Are serum inhibin concentrations new markers of placental tumours in the course of chemotherapy?

P. Pautier1,5, S. Ghione2, S. Brailly-Tabard4, C. Lhommé3, P. Morice3 and J.M. Bidart2

1 Medical Department, Gynecological Unit, 2 Clinical Biological Department and 3 Surgical Department, Institut Gustave-Roussy, 94805 Villejuif and 4 Molecular Biology and Hormonology Department, Hôpital Bicêtre, 94275 Le Kremlin-Bicêtre Cedex, France


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
BACKGROUND: The study was conducted to evaluate whether the detection of serum molecular forms of inhibin (A and B) could be useful for the diagnosis, prognosis and follow-up of placental tumours. METHODS: A total of 17 patients with hydatidiform mole (n = 13), invasive mole (n = 1) or choriocarcinoma (n = 3) were studied; serum concentrations of inhibins A and B, human chorionic gonadotrophin (HCG) and its free ß subunit (HCGß) were measured before chemotherapy (after mole evacuation for eight patients) and also during the course of chemotherapy (for 10 patients). RESULTS: After evacuation or before chemotherapy for refractory disease, serum inhibin A and B concentrations were found to be increased in 10/17 and 4/17 patients, when HCG and HCGß were high in all patients. In 10 patients with a follow-up during treatment, nine had a high concentration of inhibin A which correlated with those of HCG and HCGß. Normalization of inhibin A was faster than that of HCG and HCGß for three and six patients respectively. There was no correlation between changes of inhibin B and HCGß concentrations. CONCLUSIONS: Our results suggest that inhibins A and B are not useful markers and that HCG determination still remains the most useful marker for diagnosis and follow-up of placental tumours.

Key words: human chorionic gonadotrophin/inhibin/marker/trophoblastic disease


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Inhibins (A and B) are heterodimeric glycoprotein hormones assembled from two subunits with a common {alpha} subunit. These hormones are mainly produced by the gonads and play a critical role in the control of gamete maturation (Webb et al., 1999Go). In clinical oncology, inhibins are sensitive markers in the detection and follow-up of patients with ovarian cancers, particularly those bearing granulosa cell tumours (Lappöhn et al., 1989Go; Healy et al., 1993Go; Petraglia et al., 1998Go; Frias et al., 1999Go).

Inhibins are also produced by the placenta and fetal membranes during pregnancy (Riley et al., 1996Go, 2000Go; Fowler et al., 1998Go; Petraglia et al., 1999Go). Indeed, human placentas express inhibin ß- and {alpha}-subunit transcripts and proteins (Petraglia et al., 1991Go; McCluggage et al., 1998Go). Inhibins may be important regulators of fetal and placental development as well as being involved in the establishment of pregnancy (Riley et al., 1996Go). This probably explains why serum maternal concentrations vary according to the term of the pregnancy, declining after delivery (Wallace et al., 1997Go). Measurement of serum inhibin is useful in cases of various gestational pathologies, including pre-eclampsia, Down's syndrome and molar pregnancies (Aitken et al., 1996Go; Muttukrishna et al., 1997Go). Inhibin has been also postulated to play a role in trophoblastic molar invasion and its presence in molar trophoblast cells has been reported (Minami et al., 1993Go; McCluggage et al., 1998Go; Pelkey et al., 1999Go; Shih and Kurman, 1999Go). However, controversies exist regarding the clinical interest of measuring inhibin in patients bearing trophoblastic tumours (Yohkaichiya et al., 1989Go; Badonnel et al., 1994Go). This is due to the fact that immunoassays used in these studies detect total inhibin without differentiating the two molecular forms.

Taking advantage of the recent development of specific immunoassays for inhibin A and inhibin B (Robertson et al., 1996Go), we investigated whether molecular forms of inhibin may represent better markers than human chorionic gonadotrophin (HCG) and its free ß subunit (HCGß) for diagnosis, prognosis and follow-up of gestational trophoblastic diseases (GTD). Indeed, if HCG and HCGß are well-established sensitive markers of GTD (Schlaerth et al., 1981Go; Fan et al., 1987Go; Yedema et al., 1993Go), they have no predictive value either for prognosis or for response to chemotherapy and, moreover, their normalization after evacuation is variable over time (Bagshawe et al., 1976; Azab et al., 1988Go; Bidart et al., 1999Go).


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Serum collection
Serum samples were obtained from 17 patients who were referred to our institution for first or second line chemotherapy of a trophoblastic disease. The study included patients with a diagnosis of hydatidiform mole (n = 13, one with lung metastatis), invasive mole (n = 1) or choriocarcinoma (n = 3). The median age was 28 years old (range: 23–40) and all patients were under the same oral contraception (with 50 µg of ethinyl oestradiol). Diagnoses were reconfirmed by review of histological slides. This study was approved by the local ethics committee.

Chemotherapy consisted of a combination of dactinomycin and etoposide every week for the treatment of hydatidiform and invasive mole, and a combination of dactinomycin and etoposide every 2 weeks plus cisplatin every 4 weeks for choriocarcinoma and for methotrexate-resistant hydatidiform mole.

