Apostolos V. Tsolakis

Distribution of Obestatin and Ghrelin in Human Tissues Immunoreactive Cells in the Gastrointestinal Tract, Pancreas, and Mammary Glands

Obestatin and ghrelin are two peptides derived from the same prohormone. It is well established that ghrelin is produced by endocrine cells in the gastric mucosa. However, the distribution of human obestatin immunoreactive cells is not thoroughly characterized. A polyclonal antibody that specifically recognizes human obestatin was produced. Using this antibody and a commercial antibody vs ghrelin, the distribution of obestatin and ghrelin immunoreactive cells was determined in a panel of human tissues using immunohistochemistry. The two peptides were detected in the mucosa of the gastrointestinal tract, from cardia to ileum, and in the pancreatic islets. Interestingly, epithelial cells in the ducts of mammary glands showed distinct immunoreactivity for both ghrelin and obestatin. By double immunofluorescence microscopy, it was shown that all detected cells were immuno-reactive for both peptides. Furthermore, the subcellular localization of obestatin and ghrelin was essentially identical, indicating that obestatin and ghrelin are stored in the same secretory vesicles.

Ghrelin Immunoreactive Cells in Gastric Endocrine Tumors and Their Relation to Plasma Ghrelin Concentration

Goals Our aim was to elucidate the incidence and distribution pattern of ghrelin-immunoreactive (IR) cells in various types of human gastric endocrine tumors, and their surrounding mucosa, and relate the findings to total ghrelin concentrations in plasma. Background It has been demonstrated previously, that ghrelin-IR cells are present not only in normal human gastric oxyntic mucosa, but also in all types of enterochromaffinlike (ECL) cell carcinoids (ECL-CCs), and in mucosal regions affected by ECL cell hyperplasia. Study Forty-eight gastric endocrine tumors were included in the study: 32 type I ECL-CCs, 3 type II, 9 type III, 1 non-ECL-CC, and 3 poorly differentiated endocrine carcinomas. The tumors were analyzed immunohistochemically with antibodies raised versus chromogranin A, synaptophysin, serotonin, somatostatin, vesicular monoamine transporter 2 and ghrelin. Total ghrelin in plasma was measured in 20 patients, using a commercial radioimmunoassay kit. Results Ghrelin-IR cells were found in all types I and II ECL-CCs but in only a few cases of the other tumors. Ghrelin-IR cells were also found among the hyperplastic endocrine cells in the mucosa surrounding types I and II, where they showed diffuse, linear, nodular and adenomatoid hyperplasia patterns. In type III ECL-CCs and poorly differentiated endocrine carcinomas, only diffuse and linear ghrelin-IR cell hyperplasia was present in the oxyntic mucosa in about half of the cases, whereas the mucosa of the non-ECL-CC did not show this feature. Conclusions Despite the frequent occurrence of ghrelin-IR cells in both the neoplastic parenchyma and the oxyntic mucosa, plasma total ghrelin concentrations remained within the reference range and can therefore not be used as a clinical marker to identify ghrelin expressing ECL-CCs or ghrelin cell hyperplasia.

Obestatin/ghrelin cells in normal mucosa and endocrine tumours of the stomach

OBJECTIVE Obestatin and ghrelin are derived from the same gene and co-expressed in the same endocrine cells. Vesicular monoamine transporter-2 (VMAT-2), a marker for enterochromaffin-like (ECL) cells, is considered to be expressed in ghrelin cells. The aim was to establish if the two peptides and the transporter are co-expressed, both in normal gastric mucosa and in gastric endocrine tumours. DESIGN An immunohistochemical study was performed on gastric biopsy material and on surgical specimens from 63 patients with gastric endocrine tumours and from individuals with normal gastric mucosa. Cells displaying obestatin immunoreactivity were examined regarding co-localization with ghrelin and VMAT-2. Both single- and double-immunostaining techniques were applied. Obestatin concentration in blood was measured in a subgroup of these patients. The results were correlated to various clinico-pathological parameters. RESULTS In the normal mucosa, obestatin/ghrelin-immunoreactive cells rarely co-expressed VMAT-2. In most tumour tissue specimens, only a fraction of neoplastic cells displayed immunoreactivity to obestatin, and these cells always co-expressed ghrelin. Neoplastic obestatin-/ghrelin-IR cells invariably expressed VMAT-2, except for two ghrelinomas. The obestatin concentrations in blood were consistently low and did not correlate to clinico-pathological data. CONCLUSIONS Obestatin and ghrelin immunoreactivity always occurred in the same endocrine cells in the gastric mucosa but these cells only occasionally co-expressed VMAT-2, opposite to the findings in tumours. These results indicate that endocrine cells expressing obestatin and ghrelin mainly differ from VMAT-2 expressing cells (ECL-cells) and can develop into pure ghrelinomas. Plasma concentrations of obestatin did not correlate to cellular expression.

