Molecular and cell biological aspects of neuroendocrine tumors of the gastroenteropancreatic system

Neurons and neuroendocrine cells share a variety of common characteristics. Cell and molecular biological studies in recent years have improved our understanding of physiological and pathophysiological processes such as cellular growth, adhesion, and secretion of neuroendocrine cells. Here we review current findings from the area of basic research and from current clinical research relevant to improving the diagnosis and therapy of neuroendocrine tumors of the gastroenteropancreatic system.     

Acidic calcium pools in intraerythrocytic malaria parasites

AIMS: Angiogenesis is a complex multistep process essential for tumour growth. Vascular endothelial growth factor (VEGF) is a potent endothelial cell mitogen and vascular permeability-inducing agent. Recent studies have shown that VEGF expression is correlated to microvessel density and tumour progression. The aim of this study was to analyse VEGF expression in a series of gastrointestinal neuroendocrine tumours. METHODS AND RESULTS: Surgical specimens from 28 gastrointestinal carcinoids and 20 pancreatic endocrine tumours were examined for VEGF expression by immunohistochemistry. Intense cytoplasmic staining for VEGF was observed in several cells of the islets of Langerhans and in neuroendocrine cells of normal digestive mucosa. All midgut carcinoids showed strong VEGF expression in tumoral cells. Positive VEGF immunostaining was observed in 16 of 20 neuroendocrine pancreatic tumours but it was usually much lower than in midgut carcinoids. Western blotting analysis in eight cases identified a major band at 30-32 kDa. No correlation between VEGF expression and tumour stage was found. CONCLUSIONS: This study demonstrates that neuroendocrine cells are a major source of VEGF, particularly in midgut carcinoids. This finding suggests that the presence of VEGF may be required to maintain the differentiated state of capillary vessels in these hypervascular tumours. Such secretion, in conjunction with the other growth factors synthesized by these neuroendocrine tumours, may have an important role in tumour growth.     

Somatostatin receptors. A new way to diagnosis and therapy of neuroendocrine tumors

Verification of the presence of somatostatin receptors on neuroendocrine tumour cells opened up unique diagnostic and therapeutic possibilities. Long-acting somatostatin analogues are currently used to alleviate symptoms of excessive hormone synthesis in patients with such tumours. Radiolabelled somatostatin analogues can be used both for high specificity and high sensitivity scintigraphic localisation of such tumours and for intraoperative scintillation detection. Detailed studies in patients and in tumour cells in vitro have shown 111In-octreotide uptake to be high and retention times long in tumour tissue, and have yielded evidence of intracellular localisation of the radionuclide. These findings thus showed somatostatin receptor-mediated radiotherapy to be a possible treatment alternative after close characterisation of the individual tumour. In the future, it may be possible to use other peptide receptors (e g, growth factor receptors) according to the same principles as applied in the case of somatostatin receptors.     

Aims and objectives of medical education at the Medical School of Universidad de Concepción

This study presents the first successful use of a peptidic vector, DOTATOC, labelled with the beta-emitting radioisotope yttrium-90, for the treatment of a patient with somatostatin receptor-positive abdominal metastases of a neuroendocrine carcinoma of unknown localization. Tumour response and symptomatic relief were achieved. In addition, the new substance DOTATOC was labelled with the diagnostic chemical analogue indium-111 and studied in three patients with histopathologically verified neuroendocrine abdominal tumours for its diagnostic sensitivity and compared with the commercially available OctreoScan. In all patients the kidney-to-tumour uptake ratio (in counts per pixel) was on average 1. 9-fold lower with 111In-DOTATOC than with OctreoScan. DOTATOC could be a potential new diagnostic and therapeutic agent in the management of neuroendocrine tumours.     

Cytomegalovirus colitis mimicking a colonic neoplasm or ischemic colitis 4 years after heart transplantation

Radioimmunotherapy (RIT) seems to be a realistic option for eradication of minimal residual squamous cell carcinoma of the head and neck (HNSCC), although uptake levels of radiolabelled monoclonal antibodies (MAbs) in tumour tissue vary strongly. The aim of this study was to obtain greater insight into the factors influencing the accumulation of MAbs in HNSCC. Twenty-seven HNSCC patients were injected with radiolabelled MAb E48 or U36 and underwent surgery 2 days after injection. Radioactivity was measured in tumour biopsies taken from the surgical specimen. Uptake levels were correlated with various patient, tumour and MAb characteristics, including age, sex, site, TNM stage, volume as assesssed by computed tomography or magnetic resonance imaging, degree of differentiation, antigen expression of the tumour, the particular MAb that had been injected and the MAb dose. A stepwise regression multivariate analysis showed that tumour volume is the most significant prognostic factor (P=0. 01) for MAb uptake. In conclusion, a significantly higher MAb uptake is found in small tumours as compared to larger tumours. Therefore, RIT may be particularly effective in head and neck cancer patients when used in an adjuvant setting.     

