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Departments of Internal Medicine (R.P., D.F., W.W.d.H., S.W.J.L., L.J.H.) and Pathology (R.R.d.K.), Erasmus Medical Center, 3015 GE Rotterdam, The Netherlands; Departments of Molecular and Clinical Endocrinology and Oncology (R.P., A.F., G.L., A.C.), "Federico II" University of Naples, 80131 Naples, Italy; Department of Nuclear Medicine, European Institute of Oncology (L.B.), 20141 Milan, Italy; and Department of Endocrinological and Metabolic Sciences and Center of Excellence for Biomedical Research (D.F.), University of Genoa, 16132 Genoa, Italy
Address all correspondence and requests for reprints to: Rosario Pivonello, M.D., Ph.D., Department of Molecular and Clinical Endocrinology and Oncology, "Federico II" University, Via Sergio Pansini, 5, 80131 Naples, Italy. E-mail: rpivone{at}tin.it.
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Abstract |
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Aim: The aim of the current study was to evaluate DR and particularly D2 subtype expression in neuroendocrine tumors associated with EAS and to evaluate the in vivo effectiveness of the DA cabergoline in the treatment of EAS.
Patients and Methods: Six ACTH-secreting neuroendocrine tumors, including four lung, one pancreatic, and one thymic carcinoid, were used for the evaluation of D2 expression by immunohistochemistry. DR subtypes and D2 isoforms and number were evaluated by RT-PCR in three cases of persistent EAS after surgery. These patients were treated with cabergoline at the dose of 3.5 mg/wk for 6 months. Clinical parameters, hormonal levels, and tumor size were monitored during the treatment period.
Results: At immunohistochemistry, D2 was expressed in five (83.3%) tumors. At RT-PCR, D2 was confirmed in all three cases but at variable numbers, whereas D4 was expressed in two cases. D2long was expressed in all three cases, together with D2short in one case. A normalization of urinary cortisol levels was found in two patients (66.7%) after 3 months of treatment. However, treatment escape was demonstrated in one of these patients afterward.
Conclusion: The results of this study demonstrated that DR are expressed in neuroendocrine tumors associated with EAS and that cabergoline treatment could be effective in controlling cortisol excess in a subgroup of patients with EAS. Further studies on a larger number of patients are mandatory to confirm the usefulness of DA in EAS.
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Introduction |
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TopAbstractIntroductionPatients and MethodsResultsDiscussionReferences |
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Patients and Methods |
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TopAbstractIntroductionPatients and MethodsResultsDiscussionReferences |
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Six patients (two females and four males, 30–50 yr old) with a diagnosis of EAS associated with ACTH-secreting neuroendocrine tumors entered the study after their informed consent had been obtained. All patients were subjected to surgery for the removal of the tumor. The histological and immunohistochemical study of the tumor documented an ACTH-secreting neuroendocrine tumor in all cases: a lung carcinoid in four cases (typical in three and atypical in one) and a pancreatic well-differentiated endocrine carcinoma (pancreatic carcinoid) and a thymic carcinoid in the remaining two cases. The patients’ profile and tumor characteristics are shown in Table 1
. After surgery, a clinical, hormonal, and radiological remission of EAS was documented in three patients, whereas disease persisted in the remaining three patients (patients 1–3 in Table 1), all bearing a residual lung carcinoid (typical in two and atypical in one). In these three patients, at the diagnosis, tumor diameters were 1.8 cm (patient 1), 1.5 cm (patient 2), and 1.2 cm (patient 3). Lymph node metastases were present in two patients (patients 1 and 2), whereas no distant metastases were detected in any of the three patients. After surgery, a residual tumor was found in all three cases: tumor diameter was measurable (1.0 cm) only in one case (patient 1), where positive lymph nodes were also found, whereas in the remaining two patients residual tumors were not visible with standard imaging techniques but were clearly visualized with somatostatin receptor scintigraphy.
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DR subtype and D2 isoform (D2long and D2short) expression were evaluated by standard RT-PCR and D2 number was evaluated by quantitative RT-PCR in three cases (patients 1–3 in Table 1
), whereas D2 receptor expression and localization was evaluated by immunohistochemistry (IHC) in all six cases. The effectiveness of cabergoline treatment on clinical, hormonal, and radiological parameters was evaluated in the three cases of patients with persistent EAS after surgery (patients 1–3 in Table 1). The study was in accordance with the Helsinki Doctrine on Human Experimentation and the protocol was approved by the Local Ethical Committees.
