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Tumour biology and histopathology of neuroendocrine tumours
Best Practice & Research Clinical Endocrinology & Metabolism Vol. 21, No. 1, pp. 15–31, 2007 doi:10.1016/j.beem.2007.01.004 available online at http://www.sciencedirect.com
2 Tumour biology and histopathology of neuroendocrine tumours Gu¨nter Klo¨ppel * Professor and Director Department of Pathology, University of Kiel, Michaelisstr. 11, 24105 Kiel, Germany
The tumours of the disseminated/diffuse neuroendocrine cell system are a group of neoplasms sharing uniformly appearing cells which differ from each other in their biology, prognosis and genetics. In the lung they are called carcinoid and small/large-cell neuroendocrine carcinomas. In the gastroenteropancreatic compartment they are classified as well-differentiated neuroendocrine tumours or carcinomas and poorly differentiated neuroendocrine carcinomas. Depending on their localization these neoplasms reveal distinct phenotypes with respect to pathology, immunohistochemistry, and hormonal syndromes. Their clinical behaviour – ranging from benign and low-grade to high-grade malignancy – can be predicted on the basis of clinicopathological criteria. Currently extensive work is being performed to unravel the genetic background. Key words: neuroendocrine tumours; classification; typing; staging; prognosis.
FEATURES OF NEUROENDOCRINE CELLS The neuroendocrine cell system can be divided into cell types that form glands and others that are diffusely distributed, i.e., the disseminated/diffuse neuroendocrine system. Among the first group are the cell types that compose the adenohypophysis, the parathyroids, the paraganglia and the adrenal medulla. The second group, the disseminated neuroendocrine cell types, populate the skin, thyroid, lung, thymus, pancreas, gastrointestinal tract, biliary tract and urogenital tract. In the gastrointestinal tract and pancreas, 15 cell types producing different hormones can be distinguished.1,2 Neuroendocrine cells have uniform nuclei and abundant granular or faintly staining (clear) cytoplasm. They are either present as solid or small trabecular clusters or are dispersed among other cells and are then difficult to recognize in sections stained with haematoxylin and eosin (H&E). Immunostaining enables their exact identification. At * Tel.: þ49 431 597 3400; Fax: þ49 431 597 3462. E-mail address: [email protected]. 1521-690X/$ - see front matter ª 2007 Elsevier Ltd. All rights reserved.
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the ultrastructural level they are characterized by cytoplasmic membrane-bound dense-cored secretory granules (diameter >80 nm). They also contain small clear vesicles (diameter 40–80 nm) that correspond to the synaptic vesicles of neurons. DIAGNOSTIC AND FUNCTIONAL MARKERS There are some markers that serve as general markers and others that help to distinguish pulmonary from intestinal neuroendocrine tumours (NETs). In addition, the neuroendocrine cells express a number of peptide hormones and receptors that may also serve as markers. Here only the markers that are most relevant for the diagnosis will be discussed. Cytosolic markers Neuron-specific enolase (NSE), the g-g dimer of the glycolytic enzyme enolase, is the best-known cytosolic marker. Its reactivity is unrelated to the content of secretory granules in the cells. However, the commercially available antibodies are of limited sensitivity because of unspecific staining of certain non-endocrine tissues, such as striated muscle. Moreover, it is well known that NSE has been recognized in some nonneuroendocrine tumour tissues, such as solid-pseudopapillary neoplasms of the pancreas and serous-cystic neoplasms of the pancreas. These disadvantages, therefore, suggest that NSE positivity should be used with caution as a neuroendocrine marker reaction in tumour diagnosis. Small vesicle-associated markers Synaptophysin is an integral membrane glycoprotein (molecular weight 38,000) that occurs in presynaptic vesicles of neurons and small clear vesicles of normal and neoplastic neuroendocrine cells. It is expressed independently of the other neuroendocrine markers, notably secretory granule products. Other markers related to synaptophysin are SV2 and synaptobrevin. Large secretory granule-associated markers The chromogranins constitute a family of soluble proteins located in the matrix of secretory granules of many neuroendocrine cells. Chromogranin A, the precursor of pancreastatin, is a powerful universal marker of neuroendocrine tissues and tumours. Its expression, however, depends on the cell type and also on the number of secretory granules present in the cells. Cell membrane markers The neural cell adhesion molecule (NCAM, CD56) belongs to a group of cell-surface glycoproteins involved in direct cell–cell adhesion. It is expressed by neuronal and neuroendocrine cells.3 NCAM, however, is not only present in neuroendocrine cells but may also be expressed by non-endocrine normal tissues (renal tubules and thyroid follicle cells) and neoplastic tissues (non-small cell lung carcinomas and others).
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CDX2 The transcription factor CDX2, which belongs to the homeobox genes regulating the development of the epithelium of the small and large intestine, has proved to be a very reliable marker of all NETs arising in the midgut. In addition, some well-differentiated NETs from the lung, the pancreas and the rectum were also positive, while other NETs – in particular from the stomach, the thyroid and the paraganglia – were consistently negative.4,5 Thyroid transcription factor 1 (TTF-1) TTF-1, a homeodomain containing transcription protein of the NKx2 gene family, is expressed in poorly differentiated neuroendocrine carcinomas of the lung (85%) and also in some pulmonary carcinoids (up to 60%), but not in well and poorly differentiated NETs of the gastrointestinal tract.6,7 Somatostatin receptors Many NETs express somatostatin receptors. Five types of these receptors have been identified (e.g. SSTR1–5).8 They can be demonstrated by autoradiography9, by scintigraphic imaging (octreoscan)10, or by immunohistochemistry.11,12 The immunohistochemical expression of SSTR2, which shows a membranous pattern, correlates closely with the octreoscan signals.10 Serotonin-producing NETs and gastrinomas are more commonly positive for SSTR2/5 (up to 90%) than insulinomas are (up to 60%).11,12 Hormones The various cell types of the neuroendocrine cell system are identified by their specific cellular products, such as peptides and biogenic amines, usually acting as hormones or neurotransmitters. By means of in-situ hybridization their expression can be traced back to the gene level. Hormones that are highly specific for certain tumour localizations are luteinizing hormone (LH), follicle-stimulating hormone (FSH) and prolactin for pituitary adenomas, calcitonin for medullary thyroid carcinoma, parathyroid hormone for parathyroid adenoma, insulin and glucagon as well as pancreatic polypeptide for pancreatic tumours, serotonin and substance P for ileal and appendiceal neuroendocrine tumours, and vasoactive monoamine transporter 2 (VMAT2) for ECLomas. CLASSIFICATION PRINCIPLES The WHO classification of ‘endocrine tumours’ includes neoplasms originating from endocrine glands such as the adrenal phaeochromocytomas, the pituitary adenomas, nerve elements such as paragangliomas, ganglioneuromas and neuroblastomas, or from elements of the disseminated/diffuse endocrine system such as gastroenteropancreatic endocrine tumours. Table 1 lists the two groups of neuroendocrine cell types and the names given to the respective tumours. The neoplasms originating from the disseminated neuroendocrine cell system that are recognized by the WHO classification comprise pure endocrine tumours and mixed endocrine/exocrine tumours.8 For pure gastroenteropancreatic neuroendocrine
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Table 1. Neuroendocrine cell system and tumour classification. Gland forming Pituitary Parathyroid Paraganglia Adrenal medulla
Adenoma Adenoma/carcinoma Paraganglioma Phaeochromocytoma
Disseminated Gastrointestinal tract Endocrine pancreas Biliary tract Respiratory tract Thymus Thyroid C cells Urogenital tract Skin
Neuroendocrine tumour/carcinoma Neuroendocrine tumour/carcinoma Neuroendocrine tumour/carcinoma Carcinoid, neuroendocrine carcinoma Carcinoid Medullary thyroid carcinoma Carcinoid Merkel cell carcinoma
tumours a uniform scheme of classification identifying three categories is applied for all anatomical sites: 1. well-differentiated endocrine tumours, with benign (1.1) or uncertain behaviour (1.2) at the time of diagnosis; 2. well-differentiated endocrine carcinomas with low-grade malignant behaviour; 3. poorly differentiated endocrine carcinomas, with high-grade malignant behaviour. Because of their highly aggressive behaviour, the neoplasms of category 3 differ profoundly from those of categories 1 and 2 and require a special therapeutic approach. Conventional histopathological and cytological assessment of growth patterns and cellular features of well-differentiated neuroendocrine tumours are often of little help in predicting their functional behaviour and degree of malignancy. In contrast, poorly differentiated NETs that are composed of cells displaying severe nuclear atypia, a high mitotic index and few secretory granules are invariably high-grade malignancies. They occur frequently in the lung, but have also been observed in the oesophagus, stomach, ampulla of Vater, colon, pancreas, uterine cervix and prostate. In contrast to well-differentiated neuroendocrine neoplasms, such poorly differentiated tumours usually lack hormonal syndromes. Mixed exocrine/neuroendocrine tumours have been described particularly in the appendix as goblet-cell carcinoids. Recently, adenocarcinomas showing scattered neuroendocrine cells have been reported in increasing numbers in various locations such as the stomach, pancreas, colon and prostate. However, if truly mixed tumours are defined as neoplasms in which the exocrine and endocrine cell populations are intimately mixed and the endocrine cells comprise about one third to one half of the tumour tissue, thus clearly being distinguishable from tumours with a diffusely scattered subpopulation of neoplastic neuroendocrine cells and collision tumours, only few tumours qualify as truly mixed exocrine/neuroendocrine neoplasms.13 In general, mixed exocrine/neuroendocrine neoplasms should be kept separate from neuroendocrine tumours, because the biological behaviour of the former appears to be dictated by the differentiation of the exocrine cell compartment.