SPECT and PET in neurobiology

PET (positron emission tomography) and SPECT (single photon emission computed tomography) are isotopic methods in which the distribution is registered of radiolabelled tracers given in such small amounts that they are without effect on the organism or the organism's disposal of them. Thus, a series of important biological processes in the intact organism can be studied. The methods have been used in many disciplines but in particular for neurobiological research on the brain--e.g., the brain's regional blood circulation and mapping of the brain's functional structure. The methods have also been used in the investigation of glucose and amino acid metabolism in the brain and receptor conditions.     

Evidence for low molecular weight, non-transferrin-bound iron in rat brain and cerebrospinal fluid

Extrapolation to humans from experimental radioimmunotherapy in nude mouse xenograft models is confounded by large relative tumour size and small volume of distribution in mice allowing tumour uptake of radiolabelled antibodies unattainable in patients. Our large animal model of human tumours in cyclosporin-immunosuppressed sheep demonstrated tumour uptake of targeted radiolabelled monoclonal antibodies comparable with uptakes reported in clinical trials. Sheep immunosuppression with daily intravenous cyclosporin augmented by oral ketoconazole maintained trough blood levels of cyclosporin within the range 1000-1500 ng ml(-1). Human tumour cells were transplanted orthotopically by inoculation of 10(7) cells: SKMEL melanoma subcutaneously; LS174T and HT29 colon carcinoma into bowel, peritoneum and liver; and JAM ovarian carcinoma into ovary and peritoneum. Tumour xenografts grew at all sites within 3 weeks of inoculation, preserving characteristic morphology without evidence of necrosis or host rejection. Lymphatic metastasis was demonstrated in regional nodes draining xenografts of melanoma and ovarian carcinoma. Colonic LS1 74T xenografts produced mucin and carcinoembryonic antigen (CEA). The anti-CEA IgG1 monoclonal antibody A5B7 was radiolabelled with iodine-131 and administered intravenously to sheep. Peak uptake at 5 days in orthotopic human tumour transplants in gut was 0.027% DI g(-1) (percentage of injected dose per gram) and 0.034% DI g(-1) in hepatic metastases with tumour to blood ratios of 2-2.5. Non-specific tumour uptake in melanoma was 0.003% DI g(-1). Uptake of radiolabelled monoclonal antibody in human tumours in our large animal model is comparable with that observed in patients and may be more realistic than nude mice xenografts for prediction of clinical efficacy of radioimmunotherapy.     

Peripheral pain mechanisms

A promising new treatment for glioma involves Auger electron emitters such as 125I or 123I conjugated to deoxyuridine (IUdR). However, the presence in tumour deposits of non-proliferating cells with clonogenic potential poses a major limitation to this cycle-specific therapy. We have used multicellular tumour spheroids derived from the human glioma cell line UVW to study [125I]IUdR-targeted radiotherapy in aggregates containing cells in different proliferative states. Autoradiographic identification of labelled cells indicated that nuclear incorporation of [125I]IUdR decreased markedly with increasing size of spheroid. IUdR incorporation was maximal in the surface layer of cells and decreased with depth within spheroids. Radiopharmaceutical uptake corresponded closely to the regions of cell cycling as indicated by staining for the nuclear antigen Ki67. The uptake of drug was enhanced by increasing the duration of incubation from 52 h to 104 h. These observations suggest that significant sparing of non-cycling malignant cells would result from treatment delivered as a single injection of radiolabelled IUdR. To achieve maximal therapeutic effect. IUdR should be administered by multiple injections, by slow release from biodegradable implants or by slow-pump delivery.     

