First-line medical therapy in acromegaly generally consists of

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1 ORIGINAL ARTICLE Endocrine Care Brief Report Correlation of in Vitro and in Vivo Somatotropic Adenoma Responsiveness to Somatostatin Analogs and Dopamine Agonists with Immunohistochemical Evaluation of Somatostatin and Dopamine Receptors and Electron Microscopy Diego Ferone, Wouter W. de Herder, Rosario Pivonello, Johan M. Kros, Peter M. van Koetsveld, Ton de Jong, Francesco Minuto, Annamaria Colao, Steven W. J. Lamberts, and Leo J. Hofland Departments of Internal Medicine (D.F., W.W.d.H., P.M.v.K., S.W.J.L., L.J.H.) and Pathology (J.M.K., T.d.J.), Erasmus Medical Center, 35 GE Rotterdam, The Netherlands; Department of Molecular and Clinical Endocrinology and Oncology (R.P., A.C.) Federico II University, 83 Naples, Italy; and Department of Endocrinological and Medical Sciences and Center of Excellence for Biomedical Research (F.M.), University of Genova, 63 Genova, Italy Objective and Patients: Twenty-four pituitary adenomas from acromegalic patients (3 females, males; age range 9 65 yr) were characterized for somatostatin receptor subtype A (sst A ), dopamine D receptor (D R), GH, and prolactin (PRL) expression by immunohistochemistry, and results correlated with the in vitro and in vivo hormone responses to octreotide and quinagolide. In nine cases, GH and PRL content was further studied by immunoelectron microscopy. Results: Immunoreactivity was semiquantitatively scored as ( 5% stained cells), ( 5% stained cells), and ( % stained cells). Sst A was scored as in 3 cases, in, and in one; D R was scored as in 3 cases, in nine, and in ; GH was in 5 cases and in nine; PRL was in six cases, in 3, and in 5. Sst A was positively correlated with in vitro (P.3) and in vivo (P.6) percent GH suppression by octreotide and with the chronic suppression of IGF-I by somatostatin analogs (P.8). D R was positively correlated with in vitro percent GH (P.) and PRL (P.5) suppression by quinagolide. Electron microscopy revealed two pure somatotroph adenomas, five somatomammotrophs with a variable coexpression of GH and PRL in the same cells, and two tumors consisting of mixed cell types, which were less sensitive to quinagolide and octreotide. Conclusion: Sst A and D R are frequently coexpressed in adenomas from acromegalic patients, and immunohistochemistry may be helpful in characterizing receptor expression in pituitary adenomas to select patients responsive to different treatments. (J Clin Endocrinol Metab 93: 4 47, 8) First-line medical therapy in acromegaly generally consists of somatostatin analogs (SSAs), dopamine agonists (DAs), or combinations and should result in suppression of the elevated GH and IGF-I levels and/or significant tumor reduction (). The response to SSAs depends on the presence of a sufficiently high number of somatostatin receptors subtypes (sst 5 )onthe tumor cells. GH secretion is regulated through ligand binding of somatostatin to both sst and sst 5, whereas octreotide and lanreotide bind preferentially sst (). The GH-lowering effect by these drugs is positively correlated with the level of expression of sst mrna (3 5). DAs have been largely used for the treatment of pituitary tumors, particularly prolactinomas, and the responsiveness depends on the expression of dopamine D receptors (D R) on tumor cells. This subtype is also responsible for the paradoxical Downloaded from by guest on 6 January 9-97X/8/$5./ Printed in U.S.A. Copyright 8 by The Endocrine Society doi:./jc Received June 9, 7. Accepted January 5, 8. First Published Online January, 8 Abbreviations: DA, Dopamine agonist; D R, dopamine D receptor; EM, electron microscopy; LAR, long-acting release; PRL, prolactin; SSA, somatostatin analog; sst A, somatostatin receptor subtype A. 4 jcem.endojournals.org J Clin Endocrinol Metab. April 8, 93(4):4 47

