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Unité de Recherches sur l’Endocrinologie du Développement, INSERM, Département de Biologie, Ecole Normale Supérieure (W.-Q.L., C.B., J.-Y.P., R.R.), 92120 Montrouge; Département de Biologie Clinique (J.-M.B.) and Service de Gynécologie (P.P., C.L.), Institut Gustave Roussy, 94805 Villejuif; and Département d’Immunoanalyse, Immunotech (V.R., P.D., H.C.), 13276 Marseilles, France
Address all correspondence and requests for reprints to: Dr. Rodolfo Rey, Centro de Investigaciones Endocrinológicas, Hospital de Niños, Gallo 1330, 1425 Buenos Aires, Argentina. E-mail: rodolforey{at}infovia com.ar.
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Abstract |
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The ultrasensitive enzyme-linked immunosorbent assay has a significantly higher sensitivity than the traditional one. This resulted in the detection of low serum AMH levels, which were undetectable with the traditional assay, in several cases including one patient in whom a recurrence of a GCT had developed and two patients in whom the treatment had not been completely successful. These cases highlight the importance of the availability of a highly sensitive assay allowing evaluation with high precision of the results of treatment and to detect the recurrences of GCT at an early, preclinical stage.
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Introduction |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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The initial treatment of GCT is mainly surgical. In advanced stages or in recurrences, when the tumor is not confined to the ovaries, complete surgical removal may be difficult, and adjuvant chemotherapy and/or radiotherapy can be used. However, as GCT have low radio- and chemosensitivity, the success of the treatment is dependent on an early diagnosis of the recurrence, when complete removal of the tumor is still possible (6, 7, 8). These data highlight the importance of having a reliable biochemical marker of the disease.
GCT are hormone-secreting neoplasms; they may produce steroids, such as estradiol and progesterone, and they secrete peptide hormones, such as inhibin and anti-Müllerian hormone (AMH), also known as Müllerian inhibiting substance. Because serum levels of AMH and inhibin are increased in patients with GCT of the ovary, both hormones are used as biochemical markers of the disease (9, 10, 11, 12, 13, 14).
In a previous work we showed the reliability of serum AMH as a marker of recurrent GCT (12). We have now developed a highly sensitive assay for serum AMH determination, with the purpose of enhancing the detection of very low concentrations of AMH in the serum of patients followed up for GCT. We report here the results of serum AMH determination obtained in patients with GCT using the ultrasensitive assay and compare them with the results obtained using the traditional assay.
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Subjects and Methods |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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Serum samples from 31 female patients with a diagnosis of GCT (27 adult-type and 4 juvenile-type) were obtained from the serum bank of the Département de Biologie Clinique, Institut Gustave Roussy (Villejuif, France). Diagnoses were confirmed by evaluation of histological slides in all cases. In 16 cases, patients were only studied after bilateral ovariectomy during the remission period of the disease. In the other 15 cases, the availability of serum samples was as follows: several samples during both progressive and remission periods of the disease in bilaterally ovariectomized patients (8 cases), several samples during progressive and remission periods in unilaterally ovariectomized young patients (2 cases), several samples in patients with progressive disease despite bilateral ovariectomy and chemotherapy or radiotherapy (4 cases), and only 1 sample during progressive disease (1 case). Serum samples were also obtained from 20 prepubertal and 22 premenopausal female subjects. In all cases, serum samples had been taken for a reason independent of the present study; adults were apparently healthy blood donors and children were patients free of disorders of the hypothalamic-pituitary-gonadal axis and of chronic illnesses.