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Classification in practice Gastroenteropancreatic NETs and NETs of the respiratory system form the largest group of NETs. For the practical diagnosis of these NETs, the criteria provided by the respective WHO classification and listed in Tables 2, 3, and 4 can be used as a checklist with which to appropriately classify an individual tumour.14 Naturally this catalogue of criteria needs to be extended and refined, because in particular the group of gastrointestinal and pancreatic tumours of questionable malignant potential (‘benign or malignant behaviour possible’) is too large to be accepted in the long run in this indefinite form. As there is an increasing demand for a standard in the stratification and treatment of patients with gastroenteropancreatic NETs, the European Neuroendocrine Tumour Society (ENETS)15 recently developed guidelines which were supplemented by a proposal for a tumour/nodes/metastases (TNM) classification. This TNM staging proposal considered only the NETs of the foregut16 and will soon be joined by a classification proposal for the midgut and hindgut NETs. Apart from the TNM staging proposal, a working formulation for the grading of gastroenteropancreatic NETs based on mitotic count and Ki-67 index has been suggested. Both the TNM staging proposal and the grading system still need to be validated. NETS BY LOCALIZATION, BIOLOGY, PATHOLOGY AND PROGNOSIS Most NETs occur in the gastrointestinal tract, the pancreas (including the bile-duct system), followed by the lung, the head and neck, the thymus, the urogenital system and the skin. In 1963 Williams and Sandler17 classified the gastroenteropancreatic NETs into foregut (stomach, pancreas, duodenum and upper jejunum), midgut (lower Table 2. WHO criteria for diagnosis of neuroendocrine tumours of the lung. Typical carcinoid Well-differentiated neuroendocrine neoplasm with <2 mitoses per 2 mm2 (10 HPF), lacking necrosis, and 0.5 cm Atypical carcinoid Well-differentiated neuroendocrine neoplasm with 2e10 mitoses per 2 mm2 (10 HPF) and/or focal necrosis Large-cell neuroendocrine carcinoma Poorly differentiated neuroendocrine carcinoma (organoid nesting, palisading, rosettes, trabeculae) with high mitotic rate (11 per 2 mm2 and 10 HPF; median of 20 per 2 mm2 and 10 HPF) and necrosis (often large zones). Cytological features: large cell size, low nuclear/cytoplasmic ratio, vesicular, coarse or fine chromatin, and/or frequent nucleoli Small-cell carcinoma Poorly differentiated neuroendocrine carcinoma. Cytological features: small cells with scant cytoplasm, fine granular chromatin, absent or faint nucleoli, high mitotic rate (11 per 2 mm2 and 10 HPF; median of 80 per 2 mm2 and 10 HPF), frequent necrosis often in large zones HPF, high-power field. Modified from Travis et al (2004, Pathology and Genetics of Tumours of the Lung, Pleura, Thymus and Heart. WHO classification of tumours. Lyon: IARC Press).
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Table 3. Criteria for assessing the prognosis of neuroendocrine tumours of the gastrointestinal tract. Biological behaviour
Metastases Invasion of Histological Tumour Angioinvasion size muscularis differentiation propriaa
Benign
e
e
Benign or lowgrade malignant Low-grade malignant High-grade malignant
e
e
þ
þb
þ
þ
a b
Welldifferentiated Welldifferentiated Welldifferentiated Poorly differentiated
Ki-67 index
Hormonal syndrome
1 cma
e
<2%
ea
1e2 cm
/þ
<2%
e
>2 cm
þ
2e20%
þ
Any
þ
>20%
e
Exception: malignant duodenal gastrinomas are usually <1 cm and confined to the submucosa. Exception: benign NETs of the appendix usually invade the muscularis propria.
jejunum, ileum, appendix, caecum) and hindgut (colon, rectum) tumours, with considerable clinicopathological differences between the three groups. However, in the case of foregut tumours the usefulness of such a classification in practical diagnostic work is limited by its failure to characterize individual tumour entities with well-defined histological, hormonal and/or clinicopathological profiles. In the subsequent section the NETs are therefore classified and discussed according to their specific localizations. Not included in this discussion are the NETs of the head and neck region18–20, the thymus21,22, the urogenital system, and the skin. Respiratory tract The classification of NETs of the lower respiratory tract (i.e. the lung) includes typical carcinoid, atypical carcinoid, small-cell carcinoma and large-cell neuroendocrine carcinoma (Table 2). Typical and atypical carcinoids and large-cell neuroendocrine carcinomas make up only 2–3% of lung tumours, while small-cell carcinomas account for 15–25% of all lung malignancies. Typical carcinoids – i.e. well-differentiated NETs – are characterized by low mitotic activity (<2 mitoses per 2 mm2). They have an excellent prognosis after radical Table 4. Criteria for assessing the prognosis of neuroendocrine tumours of the pancreas. Biological behaviour Benign Benign or lowgrade malignant Low-grade malignant High-grade malignant a b c
Metastases Invasiona
Histological differentiation
Tumour Angioinvasion Ki-67 Hormonal size index syndrome
e e
e e
Well-differentiated Well-differentiated
1 cm >2 cm
e /þ
<2% <2%
/þb /þc
þ
þ
Well-differentiated
>4 cm
þ
2e20%
þc
þ
þ
Poorly differentiated
Any
þ
>20%
e
Invasion of adjacent organs (e.g. duodenum, stomach). Insulinomas. Insulinomas and other functioning tumours (e.g. glucagonomas).
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surgical treatment (90–98% 5-year survival). The diameter of these tumours is usually <3 cm, and the histological patterns range from adenoid to solid, trabecular, paraganglioid and to spindle cell-like (Figure 1). Atypical carcinoids are histologically similar to typical carcinoids (Figure 2) but have a mitotic activity of 2–10 mitoses per 2 mm2 and focal necrosis. The tumour cells do not show signs of crushing or nuclear moulding, which are characteristic of small-cell carcinoma. The 5-year survival rate is 60% after radical surgical treatment. Small-cell lung carcinoma is the most clinically distinctive type of neuroendocrine lung carcinoma. The mitotic activity in these tumours is high (>11 mitoses per 2 mm2 in 10 high-power fields). In addition, these carcinomas generally show more extensive necrosis than atypical carcinoids. The differentiation of small-cell carcinoma from large-cell neuroendocrine carcinoma is based on cell size, abundance of cytoplasm, prominent nucleoli and vesicular or coarse chromatin. The prognosis of large-cell neuroendocrine carcinomas is similar to that of smallcell carcinoma. However, while small-cell carcinomas usually respond well to chemotherapy, large-cell neuroendocrine carcinomas do not. Bronchial carcinoids, when centrally located, may lead to mechanical obstruction of the main bronchus. They may also secrete adrenocorticotropic hormone (ACTH), thereby producing a Cushing syndrome. A carcinoid syndrome due to serotonin secretion is very rare and occurs only when there are widespread metastases. Both typical and atypical carcinoids can occur in patients with multiple endocrine neoplasia type 1 syndrome (MEN1). Cushing syndrome may also be seen in small-cell carcinomas. Stomach Four types of NET can be distinguished in the stomach23,24; type 1 is by far the most frequent type (70–80%)25, whereas types 2, 3 and 4 are rare. Type 1 NETs are usually small, multifocal, broad-based, round, polypoid mucosal tumours measuring 0.5–1 cm in diameter, typically occurring in the corpus of the stomach. Histologically, these well-differentiated tumours are found in the mucosa and
Figure 1. Typical carcinoid of the lung: solid clusters of monomorphic cells with well-developed cytoplasm.
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Figure 2. Atypical carcinoid of the lung: monomorphic, densely packed cell population with some spindlecell areas.
submucosa and are composed of intensely chromogranin A-positive ECL (enterochromaffin-like) cells, best recognized by the marker VMAT2 (vesicular monoamine transporter 2).26,27 They always develop secondary to autoimmune chronic atrophic corpus gastritis (CAG), in which the loss of parietal cells leads to insufficient production of intrinsic factor (triggering pernicious anaemia) and of hydrochloric acid (causing achlorhydria). This stimulates the antral G cells to produce gastrin, causing persistent hypergastrinaemia that is assumed to promote the growth of the ECL cells of the corpus mucosa so that diffuse to micronodular ECL-cell hyperplasia develops, out of which – after a latent period of many years – the above-described multiple ECL tumours arise.28 Clinically these tumours are not associated with any hormonal symptoms and are mainly found in women. The prognosis of these tumours is good, because they are generally so small that they can be removed endoscopically.29 Regional lymph-node metastases seem to occur only in very rare cases in which the tumours are >2 cm in size and infiltrate the muscularis propria. Type 2 gastric NETs occur in association with MEN1, in the course of which a Zollinger–Ellison syndrome (ZES) has developed. Like type 1 NETs, they are usually multifocal and found in the corpus of the stomach associated with ECL-cell hyperplasia. The tumours are generally <1.5 cm and limited to the mucosa and submucosa. If angioinvasion is found or the tumour is >2 cm and/or has invaded the muscularis propria, it is likely that the tumour has metastasized, as happens in approximately 30% of cases. The genetic changes associated with MEN1 (mutations in the MEN1 gene on chromosome 11q13) in connection with gastrinoma-related hypergastrinaemia are probably the basis on which the tumour develops, because so far the development of tumours of the same type in ZES has not been confirmed. Type 3, or sporadic, NETs of the stomach are not associated with either CAG or MEN1, and show a predilection for the male sex. These tumours are solitary and do not have any special localization in the stomach. In one third of the cases the tumour is already >2 cm at the time of diagnosis. Histologically, the tumours are predominantly well differentiated and are composed of ECL cells (Figure 3). Tumours with EC (serotonin) cells or gastrin cells, by contrast, are extremely rare.24 If the tumour is >2 cm,
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Figure 3. Sporadic well-differentiated neuroendocrine carcinoma in the antrum. Preserved antral mucosa on top of the tumour, which is below the muscularis mucosae.
has invaded the muscularis propria, and angioinvasion is recognizable, it is likely that there will be metastases.29 In such cases surgical treatment is required. Type 4 gastric NETs correspond to an undifferentiated gastric carcinoma. These tumours are more common in men than in women, and there is no relationship to CAG or MEN1. At the time of diagnosis most of the tumours are already in an advanced stage (tumour diameter >4 cm) and show extensive metastases. Immunocytochemically, the tumour cells are positive for synaptophysin, with only slight focal positivity for chromogranin A. Type 4 NETs of the stomach are treated like normal gastric carcinomas. Duodenum and upper jejunum Five types of duodenal neuroendocrine tumours can currently be distinguished. These include duodenal gastrinomas, which make up approximately two thirds of all duodenal NETS, duodenal somatostatin-producing tumours, non-functioning (i.e. not causing a hormonal syndrome) serotonin-, gastrin- or calcitonin-producing tumours, poorly differentiated, predominantly ampullary neuroendocrine carcinomas and duodenal gangliocytic paragangliomas.28,30,31 Duodenal gastrinomas are either sporadic or associated with MEN1 and are combined with a ZES.32,33 In both situations these gastrinomas are usually not bigger than 1 cm (Figure 4), are located predominantly in the upper part of the duodenum, and histologically show a trabecular/pseudoglandular pattern. If associated with MEN1, they are usually multiple, in contrast to sporadic gastrinomas. In spite of their small size and the fact that they are limited to the duodenal mucosa and submucosa, at the time of diagnosis metastases are often found in the regional lymph nodes. These metastases are often much larger than the primary tumour and may erroneously be considered to be pancreatic tumours, especially if they are located close to the pancreas.34 This peculiarity is probably the reason why earlier authors spoke of primary
24 G. Klo¨ppel
Figure 4. Minigastrinoma in the duodenal mucosa (arrows). The inset shows the histology of the tumour.