2 J Clin Endocrinol Metab, April 8, 93(4):4 47 jcem.endojournals.org 43 TABLE. response DA-suppressive activity on GH in tumors of acromegalic patients (6). SSAs are more effective than DAs in lowering GH and normalizing IGF-I. However, DAs can be orally administered at lower costs (7). The additive effect in lowering GH suggests that the combination of SSAs and DAs might be useful in selected patients (7). Hyperprolactinemia in acromegaly might be caused by anatomical or functional hypothalamo-pituitary disconnection, presence of mixed GH/prolactin (PRL)-secreting tumors, or mammosomatotroph adenomas (8, 9). The availability of subtype-specific antibodies to sst and D R provides new tools to evaluate the receptor profile in those adenomas that have been unsuccessfully operated and need additional medical control (, ). In this study we characterized a series of pituitary adenomas from acromegalic patients by immunohistochemistry for GH, PRL, sst A, and D R expression. In a subgroup of these tumors, we evaluated the intracellular localization of GH and PRL granules using immunoelectron microscopy (EM) to determine which tumors coexpress sst A and D R in GH- and GH/PRL-secreting pituitary adenomas and whether the truly mixed GH/PRLsecreting adenomas represent a subgroup preferentially sensitive to DA. Immunohistochemical findings were correlated with clinical response to medical therapy as well as in vitro hormonal response to octreotide and quinagolide to establish whether the receptor expression phenotype is indicative of tailoring the adjuvant medical therapy. Patients and Methods Patients and treatments We selected 4 acromegalic patients (3 females and males, age ranging 9 65 yr, Table ) who underwent neurosurgery. In these patients, in vitro tumor response to octreotide and quinagolide as well as short- and long-term in vivo hormonal response to SSAs and/or DAs were available. Postoperative SSA treatment was continued for at least 6 months in 8 patients, whereas two were treated with DAs, and four did not receive any postoperative treatment (Table ). These latter six patients were not included in the long-term study. No patient had received radiotherapy before or during the study period. Informed consent was obtained from all patients. The acute octreotide and quinagolide tests were performed, on alternate days, as reported elsewhere (). After the tests, six patients Patient characteristics, treatment details, immunohistochemical and electron microscopy findings, and hormonal Patient no. Age/sex Therapy Dose, mg IHC score GH PRL ss A D R EM %GH OCT %GH CV %GH OCT %GH CV IGF-I a IHC score IHCscore IHC score IHC score In vitro In vitro In vivo In vivo In vivo 56/female None n.d /male OCT.3 b n.d /female LAR 3 c MIX /female LAN 6 d SM /male CAB.5 e n.d /female OCT.3 MIX /female LAR 3 n.d /male LAR SM /female LAR 3 n.d /female None n.d /female LAR n.d /male OCT.3 n.d /male LAR S /female LAR 3 n.d /female LAR 3 n.d /male LAR 3 SM /male None n.d /female None n.d /female LAR n.d /male OCT.3 S /male OCT.3 SM /female OCT.6 n.d /male LAR 3 SM /male CV.3 f n.d Downloaded from by guest on 6 January 9 IHC score:, highly positive;, positive;, negative. IHC, Immunohistochemistry; % GH OCT, percent GH suppression by octreotide; % GH CV, percent GH suppression by quinagolide; n.d., not done; OCT, sc octreotide; LAR, octreotide LAR; LAN, lanreotide Autogel; CAB, cabergoline; CV, quinagolide; MIX, mixed GH/PRL adenoma; SM, somatomammotroph adenoma; S, somatotroph adenoma. a IGF-I was scored when normalized during therapy, when reduced more than 5% but not normalized, and when reduced less than 5%. b. mg sc, three times a day. c 3 mg im, every 8 d. d 6 mg im, every 3 d. e.5 mg, orally three times a week. f.5 mg orally two times a day.

