| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
Articles |
1
Prince Henry's Institute of Medical Research, Clayton, Victoria 3168, Australia.
Departments of
2
Obstetrics and Gynecology and
3
Mathematics, Monash University, Monash Medical Centre,
Clayton, Victoria 3168, Australia.
4
Department of Biotechnology, University of Turku, Turku
SF-20500, Finland.
5
Department of Obstetrics and Gynecology, University of
Queensland, Herston, Queensland 4006, Australia.
a Author for correspondence. Fax 61 3 9550 6125; e-mail david.robertson{at}med.monash.edu.au
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
C IFMA) to replace an inhibin RIA as a diagnostic marker of
these ovarian cancers and to assess whether the C IFMA in
combination with CA125, which detects serous cancers, leads to an
improved biochemical diagnosis of all ovarian cancers.
Methods: Serum inhibin concentrations were determined in
healthy postmenopausal women (n = 165) and women with
ovarian cancers (n = 154), using an inhibin RIA and an C IFMA,
which detects inhibin forms containing the C subunit as well as the
free C subunit.
Results: The C IFMA gave a similar or better discrimination of
mucinous (90% vs 71%) and granulosa cell (100% vs 100%) cancers
compared with the inhibin RIA. Combination of CA125 and C IFMA
values by canonical variate analysis or by multiROC analysis showed
that the percentage of all ovarian cancers detected was significantly
increased compared with either CA125 or C IFMA alone.
Conclusions: The C IFMA shows a similar or better specificity
compared with the RIA, but with increased sensitivity. In combination
with CA125, the C IFMA provides an effective dual test for the
detection of the majority (90%) of ovarian cancers.© 1999
American Association for Clinical Chemistry
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
subunit (made up of three regions, Pro-, N, and C) and either the
ßA subunit (to form inhibin A) or ßB subunit (inhibin B) of
different molecular weights. The nonbiologically active free
subunit and fragments have also been identified in biological fluids,
including serum. Earlier studies (4)(5)(6)(7)(8) showed that the
measurement of serum inhibin by RIA, which detects all
C-containing inhibin forms together with free subunit, was of
value in the diagnosis and monitoring of ovarian granulosa cell and
mucinous tumors in women after menopause.
An C immunofluorometric assay
(IFMA)1
for inhibin was developed recently
(9)(10) to match the assay specificity of the
RIA, but with the advantages of increased sensitivity and speed. This
assay detects all known inhibin subunit-containing proteins. This
assay has been used to characterize inhibins in fractionated human
follicular fluid and serum from healthy men and women
(9)(10), and showed a specificity similar to the
RIA in detecting the various inhibin forms.
The aims of this study were to validate the C IFMA in application to
serum and then to compare its diagnostic accuracy with the RIA in the
detection of ovarian mucinous and granulosa cell cancers. In addition,
the value of combining inhibin measurements with that of CA125, which
readily detects serous tumors (11)(12)(13), in the detection of
all ovarian cancers was explored.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
. Nine of 11 women with granulosa cell tumors were 
50 years of
age, but two were 30–40 years of age and had undergone bilateral
salpingo-oophorectomy before the study. Serum samples were obtained
from 165 healthy postmenopausal women attending a mammography breast
screening clinic. The women studied were asymptomatic and considered
postmenopausal based on age (55 years) or increased serum
follicle-stimulating hormone (>20 IU/L). The research and ethics
committees of all participating hospitals approved the study protocol.
|
inhibin ria
Inhibin was measured with a heterologous RIA (14),
using iodinated 31-kDa inhibin as tracer and a rabbit antiserum
(no. 1989) raised against bovine 31-kDa inhibin. This assay
showed cross-reaction with inhibin, Pro-C, and Pro-N-C, but
not with N fragment, activin, or follistatin (10).