AMH assays
Serum AMH was measured using two enzyme-linked immunosorbent assays (ELISA): the traditional assay, which has been used in our laboratory for the past 5 yr (15), and a recently developed ultrasensitive assay (AMH/MIS ELISA kit,2 Immunotech-Coulter, Marseilles, France). Briefly, in the traditional assay, Immulon 2 plates (Dynatech Corp., Chantilly, VA) were coated with monoclonal antibody 10.6 (gift from Dr. R. Cate, Cambridge, MA), raised against recombinant human AMH (rhAMH) (16) by an overnight incubation, at room temperature. Sera were assayed at 1:4, 1:8, 1:16, and 1:32 dilutions in PBS containing 1% BSA. As second antibody, we used polyclonal antibody L40, consisting of an IgG fraction isolated by affinity chromatography on protein A-Sepharose from serum of a rabbit immunized with recombinant human AMH (rhAMH) (17). L40 was added to the wells at 1 µg/mL in PBS-1% BSA for 1 h at room temperature. Subsequently, an alkaline phosphatase-labeled goat anti-rabbit IgG antibody (Jackson ImmunoResearch Laboratories, Inc. West Grove, PA) was added and incubated for a further hour before the reaction was visualized with a MRX spectrophotometer (Dynatech Corp.) at 405 nm, using paranitrophenyl phosphate (Sigma Chimie, Saint-Quentin-Fallavier, France) as substrate. Results obtained using the traditional assay in 16 of these 31 patients have been previously reported (12).
The ultrasensitive AMH/MIS ELISA is also a sandwich-type assay with two
immunological steps, but using two monoclonal antibodies, 11F8 and
22A2, obtained as follows. Two female BALB/c mice were immunized with
three sc injections of 50 µg immunoaffinity-purified rhAMH produced
in Chinese hamster ovary cells (18). The first injection was made using
complete Freund’s adjuvant; for the following injections, made 3 and 4
months later, incomplete Freund’s adjuvant was used. Nine days before
death and subsequent fusion, a dose of 50 µg rhAMH was injected into
the peritoneum, and 6 days later, 10 µg rhAMH in NaCl solution were
injected by the iv route into the tail. Spleen cell suspensions were
fused with NS1 myeloma cells (four lymphocytes/one NS1 cell) in the
presence of polyethylene glycol 1000, according to Köhler and
Milstein (19). Two of the primary hybridomas (22A2 and 11F8) were
subcloned by three rounds of limiting dilution. The isotype of these
monoclonal antibodies (IgG1
) was determined using IsoStrip kit
(Roche Molecular Biochemicals, Mannheim, Germany). The
expansion of hybridomas was performed using a miniPERM Bioreactor
(Heraeus, Les Ulis, France), in Hybridoma-SFM medium
(Life Technologies, Inc., Cergy-Pontoise, France) without
serum, at 37 C in a humid atmosphere containing 5% carbon dioxide.
Monoclonal antibodies 22A2 and 11F8 were purified by protein
A-Sepharose-4 Fast-Flow chromatography (Pharmacia Biotech,
Orsay, France) from culture medium according to the method of Ey
et al. (20). Their purity was assessed on 4–20% SDS-PAGE
(Bio-Rad Laboratories, Inc., Ivry-sur-Seine, France), and
their specificity against rhAMH was determined using the traditional
ELISA and replacing polyclonal antibody L40 by either monoclonal 11F8
or 22A2.
The ultrasensitive AMH/MIS ELISA was performed as follows. Twenty-five microliters of each serum sample were incubated in duplicate for 1 h on a ready to use polystyrene plaque coated with monoclonal antibody 22A2. Subsequently, monoclonal antibody 11F8, coupled to biotin, and a streptavidin-horseradish peroxidase complex were added and incubated for 15 min. The peroxidase substrate 3,3',5,5'-tetramethylbenzidine was added, and the resulting color reaction was quantified 30 min later after stopping the reaction with 50 µL 2 N H2SO4, using the MRX spectrophotometer at 450 nm. A preparation of purified rhAMH was used to construct a standard curve ranging from 0.7–175 pmol/L.
