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Expression of somatostatin receptors, SSTR2A and SSTR5, in 108 endocrine pituitary tumors using immunohistochemical detection with new specific monoclonal antibodies
Human Pathology (2014) 45, 71–77
www.elsevier.com/locate/humpath
Original contribution
Expression of somatostatin receptors, SSTR2A and SSTR5, in 108 endocrine pituitary tumors using immunohistochemical detection with new specific monoclonal antibodies☆,☆☆ Laura Chinezu MD a , Alexandre Vasiljevic MD b,c,d , Emmanuel Jouanneau MD, PhD b,c,e , Patrick François MD, PhD f , Angela Borda MD, PhD a , Jacqueline Trouillas MD, PhD b,c,d , Gerald Raverot MD, PhD b,c,g,⁎ a
Department of Histology, University of Medicine and Pharmacy, 540139 Tirgu Mures, Romania INSERM U1028, CNRS UMR5292, Lyon Neuroscience Research Center, Neuro-oncology and Neuro-inflammation Team, University Lyon 1, F-69000 Lyon, France c Université de Lyon, Université Lyon 1, F-69372 Lyon, France d Centre de Pathologie Est, Groupement Hospitalier Est, Hospices Civils de Lyon, 69003 Lyon, France e Service de Neurochirurgie, Groupement Hospitalier Est, Hospices Civils de Lyon, 69003 Lyon, France f Service de Neurochirurgie, Hôpital Bretonneau, Centre Hospitalier Régional Universitaire de Tours, 37044 Tours cedex 9, France g Fédération d'Endocrinologie, Groupement Hospitalier Est, Hospices Civils de Lyon, 69003 Lyon, France b
Received 23 June 2013; revised 2 August 2013; accepted 2 August 2013
Keywords: Pituitary tumors; Somatostatin receptors; Immunohistochemistry; Monoclonal antibodies; Somatostatin analogs
Summary Medical treatment of endocrine pituitary tumors with somatostatin analogs depends on tumor type and somatostatin receptor (SSTR) expression. Immunohistochemical detection of these receptors using polyclonal antibodies has given conflicting results. We studied the expression of SSTR2A and SSTR5 with new procedures in 108 pituitary tumors. Using 2 new, specific monoclonal antibodies (clone UMB-1 and UMB-4), 2 fixatives (Bouin-Hollande and zinc-formalin) and 2 technical procedures (manual and automated), SSTR2A and SSTR5 expression was studied in 60 GH (growth hormone), 15 ACTH (adrenocorticotropic hormone), 23 FSH/LH (follicle-stimulating hormone/luteinizing hormone), 7 PRL (prolactin), and 3 TSH (thyroid-stimulating hormone) tumors. Only membrane staining was taken into account, and the SSTR expression was considered positive when more than 5% of the cells were immunoreactive. GH tumors were classified as GH or GH/PRL, densely or sparsely granulated, and into 3 groups according to the percentage of SSTR-immunoreactive cells (group 1: b25%; group 2: 25%-75%; group 3: N75%). Almost all GH tumors expressed SSTR2A (93%) and SSTR5 (83%) at high levels (group 3: N75%) in 52% and 37%, respectively. SSTR2A expression was significantly higher in densely than in sparsely granulated tumors. Moreover, SSTR2A was also expressed in the 3 TSH tumors
☆
Conflict of interest: The authors declare having no conflict of interest. Funding disclosures: This work was partly supported by the Sectorial Operational Programme Human Resources Development, financed by the European Social Fund and by the Romanian Government under the contract number POSDRU 80641. ⁎ Corresponding author. Fédération d'Endocrinologie du Pôle Est, Groupement Hospitalier Est-Aile A1, 59 Bd Pinel, 69003, Lyon cedex, France. E-mail address: [email protected] (G. Raverot). ☆☆
0046-8177/$ – see front matter © 2014 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.humpath.2013.08.007
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L. Chinezu et al. and weakly expressed in 26% of the FSH/LH tumors, although not in ACTH or PRL tumors. SSTR5 expression was noted in 2 of the 3 TSH tumors, in only 20% of ACTH tumors, and was absent from FSH/LH and PRL tumors. The immunohistochemical detection of SSTR is a reproducible and specific method that could help direct the choice of postoperative medical treatment. © 2014 Elsevier Inc. All rights reserved.
1. Introduction Normal pituitary and pituitary tumors express different types of somatostatin receptor (SSTR). Five SSTR subtypes have been described (SSTR1-5) and are encoded by genes localized on different chromosomes. Two forms of the SSTR2 receptor (SSTR2A and SSTR2B) are generated via alternative splicing [1-4]. The most frequent SSTRs detected in human pituitary tumors are SSTR2A and SSTR5 [4]. In acromegaly, the currently available medical treatment with somatostatin analogs (SAs) is considered the cornerstone [5]. The response to SA (octreotide and lanreotide) seems to be predicted by the expression of SSTR, predominantly SSTR2A [6-8]. In a third of the patients not responding to octreotide, the expression of SSTR5 is higher than that of SSTR2A [9]. To overcome the resistance to octreotide or lanreotide, new SAs have been developed, including pasireotide (SOM230), which binds with high affinity to both SSTR2 and SSTR5. This multireceptortargeted SA is currently under preclinical and clinical evaluation for use in the treatment of acromegaly, Cushing disease, and carcinoid tumors [10-15]. However, although significant expression of SSTR2A or SSTR5 has been described in gonadotroph tumors [16,17], so far the treatment of these tumors with SA has been disappointing. The correlation between SSTR expression and clinical results demonstrated by some authors [18] and the development of multireceptor-targeted SA suggest that the study of SSTR expression may guide the therapeutic management of these pituitary tumors. This underlines the need for a reproducible and reliable technique for SSTR detection in routine pathological practice. Studies on most cases to date have used polyclonal antibodies that were even then not routinely applied because of technical difficulties. Recently, Fischer et al [19] and Lupp et al [20] described 2 highly specific monoclonal SSTR antibodies for SSTR2A and SSTR5. Here we aimed to use these 2 antibodies to study the expression of SSTR2A and SSTR5 on a large series of all types of pituitary tumors by manual and automated immunohistochemistry (IHC).