Somatostatin and dopamine receptor expression in neuroendocrine neoplasms: correlation of immunohistochemical findings with somatostatin receptor scintigraphy visual scores

The expression of somatostatin (sstr1‐5) and dopamine (DR) receptors in neuroendocrine neoplasms (NENs) facilitates diagnosis by tumour visualization with somatostatin receptor scintigraphy (SRS) and directs towards specific treatment with peptide receptor radionuclide therapy (PRRT) with radiolabelled somatostatin analogues.

Expression of Somatostatin Receptors 1-5 and Dopamine Receptor 2 in Lung Carcinoids: Implications for a Therapeutic Role

Objective: The expression of somatostatin receptors (SSTRs) and dopamine receptor 2 (DR2) in neuroendocrine tumors is of clinical importance as somatostatin analogues and dopamine agonists can be used for their localization and/or treatment. The objective of this study is to examine the expression of the five SSTR subtypes and DR2 in lung carcinoids (LCs). Methods: We conducted a retrospective study of 119 LCs from 106 patients [typical carcinoids (TCs): n = 100, and atypical carcinoids (ACs): n = 19]. The expression of all five SSTR subtypes and DR2 was evaluated immunohistochemically and correlated to clinicopathological data. In a subgroup of cases, receptor expression was further analyzed using semiquantitative RT-PCR. Results: SSTR2A was the SSTR subtype most frequently expressed immunohistochemically (72%), followed by SSTR1 (63%), SSTR5 (40%), and SSTR3 (20%), whereas SSTR4 was negative. DR2 was expressed in 74% and co-expressed with SSTR1 in 56%, with SSTR2A in 59%, with SSTR3 in 19%, and with SSTR5 in 37% of the tumors. Receptor expression was not related to the histological subtype, tumor aggressiveness (disease extent/grading) or functionality; however, DR2 was expressed more frequently in ACs than TCs (95 vs. 70%, p = 0.017). In a subset of patients, RT-PCR findings highly suggested that the expression of SSTR2A, SSTR3, DR2, and to a lesser extent that of SSTR1 and SSTR5 is the outcome of increased gene transcription. Conclusions: The high and variable immunohistochemical expression of the majority of SSTRs along with their co-expression with DR2 in LCs provides a rationale for their possible treatment with agents that target these receptors.

Differentiated expression of somatostatin receptor subtypes in experimental models and clinical neuroblastoma

Neuroblastoma (NB) is a solid tumor of childhood originating from the adrenal medulla or sympathetic nervous system. Somatostatin (SS) is an important regulator of neural and neuroendocrine function, its actions being mediated through five specific membrane receptors. The aim of this study was to investigate the expression of the different somatostatin receptors (SSTRs) in NB tumor cells that may form targets for future therapeutic development.

Unusual Complication of a Pancreatic Neuroendocrine Tumor Presenting with Malignant Hypercalcemia

CONTEXT Hypersecretion of PTHrP is a relatively common cause of malignancy-related hypercalcemia but has only been described in a few cases of neuroendocrine tumors (NET). OBJECTIVE The aim of this case report is to describe the clinical syndrome, complex therapeutic interventions, and unusual complications caused by persistent PTHrP hypersecretion in a patient with a pancreatic NET. CASE ILLUSTRATION A 58-yr-old male patient presented with nonspecific abdominal pain and was found to have severe hypercalcemia secondary to a well-differentiated NET of the pancreas associated with extensive liver metastases. Elevated ionized calcium levels accompanied by low serum PTH and remarkably elevated PTHrP concentrations were consistent with PTHrP-related hypercalcemia that proved to be resistant to various chemotherapeutic regimens and supportive therapy. Partial control of the humoral syndrome was obtained only after the application of cytoreductive interventions and the introduction of various molecular targeted therapies. Due to persistent PTHrP action, bone disease emerged in the form of brown tumors. DISCUSSION The manifestation of paraneoplastic syndrome due to PTHrP hypersecretion, despite its rareness in NET, should be considered in the differential diagnosis of hypercalcemia in such tumors. Moreover, the appearance of bone lesions in this setting may be in the context of metabolic bone disease and could be misdiagnosed as bone metastases.

