Gastrointestinal Carcinoid Tumours

Gastrointestinal Carcinoid Tumours Histogenetic, Histochemical, Immunohistochemical, Clinical and Therapeutic Aspects

ERIK WILANDER, MONALILL LUNDQVIST KJELL OBERG

With 24 Figures and 3 Tables

GUSTAV FISCHER VERLAG· STUTTGART· NEW YORK · 1989

ERIK WILANDER, Assoc;. Prof. M. D. Endocrine Laboratory, Department of Pathology, University Hospital, S-7St 85 Uppsala (Sw~den) MONALILL LUNDQVIST, M.D .

Ludwig Institute for Cancer Research, University Hospital, S-7St 85 Uppsala (Sweden) KJELL OBERG, Clin. Assoc. Dir., Assoc. Prof. M. D. Ludwig Institute for Cancer Research and Endocrine Unit, Department of Internal Medicine, University Hospital, S-7SI 85 Uppsala (Sweden)

Acknowledgements Our work on carcinoid tumours has been supported by grants from the Swedish Medical Research Council (project No. 6817) and the Swedish Society of Medical Sciences.

CIP-Titelaufnahme der Deutschen Bibliothek Wilander, Erik: Gastrointestinal carcinoid tumours : histogenetic, histochemical, immunohistochemical, clinical and therapeutic aspects / Erik WiJander ; MonaJiJl Lundqvist ; KjeJl Oberg. - Stuttgart; New York: Fischer, 1989 (Progress in histochemistry and cytochemistry; Vol. 19, No 2) ISBN 3-437-11219-8 NE: Lundqvist, Monalill:; Oberg, Kjell:; GT

Library of Congress Card-No. 88-20469

© Gustav Fischer Verlag · Stuttgart· New York· 1989 Aile Rechte vorbehalten Gesamtherstellung: Laupp & Gobel, NehrenITiibingen Printed in Germany ISSN 0079-633-6

Contents 1

2 2.1

2.2 2.3 3 3.1 3.2

3.3 3.4 3.5 3.6

3.7 3.8 3.8.1 3.8.2 3.8.2.1 3.8.2.2 3.8.2.3 3.8.2.4 3.8.2.5 3.8.3 3.8.3.1 3.8.3.2 3.8.3.3 3.8.3.4 3.9 3.10 3.11 3.12 3.13

3.14 3.15 3.16 3.16.1 3.16.1.1 3.16.1.2 3.16.1.3

Introduction . . . . . . . . . . . . . . . . . . Normal gastrointestinal neuroendocrine cells The enterochromaffin (EC) cells Peptide hormone-producing cells . Subepithelial neuroendocrine cells Carcinoid tumours . . . . . . Short history. . . . . . . . . . Definitions and classifications . Incidence Location . . . . . . . . . . ~etastases . . . . . . . . . ~ultiple primary tumours. Second malignancies. . . . Light microscopic morphology . Routine histology . . . . . . . . Silver stains - general considerations ~asson)s stain . . . . Grimelius stain '" Sevier- ~unger stain . Davenport technique Silver stains applied to cytological specimens . Immunocytochemistry . . . . . Serotonin . . . . . . . . . . . . Serotonin in cytological samples Peptide hormones . . . . . . . . Neuroendocrine tumour markers. Electron microscopy . . . . Carcinoid tumour metastases Histogenesis of carcinoids. . Pathogenesis. . . . . . . . . Nuclear DNA measurements Carcinoid tumours in vitro Neuroendocrine cells in intestinal adenocarcinomas Clinical presentations . . The carcinoid syndrome. Flushirtg . . . . . . . . Diarrhoea . . . . . . . Respiratory symptoms

1 1

3 5 6

6 6 7

8 8 9 10 10 12 12 14 14 16 18 19 19 21 21 25 25 26 32 35 36 38

40 43 46

47 48 48 50 50

VI . Contents

3.16.1.4 3.16.1.5 3.16.2 3.16.3 3.16.4 3.16.5 3.16.6 3.16.7 3.17 3.17.1 3.17.1.1 3.17.1.2 3.17.1.3 3.17.1.4 3.17.1.5 3.17.2 3.17.3 3.18 3.18.1 3.18.2 3.18.3 3.18.4 3.18.4.1 3.18.4.2 3.18.4.3 3.18.4.43.19 3.20 4 5 6

Carcinoid heart disease . . . . . Uncommon carcinoid symptoms The ECLoma syndrome . . . . . The Zollinger-Ellison syndrome The somatostatinoma syndrome Cushing's syndrome . . . . . . . Multiple endocrine neoplasia type I . von Recklinghausen's disease and duodenal carcinoids . Diagnosis . . . . . . . . . . Circulating tumour markers. Serotonin . . . . . . . Plasma tachykinins . . . . . Pancreatic polypeptide . . . Human chorionic gonadotropin subunits. Other markers . . . . . Stimulatory tests. . . . Localizing procedures. Treatment . . . . . . . Anaesthetic considerations Hepatic artery occlusion Radiotherapy . . . . . . . Pharmacological treatment Chemotherapy . . . . Interferon treatment. . Somatostatin analogue. Other treatments . . . Survival and prognosis. Clinical conclusions . Summary .. References . . Subiect index

50 51 51 52 53 53 53 54 54 54 54 54

55 55

55 56 56 58 61 61 62 62 62 64 66 67 68 68 69 71

85

1 Introduction During the last decade one of the major research programmes at the Endocrine Laboratory of the Department of Pathology of the University Hospita1, Uppsala, Sweden, has been a study of the pathobiology of carcinoid tumours of the gastrointestinal canal. Various methods such as histological examination, silver-staining methods for identifying neuroendocrine cells, immunohistochemical techniques and electron microscopy have been used on histological material. These techniques have been supplemented with cytofluorometric nuclear DNA analyses in single tumour cells and in vitro studies of carcinoids, and the results are summarized in the following. The tumour material has been obtained through co-operation with the Endocrine Unit of the University Hospital and the Clinical Group of the Ludwig Institute for Cancer Research, which has employed modern radioimmunoassay techniques for analyzing serum and plasma levels of peptide hormones, biogenic amines and more general neuroendocrine markers in clinical and follow-up studies of patients with gastrointestinal carcinoids. These patients have been subjected to new therapeutic regimes with various antitumour agents, e. g. interferon, streptozotocin and somatostatin analogues. It has not been our ambition in this issue to try to provide a complete review of the present knowledge of gastrointestinal carcinoids, but rather to present our own results as comprehensively as possible, supplemented with some basic information and discussions. After a brief description of the gastrointestinal neuroendocrine cell system, the different aspects of carcinoid tumours will be presented.

2 Normal gastrointestinal neuroendocrine cells Intermingled with the enterocytes in the gastrointestinal mucosa, scattered neuroendocrine cells occur. These cells vary in their staining properties and ultrastructural morphology, depending on their hormonal content. Different types of neuroendocrine cells are located at different topographical sites (FEYRTER 1938; SOLCIA et al. 1981). In general the neuroendocrine cell population can be sub grouped into enterochromaffin (EC) cells producing the monoamine serotonin, sometimes in combination with a peptide hormone, and non-serotonin-producing cells. EC cells are present in all regions of the gastrointestinal mucosa, while most of the peptide-producing cells are found at specific topographical sites. The EC cells are argentaffin, while the peptide-producing neuroendocrine cells are non-argentaffin but argyrophilic or unstained with silver techniques (GRIMELIUS and WILANDER 1980). Two structurally different types of neuroendocrine cells occur in the gastrointestinal mucosa, namely open and closed cells. The closed cells are located in the body and fundic region of the stomach (the acid-secreting part), while the open cell type is found in the rest of the digestive system. The closed cells are not in contact with the luminal

2 . E. Wilander . M. Lundqvist . K. Oberg

part of the mucosal crypts, in contrast to the open cells, which are flask-shaped and whose apical part reaches the lumen of the crypt (Fig. 1). It is presumed that the closed cells respond to distension and humoral factors, while the open cells are stimulated by minor changes in the pH or composition of the luminal contents. Electron microscopically the neuroendocrine cells contain neurohormonal granules which are membranebound and are mainly located in the basal area of the cells. The structure of these hormonal granules varies with their hormone contents. These cells have therefore been subgrouped on the basis of their ultrastructural appearance and of their identified hormone content. On stimulation, the neuroendocrine cells release their hormonal products either locally or into the blood stream. Some of the neuroendocrine cells elaborate neuron-like cytoplasmic protrusions from their basal aspect, which envelop and terminate on surrounding enterocytes (neuroendocrine or exocrine), affecting their function. These cells are designated «paracrine» cells.

Fig. 1. A neuroendocrine cell of the open type in a crypt of the intestinal mucosa. The cell is flask-shaped and its apical part reaches the lumen of the crypt. Immunostaining with monoclonal serotonin antibodies. X 1080.

Gastrointestinal carcinoid tumours . 3

2.1 The enterochromaffin (EC) cells The EC cells, which represent the predominant neuroendocrine cell type, are found in the entire gastrointestinal canal (ERSPAMER 1954; SOLCIA et al. 1981). In addition to serotonin, peptide hormone immunoreactivity (substance P, leu-enkephalin and motilin) has been reported to occur in these cells (SOLCIA et al. 1984a, b). The cells can be detected with several techniques in histological sections. Of these, the argentaffin reaction of Masson (HAMPERL 1927; SINGH 1964; PORTELA-GOMES 1982), the formalininduced fluorescence technique of Falck-Hillarp (FALCK et al. 1962) and, recently, immunocytochemistry with poly clonal or monoclonal antibodies against serotonin (FACER et al. 1979; CONSOLAZIONE et al. 1981; MILSTEIN et al. 1983) appear to be the most commonly used (Figs. 2 and 3). The enterochromaffin cells also stain with the argyrophil method of Grimelius, but in addition this technique identifies other neuroendocrine cell types storing hormonal peptides (Fig.4) (LUNDQVIST and WILANDER 1984). Since the argentaffin reaction is of low chemical specificity and the formalininduced fluorescence might be due to other biogenic amines that can give yellow

Figs. 2-3. Enterochromaffin (EC) cells of the intestinal mucosa identified with the formalininduced fluorescence technique of Falck-Hillarp (Fig. 2; x 250), and with monoclonal antibodies against serotonin (Fig. 3; x 250).

4 . E Wilander . . M. Lundqvist . K. Oberg

Fig. 4. Neuroendocrine cells of small intestinal mucosa visualized with the argyrophil stain of Grimelius. X 250. Fig. 5. Mucosa of the appendix immunostained with monoclonal antibodies against serotonin. In the mucosal crypts flask-shaped neuroendocrine (Eq cells are seen. The lamina propria harbours single or small aggregates of subepithelial neuroendocrine cells which are possibly related to the peripheral nervous system. X 250.

fluorescence, the accuracy of these techniques for identification of neuroendocrine cells containing serotonin is discussed in more detail. The intensity of the cytoplasmic staining with the different techniques varies in different parts of the neuroendocrine cells. The argentaffin and the argyrophil reactions are mostly limited to the basal part of these cells, leaving the apical area unreactive. With formalin-induced fluorescence both the apical and basal parts are fluorescent, but the intensity of the reaction is more pronounced at the basal part. Application of monoclonal anti-serotonin antibodies results in strong immunostaining of almost equal intensity over the cytoplasmic structure. Comparative studies of the reactivity of EC cells with the different staining techniques have revealed that formalin-induced fluorescence plus serotonin immunoreactivity occurs in 94% of the cells, 5% are only fluorescent and 1% are only serotonin-immunoreactive. Further, an argentaffin reaction plus serotonin immunoreactivity can be seen in 90% of the cells, 2% are only

Gastrointestinal carcinoid tumours . 5

argentaffin and 7% are only serotonin-immunoreactive. The Grimelius argyrophil reaction and the immunocytochemical reaction show the best concordance, 98% of the cells being identified by both techniques, while only 1% of the cells are either argyrophil or serotonin-immunoreactive (LUNDQVIST and WILANDER 1984). The Grimelius technique is considered to be a non-discriminating method for visualization of EC cells and several other neurohormonal cells storing peptide hormones. There are indications that serotonin might also be present in non-argentaffin endocrine cells. On the other hand, the non-argentaffin cells of the small intestine store peptide hormones such as somatostatin, glicentin (enteroglucagon) and neurotensin, and these cells are either unreactive with the Grimelius argyrophil technique or relatively weakly stained (GRIMELlUS and WILANDER 1980). The different technique for identifying serotonin-storing EC cells thus yield similar but not identical results. Probably the serotonin immunocytochemical method is more sensitive than the argentaffin technique, since the serotonin immunoreactivity occupies a larger cytoplasmic area than the argentaffin reaction in the endocrine cells. The number of cells displaying formalin-induced fluorescence has been found to exceed that of serotonin-immunoreactive cells (LUNDQVIST and WILANDER 1984). The reason for this is unclear, but the finding may have been due to the presence of a small cell population containing biogenic amines other than serotonin, that can give rise to a yellow fluoresecence (BJORKLUND et al. 1968; JONSSON and SANDLER 1969; BJORKLUND et al. 1972).

2.2 Peptide hormone-producing cells In addition to EC cells, the mucosa of the stomach and intestine is endowed with other neuroendocrine cells which are the storage sites of various peptide hormones. Together they constitute the largest neuroendocrine organ of the body. The various neuroendocrine celltypes overlap in their distribution, although each cell population usually predominates in a specific region (SOLCIA et al. 1984). In the acid-secreting part (the body and fundic area) of the gastric mucosa the enterochromaffin-like (ECL) cells dominate within the neuroendocrine cell population. These cells, which are characteristically stained with the argyrophil technique of Sevier-Munger, store an unknown hormone (SEVIER and MUNGER 1965; GRIMELIUS and WILANDER 1980). The gastric antrum and duodenum are rich in EC cells, gastrin (G) cells and somatostatin (D) cells. The entire small intestine and colon are dominated by EC cells. The number of neuroendocrine cells increases in the distal part of the large intestine and in the rectum. Relatively numerous EC cells and peptide-producing neuroendocrine cells storing mainly polypeptide YY (PYY), glicentin and pancreatic polypeptide (PP) are encoun~ teredo

6 . E. Wilander . M. Lundqvist . K. Oberg

2.3 Subepithelial neuroendocrine cells Cells with tinctorial and immunocytochemical properties indicative of neuroendocrine differentation are not exclusively located in the mucosa of the gastrointestinal canal but are also present in the subepithelial stroma. In 1928 MASSON reported a finding of argentaffin cells in the lamina propria of the appendix. He concluded that these cells derived from the epithelial argentaffin (EC (Kultchitsky) cells, which by a budding process migrated from the crypts of Lieberkiihn to their final location in the lamina propria (MASSON 1928). Subepithelial neuroendocrine cells occur relatively frequently in the appendix and can be identified in most appendiceal specimens. In addition to an argentaffin and an argyrophil reaction, they display serotonin and neuronspecific enolase immunoreactivity (Fig. 5). At the electron microscopical level heteromorphous neurosecretory granules similar to those observed in the mucosal EC cells are observed. The subepithelial neuroendocrine cells occur singly or in small clusters in the deeper part of the lamina propria beneath or between the epithelial crypts (RODE et al. 1982; MILLIKIN 1983; LUNDQVIST and WILANDER 1986). Serotonin-immunoreactive cells can also be found in deeper parts in the appendix, sometimes in close contact with ganglion cells. The subepithelial neuroendocrine cells are frequently surrounded by cells with long cytoplasmic processes displaying 5-100 protein immunoreactivity. These cells have been considered to be of 5chwann cell origin. This conclusion is based on ultrastructural observations that the subepithelial neuroendocrine cells are in close contact with multiple nerve fibres and non-myelinated 5chwann cells. Although there appears to be an accumulation of subepithelial neuroendocrien cells in the appendix, similar cells also seem to be present in the small intestine. However, at that location they are very frew and occur only as single cells in the deeper part of the mucosa (LUNDQVIST and WILANDER 1984). The presence of subepithelial neuroendocrine cells in other areas of the gastrointestinal canal has been considered possible, but this question has not been not systematically analysed.

3 Carcinoid tumours 3.1 Short history The term «Karzinoide» (carcinoid) was coined by OBERNDORFER (1907). Before that time sporadic tumours with the morphology of carcinoids were described, the first probably by MERLING in 1838 (MERLING 1838, cited by ROSENBERG 1986)). The argentaffin properties of the tumours were demonstrated in 1914 by GOSSET and MASSON. Their relationship to the argentaffin cells of the intestinal mucosa (Ku1chitsky cells) was emphasized and the tumours also came to be called argentaffinomas (MASSON 1928). In the 1950s a clinical syndrome associated with carcinoids was described by several

Gastrointestinal carcinoid tumours . 7

groups and was usually referred to as the carcinoid syndrome (GRAHAME-SMITH 1972). At about the same time high concentrations of serotonin were isolated from a carcinoid tumour and it was observed that patients with the carcinoid syndrome excreted large quantities of the serotonin metabolite 5-hydroxyindole acetic acid (5-HIAA) in their urine (LEMBECK 1953; GRAHAME-SMITH 1972). During the last decade it has been evident that gastrointestinal carcinoids are multiple-hormone-producing tumours, secreting serotonin and/or various peptide hormones. These tumours are therefore morphologically heterogeneous and are characterized by special biological properties depending on their topographic location (WILLIAMS and SANDLER 1963; NOBIN et al. 1983; SOLCIA et al. 1984b; MARTENSSON et al. 1985; NORHEIM 1986). They constitute a subpopulation of human tumours within the group of malignant neoplasms of the neuroendocrine type.

3.2 Definitions and classifications WILLIAMS and SANDLER (1963) introduced a new classification of gastrointestinal carcinoid tumours into fore-gut, mid-gut and hind-gut tumours on the basis of their topographical location. This classification has an embryological basis and its aim is to gather tumours with common features into distinct groups. The fore-gut (stomach and proximal duodenum) is the area of the gastrointestinal canal which receives its blood supply from the branches of the coeliac artery, the mid-gut (distal duodenum, small intestine, ascending colon and proximal transverse colon) is the area of the intestine which is supplied by the superior mesenteric artery, and the hind-gut (distal transverse colon, descending colon and rectum) is the part of the large intestine which is mainly supplied by the inferior mesenteric artery. Such properties of carcinoid tumours as an argentaffin reaction and an ability to produce the carcinoid syndrome are almost exclusively attributable to mid-gut carcinoids, and these tumours are therefore frequently designated «classical carcinoids», while fore-gut and hind-gut carcinoids are called «non-classical» or «atypical». The term carcinoid is used here to mean a primary neuroendocrinal tumour of the digestive tract, irrespective of the presence or absence of serotonin. The designation atypical carcinoid is also used, as in this presentation, to indicate dedifferentiated carcinoids or those with a lower degree of differentiation, e. g. carcinoids in the stomach, which represent borderline tumours within the spectrum of neuroendocrine tumours from ordinary carcinoids at one end to undifferentiated neuroendocrine carcinomas at the other. The term neuroendocrine will be used in the following instead of endocrine, as endocrine cells and tumours of the gastrointestinal canal share some common structural, functional and chemical features with neurosecretory and neuronal cells in the central nervous system.

