Ki-67 is a predictor of acromegaly control with octreotide-lar independent of SSTR2 status

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1 Page 1 of 24 Accepted Preprint first posted on 7 June 2013 as Manuscript EJE Ki-67 is a predictor of acromegaly control with octreotide-lar independent of SSTR2 status and relates to cytokeratin pattern Short title: Ki-67 and response to octreotide-lar Authors: Leandro Kasuki* 1,4, Luiz Eduardo Armondi Wildemberg*¹, Leonardo Vieira Neto 1,5, Jorge Marcondes², Christina M. Takyia³, Mônica R. Gadelha¹ *LK and LEAW contributed equally to the study Affiliations: Endocrinology Unit, Clementino Fraga Filho University Hospital, Federal University of Rio de Janeiro, Rio de Janeiro, Brazil¹, Neurosurgery Unit, Clementino Fraga Filho University Hospital, Federal University of Rio de Janeiro, Rio de Janeiro, Brazil², Cell Pathology Laboratory, Biomedics Science Institute, Federal University of Rio de Janeiro³, Endocrinology Unit, Bonsucesso Federal Hospital 4, Endocrinology Unit, Lagoa Federal Hospital 5. Correspondence / Reprint requests to: Mônica R. Gadelha, MD, PhD. Neuroendocrinology Research Center Rua Professor Rodolpho Paulo Rocco, 255, sala 9F Ilha do Fundão Rio de Janeiro Brazil Zip code: Tel/Fax: mgadelha@hucff.ufrj.br Key words: Somatotropinomas, Ki-67, somatostatin receptors, somatostatin analogues. Word count: 3173 (text); 235 (abstract); 2 (tables); 3 (figure) 1 Copyright 2013 European Society of Endocrinology.

2 Page 2 of 24 Abstract Introduction: Only one study has evaluated Ki-67 as a predictor of the response to somatostatin analogues (SSA) therapy in acromegaly; however, other predictors like somatostatin receptor type 2 (SSTR2) and cytokeratin pattern expressions were not considered. Objective: To evaluate whether the Ki-67 is a predictor of octreotide LAR (OCT-LAR) response in somatotropinomas independent of SSTR2 and cytokeratin expression patterns. Methods: Protein expression was analyzed by immunohistochemistry. The percentage of cell nuclei that were immunolabeled for Ki-67 and the percentage of cells with positive SSTR2 staining was calculated. SSTR2 expression was considered high when 25%, and a cutoff of 2.3% was designated for Ki-67. Tumors were classified as densely or sparsely granulated according to the cytokeratin pattern. Results: Thirty-one somatotropinomas were studied. Fourteen patients (45.2%) were controlled with OCT-LAR therapy. The median Ki-67 LI was higher in patients not controlled with OCT-LAR than in those controlled (1.63 and 0.15, respectively, p=0.002). Higher SSTR2 expression and densely granulated tumors were correlated with control as well (p=0.04 and 0.038, respectively). There was no difference in Ki-67 levels between patients with high and low SSTR2 expression (p=0.651). After multivariate analysis, both Ki-67 and SSTR2 remained statically significant as predictors of OCT-LAR response (p=0.017 and 0.012, respectively). The Ki-67 LI was higher in sparsely than in densely granulated tumors (p=0.047). Conclusions: Ki-67 is a predictor of response to OCT-LAR in acromegaly, independent of SSTR2 expression and relates to cytokeratin patterns. 2