3 44 Ferone et al. SSR and D R in GH-Secreting Pituitary Adenomas J Clin Endocrinol Metab, April 8, 93(4):4 47 started sc octreotide treatment (Sandostatin; Novartis, Basel, Switzerland) patients long-acting SSAs: octreotide long-acting release (LAR; Sandostatin LAR) and one lanreotide (Somatuline Autogel; Ipsen, Kleve, Germany) (Table ). All patients were evaluated after 3, 6, 9, and months. The maximum dose of Sandostatin was. mg three times a day, that of Sandostatin LAR 3 mg every 4 wk, and that of Somatuline Autogel 6 mg every 8 d. Cell cultures GH-secreting pituitary adenoma tissue was collected during operation, and cells were isolated as previously described (3). A 7-h incubation without or with octreotide or quinagolide ( nm) in ml culture medium was performed in quadruplicate. Thereafter the medium was collected and stored at C until analysis. The results of each experiment were expressed as the percent suppression, compared with control untreated wells. Assays GH and PRL were determined by use of a nonisotopic, automatic chemiluminescence immunoassay system (Immulite; Diagnostic Products Corp., Los Angeles, CA). IGF-I was determined with a commercially available nonextraction immunoradiometric assay (Diagnostic Systems Laboratories, Inc., Webster, TX). Immunohistochemistry Immunohistochemistry was performed on paraffin-embedded sections (5 m) as previously described (, 4). GH, PRL, sst A, and D R immunoreactivity was semiquantitatively scored as ( 5% stained cells), ( 5% stained cells), and ( % stained cells) by three investigators. Immunoelectron microscopy Tissues were fixed in buffered, % acrolin,.4% glutaraldehyde solution (ph 7.) for4hat4candstored in M sucrose. M phosphate buffer. Samples were dehydrated up to % alcohol under lowering temperature to 35 C and infiltrated by Lowicryl. Polymerization was performed for 4 h at 35 C, followed by reverse transcription for 7 h. Semithin ( m) slides were stained with toluidin blue for orientation, and subsequently ultrathin slides were mounted on carbon-coated, formfar-covered, one-hole grids. To block aldehyde groups, the grids were incubated with.4 glycine/ M phosphate buffer (ph 7.) followed by incubation with 5% normal serum at room temperature for min. Incubation with primary antibodies included PRL (dilution :8) or GH (dilution :) for h at room temperature. Samples were rinsed with.4 M glycine. M phosphate buffer (ph 7.). Visualization was achieved by incubation of -nm colloidal, gold-labeled, goat-antimouse serum (GaMau nm, :) for hatroom temperature. After incubation, samples were rinsed with. M phosphate buffer (ph 7.)/ aquadest. Finally, the grids were contrasted using uranyl acetate and lead citrate. For EM, a Morgagni 68 D T5 D3 electron microscope (Philips, Best, The Netherlands) was used. Areas of the same cells were compared for GH and PRL positivity. Statistical analysis Statistical analysis was carried out by SPSS (version for Windows; SPSS Inc., Chicago, IL). All the data are expressed as mean SD, unless otherwise specified. The difference between percent GH and PRL in vitro suppression by octreotide and quinagolide, compared with baseline, was analyzed by the Wilcoxon matched-paired test. The test was used to calculate the association between immunohistochemistry score and the in vitro and in vivo response to SSAs and DAs. Correlation analysis was performed using Spearman s and Pearson tests. Results In vivo therapy Basal mean GH levels were (range.. to g/liter), and basal mean PRL levels, available in 8 patients, were (range.7. to g/liter). Percent GH suppression during the octreotide test ranged from 4 to 95.3%, whereas GH suppression during the quinagolide ranged from 4. to 9.9% (Table ). After longterm SSA therapy, IGF-I levels normalized in nine patients (responders), reduced more than 5% in six patients (partial responders), and reduced less than 5% in the remaining patients (poor responders). Patient treatments and dosages are reported in Table. In vitro functional studies In cultures mean GH secretion was (range 5.5. to g/liter), whereas PRL was detectable in 4 cases with a mean secretion of (range.. to g/liter). GH suppression by octreotide ranged from 8.5 to 73.7%, and quinagolide from 5.6 to 6.9% (Table ). PRL suppression by octreotide ranged from 6.4 to 73.9%, and quinagolide from. to 7.8%. Immunohistochemistry GH immunoreactivity was scored in 5 cases and in the remaining nine; PRL was scored in six cases, in 3, and in the remaining five. Sst A and D R were detected in 3 and of