Histidine decarboxylase and urinary methylimidazoleacetic acid in gastric neuroendocrine cells and tumours.

AIM: To study histidine decarboxylase (HDC) expression in normal and neoplastic gastric neuroendocrine cells in relationship to the main histamine metabolite. METHODS: Control tissues from fundus (n = 3) and corpus (n = 3) mucosa of six patients undergoing operations for gastric adenocarcinoma, biopsy and/or gastric surgical specimens from 64 patients with primary gastric neuroendocrine tumours (GNETs), as well as metastases from 22 of these patients, were investigated using conventional immunohistochemistry and double immunofluorescence with commercial antibodies vs vesicular monoamine transporter 2 (VMAT-2), HDC and ghrelin. The urinary excretion of the main histamine metabolite methylimidazoleacetic acid (U-MeImAA) was determined using high-performance liquid chromatography in 27 of the 64 patients. RESULTS: In the gastric mucosa of the control tissues, co-localization studies identified neuroendocrine cells that showed immunoreactivity only to VMAT-2 and others with reactivity only to HDC. A third cell population co-expressed both antigens. There was no co-expression of HDC and ghrelin. Similar results were obtained in the foci of neuroendocrine cell hyperplasia associated with chronic atrophic gastritis type A and also in the tumours. The relative incidence of the three aforementioned markers varied in the tumours that were examined using conventional immunohistochemistry. All of these GNETs revealed both VMAT-2 and HDC immunoreactivity, and their metastases showed an immunohistochemical pattern and frequency similar to that of their primary tumours. In four patients, increased U-MeImAA excretion was detected, but only two of the patients exhibited related endocrine symptoms. CONCLUSION: Human enterochromaffin-like cells appear to partially co-express VMAT-2 and HDC. Co-expression of VMAT-2 and HDC might be required for increased histamine production in patients with GNETs.

Malignant presacral ghrelinoma with long-standing hyperghrelinaemia.

BACKGROUND: A 57-year old man with low-back pain was found to have a 3 × 3 × 3 cm presacral neuroendocrine tumour (NET) with widespread metastases, mainly to the skeleton. His neoplastic disease responded well to peptide receptor radionuclide therapy (PRRT) with the radiotagged somatostatin agonist (177)Lu-DOTATATE. During almost 10 years he was fit for a normal life. He succumbed to an intraspinal dissemination. PROCEDURES: A resection of the rectum, with a non-radical excision of the adjacent NET, was made. In addition to computerized tomography (CT), receptor positron emission tomography (PET) with (68)Ga-labelled somatostatin analogues was used. OBSERVATIONS: The NET showed the growth pattern and immunoprofile of a G2 carcinoid. A majority cell population displayed immunoreactivity to ghrelin, exceptionally with co-immunoreactivity to motilin. Somatostatin receptor scintigraphy and (68)Ga-DOTATATE PET-CT demonstrated uptake in the metastatic lesions. High serum concentrations of total (desacyl-)ghrelin were found with fluctuations reflecting the severity of the symptoms. In contrast, the concentrations of active (acyl-)ghrelin were consistently low, as were those of chromogranin A (CgA). CONCLUSIONS: Neoplastically transformed ghrelin cells can release large amounts of desacyl-ghrelin, evoking an array of non-specific clinical symptoms. Despite an early dissemination to the skeleton, a ghrelinoma can be compatible with longevity after adequate radiotherapy.

Ki-67 Index and Solid Growth Pattern as Prognostic Markers in Small Intestinal Neuroendocrine Tumors.

BACKGROUND/AIMS: The prognostic value of histopathological grading and the growth pattern of small intestinal neuroendocrine tumors (SI-NET) is unclear. In particular, the cutoff level between grades G1 and G2 at Ki-67 index above 2% is an open issue, and both lower and higher cutoffs have been proposed. The morphological solid growth pattern (SGP) in SI-NET has been reported to be associated with worse survival. The present study investigates whether a Ki-67 index cutoff of 1% has a higher predictive power than one of 2% for disease-specific survival in SI-NET, and whether an SGP is associated with survival. PATIENTS AND METHODS: From a population-based cohort, 127 SI-NET patients with available tumor specimens were included. Medical records and pathology reports were reviewed. Tumor specimens were reexamined to confirm the diagnosis, recalculate the Ki-67 index, and assess the presence of an SGP, introducing an SGP score from 0 to 3+. RESULTS: The current grading system with a G1/G2 cutoff of 2% was more discriminative (HR 2.30; 95% CI 1.20-4.38, p = 0.012) than one with a lower cutoff of 1% (HR 1.65; 95% CI 0.95-2.87, p = 0.078) after adjustment for patient age and clinical stage. SGP score was strongly associated with clinical stage (p = 0.004) and histopathological grade (p < 0.001) but was not an independent prognostic factor for disease-specific survival in SI-NET (p = 0.122) after adjusting for age, stage, and grade. CONCLUSIONS: The present grading system of SI-NET is supported by our results. The SGP is not an independent prognostic factor for disease-specific survival in SI-NET.

Mutation screening in a Norwegian cohort with pheochromocytoma.