2. Materials and methods 2.1. Tumor samples One hundred eight pituitary tumors obtained from the Centre de Pathologie Est, Hospices Civils de Lyon, were
studied by IHC with 2 antibodies (SSTR2A and SSTR5) and 2 technical procedures (manual and automated IHC). Two fixatives were also tested: the Bouin-Hollande solution (n = 54) and the zinc-formalin solution (n = 54). The formulation of the fixatives is as follows: Bouin-Hollande solution is composed of copper acetate (25 g), picric acid (40 g), and distilled water (1 L), with the addition of 37% formaldehyde (0.1 L) just before use; zinc-formalin solution is composed of 35% formaldehyde (0.5 L), sodium chloride (90 g), zinc sulfate (30 g), and distilled water (9.5 L). The mean fixation time was 24 hours for Bouin-Hollande solution and 30 hours for zinc-formalin solution. All cases were embedded in paraffin, and the slides were stained with hematoxylin phloxine saffron (HPS) and Herlant tetrachrome. According to the recent classifications [21,22] and based on clinical and biochemical data as well as immunohistochemical reaction with antibodies against hormones (antiGH, PRL, ACTH, βFSH, βLH and βTSH), the series comprised 60 GH (pure GH, n = 39; GH/PRL, n = 21), 15 ACTH, 23 FSH/LH, 7 PRL, and 3 TSH tumors. The GH tumors were classified into densely granulated (DG; n = 32) or sparsely granulated (SG; n = 28) tumors based on HPS and Herlant tetrachrome aspect and on IHC reaction with cytokeratin, according to the criteria described by Obari and colleagues [23], for example, DG tumor when perinuclear pattern in more than 70% of cells or dot pattern in less than 8% and SG tumor when dot pattern in more than 70% of cells. The tumors with intermediate percentages were included in the DG group [23]. Eight cases, negative for cytokeratin and with no granular cells stained with HPS or Herlant tetrachrome, were classified as SG.
2.2. IHC with anti-SSTR2A and anti-SSTR5 antibodies For all 43 GH tumors fixed in Bouin-Hollande fixative, an initial manual immunostaining was performed. Nearly half of these tumors (n = 21), randomly chosen, were also tested by automated IHC to compare the technical procedures. All other tumors (n = 65) were tested using the automated procedure only. Two rabbit monoclonal antibodies were used (SSTR2A, clone UMB-1 reference 3582-1, and SSTR5, clone UMB-4 reference 3619-1; CliniSciences, Nanterre, France). Several dilutions (from 1:100 to 1:8000) were tested on normal pituitary obtained from autopsy and on tumor tissue. The optimal dilution was chosen when the membrane staining was strong and the cytoplasmic background was minimal. These optimal dilutions, which applied for all the cases and for both technical procedures, were 1:4000 for anti-SSTR2A and 1:1000 for anti-SSTR5 antibodies.
Expression of somatostatin receptors in pituitary tumors Regarding the manual IHC technique, tissue sections were dewaxed in toluene and dehydrated through a graded series of ethanol. Antigen retrieval was performed by microwave boiling in 10 mM citrate buffer at pH 6.0. After cooling the sections, an endogenous peroxidase quenching step was performed with hydrogen peroxide 3% for 5 minutes. The slides were then incubated with the primary antibodies for 1 hour at room temperature. To reveal the presence of antigens in tumor tissue, we used the labeledstreptavidin-biotin (LAB-SA) system, according to the manufacturer's instructions (Histostain-Plus Bulk kit; Invitrogen, Camarillo, CA). The final reaction product was visualized with 3,3′-diaminobenzidine (DAB Kit; Invitrogen) as a brown deposit. The nuclei were counterstained with Mayer hematoxylin. Adjacent normal pituitary tissue was used as a positive control and substitution of the primary antibody with antibody diluent as a negative control. Both controls gave satisfactory results.
73 Regarding the automated IHC technique, the Benchmark XT (Ventana Medical Systems, Tucson, AZ) was used. The differences when compared with the manual technique were as follows: (1) the dewaxing step was thermal at 72°C; (2) antigen retrieval was performed with EDTA pH 8.0 at 95 °C; (3) the incubation with primary antibodies was performed at 37°C for 32 minutes; and (4) the presence of antigens was revealed with the UltraView Universal DAB Detection Kit (Ventana), which is a biotin-free system. To evaluate the expression of SSTR, only membrane immunopositivity was considered and the final evaluation was performed mainly in the automated stained slides. The tumors were semiquantitatively scored according to the scoring system reported by Fougner and colleagues [24]. The GH tumors were classified into 3 groups, taking into account the % of immunoreactive (IR) cells but not the intensity of the staining: group 1, less than 25% of IR cells; group 2, 25% to 75% of IR cells; and group 3, more than 75% of IR cells.
