Role of Somatostatin Analogues in Oncology: European Neuroendocrine Tumour Network (Enet) February 24-27, 2000 Innsbruck, Austria (Digestion)

Role of Somatostatin Analogues in Oncology European Neuroendocrine Tumour Network (ENET) February 24–27, 2000, Innsbruc...

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Role of Somatostatin Analogues in Oncology European Neuroendocrine Tumour Network (ENET) February 24–27, 2000, Innsbruck, Austria

Guest Editor

Kjell Öberg, Uppsala

16 figures, 1 in color, and 27 tables, 2000

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Drug Dosage The authors and the publisher have exerted every effort to ensure that drug selection and dosage set forth in this text are in accord with current recommendations and practice at the time of publication. However, in view of ongoing research, changes in government regulations, and the constant flow of information relating to drug therapy and drug reactions, the reader is urged to check the package insert for each drug for any change in indications and dosage and for added warnings and precautions. This is particularly important when the recommended agent is a new and/or infrequently employed drug.

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Contents Vol. 62, Suppl. 1, 2000

001 Editorial 003 Molecular Genetics of Neuroendocrine Tumors Calender, A. (Lyon) 019 Biological and Molecular Aspects of Gastroenteropancreatic

Neuroendocrine Tumors Rindi, G.; Villanacci, V.; Ubiali, A. (Brescia) 027 Somatostatin Receptors Benali, N.; Ferjoux, G.; Puente, E.; Buscail, L.; Susini, C. (Toulouse) 033 Tumor Markers in Neuroendocrine Tumors Eriksson, B.; Öberg, K.; Stridsberg, M. (Uppsala) 039 Imaging Procedures in Neuroendocrine Tumours Ricke, J. (Berlin); Klose, K.-J. (Marburg) 045 Endoscopic Ultrasonography of Neuroendocrine Tumours Zimmer, T.; Scherübl, H.; Faiss, S. (Berlin); Stölzel, U. (Döbeln); Riecken, E.-O.; Wiedenmann, B. (Berlin) 051 Natural History of Neuroendocrine Enteropancreatic Tumors Mignon, M. (Paris) 059 Interventional Treatment of Gastrointestinal Neuroendocrine Tumours Ahlman, H.; Wängberg, B.; Jansson, S.; Friman, S.; Olausson, M.; Tylén, U.; Nilsson, O. (Göteborg) 069 OctreoTherTM: Ongoing Early Clinical Development of a

Somatostatin-Receptor-Targeted Radionuclide Antineoplastic Therapy Smith, M.C.; Liu, J.; Chen, T.; Schran, H.; Yeh, C.-M. (East Hanover, N.J.); Jamar, F. (Brussels); Valkema, R.; Bakker, W. (Rotterdam); Kvols, L. (Tampa, Fla.); Krenning, E. (Rotterdam); Pauwels, S. (Brussels) 073 Chemotherapy in the Treatment of Neuroendocrine Malignant Tumors Rougier, P.; Mitry, E. (Boulogne) 079 Hepatic Arterial Chemoembolization in the Management of Advanced

Digestive Endocrine Tumors Ruszniewski, P.; Malka, D. (Clichy) 084 Treatment of Neuroendocrine GEP Tumours with Somatostatin

Analogues – A Review Arnold, R.; Simon, B.; Wied, M. (Marburg) 092 Interferon in the Management of Neuroendocrine GEP Tumors – A Review Öberg, K. (Uppsala)

Round Table Discussion 098 Sandostatin® LAR® Leaders Meeting–European Neuroendocrine Tumour

Network ENET 108 Author and Subject Index

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Editorial

This supplement of Digestion contains the proceedings of a symposium held in February 2000 in Innsbruck, Austria. The objectives of the meeting were twofold. First to present the European Neuroendocrine Network (ENET) and second to update current knowledge in the diagnosis and treatment of neuroendocrine tumors. ENET is a newly founded organization for centers throughout Europe interested in neuroendocrine tumors. The main task of this organization is to organize common multicenter trials on different forms of neuroendocrine tumors, studies which can not be done at single centers due to a lack of patients. The other important task is to establish guidelines for histopathology, radiology and surgical procedures, and also to form a database for patients with neuroendocrine tumors. The organization is rather small but contains people interested in basic science as well as clinical research. The members of the ENET group were responsible for different presentations on neuroendocrine tumors of the gastrointestinal tract, from the genes to the clinic. During the last decade increasing knowledge in molecular genetics and tumor biology has emerged, although we do not exactly know the key molecular events behind the development of various malignant neuroendocrine tumors. At the meeting an update on somatostatin receptor expression and signal transduction were made, since somatostatin analog treatment is one of the key therapies in neuroendocrine tumors. During the meeting, tumor markers and imaging procedures were also discussed, particularly the development of somatostatin receptor scintigraphy, which is now the ‘gold standard’ for the imaging of neuroendocrine tumors, and also endoscopic ultrasonography, which has been particularly useful for small endocrine pancreatic tumors. The large part of the meeting was devoted to therapeutic considerations including surgery, tumor-targeted radiotherapy, cytotoxic treatment, embolization, somatostatin analogs and interferons. There is no doubt that both somatostatin analogs and interferons have provided significant benefit in controlling clinical symptoms and hormone levels in patients with neuroendocrine tumors, particularly carcinoids. However, we are still awaiting treatment which can cure patients with malignant metastatic disease. Tumor-targeted treatment with radioactive somatostatin analogs is still investigational but current results are rather promising. The precise dosing of the compounds has still to be established, as should the stage of disease when such treatment is to be applied. The meeting was finished by a round table discussion on the role of somatostatin analogs in oncology and also other topics brought up during the meeting by the participants. The whole round table discussion was taped and is included in this supplement. The meeting was supported by a generous educational grant from Novartis, Basel, and was excellently organized by Dr. Guido Nussbaumer. Kjell Öberg, Uppsala

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Digestion 2000;62(suppl 1):3–18

Molecular Genetics of Neuroendocrine Tumors A. Calender Department of Genetics and Cancer, Hôpital Edouard-Herriot, Lyon, France For the GENEM network (Groupe d’Etude des Néoplasies Endocriniennes Multiples)

Key Words Neuroendocrine tumors W Genetics W Multiple endocrine neoplasia W Gene W Cancer

Abstract Through insights into the molecular genetics of neuroendocrine tumors (NETs), the genes predisposing to multiple endocrine neoplasia (MEN) syndromes were identified. In MEN1, tumors occur in the parathyroids, endocrine pancreas, anterior pituitary, adrenal glands and thymic neuroendocrine tissues. The MEN1 gene encodes a putative growth-suppressor protein, menin, binding JunD, a transcriptional factor belonging to the AP-1 complex. However, new partners binding menin remain to be found. The MEN1 gene might be involved in 1–50% of sporadic NETs. Another critical mechanism involved in NETs is the deregulation of the RET-signalling pathways by oncogenic point mutations responsible for MEN2 syndromes. MEN2 refers to the inherited forms of medullary thyroid carcinoma. The RET proto-oncogene, a tyrosinekinase receptor, is activated by missense mutations occurring either in the extracellular dimerization domain or intracellular tyrosine kinase catalytic regions. In both cases the receptor is constitutionally activated in the absence of natural ligands. Endocrine tumors also belong to the clinical pattern of Recklinghausen (NF1) and von Hippel-Lindau (VHL) diseases. The genes for both

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syndromes have been characterized and provide new pathways for endocrine tumorigenesis related to G-protein physiology (NF1) and transcriptional regulation and/ or endothelial cell proliferation (VHL), respectively. Here, we propose a basic overview of recent data on genetic events leading a normal endocrine cell towards a fully malignant phenotype.

Basic Pathways Related to Genetic Predisposition

Neuroendocrine tumors (NETs) occur mainly in five independent autosomal dominant inherited syndromes for which the genetic pathways have recently been characterized. Multiple endocrine neoplasia (MEN) types 1 and 2 are the most common forms with high penetrance of various neuroendocrine proliferations. NETs were less commonly observed in von Hippel-Lindau (VHL) disease, Recklinghausen neurofibromatosis (NF1) and tuberous sclerosis (TSC). Other genetic syndromes characterized by single or multiple endocrine tumors were identified and mapped in the last decade, but the genes related to these diseases, such as Carney complex, non-MEN1 familial isolated hyperparathyroidism (FIHPT), Conn adenoma, and pituitary tumors, remain to be identified.

Dr. Alain Calender Département de Génétique et Cancer, Hôpital Edouard-Herriot, Pavillon E F–69437 Lyon Cedex 03 (France) Tel. +33 4 72 11 73 84, Fax +33 4 72 11 73 81, E-Mail [email protected]

NET Related to Inactivation of a Growth-Suppressor Gene, MEN1 MEN1 (OMIM 131100) is an inherited disease predisposing parathyroid hyperplasia/adenoma, pancreatic endocrine tumors, pituitary tumors, adrenocortical secreting or nonfunctional tumors and thymic NETs [1, 2]. Recent observations suggested that a MEN1 patient could also be affected by cutaneous or visceral (angio)lipoma and fibrosarcoma [3] and central nervous system tumors such as meningioma or ependymoma [4, 5]. The diversity of MEN1-related lesions and the embryonic origins of affected tissues suggest that the MEN1 gene might play a critical role in early embryogenesis. Larsson et al. [6] first localized the MEN1 gene in 1988 on the long arm of chromosome 11, band q13. Comparative genetic analysis of tumoral and constitutional genotypes with polymorphic DNA markers in 11q13 showed somatic loss of heterozygosity (LOH) suggesting that development of MEN1-associated tumors was a two-step process, a germline mutation affecting the first MEN1 allele, and a somatic inactivation of the unaffected allele occurring by LOH [6, 7]. This suggests that tumorigenesis in MEN1 involves loss of function of a growth-suppressor gene according to the two-hit model by Knudson [8]. The MEN1 gene was finally cloned in 1997 after 10 years of physical and genetic mapping [9, 10]. The gene spans 9 kb of the genomic DNA and contains 10 exons encoding a 610-amino acid protein, menin. The first exon and the 3) 832-bp part of exon 10 are untranslated. A 2.8-kb major MEN1 transcript was detected in all human tissues tested, including the pancreas, thymus, adrenal glands, thyroid, testis, leukocyte, heart, brain, lung, muscle, small intestine, liver and kidney [9, 10]. A large 4-kb transcript was identified in the pancreas, thymus and stomach and suggests a tissue-specific alternative promotion [10]. Menin is a nuclear protein containing two nuclear localization signals (NLSs) at codons 479–497 (NLS-1) and 588–608 (NLS-2) [11]. NLS-1 and NLS-2 have been defined by in vitro functional studies using menin deletion constructs and epitope tagging with enhanced green fluorescent protein in different subcellular fractions. Even if mostly present in the nucleus, menin was recently found to translate from the nucleus to cytoplasm during the cell cycle, the cytoplasmic transfer being observed during mitosis [12, 13]. No nuclear export signal or DNAbinding transmembrane or transactivation domains have been identified to date, even though protein structure software (PROSITE, SOPMA) predicted high hydrophobicity in the NH2-terminal half of the protein, suggesting

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at least three leucine-zipper and helix structures in the primary/secondary organization of menin [9, 10]. The murine orthologue of human MEN, Men1, was mapped to the pericentromeric region of MMU19 (murine chromosome 19) and consequently shown to have a similar organization as the human gene [14]. Two major transcripts of 3.2 and 2.8 kb, with and without intron 1, respectively, were detected in both embryonal and adult tissues. The predicted murine protein is 611 amino acids in length. Overall, it is 97% homologous to the human menin. The murine Men1 gene was subsequently cloned by 3 independent groups and the rat Men1 gene also identified [15–17]. In situ mRNA hybridization and Northern blot studies showed the pattern of Men1 expression during mouse development. The expression of Men1 was detected at gestational day 7 in the whole embryo and a strong expression in the thymus, liver, CNS and testis at day 17. Hence, Men1 expression was not only confined to endocrine organs [14, 15]. The high expression of Men1 in the testis and mainly in Sertoli cells could assess a critical function of menin in reproduction. Menin binds JunD, a transcription factor acting through the activator protein-1 (AP1) complex [18]. This interaction appeared specific and menin did not bind to other members of the AP1 family. Wild-type menin repressed transcriptional activation mediated by JunD by an in vitro cotransfection assay. Interacting regions of menin and JunD were defined by deletion mutants. Three major domains were crucial for the menin-JunD interaction, the 40 amino acids from the N-terminus of menin, and two central regions at positions 139–242 and 323– 428. Four amino acid residues at positions 139, 160, 176 and 242 should be conserved for a normal menin-JunD interaction. A putative role of the C-terminus end of menin remains controversial [19]. Menin -JunD binding required the N-terminus region of JunD, which supports the activation domain of the protein through an interaction with a co-activator, JAB1 [20]. Recently, it has been shown that menin-mediated repression of JunD transcriptional activity is relieved by a specific inhibitor of deacetylase, suggesting that deacetylation of histones could play an essential role in this pathway [19]. Lastly, menin has been shown as a true in vitro growth suppressor after overexpression in Ras-transformed NIH3T3 cells [21]. More than 300 different MEN1 germline mutations have now be identified in several independent national studies based on large series of MEN1 patients [22–27]. Figure 1 shows the spectrum of most MEN1 mutations identified in France and recurrent mutations described by other groups. Unequivocally, the mutations were spread

Calender

Fig. 1. Germline mutations in the

MEN1 gene according to the menin– JunD binding domains. Most mutations were identified in French families through the GENEM network. Mutations are represented according to the international nomenclature, i.e Val162Phe is a missense at codon 162; Arg460ter is a nonsense at codon 460; 1149del11 is a frameshift deletion beginning at nucleotide 1449. ins = Insertions; splice-mutations are in italics. The underlined mutations and black boxes at the left of the gene represent relative hotspots observed in the French series of mutations and by other groups [22– 27]. The JunD protein is represented by a bold line and the interacting regions with menin by large circles near the N terminus and the central region of the MEN1-encoded sequence. NLS1 and NLS2 designate the two nuclear localization signals identified in exon 10. The introns are not fully represented.

Molecular Genetics of Neuroendocrine Tumors


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over the entire coding and noncoding sequences of the MEN1 gene without significant clustering related to known functional domains of the protein. Approximately 70% of the MEN1 germline mutations were nonsense and (deletional or insertional) frameshift-truncating mutations. Missense substitutions and in-frame deletions/ insertions represented "20–25% of the mutations. Intronic and splice-junction mutations were reported in a few families and some of them were shown to alter RNA splicing with an abnormal exon skipping or intronic retention [28, 29]. Most mutations occurred once, while some of them were observed twice or more in apparently unrelated families [22–27]. Haplotype analysis with 11q13 polymorphic markers demonstrated that most recurrent mutations were not related to a founder effect assessing the data from two independent linkage disequilibrium studies performed before the MEN1 gene was cloned [30]. Recently, two independent groups analyzed the 11q13 haplotypes in families with recurrent mutations and showed that the same mutation occurs commonly in genetically unrelated families [31, 32]. Conversely, a common founder effect characterized by a single mutation and common haplotypes has been reported in four kindreds from Newfoundland expressing the prolactinoma variant of MEN1 (MEN1BURIN) [33]. In specific regions of the gene, hot spots involving a cytosine or a guanine might be explained by replication errors related to slippedstrand mispairing in unstable and/or repetitive motifs with an high GC content, such as the 1650del/insC in exon 10 [34]. Based on exhaustive analysis of patients with sporadic MEN1 and both their parents, Bassett et al. [24] estimated the rate of neomutations occurring in MEN1 to 10–15%. Most, if not all reported MEN1 germline mutations which alter or delete a single amino acid in humans occurred at residues highly conserved between human and rodents, suggesting thereby a functional/ pathogenic significance. No genotype-phenotype correlations have been established to date. Despite extensive studies, we and others did not succeed to find a relation between the type and location of MEN1 mutations and the clinical features of MEN1 in probands and families [22–27]. However, most patients with aggressive phenotypes share truncating mutations. Secondly, MEN1-related FIHPT, a genetic variant of MEN1, has mostly been related to missense mutations occurring between exons 3 and 7 [35, 36]. One would suggest that FIHPT-related MEN1 mutations could only be missenses and restricted to the central region of menin. Nevertheless, in MEN1, lesions occur both metachronously and synchronously and the clinical use of such data will be restricted by the

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fact that any patient with a MEN1 germline mutation might in the future develop pancreatic, pituitary and adrenal tumors [Calender et al., unpubl. observ.]. Families derived from a common ancestor and sharing common mutations and haplotypes have heterogeneous clinical expression of the disease, an observation which assesses the absence of genotype-phenotype correlation and might suggest an important role of modifier genes and/or environmental factors [37]. From a biological point of view, all truncating mutations, either nonsenses or frameshifts, affect one or both NLSs in the C-terminal region of menin. Curiously, the two NLSs were never affected by missense mutations indicating that these sequences could play a critical role in menin function and metabolism. Based on the mutation data in MEN1 patients, we could suggest two distinct mechanisms impairing the function of MEN1; the first one by deleting NLSs might affect the nuclear localization of menin and/or induce fast degradation pathway(s) of truncated protein. Our recent data indeed show that truncated forms of menin were not detectable by Western blot analysis in MEN1 patients with nonsense mutations [38]. As observed in parathyroid tumors [39], complete loss of a MEN1 allele or short truncation by nonsense mutations did not result in reduced transcription and protein expression levels, suggesting the upregulation of the wild-type allele. The second mechanism might concern the functional properties shared by internal domains of the protein that would be selectively impaired by missense or splice site mutations. In such cases, in vitro functional tests will be needed to assess the mutagenic properties of a specific amino acid substitution and distinguish it from a rare polymorphism. Around 5–10% of MEN1 families do not show germline MEN1 mutations even after full sequence analysis of exonic and intronic sequences. Clinical criteria used for the diagnosis of MEN1 are crucial and one would expect a "95% mutation detection rate when familial MEN1 was defined as a patient with three (or more) first-degree relatives with two (or more) MEN1related major lesions. Most families without MEN1 mutations display an atypical clinical pattern, with one or two lesion(s) affecting the proband and one major or uncommon MEN1-related tumor(s) in the relatives. This might also reflect the genetic heterogeneity of the syndrome or the occurrence of phenocopies which are mainly due to lesions commonly observed in the non-MEN1 population, such as pHPT and prolactinoma [40, 41]. In some MEN1 families, MEN1 mutations could also have been missed. The MEN1 gene structure is not fully known in the 5) region, and promoter(s) and/or regulatory regions

Calender

could be affected by unknown mutations. Chromosomal or intragenic rearrangements such as large germline deletions, either within or encompassing the MEN1 gene, might have been missed by PCR-based sequencing. A MEN1 deletion has recently been suggested in a Japanese pedigree by RFLP-gene dosage analysis and quantitative PCR [42] and demonstrated in a large French MEN1 family using molecular cytogenetic tools [Lespinasse et al., submitted]. Finally, the complexity and diversity of MEN1 mutations show us the need of clinical screening as a prerequisite for molecular diagnosis. In clinical practice, genetic analysis is useful to assess syndromic diagnosis of MEN1, but to date we still do not exclude the diagnosis of MEN1 when the mutation was not found. NET Related to Oncogenic Activation of RET, a Tyrosine-Kinase Membrane Receptor Germline mutations of the RET proto-oncogene encoding a transmembrane tyrosine-kinase (TK) receptor confer predisposition to clinical variants of MEN2, the inherited forms of medullary thyroid carcinoma (MTC) [43, 44]. In MEN2A (Sipple’s syndrome), MTC is associated to pheochromocytoma (30–50%) and primary hyperparathyroidism (10–20%). In MEN2B (Gorlin’s syndrome) the major clinical features are MTC, pheochromocytoma, mucosal neuromas and skeletal abnormalities associated with a marfanoid habitus and ganglioneuromatosis of the gastrointestinal tract [45]. The third variant of MEN2 was defined as familial MTC (FMTC), in which MTC occurs as the sole phenotype in 3 or more patients [46]. FMTC might be considered as MEN2A with low penetrance of pheochromocytoma. C-cell hyperplasia is the earliest lesion observed in hereditary MTC and is characterized by abnormal basal and pentagastrin-stimulated calcitonin values. MTC related to a MEN2 genetic predisposition is multifocal. Pheochromocytomas in MEN2A/B are bilateral in around 70% of cases. Two major issues, malignant evolution of MTC and cardiovascular failures due to latent pheochromocytoma, have to be considered for the prognosis of patients with germline RET mutations. MTCs in MEN2B are more aggressive and appear in young children [47]. In some FMTC, thyroid cancer is expressed only at later age and a long-term follow-up is needed in order to exclude the risk of occurrence of pheochromocytoma in a specific family [48]. The natural history of MEN2 and the risk of malignant disease underscore the need for early management of patients by surgical treatment of MTC and, when present, of pheochromocytoma. The RET gene has been assigned to chromosome 10q11–2. The c-ret protein displays an extracellular cys-


teine-rich homodimerization domain and an intracellular TK catalytic site. The distal part of the extracellular region contains a cadherin-like domain which mediates calciumdependent cell–cell adhesion [49]. The c-ret protein belongs to a multiprotein complex acting as a receptor for four related ligands, glial cell line-derived neurotrophic factor (GDNF), neurturin, artemin and persephrin, each of them acting through specific coreceptors, GFR·-1, 2, 3 and 4, respectively [50]. GFR· coreceptors interact with ligands and induce homodimerization of c-ret through the cysteine-rich region, thereby leading to the catalytic activation of TK domains. GDNF and neurturin promote the survival of a variety of neurons, and GDNF is required for the development of the enteric nervous system and kidney [51, 52]. Intracellular events after ligand binding and dimerization involve cross-phosphorylation of TK domains of dimerized RET and a signal-transducing complex consisting of Shc, Grb2 adapters and the subsequent activation of a Ras-MAP-kinase pathway [53]. RET genomic size is 60 kb and the gene contains 21 exons [54]. It is expressed in many tissues including thyroid, adrenal, neuroendocrine tissues and the developing kidney [55, 56]. The c-ret protein induces the genesis of the peripheral and central nervous system and the renal excretory tract. Nullizygote (knock-out) c-ret –/–, GDNF –/– and GFR·-1 –/– mouse strains that died soon after birth lacked neurons in the whole digestive tract and showed kidney agenesis [57– 59]. The phenotype observed in knock-out models mimicked that of the human Hirschsprung disease characterized by intestinal aganglionosis. RET mutations, including deletions, point mutations and splice-site alterations, have been observed in some autosomal dominant forms of Hirschsprung [60]. These mutations lead to loss of function and suggest that c-ret protein might be critical for differentiation, proliferation and migration of neural crest cells. The protein encoded by RET is highly expressed in human tumors of the neural crest derivatives, such as neuroblastoma, medullary thyroid carcinoma and pheochromocytoma [61]. The RET gene has also been involved in sporadic papillary thyroid carcinoma (PTC) through chromosomal rearrangements producing various types of abnormal RET/PTC fusion proteins [62]. Germline mutations of RET in MEN2 are missenses occurring either in the extracellular cysteine-rich domain or intracellular TK catalytic sites. Missense mutations in codons 609, 610, 611, 618, 620, or 634 located in the extracellular dimerization domain have been detected in 98% of MEN2A and 85% of FMTC patients [63]. The most common mutations observed in MEN2A affected codon 634, either Cys634Arg, C634Tyr and Cys634Gly


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occurring in 80–90% of patients. Codon 634 mutations, whatever the amino acid substitution may be, has been associated with a high risk of pheochromocytoma, and for Cys634Arg, of hyperparathyroidism [64, 65]. MEN2Aassociated mutations induce the formation of active RET dimers in the absence of ligand [66]. Transgenic mice expressing a Cys634Arg mutant RET allele under the control of the calcitonin gene-related protein promoter developed multifocal and bilateral MTC, as observed in humans [67]. Transgenic animals do not display pheochromocytoma. These findings provide evidence that the MEN2A mutant form of RET protein is oncogenic in parafollicular C cells. Mutations in exon 10 (codons 609, 611, 618 and 620) have been identified in 10–15% of MEN2A families and mostly those with low penetrance of pheochromocytoma and hyperparathyroidism. Rare mutations, such as 9 bp in exon 8 [68, 69] and 12 bp [70] duplications creating additional cysteine residue in exon 11, have been described in some MEN2A families. New hot spots for FMTC/MEN2A might be observed in exon 13 at codons 790 and 791 inside the intracellular TK domain. [71]. Both Leu790Phe and Tyr791Phe have been observed in large families with very low penetrance of pheochromocytoma. The molecular pathological mechanism leading to MTC must be related to a structural change in the catalytic site leading to constitutive activation and/or inappropriate binding to substrates of the intracellular signalling pathway. In MEN2B, a unique point mutation of codon 918 (exon 16) affecting the TK domain has been identified in more than 98% of patients [72]. In all cases, this mutation results in a methionine to threonine conversion (Met918Thr) and has been associated with an aggressive course of MTC. The MEN2B mutation switches the substrate specificity of the RET TK domain towards intracellular substrates, thus inducing an abnormal signalling pathway [66, 73]. More rarely, MEN2B families shared a missense mutation at codon 883 and might account for the remaining 0–5% MEN2B cases without mutations at codon 918 [74, 75]. In FMTC, mutations in exons 10 and 11 at codons 609, 611, 618, 620, and 630 are found in 60–70% of the cases [64, 65]. The latter mutations have similar pathogenic effects to those occurring at codon 634 by inducing a constitutive dimerization/activation of RET activity. Mutations in exons 13 (codons 768, 790 and 791), exon 14 (codon 804) and exon 15 (codon 891) have been described in approximately 10–20% of FMTC, depending on the series [76–80]. Exons 13–15 include the TK receptor catalytic domain and mutations and might alter both the substrate specificity and/or the TK region.

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Germline RET mutations were found in 3–5% of sporadic cases of MTC [81, 82] and represent mostly patients for whom clinical, histopathological and genetic information was insufficient at the initial screening. We have to point out that FMTC families are MEN2A with very low penetrance of pheochromocytoma, and one might suggest simplifying the classification of MEN2 into only MEN2A and MEN2B. In 80% of sporadic MTC, the Met918Thr mutation (exon 16) was found in tumoral DNA and mainly when the tumor had been microdissected [83]. When detected in tumors, the MEN2B-specific mutation might be related to the poor prognosis of MTC. In sporadic pheochromocytoma, point mutations or small deletions have been detected either at exons 9, 10, 11 or 16 in 10–20% of the tumors [84]. No RET mutations have been identified in tumoral DNA of sporadic parathyroid hyperplasia/tumors [85, 86] and digestive NETs [87]. Mutations occur de novo in 5–10% of MEN2A and 50% of MEN2B [65]. In both variants, new mutations derive mostly from the paternal allele [88, 89]. In terms of clinical use of RET screening, we might consider that direct screening of exons 8, 10, 11, 13, 14, 15, and 16 either by direct sequencing, SSCP, PCR restriction or heteroduplex techniques are now useful tools for an accurate presymptomatic diagnosis in FMTC/ MENA/MEN2B families. Genetic diagnosis in sporadic forms remain useful mainly in young patients (^50 years) and when the tumors occur bilaterally. Figure 2 shows the position of most MEN2-related RET mutations in the frame of major functional domains of the protein. Even if the risk of pheochromocytoma seemed higher with specific mutations in the RET sequence, all patients with isolated MTC must be considered as a potential MEN2A and the diagnosis of pheochromocytoma excluded. Retrospective data on 274 MEN2A cases registered in European countries have shown that pheochromocytoma occurred 2–11 years subsequent to MTC in more than 40% of these patients [90]. Predisposition for NET Related to Mutations in VHL, NF-1, and TSC1-2 Genes VHL disease is an autosomal dominant disease predisposing to renal cancers, retinal and/or cerebellar hemangioblastoma, pheochromocytoma and cystic and/or endocrine pancreatic tumors [91]. VHL-related pancreatic tumors are mostly nonfunctional cystadenoma, but 10–15% of patients with VHL could be affected by NETs derived from islet or ductal endocrine cells [92–94]. The VHL gene was cloned in 1993 on chromosome 3p35-26. [95].

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Fig. 2. Schematic overview of the RET proto-oncogene germline mutations in MEN2 and relationship with functional domains. The arrows designate the main mutations related to MEN2A and MEN2B. The figure was based on data published to date and compiled in the text.

The VHL protein interacts with the elongin family of proteins involved as regulators of transcriptional elongation [96, 97]. Other functions involving the VHL protein have been found related to the hypoxia-induced cell regulation and extracellular matrix fibronectin expression and localization which might account for new tumorigenic pathways related to VHL mutations. Histopathological data suggested that VHL-related dNET have typical neuroendocrine characteristics, most are multiple and nonfunctional and 30–40% of them demonstrate focal positivity for pancreatic polypeptide, somatostatin, glucagon and/or insulin [98]. VHL-related NET might be distinguished by: (1) the absence of primitive duodenal tumors, the tumors being restricted to the pancreas; (2) frequent nonfunctional tumors and somatostinoma; (3) a clear-cell morphology related to intracytoplasmic lipid and myelin accumulation, and (4) the frequent occurrence of microcystic adenoma around clear-cell lesions. These criteria could be helpful to decide if either the MEN1 or VHL gene must be tested in a patient with sporadic pancreatic endocrine tumors, when a familial history is suspected. In any case, the VHL gene is involved in pancreatic NET pathogenesis. Somatic mutations and allelic deletions of VHL have been observed in a significant proportion of VHL-dNET suggesting that genetic changes in the VHL gene play an important role in the genesis of both sporadic and VHL disease-associated pancreatic NET and microcytic adenoma [99]. Digestive NETs have been described in rare patients with NF1 and TSC [100–104]. Clinical features

of NF1- and TSC-related dNET include multiple tumors in the pancreas and/or duodenum with psammomatous glandular histological features and a common immunohistochemical expression of somatostatin and/or insulin. The NF1 (chromosome 17) and both TSC1 (chromosome 9) and TSC2 (chromosome 16) genes are involved in the membrane signal transduction pathway through the Gprotein-mediated activation signal of the intracellular cascade. Neurofibromin, the product of NF1, acts as a negative regulator of ras-related G proteins by disrupting the GTP-ras complex [105]. TSC2 encodes tuberin, a GTPaseactivating protein interacting with rab-5, an endosomal rap-1-related small G protein [106]. Hamartin, the product of TCS1, shows no homology to tuberin, but could interact with the TSC2 product by potential coiled-coiled domains [107, 108]. Both proteins colocalized in cytoplasm and all experimental evidence including the results of two hybrid systems suggest that TSC1- and TSC2-encoded proteins interact in a common pathway related to cytosolic small G protein-negative regulation [109].



Towards an Integrated Overview of Initiation and Progression of NETs

Major Genetic Pathways Involved in NET Initiation To find a common pathway deregulated in all subtypes of NETs will remain a challenge. At least four major mechanisms are involved based on previous data related

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to the genetic predisposition to NETs. Deregulation of mitotic and/or transcription factor activity, TK membrane signalling, transcription elongation and/or angiogenesis and small G-protein pathways have been involved as basic mechanisms of tumorigenesis. Nevertheless, MEN1 remains the most common form of genetic predisposition to NETs, and one would expect that disruption of menin function is a critical event leading to neuroendocrine cell proliferation. Tumorigenesis in MEN1 involved loss of function of menin, a growth suppressor according to the two-hit model by Knudson [8]. Nevertheless, several clinical and pathological observations suggest that the first mutation could induce an abnormal cellular state increasing the risk of chromosomal abnormalities and LOH. First, most of the endocrine tissues affected in MEN1 show hyperplasia and adenoma, or highly proliferative tumors occur later in life as monoclonal proliferations derived from a single ancestor cell [110]. Recent studies on gastrinomas have assessed the fact a monoclonal tumor derived from hyperplastic tissues was further responsible for multifocality and extrapancreatic dissemination [111]. In the same way, 11q13 allelic losses have not been found in secondary hyperparathyroidism related to renal failure or other pathogenic conditions with a hypercalcemic state, suggesting that LOH observed in MEN1 might be related to a direct pathogenic influence of MEN1 mutations on cell growth regulation [112]. Secondly, lymphoblastoid or fibroblastic cell lines derived from MEN1 patients show a low rate of chromosomal instability [113]. Alterations include abnormal rings, deletions, inversions, translocations and numeric aberrations suggesting that MEN1 belongs to the group of diseases with chromosomal instability, such as Fanconi anemia or ataxia-telangiectasia. Recent data showed in vitro evidence of premature centromere division in growing fibroblasts from MEN1 patients [114]. Thirdly, patients with MEN1 shared high rates of circulating basic fibroblast growth factor (bFGF)-like factors in the sera of MEN1 patients [115]. Recently, it was suggested that this bFGFlike activity is due to the presence of anti-bFGF-circulating autoantibodies [116]. The decrease in bFGF activity after surgical therapy of pituitary adenoma suggested that this mitogenic factor could be produced by the hyperplasic or tumoral pituitary gland, but these data have not been confirmed to date. Lastly, MEN1 also predisposed to nonendocrine tumors cosegregating with MEN1 in some families. This suggests that an alteration in menin function increases the relative risk or susceptibility to various cancers, and an exciting hypothesis might be proposed on a relation between MEN1 gene inactivation and an in-

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creased sensitivity to environmental mutagenic factors. Menin, as a partner of JunD, acts inside AP1 which has been involved in many cellular pathways in normal and stressed conditions [20]. Briefly, AP1-related regulation was involved in transcription activation or repression, apoptosis, response to external stress, mitosis and response to endogenous or exogenous growth factors. One might expect that specific target genes were involved in endocrine cells or that the menin-JunD interaction involved tissue-specific proteins acting as a coactivator or corepressor. A paradoxical observation is that JunD overexpression suppresses cell growth in NIH3T3 cells and that antisense JunD induces a mitogenic response. This suggests that JunD might be a negative regulator in AP1 and that menin, a growth-suppressor protein, binds another growth suppressor. This striking paradox assessed the fact that new partners for menin remain to be discovered and that MEN1 pathogenesis could involve several pathways other than JunD-AP1. In MEN2-related lesions, all RET mutations have been found to induce a ligand-independent constitutive activation of the c-ret protein activity [66]. Tumorigenic activity of MEN2-associated RET mutations have been confirmed in animal models, and transgenic mice expressing a MEN2A allele under the control of the calcitonin generelated protein promoter developed C-cell hyperplasia and MTC, whereas mice expressing a MEN2B allele under the dopamine ß-hydroxylase promoter expressed ganglioneuromas and renal abnormalities [67, 117]. MEN2 is a unique model in human inherited cancers characterized by genetic predisposition related to a dominantly acting oncogene. C-ret activation, either by natural ligands or MEN2 mutations, induces autophosphorylation of Tyr-905, Tyr-1015, Tyr-1062 and Tyr-1096 in the C-terminal part of the c-ret protein, which act as docking sites for Grb7/10/14, phospholipase C¯, Shc and Grb2 transduction factors, respectively, and further signalling cascades through Ras and Jun/Src kinases pathways [118–121]. Recently, it has been shown that RETMEN2A carrying a Cys634Arg (exon 11) mutation activates phosphatidyl 3-kinase (PI3K), a crucial transducer controlling mitogenesis and apoptosis [122]. Activation of PI3K activates its downstream effector, the serine/threonine AKT/protein kinase B and mutation of RET-Tyr1062, which abolishes the RET-MEN2A-transforming capacity, abrogates the RET-MEN2A-mediated PI3K activation. RET-MEN2B has also been shown to be a powerful activator of PI3K, and taken together, these data suggest that deregulation of the PI3K/AKT-related signalling pathway might be a crucial event in neural crest cell-

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derived tissue tumorigenesis and the specific clinical features observed in MEN2 syndromes [122, 123]. Again, as for MEN1, one might suggest that C-cell hyperplasia and tumoral growth in MEN2-related lesions result as a combination of at least two deregulated pathways, one activating mitosis and the second decreasing the natural process of programmed cell death. Genotype-phenotype correlations in MEN2 were clearly demonstrated for FMTC, MEN2A and MEN2B. Various RET mutations related to these variants have been shown to share different transforming capacities which correlate positively with agressivity of the phenotype [124, 125]. The low incidence of pheochromocytomas with mutations in exons 10, 13 and 14 might be explained by a tissue-specific sensitivity to RET-mediated oncogenic activation and/or variable expression of the mutated protein at the cell surface. Strinkingly, a Cys620Ser (exon 10) mutation has been observed in a family with both MEN2A and Hirschsprung disease, the latter being commonly related to inactivating mutations of RET [126]. This suggests that mutations at specific residues of TK receptor might have have both an oncogenic and a dominant-negative effect related to tissue-specific transducers of downstream signalling pathways. The relations between VHL protein and NET remain unclear. Tumorigenesis in VHL was first related to the putative role of VHL protein in the elongin complex, a trimeric transcription elongation factor [96, 97]. Such a mechanism has not been shown to be responsible for VHL disease in humans and recent functional data suggested that VHL pathogenesis could be related to the deregulation of specific growth factor expression. VHL localizes predominantly in the cytoplasm and is found also in nucleus, and a transcription-dependent nuclear-cytoplasmic trafficking is required for the normal function of VHL protein [127]. VHL protein decreased TGF-· mRNA stability [128] and repressed vasculoendothelial growth factor transcription through interaction with the Sp1 transcription factor [129]. High expression of TGF-· has been involved in renal cell cancers, a common lesion observed in VHL syndrome [130]. Negative regulation of angiogenic peptides by the VHL protein occurs under normoxic conditions and recent data have shown that VHL protein function could mainly be linked to the regulation of hypoxia-inducible mRNAs [131]. This process requires specific binding of VHL protein to molecular complexes containing elongin subunits B and C, and Cul2, a cullin exhibiting significant sequence similarity to the yeast Cdc53 protein and involved ubiquitin-dependent proteolysis [132]. Recently, it has been shown that the VHL

complex also includes Rbx1, an evolutionary conserved protein which could act as a potent activator of ubiquitin ligase, an enzyme required for ubiquitination of cyclindependent kinase inhibitors and thus involved in G1 to S cell cycle transition [133]. In conclusion, deregulation of various pathways related to growth factor activities and response to hypoxia and abnormal peritumoral angiogenesis might explain the clinical and histopathological patterns of VHL disease.



From Benign NET to Malignant Tumors Most cancers result from multistep scenarios and a major issue is now to understand the genetic events needed after MEN1, RET, VHL inactivation for the progression towards a fully malignant phenotype. Approximately 10–40% of dNET associated to MEN1 and 90– 100% of MEN2-related MTC affect the prognosis of patients upon malignant evolution. Major pathways involved in neuroendocrine tumor initiation and progression were summarized in figure 3. The p53-suppressor gene was first analyzed in a series of lung NETs, but somatic mutations have only been found in late stages of large-cell neuroendocrine and small-cell carcinoma and correlate with a high expression of the p53 protein [134, 135]. p53 mutations have never been found in either typical or atypical lung carcinoids. Nevertheless, an abnormal expression of p53 protein has been detected by immunohistochemistry in typical carcinoids of the lung and pancreatic endocrine tumors and cell lines, and the same authors observed an intracellular clustering suggesting a physiological response to the tumoral process and/or abnormal p53 processing induced by the primary tumoral event [136]. Ki-RAS, an oncogene mutated in pancreatic adenocarcinoma, shows activation or mutations in digestive carcinoid tumors [137]. Identification of p53 and/or Ki-RAS mutations in an atypical form of pancreatic lesion might be of clinical interest in the future for the clinicians helming them for differential diagnosis between an adenocarcinoma and a NET. Mutations of other oncogenes, such as HER2/NEU, C-MYC, N-MYC, N-RAS and C-JUN, have not been found in NET, but these genes were shown to be overexpressed in cell lines derived from endocrine tumors and in Ad-virus-E1A-transgenic mice presenting small intestine carcinoids [138–140]. Amplification of HER/NEU correlated with cytogenetic amplification of corresponding loci on chromosome 17q11–21 as detected by FISH and comparative genomic hybridization on NET [141]. C-myc, bcl-2, c-erb B-2, and c-jun are frequently expressed in human digestive dNETs and abnormal expression of such oncogenes might be involved

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Fig. 3. From initiation to metastatic cancer.

The figure summarizes the major events involved in tumor initiation, progression and pathogenic mechanisms involved in metastasis. MEN1 = Multiple endocrine neoplasia type 1; VHL = von Hippel-Lindau genes; NF1 = neurofibromatosis type 1 (neurofibromin); TSC1 and 2 = tuberous sclerosis genes; NGF = nerve growth factor; TGF = transforming growth factor; bFGF = basic fibroblast growth factor; VEGF = vasculoendothelial growth factor; FIHT = fragile histidine triad; PRAD-1 = parathyroid adenomarelated proteins.

in some steps of tumor progression [142]. Lastly, activating mutations of the ·-subunit of G protein (gsp oncogene) which are associated with the McCune-Albright syndrome, another mutliple endocrine disease, have not been found in endocrine tumors of the parathyroids, pancreas and pituitary gland [143, 144]. Overexpression of PRAD1 oncogene, encoding D-cyclin, has been observed in a subset of parathyroid tumors [145, 146]. In "20% of sporadic parathyroid adenomas, this deregulation results from a pericentric inv11(q13;p15) leading to the juxtaposition of the PRAD1 gene, initially located on 11q13, close to the PTH gene promoter, on 11p15. Nevertheless, no data have been reported to date on a putative relationship between PRAD1 deregulation and dNET pathogenesis. Allotyping search for specific LOH in malignant NET might be fruitful to find genes involved in tumoral progression and metastasis. Previous reports have shown significant allelic deletion on chromosomes 1p and 1q. Using a set of 1p and 1q microsatellite markers, Ebrahimi et al. [147] characterized 1p/q LOH in 10/29 dNET analyzed, and most tumors with allelic loss were aggressive lesions with hepatic metastases. The 11q13 region has also been shown to be frequently deleted in many tumors. One might suggest that even if MEN1 gene inactivation is a major process in tumor initiation, other loci localized in 11q13 could have a relevant role in progression and malignant evolution [148]. A series of 43 sporadic dNETs has been analyzed with polymorphic markers flanking the VHL gene on chromosome 3p25–26 and 33% of cases

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show clonal allelic deletion in a minimal interval centromeric to VHL [149–152]. No statistical correlation was observed between 3p loss and functional/nonfunctional types of tumors and hormonal secretion. However, allelic loss at 3p25 correlated with more clinically advanced disease, i.e. duodenal invasion, lymph node involvement and hepatic metastases. The authors suggest that 3p25 loss could be useful to identify genes involved in malignant progression and a useful marker to distinguish benign from potentially malignant dNETs. Deletions of the short arm of chromosome 3 have been related to specific genes and putative candidates, such as the fragile histidine triad which encodes a tetraphosphatase regulating DNA replication and is frequently altered in small cell lung carcinoma [153]. The proto-oncogene RON and its ligand, MSP, both implicated in the embryogenetic development of neuroendocrine derivatives, are also localized to 3p21–22 [154]. RON can acquire oncogenic potential by means of two amino acid substitutions, D1232V and M1254T, affecting highly conserved residues in the TK domain [155]. Both mutations caused RON-mediated transformation of 3T3 fibroblasts and tumor formation in nude mice. Loss of the VHL gene might also be a critical step in MEN1-related or sporadic NETs: fibronectin, an extracellular matrix component, coimmunoprecipitated with wild-type VHL protein (pVHL) but not with tumorderived pVHL mutants [156]. In vitro assembly of an extracellular fibronectin matrix by VHL–/– renal carcinoma cells was defective compared with VHL+/+ cells, and

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reintroduction of wild-type pVHL partially corrected this defect [157]. These data support a direct role of pVHL in fibronectin matrix assembly and one would expect that loss of both alleles of the VHL gene during progression of any dNET is a major event for malignant evolution and metastatic dissemination of tumors. Recent investigations have revealed a small interval of frequent deletions in 3q27 [151]. Other chromosomal regions including 11q, 3q, 16p and 22q are frequently deleted in sporadic dNETs and all these loci have been found to contain candidate genes, as MEN1 on 11q, TSC2 on 16p13 and NF2 on 22q12 [150, 157]. Strikingly, 3p-, 3q- and 22-q deleted regions were characterized as syntenic regions with loci on mouse chromosomes 9 and 16 that are deleted in insulinoma induced in a murine transgenic model using the rat insulin promoter-SV40 T antigen as a transgene [158]. Taken together, these data indicate that further fine deletion mapping of loci involved in LOH will help the characterization of candidate genes involved in different steps of dNET tumorigenesis. In a serial analysis of 12 gastrinomas and nonfunctional dNET, Muscarella et al. [159] showed that the p16/MTS1 gene on chromosome 9p21 was homozygously deleted in 42% of tumors and methylated on 5)CpG island in 58% of cases. Nevertheless, the 9p21 is not frequently involved in LOH and the authors failed to find any mutations within the sequence of p16/ MTS1. This gene codes for a negative regulatory protein acting at the G1 1 S cell cycle junction and frequent inactivation of p16/MTS1 either by deletion, intragenic mutations and aberrant mutations have been shown in 690% of exocrine pancreatic tumors and 30–40% of breast and colon cancers [160, 161]. Malignant progression of endocrine tumors could also be induced by overexpression of growth factors such as TGF·, EGF and NGF, which have been shown to stimulate growth of neuroendocrine cell lines in vitro [162, 163]. Similar effects have been shown on melanoma, parathyroid and pancreatic endocrine cell lines with VEGF and VEGF-related factors [164, 165]. Abnormal expression of different isoforms of adhesion molecules such as CD44 [166] and/or neural cell adhesion molecules [167] and/or E-cadherin [168] could account in the progression and/or malignant evolution of endocrine tumors.

Conclusions and Perspectives

In the last few years, a significant amount of clinical and fundamental data have been obtained on the physiopathogeny of NETs. Identification of the genes involved


in genetic predisposition to various types of NET allowed the characterization of new pathways involved in tumorigenesis and the individual’s risk of MEN1- and MEN2related tumors can now be assessed by simple molecular techniques available for a large number of patients. Biochemical and clinical screening programs can now be established in mutant gene carriers, optimizing the therapeutic management of patients before metabolic and/or malignant complications occur. Further issues on functional domains of menin and partners other than JunD binding the MEN1 gene-encoded protein will probably represent the most critical issue in a near future. In MEN2, new genetic insights described in this review so far represent so far one of the more demonstrative models in human inherited cancers. The molecular pathways involved in tumor initiation and progression are known and a new challenge might be the initiation of therapeutic protocols using tissue-specific gene therapy and/or modulation of TK activity by specific chemical agents. Nevertheless, preclinical identification of germline mutations in young MEN2 patients lead to prophylactic surgery and definitive recovery of the disease. Involvement of the VHL gene in NET pathogenesis helped us to understand the relations between malignancy, the regulation of peritumoral angiogenesis and the role of the extracellular matrix in metastatic dissemination of NETs. Other genes remain to be discovered, and taken together NETs, as frequently observed with uncommon and inherited forms of cancer, represent a powerful model of tumorigenesis giving us an integrated overview of what can occur as an initial event in a normal cell, leading towards a fully malignant phenotype.

Acknowledgements This review represents a compilation of bibliographic data and experimental/clinical observations obtained in the frame of GENEM (Groupe d’Etude des Néoplasies Endocriniennes Multiples), GRESZE (Groupe d’Etude du Syndrome de Zollinger-Ellison) and GETC (Groupe d’Etude des Tumeurs à Calcitonine) in France. Genetic work on MEN1 and MEN2 was supported by funds from Hospices Civils de Lyon (PHRC 97-048). Specific studies on genetic relations between MEN1 and central nervous system tumors were supported by the Association pour la Recherche contre le Cancer (ARC No. 9211). New projects related to fundamental topics on MEN1 and atypical forms of inherited NET were recently supported by ‘Comité de la Loire – Ligue national contre le Cancer’. We thank the MEN1 patients and families and our colleagues of the GENEM, GRESZE and GETC networks.


