/
control
ratio
comments
Lo et al, 1999
Hong Kong
Boston
16 (18)
23 (19)
48 (6)
46 (7)
3.0
2.0
plasma / median
plasma / 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
(65)
1.64
OVERALL
Small numbers!
J. Canick, 2003
Fetal DNA in the Maternal Circulation
Are the levels higher in Down syndrome pregnancy?
study
copies of fetal DNA/ml
plasma or serum (N)
controls
cases
case/
control
ratio
comments
Lo et al, 1999
Hong Kong
Boston
16 (18)
23 (19)
48 (6)
46 (7)
3.0
2.0
plasma / median
plasma / 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
(65)
1.64
OVERALL
Small effect?
J. Canick, 2003
Levels of Fetal DNA in Maternal Serum in Women
Carrying Male Euploid and Trisomy 21 Fetuses
fetal DNA concentration
(GE/ml)
(Lee et al, Am J Obstet Gynecol 2002;187:1217-21)
100
50
20
10
J. Canick, 2003
15
16
17
18
Gestational Age (weeks)
19
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
Second Trimester Inhibin A and
New Approaches in Prenatal Screening
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
Second Trimester Inhibin A and
New Approaches in Prenatal Screening
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
.
..
llginize tesekkur ederim.
..
..
Beni guzel ulkenize
davet
..
ettiginiz icin tesekkur ederim.
J. Canick, 2003