The RIA calibrator used was a human serum pool (designated
MMC-1) of arbitrary units. The RIA sensitivity between assays was
35–110 units/L. This assay has been used previously for
measuring inhibin in serum from postmenopausal women with ovarian
cancer (4)(5). The serum inhibin value (122
units/L) used to discriminate between control and cancer groups in the
RIA was determined previously (5). The majority (>90%) of
control samples gave nondetectable values in this RIA. The MMC-1
inhibin calibrator when assayed against the First International
Standard for inhibin A, (human recombinant, 30–34 kDa, 91/624;
National Institute of Biological Standards and Control, Potters Bar,
UK) in the C IFMA was 1.80 ng of 91/624 per unit of MMC-1 with 95%
confidence limits of 1.54 and 2.09 ng/unit.
c subunit ifma
A two-site IFMA was developed with the aim of detecting all C
subunit-containing inhibin forms in serum. The capture antibody used
was a caprylic acid IgG cut of a sheep polyclonal antibody (no. 128)
raised initially against human inhibin C subunit fusion protein
(10) and subsequently boosted with recombinant inhibin A.
The antibody used as label was a sheep polyclonal antibody (no. 41)
raised against human inhibin C subunit fusion protein
(10). The IgG fraction was immunopurified by fractionation
on a column of immobilized bovine inhibin C-subunit fusion protein
and eluted with 4 mol/L guanidine hydrochloride. The guanidine
hydrochloride was removed by gel filtration on Sephadex G25 columns
(PD-10 column; Pharmacia) and gel filtration HPLC. The IgG
fraction was concentrated by a centrifugation procedure (Centricon;
Amicon) to 1 g/L and biotinylated (4 h at room temperature)
using the biotin-isothiocyanate procedure (Wallac; Turku) in 50 mmol/L
Na2CO3 buffer, pH 9.8,
tested over a 60- to 150-fold molar excess of biotin reagent. The sheep
antisera and fusion protein were gifts from Biotech Australia P/L,
Sydney, Australia.
The assay was carried out in 96-well microtiter plates (Maxisorb;
Nunc). Wells were coated with 2 µg/well capture antibody in glycine
buffer, pH 4.4, and blocked with 50 mmol/L Tris-HCl, pH 7.5, containing
10 mg/L bovine serum albumin (BSA) for 1 day at room temperature. The
assay buffer used was 50 mmol/L Tris-HCl, pH 7.4, containing 0.154
mol/L NaCl, 1 g/L NaN3, and 5 g/L BSA (TSA-BSA)
containing 1 g/L ovine IgG. To reduce the matrix effects of
serum in the assay, we diluted the calibrator and serum samples in
serum from which the inhibin had been removed by adsorption to an
immobilized no. 41 antiserum IgG fraction. The serum was initially
recycled five times through the immunoaffinity support, although two
cycles were sufficient to remove all inhibin immunoactivity as measured
in the C IFMA. The effectiveness of the depletion process was
assessed from the low stable counts obtained in the C IFMA with
repeated cycles through the immunoaffinity support and the similar
responses observed for the depleted serum and assay buffer alone in the
C IFMA. Serum samples (100 µL) or calibrators (100 µL) and
TSA-BSA buffer (100 µL) were incubated in the antibody-coated
microtiter plate for 2 h at room temperature with shaking. The
plate was then washed, and biotinylated antibody (150 ng/well) was
added and incubated an additional 2 h at room temperature. After
the plate was washed, Eu-streptavidin (50 ng/well; Wallac) was added;
the plate was incubated for 30 min and then washed six times, and
enhancement solution (Delfia; Wallac) was added. The plates were read
on a Wallac 1234 Fluorometer. The dose–response curves were calculated
using the Multicalc program (Wallac). Where appropriate, potencies and
evidence of parallelism between serial dilutions of calibrator and
serum samples were determined using parallel line statistics as
outlined in Finney (15)
The inhibin A (91/624) preparation was used as calibrator and expressed
in terms of its nominal vial content (5 µg). The specificity of the
C IFMA had been assessed previously (10). The C IFMA
detected all the known molecular weight forms of inhibin. The detection
limit of the assay represented the mean inhibin value detected 2
SD above the zero value of the C IFMA from five experiments.
ca125
CA125 was measured by an immunoenzymometric assay (AIA-PACK CA125;
TOSOH) using a CA125 calibrator provided by the manufacturer. The
manufacturer stated that the mean serum CA125 concentration in 172
apparently healthy individuals was 12.3 kilounits/L, with an upper
limit of 33.4 kilounits/L.