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147 Ebrahimi SA, Wang EH, Wu A, Schreck RR, Passaro E Jr, Sawicki MP: Deletion of chromosome 1 predicts prognosis in pancreatic endocrine tumors. Cancer Res 1999;59:311– 315. 148 Chakrabarti R, Srivatsan ES, Wood TF, Eubanks PJ, Ebrahimi SA, Gatti RA, Passaro E Jr, Sawicki MP: Deletion mapping of endocrine tumors localizes a second tumor suppressor gene on chromosome band 11q13. Genes Chromosomes Cancer 1998;22:130– 137. 149 Chung DC, Smith AP, Louis DN, GraemeCook F, Warshaw AL, Arnold J: A novel pancreatic endocrine tumor suppressor gene locus on chromosome 3p with clinical prognostic implications. J Clin Invest 1997;100:404– 410. 150 Hessman O, Lindberg D, Einarsson A, Lillhager P, Carling T, Grimelius L, Eriksson B, Akerstrom G, Westin G, Skogseid B. Genetic alterations on 3p, 11q13, and 18q in non familial and MEN1-associated pancreatic endocrine tumors. Genes Chromosomes Cancer 1999;26:258–264. 151 Chung DC, Brown SB, Graeme-Cooke F, Tillotson LG, Warshaw AL, Jensen RT, Arnold A: Localization of putative tumor suppressor loci by genome-wide allelotyping in human pancreatic endocrine tumors. Cancer Res 1998;58:3706–3711. 152 Nikiforova MN, Nikiforov YE, Gnepp DR, Gnepp DR, Grosembacher LA, Wajchenberg BL, Fagin JA, Cohen RM: Frequent loss of heterozygosity at chromosome 3p14.2–3p21 in human pancreatic islet cell tumors. Clin Endocrinol 1999;51:27–34. 153 Sozzi G, Veronese ML, Negrini M, et al: The FIHT gene 3p14.2 is abnormal in lung cancer. Cell 1996;85:587–597. 154 Gaudino G, Avantaggiato V, Follenzi A, Acampora D, Simeone A, Comoglio RM: The proto-oncogene RON is involved in development of epithelial, bone and neuro-endocrine tissues. Oncogene 1995;11:2627–2637. 155 Santoro MM, Penengo L, Minetto M, Orecchia S, Cilli M, Gaudino G: Point mutations in the tyrosine kinase domain release the oncogenic and metastatic potential of the Ron receptor. Oncogene 1998;17:741–749. 156 Okh M, Yauch RL, Lonergan KM, Whaley JM, Stemmer-Rachamimov AO, Louis DN, Gavin BJ, Kley N, Kaelin WG Jr, Iliopoulos O: The von Hippel-Lindau tumor suppressor protein is required for proper assembly of an extracellular fibronectin matrix. Mol Cell 1998;7:959–968. 157 Onuki N, Wistuba H, Travis WD, Virmani AK, Yashima K, Brambilla E, Hasleton P, Gazda AF: Genetic changes in the spectrum of neuroendocrine lung tumors. Cancer Res 1999;85:600–607.


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Calender


Biological and Molecular Aspects of Gastroenteropancreatic Neuroendocrine Tumors G. Rindi V. Villanacci A. Ubiali Department of Pathology, University of Brescia, Spedali Civili, Brescia, Italy

Key Words Pancreas W Gut W Endocrine cells W Hormones W Immunohistochemistry W Electron microscopy W Molecular genetics W Loss of heterozygosity

Abstract Neuroendocrine tumors of the digestive tract are rare entities characterized by significant phenotype differences and traditionally considered to originate from cells of the diffuse endocrine system of the pancreas and gut. Two major categories with significant phenotype and clinical behavior differences are identified as well-differentiated and poorly differentiated tumors. Investigation on the molecular basis of tumor development points to an important role for the multiple endocrine neoplasia syndrome type-1 (MEN1) gene because of its frequent abnormality observed both in well-differentiated and poorly differentiated tumors. Other genes are possibly involved, though the available data need support from studies on larger series of tumors.

Origin

The pancreas and the mucosa of the gastrointestinal tract contain as many as 15 cell types characterized by the production of hormonal peptides or biogenic amines (table 1) [1, 2]. Together with the endocrine cells scattered in other endodermal sites such as the thyroid, lung, biliary tree and urethra, the endocrine cells of the gastroenteropancreatic tract belong to the so-called diffuse endocrine system (DES). Due to the fact that DES cells share a number of antigens with nerve elements, the term neuroendocrine is also widely used to connote such cell types and will be adopted here. It is generally accepted that transformation of DES cells may result in the development of neuroendocrine tumors. This view is supported by the experimental evidence of endocrine tumor development in transgenic mice expressing potent oncoprotein in endocrine cells of the pancreas and gut [3–6].

Diagnostic Approach

Copyright © 2000 S. Karger AG, Basel

Supported by grants from MURST (COFIN 99) and the University of Brescia.

ABC

© 2000 S. Karger AG, Basel 0012–2823/00/0625–0019$17.50/0



Neuroendocrine tumors of the gastroenteropancreatic tract are made up of tumor cells variably retaining the antigenic profile of the normal DES counterparts. The assessment of endocrine differentiation in tumors is ob-

Guido Rindi, MD, PhD Anatomia Patologica 2 Piazza Spedali Civili, 1, I–25124 Brescia (Italy) Tel. +39 030 3995 830, Fax +39 030 3995 053 E-Mail [email protected]

Table 1. Characteristics of the endocrine cells of the human gastroenteropancreatic tract

Cell

P EC D L A PP B X ECL G CCK S GIP M N

Main product

Unknown 5HT* Somatostatin GLI/pYY Glucagon PP Insulin Unknown Histamine Gastrin CCK Secretin GIP Motilin Neurotensin

Stain

Pa

Grimelius, Chg A Grimelius,PbH, Chg A, Syn Davenport, PbH, Syn PbH, Grimelius, Chg C 1 A, Syn Grimelius, Chg A 1 C, Syn Grimelius, Chg C, A, Syn Aldehyde fuchsin, Syn PbH, Grimelius Grimelius, Chg A, Syn Grimelius, Chg A, Syn

Stomach

Intestine

CF

small

f f +

+ + +

+ + +

a

An

+ + +

Ap

D

J

I

+ + + f

f + f +

f + f +

+ + +

large C

R

f + f +

f + + +

a + + +

+ + + + + f

Grimelius, Chg A

Grimelius, Chg A

+ + + + +

f f f +

Pa = Pancreas; CF = corpus fundus; An = antrum; D = duodenum; J = jejunum; I = ileum; Ap = appendix; C = colon; R = rectum; 5-HT = 5-hydroxytryptamine; + = presence of cells; f = presence of a few cells; a = presence of cells in fetus and newborn; EC = enterochromaffin; GIP = gastric inhibitory polypeptide; GLI = glucagon-like immunoreactants (glicentin, glucagon-37, glucagon-29); pYY = PP-like peptide with N-terminal tyrosine amide; PP = pancreatic polypeptide; ECL = enterochromaffin-like cell; CCK = cholecystokinin; * = substance P, neurokinins, opioids, guanylin and other peptides; PbH = lead-hematoxylin; Grimelius, Davenport = silver stains; Chg = immunoreactivity for chromogranin; 1 = heavier staining than; Syn = immunoreactivity for synaptophysin. From Rindi et al. [2].

Table 2. Cell types, most frequent sites and possible hyperfunctional syndromes of neuroendocrine tumors of the gastroenteropancreatic

tract Tumor type

Main cell type

Pa

Stomach

Intestine

CF

small

An

D Welldifferentiated

Poorly differentiated

B A PP D EC ECL G L

+ + + + +

s/i cells

+

+ +

+

+ +

+

+

Syndrome Ap

J

I

large C

R

+ +

+

+

+

+

+

+ +

+ +

+ +

+

+

+

+

+

+

+

+

PHH glucagonoma – somatostatinoma ‘carcinoid’ ‘atypical carcinoid’ ZES –

Pa = Pancreas; CF = corpus fundus; An = antrum; D = duodenum; J = jejunum; I = ileum; Ap = appendix; C = colon; R = rectum; + = presence of tumor; EC = enterochromaffin cell; ECL = enterochromaffin-like cell; PHH = persistent hyperinsulinemic hypoglycemia; ZES = Zollinger-Ellison syndrome; – = not defined; s/i = small-intermediate cells. See table 1 for main hormone product of different cell types. From Rindi et al. [2].

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Rindi/Villanacci/Ubiali

a

b Fig. 1. a Lymph node metastasis from a well-differentiated neuroendocrine carcinoma. Variable chromogranin A immunoreactivity in scattered cells. b Poorly differentiated neuroendocrine carcinoma of the duodenal ampulla.

Diffuse nuclear Ki67 immunoreactivity. Immunoperoxidase; ABC method; hematoxylin counterstain.

tained using several techniques traditionally including silver impregnation methods and electron microscopy. Such techniques are now rarely applied because of the high degree of reproducibility and the relatively low cost of immunohistochemical methods. In pathology, the diagnosis of a neuroendocrine tumor first relies on the positive assessment of general markers of neuroendocrine differentiation by immunohistochemistry [7]. Widely used are either cytosol markers such as neuron-specific enolase [8] and protein gene product 9.5 [9], or granule markers such as chromogranin A and related fragments (associated with large, dense-core vesicles) [10, 11] and synaptophysin (associated with small synaptic-like vesicles) [12–14]. Recently the identification of two isoforms of the ATP-dependent vesicular monoamine transporter (VMAT1 and VMAT2) in large, dense-core granules provided new positive tools for the diagnosis of gastric enterochromaffin-like (ECL) cell tumors [15–18]. The cell-specific characterization of neuroendocrine tumors requires hormone immunohistochemistry. In general, it is good practice to search first for the hormones expressed by the DES cells of the anatomical district where the tumor is found (table 2). The search for other hormones is justified if ‘inappropriate’ peptides or amines are reported as frequently expressed by a given tumor (e.g. gastrin in pancreatic tumors), or upon specific clinical settings (e.g. hyperfunctional syndromes).

Pathobiology of Digestive Neuroendocrine Tumors

Differentiation, Histology and Behavior

According, to the recent World Health Organization (WHO) classification, neuroendocrine tumors of the gastroenteropancreatic tract are classified as well-differentiated and poorly differentiated [19]. In general, well-differentiated tumors diffusely and intensely express the markers of neuroendocrine differentiation described above, and in particular chromogranin A (fig. 1a). Poorly differentiated tumors, on the contrary, express cytosol neuroendocrine markers together with synaptophysin, while chromogranin A may be found in rare, if any, tumor cells. Besides marker expression, significant differences between well- and poorly differentiated tumors are also appreciated at histology. Well-differentiated tumors in general display a bland histology characterized by trabecular structure, sometimes with glandular or solid areas, tumor cell monomorphism with absent or low cytological atypia and low mitotic and proliferative status as assessed for instance by nuclear Ki67 expression [1]. Poorly differentiated tumors are characterized by a prevalent solid structure with abundant necrosis, frequently central or diffuse, a high degree of cellular atypia, high mitotic index and high proliferative status by Ki67 (fig. 1b) [1]. The behavior of well-differentiated tumors may be rather unpredictable. Some degree of malignant potential


21

has to be taken in account for several types of well-differentiated neuroendocrine tumors. Notably, well-differentiated tumors are by far the largest fraction of neuroendocrine tumors of the digestive tract. However, only in the presence of proven malignant behavior, i.e. synchronous metastasis and/or invasiveness, the definition of well-differentiated neuroendocrine carcinoma or malignant carcinoid is suggested [19]. On the contrary, poorly differentiated neuroendocrine tumors are invariably malignant and for this reason are defined as poorly differentiated neuroendocrine carcinomas (PDECs). As can be seen in table 2, it is clear that well-differentiated neuroendocrine tumors of the gastroenteropancreatic tract may be significantly different with regard to the tumor cell type. However, cell typing does not provide information regarding tumor behavior unless associated with an overt clinical syndrome due to hormonal hypersecretion. Neuroendocrine tumors associated with hyperfunctional syndromes are currently defined as ‘functioning’. Neuroendocrine tumors, however, are most often not associated with any specific hyperfunctional symptom [1]. In general, tumor behavior strongly correlates with the tumor grading at histology. However, comprehensive histological tumor grading for neuroendocrine tumors of the digestive tract is missing, though it has tentatively been developed for pancreatic and gastric growths [20, 21]. For these tumors several clinicopathological parameters were also demonstrated to be useful as behavior-predicting variables [20, 21]. On the basis of such evidence, it is advisable to carefully evaluate the following variables of digestive neuroendocrine tumors: tumor size (larger tumors are more aggressive); invasion of nearby tissue (pancreas or appendix) or wall invasion beyond the submucosa; angioinvasion and invasion of perineural spaces; solid structure; necrosis; overt cell atypia; more than two mitoses in 10 microscopic high-power fields; Ki67 index of more than 100/10 high-power fields or more than 2%; loss of chromogranin A immunoreactivity, argyrophilia or hormone expression, and nuclear p53 protein accumulation. In general, the above variables should be considered altogether to assign a tentative risk class to the specific case under study. The predictive value of such parameters, however, still requires experimental proof for tumors other than those of the pancreas and the stomach.

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Genetic Background and Mechanism of Transformation

The molecular mechanism underlying the endocrine tumor development is not well understood. Indeed data have recently been accumulating; however, they often refer to tumors not well characterized in terms of tumor cell type. In the following section some information, available from the literature, is given and organized as much as possible on an anatomical and cell-type basis. Pancreatic Tumors Of particular interest are the genotype data obtained for well-differentiated tumors of the pancreas, a tumor subgroup for which extensive and relatively consistent phenotype data are present in the literature. Several genes with assessed or putative tumor-suppressor function have been found to be abnormal. Allelic deletion of the recently identified multiple endocrine neoplasia type 1 (MEN1) gene [22] on chromosome 11q13 is very frequently found in familiar [22–30] and sporadic tumors [24, 25, 29–37]. In different reports the mutation rate for sporadic cases is about 50% in gastrinomas [33, 38–40], 52% in the largest series available (21 cases) [38] and considering all referred studies (25 cases investigated). In insulinomas, 17–33% of tumors were mutated [33, 35, 41], 17% in the largest series reported of 12 cases [33], and 22% considering all referred studies (22 cases investigated). In nonfunctioning tumors the reported mutation rate is highly variable [35, 36, 38, 40], accounting for 11% in the largest series investigated (9 cases) [36] and 27% considering all studies referred (15 cases investigated). In addition, two functioning glucagonomas and one of two vipomas investigated carried mutations [36, 38, 41]. Overall such findings support the relevance of the MEN1 gene for endocrine tumorigenesis in the pancreas. Though the numbers of cases investigated is limited, the incidence of MEN1 mutation seems to be tumor-type related because it is more frequent in gastrinomas and nonfunctioning tumors as compared to insulinomas. In addition, a recent study assessing clinical behavior and different genetic abnormalities indicated a positive correlation between MEN1 gene defects and malignant phenotype in sporadic tumors [37]. The high frequency of 3p25 allelic loss has been identified in large series of pancreatic endocrine tumors suggesting the presence of a novel tumor-suppressor gene [34, 42, 43]. In addition, a positive correlation between 3p25 loss of heterozygosity (LOH) and malignant behavior have recently been identified in 22 sporadic well-differentiated pancreatic tumors [37].


Overall the data reported indicate the presence of genetic abnormality on chromosome 3p25 in 36 of 103 cases (34%), with an apparent prevalence of abnormality in nonfunctioning cases (17/37 cases, 46%) as compared to 18% of insulinomas (6/33 cases) and 26% of gastrinomas (5/19 cases). Such findings confirm the role of some unknown, potential tumor-suppressor gene on chromosome 3p25 in the genesis of pancreatic endocrine tumors with a possible prevalence for nonfunctioning cases. The p16/MTS1 or p16INK4a tumor-suppressor gene on chromosome 9p21 was demonstrated to be abnormal in 91.7% of either gastrinomas or nonfunctioning tumors in a series of 12 cases [44]. Interestingly, the tumor DNA abnormality consisted of either homozygous deletion or hypermethylation at the 5) region in the absence of mutation as assessed by SSCP. The data available so far are interesting, though require confirmation on larger series. It is believed that an abnormal p53 gene is rarely found in endocrine tumors [45]. Nevertheless high frequency of LOH for markers of the p53 locus on chromosome 17p has been described, mutations were found in 2 malignant cases and hyperexpression/accumulation of the p53 protein was reported in malignant cases [20, 34, 45, 46]. Overall, only 5 of 39 cases, 32 of which were nonfunctioning, showed some genetic abnormality. This finding confirms that an abnormal p53 gene is rarely observed in well-differentiated neuroendocrine tumors of the pancreas and possibly restricted to more aggressive, nonfunctioning cases. The DPC4/Smad4 tumor-suppressor gene located on 18q21 is frequently inactivated in pancreatic cancer [47]; however, controversial data are reported for well-differentiated endocrine tumors. Mutations were described in 55% of nonfunctioning tumors as compared to none of 16 functioning cases [48]. On the contrary, in a recent study no mutation was detected in tumors with a DPC4/Smad4 locus allelic deletion (frequency of deletion 25% for 48 cases, 16 familiar) [37]. Overall 22 of the 80 cases investigated (27%) in the reported literature showed some genetic alteration, with proven mutation in 5 of 61 (8%). Notably, the abnormal DPC4/Smad4 gene appears to be concentrated in nonfunctioning tumors [48]. ras, myc, fos, c-erbB-2 and sis oncogenes have been investigated in well-differentiated pancreatic endocrine tumors [49]. Only ras was consistently found to be overexpressed in pancreatic endocrine tumors [50–52], while fos was observed at low levels [52]. Discrepant data are available for ras mutation analysis. Most investigations report the absence of mutation in relatively large series [46, 53], though Pavelic et al. [54] reported a high frequency of

mutation in 14 cases of insulinomas, 6 of which were malignant. More recently, however, only 1 of 20 cases was reported to be mutated [34]. Overall only 10 of 90 cases investigated (11%) in the literature proved to be mutated for ras, and most of them were malignant insulinomas [54, 55]. Such findings suggest the potential role of the ras oncogene only in the development of malignant ß-cell tumors. Although limited by the relatively small numbers of investigated cases, the picture emerging from the above data depicts a complex mechanism of tumorigenesis in the endocrine pancreas, possibly involving several differnt tumor-suppressor genes. Notably, some genetic defects appear to be tumor-type restricted. This finding supports possible cell type-specific pathways of transformation and is consistent with the well-known functional and genetic differences of islet cell types. Genetic data for poorly differentiated tumors of the pancreas are missing overall, except for the reported high frequency of p53 protein overexpression/accumulation [20]. Investigations specifically addressing the genetic asset of poorly differentiated cases are needed.



Gastrointestinal Tract Tumors Information regarding neuroendocrine tumors of the gastrointestinal tract is scant, incomplete and often difficult to interpret due to the lack of consistent tumor phenotype analysis. Most data refer to neuroendocrine tumors of the stomach, a relatively well-known tumor entity for which specific clinicopathological subtypes have been identified [56]. Allelic loss for MEN1 gene markers on chromosome 11q13 is frequently detected in type-II, familial ECL cell tumors [26, 57, 58] (74% overall incidence, 23 cases investigated) and in nonfamilial cases, associated with chronic atrophic gastritis (type I) or void of specific gastric pathology (type III) [35, 58–60] (43% overall incidence, 42 cases investigated). In addition all 4 poorly differentiated carcinomas investigated showed LOH for MEN1 gene 11q13 markers [35, 58]. Recently, mutation of the RegIalfa gene was demonstrated in 3 of 5 type-I ECL cell tumors, suggesting the possible implication of a gene involved in the functional control of ECL cell growth [61]. Investigation of the human androgen receptor gene (HUMARA) on chromosome X demonstrated the clonality of type-I gastric carcinoids [61]. In addition, extensive allelic deletion of polymorphic markers on chromosome X in 2 type III and 2 PDECs suggested the potential involvement of some unknown tumor suppressor gene(s) on X chromosome [62]. Although no mutation was detected in well-differentiated

23

carcinoids of the stomach of unspecified type [45], hyperexpression accumulation of the p53 protein was demonstrated in 8/16 type-III well-differentiated tumors and in 12/16 PDECs [56]. In addition, allelic loss for p53 gene markers on chromosome 17q was detected in 4 of 9 PDECs supporting a potential role of the p53 gene in the genesis of aggressive neuroendocrine tumors of the stomach [63]. Similarly, LOH for the deleted in colorectal carcinoma (DCC) gene on chromosome 18q21 was found in 2 of 7 type-III tumors with proven malignant behavior and in 4 of 9 PDECs investigated [63]. The above data suggest that several genes are implicated in the mechanism of endocrine tumorigenesis in the stomach. The involvement of the MEN1 gene is supported by several independent studies in both ECL cell tumors and PDECs. The potential involvement of the p53 gene is suggested for PDECs only. The role of other putative genes needs to be confirmed by investigation on larger tumor series. In the small intestine, most genetic data regard the MEN1 gene and gastrin cell tumors only. Abnormal MEN1 gene is frequently observed both in familiar and sporadic duodenal gastrinomas and relative metastases, with a respective overall incidence of 18 and 37% in 63 and 46 cases investigated [27, 29, 33, 40]. Allelic loss for 11q13 MEN1 markers was reported in 3 of 9 sporadic well-differentiated tumors, ‘carcinoids’ of the jejunum and ileum [58, 59], while no p53 mutation was found in 4 cases investigated by Lohman et al. [45]. Of 9 appendiceal carcinoids, 3 showed allelic loss for MEN1 gene markers [58, 59] and no mutation for p53 was observed in 6 cases [45]. In the large intestine, 4 of 7 cases studied showed 11q13 marker LOH [58, 59], and 1 of 9 carcinoids proved to be mutated for p53 [45]. Interestingly, a study by Vortmayer et al. [64] on synchronous conventional cancer and small cell poorly differentiated neuroendocrine carcinomas of the large intestine demonstrated frequent LOH for p53, DCC and the adenomatous polyposis coli (APC) tumor-suppressor genes [64]. In the same paper, no abnormality was observed in 4 welldifferentiated neuroendocrine tumors. Such findings suggest common genetic events at the basis of colon cancer and PDEC as opposed to well-differentiated endocrine tumors of the large intestine. Overall, although scant, the genetic data available for endocrine tumors of the intestine support the potential role of the MEN1 gene in their genesis. The frequent finding of p53 gene abnormality in PDECs only supports the existence of significant genetic differences between welldifferentiated tumors and PDECs. Data for other genes need confirmation by studies on larger series.

24


Concluding Remarks

Neuroendocrine tumors of the gastroenteropancreatic tract are a group of tumors with substantial differences in terms of phenotype and behavior. For tumors of the stomach and pancreas, several clinicopathological predictors of tumor behavior and patient survival have been identified including the proliferation marker Ki67 and overexpression-accumulation of p53 protein. Further work is required for the identification of such predictors in neuroendocrine tumors of the gut. Information on the molecular basis of neuroendocrine tumors is limited overall. In general, the data available point to substantially different pathways of transformation for well-differentiated as compared to poorly differentiated tumors, with potential involvement of the p53 gene for PDECs only. The MEN1 gene appears to be involved in the genesis of both poorly differentiated and well-differentiated tumors. In the latter cases the role of the MEN1 gene is well documented and an apparent cell type-specific mechanism seems to be involved. In addition, other tumor-suppressor genes like DPC4/Smad4, p16/MTSI and other putative genes on chromosome 3p25, 9p21, 18q21, and X have been suggested to participate in the mechanism of endocrine cell transformation. It is hopeful that the solid data generated following the identification of the MEN1 gene locus [65] will be followed by similarly extensive investigations for other potentially intersting genes.


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45 Lohmann DR, Funk A, Niedermeyer HP, Häupel S, Höfler H: Identification of p53 gene mutations in gastrointestinal and pancreatic carcinoids by nonradioisotopic SSCA. Virchows Arch B Cell Pathol Incl Mol Pathol 1993;64:293–296. 46 Pellegata NS, Sessa F, Renault B, Bonato M, Leone BE, Solcia E, Ranzani NG: K-ras and p53 gene mutations in pancreatic cancer: Ductal and nonductal tumors progress through different genetic lesions. Cancer Res 1994;54: 1556–1560. 47 Hahn SA, Hoque AT, Moskaluk CA, da Costa LT, Schutte M, Rozenblum E, Seymour AB, Weinstein CL, Yeo CJ, Hruban RH, Kern SE: Homozygous deletion map at 18q21.1 in pancreatic cancer. Cancer Res 1996;56:490–494. 48 Bartsch D, Hahn SA, Danichevski KD, Ramaswamy A, Bastian D, Galehdari H, Barth P, Schmiegel W, Simon B, Rothmund M: Mutations of the DPC4/Smad4 gene in neuroendocrine pancreatic tumors. Oncogene 1999;18: 2367–2371. 49 Roncalli M, Coggi G: Oncogenes and neuroendocrine tumours; in Polak JM (ed): Diagnostic Histopathology of Neuroendocrine Tumours. London, Churchill Livingstone, 1993, pp 41– 66. 50 Chesa PG, Rettig WG, Melamed MR, Old LJ, Niman NL: Expression of p21 ras in normal and malignant human tissues: Lack of association with proliferation and malignancy. Proc Natl Acad Sci USA 1987;84:3234–3238. 51 Höfler H, Ruhri C, Putz B, Wirnsberger G, Hauser H: Oncogene expression in endocrine pancreatic tumors. Virchows Arch B Cell Pathol Incl Mol Pathol 1988;55:355–361. 52 Roncalli M, Springall DS, Varndell I, Gaitonde VV, Hamid Q, Ibrahim NBN, Grimelius L, Wilander E, Polak JM, Coggi G: Oncoprotein immunoreactivity in human endocrine tumours. J Pathol 1991;163:117–127. 53 Yashiro T, Fulton N, Hara H, Yasuda K, Montag A, Yashiro N, Straus F 2nd, Ito K, Aioshi Y, Kaplan EL: Comparison of mutations of ras oncogene in human pancreatic exocrine and endocrine tumors. Surgery 1993;114:758–763. 54 Pavelic K, Hrascan R, Kapitanovic S, Vranes Z, Beliocza M, Kruslin B, Cabrijan T: Multiple genetic alterations in malignant metastatic insulinomas. J Pathol 1995;177:395–400. 55 Pavelic K, Hrascan R, Kapitanovic S, Vranes Z, Cabrijan T, Spaventi S, Korsic M, Krizanac S, Li YQ, Stambrock P, Gluckman JL, Pavelic ZP: Molecular genetics of malignant insulinoma. Anticancer Res 1996;16:1707–1717.


56 Rindi G, Luinetti O, Cornaggia M, Capella C, Solcia E: Three subtypes of gastric argyrophil carcinoid and the gastric neuroendocrine carcinoma: A clinicopathologic study. Gastroenterology 1993;104:994–1006. 57 Bordi C, Falchetti A, Azzoni C, D’Adda T, Canavese G, Guariglia A, Santini D, Tomassetti P, Brandi ML: Aggressive forms of gastric neuroendocrine tumors in multiple endocrine neoplasia type I: Am J Surg Pathol 1997;21: 1075–1082. 58 Debelenko LV, Emmert-Buck MR, Zhuang Z, Epshteyn E, Moskaluk CA, Jensen RT, Liotta LA, Lubensky IA: The multiple endocrine neoplasia type 1 gene locus is involved in the pathogenesis of type II gastric carcinoids. Gastroenterology 1997;113:773–781. 59 Jakobovitz O, Nass D, DeMarco L, Barbosa AJA, Simoni FB, Rechavi G, Friedman E: Carcinoid tumor frequently display genetic abnormalities involving chromosome 11. J Clin Endocrinol Metab 1996;81:3164–3167. 60 D’Adda T, Keller G, Bordi C, Hofler H: Loss of heterozygosity in 11q13-14 regions in gastric neuroendocrine tumors not associated with multiple endocrine neoplasia type 1 syndrome. Lab Invest 1999;79:671–677. 61 Higham AD, Bishop LA, Dimaline R, Blackmore CG, Dobbins AC, Varro A, Thompson DG, Dockray GJ: Mutations of RegIa are associated with enterochromaffin-like cell tumor development in patients with hypergastrinemia. Gastroenterology 1999;116:1310–1318. 62 Rindi G, Alberizzi P, Candusso ME, LaRosa S, Capella C, Solcia E: Loss of heterozygosity for chromosome 17p, P53 gene, and chromosome 18q, DCC gene, in aggressive endocrine tumors of the stomach (abstract). Gastroenterology 1999;116:G2156. 63 D’Adda T, Candidus S, Denk H, Bordi C, Höfler H: Gastric neuroendocrine neoplasms: Tumour clonality and malignancy-associated large X-chromosome deletions. J Pathol 1999; 189:394–401. 64 Vortmeyer AO, Lubensky IA, Merino MJ, Wang CY, Pham T, Furth EE, Zhuang Z: Concordance of genetic alteration in poorly differentiated neuroendocrine carcinomas and associated adenocarcinomas. J Natl Cancer Inst 1997;89:1448–1453. 65 Larsson K, Skogseid B, Öberg K, Nakamura Y, Nodrenskjöld M: Multiple endocrine neoplasia type 1 gene maps to chromosome 11 and is lost in insulinoma. Nature 1988;332:85–87.



Somatostatin Receptors Naoual Benali Geraldine Ferjoux Elena Puente Louis Buscail Christiane Susini INSERM U 531, CHU Rangueil, IFR 31, Toulouse, France

Key Words Somatostatin W Receptor W Expression W Signal transduction W Biological function

Abstract Somatostatin is a neuropeptide produced by neuroendocrine, inflammatory and immune cells in response to different stimuli. Somatostatin inhibits various cellular functions including secretions, motility and proliferation. Its action is mediated by five specific somatostatin receptors (sst1-sst5) which belong to the G protein-coupled receptor family. The five receptors bind the natural peptide with high affinity but only sst2, sst5 and sst3 bind the short synthetic analogues used to treat patients with neuroendocrine tumors. The five receptors are expressed in various normal and tumor cells, the expression of each receptor being receptor subtype and celltype specific. In neuroendocrine tumors, sst2 is highly expressed whereas in advanced pancreatic adenocarcinoma as well as high-grade colorectal carcinomas, its expression is lost. Each receptor subtype is coupled to different signal transduction pathways through G protein-dependent and -independent mechanisms. The synthesis of selective agonists for each receptor and the recent development of genetic animal models with selective deletion of receptor subtype provide tools for establishing some of the biological roles of the receptors. sst1, 2 and 5 mediate inhibition of GH secretion whereas sst2 and sst5 mediate inhibition of glucagon secretion and insulin secretion, respectively.

Introduction

Somatostatin (somatotroph release-inhibiting factor), initially discovered as a hypothalamic neurohormone that inhibits growth hormone secretion, is a widely distributed peptide in both the central and peripheral nervous systems and in peripheral tissues including the endocrine pancreas, gut, thyroid, adrenals and kidneys. In addition, somatostatin is produced by inflammatory and immune cells as well as many cancer cells. In mammals, two forms of bioactive peptides, somatostatin 14 and somatostatin 28, are produced by tissue-specific proteolytic processing of a common precursor. Somatostatin acts on various targets including the brain, pituitary, endocrine pancreas, gut, adrenals, thyroid, kidney, vascular system and immune system. Somatostatin is mostly an inhibitory factor which regulates a large number of physiological functions including inhibition of endocrine and exocrine secretions, modulation of neurotransmission, motor and cognitive functions, inhibition of intestinal motility, absorption of nutrients and ions, vascular contractility and cell proliferation. Most of these effects result from the inhibitory effect of the peptide on secretion and cell proliferation [1]. Due to the short half-life of natural somatostatin peptides, many somatostatin analogues have been synthesized. Among them, octreotide (SMS 201-995), lanreotide (BIM 23014) and vapreotide have been intensively investigated and are in clinical use for the medical treatment of acromegaly and neuroendocrine tumors. These octapeptides retain amino acid residues (or substitutes) within a

Copyright © 2000 S. Karger AG, Basel

ABC

© 2000 S. Karger AG, Basel 0012–2823/00/0625–0027$17.50/0



Dr. Christiane Susini INSERM U 531, Institut Louis-Bugnard IFR 31, CHU Rangueil, F–31403 Toulouse Cedex 4 (France) Tel. +33 5 61 32 24 07, Fax +33 5 61 32 24 03 E-Mail [email protected]

Table 1. Binding affinity and signal transduction pathways coupled

to each receptor subtype sst1

sst2

sst3

Binding affinity (IC50), nM1 Somatostatin 14 0.4 0.1 Octreotide 11,000 0.3

0.3 32

Transduction pathway Adenylate cyclase Phospholipase C Tyrosine phosphatase

sst5

0.8 0.6 11,000 7.9

↓ ↑ ↑

↓ ↑ ↑

↓ ↑ ↑

↓ ↑ ↑

↓

↓ ↑

↑

↑

↑

Na+/H+ exchange

↓

↑

}

MAP kinase (ERK) Phospholipase A2

↑ ↑

↓

Ca2+ channels K+ channels

1

sst4

}

↓ ↓/↑

}

Data from Burns et al. [6].

cyclic peptide backbone involved in the biological effect of the peptide (Phe7 or Tyr7, DTrp8, Lys9 and Thr10 or Val10) and display markedly increased stability. More recently, high-affinity nonpeptide agonists have been synthesized. They display subtype-selectivity and represent useful tools to define the physiological functions of each receptor subtype [2].

Somatostatin Receptor Family

The biological effects of somatostatin are mediated by specific plasma membrane receptors that have been identified in normal and neoplastic tissues by binding studies and receptor autoradiography techniques [1, 3, 4]. Five somatostatin receptor genes have been cloned from human and mammalian libraries and designed sst1 to sst5. Human sst1-5 are encoded by 5 genes located at chromosomes 14q13, 17q24, 22q13.1, 20p11.2 and 16p13.3, respectively. Four of these genes are intronless, the exception being sst2 which is alternatively spliced to generate two isoforms named sst2A and sst2B which differ in their C-terminal sequence. The sst subtypes belong to the family of G protein-coupled receptors with seven transmembrane-spanning domains and present a high degree of sequence identity (39–57%). The sequence differences reside in the extracellular and intracellular domains and are probably responsible for their signalling specificity. They all bind somatostatin 14 and somatostatin 28 with a

28


high affinity (nM range), although with a slightly higher affinity for somatostatin 14. Only sst5 displays a 10-fold higher affinity for somatostatin 28. However, they show major differences in their affinities for analogues (table 1). Analogues exhibit a low affinity for sst1 and sst4 (61 ÌM) whereas they bind sst2 and sst5 with a high affinity, comparable to that of somatostatin 14 and bind sst3 with moderate affinity (5–30 nM) [5, 6]. Using recombinant ssts transiently or stably expressed in various eucaryote cells, the intracellular signalling pathways coupled to ssts have been extensively studied. Each receptor subtype is coupled to multiple intracellular transduction pathways via pertussis toxin-sensitive and -insensitive heterotrimeric GTP-binding (G) proteins (table 1). All five ssts are functionally coupled to inhibition of adenylate cyclase. In neuronal and neuroendocrine cells, somatostatin and analogues regulate several subsets of K+ channels causing hyperpolarization of the plasma membrane and leading to decreased Ca2+ influx through voltage-gated Ca2+ channels and consequently to a reduction in intracellular Ca2+. Expression of GIRK1 channels together with each somatostatin receptor in Xenopus oocytes demonstrates that sst2, sst3, sst4 and sst5 can couple to inward rectifying K+ channels, sst2 being the most efficiently coupled. Somatostatin can also decrease Ca2+ influx by directly inhibiting high voltage-dependent Ca2+ channels. Somatostatin receptors couple to N- and L-type voltage-dependent calcium channels in several cell types including mouse pituitary AtT-20 cells, rat insulinoma RINm5F cells and human neuroendocrine gut tumor cells. Using receptor subtype-specific analogues, it has been demonstrated that sst1, sst2 and sst5 can negatively couple to L-type voltage-dependent calcium channels [5, 7]. Somatostatin and analogues activate a number of protein phosphatases including serine/threonine phosphatases, tyrosine phosphatases and a Ca2+-dependent phosphatase [5]. Sst1, sst2, sst3 and sst4 stimulate tyrosine phosphatase activity when expressed in NIH 3T3 or CHO cells [8, 9]. Somatostatin receptor subtypes involved in the regulation of other phosphatases have not yet been identified. All five receptors stimulate phospholipase C and increase Ca2+ mobilization via both pertussis toxin-sensitive and -insensitive G protein, albeit at an agonist concentration of 11 nM [5]. The coupling of sst2, sst5 and sst3 to phospholipase C is the most efficient. For sst3, this coupling is involved in the stimulatory effect of somatostatin on guinea-pig intestinal smooth muscle cell contraction and is mediated by phospholipase C-ß3 [10]. In addi-

Benali/Ferjoux/Puente/Buscail/Susini

tion, in CHO-K1 cells expressing sst5, low concentrations of somatostatin analogues (EC50 = 0.3 nM) inhibit phospholipase C-mediated intracellular Ca2+ mobilization [11]. The MAP kinase pathway is also involved in signal transduction coupled to ssts but the modulation differs according to the receptor subtype. When expressed in CHOK1 cells, sst1 activates MAP kinase cascade [12]. Sst4 induced a Src- and Ras-dependent transient activation of Erk1/2 and a sustained activation which is mediated by proteine kinase C [13]. Conversely, sst5 inhibits MAP kinase Erk1/2 [14]. Finally, sst1 but not sst2 is negatively coupled to Na+/ + H exchanger via a pertussis toxin-insensitive G protein [5].

Expression of Somatostatin Receptor Subtypes

Somatostatin receptors are widely distributed throughout many tissues ranging from the central nervous system to the pancreas and the gut, and also in the pituitary, kidneys, thyroid, lung, inflammatory and immune cells. Using either radiolabeled somatostatin or its analogues, somatostatin receptors have also been described in a large variety of human cancers including pituitary adenomas, islet tumors, carcinoids, adenocarcinomas of the breast, prostate, ovary, kidney and colon origin, lymphomas as well as astrocytomas, neuroblastomas and medulloblastomas. Analyses of sst mRNAs demonstrate that various human tumors from neuroendocrine and gastroenteropancreatic origin, brain tumors (gliomas, meningiomas, medulloblastomas), pheochromocytomas, medullary thyroid carcinomas, prostate, lung and breast cancers express various sst mRNA, each tumor expressing more than one subtype and sst2 being the most frequently expressed [15]. In carcinoid tumors, of 87 tumors examined, approximately 85% are positive for sst2. The majority of these tumors also express sst5 with sst1, sst3 and sst4 being less abundant. The high frequency of sst2 mRNA (and probably also the presence of sst5 mRNA) found in neuroendocrine tumors allows the localization of various human tumors and metastases by somatostatin receptor scintigraphy following injection of 111In-labelled octreotide. The recent availability of polyclonal antibodies has enabled different groups to identify the sst proteins. Indeed, using sst2 antibodies, Dutour et al. [16] reported the expression of sst2 in human gliomas and meningiomas with a rich expression in both human brain tumor and peritumoral tissue, and a prominent expression in blood vessels. Immunohistochemical detection of somatostatin

Somatostatin Receptors

Table 2. Biological effect of somatostatin receptor subtypes

Inhibitory effect on Hormone and mediator secretion GH TSH Gastrin Insulin Glucagon Cytokine (IL6, IFNÁ), histamine Exocrine secretion Gastric acid Amylase Intestinal Cl – secretion Motility Gastric and intestinal relaxation Colonic contraction Cell proliferation Induction of G1 cell cycle arrest Induction of apoptosis

sst1 sst2 sst3 sst4 sst5

+

+ + + + + +

+ + +

+ + +

+ + +

+

+ +

+

+

+

receptors sst1, sst2 and sst3, using specific antibodies in 33 breast tumors, allows detection of the receptors on tumor cells but the level and the pattern of the expression of sst vary greatly between individual carcinomas, sst2 being detected at a high level in 28 tumors (85%), sst1 in 17 tumors (52%) and sst3 in 16 tumors (48%) [17]. Of 43 patients with carcinoid tumors, the presence of sst2 protein has been detected in 31 patients [18, 19]. There was a correlation with the presence of sst2 mRNA, the tracer uptake using somatostatin receptor autoradiography and the therapeutic response to somatostatin analogue treatment [18]. In contrast to that observed in neuroendocrine tumors, in advanced pancreatic adenocarcinoma as well as colorectal carcinomas of high grade, sst2 expression is lost [20]. This may confer a growth advantage in these tumors and provide one explanation for the lack of therapeutic effect of somatostatin analogues in such tumors.

Biological Effects of Somatostatin Receptor Subtypes

Much of our current knowledge on receptor subtype selectivity for biological effects of endogenous somatostatin receptors is based on results obtained with mammalian cells using selective subtype agonists or with transfected cells expressing each receptor subtype (table 2). Recently, genetic animal models with selective deletion of sst1 or sst2 receptor subtype provided valuable tools to


29

demonstrate the specificity of receptor subtype. However, due to the expression of multiple receptor subtypes in a same cell and the coupling of ssts to similar transduction pathways, we can hypothesize that the sst subtypes may act in concert with a selective temporal and spatially role. At the cellular level, the blockade of secretion by somatostatin is mediated through inhibition of Ca2+ and to a lesser degree cAMP production. Additionally, somatostatin can interfere with the exocytotic machinery by inhibiting the protein phosphatase calcineurin [21]. In the pituitary, sst2, sst5 and sst1 are involved in inhibiting growth hormone release [2, 22, 23]. In the pancreas, sst2 mediates inhibition of glucagon release, whereas sst5 is a negative regulator of insulin secretion [2]. Sst2 also mediates insulin secretion in isolated perfused human pancreas [24]. The high expression of sst1 receptors in human A pancreatic islet cells suggests that sst1 may also be involved in regulating insulin release [25]. Sst5 is involved in the inhibition of pancreatic exocrine secretion. In the stomach, sst2 contributes to the inhibition of histamine and gastrin release and the inhibition of gastric acid secretion [26]. Sst1 and sst2 mediate the inhibition of intestinal ionic secretion [21]. In human neuroendocrine gut tumor cells, somatostatin and octreotide inhibit L-type voltage-dependent calcium channels with the same amplitude suggesting that at least sst2 and sst5 may be involved in the inhibition of Ca2+ influx and thereby inhibition of tumor-produced neurotransmitters and hormone [27]. Sst3 could be involved in the stimulation of gastric and intestinal smooth muscle cell contraction and sst5 in the inhibition of colonic muscle cell contraction [10, 28]. In addition to its effect on secretion and intestinal motility, somatostatin inhibits the proliferation of normal as well as tumor cells. The antiproliferative action of somatostatin can be signalled via the five ssts which initiate pertussis toxin-sensitive G protein-dependent cell growth arrest or apoptosis according to receptor subtypes and target cells. When expressed in CHO cells, ligandactivated sst1, sst2A, sst4, and sst5 inhibit mitogenic signal of serum or growth factors as a result of hypophosphorylation of the retinoblastoma gene product (Rb) and G1 cell cycle arrest [11, 29, 30]. However, distinct signal transduction pathways are involved in the somatostatininduced G1 cell cycle arrest depending on receptor subtype. Sst1 mediates cell growth arrest through the stimulation of the tyrosine phosphatase SHP-2, activation of the Ras/MAP kinase ERK pathway and induction of the cyclin-dependent kinase inhibitor p21Waf1/Cip1 [8, 12], whereas sst5 acts by a mechanism involving a dephosphorylation cascade leading to inhibition of guanylate

30


cyclase, cGMP-dependent protein kinase G and MAP kinase ERK 1/2 [14]. The antiproliferative effect mediated by sst2 results from the activation of the tyrosine phosphatase SHP-1 and the dephosphorylation of activated growth factor receptors thus leading to the negative regulation of growth factor-induced mitogenic signalling [31]. In addition, somatostatin-activated SHP-1 induces a G1 cell cycle arrest, upregulates the cyclin-dependent kinase inhibitor p27Kip1 leading to the accumulation of hypophosphorylated Rb [32]. The antiproliferative effect of somatostatin can also result from apoptosis. Apoptosis is induced by sst3 as a result of the induction of p53 and Bax [33]. In human pancreatic cancer cells expressing mutated p53 and devoid of endogenous sst2, correction of the deficit by expression of sst2 induces an increase in cell death [34] indicating that somatostatin can induce apoptosis by p53-dependent and p53-independent mechanisms. The antiproliferative effects of somatostatin result from its actions via the endocrine pathway, but evidence exists that somatostatin can also act via an autocrine/ paracrine pathway. Immunoreactive somatostatin has been found in somatostatin receptor-positive normal and tumor cell types such as endocrine, lymphoid cells, macrophages, breast cancer cells, colonic tumor cell and additionally, somatostatin mRNA is detected in a wide variety of neuroendocrine tumors known to express somatostatin receptors [35]. Correction of the sst2 deficit in human pancreatic cancer cells by sst2 expression induces a negative autocrine loop in the absence of exogenous ligand, which is due to sst2-induced expression and secretion of endogenous sst2 ligand (somatostatin 14 and somatostatin 28). This results in inhibition of cancer cell proliferation and reversion of cell tumorigenicity in vitro and in vivo after xenografts in nude mice [36]. The somatostatin effect on tumor growth may be the result of indirect effects of the peptide resulting from the inhibition of secretion of growth-promoting hormones and growth factors which specifically regulate tumor growth. For example, the secretion of insulin-like growth factor-1 (IGF-1) which is produced by hepatocytes through GH-dependent and -independent mechanisms is negatively controlled by octreotide as a result of an effect on GH secretion and sst2 and sst5 have been demonstrated to be implicated in this effect [37]. In addition, somatostatin can decrease IGF gene expression [38]. Somatostatin also inhibits angiogenesis. Overexpression of peritumoral vascular somatostatin receptors with high affinity for somatostatin and octreotide has been reported in human primary colorectal carcinomas, small cell lung


carcinoma, breast cancer, renal cell carcinoma and malignant lymphoma. This expression appears to be independent of receptor expression in the tumor. It may reflect the presence of ssts in the venous smooth muscle cells as well as endothelial cells and may allow a vasoconstriction resulting in local hypoxia of the tumor or inhibition of endothelial cell growth and monocyte migration. Sst2, sst3 or sst5 might be involved in these effects [4, 39, 40]. Many studies have demonstrated that somatostatin has a modulatory effect on the immune system. Somatostatin and receptors are expressed in human lymphoid organs and can regulate various immune functions including lymphocyte proliferation, immunoglobulin synthesis, and cytokine production [41]. Sst2, sst3, sst4 and sst5 are expressed in human lymphoid cells and could be involved in these effects. However, the role of somatostatin in the control of the immune system in patients with neoplasms has not been established.

Agonist-Dependent Regulation of Somatostatin Receptors

As observed for other G protein-coupled receptors, somatostatin receptors are sensitive to agonist-induced internalization and sst gene regulation. The molecular mechanisms involved in these effects are poorly understood, it appears that the response of somatostatin receptors to agonist application is agonist, receptor and cell type-specific. In CHO-K1 cells, sst2–5 undergo rapid internalization (sst3 1 sst5 1 sst4 1 sst2) following the agonist but sst1 is not internalized. In contrast, in human embryonic kidney cells, a different pattern was observed.

Sst1-3 are internalized in response to somatostatin whereas sst5 is only internalized in response to somatostatin 28 and sst4 is not internalized [5, 21]. Phosphorylation of serine/threonine residues in the C-terminal domain of ssts plays an important role in the sst internalization process [5, 42, 43]. In addition, somatostatin upregulates ssts, sst1 and sst2 being the most sensitive to agonist-mediated upregulation. Somatostatin upregulates sst1 in vitro and in vivo in CHOK1 and human pancreatic cancer cells, respectively [21, 34]. Continued exposure of rat pancreatic tumors to octreotide causes upregulation of sst2 [44]. These phenomena may be important for the physiological and clinical response to somatostatin peptides.

Conclusion

Much progress has been made towards the elucidation of molecular signal transduction, the expression and regulation of ssts. However, the biological role as well as the cellular distribution of each receptor subtype is far from being completely understood. The development of specific antibodies, agonists and antagonists as well as pan somatostatin with high affinity for all sst may improve the diagnosis and therapeutic application of somatostatin. The gene knockout models will help to define the specific role of individual receptors in physiological and pathological conditions and the significance of multiple receptor subtypes in the same cell. A better knowledge of the mechanisms underlying the regulation of sst expression as well as receptor internalization could allow selective upregulation of ssts on the target cell and result in enhanced accumulation of the somatostatin analogue.

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21 Patel YC: Molecular pharmacology of somatostatin receptor subtypes. J Endocrinol Invest 1997;20:348–367. 22 Zheng H, Bailey A, Jiang MH, Honda K, Chen HY, Trumbauer ME, Van der Ploeg LH, Schaeffer JM, Leng G, Smith RG: Somatostatin receptor subtype 2 knockout mice are refractory to growth hormone-negative feedback on arcuate neurons. Mol Endocrinol 1997;11: 1709–1717. 23 Kreienkamp HJ, Akgun E, Baumeister H, Meyerhof W, Richter D: Somatostatin receptor subtype 1 modulates basal inhibition of growth hormone release in somatotrophs. FEBS Lett 1999;462:464–466. 24 Moldovan S, Atiya A, Adrian TE, Kleinman RM, Lloyd K, Olthoff K, Imagawa D, Shevlin L, Coy D, Walsh J, et al: Somatostatin inhibits B-cell secretion via a subtype-2 somatostatin receptor in the isolated perfused human pancreas. J Surg Res 1995;59:85–90. 25 Kumar U, Sasi R, Suresh S, Patel A, Thangaraju M, Metrakos P, Patel SC, Patel YC: Subtypeselective expression of the five somatostatin receptors (hSSTR1-5) in human pancreatic islet cells: A quantitative double-label immunohistochemical analysis. Diabetes 1999;48:77– 85. 26 Martinez V, Curi AP, Torkian B, Schaeffer JM, Wilkinson HA, Walsh JH, Tache Y: High basal gastric acid secretion in somatostatin receptor subtype 2 knockout mice. Gastroenterology 1998;114:1125–1132. 27 Glassmeier G, Hopfner M, Riecken EO, Mann B, Buhr H, Neuhaus P, Meyerhof W, Scherubl H: Inhibition of L-type calcium channels by octreotide in isolated human neuroendocrine tumor cells of the gut. Biochem Biophys Res Commun 1998;250:511–515. 28 Gu ZF, Corleto VD, Mantey SA, Coy DH, Maton PN, Jensi RT: Somatostatin receptor subtype 3 mediates the inhibitory action of somatostatin on gastric smooth muscle cells. Am J Physiol 1995;268:G739–G748. 29 Sharma K, Patel YC, Srikant CB: C-terminal region of human somatostatin receptor 5 is required for induction of Rb and G1 cell cycle arrest. Mol Endocrinol 1999;13:82–90. 30 Alderton F, Fan TP, Schindler M, Humphrey PP: Rat somatostatin sst2(a) and sst2(b) receptor isoforms mediate opposite effects on cell proliferation. Br J Pharmacol 1998;125:1630– 1633. 31 Bousquet C, Delesque N, Lopez F, Saint-Laurent N, Esteve JP, Bedecs K, Buscail L, Vaysse N, Susini C: sst2 somatostatin receptor mediates negative regulation of insulin receptor signaling through the tyrosine phosphatase Shp-1. J Biol Chem 1998;273:7099–7106. 32 Pages P, Benali N, Saint-Laurent N, Esteve JP, Schally AV, Tkaczuk J, Vaysse N, Susini C, Buscail L: sst2 somatostatin receptor mediates cell cycle arrest and induction of p27(Kip1). Evidence for the role of Shp-1. J Biol Chem 1999;274:15186–15193.