Serum inhibins A and B were measured using a two-site (sandwich) enzyme-linked immunoassay (ELISA; Serotec, Oxford, UK). These tests are based on specific monoclonal antibodies raised against the {alpha}, ßA and ßB subunits. Briefly, each assay was performed as follows: serum samples were first mixed with a 10% sodium dodecyl sulphate solution and incubated at 100°C for 3 min. After cooling, a hydrogen peroxide solution was added to tubes for a further incubation at room temperature. Samples were then transferred to antibody-coated microtitre plates and incubated at room temperature for 2 h (for inhibin A) and overnight (for inhibin B), according to the manufacturer's instructions. Recombinant inhibin A and inhibin B were used as standards. The inhibin A and B detection limits were 4 and 10 ng/l respectively. Serum HCG and free HCGß were measured using specific immunoradiometric assays (Cisbio International, Giff sur Yvette, France) (Ozturk et al., 1987Go). The intra- and interassay coefficients of variation were <4% and <7% for inhibin A, and <4% and <8% for inhibin B respectively.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Serum inhibin A, serum inhibin B, HCG and HCGß concentrations before starting chemotherapy are presented in Table IGo. Initial serum inhibin A was increased in 10/17 patients, including seven hydatidiform moles, one invasive mole and two choriocarcinomas. Initial serum inhibin B was increased in 4/17 patients. None except one (hydatidiform mole) of the patients had a simultaneous increase of both inhibins A and B. In contrast, all patients presented high concentrations of both HCG and free HCGß.


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Table I. Serum inhibin A, inhibin B, human chorionic gonadotrophin (HCG) and HCGß concentrations before chemotherapy
 
Serial measurement of the four biological parameters were repeated during the treatment and follow-up in 10 patients, including seven hydatidiform mole, with two patients resistant to methotrexate, one invasive mole and one choriocarcinoma. A complete response to chemotherapy was observed for the 17 patients. Figure 1Go shows that, in 10 patients with a serum follow-up during treatment, nine had a high concentration of inhibin A and a correlation was found with the decrease of HCG and HCGß. Normalization of inhibin A was faster than that of HCG and HCGß for three and six patients respectively. In contrast, there was no correlation between changes of inhibin B and HCGß serum concentrations.



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Figure 1. Serum inhibin (A and B), human chorionic gonadotrophin (HCG) and HCGß concentrations in 10 patients (patients 1, 2, 4, 6, 9, 11, 12, 14, 16 and 17) before, during and after chemotherapy.

 

    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Chemotherapy has totally transformed the prognosis of GTD. Nevertheless prognosis depends on a series of factors and some patients have a high risk tumour with poor prognosis. Hormonal follow-up by serial measurements of serum HCG and/or HCGß is reliable but has no predictive value. Furthermore, the time to obtain normalization of these markers is variable during treatment and may have an effect on the choice and/or the duration of treatment (Bidart et al., 1999Go). These observations emphasize the need for more appropriate markers. Among new potential markers, the inhibin family, including the various forms of inhibins and activins, has been proven to be useful for the detection and management of patients bearing granulosa cell tumours (Lappöhn et al., 1989Go; Petraglia et al., 1998Go). In ovarian adenocarcinoma, and particularly in mucinous tumours, inhibins display a good sensitivity but a weak specificity (Healy et al., 1993Go). The pre-operative serum inhibin A concentration is an independent prognosis parameter on the survival of post-menopausal women with epithelial ovarian carcinoma (Frias et al., 1999Go). Several observations demonstrated that trophoblast cells secrete both inhibin A and activin A, a dimer of the ß-subunit of inhibin, suggesting that those hormones may be useful in the detection and follow-up of patients with trophoblastic diseases (Petraglia et al., 1991Go; McCluggage et al., 1998Go). We recently reported that serum activin A is not a useful marker of placental tumours (Florio et al., 1998Go).

Our present observations demonstrate that the specific measurements of serum molecular forms of inhibins, namely inhibins A and B, do not seem to be of any clinical relevance in the biological survey of patients with GTD. Indeed, all our patients had elevated serum HCG and HCGß concentrations, while only 15 had elevated inhibin A, including only two patients with choriocarcinoma, and seven had a moderate increase of inhibin B, indicating that inhibins A and B are not sensitive enough markers. As is the case during pregnancy, inhibin A appears to be the major circulating form (Wallace and Healy, 1996Go). Although no serum measurement was performed before evacuation, all the patients included in our study had persistent or recurrent GTD. Moreover, three patients had choriocarcinoma, which is known to be a more aggressive disease.

In ten patients, serial determinations were performed during chemotherapy. Inhibin A was elevated in eight of these and a correlation was found between the kinetics of inhibin A and those of HCG and HCGß. However, the time to normalization was similar for the three parameters, except for five patients in whom it was faster for inhibin A. No correlation between inhibin B, HCG and HCGß was found. There was no benefit for inhibin determination before and during chemotherapy for GTD. A correlation was found between the kinetics of inhibin A and HCG during normal pregnancy: there was a peak at 9–10 weeks, coinciding with the HCG peak, and then a fall to a plateau between 15–30 weeks.

Yohkaichiya and collaborators reported the evolution of total inhibin, HCG and FSH serum concentrations before and 7–10 days after mole evacuation in six patients bearing hydatidiform mole (Yohkaichiya et al., 1989Go). Before evacuation, serum inhibins were higher than those observed during normal pregnancies at the same term. Three patients had elevated inhibin concentrations higher than those seen in the follicular phase of normal menstrual cycles 2 weeks after evacuation. Only the latter developed a persistent trophoblastic disease. But in one case, inhibin concentrations were at the upper limit of normality, which was then considered to be elevated. So, we may conclude that in this study, inhibin failed to detect a persistent disease (Wallace and Healy, 1996Go).

In conclusion, inhibin A and B do not seem to be reliable markers for persistent GTD. HCG and its free HCGß remain the most efficient and useful tumour markers for the diagnosis and follow-up after treatment of GTD.


    Acknowledgements
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
We thank Gilles Charrot for editing the manuscript.


    Notes
 
5 To whom correspondence should be addressed. E-mail: pautier{at}igr.fr Back


    References
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
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Submitted on February 27, 2001; accepted on August 13, 2001.





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