The intraassay coefficient of variation of the ultrasensitive AMH/MIS
ELISA was calculated for two different AMH concentrations by assaying
two serum samples (mean concentrations, 36 and 245 pmol/L,
respectively) 12 times within the same assay; the interassay
coefficient of variation was determined also for two different AMH
concentrations by assaying two serum samples (mean concentrations, 31
and 257 pmol/L, respectively) in duplicate in 12 independent assays
(Table 1
). Three recovery tests were
performed using 5 different serum samples, the original AMH
concentrations of which were 16.8, 14.7, 17.7, 21.4, and 25.6 pmol/L,
respectively. Ten microliters of 140 pmol/L rhAMH were added to 100
µL of each sample in the first recovery test, 5 µL 1400 pmol/L
rhAMH were added in the second, and 10 µL 1400 pmol/L rhAMH were
added in the third (Table 2). A dilution
test was performed using 3 different samples, in which AMH
concentrations before dilution were 31.28, 23.56, and 47.56 pmol/L,
respectively. Each serum sample was assayed non-diluted and diluted at
1:2, 1:4, 1:8, and 1:16 in the diluent provided with the AMH/MIS ELISA
kit (Table 3).
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C subunit
(Oxford Bio-Innovation, Oxfordshire, UK). |
Results |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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Intra- and interassay coefficients of variation showed a slightly
better performance of the ultrasensitive AMH/MIS ELISA compared with
the traditional assay (Table 1
). The most remarkable difference was the
sensitivity of the assays. The lowest AMH concentration significantly
different from the zero standard with a probability of 95% was 20
times lower using the AMH/MIS ELISA (Table 1). Results of the recovery
tests are shown in Table 2. The observed AMH concentration represented
80.2–103.6% of the expected values. The results of the dilution tests
are given in Table 3. The observed AMH concentrations reached
84.0–104.6% of those expected. Specificity was tested by assaying
samples containing known concentrations of the most AMH-related members
of a large family of growth and differentiation factors (21):
transforming growth factor-ß, inhibins A and B, inhibin pro- C
subunit, and activin A. No cross-reactivity was observed in any of the
cases (data not shown). Recovery tests performed on three serum samples
of known AMH concentration to which different amounts of inhibin B or
activin A were added showed that the observed AMH concentrations ranged
between 92–101% of the expected values.
Serum AMH in normal women and in patients with GCT
Using the ultrasensitive AMH/MIS ELISA, the serum AMH
concentration (mean ± SD) was 22.8 ± 19.6
pmol/L (range, 0.7–73.9 pmol/L) in normal prepubertal girls and
13.7 ± 18.8 pmol/L (range, 0.7–74.7 pmol/L) in normal adult
women. Immunoreactive AMH was undetectable, after bilateral
ovariectomy, in all of the serum samples obtained from 15 of 16
patients who were followed up for up to 7 yr (range, 3–81 months;
median, 23 months) after treatment of GCT and in whom no signs of
recurrent disease were observed (Table 4). In only 1 ovariectomized patient
was a value of 70 pmol/L found in 1 of 5 serum samples assayed for AMH,
even though no evidence of recurrence was detected. Serum AMH was
readily detectable in 14 of 15 patients with a recurrent GCT, including
2 juvenile GCT and 4 bilaterally ovariectomized patients in whom
routine serum samples had been assayed during follow-up when clinical
signs of a recurrence were not yet evident. In 1 case, AMH was
undetectable in the only serum sample available despite a progressive
disease. Sensitivity and positive predictive value of serum AMH for the
presence of a GCT were 93% in our series of 15 patients with a
demonstrated recurrence (Table 4). Of the 14 patients with long term
follow-up, serum AMH declined to undetectable values after successful
treatment in 8, but remained elevated in 4 others in whom the tumor
could not be completely removed and/or was not responsive to
chemotherapy. In the remaining 2 cases, unilateral ovariectomy was
performed due to the young age of the patients, and low but detectable
levels of AMH could be found in serum samples obtained after treatment.
Considering the serum samples studied only during the remission periods
of 24 bilaterally ovariectomized patients (16 of whom were in remission
during the whole follow-up period and for 8 of whom samples were
available during both progressive disease and remission), the
specificity and negative predictive value of serum AMH reached 96%
(Table 4).