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Tumor Markers in Neuroendocrine Tumors Barbro Eriksson a Kjell Öberg a Mats Stridsberg b a Section

for Endocrine Oncology, Department of Medical Sciences, and b Department of Clinical Chemistry, University Hospital, Uppsala, Sweden

Key Words Specific tumor markers W General tumor markers W Chromogranin A W Neuropeptide K W Neuron-specific enolase W Urinary 5-HIAA

Abstract Most neuroendocrine tumors produce and secrete a multitude of peptide hormones and amines. Some of these substances cause a specific clinical syndrome: carcinoid, Zollinger-Ellison, hyperglycemic, glucagonoma and WDHA syndrome. Specific markers for these syndromes are basal and/or stimulated levels of urinary 5HIAA, serum or plasma gastrin, insulin, glucagon and vasoactive intestinal polypeptide, respectively. Some carcinoid tumors and about one third of endocrine pancreatic tumors do not present any clinical symptoms and are called ‘nonfunctioning’ tumors. Therefore, general tumor markers such as chromogranin A, pancreatic polypeptide, serum neuron-specific enolase and subunits of glycoprotein hormones have been used for screening purposes in patients without distinct clinical hormonerelated symptoms. Among these general tumor markers chromogranin A, although its precise function is not yet established, has been shown to be a very sensitive and specific serum marker for various types of neuroendocrine tumors. This is because it may also be elevated in

ABC

© 2000 S. Karger AG, Basel 0012–2823/00/0625–0033$17.50/0



many cases of less well-differentiated tumors of neuroendocrine origin that do not secrete known hormones. At the moment, chromogranin A is considered the best general neuroendocrine serum or plasma marker available both for diagnosis and therapeutic evaluation and is increased in 50–100% of patients with various neuroendocrine tumors. Chromogranin A serum or plasma levels reflect tumor load, and it may be an independent marker of prognosis in patients with midgut carcinoids. Copyright © 2000 S. Karger AG, Basel

Introduction

The biochemical diagnosis of neuroendocrine tumors is very important in the clinical workup of patients. The introduction of radioimmunoassays for various peptide hormones in the middle of the 1960s increased the clinical awareness of and the ability to diagnose neuroendocrine gastrointestinal tumors. During the following decades specific radioimmunoassays were developed for various hormones and every laboratory made panels of different assays. The peptides and amines secreted by neuroendocrine tumors may serve as tumor markers not only for diagnosis but also for follow-up of treatment of these patients, and there are indications that some markers may also have prognostic implications.

Barbro Eriksson, MD, PhD Section for Endocrine Oncology, Department of Medical Sciences University Hospital S–751 85 Uppsala (Sweden) E-Mail [email protected]

Specific and General Tumor Markers

Neuroendocrine tumors are known to produce and secrete a large number of peptide hormones and amines [1, 2]. These substances give rise to various clinical syndromes related to the hormone production. These wellknown clinical syndromes include the carcinoid syndrome, Zollinger-Ellison syndrome, hyperoglycemic syndrome, glucagonoma syndrome, WDHA syndrome, and somatostatinoma syndrome. Biochemical tumor markers can be divided into specific and general markers. Specific markers for carcinoid tumors are urinary 5-HIAA, neuropeptide K, substance P or other tachykinins causing the carcinoid syndrome. Similarly, specific markers for endocrine pancreatic tumors (EPTs) include gastrin, insulin, c-peptide, pro-insulin, vasoactive intestinal polypeptide, glucagon and somatostatin. General tumor markers for both tumor types include chromogranins (Cgs), pancreatic polypeptide, HCG subunits. Already in a publication from our group in 1990, we could demonstrate that the circulating marker elevated in most cases of EPTs was CgA (94%) followed by pancreatic polypeptide (74%) [2]. Also, multiple hormone production was evident by the fact that as many as 62% had elevation of serum gastrin sometime during the clinical course of the disease considering that only 30% presented the gastrinoma syndrome initially. In a relatively recent publication from our group, different tumor markers in carcinoid tumors were looked at [3]. Urinary 5-HIAA and neuropeptide K showed quite high sensitivity in midgut carcinoid tumors, whereas in foregut and hindgut carcinoid the sensitivity of these markers was rather low. Plasma CgA on the other hand was elevated in 70–100% independent of tumor type. Sometimes patients are encountered in whom a carcinoid syndrome is strongly suspected but measurements of basal fasting levels of hormones are normal. In such cases the pentagastrin test with measurement of plasma tachykinins is quite sensitive [4], whereas in patients with recurrent ulcer disease the secretin test measuring gastrin can detect a gastrinoma in 180% of cases [5].

Chromogranins

Neuroendocrine cells contain typical secretory granules, called large dense-core vesicles based on their characteristic appearance on electron microscopy. In addition to the specific peptide hormones or neuropeptides, these granules also contain one or more Cg/secretogranin proteins [6, 7]. These belong to a unique family of secretory

34


proteins which share many biochemical properties as well as an exclusive presence in neuronal and neuroendocrine secretory granules [8, 9]. The first member of this family to be identified was CgA. Its name derives from its original discovery in the catecholamine-containing chromaffin granules of the adrenal medulla [10]. Other well-characterized members of the family are CgB and secretogranin II [9]. CgA is the best studied granin in humans. It shows the widest distribution and is present in some cells that do not express CgB or secretogranin II [6, 7]. The physiological functions of CgA has gradually been elucidated, although many questions still remain. Its ubiquitous presence in neuroendocrine tissues and its cosecretion with peptide hormones and neuropeptides makes it a suitable tissue and serum marker of neoplasms of neuroendocrine origin [11–14]. The gene encoding for CgA is located on chromosome 14 [5]. CgA is very hydrophilic with an abundance of charged, mostly acidic amino acids. It contains multiple sites with two or more adjacent basic amino acids, far exceeding the number observed in other proteins [16]. CgA may be processed at its dibasic amino acid residues into tissue-specific biologically active peptides. All members of the granin family contain multiple dibasic residues and calcium-binding sites suggesting analogous functions. CgA is most closely related to CgB [17]. Both contain a disulfide-bonded loop structure near their amino termini with a highly homologous amino acid sequence. This structure, which might have a role in directing the Cgs to the right secretory vesicles, is not present in the secretogranins [18]. The high degree of structural homology among Cgs in different mammalian species suggests conservation during evolution and this in turn implicates important biological functions [11]. Pancreastatin is one of the cleavage products of CgA corresponding to residues 248–303 of the CgA molecule [19]. Pancreastatin is a peptide which inhibits insulin and glucagon secretion from the endocrine pancreas [20, 21]. It also suppresses parathyroid hormone secretion from parathyroid cells [22]. In addition it has a regulatory effect on the secretion from non-endocrine cells including parietal cells and exocrine pancreas [23, 24]. During the last years several other peptide fragments of CgA have been described. Vasostatin is a peptide derived from the Nterminal part of CgA. In some tissues it is present as a 76-amino acid chain and in others as a 113-amino acid chain (called vasostatin I and II, respectively) [25, 26]. It has been shown to inhibit vasoconstriction of human veins [27], to inhibit PTH secretion by bovine parathyroid tissue [28], and most interesingly to stimulate cell adhesion via interaction with integrins [29]. Another

Eriksson/Öberg/Stridsberg

cleavage product, parastatin, inhibits PTH secretion [30]. Chromostatin, another fragment of CgA, has been shown to be able to inhibit the release of catecholamines by adrenal medulla cells [31]. A receptor for chromostatin has been described on chromaffin cells that binds to the peptide with high affinity [32]. The proteolytic processing of CgA probably occurs during its stay in the dense-core vesicles [8]. Depending on the type and amount of proteases present, the type and amount of the cleavage products may vary in different neuroendocrine tissues [8, 33]. The present data support that peptides derived from intragranular processing of CgA are coreleased with resident peptide hormones/ amines and exert an immediate auto- and paracrine modulatory effect on the secretory activity on neuroendocrine cells. A classical endocrine effect on distant tissues may also be possible as these fragments of CgA are also released into the circulation [11]. Quite recently it has been suggested that CgA and its fragment could play a role in the regulation of cell adhesion due to storage and rapid release from neuroendocrine cells and neurons [29].

Immunohistochemistry

CgA is found throughout the neuronal and neuroendocrine system. The endocrine cells are expressing the protein in the endocrine cells of the anterior pituitary, parafollicular C cells of the thyroid, chief cells of the parathyroids, islets cells of the pancreas and chromaffin cells of the adrenal medulla [34]. It is also present in the wide spread neuroendocrine system of the bronchial and gastrointestinal tract and of the skin (Merckel cells). CgApositive cells have also been found in the prostate. Cells that are not of neuroendocrine origin lack CgA such as follicular cells of the thyroid gland and steroid hormonesecreting cells of the adrenal cortex and the gonads. Immunohistochemical techniques to detect the presence of CgA in tumor tissues are widely used in clinical practice. A list of tumors for which CgA can serve as a tissue marker is presented in table 1. In addition to typical neuroendocrine neoplasms, tumors derived from the nervous system, such as ganglioneuroblastomas, ganglioneuroma and neuroblastoma also show immunostaining for CgA [6]. Also neuroendocrine tumors that have lost their ability to produce peptide hormones (e.g. null-cell pituitary adenomas) contain CgA-positive immunostaining. Not only tumors but also neuroendocrine cell hyperplasia, such as gastric enterochromaffin-like (ECL) cells in chronic atrophic gastritis stain positively with CgA [34].

Tumor Markers in Neuroendocrine Tumors

Table 1. CgA-positive neuroendocrine tumors by immunocyto-

chemistry Anterior pituitary tumors ACTH (Cushing) LH/FSH GH (acromegaly) TSH Prolactinoma (CgB positive!) Null cell adenoma Parathyroid tumors Medullary thyroid carcinoma Merkel cell tumor Neuroendocrine GEP tumors Carcinoids (foregut, midgut, hindgut) ECL-oma Gastrinoma Glucagonoma Insulinoma (CgB positive!) Somatostatinoma VIP-oma Nonfunctioning tumors Ectopic ACTH-producing tumors (Bronchial carcinoids, endocrine pancreatic tumors) Ganglioneuroma, neuroblastoma Pheocromocytoma Small cell lung cancer Prostate cancer

In a study performed by Borch et al. [35], plasma CgA levels correlated strongly with the endocrine cell density in the fundic mucosa (p ! 0.001) and it was suggested that the elevated CgA was derived from the ECL cells rather than gastrin-producing cells. The prolactin-producing cells of the pituitary lack CgA expression but show positive staining with CgB antibodies. The same is also true for the ß cells of the pancreas, which are sometimes negative with CgA. In agreement with this, some benign insulinomas are CgA-negative but show positive CgB staining. It should also be pointed out that several non-endocrine tumors may show positive immunoreaction for CgA in a limited number of cells, e.g. in colorectal cancer and exocrine pancreatic cancer.

Plasma and Serum Measurements

Since CgA is stored in a majority of different neuroendocrine tumors, the release to the circulation can be used as a ‘general’ marker for various neuroendocrine tumors. This is of particular interest in ‘nonfunctioning’ tumors,


35

which may lack other suitable markers. It is also of value to be analyzed when existing markers are either unstable or rapidly fluctuating, such as serotonin and catecholamine levels in plasma. The first radioimmunoassay for measurement of CgA was reported by O’Connor et al. [12] in 1983, where it was shown that most patients with established neuroendocrine tumors had elevated levels of CgA. Since then several radioimmunoassays have been developed. The problem with many of these assays is that they are not actually measuring CgA but possibly splice products such as pancreastatin, and then the results will be very different. Published results and circulating levels of CgA from different centers may vary depending on what assay they have been using. The plasma levels of CgA are elevated in various peptide-producing neuroendocrine tumors and the highest levels are noted in patients with metastatic carcinoid tumors, particularly midgut carcinoids [3]. In malignant EPT plasma CgA varies from slightly elevated to extreme levels. Plasma CgB is usually a better marker for benign insulin-producing tumors than CgA. In an earlier study, involving 44 patients with malignant carcinoid tumors, 17 patients with sporadic EPT and 11 patients with EPT as part of multiple endocrine neoplasia type 1 (MEN1), plasma level of CgA was increased in 99%, whereas CgB was increased in 88% and CgC in 6% and pancreastatin in 46% of the patients, illustrating that it is definitely superior to measure CgA compared to pancreastatin [13]. In a publication by Janson et al. [3] the relationship between tumor burden and plasma CgA levels was studied and it was shown that among patients with midgut carcinoids those with multiple liver metastases had significantly higher levels than those with only a few liver metastases or lymph node metastases only. In a previous study of midgut carcinoid patients, who had undergone surgery and after surgery small residual lymph node metastases were left, measurement of plasma CgA was compared to urinary 5-HIAA [36]. In this group of 25 patients with limited disease, all had elevated plasma CgA, whereas only 3 had elevations of urinary 5-HIAA. Similarly, in a study of plasma CgA levels in patients with MEN1, 40% of patients without other biochemical evidence of an EPT had elevation of CgA, whereas 60% of patients with biochemically unequivocal evidence of MEN1 and 100% of radiologically verified EPT had increased levels [37]. These results indicate that there is a correlation between tumor burden and plasma CgA levels in patients with neuroendocrine tumors. In the latter study, a comparison

36


was made of the sensitivity of measurements of basal plasma CgA and the meal stimulation test in detecting early involvement of the endocrine pancreas in MEN1 and it was found that the meal stimulation test is still the most sensitive method, 75 versus 60% for CgA. In a study by Bajetta et al. [38], CgA measurements seemed to be superior to urinary 5-HIAA, neuron-specific enolase and carcinoembryonic antigen as tumor markers in a large group of patients with neuroendocrine tumors (n = 127). The specificity was quite high both for urinary 5-HIAA and neuron-specific enolase (100%), whereas the sensitivity was rather low (35.1 and 32.9%, respectively). The corresponding figures for CgA were 86 and 68%. Tumor marker modifications were also studied during follow-up of patients and it was found that rises in CgA were associated with progressive disease in 83% of patients. We have made similar observations in our patient material [36, 37], where plasma CgA levels were followed together with other markers; CgA measurements are the most reliable markers in the follow-up of treatment. Furthermore, increases in plasma CgA usually precede radiological evidence of progression. Elevated plasma CgA levels are not entirely specific for a neuroendocrine tumor [39]. Slightly elevated levels have been identified in patients with non-endocrine tumors. Neuroendocrine cells are multifocally located in small nests or scattered within these tumors. Prostatic adenocarcinomas coexpress CgA and prostate-specific antigen suggesting a common malignant precursor cell [40]. Limited data suggest that prostatic adenocarcinoma containing neuroendocrine cells are more resistant to hormonal treatment. Similarly patients with colorectal adenocarcinoma containing numerous neuroendocrine cells seem to have a worse prognosis [13]. On the other hand, patients with pancreatic adenocarcinoma or non-small cell cancer whose cancer contains many neuroendocrine cells, seem to have a better prognosis [41]. In addition, there are some other pitfalls in the interpretation of CgA levels. ‘False-positive’ elevation of CgA can be seen in patients with renal impairment, liver failure, atrophic gastritis and inflammatory bowel disease [37]. Also patients receiving proton pump inhibitors have a slight increase in CgA due to gastrin-induced ECL cell hyperplasia. Furthermore, physical stress induced by exercise or somatic trauma can produce a slight elevation in CgA (about twofold) [2]. In patients with neuroendocrine tumors such as carcinoid tumors, an analysis of prognostic factors in 301 patients showed that plasma CgA is an independent pre-


dictor of bad prognosis in midgut carcinoid patients together with advanced age [3]. Independent observations that increased levels of CgA correlate with bad prognosis in different tumor diseases might indicate a role of CgA as a stimulator of growth, either the molecule itself or any of the splice products. Therefore ongoing studies analyzing splice products of CgA in carcinoid patients might reveal a correlation between some of the fragments and prognosis in those patients.

Future Prospects

CgA is for the moment the most sensitive and reliable marker both for immunohistochemistry and as a circulating marker in patients with neuroendocrine tumors. The implications that Cgs may have tumor biological effects need to be further explored. Since CgA is present in practically all neuroendocrine tumors one future application could be to use it for in vivo imaging with the PET technique.

References 1 Norheim I, Öberg K, Theodorsson-Norheim E, Lindgren PG, Lundqvist G, Magnusson A, Wide L, Wilander E: Malignant carcinoid tumors: An analysis of 103 patients with regard to tumor localization, hormone production and survival. Ann Surg 1987;206:115–125. 2 Eriksson B, Arnberg H, Lindgren PG, Lörelius LE, Magnusson A, Lundqvist G, Skogseid B, Wide L, Wilander E, Öberg K: Neuroendocrine pancreatic tumours: Clinical presentation, biochemical and histopathological findings in 84 patients. J Intern Med 1990;228:103–113. 3 Janson ET, Holmberg L, Stridsberg M, Eriksson B, Theodorsson E, Wilander E, Öberg K: Carcinoid tumors. Analysis of prognostic factors and survival in 301 patients from a referral center. Ann Oncol 1997;8:685–690. 4 Norheim I, Theodorsson-Norheim E, Brodin E, Öberg K: Tachykinins in carcinoid tumors. Their use as a tumor marker and possible role in the carcinoid flush. J Clin Endocrinol Metab 1986;63:605–612. 5 McGuigan JE, Wolfe MM: Secretin injection test in the diagnosis of gastrinoma. Gastroenterology 1980;79:1324–1327. 6 Wiedemann B, Huttner WB: Synaptophysin and chromogranins/secretogranins – Widespread constituents of distinct type of neuroendocrine vesicles and new tools in tumour diagnosis. Virchows Arch B Cell Pathol Incl Mol Pathol 1989;58:95–121. 7 Winkler H, Fischer-Colbrie B: The chromogranins A and B, the first 25 years and future perspectives. Neuroscience 1992;49:497–528. 8 Iacangelo AL, Eiden LE: Chromogranin A: Current status as a precursor for bioactive peptides and a granulogenic/sorting factor in the regulated secretory pathway. Regul Pept 1995; 58:65–88. 9 Eiden LE, Huttner WB, Mallet J, O’Connor DT, Winkler H, Zanini AA: A nomenclature proposal for the chromogranin/secretogranin proteins. Neuroscience 1987;21:1019–1021. 10 Blaschko H, Comline RS, Schneider FH, Silver M, Smith AD: Secretion of a chromaffin granule protein, chromogranin from the adrenal gland after splanchnic stimulation. Nature 1967;215:58–59.

Tumor Markers in Neuroendocrine Tumors

11 Deftos LJ: Chromogranin A, its role in endocrine function and as an endocrine and neuroendocrine tumor marker. Endocr Rev 1991; 12:181–187. 12 O’Connor DT, Burton D, Deftos LJ: Immunoreactive chromogranin A in diverse polypeptide hormone producing tumors and normal endocrine tissues. J Clin Endocrinol Metab 1983;57:1084–1086. 13 Stridsberg M, Öberg K, Li Q, Engström U, Lundqvist G: Measurements of chromogranin A, chromogranin B (secretogranin I), chromogranin C (secretogranin II) and pancreastatin in plasma and urine from patients with carcinoid tumours and endocrine pancreatic tumours. J Endocrinol 1995;144:49–59. 14 Eriksson B, Arnberg H, Öberg K, Hellman U, Lundqvist G, Wernstedt C, Wilander E: A polyclonal antiserum against chromogranin A and B – A new sensitive marker for neuroendocrine tumours. Acta Endocrinol (Copenh) 1990;122:145–155. 15 Wu HJ, Rozansky DJ, Parmer RJ, Gill BM, O’Connor DT: Structure and function of the chromogranin A gene. Clues to evolution and tissue specific expression. J Biol Chem 1991; 266:13130–13134. 16 Helman LJ, Ahn TG, Levine MA, Allison A, Cohen PS, Cooper MJ, Cohn DV, Israel MA: Molecular cloning and primary structure of human chromogranin A (secretory protein I) cDNA. J Biol Chem 1988;263:11559–11563. 17 Benedum UM, Lamouroux A, Konecki DS, et al: The primary structure of human secretogranin I (chromogranin B): Comparison with chromogranin A reveals homologous terminal domains and a large intervening variable region. EMBO J 1987;6:1203–1211. 18 Chanat E, Weiss U, Huttner WB, Tooze SA: Reduction of the disulfide bond of chromogranin B (secretogranin I) in the trans-Golgi network causes its missorting to the constitutive secretory pathway. EMBO J 1993;12: 2159–2168.

19 Iacangelo A, Fischer-Colbrie R, Koller KJ, Brownstein MJ, Eiden LE: The sequence of porcine chromogranin can serve as the precursor for the biologically active hormone pancreastatin. Endocrinology 1988;122:2339– 2341. 20 Tatemoto K, Efendic S, Mutt V, Makk HL, Feistner GJ: Pancreastatin, a novel pancreatic peptide that inhibits insulin secretion. Nature 1986;324:476–478. 21 Ishizuka J, Asada I, Poston GJ, Lluis F, Tatemoto K, Greeby GH Jr, Thompson JC: Effect of pancreastatin on pancreatic endocrine and exocrine secretion. Pancreas 1989;4:277–281. 22 Fasciotto BH, Trauss CA, Greeley GH, Cohn DV: Parastatin (porcine chromogranin A347419), a novel chromogranin A-derived peptide, inhibits parathyroid cell secretion. Endocrinology 1993;135:337–342. 23 Lewis JJ, Goldering JR, Asher VA, Modlin IM: Pancreastatin: A novel peptide inhibitor of parietal cell signal transduction. Biochem Biophys Res Commun 1989;163:667–673. 24 Schmidt WE, Siegel EG, Lamberts R, Gallwitz B, Creutzfeldt W: Pancreastatin: Molecular and immunocytochemical characterization of a novel peptide in porcine and human tissues. Endocrinology 1988;123:1395–1404. 25 Hutton JC, Hansen F, Peshavaria M: Proteolytic processing of chromogranin A in purified insulin granules. Formation of a 20 kDa N-terminal fragment (betagranin) by the concerted action of a Ca2+-dependent endopeptidase and carboxypeptidase. Biochemistry 1987;244: 457–464. 26 Drees BM, Hamilton JW: Processing of chromogranin A by bovine parathyroid secretory granules: Production and secretion of N-terminal fragments. Endocrinology 1994;134:2057– 2063. 27 Aardal S, Helle KB: The vasoinhibitory activity of bovine chromogranin A fragment (vasostatin) and its dependence of extracellular calcium in isolated segments of human blood vessels. Regul Pept 1992;41:9–18. 28 Russell J, Gee P, Liu SM, Angeletti RH: Inhibition of parathyroid hormone secretion by amino-terminal chromogranin peptides. Endocrinology 1994;135:337–342.


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29 Gasparri A, Sidoli A, Perez-Sanchez L, Longhi R, Siccardi AG, Marchisio PC, Corti A: Chromogranin A fragments modulate cell adhesion. J Biol Chem 1997;272:20835–20843. 30 Fasciotto BH, Gorr SU, De Franco DJ, Levine MA, Cohn DV: Pancreastatin, a presumed product of chromogranin-A (secretory proteinI) processing, inhibits secretion from porcine parathyroid cells in culture. Endocrinology 1989;125:1617–1622. 31 Galindo E, Rill A, Bader MF, Aunis D: Chromostatin, a 20-amino acid chromogranin chromogranin cell secretion. Proc Natl Acad Sci USA 1991;88:1426–1430. 32 Galindo E, Mendez M, Calvo S, Gonzalez-Garcia C, Cena V, Hubert P, Bader MF, Annis D: Chromostatin receptors control calcium channel activity in adrenal chromaffin cells. J Biol Chem 1992;267:407–412. 33 Deftos LJ, Gazdar AF, Hogue-Angeletti R, Mullen PS, Burton DW: Distinct patterns of chromogranin A-related species can be demonstrated in endocrine cells. Bone Miner 1990;9: 169–178.

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34 Larsson L, Alumets J, Eriksson B, Håkansson R, Lundqvist G, Öberg K, Sundler F: Antiserum directed against chromogranin A and B (CAB) is a useful marker for peptide hormoneproducing endocrine cells and tumours. Endocrine Pathol 1992;3:14–22. 35 Borch K, Stridsberg M, Burman P, Rehfeld JF: Basal chromogranin-A and gastrin concentrations in circulation correlate to endocrine cell proliferation in type-A gastritis. Scand J Gastroenterol 1997;32:198–202. 36 Eriksson B, Öberg K: Peptide hormones as tumor markers in neuroendocrine gastrointestinal tumors. Acta Oncol 1991;30:477–483. 37 Granberg D, Stridsberg M, Seensalu R, Eriksson B, Lundqvist G, Öberg K, Skogseid B: Plasma chromogranin A in patients with multiple endocrine neoplasia type 1. J Clin Endocrinol Metabol 1999;84:2712–2717.


38 Bajetta E, Ferrari L, Merlonetti A, Celio L, Procopio G, Artale S, Zilembo N, Di Bartholomeo M, Serregni E, Bombardieri E: Chromogranin A, neuron-specific enolase, carcinoembryonic antigen and hydroxyindole acetic acid evaluation in patients with neuroendocrine tumors. Cancer 1999;86:858–865. 39 Kadmon D, Thompson TC, Lynch GR, Scardino PT: Elevated plasma chromogranin-A concentrations in prostatic carcinoma. J Urol 1991;146:358–361. 40 Deftos LJ, Nakada S, Burton DW, Di Sant’Agnese PA, Cockett AT, Abrahamsson PA: Immunoassay and immunohistology studies of chromogranin A as a neuroendocrine marker in patients with carcinoma of the prostate. Urology 1996;48:58–62. 41 Sundaresan V, Reeve JG, Stenning S, Stewart S, Bleehen NM: Neuroendocrine differentiation and clinical behaviour in non-small cell lung tumors. Br J Cancer 1991;64:333–338. 42 Cryer PE, Wortsman J, Shah SD, Nowak RM, Deftos LJ: Plasma chromogranin A as marker of sympaticochromaffin activity in humans. 1991;260:E243–E246.



Imaging Procedures in Neuroendocrine Tumours J. Ricke a K.-J. Klose b a Department b Department

of Radiology, Charité Campus Virchow-Klinikum, Humboldt University, Berlin and of Radiology, Philips University, Marburg, Germany

Key Words Neuroendocrine tumour W Diagnostic imaging W Somatostatin receptor scintigraphy

Abstract Purpose: To give recommendations for diagnostic imaging workflow in suspected neuroendocrine tumours. Materials and Methods: Recommendations for imaging workflow were elaborated by consensus of researchers in neuroendocrine tumours. Results: Workflow charts are presented for: (1) nonfunctional and functional endocrine tumours of the pancreas (except insulinoma); (2) insulinoma; (3) ECL cell tumour; (4) assessment of unknown primaries in functional and nonfunctional neuroendocrine tumours of the gut; (5) assessment of metastases in functional and nonfunctional neuroendocrine tumours of the gut. Conclusions: The workflow charts facilitate diagnosis of neuroendocrine tumours as well as the design of controlled studies. Copyright © 2000 S. Karger AG, Basel

Introduction

The rarity of neuroendocrine tumours and therefor the lack of controlled studies often leave clinicians with an uncertainty of how to perform imaging procedures to

ABC

© 2000 S. Karger AG, Basel 0012–2823/00/0625–0039$17.50/0



assess the tumour localization [1–5]. In addition, neuroendocrine tumours present as an extremely heterogeneous entity [6–8]. During the years 1998 and 1999, the ENET group1 developed recommendations for imaging of neuroendocrine tumours mirroring the participant’s experiences from the fields of radiology, nuclear medicine, gastroenterology, surgery and pathology. This article summarizes the results of this consensus procedure with respect to the workflow of diagnostic imaging.

Imaging Workflow in Neuroendocrine Tumours

Figure 1 illustrates functional and nonfunctional endocrine tumours of the pancreas. Functional tumours of the pancreas usually comprise gastrinomas, insulinomas, VI-Pomas, glucagonomas, somatostatinomas and GRFomas. Since only insulinomas present different imaging properties compared to other functional neuroendocrine tumours of the pancreas [9], the workflow for insulinomas is given separately in figure 2. Nonfunctional tumours of the pancreas very much resemble functional tumours such as gastrinomas in their imaging properties. Hence, the identical workflow chart applies. 1 European Network of Neuroendocrine Tumours. Contact: Prof. K. Öberg, Department of Internal Medicine, Endocrine Oncology Unit, University Hospital, S–751 85 Uppsala (Sweden).

Dr. J. Ricke Department of Radiology, Charité Campus Virchow-Klinikum Medical Faculty, Humboldt University Berlin Augustenburgerplatz 1, D–13353 Berlin (Germany) E-Mail [email protected]

Fig. 1. Functional and nonfunctional endocrine tumours of the pancreas (without insulinoma): assessment of unknown primary.

Fig. 2. Assessment of unknown primary in suspected insulinoma.

Central diagnostic modality for the assessment of functional and nonfunctional neuroendocrine tumours of the pancreas is somatostatin receptor scintigraphy (SRS) with a sensitivity and specificity of up to 90 and 80%, respectively [10–12]. In patients with suspected gastrinoma, endoscopy should precede the diagnostic workup of the pancreas to detect potential neoplastic manifestations in the duodenum. This does specifically account for gastrinomas associated with a MEN I syndrome, showing duodenal manifestations in 80% of cases [3]. Furthermore, endoscopy enables the identification of peptic ulcers in functional gastrinomas. In patients with functional pan-

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creatic tumours and diffuse positive SRS, further diagnostic imaging may be restricted to documentation purposes, e.g. for monitoring of therapy or as part of controlled trials. For disease limited to the pancreas or in cases where tumour localization is pending but suspected in the pancreas, endosonography is regarded as the most sensitive modality [13–16]. CT and MRI show specific advantages in routine tumour staging and monitoring of therapy [9, 17, 18]. For somatostatin receptor-positive disease associated with MEN I [19], the diagnostic workup reflects established routines including MRI for recurrent hyperpara-

Ricke/Klose

Fig. 3. Assessment of ECL cell tumour.

thyroidism and tumour manifestation of the pituitary gland [20]. Primary diagnosis of hyperparathyroidism may be confirmed by cervical exploration [21]. Figure 2 illustrates the assessment of an unknown primary in suspected insulinoma. In nonmalignant insulinomas, endosonography is regarded the most sensitive modality for tumour detection [15]. However, MRI or CT may contribute additional information in a minor proportion of cases, e.g. in suspected malignancy. Insulinomas rarely express somatostatin receptors, thus limiting the sensitivity of SRS to 10–50% [9]. However, malignant insulinomas most likely show positive somatostatin receptors considerably more often. Hence, SRS is recommended for staging of confirmed or suspected malignant disease, even though controlled studies are missing. Patients with diffuse positive SRS indicating vast tumour spread do not require further workup except for documentation. The workup of localized disease associated with a MEN I syndrome should be completed by portal venous sampling considering the fact that in either malignant or nonmalignant insulinoma, multiple pancreatic manifestations are frequently seen [3, 22]. Figure 3 documents the assessment of ECL cell tumours. ECLomas are a rare tumour entity associated with the chronic atrophic gastritis, Zollinger-Ellison syndrome (ZES) and MEN I (up to 29% of ZES with MEN I present additional ECLomas) [23, 24]. These conditions are associated with high serum gastrin levels, leading to ECL formation in the gastric fundus and corpus. For local staging of ECLoma, endoscopy and endosonography of the stomach represent a sufficient workup [25]. In case of suspected malignancy, SRS is seen as the most valuable staging modality, completion of diagnosis should be gained with abdominal CT. In ECLomas associated with ZES,

accompanying gastrinomas should be identified (see fig. 1) [3, 23]. Figure 4 demonstrates the imaging workflow for the assessment of unknown primaries in functional and nonfunctional neuroendocrine tumours of the gut. In neuroendocrine tumours, patients often present with metastatic disease or clinical evidence such as flush and diarrhoea in combination with elevated serum or urinary markers [7]. To locate the primary tumour in these patients, SRS should be the central diagnostic modality. Sensitivity for the detection of neuroendocrine gut tumours of foregut, midgut and hindgut compartments is reported to be up to 90% with high specificity [10]. Specifically tumours of the hindgut show a tendency to be negative in SRS more often than others. Hence, in patients with negative SRS, an enteroclysma should follow upper and lower endoscopy during the diagnostic workup. If no diagnosis has been reached, CT of thorax and abdomen is the imaging modality of choice. Additional MIBG scintigraphy is the subject of further investigation [26]. Positive findings in SRS imply further workup following the technical recommendations given in the next chapter. In case of diffusely positive scintigraphy, further imaging should be limited to an enteroclysma when stenotic symptoms occur. Imaging for documentation purposes remains recommendable. Assessment of tumours located in the foregut depends on their location in either lung or digestive tract (stomach, duodenum, pancreas). For lung tumours, CT is the most effective imaging modality. For the other compartments, a combination of endoscopy and endosonography completed by CT of the abdomen should be performed. Except for the ascending colon, suspected midgut tumours are best visualized by abdominal CT or MRI; a

Imaging Procedures in Neuroendocrine Tumours


41

Fig. 4. Functional and nonfunctional neuroendocrine tumours of the gut (I): assessment of unknown primary.

Fig. 5. Functional and nonfunctional neuroendocrine tumours of the gut (II): assessment of metastases.

combination specifically of CT with an enteroclysma is regarded recommendable [27–29]. Due to technical limitations, the use of ileoscopy remains optional. For hindgut tumours and tumours of the ascending colon, lower endoscopy is the method of choice; completion should be gained by CT or MRI of the abdomen and, if further staging is desired, by endosonography. Finally, since hepatic lesions may occasionally not be visible in SRS, screening for neuroendocrine tumours of the gut should be completed by abdominal sonography, CT or MRI (if not already performed).

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Figure 5 illustrates the assessment of metastases in functional and nonfunctional neuroendocrine tumours of the gut. Metastatic disease in patients with confirmed neuroendocrine primary should be assessed primarily by using SRS [10–12]. Diffusely positive SRS does generally not require further imaging procedures. In negative SRS, CT screening of thorax and abdomen is the most efficient method. Localized disease identified by positive SRS may be assessed with the modality best suited for the individual location.

Ricke/Klose

Conclusions

As a consequence of the lack of controlled studies of neuroendocrine tumours, diagnostic strategies vary considerably among centres. However, if cost-effective and quality-controlled diagnostic imaging of neuroendocrine tumours is desired, standardized workflows are important. It should be considered that neuroendocrine tumours present a challenge for efficient diagnosis due to their outstanding biochemical properties such as frequent somatostatin receptor expression or hypervascularization of tumour tissue. The design of a general workflow chart for a tumour entity inevitably ignores individual considerations for a specific patient. In any case, diagnostic procedures have to be balanced with the question to be answered for the individual patient. Once the desired diagnosis has been reached, further diagnostic procedures should be avoided if no therapeutic consequences are expected. It is obvious that in clinical trials, imaging procedures may have to be used more aggressively to allow tumour documentation or further investigations. The term ‘no further investigation’ used in case of diffusely positive SRS reflects a general intention to limit

unnecessary and costly imaging procedures. However, imaging procedures are definitely needed to monitor the treatment course in the individual patient. Favourable devices for such treatment monitoring proved to be CT and MRI due to their superior documentation capabilities. Apparently, SRS has gained a central role in the diagnosis of neuroendocrine tumours, whereas tomographic procedures such as CT and MRI serve well for completion of the oncological workup and monitoring of treatment [12, 28, 30, 31]. Today, sonography is established as a cost-effective instrument during the follow-up of patients [17, 32]. Standard 18FDG PET is regarded not to be helpful for the diagnostic workup of neuroendocrine tumours. Other metabolites demonstrate promising results. However, their use is still under investigation [33, 34]. Angiography has lost its central role in the diagnosis of neuroendocrine tumours, with venous sampling for MEN I-associated insulinoma remaining as the only indication recommended [17, 22, 31]. In conclusion, efficient and successful imaging of neuroendocrine tumours demands an interdisciplinary approach combining rational and careful use of endoscopic, radiological and scintigraphic procedures.

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Imaging Procedures in Neuroendocrine Tumours

8 Wilander E, Lundquist M, Öberg K: Gastrointestinal carcinoid tumours: Histogenetic, histochemical, immunohistochemical, clinical and therapeutic aspects. Prog Histochem Cytochem 1989;19:1–87. 9 Modlin IM, Tang LH: Approaches to the diagnosis of gut neuroendocrine tumours: The last word (today). Gastroenterology 1997;112:583– 590. 10 Termanini B, Gibril F, Reynolds JC, Doppman JL, Chen CC, Stewart CA, Sutliff VE, Jensen RT: Value of somatostatin receptor scintigraphy: A prospective study in gastrinoma of its effect on clinical management. Gastroenterology 1997;112:335–347. 11 Lebtahi R, Cadiot G, Sarda L, Daou D, Faraggi M, Petegnief Y, Mignon M, Le Guludec D: Clinical impact of somatostatin receptor scintigraphy in the management of patients with neuroendocrine gastroenteropancreatic tumours. J Nucl Med 1997;38:853–858. 12 Krenning EP, Kwekkeboom DJ, Bakker WH, Breeman WA, Kooij PP, Oei HY, van Hagen M, Postema PT, de Jong M, Reubi JC, et al: Somatostatin receptor scintigraphy with [111InDTPA-D-Phe1]- and [123I-Tyr3]-octreotide: The Rotterdam experience with more than 1,000 patients. Eur J Nucl Med 1993;20:716– 731.

13 Cadiot G, Lebtahi R, Sarda L, Bonnaud G, Marmuse JP, Vissuzaine C, Ruszniewski P, Le Guludec D, Mignon M: Preoperative detection of duodenal gastrinomas and peripancreatic lymph nodes by somatostatin receptor scintigraphy. Gastroenterology 1996;111:845–854. 14 Ruszniewski P, Amouyal P, Amouyal G, Grange JD, Mignon M, Bouche O, Bernades P: Localization of gastrinomas by endoscopic ultrasonography in patients with Zollinger-Ellison syndrome. Surgery 1995;117:629–635. 15 Bansal R, Tierney W, Carpenter S, Thompson N, Scheiman JM: Cost effectiveness of EUS for preoperative localisation of pancreatic endocrine tumours. Gastrointest Endosc 1999;49: 19–25. 16 Zimmer T, Stolzel U, Bader M, Koppenhagen K, Hamm B, Buhr H, Riecken EO, Wiedenmann B: Gut endoscopic ultrasonography and somatostatin receptor scintigraphy in the preoperative localisation of insulinomas and gastrinomas. Gut 1996;39:562–568. 17 Andersson T, Erikkson B, Hemmingson A, Lindgren PG, Öberg K: Angiography, CT, MRI and ultrasonography in the detection of liver metastases from endocrine gastrointestinal tumours. Acta Radiol 1987;28:535–539.


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18 McCarthy SM, Stark DD, Moss AA, Goldberg HI: Computed tomography of malignant carcinoid disease. J Comput Assist Tomogr 1984;8: 846–850. 19 Lamberts SW, Hofland LJ, Lely AJ, de Herder WW: Somatostatin receptor expression in multiple endocrine neoplasia and in von HippelLindau disease. J Intern Med 1998;243:569– 571. 20 Bartynski WS, Lin L: Dynamic and conventional spin-echo MR of pituitary microlesions. Am J Neuroradiol 1997;18:965–972. 21 James C, Starks M, MacGillivray DC, White J: The use of imaging studies in the diagnosis and management of thyroid cancer and hyperparathyroidism. Surg Oncol Clin N Am 1999;8: 145–169. 22 Grama D, Skogseid B, Wilander E, Eriksson B, Martensson H, Cedermark B, Ahren B, Kristofferson A, Öberg K, Rastad J: Pancreatic tumours in multiple endocrine neoplasia type 1: Clinical presentation and surgical treatment. World J Surg 1992;16:611–619.

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23 Modlin IM, Esterline W, Kim H, Goldenring JR: Enterochromaffin-like cells and gastric argyrophil carcinoidosis. Acta Oncol 1991;30: 493–498. 24 Rindi G, Capella C, Solcia E: Cell biology, clinicopathological profile, and classification of gastro-enteropancreatic endocrine tumours. J Mol Med 1998;76:413–420. 25 Zimmer T, Ziegler K, Liehr RM, Stolzel U, Riecken EO, Wiedenmann B: Endosonography of neuroendocrine tumours of the stomach, duodenum, and pancreas. Ann NY Acad Sci 1994;15:425–436. 26 Taal BG, Hoefnagel CA, Valdes Olmos RA, Boot H: Combined diagnostic imaging with 131I-metaiodobenzylguanidine and 111In-pentetreotide in carcinoid tumours. Eur J Cancer 1996;11:1924–1932. 27 Semelka RC, John G, Kelekis NL, Burdeny DA, Ascher SM: Small bowel neoplastic disease: Demonstration by MRI. J Magn Reson Imaging 1996;6:855–860. 28 Sugimoto E, Lorelius LE, Erikkson B, Öber K: Midgut carcinoid tumours: CT appearance. Acta Radiol 1995;36:367–371.


29 Thiele J, Kloppel R, Schulz HG: CT-Sellink – A new method of evaluating the intestinal wall. Fortschr Geb Röntgenstr 1993;159:213–217. 30 Jetmore AB, Ray JE, Gathwright JB, McMullen K: Rectal carcinoids: The most frequent carcinoid tumour. Dis Colon Rectum 1992;35: 717. 31 Wallace S, Ajani JA, Charnsangavej C, DuBrow R, Yang DJ, Chuang VP, Carrasco CH, Dodd GD: Carcinoid tumours: Imaging procedures and interventional radiology. World J Surgery 1989;20:147–156. 32 Moertel CG: An odysee in the land of small tumours. J Clin Oncol 1987;5:1503–1522. 33 Orlefors H, Sundin A, Ahlstrom H, Bjurling P, Bergstrom M, Lilja A, Langstrom B, Öberg K, Eriksson B: Positron emission tomography with 5-hydroxytryprophan in neuroendocrine tumours. J Clin Oncol 1998;16:2534–2541. 34 Adam S, Baum R, Rink T, Schumm-Dräger PM, Usadel KH, Hör G: Limited value of 18FDG PET for the imaging of neuroendocrine tumours. Eur J Nucl Med 1998;25:79–83.

Ricke/Klose


Endoscopic Ultrasonography of Neuroendocrine Tumours T. Zimmer a H. Scherübl a S. Faiss a U. Stölzel b E.-O. Riecken a B. Wiedenmann c Departments of Internal Medicine/Gastroenterology, a Klinikum Benjamin Franklin, Freie Universität Berlin; Döbeln, and c Charité, Klinikum Rudolf Virchow, Humboldt Universität Berlin, Germany

b Krankenhaus

Key Words Neuroendocrine tumour W Insulinoma W Gastrinoma W Endoscopic ultrasonography

Abstract Neuroendocrine tumours (NETs) of the upper gastrointestinal tract are mainly located in the pancreas, stomach or duodenum. The aims of preoperative work-up are the localization of primary tumour(s), determination of local tumour invasion, of lymph node metastases and of the hormones secreted by the tumour. Endoscopic ultrasonography (EUS) offers ideal conditions to localize and stage NETs of the foregut. We report our results in localizing and staging NETs of the foregut in 40 patients examined between 1990 and 1997 by EUS, somatostatin receptor scintigraphy (SRS), computed tomography (CT), magnetic resonance imaging (MRI) and transabdominal ultrasound (US). EUS shows the highest sensitivity in localizing insulinomas compared with SRS, US, CT and MRI. US and EUS should be the first-line diagnostics if insulinoma has been proven by a fasting test. Further diagnostic procedures are unnecessary in most cases. Further diagnostics such as CT or MRI to search for distant metastases are necessary in large tumours or

ABC

© 2000 S. Karger AG, Basel 0012–2823/00/0625–0045$17.50/0



local invasive tumours. EUS shows the highest accuracy to detect or exclude pancreatic gastrinomas, but fails to detect extrapancreatic gastrinomas in about 50%. The combination of EUS and SRS gives additional information. First-line diagnostics in gastrinoma patients should be SRS and CT or MRI. If no metastases are detected, EUS should be the next preoperative imaging procedure. In nonfunctional NETs, EUS provides the best information on local tumor invasion and regional lymph node involvement. Copyright © 2000 S. Karger AG, Basel

Introduction

Neuroendocrine tumours (NETs) of the upper gastrointestinal tract are mainly located in the pancreas, stomach or duodenum. The aims of preoperative work-up are the localization of primary tumour(s), determination of local tumour invasion, of lymph node metastases and of the hormones secreted by the tumour. Functional tumours, such as insulinomas and gastrinomas, compromise approximately half of all foregut tumours. The treatment of choice is surgical removal of the tumour [1, 2].

Thomas Zimmer, MD Abteilung für Innere Medizin St. Elisabeth-Krankenhaus Koblenzer Strasse 91, D–54516 Wittlich (Germany) Tel. +49 6571 15 1201, Fax +49 6571 15 1218

Due to characteristic neuroglycopenic symptoms, insulinomas are usually diagnosed when still small, resectable and not yet metastatic. Insulinomas are located in the pancreas in almost all cases. Because of small size, localization is the main problem in the preoperative work-up for insulinomas [3]. Gastrinomas are associated with typical clinical symptoms like ulcer disease or gastroesophageal reflux disease. Gastrinomas are metastatic and multilocular in almost half of the cases at the time of diagnosis. More than half of gastrinomas are located extrapancreatic in the duodenal wall or in extraintestinal lymph nodes and are often difficult to detect [4, 5]. Nonfunctional NETs are usually diagnosed by the occurrence of liver metastases or as an incidental finding by endoscopy. In the second case, locoregional staging, especially depth of tumour infiltration into the gastrointestinal wall, is of clinical interest. Endoscopic ultrasonography (EUS) allows detailed visualization of the whole pancreas and almost all parts of the gastric and duodenal wall with high resolution. The gastrointestinal wall layers and pathological structures as small as 2–3 mm in size can be visualized by EUS. Several studies have shown that EUS is a highly sensitive imaging procedure for pancreatic endocrine tumours, detecting 80–90% of the lesions [6–9], whereas extrapancreatic tumours could be localized with lower sensitivities [10– 13]. Here we review the results obtained with EUS in diagnosis of NETs (especially insulinomas and gastrinomas) and give a guideline when to evaluate patients with NETs by EUS.

Techniques of EUS, Patients and Methods Echoendoscopes, used in the upper gastrointestinal tract, consist in most cases of side-viewing endoscopes with an ultrasonic transducer incorporated into the rigid tip of the instrument. EUS can be performed with two types of ultrasonic scanner: (1) Endoscopes equipped with a linear scanner or parallel-sector scanner (Pentax FG 32/38): A side-viewing endoscope is combined with a 5- and 7.5-MHz ultrasonic transducer fixed distal to the optics. The section of ultrasound is 105°. Scanning is performed in a plane parallel to the shaft axis of the instrument’s tip. (2) Endoscopes equipped with a radial sector scanner (Olympus GF/JF UM): A side-viewing endoscope is combined with a 7.5- and 12/20-MHz ultrasonic transducer, located distal to the side-viewing optics. The ultrasonic section of 360° is perpendicular to the shaft axis of the instrument’s tip. Patients are examined lying in the left lateral decubitus position. After introducing the echoendoscope into the descending duodenum, ultrasonic examination is performed by withdrawing the instrument.

