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 neuroen-
`docrine tumors (NETs), the genes predisposing to multi-
`ple endocrine neoplasia (MEN) syndromes were identi-
`fied. In MEN1, tumors occur in the parathyroids, endo-
`crine 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 com-
`plex. 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 syn-
`dromes. MEN2 refers to the inherited forms of medullary
`thyroid carcinoma. The RET proto-oncogene, a tyrosine-
`kinase receptor, is activated by missense mutations oc-
`curring 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 be-
`long to the clinical pattern of Recklinghausen (NF1) and
`von Hippel-Lindau (VHL) diseases. The genes for both
`
`syndromes have been characterized and provide new
`pathways for endocrine tumorigenesis related to G-pro-
`tein 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 charac-
`terized. 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) dis-
`ease, Recklinghausen neurofibromatosis (NF1) and tuber-
`ous sclerosis (TSC). Other genetic syndromes character-
`ized by single or multiple endocrine tumors were identi-
`fied and mapped in the last decade, but the genes related
`to these diseases, such as Carney complex, non-MEN1
`familial isolated hyperparathyroidism (FIHPT), Conn ad-
`enoma, and pituitary tumors, remain to be identified.
`
`ABC
`Fax + 41 61 306 12 34
`E-Mail karger@karger.ch
`www.karger.com
`
`© 2000 S. Karger AG, Basel
`0012–2823/00/0625–0003$17.50/0
`
`Accessible online at:
`www.karger.com/journals/dig
`
`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 calender@univ-lyon1.fr
`
`Roxane Labs., Inc.
`Exhibit 1015
`Page 001
`
`

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`NET Related to Inactivation of a Growth-Suppressor
`Gene, MEN1
`MEN1 (OMIM 131100) is an inherited disease pre-
`disposing parathyroid hyperplasia/adenoma, pancreatic
`endocrine tumors, pituitary tumors, adrenocortical se-
`creting or nonfunctional tumors and thymic NETs [1, 2].
`Recent observations suggested that a MEN1 patient
`could also be affected by cutaneous or visceral (angio)li-
`poma 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 so-
`matic loss of heterozygosity (LOH) suggesting that devel-
`opment of MEN1-associated tumors was a two-step pro-
`cess, a germline mutation affecting the first MEN1 allele,
`and a somatic inactivation of the unaffected allele occur-
`ring 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 tis-
`sues tested, including the pancreas, thymus, adrenal
`glands, thyroid, testis, leukocyte, heart, brain, lung, mus-
`cle, 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 promo-
`tion [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 dele-
`tion 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 DNA-
`binding transmembrane or transactivation domains have
`been identified to date, even though protein structure
`software (PROSITE, SOPMA) predicted high hydropho-
`bicity in the NH2-terminal half of the protein, suggesting
`
`at least three leucine-zipper and helix structures in the pri-
`mary/secondary organization of menin [9, 10].