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), suggesting that some tumor
tissue could not be extirpated. Fifteen days later, increasing serum
AMH levels were detected by both assays, and a progression of the tumor
was clinically confirmed. Patient B received chemotherapy for a
recurrent GCT. At 1 month of treatment, although serum AMH was below
the sensitivity level of the traditional assay, detectable levels,
suggesting the presence of residual tumor cells, were found using the
ultrasensitive AMH/MIS ELISA (Fig. 1B). Chemotherapy was continued, and
serum AMH became undetectable by 6 months of treatment. Eighteen months
later, elevated AMH levels were found in a serum sample obtained for
routine control during follow-up; a new recurrence was suspected and
subsequently confirmed by ultrasound and clinical examinations. A
retrospective comparison of AMH levels using the 2 assays was performed
in serum samples from patient C, in whom a recurrence of an adult-type
GCT had been suspected due to elevated serum AMH using the traditional
assay. When the ultrasensitive AMH/MIS ELISA was used, an elevation of
AMH levels could be detected in several serum samples obtained during
the previous 16 months, when the traditional assay still gave normal
results (Fig. 1C). The recurrence was diagnosed before any clinical
sign was evident and could be successfully removed. In patient D, serum
AMH declined after a successful surgical extirpation of the tumor; the
very low AMH levels present in serum 72 h after surgery could only
be detected with the ultrasensitive assay. No AMH was detected by any
of the assays in subsequent serum samples (Fig. 1D). In patients E and
F, bearing juvenile-type GCT, only a unilateral ovariectomy was
performed due to their young age. After surgery, although serum AMH was
undetectable with the traditional assay, it oscillated between
undetectable and low levels with the ultrasensitive AMH/MIS ELISA (Fig. 1, E and F). However, serum AMH was always within the normal range, and
this was interpreted as a result of normal secretion by the remaining
gonad. These examples corroborate the higher sensitivity of the
ultrasensitive AMH/MIS ELISA.
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Discussion |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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The results obtained in our large series of 31 GCT patients with long-term follow-up confirm the value of serum AMH in both the evaluation of treatment and the detection of recurrences of juvenile- (13) and adult-type (10, 12) GCT. Furthermore, the use of a more sensitive assay has allowed us to improve the detection of patients with circulating AMH levels, due to the recruitment of those with serum AMH levels between 0.7–14 pmol/L.
As we had previously shown in two patients (12), two new cases of GCT recurrences were suspected at an early stage due to the elevation of serum AMH in routine determinations during follow-up, before any clinical sign of the disease became evident. Moreover, using the ultrasensitive AMH/MIS ELISA, we have been able to detect low levels of serum AMH in three samples obtained from one of these patients in which no AMH could be detected with the traditional assay. Therefore, we presume that the recurrent tumor would have been suspected several months earlier if the ultrasensitive assay had been available when the earliest serum samples were taken. The ultrasensitive assay has also allowed us to detect very low serum AMH levels, suggesting the presence of residual tumor cells, 2 weeks after surgery in one patient and after the first month of chemotherapy in another patient. However, the interpretation of serum AMH levels should be performed cautiously in the first days after surgical removal of a GCT. The AMH half-life was estimated to be 48 h (25); therefore, as we show here, low levels of AMH can be detected in serum within a few days (48 h with the traditional assay and 72 h with the ultrasensitive one) after successful surgery.
Finally, the higher sensitivity of the assay also resulted in the detection of low levels of serum AMH in two young patients in whom one ovary had been preserved. Although the lowest detectable AMH levels are suggestive of a recurrence in a bilaterally ovariectomized patient, a serum AMH concentration of up to 75 pmol/L is normal in a prepubertal girl or a premenopausal woman bearing ovaries. As no data are available on normal serum AMH in women with only one gonad, the interpretation of detectable serum AMH levels in these cases may be difficult.
In conclusion, we have developed a powerful and easy to perform ELISA for AMH determination, allowing us to evaluate with high sensitivity the results of treatment and to detect recurrences of GCT at an early stage.
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Acknowledgments |
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Footnotes |
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2 AMH/MIS ELISA kit is a trademark of
Immunotech under INSERM license.
Received June 14, 1999.
Revised October 18, 1999.
Accepted October 22, 1999.
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References |
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-inhibin and estradiol. Am J Obstet Gynecol. 174:958–965.[CrossRef][Medline]
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