lymph-node gastrinomas, and why many more pancreatic gastrinomas used to be diagnosed than are today. Whereas metastasis to the regional lymph nodes occurs at an early stage, liver metastases appear to be a relatively late occurrence. In the case of pancreatic gastrinomas (see below), which are practically all sporadic and hence not associated with MEN1, liver metastases seem to occur earlier than in duodenal gastrinomas.34,35 Duodenal somatostatin-producing tumours account for approximately 15% of all duodenal NETs. Their preferential localization is in the region of the papilla of Vater or periampullary. If the muscularis propria is invaded, metastasis to the paraduodenal lymph nodes has probably occurred. Histologically, the tumours typically show a glandular pattern with psammoma bodies. They are not associated with any hormonal syndrome, but often occur in patients with neurofibromatosis type 1. In this situation a bilateral phaeochromocytoma can simultaneously develop. Non-functioning duodenal NETs usually consist of serotonin-producing cells. Occasionally there are also tumours with gastrin- or calcitonin-positive cells. The prognosis of this group of non-functioning tumours is much more favourable than ZES-associated
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gastrinomas or ampullary somatostatin-producing tumours. Metastases are not to be expected until the tumour extends beyond the submucosa. Poorly differentiated duodenal carcinomas occur primarily in the region of the papilla of Vater. They are hormonally inactive. At the time of diagnosis advanced metastasis into the regional lymph nodes and the liver has usually occurred. Duodenal gangliocytic paragangliomas occur in the vicinity of the papilla of Vater. Although the tumours are often >2 cm and infiltrate into the muscularis propria, they generally follow a benign course. Histologically, they reveal a gangliocytic component in addition to well-differentiated neuroendocrine cells, and immunocytochemically they usually express somatostatin, pancreatic polypeptide and S-100.
Appendix The NETs of the appendix are, along with ileal NETs, the most frequent GEP NETs. Their prognosis, however, is much more favourable than that of ileal NETs. They occur primarily in the tip of the appendix and almost without exception they invade the muscularis propria and, to a greater or lesser degree, the adjoining fatty tissue of the mesoappendix. Nevertheless, regional lymph-node metastases seem to be rare before the tumour reaches a size of approximately 2.0 cm. Histologically and immunocytochemically, the NETs of the appendix are comparable to the ileal NETs with their solid tumour cell formations and their positivity for serotonin and substance P. They must be distinguished from the very rare so-called goblet-cell carcinoids, which represent mixed exocrine/endocrine tumours, because the prognosis of the latter is less favourable.
Ileum These frequent NETs develop quite preferentially in the terminal ileum and occasionally in the immediately adjacent caecum, including the ileocaecal valve. Histologically, they show a solid (‘island-like’) pattern. At the time of diagnosis they are commonly >2 cm and have invaded the muscularis propria. This means that they have already metastasized to the regional lymph nodes (Figure 5). In up to 40% of cases the tumours may be multiple. Immunocytochemically, the cells contain serotonin and substance P, kallikrein and catecholamine. Approximately 20% of the patients with ileal NETs have liver metastases (Figure 6) in addition to regional lymph-node metastases. Only in these patients does a carcinoid syndrome (flush, diarrhoea and endocardial fibrosis) develop, because serotonin is inactivated by the liver and only enters the circulatory system if there are liver metastases.
Colon NETs of the colon are very rare. Histologically, they are poorly differentiated neuroendocrine carcinomas, almost all of which have already metastasized at the time of diagnosis and therefore have a poor prognosis.36 Immunocytochemically, they are generally synaptophysin-positive and may contain single serotonin- and somatostatinpositive cells.
26 G. Klo¨ppel
Figure 5. Macroscopy of a metastasizing ileal neuroendocrine carcinoma: arrows indicate the primary tumours. In the mesenterium is a large lymph-node metastasis.
Rectum Rectal NETs are much more frequent and their prognosis is much better than that of NETs of the colon. They comprise around 10% of all GEP NETs. Most appear during endoscopy as small (<1 cm) movable submucosal tumours. Metastases are likely in tumours that are 2 cm and if they invade the muscularis propria. Immunocytochemically, they are positive for glucagon, glicentin and/or pancreatic polypeptide (PP) and are usually negative for chromogranin. In this portion of the gut poorly differentiated neuroendocrine carcinomas are the exception. They have as poor a prognosis as those in the colon and other portions of the gut.
Figure 6. Liver metastasis of an ileal well-differentiated neuroendocrine carcinoma.
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Pancreas Most NETs of the pancreas are well differentiated. Of these, 50–60% are functionally active, i.e., due to largely uncontrolled secretion of insulin, gastrin, vasoactive intestinal polypeptide (VIP), glucagon, or other even rarer hormones such as ACTH or growth hormone (GH), they can induce characteristic syndromes (hypoglycaemia syndrome, Zollinger–Ellison syndrome, Verner–Morrison syndrome, glucagonoma syndrome, Cushing’s syndrome, acromegaly).37 Depending on the predominant hormone secreted, these tumours are referred to as insulinomas, gastrinomas, VIPomas, glucagonomas, etc. Pancreatic endocrine tumours without hormonal symptoms are observed more frequently than previously, though this probably does not reflect an actual increase in frequency but rather improved diagnostic methods and an increased resection rate. These non-functioning NETs are either incidental findings or are discovered along with general neoplastic symptoms, usually, however, because of local symptoms. Pancreatic neuroendocrine tumours are extremely rare in childhood, but in adults they occur in all age groups and in males and females with equal frequency. Macroscopically, they are well-demarcated, usually solitary, round tumours with a diameter of 1–4 cm, and they can occur in all parts of the pancreas. In the case of a hormonal syndrome, the hormone causing the syndrome can be detected immunocytochemically. Although the pancreatic NETs are histologically well differentiated, they are frequently malignant, with the exception of insulinomas. This hold particularly for gastrinomas, VIPomas, glucagonomas and non-functioning tumours. The most important criteria of malignancy are – apart from metastases to the regional lymph nodes and the liver or invasion of adjacent organs – a tumour size of >2 cm, angioinvasion, and proliferative activity of >2% (Figure 7).38,39 Recent studies have shown that angioinvasion plays a more important role than was assumed in the classification presented here.40 It is therefore recommended that a tumour be considered malignant if the presence of angioinvasion has been verified, even if no other criteria of malignancy are present.
Figure 7. Well-differentiated neuroendocrine carcinoma of the pancreas showing angioinvasion.
28 G. Klo¨ppel
Among the functioning tumours, insulinomas are most frequent; 95% of them are 1–2 cm in size and benign. Multiple insulinomas and insulinomas associated with MEN1 are observed in approximately 10% of the cases. Compared with solitary or sporadic insulinomas, their rate of malignancy is not increased. Histologically, in approximately 5% of insulinomas amyloid deposits are found in the tumour. Immunocytochemically, insulin and proinsulin can be detected in varying ratios, depending on the grade of differentiation of the tumour41, which is also reflected in the secretory behaviour.42 Pancreatic gastrinomas are usually >2 cm and therefore in 60% of the cases they have already metastasized at the time of diagnosis. In contrast to duodenal gastrinomas, they rarely occur in the context of MEN1.34 Included among the rare tumours with hormonal syndromes are VIPomas, glucagonomas, ACTH-producing tumours and growth-hormone-producing tumours. At the time of diagnosis most of these tumours are >2 cm and have already metastasized. This is also true of non-functioning tumours.
GENETIC PROFILES Since the traditional morphological criteria have largely been exhausted, intensive work is currently being done on the molecular characterization and differentiation of the GEP NETs. These studies have identified the gene responsible for MEN1 on chromosome 11q13, which is also mutated in up to 40% of sporadic GEP NETs.43 By means of studies of comparative genomic hybridization (CGH) and allelic loss (LOH), a large number of genomic regions with loss or gain of genetic material have been detected.44,45 In this manner changes are being identified that may play a role in tumour progression. Such studies have also confirmed that NETs in different localizations are genetically independent tumours. Hence foregut NETs often show loss of 11q. This distinguishes them from NETs of the midgut and hindgut46, which frequently show losses on chromosome 18q.47,48 Among the lung NETs, the welldifferentiated NETs (i.e. typical and atypical carcinoids) constitute a family differing from small- and large-cell neuroendocrine carcinomas, because abnormalities in genetic markers such as p53, RB loss and cyclin D1 are much more common in the poorly differentiated NETs. On the basis of these results an intensive search is currently under way for genetic markers or marker constellations that will enable us to better predict the biological behaviour of the group of GEP NETs of uncertain malignant potential.
