Second Trimester Inhibin A and New Approaches in Prenatal Screening

1
Second Trimester Inhibin A andNew Approaches in
Prenatal Screening
Jacob A. Canick, Ph.D. Women and Infants
Hospital and Brown University Providence, Rhode
Island, USA
2
Second Trimester Inhibin A andNew 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

3
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

4
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!
5
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

6
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?

7
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?

8
Where are we now?
9
Maternal Serum Markers in the 2nd
TrimesterMajor 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.

10
Serum Screening Test Performanceat a fixed 5
False Positive Rate(Dating by Ultrasound)
100

80
69
59
60
DR at 5 FPR
37
40
30
20
0
AGE AFP hCG uE3
single double triple
2nd trimester
Wald et al. 2000
11
New ApproachesAddition of Inhibin A
12
Maternal Serum Markers in the 2nd
TrimesterFurther improvement in screening for
Down syndrome
Background 1992 Maternal serum total inhibin is
elevated in Down syndrome pregnancy 1994 Materna
l 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
13
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
Inhibin B
bB subunit
14
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
15
Similarity of hCG and inhA distributions in Down
syndrome and unaffected pregnancy
16
Second Trimester Markers in 73 Down Syndrome Cases
2.00
1.91
0.74
0.61
AFP
inhA
uE3
hCG
17
Prenatal Screening Performance with Inhibin A
18
Serum Screening Test Performanceat a fixed 5
False Positive Rate (Dating by Ultrasound)
100

76
80
69
59
60
DR at 5 FPR
37
40
30
20
0
AGE AFP hCG uE3
InhA
single double triple quadruple
2nd trimester
Wald et al. 2000
19
Performance of the 2nd Trimester Quad Marker
Test in Practice
no. inhA DR FPR Study 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
20
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 Downs 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 Downs
syndrome.
21
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

22
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 Downs syndrome
• - Marker related spontaneous fetal loss

23
Serum Screening Test Performanceat a fixed 5
False Positive Rate
Wald et al. 2000
24
Mechanism?

• Genes are not located on chromosome 21
• Tissue source?
• Placenta
• Fetal membranes
• Fetus

25
Chromosome 21 Not the source of genes for serum
markers identified so far.
Marker chromosome AFP 4q hCG a 6q hCG
b 19q inhibin a 2q inhibin bA 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.

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Down Syndrome Pregnancy Fetal Underproduction /
Placental overproduction ?
Fetal origin Placental origin AFP hCG D
HEAS free a-subunit of hCG 16aOHDHEAS free
b-subunit of hCG hPL Fetoplacental
origin SP1 estriol (unconj) inhibin-A (also
total inhibin) estriol (conj) progesterone
PLGF placental GH pro-MBP NAP
(neutrophil alk phos) PAPP-A
27
Placental Inhibin A Subunit mRNA Levels
Alpha GAPDH
Beta A GAPDH
Alpha
Beta A
Control
Down syndrome
Lambert-Messerlian et al., 1998
28
New ApproachesThe Integrated Test
29
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 NTPAPP-A quad
test FULL INTEGRATED
(weeks)
Integrate results into a single risk
30
Detection rate at a fixed 5 false positive
rate according to method of screening Performance
estimates based on modeling
100
90
80
70
60
Detection rate ()
50
40
30
20
30
37
69
76
85
85
94
10
0
MA AFP triple quad combined
serum full
------ 2nd trimester ------ 1st trim --
integrated --
PPV 1140 1110 160
150 145 145 140
Wald et al, 1997, 1999, 2001
31
Detection rate at a fixed 5 false positive
rate according to method of screening Modeling
vs SURUSS ( )
100
93
89
90
83
81
80
74
70
60
Detection rate ()
50
40
30
20
30
37
69
76
85
85
94
10
0
MA AFP triple quad combined
serum full
------ 2nd trimester ------ 1st trim --
integrated --
PPV 1140 1110 160
150 145 145 140
Wald et al, 1997, 1999, 2001, 2003
32
False positive rate to achieve an 85 detection
rate according to method of screening Performance
estimates based on modeling
20
14
15
9.0
Fasle positive rate ()
10
5.0
5.0
5
1.0
0
triple quad combined serum
full
---- 2nd trim ---- 1st trim --
integrated --
PPV 1130 185
145 145 19
Wald et al, 1997, 1999, 2001
33
False positive rate to achieve an 85 detection
rate according to method of screening Performance
estimates based on modeling
20
14
15
9.0
Fasle positive rate ()
10
5.0
5.0
5
1.0
0
triple quad combined serum
full
---- 2nd trim ---- 1st trim --
integrated --
PPV 1130 185
145 145 19
Wald et al, 1997, 1999, 2001
34
False positive rate to achieve an 85 detection
rate according to method of screening Modeling
vs SURUSS ( )
20
15
10.9
Fasle positive rate ()
10
7.1
6.1
5
3.0
1.3
14
9.0
5.0
5.0
1.0
0
triple quad combined serum
full
---- 2nd trim ---- 1st trim --
integrated --
PPV 1130 185
145 145 19
Wald et al, 1997, 1999, 2001, 2003
35
False positive rate to achieve a 90 detection
rate according to method of screening modeling
vs SURUSS ( )
20
17.0
15
11.7
10.8
Fasle positive rate ()
10
5.8
5
2.8
21.5
15.2
9.9
8.8
2.2
0
triple quad combined serum
full
---- 2nd trim ---- 1st trim --
integrated --
PPV 1190 1135
190 190 120
Wald et al, 1997, 1999, 2001, 2003
36
The Integrated Test

• Advantages
• Safest and most effective screening policy
• Substantially reduces the number of amnios needed
• Preserves AFP screening for open NTDs
• 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

37
New ApproachesFetal Cells and Fetal DNA in
theMaternal Circulation Are They Useful in
Prenatal Screeningfor Down Syndrome?
38
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
39
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 761453-5) 1990 nuclea
ted 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
1651731-7) 1997 nucleated RBCs increased number
of fetal cells in blood of women with
trisomy 21 fetuses (Bianchi et al, Am J Hum
Genet 61822-9)
40
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 4181-246, 1997.
41

• Fetal DNA in the Maternal Circulation History
• cell-free DNA circulating cell-free fetal DNA is
  also present in maternal blood (Lo et al,
  Lancet 350485-7)
• 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 451747-50)
• Note that only DNA from a male fetus can be
  detected in maternal blood.

42

• Fetal DNA in the Maternal Circulation History
• cell-free DNA circulating cell-free fetal DNA is
  also present in maternal blood (Lo et al,
  Lancet 350485-7)
• 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 451747-50)
• Note that only DNA from a male fetus can be
  detected in maternal blood.

43
Fetal DNA in the Maternal Circulation Are 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)
2.0 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 OVERALL (65) 1.64
44
Fetal DNA in the Maternal Circulation Are 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)
2.0 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 OVERALL (65) 1.64
Small numbers!
45
Fetal DNA in the Maternal Circulation Are 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)
2.0 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 OVERALL (65) 1.64
Small effect?
46
Levels of Fetal DNA in Maternal Serum in
Women Carrying Male Euploid and Trisomy 21
Fetuses (Lee et al, Am J Obstet Gynecol
20021871217-21)
47

• 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.

48
Second Trimester Inhibin A andNew 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.

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Second Trimester Inhibin A andNew 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.

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