3 Page 3 of 24 Introduction Acromegaly is a chronic endocrine disease with high morbidity and mortality (1). Surgery is the treatment of choice for patients with microadenomas and macroadenomas with a high chance of a surgical cure (2). However, approximately 40 60% of patients with macroadenomas are unlikely to be cured by surgery alone (3). Somatostatin analogues (SSA) are the first treatment option in acromegalic patients for whom surgical cure is improbable and for those not cured by surgery (2). The currently available SSA can result in disease control in approximately 30% of acromegalic patients based on prospective clinical trials (4, 5). Thus, considering the present accessibility of the diverse classes of drugs and their elevated costs, it would be valuable to identify predictive markers for the patients who are more likely to respond to SSA therapy. Several predictors of SSA therapy efficacy have been evaluated, with the granulation pattern and expression of the somatostatin receptor subtype 2 (SSTR2) being the most established (6-11). Patients harboring densely granulated tumors respond better to SSA treatment than those with sparsely granulated ones (7, 12). SSTR2 expression in somatotropinomas seems to represent the best predictor of the response to SSA (9). Indeed, some studies have shown a positive correlation between the expression of SSTR2 mrna and protein and the clinical response to SSA (13-18). Ki-67 is a nuclear antigen expressed in all non-g0 phases of the cell cycle. Thus, in some studies, it has been shown to be a proliferative marker associated with tumor invasiveness or recurrence (19). Fusco et al. (20) found that a group of GH-secreting adenomas with low Ki-67 labeling index (LI) values had a better response to SSA than those with higher Ki-67 LI values. However, they did not evaluate the SSTR2 expression levels in the two groups of tumors, which might explain the difference in treatment response. No other study has evaluated Ki-67 as a predictor of the response of acromegaly to SSA therapy. 3

4 Page 4 of 24 In the current study, we aimed to evaluate whether the Ki-67 LI is a predictor of octreotide LAR (OCT-LAR) response in somatotropinomas independent of SSTR2 and cytokeratin expression pattern. Subjects and methods This study was approved by the Ethics Committee of the Clementino Fraga Filho University Hospital/Federal University of Rio de Janeiro. All the patients signed informed consent forms before study entry. Patients and tumors Thirty-one consecutive acromegalic patients who were treated with surgery but not cured were included in the study. Patients underwent operations between 2006 and The biochemical diagnosis of acromegaly was based on the current criteria (21). The exclusion criteria consisted of a history of medical treatment with SSA, dopamine agonists or GH antagonist before surgery as well as previous radiotherapy for pituitary adenoma. Magnetic resonance imaging (MRI) of the sellar region before the surgery was analyzed and the Di Chiro and Nelson formula was used to calculate tumor volume (22). Tumor invasiveness was defined according to the Knosp-Steiner criteria (23). Postsurgical evaluation Biochemical evaluation was performed 12 weeks after surgery by evaluation of oral glucose tolerance test (OGTT) and serum IGF-I levels in all subjects. Pituitary MRI was performed 3 months after the surgical procedure. Patients were considered as non-cured on the basis of the clinical picture, nadir GH levels after OGTT higher than 0.4 ng/ml, and plasma IGF-I levels higher than age-matched normal subjects. Medical therapy with OCT-LAR was started at a dose of 20 mg every 4 wk, and the dose was increased to 30 mg every 4 wk in uncontrolled patients after 3 months of therapy. Efficacy of medical therapy was evaluated at the last patient visit, and patients were 4

5 Page 5 of 24 considered uncontrolled if they had a basal GH value higher than 1.0 ng/ml and/or a plasma IGF-I level higher than age-matched normal subjects with at least 6 months of treatment with OCT-LAR at a dosage of 30 mg. Postsurgical follow-up ranged from 12 to 60 months (median 32 months). Tumor volume was not considered an endpoint in this series because the study included only post-surgical patients, which could lead to mistakes in the volume measurements due to confounding variables such as postsurgical changes. Methods Hormonal assessment Plasma GH levels were measured by a chemiluminescence assay kit (IMMULITE; Diagnostic Products Corp. Inc., Los Angeles, CA). The inter- and intra-assay coefficients of variation were 6.0 and 5.8%, respectively. The International Reference Preparation of GH was 98/574. Plasma IGF-I levels were measured by an immunoradiometric assay kit (DSL, Webster, TX, USA). The inter- and intra-assay coefficients of variation were 2.6 and 4.5%, respectively. The International Reference Preparation of IGF-I was 80/185. Immunohistochemistry The SSTR2 expression and Ki-67 labeling index (LI) were analyzed using immunohistochemistry on paraffin-embedded tissue sections as previously described (17, 24, 25). A mouse monoclonal antibody directed against the Ki-67 antigen (1:100, Dako, MIB-1 clone, cat. number M-7240) and a rabbit polyclonal antibody against SSTR2 (1:500, Gramsch Laboratories, Schwabhausen, Germany, cat. number SS-800) were used. The cytokeratin expression pattern was analyzed as previously published (26) with a mouse monoclonal antibody CAM5.2 (1:100, BD Biosciences, San Jose, CA, USA, cat. number ). 5