Implications of immediate-early gene induction in the brain following sexual stimulation of female and male rodents

Induction of immediate-early genes (IEGs), such as c-fos, has been widely used to mark the activation of brain regions following different types of sexual stimulation and behavior. A relatively common set of hormone-concentrating basal forebrain and midbrain structures in female and male rodents is activated by copulatory stimulation, in particular, stimulation of sensory nerves that innervate the penis or vagina/cervix, olfactory or pheromonal stimuli, and conditioned sexual incentives. These regions include the preoptic area, lateral septum, bed nucleus of the stria terminalis, paraventricular hypothalamus, ventromedial hypothalamus, medial amygdala, ventral premammillary nuclei, ventral tegmentum, central tegmental field, mesencephalic central gray, and peripeduncular nuclei. Regions that do not contain classic intracellular steroid receptors, such as the ventral and dorsal striatum or cortex, are also activated. IEGs have also been colocalized with cytoplasmic proteins like GnRH and oxytocin, and have been used in conjunction with retrograde tracers to reveal functional pathways associated with different sexual behaviors. Steroid hormones can also alter the ability of sexual stimulation to induce IEGs. Despite the many similarities, some differences in IEG induction between sexes have also been found. We review these findings and raise the question of what IEG induction in the brain actually means for sexual behavior, that is, whether it indicates the perception of sexual stimulation, commands for motor output, or the stimulation of a future behavioral or neuroendocrine event related to the consequences of sexual stimulation. To understand the role of a particular activated region, the behavioral or neuroendocrine effects of lesions, electrical stimulation, drug or hormone infusions, must also be known.     

The human placental renin-angiotensin system

The human placenta and related tissues are considered to be examples of the recently accepted local renin-angiotensin systems (RAS). The brain is another example of a system that is thought to be regulated independently of the kidney and the role of angiotensin within the CNS as a neural mediator has drawn considerable attention. It has been known for a long time that many of the neuroendocrine mediators and receptors are expressed in the placenta and it has been suggested that there are many parallels between the classical neuroendocrine system and the placental one. The present review summarizes information that components of the RAS are expressed in uteroplacental tissues, are regulated by endogenous substances, and have important biological functions within this reproductive system. A comparison of similarities and differences between the classical and the placental RAS may provide clues to functions in other endocrine and neuroendocrine systems. The major components of the placental RAS that are considered are renin, prorenin, angiotensin I, angiotensin II, angiotensin converting enzyme (ACE), angiotensin receptors, and angiotensinogen (renin substrate). The factors that regulate these components at the cellular and the nuclear level are described. It is concluded that prorenin via angiotensin-dependent and angiotensin-independent mechanisms influences functions within uteroplacental tissues. Some of these actions are direct and others are mediated by the release of different signalling molecules. These features are similar to many neuroendocrine systems and utilize some of the same messengers.     

Breath test diagnosis of Helicobacter pylori in peptic ulcer disease: a noninvasive primary care option

The role of chemotherapy in malignant neuroendocrine tumours is difficult to assess because of their rarity and variation in biological behaviour. We present a retrospective review of chemotherapy given to 18 patients with metastatic and one with locally advanced neuroendocrine tumours. There were eight poorly differentiated neuroendocrine tumours, six thyroid medullary carcinomas, two phaeochromocytomas, two pancreatic islet cell tumours and one undifferentiated neuroblastoma. Four patients were given 3-weekly dacarbazine, vincristine and cyclophosphamide (DOC) chemotherapy. In eight patients, this regimen was modified by substituting the dacarbazine and cisplatin and etoposide (OPEC). A further six patients were treated with dacarbazine reintroduced into the 3-weekly regimen (DOPEC). The remaining patient received cisplatin and etoposide. There were two complete responses (both with OPEC) and eight partial responses (two with DOC, three with OPEC and three with DOPEC). Five patients had stable disease and four progressed. Four received further chemotherapy on relapse, producing one complete and one partial response. The median response duration to initial chemotherapy was 10 months (range 3-34). The median survival was 12 months (range 1-42). The main toxicity was haematological, with grade 3-4 neutropenia in 12 patients; eight suffered episodes of sepsis. One death was treatment related. Other toxicity was mild although three patients discontinued vincristine with grade 2 neurotoxicity. The response rate and side effects of these three regimens appear comparable. We conclude that, although these patient numbers are small, combination chemotherapy produces an encouraging response rate (53%; 95% CI 30-75) in malignant neuroendocrine tumours, with acceptable toxicity.     