SUMMARY Most neuroendocrine tumours occur in the gastrointestinal tract and pancreas, the lung being the next most frequent location. They are either well or poorly differentiated and constitute two families rather than the two extremes of one spectrum. In addition, they reveal biological heterogeneity depending on localization and function. For the practical diagnosis, the criteria listed in Tables 2–4 can be used as a checklist with which to appropriately classify an individual tumour. Recently, a TNM staging system for foregut NETs, including a grading system, has been proposed and is currently under validation.16
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Practice points exact clinicopathological classification determines prognosis the various tumour entities have different biologies and require individual treatments the proliferative activity of an individual tumour determines it natural course and may also correlate with its responsiveness to chemotherapy
Research agenda validation of the classification protocols is needed treatment response, tumour type and tumour proliferative activity need to be documented genetic profiling of the various entities needs to be continued
REFERENCES 1. Klo¨ppel G. Neuroendokrinen Tumoren des Gastrointestinaltrakts. Der Pathologe 2003; 24: 287–296. 2. Klo¨ppel G. Tumoren des endokrinen Pankreas. Der Pathologe 2003; 24: 265–271. 3. Lloyd RV. Practical markers used in the diagnosis of neuroendocrine tumors. Endocrine Pathology 2003; 14: 293–301. 4. Erickson LA, Papouchado B, Dimashkieh H et al. Cdx2 as a marker for neuroendocrine tumors of unknown primary sites. Endocrine Pathology 2004; 15: 247–252. 5. La Rosa S, Rigoli E, Uccella S et al. CDX2 as a marker of intestinal EC-cells and related well-differentiated endocrine tumors. Virchows Archiv 2004; 445: 248–254. 6. Cai YC, Banner B, Glickman J & Odze RD. Cytokeratin 7 and 20 and thyroid transcription factor 1 can help distinguish pulmonary from gastrointestinal carcinoid and pancreatic endocrine tumors. Human Pathology 2001; 32: 1087–1093. 7. Sturm N, Rossi G, Lantuejoul S et al. Expression of thyroid transcription factor-1 in the spectrum of neuroendocrine cell lung proliferations with special interest in carcinoids. Human Pathology 2002; 33: 175–182. 8. Volante M, Bozzalla-Cassione F & Papotti M. Somatostatin receptors and their interest in diagnostic pathology. Endocrine Pathology 2004; 15: 275–292. 9. Reubi JC, Ha¨cki WH & Lamberts SW. Hormone-producing gastrointestinal tumors contain a high density of somatostatin receptors. The Journal of Clinical Endocrinology and Metabolism 1987; 65: 1127–1134. 10. Lamberts SW, Hofland LJ, van Koetsveld PM et al. Parallel in vivo and in vitro detection of functional somatostatin receptors in human endocrine pancreatic tumors: consequences with regard to diagnosis, localization, and therapy [see comments]. The Journal of Clinical Endocrinology and Metabolism 1990; 71: 566–574. 11. Kulaksiz H, Eissele R, Ro¨ssler D et al. Identification of somatostatin receptor subtypes 1, 2A, 3 and 5 in neuroendocrine tumours with subtype specific antibodies. Gut 2002; 50: 52–60. 12. Papotti M, Bongiovanni M, Volante M et al. Expression of somatostatin receptor types 1-5 in 81 cases of gastrointestinal and pancreatic endocrine tumors. A correlative immunohistochemical and reversetranscriptase polymerase chain reaction analysis. Virchows Archiv 2002; 440: 461–475. 13. Volante M, Rindi G & Papotti M. The grey zone between pure (neuro)endocrine and non-(neuro)endocrine tumors: a comment on concepts and classification of mixed exocrine-endocrine neoplasms. Virchows Archiv 2006; 449: 499–506.
30 G. Klo¨ppel *14. Klo¨ppel G, Perren A & Heitz PU. The gastroenteropancreatic neuroendocrine cell system and its tumors. The WHO classification. Annals of the New York Academy of Sciences 2004; 1014: 13–27. *15. Plo¨ckinger U, Rindi G, Arnold R et al. Guidelines for the diagnosis and treatment of neuroendocrine gastrointestinal tumours. A consensus statement on behalf of the European Neuroendocrine Tumour Society (ENETS). Neuroendocrinology 2004; 80: 394–424. *16. Rindi G, Klo¨ppel G, Ahlman H et al. TNM staging of foregut (neuro)endocrine tumors: a consensus proposal including a grading system. Virchows Archiv 2006; 449: 395–401. 17. Williams ED & Sandler M. The classification of carcinoid tumours. Lancet 1963; I: 238–239. 18. Riddle PJ, Font RL & Zimmerman LE. Carcinoid tumors of the eye and orbit: a clinicopathologic study of 15 cases, with histochemical and electron microscopic observations. Human Pathology 1982; 13: 459–469. *19. Mills SE. Neuroendocrine tumors of the head and neck: a selected review with emphasis on terminology. Endocrine Pathology 1996; 7: 329–343. 20. Ferlito A, Barnes L, Rinaldo A et al. A review of neuroendocrine neoplasms of the larynx: update on diagnosis and treatment. The Journal of Laryngology and Otology 1998; 112: 827–834. 21. Wick MR & Rosai J. Neuroendocrine neoplasms of the thymus. Pathology, Research and Practice 1988; 183: 188–199. 22. Moran CA & Suster S. Neuroendocrine carcinomas (carcinoid tumor) of the thymus. A clinicopathologic analysis of 80 cases. American Journal of Clinical Pathology 2000; 114: 100–110. 23. Bordi C, Azzoni C, D’Adda T et al. Neuroendokrine Tumoren des Magens. Der Pathologe 1997; 18: 313–321. 24. Klo¨ppel G & Clemens A. The biological relevance of gastric neuroendocrine tumors. The Yale Journal of Biology and Medicine 1996; 69: 69–74. *25. Rindi G, Luinetti O, Cornaggia M et al. Three subtypes of gastric argyrophil carcinoid and the gastric neuroendocrine carcinoma: a clinicopathologic study. Gastroenterology 1993; 104: 994–1006. 26. Eissele R, Anlauf M, Scha¨fer MKH et al. Expression of vesicular monoamine transporters in endocrine hyperplasia and endocrine tumors of the oxyntic stomach. Digestion 1999; 60: 428–439. 27. Rindi G, Paolotti D, Fiocca R et al. Vesicular monoamine transporter 2 as a marker of gastric enterochromaffin-like cell tumors. Virchows Archiv 2000; 436: 217–223. *28. Bordi C, D’Adda T, Azzoni C et al. Gastrointestinal endocrine tumors: recent developments. Endocrine Pathology 1998; 9: 99–115. 29. Rappel S, Altendorf-Hofmann A & Stolte M. Prognosis of gastric carcinoid tumours. Digestion 1995; 56: 455–462. *30. Capella C, Heitz PU, Ho¨fler H et al. Revised classification of neuroendocrine tumours of the lung, pancreas and gut. Virchows Archiv 1995; 425: 547–560. 31. Klo¨ppel G, Heitz PU, Capella C & Solcia E. Pathology and nomenclature of human gastrointestinal neuroendocrine (carcinoid) tumors and related lesions. World Journal of Surgery 1996; 20: 132–141. *32. Pipeleers-Marichal M, Somers G, Willems G et al. Gastrinomas in the duodenums of patients with multiple endocrine neoplasia type 1 and the Zollinger–Ellison syndrome. The New England Journal of Medicine 1990; 322: 723–727. 33. Pipeleers-Marichal M, Donow C, Heitz PU & Klo¨ppel G. Pathologic aspects of gastrinomas in patients with Zollinger–Ellison syndrome with and without multiple endocrine neoplasia type I. World Journal of Surgery 1993; 17: 481–488. 34. Donow C, Pipeleers-Marichal M, Schro¨der S et al. Surgical pathology of gastrinoma. Site, size, multicentricity, association with multiple endocrine neoplasia type 1, and malignancy. Cancer 1991; 68: 1329– 1334. 35. Weber HC, Venzon DJ, Lin JT et al. Determinants of metastatic rate and survival in patients with Zollinger–Ellison syndrome: a prospective long-term study. Gastroenterology 1995; 108: 1637–1649. 36. Grabowski P, Scho¨nfelder J, Ahnert-Hilger G et al. Expression of neuroendocrine markers: a signature of human undifferentiated carcinoma of the colon and rectum. Virchows Archiv 2002; 441: 256–263. 37. Klo¨ppel G, In’t Veld PA, Komminoth P & Heitz PU. The endocrine pancreas. In Kovacs K & Asa SL (eds). Functional Endocrine Pathology. 2nd ed. Boston: Blackwell, 1998, pp. 415–487. *38. Solcia E, Klo¨ppel G, Sobin LH & (In collaboration with 9 pathologists from 4 countries). In Histological typing of endocrine tumours. WHO international histological classification of tumours. 2nd ed. Berlin: Springer; 2000.
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31
39. Heymann MF, Joubert M, Nemeth J et al. Prognostic and immunohistochemical validation of the Capella classification of pancreatic neuroendocrine tumours: an analysis of 82 sporadic cases. Histopathology 2000; 36: 421–432. *40. La Rosa S, Sessa F, Capella C et al. Prognostic criteria in nonfunctioning pancreatic endocrine tumours. Virchows Archiv 1996; 429: 323–333. 41. Roth J, Klo¨ppel G, Madsen OD et al. Distribution patterns of proinsulin and insulin in human insulinomas: an immunohistochemical analysis in 76 tumors. Virchows Archiv. B: Cell Pathology 1992; 63: 51–61. 42. Creutzfeldt W, Arnold R, Creutzfeldt C et al. Biochemical and morphological investigations of 30 human insulinomas. Correlation between the tumour content of insulin and proinsulin-like components and the histological and ultrastructural appearance. Diabetologia 1973; 9: 217–231. 43. Goebel SU, Heppner C, Burns AL et al. Genotype/phenotype correlation of multiple endocrine neoplasia type 1 gene mutations in sporadic gastrinomas. The Journal of Clinical Endocrinology and Metabolism 2000; 85: 116–123. 44. Barghorn A, Komminoth P, Bachmann D et al. Deletion at 3p25.3-p23 is frequently encountered in endocrine pancreatic tumours and is associated with metastatic progression. Journal of Pathology 2001; 194: 451–458. 45. Speel EJ, Scheidweiler AF, Zhao J et al. Genetic evidence for early divergence of small functioning and nonfunctioning endocrine pancreatic tumors: gain of 9Q34 is an early event in insulinomas. Cancer Research 2001; 61: 5186–5192. 46. D’Adda T, Pizzi S, Azzoni C et al. Different patterns of 11q allelic losses in digestive endocrine tumors. Human Pathology 2002; 33: 322–329. 47. To¨nnies H, Toliat MR, Ramel C et al. Analysis of sporadic neuroendocrine tumours of the enteropancreatic system by comparative genomic hybridisation. Gut 2001; 48: 536–541. 48. Kyto¨la¨ S, Ho¨o¨g A, Nord B et al. Comparative genomic hybridization identifies loss of 18q22-qter as an early and specific event in tumorigenesis of midgut carcinoids. American Journal of Pathology 2001; 158: 1803–1808.