6 Page 6 of 24 For Ki-67, human breast carcinoma was used as a positive control. For CAM 5.2, normal kidney glomerular tissue was used as positive control, and normal human pituitary tissue was used as a positive control for SSTR2. Negative controls included omission of the primary antibody (for all) and pre-absorption of the anti-sstr2 antibody with its immunizing receptor peptide. Histomorphometry was performed using Image-Pro Plus (Media Cybernetics, Silver Spring, MD, USA) coupled to a digital camera (Evolution, Media Cybernetics) and a light microscope (Eclipse 400, NIKON, Japan). For Ki-67, high-quality images (2048 x 1536 pixels) of immunostained tumor cells from 10 representative areas with at least 1000 cells were captured using a 40x objective lens. Both immunolabeled and unlabeled nuclei were evaluated, and the percentage of positive cells (labeling index - LI) was calculated. A cut-off point of 2.3% was designated for Ki-67 LI as previously published by our group (24). The statistical analyses were also performed with a 3.0% cut-off, as suggested by the World Health Organization classification of pituitary adenomas (27). For SSTR2, high-quality images of immunostained tumor cells were randomly captured using a 40x objective lens (10 microscopical fields). Both membrane-bound and intra-cytoplasmic staining were considered. Tumors were staged according to the percentage of stained cells within the following score categories: 0 (<25% stained cells - low expression) or 1 ( 25% - high expression). This score was validated because it correlates well with real time RT-PCR expression data for SSTR2 as previously published by our group (17). Tumors were classified according to the cytokeratin expression as densely granulated, sparsely granulated or mixed forms according to previously published classification (28). Mixed tumors were considered as densely granulated for analysis, as previously suggested (28). Statistical analysis The statistical analyses were performed using SPSS version 16.0 for Windows (SPSS, Inc., Chicago, IL, USA). The results are reported as median values (minimum-maximum). The Mann- Whitney non-parametric test was used to compare numeric variables between the groups. Fisher s 6

7 Page 7 of 24 exact test or the chi-square test was used to compare frequencies between the groups according to the sample size. For multivariate analysis, a binary logistic regression was performed. A p-value less than 0.05 was considered significant. Results Patient/sample characteristics Thirty-one consecutive post-surgical acromegalic patients (19 women) were included in the study. The median age at diagnosis was 42 years (23 60). The median baseline GH level was 10.3 ng/ml ( ) and the median baseline IGF-I level was 621 ng/ml ( ). Twenty-eight out of 31 tumors were macroadenomas (90.3%), and 19 (61.2%) were invasive. Median tumor volume at diagnosis was 4.7 cm³ ( cm³). Fourteen (45.2%) tumors exhibited co-expression of GH and prolactin; the others were pure somatotropinomas. Detailed clinical, laboratorial and radiological information about each patient is provided in Supplementary Table 1. Ki-67 LI, SSTR2 expression and cytokeratin pattern in the somatotropinomas Twenty-five somatotropinomas (80.6%) expressed Ki-67, of which six (19.5%) demonstrated an LI higher than 3%, and eight presented an LI higher than 2.3% (25.8%). Examples of Ki-67 nuclear immunostaining are shown in Figure 1. There was no difference in age, gender, hormonal staining (mixed tumor or pure GH), baseline GH or IGF-I levels between patients harboring somatotropinomas with low or high Ki-67 LI (Table 1). Membrane and cytoplasmatic staining for SSTR2 was observed (Figure 1). All the somatotropinomas expressed SSTR2, with high levels of expression observed in 26 tumors (83.8%). There were no differences in age, gender, hormonal staining (mixed tumor or pure GH), baseline GH or IGF-I levels between patients harboring somatotropinomas with low or high SSTR2A 7

