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OCR for page 311
Appendix
Assessing the Validity of Biologic Markers:
Alpha-Fetoorotein
The previous chapters associated with
pregnancy issues have discussed potential
biologic markers for use in toxicity evalu-
ations during pregnancy; however, only
alpha-fetoprotein has been evaluated in
sufficient depth to allow for a rigorous
evaluation of fetal and embryonic abnor-
malities. This detailed analysis is in-
cluded to review and establish criteria
for evaluating any proposed biologic mark-
er of toxicity.
The identification of biologic markers
that indicate exposure, effect, or suscep-
tibility is a complicated process involv-
ing studies animals, refinements in labor-
atory assays, and studies in special human
populations. Moreover, even when a marker
has been validated in such studies, its
use in larger populations is not straight-
forward. This chapter discusses experi-
ence in the implementation of large pop-
ulations of alpha-fetoprotein (AFP) in
maternal serum and amniotic fluid to screen
for neural tube defects (NTDs) in fetuses.
The NTDs anencephaly and spine bifida
are among the most common and serious of
congenital malformations (Warkany, 1971~.
Both arise in the first month of pregnancy.
They are sometimes fatal and sometimes
result in lifelong handicap. During the
past decade, it has become possible to
detect the presence of neural-tube defects
311
r
during fetal life by using concentrations
of AFP as a biologic marker (Gastel et al.,
1980; Mizejewski and Porter, 1985~. Re-
viewing the use of AFP as a marker of fetal
maldevelopment provides a useful frame-
work for considering some practical as-
pects of the use of biologic markers of
effect. Biologic markers sometimes are
used primarily to assess the connection
between a particular exposure and a par-
ticular disease, and they are sometimes
used primarily to help in making personal
or public health decisions. AFP is dis-
cussed here in the latter context, but the
remarks are equally applicable to the
former context.
Anencephaly is absence of the cranial
vault with a degenerated or rudimentary
brain. The defect is incompatible with
extrauterine life; many affected infants
are spontaneously aborted, and the remain-
der are stillborn or die soon after birth.
Spina bifida is characterized by defective
closure of the spinal cord. If the spinal
cord tissue and meninges protrude through
the vertebrae, the condition is called
spine bifida cystica. The defect can occur
anywhere on the spinal column, but is most
common in the lumbar region. The protrud-
ing nervous tissue is sometimes covered
with skin. Many affected fetuses die, but
many are born alive and, depending on the
OCR for page 312
312
ll
1'~
l
APPENDIX
, j i i
1 i
~ ,
! I
~ !
JO ~
1 2 3 4 5 6 7 8910
MSAFP (Multiples of the Unaffected Median)
FIGURE A-1 Distn~ution of concentration of maternal serum alpha-fetoprote~n (MSAFT) In a population of
unaffected pregnancies (curve on left) and in a population of pregnancies in which the fetus has spine bifida
pystica (curve on right). The abscissa represents the gestational ag~corrected concentration of M:iAFP (using
the date of the last menstrual period to date specimen) plotted in terms of multiples of the (unaffected) median
(MOMs). Ike ordinate represents the frequency of MOM values with in each of the two populations. Source:
Reprinted with permission from Adams et al., 1984. Copyright 1984 by C.V. Mostar Co.
location and seriousness of the lesion,
can lead relatively normal lives, in spite
of physical handicap. Recent advances
in medical and surgical therapy have made
it possible for many severely affected
children to survive (Bamforth and Baird,
1989).
AFP was discovered about 30 years ago.
It can be detected in the maternal serum
by 29 days after conception (Bergstrand
and Czar, 1957~. It is synthesized early
by the yolk sac and liver and later almost
exclusively by the liver. In fetal serum,
the concentration reaches a peak at about
13 weeks of gestation and decreases there-
after. AFP is excreted in the urine and
therefore is found in the amniotic fluid
(Crandall, 1981~. AFP concentration is
increased in maternal serum during preg-
nancy. Some fetal conditions, including
anencephaly and spine bifida cystica,
raise the amniotic fluid and maternal serum
concentrations above normal (Bergstrand,
1986~. The finding of increased AFP in
maternal serum/amniotic fluid, or both
thus constitutes an indication of open
NTDs. (High concentrations are also found
when the fetus has a ventral wall defect
or when there are twins.) Figure A- 1 shows
the distribution of maternal serum AFP
concentrations in normal pregnancies
(curve on left) and pregnancies with spine
bifida cystica in the fetus (curve on
right).
The screening process for NTD begins
with the measurement of maternal serum
AFP during the second trimester of pregnan-
cy, preferably in week 16 of gestation.