Fig. 1 IHC detection of SSTR2A (A, C and E) and SSTR5 (B, D, and F) in GH tumors using a manual procedures for tumors fixed in BouinHollande fixative (A and B, original magnification ×40) and an automated procedure for tumors fixed in Bouin-Hollande fixative (C and D, original magnification ×40) or zinc-formalin fixative (E and F, original magnification ×40).
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The other types of tumors were classified as positive or negative based on the presence of more or less than 5% of IR cells, respectively. Two independent pathologists evaluated the results separately and scored all the pituitary tumors identically.
2.3. Statistical analysis Statistical analyses were performed using StatView 5.0 software (SAS Institute Inc., Irvine, CA, USA). Data were expressed as means ± SEM. Differences between groups were studied using the Mann-Whitney test, and differences in the proportion were studied using the Fisher exact test. P b .05 was considered significant.
Table 2
Expression of SSTR2A and SSTR5 in 60 GH tumors
Tumors
SSTR2A (%), mean ± SD
P
SSTR5 (%), mean ± SD
P
GH (n = 39) GH/PRL (n = 21) DG (n = 32) SG (n = 28)
63.8 ± 32.9 67.4 ± 32.8 78.3 ± 26.5 50.0 ± 32.7
NS
53.6 ± 36.5 50.5 ± 33.8 53.1 ± 35.1 51.8 ± 36.2
NS
.005 ⁎
NS
Abbreviation: NS, not significant. ⁎ Significant difference.
was also statistically different among the 3 groups of SSTR2A immunoreactivity (Fisher exact test, P = .027), but not among the 3 groups of SSTR5 immunoreactivity (Fig. 2). There was no significant difference between pure GH and GH/PRL tumors for either SSTR studied (Table 2).
3. Results
3.2. SSTR expression in other tumors
We compared the percentage of IR cells and staining intensity obtained using the 2 technical procedures of IHC (manual and automated) and the 2 fixatives (Fig. 1). There were no differences among the techniques (procedure and fixative) in terms of the percentage of cells IR for anti-SSTR2A antibody. However, the intensity was lower with the manual procedure than with the automated one, and the percentage of IR cells was significantly higher with the automated than with the manual procedure (manual: 48.1 ± 37.8; automated: 59.2 ± 37.1; P = .01) for anti-SSTR5 antibody. Cytoplasmic staining was considered as background and mainly observed with the manual procedure. It rendered the evaluation of specific membrane staining difficult (Fig. 1A and B).
The 3 TSH tumors highly expressed SSTR2A (N75% of the cells), and 2 of them weakly expressed SSTR5 (b25% of the cells; Table 1). SSTR2A 52%
38% 33%
Total DG 22%
SG
15% 12%
13%
12%
3%
3.1. SSTR expression in 60 GH tumors Group 1
Almost all GH tumors expressed SSTR2A and SSTR5 (93% and 83%, respectively; Table 1) at a higher level (N75%, group 3) for SSTR2A than for SSTR5 (52% and 37%, respectively). Most DG tumors (72%) had more than 75% of cells that were IR for SSTR2A, and SSTR2A expression was significantly higher (P = .0005) in DG compared with SG tumors (Table 2). Moreover, the distribution of DG and SG tumors
Group 2
SSTR5 37%
37%
27% 22% 18%
Table 1 Expression of SSTR2A and SSTR5 in the 5 types of pituitary tumors Tumor types
SSTR2A
Total
18%
DG 15%
13%
13%
SG
SSTR5
Positive (%)
Negative (%)
Positive (%)
Negative (%)
93 26 0 0 100
7 74 100 100 0
83 0 20 0 67
17 100 80 100 33
Group 1
GH (n = 60) FSH/LH (n = 23) ACTH (n = 15) PRL (n = 7) TSH (n = 3)
Group 3
Group 2
Group 3
Fig. 2 Expression of SSTR2A and SSTR5 in DG- and SG-type pituitary tumors for each group. The distribution of DG and SG among the different SSTR immunoreactivity subgroups was significantly different for SSTR2A (P b .03, Fisher exact test), but not for SSTR5.
Expression of somatostatin receptors in pituitary tumors The expression of SSTR2A and SSTR5 was found to be rare in the other tumors, and the percentage of positive cells was very low. All the FSH/LH tumors were negative for SSTR5 (Fig. 3B), and only 26% (6/23) were positive for SSTR2A (Fig. 3A). Three of the 6 positive for SSTR2A were pure FSH tumors with 40% of IR cells, and the other 3 were FSH/LH tumors with a low expression (b15% of IR cells). None of the ACTH tumors showed membrane staining for anti-SSTR2A, but 5 displayed cytoplasmic artifactual staining (Fig. 3C). Twenty percent (3/15) were positive for SSTR5 (Fig. 3D) with a low percentage of IR cells (b30%). All the PRL tumors were negative for both SSTRs (Table 1).
