Immunohistochemical Expression of Somatostatin Type 2A Receptor in Neuroendocrine Tumors

The biological behavior of neuroendocrine tumors, such as carcinoid tumors and pancreatic endocrine tumors, and pheochromocytomas has generally resulted in a good prognosis. The surgical removal of neuroendocrine tumors is often curative when the tumor is detected at an early stage. However, if such tumors have distant metastases in the liver, lung, or bone, neither anticancer chemotherapy or irradiation therapy has much of an effect. An analysis of 5-year survival rates in patients with malignant neuroendocrine tumors revealed that the survival rates are <20% when liver metastases are present (1). Neuroendocrine carcinoma, which is composed of small poorly differentiated neuroendocrine cells, is also a highly aggressive tumor with a poor prognosis (2). Capella et al. (3) proposed a new classification of the neuroendocrine tumors or the lung, pancreas, and gut. They classified neuroendocrine tumors of the pancreas, colon, and rectum as benign, benign or low-grade malignant, low-grade malignant, and high-grade malignant. High-grade malignant tumors are compatible with neuroendocrine carcinoma. Regarding metastatic pheochromocytomas, there are two types of tumors; one is a slowly progressive tumor, even with bone or lung metastases, and the other is an extremely aggressive tumor with a poor prognosis (4). There is as yet no effective treatment for either type of metastatic pheochromocytoma.

SS² has a broad spectrum of biological actions, exerts suppressive effects on multiple organs including the brain, pituitary, gut, exocrine and endocrine pancreas, adrenals, thyroid, and kidneys, and appears to be an endogenous growth inhibitor (5). SS exerts its biological effects by binding to specific high-affinity receptors, which appear in many cases to be coupled to GTP-binding proteins. SS and SS analogues effectively inhibit the proliferation of various types of cancer cells (5). All of these effects are mediated by specific high-affinity SSTRs on the target tissue (6).

Molecular cloning has revealed the presence of at least five different types of SSTRs in the rat, all of which belong to the seven-transmembrane domain receptor superfamily and are expressed in the central nervous system. They have been named SSTR1, SSTR2, SSTR3, SSTR4, and SSTR5 (7, 8). Sequence comparisons of the SSTR subtypes have revealed two subgroups of receptors in the SSTR gene family. SSTR1 and SSTR4 share common characteristics that differ from those of the subgroup containing the subtypes SSTR2, SSTR3, and SSTR5. Short peptide analogues such as octreotide demonstrate specific binding only for the subgroup consisting of SSTR2, SSTR3, and SSTR5 (6). SSTR2 is shown to be directly involved in the transduction of the antiproliferative effects (9). Although there have been investigations of the localization of SSTR by autoradiography, PCR, and in situ hybridization (10, 11, 12), few studies have been done on the cellular localization of the receptor subtype proteins in neuroendocrine tumors, especially in metastatic or highly malignant neuroendocrine tumors (13, 14). In the present study, using an anti-peptide polyclonal antibody specific for SSTR2A, we investigated the distribution of this receptor protein in various types of neuroendocrine tumors by immunohistochemistry. The presence of SSTR2 mRNA was also investigated in representative cases of neuroendocrine tumors by an in situ hybridization method. The data obtained could contribute to the assessment of the utility of SS analogues for highly malignant neuroendocrine tumors.

The majority of the specimens investigated were surgically obtained; some of the highly malignant tumors were obtained by autopsy. Autopsies were carried out within 3 h after death. Those specimens were from the files of the Department of Pathology, Tohoku University. The 62 neuroendocrine tumors examined included 16 pancreatic endocrine tumors, of which 3 had metastases (an ACTH-producing tumor with Cushing’s syndrome metastasizing to the liver and lymph nodes, a gastrin-producing tumor metastasizing to the liver, and an insulin-producing tumor with lymph node metastases); 20 carcinoid tumors, 11 from the rectum (2 had liver metastases), 2 thymus (with multiple metastases), 2 small intestine, 3 ovary, and 2 testis; 3 neuroendocrine carcinomas (primary sites were the stomach, gall bladder, and unknown origin); 2 medullary thyroid carcinomas; 2 small cell carcinomas of the lung, which were positive for chromogranin A; 15 pheochromocytomas (3 had metastases to the bone, lung, and liver); 2 neuroblastomas of undifferentiated type; and 2 ganglioneuromas. For the positive control specimens for SSTR2A, normal pancreas tissues obtained by autopsy, fixed by 10% buffered formalin, and embedded in paraffin were used.