8 Page 8 of 24 expression levels (Table 2). There was no difference in the median Ki-67 LI values between patients with high and low SSTR2 expression levels (0.81, range and 1.25, range , respectively, p=0.651) (Figure 3A). The cytokeratin pattern was analyzed in 24 tumors. Thirteen tumors (54.2%) were classified as sparsely granulated, and 11 tumors (45.8%) were classified as densely granulated (five densely granulated and six mixed forms). Examples of the observed cytokeratin staining patterns are shown in Figure 2. There were no differences in age, gender, baseline GH or IGF-I levels or SSTR2 expression levels between patients harboring sparsely and densely granulated somatotropinomas. The Ki-67 LI was higher in sparsely than in densely granulated tumors [1.63 ( ) and 0.9 ( ), respectively, p=0.047] (Figure 3B). Detailed information about individual Ki-67 LI, SSTR2A scores and cytokeratin patterns are provided in Supplementary Table 1. Correlation of Ki-67 LI, SSTR2 expression and cytokeratin pattern with tumor characteristics All tumors with a Ki-67 LI higher than 2.3% were considered invasive, while only 47.8% of the tumors with a Ki-67 LI lower than 2.3% were classified as invasive (p=0.012). The 3.0% cut-off for Ki-67 LI was also related to tumor invasiveness (p=0.037). The somatotropinomas demonstrating a high Ki-67 LI also had a greater tumor volume than those presenting with a low Ki-67 LI [9.55 cm³ ( cm³) vs 1.9 cm³ ( cm³), respectively, p=0.01] (Figure 3C). There was no correlation of SSTR2 expression or cytokeratin pattern with tumor volume or invasiveness at diagnosis. Response to octreotide LAR 8

9 Page 9 of 24 Acromegaly was controlled with OCT-LAR in fourteen (45.2%) patients. There were no patients with discordant GH and IGF-I in our series. There was no difference between age, gender, hormonal staining (mixed tumor or pure GH) and baseline GH or IGF-I levels between patients with controlled and non-controlled disease status. Acromegaly was not controlled by OCT-LAR therapy in any of the patients (n=8) with Ki- 67 LI values higher than 2.3%. Of the 23 patients with Ki-67 LI values lower than 2.3%, the disease was controlled by OCT-LAR therapy in 14 (60.9%) cases (p=0.003). The median Ki-67 LI value was higher in patients whose acromegaly was uncontrolled with OCT-LAR therapy (1.63, range ) compared with cases that were controlled with OCT-LAR therapy (0.15, range , p=0.002) (Figure 3D). Acromegaly was not controlled with OCT-LAR therapy in any of the patients (n=5) with SSTR2 expression levels lower than 25%. Of the 26 patients with SSTR2 expression levels higher than 25%, acromegaly was controlled with OCT-LAR in 14 (53.8%) cases (p=0.04). In addition, we performed a multivariate analysis through a binary logistic regression including both the SSTR2 and the Ki-67 LI as independent variables with the acromegaly disease control with OCT-LAR therapy being the dependent variable. In this multivariate analysis, both Ki-67 and SSTR2 remained statistically significant as predictors of disease control with OCT-LAR (p=0.017 and 0.012, respectively). There was a positive correlation between densely granulated tumors and response to SSA therapy. Disease control was achieved in 72.7% (eight out of 11) of the patients harboring densely granulated tumors, while only 23.1% (three out of 13) of the patients harboring sparsely granulated tumors were controlled with SSA treatment (p=0.038). There was a lower chance of disease control with OCT-LAR therapy in those patients harboring invasive tumors at diagnosis. Ten out of 12 (83.3%) of those patients whose tumors were 9