CA125 and inhibin by RIA were determined by the Monash Medical Centre
Chemical Pathology Department. C IFMA measurements were undertaken
at Prince Henry's Institute of Medical Research, Melbourne.
general statistical analyses
For statistical purposes, the patient sera were divided into five
main groups: controls, serous, mucinous, granulosa cell, and
miscellaneous. The cancers of nonovarian origin were considered
separately. In addition, the four ovarian cancer groups were combined
to form an all-ovarian cancer group. The data were normalized after log
transformation as confirmed using normal probability plots, half-normal
plots, and histograms. The data were log-transformed before analysis.
The 5th and 95th percentiles for each group were determined as the
antilogs of the 5th and 95th percentiles of the normally distributed
log-transformed data.
roc curves
The ROC curve (16)(17) for a diagnostic
test plots the sensitivity against the false-positive rate. In this
context, the sensitivity is the proportion of cancer patients who are
identified correctly by the diagnostic test, so-called true positives;
the specificity is the proportion of control patients who are
identified correctly, so-called true negatives. One minus the
specificity equals the false-positive rate. The ROC curve can be used
to select a cutoff for the diagnostic test that maximizes the
sensitivity and minimizes the false-positive rate. The areas under the
ROC curves can be used to compare the performance of the two assays.
canonical variate analysis
The data from different assays can be combined to improve the
sensitivity and specificity relative to those of the individual assays.
One approach to finding an optimal combination of several assays is
canonical variate analysis (CVA) (18). The optimal solution
chosen by CVA is the linear combination of assays that maximizes the
F-statistic of between-group variance divided by
within-group variance, where group identifies the cancer and control
patients.
multiROC analysis
The multiROC, like CVA, is a method (19) for combining
the results of multiple (two or more) assays through the derivation of
a composite test rule. For example, if two assays are available, then a
patient is classified as a cancer patient if the value for the first
assay is greater than the cutoff for that assay or the corresponding
value for the second assay is greater than its corresponding cutoff
value. The multiROC curve is drawn by constructing the ROC curve for
the first assay in the usual way, selecting the "best" cutoff for
the first assay, and then adding another curve for the second assay,
which completes the multiROC for the composite rule.
comparison of cva and multiROC
The multiROC is an ingenious method, but has some difficulties.
First, although the cutoffs for the individual assays can be
"optimally" derived from ROC curves, there is no guarantee that
those same cutoffs will be the optimal cutoffs when a composite
decision rule is formed. Second, although graphical techniques have
been proposed to aid in the formulation of a decision rule for the
multiROC, the search for the optimal rule and optimal set of cutoffs is
subjective and can become difficult as the number of assays increases.
CVA is not a panacea for these difficulties, but it does have some advantages and, therefore, is worthy of consideration: (a) the method is mathematically optimal in the sense of maximizing the F-statistic; (b) the method is objective; and (c) the method can be extended easily to multiple assays.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
c ifma in application to serumC subunit-containing proteins in serum. The inhibin standard
(WHO inhibin A reference preparation, 91/624) gave a curvilinear
response with a detection limit of 2 pg/well or 20 ng/L sample
(Fig. 1
) and an upper response >5 ng inhibin/well (data not shown).