4. Discussion Here we have studied the expression of SSTR2A and SSTR5 in 108 pituitary tumors of each type, by IHC with 2 new monoclonal antibodies. Very few studies devoted to IHC detection of SSTR in pituitary tumors have been published, mainly because of technical difficulties (Table 3). We compared results obtained by 2 procedures and 2 fixatives with regard to the expression of SSTR in GH tumors. The high optimal dilutions, higher than those recommend by Epitomics (1:4000 for SSTR2A and 1:1000 for SSTR5, instead of 1:100-250), underline the quality of these antibodies. The 2 fixatives used (Bouin-Hollande and zinc-formalin) ensured high protein conservation and membrane integrity resulting in a clear staining for both antibodies localized to the membrane. A cytoplasmic background was mainly seen using the manual
75 procedure. Formalin fixation has also given good results with the same antibodies [19,20]. Cytoplasmic staining has been attributed by some authors to the internalization mechanism, with an important role in tachyphylaxis and radiotherapeutic application of radiolabeled octapeptide SA [25]. However, considering that internalized receptors are known to be inactive [26] and only membrane localization reflects the functional status of the SSTR in the tumors and may be related to the efficacy of SA [27] and that cytoplasmic staining is related to technical procedure, we only took membrane staining into account. Our approach thus differed to that used previously using polyclonal antibodies and a manual procedure [17,27]. The IHC evaluation of the percentage of positive cells with these new highly specific antibodies was reproducible and allowed the classification of GH pituitary tumors into 3 groups according to the levels of expression of SSTR. Unlike previous studies [18-20,27], we did not consider the staining intensity in the present evaluation because it is highly subjective and could be related to technical procedure. Under our experimental conditions, SSTR2A expression was significantly higher in DG than in SG tumors, and the distribution of DG and SG tumors among the 3 groups was significantly different regarding SSTR2A but not SSTR5 immunoreactivity. Although our criteria were not the same, our results are comparable with previous studies. Thus, by comparison with DG tumors, the SG ones would appear to express lower levels of SSTR, be more often invasive macroadenomas and less responsive to SA treatment, and have a higher rate of clinical recurrence [23,28,29]. Among our large series of GH tumors, the expression of SSTR2A and SSTR5 was very high (93% and 83%,
Fig. 3 IHC detection of SSTR2A (A and C) and SSTR5 (right panels) in FSH/LH tumors (A and B, original magnification ×40) and ACTH tumors (C, original magnification ×20; D, original magnification ×40).
76 Table 3
L. Chinezu et al. Reports regarding the IHC expression of SSTR in different types of pituitary tumors
Study (no. of tumors)
Pawlikowski et al [17] (n = 18) Fougner et al [24] (n = 68) Fusco et al [30] (n = 26) Wildemberg et al [27] (n = 13) Ramírez et al [16] (n = 74) Lupp et al [20] (n = 25; 5) Our series (n = 60; 23; 15)
Antibody
Gramsch (Schwabhausen, Germany) Gramsch Gramsch Gramsch Abcam (Cambridge, MA, USA) UMB-1, UMB-4 (Epitomics, Burlingame, CA, USA) UMB-1, UMB-4 (CliniSciences, Nanterre, France)
Clonality
SSTR2A % of positive tumors
SSTR5 % of positive tumors
GH
FSH/LH
ACTH
GH
FSH/LH
ACTH
Polyclonal
ND
83
ND
ND
100
ND
Polyclonal Polyclonal Polyclonal Monoclonal Monoclonal
100 ND 92 ND 84
ND 46 ND 60 ND
ND ND ND ND 0
ND ND ND ND 100
ND 84 ND 45 ND
ND ND ND ND 80
Monoclonal
93
26
0
83
0
20
Abbreviation: ND, not determined.
respectively), in accordance with previous studies [7,18,20,26,27]. Currently available treatment of GH tumors with SA is effective in regulating hormonal hypersecretion and cellular proliferation. These effects of SA seem to be positively correlated with SSTR expression, especially SSTR2A and SSTR5 [5]. Significant levels of expression of SSTR2A and SSTR5 have been reported in nonfunctioning tumors represented in great majority by gonadotroph or FSH/LH and null cell tumors. With cytoplasmic immunostaining taken into account, the percentages obtained range from 46 to 83 for SSTR2A and 45 to 100 for SSTR5 [16,17,30]. However, with only membrane immunostaining considered [17], 17% of the tumors were positive for SSTR2A, as in our series. Expression of SSTR5 has been found in 39% of nonfunctioning tumors [17], suggesting that treatment with SA can stabilize the postoperative residual tumor [30]. However, using highly specific monoclonal antibodies, here we observed a weak expression of SSTR2A in 26% of nonfunctioning tumors and absent SSTR5 expression. This may explain the lack of significant tumor shrinkage observed with SA in nonfunctioning tumors [30]. Very few studies have been devoted to the IHC detection of SSTR in ACTH tumors. All ACTH tumors in these studies were negative for SSTR2A; however, the results are contradictory for SSTR5: indeed in one study, 4 of the 5 tumors tested were positive [20], and in another study, 3 of the 3 tested were negative [31]. In our series, only 3 of the 15 ACTH tumors expressed SSTR5, which is in accordance with recent clinical data on pasireotide (SOM230) treatment in Cushing disease, demonstrating a 20% response rate among the patients treated regardless of the SSTR5 status [32]. The correlation between SSTR5 expression and response to pasireotide now needs to be evaluated prospectively in a large cohort. Pasireotide could be a potential therapeutic option for patients with PRL tumors resistant to dopamine agonists because inhibition of PRL secretion seems to be related to SSTR5 expression [33]. However, in our study, all 7 PRL tumors were negative for both SSTRs, suggesting that
treatment of PRL tumors with pasireotide may be limited to some rare cases. Because medical treatment of pituitary tumors depends, to a certain extent, on the expression of SSTRs, a reproducible method able to detect the expression of distinct subtypes is required. Here in the present study, we have tested the IHC detection of SSTR2A and SSTR5 by 2 new monoclonal antibodies for all types of pituitary tumors using different procedures. The automated IHC procedure proved to be the most reliable and reproducible method with clear membrane staining and lack of cytoplasmic background. These monoclonal antibodies against SSTR2A and SSTR5 should now be considered for use in routine practice for pathological analysis of pituitary tumors and may prove useful in the analysis of all endocrine tumors (including, pancreas) with a view to adapting medical treatment to SSTR profile.