10 Page 10 of 24 not invasive were controlled with OCT-LAR treatment, while only four out of 19 (21.1%) patients harboring invasive tumors were controlled with medical treatment (p=0.001). Tumor volume was also greater in tumors from patients resistant to OCT-LAR treatment than in those whose disease was controlled by the treatment [7.6 cm³ ( cm³) vs 1.35 cm³ ( cm³); p=0.008]. Discussion Somatostatin analogues are currently the mainstay for the treatment of acromegaly (2). Although some studies, including a meta-analysis, indicate control rates of approximately 60% with SSA therapy (29-31), prospective clinical trials showed lower control rates with this class of drugs (approximately 30-40%) (4, 5, 32). As there is a considerable percentage of patients whose acromegaly will not be controlled by SSA, predictors of the response to this treatment are useful in clinical practice. Many possible predictors have been studied (8, 9). We did not find any difference in the baseline GH levels between patients whose disease was controlled or not controlled with OCT-LAR therapy. Although some studies report that GH levels are predictors of meeting biochemical criteria for control with SSA therapy, in a metaanalysis, this was found to be valid only for primary therapy (29). In this same study, there was no relationship between baseline GH levels and response to OCT-LAR in the secondary therapy group, as in our present study. Somatostatin analogues act by binding to SSTR. There are five known SSTR types, and all but type 4 are present in the normal pituitary and in somatotropinomas (14). The currently available SSA (lanreotide and octreotide) bind preferentially to SSTR2. Therefore, as expected, the expression of SSTR2 is one of the few predictors of patient response to SSA therapy (8, 9). Indeed, some studies have found a positive correlation between SSTR2 mrna expression and GH and IGF- I suppression during SSA therapy (13, 15). SSTR2 protein expression has also been found to be positively correlated with the clinical response to SSA (16, 18). SSTR2 expression has a high 10

11 Page 11 of 24 negative predictive value, as patients with low SSTR2 expression do not respond to SSA therapy; however, the presence of high SSTR2 expression levels is not always associated with a good response to treatment, as post-signaling regulation could be involved (10, 18). In agreement with the literature, we found that no patient with low SSTR2 expression was controlled with OCT-LAR therapy, but some patients with high SSTR2 expression were also not controlled. As mentioned above, SSTR expression has been evaluated in several studies, mainly based on detection of mrna expression (13, 14). SSTR expression was also evaluated by IHC (17, 18), which, although semi-quantitative, can be routinely used in the evaluation of pituitary adenomas because it is a widely available technique. To validate the results of IHC, we previously compared the SSTR2 mrna and protein expression levels and found a positive correlation between them (17). Ki-67 is a marker of aggressiveness in pituitary tumors. It is related to invasiveness, a low chance of surgical cure and higher rates of recurrence (19). In the current study, we also found a higher Ki-67 LI in the invasive tumors. Additionally, the tumor volume at diagnosis was higher in those somatotropinomas with a higher Ki-67 LI. Fusco et al. (20) evaluated the response to OCT-LAR treatment in 40 acromegalic patients with different Ki-67 LI values. They observed for the first time that patients with higher Ki-67 LI values were less likely to respond to SSA treatment. However, SSTR2 expression was not evaluated and the response to OCT-LAR is mainly influenced by SSTR2 expression, as emphasized by the authors. Therefore, the relationship between the low expression of Ki-67 and control of the disease through SSA treatment could have been related to a different profile of SSTR expression in the tumors with low Ki-67 LI values. Previously, no study has evaluated whether Ki-67 and SSTR2 are independent predictors of acromegaly control with SSA. There is only one study that analyzed the acute effect of 11