That measurement is the start of a multi-
stage process, which is outlined in Figure
A-2. The following discusses the various
aspects of marker validity and concen-
trate on this first stage, but the prin-
ciples apply to all stages in the screen-
ing; indeed, they apply to most medical
decision-making processes and in par-
ticular to the use of any marker of disease
or exposure (Galen and Gambino, 1975~.
Experience with the use of AFP demon-
strates the importance of determining the
OCR for page 313
ALP~4-FETOPROTEIN
Initial MSAFP*
Not Elevated Elevated
Anencep:
Ultrasonography
/ \
Diagnosis by Ultrasonography: No Diagnosis Possible
\
Amniocentesis Decision:
Does Opportunity to
Detect an Affected
Fetus Warrant Cost
and Risk of Amnio-
centesis?
Multiple Fetuses
Mistaken Ges-
tational Age
Fetal Demise
Alter
Prenatal
Care
Yes
Amniocentesis:
AFAFP** and
Karyotyping (if indicated)
Diagnosis of No Indl ation of
Affected Fetus Fetal Abnormality
Patient Decision
Plan for Induced
Special Care Abortion
at Delivery
~MSAFP = maternal serum alpha-fetoprotein
*WAFAFP = amniotic fluid alpha-fetoprotein
\`No
predictive value of a test in the general
population and developing a multistep,
multimarker screening protocol.
ASSESSING THE VALIDITY OF
BIOLOGIC MARKERS
As noted earlier, an ideal biologic
marker should be a sensitive and specific
indicator of disease or exposure and (in
the context of a specific use) should be
suitably predictive of the disease or ex-
posure. The terms "sensitive," "specif-
ic,~ and "predictive" are defined below,
followed by a discussion of how maternal
serum AFP qualifies as a marker.
"Sensitivity" refers to the ability of
a marker to indicate the presence of dis-
ease or exposure when disease or expo-
sure is present. Sensitivity is therefore
usually measured as a conditional proba-
bility, that is, the probability that the
marker will indicate disease, given the
presence of disease. In the notation of
Table A-1, the sensitivity of a marker is
P(M+ ADO. Sensitivity is therefore the
complement of the false-negative proba-
bility of the marker, P(M- +. The sen-
sitivity of markers is rarely, if ever,
perfect (1.0)—there are always false-
negatives-but a good marker will have a
sensitivity close to 1.0.
313
FIGURE A-2 Multistage prenatal detection of
neural tube defects. Source: Reprinted with
permission from Adams et al., 1984. Copyright
1984 by C.V. Mosby Co.
"Specificity" refers to the ability of
a marker to indicate the absence of dis-
ease or exposure when disease or exposure
is absent. In the notation of Table A-1,
the specificity of a marker is P(M- | Dab.
Specificity is the complement of the
false-positive probability, P(M+ IDA.
A good marker will have a specificity close
to 1.0.
TABLE A-1 Probabilities of Marker Presence and
Absence, Conditional on Disease Presence and
Absence
Marker
Positive
Negative
Total
Diseases
Present Absent
P(M+ ID+)
Pit- |D+)
P(D+)
P(M+ ID-)
P(M- I D-)
P(D-)
a p, probability; M, marker; D, disease.
Suppose that an investigator is working
with a new marker of exposure to a toxic
chemical and determines its sensitivity
and specificity from test data as pre-
sented in Table A-2. The marker has been
measured in 1,000 persons with the disease
(exposure) and 1,000 persons without the
disease. The results are encouraging:
the sensitivity is 0.95 (950/1,000), and
the specificity is 0.99 (990/1,000~.
OCR for page 314
314
There are only 5% false-negatives and
1% false-positives. Because the numbers
of exposed and unexposed persons in the
evaluation are equal, the a priori proba-
bility of exposure in the total sample
is O.S.
TABLE A-2 Cross-Classification of Marker and
Disease: Hypothetical Data from a Case-Control
Study
Marker
Disease
Present Absent
Positive
Negative
Total
950 10
50 990
1 000 1 000
, ,
Another quantity important in evaluat-
ing the validity of a marker is its predic-
tive value. Table A-3 shows the proba-
bilities of disease presence and absence,
conditional on the presence or absence
of the marker.
TABLE A-3 Probabilities of Disease Presence and
Absence, Conditional on Marker Presence and
Absence
Diseasea
Marker Present Absent Total
Positive P(D + | M + ) P(D + | M) P(M + )
Negative P(D- | M + ) P(D- | M-) P(M-)
ap, probability; M, marker; D, disease.