2 Tumour biology and histopathology of neuroendocrine tumours Gu¨nter Klo¨ppel * Professor and Director Department of Pathology, University of Kiel, Michaelisstr. 11, 24105 Kiel, Germany
The tumours of the disseminated/diffuse neuroendocrine cell system are a group of neoplasms sharing uniformly appearing cells which differ from each other in their biology, prognosis and genetics. In the lung they are called carcinoid and small/large-cell neuroendocrine carcinomas. In the gastroenteropancreatic compartment they are classified as well-differentiated neuroendocrine tumours or carcinomas and poorly differentiated neuroendocrine carcinomas. Depending on their localization these neoplasms reveal distinct phenotypes with respect to pathology, immunohistochemistry, and hormonal syndromes. Their clinical behaviour – ranging from benign and low-grade to high-grade malignancy – can be predicted on the basis of clinicopathological criteria. Currently extensive work is being performed to unravel the genetic background. Key words: neuroendocrine tumours; classification; typing; staging; prognosis.
FEATURES OF NEUROENDOCRINE CELLS The neuroendocrine cell system can be divided into cell types that form glands and others that are diffusely distributed, i.e., the disseminated/diffuse neuroendocrine system. Among the first group are the cell types that compose the adenohypophysis, the parathyroids, the paraganglia and the adrenal medulla. The second group, the disseminated neuroendocrine cell types, populate the skin, thyroid, lung, thymus, pancreas, gastrointestinal tract, biliary tract and urogenital tract. In the gastrointestinal tract and pancreas, 15 cell types producing different hormones can be distinguished.1,2 Neuroendocrine cells have uniform nuclei and abundant granular or faintly staining (clear) cytoplasm. They are either present as solid or small trabecular clusters or are dispersed among other cells and are then difficult to recognize in sections stained with haematoxylin and eosin (H&E). Immunostaining enables their exact identification. At * Tel.: þ49 431 597 3400; Fax: þ49 431 597 3462. E-mail address: [email protected]. 1521-690X/$ - see front matter ª 2007 Elsevier Ltd. All rights reserved.
16 G. Klo¨ppel
the ultrastructural level they are characterized by cytoplasmic membrane-bound dense-cored secretory granules (diameter >80 nm). They also contain small clear vesicles (diameter 40–80 nm) that correspond to the synaptic vesicles of neurons. DIAGNOSTIC AND FUNCTIONAL MARKERS There are some markers that serve as general markers and others that help to distinguish pulmonary from intestinal neuroendocrine tumours (NETs). In addition, the neuroendocrine cells express a number of peptide hormones and receptors that may also serve as markers. Here only the markers that are most relevant for the diagnosis will be discussed. Cytosolic markers Neuron-specific enolase (NSE), the g-g dimer of the glycolytic enzyme enolase, is the best-known cytosolic marker. Its reactivity is unrelated to the content of secretory granules in the cells. However, the commercially available antibodies are of limited sensitivity because of unspecific staining of certain non-endocrine tissues, such as striated muscle. Moreover, it is well known that NSE has been recognized in some nonneuroendocrine tumour tissues, such as solid-pseudopapillary neoplasms of the pancreas and serous-cystic neoplasms of the pancreas. These disadvantages, therefore, suggest that NSE positivity should be used with caution as a neuroendocrine marker reaction in tumour diagnosis. Small vesicle-associated markers Synaptophysin is an integral membrane glycoprotein (molecular weight 38,000) that occurs in presynaptic vesicles of neurons and small clear vesicles of normal and neoplastic neuroendocrine cells. It is expressed independently of the other neuroendocrine markers, notably secretory granule products. Other markers related to synaptophysin are SV2 and synaptobrevin. Large secretory granule-associated markers The chromogranins constitute a family of soluble proteins located in the matrix of secretory granules of many neuroendocrine cells. Chromogranin A, the precursor of pancreastatin, is a powerful universal marker of neuroendocrine tissues and tumours. Its expression, however, depends on the cell type and also on the number of secretory granules present in the cells. Cell membrane markers The neural cell adhesion molecule (NCAM, CD56) belongs to a group of cell-surface glycoproteins involved in direct cell–cell adhesion. It is expressed by neuronal and neuroendocrine cells.3 NCAM, however, is not only present in neuroendocrine cells but may also be expressed by non-endocrine normal tissues (renal tubules and thyroid follicle cells) and neoplastic tissues (non-small cell lung carcinomas and others).
Tumour biology and histopathology
17
CDX2 The transcription factor CDX2, which belongs to the homeobox genes regulating the development of the epithelium of the small and large intestine, has proved to be a very reliable marker of all NETs arising in the midgut. In addition, some well-differentiated NETs from the lung, the pancreas and the rectum were also positive, while other NETs – in particular from the stomach, the thyroid and the paraganglia – were consistently negative.4,5 Thyroid transcription factor 1 (TTF-1) TTF-1, a homeodomain containing transcription protein of the NKx2 gene family, is expressed in poorly differentiated neuroendocrine carcinomas of the lung (85%) and also in some pulmonary carcinoids (up to 60%), but not in well and poorly differentiated NETs of the gastrointestinal tract.6,7 Somatostatin receptors Many NETs express somatostatin receptors. Five types of these receptors have been identified (e.g. SSTR1–5).8 They can be demonstrated by autoradiography9, by scintigraphic imaging (octreoscan)10, or by immunohistochemistry.11,12 The immunohistochemical expression of SSTR2, which shows a membranous pattern, correlates closely with the octreoscan signals.10 Serotonin-producing NETs and gastrinomas are more commonly positive for SSTR2/5 (up to 90%) than insulinomas are (up to 60%).11,12 Hormones The various cell types of the neuroendocrine cell system are identified by their specific cellular products, such as peptides and biogenic amines, usually acting as hormones or neurotransmitters. By means of in-situ hybridization their expression can be traced back to the gene level. Hormones that are highly specific for certain tumour localizations are luteinizing hormone (LH), follicle-stimulating hormone (FSH) and prolactin for pituitary adenomas, calcitonin for medullary thyroid carcinoma, parathyroid hormone for parathyroid adenoma, insulin and glucagon as well as pancreatic polypeptide for pancreatic tumours, serotonin and substance P for ileal and appendiceal neuroendocrine tumours, and vasoactive monoamine transporter 2 (VMAT2) for ECLomas. CLASSIFICATION PRINCIPLES The WHO classification of ‘endocrine tumours’ includes neoplasms originating from endocrine glands such as the adrenal phaeochromocytomas, the pituitary adenomas, nerve elements such as paragangliomas, ganglioneuromas and neuroblastomas, or from elements of the disseminated/diffuse endocrine system such as gastroenteropancreatic endocrine tumours. Table 1 lists the two groups of neuroendocrine cell types and the names given to the respective tumours. The neoplasms originating from the disseminated neuroendocrine cell system that are recognized by the WHO classification comprise pure endocrine tumours and mixed endocrine/exocrine tumours.8 For pure gastroenteropancreatic neuroendocrine
18 G. Klo¨ppel
Table 1. Neuroendocrine cell system and tumour classification. Gland forming Pituitary Parathyroid Paraganglia Adrenal medulla
Adenoma Adenoma/carcinoma Paraganglioma Phaeochromocytoma
Disseminated Gastrointestinal tract Endocrine pancreas Biliary tract Respiratory tract Thymus Thyroid C cells Urogenital tract Skin
Neuroendocrine tumour/carcinoma Neuroendocrine tumour/carcinoma Neuroendocrine tumour/carcinoma Carcinoid, neuroendocrine carcinoma Carcinoid Medullary thyroid carcinoma Carcinoid Merkel cell carcinoma
tumours a uniform scheme of classification identifying three categories is applied for all anatomical sites: 1. well-differentiated endocrine tumours, with benign (1.1) or uncertain behaviour (1.2) at the time of diagnosis; 2. well-differentiated endocrine carcinomas with low-grade malignant behaviour; 3. poorly differentiated endocrine carcinomas, with high-grade malignant behaviour. Because of their highly aggressive behaviour, the neoplasms of category 3 differ profoundly from those of categories 1 and 2 and require a special therapeutic approach. Conventional histopathological and cytological assessment of growth patterns and cellular features of well-differentiated neuroendocrine tumours are often of little help in predicting their functional behaviour and degree of malignancy. In contrast, poorly differentiated NETs that are composed of cells displaying severe nuclear atypia, a high mitotic index and few secretory granules are invariably high-grade malignancies. They occur frequently in the lung, but have also been observed in the oesophagus, stomach, ampulla of Vater, colon, pancreas, uterine cervix and prostate. In contrast to well-differentiated neuroendocrine neoplasms, such poorly differentiated tumours usually lack hormonal syndromes. Mixed exocrine/neuroendocrine tumours have been described particularly in the appendix as goblet-cell carcinoids. Recently, adenocarcinomas showing scattered neuroendocrine cells have been reported in increasing numbers in various locations such as the stomach, pancreas, colon and prostate. However, if truly mixed tumours are defined as neoplasms in which the exocrine and endocrine cell populations are intimately mixed and the endocrine cells comprise about one third to one half of the tumour tissue, thus clearly being distinguishable from tumours with a diffusely scattered subpopulation of neoplastic neuroendocrine cells and collision tumours, only few tumours qualify as truly mixed exocrine/neuroendocrine neoplasms.13 In general, mixed exocrine/neuroendocrine neoplasms should be kept separate from neuroendocrine tumours, because the biological behaviour of the former appears to be dictated by the differentiation of the exocrine cell compartment.