12 Page 12 of 24 subcutaneous octreotide in acromegalic patients (33). Through univariate analysis, the authors found that SSTR2 expression and Ki-67 were related to the acute effect of octreotide in lowering GH levels. However, after a multivariate analysis, only SSTR2 expression remained a predictor of drug response. But, this study considered only the acute octreotide test, and no study has considered the effects of long-term treatment with SSA. In the present study, we confirmed that the Ki-67 LI is a predictor of acromegaly control with SSA therapy. We have also found that both SSTR2 and Ki-67 are independent predictors of disease control with SSA therapy, as we showed that there was no difference in Ki-67 LI between patients with low or high SSTR2 expression using univariate analysis. In addition, both markers remained statistically significant after multivariate analysis. Therefore, our study is the first to report that Ki-67 and SSTR2 are independent predictors of disease control with OCT-LAR treatment in acromegaly. Previous studies have demonstrated that the cytokeratin pattern of the somatotropinomas is associated with clinical and therapeutic characteristics of these tumors (7, 28). The sparsely granulated somatotropinomas are associated with a poor response to treatment with SSA (7). In this study, we found the same association. We also observed that the sparsely granulated tumors presented a higher Ki-67 LI than the densely granulated tumors, which is expected due to the known aggressiveness of these tumors. As previously mentioned, Ki-67 is a marker of cellular proliferation. Thus, it is not directly involved in the mechanism of resistance to OCT-LAR in acromegalic patients. In this study, we demonstrated that the amount of SSTR2 is not responsible for the lower control rates that are observed in patients harboring tumors with higher Ki-67 LI values. One of the possible explanations is the higher frequency of sparsely granulated adenomas between those somatotropinomas with a high Ki-67 LI, although the mechanism involved in the resistance to SSA therapy in sparsely granulated adenomas has also not been fully described yet. Probably the presence of higher Ki-67 12

13 Page 13 of 24 LI values and of the dot-like cytokeratin pattern (sparsely granulated adenomas) represent a phenotype of tumors that have alterations in the post-receptor signaling pathways involved in the response to SSA therapy. Interestingly, a low AIP expression has been observed in tumors with the same phenotype (more invasive and resistant to SSA therapy) (24, 34). Therefore, it is possible that tumors with a high Ki-67 LI present a low expression of AIP or other proteins involved in the postreceptor signaling cascade, and this could be the mechanism behind the worse response to therapy in this group of tumors, as for example, AIP has already been proved to be important in the mechanism of action of SSA (8, 35). Further studies are necessary to explain the inferior response to therapy in this subset of more aggressive tumors. In conclusion, this study indicates that Ki-67 and SSTR2 are independent predictors of the response to OCT-LAR in acromegalic patients and that the use of both markers can identify the patients who are more likely to respond to medical therapy with OCT-LAR. Additionally, Ki-67 correlates well with the cytokeratin pattern, and both can predict the therapeutic response to OCT- LAR. Disclosure statement: MRG has received grant support from Novartis Biociências S.A and Pfizer and a speaker s fee from Novartis Biociências S.A, Ipsen and Pfizer. Grants: This work is supported by grants from Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) (L.K., M.R.G.). 13

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18 Page 18 of Obari A, Sano T, Ohyama K, Kudo E, Qian ZR, Yoneda A, Rayhan N, Mustafizur Rahman M & Yamada S. Clinicopathological features of growth hormone-producing pituitary adenomas: difference among various types defined by cytokeratin distribution pattern including a transitional form. Endocr Pathol Freda PU, Katznelson L, van der Lely AJ, Reyes CM, Zhao S & Rabinowitz D. Longacting somatostatin analog therapy of acromegaly: a meta-analysis. J Clin Endocrinol Metab Murray RD & Melmed S. A critical analysis of clinically available somatostatin analog formulations for therapy of acromegaly. J Clin Endocrinol Metab Cozzi R, Attanasio R, Montini M, Pagani G, Lasio G, Lodrini S, Barausse M, Albizzi M, Dallabonzana D & Pedroncelli AM. Four-year treatment with octreotide-long-acting repeatable in 110 acromegalic patients: predictive value of short-term results? J Clin Endocrinol Metab Jallad RS, Musolino NR, Salgado LR & Bronstein MD. Treatment of acromegaly with octreotide-lar: extensive experience in a Brazilian institution. Clin Endocrinol (Oxf) Nakashima M, Takano K & Matsuno A. Analyses of factors influencing the acute effect of octreotide in growth hormone-secreting adenomas. Endocr J Kasuki L, Vieira Neto L, Wildemberg LE, Colli LM, de Castro M, Takiya CM & Gadelha MR. AIP expression in sporadic somatotropinomas is a predictor of the response to octreotide LAR therapy independent of SSTR2 expression. Endocr Relat Cancer L Chahal HS, Trivellin G, Leontiou CA, Alband N, Fowkes RC, Tahir A, Igreja SC, Chapple JP, Jordan S, Lupp A, Schulz S, Ansorge O, Karavitaki N, Carlsen E, Wass JA, Grossman AB & Korbonits M. Somatostatin analogs modulate AIP in somatotroph adenomas: the role of the ZAC1 pathway. J Clin Endocrinol Metab E