Endemic cretinism in Thailand: a multidisciplinary survey

For better understanding of the accumulation of trivalent radiometal tracers in tumours, studies of uptake of different 169Yb complexes into cultured normal (V79/4) and tumour (KTCTL-2) cells were performed. Cellular uptake of 169Yb3+ is dependent on both the metabolic activity of the cells and the nature of the ligand used. Uptake of 169Yb3+ from the citrate complex is an active cellular transport process but not tumour-specific. The 169Yb-aminopolycarboxylic acid complexes are taken up via a different, unknown mechanism, and in higher amounts by the tumour cells than by the V79/4 cell line, but the general features of uptake were principally the same with the normal and the tumour cells. Uptake of the complexes studied leads to a stable association of cellular components, which is a good premise for the therapeutic use of trivalent radiometals.     

Glutamate transporters are oxidant-vulnerable: a molecular link between oxidative and excitotoxic neurodegeneration?

Increasing evidence indicates that glutamate transporters are vulnerable to the action of biological oxidants, resulting in reduced uptake function. This effect could contribute to the build-up of neurotoxic extracellular glutamate levels, with major pathological consequences. Specific 'redox-sensing' elements, consisting of cysteine residues, have been identified in the structures of at least three transporter subtypes (GLT1, GLAST and EAAC1) and shown to regulate transport rate via thiol-disulphide redox interconversion. In this article, Davide Trotti, Niels Danbolt and Andrea Volterra discuss these findings in relation to the emerging view that in brain diseases oxidative and excitotoxic mechanisms might often operate in tight conjunction to induce neuronal damage. In particular, they review evidence suggesting a possible involvement of oxidative alterations of glutamate transporters in specific pathologies, including amyotrophic lateral sclerosis, Alzheimer's disease, brain trauma and ischaemia.     

Neuroendocrine characteristics of human Leydig cell tumours

The neuroendocrine nature of a subset of Leydig cells has already been established. The present investigation deals with neuroendocrine characteristics of Leydig tumour cells. A number of neuroendocrine and neuronal markers were demonstrated in Leydig cell tumours of 7 men aged 25-41 years. The following substances were immunocytochemically tested in Leydig tumour cells: the monoamine-synthesizing enzymes tyrosine hydroxylase and aromatic L-amino acid decarboxylase, the indoleamine serotonin, the calcium-binding protein parvalbumin, the microtubule associated protein-2, neurofilament protein 200, synaptophysin, neuron specific enolase, substance P and neuronal nitric oxide synthase (NOS). Compared to the normal interstitial cells beyond the tumours, all neoplastic cells showed a significantly weaker immunoreactivity for nerve cell markers as well as for testosterone and cyclic guanosine monophosphate (cGMP), which is usually accumulated by nitric oxide (NO). This provides evidence for a certain dedifferentiation of Leydig tumour cells. However, these results suggest that tumourous development of Leydig cells does not include loss of neuronal phenotype. Moreover, on the assumption that 'neuronal' Leydig cells exist beside 'non-neuronal' ones in normal testicular tissue, we propose the hypothesis that 'neuronal' Leydig cells can transform to tumour cells.     

Nuclear medicine approach for the detection and characterization of liver metastasis

Nuclear Medicine procedures include in vitro techniques able to detect and characterize liver metastases. Among the in vitro applications, circulating tumour markers level determination provides information about the response to therapy and the presence of active disease. While after a successful antineoplastic treatment the serum biological marker level usually falls, a progressive increase represents an alarm signal of occult/residual tumour or metastatic dissemination. However, this method is not able to identify the site of the lesion, therefore complementary imaging techniques are needed to confirm or exclude the diagnostic suspicion. The main advantages of the in vitro methods are the low cost and, consequently, the possibility of periodically checking of tumour activity. The in vivo nuclear medicine techniques are usually performed as second level test. In fact, they can play a role in detecting and characterizing hepatic metastases when radiological methods are inconclusive or when selected tumour seeking agents (i.e. radiolabelled antibodies, radioiodinated mIBG, Octreotide, etc.) can be used. Moreover, the introduction of PET procedures has provided physicians with a useful tool for the evaluation of tumour behaviour (glucose consumption, proteic, synthesis, receptor expression). A further significant diagnostic improvement is represented by the recent introduction of the multimodality co-registration of images (including CT scan, MRI, SPET, PET). The fusion imaging allows a superimposition of the functional and metabolic information to the morphological one.     