Connective tissue growth factor expression in endocrine tumors is associated with high stromal expression of α-smooth muscle actin

OBJECTIVE Complications due to fibrosis development are common in patients with well-differentiated endocrine carcinomas in the small intestine (ileal carcinoids). Connective tissue growth factor (CTGF) expression in ileal carcinoids may be related to this fibrosis development. This study aimed to examine CTGF expression in relation to local myofibroblast differentiation in a large series of ileal carcinoids and in different types of endocrine tumors. METHODS Immunoreactivity (IR) for CTGF and α-smooth muscle actin (α-SMA), a marker for myofibroblasts, was compared in serial tumor tissue sections from 42 patients with ileal carcinoids and from 80 patients with other endocrine tumors. Western blot was performed on an additional 21 patients with ileal carcinoids. RESULTS CTGF IR was present in >50% of tumor cells in all 42 ileal carcinoids and in 2 out of 14 endocrine pancreatic tumors, 4 out of 6 rectal carcinoids, and 1 out of 5 lung carcinoids. Tumors with abundant CTGF expression also displayed α-SMA IR in stromal fibroblast-like cells, whereas other endocrine tumors displayed less or no CTGF and α-SMA IR. Protein bands corresponding to full-length CTGF (36-42 kDa) were detected in protein lysates from ileal carcinoids. CONCLUSION CTGF is uniquely prevalent in ileal carcinoids when compared with most other endocrine tumor types. Immunoreactive cells are adjacent areas with increased fibrovascular stroma that express α-SMA. This supports a potential role for CTGF in myofibroblast-mediated fibrosis associated with ileal carcinoids, and indicates that CTGF should be investigated as a target for future therapy.

Expression of connective tissue growth factor and IGF1 in normal and neoplastic gastrointestinal neuroendocrine cells and their clinico-pathological significance

Connective tissue growth factor (CTGF) and IGF1 are both expressed in a variety of tumours and are involved in tumourigenesis. However, information about their expression in the gastrointestinal (GI) neuroendocrine (NE) cells and tumours is mainly limited, with the exception of midgut carcinoids where abundant CTGF expression has been demonstrated. Normal mucosa specimens from stomach and ileum, as well as tumour tissue specimens from gastric NE tumours (GNETs; n=58) and midgut NETs (n=38) were included. Immunohistochemical techniques were used to investigate the possible expression of CTGF and IGF1 in GI NE cells and tumours. The latter results were correlated with various clinico-biochemical and histopathological variables. CTGF was expressed in a proportion of NE cells of the normal GI mucosa but not in enterochromaffin-like (ECL) cells, whereas IGF1 was undetectable. CTGF was absent in the foci of ECL cell hyperplasia, and in most of the poorly differentiated carcinomas, but present in some GNETs (mainly in type III ECL cell carcinoids (ECL-CCs)) and in all but one midgut NETs. CTGF correlated with tumour stage in well-differentiated GNETs and with size larger than 1  cm but only in the subgroup of type I ECL-CCs. IGF1 was detected in the foci of ECL cell hyperplasia and in all GI NETs. These findings suggest that both CTGF and IGF1 may be involved in the neoplastic transformation of GI NE cells, whereas IGF1 may play an important role even at early stage.

Current concepts in the diagnosis and management of type 1 gastric neuroendocrine neoplasms

The vast majority of gastrin‐related gastrointestinal neuroendocrine neoplasms (GI‐NENs) develop in the context of chronic atrophic gastritis (type 1), a condition closely related to autoimmune thyroid diseases. These neoplasms are defined as gastric NENs type 1 (GNEN1) and have recently been shown to constitute the commonest GI‐NENs in a prospective study. GNEN1s are usually multiple and follow a relative indolent course, raising questions regarding the extent that such patients should be investigated and the appropriate therapeutic interventions needed. Recently, a number of consensus statements and guidelines have been published from various societies dealing with the diagnosis and management of GI‐NENs. Endocrinologists are among the many different medical specialties involved in GNEN1s diagnosis and management. However, despite recent advances, few randomized trials are available, and thus existing evidence remains relatively weak compared to other malignancies. The purpose of this review is to provide recent evidence along with currently employed modalities addressing the diagnosis, management, long‐term follow‐up and potential comorbidities of GNEN1s.