Somatostatin (SST) is known to inhibit the secretion of a wide range of hormones, exocrine glands, and gastrointestinal motility. Among other actions, SST has revealed an antiproliferative potential, reversing the impact of mitogenic signals delivered by substances such as epidermal growth factor. The actions of SST are mediated by membrane-associated receptors that comprise five distinct subtypes (termed SSTR1 to 5). Frequently multiple subtypes coexist in the same cell.
After binding their ligand, SSTR-ligand complexes undergo cellular internalisation with intracytoplasmic and intranuclear translocation. Reubi et al.1 showed that the degree of internalisation, i.e., the ratio of internalised SSTR2 to membranous SSTR2, varied greatly from one patient to the other. Although generally found in endosome-like structures, internalised SSTR2 were also identified to a small extent in lysosomes, as seen in co-localisation experiments. Very recently Waser et al.2 showed that phosphorylated SSTR2 was present in most gastrointestinal neuroendocrine tumours from patients treated with octreotide but that a striking variability existed in the subcellular distribution of phosphorylated receptors among such tumours.
Cloning of the five SSTRs has led to the development of subtype-selective ligands.3 In the era of personalised medicine and targeted therapies, SSTR profiling is an important prerequisite for successful in vivo somatostatin receptor targeting for imaging or therapeutic purposes in an individual patient. Therefore, localisation and expression of the five SSTRs in a tumour must be determined to decide whether the patient is eligible for these applications. Several methods have been used to determine the expression of SSRTs.
Tissue somatostatin receptors can be measured directly in vivo by performing a OctreoScan or 68 Ga-DOTATOC positron emission tomography/computed tomography scan. Molecular techniques such as in situ hybridisation histochemistry and autoradiography have been used in a limited number of studies.4 The former basically investigates SSTR mRNA expression in cryostat sections. The latter also utilises cryostat sections and is based on radioligands, i.e., 125I-labelled somatostatin ligands, such as octreotide. Previous studies have dealt with only some of the subtypes, therefore information is limited. The type of information obtained using these two techniques is not always comparable to that obtained with immunohistochemical analysis in formalin fixed, paraffin embedded (FFPE) tissue, in which the architecture and the cytology in the background are well preserved. In addition, the immunohistochemical technique is widely available, and faster, easier and cheaper to apply than in situ hybridisation histochemistry and autoradiography. It can be used even retrospectively on archival material.
We read with great interest the recent publication by Körner et al.5 This study was performed on neuroendocrine tumours from various gastrointestinal and extragastrointestinal sites and in a small group of non-neuroendocrine tumours. The aim of the investigation was to correlate FFPE-based immunohistochemistry using the monoclonal anti-somatostatin receptor subtype 2A antibody UMB-1 (Biotrend Chemikalien, Germany; or Epitomics, USA), with the gold standard in vitro method quantifying somatostatin receptor levels in tumour tissues. The results obtained by comparing the UMB-1 immunohistochemistry with tumoural in vitro 125I-[Tyr3]-octreotide binding site levels allowed recommendations for the use of SSTR immunohistochemistry in daily diagnostics for optimally tailored patient management.
Data on the immunohistochemical patterns of the five SSTRs in prostate cancer (PCa), its precursor high-grade prostatic intraepithelial neoplasia (HGPIN) and normal prostatic epithelium were obtained by our group in FFPE archival tissue material.6–9 Data were collected separately for the luminal/secretory and basal epithelial cells, for the latter when present, as well as for the smooth muscle cells of the stroma and for the endothelial cells (Fig. 1).
For the secretory or luminal cells, differences were found between normal, HGPIN and PCa (Fig. 2A), and between incidentally-detected and clinically-detected acinar PCa, therefore between insignificant or indolent and significant or aggressive cancers.9 Typical patterns in terms of localisation and expression of the five SSTRs in PCa with neuroendocrine differentiation,8 PCa following complete androgen ablation (CAA)6 and hormone refractory PCa7 were identified, with differences among these three groups and from untreated acinar adenocarcinoma9 (Fig. 2B).
For the basal cells in normal prostate and HGPIN, immunoreactivity was primarily detected in the cytoplasm in all the five subtypes. In subtypes 1 and 3 the mean proportions of positive cells were higher than in the other three subtypes. The proportions were higher in normal prostate compared with HGPIN. Immunoreactivity for the five SSTRs in the groups of cases with neuroendocrine tumours was similar in terms of expression and localisation to that seen in the group of untreated HGPIN. The values in the patients under complete androgen ablation were lower than in the untreated patients.