Influence of pH on the uptake of 5-fluorouracil into isolated tumour cells

To investigate the possible dependence of 5-fluorouracil (5FU) uptake in tumours on the intra- (pHi) and extracellular (pHe) pH, a pH gradient (deltapH) was imposed across the plasma membrane of ascites tumour cells in vitro, similar to that known to occur in some solid tumours in vivo, by incubation in media of PHe 5-8. A > or = 2:1 (intracellular/extracellular) accumulation of radiolabelled 5FU occurred after 5 min incubation of the cells with 0.5 mM 5FU at pHe of 5.0, 5.5 or 6.0. 5FU metabolism is slow under these conditions, and 5FU uptake was not affected by longer incubations up to 20 min, nor by the absence of a sodium gradient. pHi was estimated from the distribution of the weak acid, 5.5-dimethyl-2,4-oxazolidione ([14C]DMO) across the cell membrane. There was significant correlation between the intracellular/extracellular 5FU ratio and pHe (from pHe 6-8), deltapH and pHi (P < 0.02). Similar results were obtained with HT29 cells. Incubation with a drug that made plasma membranes permeable to H+ significantly decreased 5FU uptake in Lettre cells. The co-transport of 5FU may occur on a proton symport using the proton motive force of the deltapH.     

Role of the vascular component in growth of solid tumors: a historical review

Angiogenesis is a fundamental biological process by which new capillary blood vessels are formed. It is essential in many physiological conditions, such as embryonic development, ovulation and wound repair, and pathological ones, such as arthritis, diabetic retinopathy, and tumours. Solid tumours have angiogenesis capacity, and tumour growth and metastasis are angiogenesis-dependent. Neoplastic cell populations can grow to form a clinically evident tumour only if the host produces a vascular network sufficient to sustain tumour growth. Furthermore, the new blood vessels provide a gateway for tumour cells to enter the circulation and metastasize to distant sites. Tumour angiogenesis is essentially mediated by angiogenic molecules elaborated by tumour cells. We review here the most important literature on this topic and emphasize the crucial and paradigmatic role of this biological process and its relevance in a possible anti-angiogenic therapeutic approach to the treatment of solid tumours.     

Neuroendocrine tumours in various organ systems in a ten-year period

Neuroendocrine cells are present in various organ systems. These widely distributed cells as well as their histogenetically related tumours can produce various peptides and peptide hormones. From 1984 to 1993, 349 neuroendocrine tumours were found among 511,382 histological diagnoses at the Institute of Pathology of the Medical School of the Karl Franzens University in Graz. In 30% carcinoid of the appendix was diagnosed, in 16% carcinoid of the colorectum, in 9% carcinoid of the small intestine and in 2% duodenal carcinoid. Carcinoid of the stomach was detected in 14%. Seven of these cases showed microcarcinoidosis and five of them were combined with an adenocarcinoma. Carcinoid of the oesophagus was present in 1%, neuroendocrine pancreatic tumours in 6%. Neuroendocrine tumours of the bronchial system were found in 12%, medullary thyroid cancer in 5%. In 1% a Merkel-cell tumour was diagnosed. Other more rare localizations of neuroendocrine tumours were the uterus, ovary, breast, testes, epididymis, anal region and the upper respiratory tract.     

Gallium-67-citrate scanning of renal parenchymal malacoplakia

The purpose of this article is to review the potential role of nuclear medicine scanning, especially with 67Ga, in the presumptive diagnosis and clinical management of patients with renal parenchymal malacoplakia (RPMP), a rare disease associated with coliform bacterial infection of the kidney and characterized by chronic unresolving inflammatory infiltrates containing von Hansemann macrophages in the renal parenchyma. METHODS: Published cases of RPMP were collected from the archival literature by searching the MEDLINE database and by reviewing bibliographic references contained in articles on malacoplakia. Data on the clinical features and radiographic evaluation of patients with RPMP were extracted from the clinical case reports. RESULTS: Forty-three cases of RPMP published over the past 20 yr were identified. Ten of the 43 patients (23%) had 67Ga scanning as a component of their diagnostic evaluation. In all 10 patients, renal uptake of 67Ga was classified as intense. Two of those 10 patients had serial 67Ga scanning performed to assess response to antibiotic treatment; both patients exhibited decreased uptake or complete resolution of abnormal renal uptake over time, a finding also exhibited by our patient. CONCLUSION: Intense renal uptake of 67Ga, typically in the clinical setting of fever, progressive renal failure and nephromegaly, strongly supports a diagnosis of RPMP. In those patients receiving prolonged antimicrobial therapy for RPMP, resolution of abnormal 67Ga uptake over time may provide an objective endpoint for treatment.