Immunohistochemical detection of somatostatin receptor subtype 5 (SSTR-5) in cushing adenoma

Abstract

The actions of somatostatin hormone are mediated by specific transmembrane receptors (somatostatin receptors). These receptors are G-protein linked receptors encoded on five different chromosomes: SSTR1–SSTR5. At the protein level, SSTR-5 was found to have the highest level of expression in corticotroph adenomas [1, 2]. These data suggest that SSTR-5 may be a potential in situ target for medical therapies of adrenocorticotrophic hormone (ACTH) producing corticotroph adenomas [3]. As Cushing adenomas tend to be small and often are not identified by MR imaging [4–6], we hypothesized, based on the previous data, that SSTR-5 may be a potential target for localization of unidentified Cushing adenomas on MRI. Here, we present the results of a pilot study on immunohistochemical staining of 3 Cushing adenomas using the monoclonal SSTR-5 antibodies. Three paraffin blocks containing ACTH producing adenomatous and normal periadenomatous tissue of human pituitary were utilized. For immunohistochemistry, all mouse monoclonal anti-SSTR-5 antibodies were commercially obtained from Advanced Targeting Systems (San Diego, CA, USA). Paraffin sections were dewaxed in xylene and rehydrated through a graded series of ethanol, the antigen was retrieved in 0.01 M citrate buffer (with EDTA added) for 25 min, and the sections were incubated in 3% hydrogen peroxide for 5 min. Sections were then rinsed in TRIS buffer saline pH 7.8 twice for 3 min and blocked in mouse serum for 10 min. Sections were subsequently incubated for 30 min in mouse monoclonal SSTR-5 antibodies at 2ug/ml, rinsed in TRIS buffer saline twice for 3 min, incubated in anti-mouse IgG, biotinylated for 30 min, and rinsed again in TRIS buffer saline twice for 3 min. Sections were then incubated in strepavidin-HRP for 30 min, rinsed in TRIS buffer twice for 3 min and visualized with DAB (diaminobenzidine) for 5 min, counterstained with hematoxylin for 5 min, and cover slips were applied. The results were consistent in the 3 samples. Immunoreactivity was detected primarily in the cytoplasm and the cell membrane of the normal periadenomatous tissue. Immunopositivity was strongly correlated with ACTH-producing cells in the normal pituitary tissue as seen in slides immunostained for ACTH that had been obtained at diagnosis. Cushing adenoma was negative in all 3 samples (Fig. 1). In the literature, detection of somatostatin receptor subtypes was limited to qualitative or real-time PCR analysis of their mRNA [1, 2, 7–10]. Immunohistochemical detection and localization of somatostatin receptor subtypes has been reported in prostate tissue from patients with bladder outlet obstruction. To the best of our knowledge, there are no reports of immunohistochemical detection of SSTR-5 in ACTH-producing corticotroph adenomas. In conclusion, immunohistochemical staining was performed using monoclonal SSTR-5 antibodies on three specimens of ACTH-producing adenoma and their adjacent normal periadenomatous tissue. Our results demonstrated negative immunoreactivity for SSTR-5 in the adenomatous tissue. Studies on SSTR-5 epitopes are warranted before any trial of targeting the receptor for either localization or treatment of Cushing adenomas. W. Hassaneen (&) D. P. Cahill G. N. Fuller N. B. Levine The University of Texas, M. D. Anderson Cancer Center, Houston, TX, USA e-mail: whassaneen@mdanderson.org