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Classification in practice Gastroenteropancreatic NETs and NETs of the respiratory system form the largest group of NETs. For the practical diagnosis of these NETs, the criteria provided by the respective WHO classification and listed in Tables 2, 3, and 4 can be used as a checklist with which to appropriately classify an individual tumour.14 Naturally this catalogue of criteria needs to be extended and refined, because in particular the group of gastrointestinal and pancreatic tumours of questionable malignant potential (‘benign or malignant behaviour possible’) is too large to be accepted in the long run in this indefinite form. As there is an increasing demand for a standard in the stratification and treatment of patients with gastroenteropancreatic NETs, the European Neuroendocrine Tumour Society (ENETS)15 recently developed guidelines which were supplemented by a proposal for a tumour/nodes/metastases (TNM) classification. This TNM staging proposal considered only the NETs of the foregut16 and will soon be joined by a classification proposal for the midgut and hindgut NETs. Apart from the TNM staging proposal, a working formulation for the grading of gastroenteropancreatic NETs based on mitotic count and Ki-67 index has been suggested. Both the TNM staging proposal and the grading system still need to be validated. NETS BY LOCALIZATION, BIOLOGY, PATHOLOGY AND PROGNOSIS Most NETs occur in the gastrointestinal tract, the pancreas (including the bile-duct system), followed by the lung, the head and neck, the thymus, the urogenital system and the skin. In 1963 Williams and Sandler17 classified the gastroenteropancreatic NETs into foregut (stomach, pancreas, duodenum and upper jejunum), midgut (lower Table 2. WHO criteria for diagnosis of neuroendocrine tumours of the lung. Typical carcinoid Well-differentiated neuroendocrine neoplasm with <2 mitoses per 2 mm2 (10 HPF), lacking necrosis, and 0.5 cm Atypical carcinoid Well-differentiated neuroendocrine neoplasm with 2e10 mitoses per 2 mm2 (10 HPF) and/or focal necrosis Large-cell neuroendocrine carcinoma Poorly differentiated neuroendocrine carcinoma (organoid nesting, palisading, rosettes, trabeculae) with high mitotic rate (11 per 2 mm2 and 10 HPF; median of 20 per 2 mm2 and 10 HPF) and necrosis (often large zones). Cytological features: large cell size, low nuclear/cytoplasmic ratio, vesicular, coarse or fine chromatin, and/or frequent nucleoli Small-cell carcinoma Poorly differentiated neuroendocrine carcinoma. Cytological features: small cells with scant cytoplasm, fine granular chromatin, absent or faint nucleoli, high mitotic rate (11 per 2 mm2 and 10 HPF; median of 80 per 2 mm2 and 10 HPF), frequent necrosis often in large zones HPF, high-power field. Modified from Travis et al (2004, Pathology and Genetics of Tumours of the Lung, Pleura, Thymus and Heart. WHO classification of tumours. Lyon: IARC Press).
20 G. Klo¨ppel
Table 3. Criteria for assessing the prognosis of neuroendocrine tumours of the gastrointestinal tract. Biological behaviour
Metastases Invasion of Histological Tumour Angioinvasion size muscularis differentiation propriaa
Benign
e
e
Benign or lowgrade malignant Low-grade malignant High-grade malignant
e
e
þ
þb
þ
þ
a b
Welldifferentiated Welldifferentiated Welldifferentiated Poorly differentiated
Ki-67 index
Hormonal syndrome
1 cma
e
<2%
ea
1e2 cm
/þ
<2%
e
>2 cm
þ
2e20%
þ
Any
þ
>20%
e
Exception: malignant duodenal gastrinomas are usually <1 cm and confined to the submucosa. Exception: benign NETs of the appendix usually invade the muscularis propria.
jejunum, ileum, appendix, caecum) and hindgut (colon, rectum) tumours, with considerable clinicopathological differences between the three groups. However, in the case of foregut tumours the usefulness of such a classification in practical diagnostic work is limited by its failure to characterize individual tumour entities with well-defined histological, hormonal and/or clinicopathological profiles. In the subsequent section the NETs are therefore classified and discussed according to their specific localizations. Not included in this discussion are the NETs of the head and neck region18–20, the thymus21,22, the urogenital system, and the skin. Respiratory tract The classification of NETs of the lower respiratory tract (i.e. the lung) includes typical carcinoid, atypical carcinoid, small-cell carcinoma and large-cell neuroendocrine carcinoma (Table 2). Typical and atypical carcinoids and large-cell neuroendocrine carcinomas make up only 2–3% of lung tumours, while small-cell carcinomas account for 15–25% of all lung malignancies. Typical carcinoids – i.e. well-differentiated NETs – are characterized by low mitotic activity (<2 mitoses per 2 mm2). They have an excellent prognosis after radical Table 4. Criteria for assessing the prognosis of neuroendocrine tumours of the pancreas. Biological behaviour Benign Benign or lowgrade malignant Low-grade malignant High-grade malignant a b c
Metastases Invasiona
Histological differentiation
Tumour Angioinvasion Ki-67 Hormonal size index syndrome
e e
e e
Well-differentiated Well-differentiated
1 cm >2 cm
e /þ
<2% <2%
/þb /þc
þ
þ
Well-differentiated
>4 cm
þ
2e20%
þc
þ
þ
Poorly differentiated
Any
þ
>20%
e
Invasion of adjacent organs (e.g. duodenum, stomach). Insulinomas. Insulinomas and other functioning tumours (e.g. glucagonomas).
Tumour biology and histopathology
21
surgical treatment (90–98% 5-year survival). The diameter of these tumours is usually <3 cm, and the histological patterns range from adenoid to solid, trabecular, paraganglioid and to spindle cell-like (Figure 1). Atypical carcinoids are histologically similar to typical carcinoids (Figure 2) but have a mitotic activity of 2–10 mitoses per 2 mm2 and focal necrosis. The tumour cells do not show signs of crushing or nuclear moulding, which are characteristic of small-cell carcinoma. The 5-year survival rate is 60% after radical surgical treatment. Small-cell lung carcinoma is the most clinically distinctive type of neuroendocrine lung carcinoma. The mitotic activity in these tumours is high (>11 mitoses per 2 mm2 in 10 high-power fields). In addition, these carcinomas generally show more extensive necrosis than atypical carcinoids. The differentiation of small-cell carcinoma from large-cell neuroendocrine carcinoma is based on cell size, abundance of cytoplasm, prominent nucleoli and vesicular or coarse chromatin. The prognosis of large-cell neuroendocrine carcinomas is similar to that of smallcell carcinoma. However, while small-cell carcinomas usually respond well to chemotherapy, large-cell neuroendocrine carcinomas do not. Bronchial carcinoids, when centrally located, may lead to mechanical obstruction of the main bronchus. They may also secrete adrenocorticotropic hormone (ACTH), thereby producing a Cushing syndrome. A carcinoid syndrome due to serotonin secretion is very rare and occurs only when there are widespread metastases. Both typical and atypical carcinoids can occur in patients with multiple endocrine neoplasia type 1 syndrome (MEN1). Cushing syndrome may also be seen in small-cell carcinomas. Stomach Four types of NET can be distinguished in the stomach23,24; type 1 is by far the most frequent type (70–80%)25, whereas types 2, 3 and 4 are rare. Type 1 NETs are usually small, multifocal, broad-based, round, polypoid mucosal tumours measuring 0.5–1 cm in diameter, typically occurring in the corpus of the stomach. Histologically, these well-differentiated tumours are found in the mucosa and
Figure 1. Typical carcinoid of the lung: solid clusters of monomorphic cells with well-developed cytoplasm.
22 G. Klo¨ppel
Figure 2. Atypical carcinoid of the lung: monomorphic, densely packed cell population with some spindlecell areas.
submucosa and are composed of intensely chromogranin A-positive ECL (enterochromaffin-like) cells, best recognized by the marker VMAT2 (vesicular monoamine transporter 2).26,27 They always develop secondary to autoimmune chronic atrophic corpus gastritis (CAG), in which the loss of parietal cells leads to insufficient production of intrinsic factor (triggering pernicious anaemia) and of hydrochloric acid (causing achlorhydria). This stimulates the antral G cells to produce gastrin, causing persistent hypergastrinaemia that is assumed to promote the growth of the ECL cells of the corpus mucosa so that diffuse to micronodular ECL-cell hyperplasia develops, out of which – after a latent period of many years – the above-described multiple ECL tumours arise.28 Clinically these tumours are not associated with any hormonal symptoms and are mainly found in women. The prognosis of these tumours is good, because they are generally so small that they can be removed endoscopically.29 Regional lymph-node metastases seem to occur only in very rare cases in which the tumours are >2 cm in size and infiltrate the muscularis propria. Type 2 gastric NETs occur in association with MEN1, in the course of which a Zollinger–Ellison syndrome (ZES) has developed. Like type 1 NETs, they are usually multifocal and found in the corpus of the stomach associated with ECL-cell hyperplasia. The tumours are generally <1.5 cm and limited to the mucosa and submucosa. If angioinvasion is found or the tumour is >2 cm and/or has invaded the muscularis propria, it is likely that the tumour has metastasized, as happens in approximately 30% of cases. The genetic changes associated with MEN1 (mutations in the MEN1 gene on chromosome 11q13) in connection with gastrinoma-related hypergastrinaemia are probably the basis on which the tumour develops, because so far the development of tumours of the same type in ZES has not been confirmed. Type 3, or sporadic, NETs of the stomach are not associated with either CAG or MEN1, and show a predilection for the male sex. These tumours are solitary and do not have any special localization in the stomach. In one third of the cases the tumour is already >2 cm at the time of diagnosis. Histologically, the tumours are predominantly well differentiated and are composed of ECL cells (Figure 3). Tumours with EC (serotonin) cells or gastrin cells, by contrast, are extremely rare.24 If the tumour is >2 cm,
Tumour biology and histopathology
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Figure 3. Sporadic well-differentiated neuroendocrine carcinoma in the antrum. Preserved antral mucosa on top of the tumour, which is below the muscularis mucosae.
has invaded the muscularis propria, and angioinvasion is recognizable, it is likely that there will be metastases.29 In such cases surgical treatment is required. Type 4 gastric NETs correspond to an undifferentiated gastric carcinoma. These tumours are more common in men than in women, and there is no relationship to CAG or MEN1. At the time of diagnosis most of the tumours are already in an advanced stage (tumour diameter >4 cm) and show extensive metastases. Immunocytochemically, the tumour cells are positive for synaptophysin, with only slight focal positivity for chromogranin A. Type 4 NETs of the stomach are treated like normal gastric carcinomas. Duodenum and upper jejunum Five types of duodenal neuroendocrine tumours can currently be distinguished. These include duodenal gastrinomas, which make up approximately two thirds of all duodenal NETS, duodenal somatostatin-producing tumours, non-functioning (i.e. not causing a hormonal syndrome) serotonin-, gastrin- or calcitonin-producing tumours, poorly differentiated, predominantly ampullary neuroendocrine carcinomas and duodenal gangliocytic paragangliomas.28,30,31 Duodenal gastrinomas are either sporadic or associated with MEN1 and are combined with a ZES.32,33 In both situations these gastrinomas are usually not bigger than 1 cm (Figure 4), are located predominantly in the upper part of the duodenum, and histologically show a trabecular/pseudoglandular pattern. If associated with MEN1, they are usually multiple, in contrast to sporadic gastrinomas. In spite of their small size and the fact that they are limited to the duodenal mucosa and submucosa, at the time of diagnosis metastases are often found in the regional lymph nodes. These metastases are often much larger than the primary tumour and may erroneously be considered to be pancreatic tumours, especially if they are located close to the pancreas.34 This peculiarity is probably the reason why earlier authors spoke of primary
24 G. Klo¨ppel
Figure 4. Minigastrinoma in the duodenal mucosa (arrows). The inset shows the histology of the tumour.