The formalin-fixed, paraffin-embedded tissues were dewaxed and rehydrated. The antigen retrieval of the sections was accomplished by incubation in 0.01 m citric acid buffer (pH 6.0) and autoclaving for 5 min at 120°C, before the immunohistochemical procedures. The immunohistochemical staining for SSTR2A was carried out by the streptavidin-biotin-complex method as reported previously (15). The sheep polyclonal antibody against SSTR2A was raised against a synthetic peptide corresponding to the predicted amino acids 347–366 of the COOH terminus of rat SSTR2A (16). This peptide sequence is identical in the rat, human, and mouse SSTR2A. The working dilution of SSTR2A was 1:200. A rabbit antisheep IgG-biotinylated antibody (1:100; Vector Laboratories, Burlingame, CA) was used as the second antibody. Nuclei were slightly counterstained with hematoxylin. The specificity of the affinity-purified SSTR2A antibody has been demonstrated by Western blotting and adsorption testing using 100 μm SSTR2A peptide to block the antibody (at a dilution of 1:300) for 18h at 4°C (16). Additional controls included the omission of the primary antibody. To reveal the colocalization of immunoreactive SSTR2A and pancreatic peptide hormones, serial sections were cut, and pairs of adjacent sections were examined for SSTR2A and peptide hormones insulin, glucagon, somatostatin, pancreatic polypeptide, gastrin, and ACTH. The sources and working dilutions of these peptide hormones were as follows: insulin (1:3000; DAKO, Glostrup, Denmark), glucagon (1:3000; DAKO), somatostatin (1:3000; DAKO), pancreatic polypeptide (1:4000; DAKO), gastrin (1:3000; DAKO), and ACTH (1:4000; Incstar, Stillwater, MN).

The immunoreactive intensity of each tumor of the pancreas was compared with that of normal pancreatic islets of tissue adjacent to the tumor and evaluated as “+++” if the intensity was more than that of the islets, “++” if the same as that of the islets, “+” if less than that of the islets. “−” indicated the absence of immunoreactivity. The immunoreactivity of the tumors of other sites was compared with that of the pancreatic endocrine tumors.

On the basis of the human SSTR2 mRNA sequence cloned by Yamada et al. (17), we used four synthesized oligonucleotide probes: 48-base long oligonucleotides complementary to the bases coding for amino acids 31–46 and 237–252 (18); 359–364 (compatible with the COOH-terminus of our antibody); and 1220–1224 (untranslated region) of the human SSTR2 mRNA sequence. Those oligonucleotides were labeled with DIG with the use of a DIG oligonucleotide tailing kit (Boehringer Mannheim, Mannheim, Germany) as recommended by the manufacturer and used as a mixture. The sense probe for SSTR2 (1220–1244) was used as a negative control.

In situ hybridization was carried out for some representative tumors (5 pancreatic endocrine tumors, 5 carcinoid tumors, and 5 pheochromocytomas). The procedure was described previously (15). Briefly, deparaffinized sections were treated with 0.2 m HCl for 20 min at 37°C and then digested with proteinase K. After acetylation with 0.1 m triethanol amine solution with 0.25% acetic anhydride, the sections were prehybridized at 37°C for 1 h. Hybridization was performed for 18 h at 37°C, using DIG-labeled oligonucleotide probes at a concentration of 100 ng/ml, in a hybridization solution. The sense probe labeled with DIG was used as the control probe. After washing, the sections were processed for immunological detection. Briefly, the sections were incubated with a blocking solution and then incubated with alkaline phosphatase-labeled anti-DIG sheep IgG (Fab fragment). The visualization of the immune complex was performed using a biotin-streptavidin signal amplification system (Nichirei, Tsukiji, Tokyo) and alkaline phosphatase color reaction. Nuclei were slightly counterstained with hematoxylin.

In the normal pancreatic islets of the positive controls, the majority of islet cells showed strong immunoreactivity for SSTR2A in whole islets without immunohistochemical heterogeneity. Strong immunoreactivity was clearly detected on the cell membrane, whereas less intense staining was found in the cytoplasm (Fig. 1). The immunohistochemistry results of SSTR2A in the pancreatic endocrine tumors and the other endocrine tumors are summarized in Tables 1 and 2, respectively. Of the 16 pancreatic endocrine tumors, 15 showed SSTR2A immunoreactivity. Only one insulin-producing tumor was negative for SSTR2A. There was no distinct correlation between the expression of SSTR2A and peptide hormones produced by the tumors (Table 1). All metastatic tumors including an ACTH-producing tumor, a gastrin-producing tumor, and an insulin-producing tumor showed strong immunoreactivity for SSTR2A not only in the primary tumors but also at the metastatic sites 2(Fig. 2). Among the 20 carcinoid tumors (including 4 autopsy cases with distant metastases: 2 rectal and 2 thymic carcinoid tumors), all except the 2 autopsy cases (one rectal and one thymic carcinoid) showed moderate to strong immunoreactivity. All 3 neuroendocrine carcinomas of the stomach, gallbladder, or unknown origin obtained by autopsy with multiple metastases were positive for SSTR2A. One of two medullary thyroid carcinomas showed SSTR2A. One of two small cell carcinomas of the lung, which were composed of cells positive for chromogranin A, showed immunoreactivity for SSTR2A. Although there was immunoreactive heterogeneity, all pheochromocytomas showed immunoreactivity (Fig. 3). Interestingly, a highly malignant pheochromocytoma obtained by autopsy showed strong immunoreactivity for SSTR2 not only in the primary tumor but also in the metastatic tumors in the liver and lung (Fig. 4). This tumor was composed of small cells with an extremely high nuclear cytoplasmic ratio, diffusely positive for chromogranin A and sporadically positive for tyrosine hydroxylase, and metastasized to multiple organs. The patient with this tumor died of multiple tumor metastases 4 months after diagnosis. The other metastatic pheochromocytoma also showed strong immunoreactivity, even at metastatic sites of the lung. The neuroblastomas of undifferentiated type without ganglionic cell differentiation had no SSTR2A immunoreactivity. In contrast, the ganglionic cells of the ganglioneuroma showed strong immunoreactivity on the cell membrane (Fig. 5). Of the 62 neuroendocrine tumors, 55 (88%) showed immunoreactivity for SSTR2 (Table 2).