19 Page 19 of 24 Figure legends Figure 1 Examples of somatostatin receptor subtype 2 (SSTR2) expression and Ki-67 labeling index (LI): A Tumor with low SSTR2 expression (<25%); B Tumor with high SSTR2 expression ( 25%); C Tumor with low Ki-67 LI (<2.3%); D Tumor with high Ki-67 LI ( 2.3%). Scale bar = 500 µm. Figure 2 Examples of cytokeratin pattern expression: A Densely granulated tumor; B Sparsely granulated tumor. Scale bar = 500 µm. Figure 3 Boxplot showing the Ki-67 labeling index in: A - tumors with high or low somatostatin receptor type 2 (SSTR2); B sparsely or densely granulated tumors; D tumors from patients controlled or not with octreotide LAR therapy. Figure 3C shows the tumoral volume in patients with high ( 2.3%) or low (<2.3%) Ki-67 labeling index. * = p<

20 Examples of somatostatin receptor subtype 2 (SSTR2) expression and Ki-67 labeling index (LI): A Tumor with low SSTR2 expression (<25%); B Tumor with high SSTR2 expression ( 25%); C Tumor with low Ki-67 LI (<2.3%); D Tumor with high Ki-67 LI ( 2.3%). Scale bar = 500 µm. 254x190mm (72 x 72 DPI) Page 20 of 24

21 Page 21 of 24 Examples of cytokeratin pattern expression: A Densely granulated tumor; B Sparsely granulated tumor. Scale bar = 500 µm. 190x254mm (72 x 72 DPI)

22 Boxplot showing the Ki-67 labeling index in: A - tumors with high or low somatostatin receptor type 2 (SSTR2); B sparsely or densely granulated tumors; D tumors from patients controlled or not with octreotide LAR therapy. Figure 3C shows the tumoral volume in patients with high ( 2.3%) or low (<2.3%) Ki-67 labeling index. * = p< x190mm (72 x 72 DPI) Page 22 of 24

23 Page 23 of 24 1 Table 1 Patient characteristics according to Ki-67 labeling index (LI) Ki-67 LI>2.3% Ki-67 LI<2.3% p-value Age 42 (23-60) 38.5 (24-48) Female 50.0% 65.2% Pure GH tumor 75.0% 47.8% Treatment duration (months) 29 (9-57) 25 (12-35) Baseline GH (ng/ml) 10.3 ( ) 10.8 ( ) Baseline IGF-I (ng/ml) 618 ( ) 778 ( ) Results are presented as median (min-max)

24 Page 24 of Table 2 Patient characteristics according to somatostatin receptor subtype 2 (SSTR2) expression High SSTR2 Low SSTR2 p-value Age 43 (23-60) 40 (24-47) Female 60.0% 61.5% Pure GH tumor 40.0% 57.6% Treatment duration (months) 29 (9-57) 25 (12-35) Baseline GH 10.6 ( ) 8.2 ( ) Baseline IGF-I 622 ( ) 621 ( ) Results are presented as median (min-max)