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Original contribution
Expression of somatostatin receptors, SSTR2A and SSTR5, in 108 endocrine pituitary tumors using immunohistochemical detection with new specific monoclonal antibodies☆,☆☆ Laura Chinezu MD a , Alexandre Vasiljevic MD b,c,d , Emmanuel Jouanneau MD, PhD b,c,e , Patrick François MD, PhD f , Angela Borda MD, PhD a , Jacqueline Trouillas MD, PhD b,c,d , Gerald Raverot MD, PhD b,c,g,⁎ a
Department of Histology, University of Medicine and Pharmacy, 540139 Tirgu Mures, Romania INSERM U1028, CNRS UMR5292, Lyon Neuroscience Research Center, Neuro-oncology and Neuro-inflammation Team, University Lyon 1, F-69000 Lyon, France c Université de Lyon, Université Lyon 1, F-69372 Lyon, France d Centre de Pathologie Est, Groupement Hospitalier Est, Hospices Civils de Lyon, 69003 Lyon, France e Service de Neurochirurgie, Groupement Hospitalier Est, Hospices Civils de Lyon, 69003 Lyon, France f Service de Neurochirurgie, Hôpital Bretonneau, Centre Hospitalier Régional Universitaire de Tours, 37044 Tours cedex 9, France g Fédération d'Endocrinologie, Groupement Hospitalier Est, Hospices Civils de Lyon, 69003 Lyon, France b
Received 23 June 2013; revised 2 August 2013; accepted 2 August 2013
Keywords: Pituitary tumors; Somatostatin receptors; Immunohistochemistry; Monoclonal antibodies; Somatostatin analogs
Summary Medical treatment of endocrine pituitary tumors with somatostatin analogs depends on tumor type and somatostatin receptor (SSTR) expression. Immunohistochemical detection of these receptors using polyclonal antibodies has given conflicting results. We studied the expression of SSTR2A and SSTR5 with new procedures in 108 pituitary tumors. Using 2 new, specific monoclonal antibodies (clone UMB-1 and UMB-4), 2 fixatives (Bouin-Hollande and zinc-formalin) and 2 technical procedures (manual and automated), SSTR2A and SSTR5 expression was studied in 60 GH (growth hormone), 15 ACTH (adrenocorticotropic hormone), 23 FSH/LH (follicle-stimulating hormone/luteinizing hormone), 7 PRL (prolactin), and 3 TSH (thyroid-stimulating hormone) tumors. Only membrane staining was taken into account, and the SSTR expression was considered positive when more than 5% of the cells were immunoreactive. GH tumors were classified as GH or GH/PRL, densely or sparsely granulated, and into 3 groups according to the percentage of SSTR-immunoreactive cells (group 1: b25%; group 2: 25%-75%; group 3: N75%). Almost all GH tumors expressed SSTR2A (93%) and SSTR5 (83%) at high levels (group 3: N75%) in 52% and 37%, respectively. SSTR2A expression was significantly higher in densely than in sparsely granulated tumors. Moreover, SSTR2A was also expressed in the 3 TSH tumors
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Conflict of interest: The authors declare having no conflict of interest. Funding disclosures: This work was partly supported by the Sectorial Operational Programme Human Resources Development, financed by the European Social Fund and by the Romanian Government under the contract number POSDRU 80641. ⁎ Corresponding author. Fédération d'Endocrinologie du Pôle Est, Groupement Hospitalier Est-Aile A1, 59 Bd Pinel, 69003, Lyon cedex, France. E-mail address: [email protected] (G. Raverot). ☆☆
0046-8177/$ – see front matter © 2014 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.humpath.2013.08.007
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L. Chinezu et al. and weakly expressed in 26% of the FSH/LH tumors, although not in ACTH or PRL tumors. SSTR5 expression was noted in 2 of the 3 TSH tumors, in only 20% of ACTH tumors, and was absent from FSH/LH and PRL tumors. The immunohistochemical detection of SSTR is a reproducible and specific method that could help direct the choice of postoperative medical treatment. © 2014 Elsevier Inc. All rights reserved.
1. Introduction Normal pituitary and pituitary tumors express different types of somatostatin receptor (SSTR). Five SSTR subtypes have been described (SSTR1-5) and are encoded by genes localized on different chromosomes. Two forms of the SSTR2 receptor (SSTR2A and SSTR2B) are generated via alternative splicing [1-4]. The most frequent SSTRs detected in human pituitary tumors are SSTR2A and SSTR5 [4]. In acromegaly, the currently available medical treatment with somatostatin analogs (SAs) is considered the cornerstone [5]. The response to SA (octreotide and lanreotide) seems to be predicted by the expression of SSTR, predominantly SSTR2A [6-8]. In a third of the patients not responding to octreotide, the expression of SSTR5 is higher than that of SSTR2A [9]. To overcome the resistance to octreotide or lanreotide, new SAs have been developed, including pasireotide (SOM230), which binds with high affinity to both SSTR2 and SSTR5. This multireceptortargeted SA is currently under preclinical and clinical evaluation for use in the treatment of acromegaly, Cushing disease, and carcinoid tumors [10-15]. However, although significant expression of SSTR2A or SSTR5 has been described in gonadotroph tumors [16,17], so far the treatment of these tumors with SA has been disappointing. The correlation between SSTR expression and clinical results demonstrated by some authors [18] and the development of multireceptor-targeted SA suggest that the study of SSTR expression may guide the therapeutic management of these pituitary tumors. This underlines the need for a reproducible and reliable technique for SSTR detection in routine pathological practice. Studies on most cases to date have used polyclonal antibodies that were even then not routinely applied because of technical difficulties. Recently, Fischer et al [19] and Lupp et al [20] described 2 highly specific monoclonal SSTR antibodies for SSTR2A and SSTR5. Here we aimed to use these 2 antibodies to study the expression of SSTR2A and SSTR5 on a large series of all types of pituitary tumors by manual and automated immunohistochemistry (IHC).