The ISH of SSTR2A mRNA showed a strong reaction in the cytoplasm of the islets in the normal pancreas tissues of the positive control. The tumor cells of the pancreatic endocrine tumors (Fig. 6), carcinoid tumors, and pheochromocytomas investigated also showed a reaction in the ISH. No reaction was observed in the sections incubated with the sense probe.

It has been reported that neuroendocrine tissues and their tumors express SSTR. Earlier investigations of the tissue localization of SSTR were carried out by using SSTR autoradiography or ISH of SSTR mRNA (11, 12, 17). Reubi et al. comprehensively investigated the expression of SSTR mRNA in human tumors by using receptor autography and revealed that the presence of SSTR in most of the neuroendocrine tumors together with SS mRNA pointed toward an autocrine regulatory feedback mechanism of SS in these tissues (10). They further investigated the expression of SSTR1, SSTR2, and SSTR3 mRNA using an ISH method for 55 human primary tumors including 8 pituitary tumors, 15 gastroenteropancreatic tumors, and 6 neuroblastomas (18). The tumors were characterized as having receptors with a high affinity for the synthetic analogue octreotide (18). Janson et al. (12) reported that expression of mRNA for SSTR2 detected by ISH correlates with therapeutic outcome in patients with carcinoid tumors treated with somatostatin analogues.

The immunohistochemical localization of SSTR has been investigated in normal rat brain and spinal cord (16), neurons of the myenteric and submucosal plexuses, interstitial cells of Cajal of the intestine, and enterochromaffin-like cells of the rat stomach (19) and the rat pancreas (20). However, to our knowledge, there have been few immunohistochemical studies of SSTR2A in normal human tissues or neuroendocrine tumors. Hunyady et al. (20) reported SSTR2A in the rat pancreas, and that all acinar cells and glucagon (A)- and pancreatic polypeptide (PP)-immunoreactive cells were intensely labeled for SSTR2A, whereas no signal was detected in SS-producing cells. A very few insulin immunoreactive cells were also labeled for SSTR2A, but the signal in these cells was weaker than that in the exocrine, A, or PP cells. Our present findings revealed that the human pancreas is quite different from the rat pancreas regarding SSTR2A expression, and all islet cells in the human pancreas express SSTR2A. We also found that all but one of the pancreatic endocrine tumors expressed SSTR2A. In the other types of neuroendocrine tumors, the immunohistochemical expression of SSTR2A was also extremely high, and the expression is coincident with that of mRNA in these tumors as determined by us and also by Reubi et al. (18).

A number of somatostatin analogues have been used for the treatment of acromegaly and endocrine tumors of the gastroenteropancreatic system (such as carcinoid tumors, insulinomas, glucagonomas, and VIPomas; Refs. 5, 21, and 22). Most of these tumors and cell lines express SSTR2 (18, 23, 24), and mRNA for SSTR5 is also present in gastroenteropancreatic tumors (25). The activation of a tyrosine phosphatase and the inhibition of calcium mobilization mediated by SSTR2 and SSTR5, respectively, could be important steps in the negative mitogenic signal induced by a somatostatin analogue (25). Janson et al. (12) compared the efficacy of the expression of SSTR2 mRNA and the response to SS analogue treatment in patients with carcinoid tumors. They concluded that there was complete agreement between the presence of mRNA for SSTR2 detected by ISH and a positive therapeutic outcome. Recently, they further conducted a immunohistochemical study of SSTR2 in the above tumors and obtained the same results, concluding that the immunohistochemical method is applicable in clinical practice (14).

Although there were no cases treated with somatostatin analogues in the present study, we would like to refer to the possible utility of somatostatin analogues not only for carcinoid tumors but also for malignant neuroendocrine tumors including metastatic pheochromocytoma, neuroendocrine carcinoma, and infiltrating ganglioneuroma, for which there are no effective treatments at present. Further practical investigations of somatostatin analogues for patients with such tumors should be conducted. The presence of the other types of SSTR should also be clarified in SSTR2A-negative tumors.