Inhibin-free serum was included to reduce serum matrix effects in the
assay. Ovine IgG at 1 g/L was added to eliminate the effects of
antibody cross-linking agents in human serum. In a preliminary series,
~10% of sera showed responses that were eliminated after the
inclusion of ovine IgG.
|
Serial dilutions of a human serum pool gave a parallel response to the
dose–response curve of the inhibin standard (Fig. 1
). The detection
limit of the assay based on a response 2 SD above the controls was 2
pg/well or 20 ng/L serum. The mean precision profile of five
calibration curves is presented in Fig. 1
. The within-assay
imprecision (CV) as calculated from the reproducibility of a
serum pool (69 ± 5.5 pg/well) used as quality control in the
present studies was 4.2% (n = 4), and the between-assay CV was
8% (n = 4).
The C IFMA and CA125 were applied to serum samples from 165 healthy
postmenopausal women, 154 postmenopausal women with a variety of
ovarian cancers, and 23 women with nonovarian cancers (Table 1
).
The RIA was applied to the cancer samples only, because previous
studies (5) had shown that >90% of serum samples from
healthy postmenopausal women were nondetectable in this assay.
The data from the three assays, expressed as geometric means with 5th
and 95th percentiles, were grouped by cancer class into seven main
groups: controls, serous cancers, mucinous cancers, and granulosa cell
cancers, a miscellaneous series of ovarian cancers, an all-ovarian
cancer group, and nonovarian cancers (Table 1
). The miscellaneous
cancer group has been subgrouped into individual cancers and presented
in Table 1B
.
comparison of the various cancer groups between assays
The specificity and sensitivity of the C IFMA, RIA, and CA125
were assessed initially from the number of cancer and control values in
each assay that were detected above the 95th percentile of the
geometric mean of the control group, or in the case of the RIA, a
previously determined cutoff point (122 units/L, Table 1
). On the basis
of this approach, the C IFMA showed similar or better discrimination
characteristics compared with the RIA in the detection of mucinous
(90% vs 71%, respectively) and granulosa cell tumors (100% vs
100%). In contrast, the serous and miscellaneous tumors were better
discriminated by the CA125 method.
The C IFMA and CA125 were also assessed by ROC curve analysis. The
ROC curves are presented in Fig. 2
for the major cancer groups. The areas under the ROC curves for
each analysis and statistical comparisons of areas under the curves are
presented in Table 2
. These data in support of the results above show that at 95%
specificity, C IFMA detected 95% of mucinous and 100% granulosa
cell tumors, whereas CA125 detected 92% of serous and 79% of
miscellaneous cancers.
|
|
combination of assays for the various cancers
CVA/ROC curve and multiROC analyses were undertaken to establish
whether the combination of IFMA and CA125 assays leads to an improved
accuracy in the detection of all ovarian cancers. Application of both
methods in assessing the combination of the CA125 and IFMA led to the
detection of a high proportion of all ovarian cancers. This is
exemplified in Table 2
, where the areas under the ROC curves for the
CA125 + C IFMA combination for the all-ovarian cancer group was
significantly greater (P <0.05) by either the CVA (Fig. 3
, top) or the multiROC (Fig. 3
, bottom) method in comparison
with either assay alone. The percentage of all ovarian cancers
detected at 95% specificity by the combined assays was 89–90%
vs 80% for CA125; at 90% specificity, the percentage of ovarian
cancers detected was 91–93% vs 84% (Table 3
).
|
|
The relationship between CA125 and the C IFMA and their combination
in differentiating between controls and ovarian cancers is presented as
a scatter plot in Fig. 4
. The control group is confined to the lower left-hand quadrant,
separate from the ovarian cancer groups in the other quadrants. The
vertical line corresponds to the 95% specificity value (28.5 units/L
CA125; see Table 2
and Fig. 3
) obtained using a ROC analysis and was
chosen as the cutoff for CA125 for the multiROC decision rule. On the
basis of the multiROC graph in Fig. 3
, we decided to increase the C
IFMA value from 129 ng/L (Table 2
) to 218 ng/L, shown as the horizontal
line in Fig. 4
. Thus, the multiROC decision rule was to classify a
patient as a case if the result obtained was >28.5 units/L in the
CA125 or was >218 ng/L in the C IFMA. The diagonal line was derived
by CVA and represents the sensitivity values at 95% specificity. As
seen in Table 2
, the sensitivity values at 95% specificity of the
combined CA125 + C IFMA analyses are similar when either the CVA or
multiROC curve analysis is used.