IHC
Tumor specimens, obtained at the time of tumor excision, were fixed in formalin and embedded in paraffin for IHC study. The IHC study was performed on tissue samples according to previous reports (6, 7). Immunostaining for D2 was performed using a rabbit antihuman polyclonal antibody (Chemicon International, Temecula, CA) in a dilution of 1:500. Immunostaining for ACTH (Neomarkers, Duiven, The Netherlands; dilution 1:100), chromogranin A (Biogenix, Duiven, The Netherlands; dilution 1:100), and synaptophysin (Dako, Heverlee, Belgium; dilution 1:50) was performed, together with histology, on sequential sections. The intensity of immunostaining for ACTH, chromogranin A, synaptophysin, and D2 receptor was scored with the following semiquantitative method: –, absent; +, weak; ++, moderate; +++, strong immunostaining.
RT-PCR
Tumor specimens, obtained at the time of tumor excision, were quickly frozen on dry ice and stored in a freezer at –80 C for RT-PCR study. Standard RT-PCR was performed according to previous reports (6, 7). Quantitative RT-PCR was performed according to a previous report (8), after the isolation of total RNA, performed by using the Tri Pure Isolation Reagent kit (Roche Molecular Biochemicals, Indianapolis, IN). The detection of hypoxanthine phosphoribosyltransferase (HPRT) served as a control and was used for normalization of the D2 levels. Primer and probe sequence for HPRT has been previously described (9). Primers and probe for D2 were: 5'-GCCACTCAGATGCTCGCC-3' (forward), 5'-ATGTGTGTGATGAAGAAGGGCA-3' (reverse) and 5'-FAM-TTGTTCTCGGCGTGTTCATCATCTGC-TAMRA-3' (probe). Primers and probe for HPRT were: 5'-TGCTTTCCTTGGTCAGGCAGTAT-3' (forward), 5'-TCAAATCCAACAAAGTCTGGCTTATATC-3' (reverse), and 5'-FAM-CAAGCTTGCGACCTTGACCATCTTTGGA-TAMRA-3' (probe). Five cases of ACTH-secreting pituitary tumors were used as controls for the evaluation of the D2 number at the quantitative RT-PCR.
Therapeutic study
Cabergoline was administered at the dose of 3.5 mg/wk (0.5 mg/d), after the administration of a test dose (1 mg) during the week before starting the treatment to evaluate the tolerability for the drug. The clinical evaluation, including the scoring of the symptoms and signs, and mainly the measurement of blood pressure and serum glucose and lipid levels as well as the hormonal evaluation, including the measurement of plasma ACTH and serum and urinary cortisol levels, were performed monthly, whereas the radiological evaluation, including the measurement of tumor mass, was performed every 3 months and/or at the withdrawal of the cabergoline treatment by computed tomography or magnetic resonance imaging.
Hormone assay
Plasma ACTH levels were measured by immunoradiometric assay, whereas serum and urinary cortisol levels were measured by RIA method using commercially available kits. Both at baseline and during the follow-up evaluations, plasma ACTH and serum cortisol were measured after an overnight fasting at 0800 h in a single sample, whereas urinary cortisol levels were collected three times in the same week and the mean considered for the study. Normal ranges were 10–100 pg/ml for plasma ACTH, 50–200 µg/liter for serum cortisol and 35–135 µg/24 h for urinary cortisol.
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Results |
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In the IHC study, specific D2 immunostaining was found in five (83.3%) of the six cases, including the four lung and the thymic carcinoids. D2 immunostaining was strong and homogeneous in two lung carcinoids, moderate in one lung carcinoid, and weak and heterogeneous in one lung and in the thymic carcinoid. In the standard RT-PCR study, D2 receptor was expressed in all three cases (lung carcinoids) that were tested with this technique (both D2long and D2short in one case and only D2long in two cases), whereas D4 receptor was expressed in two of the three lung carcinoids (patients 1 and 2 in Table 1). In the quantitative RT-PCR study, a variable number of D2 was found, being relatively higher in one case (patient 1 in Table 1) and lower in two cases (patients 2 and 3 in Table 1). The results of the RT-PCR and IHC studies are summarized in Table 1. An example of the IHC results in one case of lung carcinoid (patient 4 in Table 1) is shown in Fig. 1.
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A significant inhibition of plasma ACTH, as well as serum and urinary cortisol levels, was found in two of the three (66.7%) patients with postoperative persistent EAS after 1 month, and a complete normalization of both ACTH and cortisol levels was found in these patients after 3 months of treatment with cabergoline. Conversely, no significant hormonal changes were found in the third patient, in whom, therefore, treatment was stopped after 1 month. In one of the two responsive patients, ACTH started to rise again at 4 months, whereas cortisol started to reincrease at 5 months of treatment and did not normalize despite the augmentation of the cabergoline dose from 0.5 to 1 mg/d. In this patient cabergoline treatment was stopped after 6 months of treatment. In the remaining responsive patient, ACTH and cortisol levels, despite some fluctuations, remained normal during the 6 months of treatment. Plasma ACTH and urinary cortisol levels in the three patients treated with cabergoline are shown in Fig. 2. A significant improvement of the clinical syndrome, mainly blood pressure and blood glucose levels, was found in the two responsive but not in the resistant patient. In the patient who had a treatment escape, a reworsening of the clinical syndrome was found at the 6-month follow-up. No change in tumor size was found in any of the three patients during the treatment period.