8 . E.Wilander . M .Lundqvist . K.Oberg

3.3 Incidence Carcinoids of the gastrointestinal tract constitute about 2% of all malignant tumours that are annually reported to the National Cancer Registry in Sweden. BERGE and LINELL (1976) reported that the incidence of malignant carcinoid tumours found at autopsy was 2.1 per 100,000 population. Based on 103 patients with carcinoids, we calculated the incidence of clinically significant carcinoids to be 0.7 per 100,000 population (NORHEIM et al. 1987). The carcinoid syndrome was found in 0.5 per 100,000 population. From a similar study in Ireland, BUCHANAN et al. (1986) reported an annual incidence of carcinoids of 0.32 per 100,000 population. They concluded, however, that the true incidence would be underestimated because of an absence of clinical symptoms in some patients with carcinoids and a lack of good diagnostic markers. A carcinoid tumour may be found at all ages, even in children. In young patients the appendix is the most common location and the tumours are ferquently found incidentally during an operation for acute appendicitis (ANDERSON and WILSON 1985). The age at diagnosis of patients with carcinoids of the appendix is usually between 20 an 40 years, i. e. lower than that of patients with primary carcinoids of other locations (THOMSON et al. 1979 ; DAWES et al. 1984). Carcinoid tumours of the ileum and jejunum are diagnosed at an average age of 50 to 60 years. In our own material the average age at diagnosis was 59 years. In a similar study reported in 1983 MARTENSSON et al. found that the average age at diagnosis of patients with mid-gut carcinoids was 58 years. An almost equal frequency of carcinoid tumours has been noted in males and females (DAVIES 1959). In our own study, however, there was a slight predominance of women (55%).

3.4 Location The most common location of carcinoids of the gastrointestinal tract is in the appendix, where about 50% of all these tumours are found (DAWES et al. 1984; ANDERSON and WILSON 1985). In a comprehensive study of 3,000 patients with carcinoids, ORLOFF (1971) noted that 47% of the patients had primary tumours located in the appendix and about 30% in the rest of the mid-gut. A similar distribution of the primary tumour location was reported by SANDERS and AxTELL (1964) and WILSON et al. (1970). Since a majority of appendiceal carcinoids are clinically insignificant, i. e. they cause no distinct clinical symptoms, other mid-gut carcinoid tumours predominate among patients with clinical syndromes. In our own study and a similar study from the Mayo Clinic (DAVIS et al. 1973) comprising 94 patients with carcinoid syndromes, the primary tumours were located in the small intestine (ileum, jejunum) in 72% of the patients. Onyl 1-2% of the patients had appendiceal carcinoids. Similar data hav been reported by SABBACK and O'BRIEN (1979) and HENRIKSEN et al. (1979).

Gastrointestinal carcinoid tumours . 9

3.5 Metastases The incidence of metastases from gastrointestinal carcinoids varies in different reports and is dependent on the criteria applied in the collection of the material of patients. In our own material, patients were referred for medical treatment because of the carcinoid syndrome and liver metastases, and we found liver metastases in 93% of the patients and other peripheral metastases in 31 %.Moreover, five of 103 patients had skeletal metastases and one also had a metastasis in the pituitary gland. In a less selected series reported by DAVIES (1959) the commonest site of metastases was the regional lymph nodes, followed by the liver, mesentery and peritoneum (Table 1).

Table. 1.

Percentage distribution of metastases from gastrointestinal carcinoid tumours

(DAVIES 1969). Total number of patients: 545 %

%

Lymph Liver Mesentery Peritoneum Bone Pancreas Ovary Skin

33

25 18 10 2 2 2

Omentum Brain Spleen Adrenal Mediastinum Kidney Thyroid Testis Gall bladder

1 1 1 1 Solitary cases Solitary cases Solitary cases Solitary cases

Several factors should be considered when evaluating carcinoid tumours for signs of metastases: I. The location of the primary tumour. Colonic and ileal carcinoids are considered to be more prone to produce metastases (GOODWIN 1975). About 70% of all colonic carcinoids give rise to metastases, as compared with 30--60% of ileal tumours (WILSON et al. 1970). Metastases are found in about 18% of patients with rectal carcinoids, but only in 2-5% of those with carcinoids of the appendix. II. The size of the primary tumour. The size of the primary tumour also has an impact on the development of metastases. The risk increases as a tumour becomes larger (MOERTEL et al. 1961). In patients with primary tumours with a diameter of less than 1 em, metastases were found in 6%, but among those with primary tumours measuring more than 2 em, 70% developed metastases (ZEITELS et al. 1982).

10 . E. Wilander . M. Lundqvist . K. Oberg

III. The depth of tumour penetration into the bowel wall is important for the development of metastases (HAJDU et al. 1974). Among patients whose tumours invaded the serosa, metastases were found in 69%, but no metastases were observed when the tumour was limited to the submucosa (HAJDU et al. 1974; ZEITELS et al. 1982). IV. The histological growth pattern has also been claimed to be of importance for the development of metastases. The five main growth patterns are insular [A], trabecular [B], glandular [C], undifferentiated [D] and mixed type E. The most favourable growth patterns are insular and glandular [A and C] (SOGA and KRINJI 1971). The prognostic significance of the growth pattern in patients with carcinoid tumours has been evaluated by JOHNSON et al. (1983). In a study undertaken by the Eastern Cooperative Oncology Group and comprising 138 patients, a significant difference (p < 0.001) in observed survival time was found between different tumour growth patterns. In decreasing order of median survival time in years, the growth patterns ranked as follows: Mixed insular plus glandular 4.4; insular 2.9; trabecular 2.5; mixed insular plus trabecular 2.3; undifferentiated 0.5.

3.6 Multiple primary tumours In the small intestine BERGE and LINELL (1976) found multiple primary tumours in one-third of a series of autopsy cases, a frequency which is almost similar to that reported by MARTENSSON et al. (1983). In our own material we found multiple primary tumours in 30% of the patients; however, these figures may underestimate the true frequency, as all patients had not undergone laparotomy. Carcinoids arising in the body and fundic area of the stomach, like those of the small intestine, are also often multiple.

3.7 Second malignancies Simultaneous occurrence of other malignant tumours in patients with carcinoid tumours is fairly frequent. GOODWIN (1975) and ZEITELS et al. (1982) reported the presence of accessory malignant neoplasms in 17 to 53% of their patients with carcinoids, but in our own material we only found second malignancies in 5% of the patients. A similar frequency has been reported by DAVIS et al. (1973). Most commonly the second tumour is a colonic carcinoma, and this is important to keep in mind in the follow-up of patients with carcinoids, as many symtoms may mimic those of the carcinoid tumour. In our own series of 103 cases, two patients presented with breast cancer, one with chronic myelogenic leukaemia, and one each with rectal and prostatic carcinoma. The second malignancy was diagnosed after the carcinoid tumour.

Gastrointestinal carcinoid tumours . 11

Fig. 6. Light micrograph of a section of a classical mid-gut carninoid. The tumour is built up of monomorphous tumour cells growing in an insular-like pattern. Haematoxylin-eosin. X 250. Fig. 7. A section of a rectal carcinoid, showing the trabecular growth pattern of the regular tumour cells. Haematoxylin-eosin. x 250.

12 . E. Wilander . M. Lundqvist .K. Oberg

3.8 Light microscopic morphology 3.8.1 Routine histology

Although carcinoids of the gastrointestinal canal vary in their biological and morphological features, they share some common characteristics, such as a very regular growth pattern and the fact that the tumour cells are monomorphous. As a rule there are few mitotic figures. Mostly the differential diagnosis against adenocarcinoma is obvious. The mid-gut carcinoids, arising in the small intestine and proximal colon, are surprisingly similar. They are built up of regular insular-like tumour buds which sometimes anastomose with one another (Fig. 6). However, in some cases some tumour areas may display tubular structures and cystic formations. The appendiceal carcinoids may exhibit a picture similar to that of other mid-gut carcinoids, but may also show more solid, trabecular structures or grow in small cords in a prominent fibrous stroma. The hind-gut carcinoids, especially those in the rectum, are not characteristically insular but are mainly trabecular, solid or tubular and the differential diagnosis against highly differentiated adenocarcinoma is not always clear (Fig. 7). In the fore-gut tumours mixed growth patterns are frequently observed in various areas of the tumour. All different patterns may be found in tumours of this location, including solid, insular, trabecular and ribbon-like or tubular structures. In a survey of 42 gastric carcinoids classified as proposed by SOGA (1974), 67% showed a mixed growth pattern. Fourteen per cent of the tumours displayed an atypical picture [type D] (WILANDER et al. 1984), and these tumours were mostly larger (mean 5 cm) than the others (mean 1.8 em). Atypical carcinoids were present in both the acid-secreting and antral area of the stomach (Fig. 8). Previously, some gastric tumours initially diagnosed as carcinoma of the stomach have been classified as or neuroendocrinomas after being reviewed (CHE]FEC and GOULD 1977; ROGERS and MURPHY 1979; SWEENEY and McDONNELL 1980). A spectrum of endocrine neoplasia in the stomach analogous to that observed in the bronchus has been postulated and the is considered to hold an intermediate position both in terms of histological appearance and biological aggressiveness (ROGERS and MURPHY 1979; SWEENEY and McDONNELL 1980). Thus, the differential diagnosis between a gastric carcinoid and carcinoma of the stomach might be difficult and borderline tumours occur. The situation is quite different in the mid-gut, in particular, at which location a corresponding spectrum of tumours is rarely encountered. The fibrous stroma surrounding carcinoid tumour aggregates is particularly prominent in mid-gut carcinoids. Although the reason for this is not known, it is worthy of note that in a recent study it was found that stimulation of connective tissue cell growth by peptides of the tachykinin family such as substance P and substance K occurred in vitro (NILSSON et al. 1985). Since tachykinin production from small intestinal carcinoid tumours has been reported, a biological explanation for stromal tissue proliferation in these tumours is now offered.

Gastrointestinal carcinoid tumours . 13

Fig. 8. A section of a gastric carcinoid classified as atypical (type D according to the classification of Saga 1974). The tumour cells are densely packed and have hyperchromatic nuclei. Haematoxylin-eosin. X 250. Fig.9. A section of a classical mid-gut carcinoid stained with the argentaffin technique. Both strongly silver-stained and unreactive tumour cells are seen. Masson's stain. x 250.

14 . E. Wilander . M. Lundqvist . K. Oberg

3.8.2 Silver stains - general considerations Before the immunocytochemical era, various silver stains that are useful for identifying and characterizing neuroendocrine cells were available. These stains are still in common use, often in combination with immunocytochemistry for demonstrating neuroendocrine cells and corresponding tumours (Table 2). Most of these techniques were developed empirically and the chemical background of many of them is still unknown. Several of the silver-staining techniques are simple to perform and give reproducible results that are easily evaluated.

Table. 2. Typical silver-staining properties of carcinoid tumours of various topographical locations in the grastrointestinal tract. Argentaffin reaction Gastric (body-fundic) carcinoids Antral and duodenal carcinoids Classical (mid-gut) carcinoids Rectal carcinoids

Argyrophil Argyrophil reaction reaction Sevier-Munger Grimelius

+ +

+/ +

+ + + +/-

A distinction is made between methods giving argyrophil and argentaffin reactions (GRIMELIUS and WILANDER 1980). Cells displaying an argyrophil reaction retain silver ions from the impregnation solution, but viable metallic silver only appears after a subsequent reducing process brought about by an external agent(s). Cells showing an argentaffin reaction contain one or more chemical substances which both retain silver ions (from an ammoniacal silver solution) and reduce them to metallic silver. 3.8.2.1 Masson's stain The argentaffin method has been modified by many pathologists. The best known variants are those described by HAMPERL (1927, 1932) and SINGH (1964). Recently a very simple modification has been introduced by PORTELA-GOMES (1982), where the sections are placed in a 5% ammoniacal silver solution at a temperature of 60°C for 1 hour. This modification gives reproducible results with distinct staining of the EC cells. In contrast to that of the argyrophil methods, the chemical background of the argentaffin reaction is known - the reaction product between serotonin and paraformaldehyde has been shown to cause the silver reaction (BARTER and PEARSE 1953, 1955). Electron microscopic studies have disclosed that in the Masson stain the reduced silver is formed in the secretory granules (VASALLO et al. 1971). It should be kept in mind that post-

Gastrointestinal carcinoid tumours . 15

mortem autolysis weakens the argentaffin reaction and silver staining may fail completely if the tissue is fixed later than 6 to 8 hours after death. More than seven decades ago GOSSET and MASSON (1914) found that carcinoid tumours were argentaffin and possessed the ability to reduce ammoniacal silver to metallic silver, and the term argentaffinoma was coined (Fig. 9). The argentaffin reaction is due to the presence of serotonin. However, it is not a feature of all gastrointestinal carcinoids but only of those located in the mid-gut (WILLIAMS and SANDLER 1963; WILANDER et al. 1977b). Carcinoid tumours of the small intestine, appendix and caecum are almost unexceptionally argentaffin. Often there is a higher frequency of stained cells in the superficial part of the tumour than in the deeper part. The frequency of argentaffin cells has been found to vary between 20 and 74% (mean 59%) in primary small intestinal carcinoids, and between 57 and 99% (mean 78%) in appendiceal ones (LUNDQVIST and WILANDER 1982; WILANDER et al. 1985). In studies of gastric and duodenal carcinoids, six out of 40 of the former and one out of five of the latter were found to contain argentaffin cells. However, the stained cells were few and scattered and did not constitute the majority of the tumour cell population in any of these cases (WILANDER et al. 1979, 1981). A corresponding study of rectal carcinoids revealed only one argentaffin tumour out of 12, but even in this tumour most tumour cells were not stained (WILANDER et al. 1977a). Thus, apparently, as proposed by WILLIAMS and SANDLER (1963), the argentaffin reaction is one of the major simple staining techniques differentiating mid-gut from fore-gut and hind-gut carcinoids. Since small intestinal carcinoids are mostly larger than those of the appendix, the fixation times for the tumour mass are not identical. This might explain the differences in the frequency of argentaffin cells in the tumours of these locations, but it must also be kept in mind that appendiceal carcinoids, which are biologically more benign, are built up of a more homogeneous tumour cell population. The argentaffin reaction in the carcinoids is related to the ability of the tumours to give rise to the carcinoid syndrome when they spread. Accordingly, this syndrome is frequently found in association with carcinoids of the mid-gut, mainly in the small intestine with metastatic nodules in the liver, while malignant carcinoids from other topographical areas of the digestive system very rarely give rise to the carcinoid syndrome. In spite of this the carcinoid syndrome cannot be attributed to increased serotonin production alone, but is probably the result of concomitant release of multiple active biological factors. The argentaffin staining properties characterizing carcinoids of various locations in the gastrointestinal canal are as a rule retained in their metastatic deposits. Consequently liver metastases from small intestinal carcinoids mostly display an argentaffin reaction, while those originating from more proximal or distal parts and also from endocrine pancreatic tumours do not (LINDGREN et al. 1984). This observations is of some clinical importance and emphasizes the fact that proper silver staining of the metastatic nodules gives information on the presumed site of the primary tumour.

16 . E. Wilander . M. Lundqvist . K.Oberg

3.8.2.2 Grimelius stain

The Grimelius argyrophil reaction of neuroendocrine cells of the digestive tract can be strengthend by increasing the silver nitrate concentration from 0.03 per cent (as suggested in the original method) to 0.07 per cent and the temperature of the reducing solution from 47 to 55°C (GRIMELIUS 1968; GRIMELIUS and WILANDER 1980). With this technique a large number of neuroendocrine cells can be demonstrated - glucagon and pancreatic polypeptide cells in the endocrine pancreas and the most well known neuroendocrine cell types in the gastrointestinal mucosa, except cholecystokinin (CCK), somatostatin and PYY cells. A weak argyrophil reaction can be improved by reimpregnation (GRIMELIUS and WILANDER 1980). This means that after rinsing in redistilled water the silver-stained sections are placed in another freshly prepared silver solution at room temperature for 15 minutes and then in a freshly prepared reducing solution at 55°C for 1 minute. The compositions of the silver and reducing solutions are the same as are used in the primary impregnation and reducing steps. The argyrophil reaction of Grimelius was initially developed in order to visualize the glucagon-producing A (a2) cells of the pancreatic islet tissue. It has the advantage over the previous Bodian technique that the initial silver nitrate solution is easily commercially available and stable, in contrast to the silver proteinate used in the Bodian method. Although the use of the argyrophil reaction of Grimelius for identification of the glucagon cells has nowadays been mostly superseded by immunocytochemistry with glucagon-specific antiserum, the Grimelius technique has proved to be very useful for identification of neuroendocrine differentiated cells in normal tissues and neuroendocrine tumours irrespective of their hormone content (WILANDER et al. 1977b; GRIMELIUS and WILANDER 1980). The stain is almost nondiscriminating, that is, a positive reaction can be obtained in most normal neuroendocrine cell types of the gastrointestinal canal storing peptide hormones and/or biogenic amines. Although the chemical background of the argyrophil reaction is unknown, it has been repeatedly shown at electron microscopy that the silver reaction is concentrated exclusively to the neurohormonal secretory granules, and thus the Grimelius stain can be applied on the light microscopical level to demonstrate the presence of neurohormonal granules in neuroendocrine cells and tumours which otherwise could only be detected ultrastructurally (Fig. 10). Since the Grimelius stain non-discriminately stains neuroendocrine tumours, it has developed into a routine method for identifying neuroendocrine differentiated tumours. In accordance with the above, gastrointestinal carcinoids are argyrophil (Fig. 11). A quantitative study with application of a point sampling method revealed that the mean proportion of argyrophil cells in small intestinal carcinoids was 87% (range 63-95%), and of unstained cells 13 %, while the corresponding values in appendiceal carcinoids were 97% (range 88-100%) and 3%, respectively (LUNDQVIST and WILANDER 1982;

Gastrointestinal carcinoid tumours . 17

hormon al granules. These Fig. IO. Electron microgr aph of neuroen docrine cells with round techniqu e of Grimelius. X hil granules accumulated silver particles after staining with the argyrop 16300. with the argyrop hil techniFig. 11. A section of a classical small intestinal carcinoid after staining que of Grime/ius . Most tumour cells are silver-stained. X 250.