|
Regression analysis between inhibin assays was assessed for the various
cancer groups. As seen in Table 4
, regression analyses between C IFMA and RIA showed high
correlations (r = 0.63–0.83) for mucinous and
granulosa cell cancer groups and all-ovarian cancer group with
low correlations (r = 0.31–0.5) for the other cancer
groups.
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
subunit (9)(10). Previous
studies (7)(20)(21) showed that
inhibin A was detected in 20% of mucinous tumors and 77% of granulosa
cell tumors, whereas inhibin B was detected in 60% of mucinous tumors
(21) and 100% of granulosa cell tumors. Similarly,
Pro-C, which is a monomeric inhibin subunit form, was detected
by Pro-C ELISA (22) in 90% of granulosa cell cancers and
55% of mucinous cancers (21). These findings indicate that
a replacement assay for the RIA should show a broad range of
specificity capable of detecting free subunit forms as well as
dimeric inhibin forms. The C IFMA satisfies these requirements by
detecting all known inhibin forms containing the C region of the
subunit as well as the free subunit with an assay specificity which
closely parallels the RIA (9)(10), thus
providing a similar broad specificity range. The C IFMA is likely to
detect the presence of degraded forms (9)(10)
because it detects inhibin denatured by reducing agents, whereas the
RIA does not (14).
The C IFMA shows a similar or improved detection of mucinous and
granulosa cell tumors compared with the RIA used previously. This
improvement may be attributed to the increased sensitivity of the IFMA,
in which inhibin is detected in 84% of healthy samples compared with
the limited proportion detected by the RIA.
The observation that CA125 is effective in detecting serous and
miscellaneous cancer groups, whereas inhibin is effective in detecting
the mucinous and granulosa cell cancer groups suggests that these two
analytes may provide an appropriate combined test for detecting the
majority of ovarian cancers. A combination of the CA125 and the C
IFMA by canonical variate/multiROC analysis was thus undertaken. The
data from the individual and combined assays were analyzed either on
the basis of the proportion of cancers detected above the 95th
percentile of the controls (which is used to analyze the RIA data) or
by ROC analysis. ROC analysis permits the assessment of sensitivity at
any specificity value; however, the data have been considered as an
example at 95% specificity. The analysis showed that the combination
of CA125 and C IFMA detected a significantly higher proportion of
all ovarian cancers compared with either assay alone. At 95%
specificity, that is, at a discrimination point where 5% of healthy
controls are detected, the sensitivity of cancer patients in the
all-cancer group was 89–90%, compared with 80% with CA125 or 50%
with C IFMA alone. This observation suggests that in the clinical
assessment of ovarian cancers, the combination of inhibin and CA125
increases the probability that the majority of ovarian cancers will be
detected.
It was anticipated that the combination of the two assays would lead to a synergistic response that would allow the detection of cancers with subthreshold values in the two assays. A comparison of the CVA method with the multiROC method yields a similar discrimination index, indicating that this is not the case. One explanation is that the individual assays are particularly effective in detecting their respective cancers with little overlap between cancer groups. Because no synergism was observed, a positive response in one or both assays will provide evidence of cancer without the need for an appropriate algorithm.