2. Materials and methods 2.1. Tumor samples One hundred eight pituitary tumors obtained from the Centre de Pathologie Est, Hospices Civils de Lyon, were
studied by IHC with 2 antibodies (SSTR2A and SSTR5) and 2 technical procedures (manual and automated IHC). Two fixatives were also tested: the Bouin-Hollande solution (n = 54) and the zinc-formalin solution (n = 54). The formulation of the fixatives is as follows: Bouin-Hollande solution is composed of copper acetate (25 g), picric acid (40 g), and distilled water (1 L), with the addition of 37% formaldehyde (0.1 L) just before use; zinc-formalin solution is composed of 35% formaldehyde (0.5 L), sodium chloride (90 g), zinc sulfate (30 g), and distilled water (9.5 L). The mean fixation time was 24 hours for Bouin-Hollande solution and 30 hours for zinc-formalin solution. All cases were embedded in paraffin, and the slides were stained with hematoxylin phloxine saffron (HPS) and Herlant tetrachrome. According to the recent classifications [21,22] and based on clinical and biochemical data as well as immunohistochemical reaction with antibodies against hormones (antiGH, PRL, ACTH, βFSH, βLH and βTSH), the series comprised 60 GH (pure GH, n = 39; GH/PRL, n = 21), 15 ACTH, 23 FSH/LH, 7 PRL, and 3 TSH tumors. The GH tumors were classified into densely granulated (DG; n = 32) or sparsely granulated (SG; n = 28) tumors based on HPS and Herlant tetrachrome aspect and on IHC reaction with cytokeratin, according to the criteria described by Obari and colleagues [23], for example, DG tumor when perinuclear pattern in more than 70% of cells or dot pattern in less than 8% and SG tumor when dot pattern in more than 70% of cells. The tumors with intermediate percentages were included in the DG group [23]. Eight cases, negative for cytokeratin and with no granular cells stained with HPS or Herlant tetrachrome, were classified as SG.
2.2. IHC with anti-SSTR2A and anti-SSTR5 antibodies For all 43 GH tumors fixed in Bouin-Hollande fixative, an initial manual immunostaining was performed. Nearly half of these tumors (n = 21), randomly chosen, were also tested by automated IHC to compare the technical procedures. All other tumors (n = 65) were tested using the automated procedure only. Two rabbit monoclonal antibodies were used (SSTR2A, clone UMB-1 reference 3582-1, and SSTR5, clone UMB-4 reference 3619-1; CliniSciences, Nanterre, France). Several dilutions (from 1:100 to 1:8000) were tested on normal pituitary obtained from autopsy and on tumor tissue. The optimal dilution was chosen when the membrane staining was strong and the cytoplasmic background was minimal. These optimal dilutions, which applied for all the cases and for both technical procedures, were 1:4000 for anti-SSTR2A and 1:1000 for anti-SSTR5 antibodies.
Expression of somatostatin receptors in pituitary tumors Regarding the manual IHC technique, tissue sections were dewaxed in toluene and dehydrated through a graded series of ethanol. Antigen retrieval was performed by microwave boiling in 10 mM citrate buffer at pH 6.0. After cooling the sections, an endogenous peroxidase quenching step was performed with hydrogen peroxide 3% for 5 minutes. The slides were then incubated with the primary antibodies for 1 hour at room temperature. To reveal the presence of antigens in tumor tissue, we used the labeledstreptavidin-biotin (LAB-SA) system, according to the manufacturer's instructions (Histostain-Plus Bulk kit; Invitrogen, Camarillo, CA). The final reaction product was visualized with 3,3′-diaminobenzidine (DAB Kit; Invitrogen) as a brown deposit. The nuclei were counterstained with Mayer hematoxylin. Adjacent normal pituitary tissue was used as a positive control and substitution of the primary antibody with antibody diluent as a negative control. Both controls gave satisfactory results.
73 Regarding the automated IHC technique, the Benchmark XT (Ventana Medical Systems, Tucson, AZ) was used. The differences when compared with the manual technique were as follows: (1) the dewaxing step was thermal at 72°C; (2) antigen retrieval was performed with EDTA pH 8.0 at 95 °C; (3) the incubation with primary antibodies was performed at 37°C for 32 minutes; and (4) the presence of antigens was revealed with the UltraView Universal DAB Detection Kit (Ventana), which is a biotin-free system. To evaluate the expression of SSTR, only membrane immunopositivity was considered and the final evaluation was performed mainly in the automated stained slides. The tumors were semiquantitatively scored according to the scoring system reported by Fougner and colleagues [24]. The GH tumors were classified into 3 groups, taking into account the % of immunoreactive (IR) cells but not the intensity of the staining: group 1, less than 25% of IR cells; group 2, 25% to 75% of IR cells; and group 3, more than 75% of IR cells.
Fig. 1 IHC detection of SSTR2A (A, C and E) and SSTR5 (B, D, and F) in GH tumors using a manual procedures for tumors fixed in BouinHollande fixative (A and B, original magnification ×40) and an automated procedure for tumors fixed in Bouin-Hollande fixative (C and D, original magnification ×40) or zinc-formalin fixative (E and F, original magnification ×40).