the 4 tumors, respectively. In most tumors sst A colocalized with D R. Sst A showed the highest score, namely, in 3 of 4 cases, whereas it was scored in, and in one. D R showed the highest score, namely in 3 of 4 cases, whereas it was scored in nine and in the remaining two (Table ). Mainly complete or incomplete membrane-bound as well as cytoplasmic immunoreactivity was noted in all tumors but not all cells. Figure shows, together with histology and EM, an exemplary case (patient, Table ). EM study EM studies revealed two pure somatotrophs, five somatomammotrophs with a variable expression of PRL in the same cells containing GH, and two tumors formed by mixed cell types containing either GH and PRL or single hormones (Fig. ). The in vitro GH suppression by octreotide in the two somatotrophs was significantly higher than that obtained with quinagolide, and both normalized IGF-I levels with octreotide. The two mixed tumors showed similar sensitivity to octreotide and quinagolide both in vitro and in vivo, but GH levels were suppressed less than in the pure somatotroph adenomas. SSAs did not normalize IGF-I in these patients. The five somatomammotrophs heterogeneously responded to both octreotide and quinagolide, and three of four chronically treated with octreotide normalized IGF-I, whereas in one IGF-I was reduced more than 5% (Table ). A significant association was found: between sst A score and in vitro (, 7.9; P.) and in vivo (, 6.76; P.3) GH suppression by octreotide as well as with IGF-I suppression dur- Downloaded from by guest on 6 January 9

4 J Clin Endocrinol Metab, April 8, 93(4):4 47 jcem.endojournals.org 45 FIG.. Upper panel, Immunohistochemical detection of GH, PRL, sst A, and D R in a somatotroph pituitary adenoma. A, Hematoxylin-eosin. B, Homogeneous GH immunoreactivity. C, PRL immunoreactivity in scattered cells, sections developed with 3,3 -diaminobenzidine. D, Homogeneous sst A immunoreactivity. E, Homogeneous D R immunoreactivity. F and G, Adjacent sections showing displacement of immunostaining after preabsorption of the antibodies with nm of the respective peptide antigens (no., Table ). Sections developed with New Fucsine/Naphtol AS-MX. The sections are slightly counterstained with hematoxylin. Magnification,. Lower panel, EM study in semithin sections of pituitary tumors showing the hormone content in the same granules characterizing a somatomammotroph adenoma. In this case of somatomammotroph adenoma (no. 3, Table ), immunogold particles identified GH (A C) or PRL (D F) in the same granules in the same cell. Three magnifications: 4,4 (A and D);, (B and E);, (C and F). ing SSAs (, 6.; P.), and between D R score and in vitro GH (, 5.65; P.) and PRL (, 6.37; P.4) suppression by quinagolide as well as in vitro GH suppression by octreotide (, 6.46; P.4). Moreover, sst A was positively correlated with the in vitro (P.3) and in vivo (P.6) GH suppression by octreotide (Fig., A and B) as well as with the IGF-I suppression by SSAs (P.8). Moreover, in vitro GH suppression by octreotide was positively correlated with the in vivo GH response to octreotide (P.9), which was positively correlated with IGF-I suppression by SSA treatment (P.7). D R immunoreactivity was positively correlated with the in vitro GH (P.) and PRL (P.5) suppression by quinagolide (Fig., C and D), whereas D R was not correlated with the in vivo response to quinagolide. Surprisingly, D R was also positively correlated with in vitro GH response to octreotide (P.) (Fig. E), whereas in vitro GH suppression by octreotide was positively correlated with in vitro GH suppression by quinagolide (P.3) (Fig. F). Discussion The acute bromocriptine and octreotide tests are unable to predict the long-term GH response to chronic medical therapy (, 5). PRL cosecretion and/or presence of PRL immunoreactivity in tumor specimens seem indicative of positive response to DAs or hint at combining DA and SSA in patients inadequately controlled by SSA monotherapy. However, two recent studies did not show any correlation between hyperprolactinemia and response to therapies (6, 7). In the present study, we found a positive correlation between sst A and both the in vitro and in vivo sensitivity of GH to octreotide in acromegalic patients. Sst A immunoreactivity was also correlated with control of IGF-I by chronic SSA therapy. Similarly, D R immunoreactivity positively correlated with the in vitro GH and PRL suppression by quinagolide. However, D R was not correlated with the in vivo GH response to quinagolide, suggesting