`The murine orthologue of human MEN, Men1, was
`mapped to the pericentromeric region of MMU19 (mu-
`rine 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 iden-
`tified [15–17]. In situ mRNA hybridization and Northern
`blot studies showed the pattern of Men1 expression dur-
`ing 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 interac-
`tion, 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 interac-
`tion with a co-activator, JAB1 [20]. Recently, it has been
`shown that menin-mediated repression of JunD transcrip-
`tional activity is relieved by a specific inhibitor of deace-
`tylase, 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
`
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`Fig. 1. Germline mutations in the
`MEN1 gene according to the menin–
`JunD binding domains. Most muta-
`tions were identified in French fami-
`lies 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 co-
`don 460; 1149del11 is a frameshift
`deletion beginning at nucleotide
`1449. ins = Insertions; splice-muta-
`tions are in italics. The underlined
`mutations and black boxes at the left
`of the gene represent relative hot-
`spots 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 se-
`quence. 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 muta-
`tions. Missense substitutions and in-frame deletions/
`insertions represented "20–25% of the mutations. In-
`tronic 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 reten-
`tion [28, 29]. Most mutations occurred once, while some
`of them were observed twice or more in apparently unre-
`lated 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 com-
`mon founder effect characterized by a single mutation
`and common haplotypes has been reported in four kin-
`dreds 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 slipped-
`strand 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 germ-
`line 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 correla-
`tions 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 pheno-
`types share truncating mutations. Secondly, MEN1-relat-
`ed 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
`
`fact that any patient with a MEN1 germline mutation
`might in the future develop pancreatic, pituitary and
`adrenal tumors [Calender et al., unpubl. observ.]. Fami-
`lies derived from a common ancestor and sharing com-
`mon mutations and haplotypes have heterogeneous clini-
`cal expression of the disease, an observation which as-
`sesses 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 muta-
`tions, 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 trun-
`cated 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 expres-
`sion levels, suggesting the upregulation of the wild-type
`allele. The second mechanism might concern the func-
`tional properties shared by internal domains of the pro-
`tein 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. Clini-
`cal 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) MEN1-
`related major lesions. Most families without MEN1 muta-
`tions display an atypical clinical pattern, with one or two
`lesion(s) affecting the proband and one major or uncom-
`mon 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 popula-
`tion, 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
`
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`could be affected by unknown mutations. Chromosomal
`or intragenic rearrangements such as large germline dele-
`tions, 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 prac-
`tice, genetic analysis is useful to assess syndromic diagno-
`sis of MEN1, but to date we still do not exclude the diag-
`nosis 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 encod-
`ing 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 hyperparathy-
`roidism (10–20%). In MEN2B (Gorlin’s syndrome) the
`major clinical features are MTC, pheochromocytoma, mu-
`cosal neuromas and skeletal abnormalities associated with
`a marfanoid habitus and ganglioneuromatosis of the gas-
`trointestinal 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 pheochro-
`mocytoma. C-cell hyperplasia is the earliest lesion ob-
`served in hereditary MTC and is characterized by abnor-
`mal basal and pentagastrin-stimulated calcitonin values.
`MTC related to a MEN2 genetic predisposition is multifo-
`cal. Pheochromocytomas in MEN2A/B are bilateral in
`around 70% of cases. Two major issues, malignant evolu-
`tion of MTC and cardiovascular failures due to latent
`pheochromocytoma, have to be considered for the progno-
`sis 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 calcium-
`dependent cell–cell adhesion [49]. The c-ret protein be-
`longs 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 li-
`gands and induce homodimerization of c-ret through the
`cysteine-rich region, thereby leading to the catalytic activa-
`tion 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 do-
`mains of dimerized RET and a signal-transducing complex
`consisting of Shc, Grb2 adapters and the subsequent acti-
`vation 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, neu-
`roendocrine 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. Nulli-
`zygote (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 mim-
`icked that of the human Hirschsprung disease character-
`ized 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 func-
`tion and suggest that c-ret protein might be critical for dif-
`ferentiation, 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 neu-
`roblastoma, medullary thyroid carcinoma and pheochro-
`mocytoma [61]. The RET gene has also been involved in
`sporadic papillary thyroid carcinoma (PTC) through chro-
`mosomal rearrangements producing various types of ab-
`normal 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]. MEN2A-
`associated mutations induce the formation of active RET
`dimers in the absence of ligand [66]. Transgenic mice
`expressing a Cys634Arg mutant RET allele under the con-
`trol of the calcitonin gene-related protein promoter devel-
`oped multifocal and bilateral MTC, as observed in hu-
`mans [67]. Transgenic animals do not display pheochro-
`mocytoma. 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 mu-
`tations, 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 mecha-
`nism leading to MTC must be related to a structural
`change in the catalytic site leading to constitutive activa-
`tion 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 muta-
`tion results in a methionine to threonine conversion
`(Met918Thr) and has been associated with an aggressive
`course of MTC. The MEN2B mutation switches the sub-
`strate specificity of the RET TK domain towards intracel-
`lular 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.