Other markers in combination with CA125 have been reported to improve
their diagnostic accuracy in the pretreatment evaluation of ovarian
cancers. Stenman et al. (23) reviewed the
sensitivity/specificity characteristics of several markers and
recommended CA125 and either tumor-associated trypsin inhibitor
or the gastrointestinal mucin markers CA 19-9 or CA 72.4 for mucinous
tumors. The combination of CA125 and tumor-associated trypsin inhibitor
for mucinous tumors gave 100% sensitivity at 89% specificity
(24), which is similar to that found with CA125 + C IFMA
(100% sensitivity and 90% specificity) in this study. However,
tumor-associated trypsin inhibitor is less effective in detecting
granulosa cell tumors. CA125 + CA 72.4 gave sensitivity values of 85%
and 86% and specificities of 81% and 86% for two studies examining
all ovarian cancers (25)(26). These values are
lower than the corresponding values (95–100% sensitivity and 95%
specificity) obtained in the present study. These findings suggest that
the combination of CA125 and C IFMA provides a better biochemical
diagnosis of all ovarian cancers than other available tests or
combination of tests. It should be noted that the CA125 + C IFMA
combination applies to postmenopausal women, whereas the other
combinations are also applicable to premenopausal women.
The tissue specificity of the C IFMA was assessed by examination of
a series of nonovarian cancers that gave limited detection
similar to that obtained with the RIA (5). A previous study
(5) examined the RIA inhibin concentrations in 33 benign
ovarian tumors, 11 of which (3 of 4 fibromas, 4 of 4 thecomas, and 2 of
4 mucinous cystadenomas) had increased inhibins. A corresponding group
was not assessed in the present study, but it is likely that a similar
distribution would be found with other inhibin -subunit assays. The
specificity of the assay in application to cancers of nonovarian origin
showed a low discrimination index (17%, Table 1A
). Nonetheless,
studies of a wider range and number of nonovarian cancers and other
disease processes will be needed to establish the specificity of the
assay in its clinical application.
The use of inhibin as a marker of ovarian disease is limited to women after menopause, when their serum inhibin concentrations are low. Serum inhibin concentrations produced by the ovary during the reproductive years show substantial fluctuations that would seriously compromise a preoperative assessment of any ovarian cancer. However, inhibin measurements may be useful post surgery in premenopausal ovarian cancer patients during the recovery/recurrence phase because these women undergo oophorectomy as the first stage of their treatment. Inhibin should be considered the marker of first choice for monitoring treatment of women with mucinous ovarian cancers. Studies are directed toward establishing whether there are cancer specific inhibin forms, which may be suitable during the reproductive years.
|
Acknowledgments |
|---|
|
Footnotes |
|---|
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
C-containing forms in human serum by a new ultrasensitive two-site enzyme-linked immunosorbent assay. J Clin Endocrinol Metab 1996;80:2926-2932.
The following articles in journals at HighWire Press have cited this article:
|
|
 |
|
  A. Tsigkou, D. Marrelli, F. M. Reis, S. Luisi, A. L. Silva-Filho, F. Roviello, S. A. Triginelli, and F. Petraglia Total Inhibin Is a Potential Serum Marker for Epithelial Ovarian Cancer J. Clin. Endocrinol. Metab., July 1, 2007; 92(7): 2526 - 2531. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. L. Marca and A. Volpe The Anti-Mullerian hormone and ovarian cancer Hum. Reprod. Update, May 1, 2007; 13(3): 265 - 273. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 G. E. Owens, R. A. Keri, and J. H. Nilson Ovulatory Surges of Human CG Prevent Hormone-Induced Granulosa Cell Tumor Formation Leading to the Identification of Tumor-Associated Changes in the Transcriptome Mol. Endocrinol., June 1, 2002; 16(6): 1230 - 1242. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. M. Robertson, T. Stephenson, E. Pruysers, P. McCloud, A. Tsigos, N. Groome, P. Mamers, and H. G. Burger Characterization of Inhibin Forms and Their Measurement by an Inhibin {alpha}-Subunit ELISA in Serum from Postmenopausal Women with Ovarian Cancer J. Clin. Endocrinol. Metab., February 1, 2002; 87(2): 816 - 824. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 G. P. Risbridger, J. F. Schmitt, and D. M. Robertson Activins and Inhibins in Endocrine and Other Tumors Endocr. Rev., December 1, 2001; 22(6): 836 - 858. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
) and serum pool
(
) in the 
) is the mean of five assays.