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Discussion |
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Despite the well-known DR expression and role in pituitary tumors (6, 14, 15, 16, 17, 18), the DR expression and its role in extrapituitary (ectopic) neuroendocrine tumors, and particularly in carcinoids, has never been extensively evaluated. Furthermore, despite the demonstrated DA effectiveness in the treatment of pituitary-dependent CS (6, 19), the possible DA effectiveness in the treatment of EAS has never been tested. DR represent a group of five different G protein-coupled receptors (D1–D5), mediating the various central and peripheral actions of dopamine (20). DR expression and dopamine synthesis have been demonstrated in gastrointestinal neuroendocrine tumor cell lines (5). The current study is the first demonstrating DR, particularly D2 and D4, expression in ACTH-secreting ectopic tumors, including lung, thymus, and pancreatic carcinoids, suggesting that DR may play a role in the biology of these tumors and/or form a target for treatment with DA.
Cabergoline was demonstrated to be effective in the treatment of ACTH-secreting pituitary tumors, causing CS (6). The current study demonstrates for the first time a possible effectiveness of cabergoline in the treatment of ACTH-secreting ectopic tumors, causing EAS. Short-term cabergoline treatment significantly inhibited ACTH, and consequently cortisol secretion in two of three cases with EAS due to a lung carcinoid. Hormonal suppression was maintained for a long-term follow-up in one of these two cases, with a clear-cut improvement of clinical syndrome and without any significant change in the size of the tumor. It is noteworthy that the patient not responsive to cabergoline was bearing a tumor expressing only the long isoform of D2 and the patient who experienced a treatment escape was bearing a tumor expressing the long isoform of D2 associated with D4 receptor, whereas the patient showing long-term responsiveness to cabergoline, was bearing a tumor expressing both isoforms of D2 as well as D4 receptors. Taken into consideration that three cases are not enough to draw final conclusions, it seems that the expression of short isoform of D2 and/or the coexpression of D4 may play a pivotal role in the effectiveness of DA in the treatment of carcinoid tumors associated with EAS. This hypothesis seems to be supported by similar evidence found in the treatment of pituitary tumors (6, 17). Alternatively, the responsiveness and/or the protection from treatment escape could be related to the different numbers of D2 and/or D4 expressed in the tumor. Indeed, it has to be pointed out that the patient who was long-term responsive to cabergoline treatment had a tumor with a stronger D2 at IHC and a relatively higher number of D2 at RT-PCR than the nonlong-term responsive patients.
Finally, the possible effectiveness of DA, demonstrated in the present study, and the well-described effectiveness of SA, demonstrated in previous studies, together with the demonstration of a synergistic cooperation between somatostatin receptors and DR and the potentiation of the correspondent SA and DA effectiveness in transfected cell lines (21), suggest a possible synergism in the action of the two categories of drugs in the treatment of carcinoid tumors causing EAS. This phenomenon has been indeed recently demonstrated in a case of EAS due to lung carcinoid (22).
In conclusion, DR, particularly D2 and D4, are expressed in carcinoid tumors associated with EAS, where they can mediate a therapeutic effect of DA, particularly cabergoline, in the inhibition of ACTH and cortisol secretion, and so preventing the complications of glucocorticoids excess due to EAS. These data suggest that DA might be included in the therapeutic options, especially in combination with SA, of persisting EAS, mainly for patients with occult or nonresectable tumors or patients with active disease before surgery or disease persistence after surgery, who are waiting for the definitive cure. It is important to point out that further studies on a larger number of patients are mandatory to confirm the real usefulness of DA in the management of EAS.
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Acknowledgments |
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Footnotes |
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Disclosure Statement: The authors have nothing to disclose.
First Published Online October 10, 2006
Abbreviations: CS, Cushing syndrome; DA, dopamine agonist(s); DR, dopamine receptor(s); EAS, ectopic ACTH syndrome; HPRT, hypoxanthine phosphoribosyltransferase; SA, somatostatin analog(s).
Received April 4, 2006.
Accepted September 29, 2006.
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TopAbstractIntroductionPatients and MethodsResultsDiscussionReferences |
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, patient 1; •, patient 2; 
, patient 3 in Table 1