lymph-node gastrinomas, and why many more pancreatic gastrinomas used to be diagnosed than are today. Whereas metastasis to the regional lymph nodes occurs at an early stage, liver metastases appear to be a relatively late occurrence. In the case of pancreatic gastrinomas (see below), which are practically all sporadic and hence not associated with MEN1, liver metastases seem to occur earlier than in duodenal gastrinomas.34,35 Duodenal somatostatin-producing tumours account for approximately 15% of all duodenal NETs. Their preferential localization is in the region of the papilla of Vater or periampullary. If the muscularis propria is invaded, metastasis to the paraduodenal lymph nodes has probably occurred. Histologically, the tumours typically show a glandular pattern with psammoma bodies. They are not associated with any hormonal syndrome, but often occur in patients with neurofibromatosis type 1. In this situation a bilateral phaeochromocytoma can simultaneously develop. Non-functioning duodenal NETs usually consist of serotonin-producing cells. Occasionally there are also tumours with gastrin- or calcitonin-positive cells. The prognosis of this group of non-functioning tumours is much more favourable than ZES-associated
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gastrinomas or ampullary somatostatin-producing tumours. Metastases are not to be expected until the tumour extends beyond the submucosa. Poorly differentiated duodenal carcinomas occur primarily in the region of the papilla of Vater. They are hormonally inactive. At the time of diagnosis advanced metastasis into the regional lymph nodes and the liver has usually occurred. Duodenal gangliocytic paragangliomas occur in the vicinity of the papilla of Vater. Although the tumours are often >2 cm and infiltrate into the muscularis propria, they generally follow a benign course. Histologically, they reveal a gangliocytic component in addition to well-differentiated neuroendocrine cells, and immunocytochemically they usually express somatostatin, pancreatic polypeptide and S-100.
Appendix The NETs of the appendix are, along with ileal NETs, the most frequent GEP NETs. Their prognosis, however, is much more favourable than that of ileal NETs. They occur primarily in the tip of the appendix and almost without exception they invade the muscularis propria and, to a greater or lesser degree, the adjoining fatty tissue of the mesoappendix. Nevertheless, regional lymph-node metastases seem to be rare before the tumour reaches a size of approximately 2.0 cm. Histologically and immunocytochemically, the NETs of the appendix are comparable to the ileal NETs with their solid tumour cell formations and their positivity for serotonin and substance P. They must be distinguished from the very rare so-called goblet-cell carcinoids, which represent mixed exocrine/endocrine tumours, because the prognosis of the latter is less favourable.
Ileum These frequent NETs develop quite preferentially in the terminal ileum and occasionally in the immediately adjacent caecum, including the ileocaecal valve. Histologically, they show a solid (‘island-like’) pattern. At the time of diagnosis they are commonly >2 cm and have invaded the muscularis propria. This means that they have already metastasized to the regional lymph nodes (Figure 5). In up to 40% of cases the tumours may be multiple. Immunocytochemically, the cells contain serotonin and substance P, kallikrein and catecholamine. Approximately 20% of the patients with ileal NETs have liver metastases (Figure 6) in addition to regional lymph-node metastases. Only in these patients does a carcinoid syndrome (flush, diarrhoea and endocardial fibrosis) develop, because serotonin is inactivated by the liver and only enters the circulatory system if there are liver metastases.
Colon NETs of the colon are very rare. Histologically, they are poorly differentiated neuroendocrine carcinomas, almost all of which have already metastasized at the time of diagnosis and therefore have a poor prognosis.36 Immunocytochemically, they are generally synaptophysin-positive and may contain single serotonin- and somatostatinpositive cells.
26 G. Klo¨ppel
Figure 5. Macroscopy of a metastasizing ileal neuroendocrine carcinoma: arrows indicate the primary tumours. In the mesenterium is a large lymph-node metastasis.
Rectum Rectal NETs are much more frequent and their prognosis is much better than that of NETs of the colon. They comprise around 10% of all GEP NETs. Most appear during endoscopy as small (<1 cm) movable submucosal tumours. Metastases are likely in tumours that are 2 cm and if they invade the muscularis propria. Immunocytochemically, they are positive for glucagon, glicentin and/or pancreatic polypeptide (PP) and are usually negative for chromogranin. In this portion of the gut poorly differentiated neuroendocrine carcinomas are the exception. They have as poor a prognosis as those in the colon and other portions of the gut.
Figure 6. Liver metastasis of an ileal well-differentiated neuroendocrine carcinoma.
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Pancreas Most NETs of the pancreas are well differentiated. Of these, 50–60% are functionally active, i.e., due to largely uncontrolled secretion of insulin, gastrin, vasoactive intestinal polypeptide (VIP), glucagon, or other even rarer hormones such as ACTH or growth hormone (GH), they can induce characteristic syndromes (hypoglycaemia syndrome, Zollinger–Ellison syndrome, Verner–Morrison syndrome, glucagonoma syndrome, Cushing’s syndrome, acromegaly).37 Depending on the predominant hormone secreted, these tumours are referred to as insulinomas, gastrinomas, VIPomas, glucagonomas, etc. Pancreatic endocrine tumours without hormonal symptoms are observed more frequently than previously, though this probably does not reflect an actual increase in frequency but rather improved diagnostic methods and an increased resection rate. These non-functioning NETs are either incidental findings or are discovered along with general neoplastic symptoms, usually, however, because of local symptoms. Pancreatic neuroendocrine tumours are extremely rare in childhood, but in adults they occur in all age groups and in males and females with equal frequency. Macroscopically, they are well-demarcated, usually solitary, round tumours with a diameter of 1–4 cm, and they can occur in all parts of the pancreas. In the case of a hormonal syndrome, the hormone causing the syndrome can be detected immunocytochemically. Although the pancreatic NETs are histologically well differentiated, they are frequently malignant, with the exception of insulinomas. This hold particularly for gastrinomas, VIPomas, glucagonomas and non-functioning tumours. The most important criteria of malignancy are – apart from metastases to the regional lymph nodes and the liver or invasion of adjacent organs – a tumour size of >2 cm, angioinvasion, and proliferative activity of >2% (Figure 7).38,39 Recent studies have shown that angioinvasion plays a more important role than was assumed in the classification presented here.40 It is therefore recommended that a tumour be considered malignant if the presence of angioinvasion has been verified, even if no other criteria of malignancy are present.
Figure 7. Well-differentiated neuroendocrine carcinoma of the pancreas showing angioinvasion.
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Among the functioning tumours, insulinomas are most frequent; 95% of them are 1–2 cm in size and benign. Multiple insulinomas and insulinomas associated with MEN1 are observed in approximately 10% of the cases. Compared with solitary or sporadic insulinomas, their rate of malignancy is not increased. Histologically, in approximately 5% of insulinomas amyloid deposits are found in the tumour. Immunocytochemically, insulin and proinsulin can be detected in varying ratios, depending on the grade of differentiation of the tumour41, which is also reflected in the secretory behaviour.42 Pancreatic gastrinomas are usually >2 cm and therefore in 60% of the cases they have already metastasized at the time of diagnosis. In contrast to duodenal gastrinomas, they rarely occur in the context of MEN1.34 Included among the rare tumours with hormonal syndromes are VIPomas, glucagonomas, ACTH-producing tumours and growth-hormone-producing tumours. At the time of diagnosis most of these tumours are >2 cm and have already metastasized. This is also true of non-functioning tumours.
GENETIC PROFILES Since the traditional morphological criteria have largely been exhausted, intensive work is currently being done on the molecular characterization and differentiation of the GEP NETs. These studies have identified the gene responsible for MEN1 on chromosome 11q13, which is also mutated in up to 40% of sporadic GEP NETs.43 By means of studies of comparative genomic hybridization (CGH) and allelic loss (LOH), a large number of genomic regions with loss or gain of genetic material have been detected.44,45 In this manner changes are being identified that may play a role in tumour progression. Such studies have also confirmed that NETs in different localizations are genetically independent tumours. Hence foregut NETs often show loss of 11q. This distinguishes them from NETs of the midgut and hindgut46, which frequently show losses on chromosome 18q.47,48 Among the lung NETs, the welldifferentiated NETs (i.e. typical and atypical carcinoids) constitute a family differing from small- and large-cell neuroendocrine carcinomas, because abnormalities in genetic markers such as p53, RB loss and cyclin D1 are much more common in the poorly differentiated NETs. On the basis of these results an intensive search is currently under way for genetic markers or marker constellations that will enable us to better predict the biological behaviour of the group of GEP NETs of uncertain malignant potential.