18 . E. Wilander . M. Lundqvist . K. Oberg

WILANDER et al. 1985). Since argentaffin cells are also argyrophil, the quantiative study of the silver-staining properties of the tumours disclosed three types of tumour cells in midgut carcinoids, namely: (1) cells that are both argentaffin and argyrophil, (2) cells that are argyrophil but non-argentaffin and (3) unreacative cells. In most mid-gut tumours the argentaffin cells constitute the majority of the cell population, while the argyrophil but non-argentaffin cells are in the minority. The argentaffin cells presumably store serotonin, while the non-argentaffin but argyrophil cells store other hormonal products. Thus, there is indirect evidence for a multihormonal nature of carcinoid tumours. In a study of 45 gastroduodenal carcinoids an argyrophil reaction was observed in most tumour cells in 40 cases, while in the other five - three gastric and two duodenal tumours - positively stained cells were relatively few. However, among the gastric carcinoids six displayed an atypical morphology (type D according to SOGA 1974) and in two of these tumours most tumour cells failed to stain with the argyrophil technique (WILANDER et al. 1984). Thus, there are indications that some gastric carcinoids are morphologically dedifferentiated and biologically nearer to gastric carcinomas, and in some of these tumours signs of neuroendocrine differentiation as revealed with the Grimelius silver stain tend to be less pronounced. There appears to be some controversy concerning the ability of rectal carcinoids to show silver staining with the argyrophil technique of Grimelius. Some authors claim that these tumours become silver stained without exception, whereas others have found both argyrophil and unreactive tumours. Since it was considered that the Grimelius argyrophil reaction might be of value as a supplement to routine histological examination for accurate diagnosis of these tumours, this reaction was examined in 27 rectal carcinoids. The argyrophil reaction (stronger variety) of the tumours was studied semiquantitatively (WILANDER et al. 1983). The most frequent finding was a weak silver reaction in the majority of the tumour cells and the next most frequent was a weak reaction in a minority of the tumour cells. In 16 tumours most tumour cells were stained, while in the other 11 most tumour cells were unreactive. In three tumours only few scattered silver-stained tumour cells were observed. The findings indicate that all rectal carcinoids display argyrophil cells with the slightly modified Grimelius silver stain, although the argyrophil reaction is sometimes weak and present only in a minority of the tumour cell population. Since varying staining results have been obtained in rectal carcinoids, it is critical to state whether the silver staining is performed according to the original description or whether the slight modification of the method is used. Is is clear that the Grimelius argyrophil stain is a useful supplement to the histopathologioal diagnosis of carcinoid tumours in routinely stained sections, and at present it is used in most departments of pathology in Sweden. 3.8.2.3 Sevier-Munger stain With the Sevier-Munger argyrophil stain a stronger silver reaction will occur in EC cells, ECL cells and gastric inhibitory peptide (GIP) cells of the gastrointestinal mucosa

Gastrointestinal carcinoid tumours . 19

(SEVlER and MUNGER 1965; GRIMELIUS and WILANDER 1980). This method is especially important for identifying EeL cells, since they store an unknown hormone, which means that immunocytochemical methods cannot be used. With the Sevier-Munger method EeL cells show an argyrophil but not an argentaffin reaction. In a series of gastrointestinal carcinoids a positive Sevier-Munger reaction was found in 73% of the tumours, while almost 100% were argyrophil with the Grimelius technique (WILANDER et al. 1979). In patients with atrophic gastritis and achlorhydria, proliferation of the EeL cells, mainly in the deeper part of the mucosa, is frequently encountered. This proliferation, which can be either diffuse or nodular, is considered to precede the development of infiltrating carcinoid tumours of the body and fundus of the stomach and is easily visualized after staining with the Sevier-Munger technique. Accordingly, most gastric carcinoids arising in the non-antral part of the stomach are non-argentaffin but are argyrophil with the Sevier-Munger technique. Thus, in the study of the silverstaining characteristics of gastrointestinal carcinoids the Sevier-Munger argyrophil stain provides additional information, especially in non antral gastric carcinoids.

3.8.2.4 Davenport technique A modification of the Davenport technique described by HELLERSTROM and HELLMAN (1960) can be used to identify the somatostatin cells of the pancreatic islet and gastrointestinal mucosa. At the electron microscopical level the silver grains are concentrated to the hormone granules (GRIMELIUS and STRAND 1974). The method has been applied to visualize somatostatin in carcinoids, but nowadays it is mostly considered less useful than immunocytochemistry with application of somatostatin antisera.

3.8.2.5 Silver stains applied to cytological specimens The development of new diagnostic tools such as ultrasound, computerized tomographic (CT) scans and nuclear magnetic resonance (NMR) has increased the possibility of identifying, sometimes incidentically, small nodular masses in neuroendocrine organs and also discrete metastatic lesions of primary carcinoid tumours of the gastrointesstinal tract. The question whether surgery or antitumour therapy is the more desirable for these lesions may depend on the results of clinical tests, including percutaneous fine-needle biopsy guided by ultrasound. Silver stains are currently used for identifying and caracterizing neuroendocrine tumours on histological paraffin-embedded sections. However, the accuracy of the silver-staining method as applied to cytological specimens of these tumours has only recently been evaluated. In order to get cytological material for such a study in a convenient way, imprint cytological samples have been prepared from tissue specimens obtained by percutaneous needle (2.0 mm diameter) biopsy of metastatic neuroendocrine tumours of the liver (WILANDER et al. 1985). The imprint specimens were considered to display staining properties approximately equal to those of an ordinary fineneedle biopsy, but to contain a larger number of more easily visible monolayer tumour

20 . E. Wilander . M .Lundqvist . K. Oberg

cells. Further, the staining properties of each tumour could be compared in the histopathological and cytological samples. Silver stains such as the argentaffin reaction of Masson, the argyrophil reaction of Grimelius and the Sevier-Munger stain could be evaluated (WILANDER et al. 1985). In the above study it was found that all imprints of carcinoid tumour metastases from primary small intestinal carcinoids displayed an argentaffin reaction in a variable number of cells provided the cells were fixed in 10% buffered formalin. After air-drying or other fixations (Bouin's fluid; acetone-alcohol 1:1) no argentaffin reaction was observed. A corresponding argentaffin reaction was also seen in the histological sections from the same tumours. Histological sections and cytological samples of metastases of pulmonary carcinoid tumours lacked an argentaffin reaction. As mentioned above, a prerequisite for positive staining is formalin fixation, as ~-carbolin, the presumed reaction product, is formed by condensation of serotonin and formaldehyde (BARTER and PEARSE 1953, 1955). Among neuroendocrine tumours which store peptide hormones or biogenic amines, or both, mid-gut carcinoid tumours - of which small intestinal and caecal carcinoids, in particular, behave as malignant tumours - are the only ones that characteristically store and secrete serotonin. Consequently, these tumours are almost invariably argentaffin-positive. This staining porperty is mostly preserved in their metastatic deposits. Thus, the argentaffin method applied to cytological material appears to be an accurate technique for identifying neuroendocrine tumours containing serotonin and when used on cellular samples of metastatic neuroendocrine tumours it helps to identify the primary site of the tumour. Both carcinoid tumours and malignant melanomas are members of the group of complex tumours of the neuroendocrine type that are considered to derive from the peripheral neuroendocrine cell system and to be capable of synchronous production of immunoreactive peptides, biogenic amines and melanins. A metastatic malignant melanoma in the study referred to above exhibited an argentaffin reaction that was obviously not caused by serotonin (but melanin), as the reaction was also observed in air-dried, Bouin-fixed and acetonealcohol fixed cytological specimens. The ability of melanins to reduce the silver salt solution without formalin condensation shows that an argentaffin reaction resulting from the presence of serotonin or melanins can be distinguished by the application of different fixatives to the cytological material. In contrast to the clearly positive reaction observed in the corresponding histological sections in the cytological specimens the argyrophil reaction was relatively weak or negative. The strongest positive reaction was found in tumours displaying an argentaffin reaction, while histological1y non-argentaffin but argyr~phi1 tumours were relatively weakly stained or unreactive, probably because the silver nitrate concentration in the initial solution is low (0.03-0.07 per cent); in addition, in histological sections the sites of the silver reaction - the neurohormonal granules - are exposed on the cut surface, whereas in cytological specimens the silver salt has to pass through the cell membrane. However, the argyrophil reaction of Grimelius appears to be the silver stain of choice

Gastrointestinal carcinoid tumours . 21

for identifying neuroendocrine tumour cells with an unknown hormone content in cytological material (Fig. 12). The argyrophil stain of Sevier-Munger caused a reaction in imprints of argentaffin tumour cells, but appeared less reliable. The high concentration of silver in this technique resulted in a heavy non-specific background staining reaction, which made the cytological samples difficult to interpret.

Fig. 12. Imprint cytological specimen from liver metastasis of a small intestinal carcinoid. Many tumour cells (one indicated with an arrow) display a cytoplasmic argyrophil (Grimelius) reaction. X 400.

3.8.3 Immunocytochemistry 3.8.3.1 Serotonin

Our studies on serotonin immunoreactivity in EC cells and carcinoid tumours have been mainly based on the application of a monoclonal antibody developed by CONSOLAZIONE et al. (1981). The antibody (YC5/45 HLK) was prepared from serotonin conjugated to bovine serum albumin as the immunogenic carrier protein, through a condensation reaction with formaldehyde. The antibody cross-reacts in haemagglutination tests with dopamine, serotonin, tryptamine and 5-methoxytryptamine at high

22 . E. WiJander . M. Lundqyist . K. Oberg

concentrations. The serotonin-albumin conjugate is most effective in revealing the antibody, while pure albumin is not recognized. However, in fixed preparations immunoreactivity is observed in serotonin - but not dopamine-containing cells. When the binding of antibody was tested by cross-linking various amines to serum albumin with paraformaldehyde, only the serotonin derivate was active. It was concluded that the discrimination specificity observed in immunohistochemistry is due to the paraformaldehyde fixation (MILSTEIN et al. 1983). Like that of the EC cells, the serotonin content of carcinoid tumours can be studied in histological sections with the use of various histochemical techniques such as the argentaffin reaction (PORTELA-GOMES 1982), by means of formalin-induced fluorescence according to Falck-Hillarp (FALCK et al. 1962), and immunocytochemically with antibodies against serotonin (FACER et al. 1979; CONSOLAZIONE et al. 1981; MILSTEIN et al. 1983). These techniques, as mentioned previously, stain similar but not identical neuroendocrine cells in the intestinal mucosa, but the discrepancy between the techniques for visualizing serotonin in carcinoid tumours is even more obvious. Of 14 primary small intestinal carcinoids, which were formalin-fixed and paraffinembedded, all displayed formalin-induced fluorescence, although the number of reactive cells varied in the individual cases. An argentaffin reaction was observed in all tumours except one. After application of monoclonal antibodies against serotonin, seven tumours were unreactive and seven were stained (Fig. 13). Of the latter, three cases displayed a relatively weak reaction. Thus, six tumours were argentaffin-reactive and showed formalin-induced fluorescence but no serotonin immunoreactivity. After examination of consecutive sections, it became clear that the tumour cells with an argentaffin reaction exceeded the number of serotonin-immunoreactive cells in tumours stained with both techniques, except in one case in which some areas exhibiting a strong positive reaction with serotonin antibodies failed to stain with the argentaffin reaction. In a similar study on 12 appendiceal carcinoid tumours, however, there was complete correspondence. All srotonin-immunoreactive tumours were argentaffin, and vice versa (LUNDQVIST and WILANDER 1984). The serotonin content of fore-gut carcinoid tumours was investigated in a further study (WILANDER et al. 1985). A series of 25 gastric carcinoids were stained immunocytochemically with monoclonal antibodies against serotonin and with the argentaffin reaction and examined for formalin-induced fluorescence. As well as applying the different histological and histochemical methods to consecutive sections, they were also used in identical sections. the methods were used in the following sequence: formalininduced fluorescence ~ argentaffin reaction, formalin-induced fluorescence ~ serotonin immunocytochemistry, serotonin immunoreactivity ~ argentaffin reaction and serotonin immunoreactivity ~ the modified Warthin-Starry argyrophil reaction. The latter silver stain identifies melanins (WARKEL et al. 1980). Photographs were taken from the initial staining/fluorescence and after the destainin and restaining procedure, and were compared (LUNDQVIST and WILANDER 1983).

Gastrointestinal carcinoid tumours . 23

Immunocytochemistry with monoclonal antibodies against serotonin revealed a staining reaction in a relatively large tumour cell population, but not in the majority of the cells of two gastric carcinoids, and in one gastric carcinoid there were only a few scattered immunoreactive cells. An argentaffin stain was observed in two tumours. The argentaffin stain only partly corresponded to the immunoreactivity to serotonin. One tumour displayed serotonin immunoreactivity, but consecutive tumour sections lacked argentaffin tumour cells. Application of the different techniques for identifying serotonin-storing tumour cells thus disclosed three types of tumour cells, namely: (1) serotonin-immunoreactive, nonargentaffin and non-fluoresecent cells, (2) serotonin-immunoreactive, argentaffin and fluorescent cells and (3) non-serotonin immunoreactive, argentaffin and non-fluorescent cells. The first serotonin-immunoreactive cell type was most frequently found in the tumours. In one gastric carcinoid in which argentaffin cells exceeded serotoninimmunoreactive ones a positive reaction was found with the modified W arthin -Starry reaction for demonstrating melanin. Application of the staining and restaining method to the melanin-stained gastric carcinoid showed that some but not all argyrophilic cells (modified Warthin-Starry method) were serotonin-immunoreactive. Similar results were obtained in a corresponding study of 35 bronchial carcinoids, and also in hind-gut tumours, especially rectal carcinoids. The immunocytochemical technique with use of monoclonal antibodies against serotonin has been suggested as the method of choice for identifying serotonin-containing tumours (CUELLO 1982). In our study, however, several small intestinal carcinoids displaying formalin-induced fluorescence and an argentaffin reaction failed to stain with monoclonal serotonin antibodies. Furthermore, the study of consecutive sections revealed that unidentical tumour cells were argentaffin and serotonin-immunoreactive. The reason for this discrepancy is not completely understood. Although the argentaffin reaction and the formalin-induced fluorescence are not specific, they identify serotonin. Thus, it is uncertain whether the tumour cell which is serotonin-immunoreactive but lacks a positive reaction with the other techniques really does store serotonin. However, the immunocytochemical technique with serotonin antibodies is presumably much more sensitive than the other methods. It is known that fore-gut carcinoids are usually devoid of decarboxylase for conversion of 5-hydroxytryptophan to serotonin, but the monoclonal antibody used does not cross-react with 5-hydroxytryptophan and thus the presence of this substance in the tumour cells could not explain the immunocytochemical reaction. Tumour cells that were both serotonin-immunoreactive and argentaffin also displayed formalin-induced fluorescence, as shown in identical sections, and there is strong evidence for the presence of serotonin in such cells. Argentaffin tumour cells without concomitant serotonin immunoreactivity and fluorescence are seen in gastric carcinoid tumours but are mainly found in bronchial carcinoids. In addition to serotonin, the argentaffin technique stains melanin granules and phenols (catecholamines). The modified Warthin-Starry argyrophil reaction for

24 . E. Wilander . M. Lundqvist . K. bberg

Fig. 13. A section of an infiltrating small intestinal carcinoid tumour, in which several tumour cells show serotonin immunoreactivity with monoclonal antibodies . X 250. Fig. 14. Strong neuron-specific enolase immunoreactivity in a classical carcinoid.

X

250.

Gastrointestinal carcinoid tumours . 25

melanins was positive in one gastric carcinoid with more numerous argentaffin than serotonin-immunoreactive cells. Melanin production in fore-gut carcinoids appears to be infrequent, but a few cases are on record. Carcinoid tumours can be considered to derive from cells belonging to the diffuse neuroendocrine cell system in which melanocytes are included. This means that carcinoids contain cell populations capable of synchronous production of immunoreactive peptides, biogenic amines and melanin. In accordance with this concept, in the melanin-stained gastric carcinoid some but not all argyrophilic cells (modified Warthin-Starry stain) were serotonin-immunoreactive. Immunocytochemistry with the monoclonal antibody against serotonin seems to be a sensitive and useful method for visualizing normal neuroendocrine cells storing serotonin. However, the results obtained on application of these antibodies to neuroendocrine tumour sections must be interpreted with some caution. It is suggested that the use of additional techniques increases the possibility of more accurate identification of serotonin in these tumours, since the serotonin immunoreactivity is not unquestionably absolutely specific. With regard to the argentaffin reaction the possible occurrence of melanins and catecholamines in addition to serotonin must be considered.

3.8.3.2 Serotonin in cytological samples The monoclonal antibody against serotonin has been used to visualize serotonin in cytological samples of neuroendocrine tumours (WILANDER 1985). Although the argentaffin method can be applied to such material, serotonin immunocytochemistry is probably much more sensitive. We have examined cytological material from malignant carcinoid tumours immunocytochemically with regard to the presence of serotonin. Immunoreactivity was observed in carcinoid tumour cells derived from primary small intestinal carcinoids, while bronchial carcinoid tumour cells were unreactive. Acetonealcohol fixation of the cells was a prerequisite for adequate staining. The serotoninimmunoreactive tumour cells were also argentaffin. Thus it seems that cytological specimens can be identified for the presence of serotonin with an immunocytochemical procedure. The monoclonal antibody (YC5/45HLK) applied was developed by conjugative serotonin to serum albumin as an immunogenic carrier protein, although a condensation reaction with formaldehyde, and the avidity of the antibody, increase after formaldehyde fixation of the antigen. Nevertheless, acetonealcohol fixation gave much more reliable immunostaining than air-dried, formaldehyde or Bouin-fixed cells. Probably the critical point, namely the penetration of the intracellular antigen, is only adequately obtained in the acetone-alcohol-fixed cytologic samples.

3.8.3.3 Peptide hormones During the last decade it has become evident that neuroendocrine tumours produce multiple hormones. This is also true for carcinoid tumours, and in addition to serotonin a wide variety of peptide hormonal substance have recently been identified in car-

26 . E. Wilander . M. Lundqvist . K. Oberg

cinoids. However, the occurrence of the individual peptides varies in carcinoids with respect to their site. Thus, the typical mid-gut carcinoids, which mainly store and secrete serotonin, also frequently display substance P immunoreactivity. It is possible that serotonin and substance P are stored in the same tumour cells, as has been suggested for the mucosal EC cells (AWMETS et al. 1977). Substance P is detected in the plasma of most patients with mid-gut carcinoids (EMSON et al. 1984). Chemical characterization of substance P-immunoreactive carcinoids with high pressure liquid chromatography showed that the immunoreactive material was indistinguishable from synthetic substance P (RATZENHOFER et al. 1981). Recent results indicate that substance P, which belongs to a group of peptides of the tachykinin family, is not the only substance of this family that is stored and secreted. In addition to these tachykinins, such as substance P, neuropeptide K and neurokinin A and B, various peptides, in minor cell populations, can be detected immunocytochemically; these include gastrin, gastrin releasing peptide [GRP], growth hormone releasing factor [GRF], somatostatin, enkephalin, and neuropeptide Y [NPYJ (AWMETS et al. 1978; WILANDER et al. 1979; DAYAL et al. 1980a, b; GOEDERT et al. 1980; WILANDER et al. 1981; BOSTWICH et al. 1984; DAYAL et al. 1985; MARTENSSON et al. 1985; NORHEIM 1986). Rectal carcinoids show immunoreactivity mainly wiht PYY, glucagon/glicentin, PP and somatostatin antisera, although other peptide hormones may also be present (WILANDER et al. 1977a; FIOCCA et al. 1980; TAXY et al. 1980; IWAFUCHI et al. 1986; FIOCCA et al. 1987). The tumours display immunoreactivity with one or several hormone antisera, but sometimes have no identifiable hormonal substances. It is worthy of note that serotonin, which is stored in EC cells, one of the most frequently found neuroendocrine cell types in the human rectum, is only occasionally present in rectal carcinoids and only in minor cell populations. The peptide hormones occurring in rectal carcinoids have not so far been found to cause any clinical endocrine syndromes. The fore-gut carcinoids located in the antrum of the stomach and duodenum most frequently store gastrin and somatostatin, but other hormonal substances have occasionally been identified (WILANDER et al. 1979). In the non-antral area of the stomach the ECL cell tumours predominate (CARNEY et al. 1983). They store an unknown hormone. However, minor tumour cell populations with immunoreactivity to serotonin or peptide hormones, such as PP and gastrin, may be found (WILANDER et al. 1986).