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Parts of the gastric and duodenal wall as well as the whole pancreatic area can be visualized with high resolution. Examination of the pancreatic head is performed with a scanner position in the first and second part of the duodenum. Body and tail of the pancreas are investigated with the scanner position in the stomach. A water-filled balloon at the tip of the instrument and filling of the stomach with about 400 ml of water are necessary for fluid interface between the scanner and the gastrointestinal wall. Patients Forty patients (female 23, male 17; mean age 49 years, range 8– 82) with insulinomas (13), gastrinomas (11) or gastropancreatic nonfunctional NETs (16) were prospectively examined by EUS, somatostatin receptor scintigraphy (SRS), transabdominal ultrasound (US), computed tomography (CT) and magnetic resonance imaging (MRI) at the Benjamin Franklin Hospital from 1990 until 1997. All patients were screened for hyperparathyroidism by determination of serum calcium and parathyroid hormone levels and for pituitary tumour disease by determination of serum prolactin hormone levels. The various imaging techniques were compared for their diagnostic efficacy in localizing tumours and their metastases. All investigations were carried out within 4 weeks and assessed by maximally two experienced investigators. Methods Intraoperative US was performed in patients undergoing surgery. Duodenal transilluminations were done in all gastrinoma patients. However, the latter two methods were not included for evaluation in our study protocol. EUS examinations were carried out with echoendoscopes, using an ultrasound frequency of 7.5 or 12 MHz and scanning in a plane perpendicular to the shaft axis of the endoscope (GF-UM 3/20, Olympus). The transabdominal US examinations were done using mechanical sector scanners and a sound frequency of 3.5 or 5 MHz (LSC 7000, Picker). CT examinations were performed after oral and intravenous bolus contrast application (Somatom DRH, Siemens, Erlangen, Germany). The total abdomen was examined in 8-mm and the pancreatic region in 4-mm planes. MRI examinations were done with a 1.5-Tesla (Magnetom GBSII, Siemens) in 8-mm-thick transverse sections using 3 pulse sequences. T1-weighted (SE 500/15), T2weighted (SE 2.300/90) and fast T1-weighted (GRE 160/5/80°) spinecho sequences were used. The SRS examinations were carried out after an intravenous bolus of 100–200 MBq 111In-labeled pentetreotide (Octreoscan 111, Mallinckrodt Diagnostica, Petten, The Netherlands). Planar images were recorded with a large-field-view gamma camera (Orbiter 7500, Siemens) equipped with a 360-keV parallelhole collimator. All patients underwent anterior and posterior wholebody static scintigraphy. Planar images were obtained 4, 24, and in selected cases, 48 h after injection of the radioligand. Single-photon emission computed tomography (SPECT) was performed 24 h after injection. SPECT (360° rotation in 32 min, matrix 64 ! 64), was done using a Sopha DS 7 camera (Sopha Medical, Frankfurt am Main, Germany) with a medium-energy parallel-hole general-purpose collimator; images were reconstructed with filtered back projection and Chang correction in 6.7-mm slices. Digital (planar) images were analyzed quantitatively by the region-of-interest method. Data were not corrected for transmission absorption or self-attenuation. Liver uptake was calculated from the anterior view, whereas uptakes of the spleen and kidneys were calculated from the posterior view. This technique has previously been described in detail [14, 15].

Zimmer/Scherübl/Faiss/Stölzel/Riecken/ Wiedenmann

Fig. 1. Insulinoma (*) located in the body of the pancreas imaged by EUS (a) and MRI (b). P = Pancreas, L = liver,

N = left kidney.

Results

Seventeen neuroendocrine tumour lesions were histologically verified in 13 insulinoma patients. All tumours were intrapancreatic. The mean tumour diameter was 1.7 cm. Two female patients had a malignant insulinoma with liver metastases. The primary lesion as well as the liver metastases of these 2 patients could be confirmed by biopsy. In 1 patient with evidence of multiple endocrine neoplasia type I (MENI), 5 tumours were surgically confirmed. The location of 16/17 tumours could be visualized using EUS (sensitivity 94%). Only 2/17 tumours could be localized by SRS and US (sensitivity 12%), 5/17 by CT (sensitivity 29%), and 2/16 by MRI (sensitivity 13%). Eight of 17 insulinoma lesions (47%) could only be localized by EUS. Endosonographically, 11 of 13 visualized insulinomas displayed a hypoechoic (compared to the pancreas parenchyma), homogeneous inner structure and mostly a smooth delineation (fig. 1). A total of 15 separate primary tumour lesions were histologically confirmed in 11 gastrinoma patients. Nine tumours were situated intrapancreatically (head 7, body 1, tail 1), 4 in the duodenal wall, 1 in a juxtaduodenal lymph node and 1 intrahepatically. None of the gastrinoma patients showed evidence of MENI. Four patients had two gastrinoma lesions and 7 patients had one lesion, respectively. The mean tumour diameter was 2.0 cm (pancreas 2.1 cm, duodenal 1.6 cm). Four patients had a malignant tumour with infiltration of the portal vein (1), the superior mesenteric vein (1) and liver metastases (2).

Endoscopic Ultrasonography of Neuroendocrine Tumours

13/15 tumours (sensitivity 87%) could be visualized with SRS. 12/15 tumours could be localized by EUS (sensitivity 80%) and 4/15 by US, CT and MRI (sensitivity 27%), respectively. The smallest tumours visualized by EUS were an 8-mm tumour of the duodenal wall and a 5-mm tumour in the pancreatic head. Ten of 15 gastrinoma lesions (66%) were identified only by EUS and SRS. All patients were operated and all 15 tumours could be staged by surgical and pathological examination. 83% of tumours were found to have been staged correctly by EUS. The visualized duodenal tumours were endosonographically restricted to the middle hyperechoic layer (submucosal layer) (fig. 2). Endosonographically, 8 of the 12 visualized gastrinomas displayed a hypoechoic (compared to the pancreas parenchyma), homogeneous inner structure and a smooth delineation (fig. 2). Only 3 gastrinomas with a tumour diameter of 13 cm had an inhomogeneous, hyperechoic inner structure with hypoechoic to nonechoic parts and were irregularly demarcated. A total of 21 separate primary tumour lesions were histologically confirmed in 16 patients with nonfunctional gastropancreatic NETs. Nine tumours were situated intrapancreatically (head 5, body 3, tail 1), 8 in the gastric and 4 in the duodenal wall (duodenal 2, papilla major 1, papilla minor 1). None of the patients showed evidence of MENI. The mean tumour diameter was 1.8 cm (pancreas 3.1 cm, duodenal 0.9 cm, gastric 0.7 cm). 18/21 tumours (sensitivity 86%) could be visualized with EUS. 10/21 tumours could be localized by SRS (48%), 8/21 by CT (38%), 5/20 by MRI (25%) and 6/21 by US (29%). Eleven patients were operated and 13 tumours could be staged by surgical or by pathological examina-


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Fig. 2. Gastrinoma in the head of the pancreas and in the wall of the duodenal bulb. a EUS of the pancreatic tumour. * = Tumour; Ao = abdominal aorta; Ws = vertebral column; Vc = inferior vena cava; P = pancreas. b Duodenal tumour. * = Tumour; D = duodenal bulb; → = muscular layer of the duodenal wall. c SRS with pathological hot spots projecting over the pancreatic head (*) and duodenal bulb (→). N = Kidney; L = liver; M = spleen. d Endoscopy showing a submucosal tumour of the postpyloric duodenal bulb (→).

tion. 82% of tumours visualized were staged correctly by EUS, 70% by CT, 50% by MRI and 33% by US. The visualized papilla tumours were hypoechoic and endosonographically restricted to the middle hyperechoic layer (submucosal layer). Gastric and duodenal wall tumours were also hypoechoic and mostly well demarcated. ECLomas in patients with hypergastrinemia were restricted to the mucosal or submucosal layers, duodenal tumours to the submucosal layer. Only one gastric tumour without hypergastrinemia infiltrated all gastric layers and reached the serosal layer. Small pancreatic tumours were

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hypoechoic, homogeneous and smooth delineated, but large tumours had an inhomogeneous, hyperechoic inner structure with hypoechoic to nonechoic parts and were irregularly demarcated.

Discussion

The high sensitivity of EUS in the localization of insulinomas confirms the results of other studies reporting sensitivities of 57–92% (table 1) [6–10, 16–20]. The sensi-


Table 1. Results of various studies in localizing insulinomas

Method

Sensitivity mean, %

Sensitivity range, %

US CT MRI EUS SRS Angiography Calcium provocation Portal venous sampling Intraoperative US Surgical palpation

43 (15) 32 (38) 48 (17) 75 (92) 51 (15) 54 88 83 89 82

0–63 0–73 0–100 57–92 0–53 20–86 75–100 75–100 69–100 42–100

Results in parentheses represent UKBF in localizing sporadic insulinomas in 13 patients. Data are from 6–10, 16–20.

Published results of the various imaging methods for gastrinoma localization are consistent with our results and show sensitivities of about 70% for EUS and 75% for SRS (table 2) [8–10, 23–32]. Results of EUS in detecting duodenal gastrinomas show sensitivities of only 50%. The generally small duodenal gastrinomas can be intraoperatively visualized in about 60–90% by palpation, transduodenal illumination and in all cases by direct exploration of the duodenum after duodenotomy [33, 34]. To find such small tumours, an exact surgical exploration of the duodenum, liver and pancreas must be performed in combination with intraoperative US and duodenal transillumination. Apart from localizing primary lesions, SRS is also extremely valuable for detecting further primary lesions and metastases, not revealed by CT and US [30–32].

Summary and Conclusions Table 2. Results of various studies in localizing gastrinomas

Method

Sensitivity mean, %

Sensitivity range, %

US CT MRI EUS SRS Angiography Secretin Portal venous sampling Intraoperative US Surgical palpation Endoscopic transillumination Endoscopy

22 (27) 38 (27) 31 (27) 67 (80) 74 (87) 44 83 76 52 78 70 42 (25)

6–71 4–100 20–100 40–100 48–100 0–80 54–100 17–100 26–83 42–100 64–83 38–45

Results in parentheses represent UKBF in localizing gastrinomas in 11 patients without MENI. Data are from 8–10, 23–32.

tivity of EUS thus compares favourably with that of more invasive procedures such as exploratory laparotomy with palpation and surgical US [21]. Moreover, EUS plays a special role in patients with MENI, who frequently have multiple intrapancreatic tumours that are often not detected by US, CT or other invasive procedures [22]. In contrast to gastrinomas, insulinomas are less sensitively detected by SRS. Up to 50% of all insulinomas were reported to be detected by SRS [23] (table 1). In our hands, SRS was less sensitive (12% sensitivity).

Endoscopic Ultrasonography of Neuroendocrine Tumours

EUS shows the highest sensitivity in localizing insulinomas compared with SRS, US, CT and MRI. Only invasive methods such as portal venous sampling, surgical palpation or intraoperative US reach similar sensitivities. US and EUS should be the first-line diagnostics if insulinoma has been proven by a fasting test. Further diagnostic procedures are unnecessary in most cases. If EUS fails to localize an insulinoma, despite a positive (correctly performed) fasting test, patients should be operated and be evaluated by intraoperative US and surgical palpation. Further diagnostics such as CT or MRI to search for distant metastases are necessary in large tumours or local invasive tumours. EUS shows the highest accuracy to detect or exclude pancreatic gastrinomas, but fails to detect extrapancreatic gastrinomas in about 50%. Results of EUS are comparable with SRS in gastrinoma patients, but the combination of both gives additional information. First-line diagnostics in gastrinoma patients should be SRS, CT or MRI to detect primary tumour lesion and to exclude distant metastases (e.g. liver). If no metastases are detected, EUS should be the next preoperative imaging procedure. During operation, intraoperative US, endoscopic transillumination and surgical palpation should be performed in all cases. In nonfunctional NETs, EUS provides the best information on local tumor invasion and regional lymph node involvement. In metastatic nonfunctional NETs, EUS is helpful to detect or exclude pancreatic tumours, but the findings are usually without therapeutic consequence.


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13 de Kerviler E, Cadiot G, Lebtahi R, Faraggi M, Le Guludec D, Mignon M: Somatostatin receptor scintigraphy in forty-eight patients with the Zollinger-Ellison syndrome. Eur J Nucl Med 1994;21:1191–1197. 14 Zimmer T, Ziegler K, Bäder M, Fett U, Hamm B, Riecken EO, Wiedenmann B: Localisation of neuroendocrine tumours of the upper gastrointestinal tract. Gut 1994;35:471–475. 15 Scherübl H, Bäder M, Fett U, Hamm B, Schmidt-Gayk R, Koppenhagen K, Dop M, Riecken EO, Wiedenmann B: Somatostatin receptor imaging of neuroendocrine gastroenteropancreatic tumors. Gastroenterology 1993; 105:1705–1709. 16 Bansal R, Kochman ML, Bude R, Nostrant TT, Elta GH, Thompson NW, Scheiman JM: Localization of neuroendocrine tumors utilizing linear-array endoscopic ultrasonography. Gastrointest Endosc 1995;42:76–79. 17 Meyenberger C, Bertschinger P, Zala GF, Marincek B: Endosonography in diagnosis of insulinoma. Ultraschall Med 1995;16:224–227. 18 Schumacher B, Lübke HJ, Frieling T, Strohmeyer G, Starke AAR: Prospective study on the detection of insulinomas by endoscopic ultrasonography. Endoscopy 1996;28:273– 276. 19 Pitre J, Soubrane O, Palazzo L, Chapuis Y: Endoscopic ultrasonography for the preoperative localization of insulinomas. Pancreas 1996;13:55–60. 20 De Angelis C, Repici A, Arena V, Pellicano R, Rizzetto M: Preoperative endoscopic ultrasonography in decision-making and management for pancreatic endocrine tumors: A 6-year experience. Endoscopy 1998;30(suppl 1):182– 186. 21 van Heerden AJ, Grant CS, Czako PF, Service FJ, Charboneau JW: Occult functioning insulinomas: Which localizing studies are indicated? Surgery 1992;112:1010–1015. 22 Galiber AK, Reading CC, Charboneau JW, Sheedy PF, James EM, Gorman B, Grant CS, van Heerden JA, Telander RL: Localization of pancreatic insulinoma: Comparison of pre- and intraoperative US with CT and angiography. Radiology 1988;166:405–408. 23 Krenning EP, Kwekkeboom DJ, Oei HY, de Jong RJB, Dop JF, Reubi JC, Lamberts SWJ: Somatostatin receptor scintigraphy in gastroenteropancreatic tumors: An overview of European results. Ann NY Acad Sci 1994;33: 416–424. 24 Höring G, Räth U, Rücker S, von Gaisberg U, Meincke J, Walendzik J, Dörr U, Bihl H: Somatostatinrezeptor-Szintigraphie in der Primärdiagnostik und Nachsorge bei Gastrinomen. Dtsch Med Wochenschr 1994;119:367– 374.


25 Cadiot G, Lebtahi R, Sarda L, Bonnaud G, Marmuse JP, Vissuzaine C, Ruszniewski P: Preoperative detection of duodenal gastrinomas and peripancreatic lymph nodes by somatostatin receptor scintigraphy. Gastroenterology 1996;111:845–854. 26 Modlin IM, Cornelius E, Lawton GP: Use of an isotopic somatostatin receptor probe to image gut endocrine tumors. Arch Surg 1995;130: 367–373. 27 Ruszniewski P, Amouval P, Amouval G, Grange JD, Mignon M, Bouche O, Bernades P: Localization of gastrinomas by endoscopic ultrasonography in patients with Zollinger-Ellison syndrome. Surgery 1995;117:629–635. 28 Schirmer WJ, Melvin WS, Rush RM, O’Dorisio TM, Pozderac RV, Olsen JO, Ellison EC: Indium-111-pentetreotide scanning versus conventional imaging techniques for the localization of gastrinoma. Surgery 1995;118:1105– 1113. 29 Weinel RJ, Kisker O, Joseph K, Welcke U, Zaraca F, Rothmund M: Somatostatin receptor scintigraphy in preoperative diagnosis of the site of endocrine gastrointestinal tumors. Chirurg 1994;65:849–855. 30 Alexander HR, Fraker DL, Norton JA, Bartlett DL, Tio L, Benjamin SB, Doppman JL, Goebel SU, Serrano J, Gibril F, Jensen RT: Prospective study of somatostatin receptor scintigraphy and its effect on operative outcome in patients with Zollinger-Ellison syndrome. Ann Surg 1998;228:228–238. 31 Termanini B, Gibril F, Reynolds JC, Doppman JL, Chen CC, Stewart CA, Sutliff VE, Jensen RT: Value of somatostatin receptor scintigraphy: A prospective study in gastrinoma of its effect on clinical management. Gastroenterology 1997;112:335–347. 32 Gibril F, Reynolds JC, Doppman JL, Chen CC, Venzon DJ, Termanini B, Weber C, Stewart CA, Jensen RT: Somatostatin receptor scintigraphy: Its sensitivity compared with that of other imaging methods in detecting primary and metastatic gastrinomas. Ann Intern Med 1996;125:26–34. 33 Frucht HF, Norton JA, London JF, Vinayek R, Doppman JL, Gardner JD, Jensen RT, Maton PN: Detection of duodenal gastrinomas by operative endoscopic transillumination. Gastroenterology 1990;99:1622–1627. 34 Sugg SL, Norton JA, Fraker DL, Metz DC, Pisegna JR, Fishbeyn V, Benya RV, Shawker TH, Doppman JL, Jensen RT: A prospective study of intraoperative methods to diagnose and resect duodenal gastrinomas. Ann Surg 1993;218:138–144.



Natural History of Neuroendocrine Enteropancreatic Tumors Michel Mignon Department of Hepato-Gastroenterology and Inserm U.410, Paris, France

Key Words Endocrine tumor W Pancreas W Gut W Natural history

Abstract Neuroendocrine enteropancreatic tumors (NEEPTs) include tumors developing from the pancreatic islets and the duodenal loop, and those developing from gut serotonin-secreting cells (mid-gut carcinoids). When functioning, they are rare tumors with frequently inconsistant clinical and biological characteristics. Provocative dynamic tests are very useful. Their natural history, with the exception of insulinomas and mid-gut carcinoids, is still incompletely known due to their rarity, relatively recent description, slow evolution and the absence of definitive reliable markers for prognosis. The following factors affect the natural history of NEEPTs. (1) Inclusion or not within the spectrum of multiple endocrine neoplasia type-1 (MEN1), such an integration often dramatically modifies the management strategy. In patients with MEN1, NEEPTs are often multiple, diffusely spread in the duodenopancreatic area and of small size. Definite cure by surgery is then very scarce. Mid-gut carcinoid tumors are the most protean NEEPTs. In contrast to appendiceal carcinoids, usually with a benign course, mid-gut carcinoids have a more malignant course and are very com-

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© 2000 S. Karger AG, Basel 0012–2823/00/0625–0051$17.50/0



mon indeed: local transmural invasion, early metastasis to lymph nodes and/or liver, and symptoms from hormone oversecretion. Carcinoid syndrome is usually well controlled by long-acting somatostatin analogs. (2) Disease spreading, i.e. liver and bone metastases, are the major causes of death in patients with NEEPTs. Liver metastases (LMs) are synchronous in about 75% of the patients with LMs but may develop during follow-up (about 25% of the patients with LMs were without LMs at the initial evaluation). Development of bone metastases has a very poor prognosis accompanying LM progression. Copyright © 2000 S. Karger AG, Basel

Introduction

Digestive endocrine tumors (DETs) include two main varieties of endocrine tumors: those which develop within the pancreatic islets and the duodenal loop, whether functioning or not, and carcinoid tumors essentially the serotonino-secreting tumors derived from the enterochromaffin (EC) cells. DETs are very rare, especially the duodenopancreatic tumors. Endocrine pancreatic tumors represent about 10% of the pancreatic tumors (table 1); serotonino-secret-

Prof. M. Mignon Department of Hepato-Gastroenterology Bichat – Claude Bernard Hospital, 46, rue Henri-Huchard F–75877 Paris Cedex 18 (France) Tel. +33 1 40 25 72 01, Fax +33 1 40 25 87 83, E-Mail [email protected]

Table 1. Endocrine tumors of the pancreas: anatomical, clinical and chance of cure by surgery Target organ

Main clinical symptoms

Mean tumor size, %

Main biological abnormalities

Provocative testing

Chance of definite or long-lasting cure, %

Insulinoma

Glucidic metabolism

Fasting hypoglycemia Whipple triad

85–90 small

Hyperinsulinism and raised C-peptide level

Prolonged fasting

90

Gastrinoma

Stomach (stimulation)

Peptic ulcer(s) + Diarrhea ZES

40 small

Gastric acid hypersecretion Hypergastrinemia

Secretin test

30

Vipoma

Pancreatic and biliary epithelia Intestinal epithelium Stomach (inhibition)

WDHA, i.e. Profuse aqueous diarrhea Hypokaliemia Achlorhydria1

60–100 large

Hypervipemia

Negative impact of fasting on diarrhea

20

Glucagonoma

Skin: amino acid metabolism Glucidic metabolism

Necrolytic migratory erythema Diabetes Cachexia

80–100 large

Hyperglucagonemia

Secretin test

30

Somatostatinoma

Gallbladder motility (inhibition) Pancreatic endo- and exocrine cells (inhibition) Stomach (inhibition)

Gallbladder lithiasis Diabetes Steatorrhea Achlorhydria

80–100 large

Hypersomatostatinemia

Pentagastrin or Tolbutamide or Calcium test

1

Adapted from Mignon and Jensen [2]. Verner-Morrison syndrome.

ing carcinoids (EC cell tumors) are relatively more prevalent representing 75% of DETs [1–4]. Although distinct in many respects (tables 1–3), DETs share some common characteristics [1, 2]: (1) inconstancy of typical clinical syndromes (present in no more than 50% of cases); (2) biological abnormalities, also inconstant, that will be unmasked by provocative testing; (3) integration or not within the syndrome of multiple endocrine neoplasia type 1 (MEN1), in many case, this condition modifies the management strategy; (4) DETs benefit from the most recent and sophisticated progresses in medical imagery especially somatostatin receptor scintigraphy (SRS) and endoscopic ultrasonography of the duodenopancreatic area (both these techniques have eased the development of a therapeutic strategy aiming at eradication of the tumoral process(es)), and (5) when tumoral eradication is incomplete or impossible, medical means are nowadays available that are effective at least some of the time, especially long-acting somatostatin analogs such as octreotide. Nonfunctioning endocrine tumors are relatively frequent (15–20% of endocrine pancreatic tumors). Tumors are often revealed later by non specific symptoms due to

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?


local expansion of the primary tumor or by liver metastases. Very rare DETs have been reported in the last decades [3, 4]. Tables 2 and 3 show the relevant features of those most frequently reported [3, 4]. Most prevalent are ACTHomas (about 110 cases reported, 6 personal cases), followed by PPomas (about 55 cases), GRFomas (about 50 cases exceptionally accompanied by acromegaly), neurotensinomas (about 50 cases) and parathyrinomas (about 35 cases). ACTHomas and GRFomas are often of extrapancreatic location. These rare forms of DET raise diagnostic and therapeutic problems. Diagnostic difficulties are related to the multiple hormonal production and release from these tumors, homologies between hormones from the same families leading to the mimicry of wellrecognized syndromes (for instance WDHA by neurotensinoma and Zollinger-Ellison syndrome (ZES) by CCKomas). The extreme rarity of these tumors precludes valid assessment of their natural history and their diagnostic and treatment strategies.

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Table 2. Rare forms of digestive endocrine tumors

DET type

Predominant hormone Dominant overproduction symptomatology

Prognosis

ACTHoma/corticotropinoma

ACTH → hypercortisolemia

Severe Cushing syndrome

Very poor

Ppoma/pancreatic polypeptidoma

PP → pure Ppoma (50% cases)

Pain, weight loss, watery diarrhea

Depends upon liver metastases and tumor size

Pancreatic serotoninoma/carcinoid

Serotonin

Atypical carcinoid syndrome

70% malignant 5-year survival 17%

Neurotensinoma (Ntoma)

Neurotensin in pure Ntomas

Mimick (WDHA) syndrome

Depends upon liver metastases and tumor size

Parathyrinoma (PTHoma)

PTHrP/PTH activity

Ectopic hypercalcemia S

Medium survival: 2 years 3 months (0–11 years)

GRFoma (GH releasing-factor producing tumor)

GRF (somatoliberin, somatocrin) → GH

Acromegaly + associated syndromes (ZES, Cushing ...)

Perhaps less malignant

From Delcore and Friesen [4].

Table 3. Rarer rare forms of digestive endocrine tumors

DET type

Predominant hormone overproduction

Dominant symptomatology

Prognosis

Pancreatic calcitoninoma

Calcitonin + VIP/PP

WDHA

Improved since chemo/biotherapy

Pancreatic enteroglucagonoma

Enteroglucagon

Glucagonoma syndrome

?

Pancreatic cholecystokininoma (CCKoma)

CCK

ZES-type Nl SG negative secretin test

?

Pancreatic GIPoma

GIP

WDHA

Poor

Pancreatic bombesinoma (GRPoma)

GRP-like immunoactivity

Diabetes, flush hemorragic gastritis

Liver metastases

From Delcore and Friesen [4]. SG = Serum gastrin.

Natural History Definition

Natural history can be defined as the spontaneous course of a disease. Its knowledge is important for disease management together with etiology and pathophysiology. For many neuroendocrine tumors of the duodenopancreatic area and digestive carcinoids (neuroendocrine enteropancreatic tumors, NEEPTs), the natural history of

Natural History of Neuroendocrine Enteropancreatic Tumors

the disease is not well known for the following reasons [5]: The relatively recent description of these tumors with the exception of insulinoma and midgut carcinoids. For (ZES), for example, the first description was only made in 1955 and substantial knowledge of the disease has been achieved only in the last two decades.


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Table 4. Epidemiology and pathology

Incidence n/million population

Tumors identified at surgery, %

Patients Patients with with metastases, % MEN1, %

EC cells

15

100

100

Carcinoids Gastrinoma Insulinoma VIPoma Glucagonoma Somatostatinoma Nonfunctioning Rare forms

0.5 0.8 Rare Rare Rare 0.2 ?

52–87 80–100 100 98–100 100 100 Usually

of DET

50 5–10 40 170 F70 60 25–100

Exceptional 18–24 5–10 Occasional Occasional Occasional High 0–25

Adapted from Norton [1] and Kaplan [3].

The extreme rarity of the NEEPTs (table 4). For example, the incidence of gastrinoma according to Norton [1] (table 4) is indeed estimated to approach 0.5 per million inhabitants per year. The extreme rarity of these diseases explains why rather small series were initially reported leading to heterogeneous management. However, several institutions deal with relatively large series of patients followed for long periods of time, thus offering the opportunity to appreciate the late course of the disease and particularly the frequencies of late recurrence and the diseaserelated death toll [6–8]. It is important to remember that in almost every study assessing the natural history of pancreatic endocrine tumors (PETs) or carcinoid tumors, only a small fraction of the total number of these tumors is being considered, specifically those that produced symptoms or were discovered by chance. The natural history of these small clinically silent neuroendocrine tumors (PETs and carcinoids) is largely unknown [9]. It is apparent that only a small fraction causes symptoms. These tumors are rarely detected because, until recently, a sensitive method to localize them did not exist. This may change in the future because of the recent availability and widespread use of 111In-[DPhe1]octreotide for neuroendocrine tumor localization using SRS [10–13]. The slow evolution of most of these NEEPTs as compared to exocrine tumors. ZES (gastrinoma) for instance does not escape to this peculiar disease course, a fact which encourages the medical attitude and explains, as already mentioned, the late recurrences 5–15 years after primary operation. Finally, further difficulties come from the absence of definite biological or histological markers of reliable prog-

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nostic significance. A multiparametric morphological approach has recently been suggested by Capella et al. [14] for nonfunctioning tumors of the pancreas. They use a Ki67 proliferative index of 12% and vascular and/or perineural microinvasion, which seems to accurately discriminate those patients with a poor prognosis because of the rapid evolution of tumors and, on the contrary, those with a slow evolution and a good prognosis. This has to be tested in other NEEPTs. Presently, the best prognostic element remains the presence of liver and bone metastases. For liver metastases (LMs), there are probably several subsets of patients, as suggested by our own data [15] and those of the NIH [16] in patients with ZES: one with a rapid tumoral mass evolution and, on the contrary, a group of individuals with slow evolution of the tumoral mass explaining why very long survival has been observed in many gastrinoma patients even after the development of LMs.

Factors Affecting the Natural History of NEEPTs

Once a NEEPT has been diagnosed on the basis of clinical symptoms (functioning tumors) and specific biological criteria [2, 5, 7], two main factors have to be looked for as they affect the natural course of the disease: recognition of MEN1 and disease-spread assessment, i.e. LMs and extra-abdominal metastases.

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Screening for MEN1 The MEN1 syndrome is an autosomal dominant inherited disorder characterized by the development of endocrine cell proliferation (hyperplasia or adenomas) mainly in the parathyroid glands, the endocrine pancreas/duodenum, the anterior pituitary and the adrenals. In about 60% of the patients, the syndrome is familial [17]. Screening for MEN1 is a fundamental step in NEEPT management. First, additional endocrinopathies may need specific management and may have an influence on the main tumor management. For instance, in patients with ZES, parathyroidectomy has to be performed before gastrinoma surgery. Secondly, familial screening should be considered. Thirdly, management of gastrinoma in MEN1 patients markedly differs from management of sporadic DETs. Finally, prognosis of NEEPTs might be different when MEN1 is present as seems to be the case in ZES patients. The MEN1 screening offered to patients with NEEPTs and their relatives in our department is summarized in table 5 [18]. NEEPTs can occur as part of MEN1 in a variable proportion of cases as indicated in table 4. The MEN1 gene has been located on the long arm of chromosome 11 (11q13). The gene has been cloned. The MEN1 gene probably acts as a tumor-suppressor gene, the inactivation of which gives rise to cell proliferation. The diagnosis of MEN1 is based either on the presence of MEN1-related endocrinopathies or a secondary isletcell tumor secreting peptides other than that secreted by the dominant symptomatic tumor in the patient or the presence of the latter variety of tumor or MEN1-related endocrinopathies in relatives. Primary hyperparathyroidism (HPT) is the most frequent MEN1-related endocrinopathy [19–21]. In patients with ZES, the prevalence of primary HPT in MEN1 varied from 50% to almost 100%, but the most recent data suggest that the actual prevalence reaches 90% or more. In MEN1-related HPT, the four glands are pathologic. Some authors use the term ‘hyperplasia’ and others use the term ‘multiple microadenomas’ for the same pathologic features. The diagnosis of primary HPT can be delayed by several years or can be present many years before the diagnosis of the NEEPT. Normocalcemic primary HPT may exist at the time of NEEPT diagnosis. Indeed, in a recent study by our group, using the oral calcium tolerance test, it was shown that the usual biological features of HPT, i.e. both high basal fasting calcium and iPTH levels, were lacking in 5 of 8 patients with primary HPT and ZES. Since primary HPT is most often the only associated endocrinopathy that would suggest the diagnosis of


Table 5. MEN1 screening in patients with DET

Clinical assessment of the patients and relatives Nephrolithiasis Neuroglycopenia symptoms Signs of diabetes and skin lesions Amenorrhea, galactorrhea Signs of acromegaly, Cushing’s syndrome, chiasmatic compression Systematic biological and radiological screening in the patients1 Pancreas Fasting glucose, insulin, C peptide Glucagon, VIP, somatostatin Parathyroids Basal serum-ionized calcium and PTH Oral calcium tolerance test when negative Pituitary gland Prolactin, FSH, LH, testosterone in men, estradiol in women (not done in women with normal menses) GH, IGF1 Morning cortisol and 24-hour urinary cortisol ACTH MRI (or CT scan) of the pituitary Adrenal glands CT scan of the adrenals If positive, serum and urinary K, 24-hour urinary cortisol in basal state and after dexamethasone administration (2 mg/day for 2 days) Gastric fundus Endoscopy with systematic fundic mucosal biopsies (Grimelius staining) 1

When MEN1 has been demonstrated in the patient, thymic and bronchial carcinoids should be looked for by chest CT scan in the patient and genetic screening is offered to the relatives. The biological and radiological MEN1 screening and ZES screening is offered to the relatives with positive genetic screening. When genetic screening is impossible, a reduced screening is offered to the asymptomatic relatives (gastrin, calcium, PTH, prolactin).

MEN1, an oral calcium tolerance test should be recommended systematically when typical biological features of primary HPT are lacking in any patient with ZES [22]. Specific data concerning endocrinopathies other than primary HPT in patients with NEEPTs are sparse even in ZES. In our experience with 45 patients with ZES and MEN1 [19], 31% had pituitary involvement, half of whom had increased prolactin serum levels and 35% had adrenal involvement, 13% having mild hypercorticism, the others being nonfunctioning. Symptomatic insulinomas coexisted with gastrinomas in 13% of our patients with the ZES. Due to the rare event of secondary symptomatic PET syndrome (1 insulinoma among 45 patients


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with the ZES) and the high frequency of multiple hormone elevation without any related symptoms, it has been recommended to limit screening for another PET syndrome to patients who develop new symptoms, after an initial negative screening. Severe Cushing’s syndrome, with very poor prognosis despite the administration of long-acting somatostatin analogs, is rarely observed in ZES (5%). It is due to ectopic ACTH secretion by metastases or the primary and is independent of MEN1. It should be noted that due to the prevalence of sporadic primary HPT in the population, caution must be considered when one relative of a ZES patient, especially an old one, has isolated primary HPT, particularly when it is due to a single adenoma. Caution must also be considered when a relative has an isolated adrenal mass (incidentaloma) or an isolated pituitary tumor, of which the respective prevalences could reach 2% upon examination with CT scan for the former and 10% with MRI for the latter in the general population. The association of MEN1 to NEEPTs makes the diagnostic and therapeutic management more difficult, especially as far as treatment of the tumor(s) is concerned (i.e., localization, eradication, and the chance of surgical cure). Indeed in MEN1 NEEPTs are often multiple and diffusely spread in the duodeno-pancreatic area and of small size. They can be located both in the pancreatic gland and the duodenum. Several types of endocrine tumors may coexist. In patients with ZES MEN1, it has been suggested that gastrinomas are preferentially located in the duodenum, the PETs being most often not gastrinomas, but this has not been confirmed either by us or by the NIH in Bethesda. Carcinoid tumors are the most protean NEEPTs [3]. They may present with gastrointestinal bleeding, abdominal pain, obstruction from tumor growth or tumorinduced mesenteric fibrosis or symptoms arising from tumor-secreted products or hormones, especially the carcinoid syndrome, when liver metastases have developed. In contrast to appendiceal carcinoids which usually have a benign course, small bowel, pancreatic and bronchial carcinoids have a more malignant course. Local transmural invasion, early metastasis to lymph nodes and liver, and symptoms from hormone oversecretion are common. The carcinoid syndrome is important to consider in the factors involved in determining survival in patients with carcinoid tumors. There is a clear correlation between carcinoid tumor mass and urinary 5-HIAA levels, which results from the breakdown products of serotonin

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excreted in the urine. By causing cardiac disease via a carcinoid crisis or through another mechanism, the carcinoid syndrome can be a significant cause of mortality in these patients if not adequately controlled. Therefore, similar to patients with other NEEPTs, patients with carcinoid tumors and the carcinoid syndrome have both a tumoral and hormone excess state that can contribute to mortality [9]. With the increased availability of long-acting somatostatin analogs (i.e. octreotide, lanreotide) which can control the symptoms of the carcinoid syndrome, the natural history of the carcinoid tumor per se is increasingly becoming the main determinant of long-term survival [9]. Incidence of MEN1 syndrome is exceptional in patients with midgut carcinoids [1, 5]. Disease Spread Assessment The ability of imaging modalities to localize primary NEEPTs and their metastases have been the subject of several reports [10–13], and is discussed elsewhere in these proceedings. The indications and the major interest in SRS in patients with NEEPT has been reported [13, 23]. SRS is nowadays first-line medical imagery in these patients, allowing a complete and simple evaluation of the disease spread. SRS should be mandatory in the patient’s work-up before curative surgery. Hepatic and bone metastases are the major causes of death in patients with DETs [15, 24, 25]. In patients with ZES for instance, although individual cases of patients with long-lasting survival have been observed, it is a common observation that less than half of the ZES patients with LM survive beyond 5 years and less than 30% beyond 10 years: the median survival at 5 years ranges from 17 to 53%. In contrast to LM, lymph node metastases, although as prevalent as LMs, do not seem to influence survival rate in ZES patients, at least at short term. The determinants of LM development clearly appear to be the size and location of the primary tumoral process [8, 15, 24–28]. LMs are present at the time of diagnosis (synchronous) in about 75–80% of the patients with LM but may develop during the follow-up (metachronous), although much less frequently (20–25% only of the patients without LM at the initial evaluation). From a personal series of 54 DET patients followed from 1970 to 1995 in our institution [15], including 34 ZES patients, LMs were disseminated in the both lobes in 80 % of cases. Fifty percent died after a median period of 27 (range 2–281) months after LM diagnosis. Actuarial survival rates since LM diagnosis were 46% at 5 years and

Mignon

38% at 10 years. There was no difference between patients with ZES and other types of endocrine tumors (nonfunctioning and carcinoids essentially). As suggested by the NIH report and by studies from our group, rapid growth seems to correlate with poor patient survival [15, 16]. In a first study concerning 35 patients with endocrine-type LMs (ELMs) including 20 cases of ZES, factors directly related to ELMs that could constitute major prognostic determinants for death were evaluated [29]. Among the various elements assessed during the observation period with a median duration of 35 (range 5–136) months, only rapid expansion of LMs (more than 25% within 6–12 months) appeared to be a major prognostic factor: the 5-year survival rate was 52% for evolutive ELMs versus 100% for non-evolutive ELMs. Furthermore the median interval between death and ELM mass increase was short, 6 (1–24) months. This finding is in keeping with the NIH’s observation [16]. Bone metastases (BMs) development is a very poor prognostic factor, accompanying LM progression. It has been reported to concern 12.5% of ZES patients: Among the 54 patients with ELM followed in our institution, BMs developed in 20 patients (37%): 25 and 61% in ZES and non-ZES patients, respectively [15]. The median delay for BM diagnosis was 7 (0–160) months after LM diagnosis. Seventy-five percent of these patients with BMs died versus 35% without BMs. BMs were noted in 47% of patients with LM progression versus 13% of those without LM progression. Accordingly, survival rates after diagnosis of LM progression were poorer in patients with BMs than in patients without. Survival rates after LM diagnosis did not differ with the variety of endocrine tumors, MEN1 versus non-MEN1 patients, patients with synchronous versus metachronous LM and patients with LM located in one lobe versus both lobes. BM development represents therefore a major risk factor for death. It also precludes liver surgery (hepatectomy or liver transplantation). Complete investigation of the tumoral spread is therefore mandatory before liver surgery: SRS is presently the best imaging technique to recognize extraabdominal metastases in these patients. In conclusion, in the last decades, clinical and biological knowledge of digestive endocrinology has rapidly been expanding. Taking advantage of the fantastic progress obtained in the field of digestive neurohormonal regulation, clinicians have learned to evoke overproduction of digestive hormones in a variety of clinical situations which sometimes represent the caricatural expression of the hormones’ biological properties.

The most remarkable development and refinement of hormonal radioimmunoassays and medical imagery have considerably eased the diagnostis and the management of NEEPTs, allowing diagnosis at an earlier stage. Duodenopancreatic endocrine tumors and carcinoid tumors have many similarities in their natural history. Recent studies demonstrate that many similar variables are important prognostic factors for both, such as tumor size, extent of metastases (lymph node, liver, bone), extent of tumor invasion, the presence of numerous histological features (mitotic activity, capsular invasion, nuclear atypia), flow cytometric DNA results and measures of proliferation such as Ki 67. According to Jensen [9], despite these similarities, it is important that these two groups of tumors continue to be analyzed separately rather than considered in combined series as they are in many studies. Not only are these two general groups of tumors functionally different, but they also may differ in biologic behavior in terms of metastatic patterns of spread and aggressiveness. With the increased application of molecular methods it may be possible to understand the important molecular events involved in the pathogenesis of both of these tumors. Each of these new approaches may in the future make it possible to obtain important insights into the natural histories of these two gastrointestinal endocrine tumors and the ways in which they are similar or different. It remains for the future to evaluate the currently proposed therapeutic strategies, especially those which concern the treatment of the tumoral process(es) aiming at curing the patient, at preventing the metastatic spreading of the disease or at controlling it with efficacious and costeffective medications [30].



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References 1 Norton JA: Neuroendocrine tumors of the pancreas and duodenum. Curr Probl Surg 1994;31: 81–156. 2 Mignon M, Jensen RT (eds): Endocrine Tumors of the Pancreas. Recent Advances in Research and Management. Front Gastrointest Res Basel, Karger, 1995, vol 23, pp 1–482. 3 Kaplan LM: Endocrine tumors of the gastrointestinal tract and pancreas; in Fauci AS, Brauwald E, Isselbacher KS, Wilson ID, Martin JB, Kasper DL, Hauser SL, Longo DL (eds): Harrison’s: Principles of Internal Medicine ed 14. New York, McGraw-Hill, 1999, pp 584–592. 4 Delcore R, Friesen SR: Other rare tumors of the endocrine pancreas; in Howard J, Idezuki Y, Ihse I, Prinz R (eds): Surgical Diseases of the Pancreas. Baltimore, William & Wilkins, 1998, pp 789–815. 5 Jensen RT, Norton JA: Endocrine tumors of the pancreas; in Feldman M, Scharschmidt BF, Sleisenger MH (eds): Gastrointestinal and Liver Disease, ed 6. Philadelphia, Saunders, 1998, vol 1, pp 871–894. 6 Eriksson B, Oberg K, Skogseid B: Neuroendocrine pancreatic tumors. Clinical findings in a prospective study of 84 patients. Acta Oncol 1989;28;373–377. 7 Mignon M, Cadiot G: Gastrinomas (ZollingerEllison syndrome); in Howard J, Idezuki Y, Ihse I, Prinz R (eds). Surgical Diseases of the Pancreas. Baltimore, Williams & Wilkins, 1997, pp 733–744. 8 Cadiot G, Vuagnat A, Doukhan I, Murat A, Bonnaud G, Delemer B, Thiefin G, Beckers A, Veyrac M, Proye C, Ruszniewski Ph, Mignon M: Prognostic factors in patients with Zollinger-Ellison syndrome and multiple endocrine neoplasia type I. The experience of GENEM and GRESZE. Gastroenterology 1999;116: 286–293. 9 Jensen RT: Natural history of digestive endocrine tumors; in Mignon M, Colombel JF (eds): Recent Advances in the Pathophysiology and Management of Inflammatory Bowel Diseases and Digestive Endocrine Tumors. Paris, John Libbey Eurotext, 1999, pp 192–219. 10 Kwekkeboom DJ, Krenning EP, Oei HY, Van Eyck CHJ, Lamberts SWJ: Use of radiolabeled somatostatin to localize islet cell tumors; in Mignon M, Jensen RT (eds): Endocrine Tumors of the Pancreas. Recent Advances in Research and Management. Front Gastrointest Res. Basel, Karger, 1995, vol 23, pp 298–308.

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11 Gibril F, Reynolds JC, Doppman JL, Chen CC, Termanini B, Stewart CA, Jensen RT: Does the use of octreoscanning alter management in patients with Zollinger-Ellison syndrome: a prospective study? Gastroenterology 1995;108; A194. 12 Gibril F, Reynolds JC, Doppman JL, Chen CC, Venzon DJ, Termanini G: Somatostatin receptor scintigraphy: Its sensitivity compared with that of other imaging methods in detecting primary and metastatic gastrinomas. Ann Intern Med 1996;125:26–34. 13 Lebtahi R, Cadiot G, Sarda L, Daou D, Faraggi M, Petegnief Y, Mignon M, Le Guludec D: Clinical impact of somatostatin receptor scintigraphy in the management of patients with neuroendocrine gastroenteropancreatic tumors. J Nucl Med 1997;38:853–858. 14 Capella C, La Rosa S, Solcia E: Criteria for malignancy in pancreatic tumors. Endocr Pathol 1997;8;87–90. 15 Zayène A, Cadiot G, Vuagnat A, Bonnaud G, Mignon M: Survival factors in patients with endocrine liver metastases. Gastroenterology 1997;112:A496. 16 Sutliff VE, Doppman JL, Gibril F, Venzon DJ, Fang Y, Serrano J, Jensen RT: Growth of newly diagnosed untreated metastatic. Gastrinomas and predictors of growth pattern. J Clin Oncol 1997;15:2420–2431. 17 Skogseid B, Eriksson B, Lundqvist G, Lörelius LE, Rastad J, Wide L, Wilander E, Akerström G, Oberg K: Multiple endocrine neoplasia type I: A ten-year prospective screening study in four kindreds. J Clin Endocrinol Metab 1991; 73:281–287. 18 Mignon M, Cadiot G, Rigaud D, Ruszniewski Ph, Jaı¨s P, Lehy T, Lewin MJM: Management of islet cell tumors in patients with multiple endocrine neoplasia type I; in Mignon M, Jensen RT (eds): Endocrine Tumors of the Pancreas. Recent Advances in Research and Management. Front Gastrointest Res. Basel, Karger, 1995;vol 23;pp 342–360. 19 Mignon M, Ruszniewski Ph, Podevin Ph, Sabbagh L, Cadiot G, Rigaud D, Bonfils S: Current approach to the management of gastrinoma and insulinoma in adults with multiple endocrine neoplasia type I. World J Surg 1993;17: 489–497. 20 Chanson P, Cadiot G, Murat A, on behalf of GENEM 1: Management of patients and subjects at risk for multiple endocrine neoplasia type 1: MEN1. Horm Res 1997;47:211–220.


21 Eriksson B, Arnberg H, Lindgren PG, Lörelius LE, Magnusson A, Lundqvist G, Skogseid B, Öberg K, Wide L, Wilander E: Neuroendocrine pancreatic tumors: Clinical presentation, biochemical and histopathological findings in 84 patients. J Intern Med 1990;228:103–113. 22 Cadiot G, Houillier P, Allouch A, Paillard M, Mignon M, GRESZE: Interest of the oral calcium tolerance test in the early diagnosis of primary hyperparathyroidism and multiple endocrine neoplsia type I in patients with the Zollinger-Ellsion syndrome. Gut 1996;39:273–278. 23 Cadiot G, Bonnaud G, Lebtahi R, Sarda L, Ruszniewski P, Le Guludec D, Mignon M, GRESZE. Usefulness of somatostatin receptor scintigraphy in the management of patients with Zollinger-Ellison syndrome. Gut 1997;41: 107–114. 24 Weber HC, Venzon DJ, Lin JT, Fishbein VA, Orbuch M, Strader DB, Gibril F, Metz DC, Fraker DL, Norton JA, Jensen RT: Determinants of metastatic rate and survival in patients with Zollinger-Ellison syndrome: A prospective long-term study. Gastroenterology 1995;108: 1637–1649. 25 Mignon M, Cadiot G: Natural history of gastrinoma: Lessons from the past. Ital J Gastroenterol Hepatol 1999;31(suppl 2):98–103. 26 Madeira I, Terris B, Voss M, Denys A, Sauvanet A, Fléjou JF, Belghiti J, Ruszniewski Ph: Prognostic factors in patients with endocrine tumours of the duodenopancreatic area. Gut 1998;43:422–427. 27 Howard TJ, Sawicki MP, Stabile BE, Watt PC, Passaro E Jr: Biologic behavior of sporadic gastrinoma located to the right and left of the superior mesenteric artery. Am J Surg 1993;165: 101–106. 28 Ellison EC: Forty-year appraisal of gatrinoma. Back to the future. Ann Surg 1995;222:511– 524. 29 Mignon M, Ruszniewski Ph, Hochlaf S, Sobhani I, Cadiot G: Legmann P, René E, Bonfils S, Rigaud D, Benhamou G: Do endocrine liver metastases have intrinsic evolutive features helping decision making for liver transplantation? Gastroenterology 1991;100:A290. 30 Wiedenmann B, Jensen RT, Mignon M, Modlin I, Skogseid B, Doherty G, Öberg K: Preoperative diagnosis and surgical management of neuroendocrine gastroenteropancreatic tumors: General recommendations by a consensus workshop. World J Surg 1998;22:309–318.