The focus here is on the rows, rather than
on the columns as in Table A- 1. The predic-
tive value of a positive (PVP) test for the
marker is P(D+ ~M+), that is, the probabil-
ity of the disease given a positive test.
That probability is the complement of the
false-positive probability, P(D- ~M+),
the probability that there is no disease
when there is a positive test for the mark-
er. The predictive value of a negative
(PVN) test is P(D- ~M-), which is the com-
plement of the false-negative probabili-
ty, P(D+ EMS. In applying a test for a dis-
ease or exposure, it is important to con-
sider the data both as they are presented
in Table A-3 and as they are presented in
Table A- 1. Consider the data in Table A-
APPENDIX
2 focusing on the marker (rows), rather
than on the exposure (columns). Here the
false-positive rate, the probability that
the marker indicates exposure when there
is none, is about 0.01 (10/960~. The PVP
is about 0.99 (950/960), and the PVN is
about 0.95 (990/1,040~.
Note that the data in Table A-2 have
been gathered in such a way that the number
exposed is equal to the number not exposed.
That is, the a priori probability of dis-
ease in the sample is 0.5-a typical case-
control study design. Suppose that the
a priori probability of disease in
question in the population at large is low,
about 1%. Table A-4 shows how the marker
would work in practice in this population.
The data in Table A-4 exhibit the same sen-
sitivity and specificity (0.95 and 0.99,
respectively) as the data in Table A-2,
but very different PVP. Here the PVP is
about 0.49, that is, only half the people
with positive test results will have the
disease. The sole cause of the differing
PVPs is the difference in the a priori prob-
ability of the disease in the two settings.
One setting is that of a study of the marker
where the disease is common (50%) by de-
sign, and the other setting is that of the
use of the marker in practice where the
disease is relatively infrequent. In the
example, because the disease is infre-
quent, the PVN is high (more than 0.99~.
TABLE AL Cross-ClassiBlcation of Markers and
Disease: Hypothetical Data from a Population
Study
Disease
Marker Present Absent Total
Positive 95 100
Negative 5 9,900
Total 100 10,000
195
9,905
10 100
The example reflects a common outcome
of the transition from laboratory bench
to community or clinical practice: very
good tests can perform poorly. In the clin-
ical or community setting, it is most im-
portant to know how likely it is that a posi-
tive test indicates disease truly and
how likely it is that a negative test indi-
cates the absence of disease truly. The
predictive values of tests can be increased
OCR for page 315
ALP~I-FETOPROTEIN
in two ways-by increasing the sensitivity
and/or the specificity or by choosing the
individuals or populations for testing
so that the a priori probability of the
disease or exposure is high.
In many situations, it is not possible
to change the sensitivity without changing
the specificity, and vice versa. The situ-
ation with maternal serum AFP is a case
in point. Figure A-1 shows that the AFP
distributions in normal and affected preg-
nancies overlap. If a particular concen-
tration of AFP, the "cut-point, is chosen
as an indication of the presence of an ab-
normal fetus, there will be false-posi-
tives and false-negatives, because some
normal pregnancies are associated with
higher AFP concentrations than are some
affected pregnancies. If the cut-point
is chosen so that the test is very sensi-
tive-so that nearly all affected pregnan-
cies fall above the cut-point-there will
be more false-positives, and the specifi-
city will be low. But, if the cut-point
is chosen so that nearly all normal preg-
nancies fall below, there will be more
false-negatives, and the sensitivity will
be low.
Regarding the possibilities for. in-
creasing the PVP of a test by testing only
people with a relatively high a priori
probability of exposure or disease, recall
the marked contrast in the PVP values cal-
culated from the data in Tables A-2 and
A-4. In those two examples, there was no
difference in the sensitivity or specifi-
city of the test, but only differences in
the a priori probabilities of exposure.
A major reason for disappointment in the
practical application of a test is that
it is indiscriminately applied in popula-
tions where the a priori probability of
exposure or disease is low, so the false-
positives greatly outnumber the true-
positives.
In many situations, a test with a low
PVP is applied as a screening test.
Persons with a positive result can be fol-
lowed by more definitive tests. The defin-
itive tests are usually not used as a first
step, because they are expensive and in-
vasive. The use of a screening test also
allows the use of the definitive test on
persons who have a relatively high a
315
priori probability of having the disease
or exposure of interest, which increases
the operational PVP of the definitive test
(even Definitive tests are rarely per-
fect). The currently recommended process
for screening for NTDs through the use of
maternal serum AFP testing is an example
of this approach.