3.8.3.4 Neuroendocrine tumour markers Recently neurohormonal markers have been introduced in clinical pathology to serve as tools in the differential diagnosis between carcinoids and malignant tumours of other origins. These substances which can be identified with immunohistochemical techniques by application of specific antisera, are either related to the neurohormonal secretory granules and identify proteins stored in these structures, or are not directly correlated to the granular component. In 1965 MOORE and MCGREGOR showed that a protein designated 14-3-2 appeared

Gastrointestinal carcinoid tumours . 27

during development of the brain. Further studies revealed that this protein was located in neurons and it was renamed neuron-specific protein (PICEL et al. 1976). RIDER and TAYLOR (1974) described a new immunologically distinct form of enolase in rat brain and FLETCHER et al. (1976) demonstrated that brain tissue contains three enolase isoenzymes, which they designated aa, ay and yy. The yy-enolase form was further shown to increase in concentration during development of the brain, and on structural and immunological grounds it was concluded that the yy-enolase was identical to the 14-3-2 protein (BOCK 1975; FLETCHER et al. 1976; SCHMECHEL et al. 1978b), which was then redesignated neuron-specific enolase (NSE). NSE was first considered to be located exclusively in neurons and thus to be present only in the central nervous system, but later it was also found in peripheral neuroendocrine cells (SCHMECHEL et al. 1978a; 1978b; FACER et al. 1980) and its possible use as a specific marker for tumours derived from these cells was suggested (TAPIA et al. 1981; MARANGOS et al. 1982). An investigation was undertaken to determine whether NSE immunoreactivity occurred in neuroendocrine cells of the small intestinal mucosa and in carcinoid tumours with a corresponding topographical location (LUNDQVIST et al. 1985). In addition, a comparative study on the same section was performed with regard to NSE immunoreactivity and silver staining properties of normal and neoplastic neuroendocrine cells. The NSE antiserum used was raised in sheep by repeated injections of NSE purified from human brain. The antiserum showed no cross-reactivity with aa-enolase from human brain and in an immunodiffusion experiment it gave one precipitation band with a crude brain NSE preparation. The antiserum also precipitated 125I-labelled NSE. Using an immunocytochemical method, the antiserum stained neurons, peripheral nerve bundles, pancreatic islet cells, and C cells of the thyroid gland. Sections of liver and straited muscle did not stain with the antiserum, indicating that the antiserum did not cross-react with aa- and ~~-enolases in fixed sections (PAHLMAN et al. 1984). In six out of ten formalin-fixed and paraffin-embedded sections of normal small intestinal mucosa, NSE-immunoreactive cells were observed (Fig. 14). They were located mainly in the deeper part of the crypts and displayed either homogeneous staining all over the cytoplasm or a concentrated immunoreaction in the perinuclear space leaving most of the peripheral cytoplasmic area unreacative. With the staining and restaining procedure used to characterize the NSE-immunoreactive cells in the normal intestinal mucosa, virtually all cells could be identified as neuroendocrine, as they stained with both the argyrophil and argentaffin techniques. There was a slight discrepancy, however, in that there were occasional cells with NSE reactivity but no silver reaction. The silver-staining reactions were limited to the basal part of the normal neuroendocrine cells, corresponding to the area in which the neurohormonal cytoplasmic granules are concentrated, as revealed ultrastructurally, while the immunocytochemically detected NSE was most pronounced in the perinuclear space, as NSE is a metabolic enzyme and is not related to the secretory granules.

28 . E. Wilander . M. Lundqvist . K. Oberg

In a study of 16 small intestinal carcinoid tumours that were formalin-fixed and paraffin embedded, all stained with the NSE antiserum. The intensity of the staining and the number of reactive cells varied between the individual cases. All tumours exhibited a positive reaction with the Grimelius argyrophil technique. The Masson argentaffin technique stained 14 of 16 cases. A comparative study on restained sections showed that the NSE immunoreactivity did not correlate with either the argyrophil or argentaffin reaction. Three types of cells were observed in the tumours: one which stained both with the NSE antiserum and with one or both of the silver techniques; a second type which stained with one or both silver stains but failed to react with the NSE antiserum; and a third which was only NSE-immunoreactive. Neuron-specific enolase has been demonstrated in primary tumours of neuroendocrine origin both by immunocytochemistry and by radioimmunoassay in plasma from patients with neuroendocrine neoplasia (TAPIA et al. 1981; PRINTZ et al. 1983; PRINTZ and MARANGOS 1983). Our results are in accordance with these reports. Application of the staining and restaining technique revealed that the NSE immunoreactivity of the tumour cells was not entirely correlated to the argentaffin and agyrophil reactions. It appears that the NSE immunoreactivity in these tumours reveals their neuroendocrine differentiation and funtional activity, and the silver stains show the presence of neurosecretory granules. Thus, a combination of NSE immunocytochemistry and silver stains provides good information on neuroendocrine differentiation, and for identification of neuroendocrine tumours with a low granular content, NSE immunocytochemicstry might be a more useful histological technique than silver staining. However, caution is required when interpreting a positive NSE immunoreaction, since tumours that are generally considered to be non-neuroendocrine may display a positive reaction. Furthermore, NSE may be observed in non-neuroendocrine human cell lines with use of a direct (enzymatic) and an indirect (radioimmunoassay) method. In general, neuroendocrine tumour specimens and neuroendocrine-derived tumour cell lines contain more NSE than non-neuroendocrine tumour specimens and cell lines, but some cultured haematopoietic cell lines have been shown to exhibit NSE concentrations comparable to those found, for instance, in neuroblastoma and small celIlung carcinoma cell lines (ESSCHER 1985). Thus, NSE appears not to be expressed solely in neuroendocrine tumour cells. Chromogranin comprises a family of acidic polypetides of various sizes, which constitue the major part of the soluble proteins in the neurosecretory granules of several but not all polypeptide and/or biogenic amine-storing neuroendocrine cell types (SMITH and WINKLER 1967; BLASCHKO et al. 1983; FISCHER-COLBRIE et al. 1985; O'CONNOR and DEFTOS 1986). The proteins were originally isolated from granules of the adrenal medulla. Cloned cDNAs have exhibited a chromogranin A molecule of 431 amino acids and its gene has been localized to chromosome 14 (DEFTOS et al. 1986; BENEDUM et al. 1987; MURRAY et al. 1987). The function of chromogranin A is not known, but its concentration is increased in patients with a wide variety of neuroendocrine tumours

Gastrointestinal carcinoid tumours . 29

(O'CONNOR and DEFTOS 1986). It is now presumed to be secreted by granular exocytes in neuroendocrine tissues and possibly to possess a hormonal function. Monoclonal and polyclonal antibodies have recently been developed as markers for neuroendocrine cells and tumours (WILSON and LLOYD 1984; LASSMANN et al. 1986). Examination of a monoclonal antibody to chromogranin A developed by WILSON and LLOYD (LK2H10) showed staining of the A (glucagon) cells of the pancreatic islets. Further, scattered cells were immunoreactive in the intestinal mucosa. With our restaining technique (LUNDQVIST and WILANDER 1983) these cells were found to correspond to the argyrophil (Grimelius) cells of the mucosa. The almost total accordance between the argyrophilic and chromogranin A-immunoreactive cells has led to the suggestion that chromogranin A responds to silver staining by an argyrophilic reaction, and the Grimelius silver stain, in fact, in addition to other chemical components, identify chromogranin A (Fig. 15) (WILSON and LLOYD 1984; VARNDELL et al. 1985; MALMGREN et al. 1986; RIND! et al. 1986). Small intestinal carcinoids are chromogranin A-immunoreactive. However, on the cellular level the chromogranin immunoreaction is not always identical with the argyrophil reaction (Fig. 16). Thus, some cells are both chromogranin-stained and argyrophil, while others stain with either of the two staining methods (MALMGREN et al. 1986). Gastric and duodenal carcinoids are chromogranin A-immunoreactive and also argyrophil, whereas rectal carcinoids often show only weak or negative immunostaining. In general, the monoclonal chromogranin A antibody seems to yield positive results in the same tumour tissues as the argyrophil (Grimelius) reaction. S-IOO protein is a highly acidic protein with a molecular weight of 21,000, which was first isolated from bovine brain extract by MOORE (MOORE 1965; MOORE and PEREZ 1968). The substance contains a number of molecular species, structurally built up of two polypeptide chains which associate as dimers with subunit compositions of aa, a~ and ~~. Until recently 5-100 protien was regarded as a neuro-specific protein occurring mainly in glial and 5chwann cells, but it is now known that it is also present in several non-neural cells such as melanocytes and Langerhans cells of the skin, interdigitating reticulum cells of lymphoid tissues, chondrocytes, satellite cells of the adenohypophysis, and interstitial cells of the pineal body. Immunohistochemistry has provided information on the occurrence of 5-100 protein in various human tumours and also on the distribution of the a and ~ subunits of S-1 00 protein in normal human tissues (PEREZ et al. 1970; MATUS and MUGHAL 1975; COCCHlA and MICHETTI 1981; KAHN et al. 1983). A few reports on 5-100 protein immunoreactivity in carcinoid tumours have been published, and the presence of this substance might extend the discussion on the histogenesis of these tumours (SANO et al. 1984; WILANDER et al 1985). A polyclonal antiserum to bovine brain S-100 protein (2311 Lot 113, Dako Co., Santa Barbara, California, USA), which was not specified with regard to a and ~ subunits, was applied to a series of gastrointestinal carcinoids. In 11 of 12 appendiceal carcinoid tumours S-100 protein immunoreactivity occurred.

30 . E. Wilander . M. Lundqvist . K. Oberg

Gastrointestinal carcinoid tumours . 31

Fig. 17. A section of an appendiceal carcinoid tumour after immunostaining with 5-100 protein antiserum. Immunoreactive cells with long cytoplasmic extensions are intermingled with the tumour buds. X 400.

The staining reaction was present predominantly in slender cells with long cytoplasmic processes. These cells were located mainly in the periphery of the small tumour aggregates, but could also be seen between unreactive tumour cells (Fig. 17). The S-100 protein-immunoreactive cells displayed staining both in the nucleus and in the cytoplasm. No S-100 protein-immunoreactive cells were present within or in close contact with any small intestinal carcinoid tumours, although a positive immunoreaction was observed in nerve fibres in the normal intestinal wall, and sometimes in the fibrovascular stroma surrounding the tumour aggregates. Nor was S-100 protein immunoreactivity present in any of a series of examined carcinoids of the stomach and rectum. The biphasic pattern, with neuroendocrine differentiated tumour cells in close contact or

Fig. 15. Small intestinal mucosa immunostained with monoclonal chromogranin A antibodies (a), destained, and restained with the Grimelius argyrophil stain (b). Identical neuroendocrine cells show both chromo gran in A immunoreactivity and an argyrophil reaction. X 280. Fig. 16. Illustration of chromogranin A immunoreactivity in a small intestinal carcinoid. Peroxidase-antiperoxidase stain with monoclonal antibodies. X 100.

32 . E .Wilander . M. Lundqvist . K .Oberg

intermingled with 5-100 protein-immunoreactive cells with long cytoplasmic extensions, thus seems to be a phenomenon that is restricted to appendiceal tumours among the carcinoids of the gastrointestinal tract. It is presumed that the 5-100 protein-immunoreactive cells of appendiceal carcinoid tumours are of Schwann cell origin, on the basis of their morphology and on the fact that a 5chwann cell component has been observed in carcinoid tumours of the appendix electron microscopically (AUBOCK and HOFLER 1983). A picture similar to that observed in appendiceal carcinoids has also been found after immunostaining of phaeochromocytomas and some bronchial carcinoids with 5-100 protein antiserum (EL-SALHY et al. 1986). The phaeochromocytomas develop from peripheral chromaffin cells of the sympathetic nervous system. The normal chromaffin cells are surrounded by 5-100 protein-immunoreactive satellite cells, which are considered to be closely related to 5chwann cells. Thus, it appears that in phaeochromocytomas both the chromaffin cell component and the 5chwann cell component participate in the tumour growth. It is possible that appendiceal carcinoid tumours, like phaeochromocytomas, also originate from neuroendocrine and Schwann cell complexes (RODE et al. 1982; AUBOCK and HOFLER 1983; MILLIKIN 1983; RODE et aI. 1983).

3.9 Electron microscopy Carcinoid tumours of the digestive system can nowadays be defined as neuroendocrine tumours storing peptide hormones and/or biogenic amines. These hormones, as seen ultrastructurally, are stored in cytoplasmic secretory granules, which in comparison with other intracytoplasmic organelles are relatively electron dense. Because of this and their relative abundance, they are mostly easily identified. The hormone granules vary morphologically, and at least to some extent their structure reflects the type of hormone stored. Thus, the ultrastructure of neuroendocrine tumours often displays similarities with that of the normal neuroendocrine cells producing the same hormonal substance as the tumour cells. However, the tumours sometimes contain granular struc-

Fig. 18. Electron micrograph of intestinal EC-ceU granules after immunostaining with colloidal gold (15 nm) and monoclonal serotonin antibodies. The gold particles indicate the presence of serotonin in the pleomorphic secretory granules. X 43900. Fig. 19. Electron micrograph of an argentaffin carcinoid tumour. The cytoplasm of the tumour cells contains an abundance of pleomorphic secretory granules. Osmium fixation. x 16300. Fig. 20. Electron micrograph of a carcinoid tumour of the body of the stomach. The tumour cells are seen to store hormonal granules of the EeL-cell type, with a round electron dense central core and a wide electron lucent space. Osmium fixation. x 16300.

Gastrointestinal carcinoid tumours . 33

34 . E. Wilander . M. Lundqvist . K. Oberg

tures, which is atypical compared with the expected morphology based on their hormone production (BLACK 1968; SOLCIA et al. 1979; CAPELLA et al. 1973). In typical small intestinal carcinoid tumour cells pleomorphic granules of the EC cell type predominate (Figs. 18 and 19), while fore-gut and hind-gut carcinoids mostly display round hormonal granules (BLACK 1968; LUNDQVIST and WILANDER 1982; WILANDER et al. 1977a). Carcinoids of the body and fundus region of the stomach usually show characteristic granules of the ECL cell type (Fig.20). The ultrastructurally identified secretory granules can often be traced at the light microscopical level by the occurrence of a silver-positive reaction in the histological section. It has been shown that tumour tissues obtained from argentaffin areas of small intestinal carcinoid tumours exhibit a predominance of cells with pleomorphic granules of the EC cell type. These granules accumulate silver after staining both with the argentaffin and with the argyrophil method. The size of the granules varies individually, both small and large pelomorphic granules with similar staining properties being observed (LUNDQVIST and WILANDER 1982). In areas with only argyrophilia, the granules of the tumour cells are mainly round and are covered with silver grains after application of the Grimelius argyrophil method. They do not react or are very weakly stained with the argentaffin technique. At electron microscopy argentaffin tumour cells of rectal carcinoid tumours were found to contain pleomorphic secretory granules, but solely argyrophil tumour cells of such tumours contained round granules (WILANDER et al. 1977a). Argentaffin metastatic nodules in the liver of patients with primary small intestinal carcinoid tumours possess an abundance of tumour cells with pelomorphic granules. Thus, the ultrastructure of intestinal carcinoids can to some extent be correlated to their silver-staining properties, as further emphasized by the fact that Sevier-Munger argyrophil carcinoids of the body and fundus of the stomach show an ultrastructural morphology comparable to that observed in the normal ECL cells of the gastric mucosa. Not only the morphology but also the number of hormone-secretory granules in the tumours can be traced by silver-staining methods on the light microscopical level. Thus, in a study of gastric carcinoids with atypical morphology (type D of the Soga classification), the tissues were selected in respect to the silver-staining properties of the last section from the block, and material for electron microscopy was obtained from both silver-positive (Grimelius) and silver-negative regions of the tumour tissue. Neurosecretory membrane-bound granules were clearly visible in the tumour cells of the cases examined. These granules were electron dense, round or slightly ovoid and surrounded by a limiting membrane, and were abundant in the strongly silver-positive tumour areas. Few were seen in weakly argyrophilic areas and none in silver-negative areas. Thus, the silver reaction at the light microscopical level corresponded well with the presence of secretory granules (WI LANDER et al. 1984).