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Interventional Treatment of Gastrointestinal Neuroendocrine Tumours H. Ahlman B. Wängberg S. Jansson S. Friman M. Olausson U. Tylén O. Nilsson Departments of Surgery, Radiology and Pathology at the Lundberg Laboratory for Cancer Research, Sahlgrenska University Hospital, Göteborg University, Göteborg, Sweden

Key Words Carcinoid W Endocrine pancreatic tumour W Surgical treatment W Hepatic arterial embolization W Liver transplantation

Abstract Neuroendocrine (NE) tumours of the gastrointestinal tract (carcinoids and endocrine pancreatic tumours) are rare diseases. In the presence of liver metastases these patients may suffer from disabling symptoms due to hormone overproduction. Patients with localized disease can be resected for cure and also patients with liver metastases can undergo potentially curative tumour resection. However, long-term follow-up of the latter cases indicates frequent recurrence of tumour. Using close biochemical monitoring of tumour markers combined with newer techniques for tumour visualization, these recurrences can often be diagnosed at an early stage so that repeat surgical procedures can be performed. During the last years very active surgery has been recommended for NE tumours, many of which have a relatively slow growth. Even in patients not amenable to curative liver surgery, debulking can be considered if the main tumour burden can be safely excised. The primary aim of this

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© 2000 S. Karger AG, Basel 0012–2823/00/0625–0059$17.50/0



type of treatment is palliation of hormonal symptoms. An important question is whether the aggressive treatment actually prolongs survival. No prospective studies have been performed. Such studies are hampered by the lack of strict surgical programs running over long periods and the relative rarity of NE tumours. Liver transplantation may be another treatment modality in selected cases. Copyright © 2000 S. Karger AG, Basel

Introduction

Originally, carcinoid tumours denoted all gastroenteropancreatic endocrine tumours which were subdivided according to their embryological origin into foregut carcinoids (stomach, duodenum, upper jejunum, pancreas), midgut carcinoids (lower jejunum, ileum, appendix and caecum) and hindgut carcinoids (colon, rectum) [1]. However, this classification does not accurately describe the natural history and clinical behaviour of all gut endocrine tumours. A revised classification of endocrine tumours has been proposed which takes into account tumour location, tumour size, angioinvasion, hormone production, histological grade and proliferative index [2]. Using this

Prof. Håkan Ahlman Department of Surgery, Sahlgrenska University Hospital SE–413 45 Göteborg (Sweden) Tel. +46 31 342 17 78, Fax +46 31 82 21 04 E-Mail [email protected]

Fig. 1. Characteristic morphological features of well-differentiated endocrine tumours (midgut carcinoid, MC) and aggressive neuroendocrine carcinomas (NEC). In the well-differentiated tumours, there are nests, or trabeculae, of tumour cells which are uniform in size and shape with low mitotic activity. In the NEC, tumour cells grow in solid sheets with frequent necrosis. There is considerable variation in the size of tumour cells, which display high mitotic activity. Immunocytochemical staining for endocrine markers such as chromogranin A (CgA) is strongly positive in both well-differentiated endocrine tumours and NEC. The proliferative activity, assessed by Ki67 immunostaining, is low in the well-differentiated endocrine tumours but high in the NEC.

classification, digestive neuroendocrine (NE) tumours are divided into well-differentiated tumours comprising carcinoid tumours of the gastrointestinal tract and endocrine pancreatic tumours (EPT) and poorly differentated (neuro)endocrine carcinomas (NEC or PDEC). NE tumours are usually diagnosed at a late stage when the disease is disseminated and causes hormonal symptoms. The workup of the patient includes CT, octreotide scintigraphy, biochemical screening and analysis of biopsy material. The histopathological examination aims at classifying the tumour regarding origin, biological behaviour and prognosis. While immunohistochemical staining for amine and peptide hormones may help to define the origin of the tumour, its biological behaviour has been notoriously difficult to assess. However, systematic histopathological analysis of NE tumours regarding growth pattern, degree of neuroendocrine differentiation and proliferative capac-

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ity may provide some information regarding prognosis [3]. Analysis of growth patterns includes evaluation of tumour cell growth, degree of cellular and nuclear atypia as well as presence, or absence, of necrosis. All factors are then summarized in a histological grade (G1–G3) scale. Neuroendocrine differentiation is evaluated by immunocytochemistry using antibodies against granule proteins (chromogranin A, synaptophysin) or cytosolic markers (NSE, PGP 9.5). Well-differentiated tumours are positive for all markers in a vast majority of tumour cells, while poorly differentiated endocrine tumours often lack, or have scant labeling for granule markers, while the cytosolic markers are retained. Proliferative capacity can be assessed by counting mitoses, but is more reliably estimated by analysing the number of tumour cells expressing the Ki67 antigen (fig. 1). Following a systematic histopathological analysis, NE tumours can be broadly divided

Ahlman/Wängberg/Jansson/Friman/ Olausson/Tylén/Nilsson

into well-differentiated tumours (comprising the majority of carcinoid tumours and EPT) with good or intermediate prognosis and neuroendocrine carcinomas (NEC), or poorly differentiated endocrine carcinomas (PDEC), which have a sinister prognosis. In the future, other factors such as angiogenic capacity of tumour cells and specific genetic changes may prove to be valuable tools in determining prognosis, biological behaviour and response to therapy.

Anaesthesiological Considerations

Prior to all interventional treatment of NE tumours the tumour type and hormone production must be assessed. Patients with midgut carcinoids are pretreated with longacting somatostatin analogues, e.g. octreotide (100 Ìg ! 4 s.c.), to prevent carcinoid crises during intervention. In case of a crisis reaction with bronchoconstriction and hypotension, adrenergic drugs should be avoided, since carcinoids may express adrenoceptors. Instead the surgical manipulation should be interrupted, volume substituted according to haemodynamic parameters and iterated doses of octreotide and cortisone given. Spinal anaesthesia with markedly reduced blood pressure may elicit carcinoid crises due to compensatory release of catecholamines from the adrenals in turn causing release of serotonin from the tumour [4]. For postoperative pain, epidural analgesia is preferred. Foregut carcinoids with excess production of histamine may cause an atypical carcinoid syndrome (generalized flushing, lacrimation, hypotension, cutaneous oedema, bronchoconstriction). Embolization therapy of liver metastases in these patients may be contraindicated due to noncontrollable release reactions. The cornerstone to correct diagnosis of these tumours is analysis of the main histamine metabolite, methylimidazole acetic acid (MelmAA) in urine [5]. Patients with histamine-producing tumours are optimally pretreated with a combination of somatostatin analogues, blockade of histamine receptors and cortisone. Histamine-liberating agents, e.g. morphine and tubocurarine, should be avoided. For glucagonoma and VIPoma, preoperative treatment with octreotide is usually adequate. Patients with gastrinoma maintain their medication with omeprazole for a period after removal of the tumour, since they have elevated gastric acid secretion due to hypertrophic gastric mucosa. Patients with large insulinomas may require hypertonic glucose after tumour removal and close glucose monitoring is necessary.

Intervention of Neuroendocrine Tumours

Surgical Treatment of Locoregional Disease

Surgery is the only option for curative treatment of NE tumours of the gastrointestinal tract. There is an evident correlation between the size of the primary tumour and presence of metastatic disease for lesions in certain regions, e.g. appendix and rectum [6]. Tumours !1 cm are cured by simple excision, or appendectomy, while larger or locally invasive tumours may require more extensive surgery [7]. In case of small intestinal midgut carcinoids, tumour size does not influence prognosis [8]. Two-thirds of small midgut carcinoids (!1 cm) have microscopic spread to lymph nodes and almost half of the patients have liver metastases [9]. Midgut carcinoids may be multiple and require resection of involved intestine and mesentery. The tumours are often accompanied by retroperitoneal fibrosis, or fibrotic mesenteric reaction, leading to constriction of the bowel lumen and intestinal ischaemia. The primary surgical treatment should therefore attempt sharp dissection of the high mesenteric lymph nodes to mobilize the small intestine for adequate resection in order to prevent local complications. Gastric carcinoids are divided into four subtypes: Hypergastrinemia induces hyperplasia of enterochromaffinlike (ECL) cells with subsequent progression into small multiple carcinoids in the corpus-fundus region. This condition can be associated with chronic atrophic gastritis (type I) or MEN-1 gastrinoma (type II). Type III tumours are larger solitary lesions, not associated with hypergastrinemia, and type IV tumours comprise very rare tumours, e.g. NEC. Type I lesions are fairly benign and probably best managed by endoscopic excision and follow-up. Type II lesions may require wider excision, or gastric resection. The role of antrectomy to reduce gastrin levels in order to reverse ECL cell carcinoidosis has not been fully evaluated. Type III tumours and NEC require cancer surgery when resectable [10]. The surgical management of EPT also varies with tumour type, e.g. simple enucleation of small benign insulinomas or pancreatic resection of larger, more aggressive tumours like gastrinoma, glucagonoma, VIPoma, or nonfunctional tumours. The MEN-1 pancreas is a surgical enigma, which may lead to repeat resection of successively diagnosed tumours eventually ending up with subtotal or completion pancreatectomy [11]. The surgical treatment of primary NE tumours and locoregional disease is not controversial. Treatment of patients with liver metastases is more demanding and may involve several interventional modalities. For future randomized studies of medical treatment in advanced


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disease, primary surgery is of importance to prevent local complications and correctly stage the tumour disease.

Interventional Treatment of Liver Metastases

The surgical/interventional treatment for liver metastases can be divided into four modalities: liver resection, vascular interventions, other peroperative procedures and liver transplantation. Liver Resection Anatomical liver resection can be performed for lesions situated within any of the 8 liver segments, each independently supplied by branches of the hepatic arteries and portal vein draining into one hepatic duct. For curative resections a good clearance margin is required, but for NE tumours resected for palliative reasons this margin can be narrowed and local tumour deposits can be removed by atypical resections. The extent of tumour disease is assessed by inspection, palpation and intraoperative ultrasound. The latter technique allows a good definition of liver anatomy and the relation between the tumour and the portal and hepatic venous pedicles. This is of special importance if techniques like cryo- or laser therapy are used. The most common variant in arterial anatomy is replacement of the right hepatic artery by a branch of the superior mesenteric artery. For right-sided liver tumours, preoperative angiography is therefore often valuable. For right hepatectomy it may be easy to control the right hepatic vein outside the liver rather than within the parenchyma during resection. Inflow control of the lesser omentum is usually applied by clamping. The safe duration of hepatic ischemia may exceed 1 h but can be reduced in hepatic dysfunction. Therefore the shortest possible clamping should be used, since it is associated with intestinal congestion and accumulation of toxic metabolites. For left hepatectomy the left and middle hepatic veins have their junction at variable levels, which means that they cannot always be controlled separately outside the liver. Division of the liver parenchyma most often includes the use of an ultrasonic surgical aspirator, which destroys liver cells leaving the more resistant biliary and vascular structures intact allowing exact placement of ligatures and clips. Metastasectomy of multiple deposits usually requires intermittent clamping of inflow vessels, diathermic incision around the individual lesions and blunt dissection.

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To obtain a good debulking effect, the principles of anatomical resection, wedge resection and metastasectomy are often combined. The operative mortality for elective liver surgery must be low. In most series, figures less than 5% are reported [12]. Prophylactic use of antibiotics has decreased infectious complications, but morbidity is still more frequently associated with sepsis than bleeding during surgery. For patients with multiple liver tumours, Farges and Belghiti [13] recently described the two-stage liver resection strategy. As the first step the primary tumour and the left lateral segment of the metastatic liver are removed and the right branch of the portal vein ligated. The latter procedure induces hypertrophy of the residual left liver in about 6 weeks. Thereafter, extended right-sided hepatectomy can be performed. Results Over the last decade, very active surgery has become increasingly more common as primary treatment of lowgrade NE tumours and their metastases. Curative liver surgery (= no gross residual tumour) must be considered for all patients with resectable disease. Palliative liver surgery can be considered for some patients with slow tumour growth and severe hormonal symptoms. Palliative liver resections are generally performed in patients in whom more than 90% of the tumour volume can be safely excised [14, 15]. In a series of patients with NE tumours 10 years ago, the liver resection rate with curative intent was about 10% [16, 17]. In our own consecutive series of 64 patients with the midgut carcinoid syndrome and liver metastases, 14 patients (22%) with unilateral liver lesion underwent intentionally curative liver surgery and normalized their tumour markers [18]. In recent American and French series [19–21], curative, or palliative, liver resections were performed in 40–50% of patients with NE tumours. In all referred series the mortality was low (!5%) and the complication rate below 30%. One evident problem with an active liver surgery program is that many resections are considered curative at the time of surgery but later proven not to be. With access to sensitive diagnostic tools (specific tumour markers, octreotide scintigraphy, spiral CT and MR) and careful follow-up, subclinical disease can be discovered and limited lesions re-resected [22]. If multiple metastases recur, vascular interventions, or other treatments, are at hand. In case there is no extrahepatic disease these patients may be candidates for liver transplantation. More than 10 years ago, Norton et al. [23] reported very active surgery in selected patients with advanced gas-


Vascular Interventions Markowitz [24] in 1952 first proposed hepatic vascular interventions as therapeutic procedures. The background for ischaemic treatment of NE tumours of the liver is their main blood supply from the hepatic artery [25, 26] of the tumour with maintained portal perfusion of the normal liver parenchyma. Ischaemia can be achieved by several techniques directed against the hepatic artery: ligation, selective embolization or temporary occlusion. The methods differ in completeness, distribution and duration of ischemia [27]. Ligation of the hepatic artery was clinically introduced more than 30 years ago [28]. It has largely been abandoned due to difficulties to obtain adequate ischaemia with the rich collateral blood supply to the liver, signifi-

cant mortality in end-stage patients and restricted possibilities for repeat vascular interventions. Selective embolization of the hepatic arteries causes a temporary, but complete ischaemia, since the arterial tree distal to the point of injection is filled with embolization material [29]. Contraindications are tumour burden exceeding 50% of the liver volume, occlusion of the portal vein, hyperbilirubinaemia and persistently elevated liver enzymes. Relative contraindications are contrast allergy, coagulopathies, extrahepatic tumour dominance or poor performance status of the patient [30]. In our experience, patients with tumour volumes exceeding 50% can also safely undergo this type of treatment using iterated superselective embolizations well separated in time. A similar strategy can also be followed for patients with previous hemihepatectomy and recurrent tumours in the residual liver. Immediately before embolization an arteriogram is performed to demonstrate the arterial anatomy, tumour blood flow and patency of the portal vein [31, 32]. Both absorbable and nonabsorbable embolization materials have been used, e.g. gelfoam powder and alcohol-Ivalon particles [30, 33]. The need to maintain an open route for repeat procedures excludes the choice of steel coils in the proximal arterial segment. Aiming at complete ischaemia of long duration, efforts have been directed toward smaller permanent emboli to obtain a good peripheral ischaemia without reaching the liver sinusoids [29, 34]. Chemoembolization is embolization combined with liver-targeted intra-arterial administration of chemotherapy. Hajarizadeh et al. [35] found symptomatic improvement to 5-FU in carcinoid patients with a mean duration of 2 years associated with significant tumour regression in half of the patients. Similar response rates were reported for several types of NE tumours using doxorubicin, mitomycin C and cisplatinum [36–38]; these authors reported tumour regression in one-third of their patients with a modified embolization procedure using iodized oil, doxorubicin and gelfoam particles. Some centres have reported good symptom palliation related to biochemical and clinical tumour responses in patients with the midgut carcinoid syndrome using temporary occlusion of the hepatic artery via external vessel loops (positioned during surgery) and repeat periods of applied ischaemia [39]. For long-term treatment, temporary occlusion can be applied via a port-operated tourniquet around the hepatic artery. A combination of peripheral embolization and temporary proximal occlusion can have additive effects in therapy-resistant cases. The embolization procedure is accompanied by liver pain, transient elevation of liver enzymes, nausea and late



trinoma, who all underwent major liver resections combined with other debulking procedures at tumour progression, followed by chemotherapy. On a short-term basis, good symptomatic relief and markedly reduced tumour markers were seen with this strategy. Of 42 consecutive patients with EPT treated over a 10-year period at the NIH, 17 were resected and 25 were deemed inoperable and medically treated [19]. There seemed to be a 5-year survival advantage in the surgically treated group (79%) versus the medically treated (28%) in this selected series. There was no operative mortality, but the tumour disease recurred in all surgically treated patients within 8 years. Such a delay of the tumour disease with a long medication-free interval, as also observed for midgut carcinoids [18], is a major therapeutic advantage in itself. McEntee et al. [15] from the Mayo Clinic also reported encouraging results in 37 patients treated with liver resection, including both carcinoids and EPT. They reported 17 curative resections (9 with hormonal symptoms). Eight were completely relieved of symptoms and 5 of these were alive after 2 years with no signs of residual disease. Twenty patients had palliative resection (16 with hormonal symptoms). Eight experienced symptom relief and 5 were alive after 1 year. In a review from the Mayo Clinic [20], 74 patients with metastatic NE tumours underwent liver resection including 41 patients with midgut carcinoids. The 4-year survival was 73%, even though nearly twothirds of the procedures were palliative. Symptomatic response to liver resection was seen in 90% with a mean duration of response of almost 20 months. Resection of large (110 cm) dominant lesions was clearly related to symptom relief and reduction of tumour markers. The role of resection for enhanced survival is still not proven in randomized studies.

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fever reaction. Embolization may lead to serious complications in individual patients (gallbladder ischaemia, pancreatitis, liver abscess, vascular damage and aneurysm formation, hepatorenal syndrome and hormonal crises). The mortality in the major series is less than 5% [30]. To minimize adverse reactions, several precautions can be undertaken, which include first of all an experienced interventional radiologist, but also the use of coaxial catheter systems for superselective catheterization with low risk of vascular damage, prophylactic use of octreotide, cholecystectomy at the time of primary surgery to eliminate the risk for gallbladder perforation, intravenous hydration and haemodynamic monitoring to reduce the risk for the hepatorenal syndrome [4, 27]. To monitor the outcome of ischaemic therapy, CT scan and biochemical tumour markers are used. According to our follow-up schedule these investigations are compared with preoperative results in the outpatient clinic 1 month after completion of embolization therapy [18]. It would be a therapeutic advantage if the response to ischaemia could be followed closer to embolization. For this purpose, we have tried to develop better monitoring systems. We observed bilateral tumour regression in individual patients only subjected to unilateral embolization indicating activation of systemic antitumour effects, e.g. activation of natural killer (NK) cells [40]. In one series, responsive patients developed rapid lymphocytosis with predominance of NK cells; in vitro the cytotoxic activity of isolated lymphocytes from these patients increased shortly after embolization. The observed early immunologic responses were closely correlated with late markers of the therapeutic effect [41]. MR spectroscopy of liver tumours before and after embolization with regard to energy-rich phosphates may also give information on the degree of ischaemia achieved. Results The result of ischaemic treatment of liver metastases is obviously dependent on stage of the disease. Coupe et al. [42] reported a series of 63 consecutive patients with advanced stage, of whom 30 were embolized for palliation. The survival after angiography, or angiography + embolization, was similar in both groups. Mitty et al. [43] reported long-term follow-up of 18 patients treated with embolization with prolongation of the expected survival by 2 years. Carrasco et al. [34] treated 25 patients with the carcinoid syndrome. The vast majority of patients showed both biochemical responses and tumour regression with an average duration of 1 year. Marlink et al. [44] introduced a different time schedule for embolization in

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patients with severe symptoms. Two successive embolizations were performed within 1 week leading to rapid symptom palliation. In our own study, patients with midgut carcinoids were embolized in two settings 1 month apart after primary surgery [18]. Our 40 patients could be divided into two equally large groups: responders with more than 50% tumour reduction and pronounced reduction of 5-HIAA excretion (80%) and nonresponders with less than 50% tumour reduction, and a moderate 5-HIAA reduction (30%) with clear survival advantage in the former group. In a Norwegian study the response rate to interferon (IFN) was twice as high in patients subjected to prior embolization indicating additive effects. It should be noted that IFN is a potent enhancer of NK cell cytotoxicity [45]. Reviewing our first 94 embolizations in patients with the midgut carcinoid syndrome, we met the following complications: Three patients occluded the main hepatic artery (one hepatic abscess), but had excellent symptom relief. One patient developed an aneurysm of the hepatic artery after the first embolization and a fatal hepatorenal syndrome after the second embolization. These complications together with 1 case of pancreatitis were all seen among the initial 11 embolizations before introduction of a coaxial catheter system. At the subsequent 83 embolizations only two minor complications were seen (transient renal insufficiency and cardiac arrhythmia). Peroperative Procedures Taking advantage of the high expression of somatostatin receptors by several NE tumours, attempts to develop radioguided surgery using a hand-held scintillation detector after preoperative injection of radiolabelled somatostatin analogues have been evaluated. The indications for radioguided surgery would be recurrent tumours in regions not easily investigated by other methods (e.g. neck metastases of medullary thyroid carcinoma, carcinoids and EPT), small EPT (e.g. in the MEN-1 syndrome) and as control of adequate tumour removal (e.g. carcinoid metastases in the mesenteric root after lymph node dissection). Promising results have been reported, but scintillation detection is clearly not sensitive enough to detect microscopic tumour growth or microadenomas [46]. At an advanced stage of histamine-producing tumours, ischemic liver treatment is potentially dangerous due to noncontrollable release of tumour products. In individual patients we have used cytotoxic drugs (melphalan and cisplatinum) delivered by regional hyperthermic liver perfusion. During perfusion the venous effluent from the liver with vasoactive substances was shunted from the systemic


Liver Transplantation Liver transplantation was first regarded as an optimal mode of treatment for all hepatobiliary malignancies, but the long-term results for advanced primary liver tumours were dismal with early tumour recurrence. Today there is a narrow indication for transplantation in small hepatocellular carcinomas and more unusual tumour types, e.g. fibrolamellar carcinoma and hepatoblastoma. Patients with secondary liver tumours had a very low survival rate: 12% 1 year after transplantation [54]. Better results might be expected in patients with secondary NE tumours, since metastatic disease of gastrointestinal carcinoids, or EPT, can be limited to the liver for long periods and these tumours may have slow growth potential. Many of the biogenic amines and peptides, which are produced by the

primary tumour, are metabolized by the liver. In patients with liver metastases these products are secreted directly into systemic circulation causing invalidating symptoms. When considering liver transplantation for NE tumours, the expected results with conventional treatment strategies must be weighed versus the risks with liver transplantation and subsequent immunosuppression. This is not an easy task, since a 5-year survival of 70% can be achieved in patients with midgut carcinoids and liver metastases using multimodal therapy and a 10-year survival well exceeding 50% (fig. 2) [55]. The following indications for liver transplantation in patients with NE tumours may be considered: (1) Metastatic disease limited to the liver, not accessible for curative liver surgery, including recurrent tumours in patients earlier subjected to curative liver resection and patients with severe hormonal symptoms due to liver tumours not possible to treat with other interventions, e.g. histamine-producing carcinoids. (2) Tumour progression after embolization and medical therapy in patients with no proven disease outside the liver. An upper age limit of 55 years has been suggested for liver transplantation. Some centres will not accept a tumour mass exceeding 50% of the liver volume, although no studies on NE tumour volume and outcome of transplantation are available. The overall prognosis for malignant EPT is worse than for midgut carcinoids. In our own series of EPT patients with liver metastases, the same treatment program was followed as for carcinoid tumours with much less favourable results (fig. 2). One reason for this difference was that patients with high-grade malignant tumours (NEC) were included in our series of EPT in contrast to others. NEC have a very poor prognosis and are seldom diagnosed at a resectable stage. For this tumour type, liver transplantation is contraindicated. A careful assessment of the outcome of primary surgery has been emphasized, since growth of residual tumour may accelerate during immunosuppression. Using biochemical markers, CT/MR and octreotide scintigraphy residual extrahepatic tumour was revealed in a high proportion of patients worked up for liver transplantation [56]. It therefore seems reasonable to follow a two-step strategy with histology-proven radical removal of the primary carcinoid, or EPT with tail location, followed by careful investigation of extrahepatic metastases prior to liver transplantation. EPT with head location are probably best managed with cluster transplantation (en-bloc resection of liver, pancreas and stomach). A careful workup of extrahepatic disease in these cases may include stag-



circulation avoiding vasomotor effects. Cytostatic perfusion of the isolated liver with simultaneous filtration of portal vein blood and a maintained systemic circulation were made possible by a special perfusion catheter inserted in the caval vein. The surgical technique involves isolation of the hepatic artery, portal vein and caval vein inferior and superior to the liver together with a temporary portocaval shunt that allows blood flow rates to be maintained in both the hepatic artery and portal lines [47]. Repeat hyperthermic perfusion can be very difficult to perform due to intense fibrotic reaction of the vessels used for perfusion. In elderly patients, or patients with progressive disease after previous treatment, percutaneous alcohol injections into isolated liver lesions can be performed under pharmacological blockade [48]. The volume of individual metastases is estimated ultrasonographically and the lesions are injected with equal volumes of absolute alcohol. During injection the lesions develop high echogenicity. With repeat injections marked symptom palliation related to reduced tumour markers and proven tumour necrosis can be achieved. Alternatively, the tumour can be destroyed by cryosurgical techniques, which also have the advantage that multiple lesions can be treated at a single session. Cryoprobes can also be equipped with intraoperative ultrasound for tumour localization. Modern cryoprobes can destroy tumours deep in the liver without damaging the normal overlying parenchyma [49]. Metastatic liver tumours can also be treated by interstitial laser [50] or radiofrequency therapy [51, 52] causing selective thermocoagulation. These techniques can be used intraoperatively or percutaneously. Ultrasound is usually the imaging guidance, but open thermosensitive MR systems for monitoring of coagulative effects are being developed [53].

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Fig. 2. Kaplan-Meier survival analysis in our consecutive series of patients with midgut carcinoids (MC, n = 64) and endocrine pancreatic tumours (EPT, n = 14) and liver metastases from the same time period treated with an active surgical program (resection of locoregional disease, liver surgery of unilateral liver lesions or embolization of bilateral liver lesions). The series of EPT also include NEC – left panel. Survival analysis of patients with NE tumours (n = 31) after liver transplantation in a French multicenter study – right panel. The similarities between treatment results indicate that EPT tumours are more aggressive than MC tumours. (Reproduced by permission of Br J Cancer [55]).

ing laparotomy to verify that the tumour mass can be radically removed by the cluster procedure. Results Ten years ago, Starzl’s group [57] introduced liver transplantation as treatment of NE tumours. Of the first 5 patients, 3 survived 7–34 months after surgery. Two years later another group reported upon 11 patients, of whom more than half were alive after 6 months [58]. In 1994, Bechstein and Neuhaus [59] reviewed the world literature, which at that time consisted of 30 patients with NE tumour that had undergone liver transplantation (15 EPT and 13 gastrointestinal carcinoids, 1 lung carcinoid and 1 nondefined primary carcinoid). The 1-year survival was 52%. Transplant-associated deaths occurred in half of these patients soon after transplantation; the other deaths were due to recurrent tumour. More strict criteria for selection of patients and concentration of liver transplantation to centres of excellence will probably improve these figures. Promising long-term results in selected patient series have recently been reported. The actuarial 5-year survival in a series from Hannover was almost 80% [60]. This series was dominated by patients with carcinoid disease; only 1 out of 12 patients died of tumour recurrence. Similar survival figures for carcinoid patients were recent-

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ly reported in a French multicentre study of 31 patients. In this series the 5-year survival was considerably worse for patients with EPT (!10%) (fig. 2) [61]. In both the German and French series there were several patients with correct tumour diagnoses first after transplantation, which was performed on suspicion of hepatocellular cancer. These patients underwent resection of the primary NE tumour at a second operation. For cluster procedures the prognosis for NE tumours widely surpassed other tumour diagnoses, e.g. sarcoma, hepatocellular carcinoma or cholangiocarcinoma, and reached a 5-year survival above 60% [62]. Thus, available studies indicate that patients with metastatic NE tumours confined to the liver can be offered long periods of symptom palliation by liver transplantation. In individual cases, transplantation may even offer cure.

Acknowledgements This work was supported by grants from the Swedish MRC (5220) and the Swedish Cancer Society (3911).


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17 Hughes KS, Sugarbaker PH: Resection of the liver for metastatic solid tumors; in Rosenberg SA (ed): Surgical Treatment of Metastatic Cancer. Philadelphia, Lippincott, 1987, pp 125– 164. 18 Wängberg B, Westberg G, Tylén U, Tisell LE, Jansson S, Nilsson O, Johanson V, Scherstén T, Ahlman H: Survival of patients with disseminated midgut carcinoid tumours after aggressive tumour reduction. World J Surg 1996;20: 892–899. 19 Carty SE, Jensen RT, Norton JA: Prospective study of aggressive resection of metastatic pancreatic endocrine tumours. Surgery 1992;112: 1024–1032. 20 Que FG, Nagorney DM, Batts KP, Linzl J, Kvols LK: Hepatic resection for metastatic neuroendocrine carcinomas. Am J Surg 1995; 169:36–43. 21 Dousset B, Saint-Marc O, Pitre J, Soubrane O, Houssin D, Chapuis Y: Metastastic endocrine tumors: Medical treatment, surgical resection, or liver transplantation. World J Surg 1996;20: 908–915. 22 Ahlman H, Wängberg B, Tisell LE, Nilsson O, Fjälling M, Forssell-Aronsson E: Clinical efficacy of octreotide scintigraphy in patients with midgut carcinoid tumours and evaluation of intraoperative scintillation detection. Br J Surg 1994;81:1144–1149. 23 Norton JA, Sugarbaker PH, Doppman JL, Wesley RA, Maton PN, Gardner JD, Jensen RT: Aggressive resection of metastatic disease in selected patients with malignant gastrinoma. Ann Surg 1986;203:352–359. 24 Markowitz JC: The hepatic artery. Surg Gynecol Obstet 1952;95:644–646. 25 Gelin LE, Lewis D, Nilsson L: Liver blood flow in man during abdominal surgery. II. The effect of hepatic artery occlusion on the blood flow through metastatic tumor nodules. Acta Hepatosplenol 1968;15:21–24. 26 Cho KJ, Reuter SR, Schmidt R: Effects of experimental hepatic artery embolization on hepatic function. Am J Roentgenol 1976;127: 563–567. 27 Geterud K, Tylén U, Jansson S, Stenqvist O, Tisell LE, Ahlman H: Hepatic arterial embolization in the treatment of the midgut carcinoid syndrome and other advanced endocrine tumours metastatic to the liver. J Intervent Radiol 1990;5:69–76. 28 Almersjö O, Bengmark S, Engevik L, Hafström LO, Nilsson LA: Hepatic artery ligation as pretreatment for liver resection of metastatic cancer. Rev Surg 1966;23:377–380. 29 Chuang VP, Wallace S: Hepatic artery embolization in the treatment of hepatic neoplasms. Radiology 1981;140:51–58. 30 Ajani JA, Carrasco CH, Wallace S: Neuroendocrine tumors metastatic to the liver vascular occlusion therapy. Ann NY Acad Sci 1994;733: 479–487. 31 Allison DJ, Modlin IM, Jenkins WJ: Treatment of carcinoid liver metastases by hepatic artery embolization. Lancet 1977;ii:1323– 1325.

32 Pueyo I, Jimenez JR, Herna´ndez J, Brugarolas A, Garcia-Moran M, Garcia-Muniz JI, Arroyo F: Carcinoid syndrome treated by hepatic embolization. Am J Roentgenol 1978;131:511– 513. 33 Lunderquist A, Ericsson M, Nobin A, Sandin G: Gelfoam powder embolization of the hepatic artery in liver metastases of carcinoid tumor. Radiologe 1982;22:65–70. 34 Carrasco CH, Charnsangavej C, Ajani J, Samaan NA, Richli W, Wallace S: The carcinoid syndrome: Palliation by hepatic artery embolisation. AJR 1986;147:149–154. 35 Hajarizadeh H, Ivancev K, Mueller CR, Fletcher WS, Woltering EA: Effective palliative treatment of metastatic carcinoid tumors with intra-arterial chemotherapy/chemoembolization combined with octreotide acetate. Am J Surg 1992;163:479–483. 36 Venook AP, Stagg RJ, Lewis BJ, Chase JL, Ring EJ, Maroney TP, Hohn DC: Chemoembolization for hepatocellular carcinoma. J Clin Oncol 1990;8:1108–1114. 37 Perry LJ, Stuart K, Stokes KR, Clouse ME: Hepatic arterial chemoembolization for metastatic neuroendocrine tumours. Surgery 1994; 116:1111–1116. 38 Ruszniewski P, Rougier P, Roche A, Legmann P, Sibert A, Hochlaf S, Ychou M, Mignon M: Hepatic arterial chemoembolisation in patients with liver metastases of endocrine tumors. Cancer 1993;71:2624–2630. 39 Nobin A, Månsson H, Lunderquist A: Evaluation of temporary liver dearterialization and embolization in patients with metastatic carcinoid tumor. Acta Oncol 1989;28:419–424. 40 Wängberg B, Geterud K, Nilsson O, Jansson S, Dahlström A, Tylén U, Ahlman H: Embolisation therapy in the midgut carcinoid syndrome – Just tumour ischemia? Acta Oncol 1993;32: 251–256. 41 Wängberg B, Ahlman H, Tylén U, Nilsson O, Hermodsson S, Hellstrand K: Accumulation of natural killer cells after hepatic artery embolisation in the midgut carcinoid syndrome. Br J Cancer 1995;71:617–618. 42 Coupe MO, Hodgson HJF, Hemingway A, Allison DJ: The effect of hepatic artery embolization on survival in the carcinoid syndrome. J Intervent Radiol 1989;4:179. 43 Mitty HA, Warner RRP, Neuman LH, Train JS, Parnes IH: Control of carcinoid syndrome with hepatic artery embolization. Radiology 1985;155:1623–1626. 44 Marlink RG, Lokich T, Robins SR, Clouse ME: Hepatic arterial embolization for metastatic hormone-secreting tumors: Technique, effectiveness and complications. Cancer 1990;65: 2227–2232. 45 Hanssen LE, Schrumpf E, Kolbenstvedt AN, Tausjö J, Dolva LO: Treatment of malignant metastatic midgut carcinoid tumours with recombinant human ·-2b-interferon with or without prior hepatic artery embolization. Scand J Gastroenterol 1989;24:787–795.


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46 Wängberg B, Forssell-Aronsson E, Tisell LE, Nilsson O, Fjälling M, Ahlman H: Intraoperative detection of somatostatin-receptor-positive neuroendocrine tumours using indium-IIIlabelled-DTPA-D-Phe1-octreotide. Br J Cancer 1996;73:770–775. 47 Scherstén T, Ahlman H, Wängberg B, Granérus G, Grimelius L: Hyperthermic liver perfusion chemotherapy in the treatment of the foregut carcinoid syndrome. Lancet 1991;338:568– 569. 48 Kölby L, Wängberg B, Ahlman H, Jansson S, Forssell-Aronsson E, Erickson JD, Nilsson O: Gastric carcinoid with histamine production, histamine transporter and expression of somatostatin receptors. Digestion 1998;59:160– 166. 49 Charnley RM, Doran J, Morris DL: Cryotherapy for liver metastases; a new approach. Br J Surg 1989;76:1040–1041. 50 Dowlatshahi K, Bhattacharya AK, Silver B, Motalen T, Williams JW: Percutaneous interstitial laser therapy of a patient with recurrent hepatoma in a transplanted liver. Surgery 1992;112:603–606.

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51 Bilchik AJ, Rose DM, Allegra DP, Bostick PJ, Hsueh E, Morton DL: Radio-frequency ablation: A minimally invasive technique with multiple applications. Cancer J Sci Am 1999;6: 356–361. 52 Matsui Y, Nakagawa A, Kamiyama Y, Yamamoto K, Kubo N, Nakase Y: Selective thermocoagulation of unresectable pancreatic cancers by using radiofrequency capacitive heating. Pankreas 2000;1:14–20. 53 Kettenbach J, Silverman SG, Hata N, Kuroda K, Saiviroonpurn P, Zientara GP, Morrison PR, Hushek SG, Black PM, Kikinis R, Jolesz FA: Monitoring and visualization techniques for MR-guided laser ablations in an open MR system. J Magn Reson Imaging 1998;8:933– 943. 54 Pichlmayr R: Is there a place for liver grafting for malignancy? Transplant Proc 1988;20:478– 482. 55 Johanson V, Tisell LE, Olbe L, Wängberg B, Nilsson O, Ahlman H: Comparison of survival between malignant neuroendocrine tumours of midgut and pancreatic origin. Br J Cancer 1999;80:1259–1261. 56 Frilling A, Rogiers X, Knöfel WT, Broelsch CE: Liver transplantation for metastatic carcinoid tumours. Digestion 1994;55(suppl 3):104–106. 57 Makowka L, Tzakis AG, Mazzaferro V, Teperman L, Demetris AJ, Iwatsuki S, Starzl TE: Transplantation of the liver for metastatic endocrine tumours of the intestine and pancreas. Surg Gynecol Obstet 1989;168:107–111.


58 Gulanikar AC, Kotylak G, Bitter-Suermann H: Does immunosuppression alter the growth of metastatic liver carcinoid after orthotopic liver transplantation? Transplant Proc 1991;23: 2197–2198. 59 Bechstein W, Neuhaus P: Liver transplantation for hepatic metastases of neuroendocrine tumours. Ann NY Acad Sci 1994;733:507–514. 60 Lang H, Oldhafer KJ, Weimann A, Schlitt HJ, Scheumann GFW, Flemming P, Ringe B, Pichlmayr R: Liver transplantation for metastatic neuroendocrine tumors. Ann Surg 1997; 225:347–354. 61 Le Treut YP, Delpero JR, Dousset B, Cherqui D, Segol P, Mantion G, Hannoun L, Benhamou G, Launois B, Boillot O, Domergue J, Bismuth H: Results of liver transplantation in the treatment of metastatic tumors: A 31-case French multicentric report. Ann Surg 1997; 225:355–364. 62 Allessiani M, Tzakis A, Todo S, Demetris AJ, Fung JJ, Starzl TE:. Assessment of 5-year experience with abdominal cluster transplantation. J Am Coll Surg 1995;180:1–9.



OctreoTherTM: Ongoing Early Clinical Development of a Somatostatin-ReceptorTargeted Radionuclide Antineoplastic Therapy M. Charles Smith a Jingou Liu a Tianling Chen a Horst Schran a Ching-Ming Yeh a François Jamar b Roelf Valkema c Willem Bakker c Larry Kvols d Eric Krenning c Stanislas Pauwels b a Novartis Pharmaceuticals Corporation, East Hanover, N.J., USA; b Catholic University of Louvain, Brussels, Belgium; c University Hospital Rotterdam, The Netherlands; d Moffitt Cancer Center, University of South Florida, Tampa, Fla., USA

Key Words Breast cancer W Clinical studies W DOTATOC W Neuroendocrine tumors W OctreoTherTM W Peptide radiotherapy W Small cell lung cancer W Somatostatin receptor W Yttrium-90

lung cancer. A fixed-dose regimen of 120 mCi/cycle ! 3 cycles administered with concomitant amino acid infusion has been chosen for the study. Phase I data and published literature support that this fixed dose regimen will be safely tolerated. Copyright © 2000 S. Karger AG, Basel

Abstract OctreoTherTM (90Y-DOTA-D-Phe1-Tyr3-octreotide, a.k.a. 90Y-SMT 487) consists of a somatostatin peptide analogue (Tyr3-octreotide), coupled with a complexing moiety (DOTA), and labeled with a tightly bound beta-emitter (yttrium-90). By targeting somatostatin receptor-positive tumors (as imaged by OctreaScan®) it may deliver a tumoricidal dose of radiation. Phase I clinical trials, conducted in patients with neuroendocrine tumors, established the safety and tolerability of the dose selected for further study and demonstrated the capacity of OctreoTher to deliver radiation doses to tumors that resulted in significant neuroendocrine tumor shrinkage. Novartis-sponsored phase II studies will soon begin to test the efficacy of OctreoTher in breast and small cell

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Introduction

OctreoTherTM is a Novartis development compound currently in phase I multicenter clinical trials to test its potential as a somatostatin-receptor-targeted radionuclide antineoplastic therapy. These studies are sponsored in cooperation with Mallinckrodt Medical Incorporated. The overall goal of this development program is to demonstrate the disappearance or significant shrinkage of somatostatin receptor positive solid tumors (e.g. neuroendocrine, breast or small cell lung tumors) in the absence of clinically apparent toxicity to major organ systems in patients receiving OctreoTher. Data from the Novartis-sponsored ongoing phase I study in neuroendo-

Chuck Smith, MD, PhD Novartis Pharma Corp., Bldg. 419, Room 2306, 59 Route 10 East Hanover, NJ 07936-1080 (USA) Tel. +1 973 781 8960, Fax +1 973 781 5511 E-Mail [email protected]

crine tumor patients along with recently published clinical studies not sponsored by Novartis have provided support for the choice of regimen to be tested in a multicenter phase II study. This phase II study, to begin this year at 12 centers in Australia, the United States and Europe, will examine the safety and efficacy of the chosen regimen in approximately 60 patients with breast and small cell lung cancer.

Preclinical Rationale

OctreoTher (90Y-DOTA-D-Phe1-Tyr3-octreotide, a.k.a. 487) consists of a somatostatin peptide analogue (Tyr3-octreotide), coupled with a complexing moiety (DOTA), and labeled with a tightly bound beta-emitter (yttrium-90). By targeting somatostatin receptor-positive tumors (as imaged by OctreoScan®) it may deliver a tumoricidal dose of radiation. Yttrium-90 (90Y) is a highenergy beta-emitter with a mean path length of 5 mm in tissue and a physical half-life of 64.1 h. SMT 487 binds with high affinity to somatostatin receptors (subtypes 2 and 3) and retains both its binding properties and its physiological function when labeled with 90Y. The preclinical rationale for the use of OctreoTher is based on evidence that: (1) OctreoTher accumulates in somatostatin receptorcontaining tissue or tumors. (2) Receptors remain present and able to bind OctreoTher during treatment. (3) Tumor regression was demonstrated in vivo [1]. In tumor-bearing nude mice, OctreoTher was effective in somatostatin receptor-positive tumors, a single dose both shrinking the tumor and prolonging survival. A single dose in rats caused pancreatic tumors to disappear without regrowth. No difference in tumor regression was seen between perilesional, intraperitoneal or intravenous administration of OctreoTher, so the more convenient intravenous application was selected for clinical development. 90Y-SMT

Phase I Clinical Studies of Neuroendocrine Tumors

Phase I clinical trials, conducted in patients with neuroendocrine tumors, established that following an intravenous infusion, plasma radioactivity disposition of OctreoTher was multiphasic (terminal t½ F8.6 h). Unbound radioactivity was excreted in urine. By 24 h post-

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dose, plasma radioactivity is !1% of the injected dose/l). The rapid clearance of OctreoTher from the circulation gives this radiopharmaceutical a major advantage over radiolabeled antibodies, whose long plasma half-lives (several days) cause higher levels of whole-body irradiation. OctreoTher is not significantly taken into erythrocytes. A correlation between creatinine clearance and total body radioactivity clearance was observed reflecting that the kidney is the principal drug eliminating organ and suggesting that OctreoTher is not recommended in subjects whose creatinine clearance is less than 40 ml/ min. Radioactivity exposure was mainly to kidney, spleen, urinary bladder wall, and tumors. Gamma scintigraphy with 111In-pentetreotide (OctreoScan) may be used to identify OctreoTher-binding tumors. The relative biodistribution and pharmacokinetic profile of OctreoTher was not altered by changes in the absolute peptide dose over the range of 50–500 Ìg. Notably, concomitant administration of amino acids reduced renal radioactivity uptake without altering tumor uptake in a phase I study. Cationic amino acids appear to be responsible for the ‘blocking’ of renal tubular uptake of proteins or peptides [2, 3]. A variety of other maneuvers have been attempted, so far, without success in further lowering the relative renal uptake of OctreoTher. Phase I clinical trial results support the safety and tolerability of the dose selected for further study. The organ of dose-limiting toxicity was confirmed to be primarily the kidney and secondarily the hematopoietic system. Acute radiation nephritis may appear as a syndrome of hypertension, proteinuria and anemia presenting up to a year after the causal radiation exposure [4]. Furthermore, these studies demonstrated the capacity of OctreoTher to deliver radiation doses to tumors that resulted in significant neuroendocrine tumor shrinkage. The planned phase II study will assess the safety and anti-tumor activity of OctreoTher in refractory small cell lung cancer and advanced metastatic breast cancer.

Planned Phase II Clinical Study of Small Cell Lung and Breast Cancers

Small cell lung cancer (SCLC) comprises approximately 18% of all primary lung cancer, virtually all of the tumors bearing somatostatin receptors [5–8]. Although SCLC is highly sensitive to radiation, coexistent lung tissue damage and the early occurrence of distant metastases limit the utility of external beam irradiation. Resistance

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to chemotherapy often develops early during treatment. Prognosis is correspondingly poor; approximately 95% of those afflicted succumbing to their disease, regardless of therapy or earliness of diagnosis. Hence, OctreoTher, by providing systemic, yet targeted irradiation to metastatic SCLC, should provide a much-needed, additional therapeutic modality. Breast cancer is the commonest malignancy among women. The prognosis is variable and first- and secondline therapies are well established. Approximately 70% of primary breast cancers express somatostatin receptors [5, 9–11]. Patients with advanced metastatic breast cancer, who are shown by OctreoScan to have high somatostatin receptor levels, may benefit from therapy.

(1.84 m2) would correspond to a cumulative dose of ca. 368 mCi per patient which was safe and tolerated. Some, but not all, of the administered cycles were in the presence of concomitant amino acid infusion. There was no adjustment for renal dosimetry, which was also not performed/ reported. Four patients, who all received 17,400 MBq/ m2, developed renal toxicity: 2 patients who received cumulative doses of 360 and 385 mCi, respectively, showed stable renal insufficiency; 2 other patients who received 380 and 410 mCi, respectively, showed complete renal failure (dialysis dependent). None of the 4 patients who developed renal toxicity received any concomitant amino acid infusion. Thus, all doses at or below 360 mCi administered with concomitant amino acids appear to have been tolerated safely.

Primary Study Objectives Conclusion

1 To evaluate the efficacy of OctreoTher in advanced metastatic breast cancer and refractory small cell lung cancer as measured by tumor response rate (PR + CR SWOG criteria). 2 To evaluate the safety of OctreoTher as measured by the rate of (mild/moderate/severe/life-threatening) adverse events, and serious adverse events and the monitoring of selected laboratory evaluations.

Novartis-sponsored phase II studies will soon begin to test the efficacy of OctreoTher in breast and small cell lung cancer. A fixed-dose regimen of 120 mCi/cycle ! 3 cycles administered with concomitant amino acid infusion has been chosen for the study. Phase I data and published literature support that this fixed dose regimen will be safely tolerated.

Secondary Study Objectives

1 To measure the overall survival of patients treated with OctreoTher. 2 To evaluate the effect of OctreoTher on quality of life as measured by the EURO-QOL EQ-5D. 3 To assess the frequency of tumors which are positive (tumor uptake 1 liver uptake) for OctreoScan within the screened population.

Data from Non-Novartis-Sponsored Studies in the Literature

Investigators not affiliated with Novartis have produced and conducted clinical trials with a compound, which appears to be identical to OctreoTher, called Yttrium-90 DOTATOC [12–14]. To summarize the renal toxicity reported by these investigators, for purpose of comparison, 24 patients received ^7,400 MBq/m2. This limit (7,400 MBq/m2) corresponds to 200 mCi/m2, and, using the average reported BSA of these 24 patients

OctreoTherTM: Early Clinical Development


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References 1 Stolz B, Weckbecker G, Smith-Jones P, et al: The somatostatin receptor-targeted radiotherapeutic [90YDOTA-D-Phe1,Tyr3]octreotide (90Y-SMT 487) eradicates experimental rat pancreatic CA 20948 tumours. Eur J Nucl Med 1998;25:668–674. 2 Hammond PJ, Wade AF, Gwilliam ME, et al: Amino acid infusion blocks renal tubular uptake of an indium-labelled somatostatin analogue. Br J Cancer 1993;67:1437–1439. 3 Mogensen CE, Solling K: Studies on renal tubular protein reabsorption: partial and near complete inhibition by certain amino acids. Scand J Clin Lab Invest 1977;37:477–486. 4 Maher J: Section 6: Diseases of the kidney and urinary tract, Part 8: Radiation Nephropathy; in Masary SG, Glassock RJ (eds): Textbook of Nephrology. Baltimore, Williams & Wilkins, 1989.

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5 Krenning EP, Kwekkeboom DJ, Bakker WH, et al: Somatostatin receptor scintigraphy with [111In-DPTA-D-Phe1]- and [123I-Tyr3]-octreotide: The Rotterdam experience with more than 1,000 patients. Eur J Nucl Med 1993;20: 716–731. 6 Kwekkeboom DJ, Kho GS, Lamberts SJ, et al: The value of octreotide scintigraphy in patients with lung cancer. Eur J Nucl Med 1994;21: 1106–1113. 7 O’Byrne KJ, Ennis JT, Freyne PJ, et al: Scintigraphic imaging of small cell lung cancer with [111In]pentetreotide, a radiolabelled somatostatin analogue. Br J Cancer 1994;69:762–766. 8 Bombardieri E, Crippa F, Cataldo I, et al: Somatostatin receptor imaging of small cell lung cancer by means of 111In-DPTA octreotide scintigraphy. Eur J Cancer 1995;31A:184– 188. 9 Bootsma AH, van Eijck CHJ, Schouten KK, et al: Somatostatin receptor-positive primary breast tumors: Genetic, patient and tumor characteristics. Int J Cancer 1993;54:357–362.


10 Van Eijck CHJ, Krenning EP, Bootsma A, et al: Somatostatin receptor scintigraphy in primary breast cancer. Lancet 1994;343:640–643. 11 Evans AA, Crook T, Laws SAM, et al: Analysis of somatostatin receptor subtype mRNA expression in human breast cancer. Br J Cancer 1997;75:798–803. 12 Otte A, Herrmann R, Heppler A, et al: Yttrium-90 DOTATOC: First clinical results. Eur J Nucl Med 1999;26:1439–1447. 13 Otte A, Jermann E, Behe M, et al: DOTATOC: A powerful new tool for receptor-mediated radionuclide therapy. Eur J Nucl Med 1997;24: 792–795. 14 Otte A, Mueller-Brand J, Dellas S, et al: Yttrium-90-labelled somatostatin-analogue for cancer treatment. Lancet 1998;351:417.