For the smooth muscle and endothelial cells, there were no cases with a distinct positivity in the cell membrane. Subtype 1 showed a strong immunoreactivity in the cytoplasm in the majority of the smooth muscle cells and the endothelial cells. Nuclear staining was seen only with subtypes 4 and 5. Neuroendocrine differentiation in PCa well as CAA and hormone refractory PCa did not affect SSTR expression and localisation in the in the endothelial and smooth muscle cells.
The limitation of our study was that the immunohistochemical results were not compared with data obtained with a molecular technique or with a gold standard in vitro method quantifying somatostatin receptor levels in tissues, as was in the case of the Körner paper.5 However, specificity of the antibodies used in our studies (Rabbit polyclonal anti-SSTR subtype antibodies from Chemicon International, USA) was assessed. Western blot experiments on a prostate tissue extract were performed. Western blot analysis of prostate tissue performed with the panel of five polyclonal anti-SSTR antibodies yielded single bands as previously described by Helboe et al.10
We agree with Körner et al.5 that the type of antibodies can have an influence on the result to the point that the cytoplasmic and nuclear localisation, i.e., cellular internalisation, of the SSTRs can be seen intriguing and controversial. For instance, the immunohistochemical expression at the cell level of the five SSTRs in normal and pathological prostate tissue was also investigated by Dizeyi et al.11 There were differences between our investigation and that by Dizeyi et al., probably due to the types of antibodies used (in Dizeyi's investigation the antibodies were from a private source and not commercially available).
In conclusion, our data showed that SSTR profiling in an individual patient with HGPIN and the multifaceted PCa is feasible. Even though there is no clinical application for a somatostatin-based diagnostic test for prostate pathology at present, as opposed to neuroendocrine tumour, this should be of relevance to better tailor somatostatin analogue-based diagnostic or therapeutic procedures in neoplasms other than neuroendocrine tumours.12 This is particularly important in the era of personalised medicine and targeted therapies.
1. Reubi JC, Waser B, Cescato R, et al. Internalized somatostatin receptor subtype 2 in neuroendocrine tumors of octreotide-treated patients. J Clin Endocrinol Metab
2. Waser B, Cescato R, Liu Q, et al. Phosphorylation of sst2 receptors in neuroendocrine tumors after octreotide treatment of patients. Am J Pathol
3. Hofland LJ, van der Hoek J, Feelders R, et al. Pre-clinical and clinical experiences with novel somatostatin ligands: advantages, disadvantages and new prospects. J Endocrinol Invest
4. Montironi R, Cheng L, Mazzucchelli R, et al. Immunohistochemical detection and localization of somatostatin receptor subtypes in prostate tissue from patients with bladder outlet obstruction. Cell Oncol
5. Körner M, Waser B, Schonbrunn A, et al. Somatostatin receptor subtype 2A immunohistochemistry using a new monoclonal antibody selects tumors suitable for in vivo somatostatin receptor targeting. Am J Surg Pathol
6. Mazzucchelli R, Morichetti D, Santinelli A, et al. Immunohistochemical expression and localization of somatostatin receptor subtypes in androgen ablated prostate cancer. Cell Oncol (Dordr)
7. Mazzucchelli R, Morichetti D, Scarpelli M, et al. Somatostatin receptor subtypes in hormone-refractory (castration-resistant) prostatic carcinoma. Asian J Androl
8. Morichetti D, Mazzucchelli R, Santinelli A, et al. Immunohistochemical expression and localization of somatostatin receptor subtypes in prostate cancer with neuroendocrine differentiation. Int J Immunopathol Pharmacol
9. Morichetti D, Mazzucchelli R, Stramazzotti D, et al. Immunohistochemical expression of somatostatin receptor subtypes in prostate tissue from cystoprostatectomies with incidental prostate cancer. BJU Int
10. Helboe L, Møller M, Nørregaard L, et al. Development of selective antibodies against the human somatostatin receptor subtypes sst1-sst5. Brain Res Mol Brain Res
11. Dizeyi N, Konrad L, Bjartell A, et al. Localization and mRNA expression of somatostatin receptor subtypes in human prostatic tissue and prostate cancer cell lines. Urol Oncol
12. Mazzucchelli R, Scarpelli M, Lopez-Beltran A, et al. Immunohistochemical expression and localization of somatostatin receptors in normal prostate, high grade prostatic intraepithelial neoplasia and prostate cancer and its many faces. J Biol Regul Homeost Agents