Gastrointestinal carcinoid tumours . 35

3.10 Carcinoid tumour metastases In patients with neuroendocrine tumours, liver metastases are not infrequently present at the time of admission to hospital. Even if representative biopsy specimens from the liver can verify the growth of a metastatic neuroendocrine tumour, it is not seldom difficult to predict or identify the topographical location of the primary malignancy by histopathological examination. During the last decade it has become increasingly evident that neuroendocrine tumours with differet topographical locations in the body have special characteristics, as revealed by various silver stains for neuroendocrine cells and by immunocytochemical analysis after application of antisera against peptide hormones or biogenic amines. In our studies percutaneous biopsy with the aid of ultrasonography for histopathological diagnosis of liver metastases of neuroendocrine tumours was apparently a most accurate method, since representative specimens were obtained in 20 of 21 patients [95 per cent] (LINDGREN et al. 1984). The usefulness of this technique was also demonstrated by the absence of any postoperative complications. The argyrophil reaction of Grimelius was positive in all tumours, further demonstrating that the liver metastases were of true neuroendocrine origin. The argentaffin reaction could be read in the formalin-fixed specimens, but no reproducible results were obtained after Bouin fixation. An argentaffin reaction was observed in 14 of 15 liver metastases of small intestinal carcinoids, whereas the metastases from pulmonary carcinoids and pancreatic endocrine tumours showed no argentaffin reaction. The argentaffin reaction, when present, was easily visible, although in three patients stained cells were few. One patient with an unknown primary tumour displayed liver metastases without argentaffinstained tumour cells. Strong correspondence was observed between the argentaffin reaction and immunoreactivity to the serotonin monoclonal antibodies, provided the latter staining was applied to the Bouin-fixed material. After formation fixation, the serotonin immunoreactivity was somewhat weaker and more difficult to read than the results obtained in Bouin-fixed materials. In one patient, who had a primary small intestinal carcinoid, the biopsy specimens displayed no argentaffin reaction, but after application of the monoclonal serotonin antibodies a staining reaction was observed. Thus, all immunocytochemically examined metastases of small intestinal carcinoids examined were serotonin-immunoreactive, whereas tumours of other primary sites were not. Since both the argyrophil reaction of Grimelius and immunocytochemistry with NSE antiserum are considered reliable adjuncts for identification of tumours with neuroendocrine differentiation, these two methods were applied to formalin-fixed and paraffin-embedded material from carcinoid tumour metastases of the liver obtained by percutaneous biopsy (LUNDQVIST et al. 1985). The primary tumour site was in all cases the small intestine. Of 13 tumours examined, all exhibited a relatively strong argyrophil reaction in most tumour cells, while eight biospies showed a strong positive reaction

36 . E. Wilander . M. Lundqvist . K. Oberg

with the NSE anteriserum, three were only weakly stained and two were negative. With NSE immunocytochemistry in primary small intestinal carcinoids, the staining distribution is uneven and probably depends on the functional activity and the degree of neuroendocrine differentiation of individual tumour cells. This might explain the negative staining in a few biopsies. Since the silver reaction is positive practically all over the tumour, it appears that the argyrophil reaction of Grimelius is superior to NSE immunocytochemistry for identifying carcinoid tumour in small tissue specimens obtained by percutaneous needle biopsy.

3.11 Histogenesis of carcinoids The histogenesis of intestinal carcinoids has been the subject of considerable discussion. Both epithelial and subepithelial neuroendocrine cells or their more undifferentiated progenitors have been considered as origins (GOSSET and MASSON 1914; MASSON 1928; CAPELLA et al. 1973; AUBOCK and HOFLER 1983). Regarding the possible development of carcinoids from mucosal neuroendocrine cells, preneoplastic alterations have previously been observed only in gastric carcinoids, and only in those located in the body and fundic area of the stomach. The occurrence of diffuse and/or nodular hyperplasia of mucosal neuroendocrine cells before or in association with gastric carcinoid growth is well documented and neuroendocrine tumours of the ECL-cell type are supposed to arise from proliferating Sevier-Munger-positive ECL cells in the body and fundus of the glandular stomach (BLACK and HAFFNER 1968; CAPELLA et al. 1973; SOGA et al. 1975; HODGES et al. 1981; WILANDER 1981). Concerning intestinal carcinoid tumours, the idea of development from epithelial neuroendocrine cells has been refuted by some authors. No preneoplastic state such as neuroendocrine cell hyperplasia or any connection between the carcinoid tumour and the overlying epithelium has been reported (SHERMAN et al. 1979). However, by serial sectioning of multiple carcinoids in a small intestinal specimen, it was recently observed that the number of neuroendocrine cells was increased in the mucosal crypts in the vincinity of the carcinoid tumour growth. Furthermore, direct continuity was noted between the intraepithelial neuroendocrine cells and the carcinoid aggregates. Similar observations were made in several of the tumours sectioned. The results provide strong evidence that small intestinal carcinoids originate from intraepithelial neuroendocrine cell proliferations and that when multiple small intestinal carcinoids arise they represent multiple primary tumours (Fig. 21). Patterns similar to those seen in small intestinal carcinoids have also been observed in som~ duodenal and rectal carcinoids and thus the basic questions concerning the histogenesis of most carcinoids of the digestive tract now appear to be almost resolved (LUNDQVIST and WILANDER 1986; KATO et al. 1986; KARELIN 1987; LUNDQVIST and WILANDER 1987). Within the gastrointestinal canal, the appendiceal carcinoids react differently from all

Gastrointestinal carcinoid tumours . 37

Fig. 21. Light micrograph of a small intestinal carcinoid after staining with the argentaffin method. In the intestinal mucosa there is an increased number of neuroendocrine cells in the epithelial crypts (arrow). These cells seem to proliferate and invade the surrounding stromal tissue. X 400.

other carcinoids with regard to 5-100 protein antiserum. These are the only carcinoids endowed with an abundance of 5-100 protein-immunoreactive nerve fibres which are intermingled with the tumour cells (WILANDER et al. 1985; LUNDQVIST and WILANDER 1986). Thus, appendiceal carcinoids display a characteristic diphasic pattern with neuroendocrine differentiated tumour cells in close contact with S-100 protein-immunoreactive cells carrying long cytoplasmic processes, presumably of Schwann cell origin. In their structure appendiceal carcinoids closely resemble subepithelial neuroendocrine cells: both display an argentaffin reaction and serotonin immunoreactivity, contain similar hormonal granules ultrastructurally, and show intermingling of nerve fibres and Schwann cells. For this reason the subepithelial neuroendocrine cells must be

38 . E. Wilander . M. Lundqvist . K. Oberg

seriously considered as a possible progenitor for the development of appendiceal carcinoids, or at least some of them (RODE et al. 1982; AUBOCK and HOFLER 1983; MILLIKIN 1983; RODE et al. 1983). Carcinoids of the small intestine, caecum and rectum lack 5-100 pro~ein-immunoreactive cells as an integral component. The immunohistochemical reaction of appendiceal carcinoids after application of 5-100 protein antiserum is similar to that observed in some neuroectodermal (peripheral nerve) tumours such as phaeochromocytomas, paragangliomas and chemodectomas (EL-5ALHY et al. 1986). It is possible that the appendiceal carcinoids are more closely related to these tumours than to other carcinoids of the digestive system. It is emphasized clinically that, in contrast to other carcinoids, appendiceal carcinoids mostly behave in a completely benign manner. This might be explained by their different histogenesis.

3.12 Pathogenesis The mechanisms underlying the development of carcinoids are completely unexplained, with one exception, namely that carcinoid tumours consisting of ECL cells arise in the non-antral area of the glandular stomach. In the case of these tumours a pathogenetic event is suggested. The ECL carcinoids are often multiple and are seen to grow out from proliferating mucosal ECL cells, presumably as a result of stimulation by a trophic action of gastrin (Fig.22) (LARSSON et al. 1978; RUDDELL et al. 1978; HODGES et al. 1981; CARNEY et al. 1983; HARVEY et al. 1985). The ECL-cell carcinoids occur in association with an atrophic gastritis, sometimes with pernicious anaemia. In these patients pronounced hypergastrinaemia is usually present as an effect of an inhibited negative acid feed-back mechanism (Fig. 23). Accordingly, patients with the Zollinger-Ellison syndrome, who have high serum gastrin levels and increased acid secretion and thus lack atrophic gastritis, may also develop non-antral gastric carcinoids of the ECL-cell type (BORDI et al. 1974; HARVEY et al. 1985). Moreover, administration of the drug omeprazole, which inhibits the proton pump, produces diffuse and nodular hyperplasia of the ECL cells in the acid-secreting part of the gastric mucosa in rats, and this proliferation develops into infiltrative carcinoid tumour growth when omeprazol is given in large doses for long periods (HAVU 1986). The ECL-cell hyperplasia is prevented by antrectomy prior to the drug administration, further emphasizing the stimulating effect of hypergastrinaemia on the ECL cells (HARVEY et al. 1985). It has been observed that the mean nitrate concentration in gastric juice from fasting patients with atrophic gastritis and pernicious anaemia is nearly 50 times higher than that in age-matched controls. The nitrate is essential for the formation of volatile nitrosamines, which possess carcinogenic properties, and a high concentration of nitrosamines in the achlorhydric stomach has been suggested as an important factor in the development of gastric cancer (RUDELL et al. 1978). It is therefore possible that hypergastrinaemia promotes the development of gastric carcinoids of the ECL-cell type by

Gastrointestinal carcinoid tumours . 39

Fig. 22. A section of the mucosa of the gastric body in a patient with atrophic gastritis. In the lower part of the mucosal crypts pronounced hyperplasia of neurendocrine cells (mainly EeL cells) is seen. Grimelius stain. x 250. Fig. 23. Hyperplasia of antral G cells in a patient with atrophic gastritis, hypergastrinaemia and multiple gastric carcinoids. Immunostaining with polyclonal gastrin antibodies. x 250.

40 . E. Wilander . M. Lundqvist . K. Oberg Atrophic gastritis

f'-------Increased concentration of carcinogenic nitrosoamines in the gastric juice

AChlorlhYdria

loss of intrinsic factor

t

!

Hypergastrinaemia

ECl -cell proliferation

!

ECl -cell carcinoids

Pernicious anaemia

Fig. 24. Schematic diagram of the possible pathogenesis of gastric ECL-cell carcinoids (ECLomas). inducing proliferation of the neuroendocrine cells of the mucosa, and that the tumour transformation is initiated by the presence of high concentrations of carcinogenic nitrosamines in the achlorhydric stomach. In Wistar rats oral administration of the nitroso compound N-methyl-N-nitrosa-N-nitrosoguanidine induces carcinoid tumours in the fundic portion of the glandular stomach. The tumour cells display argentaffin and/or argyrophil staining properties and ultrastructurally are loaded with secreting granules of the neurohormonal type (TAHARA et al. 1981). Thus, both hypergastrinaemia and carcinogenic nitrosamines are able to induce non-antral gastric carcinoids in experimental animals (Fig. 24). Since the frequency of carcinoids of the ECL-cell type in the body and fundus of the stomach and that of gastric cancer are increased in achlorhydria and pernicious anaemia, similarities in the pathogenetic mechanisms of these two types of tumour are possible. Against this background, it is worthy of note that in the stomach atypical carcinoids may occur which both morphologically and biologically represent borderline tumours between carcinoids and carcinomas (WILANDER 1981; WILANDER et al. 1984).

3.13 Nuclear DNA measurements Measurements of nuclear DNA in patients with carcinoid tumours may be used as a supplementary method for identification of the tumours and for assessment of effects of anti-tumour therapy on the tumour cell cycle in vivo. Carcinoid tumour specimens may occasionally be obtained at open surgery from patients with metastatic carcinoid

Gastrointestinal carcinoid tumours . 41

tumour disease during treatment, but ultrasonically guided needle (diameter 2.0 mm) biopsy material of liver tumours can be obtained by a recently described technique without postoperative complications (LINDGREN 1980, 1982; WILANDER et al. 1986). The yellow to grey tumour tissue is easily identified and is distinguishable from the brownish liver parenchyma. Such material has been used for cytofluorometric DNA measurements of single tumour cells. Briefly, the method is as follows: Multiple imprints on glass slides from the unfixed tumour biopsy are fixed in acetone-ethanol 1 :1 for 30 to 60 minutes, air dried and kept in a desiccator at 4°C until the DNA measurements are performed according to the following procedure. The slides are incubated for 1 hour at 37°C in 0.5 mg/ml DNAase solution (bovine pancreas, Dorset, UK). The cells are then stained with ethidium bromide solution (10 mg/ml) buffered with 0.1 M tris at pH 7.5: the final solution is 0.1 M in sodium chloride. The cell preparation is mounted in the staining solution. Measurements of DNA are performed in a microscope fluorometer. Frequency histograms have disclosed a diploid pattern in small intestinal and caecal carcinoids with low proliferative activity (less than 2% of the nuclei in the S-phase region) and a frequency of tetraploid cell of about 5-8%. According to the DNA values, primary tumours and their metastatic counterparts appear to show similar DNA patterns. Since carcinoids are morphologically regular and built up of tumour cells with monomorphous nuclei, the finding of diploid nuclear DNA values in these tumours is in accordance with their histological features; such values are also compatible with their clinical behaviour. The fact that diploid DNA histograms have also been observed in bronchial and rectal carcinoids indicates that this is an almost general characteristic of highly differentiated neuroendocrine tumours (BU)NDAL et al. 1983; WILANDER al al. 1984, 1986, 1987). It should also be emphasized that the so called «argyrophilic carcinomas» of the breast, which are considered to represent a variety of ordinary breast cancer but with neuroendocrine differentiation, frequently display diploid nuclear DNA values (WILANDER et al. 1984). Since carcinoid tumour metastases apparently exhibit DNA profiles similar to those observed in primary tumours without metastases, it seems that nuclear DNA measurements are of no value in the assessment of their malignant behaviour. They may, however, be useful as an adjuvant method in the differential diagnosis against adenocarcinoma, which with few exceptions is grossly aneuploid. Cytofluorometric nuclear DNA analyses in gastric carcinoids yield more irregular values, with a higher frequency of tetraploid cells and also the presence of aneuploid ones. Gastric carcinoids seem to be more heterogeneous than most intestinal «classical» carcinoids with respect to their morphology, nuclear DNA profiles and biological behaviour. These observations are in accordance with the suggestion that gastric carcinoids may represent a highly differentiated variety within a spectrum of neuroendocrine neoplasia, analogously to the corresponding variety observed in the bronchus [carcinoid-small cell carcinoma] (WILANDER et al. 1987).

42 . E. Wilander . M. Lundqvist . K. Oberg

Although intestinal carcinoid tumours have a slow rate of progression, from the point of view of the patients this type of tumour presents major therapeutic problems. First of all, the carcinoid syndrome involves symptoms that may be intractable and that may greatly impair the quality of life. Secondly, the progressive growth of the tumours and the development of metastases will eventually shorten the life span. For this reason several cytotoxic drug regimes have been tried, one of the most effective of which has been streptozotocin (STZ, marketed under the name of Zanosar®) combined with 5fluorouracil (5-FU), although this has an overall response rate of only 33%. Recently an alternative form of treatment with leukocyte interferon has been presented. This has been considered especially beneficial for patients with carcinoid syndrome, as the drug ameliorates the clinical manifestations and leads to a prompt and continuing decrease in the urinary level of 5-HIAA and the serum level of human chorionic gonadotropin subunits and pancreatic polypeptide. Biopsy specimens obtained by guided ultrasound from metastatic carcinoid tumours of the liver during STZ and 5-FU therpay did not display any distinct alterations at the light microscopical level. However, DNA measurements of tumour cell nuclei tended to show an increase in the number of tetraploid cells, with a mean value of 30%. It is unlikely that 5-FU caused any changes in the tumour cell nuclear DNA, since this substance is known to exert only antimetabolic effects (WILANDER et al. 1985). STZ is chemically a glucosamine-nitrosourea compound with widespread biological properties. It is accumulated in the pancreatic islet B-cells and has a toxic effect on these cells (TJALVE et al. 1976). For this reason, STZ is given to induce chemical diabetes mellitus in laboratory animals (RERuP 1970). Since 1967 STZ has been used in the treatment of endocrine tumours, mainly those of pancreatic islet cells and carcinoids (WEISS 1982). The side chain of STZ consists of a methyl-nitrosourea. Thus, the substance alkylates DNA and causes interstrand cross-links. The mechanism of action of STZ has been studied in malignant cells in vitro. It was found that STZ is not phase-specific and probably affects cells at all stages of cycle. However, the concentration of STZ required to cause inhibition of DNA synthesis is much higher than that needed to block the movements of cells to mitosis. The latter effect occurs in the G2 phase (BHUYAN 1970). These observations are in accordance with findings in studies in vivo on the effects of cytotoxin treatment (mainly STZ) on carcinoid tumour cells, since an increase in the number of tetraploid cells was noted in samples from the treated patients. However, the possibility cannot be ruled out that the cytotoxin treatment may have induced the establishment of tetraploid cell lines. Since regular nuclear DNA patterns are a characteristic of carcinoid tumours, the action of cytotoxin therapy on the different phases of the tumour cell cycle can be evaluated by in vivo studies on cytological specimens (WILANDER et al. 1985, 1986). Carcinoid patients who were given interferon in a dose of 3-6 X 106 IU/d for 4-30 months also displayed nuclear DNA alterations in the tumour cells. Before treatment, modal diploid DNA values were observed, but after initiation of the anti-tumour

Gastrointestinal carcinoid tumours' 43

therapy the DNA histograms were altered. The DNA values in these tumours varied, but they were mainly in the range between diploid and tetraploid. In some cases several nuclear DNA peaks were seen within the limits of the cell cycle. At light microscopy sections from interferon-treated tumours showed anisokaryosis and some hyperchromasia, and cytoplasmic vacuolization was also observed. There was an increase in the amount of newly formed collagen in the tumour cell stroma and the relative amount of tumour tissue was decreased (WILANDER et al. 1986). The mechanism of action of interferon is as unclear with regard to carcinoid tumours as it is in other situations in vivo. This is to be expected in view of the great variety of biological effects of interferon (STEWART 1979; TAYLOR-PAPADIMITRIOU 1980). To exert its antiviral activity, interferon reversibly binds to cell surface receptors, where in some way it activates cytoplasmic enzymes effecting messenger-RNA translation and protein synthesis (STIEHM et al. 1982). A similar mechanism may lead to interference with the synthesis and secretion of polypeptides and biogenic amines and amelioration of the clinical manifestations of the carcinoid syndrome observed in interferon-treated patients. Interferon influences cell growth regulation and is able to affect both specific stages and several phases of the cell cycle in vitro. Recently, antagonistic effects of interferon on actions of various growth factors have been demonstrated, suggesting that cellular proliferation may be regulated by opposition between effects of growth-promoting factors and interferon (LUNDBLAD and LUNDGREN 1981; INGLOT 1983; QUESADA and GUTIERMAN 1984; SUHADOLNIK et al. 1984). Interferon has been found to increase the nuclear DNA content in malignant carcinoid tumours in vivo, possibly by blocking the cells in various phases but mainly in the Sand G 2 phases of the cell cycle. The DNA histograms of the interferon-treated tumours were not identical. This can be explained by differences in kinetics and clonal heterogeneity in and between individual tumours. Irrespective of what causative agent(s) brings about DNA alterations of the carcinoid tumour cells, the change in the nuclear DNA histograms appears not to be correlated to any signs of progression the tumour growth, but rather to regression, as there was no increase in the size of the metastases as observed ultrasonically, the clinical symptoms were ameliorated and microscopically the relative amount of tumour tissue appeared to be decreased.