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Chemotherapy in the Treatment of Neuroendocrine Malignant Tumors Philippe Rougier Emmanuel Mitry Fédération des spécialités digestives, Service Hépato-gastroentérologie, Hôpital Ambroise-Paré, Boulogne, France

Key Words Cytotoxic treatment W Chemotherapy W Neuroendocrine tumors W Carcinoid tumors W Islet cell carcinoma

Abstract The efficacy of chemotherapy in digestive neuroendocrine tumors (NET) depends on primary site and histological differentiation. Many reports have suggested a superior activity of chemotherapy for pancreatic NET than for metastatic carcinoid tumors with response rates ranging from 40 to 60% compared to 20%. The standard chemotherapy for pancreatic NET is a combination of adriamycin and streptozocin and to a lesser extent a combination of 5FU and streptozocin. In contrast, there is no clear standard chemotherapy for carcinoid tumors and if most oncologists use a combination of 5FU and streptozocin in the case of advanced, progressive and nonresectable carcinoid tumors, the results are mostly poor and the benefit seldom counterbalances its toxicity. In these carcinoid tumors the combination of hepatic artery ischemia alternating with chemotherapy has given impressive results in one study, which, however, have never been confirmed. Tumor cell differentiation is a major prognostic factor and some reports have suggested a higher chemosensitivity for undifferentiated or

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poorly differentiated NET with tumor response rates ranging from 41 to 69% when a VP16-CDDP combination is used. This chemosensitivity is, unfortunately, as in small cell lung carcinomas, of short duration. Related to this special problem and the number of other active treatments in NET, the place of chemotherapy always has to be discussed in a multidisciplinary fashion. Surgical excision, chemoembolization, interferons and somatostatin analogues have to be emphasized and eventually combined with chemotherapy, especially in slowly growing tumors. New active chemotherapy regimens have to be tested clearly in this orphan group of tumors which does not hold much interest to the pharmaceutical companies. Copyright © 2000 S. Karger AG, Basel

Introduction

In advanced neuroendocrine tumors (NETs) surgical excision of the lesions, when possible, has always to be preferred and the role of chemotherapy is often marginal except for poorly differentiated NETs. The indications for chemotherapy have to be emphasized in a multidisciplinary way and discussed with surgeons, specialists in chemoembolization, gastroenterologists, endocrinologists

Prof. Philippe Rougier Service Hépato-gastroentérologie, Hôpital Ambroise-Paré F–92100 Boulogne (France) Tel. +33 1 49 09 53 25, Fax +33 1 49 09 53 29 E-Mail [email protected]

Table 1. Monochemotherapy in carcinoid tumors [1]

Drugs

Adriamycin 5-FU Deticene Dactinomycin Cisplatin Mitomycin C

Number of patients

OR, %

33 19 18 17 16 4

21 26 17 6 6 25

Chemotherapy of Well-Differentiated Carcinoid Tumors Duration of OR, months 3.5 3 5 – 4.5 1.5

OR = Objective response.

and medical oncologists. The aim is the total destruction of the NET even at a metastatic stage. Chemotherapy may play an adjuvant role and be indicated before and/or after surgery but also as the only treatment in some cases. Chemotherapy has also to be discussed while taking into consideration the major role played by other medical treatments like somatostatin analogs and interferons which are frequently able to control the symptoms induced by hormonal hypersecretion, especially in case of a carcinoid syndrome. These biotherapies may also decrease the tumor growth rate. The efficacy of these biotherapies and the potential toxicity of chemotherapy explain that chemotherapy is frequently used after failure of all other medical treatments or in combination with them. However, it may be beneficial for some selected cases of advanced NET, especially pancreatic or undifferentiated NETs. In the past it was difficult to have a clear idea of the chemotherapeutic efficacy in NETs because most of the reports mixed carcinoid, pancreatic NETs and other NETs like hepatic metastasis from unknown primary, as well as differentiated and undifferentiated NETs. During the past 20 years, reports have become more clear and have been separated into different kinds of NET. Diagnosis has become more precise and is facilitated by the development of immunohistochemical techniques. Beside hormone dosages, biological determination of sensitive and specific tumor markers like chromogranin A are now routinely used. Tumor responses and tumor growth rate are presently more precisely evaluated using CT scan or IMR. In this review we will successively analyze the results of chemotherapy successively in metastatic carcinoid tumors, in well-differentiated pancreatic NETs and in undifferentiated NETs.

74


Carcinoid tumors are the most frequent digestive NET and develop mainly in the mid-gut. Systemic chemotherapy is poorly active in well-differentiated metastatic carcinoid tumors. It has been used for aggressive metastatic tumors, when there is evidence of progression and after failure of all the other therapeutic options: surgical excision, chemoembolization, sandostatine analogs, and, depending on the country, after interferon [1]. There are only a few studies which have properly evaluated the chemotherapeutic efficacy in carcinoid tumors and even less which have used the WHO criteria to report its efficacy. In many studies efficacy has been reported based on mixed criteria including, besides the decrease in tumor mass with the classical 50% reduction in the product of the two largest perpendicular diameters of the main lesions, less strict criteria such as a 30% decrease in hepatomegaly below the costal edge, a clinical response when a carcinoid syndrome exists, or a biological response such as a diminution in the urinary excretion of 5-HIAA which is reported to be correlated with tumor evolution [1] but which sometimes fluctuates and requires strict collection of the total amount of urine for a period of 24 or 48 h. Single-agent chemotherapy (monochemotherapy) was mainly studied in the 1970s and there are rather few agents presently tested in NETs. The main results are reported in table 1. Only 5-FU and Adriamycin gave a response rate of 120% [1]. However, in a compilation, steptozocin gave a 44% response rate using very heterogeneous response criteria and this rate probably does not reflect the reality but a selection of positive results [2]. Chemotherapy using drug combinations (polychemotherapy) does not appear to be significantly more active (table 2) [1, 3–9] and the classical combination of 5-FU and streptozocin in two randomized studies gave only 33 and 22% response rates using non-WHO criteria. Despite this poor activity this combination is frequently used; it is as efficient and less toxic than the combination of Adriamycin and streptozocin (median survival 24 versus 16 months, p = 0.11) [8]. The combination of 5-FU, Adriamycin and streptozocin has not been studied probably for fear of toxicity; however, Moertel [1] alternated 5-FU + streptozocin and DTIC + Adriamycin with success in patients suffering from liver metastases when combined with sequential hepatic artery occlusion. The efficacy of this four-drug regimen has never been tested alone. The response duration of chemotherapy in carcinoid tumors is unpredictable and often short-lasting (3–4

Rougier/Mitry

Table 2. Main trials on polychemotherapy

in metastatic carcinoid

Studies

Protocols

Number OR of % patients

Median of survival months

Engstrom et al. [4]

5-FU + STZ versus ADR

80 81

22 21

15 11

Moertel [1]

5-FU + STZ MTX + CPM STZ + CPM

43 16 14

33 0 0

7 – –

Bukowski et al. [5]

5-FU + ADR + STZ + CPM

56

30

–

Moertel and Hanley [6]

5-FU + CPM

47

26

–

Kelsen et al. [7]

STZ + ADR

13

40

–

Haller et al. [8]

5-FU + STZ versus 5-FU + ADR

67 75

16 13

24 16 (NS)

Rougier et al. [9]

5-FU + ADR + CDDP

15

14

27

Moertel et al. [18]

VP16 + CDDP

13

0

–

OR = Objective response; 5-FU = 5-fluorouracil; STZ = streptozocin; MTX = methotrexate; CPM = cyclophosphamide; ADR = Adriamycin; CDDP = cisplatin; VP16 = etoposide.

months), but in some cases may last much longer (even years). The tolerance of these chemotherapy regimens has to be considered, and with the 5-FU-streptozocin combination there are grade-3 or 4 toxicities reported in approximately 50% of the cases and, at the beginning of the use of streptozocin, even toxic deaths were reported in 5% of the cases [3]. Presently streptozocin is better handled but may still alter renal function when cycles are repeated (cumulative toxicity) and renal function has to be checked carefully before each administration (proteinuria and creatinine). Other main toxicities of streptozocin are hepatic (cytolysis), vomiting and neutropenia or thrombocytopenia. In cases of tumor regression under chemotherapy it is important to discuss with the surgeons the possibility of an eventual secondary resection of the main metastases. This attitude seems to offer a maximal chance for the patient to control the carcinoid tumor for many years [10]. The main prognostic factors for patients receiving chemotherapy are a good general condition, age under 60 years [4] and the number of mitoses per high-power field on pathological examination [5]. The best criteria for initiating chemotherapy seem to be: young age of the patients; good pathological stage; unresectable tumors, and failure of chemoembolization or tumor extension pre-

venting locoregional treatments. In some cases a carcinoid syndrome cannot be controlled by somatostatin analogs and, in some countries, progression under interferon treatment is also a potential indication. Systemic chemotherapy has to be initiated when the general status and the main functions are good enough and very often after an observation period of many months related to the frequent slow rate of growth. For the same reason the antitumor efficacy will be judged only after 3 or even 6 months if tolerance allows administration of treatment. Dose adaptations and protocol modifications have to be done promptly to avoid undue toxicity and degradation of the quality of life of these patients who can sometimes enjoy long periods of life without any treatment. In conclusion, for carcinoid tumors, the limited efficacy of systemic chemotherapy underlines the need to restrict its use to evolutive cases and to compare its efficacy to interferons. Less aggressive treatments, i.e. somatostatin analogs or locoregional treatments, have always to be discussed before initiating chemotherapy. The 5-FUstreptozocin combination is only a ‘weak’ standard with a 10–20% response rate and functional improvement in 30–50% of the cases. DTIC and Adriamycin have a limited activity and new agents have to be tested using clear response criteria, taking into account the main prognostic

Chemotherapy in Neuroendocrine Tumors


75

factors for response and survival. Only large trials will help to find better chemotherapy regimens and new and more active strategies.

Chemotherapy of Well-Differentiated Pancreatic NETs

Pancreatic NETs are rare tumors derived from islet cells. They may be responsible for functional syndromes which often reveal NET (hypoglycemia related to insulinomas; severe peptic disease and diarrhea in case of gastrinoma, cutaneous necrolytic lesions in case of glucagonomas; severe diarrhea in case of vipomas, and diabetes and gallstones in cases of somatostatinomas). In these cases, tumor extension of the functioning pancreatic NET may be restricted to the pancreatic gland and be treated with symptomatic and antihormonal treatments followed by a surgical excision of this tumor. More and more frequently we are facing (about 70% of the cases) pancreatic NETs without any functional syndrome, even in cases of peptide secretions like calcitonin, HCG (or its subunit ·), neuron-specific enolase, chromogranine, etc., which may be useful tumor markers but not responsible for clinical manifestations. In these cases the NETs are called nonfunctioning. In rare cases the diagnosis is only supported by the histochemical characterization and no pathological secretions are detected. These cases are called nonsecreting NETs. These cases are frequently revealed by metastases, mainly hepatic metastases, or metastatic lymph nodes. These patients have a poor prognosis with a median survival ranging from 2 to 3 years. Even when metastasis exists, surgical excision is sometimes feasible and has to be discussed first [10]. When surgical excision is not possible medical or radiological treatment (chemoembolization) must be discussed according to the aggressiveness and evolution (tumor growth rate) of the NET. Chemotherapy will be discussed only in progressive tumors (increase of 125% of the main tumor masses in a follow-up period of 12 months or tumoral symptoms not treatable with other methods). The first report on the efficacy of systemic chemotherapy was made by MurrayLyon et al. [11] in 1968 showing the efficacy of streptozocine in 1 case of malignant insulinoma. This efficacy was further confirmed in a review made by Broder and Carter [3] who reported a 42% response rate with a weekly regimen which was highly toxic. This efficacy was obviously overestimated for two reasons : a bias of publications

76


Table 3. Polychemotherapy in pancreatic neuroendocrine tumors

Protocols

n

OR %

Duration of OR months

Observations

STZ + 5-FU + ADR

10

40

7

STZ + ADR [7]

25

36

22

CLZ + 5-FU [15]

44

36

11

STZ STZ + 5-FU [16]

42 42

36 63

17 (for the Randomized trial 2 arms)

Randomization STZ + ADR STZ + 5-FU CLZ [17]

36 33 33

69 45 30

18 14 17

Gastrinomas

Renal insufficiency related to CLZ

Median survival 2.2 years 1.4 years 1.5 years p ! 0.01

STZ = Streptozotocin; 5-FU = 5-fluorouracil; ADR = adriamycin; CLZ = chlorozotocin; n = number of patients; OR = objective response.

(positive experiences are more often reported than negative ones) and the use of nonstandard response criteria as discussed previously (WHO criteria were not applied) [3]. Few other drugs have been reported as having some antitumor activity. Doxorubicin (Adriamycin) gave a 20% response rate in 20 patients [12 ] and dacarbazin (DTIC) a 17% response rate in 10 patients [13]. Polychemotherapies have given better results (table 3) [14–17]. The combination of 5-FU + streptozocin is superior to streptozocin alone [16], and Moertel et al. [17] have also demonstrated that the combination of Adriamycin + streptozocin was superior to the 5-FU + streptozocin combination and chlorozotocin in terms of responses (69 vs. 45%) and overall survival (median survival 2.2 years vs. 1.4 years, p = 0.004) [17]. The combination of streptozocin and Adriamycin may be considered the standard protocol for advanced evolutive pancreatic NET [17]. It significantly prolongs survival, inhibits tumor progression and produces major shrinkage of the tumors in two thirds of the cases. Its efficacy sometimes allows secondary surgical excisions which were initially not possible and experience strongly supports the interest in a multidisciplinary approach to the management of these pancreatic NETs and the central role of surgery to obtain long-term tumor control and survival [10].

Rougier/Mitry

Table 4. Chemotherapy in poorly

differentiated neuroendocrine tumors

Trials

Protocols

Number OR of % patients

Median survival months

Moertel et al. [18]

VP16 130 mg/m2 IVCP on days 1–3 + CDDP 45 mg/m2 IVCP on days 2 and 3

18

67

19

Seitz et al. [19]

VP16 130 mg/m2 IVCP on days 1–3 + 11 CDDP 45 mg/m2 IVCP on days 2 and 3 + G-CSF 300 Ìg/m2 on days 6–13

54

1 year (survival 65%)

Mitry et al. [20]

VP16 120 mg/m2 i.v./h on days 1–3 + CDDP 100 mg/m2 on day 2

41.5

15

41

IVCP = Continuous intravenous perfusion; CDDP = cisplatin.

Chemotherapy of Undifferentiated NETs

A high tumor response rate has been reported in undifferentiated NETs and their chemosensitivity is very similar to that of small cell lung cancer. A 67% response rate was reported in 1991 by Moertel et al. [18] with a combination of etoposide (VP16) + cisplatin (CDDP). This combination was not active in differentiated NETs (15%; table 3 and 4), but was very active in undifferentiated NETs from the pancreas as well as from the digestive tract [18]. This high activity has been confirmed, in two phaseII studies conducted in France using the same type of chemotherapy with hematopoietic growth factors [19] or without hematopoietic growth factors but using a lower dose of etoposide [20].

before acceptation of this aggressive and complicated approach. The efficacy of intra-arterial hepatic chemotherapy has seldom been used and reported. In our experience we have observed a good tumor response allowing secondary resections in some cases [10, 22]; however, in 4 of 23 cases we have also observed 4 digestive hemorrhages which were severe in 2 cases and lethal in 1 case (personal data). These complications are perhaps related to the fragility of the digestive arteries in patients with carcinoid tumors, and therefore our indications for intraarterial hepatic chemotherapy of liver metastases from carcinoid tumors are restricted.

Conclusion Chemotherapy Combined with Hepatic Arterial Occlusion

Many reports, especially from Sweden, have demonstrated that hepatic artery occlusion has a therapeutic effect on liver metastasis from carcinoids; however, the duration of this positive effect is usually transitory lasting less than 1 year [1]. In a large retrospective analysis Moertel et al. [21] suggested that patients treated with a combination of arterial occlusions and systemic chemotherapy (5-FU-streptozocin alternated with Adriamycin-dacarbazin) had a 20% higher response rate and a longer tumor growth control time from 4 to 22 months (median time to progression) for pancreatic NETs and from 10 to 24 months for the carcinoid tumors. These results have never been confirmed and a comparative trial is clearly needed

Chemotherapy in Neuroendocrine Tumors

Chemotherapy has different efficacies in NETs according to the primary site and differentiation. It has a clear antitumoral efficacy in metastatic pancreatic NETs but this seems much lower in metastatic carcinoids. In case of undifferentiated NETs chemotherapy with the VP16-CDDP combination gives a major tumor response in approximately 50% of the cases with symptom improvement in most of the cases. Unfortunately these responses are of short duration. New and more active chemotherapy regimens have clearly to be discovered. Combinations or comparison of chemotherapy with other treatments such as hepatic artery ischemia, interferons, or somatostatin analogs [23], warrant study in large multicentric trials.


77

References 1 Moertel CG: An odyssey in the land of small tumors. J Clin Oncol 1987;5:1503–1522. 2 Harris AL: Chemotherapy for the carcinoid syndrome. Cancer Chemother Pharmacol 1981;5:133–138. 3 Broder LR, Carter SK: Pancreatic islet cell carcinoma. II. Results of therapy with streptozotocin in 52 patients. Ann Intern Med 1973;79: 108–118. 4 Engstrom PF, Lavin PT, Moertel CG, Folsch E, Douglass HO: Streptozocin plus fluorouracil versus doxorubicin therapy for metastatic carcinoid tumor. J Clin Oncol 1984;2:1255– 1259. 5 Bukowski RM, Johnson KG, Peterson RF, Stephens RL, Rivkin SE, Neilan B, et al: A phase II trial of combination chemotherapy in patients with metastatic carcinoid tumors. A Southwest Oncology Group study. Cancer 1987;60:2891–2895. 6 Moertel CG, Hanley JA: Combination therapy trials in metastatic carcinoid tumors and the malignant carcinoid syndrome. Cancer Clin Trials 1979;2:327–334. 7 Kelsen DP, Cheng E, Kemeny N, Magill GB, Yagoda A: Streptozotocin and Adriamycin in the treatment of APUD tumors (carcinoid, islet cell and medullary carcinoma of the thyroid). Proc Am Assoc Cancer Res 1982;23:111. 8 Haller DG, Schutt A, Dayal Y, Ryan L, Lipsitz S: Chemotherapy for metastatic carcinoid tumors: An ECOG phase II-III trial. Proc Am Soc Clin Oncol 1990;9:102.

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9 Rougier P, Oliveira J, Ducreux M, Theodore C, Kac J, Droz J-P: Metastatic carcinoid and islet cell tumors of the pancreas: A phase II trial of the efficacy of combination chemotherapy with 5-fluorouracil, doxorubicin and cisplatin. Eur J Cancer 1991;27:1380–1382. 10 Elias D, Rougier P, Lasser P, Duvillard P, Venezia P, Schlumberger M, et al: Chirurgie majeure et chimiothérapies réductionnelles dans les apudomes polymétastatiques. Ann Chir 1988;42:474–481. 11 Murray-Lyon IM, Eddelston ALWF, Williams R, Brown M, Hogbin BM, Bennett A, et al: Treatment of multiple hormone-producing malignant islet cell tumours with streptozotocin. Lancet 1968;ii:895–898. 12 Moertel CG, Lavin P, Hahn G: Phase II trial of doxorubicin therapy for advanced islet cell carcinoma. Cancer Treat Rep 1987;66:1567– 1569. 13 Altimari A, Badrinath K, Reisel H, Prinz RA: DTIC therapy in patients with malignant intraabdominal neuroendocrine tumors. Surgery 1987;102:1009–1017. 14 Eriksson B, Skogseid B, Lundqvist G, Wide L, Wilander E, Öberg K: Medical treatment and long-term survival in a prospective study of 84 patients with endocrine pancreatic tumors. Cancer 1990;65:1883–1890. 15 Bukowski RM, Tangen C, Lee R, Macdonald JS, Einstein AB Jr, Peterson R, Fleming TR: Phase II trial of chlorozotocin and fluorouracil in islet cell carcinoma: A Southwest Oncology Group study. J Clin Oncol 1992;10:1914– 1918.


16 Moertel CG, Hanley JA, Johnsson LA: Streptozocin alone compared with streptozocin plus fluorouracil in the treatment of advanced islet cell carcinoma. N Engl J Med 1980;303:1189– 1194. 17 Moertel CG, Lefkopoulos M, Lipsitz M: Streptozocin-doxorubicin, streptozocin-fluorouracil or chlorozotocin in the treatment of advanced islet-cell carcinoma. N Engl J Med 1992;326: 519–523. 18 Moertel CG, Kvols LK, O’Connell MJ, Rubin J: Treatment of neuroendocrine carcinomas with combined etoposide and cisplatin. Cancer 1991;68:227–232. 19 Seitz JF, Perrier H, Giovannini M, Monges G, Fourdan O, Barrière N, Viens P: Cancers neuroendocrines anaplasiques avancés: intérêt de l’association VP16-CDDP. Bull Cancer 1995; 82:433–434. 20 Mitry E, Baudin E, Ducreux M, Sabourin JC, Ruffié P, Aparicio T, Lasser P, Elias D, Duvillard P, Schlumberger M, Rougier P: Treatment of poorly differentiated neuroendocrine tumours with etoposide and cisplatin. Br J Cancer 1999;81:1351–1355. 21 Moertel CG, Johnson M, McKusick MA, Martin JK Jr, Nagorney DM, Kvols LK, et al: The management of patients with advanced carcinoid tumors and islet cell carcinomas. Ann Intern Med 1994;120:302–309. 22 Lasser P, Elias D, Rougier Ph, Bonvallot S, Sabourin JC, Schlumberger M, Ducreux M, Roche A: Hépatectomies pour métastases de tumeurs neuro-endocrines (à propos de 17 cas). Gastroenterol Clin Biol 1996;20:A33. 23 Öberg K: Neuroendocrine gastrointestinal tumours. Ann Oncol 1996;7:453–463.

Rougier/Mitry


Hepatic Arterial Chemoembolization in the Management of Advanced Digestive Endocrine Tumors P. Ruszniewski D. Malka Federation of Hepato-Gastroenterology, Hôpital Beaujon, Clichy, France

Key Words Digestive endocrine tumors W Carcinoid tumors W Islet cell carcinomas W Liver metastases W Chemoembolization

Abstract The management of advanced digestive endocrine tumors is often challenging. Liver metastases are usually diffuse at the time of diagnosis, and surgical resection is rarely feasible. Objective response rates with systemic chemotherapy are disappointing. Arterial hypervascularization of most liver metastases from digestive endocrine tumors argues in favor of hepatic arterial chemoembolization (HACE). It is assumed that embolizationinduced ischemia sensitizes tumor cells to cytotoxic drugs, whose tumor concentrations are increased by blood flow slowing down. The aims of HACE are: (1) to control otherwise untractable hormone-related symptoms, particularly the carcinoid syndrome (1 50% urinary 5-HIAA decrease: 57–91%) and insulinoma-related lifethreatening hypoglycemias; (2) to inhibit tumor growth (objective response rates: 33–80%; mean duration: 6– 42.5 months), and (3) to improve patients’ survival. The postembolization syndrome, usually mild and transient, is the commonest side effect. Major extrahepatic compli-

ABC

© 2000 S. Karger AG, Basel 0012–2823/00/0625–0079$17.50/0



cations are rare. In conclusion, HACE seems to be an attractive alternative treatment for diffuse (unresectable) and progressive metastases confined to the liver in patients with digestive endocrine tumors, mainly following unsuccessful systemic chemotherapy. Further studies assessing the long-term results of HACE and comparing it to other treatments, particularly systemic chemotherapy, are needed. Copyright © 2000 S. Karger AG, Basel

Digestive endocrine tumors originate mainly from the pancreas (islet cell carcinomas) or from the gut (carcinoid tumors) [1]. They can be benign or malignant and responsible for symptoms of endocrine hyperfunction [2].

Rationale for Chemoembolization

Liver metastases from digestive endocrine tumors carry a poor prognosis and are associated with the deterioration of patients’ quality of life. They are encountered in 25–90% of the patients, depending on the nature of the primary tumor, and often without extrahepatic metastases. Five-year survival rates of patients with and with-

Prof. Philippe Ruszniewski Hôpital Beaujon, Service de Gastroentérologie F–92118 Clichy Cedex (France) Tel. +33 1 40 87 53 28, Fax +33 1 42 70 37 84 E-Mail [email protected]

Table 1. Symptoms and hormonal

secretion: carcinoid syndrome

Author, year

n

Therasse, 1993 [16] Ruszniewski, 1993 [17] Clouse, 1994 [18, 19] Diaco, 1995 [20]

23 18 16 10

ADR ADR ADR ADR CDDP MMC

Sustained relief, %

5HIAA decrease 1 50%, %

100 73 90 100

91 57 69 –

ADR = Adriamycin ; CDDP = cisplatin; MMC = mitomycin C.

out metastatic carcinoid tumors are 0–40% and 75–99%, respectively [3,4]. Corresponding figures are 20 and 65% in patients with metastatic gastrinomas [5]. Unfortunately, most liver metastases are diffuse at the time of diagnosis and surgical resection is rarely feasible [6]. Intravenous chemotherapy may be used in patients with diffuse and/or progressive liver metastases. However, the rate and median duration of objective responses with systemic chemotherapy are disappointing and patients’ survival is not significantly increased [7–11]. The therapeutic strategy must be adapted to these tumors, which may progress slowly, and must begin with symptomatic treatment. Subsequently, if the tumor is progressive, or bulky (invasion of 150% of the liver), or symptomatic despite medical treatment, cytotoxic or locoregional treatment can be proposed [12]. Arterial hypervascularisation of liver metastases from digestive endocrine tumors argues in favor of chemoembolization [13]. Moreover, several arguments favor a combination of intra-arterial chemotherapy and embolization or surgical ligation of the hepatic artery: (a) certain drugs such as doxorubicin and mitomycin C, an alkylating agent, like streptozotocin, have a more active impact on anoxic cells (ischemic tumors) [14]; (b) ischemia may stimulate the increasing sensitivity of tumor cells to drug activity by increasing the number of mitotic cells, and (c) anoxia and the slowing of blood flow can increase intra-tumor drug concentrations and dwelling time in tumor cells [15]. The major aims of chemoembolization are: (1) to control symptoms (diarrhea, flushing, hypoglycemic attacks, ...), and hormonal release; (2) to inhibit tumor growth; (3) to improve patients’ survival.

80


Methods Diagnostic arteriography of the superior mesenteric artery and coeliac trunk is performed using the femoral Seldinger technique, to evaluate distribution of the hepatic arteries, portal blood flow and the number and location of liver metastases. First, an emulsion is prepared by mixing the contents of two syringes via a three-way stopcock: cytotoxic drug (adriamycin 50 mg/m2 or streptozotocin 1.5 g/ m2), dissolved in 10 ml of saline (0.9% NaCl in water) on the one hand and 10 ml of iodized oil (Lipiodol®) on the other. The mixture is then injected in the branches of the hepatic artery supplying the tumors, distally from the origin of the gastroduodenal artery. Embolization is then performed with gelatine sponge 2–3 mm particles, placed distally in the hepatic artery until a marked decrease is observed in arterial blood flow [16]. Chemoembolization courses are repeated every 3 months because of the slow natural history of endocrine tumors but intervals are adapted to tumoral response and tolerability. Intravenous hydration and antibiotics are given from 3 h before chemoembolization up to 72 h. Analgesics and antiemetics are given when necessary. Somatostatin analogues are administered in patients with the carcinoid syndrome. General anesthesia is required if streptozotocin is used because the acidity of the streptozotocin solution (pH: 3.5–4.5) can be responsible for intense pain during intra-arterial injection of the drug. Contraindications of chemoembolization are complete portal vein obstruction, hepatic insufficiency and also biliary anastomosis, which can induce infectious complications [14].

Results

Symptoms and Hormonal Secretion Carcinoid Syndrome. The efficacy of chemoembolization on the carcinoid syndrome is summarized in table 1. Islet Cell Tumors. Results are difficult to evaluate for gastrinomas because symptoms are treated by proton pump inhibitors. For insulinomas, some cases of sustained improvement in hypoglycemic attacks have been reported, particularly with streptozotocin [21, 22]. Few data are available concerning hormonal secretion.

Ruszniewski/Malka

Table 2. Variations in tumor size

(according to WHO criteria)

Author, year

n

Therasse, 1993 [16] Ruszniewski, 1993 [17] Clouse, 1994 [18, 19] Diaco, 1995 [20] Mavligit, 1993 [23]

23 18 30 10 5

Carc Carc Carc/ICC Carc ICC

Objective response rate, %

Mean duration months

Progression rate, %

35 33 78 60 80

– 21 6–8.5 42.5 18.5

12 17 – 10 –

Carc = Carcinoid tumor; ICC = islet-cell carcinoma.

Variations in Tumor Size, According to WHO Criteria The results of the available published studies are presented in table 2. Recent data concerning our experience with streptozotocin are shown in table 3 and figure 1 [22]. Overall, an objective response was achieved in 8/15 patients (53% ; median duration of 10.5 months) whatever the primary tumour (carcinoid or islet cell carcinoma). The carcinoid syndrome disappeared in 3/5 patients for 10, 11 and 17 months, respectively. Chemoembolization effectively controlled hypoglycaemic attacks (decrease of 150%) in the patient with insulinoma. The biological response was complete in 4 patients for a median duration of 7 months [22]. The hyper/iso vs. hypovascularized pattern of liver metastases, as well as their diffuse/poorly localized vs. discrete pattern has no influence on the response rates. Previous resection of the primary tumor could improve the results of chemoembolization [24, and pers. data]. No direct comparisons between chemoembolization and other therapeutic modalities, such as intravenous chemotherapy, embolization, ligation or intra-arterial chemotherapy are currently available. However, liver metastases of carcinoid tumors are poorly responsive to intravenous chemotherapy. Chemoembolization seems more effective than embolization alone [24].

Table 3. Effects of chemoembolization in 15 patients with metastat-

ic digestive endocrine tumors [from 22] Patient Type of No. tumor

Symptoms

Tumoral response

nature

response – CR (17) STAB (41) – CR (10) STAB (12) CR (11) –* cannot be evaluated1 PR (1) CR (9)

PROG STAB (10) PR (13) PROG PR (12) STAB (12) PR (9) PR (36) PROG

PR (7) STAB (30) – –

PR (7) PR (30) PR (6) PROG

1 2 3 4 5 6 7 8 9

Carc Carc Carc Carc Carc Carc Carc Carc ICC

none D D, F None D, F D, F D, F none ulcer disease

10 11

ICC ICC

12 13 14 15

ICC ICC ICC ICC

hypoglycemia nausea, abdominal pain F D none none

PROG PR (6)

Carc = Carcinoid tumor; ICC = islet-cell carcinoma; D = diarrhea; F = flushing; CR = complete response; PR = partial response; STAB = stabilization; PROG = progression. * No further evaluation (Somatostatin). Numbers in parentheses indicate the duration of response (in months). 1 Treatment by proton pump inhibitors.

Tolerance

Minor side effects are mainly the postembolization syndrome which is common, mild and transient, lasting less than a week in majority of cases: nausea and vomiting (50–70%), abdominal pain (50–60%), fever up to 38.5 ° C (30–60%) and raised transaminases (100%). Major complications are rare. Acute renal failure and bleeding peptic ulcers can be observed.

When liver metastases are resectable (localized tumors), surgery is required. If unresectable liver metastases are not progressive, symptomatic treatment is adminis-

Chemoembolization of Endocrine Tumors


Hepatic Arterial Chemoembolization: For Whom?

81

a

c

Fig. 1. a Contrast-enhanced computerized tomography in a 50-year-

old woman, showing a fundic hypervascularized mass (arrow) (corresponding to a carcinoid tumor at biopsy sample examination) with hypervascularized liver metastases. b Hepatic arterial arteriography during first course of chemoembolization, showing multiple tumoral blushes. c Contrast-enhanced computed tomography after partial gastrectomy and four courses of chemoembolization, showing objective response (with patterns suggestive of central necrosis (arrows) of liver metastases). Arrowhead = Surgical clips.

b

tered when necessary. For progressive tumors, intravenous chemotherapy and/or chemoembolization can be proposed. If metastases are confined to the liver, chemoembolization can be the first-line treatment in patients with carcinoid tumors, poorly responsive to systemic chemotherapy, while intravenous chemotherapy must be proposed at first in patients with islet cell carcinomas. If there are extrahepatic metastases, the interest of chemoembolization is scarce in patients with islet cell carcinomas, but chemoembolization can still be proposed in

82


patients with carcinoid tumors, mainly after failure of systemic chemotherapy. In conclusion, chemoembolization could be an alternative treatment for progressive liver metastases from digestive endocrine tumors, mainly following unsuccessful systemic chemotherapy. The reported results and tolerance of chemoembolization are encouraging: follow-up of these patients and additional studies will perhaps confirm the role of chemoembolization in the therapeutic strategy for patients with advanced digestive neuroendocrine tumors.

Ruszniewski/Malka

References 1 Moertel CG: An odyssey in the land of small tumors. J Clin Oncol 1987;5:1503–1522. 2 Capella C, Heitz PU, Höfler H, Solcia E, Klöppel G: Revised classification of neuroendocrine tumours of the lung, pancreas and gut. Virch Arch 1995;425:547–560. 3 Godwin JD II: Carcinoid tumors: An analysis of 2837 cases. Cancer 1975;36:560–569. 4 Zeitels J, Naunheim K, Kaplan EL, Straus F II: Carcinoid tumors : A 37-year experience. Arch Surg 1982;117:732–737. 5 Mignon M, Ruszniewski P, Haffar S, Rigaud D, René E, Bonfils S: Current approach to the management of tumoral process in patients with gastrinoma. World J Surg 1986;10:703– 710. 6 McEntee GP, Nagorney DM, Kvols LK, Moertel CG, Grant CS: Cytoreductive hepatic surgery for neuroendocrine tumor. Surgery 1990; 108:1091–1096. 7 Broder LE, Carter SK: Pancreatic islet-cell carcinoma. II. Results of therapy with streptozotocin in 52 patients. Ann Intern Med 1973;79: 108–118. 8 Moertel CG, Lefkopoulo M, Lipsitz S, Hahn RG, Klaassen D: Streptozocin-doxorubicin, streptozocin-fluorouracil, or chlorozotocin in the treatment of advanced islet-cell carcinoma. N Engl J Med 1992;326:519–523. 9 Moertel CG, Hanley JA, Johnson LA: Streptozocin alone compared with streptozocin plus fluorouracil in the treatment of advanced isletcell carcinoma. N Engl J Med 1980;303:1189– 1194.

Chemoembolization of Endocrine Tumors

10 Kessinger A, Foley JF, Lemon HM: Therapy of malignant APUD cell tumors: Effectiveness of DTIC. Cancer 1983;51:790–794. 11 Ruszniewski P, Hochlaf S, Rougier P, Mignon M: Chimiothérapie intraveineuse par streptozotocine et 5 fluoro-uracile des métastases hépatiques du syndrome de Zollinger-Ellison: étude prospective multicentrique chez 21 patients. Gastroenterol Clin Biol 1991;15:393– 398. 12 Moertel CG, Johnson CM, McKusick MA, Martin JK, Nagorney DM, Kvols LK, Rubin J, Kunselman S: The management of patients with advanced carcinoid tumors and islet cell carcinomas. Ann Intern Med 1994;120:302– 309. 13 De Baère T, Dufaux J, Roche A, Counnord JL, Berthault MF, Denys A, Pappas P: Circulatory alterations induced by intra-arterial injection of iodized oil and emulsions of iodized oil and doxorubicin: Experimental study. Radiology 1995;194:165–170. 14 Roche A: Chimio-embolisation hépatique. Bull Cancer 1989;76:1029–1037. 15 Taourel P, Dauzat M, Lafortune M, Pradel J, Rossi M, Bruel JM: Hemodynamic changes after transcatheter arterial embolization of hepatocellular carcinomas. Radiology 1994;191: 189–192. 16 Therasse E, Breittmayer F, Roche A, De Baère T, Indushekar S, Ducreux M, Lasser P, Elias D, Rougier P: Transcatheter chemoembolization of progressive carcinoid liver metastases. Radiology 1993;189:541–547. 17 Ruszniewski P, Rougier P, Roche A, Legmann P, Sibert A, Hochlaf S, Ychou M, Mignon M: Hepatic arterial chemoembolization in patients with liver metastases of endocrine tumors. A prospective phase II study in 24 patients. Cancer 1993;71:2624–2630.

18 Clouse ME, Perry L, Stuart K, Stokes KR: Hepatic arterial chemoembolization for metastatic neuroendocrine tumors: Digestion 1994; 55(suppl 3):92–97. 19 Perry L, Stuart K, Stokes KR, Clouse ME: Hepatic arterial chemoembolization for metastatic neuroendocrine tumors. Surgery 1994; 116:1111–1117. 20 Diaco DS, Hajarizadeh H, Mueller CR, Fletcher WS, Pommier RF, Woltering EA: Treatment of metastatic carcinoid tumors using multimodality therapy of octreotide acetate, intra-arterial chemotherapy, and hepatic arterial chemoembolization. Am J Surg 1995;169:523– 528. 21 Nesovic M, Ciric J, Radojkovic S, Zarkovic M, Durovic M: Improvement of metastatic endocrine tumors ot the pancreas by hepatic artery chemoembolization. J Endocrinol Invest 1992; 15:543–547. 22 Dominguez S, Denys A, Madeira I, Hammel P, Vilgrain V, Menu Y, Bernades P, Ruszniewski P: Hepatic arterial chemoembolization with streptozotocin in patients with metastatic digestive endocrine tumours. Eur J Gastroenterol Hepatol 2000;12:151–157. 23 Mavligit GM, Pollock RE, Evans HL, Wallace S: Durable hepatic tumor regression after arterial chemoembolization-infusion in patients with islet-cell carcinoma of the pancreas metastatic to the liver. Cancer 1993;72:375–380. 24 De Baère T, Lagrange C, El Kashif H, Roche A: Chimioembolisation des métastases hépatiques des tumeurs carcinoı¨des; in: Rougier P (ed): Tumeurs neuroendocrines malignes. Paris, Arnette, 1997, pp 83–91.


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Treatment of Neuroendocrine GEP Tumours with Somatostatin Analogues A Review

R. Arnold B. Simon M. Wied Department of Internal Medicine, Philipps University, Marburg, Germany

Key Words Neuroendocrine tumours W GEP tumours W Medical treatment W Octreotide W Long-acting somatostatin analogues

Abstract Background: Somatostatin and its long-acting analogues are effective in symptom control in patients with functionally active neuroendocrine GEP tumours. Several in vitro and in vivo reports suggest that they are also able to control tumour growth. Methods: Critical review of published data on the effect of long-acting somatostatin analogues on symptom and growth control in patients with metastatic neuroendocrine GEP tumours. Results: With the exception of insulinoma and gastrinoma, octreotide acetate and other long-acting somatostatin formulations are currently the therapeutic principle of first choice to control hormone-mediated symptoms. The consequences of gastric acid hypersecretion in patients with Zollinger-Ellison syndrome are best controlled by proton pump inhibitors. Available data on growth control indicate that stabilization of tumour growth seems to be the most beneficial antiproliferative effect occurring in up to 50% of patients. This effect is limited. However, it is unknown which tumour entity responds best to longacting somatostatin analogues. Conclusion: Additional

ABC

© 2000 S. Karger AG, Basel 0012–2823/00/0625–0084$17.50/0



studies in patients with known spontaneous tumour growth and avoiding a mix-up of different entities of neuroendocrine malignancies are necessary to identify subpopulations of neuroendocrine tumours which respond to long-acting somatostatin analogues in terms of longer lasting growth inhibition. Copyright © 2000 S. Karger AG, Basel

According to their secretory products released or not released into the circulation, neuroendocrine GEP tumours can be categorized into functionally active and inactive tumours. The functionally active tumours, their hormonal products and the main clinical syndromes are listed in table 1. As in the case of insulinoma, VIPoma and glucagonoma, symptoms can threaten a patient’s life and require, therefore, effective control. Somatostatin and its long-acting analogues have been proven as drugs of first choice in the reliable control of hormone-mediated symptoms. In addition, somatostatin and its analogues exert antiproliferative effects both in vitro and in vivo. Several reports suggest that they are effective in the control of growth in metastatic neuroendocrine GEP tumours. The respective data will be reviewed in this report.

Rudolf Arnold, MD, FRCP, Professor of Internal Medicine Department of Internal Medicine, Philipps University D–35034 Marburg/Lahn (Germany) Tel. +49 6421 286 6460, Fax +49 6421 286 8922 E-Mail [email protected]

Table 1. Main tumour characteristics and leading symptoms in patients with neuroendocrine GEP tumours

Tumour

Symptoms/syndrome

Main hormone/ peptide

Percent malignant

Main sites (%)

Insulinoma

Neuroglucopenia Whipple’s trias

Insulin

5–10

Gastrinoma

Zollinger-Ellison syndrome Peptic ulcer Diarrhoea Epigastric pain

Gastrin

60–90

Pancreas (30–60) Duodenum (30–40) Others (10–20)

VIPoma

Verner-Morrison syndrome Pancreatic cholera syndrome Watery diarrhoea

VIP

80–90

Pancreas (90)

Glucagonoma

Glucagonoma syndrome Necrolytic migratory erythema Diabetes mellitus Sore red tongue

Glucagon

60

Pancreas (1 90)

Somatostatinoma

Gallstones Diabetes mellitus Upper abdominal pain or functionally inactive

Somatostatin

60

Pancreas (60) Duodenum (30)

GRFoma

Acromegaly

GRF

50

Pancreas (30) Lung (50) Small intestine (10) Others (10)

Carcinoid tumour

Carcinoid syndrome or functionally inactive

Serotonin Tachykinins

90

Small intestine (70) Bronchial system (10) Pancreas (5) Others (15)

Pancreas

Because of their longer half-life, long-acting analogues as octreotide acetate (Sandostatin®), lanreotide and recently a long-lasting release (LAR) octreotide formulation have been introduced to control hormone-mediated symptoms. They represent a significant breakthrough in the symptomatic treatment of patients with functionally active neuroendocrine GEP tumours. Somatostatin exerts its cellular action through interaction with its specific heptahelical plasma membrane receptors which belong to the superfamily of G-protein-coupled membrane receptors [1]. While somatostatin-14 binds preferentially to somatostatin receptors sstr 1–3, sstr 4 binds somatostatin-14 and somatostatin-28 with equal affinities and sstr 5 somatostatin-28 with higher affinity than somatostatin-14 [2, 3]. It has been shown that sstrs are linked to intracellular signalling pathways through pertussis toxin-sensitive and pertussis toxininsensitive Gi/Go · G-protein subunits [1, 4].

The longer-acting analogues octreotide and lanreotide are modifications of the naturally occurring somatostatin14 (fig. 1). Octreotide contains four core amino acids essential for binding to somatostatin cell-surface receptors. In the lanreotide molecule, amino acid No. 7 (Phe) has been substituted by Tyr and amino acid No. 10 (Thr) by Lys (fig. 1). Size reduction and the amino acid modifications shown in figure 1 protect the molecule against enzymatic degradation and prolong the plasma half-life of octreotide to approximately 1.5 h and of lanreotide to 2.5 h vs. the 2–3 min half-life of somatostatin-14. Octreotide acetate and lanreotide bind preferentially to sstr 2 and to a lower degree to sstr 5 [1, 3]. In contrast, sstr 1 and sstr 4 do not bind octreotide acetate and lanreotide and sstr 3 display intermediate affinity towards these synthetic analogues [3]. It has been demonstrated that octreotide acetate and lanreotide mediate their effects on hormone secretion by the tumour cell and their antiproliferative effects predominantly through sstr2 [1–3, 5, 7]. In fact, most neuroendocrine tumours express sstrs. Using in

Long-Acting Somatostatin Analogues


Somatostatin and Its Analogues

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In the newly developed long-acting slow-release formulation of Sandostatin® LAR, octreotide acetate is incorporated into microspheres of the biodegradable polymer poly(D,L-lactide-co-glycolide glucose). Slow release of octreotide acetate occurs through the cleavage of the polymer ester linkage, primarily through tissue fluid hydrolysis. Administration is by intramuscular depot injection every 28 days in contrast to the subcutaneous every 8 h administration of octreotide acetate. Recommended doses of Sandostatin® LAR are 20 and 30 mg every 28 days. The respective studies demonstrate that a single dose provides a plateau serum level of octreotide for approximately 54 days after injection [9]. Since in individual patients escape has been observed between days 21 and 42, an administration interval of 28 days has turned out to guarantee consistent elevated octreotide serum concentrations providing pronounced suppression of hormone release from endocrine tumours as shown in growth hormone-secreting pituitary adenomas [9]. Lanreotide is also available in a slow release (SR) formulation obtained by incorporating lanreotide into polyactide-polyglycolide copolymer microspheres. The duration of efficacy is 2 weeks after intramuscular injection of 30 mg. Lanreotide SR has been demonstrated to reliably control symptoms in patients with carcinoid syndrome.

Control of Symptoms in Patients with Functionally Active Neuroendocrine Tumours by Long-Acting Somatostatin Analogues

Fig. 1. Structure of human somatostatin-14 and its long-acting analogues which are currently used in clinical practice.

vitro autoradiography, in situ hybridization and immunocytochemistry, all 5 sstrs have been identified in neuroendocrine GEP tumours [2, 6–8]. However, sstr expression varies from tumour to tumour and even within a specific tumour a different expression pattern can be found. It is generally accepted that tumours without sstr 2 do not respond to long-acting somatostatin analogues. Since only 50% of insulinoma express sstr 2 [7], the beneficial effect of long-acting somatostatin analogues in the prevention of hypoglycaemia occurs only in patients with sstr 2 expressing insulinomas.

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Carcinoid Syndrome Long-acting somatostatin analogues as octreotide acetate and lanreotide have been repeatedly shown to improve or even normalize diarrhoea and flushing in patients with carcinoid syndrome [10, 12–15] during both short- and long-term treatment. Symptom relief is correlated with reduction of hormonal markers. In addition, long-acting somatostatin analogues are indispensable in the treatment and prevention of bronchoconstriction and of carcinoid crisis. They have, therefore, to be administered during laparotomy or during and after embolization of liver metastases. Octreotide LAR administered at 4week intervals is equally potent in the control of flushing and of diarrhoea if compared to the subcutaneous administration route [17]. It has to be evaluated in each individual patient whether 10, 20 or 30 mg are necessary for symptomatic control. To predictably and stably reach the critical blood concentration level 11,000 mg/l, around 4 months of treatment are needed. Therefore, some patients

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Table 2. Medical control of diarrhoea in

patients with carcinoid syndrome

Cause of diarrhoea

Treatment

Hormone-associated

Octreotide Octreotide LAR Lanreotide Cyproheptadine Methysergide Odansetrone Loperamide

2 ! 50 to 3 ! 200 Ìg/daily s.c. 10–20 mg every 28 days i.m. 30 mg every 14 days i.m. 4–8 mg t.i.d. 3–4 mg b.i.d. 8 mg b.i.d. 2 mg t.i.d.