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The other types of tumors were classified as positive or negative based on the presence of more or less than 5% of IR cells, respectively. Two independent pathologists evaluated the results separately and scored all the pituitary tumors identically.
2.3. Statistical analysis Statistical analyses were performed using StatView 5.0 software (SAS Institute Inc., Irvine, CA, USA). Data were expressed as means ± SEM. Differences between groups were studied using the Mann-Whitney test, and differences in the proportion were studied using the Fisher exact test. P b .05 was considered significant.
Table 2
Expression of SSTR2A and SSTR5 in 60 GH tumors
Tumors
SSTR2A (%), mean ± SD
P
SSTR5 (%), mean ± SD
P
GH (n = 39) GH/PRL (n = 21) DG (n = 32) SG (n = 28)
63.8 ± 32.9 67.4 ± 32.8 78.3 ± 26.5 50.0 ± 32.7
NS
53.6 ± 36.5 50.5 ± 33.8 53.1 ± 35.1 51.8 ± 36.2
NS
.005 ⁎
NS
Abbreviation: NS, not significant. ⁎ Significant difference.
was also statistically different among the 3 groups of SSTR2A immunoreactivity (Fisher exact test, P = .027), but not among the 3 groups of SSTR5 immunoreactivity (Fig. 2). There was no significant difference between pure GH and GH/PRL tumors for either SSTR studied (Table 2).
3. Results
3.2. SSTR expression in other tumors
We compared the percentage of IR cells and staining intensity obtained using the 2 technical procedures of IHC (manual and automated) and the 2 fixatives (Fig. 1). There were no differences among the techniques (procedure and fixative) in terms of the percentage of cells IR for anti-SSTR2A antibody. However, the intensity was lower with the manual procedure than with the automated one, and the percentage of IR cells was significantly higher with the automated than with the manual procedure (manual: 48.1 ± 37.8; automated: 59.2 ± 37.1; P = .01) for anti-SSTR5 antibody. Cytoplasmic staining was considered as background and mainly observed with the manual procedure. It rendered the evaluation of specific membrane staining difficult (Fig. 1A and B).
The 3 TSH tumors highly expressed SSTR2A (N75% of the cells), and 2 of them weakly expressed SSTR5 (b25% of the cells; Table 1). SSTR2A 52%
38% 33%
Total DG 22%
SG
15% 12%
13%
12%
3%
3.1. SSTR expression in 60 GH tumors Group 1
Almost all GH tumors expressed SSTR2A and SSTR5 (93% and 83%, respectively; Table 1) at a higher level (N75%, group 3) for SSTR2A than for SSTR5 (52% and 37%, respectively). Most DG tumors (72%) had more than 75% of cells that were IR for SSTR2A, and SSTR2A expression was significantly higher (P = .0005) in DG compared with SG tumors (Table 2). Moreover, the distribution of DG and SG tumors
Group 2
SSTR5 37%
37%
27% 22% 18%
Table 1 Expression of SSTR2A and SSTR5 in the 5 types of pituitary tumors Tumor types
SSTR2A
Total
18%
DG 15%
13%
13%
SG
SSTR5
Positive (%)
Negative (%)
Positive (%)
Negative (%)
93 26 0 0 100
7 74 100 100 0
83 0 20 0 67
17 100 80 100 33
Group 1
GH (n = 60) FSH/LH (n = 23) ACTH (n = 15) PRL (n = 7) TSH (n = 3)
Group 3
Group 2
Group 3
Fig. 2 Expression of SSTR2A and SSTR5 in DG- and SG-type pituitary tumors for each group. The distribution of DG and SG among the different SSTR immunoreactivity subgroups was significantly different for SSTR2A (P b .03, Fisher exact test), but not for SSTR5.
Expression of somatostatin receptors in pituitary tumors The expression of SSTR2A and SSTR5 was found to be rare in the other tumors, and the percentage of positive cells was very low. All the FSH/LH tumors were negative for SSTR5 (Fig. 3B), and only 26% (6/23) were positive for SSTR2A (Fig. 3A). Three of the 6 positive for SSTR2A were pure FSH tumors with 40% of IR cells, and the other 3 were FSH/LH tumors with a low expression (b15% of IR cells). None of the ACTH tumors showed membrane staining for anti-SSTR2A, but 5 displayed cytoplasmic artifactual staining (Fig. 3C). Twenty percent (3/15) were positive for SSTR5 (Fig. 3D) with a low percentage of IR cells (b30%). All the PRL tumors were negative for both SSTRs (Table 1).