Pheochromocytomas (PHEOs) are neuroendocrine tumours, originating from chromaffin cells in the adrenal medulla. They are either sporadic or hereditary. It is important to identify the hereditary cases, so that patients and relatives with germline mutations can be offered regular surveillance. The objective of this study was the detection of pathogenic germline mutations in a cohort of Norwegian PHEO patients. Blood samples and/or formalin-fixed, paraffin-embedded tissue specimens, were collected from 60 patients who were operated upon between 1986 and 2004 at two university hospitals in Norway. DNA mutation analyses were performed successfully in the 42 blood samples and in one of the paraffin-embedded tissue specimen in VHL, RET, SDHB, SDHC, SDHD and NF1. In all, 32 different DNA variants were observed, of which 8 were classified as pathogenic (19 %), or possibly pathogenic; three in NF1, two in RET and VHL and one in SDHB. Two variants were observed in one patient, one in SDHB and one in NF1. Three of these variants are, to the best of our knowledge, new ones; two in NF1 [c.950_51insGCTGA, (p.Glu318LeufsX59) and c.1588G > A, (p.Val530Ile)] and one in VHL (c.308C > T, p.Pro103Leu). In conclusion the overall incidence of germline mutations in genes associated with familial PHEO was found to be of the same order of magnitude in the present Norwegian series as in those from other countries. Two new NF1 variants and one new VHL gene variant were detected.

Expression of cocaine- and amphetamine-regulated transcript is associated with worse survival in small bowel carcinoid tumors.

PURPOSE: Cocaine- and amphetamine-regulated transcript (CART) peptide exerts several regulatory functions acting both as neurotransmitter and hormone. We recently showed that CART is expressed in various neuroendocrine tumors, including small bowel carcinoids. The main objective of the present study was to examine whether CART expression is associated with survival in patients with small bowel carcinoid. Secondary aims were to assess whether CART expression is associated with other tumor characteristics or clinical symptoms. EXPERIMENTAL DESIGN: Specimens from 97 patients with small bowel carcinoids were examined for CART expression using immunohistochemistry. A CART score was introduced on the basis of the proportion of CART immunoreactive cells. On inclusion, specimens were examined by routine histopathologic methods and detailed clinical patient data were retrieved. The effect of CART on cell viability was assessed in vitro using two intestinal tumor cell lines. RESULTS: Expression of CART (P = 0.011) and increasing CART score (P = 0.033) were associated with worse disease-specific survival. Adjusting for age, disease stage, and tumor grade in multivariable analysis, CART expression was still associated with worse survival [Low CART HR, 5.47; 95% confidence interval (CI), 0.71-42.46; and High CART HR, 9.44; 95% CI, 1.14-78.14]. CART expression was not associated with patient age, disease stage, tumor grade, or any presenting symptom. Supporting our clinical data, we found that CART promoted tumor cell viability in vitro in two different tumor cell lines. CONCLUSION: Expression of CART in small bowel carcinoid tumors is associated with worse survival.

Cocaine- and amphetamine-regulated transcript in neuroendocrine tumors.

BACKGROUND/AIMS: Cocaine- and amphetamine-regulated transcript (CART) is an anorexigenic regulatory peptide highly expressed in the brain's appetite control centers, but also in peripheral neurons and in endocrine cells in the adrenal medulla, thyroid, pancreatic islets, and in the gastrointestinal tract. Plasma levels of CART were recently shown to be elevated in patients with neuroendocrine tumors (NETs), but the cellular sources of CART in NETs have remained unknown. The aim of the study was to establish whether CART is expressed in various types of NETs and, if so, to examine the frequency, distribution and phenotype of CART-expressing cells. METHODS: Tumor specimens from 133 NETs originating in the stomach, ileum, rectum, pancreas and thyroid were examined with immunohistochemistry and in situ hybridization. The expression of CART was quantified and the CART-expressing cells were phenotyped by double staining for established markers and hormones. RESULTS: CART-expressing tumor cells were found in the majority of the examined NETs. The expression pattern of CART was highly heterogeneous not only between tumors, but also within individual tumors. In 14% of the NETs, CART was found in a major population of the tumor cells. CONCLUSION: CART is produced in the majority of NETs, regardless of tumor origin. This likely explains the elevated levels of circulating CART in certain NETs patients, as recently described. CART could therefore prove to be a useful tool in the diagnostics of NETs not only in blood samples, but also in histopathological specimens.

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.

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.

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.

Guidelines for the management of gastroenteropancreatic neuroendocrine tumours (including bronchopulmonary and thymic neoplasms). Part I-general overview.

The incidence of neuroendocrine tumours of the gastroenteropancreatic system seems to have increased during the past decade. New diagnostic and therapeutic procedures have aroused the interest of physicians, though most see very few cases of such diseases. A group of members of the Nordic Neuroendocrine Tumour Group decided to compile some guidelines to facilitate the diagnosis and treatment of patients with these tumours. Part I of these guidelines discusses the principles of histopathology, biochemical and radiological diagnosis as well as therapeutic options.

Guidelines for the management of gastroenteropancreatic neuroendocrine tumours (including bronchopulmonary and thymic neoplasms). Part II-specific NE tumour types.

Part II of the guidelines contains a description of epidemiology, histopathology, clinical presentation, diagnostic procedure, treatment, and survival for each type of neuroendocrine tumour. We are not only including gastroenteropancreatic tumours but also bronchopulmonary and thymic neuroendocrine tumours. These guidelines essentially cover basic knowledge in the diagnosis and management of the different forms of neuroendocrine tumour. We have, however, tried to give more updated information about the epidemiology and histopathology, which is essential for the clinical management of these tumours.