Loss of bile acids

Colestyramine

1–3 ! 4 g daily

Bacterial overgrowth

Tetracycline Metronidazole plus Cephalosporin

4 ! 250 mg daily 3 ! 250 mg daily

Pancreatic insufficiency

4 ! 40,000 IU lipase daily

Consequence of somatostatin

Dose reduction

need supplemental rescue therapy with octreotide s.c. within the first 2–3 months after switching from octreotide s.c. to octreotide LAR i.m. [17]. Diarrhoea in patients with carcinoid syndrome is not always the consequence of hormone excess secretion. Sometimes diarrhoea deteriorates or even starts after surgical removal of the primary tumour in the terminal ileum, by wide segmental resection, by removal of the ileocaecal wave or by ileocolic anastomosis. These procedures result in bile acid loss and/ or increased bacterial contamination of the small intestine with subsequent deconjugation of bile acids. Diarrhoea mediated by bile acid loss should be treated with cholestyramine and diarrhoea as the consequence of bacterial contamination with antibiotics (table 2). Whether or not long-acting somatostatin analogues favourably influence onset and course of carcinoid heart disease has not been investigated systematically. Apart from long-acting somatostatin analogues and in case of insufficient symptom control, there are second-line treatment options available in the control of symptoms from carcinoid syndrome which are summarized in table 2. Verner-Morrison Syndrome Long-acting somatostatin analogues are currently the therapeutic principle of first choice to control watery, highvolume diarrhoea with the risk of loss of electrolytes and dehydration in patients with Verner-Morrison syndrome [11, 16, 18–21]. Somatostatin analogues act by inhibition of hormone release from the tumour and by direct inhibition of water and electrolyte secretion from the intestine [18]. Recommended doses are 50–200 Ìg octreotide ace-


4 ! 250 mg daily

tate s.c. t.i.d., 30 mg lanreotide SR i.m. every 14 days and 10–30 mg octreotide LAR i.m. every 28 days. Glucagonoma Syndrome The necrolytic migratory erythema, protein hypercatabolism, hypoacidonaemia and diarrhoea are the leading symptoms in patients with the glucagonoma syndrome. Significant improvement of these symptoms can be achieved by long-acting somatostatin analogues in doses as recommended for patients with Verner-Morrison syndrome [11, 22, 45]. Zollinger-Ellison Syndrome Long-acting somatostatin analogues suppress gastric acid secretion by a direct effect on the parietal cell and by inhibiting gastrin release from the tumour cell [23]. However, they have to be administered subcutaneously or intramuscularly and do not in every patient lower acid secretion sufficiently [24–26]. Therefore, long-acting somatostatin analogues cannot compete with the advantages of the currently available and even after oral intake highly effective proton pump inhibitors omeprazole, lansoprazole, pantoprazole and rabeprazole [27]. To reduce basal acid ouput !10 mval/h which ensures ulcer healing and stops diarrhoea, one or two daily doses of omeprazole or the equivalent amount of the other proton pump inhibitors are sufficient. Insulinoma Because only 50% of insulinomas have sstr 2 receptors [7], the effect of long-acting somatostatin analogues in the


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Table 3. Reported side effects during treatment with long-acting

somatostatin analogues Pain at injection site Nausea Flatulence Abdominal pain Diarrhoea Steatorrhoea Hyperglycaemia Gallstones Rash Alopecia Water intoxication

Antiproliferative strategies for the control of tumour growth in patients with metastatic neuroendocrine GEP tumours include several options: surgery with removal of the primary tumour and resection of metastases (debulking); radiology with chemoembolization; nuclear medicine with tumour-targeted radiotherapy (90Y-dota-octreotide, 111In-dota-lanreotide); medical therapy including chemotherapy, ·-interferon and long-acting somatostatin analogues.

prevention of hypoglycaemia is unpredictable in the individual insulinoma patient [28]. Another hypothesis is that only insulinomas with typical ß-granules respond to somatostatin similar to the experience with diazoxide which is ineffective in agranular tumours and those with atypical ß-granules [29]. In insulinoma patients not responding to somatostatin and its analogues, hypoglycaemia can even be aggravated through suppression of counterregulatory hormones as glucagon. Therefore, the administration of somatostatin analogues in patients with solitary insulinoma should be avoided and in patients with metastatic disease handled with caution. Side Effects of Treatment with Long-Acting Somatostatin Analogues Adverse effects of treatment with long-acting somatostatin analogues occur (table 3) but force rarely to discontinue treatment. A relevant adverse effect is the development of gallstones deriving from inhibition of cholecystokinin release which postprandially induces emptying of the gallbladder [30]. Up to 60% of patients under longterm treatment with octreotide acetate or lanreotide are believed to develop gallstones but the exact figures are unknown. Some patients develop diarrhoea after onset of treatment which according to our own experiences occurs frequently in patients after segmental resection of the terminal ileum or ileocaecal anastomosis after removal of an ileum carcinoid tumour. Here, diarrhoea is the result of bile acid loss and responds to administration of an anionbinding recin as cholestyramine. In anecdotal reports, water intoxication has been described [31]. Adverse events during treatment with octreotide acetate LAR are similar and only mild to moderate in severity [17].

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Control of Growth in Patients with Metastatic Neuroendocrine GEP Tumours by Long-Acting Somatostatin Analogues


Background Evidence for antiproliferative properties of somatostatin and its analogues derives from in vitro and in vivo studies [1, 32–38, 46]. Somatostatin can exert both cytotoxic and cytostatic actions. It has been demonstrated that it mediates arrest at the G1 phase of the cell cycle and that this effect is mediated through activation of sstr 2 and sstr5 [46]. In human breast carcinoma cells, somatostatin activates an apoptotic pathway via induction of p53 and Bax through activation of sstr 3 [46]. These data indicate that the effects of somatostatin are mediated via somatostatin receptors and that sstr 2 and sstr 5 are major players in this context. Beside these receptor-dependent effects, somatostatin exerts receptor-independent effects to influence cell growth. These include: endocrine effects to inhibit the release of circulating tumour growth-promoting humoral effectors; vascular effects with inhibition of angiogenesis and/or blood supply via peritumoural vessels and effects on immune system. In addition to these experimental data, earlier case reports describing tumour regression in patients with metastatic neuroendocrine GEP tumours support the hypothesis that somatostatin has antiproliferative properties [39, 40, 47, 48]. Recent Prospective Studies These in vitro and in vivo studies as well as the abovementioned case reports prompted several phase II trials to study the effect of the long-acting analogues octreotide and lanreotide in patients with metastatic neuroendocrine carcinoma [10, 41–44] (table 4). Different from earlier studies, these trials included patients with CT-documented tumour progression only. This is important since tumour growth in patients with metastatic neuroendocrine carcinoma is not uniform. Neuroendocrine malignancies can exhibit long phases of spontaneous tumour standstill or reveal exploding growth [49] (fig. 2). Even

Arnold/Simon/Wied

Table 4. Long-acting somatostatin

analogues in the control of growth in patients with metastatic neuroendocrine GEP tumours

Regular dose Saltz et al., 1993 [41] n = 34 Arnold et al., 1996 [10] n = 52 Di Bartolomeo et al., 1996 [42] n = 38 Ultrahigh dose Erikson et al., 1997 [43] n = 13 Faiss et al., 1999 [44] n = 30

Stabilization of tumour growth for 2–27 months in 50% No tumour regression Stabilization of tumour growth for 3–60 months in 36% No tumour regression Stabilization of tumour growth for 6–32 months in 52% Partial tumour regression in 3% Stabilization of tumour growth for 6–32 months in 70% Partial tumour regression in 5% Stabilization of tumour growth after 1 year in 36% Partial tumour regression in 3% Complete remission in 3%

Table 5. Case report: 56-year-old female patient with metastasized

neuroendocrine pancreatic carcinoma Occupation: nurse No previous serious diseases Since 1986: osteoporosis prophylaxis with oestrogen/gestagen combination 10/1995: relapsing watery diarrhoea 3/1996: colonoscopy 6/1996: heartburn, postprandial fullness 7/1996: gastroscopy 8/1996: abdominal ultrasound/CT: multiple metastases in the liver, tumour of the pancreas corpus 9/1996: inclusion into the Marburg Multicenter Trial: octreotide vs. octreotide plus ·-interferon Follow-up: stop of watery diarrhoea 1 90% decrease in size of pancreatic primary and liver metastases

Fig. 2. Schematic presentation how neuroendocrine malignancies

can grow. Notice that some tumours (→) grow so slowly that they do not meet the 25% increase according to the accepted NIH criteria of tumour progression even after 18 months. Without knowing this specific spontaneous tumour growth behaviour, the respective patient could falsely be categorized into the responder group to treatment.

spontaneous tumour regression without any treatment has been observed in the author’s institution in a female patient whose case report is summarized in table 5. Although oestrogen receptors were not investigated on the tumour since the patient refused additional fine-needle biopsy, it is likely that the oestrogen-gestagen substitution to prevent postmenopausal symptoms had trophic action on tumour growth. As indicated in figure 2, WHO criteria to assess tumour growth in oncology can be misleading if a tumour and/or its metastases increase by less than 25% within a given time period and this growth behaviour is interpreted as stable disease in response to treatment. In fact, in the patient marked with an arrow in figure 2 the same slow growth behaviour could have occurred before start of treatment. Therefore, true response to any treatment in patients with neuroendocrine carcinoma can only be assumed if the spontaneous growth behaviour prior to

treatment is recognized. Many previously published studies in patients with neuroendocrine carcinomas have not considered these specific features of neuroendocrine tumour growth. A recently published retrospective analysis concerning the effect of octreotide treatment on tumour growth in 96 patients with metastatic neuroendocrine malignancies reporting a decrease of metastases in 13%, unchanged tumour size in 63% and progressive disease in only 24% of patients is misleading since the spontaneous course of tumour growth prior to octreotide treatment has not been evaluated [50]. The studies listed in table 4 are comparable in the sense that only patients with progressive disease have been included. Contrary to earlier published data includ-



89

ing the above-mentioned NIH report [50], partial tumour regression was a rare event and stabilization of tumour growth the most favourable result occurring in 36–70% of patients. However, even stable disease is relatively shortlasting since growth inhibition was reported to last for 2– 60 months. The published reports do not answer important questions: (1) Does treatment with long-acting somatostatin analogues prolong a patient’s life? (2) What is the antiproliferative dose: low, medium, ultra-high? (3) Should octreotide be administered once per day or t.i.d.? (4) Is octreotide LAR as effective as the short-lasting formulation? (5) Which tumour responds best to treatment: slow, intermediate or fast growing tumours? (6) Do tumours from the fore-, mid- and hindgut equally respond to treatment? To answer these and other important questions, multicentre trials are mandatory to elaborate the possible benefit of treatment with long-acting somatostatin analogues which are much better tolerated than ·-interferon, chemotherapy and liver embolization. These trials should include homogeneous tumour entities (e.g. patients with endocrine pancreatic tumours only, patients with midgut tumours only, etc.) and evaluation of tumour response to treatment must be based on the spontaneous tumour growth prior to treatment. Combination Treatment of Neuroendocrine Carcinomas with Octreotide and ·-Interferon Some case reports and one prospective trial with a limited number of patients suggest that the combination

treatment of neuroendocrine tumours with octreotide acetate plus ·-interferon is superior if compared to treatment with either compound [51–53]. These data have to be confirmed by well-executed prospective studies including a higher number of patients and recognizing the above-mentioned demands. In summary: Octreotide acetate and other long-acting somatostatin analogues are currently the therapeutic principle of first choice to control flushing, wheezing, diarrhoea and bronchospasm in patients with carcinoid syndrome; diarrhoea in patients with Verner-Morrison syndrome and necrolytic migratory erythema in patients with glucagonoma syndrome. The sequelae of gastric acid hypersecretion in patients with Zollinger-Ellison syndrome can perfectly be controlled by oral proton pump inhibitors. The beneficial effect of long-acting somatostatin analogues to prevent hypoglycaemia in insulinoma patients depends on the presence of sstr 2 receptors which are found in only 50% in this tumour entity. Available data indicate that long-acting somatostatin analogues have also antiproliferative properties. Contrary to earlier case reports and to retrospective analyses, prospective studies indicate that stabilization of tumour growth seems to be the most beneficial effect. Additional studies in patients with known spontaneous tumour growth and avoiding a mix-up of different tumour entities are in progress to identify those patients and neuroendocrine malignancies which respond to long-acting somatostatin analogues in terms of growth inhibition.

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27 Metz DC, Jensen RT: Advances in gastric antisecretory therapy in Zollinger-Ellison syndrome; in Mignon M, Jensen RT (eds): Endocrine Tumours of the Pancreas. Recent Advances in Research and Management. Front Gastrointest Res. Basel, Karger, 1995, vol 23, pp 240–257. 28 Maton PN, Gardner JD, Jensen RT: Use of long-acting somatostatin analogue SMS 201995 in patients with pancreatic islet cell tumours. Dig Dis Sci 1989;34(suppl):285–291. 29 Nauck M, Stöckmann F, Creutzfeldt W: Evaluation of a euglycaemic clamp procedure as a diagnostic test in insulinoma patients. Eur J Clin Invest 1990;20:15–18. 30 Dowling RH, Hussaini SH, Murphy GM, et al: Gallstones during octreotide therapy. Metabolism 1992;41(suppl 2):22–23. 31 Halma C, Jansen JB, Janssens R, et al: Lifethreatening water intoxication during somatostatin therapy. Ann Intern Med 1987;107:518– 529. 32 Taylor JE, Bogden AE, Moreau JP, Coy DH: In vitro inhibition of human small cell lung carcinoma (NCL-H69) growth by somatostatin analogue. Biochem Biophys Res Commun 1988; 153:81–86. 33 Siegel RA, Tolscavai L, Rudin M: Partial inhibition of the growth of transplanted Dunning rat prostate tumors with the long-acting somatostatin analogue Sandostatin (SMS 201995). Cancer Res 1988;153:4651–4655. 34 Liebow C, Reilly C, Serrano M, Schally AV: Somatostatin analogues inhibit growth of pancreatic cancer by stimulating tyrosine phosphatase. Proc Natl Acad Sci USA 1989;86:2001– 2007. 35 Buscail L, Saint-Laurent N, Chastre E, Vaillant JC, Gespach C, Capella G, Kalthoff H, Vaysse N, Susini C: Loss of sst2 somatostatin receptor gene expression in human pancreatic and colorectal cancer. Cancer Res 1996;56:1823–1827. 36 Sharma K, Patel YC, Srikant CB: Subtypeselective induction of wild-type p53 and apoptosis, but not cell cycle arrest, by human somatostatin receptor 3. Mol Endocrinol 1996; 10:1688–1696. 37 Florio T, Scorziello A, Fattore M, D’Alto V, Salzano S, Rossi G, et al: Somatostatin inhibits PC C13 thyroid cell proliferation through the modulation of phosphothyrosine phosphatase activity. J Biol Chem 1996;271:6129–6136. 38 Cordelier P, Esteve JP, Bousquet C, Delesque N, O’Carrol AM, Schally AV, et al: Characterization of the antiproliferative signal mediated by the somatostatin receptor subtype sstr5. Proc Natl Acad Sci USA 1997;921:580–584. 39 Shepherd JJ, Senator GB: Regression of liver metastases in patient with gastrin-secreting tumour treated with SMS 201-995. Lancet 1986; ii:574. 40 Wiedenmann B, Räth U, Rädsch R, Becker F, Kommerell B: Tumour regression of an ileal carcinoid under the treatment with the somatostatin analogue SMS 201-995. Klin Wochenschr 1988;66:75–77.

41 Saltz L, Trochanowsky G, Buckley M, Heffernan B, Niedzwicki D, Tao Y, Kelsen D: Octreotide as an antineoplastic agent in the treatment of functional and non-functional neuroendocrine tumours. Cancer 1993;72:244–248. 42 Di Bartolomeo M, Bajetta E, Buzzoni R, Mariani L, Carnaghi C, Somma L, Zilembo N, Di Leo A and the ITMO Association: Clinical efficacy of octreotide in the treatment of metastatic neuroendocrine tumors. Cancer 1996;77: 402–408. 43 Eriksson B, Renstrup J, Iman H, Öberg K: High-dose treatment with lanreotide of patients with advanced neuroendocrine gastrointestinal tumors: Clinical and biological effects. Ann Oncol 1997;8:1041–1044. 44 Faiss S, Räth U, Mansmann U, Caird D, Clemens N, Riecken EO, Wiedenmann B: Ultrahigh dose lanreotide treatment in patients with metastatic neuroendocrine gastroenteropancreatic tumours. Digestion 1999;60:469–476. 45 Rosenbaum A, Flourie B, Chagnon S, Blery M, Modigliani R: Octreotide (SMS 201-995) in the treatment of metastatic glucagonoma: Report of one case and review of the literature. Digestion 1989;42:116–120. 46 Sharma K, Srikant CB: Induction of wild-type p53, BAX, and acidic endonuclease during somatostatin-signaled apoptosis in MCF-7 human breast cancer cells. Int J Cancer 1998;76: 259–266. 47 Kraenzlin ME, Ch’ng JC, Wood SM, Bloom SR: Can inhibition of hormone secretion be associated with endocrine tumour shrinkage. Lancet 1983;ii:1501–1504. 48 Clements D, Elias E: Regression of metastatic VIPoma with somatostatin analogue SMS 201995. Lancet 1985;i:874–875. 49 Arnold R, Neuhaus C, Benning R, et al: Somatostatin analog Sandostatin and inhibition of tumor growth in patients with metastatic endocrine gastroenteropancreatic tumors. World J Surg 1993;17:511–519. 50 Gordon PH: NIH Conference: Somatostatin and somatostatin analogue (SMS 201-995) in the treatment of hormone-secreting tumors of the pituitary and gastrointestinal tract and nonneoplastic diseases of the gut. Ann Intern 1989; 110:35–50. 51 Joensun H, Kätkä K, Kujari H: Dramatic response of a metastatic carcinoid tumour to a combination of interferon and octreotide. Acta Endocrinol (Copenh) 1992;126:184–185. 52 Janson ET, Öberg K: Long-term management of the carcinoid syndrome. Treatment with octreotide alone and in combination with alphainterferon. Acta Oncol 1993;32:225–229. 53 Frank M, Klose KJ, Wied M, Ishaque N, Schade-Brittinger C, Arnold R: Combination therapy with octreotide and ·-interferon: Effect of tumor growth in metastatic endocrine gastroenteropancreatic tumors. Am J Gastroenterol 1999;94:1382–1387.


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Interferon in the Management of Neuroendocrine GEP-Tumors A Review

Kjell Öberg Endocrine Oncology Unit, and Department of Medical Sciences, Uppsala University Hospital, Uppsala, Sweden

Key Words Interferon-alpha W Neuroendocrine GEP tumors W Combination therapy W Octreotide

Abstract Interferon has been known to be an effective antiviral and antitumor agent since the 1960s. However, a large majority of solid tumors did not respond to this kind of treatment. In early 1980s, alpha interferon was introduced in the management of classical midgut carcinoids with promising results. Since then, more than 500 patients have been treated with alpha interferon worldwide and published in the literature. The median dose has been 5 million units 3–5 times per week subcutaneously. The subjective response rate is about 60%, biochemical responses in 44% and tumor responses in 11% of the patients. There are very few randomized control trials at the moment which make survival analysis difficult but in different centers median survivals from start of treatment in malignant midgut carcinoids has been reported to be more than 3 years. The mechanisms of action of alpha interferon are direct effects on the tumor cells by inhibiting the cell proliferation via cell cycle block G1-S phase. Induction of interferon inducable genes, such as p-21, p-27, 2-5-A-synthetase, PKR, IRF-1, IRF-2. All these contribute to reduction of the growth potential of the tumor. Furthermore, alpha interferon exerts an immuno-

ABC

© 2000 S. Karger AG, Basel 0012–2823/00/0625–0092$17.50/0



modulatory effect stimulating natural killer cells, macrophages and also presents anti-angiogenetic effects. In the future, long-acting formulations of recombinant alpha interferons will come into clinical use, such as polyethylene glycosylated interferons. It will facilitate the treatment for the patients and also perhaps reduce the side effects, which are sometimes significant during treatment with alpha interferon. It might also be possible to give higher doses of alpha interferon with hopefully improved clinical efficacy. Copyright © 2000 S. Karger AG, Basel

Introduction

Interferon has been known to be an effective antiviral and antitumor agent since the 1960s [1, 2]. In the 1970s interferon or interferon inducers were introduced into clinical therapy with limited but nevertheless encouraging results. An initial limitation was attributed to the low availability of interferon for human use. Later in the 1980s large-scale recombinant interferons were produced. Many trials were subsequently initiated using sufficiently high doses of alpha interferons. In most instances, the results were disappointing. The therapeutic effect was not improved by high doses and the side effects were significant. It became clear that interferon could not be considered a conventional cytotoxic agent and it was included in

Kjell Öberg, MD, PhD Department of Medical Sciences, Uppsala University Hospital S–751 85 Uppsala (Sweden) Tel. +46 18 66 49 17, Fax +46 18 51 01 33 E-Mail [email protected]

Table 1. Interferon-alpha therapy in patients with neuroendocrine tumors

Study

Patients

Dose

Biochemical response, %

Tumor response, %

Moertel et al. [8] Schober et al. [9] Hansen et al. [1989]

27 21 19

39 56 40*;86

20 10 10*;86

Bartsch et al. [10] Välimäki et al. [11] Öberg et al. [12] Öberg et al. [13] Norheim et al. [1989]

18 8 37 21 20

IFN2a 24 MIU/m2 ! 3/week SC IFN2b 3 MIU/m2 ! 3/week SC IFN2b 5 MIU ! 8/week SC alone* or with embolization rIFN2c 2 MIU/m2 ! 12/week SC nIFN-· 3 MIU ! 7/week SC nIFN-· 6 MIU ! 7/week IM nIFN2b 5 MIU ! 3/week SC nIFN-· 6 MIU ! 7/week SC versus streptozotocin + 5FU nIFN-· ! 7/week SC rIFN2b 5 MIU ! 3/week rIFN2a 3 MIU/m2 ! 3/week versus rIFN-·2 3 MIU/m2 ! 3/week + streptozotocin + Adriamycin rIFN-·2b 2.5 MIU ! 7/week SC rIFN-·2b 5–6 MIU ! 3–5/week SC rIFN-·2b 3–6 MIU ! 3/week SC

44 50 49 53 50

0 12.5 11 0 11

0 42

0 15

25

17

0

0

60 51 71 44

18 12 27 11

Öberg and Eriksson [4] Janson et al. [14]

Biesma et al. [15] Eriksson and Öberg [16] Dirix et al. [1991] Total 1

111 22

11 571 11 383

Malignant endocrine pancreatic tumors.

the group of biological response modifiers known to interact with other soluble or cell-associated regulatory factors. So far, five interferon classes have been described. Interferon-alpha produced by leukocytes, interferon-beta produced by fibroblasts and interferon-gamma produced mainly by T-lymphocytes. The other two classes, interferon-omega and interferon-teta, are less known in clinical investigations. This chapter will only deal with interferonalpha since this has been the most evaluated interferon class in neuroendocrine GEP tumors [3, 4]. Interferon-alpha was introduced by our group in the treatment of carcinoid tumors in 1982 because of its ability to stimulate natural killer cell function and control hormone secretion, clinical symptoms and tumor growth [5]. At that time, cytotoxic agents were the only available compounds used in the treatment of malignant neuroendocrine GEP tumors [6]. Cytotoxic agents such as streptozotocin alone or in combination with 5-fluorouracil and doxorubicin have been particularly useful for the treatment of malignant endocrine pancreatic tumors but slowgrowing classical midgut carcinoids show resistance against these and other cytotoxic agents [6, 7]. Classical midgut carcinoids mostly present low proliferation capac-

ities and therefore biological treatment was one possibility. We could also demonstrate rather early on that a subset of patients with classical midgut carcinoids displayed low natural killer cell function and therefore we decided to try interferon-alpha, which was the only known compound at that time which stimulated the natural killer cell function [3]. Today, a large number of patients with neuroendocrine tumors have been treated with alpha interferon and a recombinant type of interferon, interferon-alpha 2b (Intron-A®), is registered for the treatment of malignant carcinoid tumors in many European countries.

Interferon and Neuroendocrine GEP-Tumors


Interferon Therapy of Neuroendocrine Tumors

Table 1 shows different published trials with alpha interferon in malignant neuroendocrine tumors, mostly carcinoids but also one study including endocrine pancreatic tumors [8–17]. Most of these studies were performed in the late 1980s and early 1990s. There is only a limited number of recently published trials and the results are not different from those summarized in table 1. Most trials have been using recombinant interferon-alpha eith-

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er 2a (Roferon®) or 2b (Intron-A®) but there are also some studies using human leukocyte interferon. There is essentially no difference in clinical responses between the different subtypes of alpha interferon. However, lower response rates have been reported for alpha interferon 2a, which might be related to the development of neutralizing interferon antibodies (see below). There is a limited supply of human leukocyte interferon and most therapeutic protocols today are using recombinant alpha interferons, particular 2b (Intron-A®). The recommended doses are 3– 9 MU every day or every other day, median 5 MU every other day subcutaneously. However, it is important to point out that the dose should be titrated individually taking into consideration sex, weight and age [3, 16]. As indicated in table 1, the median biochemical response rate from pooled studies is 44% and the median tumor response rate 11%. Stabilization of disease is an important tumor response in neuroendocrine tumor patients. That is noticed in median 35% of the patients. So, all together objective biochemical responses including stabilization is around 75–80% of patients. Tumor progression is noticed in about 15–20%. The duration of tumor responses is varying in different trials but from our own experience in a trial of 111 patients with malignant carcinoids, the median duration of responses was 32 months. We have noticed continuing responses in some patients for more than 15 years. There is no clear dose-response relation-ship. Therefore, optimal tolerated dose should be titrated for long-term use. As shown in table 1, in one trial (Moertel and co-workers from 1989) they tried to use high-dose treatment but they did not obtain more biochemical or tumor responses than moderate-dose treatment [8]. One can use the leukocyte count for titrating the dose of interferon aiming at reducing the number of leukocytes to below 3.0 ! 109/l. No increase in the events of infections is noticed by such a procedure. Subsequently, we have confirmed that this emperical method correlates with the induction of 2–5 A synthetase and PKR which correlates with the clinical response (see below). Another important observation has been done in a Norwegian study, which reported increased response rates after tumor reduction by embolization of liver metastases prior to the start of interferon treatment [17]. Therefore, reduction of the tumor mass may significantly improve the therapeutic results but may also indicate that interferon-alpha should be initiated earlier in the clinical course or after tumor debulking when the tumor masses are limited. Survival analysis of the patients treated with alpha interferon is still waranted. Most published studies are

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not randomized controlled studies, therefore we have to relate survival to historical controls. Such analysis done at our center shows that alpha interferon give significant survival benefit in patients with malignant midgut carcinoids compared with chemotherapy (streptozotocin plus 5-FU, p ! 0.001) but it should be pointed out that it was not a randomized trial but merely historical controls. However, in a Norwegian trial 42 patients with malignant midgut carcinoids were randomized after 1 year of treatment with interferon to continue interferon or receive either chemotherapy or somatostatin analogues [17]. After another 2 years, there was a significant survival advantage (p ! 0.0125) for patients continuing on alpha interferon. There is currently a randomized Swedish multicenter trial which is closed and data might be ready later this year where alpha interferon plus octreotide is compared with octreotide alone. In vitro studies of a neuroendocrine differentiated cell line, BON-1, has indicating a synergistic effect between the somatostatin analogue octreotide and alpha interferon. That has led to clinical trials using the combination of these two compounds. We have earlier published such a trial where patients resistant to octreotide alone, 300 Ìg/day, received interferon-alpha 3 MU 3 times/week s.c. A total of 24 patients were included and by using this combination, a complete biochemical remission was found in 18%, a partial remission in 59% and totally 77% achieved biochemical responses with a median duration of 15 months [18]. However, none of the patients demonstrated any significant reduction of tumor size on computer tomography. When interferon-alpha was withdrawn for whatever reason, immediate increase of biochemical markers and clinical symptoms were noticed but if interferon-alpha was reintroduced, significant improvement in symptoms and reduction in biochemical markers were observed. In a randomized controlled trial in 22 patients, we compared the combination of recombinant interferonalpha 2a, 2 MU/m2 3 times/week plus streptozotocin 1 g/m2 and doxorubicin 40 mg/m2, both administrated every three weeks against interferon-alpha 2a alone in the same dose [14]. There was no apparent benefit from the addition of chemotherapy to interferon-alpha. Moreover, both biochemical (25%) and objective tumor responses (7%) were lower than would have been expected for interferon alone. Furthermore, considerably toxicity was encountered: one patient died from doxorubicin-related cardiotoxicity and it is conceivable that interferon-alpha contributed to the increased sensitivity to doxorubicin. In a phase 1–2 study, in 12 patients with malignant carcinoid tumors, a combination of interferon-alpha and

Öberg

human interferon-gamma (Finnish Red Cross, Helsinki) was used. All patients were treated with interferon-alpha at doses of 5–10 MU 3–5 times/week, for a median of 22 months and showed stable or progressive disease. Interferon-gamma was then added at a daily dose of 0.5 MU s.c. After 6 months’ treatment there was one partial responder, 7 patients showed stable disease, and 3 patients had progressive disease. Half of the patients experienced subjective improvement but no significant tumor reduction was noticed [19].

Table 2. Adverse reactions in 111 patients treated with interferon-

alpha

‘Flu-like’ symptoms Weight loss Fatigue Anemia (! 110 g/l) Leukopenia (! 2.0 ! 109/l) Thrombocytopenia (! 100 ! 109/l) Hepatotoxicity Increased blood lipids Autoimmune manifestations Neutralizing IFN-abs

%

WHO grade

89 59 51 31 7 18 31 32 20 1–38*

1–2 1 1–2 1 1 1 1–2 1–2 – –

* The low figure is for human leukocyte IFN-· and the high figure for IFN-·2a.

Adverse Reactions to Interferon-Alpha

The adverse reactions of interferon-alpha therapy are listed in table 2. Flu-like symptoms are the most common and are mostly easy to handle by administration of paracetamol. This side effect is usually also short-lasting, up to 1 week. More severe side effects are chronic fatigue syndrome and mental depression which develop at various grades in about 50% of the patients. This is sometimes difficult to manage and treatment has to be stopped. Today, the new serotonin uptake inhibitors might be of benefit for treating this chronic fatigue syndrome and also mental depression. About 1/3 of the patients developed increased liver enzymes which is usually mild and one only occasionally has to withdraw the treatment. Autoimmune reaction is seen in around 15–20% of the patients and the majority are developing thyroid dysfunction, which might contribute to the fatigue that patients may experience. More severe autoimmune reactions includes the SLE syndrome and polymyocitis [20]. Patients treated with recombinant interferonalpha may develop neutralizing interferon antibodies to varying degrees with different interferon preparations, which may abrogate the antitumor response (table 3) [21]. Titres of neutralizing antibodies above 400 neutralizing units appear to be associated with an impaired antitumor effect. In such patients, a change from recombinant interferon-alpha to human leukocyte interferon may restore the antitumor effect.

Table 3. Anti-interferon antibodies developing in patients with midgut carcinoids treated with various types of interferons

Treatment

Introna A Roferon Wellferon Leukocyte IFN

Binding antibodies (immunoassay) n1

%2

1041 13 1 1

51 45 2 2

Neutralizing antibodies (bioassay) all positive

high titer3

n

%

n

%

354 11 0 0

17 38 0 0

9 8 0 0

4 28 0 0

Antibody negative n

%

100 16 44 48

49 55 98 98

All patients tested

204 29 45 49

1

The figures represent the number of patients. The proportion of patients with or without respective antibodies in percent of all patients. 3 High titer neutralizing antibodies is defined as 1 800 ‘neutralizing units’, the figure approximating the number of units interferon neutralized by 1 ml serum. The lower limit of detection was 50 ‘neutralizing units’. 4 Only patients with binding antibodies had neutralizing antibodies, because the immunoassay was routinely used to select patients for further analysis of neutralizing antibodies. 2



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Mechanism of Action of Interferon-Alpha in Carcinoid Tumors

The antitumor effect of interferon-alpha include direct and indirect mechanisms. The direct effects include antiproliferation, apoptosis, differentiation and anti-angiogenetic effects. Furthermore, interferon-alpha clearly demonstrates an immunomodulatory effect by increasing the expression of class 1 antigenes on tumor cells and induction of autoimmunity. Much progess has been made in elucidating the signal transduction pathway activated by alpha interferon. Ligand binding to specific receptors at the plasma membrane results in dimerization of receptor subunits and thereby initiates a rapid autophosphorylation of the receptor associated Janus tyrosine kinases (JAKs) JAK 1 and JAK 2. The activated JAKs in turn tyrosine-phosphorylate and activate latent cytosolic members of the Stat transcription factor family, named for their dual fuction as signal transducers and activators of transcription. Activated Stat1 and Stat2 transcription factors then associate with a 48-kD protein in multimeric complexes termed interferon-stimulated gene factor 3 (ISGF3) that translocate into the nucleus where they induce changes in gene expression, which ultimately results in the biological effect of interferon-alpha treatment. Data suggest that interferon-alpha exerts its antiproliferative effects in neuroendocrine tumor cells by inhibition of DNA synthesis and colony formation [22]. Both p21 and p27, which are upregulated by interferon-alpha, act as negative regulators of cell cycle with block of G1-S progression and as potential tumor suppressors [23, 24]. ISRE’s interferon response elements are activated in the nucleus starting transcription of several interferon-inducible genes including IRF1, IRF2, 2-5-A-synthetase and p68 kinase (PKR). Both 2-5-A-synthetase and p68 kinase (PKR) induce a degradation of mRNA which results in inhibition of proteins synthesis of various peptide hormones and growth factors. Furthermore, these enzymes have an inhibitory effect on several cyclines involved in the cell cycles. We have clearly demonstrated that induction of 2-5-A-synthetase in tumor cells in vitro correlates with clinical and biochemical responses [25]. Furthermore, staining of p68 kinase in sections from human tumors treated with alpha interferon show a correlation between staining intensity and clinical responses [26]. Another effect of interferon-alpha is induction of fibrosis within liver metastases, which has not been recorded during treatment with chemotherapy or somatostatin analogues [27]. With time, the number of tumor cells

96


decreases and they will be replaced by fibroblast without any change in tumor size recognized by ultrasonography or computer tomography. Such changes can be detected by positron emission tomography (PET) where accumulation of 11C-labelled 5-hydroxy-tryptophan correlates with metabolism of the tumor. This fibrosis might explain why a limited number of patients treated with alpha interferon show significant tumor reduction but only stabilization of the disease.

Future Prospects

Interferon-alpha has demonstrated beneficial effects in the treatment of carcinoid tumors. Several studies indicate that more than half of the patients obtain a significant reduction in tumor related endocrine symptoms, leading to improved quality of life. Around 50% of the patients achieved a significant reduction in circulating hormone levels and around 80% obtained either a biochemical response or stabilization of the disease. Patients obtaining a biochemical response may have a long-lasting response, up to several years, and stabilization of the disease may continue for more than a decade. All these results are very encouraging but of course the treatment also has significant side effects which have to be managed by dose titration, individually in each patient. In the near future, polyethylene glycosylated recombinant interferons are available with less side effects and also easier administration. The patients have to take only one injection a week and perhaps also higher dosages can be applied since the side effects are lower. By combining interferon-alpha with somatostatin analogues, even higher biochemical response rates can be obtained and the patients resistant to either drug alone may respond to the combination. Furthermore, a combination of interferon-alpha and somatostatin analogue will postpone the problem of tachyphylaxis of analogue treatment alone. In the future, further information about the biology of neuroendocrine GEP tumors should improve interferon-alpha treatment and provide the rationale for new combinations of biological agents and cytotoxic drugs. Anti-angiogenesis is a new field to explore. Many of the neuroendocrine tumors are well vascularized and IFN-· is known to exert an anti-angiogenetic effect. Alpha interferon might be combined with other anti-angiogenetic agents such as angiostatin and endostatin in the treatment of neuroendocrine GEP tumors.

Öberg

References 1 Pestka S: The human interferons. From protein purification and sequence to cloning and expression in bacteria: Before, between, and beyond. Arch Biochem Biophys 1983;221:1– 11. 2 Fleischmann WR Jr, Fleischmann CM: Mechanism of interferon's antitumor actions; Baron S, Coppenhaver DH, Dianzani F et al (eds): Interferon, Principles and Medical Application. Galveston, UTMB, 1992, pp 299–310. 3 Öberg K: The action of interferon alpha on human carcinoid tumours. Semin Cancer Biol 1992;3:35–41. 4 Öberg K, Eriksson B: The role of interferons in the management of carcinoid tumors. Acta Oncol 1991;30:519–522. 5 Öberg K, Funa K, Alm G: Effects of leucocyte interferon upon clinical symptoms and hormone levels in patients with midgut carcinoid tumors and the carcinoid syndrome. N Engl J Med 1983;309:129–133. 6 Moertel CG: An odyssey in the land of small tumors. J Clin Oncol 1987;5:1503–1522. 7 Moertel CG, Hanley JA: Combination chemotherapy trials in metastatic carcinoid tumor and the malignant carcinoid syndrome. Cancer Clin Trials 1979;2:327–334. 8 Moertel CG, Rubin J, Kvols LK: Therapy of metastatic carcinoid tumor and the malignant carcinoid syndrome with recombinant leukocyte A interferon. J Clin Oncol 1989;7:865– 868. 9 Schober C, Schuppert F, Schmoll E: Interferon alpha-2b in patients with advanced carcinoids and apudoma. Abstract 34th Ann Congr German Soc Hematol Oncol. Blut 1989;59:331.


10 Bartsch HH, Stockmann F, Arnold R: Treatment of patients with metastatic carcinoid tumors by recombinant human interferon-alpha: Results from a phase II study. J Cancer Res Clin Oncol 1990;116:305. 11 Välimäki M, Jarvinen H, Salmela P: Is the treatment of metastatic carcinoid tumor with interferon not as successful as suggested? Cancer 1991;67:547–549. 12 Öberg K, Norheim I, Lind E: Treatment of malignant carcinoid tumors with human leukocyte interferon: Long-term results. Cancer Treat Rep 1986;70:1297–1304. 13 Öberg K, Alm G, Magnusson A: Treatment of malignant carcinoid tumors with recombinant interferon-alpha-2b (Intron-A): Development of neutralizing interferon antibodies and possible loss of antitumor activity. J Natl Cancer Inst 1989;81:531–535. 14 Janson ET, Rönnblom L, Ahlström H: Treatment with alpha-interferon versus alpha-interferon in combination with streptozocin and doxorubicin in patinets with malignant carcinoid tumors: A randomized trial. Ann Oncol 1992;3:635–638. 15 Biesma B, Willemse PHB, Mulder NH: Recombinant interferon-alpha 2b in patients with metastatic apudomas: Effect on tumors and tumor markers. Br J Cancer 1992;66:880–885. 16 Eriksson B, Öberg K: An update of the medical treatment of malignant endocrine pancreatic tumors. Acta Oncol 1993;32:203–208. 17 Jacobsen MB, Hanssen LE, Kolmannskog F, Schrumpf E, Vatn MH, Bergan A: Interferon alpha-2b, with or without prior hepatic artery embolization: Clinical response and survival in midgut carcinoid patients. The Norwegian carcinoid study. Scand J Gastroenterol 1995;30: 789–796. 18 Tiensuu Janson E, Ahlström H, Andersson T: Octreotide and interferon alfa: A new combination for the treatment of malignant carcinoid tumors. Eur J Cancer 1992;28A:1647–1650.

19 Tiensuu Janson E, Kauppinen HL, Öberg K: Combined alpha- and gamma-interferon therapy for malignant midgut carcinoid tumors. Acta Oncol 1993;32:231–233. 20 Rönnblom L, Alm GV, Öberg K: Autoimmunity after alpha-interferon therapy for malignant carcinoid tumors. Ann Intern Med 1991;115: 178–183. 21 Öberg K, Alm G: The incidence and clinical significance of anti-bodies to interferon-a in patients with solid tumors. Biotherapy 1997; 10:1–5. 22 Grander D, Sangfelt O, Erickson S: How does interferon exert its cell growth inhibitory effect? Eur J Haematol 1997;59:129–135. 23 Hobeika AC, Subramaniam PS, Johnson HM: IFN· induces the expression of the cyclindependent kinase inhibitor p21 in human prostate cancer cells. Oncogene 1997;14:1165– 1170. 24 Zhou Y, Wang S, Gobl A, Öberg K: Inhibition of CDK2, CDK4 and cyclin E and increased expression of p27Kip1 during treatment with interferon-a in carcinoid tumor cells. J Biol Regul Homeost Agents 1999;13:207–215. 25 Grandér D, Öberg K, Lundqvist ML: Interferon-induced enhancement of 2)5)-oligoadenylate synthetase in mid-gut carcinoid tumors. Lancet 1990;336:337–340. 26 Zhou Y, Gobl A, Wang S: Expression of p68 protein kinase and its prognostic significance during IFN-· therapy in patients with carcinoid tumours. Cancer 1998;34:2046–2052. 27 Andersson T, Wilander E, Eriksson B: Effects of interferon on tumor tissue content in liver metastases of carcinoid tumors. Cancer Res 1990;50:3413–3415.


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Round Table Discussion Digestion 2000;62(suppl 1):98–107

Sandostatin® LAR® Leaders Meeting – European Neuroendocrine Tumour Network ENET

Dr. Rindi: I will simply read the questions for Dr. Calender first and then the replies that he wrote yesterday for these questions. Dr. Tamborani (Rome) asked whether Dr. Calender could comment just a little more extensively on the effects of adhesion molecules and the biology of pancreatic tumors. Dr. Calender replied that mutation and isoform expression, for example, of CD44 variant 6 could be associated with dissemination. And in a project it would be interesting to check the N-CAM adhesion molecules which are expressed in GEP tumors. But do they decrease during malignant progression? It is an open question. The second question was: What is Carney syndrome? Carney syndrome is an autosomal dominant inherited disease predisposing to acromegaly, Cushing syndrome, bilateral adrenocortical hyperplasia and Leydig cell testicular tumors, myxomas and skin lentiginosis. Two genes have been mapped. One seems to be the major one and is located on chromosome 2P16, but has not been cloned to date. Another question is: What is NF1? NF1 is von Recklinghausen disease, and predisposes to cutaneous neurofibromas and optic lipomas and various types of tumors such as somatostatinoma in the pancreas of less than 5% of the patients. The NF1 gene on chromosome 17 encodes a GTPase-activating protein, which is also called the GEP, and negatively regulates the transduction of signals through small G proteins. Then there was a question about the tuberous sclerosis (TSC). There are two genes, TSC1 on chromosome 9 and TSC2 on chromosome 16. They work on small G proteins and GTPases as well. Dr. Schmidt (Vienna) asked me about behavior determination: What are the pathological parameters which might be of value prior to treatment: preoperative biopsy or, most of all, intraoperative frozen section? Basically I understand that the question is, can we assess some of the markers I have illustrated? Regarding intraoperative testing, in our department in Brescia we are currently evaluating the possibility of flow cytometric analysis of tumor scrapings. This can give a very quick intraoperative answer, for instance for the diagnosis and proliferative status. This is now being done for breast cancer, but is just an experimental procedure. Perhaps in the future it can be applied to neuroendocrine tumors.

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Regarding preoperative sample analysis. Indeed there are limitations such as sampling errors and inadequacy of the sample. The amount of tissue provided to the pathologist and certainly several parameters, like the mitotic and Ki67 index, or P53 gene overexpression, can be assessed on small tumor samples before making a final decision. These are my suggestions of course. Another question came from Dr. Tamborani (Rome): Is it possible to differentiate tumors into well-differentiated and poorly differentiated before operation on the basis of plasmatic markers or different stimulation inhibition tests? The reason for this question is that I observed different responses to dioxide and somatostatin analogs in insulinoma patients. I am not aware of plasmatic or stimulation inhibition tests that are specific for poorly differentiated neuroendocrine carcinoma. That is what I know. But this depends on whether somebody has something to add, I am not sure. The next question is what happens with poorly differentiated endocrine tumors when the TSA technique is applied? The TSA technique, which is signal amplification, might enhance the capability of immunohistochemistry and will probably enable the definition of more positive cells with rather few endocrine granules in poorly differentiated neuroendocrine carcinomas. This will not change the fact that such tumor cells are indeed poorly differentiated, that is they have, for instance, rare secretory granules on electron macroscopy and can be defined as protoendocrine or poorly differentiated cells. In summary carcinomas made up of such cells will still, and with reason, be defined as poorly differentiated neuroendocrine carcinomas, even after TSA immunohistochemistry. Another question: Is there a marker for the risk of developing carcinoid heart syndrome? Many neuroendocrine tumors, for instance, gut EC cells, well-differentiated tumors, carcinoids-producing serotinin, also produce several growth factors, including basic and acidic fibroblast growth factors, which are believed to participate in the genesis of fibrosis inside and around the tumor. However, such features of tumor cells cannot be considered as markers specific for carcinoid heart disease risk. I am not aware if any such marker does exist but I hand the question to Dr. Öberg who can perhaps enlarge on this point.

Dr. Öberg: About carcinoid heart disease and risk factors, I must say that there is no direct marker which can tell you that a given patient has an increased risk of developing carcinoid heart disease, but we know from a study we performed a couple of years ago, which was published in Circulation, that there is a correlation between the serotonin and tachykinine levels. So carcinoids producing neurokinine A or neuropeptide K are more prone to develop the carcinoid heart disease. Dr. Rindi: There is a last question from Dr. Mignon (Paris): In one of your slides you indicated the possibility of serotonin secretion by enterochromaffin-like (ECL) cells, which are supposed to only secrete histamine. Has this actually been demonstrated, or is it just a hypothesis? As far as I know this is more a hypothesis than demonstrated and for sure ECL cells have machinery for amine precursor uptake that is different from that of intestinal serotonin-producing EC cells. That is the cyclo-monoamine transporter, isoform 2, is specifically abundant in ECL cells, but not vimat or the cyclomonoamide transporter, isoform 1. Vimat 1 is present in EC serotonin-producing cells. Chairman: Thank you Dr. Rindi. Are there any comments from the group at the table or any questions, comments from the audience for Dr. Rindi before he takes his leave from us this afternoon? Thank you again. And the second person whom we are going to take a little bit out of order is Dr. Ricke with questions on imaging procedures. Thank you. Dr. Ricke: I will start with some general remarks that basically reflect the questions that have been handed in. What you have seen me presenting was more or less, or explicitly, the result of a consensus procedure reflecting imaging procedures and how they are performed in a very, let’s say, clinical perspective, and since it is a consensus procedure you can imagine how hard it is to try, trans-European and trans-nationally, to imply something like that. So when you ask me for a certain method, why do they not show up in those flow charts? It is basically due to the fact that the majority of participants in those ENET meetings and the majority of people who were handed those questionnaires just don’t do it. So that was the way to reach a consensus. The first enquiry on that is basically about secretin-stimulated arteriography or calcium-stimulated arteriography for insulinoma from Dr. Knigge (Copenhagen). I would like to give the question back to the audience: how many of you perform calcium-stimulated arteriography for insulinomas in your institutions? Could you raise your hands: who is doing that on a regular basis? I have to admit it is more than I expected. But anyhow we will keep it in mind. The second remark about those work flow charts concerns, of course, the term ‘flow’. It determines it as being in continuous flow and continuous modification, and we are still working on it. I think it is a good basis to start from. Not only due to the dynamics and imaging, not only in radiology but also in nuclear medicine, a modification can be expected very soon. The second question I have regards portal venous sampling. I suppose the question arises due to sensitivities of about 70–80%. It is certainly not ‘out’ but the only recommended application for this is insulinoma in conjunction with MEN1 due to the multiplicity of tumors that are frequently seen. Here again, calcium infusion, intra-arterial calcium infusion for venous sampling for insulinoma and again I would like to give the question back: who on a regular basis performs venous sampling including calcium infusion for provocation? Is there anybody? One. So I suppose that answers the question. I personally tend to believe that evidence about this is just not published enough, but we will

think about that during the modification of the current work flow charts. Thank you. Chairman: Are there any questions from the audience or from the panel for Dr. Ricke that were not covered? Thank you very much. Now we will go back and revisit the meeting in the various sessions and try to go over the questions. So I will turn it over to Dr. Öberg in just a second. It’s my objective because our last session was the longest, it’s freshest in our minds and will probably lead to the most questions. I will try to leave the most time for the section on clinical. So we will try to move fairly quickly through the first two sessions and move to that. Dr. Öberg: I have received some questions regarding our organization. One question is: Do you have any oncologists on the board? We have several oncologist in the group of 20 members. Another question is: Do you have any endocrinologists? I can also tell you that we also have a couple of endocrinologists in this group. So we are covering different aspects of neuroendocrine tumors. Can anyone become a member? is also a question. I must tell you that we have decided to keep this group very small, just 20 members. And therefore we would like to ask people in each country to organize their own national group for neuroendocrine tumors and then they can have a representative within the ENET, otherwise the group will be too large and then it will be difficult to manage. I have a question from Dr. Toulis (Greece) about whether there are similarities between glucagonomas in rodents and man, and as you are well aware glucagonomas not only produce glucagon, they also produce the splice products of proglucagon, GLP 1, 2 and so forth. So I don’t know exactly how it looks in the rodents, perhaps Dr. Toulis can tell me. Dr. Toulis: I was impressed by the picture you showed of the patient, and I am saying that in rodents with metastatic glucagonoma there is a syndrome of aphasia and adipsia, and this is leptin-independent. I want to know if you have any idea how that operates in man: Is it because it invades the lateral hypothalamus? Is it because of oxytocin release or because of NPY? Is there any thing known about this catabolic glucagonoma syndrome which, as you indicated, is correct? Can both glucagon and GLP peptides do the same? Dr. Öberg: I must say that there are no studies that I am aware of about leptin involvement in the glucagonoma syndrome. Most of the catabolic effects are ascribed to the catabolic effect of glucagon or the propeptide. So I don’t have any information about leptin. Chairman: The other member of the first session was Dr. Susini, and so we have some questions, a couple of them are a bit clinical and one deals with somatostatin receptor status. Dr. Susini: The first question is from Dr. Pless concerning the role and importance of SST1 and SST4 for therapy. My answer is that SST1 is also very important for tumor therapy because this receptor is often upregulated in epithelial tumors, such as pancreatic, prostatic and colon tumors. This receptor is highly upregulated by somatostatin. For SST4 we have no information. Concerning the situation in prostate cancer, according to the literature there is an upregulation of the SST1 receptor. So this receptor could be an important target for tumor therapy and it would be interesting to have a specific analog for this receptor. The second question regards the antiproliferative activity of somatostatin analogs in patients with negative receptor imaging and, as Dr. Pless explained this morning, it is known that somatostatin can inhibit cell proliferation by indirect effects including inhibition of the secretion of growth factors and/or hormones, inhibition of angiogenesis, and modulation of the immune system.