VALIDITY OF MATERNAL SERUM
AFP MEASUREMENT AS A BIOLOGIC
MARKER OF NTDs
As indicated earlier, AFP is normally
present in amniotic fluid and maternal
serum, and it is present in increased con-
centrations in the presence of anencephaly
and spine bifida cystica. Measuring the
second-trimester concentration of mater-
nal serum AFP can be used to identify the
likelihood that a pregnant woman is carry-
ing a fetus with anencephaly or spine bif-
ida cystica, a ventral wall defect, or
twins; other defects can also be predicted.
These likelihoods can be used by the woman
and her physician to decide whether to bear
the risks and costs of further diagnostic
procedures. Ultrasonography can identify
multiple fetuses and fetuses with anen-
cephaly. The most common cause of in-
creased maternal serum AFP is mistaken
gestational age, which can be checked by
ultrasonography. When an increase in ma-
ternal serum AFP cannot be explained by
one of the factors assessed with ultrasono-
graphy, the likelihood of a fetus with
spine bif~da cystica or ventral wall
defect can be weighed against the cost and
risks of amniocentesis to obtain fluid
for assay for amniotic fluid AFP (see Fig.
A-2. That assay can be considered as the
definitive test for spine bifida cystica,
but also is invasive and is associated with
an increased risk of abortion.
The results of maternal serum AFP tests
are usually presented in terms of multiples
of the median (MOMs) for normal pregnan-
cies. From the specific MOM and some char-
acteristics of the mother (such as race,
geographic area, and weight), one can es-
timate the odds of having an affected fe-
tus. Rather than make a presentation in
terms of odds, for the purpose of this dis-
cussion we consider a maternal serum AFP
OCR for page 316
316
concentration of at least 2.5 MOM as "ab-
normal" and give an illustration of sen-
sitivity, specificity, and PVP. (As noted
above, the choice of a different MOM as the
"abnormal" cut-point will change the sen-
sitivity, specificity, and predictive
values of the procedure.)
The data are derived from a synthesis
of the UK Collaborative Study results (Wald
and Cuckle, 1980) and are adjusted so that
the base is a hypothetical cohort of
100,000 screened pregnant women (data on
ventral wall defects and other defects
are not included). Table A-5 puts the
data on maternal serum AFP in the format
of the other tables presented here; the
1,000 multiple pregnancies that would be
expected among 100,000 pregnancies have
been omitted from the table. The sensitiv-
ity of this test is 0.84, meaning that 16%
of babies with anencephaly or spine bifida
cystica would be missed.
TABLE A-S AFT by Presence or Absence of
Neural Tube Defects
NTD
Marker Present Absent Total
Positive
Negative
Total
334
66
400
3,254
953"
98,600
3,588
95,412
99,000
aData derived from UK Collaborative Study data.
Source: Wald and Cuckle, 1980.
The specificity is 0.97, indicating
that there are 3% false-positives. NTDs
are rare complications of pregnancy; the
false-positives greatly outnumber the
true positives, and the PVP is about 0.09.
The PVN is quite good at this stage of the
screening process, more than 0.99, and
a woman with a negative test has only a
7/10,000 risk of having a fetus with anen-
cephaly or spine bifida cystica, only about
APPENDIX
one-sixth of her a priori probability,
about 40/ 10,000 in the UK at the time
these data were obtained.
After staging through ultrasound and
amniocentesis with evaluation of amniotic
fluid AFP (see Fig. A-2), the UK data look
like those presented in Table A-6. The
sensitivity of the total screening package
for open NTD is 0.81, the specificity is
very nearly 1.0, and the PVP is almost 0.99.
In all, the process is able to detect 81%
of the fetuses affected by open NTDs at the
risk of a fairly small number of false-
positives. If one includes the risk of
fetal death due to amniocentesis (esti-
mated to be about 0.5-1.5% of the 3,588
amniocentesis performed), the net benefit
of the program is 324 fetuses with open NTDs
detected balanced against the 4 false-
positives and 10-30 fetal deaths due to
complications of amniocentesis.
TABLE AL AFP by Presence or Absence of
Neural Tube Defecta
Total Screen Present Absent Total
Positive
Negative
Total
324
76
400
4
98,596
98,600
328
98,672
99,000
aData derived from UK Collaborative Study data.
Source: Wald and Cuckle, 1980.
It is obvious that AFP screening could
be a monumental failure if stopped at the
maternal serum stage. There would be enor-
mous errors, the normal fetuses with posi-
tive test results greatly outnumbering
the affected fetuses. Therapeutic action
taken on the basis of maternal serum AFP
tests would be wrong most of the time. How-
ever, when properly used as the first stage
of a screening process, maternal serum
AFP evaluation is useful.
OCR for page 317
References
.
OCR for page 318
Representative terms from entire chapter:
serum afp