`
`Germline RET mutations were found in 3–5% of sporad-
`ic 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 pene-
`trance of pheochromocytoma, and one might suggest sim-
`plifying 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 pheochromo-
`cytoma, 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/tu-
`mors [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 spe-
`cific mutations in the RET sequence, all patients with iso-
`lated MTC must be considered as a potential MEN2A and
`the diagnosis of pheochromocytoma excluded. Retro-
`spective data on 274 MEN2A cases registered in Euro-
`pean countries have shown that pheochromocytoma oc-
`curred 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 predis-
`posing to renal cancers, retinal and/or cerebellar heman-
`gioblastoma, pheochromocytoma and cystic and/or endo-
`crine pancreatic tumors [91]. VHL-related pancreatic tu-
`mors 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 pro-
`to-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 pro-
`teins 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 local-
`ization which might account for new tumorigenic path-
`ways related to VHL mutations. Histopathological data
`suggested that VHL-related dNET have typical neuroen-
`docrine characteristics, most are multiple and nonfunc-
`tional 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 nonfunction-
`al tumors and somatostinoma; (3) a clear-cell morphology
`related to intracytoplasmic lipid and myelin accumula-
`tion, and (4) the frequent occurrence of microcystic ade-
`noma 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 pathogene-
`sis. 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 adeno-
`ma [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 glan-
`dular histological features and a common immunohisto-
`chemical 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 G-
`protein-mediated activation signal of the intracellular cas-
`cade. Neurofibromin, the product of NF1, acts as a nega-
`tive regulator of ras-related G proteins by disrupting the
`GTP-ras complex [105]. TSC2 encodes tuberin, a GTPase-
`activating 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
`
`Molecular Genetics of Neuroendocrine
`Tumors
`
`Digestion 2000;62(suppl 1):3–18
`
`9
`
`Roxane Labs., Inc.
`Exhibit 1015
`Page 007
`
`

`
`to the genetic predisposition to NETs. Deregulation of
`mitotic and/or transcription factor activity, TK mem-
`brane signalling, transcription elongation and/or angio-
`genesis and small G-protein pathways have been involved
`as basic mechanisms of tumorigenesis. Nevertheless,
`MEN1 remains the most common form of genetic predis-
`position to NETs, and one would expect that disruption of
`menin function is a critical event leading to neuroendo-
`crine cell proliferation. Tumorigenesis in MEN1 involved
`loss of function of menin, a growth suppressor according
`to the two-hit model by Knudson [8]. Nevertheless, sever-
`al 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 prolifer-
`ative tumors occur later in life as monoclonal prolifera-
`tions derived from a single ancestor cell [110]. Recent
`studies on gastrinomas have assessed the fact a monoclon-
`al tumor derived from hyperplastic tissues was further
`responsible for multifocality and extrapancreatic dissemi-
`nation [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]. Sec-
`ondly, lymphoblastoid or fibroblastic cell lines derived
`from MEN1 patients show a low rate of chromosomal
`instability [113]. Alterations include abnormal rings, dele-
`tions, 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 evi-
`dence of premature centromere division in growing fibro-
`blasts 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 bFGF-
`like activity is due to the presence of anti-bFGF-circulat-
`ing autoantibodies [116]. The decrease in bFGF activity
`after surgical therapy of pituitary adenoma suggested that
`this mitogenic factor could be produced by the hyperplas-
`ic or tumoral pituitary gland, but these data have not been
`confirmed to date. Lastly, MEN1 also predisposed to non-
`endocrine tumors cosegregating with MEN1 in some fam-
`ilies. This suggests that an alteration in menin function
`increases the relative risk or susceptibility to various can-
`cers, and an exciting hypothesis might be proposed on a
`relation between MEN1 gene inactivation and an in-
`
`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 re-
`sponse to endogenous or exogenous growth fac