Sandostatin® LAR® Leaders Meeting


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There is a question concerning the use of somatostatin analog therapy to upregulate somatostatin receptor prereceptor-mediated therapy. My answer is that upregulation of the somatostatin receptor by agonists is studied. As I told you SST1 can be highly regulated by natural peptide, and SST1 is also regulated in animal tumor models by the somatostatin analog after continued exposure to the analog, but this effect is not observed when the analog is injected 3 or 4 times/day. But we have little information about the upregulation of the somatostatin receptor by agonists. So we need more data. The next question concerns the development of specific analogs of somatostatin that have an antitumor action in different tumors. My answer is that in most of the cases each cancer cell expresses more than one subtype of somatostatin receptor. And we do not know the specific role, the special and temporary role of each receptor inside the cell. So in my opinion it will be better to have a pan-somatostatin analog, which can bind all somatostatin receptor subtypes with a high affinity. The last question concerns the role of gastrin and the molecular forms of gastrin and the promotion of exocrine pancreatic cancer growth from Prof. Mignon. My answer is that gastrin as well as the pro-form, glycine-extended form, are secreted and can act as autocrine growth factors in cancer cell models. The presence of CCK-B receptors in human pancreatic cells might indicate a role of these peptide in the development of pancreatic cancer. Recent results obtained in our laboratory with transgenic mice expressing the human CCK-B receptor are in agreement with the hypothesis. We also have a question about antibodies for somatostatin receptors usable in paraffin-fixed tissue. Many laboratories generate different somatostatin receptor antibodies but until now only a few of these are commercially available. The last question concerns the receptor type on hepatocellular carcinoma. We have no data on this type of tumor, but according to the literature there is no binding on this type of tumor. Thank you. Chairman: Are there any questions from the panel for Dr. Susini? Dr. Pauwels: May I add some information. Already 8 years ago we had the opportunity to follow-up patients over long periods with somatostatin analog therapy and to quantitate the tumor uptake. Before octreotide was started, we surprisingly found a significant difference in the uptake per gram of tumor in patients under treatment. I discussed this with quite a few people, but they did not believe it. But I am sure of it. There is a clear effect during long-term therapy and in some patients we had a clear scan after a short period. A decrease in the uptake was seen after a short period of treatment. All the patients in whom this decrease in the uptake was seen were naive to start with, I mean of the residence time, which is a combination of affinity, internalization and absence of clearance. So clearly we had the impression that there was a kind of reduction initially, but after 12 months there was an increase and statistically this was significant. Chairman: Thank you. If there are no further questions we will move on to the session on diagnostics, and Dr. Eriksson has a series of questions on tumor markers. Dr. Eriksson: Yes, I have received a lot of questions. I actually picked out some. I think I had about 20 or 25. At this point would you recommend chromogranin A as a primary measurement of outcome in clinical studies of novel therapeutic agents? My answer is, yes. Chairman: You don’t want to expand at all do you? Dr. Eriksson: Our experience so far is good in the follow-up of treatments that we recommend chromogranin A analyses strongly. Then I have a question from Dr. Graham-Smith. In the carcinoid syndrome if the symptoms are helped by somatostatin and the 5-

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HIAA4 goes down, does the chromogranin A level fall as well? Yes, it does. They usually go down in parallel. I have a question from the audience: Should the recommendations for the diagnostic considerations in carcinoid syndrome change from measuring urinary 5-HIAA to chromogranin A levels? I don’t think there is a simple answer for that. I think to establish the carcinoid syndrome we should measure and show that urinary 5-HIAA is present because that is a specific marker for the carcinoid syndrome, but then if you have the diagnosis and the follow-up of patients chromogranin A levels could very well be measured more regularly than urinary 5-HIAA. Here is another question: While following a certain patient with chromogranin A an increase happens. What is a significant increase, 50%, one time, two times? There are two answers. If, as we have done, you follow patients who had undergone surgery for carcinoids or endocrine pancreatic tumors, and you discover a slight increase in chromogranin A, we have seen that on many occasions, up to 7 ng/ ml, which is not even twice the normal value, might be significant, and we have done PET and found tumors. But then if you, have a clinical trial and use chromogranin A as a tumor marker then a significant increase cannot be defined as 50%. I mean when you do a clinical trial, a more than 25% increase in chormogranin A is progression. So that is what you would call a significant increase in that context. Another question is: Can we safely assume that chromogranin B is an alternative tumor marker for chromogranin A? We only have access to chromogranin B it says in parentheses. Well I didn’t show that yesterday, but there are a number of patients who do not have elevations in chromogranin B but have elevations in chromogranin A. Chromogranin A is elevated in many more patients and if you have such a patient who does not have an elevation in chromogranin B you will not be able to use it as a tumor marker. So chromogranin A is the most useful because it is elevated in most patients. But for instance in foregut carcinoids (insulinomas) we have patients who have chromogranin B as the best marker, but it is a small number of patients. A final question from Dr. Buchanan: What are the criteria for classifying a tumor as nonfunctioning? Should this classification be replaced by a more rational nomenclature? The criteria that we use is that a nonfunctioning tumor is a tumor for which a patient does not present any hormone-related symptoms. That is the definition that we use. I know that others have used other definitions that the tumors should not produce any hormones, but that is not correct in my mind, nonfunctioning tumors can produce chromogranin A, PP, HCG subunits, they may even produce specific markers like gastrin insulin but these perhaps do not give any symptoms. So I think the critical criteria is that the patient should not present any hormonal symptoms. I may ask the others in the panel if they agree. Chairman: Any comments? Response: Of course I agree. We have the same opinion. But I have some comments. Question: What would you say about the patient presenting an increased amount of calcitonin but not symptoms? Dr. Eriksson: That’s a nonfunctioning tumor. But you also have patients who have high levels of calcintonin with diarrhea, then it is a functioning tumor. Dr. Arnold: I agree with you. But I want to mention that there are some patients who at the beginning have nonfunctioning tumors according to your definition with measurable amounts of 5-HIAA in the urine, or chromogranin A, and later develop a carcinoid syndrome, that is also possible. My question to you is, you did not mention serotinin measurement in blood. Is this a failure or not?

Round Table Discussion

Dr. Eriksson: Well we have it available in our laboratory, in the Department of Clinical Chemistry, and we used it a long time ago, but measurements are very unreliable. You get very different levels that you cannot make any conclusion about. Response: At least in Germany serotinin can be measured by many laboratories. I frequently get patients with elevated serotinin levels because they have some kind of flushing and are transferred for exclusion of neuroendocrine tumors. This is a very tricky thing because if you do not find any tumor, a patient does not believe that he has no tumor or some hormonal-related disease because he is saying serotinin was always elevated. It is difficult to handle such a patient. Dr. Eriksson: Dr. Öberg do you have an opinion on that? Dr. Öberg: We did a study many years ago where we compared the platelet-poor plasma serotinin with urinary 5-HIAA, and I must say that the data on serotinin was not quite reliable and there were a lot of fluctuations when you followed it during the day and so on. So I am not in favor of plasma serotinin. I think you get a lot of falsepositive referrals. Response: May I say something from the aspect of tumor and cell biology, especially secretion. When we talk about functionality and nonfunctionality, we have to consider that every neuroendocrine tumor cell, as long as it is differentiated, is secreting its product, that is chromogranin, as Dr. Eriksson just said. But what happens if the tumor, depending on its state of differentiation, generates a product that makes some kind of a hormone and becomes functional, that is – you may have a small tumor and, depending on tumor individuality (some tumors just want to increase in size) it produces gastrin and then with an increasing number of metastases you see a Zollinger-Ellison syndrome. Some others have such a highly differentiated tumor that with a small tumor mass you already have so much gastrin that you have the same kind of Zollinger-Ellison syndrome. But the point is, the functionality is just defined by a hormone or peptide product that causes hypersecretion related symptoms. One thing that I want to add, when you said that chromogranin A should be a bonafide tumor marker, I think I can accept that for the initial diagnosis, but my question is, when we treat patients with somatostatin for instance, we block the secretion, although the secretory vesicles are still there, and considering that we just effect the cell cycle, not in cytotoxic context, we see a decrease in secretion and a decrease in chromogranin A, but how can we really learn anything about tumor growth? Dr. Eriksson: Well I am happy if chromogranin A goes down in a patient, even if you call it cosmetic, because as I told you yesterday, chromogranin A may be a stimulator of growth, and that may in the end effect growth. Dr. Mignon: A question for Dr. Eriksson. It seems to me that there exist some cases of patients with an elevated serotinin but not elevation of 5-HIAA because there is a lack of carboxylase in the liver, is that true? Response: Yes, that is true. Dr. Mignon: So maybe we should measure both. Response: No matter how much money you will spend, of course you can measure a lot of different things, but just for routine, if you want to get down to the minimal necessary I think 5-HIAA is more efficient. Dr. Mignon: I agree with you that there are false-positives for serotonin. Question: Dr. Eriksson there is a frequent question from clinicians in two situations. The first one is the patient with a nonfunctioning tumor, for instance liver metastases of endocrine or probably

non-endocrine origin. In this situation which determination would you advocate in the blood, or elsewhere, in the urine for instance. And the second one, if you have a patient with a functioning tumor what do you advocate in addition to the peptide, which is obviously involved, for instance gastrin in the Zollinger-Ellison syndrome. Because in many instances people ask for a lot of determinations which have limited clinical utility. Dr. Eriksson: On the first question. Do you mean that you have liver metastases of a nonfunctioning tumor with an unknown primary? Okay, because then I would measure chromogranin A, I would measure chromogranin B because that is mostly elevated in the foregut carcinoids so that would give me an idea if I am dealing with a foregut carcinoid. I would measure urinary 5-HIAA, because even though patients may be asymptomatic they may have elevation of urinary 5-HIAA and if I have that increased there is a high possibility that the liver metastases derived from a midgut carcinoid. Then of course I think I may measure calcitonin, because that is a nonfunctioning or general marker, but that would not give me a clue because then the primary could be in the lung or in the pancreas. But those are the markers that I would measure in the first run, so to speak. Then on the second question, you are talking about a metastatic patient, in this case with a functioning tumor. Question: Functioning tumor, which examinations do you ask for aside from the peptide which is involved? Dr. Eriksson: Usually in our clinical work up when we see a new patient, we have a standard battery that we use in endocrine pancreatic tumors, and that is gastrin, insulin, proinsulin, C-peptide, and plasma VIP, glucagon, PP chromogranin A and B. Those are the markers. I think that is important to know from the beginning what type of tumor, because if you have a malignant tumor there is a high likelihood that you are dealing with a patient who can have multiple syndromes. You can have an insulinoma and glucagonoma syndrome, you can have an insulinoma/gastrinoma syndrome, and that is very important to know before you start treatment. So we do the whole battery initially. And then we repeat it once per year because we know that endocrine pancreatic tumors can change their hormone profile. Chairman: I think I see two questions coming up. So we’ll take the three. Dr. Graham-Smith: Could I just return to the semantic problem of functioning and nonfunctioning. There are thousands of surgeons around the world operating on patients who present with intestinal obstruction, and they have no symptoms, no carcinoid syndrome at all, none, no flushing, no diarrhea, nothing at all. But the tumor comes back carcinoid, 5-HIAA may be slightly raised, but may not be. Now we had some very interesting stuff on somatostatin receptors and growth, and the effect of octreotide on growth of carcinoid tumors. Tell me, does anybody here treat clinically nonfunctioning carcinoid tumors with Sandostatin or one of the other somatostatin analogs in the hope that you may stabilize, or cause regression, of those tumors, because there are even more patients with nonfunctioning carcinoids by your definition than there with functioning carcinoids. And it is something that troubles me, should I be treating them in the hope that there may be some regression or stabilization of the tumor? Dr. Eriksson: If your patient shows progressive disease and the patient is octreoscan positive he will receive somatostatin analog treatment in our department. Response: I might add to that, if it is a rather rapidly progressive nonfunctioning tumor perhaps you might add interferon-·, so we combine interferon-· with somatostatin now.



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Question: You asked how we should treat this patient, yes or no? I remember having a patient with such a disease and with liver metastases and the first manifestation of the disease was in 1982. Now we have 2000. He has had this disease for 18 years and sometimes he has been treated with Sandostatin, sometimes not. I personally do not believe that he has some beneficial effect from any treatment because this is a very slowly progressive disease, extremely slowly progressive, and it is unproven whether such a patient has any benefit from treatment. If he has a metastasis then many doctors say something must happen, and he will get long-acting somatostatin analogs or perhaps also interferon. But is is not proven whether this has any benefit. That is my comment. Dr. Wiedenmann: If I understood the question correctly it was, if and when should we treat a nonfunctioning neuroendocrine tumor and I think I would – oh a carcinoid. But to follow-up on Dr. Arnold, I agree with him, we need a pre-observation period without treatment because then you really have the natural course where there is no need. But we have just finished a study in 80 therapy-naive patients and they were only treated by surgery with no other therapy. These patients were treated in a prospective randomized trial with either the somostatin analog, interferon, or the combination of the two. To make a long story short, there was no difference in antiproliferative response, looking at the monotherapies as compared to the combination of the two. To finalize, what we see is that you find 50% stable disease in functioning tumors with documented tumor progression in the therapy-naive state. So my answer would be once you have a documented progression you should treat it. Dr. Öberg: I think it is a little bit of a negative attitude just to say, if it is a nonfunctioning tumor you should not treat it. So I also agree that you should have an observation period, but as soon as you see that you have tumor progression, you also have the possibility of taking biopsies. You can look at the Ki67, you can see CD44 expression and so forth, somatostatin receptor content. So you have a lot of possibilities to determine that this is probably a tumor which you should treat. And if you have progression during the observation time you should definitely treat the patient. Dr. Toulis: I think this issue of semantics should be followed up and I would agree with the first slide by Dr. Pauwels. If a hormone is produced, or a messenger is produced this is a functioning tumor. It could be symptomless. The most interesting phenomenon we have is the so-called nonproducing tumor of the adrenal gland in which there are abnormal receptors. This tumor generates agonists that can suppress them. So we have to be very careful about what is called functioning or nonfunctioning. So you may have a product without symptoms. Perhaps some of the symptoms are not related to this product; perhaps there is some other product which cannot as yet be measured. So I would think that to begin with this tumor is functioning and symptomless, and there is a big area to be discussed. Dr. Buchanan: I produced that question for Dr. Eriksson about the nonfunctioning tumors. I think I am outside the elite 20 so I can speak my mind. I think the definition nonfunctioning is a cloak of darkness, it is ignorance really to call a tumor that produces multiple peptides and hormones, has all the machinery in it, as nonfunctioning. You do not know what these peptides or hormones are doing. If we actually get back to the mid-gut carcinoid syndrome there are hormones and there are markers, chromogranin A is probably a marker. But there are hormones and we do not know the hormonal mechanisms of the carcinoid syndrome. We do not know what causes the

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flush. We do not know what causes the diarrhea. We do not know what causes the carcinoid heart disease. We do not know what causes the lacrimation in the neuroendocrine tumors of the lung, nor the salivation. So to call tumors nonfunctioning is really retreating. They are functioning tumors. The nomenclature has to be changed. Dr. Eriksson: Do you have a suggestion? Comment: I would like to add a comment. When I was in Liverpool at the end of the 1970s, as you know Dr. Gregory was the first to isolate gastrin synthetases and published everything about the biodistribution and biological activity, he told me this story. The first time he got a gram of gastrin he injected it; an acute and huge amount. What did he observe? Blockage of acid production. In my experience, in a patient who had the highest amount of gastrin I have ever seen, I recorded a tumor-isolated progastrin. That case is famous now, it has been reported in many papers, and that patient was symptom free with no acid production. Two million fmol/ml in blood, up to 10 million before she died. Question: I have a question for Dr. Eriksson. You just said that you would treat nonfunctioning carcinoids only if the octreoscan is positive. But if you have indication of a proliferating tumor, would you also treat with octreotide, with a negative receptor status because of the antiproliferative effects, independent of the receptor status? Dr. Eriksson: If I had a carcinoid patient, nonsymptomatic, with progressing disease, then I would give the patient interferon-· in the first run. If the tumor is octreoscan negative. Question: Is there a rationale for a better anti-proliferative effect in these tumors? Negative for octreoscan? Dr. Eriksson: Well there are several studies which have shown that octreoscan is a predictive test for the sensitivity of the analogs that are available now, like Sandostatin, lanreotide and RC 160, and in most cases there is a very good correlation between the response and the presence of receptors. In general we see 25 patients per week and I make 25 decisions per week, and I pay attention to the octreoscan. If it is negative and the patient does not have symptoms I usually do not give Sandostatin in the first run, but I would give interferon, and if the patient still progresses then I may add Sandostatin. Comment: Then you give combination therapy, interferon and octreotide. Thank you. Dr. Eriksson: Yes. Dr. Waldum: I have two comments. I think everyone of you has said that these tumors are so rare. I do not think they are rare. There are a lot of tumors that will become neuroendocrine, and I think you will have a problem when designing your studies about how to define the border towards the more malignant one because this is a continuous spectrum. That is a very hard task because the results will depend on the inclusion of the tumors and this is a continuous spectrum of a large proportion of tumors. That was my first comment. And I have a question about multiple endocrine neoplasia type 1, and I think it will be to Dr. Mignon. Do you believe that the gastrinomas in these patients have the same degree of malignancy as sporadic ones? Do they develop specifically in patients with multiple endocrine neoplasia and gastrinoma? Or, do they develop in these patients because they live longer with hypergastrinemia? And if there is a difference here, would it be correct to treat the patients also with octreotide to prevent ECL-cell tumors because they will develop ECL-cell tumors with time, not only to give them proton pump inhibitors to inhibit their acid secretion. Dr. Mignon: It is a difficult question. First of all most of the ECLoma, to start with the last question, are indeed developing in those


patients with MEN1 syndrome, but we know there have been reports in the literature and one from our group, that ECL-oma can develop even in sporadic cases. Whether it is related to the amount of gastrin or to the duration of the hypergastrinemia is difficult to answer. I just want to let you know that some time ago we made comparison between 127 patients with sporadic gastrinoma and 45 patients with MEN1 Zollinger-Ellison syndrome. In the analysis of these two groups we did not find any difference between the level of gastrin and of course the level of acid secretion or other parameters; for instance results from the secretin tests were the same. So it is probably not related to the value of serum gastrin in the serum. I cannot answer the question about duration, because it is possible that those people who indeed are presenting symptoms of gastrinoma are younger than the sporadic ones. This question was already asked by somebody, so I would like to answer this problem later on if you wish. But I cannot answer in terms of duration because it is most likely, at least in our experience in Hôpital Bichat and I would like to stress this, that there is a bias in recruitment, in many many groups. For instance the Uppsala group include gastrinoma patients in their recruitment of MEN1 patients. But for us at Hôpital Bichat we have completely different recruitment mainly sporadic cases, because initially we were interested in acid-related disease, so we came to endocrinology, digestive endocrinology by the bias of acid secretion. But in our group the difference between the sporadic and the MEN1 Zollinger-Ellison patients is not as pronounced as in Uppsala, and that is probably because it was not looked for systematically in our group. So the difference for us is only 7 years. So maybe there is an increase in duration of overexposure to gastrin but I cannot really answer that question. Dr. De Angelis: In the case of a nonfunctioning tumor, if you have an increasing value of chromogranin A as the only sign of tumor progression, should you treat the patient? Dr. Eriksson: I would not rely on only one measurement of chromogranin A, I would repeat the measurement in maybe 3 months and if the level is going up at that time you would probably also then do a CT and perhaps show that the tumor is progressive on radiology too, then I would start. But not only on one chromogranin A measurement. Chairman: To some extent I have let time slip away from me a little bit and so in the area of diagnostics and imaging I would ask if there are any burning questions you have and to raise those now and when we have dealt with them then I would like to turn to the clinical issues so that we have some time for that at least in our discussion. So are there any remaining burning issues for the diagnostics and imaging folks? Dr. Mignon: I do not know if it relates to diagnosis, but I would like also to draw the attention to the problem of gastrinoma when we have a negative visualization, for instance, of granules by histopathology. That does not mean that the tumor is not secreting a peptide, because if you do as we have done (published 15 years ago in the American Journal of Physiology) and you look at the presence of the peptide using the mRNA hybridization technique, then you can show that this tumor, which was without granules, is indeed expressing the hormonal product. So before saying that the tumor is not a gastrinoma, for instance, we must be sure that it does not express gastrin by another technique. Dr. Pauwels: I have got two I think important questions about imaging, technical questions. The first, will technetium-labelled agents such as 99mTc provide cheaper but as effective imaging compared to an octreoscan? I have no experience with that compound.

The only thing I can say I participated in a multicenter trial, and I stopped including patients after my first patients, because the background in the circulation was high, the bone marrow uptake was very high, and the tumor to background contrast we obtained was that bad compared to octreoscan. I have some colleagues in the US because it is marketed in the US, who made some comparison with octreoscan and I know that they all agree that it is less effective and has less quality. Of course we are all looking for this kind of compound. Another question is: Are there guidelines for the performance of somatostatin receptor scintigraphy? Is there a need for standardized methods? I would say the best you can do is the best, but it is timeconsuming. To scan a patient with a triple-head camera, it takes our laboratory on average 3 h. So you have to afford this if you want to achieve a very good sensitivity. So I would say there is a need for standardized methods. As I said previously, we scan the patient at 4 h, why at 4 h? Because there is no bowel activity at that time. You cannot avoid bowel activity at 24 h. You cannot do your SPECT of the chest at 4 h because the background is too high, particularly in the chest. So what we recommend is to do a 4-hour scan just to see what is going on in the abdomen because you are sure that there is no bowel activity. And then at 24 h do a review, and when possible we perform SPECT, whole body SPECT, but to get enough statistics you need at least a double-, if not a triple-head camera. So this is not always possible, but when it is possible it is the best approach. Chairman: Are there any other burning questions on imaging, otherwise I am going to turn to the therapeutics section. I am going to take it, just so no one can accuse me of trying to use up the time in ducking it, I am actually going to ask Dr. Smith the one question he received first and then if that causes some discussion we will have that and then we will go back to the others. Basically I think we received about seven versions of the same question, and the question goes along the lines, why does Novartis not continue to perform a phase-II study with 90Y-DOTA octreotide in GEP tumor or in carcinoid tumor patients, but turns to breast cancer and small cell lung cancer instead? And then there are a few others too. Dr. Smith: So in considering this question over lunch and also the past few days it is not the first time I have heard it. I wanted to start with one of the other questions in the deck, which is, when will this be more widely available in Europe? And I wanted to point out that at the moment it is only available in Europe and most of the work that you saw this morning was done in Europe. But I think that was not really the intent of the question. The idea behind the question is, when will it be widely available? And here I just want to remind people that we may have a little bit of a different paradigm in mind. For us, this is a phase-I compound and we know for sure that it has a narrow therapeutic window. So what we want to do is to ask very specific scientific questions in a very careful way with a lot of structure. For us there is very little use being Novartis and the sponsors, there is very little motivation to have it widely available. We do not think that this is going to be in the long view what is best for everyone, if it is a drug, to get it out there on a market so that everybody can use it. And that the best route to that is not to make it broadly available now outside very specific trials. That said you can turn to why the shift from the neuroendocrine focus in phase-I development to phase-II development. And for that I would say that there are two different roads that we might have gone down at least, and that arguable there are advantages and disadvantages to both. To explain why we turn now to breast and small cell lung cancer, as you saw in the first slide, the goal of the program is you know the



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drug that we imagine is one that is good in multiplicity of somatostatin receptor-positive tumors. So we have decided to try to see if that is true, to test that hurdle and see if it will have a use with the significant subset of breast or small cell lung patients and to do that next. So that is the reason that we design this. Another way to go would be to specifically try to test in phase-III or phase-II trials neuroendocrine tumors, but that is not the question that we have decided to ask in the next round. Do you want me to take the other one that was in the deck about positivity? Chairman: Well there is a question of do the advanced breast cancers have a clear-cut octreoscan uptake and I suppose that would be asked of all the tumor types you are looking at. Dr. Smith: I mean, as I thought about it, the question is, have you thought that you might not find any breast or small cell lung cancer that is positive enough to be treated? And the answer is, yes, we have thought of that. The literature supports that these tumors are positive and not a small fraction of them, but what it cannot tell us is, in this condition, having been treated with chemotherapy, how many of those are greater than liver, or less than – we do not know that and that is one of the things that the study is designed to find out and why it was listed as one of the secondary objectives of the study. I would point out that from the point of view of the company, which is asking the specific question, and this is also any, it is not just the company, it is any sceptical observer who at this point might say, is this a drug for breast and small cell lung cancer? Well the literature does not give us an answer about how many patients might be eligible from that point of view, and if in the study we find that there are very few patients that are eligible from that point of view we may not fill the study, but we have our answer. I mean that is a legitimate answer to the study. No, it is not, because not that many people have that kind of positivity. So I would say that it is one of the things that we are trying to find out in profiling the drug. Chairman: I think we might comment that we have tried to set aside various, but at the moment, relatively small amounts of materials that may become available for investigator use during the year, and we will try our best to do some additional work that makes it possible for further studies in neuroendocrine tumors, but that is not the initial focus of the trials within Novartis. Obviously if we find positive results from the trials that Dr. Smith is going to undertake later this year, I think that would change the topography in terms of how we view neuroendocrine tumor studies immediately after that. But what we need is more data and wider ranging data than we have in some of the tumor types, that is what we are looking for now. But we are open to hearing from you ways that we can also be more responsive to your needs. So with that I doubt that there will be other questions that come from that, so I go to the floor for questions. First from the panel perhaps, if they have some comments. Dr. Pauwels: There have been a lot of discussions between the principle investigators and the Novartis company. It is clear that so far we have no idea of the response rate possibly because we do not know what dose to give, we have not studied the right patients. The study did not address the antitumor rate properly. But from a commercial point of view I can agree, I can accept that some people in Novartis take the decision that the window is too narrow, and that commercially they are no longer interested in it, whatever the response is. That is not my philosophy, but the only thing I can say is, okay, I agree with the position of the company. For breast cancer, as we have frequently discussed already, I have a lot of that about breast

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cancer, why? First of all the density of receptors, let us say, the residence time of the labelled peptides is very low in breast cancers. And second, as shown by Dr. Lamberts and his colleagues, it is a factor with a very bad prognosis. Now you can imagine that in breast cancers, although the deposits of your radioactivity is lower than the one you can obtain in neuroendocrine tumors, you can assume that you may have a better therapeutical response or equivalent if the tumor is more radiosensitive, but we do not know that. And I would bet that it is almost not more radiosensitive, which is not the case for the small cell lung carcinoma. So I think that the small cell tumors might be much more successful. But now the problem with the small cell is that we do not know yet what the dosage is for single dose MTD. And it may be crucial to have a single dose as high as possible in a small cell model. These are my major comments. I am pretty sure that, coming back to the neuroendocrines, if you do the right selection of patients you may have very high success rates, but in doing this you are going to deselect eventually 90% of the patients. If you want to achieve a response rate above 50, 60 or 80% you are going to deselect eventually 9 of 10 patients. Chairman: Thank you. Any other questions for Dr. Smith, or comments on this point? Question: Yes, could I just follow that up. I am rather concerned about the case selection of patients that you are going to be using in the breast cancer studies. One would assume, perhaps wrongly, that the patients who are likely to benefit from chemotherapy are likely to be patients who have negative receptors and therefore they are probably not going to be a group of patients who will benefit from your treatment. Secondly, the patients who are most likely to benefit are probably those who are going to have ER-positive tumors, or hormone-sensitive tumors, and you are going to be up against conventional standard treatments, tamoxifen, progesterone and so on, and where are you going to consider using this in the therapeutic area, in the ordering of the treatments. I am very concerned that the sort of patients that might go into this study are going to be inappropriate and will lead you to come to perhaps false or wrong conclusions which may lead you to ditch the product, and in fact deprive us in the neuroendocrine field of what is a very exiting and interesting product. Dr. Smith: Right, it’s a complicated criticism. It has two levels. One what are you doing in breast cancer and then how will that effect what you are doing in neuroendocrine cancer. In breast cancer I think it is going to depend somewhat on local practice guidelines and what IRB and ethics committees suggest. In some places it will not be possible to enrol any breast cancer patients who have not seen more than multiple therapies for metastatic disease. In other places, however, in other words the protocols will be written such that any prior therapy is allowed. So there may be a wide spectrum, and since many patients being treated for metastatic disease still do not really have a good chance at cure, and since the octreoscan suggested that this is a drug for them, you might find situations where it is permissible to treat. So there will be a variety of patients, you are probably right, they will be at the sickest end of the spectrum. How this effects the overall development of the compound is a separate question, but a legitimate one. It is not only a question of whether or not you can make a successful trial in breast cancer but whether or not it is a useful drug in breast cancer. So if it is not a useful drug in breast cancer, you know in what tumors it is a useful drug. A separate question. So I cannot say what it will do to its chances in neuroendocrine tumors.


Chairman: But I can say it is not our intention to drop the drug just because we have had just one negative experience. I think we will try to look and make sure that it has the best opportunity that we can for its development within the company. Dr. Ficoni: I am a surgeon, but for me it is difficult to think that we do not have radiotherapy among all the weapons we use in treating with GI tract neuroendocrine tumors. Why should octreotherapy work in this field? Radiotherapy is not among our weapons. And I do not understand why this kind of tumor in the GI tract should be radiosensitive. Dr. Smith: Well that is a legitimate remark. I appreciate that. In neuroendocrine tumors, while they probably have the best receptor status, there is the least reason to believe that radiotherapy will be effective in them, radiotherapy plays no role. This is hope, for example, if we can leave breast cancer aside and just talk about small cell lung cancer, which does have receptors, may be not as many as neuroendocrine tumors but they are sensitive to radiotherapy and, except for the fact that it is widely dispersed at the time of treatment and also dispersed in the lung, which is particularly sensitive to the rest of the beam therapy, radiotherapy could play a good role in small cell lung cancer. So there is a balance between these two factors. Unfortunately, I cannot name a tumor that is high in the receptor content and extremely radiosensitive, because that would be the ideal combination, but we are stuck with playing those off against each other in the various tumor types. Answer from the audience: Just to answer the question. Any tissue is actually radiosensitive if you give it enough radiation. One of the problems is that we cannot deliver enough radiation to disseminate it widely without destroying a lot of normal tissue. So that is why it is not traditionally radiosensitive. I think the clue came from Dr. Pauwels lecture about the ratio between uptake within the tumor and normal tissue, it was up to about 150 times. So as a consequence you actually have a molecular difference, which means you can actually deliver very high levels of radiation into the tumor. So that is why it works differently. And we know it works because there is a MIBG therapy, which we have not discussed, which is a standard technique, which has been licensed for many years in Europe, and has been shown to work in some of these carcinoids which have uptake of that particular product. Patients have had good clinical responses from that. So we know the method of targeting radiotherapy to these tumors does work. It is just getting the right target, at the right peptide to target and the right radioisotope. But the system works, it is just getting enough radiation into that particular tumor cell to kill it. Chairman: Thank you. Are there any more questions or comments? Question: There is scepticism about breast cancer. The point I would like to raise is that following surgery or radiotherapy, and other therapies, what are the false-positive rates of octreoscan? It has been very well known for long time that when you have an inflammatory, a septic inflammation, how can you then use octreoscan to discriminate receptor density? And following some earlier comments, if you treat with octreotide you will get a negative octreoscan and, when you stop, you will again get a positive scan. So there are a number of questionmarks if octreoscan is going to be a key measure of assessment of those who will benefit from therapy. Dr. Smith: I think there are more expert people on diagnostics than me, but I will just take the first crack at it and then turn it over. In our study, of course the patients all have histologic diagnoses of cancer, so we know that they are not false-positives when they enter the study and the measurement criteria is not octreoscan.

Question: I did not ask that. I said a false-positive octreoscan is an index that the therapy with octreotide is going to be useful. Dr. Smith: Right, so the first thing is the octreoscan is not falsepositive because there is histologic proof that they have a tumor, then in monitoring that umor it is not a false-positive because we are not monitoring with octreoscan, we are monitoring with SWOG criteria and traditional radiologic criteria. Chairman: I think, Dr. Smith, it was the choice as to whether the patient was a good patient, because we will require that they have a positive octreoscan as an inclusion criterion. The question is, is there a chance we will make an incorrect assessment and let non-somatostatin-positive patients into the trial. I do not know the answers, but. Dr. Pauwels: I can take that question. When you talk about inflammation around the tumor or inside the tumor, that may be responsible for a very high uptake and therefore be misleading in patient selection for the treatment. There is a very nice example in lung cancers: non-small cell lung cancers. They have almost no receptors. And if you look carefully at the uptake you usually get a very low uptake and a ring uptake. A second example is neuroendocrine tumors that may have a lot of inflammation in case of necrotic tumors. If you do a SPECT investigation of a necrotic tumor in the center, you see no uptake or almost no uptake. So I think that you may have some uptake in case of inflammation related to the tumor, but the bulk of the uptake has nothing to do with inflammation. In the treated patient, as I showed you this morning, there was one case where we had the opportunity to do a follow-up and in parallel do a quantitative estimation of the octreoscan uptake versus volume determination, real volume determination. And we saw parallels between both measurements. So this indicates that if you express the uptake per gram of tissue during the decrease, the shrinkage, the uptake in grams of tissue remains constant. Dr. Smith: Another challenge I think is what difference does it make? I mean if those receptors that are taking up octreoscan are sitting on an inflammatory cell that is so intimately associated with the tumor that it cannot be distinguished from the tumor mass. Now you localize radiation which has a mean distance of 5 mm, you are going to hit some tumor cells. So I do not know whether I might take that drug. Chairman: One last question for Dr. Smith and then we will let him out of the lion’s then. Dr. Ahlman: I am also a little bit disappointed that you do not go where the action is, that is in neuroendocrine tumors. You see besides being a surgeon I also run a tumor biology laboratory and over the last 2 years we have made receptor subtype-specific profiles of the neuroendocrine tumors that we operate. That means abdominal tumors, medullary thyroid carcinomas, and we have included breast carcinomas. We look into the quantitative expression of somatostatin receptors, which I think is the most important thing. Comparatively, in relative terms in carcinoids the figures in MTC would be a factor of 4, and it would be 0.8 for breast carcinoma, and you chose to go for the breast carcinoma. We can explain what is happening here with the MTC tumors because 50% of the MTC tumors lack the subtype 2 receptor, that is the reason why they get a low uptake when you look at the profile here. I also completely agree with the previous speaker that it is a question about how much you can deliver into the tumor cell. Our group I think was the first to demonstrate that the neuroendocrine tumors also express the SSTR-2 receptor, which is internalized. Today that is common knowledge. But the three first patients it was demonstrated



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in was a patient with a histamine-producing foregut carcinoid, one glucagonoma and one mid-gut carcinoid. From this mid-gut carcinoid we have established a cell line which can be transplanted into nude mice, which is an excellent model to study biokinetics. It is not only what you have as tumor burden that you can deliver to the tumor, it is also a question of how much is taken up into the liver, into the kidneys, and into the bone marrow. The problem when you see the patients clinically is that they have huge bulky metastases. For me a serious question whether it is possible to deliver octreoscan to patients with huge bulky metastases. But I would really appreciate it if that were studied instead of breast carcinoma. Chairman: Why do we not try to go through the rest of the areas, Dr. Wiedenmann do you want to take us through. Dr. Wiedenmann: I have just a few short questions and short answers. Nigel Bax (Sheffield) asked why we have not discussed MIBG scanning in both a diagnostic as well as a therapeutic context. I think the answer is that the GEP tumors are of endodermal origin and not neuroectodermal origin, and by that fact you find hardly any synthesizing machinery for the catecholamines and for that reason MIBG does not get us anywhere in GEP tumors, both in diagnostics as well as in the therapeutic context. Then Dr. Toulis asked, why have we not considered thalidomide for the treatment of those hypervascularized tumors? I think that is a very valid question and I think a lot of people move those thoughts already in their heart, but I think no one really has developed a study in this direction. Then there is a question from Nigel Bax again, and he asked what we would do with a patient with a mid-gut carcinoid and hepatic metastasis but without a carcinoid syndrome. So it would be, as we just heard, a nonfunctional tumor and I think the answers have been given previously. Dr. Bax: Some answers have been given. Let me interrupt you. May I interrupt Chairman because I am Nigel Bax and that was my question. So may I prevent you going on any further and respond. Can I take MIBG to start with? I am aware, and someone I am sure will remember the paper, that patients with carcinoid syndrome benefited symptomatically but not in terms of carcinoid tumor size as much with nonlabelled MIBG as they did with 131I-labelled MIBG. So there is undoubtedly some evidence that there is an effect and I just wondered if an expert could comment on that. Dr. Wiedenmann: If you tackle it from the detail there. There are of course some exceptions where MIBG is positive. I am just talking about the majority of cases where MIBG is negative. I hope that you agree on that. And there are a few cases that are positive. Now the interesting thing is if we go into depth with that we know that the VMA transporter takes up all those biogenic amines, among them also the catecholamines. Now the crucial question is, once the MIBG works you lack the synthesizing enzyme to make a full blown catecholamine. It really depends on the enzyme machinery you have in the tumor cell and some of the tumor cells just contain the enzymes, and that is why you have positivity to generate the product that is eventually taken up by the physical monoamine transporter. But I think that is the exception in the GEP systems and I think for that reason it is not worthwhile using MIBG. But I agree with you, if we have some other biogenic amines available in a radiolabelled situation, then in my opinion, I think this represents a very interesting alternative to what we heard about the octreotide today. Dr. Öberg: I want to comment about the use of MIBG. As you are well aware there are several publications about using both hot and cold MIBG with quite similar results. The duration of response is a

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bit shorter with cold MIBG. This has mainly been published by a group in Rotterdam and we have tried to use it in a couple of our patients not responding to other means of treatment and we have been rather disappointed. So I think that there are perhaps some problems with the method as well. It can be done better in some centers than in others. But I agree with you that about 30% of the patients might respond biochemically but very few patients respond in terms of tumor reduction. Dr. Bax: Yes, I think that was right. There were some very sick patients included in that larger study with MIBG. They were towards the end of their life anyway and failed on other treatments. Could I just go back to the other point about the treatment of the nonfunctioning tumor? Just to say, not to prolong the conversation, that unless we do actually study this area properly and get a definitive answer, we may end up committing Health Services to very large amounts of money without any real justification. And certainly my own Health Authority would be wanting the evidence before they committed large amounts of money to treating people with nonfunctioning tumors. I think we do have an opportunity to do the study in that group of patients. We have a big enough cohort across Europe and perhaps ENET and I am sure UK-NET would be addressing that in the near future. Dr. Taal: I am the one who published the paper about the radioactive MIBG. And I would just mention here that indeed the unlabelled MIBG might be effective for a small subgroup of patients. But at this moment we are doing a new study and we have already published some interesting data about the combination of first giving the unlabelled MIBG and then changing the biodistribution, and then the radioactive MIBG will be more active, more radioactive-label compound actually comes into the tumor. We have published that just in some case reports in the European Journal of Gastroenterology, and we have recently submitted a paper with more patients. Dr. Ahlman: I think it is very interesting to bring up the MIBG binding in endocrine-producing tumors. We have some recent observations and I am quite sure Dr. Wiedenmann is aware that we have used Western blots to look at the expression of VMAT1 and 2. Unfortunately the tumors are very few, but among those tumors there were a few that were exclusively VMAT2-expressing and those were the histamine-producing tumors. If you gave MIBG to those patients, those patients who were still alive, there was a positive MIBG scan. And if you look at the mid-gut carcinoids they can express both the VMAT1 and the VMAT2 protein in approximately 30% of the cases. That is actually the exact figures that Dr. Kimish found when he treated patients with radioactive MIBG and one third of the cases responded to therapy. So our proposal is actually that the mode of action of the MIBG scintigraphy and therapy operates if the tumor expresses the VMAT2 protein. Chairman: Dr. Mignon, are there any burning questions in your set? Dr. Mignon: I have indeed several questions. The first one is from Dr. Tamborani (Rome). Why are pancreatic tumors in MEN1 less malignant than in sporadic ones and occur in younger people? The second part of this question has been answered already. I would like to give some suggestions, maybe I do not satisfy you but you can complete my answers. I would say that in our experience at least which is based on more than 70 cases of Zollinger-Ellison patients with MEN1, and I speak only of Zollinger-Ellison. In MEN1, as I and several of us already eluded to this morning, a critical point is the size of the tumor, more than 2 or 3 cm in sporadic cases, in contrast to small tumors. In addition in MEN1 most of the


tumors, but not all of them, are not located in the pancreas about 40% Zollinger-Ellison MEN1 patients have tumors in the pancreas. But for the other cases the tumors are located in the duodenal submucosa, and in the lymph nodes around the duodenal pancreatic area. So it is a possibility that small size is one explanation. Now it is possible of course, and I would like to have your opinion, that MEN1 patients, who are known to have a genetic defect because this has been published by the Uppsala group and we have also observed this, may secrete or certainly do secrete several hormones. And we know that they can express several clinical syndromes, for instance, both gastrinoma and insulinoma; we have 6 patients like that. So they might produce several hormones, and why could there not be among them some inhibitory factors, somatostatin for instance, or something like that which causes the tumor to be less malignant. As you have seen, and I finished my presentation this morning by saying that the stratification proposed by Dr. Jensen for the Zollinger-Ellison syndrome into benign and malignant is exactly what we have observed also in a larger series of patients, since now we have more than 280 cases. So this is the explanation I offer you, but I would be very pleased to have your own comments on this question. Chairman: Dr. Mignon, because of the time I am going to have to go on and have just one question for each of the remaining speakers. Dr. Pauwels: I think if I have to select I would take this one. When is the optimal timing for intervention with targeted radiopeptide therapy? In other words have we to perform it when the patient still has a stable disease or progressive disease, that is the way I understand the question. Definitely, all the trials that have been done so far have exclusively included patients with progressive disease. As you know, my position is if you are convinced that there is a very high uptake, then this is the best criteria for treatment. And I would be afraid to wait and see whether the patient becomes progressive and develops some metastases that no longer express the receptors. On the other hand you have to make a balance between safety and efficacy. So I think we should wait some time and look at the long-term affect on the kidneys because in rats it has been proven now that if you have a complete response, after a period of 2 years these rats start to lose weight and they all die, and when you do biopsies fibroses are found in the kidneys. All previous studies have addressed short-term renal dysfunction. So I would be very keen to treat patients at the stage of stable disease once we have at least, I would say, a few years of follow-up to see what happens to the kidney, with the patient presently included in the ongoing trials. Chairman: Dr. Rougier has gone but he has left an able assistant to answer one question. Dr. Eriksson: Well actually the question is not so much about chemotherapy but, are there any data on the possibility of using angiogenesis inhibition in the treatment of metastatic neuroendocrine tumors in relation to this hypersecretion? Of course angiogenesis is interesting in these tumors because of their hypervascularity. There are not any studies with specific antiangion genetic factors reported in the literature as far as I know, but we have been talking about interferon-· and somatostatin analogs which are supposed to induce or inhibit angiogenesis. Dr. Arnold: There are several questions: What are the universal criteria to define the progression of disease prior to initiating therapy? The WHO criteria new lesion and there are 25% increase and the answer is, yes. The more important question is, over what period of time should progression be followed before the start of treatment? I think this is an important question because this is not defined.

According to my view, the rapidly growing tumor would be a tumor with a 50% increase in volume, say within 6 or 8 weeks, or something like that. A very slowly growing tumor is, according to my view, a tumor which increases in volume of say 25–50% within 1 year, or longer. And everything in between is a medium fast-growing tumor. But this is not defined, absolutely not defined. For me a tumor which is very slowly growing, means it needs 1 year or longer for an increase of 50% in volume. I think this tumor, or this patient, should not be treated, either by radiotherapy or cytotoxic therapy. The second question: Is the symptom-relieving carcinoid syndrome correlated with a reduction in hormone levels? This is a question from Dr. Buchanan. I would say yes. Are chromogranin A and 5-HIAA equal during follow-up of Sandostatin treatment? I would say yes. In relation to the case report presented in my lecture, it is possible to imagine some role for antiestrogen treatment in neuroendocrine tumors. This is an interesting question, but there is, according to my knowledge, no literature. Can you tell me Dr. Öberg? Dr. Öberg: Yes, there was a Greek study 10 years ago indicating that tamoxifen had an effect on mid-gut carcinoid tumors. That study was then repeated by Dr. Moertel at Mayo Clinic 2 or 3 years later, and they could not prove any effect of antiestrogen treatment. Dr. Arnold: What is known about estrogen or progesterone receptors in endocrine pancreatic tumors? I have no idea. We cannot answer this question. Which first-line treatment would be recommended in poorly differentiated neuroendocrine tumors of the pancreas. My answer is if this is a small cell tumor, then I would recommend treatment as a small cell carcinoma of the lung. Dr. Öberg: I take one of the questions and it is from Dr. Nickinen from Finland. What is the optimal dose of interferon per day and how often, daily versus three times per week? Honestly we do not know exactly what the optimal dose is, but we have some ideas. When we did the trial on induction of 2–5 A) synthethase, we correlate the amount of induction to the leukocyte count in the patients, and when we reduced the leukocyte count down to 3.0 ! 109/l we had a good induction of 2–5 A) synthethase. So that might be a thing which can be of prognostic value for the selection of those patients responding to interferon-·. We try to optimize the treatment in our patients with regard to the leukocyte count. Try to get it down below 3.0 ! 109/l. We have come to the end and it has been a really interesting 2 days here in Innsbruck. Of course this meeting could not be carried out without a lot of help from different people. So I want to start off to thank many of the people who have been involved in this. To start with I thank the secretaries who have been very helpful to me in organizing the meeting and Guido told me to start with my own secretary, Lena Hultström. I would like to take the opportunity once more to thank all the speakers, most of the speakers belong to the ENET group. But there have been some other speakers who are not members of the group. I want to thank them once more. I want to thank Novartis for continuing to sponsor this group and for helping me to put together this meeting. It has been really nice working with you. And finally, I want to really thank Dr. Guido Nussbaumer. He has been a really good guy to work with and he knows how to organize meetings. So I will use him not only within Novartis but perhaps also in other things. Not to forget former product manager, Gary Cheng, for all his help in organizing this meeting. Thank you all so much for coming all this way to listen to us.



107

Author Index

Ahlman, H. 59 Arnold, R. 84 Bakker, W. 69 Benali, N. 27 Buscail, L. 27 Calender, A. 3 Chen, T. 69 Eriksson, B. 33 Faiss, S. 45 Ferjoux, G. 27 Friman, S. 59 Jamar, F. 69 Jansson, S. 59 Klose, K.-J. 39 Krenning, E. 69 Kvols, L. 69

Liu, J. 69 Malka, D. 79 Mignon, M. 51 Mitry, E. 73 Nilsson, O. 59 Öberg, K. 33, 92 Olausson, M. 59 Pauwels, S. 69 Puente, E. 27 Ricke, J. 39 Riecken, E.-O. 45 Rindi, G. 19 Rougier, P. 73 Ruszniewski, P. 79 Scherübl, H. 45 Schran, H. 69

Simon, B. 84 Smith, M.C. 69 Stölzel, U. 45 Stridsberg, M. 33 Susini, C. 27 Tylén, U. 59 Ubiali, A. 19 Valkema, R. 69 Villanacci, V. 19 Wängberg, B. 59 Wied, M. 84 Wiedenmann, B. 45 Yeh, C.-M. 69 Zimmer, T. 45

Subject Index

Biological function 27 Breast cancer 69 Cancer 3 Carcinoid 59 – tumors 73, 79 Chemoembolization 79 Chemotherapy 73 Chromogranin A 33 Clinical studies 69 Combination therapy 92 Cytotoxic treatment 73 Diagnostic imaging 39 Digestive endocrine tumors 79 DOTATOC 69 Electron microscopy 19 Endocrine cells 19 – pancreatic tumor 59 – tumor 51 Endoscopic ultrasonography 45 Expression 27

ABC Fax + 41 61 306 12 34 E-Mail [email protected] www.karger.com



Gastrinoma 45 Gastrointestinal neuroendocrine tumors 59 Gene 3 General tumor markers 33 Genetics 3 GEP tumors 84 Gut 19, 51

Natural history 51 Neuroendocrine enteropancreatic tumors, natural history 51 – GEP tumors 84, 92 – tumors 3, 39, 45, 69, 73, 84 Neuron-specific enolase 33 Neuropeptide K 33

Hepatic arterial embolization 59 Heterozygosity, loss 19 Hormones 19

OctreoTherTM 69 Octreotide 84, 92

Immunohistochemistry 19 Insulinoma 45 Interferon-alpha 92 Islet cell carcinoma 73, 79 Liver metastases 79 – transplantation 59 Long-acting somatostatin analogues 84 Medical treatment 84 Molecular genetics 19 Multiple endocrine neoplasia 3

Pancreas 19, 51 Peptide radiotherapy 69 Signal transduction 27 Small cell lung cancer 69 Somatostatin 27 – receptor 27, 69 – – scintigraphy 39 Specific tumor markers 33 Surgical treatment 59 Urinary 5-HIAA 33 Yttrium-90 69

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