3.14 Carcinoid tumours in vitro In patients with malignant carcinoid tumours, elevated serum levels of hormones may be caused by secretion of the hormones from the tumour tissue or by mechanisms operating in other neuroendocrine tissues as a result of an abnormal hormonal state. Carcinoid tumour specimens obtained in tissue culture permit evaluation of such events (NORHEIM et al. 1987). Liver metastases from small intestinal carcinoid tumours obtained at operation or by

44 . E. Wilander . M. Lundqvisl . K. Oberg

percutaneous needle biopsy in five patients were examined in vitro. All patients had elevated serum concentrations of 5-HIAA and four out of five had elevated serum levels of tachykinin-like immunoreactivity (see below), not due to substance P at the time when the tissue specimens were taken for organ culture. The fresh tumour material was immediately placed in RPMI-1640 medium. The specimens were mechanically disintegrated into minimal fragments (if necessary trypsinated 0.25%) for 0.5-1 hour. The minced tumour was then put into Ehrlenmeyer flasks containing a bottom layer of contact inhibited glial feeder cells and RPMI -1640 medium containing 10% fetal calf serum supplemented with penicillin (100 IVl ml) and streptomycin (SO ug/ ml). The primary cultures were incubated at +37°C at normal atmospheric pressure in humidified air containing 5% CO 2, The cultures were observed at routine intervals, and half of the medium was renewed twice a week. Supernatants were stored at -70°C until assayed for tachykinin-like immunoreactivity. All tumours were kept as primary tissue cultures for up to 4.5-6 months, at which time the cells deteriorated and disappeared. The tumour cells were confined to the suspension in the form of condensed cell clusters, from which small bars of possibily proliferating tumour cells emerged (Fig. 25). However, no obvious increase in the number or size of the tumour cell aggregates was seen during the culture period. Proliferation in monolayer or established cell lines as described by others, was not observed (DEBONS-GUILLEMIN et al. 1982). During the culture period, some tumour aggregates were collected and examined by light and electron microscopy. At the light microscopical level, the tumour clusters were argentaffin and argyrophil. Electron microscopically, well preserved tumour cells with an abundance of cytoplasmic pleomorphic granules of the neurohormonal type were observed (Fig. 26). Radioimmunoassay of tachykinin immunoreactivity in the culture medium disclosed elevated levels of up to 411 pmolll (ref. <20 pmol/l) in four of the five cases mentioned above. With time, the tachykinin-like immunoreactivity decreased. High performance liquid chromatography of supernatants from carcinoid tumour cell cultures showed a clear-cut peak of immunoreactivity eluted in the position of synthetic eledoisin, and peaks in the position of synthetic neuropeptide K and neurokinin A were also seen. A correlation was observed between the plasma level of tachykinin-like immunoreactivity in the patient at the time of operation and the tumour cell secretion of this immunoreactivity. The carcinoid tumours examined had previously been found to display serotonin secretion and an ability to give rise to the carcinoid syndrome. As discussed above on page 26 patients with this syndrome may secrete active substances in addition to serotonin, among which substance P appears to be one of the most commonly produced. Substance P is considered to be synthesized and stored in some EC cells in combination with serotonin in the intestinal mucosa, and substance P immunoreactivity is frequently encountered in serotonin-producing carcinoid tumours (ALUMETs et al. 1977). Substance P belongs to the tachykinin family, a group of peptides with a common C-terminal amino acid sequence. It seems as if the peptide hor-

Gastrointestinal carcinoid tumours . 45

Fig. 25. Photograph of small intestinal carcinoid cultured in vitro. The tumour cells are in the form of free floating aggregates. X 670. Fig. 26. Electron micrograph of a small intestinal carcinoid in culture. The tumour cells display alterations such as cytoplasmic vacuolization and an increased number of hyalin antibodies, but they still show accumulations of hormonal granules of the pleomorphic type. Osmium fixation. x 6000.

46 . E. Wilander . M. Lundqvist . K. Oberg

mone secretion by mi~-gut carcinoids is even more complex than was previously thought. There are indications that the hormones secreted from mid-gut carcinoids in addition to serotonin represent a variety of peptide sequences of the tachykinin family rather than just one defined peptide. Thus, an additional set of active hormonal products must be considered in relation to the clinical picture of the carcinoid syndrome.

3.15 Neuroendocrine cells in intestinal adenocarcinomas Carcinoid tumours of the intestine are considered to originate from the neuroendocrine cells in the crypts of Liberkiihn. Nevertheless, carcinoid tumours sometimes contain cells with a content of mucous material, indicating a differentiation in the exocrine direction. On the other hand, intestinal adenocarcinomas that arise from the mucosal exocrine enterocytes sometimes contain both exocrine cells and cells with endocrine differentiation. A series of 16 intestinal adenocarcinomas (jejunal and ileal) was investigated in this respect. All tumours exhibited the morphology of ordinary adenocarcinomas, with well developed glandular or papillary structures. Staining (AB-PAS) for mucopolysaccharides gave a positive reaction in both intra- and extracellular locations in 14 of the tumours. Scattered argyrophil (Grimelius) cells were identified in three highly differentiated tumours and two of these tumours also contained a subpopulation of argentaffin cells which also stained with monoclonal serotonin antiboides. The silverand serotonin-reactive cells were intermingled with exocrine tumour cells. Examination of consecutive sections revealed that the argyrophil cells outnumbered the argentaffin ones, indicating the presence of at least two different types of neuroendocrine cells in these adenocarcinomas. Application of the different peptide hormone antisera to the tumour sections did not result in any positive reaction, but scattered lysozyme-immunoreactive tumours cells (indicating Paneth cell differentiation) were seen in two cases. One tumour containing argentaffin, argyrophil and lysozyme-immunoreactive cells was evaluated electron microscopially. Some scattered cells with large round zymogen granules of the Paneth cell type were found. Further tumour cells with neuroendocrine differentiation were observed. These cells were rich in neurosecretory granules, which in some cells were round and regular and in others pleomorphic (LUNDQVIST and WILANDER 1983). The fact that ordinary adenocarcinomas may contain cell populations with neuroendocrine differentiation is well known and has been observed, for example, in adenocarcinomas of the breast, stomach, pancreas and gonads (AZZOPARDI and POLLOCK 1963; BRODNER et al. 1980; EUSEBI et al. 1981; PARTANEN and SYR]ANEN 1981; SPORRONG et al. 1981). The malignant transformation probably results in genomic derepression and synthesis of cellular products that is not expected to occur in the tumour cells in question.

Gastrointestinal carcinoid tumours' 47

The neuroendocrine cells and the other enterocytes of the intestinal mucosa are certainly of endodermal origin and thus the maturated epithelial cells develop from precursor cells in the regenerative zone of the mucosa. Thus, the final differentiation of tumour-transformed cells may be due to some random genomic alterations induced by the carcinogenic factors that have been responsible for the development of the tumours.

3.16 Clinical presentations A majority (90%) of all carcinoids demonstrated in the necropsy series reported by BERGE and LINELL (1976) were not clinically diagnosed before death and did not give rise.to any significant clinical symptoms. On the other hand, in studies of patients with known gastrointestinal carcinoids, the majority of the patients have been found to present different clinical symptoms. In our own series of 103 patients with malignant carcinoid tumours, diarrhoea was the most frequent initial symtom [32%], followed by intestinal obstruction [25%] and flushing [22%] (NORHEIM et al. 1987). Similar frequencies have been reported by MARTENSSON et al. (1983) and HENRIKSEN et al. (1983) and HENRIKSEN et al. (1979). The initial symptoms in a patient with a small intestinal carcinoid tumour may be vague, with diffuse abdominal pain, resulting in both patient's and doctor's delay. We found that the median duration of delay between the first symptom of a carcinoid until the diagnosis was verified was one year (interquartile range 0.5-3 years). Tumours causing intestinal obstruction showed the shortest delay from the onset of the first symptoms until diagnosis (median 0.6 years), whereas patients with diarrhoea and flushing had a delay of 1.5 and 2 years, respectively. Distinct clinical syndromes have been described in patients with gastrointestinal carcinoid tumour. The most common is the carcinoid syndrome, which is preodominandy found in patients with ileal and jejunal carcinoids with liver metastases. The other four syndromes are relatively rare, and include ECL-oma syndrome, Zollinger-Ellison syndrome due to gastrin production, somatostatinoma syndrome, Cushing's syndrome due to production of ACTH or corticotropin releasing factor (CRF), and acromegaly due to tumour-secreted growth hormone releasing factor (GRF). These clinical syndromes are found in patients with predominantly fore-gut carcinoid tumours, although acromegaly has also occurred in patients with mid-gut carcinoids (ileal and jejunal) (OBERG and WIDE 1981). One patient in our own material with an ileal carcinoid presented with malignant hypertension, sweating, pallor, and a strong sensation of burning inside the body. Besides elevated concentrations of urinary 5-HIAA and plasma NPY, no other as yet identified secretory product from neuroendocrine cells was detected. After treatment the clinical symptoms disappeared and the blood pressure returned to a normal level. Patients with carcinoids of the hind-gut (distal colon and rectum) present with gastrointestinal bleeding, obstruction and/or an abdominal mass. Although these tumours

48 . E. Wilander . M .Lundqvist . K.Oberg

are known to contain several peptides such as PP, PYY or chromogranin A, none of these are known to cause any definite clinical symtoms. 3.16.1 The carcinoid syndrome

The carcinoid syndrome was originally described in 1952 by BIORCK et al. and two years later a more detailed description of the syndrome was published (THORSON et al. 1954). The syndrome includes flushing, diarrhoea, asthma, oedema, right heart lesions, pellagra-like skin lesions and occasionally also peptic ulcers and arthralgia. The term malignant carcinoid syndrome has also been used to include - and should also includeliver metastases and elevated urinary 5-HIAA levels (DAVIS et al. 1973). The occurrence of the carcinoid syndrome among patients with intestinal carcinoids has varied from 3% to 67%. The difference obviously reflects the criteria of referrals. In our own series of patients, 67% presented with the carcinoid syndrome, only patients with known metastatic disease and clinical symtoms were referred, whereas in autopsy materials such low figures as 3 % have been reported (BERGE and LINELL 1976). All patients with carcinoid syndromes arising from gastrointestinal carcinoids develop metastatic disease. The symptoms are due to humoral secretion of amines and peptides and as a rule systemic symptoms do not occur until hepatic metastases are manifested. This is probably true in the case of amines such as serotonin and 5-HTP, as the liver inactivates the amines and until the portal circulation can be bypassed, the syndrome does not develop. However, there have been several reports on cases where the syndrome has developed in the absence of hepatic metastases. Retroperiteoneal masses or multiple small tumour implants in the parietal peritoneum can drain into the systemic circulation (FELDMAN and JONES 1982). Paraaortic masses may drain directly into the systemic circulation (ROSENBERG 1984) and metastases to the ovaries can also bypass the portal circulation and produce systemic symptoms (DAVIS et al. 1973). The carcinoid syndrome occurs mainly in patients with malignant small bowel carcinoids, but it is also occasionally reported in patients with carcinoids of the stomach and appendix and also of the caecum and ascending colon. We have also observed this syndrome in a patient with a malignant melanoma and liver metastases producing serotonin.

3.16.1.1 Flushing Flushing and diarrhoea are the most common symptoms of the carcinoid syndrome. Different types of flushing have been described (GRAHAME-SMITH 1972). Most often there is a diffuse erythematous flush affecting not only the normal flushing area but also the skin of the back, abdomen and palms, and lasting for 2-5 minutes. Another, more longer-lasting flush may occur, including dilated facial veins, telangiectases, watering eyes and conjunctival suffusion. Patients with malignant gastric carcinoids and excessive histamine release sometimes display a bright red patchy flush (geographical). A fourth

Gastrointestinal carcinoid tumours . 49

type of flushing is seen in patients with bronchial carcinoids; this is a bluish red flush lasting for hours, associated with swelling of the facial skin, lacrimation, hypotension and palpitations. Precipitating factors of the carcinoid flush include eating, especially spicy food, hot drinks, exercise, alcohol, excitement, sexual intercourse, defaecation and postural changes. The most severe spontaneous flushing attacks occur in the morning when the patient is standing up, washing, or having breakfast. Surgery and infections can provoke severe life-threatening attacks, i. e. carcinoid crisis, including severe vasodilation and hypotension. Several mediators of the carcinoid flush have been proposed. Initially it was thought that 5-HT was the main mediator, but no correlation between flush and the excretion of urinary 5-HIAA has been found (ROBERTSON et al. 1962; LEVINE and S]OERDSMA 1963). OATES and colleagues (1964) reported that bradykinin might be an important mediator, but other authors have failed to demonstrate any increase in bradykinin after spontaneous or stimulated flushes (LUCAS and FELDMAN 1986). Both catecholamines and alcohol are known to provoke flush reactions (ADAMSSON et al. 1969) and it has been postulated that alcohol indirectly releases norepinephrine, which might then stimulate a release of kallikrein from the tumour. Such alcohol-induced flushing can be counteracted by an alpha-blocking drug such as phentolamine (ADAMSSON et al. 1969). Other proposed mediators are prostaglandins E and F2 alpha. However, a review of their roles suggested that they are unlikely to be major mediators of flushing or diarrhoea (METZ et al. 1981). We have very recently observed increased circulating concentrations of multiple tachykinins, i. e. substance-P-related peptides, in patients with carcinoid tumours (NORHEIM et al. 1984; THEODORSON-NoRHEIM et al. 1985). Release of tachykinins (neuropeptide K [NPK] and neurokinin A [NKA] has been demonstrated during spontaneous, alcohol-induced and pentagastrin-stimulated flush in patients with malignant carcinoid tumours (N ORHEIM et al. 1986). Pentagastrin is known to stimulate flush and release of serotonin in carcinoid patients (FRoLIcH et al. 1978; AHLMAN et al. 1985) and it has also been shown to stimulate histamine release from gastric carcinoids (ROBERTS et al. 1983). The pentagastrin- induced flush could not be counteracted by pretreatment with a 5-HT2 receptor blocking agent such as Ketanserin®, which abolished the gastrointestinal symptoms (AHLMAN et al. 1985). This might imply that it is unlikely that serotonin plays a role in the carcinoid flush. Tachykinins are a new family of substanceP-related peptides, that are known to produce vasodilation, lacrimation, palpitation and bronchoconstriction (HUA et al. 1984). The release of tachykinins after pentagastrin injections is very rapid and short-lasting, with a peak level within 3 minutes, which fits very well with the clinical signs of the typical short-lasting carcinoid flush (N ORHEIM et al. 1986). Furthermore, infusion of tachykinins in man resulted in a typical flushing attack (KANTos et al. 1964). A subcutaneous injection of the somatatostatin analogue SMS 201-995, which completely inhibits the tachykinin release, also abolished the

50 . E. Wilander . M. Lundqvist . K. Oberg

carcinoid flush. The final proof that tachykinins are involved in the carcinoid flush cannot be produced until tachykinin receptor blockers can be tested in man. 3.16.1.2 Diarrhoea This symptom is almost as common as flushing and the diarrhoea may not be completely associated with a flushing attack, suggesting that the mediators are different. Watery stools, accompanied by colicky abdominal pain and urgency of defaecation, are common. 5-HT stimulates the small bowel motility and secretion GAFFE 1979), and 5HT2 receptor blockers might abolish the diarrhoea without affecting flushing (AHLMAN et al. 1985). Steatorrhoea occurs more rarely, but signs of malabsorption can be found in patients with carcinoid tumours. The malabsorption might be due to obstruction of the lymphatics. Abdominal pain is very common in patients with carcinoid tumours and might be caused by intestinal stenosis due to tumour or fibrotic reactions (PECK et al. 1983). Liver metastases may also give rise to abdominal pain through stretching of the liver capsule and necrosis of the metastases. 3.16.1.3 Respiratory symptoms Asthma-like episodes occur less frequently than both flushing and diarrhoea, but are often associated with flushing attacks. Hyperventilation is the most common symptom, but a true bronchospasm has also been found. The aetiological agent of such bronchospasm is not yet established, but it is known from animal studies that tachykinins cause bronchospasm (KANTos et al. 1964; HUA et al. 1984). We have been able to relieve such bronchospasm ain patients with carcinoid tumours by injecting the somatostatin analogue SMS 201-995, a blocker of tachykinin release. Wheezing could be alleviated by an isoprotenerol inhaler, but obviously beta receptor stimulators should not be given systemically, as they might worsen the carcinoid syndrome. 3.16.1.4 Carcinoid heart disease Patients with the carcinoid syndrome develop a characteristic heart disease, which has been reported to occur in 19-53% of the patients (ROBERTS and S]OERDSMA 1964; T ORNEBRANDT et al. 1986). In a majority of the patients the endocardial fibrous lesions that are considered to be pathognomonic for carcinoid heart disease involve the cavities and valves of the right heart (ROBERTS and SJOERDSMA 1964). Only a small number of patients whith typical left heart changes have been reported. The pathogenesis of the carcinoid heart lesion is not yet elucidated. In a recent study we investigated 68 patients prospectively with cardiac ultrasound and correlated abnormal findings with circulating levels of tachykinins and with urinary excretion of 5-hydroxy-indoleacetic acid (5HIAA). The patients with the most pronounced right heart dies ease had significantly higher (p < 0.01) plasma levels of the tachykinins, NPK and substance P, and also significantly greater (p < 0.001) urinary excretion of 5-HIAA. We also found that the

Gastrointestinal carcinoid tumours· 51

frequency of cardiac involvement was higher than previously reported 66% (LUNDIN et a1. 1988).