that the in vivo sensitivity to DAs might be affected by other mechanisms. Indeed, heterogeneity as well as coexpression of other specific receptor subtypes might influence the activity of sst and D R. For example, a relative higher expression of sst 3 or sst 5 may promote an antiproliferative effect of SSAs despite the lack in controlling hormone secretion (8, 9). Octreotide and lanreotide bind predominantly to sst, whereas quinagolide and cabergoline bind predominantly to D R. Emerging data have demonstrated heterodimerization of somatostatin receptors and DRs (). In support of this hypothesis is the finding that D R was positively correlated with in vitro GH sensitivity to octreotide. Moreover, in vitro GH suppression by octreotide positively correlated with in vitro GH suppression by quinagolide. PRL staining did not correlate with D Rorthe acute in vivo or in vitro sensitivity of both GH and PRL to quinagolide. By EM study, we have identified two pure somatotroph ad- Downloaded from by guest on 6 January 9

5 46 Ferone et al. SSR and D R in GH-Secreting Pituitary Adenomas J Clin Endocrinol Metab, April 8, 93(4):4 47 A C E in vitro % GH suppression by octreotide in vitro % GH suppression by quinagolide in vitro % GH suppression by octreotide r,583 p,3 r,84 p, r,495 p,4 3 sst IHC score D IHC score B D F in vivo % GH suppression by octreotide in vitro % PRL suppression by quinagolide in vitro %GH suppression by octreotide r,59 p,6 r,754 p,5 7 sst IHC score D IHC score in vitro %GH suppression by quinagolide 8 3 r,546 p,3 Downloaded from by guest on 6 January 9 D IHC score FIG.. Box plots show the median, interquartile range, outliers (E, cases with values between.5 and three box lengths from the upper or lower edge of the box), and extreme cases (*, cases with values more than three box lengths from the upper or lower edge of the box) of individual variables. The box length is the interquartile range. A, Positive correlation between the percentage of GH suppression by octreotide in vitro and sst A immunohistochemistry (IHC) score. B, Positive correlation between the percentage of GH suppression by octreotide in vivo and sst A immunohistochemistry score. C, Positive correlation between the percentage of GH suppression by quinagolide in vitro and D R immunohistochemistry score. D, Positive correlation between the percentage PRL suppression by quinagolide in vitro and D R immunohistochemistry score. E, Positive correlation between the percentage of GH suppression by octreotide in vitro and D R immunohistochemistry score. F, Positive correlation between the percentage of GH suppression by octreotide in vitro and the percentage of GH suppression by quinagolide in vitro.

6 J Clin Endocrinol Metab, April 8, 93(4):4 47 jcem.endojournals.org 47 enomas, five somatomammotrophs, and two truly mixed GH/ PRL tumors. Although the number of cases is too low to draw definitive conclusions, the mixed tumors seem more resistant to SSAs, whereas almost no difference in sensitivity to octreotide and quinagolide was observed in somatotroph and somatomammotroph tumors. This might be due to the lower density of sst in mixed tumors or an interfering role of other receptor subtypes. In conclusion, immunohistochemistry is a useful method to characterize receptor expression in pituitary adenomas to optimize postoperative medical therapies. Receptor characterization can be used for studying the mechanisms regulating the different responses to therapy, particularly when new compounds with specific receptor binding profiles become available in the near future. Acknowledgments Address all correspondence and requests for reprints to: Diego Ferone, M.D., Ph.D., Department of Endocrinology and Medical Sciences, University of Genova, Viale Benedetto XV, 6, 63 Genova, Italy. ferone@unige.it. Current address for D.F.: Department of Endocrinological and Medical Sciences and Center of Excellence for Biomedical Research, University of Genova, Genova, Italy. Disclosure Statement: D.F., W.W.d.H., R.P., J.M.K., P.M.v.K., T.d.J., F.M., A.C., S.W.J.L., and L.J.H. have nothing to declare. References. Colao A, Martino E, Cappabianca P, Cozzi R, Scanarini M, Ghigo E, A.L.I.C.E. Study Group 6 First-line therapy of acromegaly: a statement of the A.L.I.C.E. (Acromegaly primary medical treatment Learning and Improvement with Continuous Medical Education) Study Group. J Endocrinol Invest 9:7. Moller LN, Stidsen CE, Hartmann B, Holst JJ 3 Somatostatin receptors. 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