SUMMARY Most neuroendocrine tumours occur in the gastrointestinal tract and pancreas, the lung being the next most frequent location. They are either well or poorly differentiated and constitute two families rather than the two extremes of one spectrum. In addition, they reveal biological heterogeneity depending on localization and function. For the practical diagnosis, the criteria listed in Tables 2–4 can be used as a checklist with which to appropriately classify an individual tumour. Recently, a TNM staging system for foregut NETs, including a grading system, has been proposed and is currently under validation.16
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Practice points exact clinicopathological classification determines prognosis the various tumour entities have different biologies and require individual treatments the proliferative activity of an individual tumour determines it natural course and may also correlate with its responsiveness to chemotherapy
Research agenda validation of the classification protocols is needed treatment response, tumour type and tumour proliferative activity need to be documented genetic profiling of the various entities needs to be continued
REFERENCES 1. Klo¨ppel G. Neuroendokrinen Tumoren des Gastrointestinaltrakts. Der Pathologe 2003; 24: 287–296. 2. Klo¨ppel G. Tumoren des endokrinen Pankreas. Der Pathologe 2003; 24: 265–271. 3. Lloyd RV. Practical markers used in the diagnosis of neuroendocrine tumors. Endocrine Pathology 2003; 14: 293–301. 4. Erickson LA, Papouchado B, Dimashkieh H et al. Cdx2 as a marker for neuroendocrine tumors of unknown primary sites. Endocrine Pathology 2004; 15: 247–252. 5. La Rosa S, Rigoli E, Uccella S et al. CDX2 as a marker of intestinal EC-cells and related well-differentiated endocrine tumors. Virchows Archiv 2004; 445: 248–254. 6. Cai YC, Banner B, Glickman J & Odze RD. Cytokeratin 7 and 20 and thyroid transcription factor 1 can help distinguish pulmonary from gastrointestinal carcinoid and pancreatic endocrine tumors. Human Pathology 2001; 32: 1087–1093. 7. Sturm N, Rossi G, Lantuejoul S et al. Expression of thyroid transcription factor-1 in the spectrum of neuroendocrine cell lung proliferations with special interest in carcinoids. Human Pathology 2002; 33: 175–182. 8. Volante M, Bozzalla-Cassione F & Papotti M. Somatostatin receptors and their interest in diagnostic pathology. Endocrine Pathology 2004; 15: 275–292. 9. Reubi JC, Ha¨cki WH & Lamberts SW. Hormone-producing gastrointestinal tumors contain a high density of somatostatin receptors. The Journal of Clinical Endocrinology and Metabolism 1987; 65: 1127–1134. 10. Lamberts SW, Hofland LJ, van Koetsveld PM et al. Parallel in vivo and in vitro detection of functional somatostatin receptors in human endocrine pancreatic tumors: consequences with regard to diagnosis, localization, and therapy [see comments]. The Journal of Clinical Endocrinology and Metabolism 1990; 71: 566–574. 11. Kulaksiz H, Eissele R, Ro¨ssler D et al. Identification of somatostatin receptor subtypes 1, 2A, 3 and 5 in neuroendocrine tumours with subtype specific antibodies. Gut 2002; 50: 52–60. 12. Papotti M, Bongiovanni M, Volante M et al. Expression of somatostatin receptor types 1-5 in 81 cases of gastrointestinal and pancreatic endocrine tumors. A correlative immunohistochemical and reversetranscriptase polymerase chain reaction analysis. Virchows Archiv 2002; 440: 461–475. 13. Volante M, Rindi G & Papotti M. The grey zone between pure (neuro)endocrine and non-(neuro)endocrine tumors: a comment on concepts and classification of mixed exocrine-endocrine neoplasms. Virchows Archiv 2006; 449: 499–506.
30 G. Klo¨ppel *14. Klo¨ppel G, Perren A & Heitz PU. The gastroenteropancreatic neuroendocrine cell system and its tumors. The WHO classification. Annals of the New York Academy of Sciences 2004; 1014: 13–27. *15. Plo¨ckinger U, Rindi G, Arnold R et al. Guidelines for the diagnosis and treatment of neuroendocrine gastrointestinal tumours. A consensus statement on behalf of the European Neuroendocrine Tumour Society (ENETS). Neuroendocrinology 2004; 80: 394–424. *16. Rindi G, Klo¨ppel G, Ahlman H et al. TNM staging of foregut (neuro)endocrine tumors: a consensus proposal including a grading system. Virchows Archiv 2006; 449: 395–401. 17. Williams ED & Sandler M. The classification of carcinoid tumours. Lancet 1963; I: 238–239. 18. Riddle PJ, Font RL & Zimmerman LE. Carcinoid tumors of the eye and orbit: a clinicopathologic study of 15 cases, with histochemical and electron microscopic observations. Human Pathology 1982; 13: 459–469. *19. Mills SE. Neuroendocrine tumors of the head and neck: a selected review with emphasis on terminology. Endocrine Pathology 1996; 7: 329–343. 20. Ferlito A, Barnes L, Rinaldo A et al. A review of neuroendocrine neoplasms of the larynx: update on diagnosis and treatment. The Journal of Laryngology and Otology 1998; 112: 827–834. 21. Wick MR & Rosai J. Neuroendocrine neoplasms of the thymus. Pathology, Research and Practice 1988; 183: 188–199. 22. Moran CA & Suster S. Neuroendocrine carcinomas (carcinoid tumor) of the thymus. A clinicopathologic analysis of 80 cases. American Journal of Clinical Pathology 2000; 114: 100–110. 23. Bordi C, Azzoni C, D’Adda T et al. Neuroendokrine Tumoren des Magens. Der Pathologe 1997; 18: 313–321. 24. Klo¨ppel G & Clemens A. The biological relevance of gastric neuroendocrine tumors. The Yale Journal of Biology and Medicine 1996; 69: 69–74. *25. Rindi G, Luinetti O, Cornaggia M et al. Three subtypes of gastric argyrophil carcinoid and the gastric neuroendocrine carcinoma: a clinicopathologic study. Gastroenterology 1993; 104: 994–1006. 26. Eissele R, Anlauf M, Scha¨fer MKH et al. Expression of vesicular monoamine transporters in endocrine hyperplasia and endocrine tumors of the oxyntic stomach. Digestion 1999; 60: 428–439. 27. Rindi G, Paolotti D, Fiocca R et al. Vesicular monoamine transporter 2 as a marker of gastric enterochromaffin-like cell tumors. Virchows Archiv 2000; 436: 217–223. *28. Bordi C, D’Adda T, Azzoni C et al. Gastrointestinal endocrine tumors: recent developments. Endocrine Pathology 1998; 9: 99–115. 29. Rappel S, Altendorf-Hofmann A & Stolte M. Prognosis of gastric carcinoid tumours. Digestion 1995; 56: 455–462. *30. Capella C, Heitz PU, Ho¨fler H et al. Revised classification of neuroendocrine tumours of the lung, pancreas and gut. Virchows Archiv 1995; 425: 547–560. 31. Klo¨ppel G, Heitz PU, Capella C & Solcia E. Pathology and nomenclature of human gastrointestinal neuroendocrine (carcinoid) tumors and related lesions. World Journal of Surgery 1996; 20: 132–141. *32. Pipeleers-Marichal M, Somers G, Willems G et al. Gastrinomas in the duodenums of patients with multiple endocrine neoplasia type 1 and the Zollinger–Ellison syndrome. The New England Journal of Medicine 1990; 322: 723–727. 33. Pipeleers-Marichal M, Donow C, Heitz PU & Klo¨ppel G. Pathologic aspects of gastrinomas in patients with Zollinger–Ellison syndrome with and without multiple endocrine neoplasia type I. World Journal of Surgery 1993; 17: 481–488. 34. Donow C, Pipeleers-Marichal M, Schro¨der S et al. Surgical pathology of gastrinoma. Site, size, multicentricity, association with multiple endocrine neoplasia type 1, and malignancy. Cancer 1991; 68: 1329– 1334. 35. Weber HC, Venzon DJ, Lin JT et al. Determinants of metastatic rate and survival in patients with Zollinger–Ellison syndrome: a prospective long-term study. Gastroenterology 1995; 108: 1637–1649. 36. Grabowski P, Scho¨nfelder J, Ahnert-Hilger G et al. Expression of neuroendocrine markers: a signature of human undifferentiated carcinoma of the colon and rectum. Virchows Archiv 2002; 441: 256–263. 37. Klo¨ppel G, In’t Veld PA, Komminoth P & Heitz PU. The endocrine pancreas. In Kovacs K & Asa SL (eds). Functional Endocrine Pathology. 2nd ed. Boston: Blackwell, 1998, pp. 415–487. *38. Solcia E, Klo¨ppel G, Sobin LH & (In collaboration with 9 pathologists from 4 countries). In Histological typing of endocrine tumours. WHO international histological classification of tumours. 2nd ed. Berlin: Springer; 2000.
Tumour biology and histopathology
31
39. Heymann MF, Joubert M, Nemeth J et al. Prognostic and immunohistochemical validation of the Capella classification of pancreatic neuroendocrine tumours: an analysis of 82 sporadic cases. Histopathology 2000; 36: 421–432. *40. La Rosa S, Sessa F, Capella C et al. Prognostic criteria in nonfunctioning pancreatic endocrine tumours. Virchows Archiv 1996; 429: 323–333. 41. Roth J, Klo¨ppel G, Madsen OD et al. Distribution patterns of proinsulin and insulin in human insulinomas: an immunohistochemical analysis in 76 tumors. Virchows Archiv. B: Cell Pathology 1992; 63: 51–61. 42. Creutzfeldt W, Arnold R, Creutzfeldt C et al. Biochemical and morphological investigations of 30 human insulinomas. Correlation between the tumour content of insulin and proinsulin-like components and the histological and ultrastructural appearance. Diabetologia 1973; 9: 217–231. 43. Goebel SU, Heppner C, Burns AL et al. Genotype/phenotype correlation of multiple endocrine neoplasia type 1 gene mutations in sporadic gastrinomas. The Journal of Clinical Endocrinology and Metabolism 2000; 85: 116–123. 44. Barghorn A, Komminoth P, Bachmann D et al. Deletion at 3p25.3-p23 is frequently encountered in endocrine pancreatic tumours and is associated with metastatic progression. Journal of Pathology 2001; 194: 451–458. 45. Speel EJ, Scheidweiler AF, Zhao J et al. Genetic evidence for early divergence of small functioning and nonfunctioning endocrine pancreatic tumors: gain of 9Q34 is an early event in insulinomas. Cancer Research 2001; 61: 5186–5192. 46. D’Adda T, Pizzi S, Azzoni C et al. Different patterns of 11q allelic losses in digestive endocrine tumors. Human Pathology 2002; 33: 322–329. 47. To¨nnies H, Toliat MR, Ramel C et al. Analysis of sporadic neuroendocrine tumours of the enteropancreatic system by comparative genomic hybridisation. Gut 2001; 48: 536–541. 48. Kyto¨la¨ S, Ho¨o¨g A, Nord B et al. Comparative genomic hybridization identifies loss of 18q22-qter as an early and specific event in tumorigenesis of midgut carcinoids. American Journal of Pathology 2001; 158: 1803–1808.