4. Discussion Here we have studied the expression of SSTR2A and SSTR5 in 108 pituitary tumors of each type, by IHC with 2 new monoclonal antibodies. Very few studies devoted to IHC detection of SSTR in pituitary tumors have been published, mainly because of technical difficulties (Table 3). We compared results obtained by 2 procedures and 2 fixatives with regard to the expression of SSTR in GH tumors. The high optimal dilutions, higher than those recommend by Epitomics (1:4000 for SSTR2A and 1:1000 for SSTR5, instead of 1:100-250), underline the quality of these antibodies. The 2 fixatives used (Bouin-Hollande and zinc-formalin) ensured high protein conservation and membrane integrity resulting in a clear staining for both antibodies localized to the membrane. A cytoplasmic background was mainly seen using the manual
75 procedure. Formalin fixation has also given good results with the same antibodies [19,20]. Cytoplasmic staining has been attributed by some authors to the internalization mechanism, with an important role in tachyphylaxis and radiotherapeutic application of radiolabeled octapeptide SA [25]. However, considering that internalized receptors are known to be inactive [26] and only membrane localization reflects the functional status of the SSTR in the tumors and may be related to the efficacy of SA [27] and that cytoplasmic staining is related to technical procedure, we only took membrane staining into account. Our approach thus differed to that used previously using polyclonal antibodies and a manual procedure [17,27]. The IHC evaluation of the percentage of positive cells with these new highly specific antibodies was reproducible and allowed the classification of GH pituitary tumors into 3 groups according to the levels of expression of SSTR. Unlike previous studies [18-20,27], we did not consider the staining intensity in the present evaluation because it is highly subjective and could be related to technical procedure. Under our experimental conditions, SSTR2A expression was significantly higher in DG than in SG tumors, and the distribution of DG and SG tumors among the 3 groups was significantly different regarding SSTR2A but not SSTR5 immunoreactivity. Although our criteria were not the same, our results are comparable with previous studies. Thus, by comparison with DG tumors, the SG ones would appear to express lower levels of SSTR, be more often invasive macroadenomas and less responsive to SA treatment, and have a higher rate of clinical recurrence [23,28,29]. Among our large series of GH tumors, the expression of SSTR2A and SSTR5 was very high (93% and 83%,
Fig. 3 IHC detection of SSTR2A (A and C) and SSTR5 (right panels) in FSH/LH tumors (A and B, original magnification ×40) and ACTH tumors (C, original magnification ×20; D, original magnification ×40).
76 Table 3
L. Chinezu et al. Reports regarding the IHC expression of SSTR in different types of pituitary tumors
Study (no. of tumors)
Pawlikowski et al [17] (n = 18) Fougner et al [24] (n = 68) Fusco et al [30] (n = 26) Wildemberg et al [27] (n = 13) Ramírez et al [16] (n = 74) Lupp et al [20] (n = 25; 5) Our series (n = 60; 23; 15)
Antibody
Gramsch (Schwabhausen, Germany) Gramsch Gramsch Gramsch Abcam (Cambridge, MA, USA) UMB-1, UMB-4 (Epitomics, Burlingame, CA, USA) UMB-1, UMB-4 (CliniSciences, Nanterre, France)
Clonality
SSTR2A % of positive tumors
SSTR5 % of positive tumors
GH
FSH/LH
ACTH
GH
FSH/LH
ACTH
Polyclonal
ND
83
ND
ND
100
ND
Polyclonal Polyclonal Polyclonal Monoclonal Monoclonal
100 ND 92 ND 84
ND 46 ND 60 ND
ND ND ND ND 0
ND ND ND ND 100
ND 84 ND 45 ND
ND ND ND ND 80
Monoclonal
93
26
0
83
0
20
Abbreviation: ND, not determined.
respectively), in accordance with previous studies [7,18,20,26,27]. Currently available treatment of GH tumors with SA is effective in regulating hormonal hypersecretion and cellular proliferation. These effects of SA seem to be positively correlated with SSTR expression, especially SSTR2A and SSTR5 [5]. Significant levels of expression of SSTR2A and SSTR5 have been reported in nonfunctioning tumors represented in great majority by gonadotroph or FSH/LH and null cell tumors. With cytoplasmic immunostaining taken into account, the percentages obtained range from 46 to 83 for SSTR2A and 45 to 100 for SSTR5 [16,17,30]. However, with only membrane immunostaining considered [17], 17% of the tumors were positive for SSTR2A, as in our series. Expression of SSTR5 has been found in 39% of nonfunctioning tumors [17], suggesting that treatment with SA can stabilize the postoperative residual tumor [30]. However, using highly specific monoclonal antibodies, here we observed a weak expression of SSTR2A in 26% of nonfunctioning tumors and absent SSTR5 expression. This may explain the lack of significant tumor shrinkage observed with SA in nonfunctioning tumors [30]. Very few studies have been devoted to the IHC detection of SSTR in ACTH tumors. All ACTH tumors in these studies were negative for SSTR2A; however, the results are contradictory for SSTR5: indeed in one study, 4 of the 5 tumors tested were positive [20], and in another study, 3 of the 3 tested were negative [31]. In our series, only 3 of the 15 ACTH tumors expressed SSTR5, which is in accordance with recent clinical data on pasireotide (SOM230) treatment in Cushing disease, demonstrating a 20% response rate among the patients treated regardless of the SSTR5 status [32]. The correlation between SSTR5 expression and response to pasireotide now needs to be evaluated prospectively in a large cohort. Pasireotide could be a potential therapeutic option for patients with PRL tumors resistant to dopamine agonists because inhibition of PRL secretion seems to be related to SSTR5 expression [33]. However, in our study, all 7 PRL tumors were negative for both SSTRs, suggesting that
treatment of PRL tumors with pasireotide may be limited to some rare cases. Because medical treatment of pituitary tumors depends, to a certain extent, on the expression of SSTRs, a reproducible method able to detect the expression of distinct subtypes is required. Here in the present study, we have tested the IHC detection of SSTR2A and SSTR5 by 2 new monoclonal antibodies for all types of pituitary tumors using different procedures. The automated IHC procedure proved to be the most reliable and reproducible method with clear membrane staining and lack of cytoplasmic background. These monoclonal antibodies against SSTR2A and SSTR5 should now be considered for use in routine practice for pathological analysis of pituitary tumors and may prove useful in the analysis of all endocrine tumors (including, pancreas) with a view to adapting medical treatment to SSTR profile.
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