3.16.1.5 Uncommon carcinoid symptoms Pellagra-like skin lesions are found in a small number of patients with carcinoid syndrome and long-standing advanced disease. The symptom may be reversed by administration of vitamin B6 . Paraneoplastic polyneuropathy has also been described in rare cases, as has myopathy (SWASH et a1. 1975). In one single patient we have observed hypog1ycaemia and in a similar case reported in the literature it was found that the tumour produce an insulin-like molecule called «non-suppressible insulin-like activity» [NSILA] (MODHI and NICOLlS 1984). Acromegaly has been noted in patients with gastrointestinal carcinoid tumours and the carcinoid syndrome (DABEK 1974; FELDMAN et a1. 1975). Most frequently it has been observed in patients with bronchial carcinoids, gastro-duodena1 carcinoids or endocrine pancreatic tumours. In some of these cases ectopic production of GRF has been reported. In our own study of 33 consecutive patients with carcinoid tumours, we found disturbed regulation of serum growth hormone in as high a proportion as 52 % (OBERG et a1. 1985). However, only four patients had clinically overt acromegaly. In those patients we have since observed an increased circulating level of GRF. 3.16.2 The ECLoma syndrome

There is a distinct clinico-pathologica1 condition in which carcinoid tumours of the body and fundus of the stomach are involved (CARNEY et a1. 1983; HARVEY et a1. 1985). In homology with the terminology used for syndromes caused by some other hormoneproducing neuroendocrine tumours of the gastrointestinal canal, this syndrome may be called the ECLoma syndrome, as the gastric tumours involved are built up of cells of the ECL-cell type. The features of the ECLoma syndrome are carcinoid tumours often multiple - located in the body and fundus of the stomach, atrophic gastritis, achlorhydria and hypergastrinaemia, often in association with pernicious anaemia. The initial event in the development of all the clinico-patho10gical signs is apparenty1 atrophic gastritis of the acid-secreting area of the stomach, resulting in parietal cell loss and achlorhydria. The neutral pH of the gastric juice initiates increased secretion of gastrin due to inhibition of a negative acid feed-back mechanism, and the serum gastrin concentration rises. In accordance with this, hyperplasia of the antral G cells is often seen in these patients (POLAK et a1. 1973). The parietal cell loss leads to decreased production of intrinsic factor and failure of vitamin B12 absorption in the distal small intestine - a situation promoting the development of pernicious anaemia. Further, a diffuse or nodular hyperplasia of the ECL cells in the deeper part of the gastric body mucosa is frequently seen before or in association with the growth of the ECL-cell carcinoids (BLACK and HAFFNER 1968; SOGA et a1. 1975 j CAPELLA et a1. 1980;

52 . E. Wilander . M. Lundqvist . K. Oberg

WILANDER 1980; HODGES et al. 1981; WILANDER 1981). It is presumed that the ECLcell hyperplasia represents a preneoplastic state and precedes the development of the carcinoids. ECL-cell hyperplasia of the fundus mucosa is frequently encountered in atrophic gastritis with achlorhydria. Gastrin, the concentration of which is elevated in the serum of most patients with achlorhydria, is a trophic hormone for the body and fundus of the stomach and, probably by a direct mechanism, stimulates ECL-cell proliferation (RUBIN 1973; BORD! et al. 1975; LARSSON et al. 1978; WILANDER 1980; HODGES et al. 1981). Thus, patients with atrophic gastritis and achlorhydria run an increased risk of developing Eel-cell carcinoids. Sporadic case reports on gastric carcinoids with such features have been published since 1937 (CARNEY et al. 1983). In 1978 LARSSON et al. suggested that the hypergastrinaemia is instrumental in bringing about neoplasia of the ECL cells and in 1980 it was considered that achlorhydria, occasionally in combination with pernicious anaemia, occurs in a higher frequency than has been previously believed and represents the most distinct condition associated with these tumours (WILANDER 1980). A comprehensive review of the ECLoma syndrome was published by CARNEY et al. (1983). From a scrutiny of the literature and adding their own cases, they collected a total number of 61 cases. In this series 69% of the patients presented with multiple tumours and 49% of the cases were associated with mucosal argyrophil endocrine cell hyperplasia. Concomitant pernicious anaemia was observed in 56%. Hypergastrinaemia was found in all patients in whom serum gastrin measurements were performed (8 cases). Next to the classical carcinoid syndrome, the ECLoma syndrome appears to be the most frequent clinico-pathological manifestation in gastrointestinal carcinoids, despite the fact that it is limited to carcinoid tumours of the gastric body and fundus. In two studies employing endoscopic screening of a large number of patients with pernicious anaemia, occasional gastric carcinoids were found in 1-4 % of the cases, and thus patients with pernicious anaemia run an increased risk of developing not only gastric carcinoma but also gastric carcinoids (STOCKBRUGGER et al. 1983; BORCH and LIEDBERG 1984; BORCH 1985; BORCH et al. 1985). 3.16.3 The Zollinger-Ellison syndrome

This syndrome was originally described by ZOLLINGER and ELLISON in 1955 in two patients with recurrent peptic ulceration, marked gastric acid hypersecretion and islet cell tumours. In subsequent studies large amounts of gastrin were found both in the serum and in tumours in the patients with the Zollinger-Ellison syndrome (MCGUIGAN and TRUDEAU 1969). The majority of the patients with this syndrome have a pancreatic endocrine tumour (21-87%) (ELLISON and WILSON 1964; CAMERON and HOFFMAN 1974; STAGE and STADIL 1974). However, in some materials as many as 23% of the patients had a duodenal carcinoid producing gastrin (CAMERON and HOFFMAN 1974).

Gastrointestinal carcinoid tumours . 53

Some tumour-free patients can also show hyperplasia of the gastric antral G cells with clinical symptoms indistinguishable from those of patients with tumours. Not seldom the clinical syndrome is recognized but no tumours are found despite thorough investigation. The most frequent symptoms are gastritis [50-90%], alone or combined with diarrhoea [20-50%] (JENSEN et al. 1983), and abdominal pain. Radiological and endoscopic studies reveal multiple gastrointestinal ulcers in up to 52% of the patients. The diagnosis of Zollinger-Ellison syndrome is verified by a finding of an elevated serum gastrin level combined with increased gastric acid output, both basal and stimulated. The diagnosis can be further confirmed by a secretin stimulatory test (MCGUIGAN and WOLFE 1980). There ist no test which discriminates between gastrinomas of duodenal and pancreatic origin. A duodenal gastrinoma can often be verified by endoscopy and/ or a barium enema. In exceptional cases portal vein catheterization with measurement of the gastrin concentration in different parts of the veins draining the pancreas and duodenum might be helpful.

3.16.4 The somatostatinoma syndrome

Carcinoids associated with diabetes mellitus, steatorrhoea and cholelithiasis together with elevated circulating levels of plasma somatostatin constitute the somatostatinoma syndrome (KREJS et al. 1979). Originally these tumours were described in the endocrine pancreas, but occasionally the syndrome has been found in patients with carcinoids of gastro-duodenal origin, or with pulmonary carcinoids or thymic carcinoids (PENNMAN et al. 1980). The initial symptoms are often rather diffuse and the tumours can become very large before they are diagnosed. The demonstration of an increased plasma somatostatin level is essential for the diagnosis.

3.16.5 Cushing's syndrome

Cushing's syndrome has been reported in patients with pulmonary, gastro-duodenal and pancreatic endocrine tumours. A majority of the tumours in these patients produce ACTH (IMuRA 1980), but some tumours also produce CRF. The clinical symptoms are similar to those associated with pituitary tumours producing ACTH, but the clinical course is often more rapid in patients with ectopic ACTH production and the patients more often show increased pigmentation.

3.16.6 Multiple endocrine neoplasia type I

Among the carcinoids that may be involved in multiple endocrine neoplasia type I (MEN-I), those of the bronchus are the most common, but carcinoids at other sites have also been reported (EBERLE and GRUN 1981). The MEN-I syndrome can include

54 . E. Wilander . M. Lundqvist . K. Oberg

lesions of the anterior pituitary gland, hyperparathyroidism and pancreatic islet tumours, as well as hyperplasia of the adrenal cortex. 3.16.7 von Recklingshausen's disease and duodenal carcinoids Carcinoi'ds of the ampulla of Vater and duodenum constitute only 2 % of all gastrointestinal carcinoids. Eight cases associated with von Recklinhausen's disease have been reported (HOUGH et a1. 1983). Somatostatin was present in the duodenal carcinoids.

3.17 Diagnosis 3.17.1 Circulating tumour markers

3.17.1.1 Serotonin Qualitative and quantitative measurement of the urinary excretion of 5-HIAA, the major metabolite of serotonin, has been the principal laboratory test for the diagnosis of serotonin overproduction by carcinoid tumours (GRAHAME-SMITH 1972). Measurement of serotonin in whole blood, serum or platelets is also used occasionally to detect excess serotonin produced by such tumours (GRAHAME-SMITH 1972). In a recent study by FELDMAN (1986), increased urinary excretion of 5-HIAA wasfound in a series of 75 patients with the carcinoid syndrome, and 64 displayed elevated concentrations of serotonin both in the urine and platelets. Urinary 5-HIAA seemed to be the most valuable marker for carcinoid tumours, but by adding measurement of urinary serotonin, a further six out of seven patients could be correctly diagnosed. Among 103 patients with carcinoid tumours, the majority of whom had the carcinoid syndrome, we found an elevated urinary 5-HIAA level in 88%. This increase was mainly found in patients with mid-gut carcinoids, but also in some patients with a carcinoid of the bronchus. In patients with mid-gut carcinoids there was a fairly good correlation between the urinary 5-HIAA level and the size of the tumour mass.

3.17.1.2 Plasma tachykinins The second most important markers for carcinoid tumours are plasma tachykinins. Among our patients NPK was elevated in 66% (NORHEIM et a1. 1984; NORHEIM 1986). Patients with fore-gut or hind-gut carcinoids did not show such an increase and determination of tachykinins might be an aid in differentiating between carcinoids of different origin. NPK is more useful as a circulatory marker for carcinoids of mid-gut origin than the closely related substance P, a member of the same peptide family. NPK is a larger peptide (31 amino acids) than substance P (11 amino acids) and has a longer halflife in the circulation.

Gastrointestinal carcinoid tumours . 55

3.17.1.3 Pancreatic polypeptide Another important marker is the serum or plasma concentration of pancreatic polypeptide, which was elevated in 60% of our patients (OBERG et al. 1981). Normally PP cells are found in the pancreas and colonic mucosa, but ectopic production of this polypeptide has been found in patients with bronchial carcinoids. Extremely elevated serum PP levels have been noted in patients with bronchial carcinoids. Although patients with mid-gut carcinoids display elevated serum PP, no PP has been found in the tumours. The increased circulating levels of PP might be due to stimulation of normal PP cells in the pancreas or colon.

3.17.1.4 Human chorionic gonadotropin subunits The concentration of human chorionic gonadotropin (HCG)-a subunits in the serum has been found to be elevated in up to 69% of patients with malignant carcinoid tumours (OBERG and WIDE 1981). Interestingly, the highest HCG-a levels were noted in patients with bronchial or rectal carcinoids. A small number of patients also have elevated HCG-~ levels and these patients have a worse prognosis, with more aggressive disease and a poorer response to therapy.

3.17.1.5 Other markers Very recently a new marker for endocrine tumours has been explored. A novel protein has been isolated from porcine and human pituitary gland designated IR-7 B2 or APPG (SUZUKI et a1.1986). In a preliminary study, 64% of a series of patients with midgut carcinoid tumours were found to display elevated plasma levels of IR-7 B2. In a recent study by our group, we observed that 89% of patients with carcinoid tumours and concomitantly elevated serum HCG subunits also showed significantly increased plasma concentrations of IR-7 B2. Chromogranin A has recently been reported to be a marker for neuroendocrine tumours (O'CONNOR and DEFTOS 1986). We have developed a polyclonal antibody against chromogranin A plus B which has proved to be an excellent marker for different neuroendocrine tumours. All patients with carcinoid tumours had elevated plasma concentrations of this antibody (mean 6.545 ± 1.437 ng/ml) compared with those in healthy controls (mean 89.6 ± 15.7 ng/ml). Motilin is normally present in enteric nerves. We have found that in 52% of patients with mid-gut carcinoids and the carcinoid syndrome the plasma motilin level was increased (OBERG et al. 1987). However, no motilin was found within the tumour either by extraction or immunocytochemistry. It is possible that motilin was released from motilin-containing cells of the small intestine and not from the tumour cells and that motilin may contribute to a vicious circle including diarrhoea in patients with carcinoid tumours. There was a good correlation between the severity of diarrhoea and plasma motilin.

56 . E. Wilander . M. Lundqvist . K. Oberg

At present the urinary concentration of 5-HIAA is still the most important marker for gastrointestinal carcinoids, but in the near future plasma chromogranin A + B may be even more valuable. Plasma tachykinins are very useful markers in patients with midgut carcinoid tumours and the carcinoid syndrome, and PP and HCG-a subunits can be used as further aids in establishing a correct diagnosis (Table 3). Table. 3.

Circulating tumour markers in carcinoids. Gastrointestinal carcinoids

Marker

Fore-gut

Mid-gut

u-S-HIAA p-Chromogranin A p-APPG (IR-7B2) p-NPK s-PP s-HCG-a s-HCG-p s-gastrin p-somatostatin p-neurotensin

+ +++ ++ (+) ++ ++ + ++ + +

+++ +++ +++ ++ + + +

+++ ++

+

Hind-gut

+++ ++ + + (+) (+)

very useful useful may be useful useless

3.17.2 Stimulatory tests

In the majority of patients with symptoms of carcinoid syndrome, the ZollingerEllison syndrome or other clinically distinct syndromes associated with carcinoids, the basal serotonin level, urinary 5-HIAA or other markers of carcinoids are positive. In a few patients without metastases or with local metastases and/or very small non-detectable liver metastases, a stimulatory test can be beneficial. The most important test for mid-gut carcinoids is the pentagastrin stimulatory test (see above). For patients with Zollinger-Ellison syndrome, a secretin test might be of help for the diagnosis (see above). For early detection of endocrine tumours in persons with the MEN-I trait, we have developed a specific meal stimulatory test (SKOGSEID et al. 1987). 3.17.3 Localizing procedures

Endoscopy and different radiological examinations are important tools for localizing gatrointestinal carcinoid tumours and their metastases. For detection of liver metas-

Gastrointestinal carcinoid tumours . 57

tases, ultrasound investigation (Fig. 27) and CT scans (Fig. 28) are the most useful. An advantage of ultrasound is that guided biopsies of the lesions can be performed. For localization of the primary tumour(s) a barium enema can give some information (Fig. 29), but this is negative in most cases. Angiography of the coeliac axis, including

Fig. 27. Utrasound investigation demonstrating a liver metastasis.

58 . E .Wilander . M. Lundqvist . K. Oberg

mesenteric arteries, might indicate the location of the primaries (Fig. 30). In exceptional cases catheterization of the portal and mesenteric veins with sampling for assay of plasma serotonin can be helpful in the localizing procedure. Recently scanning with 1131 metaiodobenzylguanidine has been reported (FISHER et al.1984; FELDMAN et al. 1986). Phaechromocytomas have been localized by this method, which has now been developed to detect carcinoid tumours. Endoscopy is important for the detection not only of rectal, sigmoidal and colonic carcinoids, but also of those of the stomach and duodenum.

3.18 Treatment Surgical excision has been considered the treatment of choice for carcinoid tumours, since most of the tumours are slow-growing (MARTENSSON et al. 1983). In patients with localized tumours, local removal is all that is necessary, at least for those under 2 cm in diameter. More extensive resection is necessary for tumours measuring 2 cm or more. In patients with metastases confined to the regional nodes or with regional invasion, it is worth trying to resect all visible disease. If the tumour is not resectable, attempted

Fig. 28. CT-scan of the liver showing multiple liver metastasis in the right liver lobe.

Gastrointestinal carcinoid tumours . 59

Fig. 29. Barium enema of the small intestine demonstrating a typical rosett formation of the small bowel.

60 . E. Wilander . M. Lundqvist . K.Oberg

Fig. 30. Angiography of the superior mesenteric vein showing arterio-venous anastomoses typical for a carcinoid tumour of the small bowel.

Gastrointestinal carcinoid tumours' 61

debulking can lead to apparent improvement of clinical symptoms. Areas of small bowel obstruction will need to be bypassed irrespective of the resectability. Even if the patient has liver metastasis, surgery has to be considered the first therapeutic option. If metastases are confined to one lobe of the liver and the patient has severe symptoms from local disease or carcinoid syndrome, an attempt to resect the secondaries is worth while. Surgical resections may also be of help after inital conservative treatment has reduced the number or size of metastic tumours. Reduction of tumour masses is important to perform before treatment with biological response modifiers. 3.18.1 Anaesthetic considerations

Anaesthesia for patients with carcinoid syndrome needs to be given careful consideration. Severe blood pressure variations and flushing may occur during surgery, even in patients with a non-metastatic carcinoid. Furthermore, bronchial constriction may develop both during operation and postoperatively. Special care should be taken when giving nerve blockers, acetylcholine, curare and morphine. Catecholamines should not be used to treat hypotension (MASON and STEANE 1976; JONES and KNIGHT 1982). Most of the intraoperative and postoperative problems in patients with the carcinoid syndrome can nowadays be managed by infusion of the new somatostatin analogue SMS 201-995. This should be infused intravenously in a dose of 50 f,lg/h starting at least half an hour before surgery. The mechanisms of actions of somatostatin and its analogues are probably blockade of the release of mediators from the tumour and probably also blockade of the peripheral effects of these mediators. 3.18.2 Hepatic artery occlusion

Patients with carcinoid syndrome usually present with multiple liver metastases of varying size involving both lobes of the liver. Other approaches than surgery are needed in such patients. Most of the oxygen supply to the secondaries is arterial (90%), compared with only 50% in normal liver. Hepatic artery ligation and/or embolisation has been performed in several centres. After occlusion of the hepatic artery, objective tumour regression can be observed and the urine concentration of 5-HIAA falls rapidly to a normal or slightly elevated level (MoERTEL 1983). However, in several series the median duration of the response has only been about five months; in one series reported by BENGMARK et al. (1983) it was 14 months. Among 25 patients treated with embolisation by CARRASCO et al. (1986), 87% responded, with a median duration of response of 11 + months. Such a procedure can be repeated several times and can maintain clinical remissions for prolonged periods of time. Mortality rates as high as 22% have been reported after hepatic artery ligation (MoERTEL 1983), the causes of death include hepatic necrosis, hepato-renal syndrome, liver abscesses, carcinoid crises, gall bladder necrosis, cardiovascular collapse and septicaemia. Almost all patients develop high fever

62 . E. Wilander . M. Lundqvist . K. Oberg

and the serum concentrations of liver transaminases are grossly elevated, with only minimal increases in bilirubin and alkaline phosphatase. Collaterals can develop after ligation of the hepatic artery and more than 20 different accessory arteries have been observed (MICHELS 1960). Embolisation via the hepatic artery has a theoretical advantage of blocking the many smaller intrahepatic blood vessels, plus the fact that it is repeatable if the tumour revascularises (ALLISON et al. 1977). Hepatic artery ligation with cytotoxic drug perfusion has also been tried, but there is no evidence that this is superior to devascularisation alone. Transient dearterialisation has been performed in an attempt to overcome the problem of collateral blood supply and the possibility of acute hepatic necrosis. Accessory arterial supplies are ligated and a sling is passed around the hepatic artery so that it can be occluded temporarily a few days after the operation (BENGMARK et al. 1983). However, the peri operative death rate is considerable, 12.5 %, without any significant benefit in the survival. In our own opinion embolisation of the liver arteries is preferable to hepatic artery ligation. This procedure should not be the sole form of treatment, but should be combined with other conservative treatment such as administration of somatostatin analogues or interferons, or chemotherapy.

3.18.3 Radiotherapy

The results of the use of radiotherapy in carcinoid patients have been controversial. In a study by KEANE et al. (1981) of patients treated with whole abdominal irradiation to 2,000-2,500 rad, no objective response was noted. Two patients developed intractable diarrhoea and died. In contrast, SAMLOWSKI et al. (1986), observed an objective response in 25% of patients with metastatic carcinoid tumours. The best response was found in patients receiving the highest doses of radiotherapy (> 29 Gy). Patients who exhibited the carcinoid syndrome appeared to respond less frequently to radiotherapy than those who did not. Although the series was small, the difference in radiosensitivity between patients with and without carcinoid syndrome might be explained by the observation that serotonin is radioprotective (BARNES and LOWMAN 1971; DEANOVIC et al. 1971). At present, radiotherapy is indicated for treatment of bone secondaries for symtomatic relief.

3.18.4 Pharmacological treatment (conservative treatment)

3.18.4.1 Chemotherapy

The somewhat indolent and varied biological behavior of carcinoid tumours, coupled with the limited efficacy of currently available antineoplastic drugs, demands careful selection of patients for conservative treatment. Symptoms that significantly interfere with daily activities, such as flushing and diarrhoea, represent one indication for chemotherapy or other conservative treatment. The development of one of the poor

Gastrointestinal carcinoid tumours . 63

prognostic signs such as liver metastases and/ or carcinoid heart disease might further sugget chemotherapy or other conservative treatment. Numerous drugs have been tried as single agents or in combinations and in different doses. 3.18.4.1.1 Single agent chemotherapy 5-fluorouracil

In a study by the Eastern Cooperative Oncology Group, 5-fluorouracil 500 mg/m2 given daily for five days every five weeks gave an objective response in 18% of the patients (MOERTEL 1975). A similar result has been obtained at the Mayo Clinic (KVOLS 1986). Doxorubicin

In three separate trials of doxorubicin given at a dose of 60 mg/m2 every two to four weeks, an objective response has been seen in about 21 % of the patients (ENGSTROM et al. 1983; MOERTEL 1983; K VOLS 1986).

Dacarabazine

KESSINGER et al. (1983) reported objective improvement lasting for a year in one patient and subjective improvement in another patient, both of whom were treated with dacarbazine. Actinomycin D

In an early study, objective response were noted in 3/5 patients with carcinoid tumours (DOLLINGER and GOLBEY 1967), but in a later study the figures were less encouraging, only 1/17 patients showing a response (VAN HAZEL et al. 1983). Cisplatinum

Among 15 patients treated with cisplatinum intravenously in doses of 45-90 mg/m 2 at the Mayo Clinic, only one exhibited an objective response (7%). The response was also short-lasting, 3.5 months (MOERTEL et al. 1986). Streptozotocin

This nitroso-urea antibiotic has been found to have a significant effect in the treatment of endocrine pancreatic tumours, which are closely related to gastrointestinal carcinoids. In a study at the Mayo Clinic, MOERTEL (1983) reported objective regression in six out of nine patients with carcinoid tumours treated with STZ. Other authors have also observed an anti tumour effect of this drug (STOLINSKY et al. 1972), but SCHEIN et al. (1974) found no response among eight patients. 3.18.4.1.2 Combination chemotherapy

MENGEL and SHAFFER (1973) reported responses in six of 11 patients with carcinoid tumours treated with a combination of cyclophosphamide plus methotrexate. Later investigators at the Mayo Clinic failed to observed any single response to this comb ina-

64 . E. Wilander . M. Lundqvist . K. Oberg

tion (MOERTEL et al. 1984). Using a combination of 5-fluorouracil and STZ, MOERTEL (1975) noted a response in about 6/9 patients with carcinoid tumours. CHERNICOFF et al. (1979) reported that 4/10 carcinoid patients responded to this same regime. In a randomised multi-institutional trial undertaken by investigators of the Eastern Cooperative Oncology Group, the objective response rate was 33% for a combination of STZ and 5-fluorouracil, compared with 26% among patients treated with cyclophosphamide plus STZ (MOERTEL and HANLEY 1979). The response rates were more favourable in patients with documented carcinoid syndrome and in those with a better performance status. It was also noted that with both regimes the response rates were significantly higher for mid-gut carcinoids than for carcinoids of pulmonary or unknown origin. In a study by the Eastern Cooperative Oncology Group, STZ was given at an inverval of 10 weeks instead of five weeks in an attempt to reduce anorexia, nausea and vomiting. 5-fluorouracil was still given daily for 5 days every five weeks. The study was randomised, a second treatment arm being single agent doxorubicin 60 mg/m 2 given intravenously on a monthly basis. The response rates were 23% for both regimes. The median duration of response was 26 weeks for doxorubicin and 31 weeks for the combination (ENGSTROM et al. 1984). In a recent study in which STZ was adminstered in a weekly schedule with a single infusion of doxorubicin, objective responses were noted in 4/10 patients with carcinoid tumours (KELSEN et al. 1982). A four-drug regime consisting of 5-fluorouracil, STZ, doxorubicin and cyclophosphamide did not appear to have any obvious therapeutic advantage, 7/20 patients (35%) showing an objective response (BUKOWSKI et al. 1983). Our own experience of a combination of STZ and 5fluorouracil is that among 31 patients with malignant mid-gut carcinoid tumours, objective responses occurred in only 10%. The responses were short-lasting, with a median duration of 3 months (OBERG et al. 1987). It is concluded from the results of chemotherapy in patients with carcinoid tumours that no ideal combination of chemotherapeutic agents is available at present. The objective response rates are lower than in other neuroendocrine tumours, such as endocrine pancreatic tumours, and the duration of the responses is shorter. The best combination of cytotoxics seems to be STZ plus 5-fluorouracil or doxorubicin, to which an objective response occurs in about 30-40%. The toxicity of chemotherapy is considerable; the adverse effects include severe vomiting, nausea, and renal and hepatic failure in patients treated with STZ, and a combination with doxorubicin adds hair loss, anaemia, leukopenia and thrombocytopenia and also an increased risk of heart complications.

3.18.4.2 Interferon treatment Interferons belong to the system of biological response modifiers with documented effects on tumour cell growth and the immune system (PFEFFER 1987; TAMM et al. 1987). Treatment with human leukocyte interferon was started in our hospital in 1982 and in a subsequent preliminal)' study (OBERG et al. 1983) we found effects on hormone

Gastrointestinal carcinoid tumours . 65

levels and on clinical symptoms in patients with malignant carcinoid tumours. Since then three separate studies have been carried out with similar results. The first one was a long-term study in 36 patients with carcinoid tumours (primary tumours: 29 patients had a mid-gut carcinoid, four pulmonary, one rectal, one ovarian and one unlocalized). Thirty-two of 36 patients had liver metastases and 19/36 had received cytotoxic treatment previously. The patients were given 3-6 MU of human leukocyte interferon s.c. daily. An objective response was achieved in 17/36 patients (47%), 14/29 patients with mid-gut carcinoids (48%) and 3/4 patients with pulmonary carcinoids (75%). The disease remained stable in a further 14 patients (39%) and progressed in five (14%). In 16 of the 17 patients with an objective response there was a significant decrease in tumour markers and in four of these the tumour size was significantly reduced. Two patients showed complete remission. The median duration of the objective response was 34 months and the median duration of stable disease was 25 months (OBERG et al. 1986). The second study was a randomised controlled study comparing human leukocyte interferon (6 MU s.c./d) with STZ 1.5 g/m 2 three times a week every 3 weeks + 5fluorouracil 400 mg/m 2 three times a week every 3 weeks in patients with malignant mid-gut carcinoid tumours. Twenty patients with mid-gut carcinoids and liver metastases were included in the study. Ten patients received interferon and ten chemotherapy. After six months' treatment objective responses were seen in five of the ten interferon patients (50%) but in none of those given chemotherapy. Stable disease was observed in 50% of both groups; the disease progressed in five of the patients on chemotherapy but in none of the interferon group. A chi-square analysis of the difference in the proportions of responses in the two groups showed that interferon was significantly more effective than chemotherapy (p = 0.0067) (to be published). In the third study 20 patients with carcinoid tumours were treated with recombinant interferon alpha 2-b (Intron-A) 6 MU three times a week. Eighteen of the patients had malignant mid-gut carcinoids and two had metastatic lung carcinoids. After six months' treatment, 11 of the 20 patients (53%) showed an objective response, in six patients the disease remained stable and in three it progressed (to be published). In a similar study by DOLVA et al. in Oslo, the same rate of objective response to recombinant interferon alpha 2-b was noted among 12 patients with malignant mid-gut carcinoid tumours, i.e. seven out of 12. In two patients complete remission occurred (to be published). In these latter studies about 70-80% of the carcinoid patients experienced alleviation of their clinical symptoms and an improved quality of life, simultaneously with a reduction of the concentration of circulating hormones. There does not seem to be any difference in efficacy between human leukocyte interferon alpha and recombinant interferon. However, we have noticed in a recent study that when patients developed neutralising antibodies against interferon alpha 2-b (Intron-A), the previously recognised objective responses ceased and the disease began to progress. About 15% of the patients receiving recombinant interferon alpha 2-b developed neutralising antibodies.

66 . E. Wilander . M. Lundqvist . K. Oberg

The adverse reactions to interferon treatment are less severe than with chemotherapy and mainly consist in «flu-like» symptoms in the first 3-5 days and mild to moderate leukopenia, anaemia, weight loss and moderate fatigue. Autoimmune phenomena such as thyroid autoantibodies occurred in about 15% of the patients and in one single patient the SLE syndrome developed. It is important to note that the doses used in all these studies represented «low dose therapy», i. e. doses of 3-6 MU/day s.c. every day or three times a weak. When patients are receiving higher doses, such as 20 or 25 MU/ day, they develop more severe side-effects, including mental confusion and severe lethargia. 3.18.4.3 Somatostatin analogue Somatostatin is a hormone that inhibits the release of numerous peptides, such as growth homones, insulin, glucagon and gut peptides (REICHLIN 1983). Native somatostatin has been reported to be effective in blocking the carcinoid flush induced by pentagastrin and in controlling other symptoms associated with the carcinoid syndrome (THULIN et al. 1978). However, the therapeutic application of the compound is limited by its short half-life, which means that continuous intravenous infusion is required. An analogue of somatostatin with eight amino acids rather than 14 has been synthesized and reported to be more specific, potent and longer acting in its inhibitory effects (BAUER et al. 1982). In one study, 21 patients with histologically proved metastatic carcinoid tumours and the carcinoid syndrome received the drug SMS 201-995 in a dose of 150 J.lg three times a day. In all patients flushing and diarrhoea associated with the syndrome were promptly relieved. In 15 of the 21 patients (71 %) the urinary excretion of 5-HIAA decreased significantly. The median duration of the biochemical response was more then 9 months. Adverse effects were few and the most pronounced were steatorrhoea and malabsorption (KVOLS et al. 1986). We have also tried SMS 201-995 in 20 patients with malignant carcinoid tumours, but our patients received a low dosage, 50 J.lg twice a day, and objective rsponses were noted in only 30%. The dose was increased after six months of observation and the frequency of objective responses subsequently increased. The dose of this analogue SMS 201-995 seems to be important for its effect and the optimal dose seems to be 100--150 J.lg 2-3 times/day s.c. In some of the patients in our study diarrhoea and malabsorption occurred, which might have led to withdrawal of the therapy. Another newly observed sideeffect is hypocalcaemia, probably due to malabsorption and to an inhibitory effect of the analogue on calcium and vitamin D absorption from the gut mucosa. The drug has shown very beneficial effects in acute situations, postoperatively, and in so called «carcinoid crises» (KVOLS et al. 1985). By intravenous infusions of 50 J.lg/h, we have been able to alleviate such carcinoid crises on four occasions. The drug may be lifesaving in such situations. For long-term management of patients with the carcinoid syndrome, SMS 201-995 may be attended with the problem of tachyphylaxis, so that the dose has to be steadily increased to keep the patient in good clinical condition.

Gastrointestinal carcinoid tumours . 67

Our current treatment programme for patients with malignant carcinoid tumours and the carcinoid syndrome mainly consits in administration of alpha interferon, as it has shown inhibitory effects on both the hormone production and tumour cell growth. If the patient does not respond to interferon, or if the treatment is not sufficient to relieve the clinical symptoms, the somatostatin analogue 201-995 is added. In view of the tendency for the sensitivity to both alpha interferon and SMS 201-995 to decline during long-term treatment, it is appropriate to alternate between these two drugs; in this way good control of the clinical symptoms and tumour growth can be achieved for extended periods of time. In the acute situation, the somatostatin analogue 201-995 is the drug of choice, adimistered as an intravenous infusion. At any time during the long-term management of carcinoid patients, embolisation or surgical procedures can be added to further control the clinical symptoms. However, it is very important to consider the possible adverse effects of any therapy in relation to the severity of the clinical symptoms of the patients.

3.18.4.4 Other treatments Parachlorophenylalanine has been shown to relieve diarrhoea and to reduce the urinary excretion of 5-HIAA, but it rarely helps flushing (SJOERDSMA et al. 1970). The side-effects of this agent, predominantly hypersensitivity reactions and psychiatric disturbances, make it intolerable for long-term clinical use. Cyproheptadine, a histamine HI antagonist, has given objective improvement in one study, where 2/12 patients showed regression of the carcinoid tumour (HARRIS and SMITH 1982). These findings need to be confirmed in further studies. Tamoxifen, a synthetic anti-oestrogen, has been reported to alleviate the symptoms in solitary patients with the carcinoid syndrome (STATHOPOULOUS et al. 1981). These reports concern single cases and in a study from the Mayo Clinic comprising 16 patients with metastatic carcinoid tumours, no evidence of objective improvement was noticed. These patients received 20-40 mg of tamoxifen for 8 weeks. The median length of time to progression was 10 weeks and the disease had progressed in all patients by 24 weeks (MoERTEL et al. 1984). Other agents that can produce symptomatic relief in the carcinoid syndrome include adrenergic blocking agents such as clonidine, phenoxybenzamine, phentolamine and propranolol. Inhibition of serotonin synthesis may be achieved by giving the patient alpha-methyldopa, 5-fluorotryptophan or parachlorophenylalanine. Peripheral serotonin antagonists include cyproheptadine, methysergide and ketanserin. Histamine H2 receptor antagonists, such as cimetidine, can also give symptomatic relief and are especially indicated in patients with fore-gut carcinoids with gastrin production and the Zollinger-Ellison syndrome. These patients should also be considered for treatment with the new agent omeprazol, which blocks the hydrochloric acid production in the parietal cells of the gastric mucosa. Corticosteroids and chlorpromazine have been used

68 . E. Wilander . M. Lundqvist . K. Oberg

for treatment of diarrhoea and an especially useful antidiarrhoeal agent is loperamide. Verapamil has been reported to alleviate diarrhoea in some patients with the carcinoid syndrome.

3.19 Survival and prognosis Carcinoid tumours are neoplasms which progress relatively slowly. However, the estimated median survival without therapy in patients who present with liver metastases is 2.5 years (MOERTEL et al. 1961). In a study by MARTENSSON et al. (1983), the fiveyear survival among patients without metastases was 80%, among those with local lymph node metastases it was 67%, and among patients with liver metastases 43%. DAVIS et al. (1973) reported that patients with the carcinoid syndrome showed a median survival from the onset of flushing of 38 months, and from the first recognition of an elevated 5-HIAA level in the urine of 23 months. Urinary 5-HIAA has been suggested to be of prognostic value; thus in patients with an initial concentration of 10-49 mg/24 h, the medial survival time was found to be 29 months, at 50-149 mg124 h it was 21 months and at a concentration higher than 150 mg/24 h it was 13 months. In patients with clinical evidence of carcinoid heart disease, the median survival was further reduced to 11 months. In our own study of 103 patients with carcinoid tumour, the median survival was 8.3 years from the first symptom of flush and diarrhoea and there was a five-year survival of 70% (N ORHEIM et al. 1987). Thirty-nine of the 103 patients died during the observation period; of these, 44% died of tumour progression and 36% of cardiac insufficiency. Our five-year survival data were considerably improved compared with those of earlier studies. The reason for such an improvement is difficult to evaluate, as no control group was followed up in our own study, but it may have been due to our multistep form of treatment including interferon, somatostatin analogue, liver embolisation and surgery. Earlier diagnosis and institution of treatment might also be an important factor for prolonged survival of patients with malignant carcinoid tumours and the carcinoid syndrome.

3.20 Clinical conclusions The majority of all gastrointestinal carcinoid tumours are clinically benign, and surgery can cure all these patients provided the tumour is less than 2 cm in diameter. The tumours of the mid-gut origin are prone to give rise to the carcinoid syndrome and when the patient has developed liver metastases and the carcinoid syndrome, other forms of treatment have to be considered. The ultimate treatment has probably not yet been developed, but significant alleviation of clinical symptoms and improvement of the quality of life have been achieved by the use of alpha interferons and a new somatostatin

Gastrointestinal carcinoid tumours . 69

analogue SMS 201-995. The effects of alpha interferons might be partly exerted through inhibition of oncogene expression, DNA replications, protein synthesis and reduction of autocrine growth factors for the tumours. A similar reduction of tumour growth factors might be effected by the somatostatin analogue. When the mechanisms underlying the growth of these neuroendocrine tumours has been elucidated and growth factors have been characterized, specific blockers of such growth factors and also specific growth factor receptor blocking agents might come into clinical use.

4 Summary The increased knowledge of the pathobiology of gastrointestinal carcinoid (neuroendocrine) tumours and the improved therapeutic possibilities have brought a demand for more precise diagnosis. Although the carcinoid tumours can often be tentatively recognized in routinely processed microscopic slides, their more accurate identification reguires additional diagnostic procedures. General neuroendocrine markers such as the argyrophil reaction of Grimelius and immunohistochemistry with application of antibodies against chromogranin A and of neuron-specific enolase are discriminatory staining methods which are used to reveal the neuroendocrine origin of almost all highly differentiated carcinoid tumours of the gastrointestinal tract. Mid-gut carcinoids, which predominate among these tumours almost unexceptionally contain serotonin. This biogenic amine can be demonstrated by the argentaffin reaction of Masson, serotonin immunoreactively or by formalin-induced fluorescence. The characteristic staining pattern of mid-gut carcinoids is almost invariably preserved in the metastatic deposits and consequently the staining methods for identifying serotonin can also be used on metastases to reveal a primary mid-gut carcinoid. The enterochromaffin-like (ECL) cell carcinoids of the body and fundic area of the stomach often seen in association with pernicious anaemia are argyrophil with the Sevier-Munger silver stain. Other neuroendocrine tumours, viz. antral, duodenal and rectal carcinoids should be studied by a battery of relevant peptide hormone antisera for adequate diagnosis. During the last decade new peptide hormones have been found in circulation in patients with carcinoid tumours, but serotonin and urina~y 5-HIAA are still the most important markers for carcinoids of the mid-gut origin. Other clinically useful tumour markers are chromogranin A + B, pancreatic polypeptide, human chorionic gonadotropin alpha and beta subunits. For localizing procedures, angiography is the most reliable investigative method for primary tumours in the gut, whereas CT -scan and ultrasound investigations are good for detection of liver metastases. During the last five years, the therapy for malignant carcinoid tumours has been considerably improved. Chemotherapy has only revealed objective response rates in about 10 - 30% of the patients giving median survivals from start of therapy of about 10 months. Recently treatment with alpha interferons and the new somatostatin analogue

70 . E. Wilander . M. Lundqvist . K. Oberg

octreotide have given objective responses in 50-75% of patients with malignant midgut carcinoid tumours. These patients have now a median survival from start of therapy of 70 months when treated with alpha interferons. In the future new therapies will corne into use such as monoclonal antibodies and perhaps also agents blocking different growth factors.

Gastrointestinal carcinoid tumours . 71

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